The development of the gubernaculum and inguinal closure in the marsupial Macropus eugenii (original) (raw)

J Anat. 2002 Sep; 201(3): 239–256.

Douglas Coveney

1Department of Zoology, The University of Melbourne, Victoria 3010, Australia

Geoffrey Shaw

1Department of Zoology, The University of Melbourne, Victoria 3010, Australia

John M Hutson

2Department of Pediatrics, The University of Melbourne, Victoria 3010, Australia

3Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Victoria 3052, Australia

Marilyn B Renfree

1Department of Zoology, The University of Melbourne, Victoria 3010, Australia

1Department of Zoology, The University of Melbourne, Victoria 3010, Australia

2Department of Pediatrics, The University of Melbourne, Victoria 3010, Australia

3Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Victoria 3052, Australia

Correspondence Dr D. Coveney, Department of Zoology, The University of Melbourne, Victoria 3010, Australia. Tel. + 61 38344 6259; fax: + 61 39418 1719; e-mail: ua.ude.bleminu.ygolooz@yenevoc.d

Copyright © Anatomical Society of Great Britain and Ireland 2002

Abstract

This study reports the developmental anatomy of testicular descent and inguinal closure of the tammar wallaby (Macropus eugenii) from birth to maturity. In females the ovary migrated caudally between days 10 and 20 after birth. The gubernaculum differentiates into the round ligament in the abdomen and extra-abdominally as the ilio-marsupialis muscle of the mammary glands. In males the testes migrated to the internal inguinal ring by day 20 post partum (pp), coinciding with the enlargement of the gubernaculum, and from the internal inguinal ring to the scrotum between days 20 and 65 pp. During descent there was an increase in the hyaluronic acid concentration in cells of the gubernaculum and scrotum. Development of the cremaster muscle began by day 10 pp on the periphery of the gubernaculum and its basic structure was completed by day 60 pp. After descent the inguinal canal closed between days 50 and 60 pp, but a small irregular lumen persisted, somewhat similar to that seen in the congenital scrotal hydrocoele of humans. Tammars have a hopping mode of locomotion and, like humans, are essentially bipedal. We suggest that inguinal closure evolved in these two species because their upright posture may otherwise lead to a high incidence of inguinal hernias.

Keywords: evolution, hyaluronic acid, pouch, processus vaginalis, scrotum, tammar wallaby, testicular descent

Introduction

In all mammals the testis forms in the abdominal cavity, and in most the testis migrates caudally into the scrotum: although in some species the adult testis is situated within the abdominal cavity as in the elephant, or in the inguinal canal as in the hedgehog (Carrick & Setchell, 1977). The driving force for the evolution of the scrotal testis and the pampiniform plexus may have been the cooler temperatures that resulted from an external position and counter-current heat exchange system (Bedford, 1978). The cooler temperatures may provide a selective advantage through a decrease in the mitochondrial mutation rate in spermatogonia within the testis (Short, 1997).

In 1762 John Hunter gave the first account of testicular descent and structure of the gubernaculum, so named because he realized it was ‘the rudder of the testis’. The anatomy of the gubernaculum and the cremaster muscle differs between large mammals and smaller rodents (Wensing, 1986), but its development appears to be similar in most eutherian species. The gubernaculum first develops as a mesenchymatous cord, connecting the distal portion of the gonad and mesonephros to the ventro-lateral aspect of the abdominal wall (Backhouse, 1964; Gier & Marion, 1969). The gubernaculum at this stage does not continue through to the developing genital folds (the future labia and scrotum), but terminates between the external and internal inguinal rings (Heyns, 1987; Fallat et al. 1992; Lam et al. 1998). The processus vaginalis develops as a small invagination of the peritoneum into the ventral aspect of the gubernaculum in the indifferent stage of the male and female reproductive tracts (Gier & Marion, 1969). Further development in the fetus results in the sexually dimorphic changes in the structure of the gonads, gubernaculum, processus vaginalis, blood vessels and urogenital ducts that result in the male and female phenotype. Testicular descent requires the male-specific development of the gubernaculum and the elongation of the processus vaginalis, vas deferens and spermatic vessels.

Testicular descent occurs in two distinct phases, transabdominal migration followed by inguinoscrotal migration (Hutson, 1985; Hutson et al. 1997). During transabdominal migration the gubernaculum swells and the testes migrate from a location just below the kidneys to the internal inguinal ring (Hutson, 1985). The gubernacular swelling reaction occurs in all eutherian mammals that have ‘descended testes’ during adult life, including rodents (Gier & Marion, 1969; Wensing, 1986), cows (Hullinger & Wensing, 1985), pigs (Backhouse & Butler, 1960; Backhouse, 1964; Wensing, 1968), dogs (Gier & Marion, 1969), horses (Bergin et al. 1970) and humans (Wyndam, 1943; Heyns, 1987). The swelling of the gubernaculum results in testis migration to a position near the internal inguinal ring, and in the process it dilates the inguinal canal and scrotum to accommodate the testis (Williams & Hutson, 1991). During inguinoscrotal migration there is significant regression of the gubernaculum and migration into the scrotum, directing the testis in its descent (Hutson, 1985). During this phase, the processus vaginalis elongates into the mesenchyme of the gubernaculum, hollowing out the inguinal canal (Clarnette & Hutson, 1999).

Unlike in any other mammal so far described, in humans the inguinal canal closes after testicular descent. Failure of this process predisposes the individual to inguinal hernia (Clarnette & Hutson, 1999). Inguinal closure appears to be the result of controlled loss of the epithelium of the processus vaginalis (Hutson et al. 2000). When hernial sacs were induced to fuse in vitro epithelial cells were transformed into fibroblast-like mesenchymal cells that migrated into the underlying extracellular matrix, accompanied with an expression of the mesenchymal marker vimentin, and loss of expression of the epithelial cell marker cytokeratin (Hutson et al. 2000). Furthermore, F-actin arrangement changed from parallel to perpendicular (stress fibres) in relation to the plasma membrane during the culture period. The increase in vimentin and loss of cytokeratin and F-actin rearrangement is characteristic of epithelial–mesenchymal transformation (Cook et al. 2000).

