Expression of constitutively active Notch1 in male genital tracts results in ectopic growth and blockage of efferent ducts, epididymal hyperplasia and sterility

The Notch signaling pathway is involved in a variety of developmental processes. Here, we characterize the phenotypes developing in the reproductive organs of male transgenic (Tg) mice constitutively expressing the activated mouse Notch1 intracellular domain (Notch1(intra)) under the regulatory control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). Tg expression was detected in testis, vas deferens and epididymis by Northern blot analysis. In situ hybridization with a Notch1-specific probe lacked sensitivity to detect expression in normal-appearing cells, but demonstrated expression in hyperplastic epithelial cells of the vas deferens, epididymis and efferent ducts. Tg males from three independent founder lines were sterile. Histological analysis of reproductive organs of young Tg males (postnatal ages 8 and 21) showed no difference compared to those of non-Tg males. In contrast, in adult Tg mice from day 38 onwards, the efferent ducts, the vas deferens and most epididymal segments revealed bilateral epithelial cell hyperplasia with absence of fully differentiated epithelial cells. Electron microscopy confirmed the uniformly undifferentiated state of these cells. Immunohistochemistry with anti-PCNA antibody also revealed enhanced proliferation of Tg epididymis. In adult Tg testis, the different generations of germ cells of seminiferous tubules appeared normal, although some tubules were highly dilated and revealed an absence of early and/or late spermatids. The epithelial cells of the Tg tubuli recti and rete testis were not abnormal, but the rete testis was highly dilated and contained numerous spermatozoa, suggesting a downstream blockage. Consistent with a blockage of efferent ducts often seen at the rete testis/efferent duct interface, spermatozoa were absent in epididymis of all adult Tg mice and in all highly hyperplastic efferent duct tubules of these Tg mice. Such a blockage was visualized by injection of Evans blue dye into the rete testis lumen. Finally, the presence of ectopic hyperplastic efferent duct tubules was observed within the testicular parenchyma itself, outside their normal territory, suggesting that Notch1 signaling is involved in the establishment of these borders. This phenotype seems to represent a novel developmental defect in mammals. Together, these results show that constitutive Notch1 signaling significantly affects the development of male reproductive organs.     

Distribution of immune cells in the epididymis of the aging Brown Norway rat is segment-specific and related to the luminal content.

Remarkable changes occur during aging in the testis and epididymis of the Brown Norway rat. A dramatic increase in the number of halo cells, which are present in the epididymal epithelium and originate from the immune system, is found in animals of increasing age. Halo cells have been postulated to be either lymphocytes or monocytes. We hypothesized that halo cells are a mixture of different immune cells and that their relative composition changes with age. To verify this hypothesis, markers for helper T lymphocytes, cytotoxic T lymphocytes, B lymphocytes, and monocytes-macrophages were used to identify the major categories of immune cells in the epididymides of Brown Norway rats ranging in age from 3 to 24 mo. The numbers of immunocompetent cells in the epididymis were determined in relation to age, epididymal segment, and luminal content. We found that monocytes, helper T lymphocytes, and cytotoxic T lymphocytes belong to the population of halo cells. In addition, a segment-specific increase with age in the number of these immune cells was noted. Finally, we report a segment-specific recruitment of cytotoxic T lymphocytes and monocytes-macrophages in the epididymal epithelium of aged rats whose epididymal lumen contained few spermatozoa. We postulate that accumulation of damaged epithelial cells and antigens of germ cell origin, leaking through a dysfunctional blood-epididymis barrier, may contribute to the active recruitment of immune cells with age.     

