Influence of rat seminiferous tubules on Leydig cell testosterone production in vitro

The interaction of seminiferous tubules and Leydig cells was investigated in the rat in vitro. Crude collagenase-dispersed, or Percoll-purified, Leydig cells were incubated together with seminiferous tubules at different stages of the cycle, isolated by transillumination-assisted microdissection. The testosterone production was measured. Seminiferous tubules inhibited testosterone production of crude Leydig cell preparations, but induced a clear stimulation (30–100%) in Percoll-purified cells. The stimulation was maximal at stages VII and VIII of the cycle, significantly higher than at stages II–VI (P < 0.05). The stimulation by seminiferous tubules was observed both in basal and hCG-stimulated testosterone production. The effect was independent of FSH or GnRH action. These results demonstrate the presence of paracrine regulatory interaction between seminiferous tubules and Leydig cells, and are in agreement with the concept of a perferential androgen requirement of stages VII and VIII of the cycle.     

Stage-specific inhibition of interstitial cell testosterone secretion by rat seminiferous tubules in vitro

The stage-specific influence of the secretions from rat seminiferous tubules on the LH-stimulated testosterone production by rat Leydig cells in vitro was studied. The spent media from incubated seminiferous tubules (SMST) from stages VII-VIII of the seminiferous epithelial cycle caused about 50% inhibition of the LH-dependent testosterone production by a crude preparation of rat interstitial cells. The SMST from other stages had no effect on testosterone production. Mixed tubules of unidentified stages gave an intermediate response. When SMST from ten different stages of the seminiferous wave were compared, the most pronounced inhibitory activity was found in stages VI and VIII–XI, while SMST from stages I, VII and XIII–XIV had no inhibitory effects on interstitial cell testosterone production. No stimulation was found in this system. Prolonged incubation of the interstitial cells with SMST from stages VIII–XI resulted in loss of inhibitory activity after 12 h of incubation. Maximum inhibitory activity was noted after 3 h of incubation.

The inhibitory activity of the SMST from stages VIII–XI was retained after prolonged dialysis, and was unchanged after heating the medium at 60°C for 1 h. The activity did not seem to be due to the presence of proteolytic enzymes, since it was not influenced by addition of protease inhibitors. SMST from stages VIII–XI had no effect on the metabolism of [³H]testosterone added to the interstitial cell preparations. No inhibitory effect was observed when Leydig cells were incubated with dibutyryl cAMP instead of LH, suggesting an early influence on the LH-receptor-adenylate cyclase chain of events. A gradual increase in the inhibitory action of SMST from stages VIII–XI was noted with increasing age of the animals. When Percoll-purified Leydig cells were used, no inhibitory activity was observed with any of the media tested. In contrast, stimulation of Leydig cell testosterone production resulted with all kinds of SMST tested. The different results with crude interstitial cells and purified Leydig cells may indicate either that other cells (macrophages?) are involved in the inhibitory activity on the crude cells, or that purified cells represent only a subpopulation of the Leydig cells.     

Characterization of a nuclear receptor for testosterone in seminiferous tubules of mature rat testes.

A specific androgen receptor could be demonstrated in the nuclear and cytoplasmic fractions of testicular tissue of mature hypophysectomized rats, either in vivo after injection of testosterone or in vitro after incubation of testis tissue with testosterone. Using agar-gel electrophoresis this receptor could be distinguished from the testicular transport-like protein for androgens (androgen binding protein = ABP). After in vivo administration of testosterone the steroid bound to the receptor in mature rat testis was mainly unmetabolized testosterone. After dissection of testis tissue the larger part of the receptor was shown to be present in the seminiferous tubules. The amount of exogenous testosterone that could be bound per mg of protein in the nuclear extract increased gradually during 20 days after hypophysectomy. Some characteristics of the receptor in the nuclear extract and of ABP were compared : the receptor was more sensitive to temperature increases than ABP; the steroid dissociated more slowly from the receptor than from ABP; cyproterone acetate showed almost no effect on the binding of dihydrotestosterone to ABP, but did compete for the receptor binding sites in the nuclear extract.     

The restricted penetration of iodinated rat FSH and LH into the seminiferous tubules of the rat testis.

The penetration of 125I-iodinated rat follicle-stimulating hormone (FSH; labelled by three different techniques) and luteinizing hormone (LH) through the walls of the seminiferous tubules of the rat testis has been studied by injecting the labelled hormone into rats with the efferent ducts of one testis ligated 16 h before the collection of samples of blood and tissues. The concentration of trichloracetic acid-precipitable and immunoprecipitable radioactivity was measured in blood plasma and rete testis fluid and calculated for the total secreted fluid retained in the testis by the ligature, and for the additional tubular fluid from the ligated testis, separated by centrifugation after decapsulating the testis and dispersing the cells. Very little intact hormone penetrated into the testicular fluids, even 16 h after injection of the labelled hormone, and the volume of distribution in the unligated testis of the trichloracetic acid-precipitable radioactivity was only slightly greater than that for markers known to be confined to the extracellular interstitial fluid. This suggests that the labelled hormones do not penetrate readily through the walls of the semiferous tubules into their lumina. Injected inorganic iodiide and trichloracetic acid-soluble 125I-circulating after the injection of iodinated hormones penetrated more rapidly into the tubules, but had not reached equilibrium between the testicular fluids and blood plasma 16 h after injection. Labelled FSH was reasonably stable in the circulation after injection, but 80% of the 125I was not protein-bound 16 h after injection of labelled LH.     