As in eutherians, marsupial mammals have descended testes. This is clearly evident in the macropodids (the kangaroos and wallabies) where the scrotum is particularly pendulous (Tyndale-Biscoe & Renfree, 1987). Testicular descent appears to be similar to that of eutherian mammals in many aspects, but the anatomy and timing differ (Hutson et al. 1988; Griffiths et al. 1993). As a consequence of the highly altrical young, most sexual development occurs post partum (pp). The testes form seminiferous cords by day 2 pp, and transabdominal migration occurs between day 10 and 25 pp. In this phase the gubernaculum swells as the testis migrates to the internal inguinal ring (Hutson et al. 1988; Renfree et al. 1996). During inguinoscrotal descent, between day 25 and 60 pp, the gubernaculum regresses, as the testis migrates into the scrotum (Hutson et al. 1988; Renfree et al. 1996). Unlike in eutherian mammals, the scrotum develops cranially to the phallus (Tyndale-Biscoe & Renfree, 1987) and its development is independent of testicular hormones (Shaw et al. 1988, 1990; Renfree et al. 1995, 2001). Furthermore, the scrotum, gubernaculum and processus vaginalis are sexually dimorphic by day 0 pp, before the differentiation of the testis (Renfree & Short, 1988; O et al. 1988) or the production of testicular androgens (Renfree et al. 1992).

Marsupial and eutherian mammals diverged from the therian lineage around 100 million years ago (Archer & Clayton, 1984). As scrotal anatomy and development differs substantially within and between these two mammalian subclasses, the scrotal position of the testes may have evolved after their divergence. This study reports the developmental anatomy of testicular descent and inguinal closure in the tammar from the day of birth to maturity.

Materials and methods

Animals

Tammar wallabies of Kangaroo Island origin were held in open grassy yards in our breeding colony in Melbourne, Victoria, Australia. Food was supplemented with lucerne cubes, oats and fresh vegetables. Removal of the pouch young (RPY) during the breeding season reactivates the diapausing embryo and birth occurs after a 26.5 ± 0.4 day gestation (Renfree et al. 1989). Animals were checked daily for birth from day 24 RPY and the day when the young was first observed was termed day 0 pp. The pouch young were examined with an otoscope to determine sex by the presence of scrotal bulges (male) or mammary primordia (females) (O et al. 1988). Older pouch young were collected from animals culled on Kangaroo Island and ages were determined from headlength (Poole et al. 1991).

All animal handling and experiments conformed to the Australian National Health & Medical Research Council 1997 guidelines and were approved by institutional animal experimentation ethics committees.

Morphology of the gubernaculum testis and inguinal canal

Adult males (n = 10) were killed by overdose of pentobaritone (60 mg kg−1). The skin was removed from the abdominal wall and inguinal canal, exposing the abdominal muscles and external spermatic fasciae. An incision was made in either the tunica vaginalis (n = 5) or through the abdominal muscles (n = 5), exposing the testis or internal inguinal ring, respectively, and the anatomy was photographed. Patency of adult inguinal canals was investigated using a 1-mm blunt probe or by injection of trypan blue through the internal inguinal ring or into the scrotal cavity through the tunica vaginalis.

Male young of the ages of d 0 (n = 3), d 10 (n = 3), d 20 (n = 3), d 25 (n = 1), d 30 (n = 1), d35 (n = 1), d 40 (n = 2), d 50 (n = 2), d60 (n = 2), d 70 (n = 2), d 80 (n = 3), d 90 (n = 1) and female young of the ages of d 0 (n = 3), d 10 (n = 3), d 20 (n = 3) were fixed in 10% neutral buffered formalin (NBF), decalcified in 10% formic acid, paraffin embedded, either transversally or sagittally serially sectioned (Fig. 1) at 8 µm and stained with Harris' haematoxylin and Putt's eosin. The inguinal canals of older males of the ages of d 100 (n = 3), d 150 (n = 2), d 200 (n = 2) and adult (n = 2) were dissected out, fixed in 10% NBF, paraffin embedded, transversally serially sectioned at 8 µm and stained with Harris' haematoxylin and Putt's eosin.

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The plane of histological sections in Figs 3, ​5, ​6 and ​8 and in the two-dimensional reconstructions in Fig. 4. Sections were taken in a sagittal plane and the area of the gonad, mesonephros, gubernaculum and scrotum photographed.

As epithelial loss, via an epithelial–mesenchymal transition, occurs during inguinal closure in humans (Hutson et al. 2000), the presence or absence of the processus vaginalis epithelium and co-localization of intermediate fibres were assessed throughout development. Inguinal canals of male pouch young of the ages of d 20 (n = 1), d 30 (n = 1), d 40 (n = 2), d 50 (n = 3), d 60 (n = 2), d70 (n = 2), d 90 (n = 1), d 100 (n = 2), d 150 (n = 2) and d 300 (n = 1) were stained for cytokeratin. Inguinal canals of male pouch young of the ages of d 40 (n = 1), d 50 (n = 3), d 60 (n = 1) and d 100 (n = 2) were fixed for double cytokeratin and vimentin immunofluorescence.

Male pouch young of the ages of d 0, d 10, d 20, d 25, d 30, d 35, d 40 and d 50 (n = 1 in each age group) were fixed in 10% NBF for glycosaminoglycan and hyaluronic acid assay.

