Effects of human recombinant luteinizing hormone and follicle-stimulating hormone in patients with acquired hypogonadotropic hypogonadism: study of Sertoli and Leydig cell secretions and interactions

Experimental data suggest that FSH-stimulated Sertoli cells can enhance LH-induced Leydig cell testosterone (T) production. The function of Leydig and Sertoli cells can be selectively studied by using recombinant human LH (rhLH) and recombinant human FSH (rhFSH) in patients with complete gonadotropin deficiency. The aim of the present study was to assess the secretion of testicular T, estradiol (E2), and inhibin B and the physiological relevance of the Sertoli-Leydig cell interaction in man. For that purpose, six patients with acquired complete hypogonadotropic hypogonadism received the following treatments for three periods of 1 month in a random order: 1) rhLH, 900 IU/day sc; 2) rhFSH, 150 IU/day sc; and 3) combined rhLH/rhFSH treatments. Each treatment period was separated by a washout period of 15 days. Plasma LH, FSH, T, E2, and inhibin B were measured before and every 10 days during each treatment. During rhLH administration, mean plasma LH levels rose significantly from 0.4 +/- 0.2 IU/L to 11.7 +/- 1.2 IU/L (P < 0.01) and plasma FSH levels did not change. rhFSH administration induced a significant increase in plasma FSH levels (from 0.5 +/- 0.4 to 12.1 +/- 1.4 IU/L; P < 0.01), whereas mean plasma LH levels remained low. Mean plasma E2 levels were unchanged during rhFSH treatment, but they increased significantly during rhLH from 22 +/- 4 to 54 +/- 8 pmol/L (P < 0.01) and during rhLH plus rhFSH administration. rhFSH treatment induced a sustained elevation of mean plasma inhibin B levels from 58 +/- 13 to 175 +/- 25 pg/mL (P < 0.01), similar to the increase occurring during rhFSH plus rhLH administration. In contrast, mean plasma inhibin B levels did not increase during rhLH administration. Finally, a similar and significant increase in mean plasma T levels occurred during both rhLH and rhLH plus rhFSH treatment from 0.9 +/- 0.3 to 5.4 +/- 0.7 nmol/L (P < 0.01) and from 1.0 +/- 0.4 to 6.0 +/- 0.9 nmol/L (P < 0.01), respectively. In contrast, during rhFSH treatment mean plasma T levels remained unchanged when compared with baseline. In conclusion: 1) the increase of plasma E2 induced by rhLH and the absence of effect of rhFSH confirm that Leydig cells are the major site of testicular E2 production in man; 2) the secretion of inhibin B is increased by rhFSH and not by rhLH, and, thus, Sertoli cells seem to be the main source of inhibin B production; and 3) the increase of plasma T induced by rhLH is not enhanced by rhFSH. These results suggest that the stimulatory effect of FSH on Leydig cell steroidogenesis by a Sertoli cell paracrine factor does not seem to play a major physiologic role in man.     

Gonadotrophins, testosterone and spermatogenesis in neonatally irradiated male rats: evidence for a role of the Sertoli cell in follicle-stimulating hormone feedback

Peripheral concentrations of FSH in the male rat seem to be regulated in part by a protein hormone, inhibin, which originates from the testes. In an attempt to ascertain which type of testicular cell secretes inhibin, groups of male rats were irradiated prenatally or on days 4, 6 or 8 of postnatal life, and killed at 21, 51 or 81 days of age together with castrated and intact controls. The concentrations of FSH and LH in the pituitary gland, and FSH, LH and testosterone in the plasma were estimated for each animal, and the numbers of each class of intratubular cell in the testes were calculated. Rats irradiated neonatally had fewer Sertoli cells than controls at all ages studied, while the numbers of Sertoli cells in rats irradiated prenatally were higher than those in controls on day 21. The number of spermatogenic cells was usually decreased in rats irradiated postnatally. In the rats irradiated prenatally normal numbers of spermatogenic cells were found at day 51. Numbers of spermatogenic cells were significantly correlated with the number of Sertoli cells at the ages of 51 and 81 days. The concentrations of FSH in the plasma usually increased in the postnatally irradiated animals on days 21 and 51, but not on day 81; prenatal irradiation did not result in altered levels of FSH at any age. Peripheral levels of LH and testosterone were not affected by irradiation. The concentration of FSH in the plasma was negatively correlated with the number of Sertoli cells in all age groups, whereas significant correlations between the level of FSH and the number of spermatogenic cells were only found at days 51 and 81. It is concluded from these data that the Sertoli cell is the most likely source of inhibin.     

