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* This work was supported in part by grant-in-aids for scientific research on priority areas (B), scientific research (A), and exploratory research from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology in Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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.
proteins extracted from acrosome-reacted sperm withn-octyl-β-d-glucopyranoside
soluble proteins released by A23187-inducedacrosome reaction of sperm including acrosomal components
proteins on the plasma and outer acrosomalmembranes fused during the acrosome reaction
Hyaluronic acid, a polymer consisting of repeating disaccharide units of N-acetyl-d-glucosamine andd-glucuronic acid, is one of the most common glycosaminoglycans present in the extracellular matrix of connective tissues (
). Because the structural and functional components in the extracellular matrix maintain the tissue architecture, hyaluronic acid is implicated in many physiological processes, including cell migration, proliferation, and differentiation. Hyaluronidase, which hydrolyzes hyaluronic acid to oligosaccharides, has been identified in mammalian, insect, and bacterial species (
To explore the role of PH-20 in vivo, using homologous recombination we have produced male mice carrying a disruptive mutation in the PH-20 gene. The mice lacking PH-20 are still fertile, providing evidence that PH-20 is not essential for the penetration of sperm through the cumulus cell layer. Moreover, SDS-polyacrylamide gel electrophoresis (PAGE) in the presence of hyaluronic acid demonstrates the existence of other hyaluronidase(s) except PH-20 in mouse sperm. In particular, a hyaluronic acid-hydrolyzing protein with a size of ∼55 kDa is abundantly present in soluble proteins released byA23187-induced acrosome reaction of sperm, including the acrosomal components. Thus, the process governing sperm penetration of the cumulus cell layer needs to be reassessed at least in the mouse model.
To elucidate the functional role(s) of PH-20 in fertilization, the mouse PH-20 gene was disrupted in ES cells by homologous recombination using a targeting construct containing neo and HSV-TK expression cassettes (Fig.1A). A part of exon 2 (E2) coding for the hyaluronidase domain in the PH-20 gene was replaced by the neo cassette. Two of five independent ES cell clones carrying a mutated allele generated chimeric mice transmitting the allele to progeny. The genotypes of wild-type (Ph20+/+), heterozygous (Ph20+/−), and homozygous (Ph20−/−) mice for the null mutation of thePH-20 gene were determined by Southern blot analysis of genomic DNA (Fig. 1B). Northern blot analysis of testicular RNA demonstrated the absence of PH-20 mRNA in thePh20−/− mouse (Fig. 1C). The level of the PH-20 mRNA in the Ph20+/−mouse was ∼50% of that in the wild-type mouse. Moreover, protein extracts of cauda epididymal sperm fromPh20−/− mice completely lacked an immunoreactive 52-kDa protein corresponding to PH-20 when affinity-purified anti-mouse PH-20 antibody was used as a probe (Fig.1D). These data conclusively show the absence of PH-20 inPh20−/− mouse sperm.
Male and female Ph20−/− mice were normal in behavior, body size, and health condition. Morphological analysis demonstrated no significant difference of the shapes, numbers, and sizes of testicular germ cells and epididymal sperm amongPh20+/+, Ph20+/−, and Ph20−/− mice (data not shown). Both the formation of copulation plugs in mated female mice and the motility of cauda epididymal sperm were also normal in the heterozygotes and homozygotes. Moreover, malePh20−/− mice showed normal fertility and produced an average litter size (means ± S.E. = 13.8 ± 0.4, 13.6 ± 1.4, and 12.2 ± 0.8 for 5, 5, and 21 litters inPh20+/+, Ph20+/−, and Ph20−/− mice, respectively). Essentially similar results were obtained in the mouse lines derived from two independent ES clones. Therefore, these results provide evidence that PH-20 is not essential for fertilization at least in the mouse. FemalePh20−/− mice also exhibited normal fertility.
