A glycosylphosphatidylinositol (GPI)-anchored hyaluronidase, PH-20, on the sperm surface has long been believed to assist sperm penetration through the cumulus mass surrounding the eggs. However, mouse sperm lacking PH-20 were still capable of penetrating the cumulus mass despite a delayed dispersal of cumulus cells. Intriguingly, a 55-kDa hyaluronan-hydrolyzing protein was abundantly present in wild-type and PH-20-deficient mouse sperm. In this study, we purified the 55-kDa mouse protein from soluble protein extracts released from epididymal sperm by acrosome reaction and identified as a hyaluronidase, Hyal5.

Fertilin, a heterodimeric protein complex composed of ␣ (ADAM1) and ␤ (ADAM2) subunits on the sperm surface, is believed to mediate adhesion and fusion between the sperm and egg plasma membranes. Here we have shown that mutant male mice lacking ADAM1b are fertile and that the loss of ADAM1b results in no significant defect in sperm functions such as migration from the uterus into oviduct, binding to egg zona pellucida, and fusion with zona pellucida-free eggs. ADAM1b-deficient epididymal sperm showed a severe reduction of ADAM2 on the cell surface, despite the normal presence of ADAM2 in testicular germ cells. The appearance of ADAM1b and ADAM2 on the sperm surface depended on formation and abundance of ADAM1b/ADAM2 fertilin in testicular germ cells. These results suggest that mouse ADAM1b/ADAM2 fertilin may play a crucial role not in the sperm/ egg fusion but in the appearance of these two ADAMs on the sperm surface.

Although sperm entry into the oocyte-cumulus complex and subsequent sperm penetration through the cumulus matrix to reach the oocyte zona pellucida are essential for mammalian fertilization, the molecular mechanism remains controversial. Previously, we have shown that mouse sperm lacking SPAM1 are capable of penetrating the cumulus matrix despite a delayed dispersal of cumulus cells. We also have identified another sperm hyaluronidase, HYAL5, as a candidate enzyme involved in sperm penetration through the cumulus. In the present study, we produced HYAL5-deficient mice to uncover the functional roles of HYAL5 and SPAM1 in fertilization. The HYAL5-deficient mice were fully fertile and yielded normal litter sizes. In vitro fertilization assays demonstrated that HYAL5-deficient epididymal sperm is functionally normal. We thus conclude that HYAL5 may be dispensable for fertilization. Comparative analysis among wild-type, HYAL5-deficient, and SPAM1-deficient epididymal sperm revealed that only SPAM1 is probably involved in sperm penetration through the cumulus matrix. Notably, the loss of SPAM1 resulted in a remarkably increased accumulation of sperm on the surface or outer edge of the cumulus. These data suggest that SPAM1 may function in sperm entry into the cumulus and sperm penetration through the cumulus matrix.

Mouse spermatogenic cells are known to contain at least two isoforms of cytoplasmic poly(A)-binding proteins, PABPC1 and PABPC2 (previously known as PABPT). In this study, we have characterized PABPC1 and PABPC2. PABPC2 was present in pachytene spermatocytes and round spermatids, whereas elongating spermatids still included PABPC1. These two proteins are capable of binding mRNA poly(A) tails nonspecifically and of directly associating with each other and with several translational regulators, including EIF4G1, PAIP1, PAIP2, and PIWIL1 (previously known as MIWI). Moreover, both PABPC1 and PABPC2 exhibited the ability to enhance translation of a reporter mRNA in vitro. Despite these similarities, PABPC2 was distinguished from PABPC1 by the absence of PABPC2 in actively translating polyribosomes of testicular cells. PABPC1 was distributed in polyribosomes and in translationally inactive messenger ribonucleoprotein particles. Most importantly, PABPC2 and PIWIL1 were noticeably enriched in the chromatoid body of round spermatids. These results suggest that PABPC2 may function in translational repression during spermatogenesis.

Mammalian fertilization requires sperm to penetrate the cumulus mass and egg zona pellucida prior to fusion with the egg. Although sperm penetration through these physical barriers is essential, the molecular mechanism has not yet been completely elucidated. In addition to sperm motility, hyaluronan-hydrolyzing and proteolytic enzymes of sperm have been suggested to participate in the penetration events. Here we focus on the functional roles of hyaluronidase and protease in sperm passage through the cumulus mass and zona pellucida.

14The elongation of poly(A) tails in cytoplasm is essential for oogenesis and early

The effects of various proteolytic enzymes on the high molecular weight protein (connectin) present in a direct sodium dodecyl sulfate extract of myofibrils from chicken breast muscle were studied in detail. To keep the high molecular weight proteins intact, myofibrils had to be prepared in the presence of EGTA. Trypsin, chymotrypsin, papain, and nagarse readily hydrolyzed connectin (doublet band of titin) and the band 3 protein (N2-line protein). Pepsin did not attack connectin, but digested the band 3 protein and myosin. Calcium-activated neutral proteinase hydrolyzed the band 3 protein, leaving connectin intact. On the other hand, serine protease digested connectin but not the band 3 protein.

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