Testicular gonadotrophins and their receptors in human cryptorchidism as revealed by immunohistochemistry and radioreceptor assay

The localisation of endogenous FSH and LH was studied in 4 inguinal adult human testes by the immunoperoxidase technique utilising antisera against the beta-subunits of human FSH and LH. The content of available FSH and LH receptors was determined by radioreceptor assay. The Sertoli cells and about 10% of cells in the intersitium the Leydig cells, possibly the testicular macrophages, were similarly FSH-positive in cryptorchidism and control testes. The FSH receptor levels per testis were significantly lower in cryptorchidism than in control testes. Also the localisation of LH in Leydig cells in cryptorchidism was similar to the control testes, but the LH receptor level was significantly lower. These data bring further evidence for Leydig and Sertoli cell malfunction in the inguinal human testis.     

Complete Sertoli cell proliferation induced by follicle-stimulating hormone (FSH) independently of luteinizing hormone activity: evidence from genetic models of isolated FSH action

Defining the gonadal effects of FSH distinct from those of LH remains difficult. We have characterized and compared the level of Sertoli and germ cell development in three mouse models recently created to isolate FSH activity from LH effects. Two models used LH-deficient hypogonadal (hpg) mice to selectively study either pituitary-independent transgenic (tg) FSH or ligand-independent activated tg FSH receptor (FSHR(+)) expression, and the third model used LH receptor (LHR)-deficient mice to isolate and examine endogenous mouse FSH effects. Stereological evaluation revealed tg-FSH or tg-FSHR(+) activity significantly increased total Sertoli cell numbers per testis in both hpg models relative to control hpg testes. Furthermore, tg-FSH dose-dependently restored hpg Sertoli cells to wild-type (wt) (non-hpg) levels, and LHR-/- testes also exhibited wt Sertoli numbers. Spermatogonial proliferation and meiotic development were enhanced by tg-FSHR(+) or tg-FSH. Despite producing normal Sertoli numbers, isolated tg-FSH activity only increased total spermatogonia and spermatocyte populations to 57 and 44% of wt, which was comparable to spermatogonia and spermatocyte numbers observed in LHR-null testes (45 and 34% of wt). Selective FSH activity initiated round spermatid formation in all three models. However, elongated spermatid formation was detected in tg-FSH and tg-FSHR(+) hpg testes but not in LHR-/- testes, which may reflect even lower intratesticular testosterone levels in LHR-null compared with hpg testes. FSH increased round and elongated spermatid numbers in hpg testes to 16 and 6% of wt without altering intratesticular testosterone levels, but failed to produce spermatozoa demonstrating the inability of FSH to complete spermatogenesis. These findings revealed that full Sertoli cell proliferation can be accomplished by FSH activity without LH requirement, and although postnatal mitotic and meiotic germ cell development can be promoted by FSH alone, LH-mediated effects remain a critical determinant for initiating the full complement of germ cells and final stages of postmeiotic development.     

Differences in the regulation of steroidogenesis and tropic hormone receptors between the scrotal and abdominal testes of unilaterally cryptorchid adult rats

