Demonstration and partial characterization of cytosol receptors for testosterone.

Androgen uptake was investigated in several peripheral organs after administration of (1,2,6,7 minus -3H)testosterone to castrated male rats. The animals were killed after 30 min, the organs were taken out, and the radioactivity was determined after tissue combustion. A relatively high accumulation of androgen was found in pancreas, adrenals, spleen, thigh muscle, kidneys, and liver in addition to the classical androgen target organs coagulation glands, seminal vesicles, prostate, preputial glands, and harderian glands. In a second serier of experiments, nuclear and cytosol fractions were prepared from prostate, seminal vesicles, coagulation glands, preputial glands, spleen, submaxillary glands, kidneys, and pancreas from castrated male rats give (1,2,6,7 minus -3H)testosterone, and these fractions were then characterized by thin-layer and radio-gas chromatography with respect to their patterns of labeled steroids. Only prostate and seminal vesicles were found to contain significant amounts of nuclear 5alpha-(-3H)dihydrotestosterone. The major nuclear androgen was (-3H)testosterone that was the only detectable labeled steroid in coagulation glands, preputial glands, and spleen and that constituted 70% or more of the nuclear radioactivity in seminal vesicles, submaxillary glands, kidneys, and pancreas. These results indicate that testosterone itself may be the predominant active androgen principle in vivo in most androgen target organs and that conversion to 5alpha-dihydrotestosterone is generally not a prerequisite for androgen activity. Using an ultrasensitive micromodification of isoelectric focusing (cf. M. Katsumata and A. S. Goldman (1974), Biochem. Biophys. Acta 359, 112. It was possible to show that cytosol from kidney; submaxillary gland, thigh muscle, and levator ani muscle and nuclei from kidney and submaxillary gland contained androgen-binding proteins with pI's in the region 4.6-5.1 ("4.6 minus 5.1 Complex"). This complex also formed in vitro after incubation of (1,2,6,7 minus -3H)testosterone with cytosol from kidney and submaxillary gland. (1,2,6,7 minus -3H)Testosterone was bound with high affinity to receptor proteins in cytosol from both kidney, submaxillary gland, and thigh muscle with dissociation constants of 5.0 x 10 minus -12 M (kidney), 3.3 x 10 mi;nus -11 M and 4.1 x 10 minus -10 M (two types of binding sites, submaxillary gland), 2.4 x 10 minus -12 M (thigh muscle) and 1.9 x 10 minus -12 M (levator ani muscle). The number of binding sites was in all cases between 1 and 20 fmol/mg of protein. On the basis of these results the hypothesis is presented that a common class of testosterone receptors is present in most organs and that these receptors can be detected both in vivo and in vitro provided methods sensitive enough are utilized.     

Pneumadin in the ventral prostate and seminal vesicles of estradiol-treated rats: RIA and immunocytochemical studies

Pneumadin (PNM) is a decapeptide (the rat peptide: Tyr-Gly-Glu-Pro-Lys-Leu-Asp-Ala-Gly-Val-NH2) isolated from mammalian lungs. Human and rat PNM differ only by substitution of one amino acid--Tyr/Ala. PNM evokes an antidiuretic effect via a potent stimulation of arginine-vasopressin (AVP) release. By means of recently established, highly specific RIA method, high concentration of PNM had been found in the rat ventral prostate. Castration resulted in a profound drop in PNM concentration, an effect prevented by testosterone replacement. The present studies were aimed at investigating the effect of prolonged estradiol administration on PNM concentration, content and localization in the prostate and seminal vesicles of the rat. Depo estradiol (estradiolum valerianicum) administration to adult male rats resulted in a notable atrophy of ventral prostate and seminal vesicles. During the entire experiment (till day 30 after administration), PNM concentration in ventral prostate was similar to that seen in intact animals, while peptide content per gland was markedly lowered. PNM immunostaining was observed in prostate epithelium of estradiol-treated rats and its localization resembled that observed in intact animals. Nearly 40 times lower PNM concentration than in ventral prostate was found in seminal vesicles. In contrast to prostate, on days 20 and 30 of estradiol treatment PNM concentration in seminal vesicles was higher than in intact rats. However, due to profound seminal vesicle atrophy, PNM content per entire gland was notably lowered in estradiol-injected rats. By immunocytochemistry, PNM-immunoreactive substances were not found in seminal vesicles of either intact or estradiol-administered rats. High PNM concentration in the rat prostate suggests its important role in the function of the gland.     

