Rat prostatic inhibin: less than 35 kDa protein(s).

An attempt was made to purify and physicochemically characterize the inhibin-like activity of rat ventral prostate based on the technique of immunosorption. It was possible to partially purify prostatic inhibin by affinity chromatography and the molecular size of inhibin-like material was shown to be less than 35 kDa.

Synthesis of noncollagenous extracellular matrix proteins during development of mineralized nodules by rat periodontal ligament cells in vitro.

To characterize the mineralized nodules produced by rat periodontal ligament (PDL) cells in vitro, we have studied the synthesis and distribution of mineralized tissue proteins at various stages of nodule formation. PDL cells were obtained from coagulum in the socket at 2 days after tooth extraction and cultured in Dulbecco's Modified Eagles Medium (DMEM) containing 10% fetal bovine serum and antibiotics. Confluent cells were grown in the presence of ascorbic acid (50 micrograms/ml), dexamethasone (5 microM), and beta-glycerophosphate (10 mM) for 3 weeks. Four stages showing distinct morphological characteristics during development of mineralized nodules were identified. Protein synthesis and deposition of proteins into the matrix were studied during these stages by metabolic labeling with [35S]methionine for 24 hours. Large quantities of SPARC (secreted protein, acidic and rich in cysteine) were synthesized by confluent cells but decreased during the progress of mineralized nodule formation. Two forms of osteopontin (OPN) (67 kDa and 61 kDa) were synthesized in comparable quantities by confluent cells; OPN and bone sialoprotein (BSP) were induced by dexamethasone and represented the major proteins in the mineralized matrix. The 67 kDa form of OPN was the predominant species in the mineralized matrix. Both OPN and BSP were localized by immunogold electron microscopy on globular as well as fused electron-dense structures at sites of tissue mineralization.

Evidence for up-regulation of epidermal growth-factor receptors on rat periodontal ligament fibroblastic cells associated with stabilization of phenotype in vitro.

This study sought to understand the role of epidermal growth factor receptor (EGF-R) in periodontal ligament (PDL) fibroblasts. Rat PDL fibroblastic cells and ROS 17/2.8 cells (highly differentiated osteoblastic osteosarcoma cells) were cultured and treated with transforming growth factor-alpha (TGF-alpha), EGF, dexamethasone (Dex) or a combination of EGF and Dex. Alkaline phosphatase (ALP) activity, an early differentiation marker for mineralized tissue-forming cells, was measured using p-nitrophenylphosphate as a substrate. For Scatchard analysis of [125I]-EGF binding, cells were incubated in Dulbecco's modified Eagle's medium containing 0.2% bovine serum albumin and 0-64 ng/ml of [125I]-EGF for 4 h at 4 degrees C. Also, the synthesis of EGF-R protein and the expression of mRNA for EGF-R were measured by immunoprecipitation and Northern blot analysis, respectively. Untreated PDL fibroblastic cells showed a gradual increase in spontaneous ALP activity from 32.4 U/10(6) cells at 2 days to 49.6 U/10(6) cells at 7 days of culture. ALP activity was further increased to 70.8 U/10(6) cells at 7 days after treatment with Dex, whereas EGF treatment reduced it to 19.4 U/10(6) cells. Culture of PDL fibroblastic cells in the presence of a combination of Dex and EGF decreased the Dex-induced ALP activity from 70.8 U to 41.8 U/10(6) cells at 7 days. A similar inhibitory effect on ALP activity was found after treatment with TGF-alpha. In contrast, ROS cells maintained a high ALP activity (1748 U/10(6) cells) throughout culture, unaffected by EGF. Scatchard analysis demonstrated that PDL fibroblastic cells have both high- and low-affinity forms of EGF-R, while ROS cells did not have any detectable EGF-R. Treatment of PDL cells with Dex for 2 days decreased the synthesis of EGF-R protein, the expression of EGF-R mRNA and the number of EGF-R. In contrast, EGF treatment increased the expression of EGF-R mRNA. These data suggest that PDL fibroblastic cells express numerous EGF-R, but the number decreases during their differentiation into mineralized tissue-forming cells under the influence of Dex. Thus, EGF-R may function in the stabilization of phenotype in PDL fibroblastic cells.

Effect of cyproterone acetate on structure and function of rhesus monkey reproductive organs.

