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Gamete Transport and Fertilization

Richard E. Jones PhD, Kristin H. Lopez PhD, in Human Reproductive Biology (Fourth Edition), 2014

Sperm Passage through the Cumulus Oophorus

The ovulated ovum is surrounded by the cumulus oophorus, which is a sphere of loosely packed follicle cells (Figure 9.4). Appropriately, cumulus oophorus means “egg-bearing little cloud.” As a sperm enters the cumulus oophorus, the enzyme hyaluronidase on the sperm head dissolves hyaluronic acid, a major component of the cementing material found between the cells of the cumulus oophorus as well as between other cells in the body. Enzymatic dissolution of hyaluronic acid allows the swimming sperm to penetrate the spaces between cells of the cumulus oophorus and to reach the zona pellucida.

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Toxicologic pathology of the reproductive system

Pralhad Wangikar, ... Subrahmanyam Vangala, in Reproductive and Developmental Toxicology, 2011

Ovary

The ovaries during diestrus show increased numbers of large follicles with a single antral cavity filled with follicular fluid. Increased numbers of atretic follicles are also seen during this phase. Currently formed corpora lutea from previous ovulation which attends the maximum size is the characteristic marker of diestrus. The luteal cells show foamy, eosinophilic cytoplasm and infiltration of fibrous tissue may also be seen at this stage. During proestrus graffian follicles are present at the surface area of the ovary. Most of the follicles are without the cumulus oophorus. The corpora lutea shows degenerative changes characterized by vacuoles in the cytoplasm and increased apoptotic cells. The fibrous tissue formation is evident at the central part. During estrus the degenerating follicles show apoptotic granulosa cells. Both newly formed and degenerating corpora lutea are seen at this stage. Newly formed corpora lutea shows basophilic, small and spindle-shaped luteal cells with capillary formation. The central fluid-filled cavity retained from the follicular stage is seen occasionally. Previously formed corpora lutea shows advanced degenerative process including fibrosis. During metestrus many growing follicles along with atretic follicles are seen. The newly formed corpora lutea contains the fluid-filled cavity of various sizes; the luteal cells are still basophilic with large nuclei. Previously formed corpora lutea shows advanced fibrosis.

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Reproductive and Endocrine Toxicology

A.R. LaBarbera, in Comprehensive Toxicology, 2010

11.17.6.3 Fertilization and Implantation

It is necessary for the gametes to be delivered to the fallopian tubes in a fertilizable state. In addition to causing, directly or indirectly, a resumption of meiosis in the oocyte, the LH surge causes the cumulus oophorus cells surrounding the oocyte to secrete hyaluronic acid, which results in cumulus expansion in order to facilitate penetration by spermatozoa. After ovulation, the cumulus–oocyte complex is transported along the surface of the ovary and through the ostium, a process that requires several minutes and is facilitated by the beating of the cilia lining the fimbria. Fertilization, the fusion of a spermatozoon and an oocyte arrested in the second meiotic division, takes place in one of the fallopian tubes (Figure 8).

Figure 8. Ovulation, fertilization, and implantation. 1, metaphase II oocyte with surrounding cumulus; 2, fertilization; 3, two pronuclear stage zygote; 4, first mitotic division; 5, two-cell preembryo; 6, morula; 7, late morula; 8, blastocyst; 9, implantation. From Langman, J. Medical Embryology; Williams and Wilkins: Baltimore, MD, 1975; p 30.

Spermatozoa are ejaculated into the vagina near the external os of the cervix during coitus, which usually occurs within 10 min of intromission. The alkaline semen buffers the acidic (pH 5) vaginal fluid; this facilitates capacitation and hyperactivation, during which sperm become hypermotile. The seminal plasma coagulates, keeping the sperm in the cervix until they become hypermotile; fibrinolysin from the prostate in the ejaculate causes liquefaction within 20–30 min; maximum motility is achieved in about 1 h. Sperm migrate through the cervical mucus and travel about 2–3 mm min−1, aided by uterine contractions; the first sperm reaches the fallopian tube 5 min after ejaculation. Less than 200 spermatozoa are present in the fallopian tube at one time; they can remain intact for 24–60 h.

Contractions of the oviductal muscles direct the cumulus–oocyte complex into the ampulla of the fallopian tube; it remains there for 3 days; the ampullary–isthmic sphincter remains contracted. Oocytes remain fertile for 15–18 h after ovulation; sperm remain fertile for 24 h after ejaculation. Hyaluronidase on the outer surface of the acrosome of the spermatozoon facilitates migration through the cumulus–oocyte complex. When a spermatozoon encounters the zona pellucida, it undergoes the acrosome reaction. This reaction, which requires high calcium, entails breakdown of the acrosomal membrane. Acrosin, a proteolytic enzyme, is released, permitting penetration of the spermatozoon through the zona pellucida. The sperm head membrane binds to the sperm receptor, which is followed by fusion with the oolemma. Microvilli on the oocyte surface surround the sperm head and the oocyte undergoes the cortical reaction in which cortical granules are mobilized. The zona pellucida hardens and no other spermatozoa can penetrate the oolemma. The oocyte nucleus completes maturation to yield the female pronucleus and the second polar body; the sperm nucleus forms the male pronucleus.

The zygote is kept in the fallopian tube for about 3 days due to spastic contractions of the estrogen-dominated isthmus; as progesterone increases, muscle tone decreases. In the fallopian tube, the zygote undergoes cleavage division (1 cell to 8 cell), compaction, and blastocyst formation. The inner cell mass becomes the fetus and the outer cells become the placenta and fetal membranes.

Approximately 7 days after fertilization, the blastocyst bursts from the zona pellucida, termed hatching, and implants in the wall of the uterus, termed nidation. Implantation occurs only in an endometrium previously conditioned by progesterone during the luteal phase of the menstrual cycle. Chorionic gonadotropin from the blastocyst stimulates the corpus luteum to continue secreting progesterone. Progesterone antagonists such as RU486 block implantation. The blastocyst attaches at the embryonic pole to the wall of uterine fundus; trophoblast cells, aided by proteolytic enzymes, invade through endometrial epithelium into the endometrial stroma. Stromal cells enlarge and become transcriptionally active, termed decidualization, and surround the blastocyst.

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Systems Toxicologic Pathology

Daniel G. Rudmann, George L. Foley, in Haschek and Rousseaux's Handbook of Toxicologic Pathology (Third Edition), 2013

2.1 The Ovary

The surface of the ovary is covered by a single layer of mesothelium (germinal epithelium). Depending on its location, this specialized peritoneal mesothelium may be squamous, cuboidal, or columnar. Beneath the mesothelium is a thin layer of connective tissue called the tunica albuginea or lamina propria. The parenchyma below the tunica albuginea can be divided into two poorly demarcated zones. The outer zone, or cortex, contains the oocytes, follicles, corpora lutea, interstitial glands, and other glandular structures embedded in a highly cellular compact stroma (Figure 60.1). The inner zone, or medulla, contains larger blood and lymph vessels, interstitial glands, and rudimentary epithelial structures, such as rete ovarii and medullary cords, in loosely arranged connective tissue. Nerves and blood vessels enter the ovarian parenchyma at the center of the median pole or hilus.

FIGURE 60.1. Ovary, rat, normal ovary. Numerous corpora lutea and follicles give the ovary a grape-like appearance. 15×, H&E.

In healthy sexually mature animals, follicles in different stages of development are present. Most of these will undergo atresia sometime during maturation. The primordial follicle (Figure 60.2) is often located immediately beneath the tunica albuginea. It is the least developed follicle, and consists of an oocyte surrounded by a single layer of squamous epithelial (follicular) cells. It represents the resting stage of the oocyte, and is present during fetal life. The first stage of follicular growth is the recruitment and development of a cohort of primordial follicles into primary follicles. As the oocyte develops, it rapidly increases in size from approximately 15 μm to 100 μm in diameter and there is a proliferation and transformation of follicular cells, from flattened to columnar, surrounding the oocyte. The next phase of follicular growth consists of further proliferation and differentiation of the single layer of follicular cells into multiple layers of granulosa cells and the formation of the zona pellucida, a glycoprotein coat surrounding the oocyte. The follicle is now designated as a secondary follicle (Figure 60.2). As the follicle continues to grow, multiple fluid-filled spaces appear, and it is now called a vesicular follicle. When the cystic spaces become confluent, forming a single large space called an antrum, the follicle is called a tertiary follicle. At this stage, the oocyte with attached granulosa cells becomes eccentrically located in the follicle; the mass of granulosa cells enclosing the oocyte projects into the antrum forming a hillock called the cumulus oophorus (Figure 60.2). The granulosa cells surrounding the oocyte are called the corona radiata and the fluid in the antrum, the liquor folliculi. The term “Graafian follicle” or preovulatory follicle is used to denote a tertiary follicle during preovulatory growth.

FIGURE 60.2. Ovary, rat, normal follicle maturation. Primary (∗) and secondary (∗∗) follicles (A); tertiary follicles (B); and a Graafian follicle without (C) and with (D) a central oocyte. 100×, (B) 50×, (C and D) 75×. H&E.

During follicular development, the stromal cells encapsulating the follicle also undergo morphological changes. The elongated fibroblast-like stromal cells which form concentric layers around the developing follicle are called theca cells (Figure 60.2). Since the granulosa cells of a follicle are avascular, they rely on the vasculature of the theca. At later stages of follicular development, the theca is further divided into the theca interna and theca externa. Cells in the theca interna become polygonal in shape, with vacuolated cytoplasm and vesicular nuclei. These cells further hypertrophy as proestrus approaches, and are believed to be the major site of sex steroid production. The cells of the theca externa maintain their fibroblast-like morphology. The theca externa contains contractile elements that are believed to assist in the process of ovulation.

Degeneration of the follicle, known as atresia, occurs most commonly with 200- to 400-μm diameter follicles. Consequently, the appearance of atretic follicles varies depending on the stage of development at which atresia occurs. The earliest noticeable changes, including nuclear pyknosis and karyorrhexis, frequently occur in, but are not limited to, the granulosa cells immediately adjacent to the lumen in tertiary follicles and just external to the corona radiata of vesicular follicles. In the tertiary follicle, necrotic cells slough from their attachment, resulting in a greatly reduced cumulus oophorus; this sloughing allows the ovum, covered by the zona pellucida, to float in the follicular liquor. The zona pellucida and ovum also undergo degenerative and necrotic changes followed by complete degeneration, necrosis, and disappearance of granulosa cells. There is no inflammatory response during the process of atresia. Atresia of follicles at earlier stages of development is not as conspicuous as that of vesicular and tertiary follicles, due to their relatively small size.

