Protease activity involvement in the passage of mammalian sperm through the zona pellucida

The interaction between acrosome-reacted sperm and zona pellucida proteins is not yet fully understood. Serine protease acrosin and its zymogen proacrosin have been proposed to fulfill this function due to their capacity to bind zona pellucida glycoproteins. However, the molecular mechanism underlying this interaction has been merely speculative. Here we show that fucoidan (a sulfated polysaccharide) and solubilized zona pellucida glycoproteins, but not soybean trypsin inhibitor, are able to detach bound spermatozoa, which suggests that live sperm binds to the zona pellucida in a non-enzymatical way. Interestingly, mild proteolytic digestion with acrosin or trypsin does not modify the structure of the zona pellucida, but rather results in fewer spermatozoa binding to the zona. These results agree with a model where the active site of acrosin digests the zona pellucida and binds through the polysulfate-binding domain through a three-dimensional zona structure rather than a single ligand.

Modulation of spermatozoon acrosome reaction

Spermatozoon acrosome reaction is an exocytotic event of the utmost importance for the development of mammalian fertilisation. Current evidence shows that the triggering of the acrosome reaction (AR) could be regulated by the action of diverse compounds, namely, metabolites, neurotransmitters and hormones. The aim of the present review is to describe the modulating effects of several compounds that have been classified as inductors or inhibitors of acrosome reaction. Among AR inductors, it is necessary to mention progesterone, angiotensin II, atrial natriuretic peptide, cathecolamines, insulin, leptin, relaxin and other hormones. Regarding the inhibitors, oestradiol and epidermal growth factor are among the substances that retard AR. It is worth mentioning that gamma-aminobutyric acid, a neurotransmitter known to be an inhibitor in the central nervous system, has been shown to induce AR. The multiple hormones located in the fluids of the female reproductive tract are also likely to act as subtle regulators of AR, constituting a fundamental aspect for the development of successful fertilisation. Finally, it is necessary to emphasise that the study of regulation exerted by hormones and other compounds on AR is essential for further understanding of mammalian reproductive biology, especially spermatozoon physiology.

Evaluation of acrosomal status and sperm viability in fresh and cryopreserved specimens by the use of fluorescent peanut agglutinin lectin in conjunction with hypo-osmotic swelling test

OBJECTIVE: In this study, we evaluated whether the hypo-osmotic swelling test (HOST) can be used as a vital marker in combination with peanut agglutinin (PNA) - labeling in fresh and cryopreserved spermatozoa. MATERIALS AND METHODS: Human sperm populations were exposed to a hypo-osmotic medium for 60 minutes, and then incubated in a 1 µg/mL solution of the fluorescent dye Hoescht 33258 (H33258) for 10 minutes. Excess stain was removed by washing in phosphate-buffered saline (PBS) solution, and the pellet was resuspended in 100 µL of culture medium. Twenty microliters of this solution were subsequently smeared on a microscope slide, and fixed in ice-cold methanol to permeabilize the sperm membranes. The fixed smears were finally incubated in a 40-µg/mL FITC-PNA solution for 20 minutes. Simultaneous assessment of acrosome and viability scores was done in a fluorescent microscope equipped with appropriate filters and phase contrast illumination. The same slide was examined for FITC-PNA labeling, tail swelling, and for Hoechst-33258 staining by interchanging the filters and phase contrast optics. RESULTS: In fresh specimens, HOST was found to provide viability assessments comparable to those obtained using the H33258 method (r = 0.95). However, the results of HOST and H33258 were not correlated in cryopreserved specimens (r = 0.22). There was no alteration of PNA-labeling due to the HOST or H33258. CONCLUSIONS: FITC-PNA labeling in conjunction with the visualization of the morphological change induced by exposure to hypo-osmotic solution provides a simple but effective method for establishing the state of acrosomal membrane and viability in fresh human spermatozoa, but this technique is not reliable for cryopreserved ones.

A carbohydrate-based mechanism of species recognition in sea urchin fertilization

In the present review, we describe a systematic study of the sulfated polysaccharides from marine invertebrates, which led to the discovery of a carbohydrate-based mechanism of sperm-egg recognition during sea urchin fertilization. We have described unique polymers present in these organisms, especially sulfated fucose-rich compounds found in the egg jelly coat of sea urchins. The polysaccharides have simple, linear structures consisting of repeating units of oligosaccharides. They differ among the various species of sea urchins in specific patterns of sulfation and/or position of the glycosidic linkage within their repeating units. These polysaccharides show species specificity in inducing the acrosome reaction in sea urchin sperm, providing a clear-cut example of a signal transduction event regulated by sulfated polysaccharides. This distinct carbohydrate-mediated mechanism of sperm-egg recognition coexists with the bindin-protein system. Possibly, the genes involved in the biosynthesis of these sulfated fucans did not evolve in concordance with evolutionary distance but underwent a dramatic change near the tip of the Strongylocentrotid tree. Overall, we established a direct causal link between the molecular structure of a sulfated polysaccharide and a cellular physiological event - the induction of the sperm acrosome reaction in sea urchins. Small structural changes modulate an entire system of sperm-egg recognition and species-specific fertilization in sea urchins. We demonstrated that sulfated polysaccharides - in addition to their known function in cell proliferation, development, coagulation, and viral infection - mediate fertilization, and respond to evolutionary mechanisms that lead to species diversity.

