Luteinizing hormone receptor-mediated effects on initiation of spermatogenesis in gonadotropin-deficient (hpg) mice are replicated by testosterone

Testosterone (T) is an absolute requirement for spermatogenesis and is supplied by mature Leydig cells stimulated by LH. We previously showed in gonadotropin-deficient hpg mice that T alone initiates qualitatively complete spermatogenesis bypassing LH-dependent Leydig cell maturation and steroidogenesis. However, because maximal T effects do not restore testis weight or germ cell number to wild-type control levels, additional Leydig cell factors may be involved. We therefore examined 1). whether chronic hCG administration to restore Leydig cell maturation and steroidogenesis can restore quantitatively normal spermatogenesis and testis development and 2). whether nonandrogenic Leydig cell products are required to initiate spermatogenesis. Weanling hpg mice were administered hCG (0.1-100 IU i.p. injection three times weekly) or T (1-cm subdermal Silastic implant) for 6 weeks, after which stereological estimates of germinal cell populations, serum and testicular T content, and testis weight were evaluated. Human CG stimulated Leydig cell maturation and normalized testicular T content compared with T treatment where Leydig cells remained immature and inactive. The maximal hCG-induced increases in testis weight and serum T concentrations were similar to those for T treatment and produced complete spermatogenesis characterized by mature, basally located Sertoli cells (SCs) with tripartite nucleoli, condensed haploid sperm, and lumen development. Compared with T treatment, hCG increased spermatogonial numbers, but both hCG and T had similar effects on numbers of spermatocytes and round and elongated spermatids per testis as well as per SC. Nevertheless, testis weight and germ cell numbers per testis and per SC remained well below phenotypically normal controls, confirming the involvement of non-Leydig cell factors such as FSH for quantitative normalization of spermatogenesis. We conclude that hCG stimulation of Leydig cell maturation and steroidogenesis is not required, and that T alone mostly replicates the effects of hCG, to initiate spermatogenesis. Because T is both necessary and sufficient for initiation of spermatogenesis, it is likely that T is the main Leydig cell secretory product involved and that additional LH-dependent Leydig cell factors are not essential for induction of murine spermatogenesis.     

Effects and interactions of LH and LHRH agonist on testicular morphology and function in hypophysectomized rats

The effects of single or combined daily treatment with an LHRH agonist and low or high doses of LH upon the testes of adult hypophysectomized rats were studied for up to 2 weeks in which changes in testicular histology, particularly the interstitial tissue, were examined by morphometry and related to functional assessment of the Leydig cells in vivo and in vitro. Compared to saline-treated controls, LHRH agonist treatment did not alter testis volume or the composition of the seminiferous epithelium or any of the interstitial tissue components although serum testosterone and in-vitro testosterone production by isolated Leydig cells were significantly reduced. With 2 micrograms LH for treatment, testis volume was increased, spermatogenesis was qualitatively normal, total Leydig cell volume was increased, serum testosterone values were initially elevated but subsequently declined and in-vitro testosterone production was enhanced. Testis volume with 20 micrograms LH treatment was unchanged compared to saline treatment, the seminiferous epithelium exhibited severe disruption but total Leydig cell volume was greatly increased due to interstitial cell hyperplasia. This group showed elevated serum testosterone concentrations and major increases in testosterone production in vitro. Treatment with LHRH agonist with either dose of LH resulted in reduced testis volume, moderate to very severe focal spermatogenic disruption and increased total Leydig cell volume although serum testosterone values and in-vitro testosterone production were markedly reduced compared to control rats. It is concluded that, in the absence of the pituitary, LHRH agonist fails to disrupt spermatogenesis and the previously described antitesticular action of LHRH agonists in intact rats is therefore dependent upon the presence of LH, which alone or in combination with LHRH agonist, may focally disrupt spermatogenesis in hypophysectomized rats whereas the Leydig cells undergo hyperplasia. The findings show that impairment of spermatogenesis is accompanied by alterations of the interstitial tissue and suggest that communication between these two compartments is involved in the regulation of testicular function.     