Rats were made unilaterally cryptorchid by cutting the gubernaculum testis at birth. At 100 days age, the rats were injected with 600 IU/kg hCG. A biphasic testosterone response was seen in the scrotal (Scr) testis in response to hCG, with maxima at 1 h and 3 days after injection. The acute peak of testosterone was of similar magnitude in the abdominal (Abd) testis, but the secondary peak was not present. The response of testicular progesterone concentration to hCG stimulation showed a maximum at 1 day in both gonads, but it was 10- to 20-fold higher (P less than 0.01) in the Abd testis. The content of LH, FSH, and PRL receptors per testis was decreased on the Abd side. After hCG injection, the loss of available LH receptors was faster in the Abd testis. Likewise, the recovery of binding was faster in the Abd testes; at day 10 of the experiment, it was 102 +/- 5% of the starting levels compared to 52 +/- 4% on the Scr side (P less than 0.01). hCG did not affect FSH binding of the Scr testes, but induced a transient drop of 25-35% on day 1 on the Abd side (P less than 0.05). Thereafter, on days 3-10, the FSH binding of the Abd testes was 20-40% higher than on the Scr side (P less than 0.05-0.01). In PRL binding, similar heterologous down-regulation of 50-80% was found in both testes between 12-24 h. Thereafter, the Abd testis PRL receptors showed a transient elevation of 25-70% (P less than 0.05-0.01) on day 3, which was not seen in the Scr testes. In conclusion, the Abd testis displays a dramatically enhanced blockade of C21 steroid side-chain cleavage upon gonadotropin stimulation. The kinetics of changes in testicular LH receptors after hCG stimulation is faster in the Abd testis. Only Abd testes displayed hCG-induced changes in FSH binding and transient up-regulation of PRL receptors. The altered tropic regulation of Leydig and Sertoli cells of the Abd testis are indicative of direct functional changes in these cells in the elevated intra-Abd temperature and/or of changes in the paracrine component of testicular regulation.     

Ageing, testicular tumours and the pituitary-testis axis in dogs

Dogs of different ages without testicular diseases were evaluated to study possible age-related changes in hormone concentrations in serum. Dogs with testicular tumours were also investigated to study the relation between tumour type and hormone concentrations; in this study, dogs with Sertoli cell tumours, Leydig cell tumours and seminomas were included. We measured testosterone, oestradiol, LH, FSH and inhibin-like immunoreactivity concentrations in peripheral venous and testicular venous blood of these animals. In normal dogs there appeared to be no age-related changes in the concentrations of the investigated hormones, except for a significant age-related decrease in oestradiol concentrations in testicular venous blood (P<0.02). Dogs with a Sertoli cell tumour had greater oestradiol concentrations and inhibin-like immunoreactivity in both peripheral and testicular venous blood than did dogs without a neoplasm (P<0. 05). Testosterone concentrations were reduced in dogs with Sertoli cell tumours, as were FSH and LH. Feminisation occurred in eight of 13 dogs with a Sertoli cell tumour and in two of 14 dogs with a Leydig cell tumour; it was accompanied by a significantly greater oestradiol concentration than in normal dogs and in dogs with Sertoli cell tumours without signs of feminisation. Dogs with a Leydig cell tumour had greater concentrations of oestradiol and inhibin-like immunoreactivity in both peripheral venous and testicular venous blood than did dogs without a neoplasm (P<0.05). The testosterone concentration in testicular venous blood of these dogs was lower than that in dogs with normal testes. The concentration of LH in peripheral venous blood was also reduced (P<0. 05). Hormone concentrations in dogs with a seminoma were not different from those in normal dogs. It was concluded that seminomas are not endocrinologically active. In contrast, both Sertoli cell tumours and Leydig cell tumours can cause increased oestrogen production leading to signs of feminisation. These tumours also have considerable amounts of inhibin-like immunoreactivity, but only in Sertoli cell tumours does this result in a reduction in FSH concentrations, suggesting that Sertoli cell tumours secrete dimeric inhibin, whereas Leydig cell tumours presumably produce loose alpha-subunits that cross-react in the inhibin assay but are not biologically active.     

The influence of luteinizing hormone on the Leydig cells of cryptorchid rat testes

The influence of circulating LH levels on Leydig cells from cryptorchid adult rats was examined after ablation of the pituitary. After 2 weeks cryptorchidism, serum FSH and LH levels rose 2-fold while serum testosterone (T) remained unchanged. Leydig cells were hypertrophied and showed an increased response to in vitro hCG stimulation. Two weeks after hypophysectomy (hypox), serum hormone levels (LH, FSH and T), Leydig cell size, cytoplasm, organelle content and in vitro T production were all dramatically reduced. However, when hypophysectomy was combined with cryptorchidism (hypox/crypt), there was an increase in Leydig cell size, compared to hypophysectomy alone, in the presence of very low levels of serum FSH, LH and T. Compared to the hypophysectomised state, the mitochondria were larger and the cytoplasm contained more smooth endoplasmic reticulum. The response of the hypox/crypt testes to in vitro hCG stimulation, though significantly less than the cryptorchid testes, was significantly greater than the hypox testes. These results demonstrate that the changes observed in the Leydig cell after cryptorchidism can occur in the absence of peripheral pituitary hormones and are consistent with the hypothesis that a local feedback loop exists within the testis.     