As mentioned previously, only a limited number of developed follicles reach the stage of preovulatory follicles and ovulate. After ovulation, both the thecal cells and the retained granulosa cells luteinize and become the luteal cells of the newly formed corpus luteum (Figure 60.3). Histologically, luteinization is characterized by both hyperplasia and hypertrophy of luteal cells, and by vascular proliferation. This often results in a corpus luteum larger than the mature follicle. Changes observed in the cytoplasm include an initial increased acidophilia, followed by a foamy appearance and, finally, by an accumulation of vacuoles.

FIGURE 60.3. Ovary, rat, corpora lutea (CL) types. (A) Normal eosinophilic (Eos) CL, (B) mixed esosinophilic and basophilic CL, and (C) basophilic (Baso) CL. 50×, H&E.

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Embryo Transfer and Other Assisted Reproductive Technologies

Henrik Callesen, ... Torben Greve, in Veterinary Reproduction and Obstetrics (Tenth Edition), 2019

Technique

OPU is generally performed in cattle under epidural anaesthesia (occasionally combined with sedation) and in mares after sedation and analgesia. Basically, the technique requires a 5 or 7.5 MHz transducer incorporated into a needle guide that can be inserted into the vagina of a cow or mare. Various models of needle guide and aspiration system have been developed, often based upon the different available ultrasound machines. The ovary is grasped transrectally by the hand and gently pulled caudally towards the transducer head (Fig. 44.16). When a suitable follicle is positioned correctly in relation to the monitor's puncture line, an aspiration needle is inserted into the follicular cavity. Its content is aspirated and the follicular cavity is curetted and/or flushed several times with modified PBS to create turbulence (Pieterse et al. 1991, Brück et al. 1992).

Within the follicular cavity, the oocyte is surrounded by many layers of cumulus cells, which together form the so-called cumulus oophorus. Once detached from the follicular wall, the oocyte surrounded by cumulus cells is termed the cumulus oocyte complex (COC). Flushing the follicular cavity requires that the follicle's diameter is at least 5 mm in order to avoid the COCs being flushed backwards and forwards within the dead space of the aspiration needle; this will result in a considerable decrease in the number of cumulus cells surrounding the oocyte (Brück et al. 1997). Alternatively, a double lumen needle may be used (Bracher et al. 1993, Cook et al. 1993, Duchamp et al. 1995). Oocyte recovery rates improve with increasing needle diameter, bevel length, and vacuum pressure, but the latter also increases the proportion of denuded oocytes (Bols et al. 1997, Fry et al. 1997). Because fertilisation rates and subsequent developmental competence of denuded oocytes are reduced (Tanghe et al. 2002), a compromise in the vacuum pressure must be sought.

Recovery rates vary with species, individual animals, follicular maturity, and experience of the person performing the aspiration (Merton et al. 2003). In cattle, twice-weekly follicular aspirations at 3- to 4-day intervals prevent a dominant follicle being selected, and this has proved to be an efficient regimen for repeated follicular aspirations with a yield of two to eight COCs from each aspiration session (Merton et al. 2003, Kruip et al. 1994, Broadbent et al. 1997, Garcia & Salaheddine 1998). In mares, oocyte recovery rates from subordinate follicles are approximately 30% to 50% and 70% to 80% from preovulatory follicles after hCG treatment (reviewed by Hinrichs 2016 and Bøgh 2003).

In the early OPU studies in cattle, no hormonal stimulation was used, but subsequent studies have shown that pretreatment with FSH over 2 to 3 days, followed by follicular aspiration 1.5 to 2 days after the last FSH treatment, the so-called ‘coasting’, may yield a larger number of oocytes with improved developmental competence (Blondin et al. 2012). Today, in some commercial settings, often depending on practical and economical considerations, hormonal pretreatment is routinely used.

Transvaginal oocyte recovery can be performed also on pregnant cows and mares as long as the ovaries are still accessible by rectal palpation, i.e., during the first trimester, with no effect on the ongoing pregnancy (Meintjes et al. 1995a, Meintjes et al. 1995b, Goudet et al. 1998, Cochran et al. 2000, Chastant-Maillard et al. 2003).

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Reproductive Physiology of Male Animals

Timothy J. Parkinson, in Veterinary Reproduction and Obstetrics (Tenth Edition), 2019

Anatomy of the Male Reproductive System

Testis, Spermatic Cord, and Scrotum

The testes of all domestic male animals are located at the inguinal region within a scrotum. In the bull and the ram this structure is pendulous and has an elongated neck**,** but in most other domestic species the scrotum is closely applied to the inguinal region. The scrotum consists of a skin pouch overlying various fibroelastic and muscular layers, of which the most prominent is the tunica dartos (Fig. 2.2). The dartos layers are confluent between the testes, where they form the intertesticular septum. In the boar the external spermatic fascia is also prominent. The testis itself is surrounded by two layers of peritoneum, which are formed during its descent into the scrotum, as a single outpouching of the parietal peritoneum, through the inguinal canal. The outer layer of peritoneum, the processus vaginalis (tunica vaginalis reflexa), is reflected on to the testis to form the serous outer layer of that organ, the tunica vaginalis propria. Accompanying this outpouching of peritoneum through the inguinal canal is a diverticulum of the internal abdominal oblique muscle, which inserts on to the cremasteric fascia and the vaginal tunics. This muscle, the cremaster, raises or lowers the testis in response to the immediate environmental temperature or noxious stimuli.

The capsule, or tunica albuginea, of the testis is composed principally of fibrous tissue but has a smooth muscle component whose function is largely unknown. Overlying the capsule is the tunica vaginalis propria. The main blood vessels of the testis are distributed over the surface of the tunica albuginea, before penetrating the capsule to supply the testicular parenchyma; the innervation of the testis is mainly confined to the periphery, and little nervous tissue is found in its substance. The substance of the testis (Fig. 2.3) is composed of two main tissues: seminiferous tubules and interstitial tissue. Each seminiferous tubule is a highly convoluted, unbranched tube that opens at both ends into collecting tubules and then into the rete testis. The seminiferous tubules are limited by a basement membrane, which is partially surrounded by contractile myoid cells. Within the tubule the seminiferous epithelium is composed of two main cell lines: somatic Sertoli cells and the sperm-producing germinal cell lines. Interstitial tissue, which consists of steroid-producing Leydig cells, blood vessels, and lymphatics, exhibits much variation in its quantity and morphology between species. For example, Leydig cells in the ram occur in small clusters around blood vessels, interspersed in relatively large lymphatics, whereas the interstitial tissue of the boar has densely packed Leydig cells and small lymphatics (Fawcett 1973).

At the ends of each seminiferous tubule, there is a transitional zone between spermatogenic tissue and the rete testis. In ungulates and carnivores the rete testis is in the centre of the testis within the fibrous matrix of the mediastinum, although its position differs in rodents and primates. The number of seminiferous tubules opening into each of the transitional zones varies between species, as does the number of channels comprising the rete (Setchell & Breed, 2006). The rete testis consists of a network of interconnecting channels with a simple epithelial lining that moves the sperm into the epididymis. The rete opens into the epididymis via 13 to 20 efferent ducts (Hemeida et al. 1978). These tubules are initially relatively straight but have a long convoluted section before opening into the epididymal duct. Fluid resorption occurs in both rete testis and efferent ducts.

The epididymis is a single, highly convoluted tube into which the vasa efferentia drain the seminiferous tubules. Grossly, the epididymis appears as an approximately cylindrical organ, which is divided into a prominent head that is situated close to the suspension of the testis from the spermatic cord, a smaller, medially situated body, and a distended tail that is continuous with the ductus deferens (vas deferens). The muscular wall of the epididymal duct moves sperm through its lumen by peristalsis so that, during their passage through the epididymis, sperm, which are immature on release from the testis, undergo final maturation. The tail of the epididymis also acts as a reservoir for fully mature sperm, which becomes turgid with the accumulation of stored sperm in sexually active animals. In domestic animals the epididymis is closely applied to the exterior of the testis, whereas in rodents and primates the connection is much looser.

The vas deferens is a relatively thick-walled, muscular tube that acts as both a reservoir for sperm and the means of their conduction between the epididymis and the penis. It is situated mediocaudally within the spermatic cord, in a small diverticulum of peritoneum. In addition to the vas deferens the spermatic cord also contains the arteries, veins, and nerves supplying the testis, all of which are contained within the peritoneal vaginal tunics. Together, these structures form the spermatic cord. The spermatic sac includes the spermatic cord, the internal spermatic fascia, cremaster muscle, and cremasteric fascia. The cremaster muscle is situated on the opposite side of the sac to the vas deferens (i.e., on the cranio–lateral surface). The vasa deferentia enter the abdomen through the inguinal canals, from where they run in a caudal direction to join the pelvic urethra where the latter organ joins the neck of the bladder.

A number of short ligaments exist between the various structures within the scrotum, as shown in Fig. 2.4. The proper ligament of the testis joins the ventral pole of the testis to the tail of epididymis, which is also joined to the vaginal tunic by the caudal ligament of the epididymis. These ligaments are derived from the gubernaculum. Finally, on the external surface of the vaginal tunic, the scrotal ligament joins the tunic to the scrotal fascia.

Blood and Nervous Supply to the Testis

The testes are supplied with blood through the spermatic arteries, which arise from the caudal aorta close to the renal arteries. In the domestic species these arteries pass through the inguinal canal, enclosed in peritoneum, forming a major component of the spermatic cord. In animals with scrotal testes the spermatic artery becomes highly convoluted from the point at which it passes through the inguinal canal, although the degree of convolution is less if the scrotum is inguinal than if pendulous. In the bull the spermatic artery in the spermatic cord is approximately 5 m in length (Setchell & Breed 2006). The artery does not divide until it reaches the testis (Setchell & Breed 2006), at which point it divides into major distributing arteries that run over the surface of the testis before descending into its parenchyma.