Temporal changes in rat Leydig cell function after the induction of bilateral cryptorchidism

Adult rats were made bilaterally cryptorchid and studied at intervals of 3, 7, 14 or 21 days to study temporal changes in Leydig cell function. Serum FSH and LH levels were measured and the cross-sectional area of the Leydig cells assessed by morphometry. The function of the Leydig cells was judged by the binding of 125I-labelled hCG to testicular tissue in vitro and the testosterone response of the testis to hCG stimulation in vitro. By 3 days after cryptorchidism, the binding of labelled hCG to testicular tissue was significantly decreased compared to that of controls, but the testes were able to respond to hCG stimulation in vitro. At 7, 14 and 21 days after cryptorchidism, an enhanced testosterone response was observed and the size of the Leydig cells was significantly greater than that of the controls, which indicated increased secretory activity by the cryptorchid testis. Although serum FSH levels were significantly elevated after 3 days of cryptorchidism, serum LH levels did not rise until 7 days, thereby suggesting that the loss of receptors is unlikely to result from down-regulation by LH. The reduced testosterone response of the cryptorchid testis in vivo to low doses of hCG and the enhanced response at high doses are probably related to the reduced blood flow to the cryptorchid testis and the decreased sensitivity of the Leydig cells induced by LH/hCG receptor loss.     

Leydig cell number and function in the adult cynomolgus monkey (Macaca fascicularis) is increased by daily hCG treatment but not by daily FSH treatment

Daily treatment of adult cynomolgus monkeys with 450 i.u. hCG for 16 days resulted in a significant 163% increase in the number of Leydig cells, and a 9-fold rise in plasma testosterone concentrations. The number of proliferating Leydig cells was very low, even after 16 days of treatment with hCG. Daily FSH administration (2 injections of 15 i.u. per day) did not have any effect on the number of Leydig cells or plasma testosterone values. It can be concluded, therefore, that in adult cynomolgus monkeys daily hCG treatment results in an increase in the number of Leydig cells, which is mainly caused by the differentiation of precursor cells. Since plasma testosterone concentrations were increased to an even higher extent, the steroid production per Leydig cell was also stimulated.     

Testicular blood flow and microcirculation in rats after treatment with ethane dimethyl sulfonate

The effects of ethane dimethyl sulfonate (EDS) on total testicular blood flow, microcirculation, and the testicular interstitial fluid volume (IFV) in rats were studied. In agreement with previous studies, treatment of control rats with human chorionic gonadotropin (hCG) induced an increase in IFV and total testicular blood flow as measured with radioactive microspheres. These effects of hCG were completely abolished in rats pretreated with EDS; in EDS-treated rats not receiving any hCG, there were decreases in IFV when compared with untreated control rats. Furthermore, the pulsatile pattern of testicular microcirculation registered with laser-Doppler flowmetry was abolished after EDS treatment, and this effect was not influenced by hCG treatment. The hCG-induced increase in IFV is associated with an increased accumulation of polymorphonuclear leukocytes locally in the testis, but this accumulation of leukocytes was not observed in rats pretreated with EDS. It was concluded from the present study that hCG-induced changes in total testicular blood flow and testicular microcirculation require functionally intact Leydig cells.     

Histophysiological changes of the testicular tissue due to busulphan administration in the wild Indian house rat (Rattus rattus)

The results of the present study indicate the antispermatogenic activity of Busulphan or Myleran (1,4-dimethane-sulphonoxy butane) on the testicular tissue of adult male Indian house rat, Rattus rattus. Single oral dose of Busulphan (10 mg/Kg body weight) was administered and its activity was noticed at 10, 40, 70 and 100 days of posttreated animals. Histological observation and quantitative histological study indicates no major alteration in the relative percentages of primary spermatocytes, spermatid and Sertoli cells at 10 days of posttreatment. But there was a gradual decrease in the seminiferous tubular diameter at 40 and 70 days of post treated groups. However, the Leydig and Sertoli cells morphology and number remained normal in all the treatment groups. At 40 days, the normal cellular associations in all the tubules were disrupted. The tubules constituted only spermatogonia, Sertoli cells and some zygotene spermatocytes. At 70 days, repopulation of Type A, Type B spermatogonia, resting and zygotene spermatocytes occurred at this stage. The tubules were still devoid of pachytene spermatocytes, spermatid and spermatozoa. At 100 days, active spermatogenesis was observed in majority of the tubules. The various types of germ cell population were regaining towards normalcy. Histochemical studies clearly revealed that due to busulphan administration there was no major alteration in the intensities of some key enzymes (i.e. delta5 3beta-HSDH and 17beta-HSDH) involved in the biosynthesis of steroid hormones. Only the acid phosphatase activity was slightly depressed within the 40th and 70th days of posttreatment. Sudanophilic lipid materials increased in the interstitium of all the busulphan post treated groups. The changes which were noticed due to busulphan treatment regained normalcy at 100 days of post treated animals. The mode of action of Busulphan on the testicular tissue of adult Indian house rat (Rattus rattus) has been pointed out and discussed.     

