Activation of saturniid moth sperm by a secretion of the male reproductive tract

Whereas sperm under storage in the seminal vesicles of male Saturniid moths are essentially motionless, they become vigorously motile when ejaculated into the spermatophore. This activation is provoked by a certain secretion of the male's ‘common duct’. The amount of activator in the secretion of a male Antheraea pernyi is prodigious: an extract of a single common duct is sufficient to induce motility in the sperm of nearly half a million male moths. Activating substances could not be recovered from any other tissues or organs of either developing or mature male moths. Nevertheless, substances capable of activating pernyi sperm were obtained from the common ducts of diverse Lepidoptera, and even from the accessory glands of two Orthoptera.Provided that the dilution was not excessive, activated silkmoth sperm in vitro remained optimally motile in a wide variety of solutions having a pH between 5·8 and 7·8 and an osmolarity between 80 and 190 milliosmols. Motility was enhanced in the presence of added protein, but was completely inhibited by dilute concentrations of heavy metal cations. Motility was normal under anaerobic conditions, and even in an atmosphere of carbon monoxide. Apparently the motility of silkmoth sperm can be sustained by glycolysis alone.     

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.     

Proton secretion in the male reproductive tract: involvement of Cl--independent HCO-3 transport

The lumen of theepididymis is the site where spermatozoa undergo their final maturationand acquire the capacity to become motile. An acidic luminal fluid isrequired for the maintenance of sperm quiescence and for the preventionof premature activation of acrosomal enzymes during their storage inthe cauda epididymis and vas deferens. We have previously demonstratedthat a vacuolar H⁺-ATPase[proton pump (PP)] is present in the apical pole of apical and narrow cells in the caput epididymis and of clear cells in thecorpus and cauda epididymis and that this PP is responsible for themajority of proton secretion in the proximal vas deferens. We now showthat PP-rich cells in the vas deferens express a high level of carbonicanhydrase type II (CAII) and that acetazolamide markedly inhibits therate of proton secretion by 46.2 ± 6.1%. The rate ofacidification was independent ofCl and was stronglyinhibited by SITS under both normal andCl-free conditions (50.6 ± 5.0 and 57.5 ± 6.0%, respectively). In the presence ofCl,diphenylamine-2-carboxylate (DPC) had no effect, whereas SITS inhibitedproton secretion by 63.7 ± 11.3% when applied together with DPC. In Cl-freesolution, DPC markedly inhibited proton efflux by 45.1 ± 7.6%,SITS produced an additional inhibition of 18.2 ± 6.6%, and bafilomycin had no additive effect. In conclusion, we propose that CAIIplays a major role in proton secretion by the proximal vas deferens.Acidification does not require the presence ofCl, but DPC-sensitiveCl channels mightcontribute to basolateral extrusion ofHCO₃ underCl-free conditions. Theinhibition by SITS observed under both normal andCl-free conditionsindicates that aCl/HCO₃exchanger is not involved and that an alternativeHCO₃ transporter participates in proton secretion in the proximal vas deferens.

     

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.     

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.     

Distribution of the vacuolar H+ atpase along the rat and human male reproductive tract

Luminal acidification in parts of the male reproductive tract generates an appropriate pH environment in which spermatozoa mature and are stored. The cellular mechanisms of proton (H+) secretion in the epididymis and the proximal vas deferens involve the activity of an apical vacuolar H+ ATPase in specialized cell types, as well as an apical Na+/H+ exchanger in some tubule segments. In this study we used Western blotting and immunocytochemistry to localize the H+ ATPase in various segments of the male reproductive tract in rat and man as a first step toward a more complete understanding of luminal acidification processes in this complex system of tissues. Immunoblotting of isolated total cell membranes indicated a variable amount of H+ ATPase in various segments of the rat reproductive tract. In addition to its known expression in distinct cell types in the epididymis and vas deferens, the H+ ATPase was also localized at the apical pole and in the cytoplasm of epithelial cells in the efferent duct (nonciliated cells), the ampulla of the vas deferens and the ventral prostate (scattered individual cells), the dorsal and lateral prostate, the ampullary gland, the coagulating gland, and all epithelial cells of the prostatic and penile urethra. Both apical and basolateral localization of the protein were found in epithelial cells of the prostatic ducts in the lateral prostate and in periurethral tissue. Only cytoplasmic, mostly perinuclear localization of the H+ ATPase was found in all epithelial cells of the seminal vesicles and in most cells of the ventral prostate and coagulating gland. No staining was detected in the seminiferous tubules, rete testis, and bulbourethral gland. In human tissue, H+ ATPase-rich cells were detected in the epididymis, prostate, and prostatic urethra. We conclude that the vacuolar H+ ATPase is highly expressed in epithelial cells of most segments of the male reproductive tract in rat and man, where it may be involved in H+ secretion and/or intracellular processing of the material endocytosed from the luminal fluid or destined to be secreted by exocytosis.     

