Identification of the uterine factor(s) which induce the acrosome reaction in buffalo (Bubalus bubalis) spermatozoa (original) (raw)
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International Journal of Andrology, 2002
In this study, we investigated the effect of heparin and calcium ionophore A23187 on in vitro induction of buffalo sperm acrosome reaction (AR). Two methods for detection of the AR and viability were employed. Fluorescein isothiocyanate-conjugated Arachis hypogea agglutinin (FITC-PNA) was used as a vital stain in combination with ethidium homodimer-1 (EthD-1) to determine the acrosome status of viable spermatozoa. In another experiment, trypan blue replaced EthD-1 to differentiate live and dead spermatozoa having undergone AR. The results from the two methods were significantly correlated (r > 0.9). Four different staining patterns were found in both methods. The FITC-PNA intensely labels the acrosome region of acrosome-intact spermatozoa. EthD-1 and trypan blue stained red and blue at the post-acrosomal region of dead spermatozoa, respectively. Spermatozoa incubated with heparin showed a significant increase ( p < 0.05) in the percentage of live acrosome-reacted sperm after 30 min incubation period. This trend continued and was significantly different over the entire incubation period when compared with the control group at the same interval. In the ionophoretreated group, the proportion of changes in live acrosome-intact and live acrosomereacted spermatozoa was statistically significantly different ( p < 0.001) when compared with those treated with heparin at the same interval. The AR occurred sooner and to a greater extent when incubated with the ionophore but at 5 h of incubation the percentage of false acrosomal reaction was significantly higher than those in the control and heparin-treated groups. The results in this study indicated that the in vitro induction of AR by heparin and calcium ionophore evaluated by both methods could be used to assess sperm fertilizing capacity for in vitro fertilization of this species.
Background: Endocrine disruptors are exogenous substance, interfere with the endocrine system, and disrupt hormonal functions. However, the effect of endocrine disruptors in different species has not yet been elucidated. Therefore, we investigated the possible effects of 17ß-estradiol (E2), progesterone (P4), genistein (GEN) and 4-tert-octylphenol (OP), on capacitation and the acrosome reaction in bovine, mouse, and porcine spermatozoa. In this in vitro trial, spermatozoa were incubated with 0.001-100 μM of each chemical either 15 or 30 min and then assessed capacitation status using chlortetracycline staining. Results: E2 significantly increased capacitation and the acrosome reaction after 30 min, while the acrosome reaction after 15 min incubation in mouse spermatozoa. Simultaneously, capacitation and the acrosome reaction were induced after 15 and 30 min incubation in porcine spermatozoa, respectively. Capacitation was increased in porcine spermatozoa after 15 min incubation at the lowest concentration, while the acrosome reaction was increased in mouse spermatozoa after 30 min (P <0.05). E2 significantly increased the acrosome reaction in porcine spermatozoa, but only at the highest concentration examined (P <0.05). P4 significantly increased the acrosome reaction in bovine and mouse spermatozoa treated for 15 min (P <0.05). The same treatment significantly increased capacitation in porcine spermatozoa (P <0.05). P4 significantly increased capacitation in mouse spermatozoa treated for 30 min (P <0.05). GEN significantly increased the acrosome reaction in porcine spermatozoa treated for 15 and 30 min and in mouse spermatozoa treated for 30 min (P <0.05). OP significantly increased the acrosome reaction in mouse spermatozoa after 15 min (P <0.05). Besides, when spermatozoa were incubated for 30 min, capacitation and the acrosome reaction were higher than 15 min incubation in E2 or GEN. Furthermore, the responsiveness of bovine, mouse and porcine spermatozoa to each chemical differed. Conclusions: In conclusion, all chemicals studied effectively increased capacitation and the acrosome reaction in bovine, mouse, and porcine spermatozoa. Also we found that both E2 and P4 were more potent than environmental estrogens in altering sperm function. Porcine and mouse spermatozoa were more responsive than bovine spermatozoa.