Antigenic homogeneity of male Müllerian gland (MG) secretory proteins of a caecilian amphibian with secretory proteins of the mammalian prostate gland and seminal vesicles: evidence for role of the caecilian MG as a male accessory reproductive gland

Whereas in all other vertebrates the Müllerian ducts of genetic males are aborted during development, under the influence of Müllerian-inhibiting substance, in the caecilian amphibians they are retained as a pair of functional glands. It has long been speculated that the Müllerian gland might be the male accessory reproductive gland but there has been no direct evidence to this effect. The present study was undertaken to determine whether the caecilian Müllerian gland secretory proteins would bear antigenic similarity to secretory proteins of the prostate gland and/or the seminal vesicles of a mammal. The secretory proteins of the Müllerian gland of Ichthyophis tricolor were evaluated for cross-reactivity with antisera raised against rat ventral prostate and seminal vesicle secretory proteins, adopting SDS-PAGE, two-dimensional electrophoresis and immunoblot techniques. Indeed there was a cross-reaction of five Müllerian gland secretory protein fractions with prostatic protein antiserum and of three with seminal vesicle protein antiserum. A potential homology exists because in mammals the middle group of the prostate primordia is derived from a diverticulum of the Müllerian duct. Thus this study, by providing evidence for expression of prostatic and seminal vesicle proteins in the Müllerian gland, substantiates the point that in caecilians the Müllerian glands are the male accessory reproductive glands.     

Male reproductive effect of nickel sulphate in mice

Nickel sulphate was administered orally to adult male mice at dose level of 5 and 10 mg/kg body weight (5 days per week) for 35 days. There was no change in body weight. However a significant decrease in absolute and organ-to-body weight ratios of testes, epididymides, seminal vesicles and prostate gland was observed. The sperm abnormality, associated with decrease in sperm motility and sperm count was also observed. Significant alterations in the activities of marker testicular enzymes, viz. sorbitol dehydrogenase (decreases), lactate dehydrogenase (increases) and -glutamyl transpeptidase (increases) associated with histopathological changes in testes, epididymides and seminal vesicles, were also observed. Accumulation of nickel in testes, epididymides and seminal vesicles was also observed. The study reveals that the oral exposure to nickel may affect the histology of testes, epididymides, seminal vesicles and sperms morphology. These testicular and spermatotoxic changes may be responsible for observed male mediated developmental toxic effects.     

Carcinogenesis in accessory sex organs of rats induced by 3,2′-dimethyl-4-aminobiphenyl: response to androgen deprivation

 It is generally accepted that early human prostate cancers reveal higher androgen dependency than do advanced ones. In the present study, we examined whether the animal model of prostate cancer has already lost androgen dependency at the early stages of carcinogenesis. At experimental week 46, androgen deprivation was induced in rats and the incidences of atypical hyperplasia and cancer were examined in the ventral, dorsolateral prostate, coagulating glands, and seminal vesicles. Androgen deprivation significantly lowered the incidence of atypical hyperplasia in all four organs. As for the incidence of cancer, no significant differences were observed in the coagulating glands and seminal vesicles. Regarding atypical hyperplasia, androgen deprivation significantly decreased the proliferative cell nuclear antigen labeling index in the coagulating gland and seminal vesicles. The presence of cancer was also decreased in the coagulating gland but not in the seminal vesicles. With control group specimens, more intense staining of androgen receptor was observed in atypical hyperplasias than in cancers. Compared with the atypical hyperplasias, the cancers revealed low androgen dependency at the early stages of carcinogenesis. The cancers in the seminal vesicles also revealed higher androgen independency than did those in the coagulating gland. Accepted: 6 May 1997     

Sexual selection affects the sizes of the mammalian prostate gland and seminal vesicles

The accessory reproductive glands of male mammals contribute the bulk of the secretions in which spermatozoa are transported to the female tract during copulation. Despite their morphological diversity,and the chemical complexity of their products,little is known about the possible effects of sexual selection upon these glands in mammals. Here we consider the seminal vesicles and prostate glands in a sample of 89 species and 60 genera representing 8 Orders of mammals. The sizes of the accessory glands are analysed in relation to body weight and testes weight. Both the seminal vesicles size and prostate size (corrected for body weight) correlate positively with relative testes size in this sample; this finding remains highly significant after application of procedures to correct for possible phylogenetic biases in the data set. The accessory reproductive glands are also significantly larger in those mammals which have large relative testes sizes,and in which the likelihood of sperm competition is greatest. These results support the hypothesis that sexual selection has played an important role in the evolution of the mammalian prostate gland and seminal vesicles.     

