Epididymal SPAM1 is a marker for sperm maturation in the mouse

Sperm adhesion molecule 1 (SPAM1), is a glycosyl phoshatidylinositol-linked sperm membrane protein that is dually expressed in testis and epididymis. Epididymal SPAM1 is secreted in all three regions of the epididymis in all mammalian species studied, including humans. It shares the same molecular mass and neutral hyaluronidase activity as the testicular and sperm isoforms that are responsible for the penetration of the cumulus during fertilization. Using wild-type (W/T) sperm and those from mice homozygous for either a null (Spam1-/-) or mutant Spam1 allele, which results in decreased mRNA and protein, we demonstrate that sperm binding of epididymal SPAM1 occurs in vitro after exposure to W/T sperm-free epididymal luminal fluid (ELF). Binding or adsorption that occurred after incubation at room temperature or 32 degrees C was detected immunocytochemically and confirmed quantitatively using flow cytometry. The localization of SPAM1 on the plasma membrane of Spam1-null sperm mimicked that seen in the W/T. The remarkable increase in binding on W/T caudal sperm indicates that they are not fully saturated with SPAM1 during storage, and suggests that uptake of epididymal SPAM1 in vivo augments testicular SPAM1. Spam1-null sperm exposed to W/T ELF for 45-60 min during in vitro capacitation to allow epididymal SPAM1 binding showed a highly significant (P < 0.001) increase in cumulus penetration after 6-7 h compared to those incubated in ELF from null males. Similarly, the number of cumulus-free oocytes was also highly significantly greater (P < 0.001) than that for sperm capacitated in W/T SPAM1-antibody-inhibited ELF. Because epididymal SPAM1 uptake significantly increases cumulus penetration, we conclude that it is a marker of sperm maturation.     

SPAM1 (PH-20) protein and mRNA expression in the epididymides of humans and macaques: utilizing laser microdissection/RT-PCR

Background  

The Sperm Adhesion Molecule 1 (SPAM1) is an important sperm surface hyaluronidase with at least three functions in mammalian fertilization. Previously our laboratory reported that in the mouse, in addition to its expression in the testis, Spam1 is synthesized in the epididymis where it is found in membranous vesicles in the principal cells of the epithelium in all three regions. Since SPAM1 is widely conserved among mammals the aim of the study was to determine if its expression pattern in the epididymis is conserved in rodents and primates.     

Rat sperm 2B1 glycoprotein (PH20) contains a C-terminal sequence motif for attachment of a glycosyl phosphatidylinositol anchor. Effects of endoproteolytic cleavage on hyaluronidase activity

Rat sperm 2B1 antigen (the orthologue of guinea pig sperm PH20) is a plasma membrane-bound glycoprotein that is endoproteolytically cleaved during passage through the epididymis and subsequently migrates from the tail to the acrosomal domain during capacitation. Unlike guinea pig PH20, however, sperm surface 2B1 is insensitive to phosphatidylinositol phospholipase C, nor is it known how endoproteolytic cleavage affects its hyaluronidase activity. In this investigation we have expressed 2B1 cDNA in Chinese hamster ovary cells; we have shown that it contains an internal sequence motif for attachment of a glycosyl phosphatidylinositol (GPI) anchor and that cleavage from a single- into a two-chain molecule causes a significant shift in the optimum pH for hyaluronidase activity. Functionally, these results suggest that 1) 2B1 glycoprotein on rat spermatozoa is attached to the plasma membrane via a GPI anchor and that this is an important factor in its ability to migrate from the tail to the acrosomal domain during capacitation; and 2) endoproteolytic cleavage of 2B1 serves to optimize its hyaluronidase activity immediately before fertilization, thereby facilitating penetration of spermatozoa through the cumulus oophorus.     

Investigating the role of murine epididymosomes and uterosomes in GPI-linked protein transfer to sperm using SPAM1 as a model

