Carbohydrates and fertilization in animals

A frequently used mechanism for sperm-egg recognition in many species involves complementary protein-carbohydrate interaction. The usual paradigm includes complex glycoconjugates in reproductive tract fluids or on the eggs which are recognized by carbohydrate-binding proteins on the sperm surface. Various glycoconjugates are utilized in the steps of sperm capacitation, sperm binding to the egg extracellular matrix and vitelline membrane and induction of the acrosome reaction. Several types of complex glycoconjugates are involved in these processes, including proteoglycans, lactosaminoglycans, sulfated fucose-containing glycoconjugates, and glycoproteins. There appear to be some structural similarities between active glycoconjugates; they are large in molecular weight and complex, and they are often sulfated, fucosylated, and attached to a protein through serine or threonine residues. In some species, the protein core of the glycoconjugates also participates in the interaction by limiting the binding of carbohydrates to sperm only of the relevant species, likely by providing the proper steric arrangement for the interaction. In other cases the protein core seems to serve more as a crosslinker of the carbohydrate moieties. This review discusses the types of glycoconjugates implicated in fertilization and the complementary lectin-like proteins found on sperm.     

Comparison of the ability of progesterone and heat solubilized porcine zona pellucida to initiate the porcine sperm acrosome reaction in vitro

It has been previously shown that progesterone can initiate the acrosome reaction (AR) of capacitated human and hamster sperm in vivo. We report here that progesterone can initiate a morphologically normal AR in porcine sperm that have undergone capacitation in a Hepes-buffered medium in vitro. In addition, we have compared the abilities of progesterone and heat-solubilized porcine zona pellucida (zona) to initiate the porcine sperm AR. Capacitated porcine sperm were treated with 1 m?g/ml progesterone, 150 m?g/ml porcine zona, or solvent control for 10 min. After treatment, sperm were incubated with the supravital dye Hoechst 33258, fixed and the acrosomal status determined in the previously viable sperm by fluorescence microscopy with fluorescein isothiocyanate-labeled Pisum sativum agglutini (FITC-PSA). There was no significant difference between the percentage of AR initiated by zona compared to that initiated by progesterone. In order to determine whether there was a synergistic interaction between the two AR initiators, both were added simultaneously to capacitated porcine sperm at optimal (1 m?g/ml progesterone, 150 m?g/ml zona) and suboptimal (75 ng/ml progesterone and 75 m?g/ml zona) concentrations. Simultaneous addition of the two AR-initiators at the two concentrations stimulated an additive AR-initating response, rather than a synergistic one. Several possible explanations for the additive results are discussed. © 1994 Wiley-Liss, Inc.     

Cell biology and functional dynamics of the mammalian sperm surface

Theriogenology has now a 40-year rich history on covering sperm biological aspects with a special emphasis on farm and husbandry animals. The major and most influential of these contributions will be placed into an evolutionary perspective of ongoing and intriguing progresses made in this field. Although many molecular details have been published, it is more the aim of this contribution to provide a guide through the main established aspects and concepts of sperm surface biology and refer only to major molecular players and mechanisms involved in sperm physiology. Those interested in more molecular details and in-depth knowledge can easily access the most relevant literature which is included here for reference purposes. With this approach, a logical and easy to follow buildup can be made of the general picture of sperm surface dynamics and of the ergonomics of sperm physiology and their function in mammalian fertilization. Understanding the ins and outs of sperm surface biology and the dynamics thereof, might challenge future researchers to design novel generation of better sperm-handling procedures. This could be beneficial for assisted reproductive technology and animal breeding industries.     

