Endocrine status of the testicular feminized male (TFM) rat

The plasma levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T), androstenedione (A), dihydrotestosterone (DHT), corticosterone and corticosterone binding globulin (CBG) were determined in 160- and 350-day testicular feminized male (tfm) and normal littermate (NL) rats. In the younger group the concentrations of T, A, DHT and 5α-androstan-3α,17β-diol (Adiol) were also determined in testis tissue.Tfm rats showed a greatly elevated plasma LH indicating lack of androgen feedback. Plasma FSH, however, was normal in both age groups, suggesting that inhibin production from tfm testes was relatively unaffected.Intratesticular concentrations of A were greatly elevated in the tfm animals at both ages whilst the testicular levels of T were highly reduced when compared to NL rats. This indicates a gonadal deficiency of the 17β-hydroxy steroid dehydrogenase (17β-HSD). Furthermore, the relatively low T: DHT ratio and high concentrations of Adiol in the tfm testes confirmed previous reports that the high 5α-reductase typical for the immature rat testis is maintained into adulthood in rats with the tfm condition. A considerable degree of peripheral conversion (A → T) probably helps to maintain normal or supranormal plasma levels of T. Gonadectomy rendered all plasma androgens undetectable, indicating that the adrenal contribution was negligible.Increasing age (350 days) was associated with a marked increase in circulating androgens in tfm rats. This is probably the reason for the observed significant reduction in circulating LH in this age group when compared to the 160-day animals. The aging tfm rat is predisposed to testicular tumors which, on the basis of histology, specific [¹²⁵I]hLH binding and in vitro responsiveness to hCG, appear to be of Leydig cell origin.Microflow fluorometry (MFF) of tfm testis cell suspensions revealed the presence of haploid cells, suggesting that meiosis proceeds to a limited extent.Plasma corticosterone levels in the tfm rat were normal although plasma CBG levels were highly significantly elevated at both ages when compared to the levels in NL rats. We suggest that the adrenal hyperplasia observed in tfm rats is secondary to reduced corticosterone production and diminished free corticosterone in the circulation.     

Androgens alter brain catecholamine content and blood pressure in the testicular feminized male rat

Androgens interact with catecholamines in the central nervous system (CNS) to regulate many physiological processes including blood pressure (BP). To test the hypothesis that testosterone (T) and 5a-dihydrotestosterone (DHT) modulate CNS catecholamines and BP through androgen receptor (AR)-dependent and independent mechanisms, we used the testicular feminized male (Tfm) rat. Females that carry the AR mutation (Tfm mutation) on the X chromosome were bred with spontaneously hypertensive rat (SHR) males. The normal AR male and Tfm offspring were divided into groups: control, castrated, castrated, and T or (DHT) replacement. In both AR normal and Tfm males, BP was reduced by castration, but T restored BP in both groups. In the amygdale, castration decreased dopamine (DA) in both strains and both T and DHT restored it. In the bed nucleus of the stria terminalis castration increased DA which was further increased by DHT and reduced to normal by T in both strains. In the frontal cortex, castration reduced DA content in both strains but only T restored it to normal in SHR but not in Tfm. Brain norepinephrine (NE) content showed a significant strain effect for the preoptic area (POA), but no treatment effect. Although castration did not change NE in the amygdala or POA in either strain, both T and DHT increased NE in the Tfm castrates. Blood pressure was influenced by T manipulation and correlated most significantly with DA content in the amygdala, frontal cortex, and stria terminalis. These data demonstrate an action of androgen on brain catecholamines and BP, which is independent of the classic androgen receptor.     

Pituitary regulation of Leydig cell function in the adult male rat.

The effects of hypophysectomy on serum testosterone, 125I-labelled hCG binding to testicular membranes and on testicular responsiveness were studied in adult rats. Serum testosterone decreased rapidly over the first 6 h after hypophysectomy. LH receptors were determined (pmol/testis) by measuring the specific binding of 125I-labelled hCG in membrane preparations of testes of rats hypophysectomized 1, 2, 3, 6, 9, or 15 days earlier. Hypophysectomy did not result in a decrease in 125I-labelled hCG binding on day 1 but this had decreased to 40% of that in intact controls by day 2. A gradual decline was found between days 2 and 6 at which time hCG binding had decreased to 15%. No further decrease occurred between days 6 and 15. Scatchard analysis indicated that the decline in hCG binding was due to a decreaffinity. FSH, testosterone, dihydrotestosterone, and oestradiol were unable to prevent the decline in hCG binding. Although serum testosterone, testicular testosterone content, and 125I-labelled hCG binding decreased rapidly after hypophysectomy, testicular responsiveness to LH was biphasic. The intraperitoneal administration of 25 microgram LH 2 h before decapitation increased testosterone in the circulation to a greater extent extent in animals hypophysectomized for 1 day than in intact controls while hCG binding affinities and capacities had not changed. Two or three days after hypophysectomy testicular responsiveness to LH was similar to that of intact controls even though hCG binding in hypophysectomized animals had decreased to 40 and 28% of intact controls respectively. It is concluded that (1) the testis is dependent on anterior pituitary hormones for maintenance of testicular LH receptors and testosterone secretion, (2) FSH, testosterone, dihydrotestosterone, or oestradiol cannot prevent the decline in testicular LH receptors resulting from hypophysectomy, and (3) steroidogenic capacity of the testis persists significantly longer than the hCG binding capacity of the testis.     

