用原位杂交法研究兔附睾蛋白BE－20的基因表达

兔附睾尾液经ＨＰＬＣ纯化后，获得分子量为２０ｋＤ的附睾特异蛋白，命名为ＢＥ－２０。ＢＥ－２０的抗体具有明显抑制人精子穿透去透明带仓鼠卵的能力。已获得编码ＢＥ－２０的０．５ｋｂｃＤＮＡ片段，命名为ＲＥＤ－２０。用地高辛标记此片段，与兔睾丸、附睾、输精管和肝组织进行原位杂交，观察ＢＥ－２０在各种组织中的定位和表达。采用杂交前热变性与不用热变性组织两种方法进行原位杂交。杂交信号均为紫褐色的颗粒，分布在附睾尾的主细胞和近端输精管的上皮细胞的胞质内，表明胞质内ｍＲＮＡ与地高辛标记ＲＥＤ－２０ｃＤＮＡ探针杂交。组织经热变性后，附睾尾和近端输精管上皮细胞核出现强阳性反应，表明核内的同源ＤＮＡ与地高辛标记的ＲＥＤ－２０ｃＤＮＡ探针杂交。但在睾丸和肝脏内未见杂交信号，提示ＢＥ－２０是附睾尾和近端输精管上皮细胞合成的蛋白质。经分析证明，此蛋白是一种细胞外的蛋白酶抑制剂，可能与防止精子过早获能有关，它是否能用于制备抗生育疫苗有待深入的研究。     

Cytotoxic phenotype of intra-epithelial lymphocytes in normal and cryptorchid human testicular excurrent ducts

BACKGROUND: Most testicular and epididymal lymphocytes express T-cell markers, but their cytotoxic potential and activation status have not been reported. In this study, distribution of the cytotoxic cells was compared between normal and cryptorchid testes stratified into two groups: the first with complete absence of germ cells [Sertoli cell-only (SCO)] and the second with arrested spermatogenesis (SCA). METHODS: Immunohistochemistry for the T-lymphocyte marker CD3 and cytotoxic markers CD8, TIA-1 and granzyme B was performed on paraffin-embedded sections. RESULTS: The number of CD8+ and CD3+ intra-epithelial lymphocytes (IELs) increased distally throughout the normal epididymis. TIA-1 immunostaining revealed that a significant proportion of IELs exhibited cytotoxic potential, whereas granzyme B staining disclosed a subpopulation of activated cytotoxic lymphocytes (CTLs). TIA-1/CD8 and granzyme B/CD8 double immunostaining revealed that the vast majority of TIA-1+ and granzyme B+ cells were CD8+. The proportion of activated granzyme B+ lymphocytes increased distally throughout the normal epididymis. The number of TIA-1+ and granzyme B+ intra-epithelial and stromal lymphocytes was significantly increased in the normal as opposed to the SCO cryptorchid epididymis and proximal vas deferens. CONCLUSIONS: These results suggest that exposure of the testicular excurrent ducts to spermatozoa or immature germ cells triggers the activation and recruitment of CTLs. Cytotoxic granule effector mechanisms may contribute to the immunological barrier preventing the immune response to spermatozoa in testicular ducts.     

Ultrastructural study of the epididymis and the vas deferens and electrophoretic profile of their luminal fluid proteins in the lizard Mabuya carinata

The light microscopy, histochemical and TEM studies of the epididymis and the vas deferens revealed the presence of PAS positive secretory granules in the epithelial cells lining the lumen of these organs. One dimensional SDS gel electrophoretic pattern of luminal fluid proteins and the total protein content of the testis, three regions of the epididymis and the vas deferens of the lizard, Mabuya carinata were studied during breeding and nonbreeding season of the reproductive cycle. During breeding season, 25 protein bands in the testicular luminal fluid, 26 in the anterior epididymal luminal fluid and 28 in the middle and posterior epididymal luminal fluid were found. Ten new protein bands appeared in the anterior epididymal region whereas five new protein bands appeared in the middle region of the epididymis indicating regional difference in protein secretions of the epididymis. Vas deferens luminal fluid showed the highest number of protein bands (32) and the highest total protein content (9.07 mg/ml) compared to the testis and the epididymis. Four new protein bands appeared in the vas deferens. Number of protein bands in the luminal fluids of testis, epididymis and the vas deferens were significantly reduced during nonbreeding season compared to those of the breeding season. Consistent with the decrease in the number of protein bands, there was a significant reduction in the total protein concentration in all the tissue samples during nonbreeding season. The results indicate seasonal differences in number of proteins secreted and quantity of proteins in the luminal fluid of male reproductive tract of M. carinata. This is the first study in reptiles revealing appearance of new proteins in epididymis, and vas deferens by conducting simultaneous electrophoretic profile of testicular, epididymal and vas deferens luminal contents.     