The role of the seminal vesicles, coagulating glands and prostate glands on the fertility and fecundity of mice.

Female mice were allowed to mate with males which had been sham-operated (Group 1); had seminal vesicles, coagulating glands and ventral and dorsal prostate glands removed (Group 2); had the seminal vesicles removed (Group 3); had the coagulating glands removed (Group 4) or had the ventral and dorsal prostate gland removed (Group 5). The pregnancy rate was normal in Groups 1 and 4, severely reduced in Groups 2 and 3 and less so in Group 5. Litter size was reduced in Groups 2 and 3 but not in Group 5. It is suggested that the seminal vesicles and possibly the prostate glands are important in the production of young in mice.     

Effect of feed restriction on Hershberger and pubertal male assay endpoints

BACKGROUND: The Hershberger and male pubertal onset assays have been identified as possible Tier I screening tests to detect endocrine‐active compounds (EACs). Both tests rely on changes in reproductive and/or accessory sex gland (ASG) weights in young animals. Because chemical treatment may affect growth rate, the relationship between body weight and reproductive/ASG weights was examined using feed restriction (FR) to produce a targeted 10% decrease in body weight. METHODS: In the male pubertal onset assay, 23‐day‐old rats (12/group) were given ad lib feed or FR until euthanized at 45, 49, 52, 56, or 59 days of age. Despite a 10% body weight differential, pubertal onset was not significantly delayed and testes weights were conserved. Absolute prostate, ventral prostate, seminal vesicle, epididymides, and liver weights were decreased by FR. Relative weights for the prostate, ventral prostate, and seminal vesicles were similar to controls, but relative epididymides and liver weights still exhibited FR‐mediated changes. In the Hershberger assay, male rats (12/group) castrated at 36 days of age were given ad lib feed or FR in the presence or absence of testosterone propionate (T) from 46–55, 50–59, or 56–65 days of age. At 56, 60, and 66 days of age, rats were euthanized. In untreated animals, FR did not alter absolute ventral prostate, seminal vesicles, or Cowper's gland weights; however, absolute and relative weights of the levator ani‐bulbocavernosus muscles (LABC) were affected. In T‐treated animals, absolute organ weights (the ventral prostate, seminal vesicles, LABC, and glans penis) were relatively insensitive to FR. The weight of the Cowper's gland was affected only at 66 days of age. RESULTS: These data show that reproductive and ASG organ weight endpoints in the Hershberger and male pubertal onset assays can be influenced by FR levels that produce a 10% change in terminal body weight. CONCLUSIONS: The establishment of objective criteria for a positive or negative result is problematic due to the confounding effects of body weight on some endpoints. Furthermore, a 10% decrease in body weight seems to be excessive as a requirement for high‐dose toxicity in these assays due to possible indictment of agents that are not EACs, as well as potential masking of EAC effects coincident with body weight changes. Minimally, caution must be used in interpreting assay results in the presence of a 10% body weight change, recognizing the possible confounding effects of this degree of growth suppression. Birth Defects Res B 68:363–374, 2003. © 2003 Wiley‐Liss, Inc.     

Expression of nerve growth factor (NGF), and its receptors TrkA and p75 in the reproductive organs of the adult male rats

Immunolocalization of nerve growth factor (NGF) and its receptors, TrkA and p75 in the reproductive organs of adult male rats was investigated. Sections of the testis, efferent duct, epididymis, deferent duct, seminal vesicle, coagulating gland and prostate of adult male rats were immunostained by the avidin-biotin-peroxidase complex methods (ABC). NGF was expressed in Leydig cells, primary spermatocytes and pachytene spermatocytes in the testis. TrkA only immunoreacted to elongate spermatids and p75 showed positive immunostaining in the Sertoli cells, Leydig cells, the pachytene spermatocytes and elongate spermatids. Immunoreactions for NGF and its two receptors were detected in epithelial cells of efferent duct, deferent duct and epididymis. In addition, immunoreactions for NGF and its two receptors were also observed in columnar secretory epithelium lines of the seminal vesicles, prostate and coagulating gland. These results suggest that NGF is an important growth factor in gonadal function of adult male rats.     