Evaluation of the acrosomal status in Lama glama sperm incubated with acrosome reaction inducers
Animal Reproduction Science, 2015
The objectives of this study were to evaluate the effect of different acrosome reaction (AR) inducers on viability and acrosomal status in llama spermatozoa, by using the FITC-PNA/PI technique and evaluate if there is a positive correlation between the FITC-PNA/PI and the Coomassie blue (CB) staining techniques. After incubating twenty ejaculates in 0.1% collagenase the centrifuged pellets were resuspended in TALP-BSA medium. An aliquot was sonicated to remove the acrosomal content (positive control). The rest of the sample was incubated for 3 h at 38 • C with 5% CO 2 and 100% humidity. Treatments: Three aliquots were further incubated 1 h with one of the following AR inducers: calcium ionophore, ionomycin or progesterone. Controls: One without inducers and the other, incubated with dimethyl sulfoxide (vehicle of the inducing agents). Acrosomes were evaluated at time 0 and after 4 h incubation. Calcium ionophore was the most potent agent for inducing the AR (67.2 ± 14.4% live + dead AR sperm) (P < 0.05). These samples showed no motility and viability was very low (0-30%). Both ionomycin and progesterone presented significantly higher (P < 0.05) percentages of total AR sperm than the controls, but had similar percentages of dead reacted sperm to the controls. A positive correlation was observed between the intact acrosome FITC-PNA/PI pattern (live + dead sperm) and the acrosome-present CB pattern (r = 0.64; P = 0.000) in all the evaluated samples. Conclusions: the FITC-PNA/PI technique simultaneously evaluates viability and acrosomal status in llama spermatozoa and calcium ionophore could be used as a control of AR.
Evaluation of boar and bull sperm capacitation and the acrosome reaction using flow cytometry
Animal Reproduction Science, 2021
Flow cytometry can be used to evaluate many sperm attributes and Dr. Duane Garner was influential in developing assays to understand sperm physiology and function. We review some of Dr. Garner's work and describe experiments that evaluate sperm capacitation using Dr. Garner's philosophy. In exploratory experiments, boar sperm were cryopreserved in lactose egg yolk (LEY) or Beltsville Freezing Extender 5 (BF5) and incubated in one capacitating medium. In another experiment, frozen-thawed bull sperm were incubated in TALP-Ca or CFDM1 capacitating media. In both experiments, sperm viability and capacitation were evaluated using multiple probes. Boar sperm frozen in LEY had greater survival rates (38%) than sperm frozen in BF5 (22%; P < 0.05) but did not capacitate as effectively as sperm in BF5 (P < 0.05). In Experiment 2, bull sperm survived to a greater extent when incubated in TALP-Ca than in CFDM1 (P < 0.05) and had greater capacitation for most parameters (P < 0.05). Of particular interest, 77% of sperm incubated in TALP-Ca had activated second messenger systems involved in capacitation, compared with < 5% of sperm incubated in CFDM1. The results indicate different freezing and capacitating media induce different responses to sperm capacitation and functions. If only sperm viability and acrosomal integrity were evaluated, these results would be interpreted very differently. Dr. Garner's philosophy of evaluating multiple sperm parameters was an impetus to determine unique treatment differences which help in understanding sperm capacitation, and design further experiments to determine how media content causes sperm physiology differences.
Reproduction, 1974
Epididymal spermatozoa of the golden hamster can undergo the acrosome reaction and subsequently penetrate eggs in vitro in a variety of heat-pretreated blood sera . This report describes the effect of varying sperm concentration on the acrosome reaction and sperm penetration of eggs in vitro using heat-pretreated (60\s=deg\C for 1 hr) blood sera. The relationship between sperm concentration and occurrence of the acrosome reaction was examined in the blood sera of five mammalian species, including baboon, chimpanzee, hamster, rhesus monkey and rabbit (rabbit anti-hamster sperm-immune serum). Two additional experiments examined the relationship between sperm concentration and capacitation, i.e. the capacity, as classically defined, of spermatozoa to penetrate eggs (Text- ).