Specific expression of heat shock protein HspA2 in human male germ cells

In the mouse, the heat shock protein 70-2 (Hsp70-2) has been found to play a critical role in spermatogenesis. The HspA2 gene is the human homologue of the murine Hsp70-2 gene with 91.7% identity in the nucleotide coding sequence. We examined the expression of HspA2 in human tissues. To detect HspA2 expression, antiserum 2A that was raised against mouse Hsp70-2 and that cross-reacted with human HspA2 protein expressed in Escherichia coli was used. The results of Western blotting indicate that significant HspA2 expression occurs in testes with normal spermatogenesis, whereas only a low amount of HspA2 was expressed in testis with Sertoli cell-only syndrome. Only a small amount of HspA2 was detected in breast, stomach, prostate, colon, liver, ovary, and epididymis. Immunoreactivity to HspA2 was present in spermatocytes and spermatids in the testes with normal spermatogenesis, while immunoreactivity to HspA2 in testis with Sertoli cell-only syndrome was remarkably decreased or inconspicuous over the entire cell. These results demonstrate that the HspA2 protein is highly expressed in human male specific germ cells, suggesting that HspA2 protein may play a specific role during meiosis in human testes as found in the murine model.     

Embryonal remnants in inguinal hernia sacs

Inguinal herniorrhaphy is the most common general surgical procedure performed on the neonate or young pediatric patient. The vas deferens and epididymis are vulnerable to damage, including transection during inguinal exploration or hernia repair. Occasionally the surgical pathologist encounters glandular or tubular epithelial-lined structures in hernia sac tissue. Significant medicolegal implications arise when embryonal remnants are mistakenly identified as true vas deferens or epididymis. This study evaluates the incidence and morphology of these embryonal remnants in hernia sacs from patients of The Children's Hospital, Denver, CO. Embryonal remnants were found in 1.5% of 599 hernia sacs from 427 consecutively operated males aged 37 weeks gestation to 19 years. True vas deferens and epididymis were each identified once for an incidence of 0.33%. The remnants resemble either vas deferens or epididymis. The average remnant diameter is 0.17 mm, and did not change significantly with age. Remnants are surrounded by varying amounts of condensed mesenchyme, trichrome-negative for muscle. The testes and vasa differentia from 32 autopsy cases, ranging in age from 26 weeks gestation to 7 years of age, were used to evaluate normal development and morphology of the vas deferens, epididymis and embryonal remnants. The vas deferens is well developed by 26 weeks gestation. The surrounding smooth-muscle coat does not stain with trichrome until 32 weeks gestation. The vas deferens increases in diameter in a linear fashion during gestation, and continues to increase in diameter in the postnatal period. The vas deferens at 4 months of age is 1.2 to 1.4 mm in diameter; this is also the age of highest incidence of bilateral herniorrhaphies and presence of remnants.     

Mucin gene expression in human male urogenital tract epithelia

BACKGROUND: Mucins are large, hydrophilic glycoproteins that protect wet-surfaced epithelia from pathogen invasion as well as provide lubrication. At least 17 mucin genes have been cloned to date. This study sought to determine the mucin gene expression profile of the human male urogenital tract epithelia, to determine if mucins are present in seminal fluid and to assess the effect of androgens on mucin expression. METHODS AND RESULTS: Testis, epididymis, vas deferens, seminal vesicle, prostate, bladder, urethra and foreskin were assessed for mucin expression by RT-PCR (for 14 mucin genes) and immunohistochemistry (nine antibodies for five mucins). Epithelia of the vas deferens, prostate and urethra expressed the greatest number of mucins, each with mRNA for between 5 and 8 mucins. Except for MUC20 in epididymis, mRNA for MUC1 and MUC20, both membrane-associated mucins, was detected in all tissues analysed. By comparison, MUC6 was more restricted in expression, being primarily detected in seminal vesicle. MUC1, MUC5B and MUC6 were detected in seminal fluid samples by immunoblot analysis. Androgens had no effect on mucin expression in cultured human prostatic epithelial cells. CONCLUSIONS: Each region of urogenital tract epithelium expressed a unique mucin gene repertoire. Secretory mucins are present in seminal fluid, and androgens do not appear to regulate mucin gene expression in prostatic epithelial cells in culture.     