The testis is drained by an anastomosing plexus of veins (the pampiniform plexus) that arise in the tunica albuginea and return to the spermatic cord through the inguinal canal and thence to the caudal vena cava. Initially, many veins are identifiable in the plexus, but as the plexus ascends the spermatic cord, fewer and fewer collateral branches are apparent until a few main veins penetrate the inguinal canal. These finally join, as a single vein, with either the caudal vena cava or renal vein (Setchell & Breed 2006). In the bull, veins of different sizes are present in the plexus: large veins running parallel to each other and surrounding the spermatic artery, smaller veins with a less organised architecture, and tiny veins in or closely applied to the walls of the spermatic artery. All these veins communicate with one another (Hees et al. 1984), and there is also evidence of arteriovenous anastomoses within the spermatic cord.

The spermatic artery is therefore in very intimate contact with the pampiniform plexus. This complex vascular anatomy fulfils several functions. The extension of the length of the spermatic artery results in the arterial pulse being almost completely eliminated by the time the artery reaches the testis (Waites & Moule 1960). In the rat (Maddocks & Setchell 1988) the pulse is reduced from 34 mm Hg at the proximal end of the spermatic cord to 6 mm Hg at the testicular end. It appears that a pulsatile arterial blood supply to the testis is incompatible with normal spermatogenesis. Second, spermatogenesis is more efficient at temperatures lower than the mammalian core body temperature. The close apposition of artery and veins allows heat exchange to occur between spermatic artery and vein such that the temperature in the testis is several degrees lower than the core body temperature. Third, it is possible that some countercurrent exchange of small molecules such as testosterone may occur between spermatic vein and artery, although the importance of such transfers remains to be established.

The nervous supply of the testis is derived from the thoracolumbar sympathetic outflow, whose visceral motor fibres innervate the smooth muscle of the testicular arterioles and of the testicular and epididymal capsules. These fibres and their accompanying visceral sensory fibres run in the spermatic cord. The scrotum has both visceral and somatic innervation, which is derived from nerves that pass through the inguinal canal, and arise as branches of the pudendal nerve. A further prominent feature of the innervation of the scrotum is the motor supply to the cremaster muscle and dartos. However, as might be expected from the interspecies variation in anatomy of the scrotum, there is also considerable variation in the detail of its nervous supply.

Accessory Glands

The accessory glands include the ampullae, prostate, vesicular glands, and bulbourethral (or Cowper's) glands. There is much variation between the anatomy of the accessory glands in the different species, which is summarised in Table 2.1.

Structure and Function of Spermatozoa

Spermatozoa are divided into three main segments: the head, midpiece, and tail (Fig. 2.5). The head consists of little other than the condensed nucleus and the overlying acrosome. Of the other enzymes contained within the acrosome, the main two are acrosin and hyaluronidase. During the acrosome reaction the outer acrosomal membrane fuses with the sperm membrane, under the control of intra- and extracellular calcium, whereupon exocytosis of the contents of the acrosome occurs (Harrison & Roldan 1990). The main functions ascribed to the acrosomal enzymes are dispersal of the cumulus oophorus and local lysis of the zona pellucida. The inner membrane of the acrosome is relatively stable and remains intact after the acrosome reaction has occurred; some of the acrosomal enzymes are probably bound to the inner acrosomal membrane (Ferrer et al. 2012). Penetration of the zona pellucida and fusion with the oolemma are both receptor-mediated events, with specific areas of the sperm head binding to target components of the oocyte (Wassarman 1990). The classical proposed sequence of acrosome reaction and fertilisation is that the acrosome reaction is initiated in the cumulus oophorus and that the action of the acrosomal enzymes plus the hypermotility of the capacitated sperm propels the sperm through the cumulus until it binds with the zona pellucida (Jin et al. 2011). However, there is evidence for zona-induced acrosome reaction in some species, whereas in others, induction of the acrosome reaction by the cumulus, rather than by the zona, may be required for successful fertilisation (Gadella 2013). Moreover, there is now evidence that acrosin, apart from any role it has in dispersal of the cumulus or zona, is also involved in the binding between the sperm head and the oolemma (Mao & Wang 2013).

The midpiece and tail of the sperm may be considered to form a single functional entity. The tail itself consists of a central axoneme, which, in the region of the midpiece, is sheathed in a helix of mitochondria (Bedford & Hoskins 1990, Amaral et al. 2013). Sperm metabolise simple molecules, principally sugars and their derivatives (e.g., fructose, glucose, mannose, and pyruvate), by both aerobic and anaerobic pathways to provide energy for motility and the maintenance of ionic gradients across membranes (Florman & Sulcibella 2006). Forward motility of sperm results from coordinated waves of flagellar bending progressing from the neck along the length of the tail. Bending of the tail occurs as the result of forces generated between adjacent peripheral doublets of the axoneme (Satir et al. 1981). The dynein arms of the doublet, which in the resting state are bound to the adjacent doublet, unbind, elongate, and then bind to a new site further along the filament. The unbinding process, which is the adenosine triphosphate (ATP)-using step, is then repeated, resulting in a progressive bending of the flagellum. The doublets on one side of the axoneme work in opposition to each other, providing the alternating beat of the tail. After capacitation the rate and amplitude of the flagellar beat greatly increases, and the rate of energy usage by the sperm is correspondingly elevated (Florman & Dulcibella 2006). The motility of the cell itself probably has little role in the movement of spermatozoa through the cervix and uterus, for this is accomplished mainly through contractions of the female genital tract (Hunter 1980). However, passage through the uterotubal junction and within the oviduct does require sperm motility, whereas the enhanced, whiplash motility of the capacitated sperm is necessary for penetration of the cumulus and zona pellucida.

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Hormonal Control of Reproduction in the Female

H. Maurice Goodman, in Basic Medical Endocrinology (Fourth Edition), 2009

Female Reproductive Tract

Ovaries

The adult human ovaries are paired, flattened ellipsoid structures that measure about 5 cm in their longest dimension. They lie within the pelvic area of the abdominal cavity attached to the broad ligaments that extend from either side of the uterus by peritoneal folds called the mesovaria. Both the gamete producing and hormone producing functions of the ovary take place in the outer or cortical portion. It is within the ovarian cortex that the precursors of the female gametes, the oocytes, are stored and develop into ova (eggs). The functional unit is the ovarian follicle, which initially consists of a single oocyte surrounded by a layer of granulosa cells enclosed within a basement membrane, the basal lamina, that separates the follicle from cortical stroma. When they emerge from the resting stage, follicles become ensheathed in a layer of specialized cells called the theca folliculi. Follicles in many stages of development are found in the cortex of the adult ovary along with structures that form when the mature ovum is released by the process of ovulation. Ovarian follicles, in which the ova develop, and corpora lutea derived from them are also the sites of ovarian hormone production. The inner portion of the ovary, the medulla, consists chiefly of vascular elements that arise from anastomoses of the uterine and ovarian arteries. A rich supply of unmyelinated nerve fibers also enters the medulla along with blood vessels (Figure 13.1).

Figure 13.1. Drawing of a human ovary showing the progression of the various stages of follicular and luteal development. (From Netter, F.H. (1997) Atlas of Human Anatomy, 2nd ed., plate 349. Novartis, Hanover, NJ.)

Folliculogenesis

In contrast to the testis, which produces hundreds of millions of sperm each day, the ovary normally produces a single mature ovum about once each month. The testis must continuously renew its pool of germ cell precursors throughout reproductive life in order to sustain this rate of sperm production, whereas the ovary needs to draw only upon its initial endowment of primordial oocytes to provide the approximately 400 mature ova ovulated during the four decades of a woman’s reproductive life. Although ovulation, the hallmark of ovarian activity, occurs episodically at 28-day intervals, examination of the ovary at any time during childhood or the reproductive life of a mature woman reveals continuous activity with multiple follicles at various stages in their life cycle.

Folliculogenesis begins in fetal life. Primordial germ cells multiply by mitosis and begin to differentiate into primary oocytes and enter meiosis between the eleventh and twentieth weeks after conception. Primary oocytes remain arrested in prophase of the first meiotic division until meiosis resumes at the time of ovulation, and is not completed until the second polar body is extruded at the time of fertilization, which may be more than four decades later for some oocytes. Around the twentieth week of fetal life there are about 6 to 7 million oocytes available to form primordial follicles, but the human female is born with about only 300,000 to 400,000 primordial follicles in each ovary. Oocytes that fail to form into primordial follicles are lost by apoptosis, and many primordial follicles are also lost during fetal life in a process called atresia. The vast majority of primordial follicles remain in a resting state for many years. By some seemingly random process, perhaps because they are relieved of inhibition or are activated by still unknown factors, some follicles enter into a growth phase and begin the long journey toward ovulation, but the vast majority become atretic and die at various stages along the way. This process begins during the fetal period and continues until menopause at around age 50, when all the follicles are exhausted.

As primordial follicles emerge from the resting stage the oocyte grows from a diameter of about 20 μm to about 100 μm and a layer of extracellular mucopolysaccharides and proteins called the zona pellucida forms around it (Figure 13.2). Granulosa cells change in morphology from squamous to cuboidal. This early step in the process is estimated to require more than 120 days. Growth of primary follicles is accompanied by migration and differentiation of mesenchymal cells to form the theca folliculi. Its inner layer, the theca interna, is composed of secretory cells with an extensive smooth endoplasmic reticulum characteristic of steroidogenic cells. The theca externa is formed by reorganization of surrounding stromal cells. At this time also a dense capillary network develops around the follicle. The oocyte completes its growth by accumulating stored nutrients and the messenger RNA and protein synthesizing apparatus that will be activated upon fertilization. As the follicle continues to grow, granulosa cells increase in number and begin to form multiple layers. The innermost granulosa cells are in intimate contact with the oocyte through cellular processes that penetrate the zona pellucida and form gap junctions with its plasma membrane. Granulosa cells also form gap junctions with each other and function as nurse cells supplying nutrients to the oocyte, which is separated from direct contact with capillaries by the basal lamina and the granulosa cells.

Figure 13.2. Development of ovarian follicles. A. Primordial follicle. B. Primary follicle, the ovum has grown to full size, and there has been some proliferation of granulosa cells. C. Secondary follicle. Follicular cells have multiplied, and the theca intern is apparent. D. Early antral follicle. Granulosa cells have proliferated further, the theca has thickened, and a small amount of fluid has begun to appear. The follicle is now sensitive to FSH. E. Mature preovulatory follicle. Continued accumulation of fluid and further proliferation of granulosa cells have increased the follicle diameter some twentyfold. (Redrawn from Erickson, G.F. (1995) Endocrinology and Metabolism, 3rd ed., 973–1015. McGraw Hill, New York, with permission of the McGraw-Hill Companies.)