A low dose of Cyproterone acetate (CPA; 1 mg/kg body weight/day for 70 days) was administered to adult male rhesus monkeys to assess its effects on testicular and epididymal structure and function in a nonhuman primate species. CPA caused extensive degenerative changes in morphology of seminiferous, efferent duct, and epididymal epithelia, including decrease in diameter of seminiferous and epididymal tubules and their lumen, height of epididymal epithelium, and an increase in intertubular connective tissue. The protein profile of spermatozoa showed alterations during their epididymal transit in control and CPA-treated monkeys. In CPA-treated animals, 19 polypeptides were acquired and nine were eliminated during epididymal transit in contrast to acquisition of 12 and loss of 14 polypeptides in control animals. Treatment with CPA also resulted in the appearance of 14 new polypeptides in epididymal cytosol and luminal fluid, probably of lysosomal origin. The protein pattern of caput and cauda epididymal tubule cytosol, maintained in organ culture and exposed to 100 microM CPA for 3 days, showed absence of eight polypeptides. These results indicate that even at the low dose used in this study, CPA has caused spermatogenic arrest, degenerative changes in the epididymal structure, and alterations in epididymal and sperm protein profile. Suppression of serum testosterone levels indicates the need for androgen supplementation if CPA is to be used for male contraception.

In vitro formation of mineralized nodules by periodontal ligament cells from the rat.

The purposes of this study were to determine whether periodontal ligament (PDL) cells are capable of producing mineralized nodules in vitro and to analyze ultrastructural features of the nodules. Rat PDL cells were obtained from coagulum in the socket at 2 days after tooth extraction and cultured at confluence in standard medium containing Dulbecco's Modified Eagle's Medium supplemented with 10% FBS and antibiotics. To test mineralized nodule formation, cells were further cultured for an additional 3 weeks in the standard medium containing (1) ascorbic acid (50 micrograms/ml) and sodium beta-glycerophosphate (10 mM), (2) ascorbic acid, sodium beta-glycerophosphate, and dexamethasone (5 microM), or (3) ascorbic acid alone. Cells were then fixed in 2.5% glutaraldehyde, postfixed in 1% OsO4, and prepared for light and electron microscopy. Three-dimensional nodules containing mineralized matrices were formed only when the cells were cultured in the presence of ascorbic acid and dexamethasone. They were composed of multilayered fibroblasts (up to 13 layers), and highly organized collagen fibrils with 64 nm cross-banding patterns between the cell layers. The fibroblasts in the nodules exhibited an elongated shape with a high degree of cytoplasmic polarity throughout the nodule, and have the morphological features of PDL fibroblasts as seen in vivo. Mineral deposition with needle-like crystals was initiated on collagen fibrils located in intercellular spaces of the upper cell layers and became increasingly heavier towards the bottom half of the nodules. X-ray microanalysis and electron diffraction analysis confirmed that mineral deposition contained calcium and phosphate in the form of immature hydroxyapatite.(ABSTRACT TRUNCATED AT 250 WORDS)

Steroid metabolism by germ cells and spermatozoa in men after vasoepididymostomy.

The ability of germ cells (spermatocytes and spermatids) and spermatozoa present in human ejaculate to metabolize steroids was studied in men with obstructive infertility who had undergone vasoepididymostomy as corrective surgery. Steroid metabolism by spermatozoa in men who had undergone vasovasostomy was also investigated. Germ cells converted testosterone mainly to androstenedione. In addition to androstenedione, dihydrotestosterone and androstanediols were also formed in incubations using spermatids. Both types of germ cells converted estradiol to estrone. Spermatozoa from subjects who had undergone vasoepididymostomy or vasovasostomy converted testosterone to androstenedione as in normal men, while spermatozoa from infertile subjects converted testosterone mainly to dihydrotestosterone. Seminal fluid, free of germ cells, did not show steroid-metabolizing capability.

PIP: Metabolism of testosterone and estradiol by primary spermatocytes, spermatids and spermatozoa of 6 fertile men, 6 men infertile due to immobile sperm, 8 men who had vasovasostomy, and 11 men who had vasoepididymostomy because of obstruction, was studies by thin layer chromatography. Germ cells were collected at 3-month intervals after surgery, and separated by Percoll gradients. Results were reported as percentages of total counts in substrates and products. Germ cells of normal and post-operative subjects converted testosterone primarily to androstenedione, and their spermatids also formed dihydrotestosterone and androstanediols. Spermatozoa and spermatids also formed estrone from estradiol. Spermatozoa from infertile men primarily produced dihydrotestosterone from testosterone.

In vitro organ culture of rhesus monkey epididymal tubules.

The caput and cauda epididymal tubules of rhesus monkey were cultured for 5 days using a simple organ culture system. The viability of the tubules in culture was established by assessing: (a) the histology of the epididymis; (b) motility and viability of spermatozoa; and (c) scanning electron microscopic morphology of spermatozoa before and at the end of culture. The efficacy of the culture was evaluated by introducing the antiandrogen, cyproterone acetate, into the culture medium. Cyproterone acetate caused degenerative changes in the histology of the epididymis and coiling of the epididymal spermatozoa which may be due to alterations in epididymal milieu.