ProCNP and CNP are expressed primarily in male genital organs

Lack of knowledge about the cellular origin of C-type natriuretic peptides (CNP) in the body has hampered the understanding of their biology. We examined the tissue specific expression of proCNP and CNP in the pig. The concentration of the CNP precursor, proCNP, was measured in extracts of 32 different tissues using a newly developed RIA. In 22 tissue extracts, we also measured CNP using a commercial RIA. In selected tissues, CNP mRNA was quantified by PCR, and the cellular CNP and proCNP localization was visualized by immunocytochemistry. Extracts from selected tissues were examined by gel chromatography. The highest peptide concentrations were found in extracts from the epididymis, seminal vesicles and prostate. CNP mRNA in the seminal vesicles and epididymis was 125-fold higher than in the other tissues examined. Gel chromatography showed that a CNP-53-like peptide is the dominant CNP tissue-form. Immunocytochemistry confirmed the pattern of peptide expression measured by RIA. In conclusion most proCNP-derived peptides are synthesized in epithelial cells in the epididymis, the prostate gland and in the seminal vesicles. The expression in male genital organs suggests a role of CNP in reproduction.     

Influence of dietary selenium on metabolism of cadmium and mercury in reproductive tissues of rats

Dietary selenium supplementation for rats resulted in a greater deposition of ¹⁰⁹Cd in testis, but caused decreased deposition of the isotope in seminal vesicles, epididymis and prostate gland. In contrast, dietary selenium caused increased deposition of ²⁰³Hg in seminal vesicles and prostate gland but drastically reduced the levels of this radioisotope in testis and epididymis. Selenium diverted the binding of ¹⁰⁹Cd in cytosols in testis, seminal vesicles, epididymis and prostate gland, but had minimal effects on the binding of ²⁰³Hg in these reproductive organs. Selenium deficiency caused increased excretion of ¹⁰⁹Cd in feces and urine, and increased excretion of ²⁰³Hg in urine of rats. The biological half-lives of the two radioisotopes in the −Se and +Se rats were calculated to be, respectively, 202 and 219 days for ¹⁰⁹Cd, and 2 and 6 days for ²⁰³Hg.     

Localization,metabolism, and binding of estrogens in the male rat

Estrogen assimilation by male Wistar rats was examined in these studies in several accessory sex organs (seminal vesicles and anterior, dorsal, lateral, and ventral prostates) as well as in a variety of nonaccessory sex organs. When [³H]estradiol was injected into intact 3- to 4-month-old rats in a pulse dose, no selective accumulation of radioactivity recovered as estradiol was found in the accessory sex glands when compared to other organs. This was due at least in part to the metabolism of estradiol to estrone and to the relatively low concentration of high affinity estrophilic molecules in the accessory sex organs. The order for the rate of formation of estrone from estradiol in tissues obtained from intact animals was ventral prostate > lateral and dorsal prostate > anterior prostate and seminal vesicles. Steroid specificity studies for cytosol estradiol binding by the ventral prostate and seminal vesicles revealed that estrophilic molecules exist in these organs. Based on Scatchard plot analyses in 24-h castrates, the number of available estradiol binding sites was too low in the ventral prostate to quantify accurately, but the seminal vesicles contained distinctly more estrophilic activity than the ventral prostate. The affinity for the seminal vesicle cytosol estradiol-estrophile binding exceeded that quantified for the seminal vesicle dihydrotestosterone-androphile reaction while the number of estradiol binding sites was less than that quantified for dihydrotestosterone. In relation to the accessory sex organs of other species, the rat seminal vesicles have a relatively small amount of cytosol estrophile. The findings that the seminal vesicles catabolize less estradiol and contain significantly more estrophilic activity than the ventral prostate is consistent with and offers insight into the noted estrogenic sensitivity of the seminal vesicles and lack thereof in the rat ventral prostate. With aging of the rat from 3–4 months to 22–26 months, the affinity of the seminal vesicle estradiol-estrophile interaction was unchanged but the number of binding sites increased significantly.     