The effect of prenatal treatment with busulfan on in vitro androgen production by testes from rats of various ages

Treatment of rats with busulfan in utero severely depletes the germ cell population of the seminiferous tubules. These studies have examined the in vitro capacity of testicular tissue and Leydig cells from such testes to secrete androgens. Leydig cells were identified by staining for 3 beta-hydroxy steroid dehydrogenase. Rats were studied at several ages to identify any developmental changes in the androgen-secreting capacity of control and treated gonads. At 30 days of age, no effect of treatment on serum androgen was found. At 60 and 90 days of age, treatment caused decreased androgen and increased LH content of the serum. At 12, 30, 60, and 90 days of age, the amount of androgen secreted per milligram of testicular tissue in response to LH was higher in busulfan-treated rats. Leydig cells from 60- and 90-day-old rats which had received busulfan were also hyperresponsive to LH. It was concluded that Leydig cells from testes essentially devoid of germ cells were hyperresponsive to LH. Serum androgen levels were decreased yet androgen production per Leydig cell was increased. A possible explanation of this apparent paradox is that busulfan treatment resulted in decreased numbers of Leydig cells in the gonads.     

Development of hCG sensitivity in the testis of the immature rat

The activity of the enzyme alcohol dehydrogenase has been used as a marker to follow the maturation of the Leydig cell in the testis of the rat between birth and the 24th day of life. ADH activity in control rats and in rats that have had daily injections of hCG from birth onwards indicates that the premature Leydig cell is insensitive to exogenous hCG and acquires its hormone sensitivity at the end of the 2nd week of life. Alcohol dehydrogenase activity can be enhanced by db-cyclic AMP or theophylline during the entire hCG insensitive period. ¹²⁵I-hCG is specifically bound to testicular tissue of 10, 15, and 20 day old rats.We therefore conclude that in spite of its hCG insensitivity, the infantile Leydig cell possesses both the specific hCG receptor and the enzyme adenyl cyclase, both of which are prerequisites for polypeptide hormone action.     

Determination ob biologically active LH in the serum of male rheusus monkeys (Macaca mulatta)

The in-vitro bioassay for LH, using mouse Leydig cells, has been modified for the direct measurement of serum LH in the male rhesus monkey. Validation of the assay demonstrated good reliability in terms of accuracy, precision and sensitivity (1.5 mi.u./ml). Basal LH concentrations in laboratory-maintained monkeys with and without anaesthesia were not significantly different from those in free-ranging, feral monkeys. LH-RH (50 micrograms i.v.) elicited a 30-fold increase in LH concentrations after 30 min. LH levels in castrated adult monkeys were approximately 50 times the normal levels. Intact and castrated juvenile males had only very low LH levels. LH from the serum of an adult male castrate was further characterized by Sephadex G100 column chromatography. The in-vitro bioassay provides a preferable alternative to the heterologous radioimmunoassay method for the routine determination of LH in the rhesus monkey.     

Gonadotrophin-induced accumulation of 'interstitial fluid' in the rat testis.

'Interstitial fluid' containing high levels of testosterone (60-250 ng/ml) was recovered from the testes of rats, the amounts increasing with increase in age and testis weight. Injection of 170 i.u. hCG/kg resulted 20 h later in significant increases in interstitial fluid and its testosterone content (300-800 ng/ml). In immature rats this effect of hCG was dose-dependent and time-related and the accumulated fluid contained high levels of potassium and phosphate; levels of sodium, calcium and protein were similar to those in serum. At 20 h after injection of hCG, other testicular changes were (1) increased 'adhesiveness', (2) reduced in-vitro binding of 125I-labelled hCG, and (3) an hCG-induced increase in the testis:blood ratio of hCG in vivo.     