Evaluation of the ability of a new long-acting androgen ester to maintain accessory gland function in castrated rhesus monkey.

The ability of 40 mg of milled suspension of a new long-acting androgen ester (20 Aet-1) to restore and maintain accessory gland function was compared with that of testosterone enanthate (TE) in castrated adult rhesus monkeys. Castration did not abolish the ejaculatory response since only two animals did not void semen in the postcastration period. A single intramuscular injection of 40 mg of these compounds stimulated accessory gland function at levels lower than in the pretreatment period. 20 Aet-1-induced stimulation of prostatic acid phosphatase activity never exceeded control levels unlike that induced by testosterone enanthate which caused hyperstimulation on day 21 of drug treatment. In terms of support of accessory gland function, 20 Aet-1 would appear to offer hope of being a successful androgen for supplementation therapy in male animals.

Alterations in protein profile of rat spermatozoa during maturation.

Alterations in the polypeptide pattern of rat spermatozoa during epididymal transit were studied by SDS-PAGE and compared with that of epididymal cytosol and luminal fluid. The total number of cytosol and luminal fluid polypeptides increase from caput to cauda epididymidis but sperm associated polypeptides decrease during epididymal transit. Changes in polypeptide pattern of spermatozoa are due to their acquisition, loss or modification. Spermatozoa acquire seven polypeptides, of which six are acquired in corpus (MW 16.5, 38, 41, 72, 75 and 100 Kdal) and one (MW 28.5 Kdal) in cauda epididymidis. Spermatozoa lose one polypeptide of MW 72.5 Kdal in caput and two polypeptides of MW 70 and 115 Kdal in cauda epididymidis. Four polypeptides of MW 18.5, 19.5, 64 and 67.5 Kdal disappear from cauda spermatozoa without appearing in the luminal fluid. Polypeptide of MW 62.5 Kdal is observed only in spermatozoa and luminal fluid from cauda epididymidis.

Inhibition of spermatozoa maturation in rhesus monkey by cyproterone acetate.

The morphology of spermatozoa from the initial segment, caput, corpus and cauda epididymides of normal and cyproterone acetate-treated animals was studied using light and scanning electron microscopy to understand the changes taking place during spermatozoa maturation. A progressive and significant decrease in the percentage of spermatozoa that retained the cytoplasmic droplet and a shift in its position from proximal end of the midpiece to its distal end were seen during epididymal transit; these events were inhibited in cyproterone acetate-treated animals. A large percentage of spermatozoa from the initial segment and the caput epididymides showed coiling of the spermatozoa tail which involved the midpiece, principal piece and the endpiece. The percentage of spermatozoa that showed the coiled tail decreased; a gradual straightening of the spermatozoa tail with less complex types of coiling was also seen during epididymal transit. Cyproterone acetate reversed these changes occurring during maturation. These results indicate that spermatozoa maturation in the rhesus monkey, occurring between the corpus and cauda epididymides, is an androgen-dependent event.

Effect of two new androgen esters on serum levels of testosterone in castrated rhesus monkey.

The pharmacokinetics of two new androgen esters were tested in castrated male rhesus monkeys. A single injection of 40 mg of 20 Aet-2 (testosterone-trans-4-n-pentyl cyclohexyl carboxylate) increased serum testosterone to three times the castrate levels and was maintained nearly at this level until day 182. When given as four separate injections of 20 mg each, 20 Aet-2 increased serum testosterone within 24 hours and the peak levels were attained on day 2 (34.6 +/- 6.20 nmol/L) which was more than five times the levels obtained by 40 mg 20 Aet-2 given as a single injection. This was followed by a decrease in serum testosterone until day 98 when the levels were similar to that in animals given a single injection of 20 Aet-2. Administration of 40 mg of 3 Ad (testosterone-cis-3-(n-hexyl) cyclobutane carboxylate) given as a single injection increased serum testosterone levels to reach a peak level (47.4 +/- 4.64 nmol/L) on day 5 followed by a gradual decrease to castrate levels by day 70. The profile of serum testosterone in animals injected with 80 mg of 3 Ad as four separate injections was similar to that in animals given a single dose of 40 mg of 3 Ad. The data suggest a correlation between the bioavailability of the drug and the formulation of the vehicle. The possible effect of hydrolysis rate on pharmacokinetics of the drugs is discussed.

Histology of the epididymis in men with obstructive infertility.