Sperm uptake of glycosyl phosphatidylinositol (GPI)-linked proteins from luminal fluids has been shown to occur in male and estrous female reproductive tracts. In males, this is attributed to membranous vesicles secreted into the epididymis and prostate. While epididymosomes have been characterized, there have been no reports of the presence of vesicles in female luminal fluids. Here we report the presence of vesicles, characterized as "uterosomes," in the murine estrous female reproductive fluid; and use Sperm Adhesion Molecule 1 (SPAM1/PH-20), a well-known hyaluronidase found in male and female fluids, as a model to investigate vesicle-mediated GPI-linked protein transfer to sperm. Epididymosomes and uterosomes isolated after ultracentrifugation of epididymal (ELF) and uterine luminal fluid (ULF) were analyzed by electron microscopy and shown to be approximately 10-70 and approximately 15-50 nm in diameter. The structural integrity of uterosomes was confirmed by their resistance to hypo-osmotic and freeze/thaw stresses; and immunogold labeling localized SPAM1 to their outer membrane surface, as was the case for epididymosomes. SPAM1 was acquired by caudal sperm during incubation in epididymosomes and uterosomes; uptake was abolished when the GPI anchor was enzymatically cleaved. Sperm analyzed by confocal and transmission electron microscopy (TEM) after incubation in fluorescently labeled vesicles revealed the label on the membrane over the acrosome and midpiece of the flagella, where SPAM1 normally resides. High magnification TEM images demonstrated vesicles juxtaposed to the sperm plasma membrane potentially transferring SPAM1. Taken together, these results implicate vesicular docking as the mechanism of vesicle-mediated GPI-linked protein transfer to sperm from murine reproductive fluids.     

p53 independent,region‐specific epithelial apoptosis is induced in the rat epididymis by deprivation of luminal factors

Luminal testicular factors are known to be important for the regulation of the epididymal epithelium. The present study was undertaken to test the hypothesis that complete deprivation of luminal factors by efferent duct ligation (EDL) would induce apoptosis in the epididymal epithelium, as does removal of trophic factors from other cell types. Additionally, experiments were performed to determine whether the apoptosis detected was p53 dependent or independent. Apoptosis detection was by terminal deoxynucleotidyl‐mediated deoxyuridine triphosphate‐biotin nick‐end labeling and by DNA fragmentation studies. EDL caused loss of testicular luminal contribution in zone 1A of the rat epididymis (proximal initial segment) within 6 hr and induced epithelial apoptosis within 12 hr of the efferent duct obstruction. The wave of apoptosis in zone 1A was completed by three days after EDL and was followed by a much smaller wave in zone 1B which peaked three days after EDL. Significant apoptosis was not detected in any epididymal region distal to the initial segment for periods as long as 15 days after EDL. p53, a key apoptotic‐pathway molecule in many tissues and conditions was tested by immunohistochemical and Western blot techniques and was not upregulated in the initial segment epithelium within the time cells were undergoing apoptosis and well before the wave of apoptosis was complete. It was concluded that epithelial apoptosis in the initial segment of the rat epididymis is induced by deprivation of luminal testicular factors, is localized to the proximal and middle initial segment, and is p53 independent. Mol. Reprod. Dev. 53:188–197, 1999. © 1999 Wiley‐Liss, Inc.     

Immunohistochemical localization of epididymal secretory glycoprotein EP1 in the adult male chimpanzee.

Proteins, synthesized by the epididymal epithelium, are secreted sequentially into the lumen of the ducts epididymis where they effect sperm maturation and enable functional motility and fertilizing capacity. EP1 is a major secretory glycoprotein of chimpanzee (Pan troglodytes) epididymis. The epididymal duct exhibits diverse histology (Smithwick & Young, 1997). Epithelia I-V of the efferent ducts show no characteristic anti-EP1 binding. The densest granules of anti-EP1 reaction product appear in epithelium VI adjacent to the basal lamina in the infranuclear region of the principal cells (PCs), in the cytoplasm of the apical half of the PCs, and in the perinuclear and perivacuolar cytoplasm of the basal cells. In epithelia VII-XIV of the ductus epididymis proper, anti-EP1 binding decreases distally and is localized in the cytoplasm of the PCs and basal cells, among the stereocilia of the luminal border, within various microvillar borders, and in the luminal fluid. Therefore, EP1 appears to be synthesized and secreted primarily in the caput region of the ductus epididymis and may be reabsorbed nonselectively across epithelia with apical microvilli, including the non-ciliated cells of efferent ducts, the distal corpus and cauda of the ductus epididymis, and the proximal ductus deferens.     

Mouse Hyal3 encodes a 45- to 56-kDa glycoprotein whose overexpression increases hyaluronidase 1 activity in cultured cells

