Lipids of Drosophila: a newly detected lipid in the male.

A thin-layer chromatographic analysis of Drosophila melanogaster revealed a lipid to be found almost exclusively in the adult male. The compound or class of compounds having an R(F) close to that of methyl oleate is present in substantial amounts and is located predominantly in the ejaculatory bulb. It appears from genetic studies that the formulation of the lipid is not mediated by the Y chromosome.

Cyclic guanosine 3'-5'-monphosphate. High levels in the male accessory gland of Acheta domesticus and related crickets.

Guanosine 3',5'-monophosphate (cyclic GMP) was found in the accessory gland of reproductively mature male house crickets (Acheta domesticus (L.)) up to the exceptionally high level of 500 pmol/mg protein (10(-4) mol/kg wet weight). The identity of cricket cyclic GMP was confirmed by enzymatic and spectral analysis. A survey of 10 closely related species of Orthoptera indicated that high levels of cyclic GMP in the accessory gland occur in the subfamily Gryllinae, to which A. domesticus belongs. In these crickets, cyclic GMP in the accessory gland increases together with protein content during two weeks after the final molt. Levels are not augmented by dissection, and are independent of the presence of sperm in the seminal vesicles and of the production of spermatophores by the gland. The function of cyclic GMP in the accessory gland is not yet understood.

On the androgen microenvironment of maturing spermatozoa.

Adult anesthetized male rats were submitted to in vivo micropuncture of the seminiferous and epididymal tubules and reproductive tract vasculature to obtain fluids for analysis of testosterone, 5 alpha-dihydrotestosterone, and androgen-binding protein (ABP). Androgen and ABP concentrations were determined by RIA. The highest concentrations of testosterone (73.14 +/- 5.12 ng/ml) were in testicular interstitial fluid. A significant downhill concentration gradient exists between testosterone concentrations in testicular interstitial fluid and seminiferous tubule fluid (50.24 +/- 2.26 ng/ml); another significant decrease occurs between seminiferous tubule fluid and rete testis fluid (17.85 +/- 2.11 ng/ml). 5 alpha-Dihydrotestosterone concentrations were highest in intraluminal caput epididymidal fluids (58.73 +/- 6.48 ng/ml) as were ABP concentrations (33.30 +/- 2.40 mu leq/microliter). Intraluminal sperm concentrations were also determined, and from these data, fluid reabsorption by the efferent ducts and epididymal tubules were calculated. Eighty-nine percent of the fluid leaving the testis is reabsorbed between the rete testis and caput epididymidis, and 96% is reabsorbed between rete and cauda. It was calculated that large losses of androgen and ABP also occur from the lumen of the excurrent duct system. These losses may be due to metabolism, diffusion from the lumen, or uptake by cells.

Prostaglandins masculinize the mouse genital tract.

The masculinizing effects of prostaglandins (PGS) PGE2 and PGF2 alpha on mouse fetal genital tract differentiation were studied both in vivo and in vitro. Prenatal exposure to PGE2 and PGF2 alpha on days 11-17 of gestation (the critical period of the differentiation) increased the anogenital distance of the female fetuses in a dose-dependent manner. PGE2 also increased the anogenital distance of male fetuses in the presence of an inhibitor of testosterone synthesis, namely estradiol (2 mg/kg.day), and in the androgen-insensitive Tfmy males. Internally, PGE2 induced the epididymal duct in the females, estrogen-exposed males, and Tfmy males. However, no other changes were noticed in the internal genital tract of these fetuses. To avoid the problems associated with the placental transfer of any external agent, we also studied the effect of PGE2 in an in vitro system. Female genital ducts on day 13 of gestation were cultured in the presence and absence of different concentrations of PGE2 for a total of 6 days. PGE2 at doses 0.2 and 1 microgram/ml induced and stimulated the Wolffian and epididymal ducts. Thus, PGs appear to have a masculinizing role in androgen-induced sexual differentiation.

Immunoreactive somatostatin in male reproductive system in humans.