Photoperiodic modulation of testicular LH receptors in the bank vole (Clethrionomys glareolus)

The temporal changes in testicular binding of 125I-labelled hCG in juvenile bank voles (18 days of age, born and reared in a 18L:6D photoperiod) exposed to a long (18L:6D, Group L) or short (6L:18D, Group S) photoperiod for 0, 3, 7, 14 and 42-56 days were investigated. During testicular maturation, in Group L, there was a slight initial decrease in LH receptor numbers per testis followed by a marked prepubertal rise during the initial phase of rapid testicular growth after which a decrease took place. In Group S, during testicular regression, the temporal changes in LH receptor numbers per testis resembled those of Group L except that the corresponding increase in hCG binding during the initial week was considerably less marked and the receptor numbers remained thereafter at a significantly lower level than in Group L. Leydig cell count indicated that the observed changes in LH receptors per testis were due to changes in the number of Leydig cells as well as in LH receptors per Leydig cell. The present results indicate, that (1) photoperiod is an important modulator of testicular LH receptor numbers in this species, (2) photoperiod or age has no significant effect on the binding affinity of LH receptors, (3) short photoperiods arrest the induction of LH receptors as well as the increase in Leydig cell numbers associated with normal testicular maturation, and (4) changes in LH receptor numbers per testis correlate well with the photoperiod-induced changes in androgen biosynthesis, spermatogenesis and Leydig cell morphology observed in our previous studies.     

Pubertal development of the boar: age-related changes in testicular morphology and in vitro production of testosterone and estradiol-17 beta

The responsiveness of testicular tissue, in terms of testosterone (T) and estradiol-17 beta (E2) production, to human chorionic gonadotropin (hCG) stimulation in vitro was assessed during pubertal development of the boar. A morphometric investigation was conducted concurrently to quantitate Leydig cell and seminiferous tubule changes in the testes of developing boars. Testicular volume percentage of seminiferous tubules increased from 36% at 40 days of age to a maximum of 72% at 190 days of age. Increases in tubular diameter were from 65 micrometers at 40 days of age to 236 micrometers at 250 days of age. Testicular volume percentage of Leydig cells decreased from 40% at 40 days of age to 10% at 250 days of age. Leydig cell number increased rapidly to 130 days of age, remained constant through 160 days, and then increased steadily to 220 days of age. Volume per Leydig cell changed little from 40 to 130 days of age, increased by 75% at 160 days, and declined thereafter. Total Leydig cell weight increased steadily from 40 to 160 days of age and then declined slightly. The capacity of Leydig cells for T production and testicular tissue for E2 production was greatest (P less than 0.05) after hCG stimulation in boars that were 130 and 160 days of age. In addition, sensitivity, as judged by the regression coefficient of T or E2 production per Leydig cell on log dosage of hCG was greater (p less than 0.05) for T at 130 days of age and for E2 at 160 days of age. The data presented support the hypothesis that one factor in pubertal development of boars is an increased capacity and sensitivity of the testes to gonadotropin stimulation.     

Comparative protective actions of gonadotrophins and testosterone against the antispermatogenic action of ethane dimethanesulphonate

After a single dose of ethane dimethanesulphonate (EDS) (75 mg/kg) to rats the prolonged antispermatogenic action is due to a temporary elimination of the functional Leydig cell population. Replacement therapy with testosterone propionate (3 mg/day) maintains the spermatogenic epithelium but the EDS effect develops when hormone treatment is discontinued. In contrast, a short treatment with hCG (10-100 i.u./day) or LH (714 micrograms/day), starting before the EDS dose, permanently protects the spermatogenic epithelium. FSH treatment was completely ineffective. Although histological protection of spermatogenesis appeared complete with testosterone or hCG, effects on fertility remained but over different periods of time. Antispermatogenic and antifertility effects were produced in mice using much higher doses of EDS (5 X 250 mg/kg) but there was no protection from androgen or hCG. It is suggested that EDS binds to Leydig cells irreversibly, interfering with the action of gonadotrophin. At the dose level used the evidence suggests that the degree of reaction renders most of the Leydig cell population non-viable. A direct cytotoxic effect of the compound upon the spermatogenic epithelium might account for the inability of testosterone or hCG alone or in combination to maintain fertility at normal levels.     

