The cDNA structure and expression analysis of the genes for the cysteine proteinase inhibitor cystatin C and for beta 2-microglobulin in rat brain.

Tissue patterns of gene expression were analyzed by measuring mRNA levels and incorporation of radioactive amino acids for cystatin C and beta 2-microglobulin, the two extracellular proteins in the brain with the highest ratio of concentration in cerebrospinal fluid over that in blood plasma. The primary structure of rat cystatin C mRNA from choroid plexus was determined by nucleotide sequencing of cloned cDNA and the tissue patterns of gene expression were analysed by RNA blot analysis and in situ hybridization. Cystatin C was found to be composed of 120 amino acids and to contain a potential site for N-linked glycosylation.

Follitropin (FSH) stimulation of inhibin biological and immunological activities by seminiferous tubules and Sertoli cell cultures from immature rats.

Seminiferous tubules from 20-day-old rats were isolated by mechanical dissection and the conditions to produce optimal inhibin secretion over a 5-day period of culture were established. Inhibin was measured by a specific heterologous radioimmunoassay and by an in vitro bioassay using rat pituitary cells in culture. The tubule production of biologically and immunologically active inhibin was stimulated by ovine follitropin (FSH); however, the ratio of biological to immunological (B:I) activity fell significantly with increasing dose.

Epidermal growth factor increases inhibin synthesis by isolated segments of rat seminiferous tubules.

The effect of epidermal growth factor (EGF) on the production of immunoreactive inhibin by adult rat isolated seminiferous tubules in vitro has been investigated. EGF (0.1-1000 ng/ml) added to cultures of seminiferous tubules from adult rats caused a dose-dependent increase in inhibin content in the tubules without changing the amount secreted into the media.

Effect of testosterone on serum immunoactive inhibin concentrations in intact and hypophysectomized male rats.

Intact and hypophysectomized rats were treated with graded doses of testosterone via subcutaneous Silastic implants over a 13-week period. Serum inhibin concentrations fell 50% (P less than 0.001) after 2 weeks of hypophysectomy, remaining suppressed at this level for 13 weeks.

Immuno- and bioactive inhibin and inhibin alpha-subunit expression in rat Leydig cell cultures.

The rise in serum immunoactive inhibin levels in male rats following hCG stimulation raised the possibility that Leydig cells may produce inhibin. This study therefore evaluated whether Percoll-purified Leydig cells from adult male rats synthesized and secreted inhibin in vitro as measured by Northern blot analysis, radioimmunoassay and in vitro bioassay. Northern blot analysis demonstrated the presence of alpha-inhibin subunit mRNA in the Leydig cell and inhibin bioactivity was detected in Leydig cell culture media.

Inhibin secretion is influenced by Leydig cells: evidence from studies using the cytotoxin ethane dimethane sulphonate (EDS).

Adult male rats given a single intraperitoneal injection of the Leydig cell cytotoxin ethane dimethane sulphonate (EDS) show a significant decrease in testosterone from 7 to 14 days, and elevation of serum FSH and LH levels commencing 7 days after treatment, returning to normal at 28 days for LH and 49 days for FSH. A significant rise in serum inhibin levels was seen at day 14 after EDS treatment with levels returning to normal at day 49. In a second series of experiments, silastic implants of testosterone, either 2.

Effect of ethane dimethane sulphonate on proopiomelanocortin (POMC) mRNA and POMC-derived peptides in the rat testis.

Recent studies have shown that both proopiomelanocortin (POMC)-derived peptides and a range of POMC gene transcripts are present in the testis. Previous immunocytochemical studies have reported immunoreactive (ir)-beta-endorphin (EP) and ir-ACTH to be localized in the Leydig cells, and ir-NacEP in spermatogonia and primary spermatocytes. In the present study, we have further examined the hypothesis that testicular Leydig cells are the principal site of synthesis of these peptides, by determining the effects of the administration of the cytotoxic drug ethane dimethane sulphonate (EDS) which selectively destroys the Leydig cells of the testis.

The involvement of Leydig cells in the regulation of inhibin secretion by the testis.

The stimulation of Leydig cells by the administration of a single injection of 100 IU human CG (hCG) to adult male rats caused a significant biphasic stimulation of serum testosterone levels at 2 h and 3 days after injection. Serum immunoreactive (IR)-inhibin levels were elevated by 6 h and peaked at 24 h after hCG, then progressively declined thereafter. The removal of Leydig cells in vivo by the injection of the cytotoxic drug ethane dimethane sulfonate (EDS) causes a significant decrease in serum testosterone levels within 4 days, which is sustained 1 and 2 weeks after EDS.

In vivo and in vitro production of inhibin by cryptorchid testes from adult rats.