Maturation of spermatozoa in the epididymis of the Chinese hamster

Chinese hamster spermatozoa gain their ability to move when they descend from the testis to the distal part of the caput epididymis, but it is not until they enter the corpus epididymis that they become capable of fertilizing eggs. The maturation of the spermatozoa proceeds as they further descend the tract and perhaps continues even in the vas deferens. During transit between the distal caput and proximal cauda epididymides, small membrane-limited vesicles (and tubules) appear on the plasma membrane over the acro somes of the spermatozoa. The number of vesicles appearing on the sperm brane reaches a maximum when the spermatozoa are in the proximal cauda epididymis. It declines sharply in the distal cauda epididymis. Spermatozoa in the vas deferens are free of the vesicles. The origin, chemical nature, and functional role of the vesicles that appear on the sperm surface during epididymal transit must be the subject of further investigation.     

Members of the heat-shock protein 70 family promote cancer cell growth by distinct mechanisms

Whereas the stress-inducible heat-shock protein 70 (Hsp70) has gained plenty of attention as a putative target for tumor therapy, little is known about the role of other Hsp70 proteins in cancer. Here we present the first thorough analysis of the expression and function of the cytosolic Hsp70 proteins in human cancer cells and identify Hsp70-2, a protein essential for spermatogenesis, as an important regulator of cancer cell growth. Targeted knock-down of the individual family members by RNA interference revealed that both Hsp70 and Hsp70-2 were required for cancer cell growth, whereas the survival of tumorigenic as well as nontumorigenic cells depended on Hsc70. Cancer cells depleted for Hsp70 and Hsp70-2 displayed strikingly different morphologies (detached and round vs. flat senescent-like), cell cycle distributions (G2/M vs. G1 arrest) and gene expression profiles. Only Hsp70-2 depletion induced the expression of macrophage inhibitory cytokine-1 that was identified as a target of P53 tumor-suppressor protein and a mediator of the G1 arrest and the senescent phenotype. Importantly, concomitant depletion of Hsp70 and Hsp70-2 had a synergistic antiproliferative effect on cancer cells. Thus, highly homologous Hsp70 proteins bring about nonoverlapping functions essential for cell growth and survival.     

Aquaporin 9 (AQP9) Localization in the Adult Dog Testis Excurrent Ducts by Immunohistochemistry

Aquaporins (AQPs) are small, intrinsic membrane proteins that are present in many cell types involved in fluid transport. AQP9 is a major apical water channel that is expressed throughout the efferent ducts, epididymis, and vas deferens, as well as in other regions of the human and rodent male reproductive tract. The target of this study was to examine the expression of AQP9 in epithelial cells in the adult dog efferent ducts, epididymis, and vas deferens. Samples of dog male reproductive tract comprising fragments of the testis; initial segment, caput, corpus, and cauda of the epididymis; and vas deferens were obtained from eight adult mongrel dogs. Immunohistochemistry and Western blotting procedures were used to show AQP9 localization and distribution. AQP9 expression was not detected either in dog seminiferous tubules or rete testis. However, apical labeling for AQP9 was detected in the different regions of epididymis and vas deferens, with the reaction being less intense in the caput epididymis. Thus, AQP9 is abundantly expressed in dog male reproductive tract, in which it is an important apical pathway for transmembrane flow of water and neutral solutes. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

Vasectomy review: Sequelae in the human epididymis and ductus deferens

Following vasectomy, spermatogenesis continues, the human epididymis and ductus deferens may distend and leak, and the extravasated spermatozoa stimulate formation of a sperm granuloma. Granulomas may occur at 60% of vasectomy sites and are usually asymptomatic and relieve intraluminal pressure. About 3–5% of patients experience pain. Intraluminal phagocytosis may explain why some reproductive tracts become depleted of spermatozoa. Distension of the epididymis is common after vasectomy and may lead to granuloma formation there. Up to 6% of patients have symptoms, but many with epididymal changes have no discomfort. Most episodes of painful epididymitis and granulomas resolve with conservative treatment, but <1% require vasectomy reversal or, if this is ineffective, excision of the epididymis and obstructed ductus deferens. © 1996 Wiley-Liss, Inc.     

Localization of CuZn-superoxide dismutase in the human male genital organs.