Flow cytometric analysis of mouse sperm using monoclonal anti-sperm antibody A-1

We have previously prepared an anti-mouse sperm monoclonal antibody (A-1) which inhibited sperm penetration into the egg zona pellucida. By indirect immunofluorescence (IIF), the A-1 antibody was shown to recognize an antigen localized in the acrosomal area of sperm. This antibody bound negligibly to fresh sperm, while binding to methanol-fixed sperm was almost complete. After methanol fixation, no sperm that penetrated into the zona were immunoreactive for this antibody. In the present study we examined the localization and fate of A-1 antigen during the acrosome reaction by IIF and flow cytometry (FCM). Cauda epididymal sperm were treated with either calcium ionophore A23187 or zona solution, immunostained indirectly, and subjected to FCM. Treatment with A23187 reduced the percentage of immunoreactive sperm to 59% from the 80% obtained in the untreated sperm. The treatment also reduced the average fluorescence intensity per fluorescence-positive spermatocyte to 65 channels, while this intensity was 89 channels in the untreated sperm. A similar result was obtained from treatment with zona solution. The proportion of sperm that was immunoreactive with A-1 antibody was reduced to 55% by incubation in zona-containing media from the 80% obtained in zona-free media. On the other hand, neither A23187 nor the zona solution affected the immunoreactivity or the fluorescence intensity of caput epididymal sperm, while the A-1 antigen was present in both the immature sperm from the caput epididymis of adult mice and in the mature sperm from the cauda epididymis of the same mice. These findings suggest that the intramembrane antigen recognized by the A-1 monoclonal antibody is released from sperm as a result of the acrosome reaction. © 1994 Wiley-Liss, Inc.     

Modulation of spermatozoa and zona pellucida properties by the soluble acrosome reaction-inducing factor of the ovulated egg-cumulus complex

Three sources of hamster periovulatory fluids (± heat inactivation at 56°C), with bovine serum albumin (BSA) as control, were tested for effects on penetration of three classes of eggs by hamster sperm precapacitated in BSA. These fluids were a soluble extract of cumulus oophorus fluid (COF) from the ovulated hamster egg-cumulus complex, serum, and follicular fluid. Egg types were ovulated, salt-stored (ovulated), and follicular. In both COF and serum, there were significant differences among egg types in mean penetration, and significant effects of fluid addition. In contrast, there was no effect of follicular fluid and no differences between follicular and stored eggs. For the follicular eggs (combined data, normalized, ranked), patterns of response to the three factors (± heating) were different: only unheated COF and heated serum increased penetration significantly above BSA control levels (average rank 20.2, 41.4, 38, for BSA, COF (unheated), serum (heated), respectively). This indicated that the active component in COF was heat labile, not present in either serum or follicular fluid, and, therefore, of oviductal origin. Oviduct and/or COF exposure of eggs and sperm was tested for effects as an acrosome reaction inducing factor (ARIF) for acrosome reactions (AR; zonabound and free-swimming sperm) and on sperm:zona binding and penetration. The COF ARIF for free-swimming sperm AR was heat stable. Penetration of follicular eggs increased after incubation in COF prior to sperm addition, but a greater response occurred when COF was added to eggs with sperm. In kinetic experiments, 25 min following sperm attachment, follicular eggs had lost 41% of initially bound sperm, vs. 23% for ovulated eggs, and had only 16 AR sperm/egg, vs. 26 for ovulated. Follicular eggs incubated in COF (then washed three times) had the same number of bound AR sperm as ovulated eggs. Acid solubilized zona pellucida (ASZP) from ovulated eggs was more effective as an ARIF per zona than ASZP from follicular eggs. Zonae of follicular eggs, as eivdenced by dissolution times in β-mercaptoethanol (β-MEOH), were not “harder” than those of ovulated eggs. There were differences in lectin binding antigens on zonae of both fresh and stored, follicular and ovulated, eggs. We conclude that multiple biological factors orchestrate sperm:egg interactions in the ampulla. Our data are consistent with the presence of at least three effective components: (1) the oviductal lectin-binding antigen (ZPO or oviductin), (2) an additional heat-labile component, and (3) the heat-stable ARIF for free-swimming sperm. © 1994 Wiley-Liss, Inc.     