Operation of the follicle-stimulating hormone (FSH)–inhibin B feedback loop in the control of primate spermatogenesis

This paper reviews our current understanding of the function and operation of the follicle-stimulating hormone (FSH)–inhibin feedback loop in the male rhesus monkey (Macaca mulatta). Inhibin B is the major testicular inhibin in the monkey, and the pattern of secretion of this hormone during postnatal development is temporally coupled to that of gonadotropin. Inhibin B secretion by the Sertoli cell is stimulated by FSH and inhibited by luteinizing hormone (LH), the latter presumably acting via Leydig cell production of testosterone (T). The dynamics of the FSH–inhibin B feedback loop in the adult monkey is revealed following unilateral orchidectomy (UO). Interestingly, a sustained, 50% deficit in inhibin B secretion occurs after UO and this persistent error signal, in turn, results in elevated concentrations of FSH in the circulation. The elevated secretion of FSH appears to be the principal drive for the increased sperm output by the remaining testis. Available data for the functioning of the FSH–inhibin B feedback loop in the human male are placed in perspective, and a model for the negative feedback regulation of sperm number in primates is proposed.     

Inhibition of testicular and somatic development after treatment of the postnatal rat with the Leydig cell cytotoxic ethylene dimethanesulphonate

The Leydig cell cytotoxic ethylene dimethanesulphonate (EDS) was administered s.c. daily (50 mg/kg) to male rats aged 5-16 days. Apart from loss of weight and that the eyelids unfused earlier, no gross toxicity was observed during treatment. On day 17 testis weights, serum testosterone concentrations, testicular serum testosterone content and 125I-labelled human chorionic gonadotrophin (hCG) binding to testicular homogenates were reduced. Serum LH and FSH concentrations were elevated. The testes did not recover from EDS treatment and at 63 and 120 days were minute (less than 2% of control), and the prostate and seminal vesicles were small although not completely atrophied. In addition, body weights were substantially reduced. Serum and testicular testosterone and 125I-labelled hCG binding to testicular homogenates were reduced but not absent. Serum LH and FSH concentrations were increased. Light microscopy of the adult testes showed that EDS treatment inhibited the development of the seminiferous tubules. Most of the tubules were devoid of germ cells and Sertoli cells were rare. Occasionally tubules also contained spermatogonia and spermatocytes but no signs of spermiogenesis. The testes were composed mainly of closely packed interstitial tissue with no lymphatic space. The interstitial cells resembled Leydig cells and stained for 3 beta-hydroxysteroid dehydrogenase. Histochemically identified Leydig cells were absent during treatment but reappeared when treatment was withdrawn.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of pulsatile and continuous luteinizing hormone (LH) infusions on testosterone responses to LH in rams actively immunized against gonadotropin-releasing hormone

Yearling rams actively immunized against GnRH were used as a hypogonadotropic model for studies of the significance of the pulsatility of LH secretion in determining the trophic actions of the hormone on testicular steroidogenesis. GnRH-immunized rams, in which testicular regression was complete, were infused iv for 12-20 days with ovine LH (NIDDK oLH 24) in three different regimens, delivering a total daily dose of 60 micrograms/100 kg: 1) 1-min pulses of 5 micrograms/100 kg every 2 h (low amplitude, high frequency), 2) 1-min pulses of 30 micrograms/100 kg every 12 h (high amplitude, low frequency), or 3) continuous infusion of 2.5 micrograms/100 kg.h. Serum testosterone levels and acute responses to LH challenges were monitored at intervals throughout the infusion periods. Acute responses to LH were evaluated in terms of the area under the curve for serum testosterone vs. time after LH and the lag time between the infusion of LH and attainment of maximum serum testosterone levels. At the beginning of the experiments, serum testosterone was at castrate values, and testosterone responses to LH were of low magnitude with a long lag time. LH infusion in the low amplitude, high frequency regimen consistently increased the magnitude and decreased the lag time of acute responses to LH; these effects were significant by the sixth day of treatment and persisted for the duration of the experiments. This regimen also had positive effects on morphological features of testes and Leydig cells. Infusion of the high amplitude, low frequency regimen, however, had neither of the positive effects on responsiveness to LH, but did seem to improve testicular and Leydig cell morphology. Continuous infusion of LH also increased the magnitude and decreased the lag time of responses to low amplitude pulses of LH, at least as well as the high frequency infusion regimen did. These results suggest that the high frequency, low amplitude pattern of LH secretion characteristic of reproductively active animals has trophic actions on the testes, increasing their responsiveness to acute gonadotropic stimulation, but the pulsatility of that pattern of LH secretion is not necessary for its trophic actions. The efficacy of high frequency LH secretion may depend only on the elevation of basal or mean LH concentrations, rather than on the low amplitude peaks or the dynamic changes in LH concentrations to which the testes are exposed.     