Cytokeratin Immunofluorescence

Inguinal canals were fixed in 4% PFA for 24 h at 4 °C, washed in 0.01 m PBS (pH 7.4), stored in 30% sucrose dissolved in 0.01 m PBS (pH 7.4) for 1–7 days at 4 °C, frozen in Tissue-Tek OCT (Miles Laboratories, Melbourne, Victoria, Australia) and stored at −80 °C until sectioning. Tissue sections were cut at 10 µm on a Reichert-Jung 3 CM3000 cryostat (Leica, Hawthorn East, Victoria, Australia) and mounted on superfrost slides (Grale Scientific, Ringwood, Victoria, Australia). Tissue sections were air-dried for at least 1 h at room temperature, washed in 0.01 m PBS (pH 7.4), post-fixed in ice cold 100% acetone and then washed in 0.01 m PBS (pH 7.4). Sections were then incubated with 0.5% BSA dissolved in 0.01 m PBS (pH 7.4) for 30 min at room temperature to decrease non-specific binding.

Two antibodies were used to immunolocalize cytokeratin in the tammar tissue. Antibody 5.2 CAM is a monoclonal mouse anti-human cytokeratin antibody (Cat. no. 349205; Becton-Dickinson, Victoria, Australia). 5.2 CAM cross-reacts with the cytokeratin subunits; 50 kDa (cytokeratin 8), 43 kDa (cytokeratin 18) and 39 kDa (cytokeratin 19), which are found in most epithelial cells (Makin et al. 1984). Antibody WSS is a polyclonal rabbit anti-bovine cytokeratin antibody (Cat. No. Z0622; DAKO (Australia), Botany, NSW, Australia) that was raised against cytokeratin subunits; 58 kDa (cytokeratin 5), 56 kDa (cytokeratin 11), 52 kDa (cytokeratin 8), 60 kDa (cytokeratin 4), 51 kDa (cytokeratin 14–15), and 48 kDa (cytokeratin 16). Antibody WWS recognizes a wide variety of cytokeratin subunits from a number of species including mouse, rat and human.

The 5.2 CAM and WWS antibodies were used undiluted and at a dilution of 1 : 800, respectively. A stock solution of 0.1% BSA dissolved in 0.01 m PBS (pH 7.4) was used to dilute all reagents. Tissue sections were incubated with primary antibodies for 24 h at 4 °C. After primary antibody incubation, sections were washed in 0.01 m PBS (pH 7.4) and incubated with secondary antibody at a dilution of 1 : 400 (Goat anti-mouse immunoglobulins/biotinylated or Goat anti-rabbit immunoglobulins/biotinylated; DAKO (Australia)) for 1 h at room temperature. Sections were then washed in 0.01 m PBS (pH 7.4) and incubated with avidin–biotin complex at a dilution of 1 : 400 (Vectastain ABC Kit; Vecta Laboratories, Camperdown, NSW, Australia) for 30 min at room temperature to amplify the final protein signal. Sections were then washed in 0.01 m PBS (pH 7.4) and incubated with streptavidin-Alexafluor 488 (Molecular Probes, Sydney, NSW, Australia) at a dilution of 1 : 400 for 40 min at room temperature. Sections were then washed in 0.01 m PBS (pH 7.5) and counter stained with propidium iodide (0.25 µg mL−1) (Sigma-Aldrich, Castle Hill, NSW, Australia) for 10 min at room temperature. After incubation the sections were washed in 0.01 m PBS (pH 7.4), mounted with Mowiol/DABCO antifade media, coverslipped and stored in the dark at 4 °C for a least 2 h before viewing. Sections were examined using a BioRad confocal laser microscope system with excitation laser wavelengths of 488 nm and 543 nm.

Double cytokeratin and vimentin immunofluorescence

Sections stained for cytokeratin with the WWS cytokeratin antibody and streptavidin-Alexafluor 564, as described above, were washed in 0.01 m PBS (pH 7.4) and incubated with mouse anti-vimentin antibody at a dilution of 1 : 30 for 1 h at room temperature. Slides were then washed in 0.01 m PBS (pH 7.4), incubated in goat anti-mouse immunoglobulins/FITC at a dilution of 1 : 200 (DAKO (Australia)) for 1 h at room temperature, washed in 0.01 PBS (pH 7.4). Sections were then mounted and examined using confocal microscopy as described above.

The vimentin antibody used was a monoclonal mouse anti-porcine Vimentin (Clone V9; DAKO (Australia)) that recognizes the 57-kDa intermediate filament protein present in cells of mesenchymal origin. This antibody shows wide species cross-reactivity, recognizing vimentin in primates, rodents and birds, and proved to be highly effective in this marsupial.

Glycosaminoglycans and hyaluronic acid

Male pouch young were fixed in 10% NBF, decalcified in 10% formic acid, paraffin embedded, sectioned at 8 µm and placed on two sets of superfrost slides (Grale Scientific, Ringwood, Victoria, Australia) for control and hyaluronidase digestion. Tissue sections were then deparaffinized and hydrated in distilled H2O before incubating with either hyaluronidase (1500 I.U.; Fisons Pharmaceuticals, Sydney, NSW, Australia) 1 : 5 dilution in 0.01 m PBS (pH 7.4) or with 0.01 m PBS (pH 7.4) alone for 1 h at 37 °C. After incubation, sections were washed in running water for 5 min, incubated in 3% acetic acid for 3 min, then counterstained in Alcian Blue solution (pH 2.5) for 30 min at room temperature. Sections were then washed in running water for 2 min and rinsed in distilled H2O before incubating for Nuclear-Fast red for 5 min. Slides were rinsed in running water, dehydrated, mounted in DPX (Grale Scientific) and coverslipped.