Paracrine regulation of testicular function

Data from several experimental approaches have been reviewed and the findings clearly indicate the existence of multiple interactions between testicular cells and the potential role of these interactions in the paracrine control of testicular functions. Both testicular interstitial fluid and spent media from cultured Sertoli cells had an acute steroidogenic effect on Leydig cells, and this effect is not species specific. The secretion of this steroidogenic factor(s), which is probably a protein, is enhanced by previous FSH treatment of Sertoli cells. Coculture for 2-3 days of pig Leydig cells with homologous or heterologous Sertoli cells enhances Leydig cell specific functions (hCG receptor number and hCG responsiveness) and induces Leydig cell hypertrophy. A similar but less pronounced trophic effect is seen when Leydig cells are cultured with spent media from Sertoli cells cultured in the presence of FSH and high concentrations of insulin, but the spent media from Sertoli cells cultured in the absence of these two hormones inhibits Leydig cell specific functions. Somatomedin-C might play an important role in the positive trophic effect of Sertoli cells on Leydig cells, since this peptide is secreted by Sertoli cells and it has trophic effects on the specific function of Leydig cells. Moreover, Sertoli cells, probably through a diffusible factor and cell-to-cell contacts, control the multiplication, meiotic reduction and maturation of germ cells. In turn, the activity of Sertoli cells is modulated by the stage of neighbouring germ cells. Thus, if a normal Sertoli cell function (which depends not only on FSH but also on Leydig and myoid cell secretory products) is an absolute requirement for germ cell multiplication and maturation, these cells, in turn, cyclically regulate Sertoli cell function and through these cells the size and probably the function of Leydig cells.     

Influence of a prolonged tamoxifen administration on steroidogenesis by incubated rat testes

Tamoxifen was administered i.m. for 9 days to adult male rats in a daily dose of 100 micrograms or 1 mg. The treatment resulted in a significant reduction of the plasma levels of testosterone and LH, without modification of the plasma levels of FSH and of the testes weight. Upon incubation, the testes from the tamoxifen-treated rats produced less testosterone and 7 alpha-hydroxytestosterone, but metabolized [4-14C]testosterone in the same way as the control animals. Small doses of hCG (0.5 i.u. for 9 days) were unable to modify the tamoxifen effect on the testicular function, while tamoxifen significantly inhibited the increase of the plasma levels of testosterone induced by the administration of moderate doses of hCG (1.5 i.u. or 2.5 i.u. for 9 days) to hypophysectomized rats. Tamoxifen treatment, however, did not modify significantly the reactivity of the testes towards high doses of hCG (10 i.u.), administered either 2 h before sacrifice or for 9 days. It is concluded that a prolonged administration of tamoxifen in the rat has, besides an indirect effect resulting from a decrease of the LH levels, a direct inhibitory influence on the testicular testosterone formation, which can be reversed by high doses of hCG.     

Multiple regulation of testicular steroidogenesis

One single injection of ethylene dimethane sulfonate (EDS) to mature rats causes specific degeneration of testicular Leydig cells which is complete after 3 days. At this time no steroidogenic activities can be detected, indicating that Leydig cells are the source of steroids. The mechanism of this cytotoxic effect of EDS has been investigated with isolated cells. Extensive protein alkylation has been shown to occur in Leydig cells, Sertoli cells and hepatocytes. Steroid production by Leydig cells is always inhibited by EDS, but cytotoxic effects of EDS could only be demonstrated in Leydig cells from mature rats or tumour tissue and not in Leydig cells from immature rats. A new population of Leydig cells develops during the next 2-5 weeks after EDS treatment. In hypophysectomized rats this repopulation only occurs when hCG is given daily. FSH has no effects. The proliferative activity in the interstitial tissue increases within 2 days after administration of hCG or EDS and there are indications that LH and locally produced factors are involved in the proliferation of Leydig cells or Leydig cell precursor cells. Inhibition of cAMP production with inhibitors of adenylate cyclase results in an enhancement of the LH-stimulated steroid production similar to that observed with an LHRH agonist and phospholipase C (PLC). Since the effects of LHRH and PLC on protein phosphorylation and steroid production are similar and different from LH or active phorbol esters, it is proposed that LHRH and PLC may stimulate steroid production via liberation of calcium from a specific intracellular pool. Sterol carrier protein2 (SCP2) which is specifically localized in Leydig cells and regulated by LH probably plays a role in the delivery of cholesterol to the mitochondria although the mechanism of this carrier function is not clear. The results indicate that regulation of Leydig cell development and the steroidogenic activities by gonadotrophins and locally produced factors occur via different transducing systems and regulatory pathways.     