Two-dimensional electrophoresis of proteins in principal cells, spermatozoa, and fluid associated with the rat epididymis

Spermatozoa, fluids, and principal cells from different regions of the epididymis were characterized by two-dimensional electrophoresis. Rete testis fluid was collected after 36-h efferent duct ligation, and cauda epididymal fluid was collected by retrograde perfusion through the vas deferens. Spermatozoa were collected after their exudation from minced caput and corpus epididymal tissue. Principal cells were recovered after enzymatic disaggregation and centrifugal elutriation of epididymides. Two-dimensional polyacrylamide gel electrophoresis was used to prepare protein profiles of all samples. Comparison of the proteins found in rete testis fluid versus those found in cauda epididymal fluid revealed a dramatic change in composition, including the loss, addition, or retention of specific proteins as well as changes in the relative concentrations of certain proteins. Prominent cauda epididymal fluid proteins, possibly contributed by the epididymal epithelium, were detected at 16, 23, and 34 kDa. After epididymal transit, a considerable decrease was observed in the number of aqueous-soluble sperm proteins. Differences in the protein composition of epididymal epithelial principal cells from the caput versus corpus epididymidis were also noted, suggesting that functional differences exist for these epididymal regions. Of particular interest was the occurrence of a prominent protein of approximately 20-23 kDa found in all sperm samples, in fluids, and in caput and corpus principal cells. However, this protein was absent in cauda epididymal sperm after 36-h efferent duct ligation. The rapid loss of this protein from sperm after efferent duct ligation suggests that this surgical intervention may affect spermatozoa residing within the epididymis.     

Influence of rete testis fluid deprivation on the kinetic parameters of goat epididymal 5 alpha-reductase

A surgical technique to cannulate the rete testis of the goat was utilized to examine the effects of rete testis fluid (RTF) deprivation on the enzymatic activity of epididymal 5 alpha-reductase. Kinetic techniques were used to determine whether the regional enzymatic effect of RTF deprivation is to decrease the apparent number of 5 alpha-reductase active sites or the catalytic activity of each active site within the epididymal epithelium. Paired comparisons of (Vmax)app and (Km)app values between control and RTF-deprived epididymides indicated that RTF deprivation affected the value of (Vmax)app with no apparent change in the values of (Km)app in caput, corpus, and cauda epididymal regions. We conclude that RTF deprivation in the goat epididymis for 7 days results in a decreased number of apparent 5 alpha-reductase active sites within the epididymal epithelium.     

Expression and localization of PMCA4 in rat testis and epididymis

It has recently been shown in mice that the plasma membrane Ca²⁺-ATPase isoform 4 (PMCA4) is essential for sperm fertilization capacity. We analyzed whether sperm PMCA4 is formed in the rat during spermatogenesis or is synthesized in the epididymis and transferred onto sperm during sperm maturation. We could show that PMCA4 is conserved in sperm from testis to epididymis. In testis, PMCA4 mRNA was restricted to spermatogonia and early spermatocytes, while the PMCA4 protein was detected in spermatogonia, late spermatocytes, spermatids and in epididymal sperm. In epididymis PMCA4 mRNA was localized in basolateral plasma membranes of epithelial cells of the caput, corpus and cauda epididymidis. In contrast, the protein was only detectable in the epithelial cells of the caput, indicating that PMCA4 mRNA is only translated into protein in caput epithelium. In the epididymal corpus and cauda, PMCA4 mRNA and protein, respectively, was localized and in peritubular cells. Furthermore, we detected an identical distribution of PMCA4a and b splice variants in rat testis, epididymal corpus and cauda. In the caput epididymidis, where PMCA4 is located in the epithelium splice variant 4b was more prominent. Further experiments have to clarify the functional importance of the differences in the PMCA4 distribution.     

Seasonal changes in epididymis and the effects of FSH and testosterone in the spiny-tailed lizard,Uromastix hardwicki

Seasonal changes in epididymal weight and histology were studied in relation to testicular function in the adult spiny-tailed lizard, Uromastix hardwicki, over a period of 1 year. The eipdidymal weights, tubular diameter, and epithelial height increased in March, reaching a peak in April. This peak coincided with sperm maturation, elevated plasma testosterone levels, and release of sperm into the epididymis. The epididymal weights decreased in May following a sudden regression of the testis early in the month. The epididymal weights decreased further during June and remained low until February. The diameter of the duct and the height of the epithelial cells also decreased in May and the epididymal epithelium maintained a low histological profile from June to February. The fall testicular recrudescence was not accompanied by a change either in the weight or the histological structure of the epididymis. Administration of oFSH (0.1 mg) daily for 7 days during the sexually quiescent period induced a significant increase in the weight of the epididymis and epithelial height of the duct. Administration of testosterone alone, (2.0 mg) daily for the same period and under identical conditions, did not induce a change in the weight of epididymis or its histology. A possible permissive role of gonadotrophin in the hormonal regulation of the lizard epididymis has been suggested.     