Effects of flutamide or of supplementation with testosterone in prepubertal male rats prenatally treated with busulfan

Male rats treated prenatally with busulfan in order to render them aspermatogenic were treated between 23 and 38 days of age either with testosterone, or flutamide. In such aspermatogenic rats, testosterone supplementation stimulated the growth of accessory sex organs, markedly decreased the secretion of gonadotrophins (by 61% for LH and 83% for FSH), decreased testicular weight (-60%) as well as all parameters relating to testicular histology. Flutamide treatment decreased the weight of accessory sex organs, stimulated the secretion of both LH (+200%) and FSH (+63%) by inhibition of the negative feedback of testosterone; endogenous testosterone secretion was increased by 363%. Testicular weight was increased by 44% and the total volume and the cytoplasmic and nuclear area of Leydig cells were increased by 254 and 28%, respectively, but their number per testis was unchanged. The number of Sertoli cells per testis was increased by 49%, but their nuclear area was not modified. In aspermatogenic prepubertal rats, the large increase in plasma FSH demonstrated that FSH release was partly under the control of inhibin, secreted by Sertoli cells when germinal cells were present. Nevertheless, FSH levels after testosterone (inhibition) or flutamide treatment (stimulation) clearly demonstrated that, in the growing rat, FSH secretion is also partly dependent on the negative feedback of testosterone. In the absence of germ cells, FSH was able to re-initiate Sertoli cells mitoses, but not their functional activity. LH secretion was solely controlled by the negative feedback of androgens and, even in the absence of germinal cells, Leydig cells were able to respond to LH stimulation by an increase in testosterone secretion.     

Activating mutations in the luteinizing hormone receptor gene: a human model of non-follicle-stimulating hormone-dependent inhibin production and germ cell maturation

CONTEXT: Familial male-limited precocious puberty is a dominant autosomal genetic disease caused by activating LH receptor gene mutations, clinically expressed only in males. In preliminary studies, in addition to the expected testosterone increase, we found high inhibin B levels before the age of normal puberty. OBJECTIVES: The objective of the study was to assess the cellular origin of serum inhibin thanks to testis section immunostaining. MAIN OUTCOME MEASURES: Serum testosterone, gonadotropin, inhibin B, pan-alphaC-inhibin, and anti-Mullerian hormone levels were measured. Immunostaining was performed using specific anti-alpha- and anti-beta-subunit antibodies. SUBJECTS AND METHODS: Five boys from three families (mutation M398T or I542L) were investigated at onset (2-6 yr), on ketoconazole treatment, and at adolescence. Testis biopsies were performed in three subjects before the disease was fully characterized. RESULTS: The high testosterone levels were suppressed by ketoconazole. Anti-Mullerian hormone levels were inversely related to testosterone: low at diagnosis, elevated after testosterone suppression. Despite FSH suppression, inhibin B and pan-alphaC-inhibin levels were high from clinical onset to adolescence. Biopsy specimens showed normal Sertoli cell complement and germ cell maturation until the spermatocyte II stage. Sertoli and Leydig cells displayed positive inhibin alpha-subunit immunostaining. Only Leydig cells and spermatogonia stained positively for the inhibin betaB-subunit. CONCLUSIONS: Familial male-limited precocious puberty is a unique model of inhibin B secretion, demonstrating that Leydig cells can produce significant amounts of the dimeric molecule. Our results also suggest that the pubertal FSH rise is not required for full expression of the two inhibin B genes and for the initiation of germ cell maturation.     