Somatostatin-like immunoreactivity is distributed widely in humans; the highest concentration is in the central nervous system, gastrointestinal tract, and pancreas. To determine if somatostatin is present in the male reproductive system, we analyzed human testis, epididymis, prostate, and semen. Somatostatin-like immunoreactivity was detectable in acid extracts of human testis, epididymis, and prostate (n = 6 each) in concentrations of 4.0 +/- 1.4 (+/- SD), 14.7 +/- 3.2, and 27.5 +/- 5.1 pmol/g wet wt, respectively. Considerable amounts of immunoreactive somatostatin also were detectable in semen; the mean value was 3.8 +/- 1.3 nmol/L (n = 6). This value was 200-fold higher than that in peripheral plasma. The somatostatin immunoreactivity in these tissues was characterized by gel filtration chromatography. Two peaks of somatostatin immunoreactivity, one coeluting with somatostatin-14 and the other with somatostatin-28, were found in the testis, epididymis, prostate, and hypothalamus. The amounts of the two sizes were nearly equal in the testis; somatostatin-14 predominated in the epididymis, prostate, and hypothalamus; whereas only somatostatin-28 was detected in semen. The presence of somatostatin in the male reproductive system suggests that somatostatin may play a role in the regulation of reproductive function in men.

Identification of epidermal growth factor-like activity in human male reproductive tissues and fluids.

Epidermal growth factor-like activity (EGF-LA) has been detected in human seminal plasma in concentrations of 5-150 ngeq/ml (36.4 +/- 2.1, mean +/- SEM), using a heterologous RRA with murine EGF. The samples were obtained from normal, subfertile and azoospermic men, aged 21-50 yr. No correlation was found between EGF concentration and age of donor, sperm count, sperm motility, or period of sexual abstinence before sample collection. High performance liquid chromatography of the seminal plasma resulted in a main peak of EGF-LA which eluted at 29% acetonitrile, compared to 33% for murine EGF. Microsomal membranes were prepared from several tissues from the human male reproductive tract and were tested for their ability to bind radioiodinated murine EGF. Specific EGF binding activity was detectable in testicular membrane preparations but was not detectable in membranes prepared from human prostate, seminal vesicle, epididymis, or spermatozoa. Endogenous EGF-LA was detectable in human testis, seminal vesicle, prostate, and epididymis.

Absence of relaxin immunostaining in the male reproductive tracts of the rat and mouse.

By use of the biotin-avidin immunohistochemical method and a homologous antiserum as the primary antiserum, relaxin immunostaining was absent in the testes, prostate, seminal vesicles, and epididymides of the rat. Relaxin immunostaining was also lacking when anti-porcine relaxin serum was employed as the primary antiserum. Furthermore, immunohistochemical studies for relaxin localization in the reproductive tract of the male mouse using both anti-rat and anti-porcine relaxin sera also revealed an absence of the hormone in the reproductive system of this species. Although this study suggests that immunoreactive relaxin is absent in the male reproductive tracts of both the rat and mouse, it raises some questions concerning the reports in the literature of the presence of relaxin-like substances in the male reproductive tracts of other species. These reports are discussed in relation to our current results.

Localization of metallothionein in the genital organs of the male rat.

We studied the immunohistological localization of metallothionein (MT), a low molecular weight metal binding protein, in male rat genital organs (testis, epididymis, ejaculatory duct, seminal vesicle, coagulating gland, and prostate) by use of the avidin-biotin-peroxidase complex method. MT concentrations in testis, seminal vesicle, and prostate ranged from 15-30 micrograms/g tissue. In testis, seminiferous tubules with mature spermatozoa exhibited weak MT staining, whereas the tubules containing differentiating spermatogenic cells but not containing spermatozoa showed strong MT staining. No MT immunostaining was observed in Leydig cells. In growing rat testes, the pattern of MT immunostaining was found to change with development: MT was found in supporting cells only on Day 7, spermatogonia adjacent to basement membrane on Day 14, and spermatocytes localized in the central part of the tubules on Day 21. Strong MT immunostaining in the basal cells was a common feature in other genital tissues, except the ductus efferentes. In prostate, the strongest MT staining was found in the lateral lobe, and MT was localized in apocrine secretions in the dorsal lobe. The present results suggest a close association of MT with cell proliferation and differentiation, as well as possible involvement of MT in supply or storage of zinc ions.