Capacitation of Bovine Spermatozoa by Oviduct Fluid1
Biology of Reproduction, 1989
Oviduct fluid collected from chronically cannulated oviducts of he(fers was evaluated for its effect on capacitazion of bovine sperm in vitro. Capacitation was determined by the ability of sperm to fertilize bovine oocytes in vitro and to undergo an acrosome reaction (AR) upon exposure to lysophosphatidylcholine (LC). After incubation of sperm with 0-25% (v/v) estrual oviduct fluid (collected ± 1 day from estrus) for 4 h, addition of LC (100 pg/ml) for an additional 025 h resulted in an increasing percentage of acrosome-reacted sperm as the concentration of oviduct fluid increased. Sperm incubated 4 h with 25% estrual oviduct fluid fertilized more oocytes than sperm incubated in medium alone (p<O.O5) but was not different from sperm incubated with 10 p.g/ml heparin (p>'O.05). Glucose inhibited the ability of LC to induce ARs in sperm incubated 4 h with heparin or estrual oviduct fluid. Incubation of sperm with 25% oviduct fluid collected at various days over the estrous cycle demonstrated that peak capacitasing activity was found at estrus but was also present ± 1 day from estrus. The active capacitating factor in oviduct fluid was found to be heat stable. In addition, when extraction procedures were applied in sequential order, oviduct fluid capacitating activity was resistant to protease digestion, precipitable by ethanol, size-excluded by Sephadex G-25, and destroyed by nitrous acid. These results suggest that a heparin-like glycosaminoglycan from the oviduct is a potential in vivo capacitating agent in the bovine.
Relationship of in Vitro Acrosome Reaction to Sperm Function: An Update
Understanding the mechanisms by which the acrosome reaction is regulated is central to models of fertilization. This article reviews the relationship of the acrosome reaction detected in vitro to sperm function. Proteolytic enzymes in the acrosome digest through the zona pellucida allowing for sperm-oolemma fusion. When this process is impaired, either by lack of an acrosome or acrosomal dysfunction, fertility can be compromised. Optical microscopy and staining with different fluorescent lectins that bind to acrosomal membranes is the method of choice for acrosomal evaluation. Because acrosomal loss can be a result of sperm death, this test should be used in in conjunction with an assay to monitor sperm viability. Different stimulants, such as phosphodiesterase inhibitors, drugs and toxins have been investigated in their ability to affect the sperm ability to undergo in vitro acrosome reaction. Sperm acrosomes are also sensitive to the freezing-thawing process and strategies have been described to minimize cryodamage. The assessment of the acrosome has been shown to be a stable parameter of sperm function and a valid tool to predict the fertilizing potential of human spermatozoa. The acrosome reaction following ionophore challenge (ARIC) is an in vitro assay with good predictability of the sperm's fertilizing potential for assisted conception techniques including intrauterine insemination and conventional in vitro fertilization. The AR determination has been also used as an important biomarker in studies involving drugs and toxins. Recently, novel aspects of sperm-oocyte fusion have been depicted in humans involving glycoproteins present in the zona pellucida and the female reproductive tract.
Human Reproduction, 1993
We studied the effect of media composition on sperm capacitation, using Biggers-Whitten-Whittingham (BWW) medium, Ham's-FlO and a modified Tyrode's medium (HSM) supplemented with bovine serum albumin (BSA) or fetal cord serum (FCS). We evaluated the effect of chemical environment and protein supplementation on the sperm motion parameters of curvilinear velocity and linearity, and on the ability of incubated spermatozoa to undergo follicular fluid induced acrosome reaction. Neither chemical composition nor protein supplementation of capacitation media greatly affected motion parameters after 2 h incubation. Furthermore, chemical composition had only a small effect on the ability of spermatozoa to undergo the acrosome reaction upon exposure to follicular fluid. A higher proportion of spermatozoa underwent acrosome reaction after incubation in HSM (8% control (C); 28% follicular fluid) than in BWW (8% C, 17% follicular fluid) or Ham's F-10 (6% C, 19% follicular fluid). By contrast, protein source proved critical in determining acrosome reaction inducibility. Spermatozoa incubated hi BSA-supplemented media showed a 4-fold increase in acrosomal discharge when exposed to follicular fluid (6% C, 22% follicular fluid) compared to controls while spermatozoa incubated in FCS were unable to undergo acrosome reaction (6% C, 6% follicular fluid). Simultaneous addition of FCS to BSA capacitation medium blocked acrosome reaction inducibility and the late addition of BSA, after sperm incubation in FCS, did not facilitate acrosome reaction. We propose that an inhibitor of sperm capacitation is present in FCS and therefore, the selection of optimum incubation conditions for spermatozoa may be of critical importance when evaluating or treating infertile patients.