Metalloproteinase activities expressed during development and maturation of the rat prostatic complex and seminal vesicles.

The objective of this study was to characterize proteinase activities expressed during development and maturation of the prostate gland and seminal vesicles of the rat by using gelatin-and casein-containing SDS polyacrylamide gel zymography. The prostatic complexes of 2- and 10-day-old animals and the individual lobes of the prostate (ventral, dorsolateral, and anterior [coagulating gland]) and the seminal vesicles of 15-day-old animals expressed prominent gelatinolytic activities of approximately 64, 71, and 76 kDa. These activities had properties of metalloproteinases; i.e., they were stimulated by Ca2+ and inhibited by EDTA and EGTA. They were greatly diminished by 52 days of age (immediately postpuberty) and were not detected in the dorsal lobe of the adult. Less active gelatinolytic proteinases with molecular masses of approximately 34 and 43 kDa were expressed in the developing prostatic complexes and individual lobes and seminal vesicles, but they were not detected in postpubertal animals. Weak gelatinolytic activities of 82, 85, and 89 kDa were found in the prostatic complexes; these activities were greatly diminished in all prostate lobes with sexual maturation but were expressed in the seminal vesicles at all ages. A large-molecular-mass Ca(2+)-independent proteinase of 130 kDa or greater was first detected in the dorsolateral prostate at 21 days of age. This activity was expressed in both the lateral and dorsal lobes of the adult but was greater in the lateral lobe. Proteinase activities of about 22 and 26 kDa that were not stimulated by Ca2+ were detected in the ventral prostate at 15 days of age by means of both gelatin and casein gels.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mouse seminal vesicle shape mutation is allelic with Fgfr2

The mouse seminal vesicle shape (svs) mutation is a spontaneous recessive mutation that causes branching morphogenesis defects in the prostate gland and seminal vesicles. Unlike many other mutations that reduce prostatic and/or seminal vesicle branching, the svs mutation dramatically reduces branching without reducing organ growth. Using a positional cloning approach, we identified the svs mutant lesion as a 491 bp insertion in the tenth intron of Fgfr2 that results in changes in the pattern of Fgfr2 alternative splicing. An engineered null allele of Fgfr2 failed to complement the svs mutation proving that a partial loss of FGFR2(IIIb) isoforms causes svs phenotypes. Thus, the svs mutation represents a new type of adult viable Fgfr2 allele that can be used to elucidate receptor function during normal development and in the adult. In the developing seminal vesicles, sustained activation of ERK1/2 was associated with branching morphogenesis and this was absent in svs mutant seminal vesicles. This defect appears to be the immediate downstream effect of partial loss of FGFR2(IIIb) because activation of FGFR2(IIIb) by FGF10 rapidly induced ERK1/2 activation, and inhibition of ERK1/2 activation blocked seminal vesicle branching morphogenesis. Partial loss of FGFR2(IIIb) was also associated with down-regulation of several branching morphogenesis regulators including Shh, Ptch1, Gli1, Gli2, Bmp4, and Bmp7. Together with previous studies, these data suggest that peak levels of FGFR2(IIIb) signaling are required to induce branching and sustain ERK1/2 activation, whereas reduced levels support ductal outgrowth in the prostate gland and seminal vesicles.     

Components of human split ejaculates. II. Enzymes and proteinase inhibitors.

Lysozyme, alpha-amylase, neutral proteinase and plasminogen activator were most concentrated in the initial portion of the ejaculate that consists mostly of Cowper's gland and prostate gland fluids as well as spermatozoa. The concentration of the high molecular weight proteinase inhibitors, alpha1-antitrypsin and alpha1X-antichymotrypsin, was essentially unaltered throughout the ejaculate fractions, although their absolute amounts showed an increase towards the final fraction. By contrast, the total inhibitory activity towards pancreatic trypsin was highest both in concentration and amount in the last fraction, thus indicating that the seminal vesicles are its primary source. Plasminogen, prothrombin, Factor XIII, and the proteinase inhibitors antithrombin III, alpha2-macroglobulin, inter-alpha-trypsin inhibitor and C1S-inactivator could not be detected immunochemically in whole ejaculates, and indicates the dissimilarity between the coagulation/liquefaction processes of semen and blood.     