Distribution of soltriol [1,25(OH)2-vitamin D3] binding sites in male sex organs of the mouse: an autoradiographic study.

After injection of [3H]-1,25(OH)2-vitamin D3 (soltriol), nuclear labeling is found in Sertoli cells of testes, being highest at the stage of spermiosis, in epithelium of efferent ductules and caput epididymidis and in connective tissue cells of epididymis, in lamina propria and muscular sheath of deferent duct, and in epithelium and muscular sheath of dorsal and ventral prostate of the mouse. This labeling pattern is characteristic for [3H]-soltriol and differs from that for [3H]-dihydrotestosterone and [3H]-estradiol, although with overlap. The nuclear labeling with [3H]-soltriol suggests an action of the hormone on certain processes during spermatogenesis, on sperm maturation, on epididymal fluid resorption, and on secretion and transport of spermatozoa.

Immunohistochemical localization of androgen receptors with mono- and polyclonal antibodies to androgen receptor.

Rat, human, and mouse tissues were stained immunohistochemically using mono- and polyclonal androgen receptor antibodies. Monoclonal antibodies were raised in rats and used to stain human and mouse tissues; polyclonal antibodies were raised in rabbits and used to stain rat tissues. Frozen tissue sections were incubated with the appropriate androgen receptor antibody and staining was completed by the indirect avidin-biotin peroxidase method. A comprehensive survey of rat and mouse tissues was performed. Antibody staining was found exclusively in the nucleus of certain specific cell types, suggesting that the androgen receptor is a nuclear protein. All male sexual organs in the rat showed strong positive nuclear staining for androgen receptor. Weaker positive reactions were seen in kidney, liver, adrenal cortex and pituitary gland. Furthermore, positive staining for androgen receptor was exhibited in skeletal, cardiac and smooth muscle cells, and central nervous tissue. Female reproductive organs also contained androgen receptor-positive cells. The spleen was found to be the only organ examined which did not stain for androgen receptor. The monoclonal antibody could also demonstrate androgen receptor-positive cells in a human prostatic cancer and in a prostate with benign hyperplasia. These data demonstrate the use of antibodies in revealing cellular/subcellular distribution of androgen receptor in target tissues.

Hypophysectomy of the tammar wallaby, Macropus eugenii: surgical approach and general effects.

A technique of hypophysectomy and regimes of pre- and post-operative care were developed for the tammar wallaby, Macropus eugenii, to a stage when animals can survive the operation with little apparent stress. Thyroid and adrenal gland weights declined after hypophysectomy, especially within the first 20-30 days. Changes in the adrenal cortex after hypophysectomy suggested that this region may have a zonal organization different from that in eutherian mammals. The reproductive tracts of males and females lost weight rapidly after hypophysectomy. Eleven plasma parameters were studied for the effects of hypophysectomy. There was a reduction in sodium and chloride and a tendency to higher potassium levels, reflecting inadequate adrenal cortical function. Calcium and total protein values remained unaffected, but inorganic phosphate, glucose and cholesterol were depleted, and a less significant depletion in blood urea, nitrogen and uric acid was evident.

Effects of testosterone and prolactin or growth hormone on the accessory sex organs of castrated mice.

In 5-day experiments, neither bovine prolactin (300 or 100 i.u./kg) nor ovine growth hormone (25 i.u./kg) alone significantly enhanced accessory sex organ weights in the castrated mouse. Seminal vesicle weights, and to a lesser extent anterior prostate gland weights, were augmented by the simultaneous injection of testosterone (1-5 mg/kg) daily plus prolactin or growth hormone. The effect was greater than that produced by testosterone alone. The levels of fructose in accessory sex organs used to indicate androgenic activity were similar in castrated mice receiving testosterone alone or in combination with prolactin or growth hormone. Prolactin alone did not influence uptake of (3H)testosterone by the seminal vesicles or anterior prostate gland over a 5 min period in vivo.

Identification and distribution of peripheral benzodiazepine binding sites in male rat genital tract.