Results

Adult inguinal canal

In males the internal inguinal rings were situated in the postero-ventral abdominal wall and the inguinal canals passed lateral to the epipubic bone (a pair of bones running antero-laterally from the centre of the pubis in marsupials) to the scrotum. The cremaster formed a broad strip below the internal inguinal ring and more posteriorly it surrounded the canal (Fig. 2). The cremaster muscle entered the scrotum and attached to the tunica vaginalis, but at this connection the muscle was only present on the medial, lateral and dorsal aspects of the canal. A thin membrane attached to the postero-ventral abdominal wall and extended dorsally to cover the internal inguinal ring (Fig. 2a,f). The spermatic vessels and vas deferens passed under this membrane to enter the inguinal canal. A small 1-mm blunt probe could pass to the midpoint of the canal, but no further. However, injected dye was able to pass though a small lumen in the canal in both directions.

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The anatomy of the adult male inguinal canal. (a) A section of the inguinal canal at the level of the internal inguinal ring, showing the vas deferens (v) and spermatic vessels (s) lying underneath a membrane (m) that covers the entrance of the canal. (b) A section just distal to the inguinal ring, in which the thin processus vaginalis (pv) surrounds the vas deferens. The spermatic vessels are in a retro-peritoneal position and not within the processus vaginalis. The well developed cremaster muscle (c) runs along the dorsal aspect of the inguinal canal. (c) A section of the inguinal canal at a more posterior position, showing the vas deferens and pampiniform plexus (pp) situated in a larger processus vaginalis. (d) A section of an inguinal canal just proximal to the scrotum and testis, in which the vas deferens and large pampiniform plexus are situated within the processus vaginalis. At this level the cremaster muscle evenly surrounds the inguinal canal. (e) A schematic reconstruction of the adult inguinal canal showing the level of transverse sections represented in figures (a) to (d). (f) The internal inguinal ring of an adult male, showing the vas deferens and spermatic vessels entering the inguinal canal underneath a membranous flap that covers the internal inguinal ring. Scale bars represent 2 mm in a, b, c and d, and 5 mm in f.

The gonad, gubernaculum, cranial suspensory ligament and processus vaginalis

The undifferentiated testis of day 0 males was situated on the medial side of the mesonephros as a ridge of somatic and germ cells (Fig. 3a,b). The mesonephros attached to the dorsal abdominal musculature and extended the entire length of the abdominal cavity (Fig. 3a,b). The Wolffian and Müllerian ducts were present extending along the lateral aspect of the mesonephros. The gubernaculum consists of three contiguous structures at this age. The plica gubernaculi, the strand that attaches to the mesonephric–gonadal complex, was surrounded by the processus vaginalis (Figs 3a–c and ​4b). The pars vaginalis gubernaculi surrounded the processus vaginalis. The pars infravaginalis gubernaculi was distal to the processus vaginalis and continuous with the pars vaginalis gubernaculi (Figs 3 and ​4b). The plica gubernaculi consisted of a cord of mesenchyme attached to the distal portion of the mesonephros and gonad and extended through the inguinal canal connecting to the pars infravaginalis gubernaculi. The pars infravaginalis gubernaculi passed ventro-medially, lateral to the epipubic bone, and into the small scrotal bulge, in which it expanded (Fig. 3b,c). The pars infavaginalis gubernaculi was clearly identifiable as a mass of densely staining mesenchymal cells against the lighter staining and more dispersed mesenchyme of the surrounding tissue (Fig. 3a-c). The processus vaginalis was present as a thin, deep cavity in the mesenchyme of the gubernaculum extending from the abdominal wall, past the external inguinal ring, terminating at the level of the epipubic bone (Fig. 3a). The processus vaginalis formed a crescent-moon-shaped cavity in cross-section and had a squamous epithelium similar to that of the peritoneum. The abdominal musculature terminated at the internal inguinal ring, and muscle cells were completely absent from the three parts of the gubernaculum. The cranial suspensory ligament was present and consisted of a cord of mesenchyme attached to the anterior surface of the mesonephros and gonad and connected to the psoas muscle, posterior to the developing metanephros.

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A series of sagittal sections from lateral to medial (left to right) of male pouch young of the ages of day 0 (a–c), day 10 (d–f) and day 20 (g–i). (a) A lateral section of a day 0 male showing the testis (t) and mesonephros (m) situated high in the abdomen. The Müllerian duct (md) is situated at the posterior aspect of the mesonephros. A thin plica gubernaculi (pl) is present, surrounded by the processus vaginalis and pars vaginalis gubernaculi. The processus vaginalis teminates beside the epipubic bone (e). The scrotal bulges (s) are prominent. (b) A section showing the continuous gubernacular structure extending from the mesonephros to the scrotum. The plica gubernaculi is attached to the mesonephros and gonad and extends into the inguinal canal. The pars infravaginalis gubernaculi (pi) expands with the mesenchyme of the scrotum. (c) A medial section of a day 0 male showing the pars infravaginalis gubernaculi as a dark mass of mesenchyme within the scrotum. (d) A lateral section of a day 10 male showing the testis attached to the mesonphros situated within the abdominal cavity. The plica gubernaculi is attached to the mesophros and is surrounded by the processus vaginalis (pv). (e) A section showing the cremater muscle (c) developing on the dorsal aspect of the pars vaginalis gubernaculi (pr). The processus vaginalis is well developed and extends past the epipubic bone. (f) A medial section showing the pars infravaginalis gubernaculi within the scrotum. (g) A lateral section of a day 20 male in which the testis and regressing mesonephros are situated at the internal inguinal ring. The plica gubernaculi is larger than that of a day 10 male and contains the Wolffian duct (wd). (h) A section showing the processus vaginalis at the neck of the scrotum. The cremaster muscle is developing on the dorsal aspect of the pars vaginalis gubernaculi. (i) The pars infravaginalis gubernaculi is situated within the scrotum and the cremaster is developing on its dorsal aspect. Scale bars represent 500 µm.