Follicular development continues with further proliferation of granulosa cells and gradual elaboration of fluid within the follicle. Follicular fluid is derived from blood plasma and contains plasma proteins, including hormones, and various proteins and steroids secreted by the granulosa cells and the ovum. Accumulation of follicular fluid brings about further enlargement of the follicle and the formation of a central fluid-filled cavity called the antrum. Follicular growth up to this stage is independent of pituitary hormones, but without support from follicle stimulating hormone (FSH; see Chapters 2 and 12) further development is not possible and the follicles become atretic. Any follicle can be arrested at any stage of its development and undergo the degenerative changes of atresia. Atresia is the fate of all the follicles that enter the growth phase before puberty, and more than 99% of the 200,000 to 400,000 remaining at puberty. The physiological mechanisms that control this seemingly wasteful process are poorly understood.

In the presence of FSH antral follicles continue to develop slowly for about two months until they reach a critical size. About 20 days before ovulation a group or cohort of six to 12 of these follicles enters into the final rapid growth phase, but in each cycle normally only one survives and ovulates, while the others become atretic and die (Figure 13.3). The surviving follicle has been called the dominant follicle because it may contribute to the demise of other developing follicles. As the dominant follicle matures, the fluid content in the antrum increases rapidly, possibly in response to increased colloid osmotic pressure created by partial hydrolysis of dissolved mucopolysaccharides. The ripe, preovulatory follicle reaches a diameter of 20 to 30 mm and bulges into the peritoneal cavity. At this time it consists of about 60 million granulosa cells arranged in multiple layers around the periphery. The ovum and its surrounding layers of granulosa cells, the corona radiata, are suspended by a narrow bridge of granulosa cells (the cumulus oophorus) in a pool of more than 6 ml of follicular fluid. At ovulation a point opposite the ovum in the follicle wall, called the stigma, erodes and the ovum with its accompanying granulosa cells (the cumulus oophorus complex) is extruded into the peritoneal cavity in a bolus of follicular fluid (Figure 13.4).

Figure 13.3. Follicular development at various stages of a woman’s life. (Adapted from McGee, E.A. and Hsueh, A.J.W. (2000) Endocr. Rev.21: 200–214, with permission of the Endocrine Society.)

Figure 13.4. Ovulation in a rabbit. Follicular fluid, granulosa cells, some blood, and cellular debris continue to ooze out of the follicle even after the egg mass has been extruded. (From Hafez, E.S.E. and Blandau, R.J. (1969) Gamete transport-comparative aspects. In The Mammalian Oviduct, Hafez, E.S. E. and Blandau, R.J., eds. University of Chicago Press, Chicago.)

Following ovulation there is ingrowth and differentiation of the remaining mural granulosa cells, thecal cells, and some stromal cells, which fill the cavity of the collapsed follicle to form a new endocrine structure, the corpus luteum. The process by which granulosa and thecal cell are converted to luteal cells is called luteinization (meaning yellowing) and is the morphological reflection of the accumulation of lipid. Luteinization also involves biochemical changes that enable the corpus luteum to become the most active of all steroid-producing tissues per unit weight. The corpus luteum consists of large polygonal cells containing an extensive smooth endoplasmic reticulum and smaller steroid secreting cells thought to be derived from the theca interna. Its metabolic needs are served by a rich supply of fenestrated capillaries. Unless pregnancy ensues, the corpus luteum regresses after two weeks, leaving a scar on the surface of the ovary.

Oviducts and Uterus

The primitive müllerian ducts that develop during early embryonic life give rise to the duct system that in primitive animals provides the route for ova to escape to the outside (Figure 13.5). In mammals these tubes are adapted to provide a site for fertilization of ova and nurture of embryos. In female embryos the müllerian ducts are not subjected to the destructive effects of the antimüllerian hormone (seeChapter 12) and, instead, develop into the oviducts, uterus, and upper portion of the vagina. Unlike the development of the sexual duct system in the male fetus, this differentiation is independent of gonadal hormones.

Figure 13.5. Uterus and associated female reproductive structures. The right side of the figure has been sectioned to show the internal structures. Insert on the left shows surface anatomy. (Adapted from Netter, F.H. (1997) Atlas of Human Anatomy, 2nd ed., plate 346. Novartis, Hanover, NJ.)

The paired oviducts (fallopian tubes) are a conduit for transfer of the ovum to the uterus (see Chapter 14). The proximal end comes in close contact with the ovary and has a funnel-shaped opening, the infundibulum, surrounded by finger-like projections called fimbriae. The oviduct, particularly the infundibulum, is lined with ciliated cells whose synchronous beating plays an important role in egg transport. The lining of the oviduct also contains secretory cells whose products provide nourishment for the zygote (fertilized ovum) in its three- to four-day journey to the uterus. The walls of the oviducts contain layers of smooth muscle cells oriented both longitudinally and circularly.

Distal portions of the müllerian ducts fuse to give rise to the uterus. In the nonpregnant woman the uterus is a small, pear-shaped structure extending about 6 to 7 cm in its longest dimension. It is capable of enormous expansion, partly by passive stretching and partly by growth, so that at the end of pregnancy it may reach 35 cm or more in its longest dimension. Its thick walls consist mainly of smooth muscle and are called the myometrium. The secretory epithelial lining is called the endometrium and varies in thickness with changes in the hormonal environment, as discussed later. The oviducts join the uterus at the upper, rounded end. The caudal end constricts to a narrow cylinder called the uterine cervix, whose thick wall is composed largely of dense connective tissue rich in collagen fibers and some smooth muscle. The cervical canal is lined with mucus-producing cells and usually is filled with mucus. The cervix bulges into the upper reaches of the vagina, which forms the final link to the outside. The lower portion of the vagina, which communicates with the exterior, is formed from the embryonic urogenital sinus.

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Reproductive Physiology of Male Animals

Timothy J. Parkinson, in Veterinary Reproduction and Obstetrics (Tenth Edition), 2019

Structure and Function of Spermatozoa

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Systems Toxicologic Pathology

Daniel G. Rudmann, George L. Foley, in Haschek and Rousseaux's Handbook of Toxicologic Pathology (Third Edition), 2013

2 Structure, Function, and Cell Biology

Before a toxicologic pathologist can effectively complete an accurate, thorough evaluation and safety assessment of female reproductive study data, he or she needs to be very familiar with normal anatomy and histology as well as the endocrinology associated with the different species used in preclinical studies. The estrous cycle progresses in waves; for example, in the rat it takes approximately 50 days for a primordial follicle to develop to a tertiary or antral follicle and ovulate, and most of these follicles undergo atresia before ovulation. There are also several key species differences especially in the characteristics of the estrous cycle. These are described for the rat, Beagle dog, and cynomolgus monkey later in this section. A summary of the differences between the rat, Beagle dog, and cynomolgus monkey with regard to sexual development and reproductive cycle characteristics is given in Table 60.3.

TABLE 60.3. Differences between Sexual Development and Reproductive Cycle Characteristics of the SD Rat, Beagle Dog, and Cynomolgus Monkey

Empty Cell	SD rat	Beagle dog	Cynomolgus monkey
Sexual maturity	8 weeks	Approximately 1.5 years	Approximately 4 yrs
Senescence (S) or menopause (M)	> 9 months (S)	Not applicable (S)a	> 20 years (M)
Cycle length	4 days	3.5–13 months	28 days
Seasonal influence	None	None	Yes, and social influences

While cycle length can be prolonged in older female Beagle dogs, true senescence is not described.

This section will summarize some of the more pertinent information from recent journal articles and other sources, focusing on the normal reproductive system of the virgin, adult female in a specific preclinical animal model system. The embryologic and prepubescent development of the female reproductive system will not be described except as it pertains to the female rat pubertal assay, and in cases where it is critical to add perspective to comparative mammalian physiology and endocrinology. The focus of the description will be for the rat (Sprague-Dawley and Wistar strains), Beagle dog, and cynomolgus monkey, which are by far the most common species used in toxicology studies; however, the minipig will also be discussed because of its emergence as a toxicology model for dermal and medical device testing (see The Use of Minipigs in Non-Clinical Research, Chapter 13).

The ovary is the most dynamic and complicated female reproductive organ with several common characteristics between species. For this reason, we describe the ovary in general first and then add species-specific differences in our detail of the estrous cycles of the rat, Beagle dog, and cynomolgus monkey. For each of these estrous cycle descriptions, we first highlight some of the species-specific characteristics of the female reproductive tract. Then, stage by stage, we describe the histologic changes in these tissues. It is important to note that we do not include the mammary gland in our descriptions because it is covered by a separate chapter in this book (Mammary Gland, Chapter 61). However, the mammary gland should be considered part of female reproductive tract assessment when studying a test agent’s potential to modulate the homeostasis of the female reproductive system. Descriptions of the clitoral glands are reserved for the neoplasia section of this chapter, since these structures are generally only examined in the lifetime carcinogenicity bioassay in rodents.

2.1 The Ovary

FIGURE 60.1. Ovary, rat, normal ovary. Numerous corpora lutea and follicles give the ovary a grape-like appearance. 15×, H&E.

FIGURE 60.3. Ovary, rat, corpora lutea (CL) types. (A) Normal eosinophilic (Eos) CL, (B) mixed esosinophilic and basophilic CL, and (C) basophilic (Baso) CL. 50×, H&E.

2.2 The Histology of the Rat Ovary during Prepubertal and Pubertal Development

The Endocrine Disrupter Screening Program (EDSP) test guidelines released in 2009 by the US EPA included a female pubertal assay as part of the risk assessment paradigm for endocrine-active chemicals. The assay requires administration of a test agent by oral gavage to female rats from postnatal day (PND) 22 through PND 42 or 43, followed by the evaluation of multiple endpoints including reproductive tissue histology. This period of female rat development includes the onset of estrus (PND 29–38) and vaginal patency (PND 33). Because the toxicologic pathologist may be required to assess the histologic features of the reproductive tract from rats used in this assay, we include a brief outline of the normal histology of the peripubertal reproductive tract.

