Localization of sulfated glycoprotein-2 (clusterin) on spermatozoa and in the reproductive tract of the male rat

Sulfated glycoprotein-2 (SGP-2) is one of the major proteins secreted by rat Sertoli cells and epididymal cells in culture. The disulfide-linked dimeric protein secreted by Sertoli cells and found in seminiferous tubule fluid is composed of monomers of Mr 47 000 and 34 000 whereas the epididymal protein exhibits monomers of Mr 40 000 and 29 000. When both forms were chemically or enzymatically deglycosylated, they yielded proteins of similar molecular weight. No modification of the higher molecular weight testicular form by epididymal cells or fluids could be detected in incubation media. SGP-2 mRNA was localized in epididymal epithelium by in situ hybridization. Northern blot analysis indicated the testicular and epididymal mRNAs were of similar size. These findings suggest that the two forms of the protein occur because of tissue-specific post-translational modifications. The detergent-extracted protein from washed testicular spermatozoa is of the higher molecular weight form while epididymal sperm carry the lower molecular weight form. Immunohistochemical evidence suggests that the testicular form is removed prior to the initial segment of the epididymis and the epididymal form is applied in the proximal caput epididymidis. SGP-2 was immunolocalized to the sperm membrane at the ultrastructural level and was distinctly different from the immunolocalization of outer dense fiber proteins and fibrous sheath proteins.     

The distribution of rat testibumin in the male reproductive tract

A glycoprotein, designated CMB-1, has been identified in media from Sertoli cell-enriched cultures that increases in concentration in response to follicle-stimulating hormone (FSH) and testosterone. Subsequent studies indicated that CMB-1 is immunologically related to albumin and alpha-fetoprotein and is concentrated in the luminal compartment of the testis in adult rats. Thus, CMB-1 was termed testibumin. The goal of the present study was to determine the concentrations of this protein in testes, epididymides, and serum of normal rats between 10 and 180 days of age and to compare them to rat androgen-binding protein (rABP). Testibumin concentration in rat testes increased with age and peaked at Day 60; thereafter, unlike rABP, its concentration declined, reaching a plateau by 150 days of age. Testibumin concentration in the epididymal compartment also increased with age and peaked at Day 90; thereafter, its concentration remained relatively unchanged. Unlike rABP, which accumulates in the caput epididymis, testibumin did not accumulate preferentially in any particular region of the epididymis. In spite of the marked changes of testibumin concentration in the male reproductive tract, the levels in blood remained relatively constant between 10 and 180 days of age. In adult male and female rats, the serum concentrations of testibumin were similar. Following orchiectomy, serum testibumin concentration decreased by 50% with an apparent t1/2 of approximately 8 h. The presence of immunoreactive macromolecules in other species that share epitopes with rat testibumin was also investigated. Material in human sera and extracts of human and monkey testes cross-reacts with rat testibumin. After [35S]methionine was added to the primary Sertoli cell-enriched cultures, anti-testibumin antiserum selectively immunoprecipitated a radiolabeled protein with the same electrophoretic mobility as purified testibumin on sodium dodecyl sulfate (SDS)-polyacrylamide gels. We conclude that 1) rat testibumin is synthesized and secreted by Sertoli cell-enriched cultures; 2) the relative concentrations and distribution of testibumin in testis, epididymis, and serum of the rat as a function of age are strikingly different from those of rABP; 3) rat testibumin shares epitopes with proteins in human serum and testicular extracts of monkey and man.     

Coccidioidomycosis of the male reproductive tract

Coccidioides immitis infection of the male reproductive tract is a rare entity that can evade diagnosis and pose a dilemma in management. Initially, patients are often evaluated for malignancy or other infections such as tuberculosis. In the past, surgery was the only management option for C. immitis infection of the male reproductive tract, but azole therapy now provides an adjunct or an alternative. We describe two patients who received azole therapy for C. immitis infection of the male reproductive tract. One received fluconazole for prostatic disease, while one received surgery followed by itraconazole for testicular disease. After 12 months of therapy, both remain asymptomatic and have decreased antibody titers against C. immitis.Disclaimer: The Views expressed herein are those of the authors do not reflect the official policy or position of the Department of the Air Force, Department of the Army, Department of Defence, or the U.S. Government.     