In the present study we identified and characterized the distribution of high-affinity peripheral benzodiazepine binding sites (PBzS) in male rat vas deferens (whole, and prostatic and epididymal portions), prostate, seminal vesicles, and Cowper's glands. [3H]PK 11195, an isoquinoline carboxamide derivative, was used as a radioligand specific for PBzS. Scatchard analysis of saturation curves of [3H]PK 11195 binding in the whole vas deferens, the prostatic and epididymal portions of the vas deferens, the prostate, the seminal vesicles, and Cowper's glands yielded mean maximal numbers of binding sites of 1211 +/- 158, 1012 +/- 311, 1451 +/- 156, 1805 +/- 86, 865 +/- 51, and 2251 +/- 135 fmol/mg protein, respectively. The equilibrium dissociation constant values ranged between 1 and 3 mM in all the above tissues. The ability of various drugs to displace the specific binding of [3H]PK 11195 from PBzS in Cowper's gland membranes was also tested. The inhibition constants for Ro 5-4864, diazepam, and PK 11195 were 28, 330, and 4 nM, respectively, whereas clonazepam, Ro 15-1788, and testosterone were inefficient in displacing [3H]PK 11195. The presence of high densities of PBzS in the male genital tract suggests a functional role in these hormone-dependent organs.

Identification of cytoplasmic estrogen receptors in the accessory sex organs of the rabbit and their comparison to the cytoplasmic estrogen receptor in the epididymis.

Estrogen receptors are present in cytosol prepared from the accessory sex organs (vesicular gland, proprostate, prostate, bulbourethral gland) of sexually immature and of sexually mature rabbits. The receptor in these organs from animals of both age groups has a sedimentation coefficient of 8-10S on low ionic strength (0.01 M KCl) sucrose gradients. Under high ionic strength (0.4 M KCl) conditions, the receptor sediments at approximately 4S. The cytoplasmic estrogen receptor from the epididymis shows age-dependent changes in its sedimentation coefficient. It is 8S under low ionic strength conditions when prepared from immature rabbits and 4S under identical conditions when prepared from sexually mature animals. Although the dissociation constant of the cytoplasmic estrogen receptor in the immature and mature epididymis and accessory sex organs remains constant during development (approximately 0.1 nM), the number of available cytoplasmic estrogen binding sites declines from about 160 fmoles/mg cytosol protein in the immature rabbit to about 40 fmoles/mg cytosol protein in the adult animal. The estrogen receptor in the accessory sex organs is highly specific, the relative affinities of various potential competitors being: estradiol and estrone = 1, diethylstilbestrol = 0.3, estriol = 0.2, tamoxifen = 0.08, testosterone = 0.0004 and 5 alpha-DHT = 0.00005. Changes with age in the physicochemical characteristics of the estrogen receptor and in the concentration of binding sites suggest that the estrogen receptor may be involved in the development and physiological regulation of the male reproductive tract.

Pregnancy proteins in seminal plasma, seminal vesicles, preovulatory follicular fluid, and ovary.

A number of proteins previously thought to be specific for the placenta or pregnancy have been identified in the fluids bathing both the oocyte and the sperm. In many cases their concentrations in follicular fluid and seminal plasma greatly exceeded those in the serum of nonpregnant women or men, and sometimes they even exceeded the levels in pregnancy sera. We report here the occurrence of PP5, PP12, PP14 and PAPP-A in follicular fluid and seminal plasma. In follicular fluid, the levels of PP5, PP12, and PAPP-A correlate with the estrogen concentration of the same fluid, and the PP12 and PAPP-A levels also bear a positive correlation to the progesterone concentration. The levels of PP12 and PAPP-A increase as the follicle grows, as do the levels of many steroid hormones. Therefore, the apparent correlations observed may be merely coincidental. However, circumstantial evidence from other reproductive organs indicates that the synthesis of PP12 and PAPP-A is stimulated by progesterone. Results of immunohistochemical staining show that PP12 and PAPP-A are localized in the luteinized granulosa cells and the corpus luteum. Previous studies indicate that PP5 and PAPP-A inhibit the action of proteolytic enzymes plasmin and elastase, which are believed to be involved in the mechanisms of ovulation. The study of the significance of these various placental proteins for human reproduction is only at its beginning. Clearly, elucidation of their function is the key to a more fundamental understanding of their role in the events governing ovulation and implantation.