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Two-dimentional reconstructions of (a) a day 0 female, (b) a day 0 male, (c) a day 10 male, (d) a day 20 male, and (e) a day 30 male. Scale bar represents 1 mm. o = ovary; t = testis; m = mesonephros; g = gubernaculum; pl =plica gubernaculi; pr =pars vaginalis gubernaculi; pi =pars infravaginalis gubernaculi; pv = processus vaginalis; v = vas deferens; ep = epididymis; s = scrotum; mp = mammary primordium.

The undifferentiated ovary of day 0 females was situated on the medial side of the mesonephros as a ridge of somatic and germ cells (Fig. 5a,b). As in males, the mesonephros was well developed, extending the entire length of the abdominal cavity (Fig. 5a,b). The Müllerian and Wolffian ducts were present, extending along the lateral aspect of the mesonephros. The gubernaculum was poorly developed compared to that of the day 0 male (Fig. 4a,b). It consisted of a very thin cord of mesenchyme, devoid of musculature, that attached to the mesonephros and gonad, and extended past the internal inguinal ring, passing ventro-medially laterally to the epipubic bone, connecting to the tissues underlying the mammary primordia (Figs 4a and 5a,b). The gubernacular mesenchyme was not easily identifiable from the surrounding tissue and consisted of 5–6 cell layers orientated in a ventro-medial direction. The processus vaginalis (canal of Nuck) was present as a short and narrow diverticulum in the gubernaculum mesenchyme at the level of the abdominal wall (Figs 4a and ​5a). Unlike the processus of day 0 males, it only extended a short distance, terminating before the external inguinal ring (Fig. 4a,b). The cranial suspensory ligament was clearly identifiable, consisting of a thin cord of mesenchymal cells that attached to the proximal mesonephric–gonadal complex and connected to the psoas muscle at the lower pole of the metanephros.

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A series of sagittal sections from lateral to medial (left to right) of a female pouch young at day 0 (a,b), day 10 (c,d) and day 20 (e,f). (a) A lateral section of a day 0 female showing the ovary (o) and mesonephros (m) situated high in the abdomen. The gubernaculum (g) is attached to the posterior portion of the mesonephros, ovary and Müllerian duct (md) and extends into the abdominal wall. The processus vaginalis (pv) is present as a small cavity in the gubernaculum at the level of the abdominal wall. (b) A medial section of a day 0 female showing the gubernaculum extending from the abdominal wall to the dorsal aspect of the mammary primordia (mp). (c) A lateral section of a day 10 female showing the mesonephros, Müllerian and Wolffian ducts (wd) situated within the abdominal cavity. The gubernaculum extends past the abdominal wall, but the processus vaginalis teminates before the external inguinal ring. (d) A medial section of a day 20 female showing the attachment of the gubernaculum to the developing Müllerian duct. (e) A lateral section of a day 20 female, showing a well-developed ovary attached to a regressing mesonephros. The gubernaculum extends past the external inguinal ring, but the processus vaginalis remains small terminated within the inguinal canal. (f) A medial section of a day 20 female in which the gubernaculum is in close opposition with the Müllerian duct and extends past the abdominal musculature. Scale bars, 500 µm.

By day 10 the testis was well developed and had clearly identifiable seminiferous cords, Sertoli cells and germ cells (Fig. 3d). The testis was an ovoid shape attached to the mesonephros via the developing mesorchium (Fig. 3d). The mesonephros and testis were still situated high in the abdomen (Fig. 3c,d). The gubernaculum had increased in size, but still consisted of densely packed dark staining mesenchyme that extended from the distal portion of the mesonephros and testis to the scrotum (Fig. 3d–f). The processus vaginalis had extended along the length of the gubernaculum, terminating at the neck of the scrotum (Figs 3d,e and ​4c). The pars vaginalis gubernaculi had increased in thickness and the cremaster muscle was developing on its dorsal aspect, adjacent to the mesenchymal bridge connecting the pars vaginalis gubernaculi with the plica gubernaculi (Fig. 3e). The longitudinal muscle fibres extended along the pars vaginalis gubernaculi and terminated before the fundus of the processus vaginalis, not extending into the scrotum. The scrotal bulges had increased in size and had begun to fuse, forming the single scrotum. The scrotal mesenchyme comprised loose matrix, within which the denser mesenchyme of the gubernaculum was clearly distinguishable (Fig. 3f). The cranial suspensory ligament had regressed and was completely absent at the anterior of the mesonephros.

The ovary in day 10 females had developed a medulla and cortex with clearly identifiable germ cells. The gubernaculum was well developed and attached to the mesonephros and developing Müllerian ducts extending through the internal inguinal ring connecting to the mammary primordia (Fig. 5c,d). A thin layer of muscle, the developing ilio-marsupialis, was present on the dorsal, medial and lateral aspects of the gubernaculum just distal to the abdominal musculature extending to the level of the epipubic bone. The processus vaginalis was present in the mesenchyme of the gubernaculum and appeared to be increased in length from that of a d 0 female, but still terminated before the external inguinal ring (Fig. 5c). Therefore, this apparent increase in length was due to the thickening of the surrounding musculature and not due to active invasion of the processus vaginalis. The cranial suspensory ligament was present as a thin cord of mesenchyme attached to the distal portion of the mesonephros and extended to the psoas muscle below the metanephros.

