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Relocalization of Izumo and beta1-integrin during spontaneous acrosome reaction
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Abstracts / Journal of Reproductive Immunology 94 (2012) 5–130
governing female sperm selection are not understood. Anti sperm antibodies (ASAs) have been reported in both females and males of a number of species and have been shown to inhibit sperm function. The present study tested the hypothesis that female ASAs play a role in post copulatory sperm selection in the red jungle fowl. Virgin hens were initially screened for presence of ASAs using indirect ELISA. Mean serum IgM and IgY reactivity was significantly higher against particular males’ sperm relative to others (p < 0.001). IgA reactivity was undetectable. To examine the influence that these serum ASAs have on sperm function, semen was incubated with virgin hen serum or foetal bovine serum at 41 degrees C (female n = 26, male n = 13) for periods up to one hour. In all cases, hen serum incubations resulted in higher rates of sperm agglutination (p < 0.001) and reduced sperm motility (p < 0.001) relative to bovine serum (acting as a cross-species control). The level of agglutination was dependent upon the identity of both the female and the sperm i.e. some pairs resulted in higher agglutination rates than others (p < 0.01). We next sought to determine whether presence of agglutinating ASAs is able to influence sperm transit through the female tract in vivo. Using a paired design, two different females were artificially inseminated with semen from one male (total female n = 26, total male n = 13). Subsequent eggs were collected and the number of sperm holes (result of hydrolysis) at the egg perivitelline layer (PVL) was determined. The number of sperm reaching the PVL differed significantly between female pairs, and the rate of agglutination was predictive of PVL count (negative correlation). Our findings indicate that virgin females posses ASAs able to differentiate between individual males within a given population. As a consequence, sperm transit through the female tract can be selectively inhibited. This interaction may provide a mechanism for post copulatory sperm selection in polyandrous species. doi:10.1016/j.jri.2012.03.379 P 078 Relocalization of Izumo and beta1-integrin during spontaneous acrosome reaction K. Dvorakova-Hortova 1,∗ , K. Vesela 1 , N. Sebkova 1,2 1 2
Faculty of Science, Zoology, Prague 2, Czech Republic Faculty of Science, Cell Biology, Prague 2, Czech Republic
Sperm is capable to fertilize the ova after successful capacitation and acrosomal reaction (AR). It has been recently shown in mice that sperm undergo AR by passing through cumulus cells, further they can bind to zona pellucida, and consequently fertilize the egg. During AR relocalization of primary fusion protein IZUMO, a member of IgSF, into equatorial segment is crucial for sperm-egg fusion. There is a high rate of spontaneous acrosomal reaction in rodents,with up to 60% in promiscuous species. Moreover, in field mice (Apodemus) CD46 protein, which is predicted to be responsible for acrosome integrity via it’s interaction with integrins, is not translated. The aim was to find out, whether the relocalization of IZUMO and
also beta-integrins, happens during physiological spontaneous AR, or whether, it occurs only in sperm withzona pellucidaor cumulus cells induced AR. Therefore, whether spontaneous acrosome reacted sperm are able to fertilize the egg. Immunofluorescent detection of selected proteins at specific times of sperm capacitationin vitroduring spontaneous and calcium ionophore induced AR was monitored. The state of acrosome was visualized by PNA labelling. Our results show that during spontaneous AR there is a clear IZUMO relocalization from the acrosomal membrane to the equatorial and post-acrosomal segment. Moreover, at the same time, also relocalization of beta1-integrin occurs out of the subcortical acrosome cytoskeleton over the entire sperm head, even in the absence of CD46. Therefore, in conclusion, spontaneously acrosome reacted sperm have same fertilizing potential as those after induced AR. Findings, that crucial molecular changes essential for sperm-egg fusion represented by dynamics of Izumo and integrins happening also during spontaneous AR are vital for novel understanding of fertilization in mice. Moreover, this may represent a unique mechanism how to accelerate fertilizing process in highly promiscuous environment under selective pressure of intra-specific sperm competition. Financial support: GAUK 304911, GACR P506/12/1046 doi:10.1016/j.jri.2012.03.380 P 079 Vacuolar ATPase subunit a2 expression is associated with normal spermatogenesis in mice B.K. Kolli ∗ , M. Jaiswal, A. Gilman-Sachs, K. Beaman Rosalind Franklin University of Medicine and science, Microbiology and Immunology, North Chicago, United States PROBLEM: We have previously observed that the a2 subunit of vacuolar ATPase (a2V-ATPase) plays a critical role in successful pregnancy outcome. It is also abundantly expressed in mature but not in immature sperm. A2VATPase is thought to be expressed in developing tissues and has been related to lymphocytic apoptosis. Increased apoptosis has also been observed to be associated with spermatogenesis in normal males. Here, we explore the involvement of a2V-ATPase in apoptosis in mice testis in vivo. METHODS: A single dose of ATP6V0A2 (a2V-ATPase) siRNA (1 nmole/testis) was injected into the male testis of Balb/c mice through the transverse incision at the abdominal wall. Control mice received scrambled siRNA. The expression of a2V-ATPase and the apoptotic genes, bax and bcl2 was evaluated in the testis by real-time PCR and Western blot after 48 h of treatment. RESULTS: Real-time PCR analysis showed that a2VATPase gene expression decreased significantly in the testis recovered from siRNA injected mice compared to that in the testis from control. Western blot analysis confirmed the knock-down of a2V-ATPase. In addition, significant differences in the expression of the pro- and anti-apoptotic markers were observed in the testis of the control and the a2V-ATPase SiRNA injected mice. While the pro-apoptotic