To examine whether the absence of PH-20 affects the process of sperm penetration through the layer of cumulus cells, in vitrofertilization analysis was carried out using capacitated cauda epididymal sperm. We have categorized the status of the mass of cumulus cells surrounding an egg into four patterns (patterns a, b, c, and d) to monitor successive dispersal of cumulus cells from the mass (Fig.2A). When the cumulus cell mass was incubated in the absence of sperm, cumulus cells were spontaneously broken away from the mass. However, the eggs were still associated with many cumulus cells (patterns a and b) in the masses even after the incubation for 6 h (Fig. 2B). Ph20+/+ mouse sperm readily dispersed cumulus cells from the mass, and ∼70% of the eggs completely lost cumulus cells (pattern d) 3 h after insemination. As compared with the wild-type mouse, thePh20−/− mouse showed a remarkable delay in the dispersal of cumulus cells from the mass. The ratio of the patternd cumulus cell mass was less than 20% in thePh20−/− mouse at 3 h after insemination. Thus, the delayed dispersal of cumulus cells may reflect the reduced ability of Ph20−/− mouse sperm to hydrolyze hyaluronic acid in the extracellular matrix of cumulus cells.
As shown in Fig. 3A, anin vitro fertilization assay confirmed the fertility ofPh20−/− mouse sperm. AlthoughPh20+/+ and Ph20−/−mouse sperm equally fertilized the eggs 3 h after insemination, the rate of fertilization in Ph20−/− mouse sperm was significantly lower than that inPh20+/+ mouse sperm solely at the early stages (1 and 2 h) after insemination. To verify the delayed fertilization of Ph20−/− mouse sperm, eggs with or without associated cumulus cells were inseminated with an equally mixed suspension of Ph20+/+ and Ph20−/− mouse sperm. The fertilized eggs with male and female pronuclei were then developed into embryos in vitro, and the genotype of each of the embryos was assessed by PCR. Two DNA fragments with the sizes of 254 and 145 nucleotides, which were PCR-amplified from the null-mutated and wild-type alleles, respectively, were detected (Fig. 3B). When the cumulus-intact eggs were used, Ph20−/− mouse sperm were approximately three times slower to fertilize the eggs thanPh20+/+ mouse sperm (Fig. 3C). In the cumulus-free eggs, the fertilization rate inPh20−/− mouse sperm was still slow but close to that in Ph20+/+ mouse sperm. These data imply that the reduced fertilization rate in Ph20−/−mouse sperm may be due to the delay of the sperm penetration through the cumulus cell layer.
Despite the time delay, Ph20−/− mouse sperm are indeed capable of penetrating the layer of cumulus cells. This fact raises a possibility that other hyaluronidase(s) besides PH-20 may be present in mouse sperm and may participate in the sperm penetration through the cumulus layer, possibly in cooperation with PH-20. To ascertain this possibility, acrosome-intact sperm were extracted withn-octyl-β-d-glucopyranoside, and total hyaluronidase activities in the sperm protein extracts were measured (Fig. 4A). The activities in the Ph20+/− and Ph20−/− mice were found to be ∼80 and 40% of that in the Ph20+/+ mouse, respectively. SDS-PAGE in the presence of hyaluronic acid revealed thatPh20+/+, Ph20+/−, and Ph20−/− mouse sperm all contain a hyaluronic acid-hydrolyzing protein(s) with an approximate size of 55 kDa (Fig.4B). A relatively sharp band of an enzymatically active 52-kDa protein was completely absent only in thePh20−/− mouse. Consequently, the 52-kDa protein corresponds to PH-20, which is consistent with the experimental data obtained by Western blot analysis (Fig. 1D).
To examine the location of a 55-kDa protein(s) and PH-20 exhibiting hyaluronidase activity in sperm, four protein fractions (AI, AR, SPA, and MPA) were prepared from epididymal sperm ofPh20+/+ and Ph20−/−mice and analyzed by Western blotting using affinity-purified anti-mouse PH-20 antibody (Fig. 4C). Affinity-purified antibody against an acrosomal proacrosin-binding protein, sp32 (
), was also used as a control. In the Ph20+/+mouse, only the SPA fraction contained no PH-20, whereas the 55-kDa protein was abundantly present in the SPA fraction. In addition, PH-20 was absent in the AI, AR, and MPA fractions from thePh20−/− mouse. These data clearly demonstrate the presence of other hyaluronidase(s) besides PH-20 presumably within the acrosome of mouse sperm. It should be noted that a very small amount of the hyaluronidase activity resulting from the 55-kDa protein(s) is also found in AR fraction. This may be due to the presence of contaminating acrosome-intact sperm in the AR fraction, because only 80–90% of sperm were acrosome-reacted by calcium ionophore A23187 under the conditions employed in the present study. Indeed, acrosomal protein sp32 is slightly but significantly detectable in the AR fraction (Fig. 4C).