Differential sensitivity of progesterone- and zona pellucida-induced acrosome reactions to pertussis toxin

Pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins) have previously been shown to mediate the zona pellucida-induced acrosome reaction in mammalian sperm. In this study we compared the inhibitory effect of pertussis toxin on the zona-induced acrosome reaction in human spermatozoa with that on the reaction induced by progesterone, another physiological acrosome reaction-promoting stimulus associated with the ovulated oocyte. Up to the concentration of 1 μg/ml, pertussis toxin did not produce any direct effects on the acrosome reaction frequency nor did it influence sperm movement and viability. However, preincubation of spermatozoa with the toxin at a concentration of 100 ng/ml completely abolished the increase in the acrosome reaction frequency upon subsequent exposure to solubilized zona pellucida material. In contrast, the same treatment did not impair the ability of spermatozoa to initiate the acrosome reaction in response to progesterone. Moreover, the preincubation with pertussis toxin did not modify the changes in the intracellular concentration of calcium ions occurring after progesterone addition. These data suggest that different physiological stimuli may utilize different signal transduction pathways to induce the human sperm acrosome reaction. © 1993 Wiley-Liss, Inc.     

The extracellular protein coat of the inner acrosomal membrane is involved in zona pellucida binding and penetration during fertilization: characterization of its most prominent polypeptide (IAM38)

A consequence of the acrosome reaction is to expose the inner acrosomal membrane (IAM), which is a requirement for the sperm's ability to secondarily bind to and then penetrate the zona pellucida (ZP) of the mammalian oocyte. However, the proteins on the IAM responsible for binding and presumably penetrating the zona have not been identified. This issue can be resolved if direct information is made available on the composition of the IAM. For this purpose, we devised a methodology in order to obtain a sperm head fraction consisting solely of the IAM bound to the detergent-resistant perinuclear theca. On the exposed IAM surface of this fraction, we defined an electron dense protein layer that we termed the IAM extracellular coat (IAMC), which was visible on sonicated and acrosome-reacted sperm of several mammalian species. High salt extraction removed the IAMC coincident with the removal of a prominent 38 kDa polypeptide, which we termed IAM38. Antibodies raised against this polypeptide confirmed its presence in the IAMC of intact, sonicated and acrosome-reacted sperm. By immunoscreening of a bovine testicular cDNA library and sequencing the resulting clones, we identified IAM38 as the equivalent of porcine Sp38 [Mori, E., Kashiwabara, S., Baba, T., Inagaki, Y., Mori, T., 1995. Amino acid sequences of porcine Sp38 and proacrosin required for binding to the zona pellucida. Dev. Biol., 168, 575-583], an intra-acrosomal protein with ZP-binding ability, whose precise localization in sperm was unknown. The blockage of IVF at the level of the zona with anti-IAM38 antibodies and the retention of IAM38 after sperm passage through the zona support its involvement in secondary sperm-zona binding. This study provides a novel approach to obtain direct information on the peripheral and integral protein composition of the IAM for identifying other candidates for sperm-zona interactions.     