In males by day 20 the mesonephros had regressed, only persisting as a network of tubules adjacent to the testis (Fig. 3g). The testis had descended across the abdominal cavity and was situated at the internal inguinal ring (Figs 3g and ​4d). The plica gubernaculi had increased in size to about the same size of the testis (Figs 3g,h and ​4d). There appeared to be a decrease in cell density of the plica gubernaculi reflected by the lighter staining mesenchyme (Fig. 3g). Although the gubernaculum increased distal to the external inguinal ring, the greatest increase in size occurred at the level of the internal and external inguinal rings (Fig. 4d). The processus vaginalis increased in size as a result of the enlarged plica gubernaculi, but still terminated at the neck of the scrotum (Figs 3g and ​4d). The majority of the cremaster muscle was still on the dorsal aspect of the pars vaginalis gubernaculi, but thin layers of muscle cells were present on the lateral and medial aspects (Fig. 3g–i). The cremaster had also increased in length on the dorsal aspect, extending past the fundus of the processus vaginalis at the neck of the scrotum (Fig. 3i).

By day 20 the ovary of female young had migrated caudally, but was situated relatively higher than the testis of a day 20 male. The plica gubernaculi was a long and thin cord, attaching the Müllerian duct and terminating at the internal inguinal ring (Fig. 5e,f). The developing ilio-marsupialis muscle had increased in length from that of a d 10 female, extending from the external inguinal ring, past the epipubic bone and terminating near the dorsal aspect of the mammary primordia. The processus vaginalis was very small and only extended a short distance into the internal inguinal ring (Fig. 5e,f). The cranial suspensory ligament was still present as a cord of mesenchymal cells. It attached to the proximal portion of the ovary and regressing mesonephros and extended to the psoas muscle, below the metanephros.

At day 30 the testis had entered the inguinal canal, carrying with it the Wolffian duct, regressing mesonephros and the spermatic vessels (Fig. 6b). The Wolffian duct was connected to the gubernaculum and extended from the base to the top of the testis (Fig. 6a–c). The spermatic vessels exited the peritoneum before entering the inguinal canal and connected to the testis at the same level as the Wolffian duct. The plica gubernaculi consisted of dispersed mesenchymal cells, which attached to the Wolffian duct and testis and extended into the base of the scrotum (Figs 4e and ​6c). The processus vaginalis had extended into the scrotum (Figs 4d and ​6c). The scrotum had increased in size with a stroma consisting of highly dispersed mesenchymal cells (Fig. 6c). The developing cremaster muscle was mainly on the dorsal aspect of the pars vaginalis gubernaculi, extending into the dorsal portion of the pars infravaganalis gubernaculi within the scrotum.

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A series of sagittal sections from lateral to medial (left to right) of male pouch young at day 30 (a–c), day 40 (d–f) and day 50 (g–i). (a) A lateral section of a day 30 male in which the testis (t) is situated within the processus vaginalis (pv) just distal to the epipubic bone (e). The Wolfian duct is differentiating into the vas deferens (v) and epididymis (ep). (b) A section of a day 30 male showing the relative position of the testis and scrotum (s). (c) A medial section of a day 30 male showing the plica gubernaculi (pl) attached to the epididymis and extending into the scrotum. The processus vaginalis also terminates with the scrotum. (d) A lateral section of a day 40 male in which the well-developed testis is situated within the processus vaginalis. The cremaster muscle (c) is developing on the dorsal aspect of the inguinal canal. (e) The processus vaginalis, plica gubernaculi, pars vaginalis gubernaculi (pr) and the cremaster are present within the scrotum. (f) The pars infravaginalis gubernaculi is present with the scrotum situated distal to the processus vaginalis. (g) A lateral section of a day 50 male, in which the testis is situated at the neck of the scrotum. The vas deferens and pampiniform plexus (pp) are situated within the inguinal canal. The plica gubernaculi is attached to the epididymis and extends a short distance into the scrotum. (h) A section showing the processus vaginalis and plica gubernaculi with the scrotum.(i) The pars infravaginalis gubernaculi is present within the scrotum below the processus vaginalis. Scale bars, 1 mm.

By day 40 the testis had descended further and the distal portion of the epididymis was at the neck of the scrotum (Fig. 6d). The mesonephros had completely regressed and only the vas deferens and epididymis remained. The epididymis was developing in the mesenchyme of the gubernaculum as numerous epididymal coils. The plicae gubernaculi attached to the epididymis and extended a short distance to the base of the scrotum (Fig. 6d–f). The processus vaginalis terminated in the centre of the scrotum (Fig. 6e). The cremaster extended dorsally, as a strip, close to the internal inguinal ring and surrounded the canal more caudally.

By day 50 the testis was at the neck of the scrotum and the first signs of the pampiniform plexus could be identified as a network of blood vessels proximal to the testis (Fig. 6g,h). The epididymis was well developed and its distal portion was within the scrotum. The gubernaculum attached to the distal portion of the epididymis and extended a short distance to the base of the scrotum (Fig. 6g,h). The processus vaginalis terminated at the base of the scrotum just before the tip of the gubernaculum.

By day 70 the testis was situated in the base of the scrotum and the basic structure of the inguinal canal was complete. A membranous fold covered the internal inguinal ring, which the vas deferens and spermatic vessels passed under to enter the canal. The processus vaginalis had an hourglass shape. Below the internal inguinal ring it constricted to a small cavity only large enough to allow the vas deferens to pass. The processus vaginalis then increased in diameter more caudally, allowing the pampiniform plexus to expand out of the peritoneum and into the canal. The cremaster near the internal inguinal ring extended latero-dorsally from the canal and caudally extended as a thin layer surrounding the canal, but was absent from the ventral aspect within the scrotum.

After day 70 the basic structure of the inguinal canal remains the same, but there is substantial growth of the canal as a whole. The spermatic vessels increase in length and the pampiniform plexus expands. The pars vaginalis gubernaculi differentiates into the internal spermatic fasciae and the processus vaginalis persists as the tunica vaginalis surrounding the testis. The cremaster increases in thickness surrounding the inguinal canal near the scrotum and forms a muscular strip in the anterior of the canal.