Effects of breeding season and mating on total number and distribution of spermatozoa in the epididymis of the brown marsupial mouse, Antechinus stuartii

Changes in the number and distribution of spermatozoa in the epididymis of the adult brown marsupial mouse were examined during July/August in mated and unmated males. The effects of mating on epididymal sperm populations were studied in 2 groups of males each mated 3 times and compared with the number and distribution of spermatozoa in the epididymides of 4 unmated control groups. One testis and epididymis were removed from each animal (hemicastration) either before or early in the mating season to provide information on initial sperm content and distribution. The contralateral side was removed later in the mating season to examine the effects of mating or sexual abstinence on epididymal sperm distribution. Epididymal sperm number peaked in both the distal caput and distal corpus/proximal cauda epididymidis in late July. The total number of spermatozoa, including those remaining in the testis, available to each male at the beginning of the mating season in early August was approximately 4.4 x 10(6)/side. Although recruitment of spermatozoa into the epididymis from the testis continued until mid-August, sperm content of the epididymis reached a peak of about 3.5 x 10(6)/epididymis in early August. At this time approximately 0.9 x 10(6) spermatozoa remained in the testis which had ceased spermatogenic activity. Throughout the mating season, epididymal spermatozoa were concentrated in the distal corpus/proximal cauda regions of the epididymis and were replenished by spermatozoa from upper regions of the duct. Relatively few spermatozoa were found in the distal cauda epididymidis, confirming a low sperm storage capacity in this region. A constant loss of spermatozoa from the epididymis, probably via spermatorrhoea, occurred throughout the mating season and very few spermatozoa remained in unmated males in late August before the annual male die-off. Mating studies showed that an average of 0.23 x 10(6) spermatozoa/epididymis were delivered per mating in this species, but the number of spermatozoa released at each ejaculation may be as few as 0.04 x 10(6)/epididymis when sperm loss via spermatorrhoea is taken into account. We suggest that the unusual structure of the cauda epididymidis, which has a very restricted sperm storage capacity, may function to limit the numbers of spermatozoa available at each ejaculation and thus conserve the dwindling epididymal sperm reserves in order to maximize the number of successful matings which are possible during the mating season.     

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.     

Zika virus persistence in the male macaque reproductive tract

Zika virus (ZIKV) is unique among mosquito-borne flaviviruses in that it is also vertically and sexually transmitted by humans. The male reproductive tract is thought to be a ZIKV reservoir; however, the reported magnitude and duration of viral persistence in male genital tissues vary widely in humans and non-human primate models. ZIKV tissue and cellular tropism and potential effects on male fertility also remain unclear. The objective of this study was to resolve these questions by analyzing archived genital tissues from 51 ZIKV-inoculated male macaques and correlating data on plasma viral kinetics, tissue tropism, and ZIKV-induced pathological changes in the reproductive tract. We hypothesized that ZIKV would persist in the male macaque genital tract for longer than there was detectable viremia, where it would localize to germ and epithelial cells and associate with lesions. We detected ZIKV RNA and infectious virus in testis, epididymis, seminal vesicle, and prostate gland. In contrast to prepubertal males, sexually mature macaques were significantly more likely to harbor persistent ZIKV RNA or infectious virus somewhere in the genital tract, with detection as late as 60 days post-inoculation. ZIKV RNA localized primarily to testicular stem cells/sperm precursors and epithelial cells, including Sertoli cells, epididymal duct epithelium, and glandular epithelia of the seminal vesicle and prostate gland. ZIKV infection was associated with microscopic evidence of inflammation in the epididymis and prostate gland of sexually mature males, pathologies that were absent in uninfected controls, which could have significant effects on male fertility. The findings from this study increase our understanding of persistent ZIKV infection which can inform risk of sexual transmission during assisted reproductive therapies as well as potential impacts on male fertility.     