Cell-cell interaction underlies formation of fluid in the male reproductive tract of the rat

The epithelia lining the epididymides of many species consists of several cell types. We have provided evidence that the basal cells are essential to the integrated functions of the epithelium. Basal cells, but not principal cells, and other cells in the epididymis express TRPC3 and COX-1. We have isolated basal cells from intact rat epididymis using antibody-coated Dynabeads and subjected them to whole-cell patch-clamp measurement of nonselective cation channel activity, a feature of TRPC3 protein, and Fluo-3 fluorescence measurement of intracellular Ca2+ concentration. The results show that a nonselective cation current blockable by La3+ (0.1 mM), Gd3+ (0.1 mM), or SKF96365 (20 microM) could be activated by lysylbradykinin (200 nM). In cells loaded with Fluo-3, addition of lysylbradykinin (100 nM) caused a sustained increase of intracellular Ca2+. This effect was blocked by Gd3+ (0.1 mM) or SKF96365 (20 microM) and was not observed in Fluo-3-loaded principal cells. Stimulation of basal cell/principal cell cocultures with lysylbradykinin (200 nM) evoked in principal cells a current with CFTR-Cl- channel characteristics. Isolated principal cells in the absence of basal cells did not respond to lysylbradykinin but responded to PGE2 (100 nM) with activation of a CFTR-like current. Basal cells, but not principal cells, released prostaglandin E2 when stimulated with lysylbradykinin (100 nM). The release was blocked by SKF96365 (20 microM) and BAPTA-AM (0.05 or 0.1 mM). Confluent cell monolayers harvested from a mixture of disaggregated principal cells and basal cells responded to lysylbradykinin (100 nM) and PGE2 (500 nM) with an increase in electrogenic anion secretion. The former response was dependent on prostaglandin synthesis as piroxicam blocked the response. However, cell cultures obtained from principal cells alone responded to PGE2 but not to bradykinin. These results support the notion that basal cells regulate principal cells through a Ca2+ and COX signaling pathway.     

Effect of mating on sperm distribution in the reproductive tract of the male rat

Extragonadal sperm reserves in male rats were measured in different regions of the genital tract before and subsequent to normal ejaculation. In sexually rested rats, the sperm count (million spermatozoa for the paired organs) in different regions was: distal vas, 18; proximal vas, 9.8; cauda epididymidis, 229; caput + corpus epididymidis, 154. Following mating, the sperm count was reduced in the proximal and distal vas deferens and in the cauda epididymidis. The reproductive tract of mated females was found to contain 29% (no copulatory plug) or 59% (with copulatory plug) of the estimated mean ejaculate, which was estimated from the difference between the sperm counts in the sexually rested rat and following ejaculation. It is concluded that in the rat the immediate source of spermatozoa for ejaculation is the cauda epididymidis, with a smaller contribution arising from the vas deferens.     

Epididymal protease inhibitor (EPPIN) is differentially expressed in the male rat reproductive tract and immunolocalized in maturing spermatozoa

EPPIN (epididymal protease inhibitor; SPINLW1), an antimicrobial cysteine‐rich protein containing both Kunitz and whey acidic protein (WAP)‐type four disulfide core protease inhibitor consensus sequences, is a target for male contraception because of its critical role in sperm motility. Here, we characterized EPPIN's expression and cellular distribution in rat tissues and its in vivo regulation by androgens in the epididymis. EPPIN (mRNA and protein) was abundantly expressed in the rat testis and epididymis; we also found that the vas deferens, seminal vesicles, and brain were novel sites of EPPIN expression. PCR studies demonstrated that in addition to Sertoli cells, spermatogenic cells expressed Eppin mRNA. EPPIN was immunolocalized in Sertoli cells and spermatogenic cells (pachytene spermatocytes and round and elongated spermatids) and in epithelial cells and spermatozoa from efferent ductules and epididymis. EPPIN staining was observed on the middle and principal pieces of the flagellum of testicular spermatozoa. Epididymal spermatozoa had more intense EPPIN staining on the flagellum, and the EPPIN staining became apparent on the head and neck regions. This suggested that the EPPIN found on maturing spermatozoa was secreted primarily by the epithelial cells of the epididymis. Surgical castration down‐regulated EPPIN expression levels (mRNA and protein) in the caput and cauda epididymis, an effect reversed by testosterone replacement. Altogether, our data suggested that EPPIN expression in rats is more widespread than in humans and mice, and is androgen‐dependent in the epididymis. This species could be used as an experimental model to further study EPPIN's role in male fertility. Mol. Reprod. Dev. 79: 832–842, 2012. © 2012 Wiley Periodicals, Inc.     

Pkd1 is required for male reproductive tract development

Reproductive tract abnormalities and male infertility have higher incidence in ADPKD patients than in general populations. In this work, we reveal that Pkd1, whose mutations account for 85% of ADPKD cases, is essential for male reproductive tract development. Disruption of Pkd1 caused multiple organ defects in the murine male reproductive tract. The earliest visible defect in the Pkd1^?/? reproductive tract was cystic dilation of the efferent ducts, which are derivatives of the mesonephric tubules. Epididymis development was delayed or arrested in the Pkd1^?/? mice. No sign of epithelial coiling was seen in the null mutants. Disruption of Pkd1 in epithelium alone using the Pax2-cre mice was sufficient to cause efferent duct dilation and coiling defect in the epididymis, suggesting that Pkd1 is critical for epithelium development and maintenance in male reproductive tract. In-depth analysis showed that Pkd1 is required to maintain tubulin cytoskeleton and important for Tgf-β/Bmp signal transduction in epithelium of male reproductive tract. Altogether, our results for the first time provide direct evidence for developmental roles of Pkd1 in the male reproductive tract and provide new insights in reproductive tract abnormalities and infertility in ADPKD patients.     