Adult male rats were made bilaterally cryptorchid for 3, 7, 10, and 14 days, and the peripheral serum, testicular vein serum, and interstitial fluid levels of inhibin were measured by RIA, and compared with values obtained in intact animals. The levels of inhibin in peripheral serum, testicular vein, and interstitial fluid were significantly elevated (P less than 0.01) after 3 days of cryptorchidism but declined significantly thereafter.

Differential effects of the destruction of Leydig cells by administration of ethane dimethane sulphonate to postnatal rats.

Ethane dimethane sulphonate (EDS) is a cytotoxic drug that selectively destroys Leydig cells in adult testes. This study has examined the effect of a single injection of EDS on the Leydig cell populations present in the testes of rats aged 5, 10, or 20 days. Microscopic examination of the tissue demonstrated that the fetal Leydig cell population was destroyed at all ages, but that subsequent development of the adult population of Leydig cells was not affected.

The effect of insulin on inhibin production in isolated seminiferous tubule segments from adult rats cultured in vitro.

The effect of insulin and its interaction with intracellular messenger systems on in vitro inhibin production by adult rat isolated seminiferous tubules has been investigated using a recently developed inhibin radioimmunoassay (RIA). Seminiferous tubule segments (5 cm) from intact adult rats were exposed to insulin (0.05-5000 ng/ml) for 2 days of culture.

Stage-specific inhibin secretion by rat seminiferous tubules.

The endogenous inhibin content was measured by radioimmunoassay in segments of seminiferous tubules at specific stages of the spermatogenic cycle isolated by transillumination-assisted dissection as described by Parvinen (1982). In addition, the immunoactive inhibin production in response to FSH stimulation was investigated. The inhibin content was greatest in tubules at stages II-VI of the cycle and was lowest at stages VII-VIII.

Inhibin and activin regulate [3H]thymidine uptake by rat thymocytes and 3T3 cells in vitro.

[3H]Thymidine incorporation by adult rat thymocytes, in the presence of phytohaemagglutinin (PHA), was stimulated by bovine inhibin (ED50 0.7 nM), and inhibited by bovine activin (ID50 0.4 nM) and porcine transforming growth factor-beta (TGF-beta) (ID50 4 pM); inhibin opposed the actions of activin and TGF-beta.

In vitro synthesis and release of inhibin in response to FSH stimulation by isolated segments of seminiferous tubules from normal adult male rats.

The production of inhibin by isolated segments of seminiferous tubules from adult male rats cultured in vitro was investigated using a heterologous specific radioimmunoassay. Increasing lengths of tubules (5, 10, 20 and 40 cm) maintained in culture for 4 or 5 days produced increasing amounts of inhibin in vitro. A dose-dependent increase in inhibin production was observed after stimulation with ovine follicle-stimulating hormone (FSH)-s17 (0.

Effect of unilateral cryptorchidism on the intertubular tissue of the adult rat testis: evidence for intracellular changes within the Leydig cells.

Adult male rats were made unilaterally cryptorchid for 1, 2 or 4 weeks, and the morphological response of the Leydig cells was then studied using morphometric assessment of total Leydig cell volume and number per testis in abdominal and scrotal testes. Serum hormone levels were measured and the steroidogenic properties of isolated Leydig cells were evaluated by in-vitro stimulation with hCG and interstitial fluid (IF) obtained from normal rat testes. Total Leydig cell volume and number per testis were not altered in abdominal vs scrotal testes, although the volume of the abdominal testis was 46, 29 and 21%, respectively, of the volume of the contralateral scrotal testis after 1, 2 and 4 weeks.

Alterations in mitogenic and steroidogenic activities in rat testicular interstitial fluid after administration of ethane dimethane sulphonate.

A single dose of EDS was given to mature male rats and interstitial fluid (IF) was collected to determine the potency of mitogenic and steroidogenic activities therein. The potency of the factor stimulating testosterone secretion in vitro by Percoll-purified Leydig cells was significantly elevated 2 weeks after EDS, whilst the potency of mitogenic activities (stimulation of DNA synthesis by BALB/c 3T3 cells) was not elevated until 4 weeks after EDS treatment. This study suggests that two separate factors, one with mitogenic and the other steroidogenic activity, may be involved in the response of Leydig cells after EDS administration.

Stimulation of interstitial cell growth after selective destruction of foetal Leydig cells in the testis of postnatal rats.

Five-day-old male rats received a single treatment of ethane dimethanesulphonate (EDS), and the response of the testis on days 6-10 and 21 was examined by light microscopy and morphometry, supplemented by measurement of peripheral testosterone levels. One day after treatment, foetal Leydig cells degenerated, showing fragmentation, condensation and nuclear pyknosis. Macrophages phagocytosed the foetal leydig cells resulting in their disappearance by day 7.

