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Steroid receptor coactivator-3 (SRC-3) is a coactivator of nuclear receptors in the SRC family as assayed in vitro. Here, we show that mouse SRC-3 is expressed in a tissue-specific fashion and distributed mainly in the oocytes, mammary glands, hippocampus, olfactory bulb, smooth muscle, hepatocytes, and vaginal epithelium. Genetic disruption of SRC-3 in mice results in a pleiotropic phenotype showing dwarfism, delayed puberty, reduced female reproductive function, and blunted mammary gland development. Hormonal analysis indicates that SRC-3 plays a role in both the growth hormone regulatory pathway and the production of estrogen, which may explain the observed phenotypes. These results suggest that the physiological role of SRC-3 is different from that of SRC-1 and prove the diversity among coactivator family members.

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We have previously reported the occurrence and partial characterisation of an alpha-D-mannosidase activity on plasma membranes of rat, mouse, hamster and human spermatozoa. A soluble isoform of the rat sperm surface mannosidase was purified and polyclonal antibody raised. Since several reports have suggested that mannosyl residues on the rat, mouse and human zona pellucida may be involved in sperm-zona binding, studies were undertaken to examine the receptor-like role of mannose-binding molecules on rat spermatozoa. Sprague-Dawley rats (25-30-days old) were superovulated and eggs collected from the oviduct were treated with 0.3% hyaluronidase to remove the cumulus cells. Spermatozoa, collected from the cauda epididymis were capacitated for 5 h at 37 degrees C in 5% CO2 in air. The sperm-zona binding assay was performed in the presence of increasing concentrations of several sugars as well as preimmune and immune (anti-mannosidase or anti-mannose binding protein) IgG. Data from these studies show that: (1) significantly fewer sperm bound per egg in the presence of competitive inhibitors of mannosidase; (2) among the sugars examined, D-mannose was the most potent inhibitor causing 70% reduction in the number of sperm bound per egg; (3) anti-mannosidase or anti-mannose binding protein (but not preimmune) IgG showed a dose-dependent reduction in the number of sperm bound per egg; (4) anti-mannosidase IgG (but not anti-mannose binding protein IgG) showed a dose-dependent inhibition of sperm surface mannosidase activity; (5) the competitive inhibitors of mannosidase or the immune IgG had no effect on sperm motility or the sperm acrosome reaction. These result suggest that mannose-binding molecule(s) such as alpha-D-mannosidase or mannose-binding protein on the spermatozoa may recognise mannosyl residues on zona pellucida, and play a receptor-like role in sperm-egg interaction in the rat.

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The zona pellucida (ZP), the extracellular glycocalyx that surrounds the oocyte, is well known to mediate homologous gamete interaction. In a previous study from our laboratories, we reported the qualitative characterization of the rat ZP. The ZP in this species, like the mouse, hamster, and human, was found to contain three glycoproteins, namely rZP1, rZP2, and rZP3 (Araki et al. [1992] Biol Reprod 46:912-919). In this study, cDNAs encoding whole rat ZP major components have been isolated and characterized. A rat ovary cDNA library was screened with the mouse ZP3 and ZP2 cDNA probes, respectively. For rZP1 cDNA cloning, cDNAs generated using reverse transcriptase-polymerase chain reaction and rapid amplification of 5' and 3' cDNA ends, were isolated and sequenced. The rZP3 cDNA showed 1338 bp with a coding region containing 1272 bp, that translates into 424 amino acids. The total translation of rZP3 peptide has a molecular weight of 45,820, containing six potential N-glycosylation sites and 75 Ser/Thr residues, possible O-glycosylation sites. The amino acid sequence derived from the cDNA sequence shares high sequence homologies to mouse (90%), hamster (78%), and human (65%) ZP3 (ZPC) glycoproteins, indicating that the rat and mouse ZP3 have quite a conserved amino acid sequence, including the potential glycosylation sites. The total transcript of the rZP2 was 2154 nucleotides and the largest open reading frame was 695 amino acids. This would translate into a protein of 78.4 kDa. In the case of rZP1, the cDNA clone consisted of 1960 bp, and the coding region contained 1851 bp translating into 617 amino acids. Significant homologies were observed between rZP2 and ZPA family from various mammalian species. The rZP1 also showed a sequence homology to mouse ZP1, known as a mouse orthologue of ZPB family, suggesting that the rZP2 and rZP1 are members of ZPA and ZPB families, respectively. The message distributions for each zona components were limited within the ovary and the signal was detectable in the growing oocytes. The present results will further our understanding of the structure of rat zona components and lead to a better understanding of species-specificity observed during sperm-egg interaction.

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