Porcine sperm zona binding ability as an indicator of fertility

The escalated use of artificial insemination in swine has increased the importance of determining fertility of a semen sample before it is used. Multiple laboratory assays have been developed to assess fertilizing potential but they have yielded inconsistent results. This experiment sought to determine the relationship between in vitro competitive zona binding ability and in vivo fertility based on heterospermic inseminations and paternity testing. The zona pellucida binding ability and fertility of sperm from 15 boars was assessed by comparing sperm from one boar with sperm from other individual boars in a pairwise fashion using four ejaculates. The relationship of zona binding ability to the mean number of piglets sired per litter for each boar as well as historic fertility data (litter size and farrowing rate) was assessed. The in vitro competition assay consisted of labeling sperm from each boar of the pair with a different fluorophore and incubating an equal number of sperm from each boar in the same droplet with porcine oocytes. The competitive assay was highly effective in ranking boars by zona binding ability (R2=0.94). Paternity testing using microsatellite markers was used to determine the mean number of piglets sired per litter for each boar during heterospermic inseminations. The pairwise heterospermic insemination assay was effective in ranking boar fertility (R2=0.59). Using historical data from these boars, average litter size and farrowing rate were correlated (r=0.81, p<0.001). However, zona binding ability was not significantly correlated with historic farrowing rate data or historic average litter size. Boar sperm zona binding ability was also not correlated significantly with the mean number of piglets sired per litter following heterospermic insemination. But the number of piglets sired by each boar was related to a combination of zona binding ability, sperm motility, normal morphology, acrosomal integrity, and the presence of distal droplets (R2=0.70). These results suggest that zona binding ability is not an accurate predictor of fertilizing ability when used alone; however, when coupled with other sperm assessments, fertility may be predicted successfully.     

Hemizona assay for measuring zona binding in the lowland gorilla.

The hemizona assay is a diagnostic test used to evaluate the binding potential of spermatozoa to zonae pellucida and has been used to predict fertilization potential in the human. In this study, frozen-thawed gorilla spermatozoa were coincubated with human hemizonae to evaluate tight binding and to assess the use of human zonae in evaluating sperm fertility. Matching hemizonae were incubated with human sperm to serve as a control. For evaluation of binding studies in a homologous system, matching halves of gorilla hemizonae were coincubated with both gorilla and human sperm. Whole, intact zonae of both human and gorilla oocytes were also coincubated with heterologous sperm to determine if penetration into the perivitelline space could occur. This study found that gorilla sperm bound well to both gorilla and human hemizonae, with a mean of 112.5 and 81.0 tightly bound sperm, respectively. Human sperm also bound to gorilla (mean 229.5) and human (mean 236.5) hemizonae. Following incubation with intact gorilla zonae, motile human sperm were found within the perivitelline space. However, gorilla sperm were not visible within the perivitelline space of nonviable human oocytes. These findings demonstrate that the zonae of nonviable human oocytes can be used to assess sperm binding of gorilla sperm. Studies continue for optimizing assay condition and correlation of findings with the fertility potential of gorilla sperm.     

Effect of bovine viral diarrhoea virus biotypes on adherence of sperm to oocytes during in-vitro fertilization in cattle

Bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus, is one of the most important pathogens of dairy cattle; it can cause several clinical syndromes, ranging from subclinical to severe disease. The objectives of the current studies were to assess the effects of two biotypes of BVDV on sperm attachment to the zona pellucida (ZP) of oocytes and on fertilization rate in bovine in vitro fertilization (IVF). In two experiments, sperm at two concentrations (10⁵ and 10⁶/mL) and oocytes were incubated with 10⁶ TCID₅₀/mL cythopatic (CP) or noncythopatic (NCP) BVDV. In the first experiment, with the lower sperm concentration (10⁵/mL), male and female gametes were infected with CP or NCP BVDV, whereas in the second experiment, the sperm concentration was 10⁶/mL, and sperm and oocytes were also infected with CP or NCP BVDV. The number of sperm attached to the ZP and the fertilization rate were evaluated with fluorescence microscopy on the ZP of fertile and infertile oocytes. In the first experiment, compared to the control group (n = 97), oocytes infected with CP BVDV and incubated at the lower (10⁵/mL) sperm concentration positively affected sperm attachment (n = 123) to the ZP of fertile oocytes (P < 0.05). In comparison with the control group (n = 115), sperm infected with CP BVDV negatively affected sperm binding (n = 93) to the ZP of infertile oocytes (P < 0.05). In the second experiment (10⁶ sperm/mL), for both fertile and infertile oocyte groups, sperm attachment in the control group was very high and deemed uncountable. However, in treated groups, the number of sperm attached to the ZP was countable. Only sperm infected with CP BVDV negatively affected sperm binding capacity (n = 81) to the ZP of fertile oocytes (P < 0.05). Although CP and NCP BVDV significantly reduced the fertilization rate of oocytes incubated with a higher sperm concentration, with the lower sperm concentration, only NCP BVDV significantly diminished fertilization rate with contaminated sperm and oocytes (P < 0.05). In conclusion, this study supported the detrimental impacts of sperm or ooctyes infected with CP or NCP BVDV on sperm attachment to the ZP of bovine oocytes and on fertilization rate during bovine IVF.     