Inguinal closure

All epithelial cell types tested were positive for cytokeratin, whilst mesenchymal and endothelial cell types had no staining (Table 1). Vimentin was localized in mesenchymal cell types, but was not detected in epithelial and endothelial cell types (Table 1).

Table 1

Tissue specificity of cytokeratin and vimentin immunostaining with tammar wallaby tissue

Tissue Cytokeratin Vimentin
Peritoneum + −
Processus vaginalis epithelium + −
Tunica vaginalis + −
Testis:
Surface epithelium + −
Sertoli cells − +
Tunica albuginea − +
Interstitium − +
Vas deferens:
Lumen epithelium + −
Surface epithelium + −
Mesenchyme − +
Spermatic vessels:
Vascular endothelium − −
Mesenchyme − +
Surface epithelium + −

At day 20 the processus vaginalis epithelium was clearly identifiable on the surface of the plicae and pars vaginalis gubernaculi (Fig. 7a). By day 40 the testis, blood vessels, epididymis and vas deferens were surrounded by the processus vaginalis and the epithelial lining of the processus was evident with no obvious signs of epithelial loss (Fig. 7b). Between day 50 and 60 there was a decrease in the diameter of the canal in which the processus vaginalis tightly surrounded the vas deferens (Fig. 7c,d). However, there was no obvious loss of the processus vaginalis epithelium. The small cavity that surrounds the vas deferens is present at day 100 (Fig. 7e) and persists into adult life. Vimentin and cytokeratin were not co-localized in any of the tissues or the epithelium of the processus vaginalis in the animals tested between day 40 to day 100.

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Cytokeratin immunolocalization (green fluorescence) on male inguinal canals (transversely sectioned) which were counter stained with propidium iodide (red fluorescence) that stains nuclei. (a) The inguinal canal of a day 20 male in which cytokeratin was immunolocalized to epithelium of processus vaginalis (pv). The plica gubernaculi (pl) is situated within the processus vaginalis. (b) An inguinal canal from a day 40 male, in which the testis (t), epididymis (ep) and vas deferens (v) are surrounded by the processus vaginalis epithelium. (c) At day 50 the processus vaginalis epithelium surrounds most of the enlarged spermatic vessels (sv) and vas deferens. (d) An inguinal canal of a day 60 male, in which the processus vaginalis only surrounds the vas deferens. (e) An inguinal canal of a day 100 male showing the processus vaginalis epithelium persisting only arround the vas deferens. Scale bars, 500 µm.

Glycosaminoglycans

The gubernaculum of day 0 males did not stain with Alcian blue; however, the mesenchyme of the scrotum was highly positive (Fig. 8a). Hyaluronidase incubation significantly decreased Alcian blue staining within the scrotal mesenchyme (Fig. 8b). By day 10, both the gubernaculum and the scrotum mesenchyme were positive for Alcian blue, which was decreased by incubation with hyaluronidase (Fig. 8c,d). The level of Alcian blue staining within the gubernaculum and scrotum increased by day 25 and appeared to remain at a constant level up to day 40 (Fig. 8e,g). Incubation with hyaluronidase significantly decreased Alcian blue staining over this time period (Fig. 8f,h). Staining of bone by alcian blue was not affected by hyaluronidase incubation.

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Sagittal sectioned pelvic regions from male pouch young at days 0 (a,b), 10 (c,d) and 35 (e,f). (a) A control day 0 male stained with alcian blue, which stains glycosaminoglycans. Alcian blue staining is very weak in the gubernaculum (g), but stronger in the mesenchyme of the scrotum (s). (b) An adjacent section which has been incubated with hyaluronidase before alcian blue staining shows reduced staining in the scrotum and gubernaculum. (c) A control treated section from day 10 male showing strong alcian blue staining in both the gubernaculum and the scrotum, which is reduced by hyaluronidase treatment (d). (e) The strong alcian blue staining of both the gubernaculum and scrotum at day 35 is substantially reduced by hyaluronidase (f). Scale bars represent 500 µm. pv = processus vaginalis.

Discussion

In the tammar wallaby the inguinal canal substantially closes after the testis descends into the scrotum. This is the first report of inguinal closure in any mammal other than man.

The gubernaculum, its muscle and the processus vaginalis are sexually dimorphic by the day of birth in the developing wallaby (O et al. 1988) and the differences become greater after birth. In females the gubernaculum differentiates into the round ligament within the abdomen and extra-abdominally the ilio-marsuipialis muscle develops on its periphery. In males the gubernaculum induces the descent of the testis and only persists as the external spermatic fasciae. The cremaster muscle also develops on the periphery of the pars vaginalis gubernaculi.

The ilio-marsupialis muscle is unique to marsupials and no homologous structure is present in female eutherian mammals. It is a broad band of striated muscle that attaches to the pelvis and extends ventrally, penetrating deep into the mammary glands (Tyndale-Biscoe & Renfree, 1987). Its function appears to differ between pouched and pouchless marsupials. The ilio-marsupialis in pouchless marsupials provides mechanical support for the attached young, which can in some species weigh up to 47% of the mother's body weight (Griffiths & Slater, 1988). In pouched marsupials, the ilio-marsupialis is less well developed, but may help in gland contraction to express milk during suckling (Griffiths & Slater, 1988) and it carries the genitofemoral nerve that innervates the mammary gland (Renfree, 1979).

By day 10 the cremaster muscle in males and ilio-marsupialis muscle in females develop on the periphery of the extra-abdominal gubernaculum, well after the differentiation of the testis. However, the sexually dimorphic development of these muscles does not appear to be dependent on gonadal hormones as male pouch young treated with the anti-androgen, flutamide, or oestrogen developed cremaster muscles (Lucas et al. 1997; Shaw et al. 1988; Coveney et al. 2002a, 2002b). In contrast, the gubernaculum is sexually dimorphic before testis differentiation, and like the ilio-marsupialis and cremaster muscles it attaches either to the mammary gland or to the scrotum. Thus the sexually dimorphic development of the gubernaculum may direct the differentiation of these muscles.