Thyrotropin-releasing hormone and a homologous peptide in the male rat reproductive system

TRH and a TRH-like peptide have been shown to occur throughout the male rat reproductive system by TRH radioimmunoassay, SP-Sephadex C25 cation exchange chromatography, high pressure liquid chromatography and parallel line analysis. The total concentration of TRH and TRH-like peptide was highest in the prostate followed by the testis, cauda epididymis and seminal vesicles. Dilution curves for extracts of prostate, testis and seminal vesicles were parallel with TRH while the corresponding curve for epididymis was nonparallel.     

Isolation of non-heparin-binding and heparin-binding proteins of boar prostate

Proteins of boar prostate secretion were separated by affinity chromatography on heparin-polyacrylamide to non-heparin-binding (H) and heparin-binding (H+) protein fractions. H- and H+ fractions were then subjected to RP HPLC. Elution profiles of H-and H+ fractions of prostate secretion were compared with those of seminal plasma and the amounts of corresponding proteins were compared. Besides, the isolated proteins were characterized by SDS-PAGE. In the H- fraction of prostate secretion, PSP I and PSP II spermadhesins and in the H+ fraction AQN 2 and AWN 1 spermadhesins were found in substantially lower amounts than in seminal plasma. On the contrary, beta-microseminoprotein was identified in abundant amounts both in H- and H+ fractions of boar prostate secretion. AQN 2 and AWN 1 spermadhesins were proved by their antibodies. Some seminal plasma proteins originating mainly in seminal vesicles could also be secreted by the prostatic gland. beta-Microseminoprotein was found to be produced mainly by the prostate.     

Anatomic,Histologic and Certain Enzymatic Studies on the Male Genital Organs of Hemiechinus auritus collaris Gray,the Indian Long Eared Hedgehog

Anatomically the male reproductive organs of Hemiechinus auritus collaris are of considerable interest and present a unique pattern of arrangement of different glands. The testes are inguinal and a true scrotum is absent. A pair of accessory glands, the seminal vesicles, are situated dorsal to the bladder. A pair of obviously lobulated glands, ventral to the bladder, represent the internal prostate and a pair of compact glands situated outside the pelvis in para-anal position the external prostate. The Cowper's gland and the gland of ampulla are absent. The present studies also concern enzymes of the phosphatase group, including both specific and non-specific phosphatases, in the testes and the sex accessory glands during both active and inactive periods in this insectivore.     

Fertilization-promoting peptide in reproductive tissues and semen of the male marmoset (Callithrix jacchus)

Fertilization-promoting peptide (FPP) is present in the prostate gland and semen of some mammals, and has been shown to enhance the fertilizing ability of both epididymal mouse and ejaculated human spermatozoa. The novel peptide may prove of importance for the treatment of some cases of male infertility, and a suitable animal model would be useful to test this hypothesis. To this end, we examined reproductive tissues and semen of the male marmoset for the presence of FPP. Peptides were extracted from seminal plasma, testes, prostate, and bulbourethral glands of intact and castrated male marmosets. The peptides were identified by ion-exchange chromatography followed by radioimmunoassay. The mean concentration of FPP immunoreactivity in semen from intact males was 58.7 nM (SE ± 9.9 nM, n = 10), and anion-exchange chromatography revealed FPP as the only immunoreactive peptide present. Analysis of tissues revealed that FPP in semen was likely to be derived from the prostate gland, which contained this peptide as the major source of immunoreactivity (10.86 pmol/gland; SE ± 4.39 pmol/gland, n = 4). Only low concentrations of FPP were detectable in the bulbourethral glands, and the peptide was undetectable in the testis. Surprisingly, FPP was readily detectable in the seminal plasma from one castrated marmoset and was present in the prostate gland from 3 castrates at levels which did not differ significantly from those in intact animals (5.47 pmol/gland, SE ± 1.64 pmol/gland, n = 3). Plasma testosterone measurements indicated that residual circulatory androgens remained after castration, which may be consistent both with the maintenance of mating behavior and the presence of prostatic FPP. We conclude that FPP is present within the prostate gland and seminal plasma of the marmoset at concentrations consistent with a role in male fertility in this species. Mol. Reprod. Dev. 47:113–119, 1997. © 1997 Wiley-Liss, Inc.