A guanine nucleotide-binding regulatory protein in human sperm mediates acrosomal exocytosis induced by the human zona pellucida.

Guanine nucleotide-binding regulatory proteins play key intermediary roles in regulating zona pellucida-mediated acrosomal exocytosis in mouse and bull sperm. Since human sperm possess a Gi-like protein and undergo the acrosome reaction in response to the human zona pellucida, we investigated whether this G protein plays a regulatory role in this exocytotic process. Zonae pellucidae isolated from eggs that had been inseminated but had shown no signs of fertilization after retrieval for in vitro fertilization and embryo transfer were pooled into groups of greater than or equal to 50 in order to reduce variability in biological responses due to the possible presence of ZP that had undergone modifications associated with the polyspermy block. Acid-solubilized zonae pellucidae were incubated with capacitated sperm, and the sperm then assessed for the acrosome reaction using both the P. sativum agglutinin and chlortetracycline fluorescence assays; both assays gave similar results. Sperm incubated with solubilized zonae pellucidae at a final concentration of 2, 4, or 6 ZP/microliter underwent acrosomal exocytosis to a similar extent as compared with A-23187. Sperm were incubated with 1 microgram/ml pertussis toxin during capacitation to functionally inactivate the Gi-like protein. Pertussis toxin treatment of sperm did not affect sperm motility and the ability of the cells to bind to structurally intact zonae pellucidae. Pertussis toxin, however, completely inhibited the percentage acrosome reactions induced by solubilized zonae pellucidae. By contrast, the A-23187-induced acrosome reaction was insensitive to PT treatment. Pertussis toxin inhibition of the zona pellucida-induced acrosome reaction occurred in a concentration-dependent manner with maximal effects observed at 100 ng/ml PT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of a soluble acrosome reaction-inducing factor, different from serum albumin, associated with the ovulated egg-cumulus complex.

Soluble extracts of the ovulated hamster egg-cumulus complex (ECC) were tested on capacitated sperm for activity in inducing the physiological acrosome reaction (AR). Evidence for occurrence of the physiological AR included enhanced sperm penetration of intact homologous zonae pellucidae as well as induction of AR in nonattached and in zona-bound sperm following a brief coincubation with test compound. Since hamster serum albumin, a major protein of hamster body fluids, also induces spontaneous ARs under certain conditions, it was used as one of the comparators for the acrosome reaction inducing factor (ARIF; Westrick et al., Biol Reprod 32 [Suppl 1]. 213, 1985) activity in the ECC. Sperm exposure to concentrations of the soluble ECC extract ranging from 0.04 to 0.2 mg protein/ml significantly increased penetration of salt-stored zonae by 36%, mean numbers of penetrating sperm by 90%, ARs in nonattached sperm by 65%, and ARs in zona-bound sperm by 102%. Hamster serum albumin added after completion of capacitation had no significant effect on these parameters. We conclude that 1) the ovulated ECC contains a soluble ARIF that augments zona-induced ARs and sperm penetration and 2) the ARIF is not serum albumin.     

