Shin-ichi Kashiwabara

Acrosin Accelerates the Dispersal of Sperm Acrosomal Proteins during Acrosome Reaction

Using homologous recombination, we have previously produced male mice carrying a disruptive mutation (Acr −/−) in the acrosin gene. AlthoughAcr −/− mouse sperm lacking the acrosin protease activity still penetrated the zona pellucida and fertilized the egg, the mutant sperm exhibited a delay in penetration of the zona pellucida solely at the early stages after insemination. To further elucidate the role of acrosin in fertilization, we have examined the involvement of acrosin in the acrosome reaction of sperm using theAcr −/− mutant mice. When the ability of sperm to adhere (attach) and bind to the zona pellucida of cumulus-free eggs was assessed in vitro, no significant difference was observed among Acr +/+,Acr +/−, and Acr −/−mouse sperm. Immunocytochemical analysis demonstrated that the release of several acrosomal proteins from the acrosome ofAcr −/− mouse sperm was significantly delayed during the calcium ionophore- and solubilized zona pellucida-induced acrosome reaction, despite normal membrane vesiculation. These data indicate that the delayed sperm penetration of the zona pellucida in the Acr −/− mouse results from the altered rate of protein dispersal from the acrosome and provide the first evidence that the major role of acrosin is to accelerate the dispersal of acrosomal components during acrosome reaction.

Mouse sperm lacking cell surface hyaluronidase PH-20 can pass through the layer of cumulus cells and fertilize the egg

The function of glycosylphosphatidylinositol-anchored sperm hyaluronidase PH-20 in fertilization has long been believed to enable acrosome-intact sperm to pass through the layer of cumulus cells and reach the egg zona pellucida. In this study, we have produced mice carrying a null mutation in the PH-20 gene using homologous recombination. Despite the absence of sperm PH-20, the mutant male mice were still fertile. In vitro fertilization assays showed that mouse sperm lacking PH-20 possess a reduced ability to disperse cumulus cells from the cumulus mass, resulting in delayed fertilization solely at the early stages after insemination. Moreover, SDS-PAGE of sperm extracts and subsequent Western blot analysis revealed the presence of other hyaluronidase(s), except PH-20, presumably within the acrosome of mouse sperm. These data provide evidence that PH-20 is not essential for fertilization, at least in the mouse, suggesting that the other hyaluronidase(s) may play an important role in sperm penetration through the cumulus cell layer and/or the egg zona pellucida, possibly in cooperation with PH-20, although the importance of sperm motility cannot be neglected.

Mammalian Copper Chaperone Cox17p Has an Essential Role in Activation of Cytochrome c Oxidase and Embryonic Development

ABSTRACT Cox17p is essential for the assembly of functional cytochrome c oxidase (CCO) and for delivery of copper ions to the mitochondrion for insertion into the enzyme in yeast. Although this small protein has already been cloned or purified from humans, mice, and pigs, the function of Cox17p in the mammalian system has not yet been elucidated. In vitro biochemical data for mammalian Cox17p indicate that the copper binds to the sequence -KPCCAC-. Although mouse embryos homozygous for COX17 disruption die between embryonic days E8.5 and E10, they develop normally until E6.5. This phenotype is strikingly similar to embryos of Ctr1(−/−), a cell surface copper transporter, in its lethality around the time of gastrulation. COX17-deficient embryos exhibit severe reductions in CCO activity at E6.5. Succinate dehydrogenase activity and immunoreactivities for anti-COX subunit antibodies were normal in the COX17(−/−) embryos, indicating that this defect was not caused by the deficiency of other complexes and/or subunits but was caused by impaired CCO activation by Cox17p. Since other copper chaperone (Atox1 and CCS)-deficient mice show a more moderate defect, the disruption of the COX17 locus causes the expression of only the phenotype of Ctr1(−/−). We found that the activity of lactate dehydrogenase was also normal in E6.5 embryos, implying that the activation of CCO by Cox17p may not be essential to the progress of embryogenesis before gastrulation.