Abstracts / Journal of Reproductive Immunology 94 (2012) 5–130
governing female sperm selection are not understood. Anti sperm antibodies (ASAs) have been reported in both females and males of a number of species and have been shown to inhibit sperm function. The present study tested the hypothesis that female ASAs play a role in post copulatory sperm selection in the red jungle fowl. Virgin hens were initially screened for presence of ASAs using indirect ELISA. Mean serum IgM and IgY reactivity was significantly higher against particular males’ sperm relative to others (p < 0.001). IgA reactivity was undetectable. To examine the influence that these serum ASAs have on sperm function, semen was incubated with virgin hen serum or foetal bovine serum at 41 degrees C (female n = 26, male n = 13) for periods up to one hour. In all cases, hen serum incubations resulted in higher rates of sperm agglutination (p < 0.001) and reduced sperm motility (p < 0.001) relative to bovine serum (acting as a cross-species control). The level of agglutination was dependent upon the identity of both the female and the sperm i.e. some pairs resulted in higher agglutination rates than others (p < 0.01). We next sought to determine whether presence of agglutinating ASAs is able to influence sperm transit through the female tract in vivo. Using a paired design, two different females were artificially inseminated with semen from one male (total female n = 26, total male n = 13). Subsequent eggs were collected and the number of sperm holes (result of hydrolysis) at the egg perivitelline layer (PVL) was determined. The number of sperm reaching the PVL differed significantly between female pairs, and the rate of agglutination was predictive of PVL count (negative correlation). Our findings indicate that virgin females posses ASAs able to differentiate between individual males within a given population. As a consequence, sperm transit through the female tract can be selectively inhibited. This interaction may provide a mechanism for post copulatory sperm selection in polyandrous species. doi:10.1016/j.jri.2012.03.379 P 078 Relocalization of Izumo and beta1-integrin during spontaneous acrosome reaction K. Dvorakova-Hortova 1,∗ , K. Vesela 1 , N. Sebkova 1,2 1 2
Faculty of Science, Zoology, Prague 2, Czech Republic Faculty of Science, Cell Biology, Prague 2, Czech Republic
Sperm is capable to fertilize the ova after successful capacitation and acrosomal reaction (AR). It has been recently shown in mice that sperm undergo AR by passing through cumulus cells, further they can bind to zona pellucida, and consequently fertilize the egg. During AR relocalization of primary fusion protein IZUMO, a member of IgSF, into equatorial segment is crucial for sperm-egg fusion. There is a high rate of spontaneous acrosomal reaction in rodents,with up to 60% in promiscuous species. Moreover, in field mice (Apodemus) CD46 protein, which is predicted to be responsible for acrosome integrity via it’s interaction with integrins, is not translated. The aim was to find out, whether the relocalization of IZUMO and
also beta-integrins, happens during physiological spontaneous AR, or whether, it occurs only in sperm withzona pellucidaor cumulus cells induced AR. Therefore, whether spontaneous acrosome reacted sperm are able to fertilize the egg. Immunofluorescent detection of selected proteins at specific times of sperm capacitationin vitroduring spontaneous and calcium ionophore induced AR was monitored. The state of acrosome was visualized by PNA labelling. Our results show that during spontaneous AR there is a clear IZUMO relocalization from the acrosomal membrane to the equatorial and post-acrosomal segment. Moreover, at the same time, also relocalization of beta1-integrin occurs out of the subcortical acrosome cytoskeleton over the entire sperm head, even in the absence of CD46. Therefore, in conclusion, spontaneously acrosome reacted sperm have same fertilizing potential as those after induced AR. Findings, that crucial molecular changes essential for sperm-egg fusion represented by dynamics of Izumo and integrins happening also during spontaneous AR are vital for novel understanding of fertilization in mice. Moreover, this may represent a unique mechanism how to accelerate fertilizing process in highly promiscuous environment under selective pressure of intra-specific sperm competition. Financial support: GAUK 304911, GACR P506/12/1046 doi:10.1016/j.jri.2012.03.380 P 079 Vacuolar ATPase subunit a2 expression is associated with normal spermatogenesis in mice B.K. Kolli ∗ , M. Jaiswal, A. Gilman-Sachs, K. Beaman Rosalind Franklin University of Medicine and science, Microbiology and Immunology, North Chicago, United States PROBLEM: We have previously observed that the a2 subunit of vacuolar ATPase (a2V-ATPase) plays a critical role in successful pregnancy outcome. It is also abundantly expressed in mature but not in immature sperm. A2VATPase is thought to be expressed in developing tissues and has been related to lymphocytic apoptosis. Increased apoptosis has also been observed to be associated with spermatogenesis in normal males. Here, we explore the involvement of a2V-ATPase in apoptosis in mice testis in vivo. METHODS: A single dose of ATP6V0A2 (a2V-ATPase) siRNA (1 nmole/testis) was injected into the male testis of Balb/c mice through the transverse incision at the abdominal wall. Control mice received scrambled siRNA. The expression of a2V-ATPase and the apoptotic genes, bax and bcl2 was evaluated in the testis by real-time PCR and Western blot after 48 h of treatment. RESULTS: Real-time PCR analysis showed that a2VATPase gene expression decreased significantly in the testis recovered from siRNA injected mice compared to that in the testis from control. Western blot analysis confirmed the knock-down of a2V-ATPase. In addition, significant differences in the expression of the pro- and anti-apoptotic markers were observed in the testis of the control and the a2V-ATPase SiRNA injected mice. While the pro-apoptotic