Role and regulation of sperm gelsolin prior to fertilization

To acquire fertilization competence, spermatozoa should undergo several biochemical changes in the female reproductive tract, known as capacitation. The capacitated spermatozoon can interact with the egg zona pellucida resulting in the occurrence of the acrosome reaction, a process that allowed its penetration into the egg and fertilization. Sperm capacitation requires actin polymerization, whereas F-actin must disperse prior to the acrosome reaction. Here, we suggest that the actin-severing protein, gelsolin, is inactive during capacitation and is activated prior to the acrosome reaction. The release of bound gelsolin from phosphatidylinositol 4,5-bisphosphate (PIP(2)) by PBP10, a peptide containing the PIP(2)-binding domain of gelsolin, or by activation of phospholipase C, which hydrolyzes PIP(2), caused rapid Ca(2+)-dependent F-actin depolymerization as well as enhanced acrosome reaction. Using immunoprecipitation assays, we showed that the tyrosine kinase SRC and gelsolin coimmunoprecipitate, and activating SRC by adding 8-bromo-cAMP (8-Br-cAMP) enhanced the amount of gelsolin in this precipitate. Moreover, 8-Br-cAMP enhanced tyrosine phosphorylation of gelsolin and its binding to PIP(2(4,5)), both of which inactivated gelsolin, allowing actin polymerization during capacitation. This actin polymerization was blocked by inhibiting the Src family kinases, suggesting that gelsolin is activated under these conditions. These results are further supported by our finding that PBP10 was unable to cause complete F-actin breakdown in the presence of 8-Br-cAMP or vanadate. In conclusion, inactivation of gelsolin during capacitation occurs by its binding to PIP(2) and tyrosine phosphorylation by SRC. The release of gelsolin from PIP(2) together with its dephosphorylation enables gelsolin activation, resulting in the acrosome reaction.     

Comparison of sperm binding potential of uninseminated, inseminated-unfertilized, and fertilized-noncleaved human oocytes under hemizona assay conditions.

In this study, human oocytes obtained after ovarian hyperstimulation for in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT) were utilized to evaluate sperm/zona pellucida binding potential. Three groups of oocytes were evaluated: 1) uninseminated; 2) inseminated-unfertilized; and 3) fertilized-uncleaved. All oocytes had undergone germinal vesicle breakdown at the time of retrieval and were salt-stored (pH 7.2) for not more than 30 days. Sperm binding was recorded under hemizona assay (HZA) conditions using spermatozoa from eight fertile men (HZA control) and from 1) four teratozoospermic (HZA test) and 2) four normozoospermic (HZA test) infertile men. First, the mean numbers (+/- SD) of sperm tightly bound for fertile controls and teratospermic men to hemizonae from uninseminated oocytes were 69.7 +/- 16 and 14.5 +/- 7, respectively (P = 0.02). Likewise, hemizonae from uninseminated oocytes bound 102.0 +/- 19 and 114.0 +/- 28, respectively, for fertile controls and normospermic men (P = 0.5). Second, hemizonae obtained from inseminated-unfertilized IVF oocytes bound 44.2 +/- 12 and 19.7 +/- 6 for fertile controls and teratospermic men, respectively (P = 0.02). This category of oocytes bound 100.5 +/- 7 and 108.5 +/- 11 sperm, respectively, for fertile controls and normospermic semen (P = 0.3). Third, HZA results of fertilized but uncleaved oocytes showed a mean number of tightly bound sperm of 6.0 +/- 4 compared with 65.0 +/- 1 in control, uninseminated oocytes using fertile sperm. These results demonstrate that uninseminated and inseminated-unfertilized human oocytes, salt-stored under controlled pH conditions, give reliable information regarding sperm binding potential under HZA conditions.     