Transabdominal migration of the gonads in the tammar occurs between day 10 and day 20 pp followed by inguinoscrotal descent between day 20 and 60 pp (Hutson et al. 1988; Renfree et al. 1996). During this phase in the tammar there is a significant increase in the bulk of the gubernaculum and scrotum, which is associated with an increase in the concentration of glycosaminoglycans. A significant proportion of the total glycosaminoglycan concentration consists of hyaluronic acid. Glycosaminoglycans are strongly hydrophilic, inflexible polysaccharide chains that occupy a large volume in the extracellular matrix (Alberts et al. 1994). Gubernacular swelling is distinctive in all eutherian mammals that exhibit testicular descent and appears to be the result of hyperplasia and hypertrophy of the gubernacular mesenchyme, via a rapid increase in cell proliferation, water binding capacity and glycosaminoglycan content (Backhouse, 1964; Wensing, 1973; Heyns et al. 1986, 1990; Fentener van Vlissingen et al. 1989).

One of the most interesting aspects of descent in the tammar is that the inguinal canal goes through a process of closure after the testis has reached the scrotum. Inguinal closure is an uncommon developmental process among mammalian species that have descended testes and until now was thought to be confined to humans. In humans closure is the result of the obliteration of the processus vaginalis epithelium, leaving the underlying mesenchyme to fuse around the spermatic cord (Cook et al. 2000; Hutson et al. 2000). In the tammar, the testis is situated within the scrotum by day 60 (Hutson et al. 1988; Renfree et al. 1996) at which time the inguinal canal has closed. Inguinal closure is the result of a reduction in the diameter of the processus vaginalis, only allowing the vas deferens to pass through. This reduction in processus vaginalis size is not the result of epithelial–mesenchymal transformation of the processus vaginalis epithelium since there is no significant co-localization of cytokeratin or vimentin intermediate filaments. Although the inguinal canal is essentially closed, and the testis cannot pass back into the abdomen, a small lumen persists that fluid can pass through. The degree of closure in the tammar is similar to that seen in humans with scrotal hydrocoeles, in which a small lumen persists along the length of the canal allowing peritoneal fluid to fill the scrotum. Unlike scrotal hydrocoeles in humans, significant amounts of peritoneal fluid are never present in the tunica vaginalis of the wallaby. This may be due to the development of an alternative mechanism to prevent fluid entry into the scrotum. By day 70 a membranous flap or valve has developed that covers the internal inguinal ring and we suggest that this prevents coelomic fluid from entering the scrotum via the processus vaginalis.

As in humans, if inguinal closure fails in the tammar it predisposes the affected animal to inguinal hernia. Inguinal closure in the tammar is androgen dependent and treatment with the anti-androgen, flutamide, results in 33–75% incidence of inguinal hernia depending on time and duration of treatment (Lucas et al. 1997; Coveney et al. 2002a). The release of calcitonin gene related peptide (CGRP) from the CGRP neurones of the genitofemoral nerve have been implicated in this process of inguinal closure. In humans, patients with androgen-insensitive syndrome or abnormalities in the development of the GFN are linked with patent inguinal canals and disorders such as inguinal hernias and scrotal hydrocoeles (Atwell, 1961, 1962; Clarnette & Hutson, 1996). Furthermore, CGRP induces processus vaginalis epithelial obliteration and fusion of human hernia sacs in culture, suggesting an important role for CGRP in this process (Hutson et al. 2000). There is evidence that the GFN and CGRP may be involved in inguinal closure in the tammar. CGRP is present within the GFN cell bodies during the period of inguinal close (Coveney et al. 2002a). CGRP is expressed in a sexually dimorphic pattern, with more CGRP cell bodies in males than in females (Coveney et al. 2002a). This sexual dimorphism is androgen dependent as males treated with flutamide have significantly fewer CGRP cell bodies, suggesting that the high incidence of inguinal hernia in these animals may be the result of decreased CGRP exposure (Coveney et al. 2002a).

Although extra-abdominal scrotal testes appear to have evolved separately within the Class Mammalia, the initial development of the testis and associated structures are similar. In all mammals with testicular descent the cranial and caudal ligaments suspend the mesonephros and gonad in the abdominal cavity. The formation of the processus vaginalis as an invagination of the peritoneum into the gubernaculum mesenchyme is similar. Furthermore, swelling of the gubernaculum via an increase in glycosaminoglycans and water is similar in both subclasses. Interestingly, initial comparative research on the hormonal control of gubernacular swelling in marsupials and eutherians has revealed no significant differences, but further studies are needed to confirm this (Renfree et al. 2001). The existence of primary testicond eutherian mammals, such as the elephant, which completely lack a gubernaculum during development (Gaeth et al. 1999), suggests that initial caudal migration may have evolved after marsupials and eutherians diverged. In most mammals, once the testis had moved into the scrotum, the inguinal canal remained open. With the development of an upright posture, there would have been a significant selective pressure to prevent herniation of the gut through the narrow inguinal canal. We suggest that inguinal closure in macropodid marsupials has evolved as a result of the upright mode of locomotion and the need to prevent inguinal hernias.

Acknowledgments

We thank Deidre Mattiske and Patrick Jackson for help in field animal collection. Animals were collected under permits from South Australian National Parks and Wildlife, and held under permit number RP-95-088 of the Department of Natural Resources and Environment, Victoria. This study was funded by grants from National Health and Medical Research Council of Australia, grant number 980779.

References

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