Retinol-induced stage synchronization in seminiferous tubules of the rat

Vitamin A deficiency (VAD) in rats causes a progressive germ cell depletion and cessation of spermatogenesis resulting in seminiferous tubules which contain only Sertoli cells, spermatogonia and a small number of preleptotene spermatocytes. Spermatogenesis can be rapidly restored by the administration of retinol. Our preliminary studies suggested a partial stage synchronization of many seminiferous tubules in VAD male rats subsequently treated with retinol. To confirm this observation and to achieve a better synchronization, VAD rats received 2 SC injections of retinol suspended in sesame oil followed by daily oral administrations of 0.5 mg of retinol. In all rats an almost perfect synchronous stage development of seminiferous tubules evolved in a predictable manner and was maintained through 2 spermiations.     

Stage-dependent secretion of ABP by rat seminiferous tubules

The secretion rate of the Sertoli-cell-specific androgen-binding protein (ABP), has been studied in isolated rate seminiferous tubules, where the stages of the spermatogenic cycle have been identified by a transillumination method. The secretion of ABP was highest in stages VII-XII, as determined by steady-state polyacrylamide-gel electrophoresis as well as by radioimmunoassay. More specifically, when the sensitive RIA technique permitted the assay of ABP secretion from a total amount of 10-30 mm of isolated tubules, it was found that maximal ABP secretion occurred at stages VIII-XI and minimal at stages IV-V. The present results show that the secretory activity of the Sertoli cells (ABP) is influenced by the type of germ cell at each cell association. It is postulated that the variation is Sertoli-cell secretory activity is of importance for the normal maintenance of spermatogenesis.     

Inhibin from cultures of rat seminiferous tubules.

Medium from cultures of mature rat seminiferous tubules contained a substance which suppressed, in a dose-related manner, the luteinizing hormone releasing hormone (LH-RH)-stimulated secretion of FSH by cultured rat pituitary cells. The secretion of LH was suppressed to a lesser extent and the basal secretion of both LH and FSH was inconsistently affected. Gel filtration on Sephadex G-100 did not fractionate the activity. The active material did not inhibit the secretion of TSH or destroy LH-RH and the activity was not due to testosterone or oestradiol in the medium. Control media from liver cultures were inactive. It is concluded that inhibin is present in media from cultures of rat seminiferous tubules.     

Mechanism of action of the factor(s) secreted by rat seminiferous tubules and inhibiting interstitial cell testosterone production in vitro

Rat seminiferous tubules secrete a factor which inhibits LH-dependent steroidogenesis by interstitial cells. The inhibitory activity was found to be specific for the testes, as cytosols from other rat tissues such as the kidney, heart, spleen, liver and epididymis had no significant effect on testosterone production by interstitial cells. Preliminary characterization by Ultrogel AcA 44 gel chromatography demonstrated that the active substance in SMST has a molecular weight between 40-50 kD. Spent medium from incubation of seminiferous tubules (SMST) caused a dose-dependent inhibition of LH-or cholera toxin-stimulated in vitro testosterone production by rat interstitial cells. However, SMST failed to inhibit forskolin-stimulated steroidogenesis. The effect of SMST was not altered by pre-incubating the cells with the sulfhydryl reagent, N-ethylmaleimide. Considering the proposed mode of action of these modulators of adenylate cyclase activity, the present studies suggest that a high molecular weight testis specific factor acts through the guanine nucleotide-binding stimulatory regulatory protein of the adenylate cyclase complex to inhibit LH-dependent testosterone production by Leydig cells.     

Testosterone metabolism in isolated cells of rat and ram seminiferous tubules]

Aspermatogenic seminiferous tubules were obtained from adult Wistar rats treated with Busulfan at the 20th day of foetel life. The isolated tubules converted 14C testosterone (T) into androstenedione (delta4), 3,5%) 5 alpha-dihydrotestosterone (DHT), (1%) ANd 5 alpha-androstane-3 beta, 17 beta-diol at unit gravity and incubated with 14C-T. Only delta was produced (2%). Using a similar technique the same result was obtained when pure preparation of round spermatids and primary spermatocytes from ram testis were incubated with 14C-T. From these experiments, we conclude that 5 alpha-reductase activity is present only in Sertoli cells.     