Failure of normal Leydig cell development in follicle-stimulating hormone (FSH) receptor-deficient mice,but not FSHbeta-deficient mice: role for constitutive FSH receptor activity

Previous studies have suggested that FSH may be involved in regulation of Leydig cell function. We have examined this directly using two mouse models with null mutations in either the FSH beta-subunit (FSHbetaKO mice) or the FSH receptor (FSHRKO mice). Circulating LH levels were normal in adult FSHbetaKO mice, but were significantly increased in FSHRKO mice. Intratesticular testosterone levels increased normally in FSHbetaKO mice from birth to adulthood, whereas testosterone levels in FSHRKO mice failed to increase normally after puberty and were significantly reduced in adult animals. This was associated with reduced levels of mRNA encoding cytochrome P450 side-chain cleavage, 3beta-hydroxysteroid dehydrogenase type VI, and steroidogenic acute regulatory protein in FSHRKO mice. Leydig cell number was normal in FSHbetaKO mice during development, but in FSHRKO mice Leydig cell number increased slowly after puberty and was significantly reduced in the adult animal. Transfection studies showed that the FSHR exhibits constitutive activity in the absence of agonist stimulation. The results indicate, therefore, that Sertoli cells regulate the development of Leydig cell number and that constitutive activity within the FSHR is sufficient to stimulate this process. The presence of the hormone itself is not required when circulating LH levels are adequate.     

Influence of neonatal hemicastration on in-vitro secretion of inhibin, gonadotrophins and testicular steroids in male rats

Pituitary secretion of FSH in male animals is regulated, at least partly, by a protein hormone, inhibin, which is produced by Sertoli cells in the testes. To establish at which age the role of testicular inhibin in the regulation of FSH secretion becomes apparent, groups of male rats were hemicastrated or sham-operated on day 1 of life and pituitary and testicular function were investigated in vitro at 21, 42 or 63 days of age. Testis weights were increased in hemicastrated rats at all ages studied. Peripheral concentrations of gonadotrophins generally showed a good correlation with the concentrations of FSH and LH measured in the medium of hemipituitary glands which were incubated in vitro at 37 degrees C in the absence or presence of LH-releasing hormone. Peripheral testosterone concentrations in hemicastrated animals were not significantly different from those in sham-operated rats at all ages studied. Steroid production by Leydig cells in vitro was not significantly influenced by hemicastration. The secretion of inhibin by Sertoli cells from 21-day-old hemicastrated rats was decreased while Sertoli cells from 42- and 63-day-old hemicastrated animals secreted slightly but not significantly more inhibin than Sertoli cells from sham-operated rats. It is concluded that although compensatory increases of testosterone and inhibin production at later ages make it difficult to draw conclusions about the relative importance of inhibin in the feedback regulation of FSH secretion at different ages, it is likely that inhibin plays a role in the feedback of FSH in immature, rather than in mature male rats.     

In vitro stimulation of adenosine 3',5'-monophosphate in unilateral undescended testes of humans by follicle-stimulating hormone and luteinizing hormone.

The in vitro effects of FSH and LH on cAMP generation by unilateral undescended testes and contralateral descended testes were investigated. Biopsy tissue was preincubated at 37 C for 5 min in 1 ml Krebs-Ringer bicarbonate buffer (KRB) containing 10 mM aminophylline. Half of each specimen was frozen in liquid nitrogen to assess basal levels of cAMP. The other half was incubated for 20 min at 37 C in 1 ml fresh KRB containing aminophylline and FSH (10 microgram) or LH (10 microgram). The mean concentration of cAMP in the descended testes was 1.03 +/- 0.04 pmol/mg protein, whereas in the undescended testes the level was 1.98 +/- 0.07 pmol/mg protein (P less than or equal to 0.01). After the 20-min incubation period with FSH the concentrations of cAMP in the descended and undescended testes were 10.85 +/- 0.86 pmol/mg protein and 11.21 +/- 1.1 mol/mg protein, respectively. Incubation with LH resulted in the generation of equivalent levels of cAMP in both the descended (2.76 +/- 0.30 pmol/mg protein) and undescended (3.0 +/- 0.17 pmol/mg protein) testes. Although marked histological differences were observed in the germinal epithelium, the appearance and numbers of Leydig and Sertoli cells were comparable. The data suggest that the Sertoli cell is the primary receptor for FSH. Leydig and Sertoli cell functions of the undescended testes, at least in terms of cAMP generation, are similar to normal descended testes.     