At PND 20–21, early antral follicles are present (Figure 60.4A) and the ovary has atresia of secondary and tertiary follicles (early to mid-stage) (Figure 60.4B), which progress to confluent expanses of apoptotic granulosa cells by PND 22 (Figure 60.4C). By PND 27–28, there is a dramatic increase in necrotic ova within the ovarian medulla (Figure 60.4D–E). Also, the outer cortex with larger, antral follicles is clearly distinguished from the center of the medulla, which contains atretic follicles (Figure 60.4E). Corpora lutea (CL) first appear between PND 28 and 38, and the numbers of necrotic ova and atretic follicles are decreased (Figure 60.4F). By PND 38, rats have several ovulatory follicles and 3–6 CL per section of ovary. At PND 43, 4–10 CL are present and atretic follicles are in lower numbers (1–4 per section) (Figure 60.4G).

FIGURE 60.4. Ovary, rat, PND 21. (A) Early antral follicles are present (arrowheads). (B) PND 21, atresia of secondary and tertiary follicles (early to mid-stage). (C) PND 22, confluent expanses of apoptotic granulosa cells by PND 22. (D) PND 26 (arrowheads). (E) PND 28, necrotic ova within the ovarian medulla; large antral follicles (arrow) are clearly distinguished from the center of the medulla which contains atretic follicles. (F) PND 37, corpora lutea (CL) present and number of necrotic ova and atretic follicles are decreased. (G) PND 43, several ovulatory follicles CL present; fewer atretic follicles (arrowheads). (A, E, F) 25×, (B, C) 200×, (D) 50×, (G) 12.5×. H&E.

Photograph courtesy of Drs Cathy Picut and Amera Remick.

2.3 The Histology of the Rat Female Reproductive Tract during the Estrous Cycle

For any toxicologic pathologist, a strong understanding of the normal histologic changes that occur in the rat reproductive tract during the estrous cycle is fundamental. Toxicology studies using the sexually mature female rat are probably the first and best opportunity for a pathologist to assess the potential reproductive effects of test agents in man. Table 60.4 lists what we consider are the best distinguishing characteristics of each stage. We believe that the most useful histologic criteria for differentiating among the various stages of estrous occur in the vagina (Figure 60.5). Note that the most common rat strains used (Sprague-Dawley and Wistar) have a similar estrous cycle, and our descriptions of the cycle will be limited to the Sprague-Dawley (SD) and Wistar. The information provided below will be a compilation of our personal experience, the literature, and the results from a detailed estrous cycle study completed at Eli Lilly in the late 1990s with the Crl:CD (SD) IGS BR Sprague-Dawley rat.

TABLE 60.4. Key Vaginal Histologic Criteria for the Rat Estrous Dycle

Stage	Histology
Proestrus	Mucified cuboidal epithelium overlying a cornified layer
Estrus	Stratified epithelium with keratin loosely associated or sloughed into lumen
Metestrus (Diestrus I)	Apoptosis of vaginal epithelial cells
Diestrus (Diestrus II)	Increased mitoses of vaginal epithelial cells

FIGURE 60.5. Vagina, rat, changes with estrous cycle. Proestrus (Early and Late P): Early in proestrus, the vagina is lined by four to eight layers of plump epithelial cells that have a mucoid character to their cytoplasm. Later in proestrus the vaginal epithelium is thicker (up to 10 layers) and overlies a thin layer of cornified cells. Estrus (E): There is no superficial vacuolated layer, the epithelium is approximately six to eight layers thick and has characteristic plaques of cornified epithelium. Diestrus I or metestrus (DI): The vagina has a non-cornified epithelium that is four to six layers in thickness with intraepithelial and luminal leukocytes. Diestrus II (DII): The vagina is thinner, only three or four cell layers thick, non-cornified, and has prominent intraepithelial leukocytes. 80×, H&E.

The cycle of the SD and Wister rat is usually 4 days in length; however occasionally rats will have 5-day cycles or will switch between 4- and 5-day cycles. SD and Wistar rats start entering senescence at 5–6 months old, which alters the cycle, usually prolonging the portions of the cycle under estradiol influence (late diestrus and proestrus). As mentioned above, the vagina is very helpful for female reproductive tract staging and in some cases vaginal morphology may indicate a slightly later stage of estrous than the uterus. In those cases it is best to use the vagina as the definitive indicator of estrous stage, because it changes the quickest in response to the addition or withdrawal of trophic factors. Note that histologic samples of rat vagina should be collected from the anterior vagina since the posterior third of the vaginal mucosa is always covered by a thick, cornified stratified epithelium. While the vagina is very useful in staging, it is important to emphasize that all reproductive tissues should be examined as a unit to evaluate for a potential effect of a test agent. Also, trimming and sectioning should be standardized to protect bias, especially in the case of the ovary (Figure 60.6). It is important to realize that in some cases the only treatment effect may be a lack of synchrony between the histologic appearance of the ovary, uterus, and vagina. This asynchrony will only be apparent if the tissues are examined as a unit.

FIGURE 60.6. Ovary, rat, effects of section. Because the section was not optimally processed, the top ovary (A) compared to the bottom (B) appears to have a lower number of follicles and CL. 15×, H&E.

A high level of proficiency in female reproductive tract staging can be attained quickly with a little practice at recognizing the cyclical changes in the ovary, uterus, and vagina of the young, sexually mature female rat. While Table 60.4 is an excellent quick reference, it is important to have an understanding of the rat-specific characteristics of all female reproductive tissues as well as the details of tissue changes for each stage of the rat estrous cycle. Note that while we will not describe histologic changes in the cervix, generally cervical changes mirror vaginal changes during the rat estrous cycle.

In the rat, the paired ovaries, suspended from the dorsal abdominal wall by the mesovarium, are globose. During each estrous cycle, several follicles and corpora lutea may develop. These structures often protrude from the ovarian surface, thus giving rise to the grape-like appearance of the ovary at necropsy. In rats the ovarian theca interna persists as the interstitial gland, which often contains degenerated zona pellucida in the center. The interstitial gland eventually breaks up into small groups of cells that are scattered in the medulla.

The rat has a duplex uterus with two long and straight uterine horns and separate uterine bodies joined externally at their cervical ends but with two independent cervical openings. The uterus is composed of an innermost mucosa, the endometrium, a middle muscular layer, the myometrium, and an outer serosal layer, the perimetrium (Figure 60.7). The endometrium is composed of a surface epithelium, endometrial glands, and the lamina propria. A single layer of columnar epithelial cells lines the lumenal surface and endometrial glands. The lamina propria is composed of abundant small stromal cells, migrating lymphocytes and polymorphonuclear leukocytes, and vascular spaces within a framework of connective tissue. The myometrium is composed of inner circularly and outer longitudinally arranged bundles of smooth muscle cells. The perimetrium is composed of a single layer of mesothelial cells which overlies a thin layer of connective tissue.

FIGURE 60.7. Uterus, rat, normal. The uterus is composed of an innermost mucosa, the endometrium; a middle muscular layer, the myometrium; and an outer serosal layer, the perimetrium. 60×, H&E.

The vagina consists of an inner layer, the mucosa, a middle layer, the muscularis, and an outer layer, the adventitia (Figure 60.8). The mucosa is composed of a stratified squamous epithelium and its underlying lamina propria. The muscularis is composed of smooth muscle fibers arranged more or less circularly in the inner layer and longitudinally in the outer layer. The adventitia is composed of a thin layer of connective tissue.

FIGURE 60.8. Vagina, rat, estrus. Cornified material is sloughed into the vaginal lumen; later in estrus there may be very little cornified debris left. (A) 12×, (B) 60×. H&E.

On the day of estrus, the ovary usually has four sets of corpora lutea. The new, small basophilic partially luteinized corpora lutea (CL) are from the most recent ovulation event, basophilic CL from the last cycle are now beginning to appear eosinophilic, eosinophilic corpora lutea present from the previous cycles show degenerative changes, and old shrunken CL remain from several cycles ago (Figure 60.9). Dependent on section, one may observe an ovulatory fossa with hemorrhage, mitotic activity, and luteinizing granulosa cells, and oviducts, if present, may have ova or cumulus oophorous cells. The uterus has extensive apoptosis of both the luminal and glandular epithelium, and there is stromal edema and eosinophil infiltrates (Figure 60.9). The uterine epithelial cells are crowded and tall, and the uterine myometrium is thick. The vagina is approximately six to eight layers thick, and has characteristic plaques of cornified epithelium that are sloughed into the vaginal lumen early in estrus and cleared quickly; therefore, late on the day of estrus very little cornified debris may be left (Figures 60.5, 60.8).

FIGURE 60.9. Ovary and uterus, rat, estrus. The uterus has a thick endometrium and myometrium (A, 12×) and there is apoptosis of both luminal and glandular epithelial cells with endometrial inflammatory cell infiltrates (B, 120×). The ovary has four sets of corpora lutea (C, 10×) and large antral follicles (D, 120×). The new, small basophilic partially luteinized CL are from the most recent ovulation event, basophilic CL from the last cycle are now beginning to appear eosinophilic, eosinophilic corpora lutea present from the previous cycles show degenerative changes, and old shrunken CL (E, 150×) remain from several cycles ago. H&E.

On the day of metestrus (sometimes referred to as diestrus I), three sets of corpora lutea are present: a partially filled basophilic CL with luteolytic changes, an eosinophilic CL from the previous cycle, and old, shrunken CL from past cycles (Figure 60.10). The vagina has a non-cornified epithelium that is four to six layers in thickness, with intraepithelial and luminal leukocytes (Figure 60.5). The uterus still has apoptosis but only in the luminal epithelial cells (Figure 60.6). The uterine epithelial cells lining both the lumen and the glands are shorter and more distinct. The uterine myometrium is thinner (Figure 60.10).

FIGURE 60.10. Uterus and ovary, rat, metestrus (diestrus I) and diestrus (II) (A–D). Metestrus: the uterus (A,B) has apoptotic cells but only in the luminal epithelial cells, uterine epithelial cells lining both the lumen and the glands are shorter and more distinct, and the uterine myometrium is thinner. In the metestrus ovary (C, D), three sets of corpora lutea are present: a partially filled basophilic CL with luteolytic changes (∗), an eosinophilic CL from the previous cycle, and old, shrunken CL from past cycles. Diestrus (or diestrus II): The uterus is quiescent with a short columnar lining and glandular epithelium and also has a thinner myometrium. In the ovary, there are three sets of corpora lutea: a filled basophilic CL, an eosinophilic CL with luteolysis, and old corpora lutea from past cycles. (A) 20×, (B) 120×, (C) 15×, (D) 30×. H&E.