Morphology and immunolocalization of aquaporins 1 and 9 in the agouti (Dasyprocta azarae) testis excurrent ducts

This study investigated the morphology and immunoexpression of aquaporins (AQPs) 1 and 9 in the rete testis, efferent ducts, epididymis, and vas deferens in the Azara’s agouti (Dasyprocta azarae). For this purpose, ten adult sexually mature animals were used in histologic and immunohistochemical analyses. The Azara’s agouti rete testis was labyrinthine and lined with simple cubic epithelium. Ciliated and non-ciliated cells were observed in the epithelium of the efferent ducts. The epididymal cellular population was composed of principal, basal, apical, clear, narrow, and halo cells. The epithelium lining of vas deferens was composed of the principal and basal cells. AQPs 1 and 9 were not expressed in the rete testis. Positive reaction to AQP1 was observed at the luminal border of non-ciliated cells of the efferent ducts, and in the peritubular stroma and blood vessels in the epididymis, and vas deferens. AQP9 was immunolocalized in the epithelial cells in the efferent ducts, epididymis and vas deferens. The morphology of Azara’s agouti testis excurrent ducts is similar to that reported for other rodents such as Cuniculus paca. The immunolocalization results of the AQPs suggest that the expression of AQPs is species-specific due to differences in localization and expression when compared to studies in other mammals species. The knowledge about the expression of AQPs in Azara’s agouti testis excurrent ducts is essential to support future reproductive studies on this animal, since previous studies show that AQPs may be biomarkers of male fertility and infertility.     

Reproduction in the male sub-Antarctic fur seal Arctocephalus tropicalis

The reproductive tracts of male sub-Antarctic fur seals Arctocephalus tropicalis ( n = 123), taken at Gough Island (40° 20'S, 9° 54'W) between November 1977 and October 1978, were examined. The presence of spermatozoa in the epididymal tubules showed that all males ≥4 years old had reached puberty, and the marked slower increase in mean testis weight and baculum length suggested that sexual (social) maturity was approached by 8-year-olds. Full adulthood was attained at 10–11 years of age based on the peak in mean testis and prostate weights, and mean baculum length. Secondary sexual characteristics were only fully developed in males ≥9 years old. A significant decline in mean testosterone concentration, and in testis, epididymis and prostate weights showed that adult males were reproductively quiescent during winter from May to July when seminiferous and epididymal tubules had lowest mean diameters with no spermatozoa present. Both the mean plasma testosterone concentration and mean testis weight peaked twice during the austral summer. The first peak coincided with the breeding season, and the second peak with the moulting period when adult males were impotent. Photoperiodic cueing might explain this seasonal trend.     

Direct injection of foreign DNA into mouse testis as a possible in vivo gene transfer system via epididymal spermatozoa.

We have attempted to transfect testicular spermatozoa with plasmid DNA by direct injection into testes to obtain transgenic animals [this technique was thus termed "testis-mediated gene transfer (TMGT)"]. When injected males were mated with superovulated females 2 and 3 days after injection, (i) high efficiencies (more than 50%) of gene transmission were achieved in the mid-gestational F0 fetuses, (ii) the copy number of plasmid DNA in the fetuses was estimated to be less than 1 copy per diploid cell, and (iii) overt gene expression was not found in these fetuses. These findings suggest the possibility that plasmid DNA introduced into a testis is rapidly transported to the epididymis and then incorporated by epididymal spermatozoa. The purpose of this study was to elucidate the mechanism of TMGT by introducing trypan blue (TB) or Hoechst 33342 directly into testis. We found that TB is transported to the ducts of the caput epididymis via rete testis within 1 min after testis injection, and TB reached the corpus and cauda epididymis within 2-4 days after injection. Staining of spermatozoa isolated from any portion of epididymis was observed 4 days after injection of a solution containing Hoechst 33342. Injection of enhanced green fluorescent protein (EGFP) expression vector/liposome complex into testis resulted in transfection of epithelial cells of epididymal ducts facing the lumen, although the transfection efficiency appeared to be low. In vivo electroporation toward the caput epididymis immediately after injection of EGFP expression vector into a testis greatly improved the uptake of foreign DNA by the epididymal epithelial cells. PCR analysis using spermatozoa isolated from corpus and cauda epididymis 4 days after injection of a DNA/liposome complex into testis revealed exogenous DNA in these spermatozoa even after treatment with DNase I. These findings indicate that exogenous DNA introduced into tesits is rapidly transported to epididymal ducts via the rete testis and efferent ducts, and then incorporated by epithelial cells of epididymis and epididymal spermatozoa.     

