Exocytosis in mammalian spermatozoa (the acrosome reaction) is a process essential for fertilization. Both progesterone and zona pellucida induce exocytosis in spermatozoa, which may encounter both during penetration of the oocyte's vestments. When mouse spermatozoa were exposed first to progesterone and then to zona pellucida, exocytosis was enhanced to a greater degree than that seen when the agonists were presented together or in the inverse order, which suggests that the steroid exerts a priming effect. Progesterone similarly primed the generation of intracellular messengers evoked by zona pellucida. The effects triggered by progesterone were mimicked by gamma-aminobutyric acid (GABA) and were blocked by bicuculline, which indicates that the steroid acts on a GABAA receptor.

A number of avian species are difficult to sex morphologically, especially as nestlings. Like other avian species, many species of Falconiformes are sexually monomorphic. Therefore, it is desirable that new methods based on DNA analysis are established in Falconiformes and other sexual monomorphic species. We identified sex in Falconiformes by two alternative methods. First, we used a sexing method based on the intronic length variation between CHD1W and CHD1Z using primers flanking the intron. In this method, two species of Falconidae could be identified for sexing. However, six species of Accipitridae could not, because they have few length variations. The second method used was based on differences in sequences between CHD1W and CHD1Z. From sequence analysis, a 3'-terminal mismatch primer on point mutation conserved among Falconiformes was designed, and identification of sex with the amplification refractory mutation system (ARMS) was performed. This method could identify sex in all species tested. In addition, because the 3'-terminal mismatch primer was designed on a point mutation conserved among Falconiformes, ARMS with these primers may identify sex in all Falconiformes. These are simple and rapid sexing methods, since only polymerase chain reaction (PCR) and agarose electrophoresis are required. In conclusion, sex identification by an alternative PCR approach based on intronic length variation and on differences in sequences between CHD1W and CHD1Z proved applicable to and useful for Falconiformes.

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We tested the involvement of protein tyrosine kinase and G-protein transducing pathways in the formation of diacylglycerol (DAG) during exocytosis in mouse spermatozoa. In capacitated spermatozoa, stimulation with solubilized zona pellucida (ZP) or progesterone led to the formation of DAG and to exocytosis of the acrosomal granule. Stimulation of DAG formation and exocytosis by ZP were inhibited in a concentration-dependent fashion by pre-exposure to tyrphostin A48, a protein tyrosine kinase inhibitor. These ZP-induced responses were also reduced in a concentration-dependent manner by prior incubation with pertussis toxin, a G-protein (Gi class) inhibitor. On the other hand, generation of DAG and exocytosis triggered by progesterone were inhibited if spermatozoa were preincubated with different concentrations of tyrphostin A48, but were not affected by pre-exposure to pertussis toxin. Progesterone acts on at least two novel surface receptors, one being a gamma-aminobutyric acid (GABA) type A (GABAA)-like receptor. Transducing mechanisms coupled to this receptor were tested directly by stimulating spermatozoa with GABA. Treatment of capacitated spermatozoa with GABA resulted in DAG formation and exocytosis. These responses were not seen when cells were preincubated with tyrphostin A48. Pertussis toxin, however, did not affect the generation of DAG and exocytosis triggered by GABA, in agreement with results obtained using progesterone. Taken together, these results indicate that DAG formation during acrosomal exocytosis is differentially regulated by transducing pathways activated by oocyte-associated agonists.

The aim of this study was to elucidate the relationship between protein tyrosine phosphorylation state and sperm characteristics in frozen-stored spermatozoa of Japanese Black bulls. The spermatozoa were washed with PBS containing polyvinyl alcohol and then incubated with cell-permeable cAMP analog cBiMPS to induce flagellar hyperactivation. Before and after incubation, the spermatozoa were used for immunodetection of tyrosine-phosphorylated proteins, assessment of morphological acrosome condition and evaluation of motility. In bulls whose frozen-stored spermatozoa were classified as having a high-grade acrosome condition before incubation, sperm tyrosine-phosphorylated proteins, including the 33-kDa tyrosine-phosphorylated SPACA1 protein, were localized in the anterior region of the acrosome and equatorial subsegment. The immunodetection level of the 41- and 33-kDa sperm tyrosine-phosphorylated proteins in the Western blots and the immunofluorescence of tyrosine-phosphorylated proteins and SPACA1 proteins in the anterior region of the sperm acrosome were lower in bulls whose frozen-stored sperm were classified as having a low-grade acrosome condition. On the other hand, after incubation with cBiMPS, immunodetection levels of at least 10 tyrosine-phosphorylated proteins increased in the connecting and principal pieces of spermatozoa, coincident with the induction of flagellar hyperactivation. Many of the spermatozoa also exhibited detection patterns similar to those of boar hyperactivated spermatozoa. These results are consistent with the suggestion that immunodetection levels of tyrosine-phosphorylated proteins are valid markers that can predict the level of tolerance to frozen storage and the potential to undergo cAMP-dependent hyperactivation for the spermatozoa of individual Japanese Black bulls.