Effect of cryptorchidism on steroidogenic and mitogenic activities in rat testicular interstitial fluid.

There was a significant (P less than 0.05) and consistent increase in the potency of steroidogenic stimulatory activity (testosterone production by purified Leydig cells in vitro) in testicular interstitial fluid of the cryptorchid compared to the scrotal testis from 1 to 4 weeks after the induction of unilateral cryptorchidism. In contrast, the level of mitogenic activity [( 3H]thymidine incorporation into 3T3 cells) was not significantly different between interstitial fluid from cryptorchid and scrotal testes for up to 4 weeks after surgery.

Variation in the effect of a nongonadotropic Leydig cell stimulating factor in testicular interstitial fluid after exposure of the testis to a single episode of heat treatment.

The activity of a nongonadotropic factor present in rat testicular interstitial fluid and capable of stimulating Leydig cell testosterone production was measured at intervals for 8 weeks after exposure of the testis to a single episode of heat treatment (43 C for 15 minutes). The activity of this factor was determined using a Leydig cell bioassay and the testicular albumin space (a measure of the interstitial fluid volume) was determined after the injection of 125I-labeled bovine serum albumin. Additionally, the response of interstitial cells to hCG stimulation was measured at each time point after heat treatment.

Morphometric analysis of the components of the neonatal and the adult rat testis interstitium.

Leydig cells in the foetal rat testis are still present at birth and it has been hypothesized that they commence to degenerate immediately after birth, based on the decrease in their volume density (v/v%) with age. In this study the interstitium of the rat testis was studied quantitatively at 1, 5, 10, 15, 20 and 90 days after birth: the latter are considered to be adults. The absolute volumes of connective tissue cells and blood vessels increased with age.

Intragonadal control mechanisms.

On the weight of the evidence presented above, it is concluded that regulation at a local, intragonadal level is an integral part of the overall regulation of gonadal function in both sexes. The interaction between cells within a gonad extends beyond the same cell type to include germ cell-somatic cell interactions as well. We believe this local interaction between cell types facilitates the differing requirements of the various developmental stages of germ cells within the gonad, which would not be possible by simply varying the afferent pituitary hormone supply.

Influence of the cryptorchid testis on the regeneration of rat Leydig cells after administration of ethane dimethane sulphonate.

Leydig cells were selectively eliminated from the testis by treatment with the cytotoxin, ethane dimethane sulphonate. Rats were then made unilaterally cryptorchid and the morphological and functional response of the interstitial tissue in abdominal compared with scrotal testes was examined for up to 8 weeks. Regeneration of new Leydig cells occurred more rapidly in the interstitial tissue of abdominally cryptorchid testes compared with the interstitial tissue of contralateral scrotal testes.

The interaction of hCG, hydroxysteroids and interstitial fluid on rat Leydig cell steroidogenesis in vitro.

Rat testicular interstitial fluid and hydroxycholesterol both stimulated testosterone production by isolated Leydig cells in vitro in a dose-dependent manner, but the dose-response lines were not parallel. The addition of cycloheximide blocked the stimulation by interstitial fluid but not that of hydroxycholesterol. Use of the compounds SU 10603 and cyanoketone (which inhibit 3 beta-hydroxysteroid dehydrogenase and 17 alpha-hydroxylase respectively) or aminoglutethimide (which acts on the cholesterol side-chain cleavage enzyme) showed that the stimulatory factor(s) in interstitial fluid stimulated steroidogenesis at the cholesterol side-chain cleavage enzyme, before the conversion of pregnenolone.

Percoll-gradient separation of Leydig cells from postnatal rat testes.

The characteristics of the fetal and adult populations of Leydig cells from postnatal rat testes were compared by Percoll gradient centrifugation. A single peak of hCG binding, due to the presence of fetal Leydig cells, was obtained after purification of intertubular cells from 8-day-old animals. Two peaks of specific hCG binding were obtained after purification of intertubular cells from 15-day-old rats: it was confirmed by autoradiographic techniques that the hCG was bound by adult-like Leydig cells in one peak and fetal Leydig cells in the other.

Gestational changes in prostaglandin production by ovine fetal trophoblast cells.

The effect of gestational age on the synthesis of prostaglandins (PG) by isolated preparations of uninucleate and binucleate cells of the ovine placentome has been investigated. PG synthesis by the cells was dependent upon cell number in a linear manner, and was significantly inhibited by indomethacin, but not affected by the addition of exogenous arachidonic acid. The net output of PG by the cells increased progressively with increasing gestational age of the ewe from 35 to 145 days, particularly after 100 days' gestation.