Sperm capacitation induces an increase in lipid rafts having zona pellucida binding ability and containing sulfogalactosylglycerolipid

Sperm gain full ability to bind to the zona(e) pellucida(e) (ZP) during capacitation. Since lipid rafts are implicated in cell adhesion, we determined whether capacitated sperm lipid rafts had affinity for the ZP. We demonstrated that lipid rafts, isolated as low-density detergent resistant membranes (DRMs), from capacitated pig sperm had ability to bind to homologous ZP. This binding was dependent on pig ZPB glycoprotein, a major participant in sperm binding. Capacitated sperm DRMs were also enriched in the male germ cell specific sulfogalactosylglycerolipid (SGG), which contributed to DRMs-ZP binding. Furthermore, SGG may participate in the formation of sperm DRMs due to its interaction with cholesterol, an integral component of lipid rafts, as shown by infrared spectroscopic studies. Since sperm capacitation is associated with cholesterol efflux from the sperm membrane, we questioned whether the formation of DRMs was compromised in capacitated sperm. Our studies indeed revealed that capacitation induced increased levels of sperm DRMs, with an enhanced ZP affinity. These results corroborated the implication of lipid rafts and SGG in cell adhesion and strongly suggested that the enhanced ZP binding ability of capacitated sperm may be attributed to increased levels and a greater ZP affinity of lipid rafts in the sperm plasma membrane.     

Complexin I is required for mammalian sperm acrosomal exocytosis

Regulated exocytosis in many cells is controlled by the SNARE complex, whose core includes three proteins that promote membrane fusion. Complexins I and II are highly related cytosolic proteins that bind tightly to the assembled SNARE complex and regulate neuronal exocytosis. Like somatic cells, sperm undergo regulated exocytosis; however, sperm release a single large vesicle, the acrosome, whose release has different characteristics than neuronal exocytosis. Acrosomal release is triggered upon sperm adhesion to the mammalian egg extracellular matrix (zona pellucida) to allow penetration of the egg coat. Membrane fusion occurs at multiple points within the acrosome but how fusion is activated and the formation and progression of fusion points is synchronized is unclear. We show that complexins I and II are found in acrosome-intact mature sperm, bind to SNARE complex proteins, and are not detected in sperm after acrosomal exocytosis (acrosome reaction). Although complexin-I-deficient sperm acrosome-react in response to calcium ionophore, they do not acrosome-react in response to egg zona pellucida proteins and have reduced fertilizing ability, in vitro. Complexin II is present in the complexin-I-deficient sperm and its expression is increased in complexin-I-deficient testes. Therefore, complexin I functions in exocytosis in two related but morphologically distinct secretory processes. Sperm are unusual because they express both complexins I and II but have a unique and specific requirement for complexin I.     

Bovine sperm acrosome reaction induced by G protein-coupled receptor agonists is mediated by epidermal growth factor receptor transactivation

We have previously demonstrated the presence of active epidermal growth factor receptor (EGFR) and its involvement in sperm capacitation and the acrosome reaction; however, the mechanism of EGFR activation was not clear. We show here that the sperm EGFR can be transactivated by angiotensin II or by lysophosphatydic acid, two ligands which activate specific G-protein-coupled receptors (GPCR), or by directly activating protein kinase A using 8Br-cAMP. This transactivation occurs in noncapacitated sperm and is mediated by PKA, SRC and a metalloproteinase. We also show that the EGFR is activated in sperm incubated under in vitro capacitation conditions, without any added ligand, but not in bicarbonate-deficient medium or when PKA is blocked. Despite the fact that EGFR is activated in capacitated sperm, this state is not sufficient to induce the acrosome reaction. We conclude that the EGFR is stimulated during capacitation via PKA activation, while further activation of the EGFR in capacitated sperm is required in order to induce the acrosome reaction. The acrosome reaction can be induced by GPCR via the transactivation of the EGFR by a signaling pathway involving PKA, SRC and metalloproteinase and the EGFR down-stream effectors PI3K, PLC and PKC.     