Adrenocorticotropic hormone directly stimulates testosterone production by the fetal and neonatal mouse testis

Adult Leydig cell steroidogenesis is dependent on LH but fetal Leydig cells can function independently of gonadotropin stimulation. To identify factors that may be involved in regulation of fetal Leydig cells expressed sequence tag libraries from fetal and adult testes were compared, and fetal-specific genes identified. The ACTH receptor [melanocortin type 2 receptor (Mc2r)] was identified within this fetal-specific group. Subsequent real-time PCR studies confirmed that Mc2r was expressed in the fetal testis at 100-fold higher levels than in the adult testis. Incubation of fetal or neonatal testes with ACTH in vitro stimulated testosterone production more than 10-fold, although ACTH had no effect on testes from animals aged 20 d or older. The steroidogenic response of fetal and neonatal testes to a maximally stimulating dose of human chorionic gonadotropin was similar to the response shown to ACTH. The ED(50) for ACTH, measured in isolated fetal and neonatal testicular cells, was 5 x 10(-10) M and the lowest dose of ACTH eliciting a response was 2 x 10(-11) M. Circulating ACTH levels in fetal mice were around 8 x 10(-11) M. Neither alpha-MSH nor gamma-MSH had any effect on androgen production in vitro at any age. Fetal testosterone levels were normal in mice that lack circulating ACTH (proopiomelanocortin-null) indicating that ACTH is not essential for fetal Leydig cell function. Results show that both LH and ACTH can regulate testicular steroidogenesis during fetal development in the mouse and suggest that fetal Leydig cells, but not adult Leydig cells, are sensitive to ACTH stimulation.     

Targeted disruption of luteinizing hormone beta-subunit leads to hypogonadism, defects in gonadal steroidogenesis, and infertility

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) act on gonadal cells to promote steroidogenesis and gametogenesis. Clarifying the in vivo roles of LH and FSH permits a feasible approach to contraception involving selective blockade of gonadotropin action. One way to address these physiologically important problems is to generate mice with an isolated LH deficiency and compare them with existing FSH loss-of-function mice. To model human reproductive disorders involving loss of LH function and to define LH-responsive genes, we produced knockout mice lacking the hormone-specific LHbeta-subunit. LHbeta-null mice are viable but demonstrate postnatal defects in gonadal growth and function resulting in infertility. Mutant males have decreased testes size, prominent Leydig cell hypoplasia, defects in expression of genes encoding steroid biosynthesis pathway enzymes, and reduced testosterone levels. Furthermore, spermatogenesis is blocked at the round spermatid stage, causing a total absence of the elongated spermatids. Mutant female mice are hypogonadal and demonstrate decreased levels of serum estradiol and progesterone. Ovarian histology demonstrates normal thecal layer, defects in folliculogenesis including many degenerating antral follicles, and absence of corpora lutea. The defects in both sexes are not secondary to aberrant FSH regulation, because FSH levels were unaffected in null mice. Finally, both male and female null mice can be pharmacologically rescued by exogenous human chorionic gonadotropin, indicating that LH-responsiveness of the target cells is not irreversibly lost. Thus, LHbeta null mice represent a model to study the consequences of an isolated deficiency of LH ligand in reproduction, while retaining normal LH-responsiveness in target cells.     