Aminopeptidase-B in the rat testes: isolation, functional properties and cellular localization in the seminiferous tubules

An aminopeptidase of the B-type, with an apparent M_r 72 000 and pI = 4.9, was isolated from rat testes and characterized. The enzyme was able to remove only Arg and/or Lys residues from -amino acid β-naphthylamide derivatives and from the N-terminus of several peptides. No cleavage occurred in the case of Arg-Pro bonds as found in bradykinin and substance P. The enzyme was sensitive to cysteinyl reagents and to aminopeptidase inhibitors, such as bestatin, amastatin and arphamenines A and B. The aminopeptidase activity, tested with -Arg β-naphthylamide and with Arg⁰-Met-enkephalin as substrates, was inhibited by o-phenanthroline, and restored by Zn²⁺ suggesting its metallopeptidase character. The partial characterization of an aminopeptidase-B activity in rat brain cortex identified a protein which is biochemically and immunologically related to the testis enzyme. By immunohistochemistry, the aminopeptidase-B was found to be particularly abundant in the seminiferous tubules at late stages of spermatogenesis and was clearly detected in a restricted area of elongated spermatids. Remarkably, the enzyme was observed to concentrate massively in the residual bodies. Since this aminopeptidase-B was able in vitro to trim out N-terminal Arg and/or Lys residues from peptides mimicking processing intermediates, it is proposed that this enzyme may be involved in propeptide and proprotein processing mechanisms in the course of spermatid differentiation.     

Observations on the binding of androgens by rat testis seminiferous tubules and testis extracts

Isolated seminiferous tubules from recently hypophysectomized adult rats rapidly bound ³H-testosterone (T). Analysis of Scatchard plots of the data indicated the existence of at least two androgen binding proteins (ABP), one of which had a high affinity for T and dihydrotestosterone (DHT). The binding capacity of the high affinity ABP was greatly decreased in tubules from regressed hypophysectomized rats, whereas that of the low affinity ABP was increased. Extracts were prepared from homogenates of seminiferous tubules or from whole testes of normal, cryptorchid and hypophysectomized rats, and endogenous androgens were removed from the high speed supernatant fractions by adsorption onto dextran-coated charcoal. Assays for the high affinity ABP (K_d of approximately 10^?9 M) indicated relatively high binding capacity in testis extracts from normal or cryptorchid testes, but very low capacities in testis extracts from regressed hypophysectomized rats. In vivo treatment of hypophysectomized animals with follicle stimulating hormone (FSH) increased ABP capacities in testis extracts within 24 h after hormone administration, whereas treatment with luteinizing hormone was less effective. The ABP was shown to be specific for T and DHT. After ligation of the efferent ducts from the testis, ABP of high binding capacity was observed in extracellular extracts. The data are discussed in relation to the hypothesis that Sertoli cells produce ABP and secrete this protein into tubular fluid under the regulation of FSH. A large portion of the high affinity ABP found in testicular extracts is derived from that present in tubular fluid. The remainder is from the cells of the seminiferous tubules, and little or none is in interstitial cell preparations.     

Characterization of the androgen receptor in nuclei of seminiferous tubules of the mature male rat based upon radioimmunoassay of endogenous levels of bound testosterone.

A macromolecular testosterone complex which was salt-extracted from nuclei isolated from seminiferous tubules of the adult male rat was investigated by quantifying the testosterone directly by RIA. The steroid-binding capacity of the complex was destroyed by heating at both 12 and 50 C, suggesting the presence of an extremely thermolabile complex. This tubular complex was shown to be similar to the radioactively labeled androgen receptor isolated from ventral prostatic nuclei by their coelution during adsorption chromatography, by their coleution during ion exchange chromatography, their similar migration rate (4-4.46S) on both sucrose and glycerol density gradients, and their similar slow dissociation at 0 C. These observations indicate that the tubular complex contains a high affinity androgen receptor and suggest that the direct measurement of testosterone in this complex by RIA may be useful in identifying its cellular distribution.     

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. However, after continuous stimulation with EGF for periods in excess of 5 days, an increase in inhibin secretion was observed. In the presence of 10 and 100 ng FSH/ml, EGF (10 ng/ml) produced a further increment in the inhibin content of the tubules, but this effect was not found with FSH concentrations of 500 or 1000 ng/ml. EGF also increased the tubule content of inhibin after the addition of 100 micrograms dibutyryl cyclic AMP/ml but no effect of EGF was observed on the FSH- or dibutyryl cyclic AMP-induced secretion of inhibin into the medium. The effect of EGF on inhibin content in the tubules was partially suppressed by the addition of 4 beta-phorbol-12 beta-myristate-13 alpha-acetate (20 ng/ml). Insulin (1-100 ng/ml) decreased basal inhibin secretion without changing the inhibin content of tubules and this effect was antagonized by EGF (10 ng/ml) with insulin doses of 1-50 ng/ml whereas, at 100 ng/ml, the effect of EGF on tubule inhibin content was reversed. The addition of EDTA (2 mmol/l) resulted in an inhibition of basal and EGF-induced inhibin production. These data demonstrate a stimulatory effect of EGF on inhibin production by isolated seminiferous tubules which is inhibited by insulin and phorbol esters, both stimulators of protein kinase C activity.