Hyaluronidases are enzymes that mediate the breakdown of hyaluronan(HA), a large polysaccharide abundant in the extracellular matrixof vertebrate tissues. Six genes have been predicted to encodehyaluronidases in humans, but the protein products of only SPAM1,HYAL1, and HYAL2 have been characterized. We have now expressedthe mouse Hyal3 gene product, hyaluronidase 3 (Hyal3), in BabyHamster Kidney (BHK) cells and demonstrated the presence ofmultiple forms of Hyal3 ranging from 45 to 56 kDa in expressionlysates. Complete and partial digestions of the expressed proteinwith PNGase F showed three N-linked oligosaccharides accountedfor all forms of Hyal3 detected in expression lysates. Mostof these oligosaccharides were Endo H sensitive, indicatingthat they were high mannose or hybrid N-linked oligosaccharides.Subcellular fractionation of Hyal3-expressing BHK cells by densitygradient centrifugation revealed most Hyal3 in a low-densityvesicular population. Low levels of Hyal3 were detected in higherdensity vesicles, but no colocalization with the late endosomal/lysosomalmarker Lamp1 was found by immunofluorescence microscopy. BHKcells stably expressing Hyal3 had increased acid-active hyaluronidaseactivity, but no such activity was detected when Hyal3 was transfectedinto Hyaluronidase 1 (Hyal1)-deficient fibroblasts. Overexpressionof Hyal3 in BHK cells increased the Hyal1 protein and mRNA levels,suggesting that the increased hyaluronidase activity in thesecells was due to Hyal1 rather than Hyal3. The results indicatethat Hyal3 overexpressed in cultured cells lacks intrinsic hyaluronidaseactivity and that Hyal3 may contribute to HA metabolism by augmentingthe activity of Hyal1.     

Effect of swainsonine on rat epididymal glycosidases

Each epididymis of control and swainsonine-fed rats (5 micrograms/ml drinking water) was divided into 5 segments, and tissue, spermatozoa and sperm-free supernatants were prepared from each segment. When levels of 3 lysosomal glycosidases and total protein were determined, the proximal cauda contained the greatest concentration of glycosidase. The specific-activity profile for beta-glucuronidase and beta-galactosidase was similar in swainsonine-fed and control rats. However, the concentration of alpha-D-mannosidase in tissue of all segments was significantly greater in swainsonine-fed rats than in age-matched controls. Enzyme activity for alpha-D-mannosidase after swainsonine treatment was significantly greater in spermatozoa from the caput, than in spermatozoa from the corpus and the cauda epididymidis. Since the alpha-D-mannosidase activity was optimal at pH 4.5 and studies with highly specific antibody to lysosomal alpha-D-mannosidase immunoprecipitated all of the alpha-D-mannosidase present in detergent extracts of epididymal tissue, spermatozoa, and sperm-free supernatant, the enzyme studied is of lysosomal origin.     

A porcine homolog of the major secretory protein of human epididymis, HE1, specifically binds cholesterol.

A porcine homolog of the major secretory protein of human epididymis, HE1, was for the first time purified from the porcine cauda epididymal fluid. The HE1 homolog was secreted into the epididymal fluid as a 19-kDa glycoprotein, whose sugar moiety was gradually processed to form a 16-kDa protein during transit through the epididymis. The HE1 homolog mRNA was detected only in the caput and corpus epididymis among the porcine tissues examined. The purified HE1 homolog specifically bound cholesterol with high affinity (Kd=2. 3 microM). The binding stoichiometry was determined to be 0.94 mol/mol, suggesting that 1 mol of cholesterol binds to 1 mol of the protein. It was also found that the HE1 homolog is a major cholesterol-binding protein in the porcine epididymal fluid. The possibility that the HE1 homolog is involved in the regulation of the lipid composition of the sperm membranes during the maturation in epididymis is discussed.     

Binding of a 15 kDa glycoprotein from spermatozoa of boars to surface of zona pellucida and cumulus oophorus cells.

A highly purified 15 kDa glycoprotein isolated from ejaculated spermatozoa was used to raise antisera in female rabbits. An indirect immunofluorescence technique was used to detect the antigen in the seminal vesicle tissue and on the acrosomes of ejaculated, native and capacitated, boar spermatozoa. No immunoreactivity was detected on cells of the seminiferous tubules (spermatogonia, spermatocytes, and spermatids), on spermatozoa in the ductus epididymis and in cells of the epididymal and testicular tissues. These observations support the view that the 15 kDa protein is produced in the seminal vesicle secretory epithelium, and is attached to the sperm plasma membrane during the exposure of spermatozoa to seminal vesicle compounds. The observations that the antigen remained on the acrosome of ejaculated spermatozoa after capacitation and blocked sperm-oocyte binding in vitro suggest that the antigen plays a role in sperm-egg interactions. The strong immunoreactivity exhibited by cumulus cells after incubation of antisera with the porcine egg surrounded by cumulus cells shows the possible importance of the 15 kDa glycoprotein for contact of spermatozoa with cells of the cumulus oophorus surrounding the egg.     

