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Progesterone and the zona pellucida activate different transducing pathways in the sequence of events leading to diacylglycerol generation during mouse sperm acrosomal exocytosis.

ABSTRACT: 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.

SUBMITTER: Murase T

PROVIDER: S-EPMC1218029 | biostudies-other | 1996 Dec

REPOSITORIES: biostudies-other

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Project description:Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.

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Progesterone and the zona pellucida activate different transducing pathways in the sequence of events leading to diacylglycerol generation during mouse sperm acrosomal exocytosis.

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