On the day of diestrus (sometimes referred to as diestrus II), there are three sets of corpora lutea: a filled basophilic CL, an eosinophilic CL with luteolysis, and old corpora lutea from past cycles. The filled basophilic CL has luteal cells that are larger with very prominent nucleoli, some containing vacuoles (lipid droplets) in their cytoplasm (Figure 60.10). The vagina is thinner, only three or four cell layers thick, non-cornified, and has prominent intraepithelial leukocytes. Mitoses may be observed in the basal layer (Figure 60.5). The uterus is quiescent, with a short columnar lining and glandular epithelium and a thinner myometrium (Figure 60.10).

On the day of proestrus, the ovary has mature eosinophilic corpora lutea, filled basophilic corpora lutea without luteolysis, and old shrunken CL. Large preovulatory follicles are often present in the ovary at this stage of the estrous cycle (Figure 60.11). Interstitial glands will be the most prominent during proestrus. The uterus has a dilated lumen, stromal edema, a hypertrophied myometrium, and mitoses in luminal and glandular epithelial cells (Figure 60.11). Eosinophils in the stroma are most prominent in this stage. Uterine weights are often increased during proestrus, and appear larger at necropsy because of intraluminal fluid. Early in proestrus, the vagina is lined by 4–8 layers of plump epithelial cells that have a mucoid character to their cytoplasm. Later in proestrus the vaginal epithelium is thicker (up to 10 layers) and overlies a thin layer of cornified cells (Figure 60.5).

FIGURE 60.11. Uterus and vagina, rat, proestrus. The uterus has a dilated lumen (A), stromal edema, a hypertrophied myometrium, and mitoses in luminal and glandular epithelial cells (B). Eosinophils in the stroma are most prominent in this stage. Early in proestrus, the vagina is lined by four to eight layers of plump epithelial cells that have a mucoid character to their cytoplasm (C). Later in proestrus the vaginal epithelium is thicker (up to 10 layers) and overlies a thin layer of cornified cells (D). (A) 10×, (B) 80×, (C, D) 125×. H&E.

2.4 The Histology of the Dog Female Reproductive Tract during the Estrous Cycle

The estrous cycle of the dog is very different from the rat. It is markedly longer, more variable in phase length (3.5–13 months), and has a normal “anestrus” phase (for review, see Chandra and Adler, 2008). Evaluating the reproductive tract in dog toxicology studies can be challenging because young, peripubertal dogs may be used in toxicology studies and numbers in test groups are small (generally three or four) (Figure 60.12). An accurate phasing of the dog estrous cycle will assist in assessing test-agent effects on synchronization of the reproductive events in the female dog (bitch) reproductive tract. In fact, in the past several decades, the Beagle dog as an animal model has been a very useful predictor of the effects of treatments targeted at human contraception.

FIGURE 60.12. Ovary and uterus, cynomolgus monkey and dog. Immature. Ovaries and uteri are small grossly (not shown). (A) Ovary: there are no corpora lutea and follicles are often immature (50×). (B, C) Uterus: the uterine endometrium and myometrium is thin and endometrial glands are quiescent (6× and 20×). H&E.

Dog ovaries are irregularly ellipsoid. Several follicles and corpora lutea may develop during each cycle, and polyovular follicles (follicles with two or more oocytes) commonly occur in young bitches. Like the rat, these structures often protrude from the ovarian surface, thus giving rise to the grape-like appearance of the ovary. Dog ovaries also have interstitial glands or granulosa cell cords derived from atretic follicles and stromal cells. Granulosa cells of canine follicles begin to luteinize with extensive infoldings of the granulosa lining prior to ovulation. It is important to note that the infolded luteinized preovulatory follicles in the dog are normal but can often be misinterpreted as abnormal. Unique to dogs at all phases of the reproductive cycle are the presence of invaginated cord-like proliferations of the surface epithelium that extend through the tunica albuginea into the superficial cortex. These structures have been termed subsurface epithelial structures (SES), and are most numerous during anestrus. The SES are hormonally responsive and commonly become cystic in aged dogs. Their functional significance is not known. The dog has a bicornuate uterus with a short body and long and straight uterine horns. The two separate uterine horns open into one common cervical canal. Otherwise, the general histology of the uterus and vagina are comparable to the rat.

Estrus and proestrus phases together account for 1–3 weeks of the estrous cycle in dogs. At estrus, the ovary has large pre-ovulatory follicles with luteinization of the granulosa cell layers (Figures 60.13, 60.14). The uterus has myometrial hypertrophy, stromal cell proliferation, and stromal congestion, edema, and hemorrhage (Figure 60.14). The surface and glandular epithelium are increased in height, and there are numerous mitotic figures. The vagina is the thickest during estrus, and heavily cornified (Figures 60.13, 60.14). The stroma and smooth muscle are hyperplastic, and there is both stromal congestion and edema.

FIGURE 60.13. Ovary, dog, estrous cycle. Typical morphologic features of the ovary during different stages of estrus. Anestrus (A): the ovary appears quiescent with one or two sets of old CL. Proestrus (P): there is a gradual increase in ovarian follicular development and large antral follicles with thick granulosa cell layers are evident. Estrus (E): the ovary has large pre-ovulatory follicles with luteinization of the granulosa cells. Diestrus (D): the ovary has new corpora lutea. 10×, H&E.

Photograph courtesy of Dr Sundeep Chandra.

FIGURE 60.14. Uterus and vagina, dog. Vaginal changes as described in Figure 60.12. In anestrus (A), the uterine surface and glandular epithelium are lined by low columnar cells with basally situated nuclei and the vaginal epithelium is thin and underlying stroma compact. In proestrus (P), the uterine mucosa becomes more vascular and edematous with occasional small focal areas of erythrocyte extravasation in the zona compacta. The surface and glandular epithelium increases in height and numerous mitotic figures are present. The vagina becomes thicker and cornified. The stroma and smooth muscle also start to thicken and there is both stromal congestion and edema. In estrus (E), the uterus has myometrial hypertrophy, stromal cell proliferation, and stromal congestion, edema, and hemorrhage. The surface and glandular epithelium are increased in height and there are numerous mitotic figures. The vagina is thick and heavily cornified. In diestrus (D), luminal glands are proliferative and straight but abruptly become tortuous towards the basement membrane. The glandular lumina may contain pink secretions; stromal edema and congestion are decreased. The upper gland and luminal epithelial cells progressively develop more foamy cytoplasm as a consequence of the influence of progesterone. The vagina is much thinner (three or four cell layers) and loses it cornification. 50×, H&E.

Photograph courtesy of Dr Sundeep Chandra.

Metestrus or diestrus is up to 100 days in length in the dog. At metestrus the ovary has new corpora lutea (Figures 60.13, 60.14). Luminal glands are proliferative and straight and abruptly become tortuous towards the basement membrane (Figure 60.14). Often the glandular lumina contain pink secretions and the stromal edema and congestion are markedly decreased. The upper gland and luminal epithelial cells progressively develop more foamy cytoplasm as a consequence of the influence of progesterone. The vagina becomes much thinner (three or four cell layers) and loses its cornification (Figures 60.13, 60.14). Because of the long life of the corporal lutea, pseudocyesis (false or pseudopregnancy) with or without clinical signs is relatively common in the dog. In pseudocyesis, dog mammary glands will demonstrate alveolar lobular hyperplasia and increased secretions which could be interpreted as a treatment-related effect in a toxicology study. Also, the uterus has a hyperplastic endometrium and may have implantation-like areas without the fetal contribution.

Anestrus is the most variable period in the dog, its length partially dependent on the breed. This stage can range from 1 to 6 months in length. During anestrus, the ovary appears quiescent with one or two sets of old CL (Figure 60.13). The uterine surface and glandular epithelium are lined by low columnar cells with basally situated nuclei, and the vagina remains three or four cell layers thick (Figures 60.13, 60.14). The vagina epithelium is at its thinnest, and remains non-cornified (Figures 60.13, 60.14).

At proestrus, there is a gradual increase in ovarian follicular development, and large antral follicles with thick granulosa cell layers are evident (Figure 60.13). Often there are old CL composed of small foci of vacuolated cells and macrophages with intracytoplasmic lipofuscin. During proestrus, the uterine mucosa becomes more vascular and edematous with occasional small focal areas of erythrocyte extravasation in the zona compacta. The surface and glandular epithelium increases in height, and numerous mitotic figures are present (Figures 60.13, 60.14). The vagina becomes thicker and cornified (Figures 60.13, 60.14). The stroma and smooth muscle also start to thicken, and there is both stromal congestion and edema.

2.5 The Histology of the Cynomolgus Monkey Female Reproductive Tract during the Menstrual Cycle

The cynomolgus monkey has a menstrual cycle (and endocrinology) that is very similar to the human. However, as was the case for the dog, assessment of the female reproductive system can be challenging because the numbers of animals used in toxicology studies are small (usually three or four) and animals are often pre- or peripubertal (Figure 60.12). In the past couple of decades, the attention given to the monkey model has heightened because of the large influx of biologics now in preclinical development. Many of these biologics are not amenable to study in rodents, and the cynomolgus monkey has become a key animal model of human relevance. As a result, the use of sexually mature cynomolgus monkey is now more common, and it is important for the toxicologic pathologist to be familiar with this model system.

The cynomolgus monkey ovaries are amygdaloid in shape and found in the pelvic cavity. The ovary has multiple primary follicles that develop with each cycle, but usually only one follicle will become the preovulatory follicle, resulting in a single protrusion from one ovarian surface. Degenerating primary oocytes are likely the origin of calcified foci found in the ovarian cortex of cynomolgus monkeys. The calcified areas may be multiple and/or bilateral. Buse et al. (2008) presents an extensive review of the cynomolgus monkey ovary.

In the sexually mature cynomolgus monkey, the cervix has a stratified squamous exocervix, squamocolumnar junction (SCJ) and transformation zone, and glandular endocervix with prominent colliculi. The stratified squamous epithelium of the exocervix changes to tall columnar glandular epithelium at the SCJ. In contrast, the cervical mucosa is atrophic in the sexually immature cynomolgus monkey and the SCJ is not distinct. The cervical epithelium in macaques is highly responsive to estrogens. Estrogen stimulation results in marked keratinization of the exocervical epithelium and thickening of the stratified squamous epithelium near the SCJ, and squamous metaplasia and hypertrophy of the endocervical glands.