Expression Analysis of Six Paralogous Human Hyaluronidase Genes Clustered on Chromosomes 3p21 and 7q31

Two new members of a family of putative hyaluronidase genes involved in glycosaminoglycan catabolism have been identified and mapped by FISH and YAC library screening to chromosome 7q31.3. One of these (HYALP1) is an expressed pseudogene with mutations in the genomic DNA and cDNA. The six members of the hyaluronidase family are grouped into two tightly linked triplets on human chromosomes 3p21.3 (HYAL1, HYAL2, and HYAL3) and 7q31.3 (HYAL4, SPAM1 (PH-20), and HYALP1). This arrangement could arise by an ancient cluster formation, followed by a more recent cluster block-duplication. All of the hyaluronidase genes have unique tissue-specific expression patterns as determined by Northern blot analysis of 23 human tissues. HYAL1, HYAL2, and HYALP1 are widely expressed, but HYAL3 is differentially expressed in bone marrow and testis, while HYAL4 is differentially expressed in placenta and skeletal muscle. SPAM1 (PH-20) was detectable only in testis by Northern blot as previously reported, but was detectable in fetal and placental cDNA libraries by PCR, suggesting a possible role for this gene during embryonic development.     

Immunolocalization of a 25-kilodalton protein in mouse testis and epididymis.

We have recently observed that a polyclonal antibody raised against a mouse epididymal luminal fluid protein (MEP 9) recognizes a 25-kDa antigen in mouse testis and epididymis [Rankin et al., Biol Reprod 1992; 46:747-766]. This antigen was localized by light and electron microscopic immunohistochemistry. The immunoreactivity in the testis was found in the residual cytoplasm of the elongated spermatids, in the residual bodies, and in the cytoplasmic droplets of spermatozoa. In the epididymis, the epithelial principal cells were stained from the distal caput to the distal cauda. Immunogold labeling in the principal cells showed diffuse distribution without preferential accumulation in either the endocytic or the secretory apparatus of the cells. In the epididymal lumen, the immunoreactivity was restricted to the sperm cytoplasmic droplets. No membrane-specific labeling was observed in luminal spermatozoa, cytoplasmic droplets, or isolated sperm plasma membranes. Three weeks after hemicastration or severance of the efferent ducts, a normal distribution of the immunoreactive sites was found in the epididymis. Immunoreactivity, was also detected in the epididymal epithelium of immature mice as well as in that of XXSxr male mice having no spermatozoa in the epididymis. These results suggest that the immunoreactivity seen in the principal cells originates from synthesis rather than endocytosis of the testicular protein from disrupted cytoplasmic droplets. Furthermore, these results suggest that the 25-kDa protein is synthesized independently by both testis and epididymis.     

VEGF和Flt-1在精索静脉曲张大鼠附睾中的表达变化

目的研究血管内皮生长因子(VEGF)及其受体Flt-1在实验性左侧精索静脉曲张(ELV)大鼠附睾组织中的表达变化,探讨它们与精索静脉曲张(VC)的关系及致男性不育的病理生理学机制。方法建立青春期雄性SD大鼠ELV模型,采用免疫组化SP法检测ELV及对照组附睾中VEGF和Flt-1的表达。结果 VEGF和Flt-1蛋白在大鼠附睾中均有表达,并具有细胞和区域特异性。ELV4周时双侧附睾中VEGF蛋白的表达明显上调(P<0.01),8周时则显著下降(P<0.01);而Flt-1蛋白在ELV4周左侧显著下降(P<0.01),右侧未见明显差异(P>0.05),8周组均显著下降(P<0.01)。结论 ELV引起大鼠附睾中VEGF和Flt-1表达量发生变化,可能影响精子的成熟,是VC引起男性生育力下降甚至不育的原因之一。     

Specific distribution of an epididymal 50 kDa protein revealed by an anti-Mos protein monoclonal antibody.

An anti-Mos protein monoclonal antibody, 4A6, was used to investigate the distribution of the antigen in the epididymis, in which the c-mos gene is reportedly expressed. The 4A6-reactive antigen was found on the basement membrane and luminal surface of the epithelial cells in the caput epididymis of BALB/c male mice as well as in the proximal corpus epididymis, the cauda epididymis, and the vas deferens. The 4A6 antigen was also found on the luminal surface of the epithelial cells in the epididymis of male germ cell-deficient C57BL/6J-Wv/Wv mice. This confirmed that the 4A6 antigen does not derive entirely from the testicular c-Mos protein but is synthesized in the epididymis. Western blot analysis revealed that the molecular weight of the epididymal 4A6 antigen was 50 kDa, which is unusually high for the c-Mos protein. With its specific distribution in the epididymis, the protein should play a specific role in functions of the epididymis.