Acrosin biosynthesis in meiotic and postmeiotic spermatogenic cells

It has been widely accepted that mammalian sperm acrosin is first synthesized only in the postmeiotic stages of spermatogenic cells. In this study, we carried out Northern blot analysis of RNAs prepared from purified populations of mouse spermatogenic cells. The acrosin mRNA was obviously found in meiotic pachytene spermatocytes, and the mRNA content markedly increased in postmeiotic round spermatids. Also, the acrosin mRNA in pachytene spermatocytes was functionally associated with polysomes. These results provide evidence that acrosin biosynthesis is already started in meiotic cells and continues through the early stages of spermiogenesis.

Primary structure of human proacrosin deduced from its cDNA sequence

cDNA clones encoding proacrosin, the zymogen of acrosin, were isolated from a human testis cDNA library by using a fragment of boar acrosin cDNA as a probe. Nucleotide sequencing of the longest cDNA clone has predicted that human proacrosin is synthesized with a 19‐amino‐acid signal peptide at the N‐terminus. The cleavable signal sequence is followed by a 23‐residue segment corresponding to the light chain and then by a 379‐residue stretch that constitutes the heavy chain containing the catalytic site of the mature protease. The C‐terminal portion of the deduced sequence for the heavy chain is very rich in proline residues, most of which are encoded by a unique repeat of CCCCCA. The active‐site residues including histidine, aspartic acid, and serine are also predicted to be located at residues 69, 123, and 221, respectively.

Mouse Sperm Lacking ADAM1b/ADAM2 Fertilin Can Fuse with the Egg Plasma Membrane and Effect Fertilization

Fertilin, a heterodimeric protein complex composed of α (ADAM1) and β (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.

Functional Roles of Mouse Sperm Hyaluronidases, HYAL5 and SPAM1, in Fertilization

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.

A homologue of pancreatic trypsin is localized in the acrosome of mammalian sperm and is released during acrosome reaction.

We have identified cDNA and genomic clones encoding a homologue of pancreatic trypsin, termed TESP4, as a candidate protein involved in the sperm penetration of the egg zona pellucida in mouse. The deduced amino acid sequence indicates that TESP4 is 90% identical to pancreatic trypsin. Analysis of Northern blotting and reverse transcriptase-polymerase chain reaction reveals that the mouse TESP4 gene is ubiquitously expressed in all tissues tested, including the pancreas and testis, and the transcript is present in the haploid stages of male germ cells. Moreover, immunochemical analysis of mouse cauda epididymal sperm using an affinity-purified antibody against bovine pancreatic trypsinogen shows that TESP4 is localized only in the sperm acrosome and is released during the acrosome reaction induced by calcium ionophore A23187. These findings may open a new point of view regarding the molecular mechanisms of the sperm/egg interactions, including the sperm penetration of the egg zona pellucida.

Characterization of Two Cytoplasmic Poly(A)-Binding Proteins, PABPC1 and PABPC2, in Mouse Spermatogenic Cells

Abstract 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.

p-Aminobenzamidine-sensitive acrosomal protease(s) other than acrosin serve the sperm penetration of the egg zona pellucida in mouse

It has been reported that a significant delay in protein dispersal from the acrosomal matrix is observed in wild-type sperm by adding p-aminobenzamidine, a trypsin/acrosin inhibitor, to the incubation medium. The pattern of this delayed release was similar to that of the acrosin-deficient mutant mouse sperm (Yamagata et al., J. Biol. Chem., 273, 10470-4, 1998). In the present study, no further delay in protein dispersal was found when the acrosin-deficient sperm were treated with p-aminobenzamidine, indicating that among the p-aminobenzamidine-sensitive protease(s) only acrosin may function to accelerate this process. Although the acrosin-deficient sperm penetrated the zona pellucida (Baba et al., J. Biol. Chem., 269, 31845-9, 1994), the addition of p-aminobenzamidine to the fertilisation medium caused a significant inhibition of fertilisation in vitro. This indicates that there is a p-aminobenzamidine-sensitive protease(s) other than acrosin participating in the zona penetration step. Indeed, we demonstrated that a non-acrosin protease with a size of 42 kDa was present in the supernatant of the acrosome-reacted sperm suspension. The enzyme was inhibited by p-aminobenzamidine, diisopropyl fluorophosphate and N alpha-tosyl-L-lysine chloromethyl ketone, and was apparently activated by acrosin.