This study demonstrates both a partial contribution of mouse PH-20 toward the sperm penetration through the layer of cumulus cells (Figs. 2 and 3), and the presence of other hyaluronidase(s) besides PH-20 in mouse sperm (Fig. 4). PH-20 has long been thought to be the sole hyaluronidase involved in sperm penetration through the cumulus cell layer, because other sperm hyaluronidases have not been characterized well. Our results indicate that a 55-kDa protein exhibiting hyaluronidase activity is abundantly released by calcium ionophore-induced acrosome reaction presumably from the acrosome (Fig.4). If the acrosome reaction does not occur until acrosome-intact sperm reach the egg ZP, the 55-kDa hyaluronidase may be functional in the sperm/ZP interactions, including secondary sperm-ZP binding. It is also speculated that the motility may be the most important factor in the sperm penetration of the cumulus layer, if hyaluronidases other than PH-20 are absent on the sperm membrane. Thus, the other sperm hyaluronidases remain to be characterized.
Mice carrying either of two Robertsonian translocations on chromosome 6, Rb(6.16) and Rb(6.15), have been reported as showing a significant transmission ratio distortion in the progeny (transmission ratios of 3.6:1 and 2.4:1, respectively) (
). The gene encoding sperm adhesion molecule 1, Spam1, identical to PH-20, is a candidate gene involved in the sperm dysfunction leading to transmission ratio distortion in the Rb(6.16) and Rb(6.15) mice (
). In the present study, the impaired ability of Ph20−/− mouse sperm to fertilize cumulus-intact eggs in vitro (Fig. 3) is apparently consistent with the dysfunction of Rb-bearing mouse sperm. However, Ph20−/− male mice produce a normal average litter size, in contrast with the Rb homozygous mice. These data imply that the dysfunction of Rb-bearing mouse sperm may not be ascribed solely to the reduced amount of sperm Spam1/PH-20.
The human and mouse genomes have been reported to possess six hyaluronidase-like genes, each three genes of which form a cluster on the chromosomes (
). As far as we have examined, the mouse HYALP1and PH-20/SPAM1 genes are both expressed exclusively in testicular tissues, whereas expression of the mouse HYAL4gene in the testis is barely detectable. We have also found the presence of an additional hyaluronidase-like gene (tentatively termedHYAL5) that is localized almost 100 kbp away from thePH-20/SPAM1 gene on the mouse chromosome 6.
), although no direct evidence has been provided that these mutations are responsible for the reduction in the hyaluronidase activity and gene expression. Even so, other point mutation(s) that affect the enzyme activity may occur in theHYALP1 and HYAL5 genes because of the localization adjacent to the PH-20/SPAM1 gene on chromosome 6. Thus, additional experiments concerning possible mutations in the HYALP1 and HYAL5 genes would seem to be required. Moreover, it is important to ascertain whether these gene products are indeed present in mouse sperm.
The reduced rate of Ph20−/− mouse sperm inin vitro fertilization with cumulus-intact eggs (Fig. 3) appears to be ascribable solely to the delayed penetration through the layer of cumulus cells. However, there is a possibility that an incomplete interaction between Ph20−/− sperm and egg ZP, because of the absence of PH-20, may result in the reduced fertilization rate. Our preliminary experiments indicate that no significant difference of ability to bind cumulus-free, ZP-intact eggs 30 min after insemination was observed amongPh20+/+, Ph20+/−, and Ph20−/− mouse sperm (numbers of sperm bound to ZP/egg = 8.8 ± 1.6, 11.3 ± 1.8, and 11.5 ± 2.2, respectively). These data appear to weaken the possible importance of PH-20 in the sperm/ZP interactions, although we have not yet examined the effects of mouse sperm lacking PH-20 on the secondary sperm-ZP binding using “live” acrosome-reacted sperm.
We thank Dr. P. Primakoff for the kind gift of recombinant mouse PH-20.