Proacrosin-acrosin activity in capacitated and acrosome reacted sperm from cryopreserved bovine semen

Acrosin activity is associated with normal fertility in human and bovine spermatozoa. The aim of the study was to determine the variation of the enzyme activity in the proacrosin-acrosin system in capacitated and acrosome recated cryopreserved bovine sperm. Enzyme activity was assessed spectrophotometrically using N-alpha-benzoyl-DL-arginine p-nitroanilide (BAPNA) as specific substrate for acrosin at pH 8. Capacitation with heparin and quercitin failed to induce conversion of proacrosin to acrosin. An increase in acrosin activity produced by the presence of progesterone, in a dose-dependent manner, was related with the induction of true acrosome reaction. The total level of acrosin activity registered showed that 96 per cent of acrosin of capacitated sperm samples and control is present in the zymogen form. Moreover, progesterone is capable of duplicating the level of active enzyme, indicating that enzyme activity changes are related to acrosome reaction, suggesting that only a minor proportion of the total of proacrosin-acrosin system is required in the exocytotic process on cryopreserved bovine sperm.

The acrosomic reaction in stallion spermatozoa: inductive effect of the mare preovulatory follicular fluid

In the female genital tract, spermatozoa must undergo capacitation and acrosome reaction prior to fertilization. A number of factors may induce physiological acrosome reaction assayed in vitro. The aims of this study are to determine the inductive effect of the preovulatory follicular fluid on the sperm acrosomal status in the equine, once some characteristics of the follicular fluid during folliculogenesis had been evaluated. The spermatozoa were obtained from cauda epididymes of adult stallion. Follicular fluid was taken from mare ovarian follicles classified according to their diameter. In these fluids, total protein, progesterone, estradiol and osmolarity were determined. Afterwards, the effect of preovulatory follicular fluid (50) upon induction of the acrosomic reaction in stallion capacitated spermatozoa was assayed. Results show that during folliculogenesis the ratio progesterone/estrogen is below 1. In large preovulatory follicles, there is a sharp increase of progesterone, reaching a ratio progesterone/estrogen close to 4. Protein concentration and osmolarity increase together with follicular development, being osmolarity very high at the preovulatory stage. Follicular fluid--in vitro--increases the percentage of spermatozoa with acrosome reaction, maintaining high rates of vitality and motility. The characteristics of follicular fluid undergo dynamic changes during the folliculogenesis, such as steroid level, protein concentration and osmolarity. These events may play a role in the reproductive process in vivo, considering that in vitro the follicular fluid is a very effective inductor of the acrosome reaction, with optimum levels of vitality and motility.

Surface protein changes in goat spermatozoa during capacitation.

Polypeptides of goat sperm surface before and after capacitation were examined by radiolabelling and immunologically using polyclonal antisera. Radioiodination revealed five protein bands having mol wt of 14.8, 72.4, 81, 100 and 128 kDa in uncapacitated ejaculated spermatozoa and only three bands of 23.4, 27 and 72.4 KDa in capacitated spermatozoa. The protein band with mol wt 72.4 kDa was only feebly iodinated in uncapacitated sperm surface but in capacitated spermatozoa it was heavily labelled. Western blot analysis of detergent-extracted proteins using gamma-globulin fraction of antisera raised against purified goat sperm plasma membrane revealed six antigens (17.8, 29.1, 33.4, 45.6, 85.1, 123.2 kDa) in uncapacitated spermatozoa, four (26, 32.1, 40.1, 45.6 kDa) in capacitated spermatozoa and only one (45.6 kDa) in acrosome-reacted spermatozoa. High mol wt proteins were more numerous on the surface of uncapacitated spermatozoa while the capacitated spermatozoa had relatively low mol wt proteins. An apparent effect of capacitation is the metabolism and reorganisation of proteins on goat sperm surface. Polypeptides on capacitated sperm surface revealed through radiolabelling and polyclonal antisera may have a likely receptor(s) role in the recognition and binding to homologous zona pellucida during fertilization.

Capacitaçäo espermática em ruminantes / Sperm capacitation in ruminants

A autora apresenta uma revisão de literatura da capacitação dos espermatozóides de ruminantes, relatando os prováveis mecanismos fisiológicos que estão envolvidos no processo e os agentes que provocam a capacitação in vitro dos espermatozóides.

Role of estradiol in the capacitation and acrosome reaction of hamster epididymal spermatozoa in the isolated uterus of mice incubated in vitro.

The site of sperm capacitation, the agents and mechanisms causing capacitation and acrosome reaction (AR) in vivo are not well understood. The female reproductive tract has been reported to play a key role during capacitation and AR. Some experiments were carried out on the capacitation and AR of hamster epididymal spermatozoa in the estrogen and progesterone dominated uterus (estrous and diestrous respectively) albino mice, incubated in TALP without calcium and BSA. Also the effect of estradiol (200 micrograms/ml) supplemented to TALP, on capacitation and AR was examined. Capacitation and AR of hamster spermatozoa incubated in the isolated uterus of both estrous and diestrous mice were significantly (P < 0.05) higher in the presence of exogenous estradiol than that in its absence. Acrosome shedding occurred earlier i.e. at 3rd hour as compared to the in vitro studies where it occurred at 5th hour. The present study thus reveals that uterus of both estrogen and progesterone dominated mice play an important role in the induction of capacitation and AR. The addition of estradiol might have the influence on the synthesis of uterine proteins of mice which might be important for capacitation and AR.