Sialomucin complex (Muc4) expression in the rat female reproductive tract

Previous studies in our laboratory demonstrated the presence of sialomucin complex (SMC)/Muc4 covering the rat uterine luminal epithelium. SMC/Muc4 expression in the uterus is regulated by estrogen and progesterone and lost at the time of receptivity. In contrast to this hormonal regulation at the uterine luminal surface, SMC/Muc4 in the uterine glandular epithelium, oviduct, cervix, and vagina was constitutively expressed at all stages of the estrous cycle. Furthermore, SMC was expressed in the cervix and vagina of the ovariectomized rat, even though it is not found in the uterine luminal epithelium. Both soluble and membrane-bound forms of SMC were present in these tissues. Immunohistochemical analyses showed distinctive localization patterns of SMC in the various tissues during the estrous cycle. Moreover, the previously unreported expression of SMC/Muc4 in the isthmus, ampulla, and infundibulum of the oviduct suggests potential functions in gamete development. These results indicate that SMC/Muc4 is expressed in most tissues of the female reproductive tract, in which it may have multiple functions. However, hormonal regulation appears to be restricted to the uterine luminal epithelium.     

Production and exocrine secretion of LHRH-like material by the male rat reproductive tract

Luteinizing hormone-releasing hormone (LHRH)-like immunoreactivity was localized in the male reproductive system of the rat. Epithelial cells of the epididymus, seminal vesicles and coagulation gland showed a strong reaction to anti-LHRH serum. Also the epithelia of the ductus deferens and the prostate gland appeared to be immunoreactive, albeit to a lesser extent. The LHRH-like substances are most likely secreted into the male tract, as can be concluded from the observation that the secretion product in the lumina of the seminal vesicles, coagulation gland and prostate gland was also immunopositive. The functional significance of these phenomena is discussed. No immunostaining was obtained with antisera to FSH, LH or beta-hCG.     

Identification and characterization of Wfdc gene expression in the male reproductive tract of the rat

WFDC (Whey Acidic Protein Four Disulfide Core)-containing proteins have been reported in many species, yet they remain uncharacterized in the rat. In this study, we report the identification and characterization of four rat Wfdc genes, Wfdc6a, Wfdc8, Wfdc11 and Wfdc16. Their expression profile in a variety of tissues including the male reproductive tract is analyzed. Wfdc8, Wfdc11 and Wfdc16 expression is confined to the epididymis, while Wfdc6a is expressed widely. Since gene expression in the male reproductive tract is largely androgen-dependent, Wfdc expression was analyzed in the developing (20-60-day-old) and castrated rats. Their expression pattern in developing rats does not correlate with changes in testosterone. Wfdc genes are, however, down-regulated in castrated adult rats, indicating that their dependence on androgens for expression is more pronounced in the adult than in the developing rat. To test the anti-microbial potential of WFDC8, a recombinant WFDC8 C-terminal protein was produced, which exhibited potent anti-bacterial activity against Eschericia coli. Induction of anti-microbial genes is one of the responses during infections in many organ systems. To determine if WFDCs form the components of male reproductive tract innate immunity, Wfdc8 expression pattern was observed in rats challenged with lipopolysaccharide (LPS). For the first time we report the induction of Wfdc8 gene expression in LPS-treated rats, indicating their contributions to the innate immune functions of the male reproductive tract.     

Morphology and development of the male reproductive tract in Callinectes danae (Crustacea: Brachyura)

The aim of this study was to characterize the morphology and function of each section of the reproductive system of male Callinectes danae, as well as the stages of reproductive development and their relation to secondary sexual characteristics. Development of their reproductive system begins after completion of the pubertal moult. The growth of the gonopodium showed negative allometry for both juveniles and adults. The reproductive system is divided into portions with different functions. There is a germinal zone in the testes containing spermatogonia, a zone of maturation containing spermatocytes, spermatids or spermatozoa, as well as a collecting duct, which carries spermatozoa to the vas deferens. There are two matrices in the anterior vas deferens that initiate the separation of spermatozoa groups, one composed of polysaccharide acids (matrix I) and another consisting of neutral polysaccharides (matrix II). In the median vas deferens, the matrix II forms an acellular capsule, which forms the spermatophores. In the posterior vas deferens, the matrices are accumulated, initially with a granular texture and are homogenous for the final portion. The ejaculatory duct and penis have muscle lining to expel the spermatophores at copulation. Even after copulation, males retain a stock of spermatophores, allowing copulation with other females.