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.     

Abnormal secretion of reproductive hormones and antioxidant status involved in quinestrol-induced reproductive toxicity in adult male rat

This study aimed to evaluate the effects of quinestrol, a synthetic oestrogen homologue with reproductive toxicity, on the secretion of reproductive hormones and antioxidant status in adult male rat. Our results showed that quinestrol exposure significantly decreased the weight of the testis, epididymides, seminal vesicle, and prostate, as well as the sperm counts in the cauda epididymis of rats. Quinestrol significantly reduced the size of seminiferous tubules and the total number of spermatogenic cells. Serum testosterone, follitropin, and lutropin were also significantly reduced in a dose-related manner after quinestrol exposure. Meanwhile, the activity of superoxide dismutase, glutathione peroxidase, and total antioxide capacity significantly decreased, whereas the malondialdehyde and nitric oxide concentrations significantly increased in the testes. These findings revealed that endocrine disorders of reproductive hormones and oxidative stress may be involved in reproductive toxicity induced by quinestrol in adult male rats.     

Stimulation of rat pancreatic exocrine secretion by urocortin and corticotropin releasing factor

Neural and hormonal mechanisms control pancreatic secretion. The effects of the corticotropin releasing factor (CRF) related neuropeptide urocortin (UCN) on pancreatic exocrine secretion were examined. In anesthetized male rats, pancreatic secretion volume and total protein were assayed. UCN increased pancreatic secretory volume and protein secretion and potentiated cholecytokinin-stimulated protein secretion. Astressin, a non-specific CRF receptor antagonist, inhibited UCN-stimulated protein output while CRF(2) receptor antagonist, antisauvagine-30, was without effect. Atropine, but not subdiaphragmatic vagotomy, inhibited UCN-mediated secretion. In acinar cells, UCN did not stimulate release of amylase nor intracellular cAMP. UCN is a pancreatic exocrine secreatagogue with effects mediated through cholinergic intrapancreatic neurons.     

Sulfated glycoprotein-1 (saposin precursor) in the reproductive tract of the male rat

Sulfated glycoprotein-1 is one of the major protein secretion products of rat Sertoli cells in culture. This 70,000 Mr protein shares substantial sequence similarity with human prosaposin, the precursor of lysosomal saposins. Saposins are known to enhance the activity of lipid modifying enzymes presumably by solubilizing the lipids. We report here the immunolocalization of sulfated glycoprotein-1 in the cells and fluid of the male reproductive tract. The protein is present in secondary lysosomes of Sertoli cells and also in the luminal fluid of seminiferous tubules and epididymis. The highest concentrations of the protein are in seminiferous tubule fluid and rete testis fluid, while relatively low amounts are found in cauda epididymal fluid and serum. Sulfated glycoprotein-1 is believed to be involved in degradation of lipids in residual bodies and may also assist in modification of membrane lipids during sperm maturation.     

Aquaporin 9 expression along the male reproductive tract

Fluid movement across epithelia lining portions of the male reproductive tract is important for modulating the luminal environment in which sperm mature and reside, and for increasing sperm concentration. Some regions of the male reproductive tract express aquaporin (AQP) 1 and/or AQP2, but these transmembrane water channels are not detectable in the epididymis. Therefore, we used a specific antibody to map the cellular distribution of another AQP, AQP9 (which is permeable to water and to some solutes), in the male reproductive tract. AQP9 is enriched on the apical (but not basolateral) membrane of nonciliated cells in the efferent duct and principal cells of the epididymis (rat and human) and vas deferens, where it could play a role in fluid reabsorption. Western blotting revealed a strong 30-kDa band in brush-border membrane vesicles isolated from the epididymis. AQP9 is also expressed in epithelial cells of the prostate and coagulating gland where fluid transport across the epithelium is important for secretory activity. However, it was undetectable in the seminal vesicle, suggesting that an alternative fluid transport pathway may be present in this tissue. Intracellular vesicles in epithelial cells along the reproductive tract were generally poorly stained for AQP9. Furthermore, the apical membrane distribution of AQP9 was unaffected by microtubule disruption. These data suggest that AQP9 is a constitutively inserted apical membrane protein and that its cell-surface expression is not acutely regulated by vesicular trafficking. AQP9 was detectable in the epididymis and vas deferens of 1-wk postnatal rats, but its expression was comparable with adult rats only after 3--4 wk. AQP9 could provide a route via which apical fluid and solute transport occurs in several regions of the male reproductive tract. The heterogeneous and segment-specific expression of AQP9 and other aquaporins along the male reproductive tract shown in this and in our previous studies suggests that fluid reabsorption and secretion in these tissues could be locally modulated by physiological regulation of AQP expression and/or function.