We performed an immunohistochemical analysis using a polyclonal antibody to determine the localization of CuZn-superoxide dismutase (SOD), a scavenger of superoxide anion radicals, in the human male genital organs. In the testis, intense immunoreactivity of CuZn-SOD was shown in both the cytoplasm and nucleus of the spermatogonia of the seminiferous tubules. Spermatocytes, further differentiated germ cells and Sertoli cells showed no or weak immunoreactivity. In the ductus epididymis, the principal cells showed no or weak immunoreactivity except for the stereocilial region, while the basal cells showed relatively intense immunoreactivity. In the ductus deferens, the prostate and the seminal vesicles, columnar and cuboidal epithelia showed CuZn-SOD immunoreactivity. The immunoreactivity was more intense in the epithelia of the ductus deferens than in the prostate or the seminal vesicles. Basal cells in the prostate also showed intense immunoreactivity. Collectively, the present immunohistochemical results suggest that CuZn-SOD in the male genital organs is localized where it could play an important role in cell differentiation, including spermatogenesis. The CuZn-SOD could also play a role in local defence mechanisms against tissue damage mediated through superoxide anion radicals, as well as in providing SOD to the seminal plasma.     

Morphology of the male reproductive duct system of Caiman crocodilus (Crocodylia, Alligatoridae)

The male reproductive duct system of Caiman crocodilus in different reproductive stages was studied using light and electron microscopy, to determine whether shared morphological features exist between Crocodylia and Aves, in concurrence with the Archosauria hypothesis. The sexual duct system of Caiman crocodilus is constituted of the rete testis, ductuli efferentes, ductuli epididymides, ductus epididymidis, and ductus deferens. The morphology and histochemical properties of these ducts suggest their involvement in seminal fluid production and/or its modification. Three types of non-ciliated cells were identified along the duct system. 1. The noncliated cells of the ductuli efferentes contain electron dense worm-like structures and coated vesicles, both related to absorptive processes, as has been suggested in Aves. 2. The non-ciliated cells of the ductuli epididymides have apical electron dense granules suggesting a secretory role, and 3. The non-ciliated cells of the ductus epididymidis and ductus deferens did not exhibit conspicuous storage of secretory material, but have a prominent rough endoplasmic reticulum content indicating active protein production. The occurrence of abundant secretory granules in the epithelial cells of the distal ductus deferens during non-reproductive stages suggests its participation in the removal of luminal debris when the reproductive season ends. Additional ducts were observed running along the ductus deferens; they shared morphological characteristics with the ductuli epididymides. The maximum diameter and therefore the greatest sperm accumulation of the excurrent ducts were observed during the initial testicular regression. The comparative analysis suggests that the male reproductive system of the Crocodylia exhibits structural characteristics nearer to those of Aves than to Lepidosauria, Testudines, and Mammalia, additional data that support an Aves and Crocodylia sister group relationship.     

Live birth resulting from in-office vas deferens sperm retrieval combined with natural-cycle insemination

In-office retrieval of spermatozoa from the vas deferens wassuccessfully combined with intra-uterine insemination of thewife during her natural cycle in the case of a man with azoospermiadue to a non-correctable obstruction of the vas deferens. Thecombined cost of both procedures (sperm retrieval and intra-uterineinsemination with processed spermatozoa) was less titan $1000.The cost and convenience compare quite favourably with thatof epididymis sperm aspiration combined with in-vitro fertilization,which is the currently recommended alternative treatment insuch cases. These alternative therapies are compared and discussedrelative to their advantages and disadvantages. Our techniquefor In-office vas deferens sperm retrieval is also presented.     

Specific expression of the mRNA for 25 kDA heat-shock protein in the spermatocytes of mouse seminiferous tubules

 The 25 kDa heat-shock protein (Hsp25) is a member of the family of small heat-shock proteins. We investigated the expression and cellular localization of Hsp25 mRNA in the testis of adult and developing mice using Northern blotting and in situ hybridization techniques. In the early postnatal days, i.e., before the onset of spermatogenesis, no Hsp25 mRNA was detected in the testis. At around 10 days postpartum, Hsp25 mRNA began to be expressed in the testis in coincidence with the onset of the first wave of spermatogenesis and increased in amount progressively toward adulthood. Throughout the testis development, the signal for Hsp25 mRNA was localized exclusively to germ cells and was not detected in Sertoli or interstitial cells. The testis of W/Wv mutant mice, which lack the germ cell line, exhibited no Hsp25 mRNA expression. In the testis of normal adult mice, the abundance of Hsp25 mRNA differed among the seminiferous tubules in different stages of spermatogenesis. The most intense signal for Hsp25 mRNA was localized to the spermatocytes at leptotene, zygotene and early pachytene phases, which are present in the tubules of stages I–III and IX–XII. The signal decreased in intensity in the late pachytene and diplotene spermatocytes and was not detected in spermatids. Spermatogonia were also devoid of the signal. These results suggested that Hsp25 plays some specific role in the meiotic prophase of the testicular germ cell. Accepted: 27 Oct 1998