Regulation of the sperm EGF receptor by ouabain leads to initiation of the acrosome reaction

The sperm acrosome reaction occurs after the binding of the capacitated sperm to the egg zona pellucida. This study describes a novel mode of regulation of the sperm epidermal growth factor receptor (EGFR) under physiological conditions and its relevance to the acrosome reaction. Ouabain, a known Na/K ATPase blocker is present in the blood and in the female reproductive tract. We show here that physiological concentrations (nM) of ouabain enhance phosphorylation of EGFR on tyr-845, stimulate Ca²⁺ influx and induce the acrosome reaction in sperm. These effects could be seen only in the presence of very low concentrations of EGF (0.1 ng/ml or 0.016 nM) added together with nano-molar ouabain. Phosphorylation, Ca²⁺ influx, and the acrosome reaction are inhibited by an EGFR blocker, suggesting that trans-activation of the EGFR is involved. Moreover, our data revealed that protein kinase A and the family of tyrosine kinase, SRC, shown before to be involved in EGFR activation in sperm, mediate the acrosome reaction induced by ouabain. Ouabain alone (without EGF) at relatively high concentration (10 µM) could enhance EGFR phosphorylation, Ca²⁺ influx and acrosome reaction, and these processes were inhibited by EGFR blockers. Moreover, we show here that PKA and SRC family are involved in the activation of EGFR by 10 µM ouabain, further demonstrating that ouabain induces the acrosome reaction by a mechanism mediated by the trans-activation of EGFR. In conclusion, this study describes an interesting regulatory path of EGFR by physiological concentrations of ouabain and EGF found in the female reproductive tract. Neither of these compounds can activate the EGFR alone at such low physiological levels; however, when both are present, the interaction of ouabain with the Na/K ATPase leads to the priming of the EGFR, which undergoes its full activation by EGF.     

Subzonal insemination with a single spermatozoon using manipulation assisted sperm adhesion onto the ooplasmic membrane in mouse ova.

A simple and successful method of microinjection of a single spermatozoon under the zona pellucida of a mouse oocyte has been developed. A characteristic of this method is that the tip of the sperm injection needle pierces the zona pellucida without touching the ooplasmic membrane. All the ova (277) used for this series of experiments had normal morphology after the injection procedure. Spermatozoa preincubated in culture medium for capacitation and those treated with ionophore A23187 for induction of acrosome reaction were used. In combination with some of these injections, a manipulation assisting the adhesion of the sperm head onto the ooplasmic membrane was employed. The fertilization rate (67.3%) of the ova injected with the ionophore-treated sperm using the sperm-adhesion treatment was significantly higher (P less than 0.005) than that obtained by the injection of the preincubated sperm without applying the adhesion treatment (23.6%). All three of the recipients that received the 24 fertilized ova became pregnant and gave birth to 11 offspring (45.8%). The inseminations performed with the sperm-adhesion treatment using the immotile sperm from the preincubated population and/or those from the ionophore-treated population did not result in fertilization in any case. These results suggest that the fertilization rate of subzonal insemination with motile ionophore-treated sperm can be improved by applying the sperm-adhesion treatment and that sperm motility might be involved in the establishment of fertilization, even after the adhesion of the sperm head with the mouse ovum membrane.     

Understanding fertilization through intracytoplasmic sperm injection (ICSI)

Since the establishment of in vitro fertilization, it became evident that almost half of the couples failed to achieve fertilization and this phenomenon was attributed to a male gamete dysfunction. The adoption of assisted fertilization techniques particularly ICSI has been able to alleviate male factor infertility by granting the consistent ability of a viable spermatozoon to activate an oocyte. Single sperm injection, by pinpointing the beginning of fertilization, has been an invaluable tool in clarifying the different aspects of early fertilization and syngamy. However, even with ICSI some couples fail to fertilize due to ooplasmic dysmaturity in relation to the achieved nuclear maturation marked by the extrusion of the first polar body. More uncommon are cases where the spermatozoa partially or completely lack the specific oocyte activating factor. In this work, we review the most relevant aspects of fertilization and its failure through assisted reproductive technologies. Attempts at diagnosing and treating clinical fertilization failure are described.