Follicle-stimulating hormone induction of Leydig cell maturation

The effects of FSH on the testes of immature hypophysectomized rats were investigated by comparing functional changes in Leydig cells with changes in their number and morphological appearance. Rats were treated twice daily for 7 days with 0.5 ml saline vehicle, 10 micrograms rat FSH, or 20 ng ovine LH (an equivalent amount of LH known to contaminate the FSH preparation). FSH treatment caused a significant increase in testis weight and stimulated more advanced spermatogenic activity compared to saline or LH treatment. Morphometric analysis of glutaraldehyde perfusion-fixed testes showed no significant increase in Leydig cell number after LH treatment [saline, 4.63 +/- 0.14 million cells; LH, 6.38 +/- 0.55 million mean +/- SE)], but a significant (P less than 0.001) increase after FSH treatment (11.55 +/- 0.79 million). FSH, but not LH or saline, treatment resulted in Leydig cell hypertrophy and ultrastructural features identical to those of adult Leydig cells, these changes being reflected by enhanced hCG- and LHRH agonist-stimulated testosterone production in vitro by whole testes or dispersed interstitial cells. FSH and LH treatment caused minor but significant decreases in LH receptor numbers on dispersed interstitial cells compared to saline treatment. LHRH receptor numbers on interstitial cells were significantly increased only by FSH treatment. It is suggested that since FSH acts only on the seminiferous epithelium, then the hypertrophy, hyperplasia, and functional enhancement of Leydig cells after FSH treatment may be mediated by the secretion of one or more factors from the seminiferous tubules, providing additional evidence to support the view that gonadotropic regulation of testicular function is modulated by local interactions between the seminiferous tubules and the interstitial tissue.     

Stimulation of serum inhibin concentrations by gonadotropin-releasing hormone in men with idiopathic hypogonadotropic hypogonadism

Inhibin is a gonadal hormone thought to be important in FSH regulation. We investigated the effects of the hypogonadotropic state and subsequent GnRH-induced increases in gonadotropin levels on inhibin secretion. Serum levels of inhibin, LH, FSH, and testosterone (T) as well as sperm concentrations were measured in 5 men with idiopathic hypogonadotropic hypogonadism (IHH) before (baseline) and during 8 weeks of GnRH therapy (5 micrograms, sc, every 2 h). Baseline and peak inhibin levels were compared to those in a group of 19 normal men. Before GnRH administration, the mean serum inhibin level was significantly lower in the IHH men than in the normal men [166 +/- 56 (+/- SE) vs. 588 +/- 30 U/L; P less than 0.001]. Serum inhibin levels rose after 1 week of GnRH therapy (P less than 0.05) and remained higher than the baseline level thereafter. The mean peak inhibin level during GnRH administration was lower than the mean value in normal men (485 +/- 166 vs. 588 +/- 30 U/L; P less than 0.05). Serum LH and FSH levels rose promptly to the midnormal range or slightly above it. Serum T levels did not significantly increase until 4-5 weeks of GnRH administration and remained in the low normal range. All IHH men were azoospermic throughout the study. These data are consistent with the hypothesis that inhibin is produced by the testis under gonadotropin control. They also suggest the possibility of defective Sertoli and Leydig cell function in men with IHH, since the men's serum inhibin and T levels did not rise to the same extent as did their normalized serum gonadotropin levels during GnRH administration.     

Use of a pulsed infusion of luteinizing hormone releasing hormone to mimic seasonally induced endocrine changes in the ram.

Adult Soay rams were housed indoors under natural lighting during the spring non-mating season when gonadotrophin secretion was low. Four animals received small doses (100 ng or 500 ng) of synthetic LH releasing hormone (LH-RH) infused into the jugular vein by a mechanical device for 60 s every 2 h for 33-57 days: two other rams acted as controls. The prolonged treatment with LH-RH resulted in growth of the testes and the development of the sexual skin flush; these effects were lost when treatment stopped. The plasma concentrations of LH, FSH and testosterone were low at the beginning; each short infusion of LH-RH resulted in a transitory increase in the level of LH and testosterone while the concentration of FSH was only marginally affected. After prolonged treatment with 500 ng pulses of LH-RH the plasma concentrations of all three hormones were permanently raised. The response to the individual injections of LH-RH was also modified, the peak in LH being reduced in amplitude but more prolonged while the FSH and testosterone responses were both enhanced. When the pulsed infusion was stopped the concentration of LH and testosterone declined rapidly while the decline in FSH levels took many days. These endocrine changes induced by the pulsed infusion are comparable to those that occur naturally in the ram during testicular redevelopment before the mating season.