The menstrual cycle of the cynomolgus monkey is divided into four major phases: the follicular (or proliferative) phase, the luteal (or secretory) phase, the menstrual phase, and the regenerative phase (Figures 60.15–60.17) For a detailed description of these phases, the reader is referred to the review by Van Esch and colleagues (2008a). The regenerative phase is not included in some descriptions, resulting in a three phase menstrual cycle. Cynomolgus monkey endometrial changes are similar to those of women, with menstrual discharge toward the end of each reproductive cycle. The follicular or proliferative stage follows menses. During the follicular (proliferative) phase, the superficial and glandular epithelium, the stroma, and the endometrial vasculature are in varying degrees of physiological proliferation (Figures 60.16, 60.17B). In effect, the endometrium thickens, spiral arteries develop, and the complexity of the uterine glands increases (Figure 60.16). By day 6–8, the dominant ovarian follicle can be identified and will ovulate following the LH surge (Figure 60.15).

FIGURE 60.15. Ovary, monkey. Ovarian histology during the menstrual cycle of cynomolgus macaques. Clockwise from upper left, the ovaries shown are from the early follicular phase (developing follicles and the corpus luteum of the prior cycle); late follicular phase (dominant follicle); ovulation (corpus hemorrhagicum); and luteal phase (corpus luteum). 10×, H&E.

Photograph courtesy of Dr Mark Cline.

FIGURE 60.16. Uterus, monkey, uterine histology across the menstrual cycle of cynomolgus macaques. Stages shown from left to right are: (A) regenerative phase; (B) follicular phase; (C) periovulatory/early luteal phase; (D) luteal phase; and (E) early menstrual phase. 25×, H&E.

Photograph courtesy of Dr Mark Cline.

FIGURE 60.17. Uterus, monkey, uterine histology of cynomolgus macaques. (A) Quiescent/atrophic; (B) follicular phase (note mitoses and pseudostratification); (C) luteal phase (subnuclear vacuolation; (D) menstrual phase (hemorrhage, apoptosis). 100×, H&E.

Photograph courtesy of Dr Mark Cline.

Following ovulation, the secretory or luteal phase occurs; the hallmark of ovulation is the appearance of subnuclear vacuoles (Figure 60.16C). The luteal phase lasts approximately 13–14 days, and is marked by endometrial glands secreting a glycogen-rich material. The glands become coiled and saccular, giving a saw-tooth appearance. The spiral arteries become coiled. Implantation typically occurs on day 21–23. If pregnancy is not established, the spiral arteries constrict with declining progesterone levels of the regressing corpus luteum, there is massive apoptosis of endometrial glands (Figure 60.16D), and menses occurs. The postpartum uterus of monkeys may have prominent hyalinized myometrial arteries that should not be confused with a toxicologic finding. Cervical intraepithelial neoplasia, endometriosis, ectopic growth of endometrial tissue outside the uterus (endometriosis), and adenomyosis (extension of endometrium into the subjacent myometrium) are reported across the primate order as spontaneous findings.

2.6 The Minipig in Toxicology Studies

Of special note is the fairly recent increase in use of the Göttingen, Yucatan, and Troll miniature pigs (minipig) for dermal and medical device toxicity testing (see The Use of Minipigs in Non-Clinical Research, Chapter 13). There are very few published data on spontaneous or experimentally induced female reproductive changes in any species of minipig, and, to our knowledge, there are no detailed descriptions of the histologic and hormonal changes that occur in the minipig during the estrous cycle.

The female reproductive system of the minipig has a bicornuate uterus with tortuous fallopian tubes, and is similar to humans in terms of uterine histology and estrous cyclicity. However, unlike humans and cynomolgus monkeys, the minipig does not menstruate. In Figure 60.18, the morphologic changes in the uterus and vagina during the normal estrous cycle are shown; however, please note that hormonal levels were not measured in sampled minipigs and the staging was based on literature descriptions.

FIGURE 60.18. Uterus and vagina, minipig, estrous cycle. Estrus (A, B), diestrus I (C, D), diestrus II (E, F), proestrus (G, H). 20×, H&E.

Estrus: (A) the uterine mucosa has increased apoptosis and vacuolation in the columnar surface and glandular epithelium, and neutrophils and eosinophils are present in the subepithelial stroma. (B) The vaginal mucosa has non-degenerate squamous epithelium without luminal debris.

Metestrus: (C) the uterine mucosa has a shorter surface epithelium, abundant eosinophilic cytoplasm in the glandular epithelium, and minimal inflammatory aggregates in the subepithelial stroma; (D) the vaginal mucosa has minimal keratinocyte degeneration and some luminal cellular debris.

Diestrus: (E) the uterine mucosa is largely similar to that in metestrus as described above, with increased tortuosity of endometrial glands; (F) the vaginal mucosal epithelium shows abundant keratinocyte degeneration and increased luminal debris (as in D).

Proestrus: (G) the uterus has a thicker surface epithelium with mitotic figures, endometrial glands with a short (cuboidal) basophilic epithelium, and infiltrating neutrophils and eosinophils within the subepithelial stroma; (H) the vaginal mucosa has a non-degenerate squamous epithelium with increased stratum basale.

Photograph courtesy of MPI, Dr Chidozie J. Amuzie.

The most well-described spontaneous histologic findings are in the uterus of pet minipigs (Vietnamese pot-bellied pigs [_Sus scrofa_]), where a high prevalence of cystic endometrial hyperplasia and smooth muscle neoplasms has been observed. In these studies, the average age of the minipigs with these uterine histologic changes was approximately 10 years of age for the minipigs. Generally, minipigs used for toxicology studies are much younger in age (approximately 3 months of age at study start), so uterine histologic findings are unlikely to be encountered in minipigs used for toxicologic research.

2.7 The Endocrinology of the Estrous Cycle

While a strong fundamental understanding of the anatomy and histology of the female reproductive system is critical for the toxicologic pathologist, the ability to interpret the gross and microscopic changes requires an understanding of the corresponding cellular and endocrinological connections between structure and function. Following input from the cerebral cortex, the hypothalamus secretes gonadotropin-releasing hormone into the hypophyseal portal circulation, which stimulates the anterior lobe of the pituitary to release gonadotropins. Subsequent to gonadotropin stimulation, the ovary synthesizes and releases sex steroid hormones. The levels of circulating sex steroid hormones in turn affect the activity of the hypothalamus and pituitary in the regulation of reproductive function in either a stimulatory (positive) or an inhibitory (negative) manner, depending on the stage of the cycle (Figure 60.19). In addition to steroidogenesis, ovarian tissues are capable of making a large number of growth hormones and cytokines. Ovarian hormones and growth factors exert stimulatory and inhibitory effects both locally (e.g., adjacent follicles) and systemically. In non-rodents and women, the adrenal cortex also produces sex hormones; therefore, the adrenal cortex should be evaluated with the reproductive tract in order to correctly interpret changes found in toxicologic studies.

FIGURE 60.19. Schematic drawing of hormonal regulation of reproduction (+, stimulatory; −, inhibitory).

Figure reproduced from Handbook of Toxicologic Pathology, 2nd Ed. W. M Haschek, C. G. Rousseaux and M. A. Wallig, eds. (2002) Academic Press, Fig. 9, p. 863, with permission.

2.8 Hormonal Events during a Given Reproductive Cycle

Between puberty and senescence, the reproductive cycle is manifested by changes of the reproductive organs as described in the previous section. The cyclic changes within the ovary are mainly regulated by luteinizing hormone (LH) and follicle-stimulating hormone (FSH), whereas the cyclic changes of the uterus and vagina are dependent on ovarian sex steroids. Figure 60.19 provides a relational diagram for the HPO axis. The easiest way to conceptually visualize the HPO axis is as a chemical equation in a cyclic equilibrium composed of a precursor pool of gonadotropins (LH, FSH) stimulating the ovary (also the adrenal in the non-rodent) to synthesize steroidal hormones, which then in turn modulate the physiology of the reproductive end organs (uterus, vagina, cervix, and mammary glands).

Each cycle begins with follicular growth and maturation, followed by ovulation, and the subsequent formation and regression of the corpus luteum. Although the recruitment and growth of a primordial follicle to the stage of an early tertiary follicle occurs spontaneously and does not require hormonal stimulation, the presence of FSH and LH is necessary for continued follicular maturation to the stage of a preovulatory follicle. It has been estimated that it takes 50 days for a primordial follicle to develop into a preovulatory follicle in the adult rat. Selection of follicles to continue on to the preovulatory stage depends on follicles having granulosa cells able to express the necessary gonadotropin receptors at the time of elevated gonadotropin levels. Most follicles undergo atresia and do not reach the preovulatory stage.

LH and FSH are glycoprotein hormones secreted from the anterior pituitary. In polyestrous non-seasonal breeding animals such as the rat, LH and FSH concentrations increase shortly after the end of the preceding cycle. FSH stimulates the proliferation of granulosa cells and induces receptors for LH on these cells. LH stimulates thecal cells to secrete androstenedione, which serves as a precursor for estrogen and androgen synthesis by granulosa cells through the catalytic function of aromatase.

The estrogen and androgen secreted from growing follicles promotes cell proliferation and maturation in both the uterus and the vagina. Under the influence of estradiol and androgens, the stromal fibroblasts and epithelial cells of the endometrium proliferate and increase in size. Estrogen and androgen also promote the synthesis of actomyosin and glycogen in smooth muscle cells, resulting in hypertrophy of the myometrium. In addition, estrogen primes the uterus for its response to progesterone. Polymorphonuclear leukocyte migration in the uterus, which may be causally related to intercellular edema and hyperemia, is also attributed to the effect of estrogen. In the vagina, estrogen not only stimulates cell proliferation but also induces epithelial cornification.

In the mature preovulatory follicle, a surge of LH results in ovulation with subsequent formation of the CL. The newly formed corpus luteum incorporates both granulosa and thecal cells, and is capable of secreting progesterone for only a certain period of time. Continued function of the corpus luteum requires stimulation by luteotrophic hormones such as LH and prolactin. Prolactin secretion in rats is stimulated by cervical stimulation. The ovarian steroid hormones, including estrogens, progestins, and androgens, exert their influence on the uterus and vagina, and other parts of the body, through the circulatory system.

In primates and dogs, ovulation occurs spontaneously. In the rat, ovulation is also spontaneous and coincides with the preovulatory LH surge that is under photoperiod control. In rabbits and other induced ovulators, the LH surge does not occur until cervical neurons are stimulated by mating or other means to activate the release of luteinizing hormone-releasing hormone (LHRH).