Focal disruption of spermatogenesis in the testis of adult rats after a single administration of human chorionic gonadotrophin

Summary The effect of a single injection of 100 i.u. human chorionic gonadotrophin (hCG) upon the morphology of the rat testis was studied by light and electron-microscopy from 12–48 h after treatment. Twelve hours after injection of hCG, emigration of leukocytes occurred across the intertubular blood vessels and, both at this time and at 24 h, infiltrations of leukocytes were observed within the extracellular tissue spaces. Furthermore, 12 h after hCG, the seminiferous epithelium showed focal disruption of spermatogenesis involving germ cell degeneration and pyknosis. Focal damage to the seminiferous epithelium persisted at 24 h and 48 h after injection of hCG, the affected seminiferous tubules showing failure of spermiation, accumulation of extracellular vacuoles and degeneration or partial loss of spermatogonia and primary spermatocytes. The observations show that, after stimulation of the Leydig cells with hCG, the intertubular tissue exhibits an inflammatory-type response and this is associated with focal tissue destruction in the seminiferous tubules. It is concluded that a high dose of hCG exerts anti-spermatogenic effects upon the testis and raises the possibility of unexpected interference with tests of normal Leydig cell function in both laboratory and clinical investigations.     

An early steroidogenic defect in hormone-induced Leydig cell desensitization

To define the nature of the lesion of the early steroidogenic pathway (prior to pregnenolone formation) in gonadotropin-induced desensitization of rat testicular Leydig cells, we evaluated cholesterol side-chain cleavage activity in isolated mitochondria by measurement of pregnenolone synthesis and [14C]isocaproic acid formation from [26-14C]cholesterol. The enzyme activity was shown to be reduced after in vivo treatment with 10 micrograms hCG when compared to that of mitochondria from control animals only when measured in the presence of limiting NADPH concentrations (100 microM). Sonication of mitochondria from control and hCG-treated rats caused complete loss of cholesterol side-chain cleavage activity. When acetone-powdered adrenal cell mitochondria were employed as the source of the enzyme, the addition of sonicated Leydig cell mitochondria from control and hCG-treated animals caused the same differences as those observed with intact Leydig cell mitochondria in the presence of low concentration of NADPH. The Km value of the adrenal enzyme for NADPH incubated with Leydig cell mitochondria increased from 0.111 mM in control to 0.37 mM after hCG, with no changes in Vmax. Moreover, cholesterol side-chain cleavage activity of adrenal mitochondria assayed in the presence of 100 microM cholesterol was progressively inhibited by increasing amounts of acetone powder from Leydig cell mitochondria of control and hCG-treated rats, with ID50 of 500 and 280 micrograms protein, respectively. The inhibiting factor was not a lipid or steroid but a heat-labile protein, with an approximate Stokes radius of 4.8 nm and an isoelectric point of 5.05 +/- 0.23 SD (n = 8). The inhibitory effect was confined to the Leydig cell mitochondrial membrane, and was not related to changes in oxidative phosphorylation. NADPH was not directly oxidized or immobilized by the mitochondrial factor, and this inhibiting substance was not adsorbed on 2',5' ADP-Sepharose 4B. These results have demonstrated that a heat-labile inhibiting protein factor is present in mitochondria from normal Leydig cells and is markedly activated or increased by hCG treatment. This substance that competitively modulates cholesterol side-chain cleavage activity could contribute to the early steroidogenic lesion, and also serve as an endogenous modulator of steroid hormone biosynthesis.     

The Capacity of Testicular Cells of the Postnatal Rat to Reorganize into Histotypic Structures

Cell reorganization experiments in vitro were performed with dissociated rat testes at different ages of postnatal development namely, newborn, 8–10, 18–25, 35–40, and 90 days. Only newborn and juvenile rat testicular cells reassociated into testicular-like organization in rotation culture. Puberal and adult rat testicular cells show morphogenetic organization when they were deprived of germ cells by busulphan pretreatment. A factor present in testicular tissue of puberal and adult rats inhibits reorganization. The inhibitor is confined to the spermatic cell fraction in the testis.