The histology of different regions of human epididymis in men undergoing vasoepididymostomy to correct epididymal obstruction was studied. The data indicate major degenerative changes in intertubular connective tissue and in the epididymal epithelium. These include increase in connective tissue thickness and its infiltration by leucocytes in some cases, decrease in tubular diameter, degeneration and/or vacuolation of cytoplasm of nonciliated cells of efferent duct and principal cells of epididymis and presence of multinucleate giant cells in the epididymal lumen. These histological abnormalities are discussed in relation to the role such epididymis can play in sperm maturation following vasoepididymostomy.

Effect of non-aromatizable androgens on testicular and accessory gland functions in rhesus monkeys (Macaca mulatta).

Adult male rhesus monkeys were injected intramuscularly 100 micrograms, 1000 micrograms 5 alpha-androstane-3 alpha, 17 beta-diol or 100 micrograms dihydrotestosterone (DHT) per day for 70 days. A decrease in seminiferous tubular diameter was seen in treated animals. Androstanediol treatment disrupted spermatogenesis in most tubules. Sperm motility decreased within 40 days and by Day 70 non-motile spermatozoa were seen in 2 animals of each group treated with androstanediol. DHT treatment also decreased sperm motility progressively from Day 40. Both androgens caused retention of the cytoplasmic droplet and an increase in coiling of the tail of spermatozoa. Seminal fructose was decreased by Day 40 (1000 micrograms androstanediol) or Day 70 (100 micrograms androstanediol and 100 micrograms DHT). Seminal glycerophosphocholine (GPC) and acid phosphatase levels decreased by Day 70 in all treatment groups. Both steroids decreased circulating concentrations of testosterone without altering FSH or oestradiol values.

Effect of dihydrotestosterone on ultrastructural changes in rhesus monkey spermatozoa.

Dihydrotestosterone (DHT) was given at 10, 100 or 1000 micrograms per day for 70 days to adult male rhesus monkeys. Spermatozoa, collected by electroejaculation on days 21, 41 and 71 of treatment, were processed for spermiogram and Transmission Electron Microscopy. DHT, at all doses increased the number of spermatozoa showing coiled tails and the degree of coiling as well as ultrastructural changes. In the 10 micrograms group, on day 20 of treatment coiling of sperm tail was seen while 100 micrograms DHT induced additional changes like displacement of midpiece, loosening of plasma membrane over head region and increase in electron density of acrosomal region. Similar changes were seen only on day 40 in animals treated with 100 micrograms DHT. By day 40 - 70 of treatment, spermiophagy by macrophages was seen in all groups.

Pharmacokinetics and pharmacodynamics of a new long-acting androgen ester: maintenance of physiological androgen levels for 4 months after a single injection.

The pharmacokinetics and pharmacodynamics of testosterone trans-4-n-butylcyclohexyl carboxylate (Code name: 20 Aet-1), a new long-acting androgen ester, were evaluated in castrated adult rhesus monkeys and compared with those of testosterone enanthate (TE). A single intramuscular injection of 40 mg of 20 Aet-1 returned serum testosterone (T) to within or close to the diurnal physiological range for 80-136 days. In contrast, a similar dose of TE increased serum T to supraphysiological levels and the response evoked was of short duration. The ratio of T to dihydrotestosterone (T/DHT) ratio in monkeys treated with 20 Aet-1 was comparable to that found in control animals while in TE-treated animals, it was highly elevated. Serum estradiol (E2) elevation by 20 Aet-1 was also of smaller magnitude compared to TE. 20-Aet-1 suppressed LH levels from day 5 until day 115. The levels of LH on day 115 were 45.8% lower compared to the levels on day 13 post-castration. TE suppressed LH levels from day 1-7 post-injection. The values on day 7 were 76.6% lower compared to values on day 13 post-castration. Thus, TE-induced suppression of LH was of shorter duration, but of greater magnitude compared to the effect caused by 20 Aet-1. Similarly, FSH was suppressed for a longer duration (days 21-74) by 20 Aet-1 than by TE. The results indicate that the new testosterone ester has highly favourable pharmacokinetic properties and may prove to be the androgen of choice for supplementation therapy in contraceptive regimens.

Effect of dihydrotestosterone on testicular and accessory gland function in male rhesus monkeys.

Pituitary, testicular and accessory gland functions were assessed at intervals of 10-20 days in adult male rhesus monkeys given 10, 100 or 1000 micrograms dihydrotestosterone (DHT). Circulating levels of LH and testosterone were suppressed. Ejaculated spermatozoa showed morphological abnormalities and decrease in motility but sperm counts were unaffected. Seminal fructose was decreased in animals receiving DHT. Sexual behaviour was maintained in these animals.