Immunohistochemical demonstration of androgen-binding protein in the rat prostatic gland.

Androgen-binding protein (ABP) is one of the best-characterized products of synthesis by the Sertoli cells in the rat. Although the exact physiological role of ABP remains to be determined, it has been widely used to study Sertoli cells and testicular function in this species. Since this protein is the principal carrier for testosterone in rat testis and epididymis, we decided to investigate ABP immunoreactivity (ABP-I) in androgen-dependent organs, including testicle, epididymides, prostate, and seminal vesicles. The location of ABP was investigated by immunohistochemistry using specific antisera against rat ABP. As previously described in the testis, rat ABP-I was identified in the seminiferous tubules within the cytoplasm of the Sertoli cells and the tubular luminae. The epididymis showed ABP-I only in epithelial cells of the proximal caput. We demonstrated ABP-I in the apical portions of epithelial cells of the rat prostate. Short-term castration and/or ligation of the efferent ducts did not suppress prostatic ABP-I. ABP-I was not present in seminal vesicles of control rats nor under any of the experimental conditions used throughout this study. The results also indicate the presence of ABP-I in prostatic epithelium, probably because of a mechanism similar to that described in epididymis. Our data support and enhance the concept that ABP may serve as a transmembrane carrier protein for androgens in androgen target organs in the male reproductive tract.     

Association of increased type I collagen expression and relative stromal overgrowth in mouse epididymis neonatally exposed to diethylstilbestrol

The aim of this study was to investigate the molecular changes that underlie morphological changes in the epididymis following neonatal exposure to potent synthetic estrogen, namely diethylstilbestrol (DES). Newborn male mice were subcutaneously injected with DES or endogenous estrogen, namely 17 beta-estradiol (E2) (5 microg/mouse/day), for the first 5 days. At the age of 2, 4, and 8 weeks, epididymides of the mice were dissected. Characteristic morphological abnormality, such as relative stromal overgrowth, was observed at the age of 2 weeks in the epididymis of DES-treated mice, but not in E2-treated mice. Microarray and real-time RT-PCR analyses revealed that the expression levels of procollagen type I alpha 1 (col1a1) and col1a2 genes were markedly upregulated at the age of 2 weeks in the epididymis of DES-treated mice in comparison with the control. Western blot analysis revealed that type I collagen protein expression level in epididymis of DES-treated mice was elevated at the age of 2 weeks. In situ hybridization analysis revealed that the signals of col1a1 mRNA were detected similarly throughout the stromal tissue of epididymis at the age of 2 weeks in control and DES- and E2-treated mice. The gene expression level of epididymal type III collagen (col3a1), which is found in many stromal connective tissues as well as type I collagen, did not change at the age of 2 weeks in all groups. These results suggest that the increased type I collagen expression is associated with the relative stromal overgrowth in the epididymis of DES-treated mice.     