Distribution of galanin immunoreactivity in the genitourinary tract of man and rat.

Galanin has been shown to be present in substantial quantities in the human and rat genitourinary tract by radioimmunoassay and immunocytochemistry. The highest concentrations measured by radioimmunoassay were found in the human vas deferens, corpus cavernosum and spongiosum and in the vagina and cervix. In man gel chromatographic analysis showed two molecular forms. The earlier eluting peak was different from porcine galanin standard. There was only one molecular form in the rat which emerged in an earlier position than the porcine standard. Galanin immunoreactive nerve fibres demonstrated in the genitourinary tract were found both in man and rat. They were found within smooth muscle and in close relationship to blood vessels. The presence and distribution of galanin in the genitourinary system suggest the possibility that this neuropeptide could play a role in the regulation of smooth muscle tone, blood flow and motility.

Peptide histidine methionine (PHM) and the human male genitalia.

Peptide histidine methionine-like immunoreactivity (PHM-IR) has been demonstrated to be present in the human penis both by radioimmunoassay and immunohistochemistry with particularly high levels in the corpus cavernosum and vas deferens. In the cavernosa, PHM-IR has been localised entirely, in nerves around arteries. High performance liquid chromatography indicated that this PHM-IR co-eluted with synthetic PHM but not porcine PHI. The presence of PHM-IR in the penis suggests that this neuropeptide may play a functional role in penile function.

Neuropeptide Y in the human male genital tract.

Neuropeptide Y (NPY) was found in high concentrations in the male genital tract. NPY levels were highest in the seminal vesicles, prostate, corpus cavernosum and vas deferens, where large numbers of immunoreactive nerve fibres were detected. Considerable quantities were also found in the epididymis and spongiosum. Lower concentrations were found in the glans penis, testis and foreskin. The presence of a large number of nerves containing NPY suggest that this active neuropeptide may play a role in control of genital function.

Somatostatin-14 and -28 in the male rat reproductive system.

Somatostatin-14 (SRIF-14) has been shown to occur throughout the male rat reproductive system by SRIF radioimmunoassay, Sephadex G-50 exclusion chromatography, and parallel line analysis. SRIF-28 was found only in the epididymis. The highest concentrations of total SRIF-like immunoreactivity (SLI), representing the combined concentrations of SRIF-14 and SRIF-28, were measured in the prostates of 1 1/2- and 3-month-old Sprague-Dawley rats. The levels of SLI in prostates from 9-month-old males were about 10% that of the younger animals. Dilution curves for extracts of all reproductive tissues were parallel with synthetic SRIF-14.

Effect of antiserum to luteinizing hormone (LHAS) on the physiology of the epididymis and accessory glands in the ablino rat.

PIP: Rabbit antiserum to ovine LH (LHAS) was injected subcutaneously in dosages of .1 ml/day or .2 ml/day for 5 days to determine its effects on the physiology of the epididymis and accessory glands of intact, adult male rats. The weight of the vas deverens decreased significantly with .1 ml LHAS when compared with intact controls (p less than .05), though the higher dose did not further decrease the weight. The weight of the testes was not affected by either dose of LHAS. Castration caused a marked decrease in the weights of all the accessory glands. Administration of .2 ml of LHAS resulted in a significant reduction in the weights of the dorsolateral prostrate, coagulating glands, seminal vesicles, and Cowpers glands compared with intact controls (p. less than .05), and the weights were comparable with those in castrate controls. The secretory activity of the accessory glands was decreased with administration of .2 ml of LHAS. The content of sialic acid in the caput and cauda epididymis decreased to castrate levels after treatment with .1 ml of LHAS. The content of sialic acid in the vas deferens was significantly reduced (p less than .01), and sialic acid in the Cowpers glands was markedly decreased (p less than .05), in all 3 experimental groups.^ieng