In the newly formed corpus luteum, progesterone secretory activity seems to be autonomous and, in most species, does not require luteotrophic factors. For continued function, luteotrophic hormones from the pituitary are required; LH is considered to be the most important luteotrophic factor in most species. However, in rats prolactin has been identified as the luteotrophic factor, whereas in rabbits estrogen is the only known luteotrophic factor.

The functional life of the corpus luteum varies among animal species. In the rat, it functions only for the first 2 days after formation. If cervical stimulation takes place, prolactin continues to be secreted by the pituitary gland and the functional life of corpora lutea is prolonged with continued progesterone secretion. If implantation does not take place, the life of corpora lutea is terminated 12–14 days after formation. The period during which the corpus luteum is functional is known as pseudopregnancy.

In dogs, luteinization of the preovulatory follicles is accompanied by progesterone secretion prior to ovulation. This is most likely due to the long interval between the LH surge and ovulation. The functional life of corpora lutea in dogs is approximately 60 days and is not affected by mating or implantation, and both LH and prolactin are required for their maintenance. Due to the long luteal lifespan in dogs, pseudopregnancy is common but not always clinically apparent.

In primates, the corpus luteum has a defined functional lifespan that is unaffected by mating or mechanical stimulation of the cervix. A continuous low level of LH is required to maintain the corpus luteum. If pregnancy occurs, the embryonic trophoblast secretes chorionic gonadotropin as early as 8–9 days after fertilization, thus maintaining the corpus luteum throughout early pregnancy.

Progesterone inhibits cell division and maturation but promotes secretion by the endometrial glands. With prior estrogen priming, it can also cause the endometrial stromal cells to change from the small inactive form with fusiform nuclei to large cells with ovoid nuclei. Progesterone causes myometrial cells to become hypertrophic with prominent myofibrils. In the vagina, when estrogen is also present, progesterone induces mucification by increased production and intracytoplasmic accumulation of sialic acid. In primates, corpora lutea secrete estrogen as well as progesterone.

At the end of its functional life, the corpus luteum regresses (luteolysis). The only clearly demonstrated luteolytic factor is prostaglandin F (2-alpha), produced by the endometrium. Experimental data indicate that this prostaglandin is responsible for luteolysis in large domestic species such as cattle and sheep, and in pseudopregnant rodents such as rat and hamster. Prostaglandin F 2α has also been demonstrated to be luteolytic in rhesus monkeys.

2.9 Regulation of Hormonal Secretion

As indicated earlier, the regulation of hormonal secretion is complex and involves many modulators that exert positive (stimulatory) or negative (inhibitory) feedback control. Readers are referred to a standard textbook of endocrinology for a complete discussion.

LH and FSH secretion by gonadotrophs in the anterior pituitary is initiated by a rhythmic pulsatile secretion of LHRH from the hypothalamus into hypothalamo-hypophyseal portal vasculature. LHRH is assumed to be the only gonadotropin-releasing hormone responsible for the release of both LH and FSH, although some investigators believe a different hormone may specifically control the release of FSH. The pulsatile release of LHRH is essential for normal LH and FSH secretion from the pituitary. The continuous presence of LHRH will at first stimulate and later desensitize secretion of LH and FSH due to a downregulation of receptors.

Each LH peak is preceded by an LHRH peak, but not every LHRH peak is followed by an LH peak. When LHRH release is not followed by LH release, the pulse may serve as a self-priming signal by increasing the number of LHRH receptors in the pituitary; this requires the presence of a small amount of estrogen in the serum.

Ovarian hormones modulate both the amplitude and frequency of LHRH pulses. Experimental data suggest that estradiol suppresses LH pulse amplitude but not the frequency, while progesterone primarily suppresses frequency. At the beginning of a reproductive cycle, the secretion of LH is inhibited by estrogen secreted from growing follicles. This negative feedback control affects LH release but not LH synthesis, which enables the pituitary to accumulate enough LH for the preovulatory surge.

Similar to their effect on LH secretion, ovarian steroids also affect FSH secretion, and this effect depends on the stage of the cycle and the hormonal status of the animal. During late estrus through early proestrus, estradiol has an inhibitory effect on FSH secretion. This inhibition is potentiated by the presence of progesterone. However, this negative feedback changes just prior to the preovulatory surge of LH. Again it seems that estrogen is the dominant force, acting directly on the hypothalamus, whereas progesterone potentiates the effect by stimulating FSH synthesis. The secretion of FSH is inhibited by the peptide hormone, inhibin, which is produced by the granulosa cells of growing follicles.

When follicles approach their final stage of maturity, the output of steroid hormones increases prior to ovulation. As concentrations of these hormones reach a certain level, their effects on the regulation of LHRH secretion also change from inhibitory to stimulatory. This is the basis of the preovulatory surge of LHRH and subsequent LH and FSH secretion. The preovulatory surge of LHRH induces a cascade of events within the hypothalamus that is necessary for normal ovulation to proceed. The mechanism responsible for this may involve estrogen, which augments the number of pituitary binding sites for LHRH, increases the responses of the pituitary to hormone binding, and directly promotes LHRH secretion from the hypothalamus. An alternative possibility is that estradiol affects norepinephrine activity, which in turn stimulates LHRH secretion. Estradiol may also contribute by inducing and maintaining LHRH receptors in conjunction with LHRH in the pituitary. The effect of progesterone on LHRH secretion is less clear, but is believed to be similar to that of estrogen.

Neurons controlling the secretion of LHRH also receive innervation, capable of either stimulation or inhibition, from other neural sites. Accordingly, the reproductive cycle adjusts to changes in light cycle, seasonal variation, and other physical (cervical stimulation) or olfactory (pheromone) stimuli. LHRH has been found in extrahypothalamic neurons in different regions of the limbic system of the cerebrum, and has been implicated in the regulation of reproductive behavior.

Factors involved in termination of the gonadotropin surge may include downregulation of LHRH receptor numbers and subsequent loss of sensitivity of the pituitary gonadotrophs. During the luteal phase, estrogen acts in concert with progesterone to inhibit LH synthesis and release.

Unlike the LH release, prolactin secretion is normally inhibited by the presence of dopamine from the hypothalamus. At the height of prolactin secretion, the level of dopamine is lowest and that of thyroid-releasing hormone (TRH) is highest, indicating that TRH may also play a role in the release of prolactin. Estradiol stimulates prolactin release prior to ovulation by acting through the pituitary to decrease the dopamine inhibitory effect.

Several growth factors have been identified in the ovary. These factors are large protein molecules with molecular weights of several to tens of thousands. The interaction between these factors and gonadotropins is believed to be vital for normal follicular growth and luteinization. Although the precise mechanisms of action need to be investigated, the participation of these molecules in reproductive function has been postulated as follows. Epidermal growth factor and insulin-like growth factor may assist in the induction of granulosa and thecal cell proliferation, and are regarded as initiation factors. The insulin-like growth factor may also be required for continuation of cellular proliferation. Epidermal growth factor has been localized to oocytes, pre-granulosa cells, and ovarian stroma. The granulosa cell is believed to be a major source of insulin-like growth factor. A growth factor from thecal cells, transforming growth factor alpha, may also contribute to both the initiation and maintenance of ovarian cellular proliferation. Vascular proliferation during follicular growth and corpus luteum formation is attributed, in part, to an angiogenic factor from follicular fluid called fibroblastic growth factor. Transforming growth factor beta, also produced by thecal cells, may inhibit cell proliferation and promote differentiation and transformation of granulosa cells to luteal cells during luteinization. TGF-β1 has also been localized in areas of corpora lutea. Growth factor secretion by ovarian cells has been reported to vary depending on the stage of estrous and the stage of follicular development. These factors may also be involved in the regulation of cyclic changes that occur in the remainder of the reproductive tract. Many other cytokines are reported to be both locally produced by ovarian structures as well as having regulatory effects on follicular development, atresia, and luteal function. An example of one such cytokine interaction is tumor necrosis factor alpha (TNF-α), which has been reported to play an interactive role with Fas ligand in the induction of atresia.

Control of the female reproductive cycle is complex, and comprises a cascade of events emanating from the hypothalamus and extending to the pituitary, ovary, uterus, and vagina, with stimulatory or inhibitory regulation by the ovarian steroids. Any disruption in this cascade of hormonal events may result in functional and structural alterations. If a xenobiotic has a direct effect on a single steroid pathway, the biological response in the animal model may be relatively straightforward. However, in this author’s experience, xenobiotics often have multiple effects on steroid synthesis pathways or may alter differentially, based on end organ, the response of the nuclear hormone receptors for the steroids. Ovarian steroid nuclear hormone receptors are complicated intracytoplasmic receptors that are influenced by tissue and binding pocket specific corepressor or costimulating proteins. Pharmaceutical companies have actually taken advantage of this characteristic of steroid receptors and designed therapeutics with the advantageous properties of a hormone while eliminating or minimizing the unwanted effects of the hormone. A classic example of this type of therapeutic is the Selective Estrogen Receptor Modulator (SERM).

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Hormones and Reproductive Cycles in Bats

Amitabh Krishna, Kunwar P. Bhatnagar, in Hormones and Reproduction of Vertebrates: Mammals, 2011

6.2.1.2 Pattern II: Unilateral follicular development and ovulation

With the exception of vespertilionid and rhinolophid bats, the majority of the bats show a brief period of estrus with mating, ovulation, and fertilization occurring in quick succession. During each cycle, a single large Graafian follicle (about 350 μm in diameter) develops that contains a small cumulus oophorus, large antrum, and two to three layers of membrane granulose. The ICs are generally few and not very prominent. Endocrine activity of the ovary is restricted to thecal cells and granulosa layers. These bats are generally monotocous or monovular and mono- or polyestrous. The majority of these species breed twice each year and have both ovaries functional (symmetric dominance). As bats find it difficult to successfully carry more than a single conceptus, these bats exhibit alternation of successive ovulation between two ovaries. The postpartum estrus always occurs in the ovary contralateral to that which supported the first or earlier pregnancy of the breeding season.

Many monovular bats have only one functional ovary. In dextral dominant molossid bats, the ovary contains numerous small follicles at different stages of development and atresia. A single large Graafian follicle develops before ovulation (350 μm in diameter). The left ovary contains numerous follicles up to 300 μm in diameter, but no Graafian follicles or CL. Hypertrophied ICs are prominent in the left ovary, show steroidogenic enzymes, and appear to perform only endocrine functions (Jerrett, 1979).

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