Immunolocalization of anti-agglutinin for spermatozoa in boars

A boar "anti-agglutinin," which inhibits head-to-head agglutination of spermatozoa, has been identified as a 25-kDa sialoprotein contained in epididymal and seminal plasma. This study was conducted to determine the location of the anti-agglutinin on spermatozoa and in various organs, including epididymides, by indirect immunofluorescence and Western blotting techniques. Ejaculated boar spermatozoa were washed and subjected to immunocytochemical observation. Epididymal plasma was recovered from three different regions of epididymides and subjected to sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting. Twelve kinds of organs (testis, epididymis, seminal vesicle, prostate, heart, liver, kidney, spleen, stomach, small intestine, lung, and muscle) were recovered from boars. The unilateral epididymides were fixed, cut into 10-microm frozen sections, and subjected to immunohistochemical observation. The other organs were homogenized and used for SDS-PAGE and Western blotting. Immunocytochemical observations revealed that the antiserum strongly recognized the acrosomal region and equatorial segment on unfixed and methanol-fixed spermatozoa. Immunohistochemical observations revealed that the epithelia of the epididymal ducts were recognized by the antiserum mainly in the corpus epididymides. Moreover, the antiserum reacted with the luminal contents of the corpus and cauda epididymides. However, no specific reaction was detected in the caput epididymides. Western blotting showed that the antiserum selectively recognized a band of the anti-agglutinin in the corpus and cauda epididymal plasma, although no band was detected in the caput epididymal plasma. In the extracts from various organs, the single band was detected in the corpus and cauda epididymides at the same mobility as the anti-agglutinin, but not in the other organs. Based on these results, the following matters concerning the anti-agglutinin are discussed: (1) the importance of its association with the acrosome of spermatozoa in inhibiting sperm head-to-head agglutination; (2) its origin in the epididymis; and (3) its tissue specificity.     

Evidence for the presence of oxytocin in the ovine epididymis

The testes of several species contain oxytocin and/or neurophysin, but the content or localization of oxytocin in epididymal tissue has not been studied. The present study was undertaken to localize oxytocin and neurophysin in epididymal tissue of the ram, and to quantify oxytocin in the ductus epididymidis and fluids entering and leaving the ductus epididymidis. Neurophysin was not detected in the epididymis; thus, synthesis of oxytocin by the epididymis is unlikely. Immunohistochemical localization of oxytocin was confined to the epithelium and capillaries. Oxytocin immunostaining was most intense for epithelium of the caput and declined in corpus and cauda regions. However, based on radioimmunoassay, no difference in oxytocin concentration was detected among regions of the epididymis. Since rete testis fluid entering and cauda epididymal fluid leaving the epididymis contained at least fourfold more oxytocin than testicular venous plasma, it was concluded that regional differences in epithelial concentration of oxytocin may have been masked by oxytocin contained in the luminal fluid. It was concluded further that the epididymis of the ram does not synthesize oxytocin, but about 22 ng/day enters the epididymis in rete testis fluid. Most of this luminal oxytocin apparently is absorbed by the epithelium of the caput epididymidis, with additional adsorption in the corpus and cauda. Although a role for oxytocin in ductal contractility cannot be excluded, it is more likely that the luminal oxytocin influences epithelial or sperm function.     

Analyses of epididymal sperm surface antigens by monoclonal antibodies against hamster spermatozoa

BALB/c mice were immunized with spermatozoa from cauda epididymides of hamsters and the immune spleen cells were fused with mouse myeloma cells (P3U1). Seven hybridomas (GHS-1,-2,-3,-4,-5,-6, and -7) that produced monoclonal antibodies (Mabs) binding to the epididymal spermatozoa were established. Three Mabs (GHS-3,-4, and -6) were IgM and the other four were IgG1. All Mabs reacted to hamster spermatozoa from cauda epididymides but none of the Mabs except GHS-5 and -7 reacted to spermatozoa in testis. GHS-5 and -7 Mabs bound to the acrosome region of spermatozoa in both testis and epididymis. The antigens corresponding to GHS-2, -4, and -6 Mabs appeared to be excreted from epithelial cells of caput epididymis, while those to GHS-1 and -3 Mabs seemed to be produced in cauda epididymis. Both groups of the antigens bound to the surface of spermatozoa during their epididymal transit. Immunoblotting analyses of epididymal fluid showed that the antigen epitopes corresponding to GHS-1,-2,-3,-4, and -6 Mabs were distributed to multiple components with different molecular weights ranging from over 100 to 25 kd. The distribution patterns of the epitopes corresponding to GHS-1 and -3 Mabs and GHS-2,-4, and -6 Mabs were very similar, respectively, but each group pattern was quite different from each other. GHS-5 Mab reacted to a component of sperm extract with a molecular weight of around 94 kd, while GHS-7 Mab failed to recognize any components transblotted.