Growth hormone secretion by individual somatotropes of the testicular feminized rat

To investigate the cellular mechanisms underlying the unique GH secretory apparatus of the androgen-resistant testicular feminized (Tfm) rat we employed a reverse hemolytic plaque assay to assess GH secretion by individual cells from normal male, normal female, and Tfm rats. Acutely dispersed pituitary cells were incubated for 90 min with GH anti-serum in the presence of medium alone, 0.01, 0.1, 1, 10, or 100 nM GHRH, or 3 microM forskolin after which hemolytic plaques were developed over an additional 30 min. Body weights of the Tfm rats [318 +/- 7 g (mean +/- SEM)] were intermediate between intact males (372 +/- 18 g) and females (218 +/- 7 g). The total number of cells recovered from dispersion of Tfm rat pituitaries [3.20 +/- 0.42 X 10(6) (mean +/- SEM)] was greater than that from males (1.43 +/- 0.12 X 10(6); P = 0.001), but not distinguishable from that from females (2.31 +/- 0.30 X 10(6); P = 0.06). However, the absolute population of recovered somatotropes from the Tfm animals (1.24 +/- 0.22 X 10(6) exceeded both male (0.56 +/- 0.10 X 10(6); P = 0.002) and female (0.80 +/- 0.14 X 10(6); P = 0.046) values. Mean basal and maximal GH plaque areas were greater for cells from male rats than for those from either female or Tfm rats (P less than 0.05) regardless of whether GHRH or forskolin was used as the secretagogue. Plaque areas from female and Tfm cells were indistinguishable under all study conditions. These data suggest that a deficiency of androgen receptors prevents establishment of the greater GH secretory capacity of individual somatotropes characteristic of the adult male rat. This androgen receptor-dependent modulation of GH secretory capacity appears to occur at a step distal to the GHRH receptor. The data also suggest that an increase in the absolute population of somatotropes is an additional consequence of androgen receptor deficiency. This combination of individual somatotropes, each possessing a GH secretory capacity similar to that of cells from normal females, but present in greater absolute numbers, may explain the intermediate values found during previous studies of the Tfm rat GH axis which were based on assessment of large mixed populations of pituitary cells.     

Leydig cell responsiveness with germinal cell resistance to gonadotropin therapy in Kallman's syndrome.

Patient's with Kallman's syndrome have been divided into gonadotropin-sensitive gonadotropin-resistant types. This has been based on the testosterone response of the Leydig cells to human chorionic gonadotropin (HCG) and the resultant sexual characteristics. Whether the germinal epithelium is similarly sensitive has not been previously assessed. The present study was set up to see if a male with anosmia and hypogonadotropic hypogonadism could be made fertile by treating with human menopausal gonadotropins (HMG) in combination with HCG, a regime previously found effective in other types of hypogonadotropic hypogonadism.The patient was previously shown to be HCG-responsive by the induction of secondary sexual characteristics following gonadotropin therapy. This was confirmed by measuring serum testosterone levels before and after the administration of HCG. Therapy with HMG, 75 IU intramuscularly, and HCG, 2,000 intramuscularly three times a week, was started. After six months, despite perfectly normal secondary sexual characteristics and near normal-sized testes, he still showed azoospermia. His HMG was increased to 150 intravenously thrice weekly. After an additional two months of therapy, his count was still zero.A testicular biopsy was performed and disclosed Leydig cell hyperplasia but very little active spermatogenesis. Although this man was gonadotropin-sensitive as far as his Leydig cells are concerned, his germinal epithelium was resistant. Thus, HCG sensitivity does not ensure fertility.     

Pulmonary sarcoidosis associated with Leydig cell testicular neoplasm

The first case of association between Leydig cell testicular tumor and sarcoidosis is reported. From a review of the literature, this is the ninth case of association between a testicular tumor and Besnier's disease. Lung biopsy should always be performed in patients with testicular cancer when retroperitoneal lymph node involvement cannot be demonstrated in order to avoid unnecessary antineoplastic chemotherapy.     

Do testicular opiates regulate Leydig cell function?

beta-Endorphin is believed to be synthesized in testicular Leydig cells. To gain more information about the role of this and other endogenous opioid peptides in the testis, opiate antagonists (naloxone and nalmefene, 100 micrograms/testis) were administered intratesticularly to hemicastrated adult rats. Leydig cell function was evaluated by measurement of serum testosterone and testosterone production in vitro. Estimation of androgen binding protein (rABP) was used as an index of Sertoli cell function. Serum testosterone was reduced significantly by intratesticular administration of naloxone and nalmefene in treated animals. Systemic administration of these antagonists had no effect at the doses used. Testes from treated animals incubated in vitro with or without hCG produced significantly less testosterone than vehicle-treated control testes. Hemicastration reduced rABP synthesis and secretion; however, treatment with opiate antagonists did not alter the amount of this protein in the serum or epididymides of these rats. These observations suggest that endogenous testicular opiates modulate testosterone secretion by Leydig cells.     

Leydig cell responsiveness to single and repeated human chorionic gonadotropin administration.

A single im injection of 1500 IU hCG significantly increased plasma testosterone levels for at least 96--120 h in normal men (n = 7), patients with isolated gonadotropin deficiency (n = 6), and boys with delayed puberty (n = 7); the maximum values [1315 +/- 309, 370 +/- 177, and 963 +/- 249 ng/100 ml (mean +/- SD), respectively] were achieved after 72 h in each group. Repeated daily injections of 1500 IU hCG for 3 days increased plasma testosterone levels in the same subjects at 72 h after the start to levels (1342 +/- 412, 407 +/- 199, and 1052 +/- 449 ng/100 ml, respectively) similar to those found in the single dose experiment. The levels achieved at 24 and 48 h also did not differ significantly in the two experiments. The data indicate the lack of additional leydig cell stimulation by repeated hCG injections given within 48 h after a single dose.     

Adult rat Leydig cell cultures: minimum requirements for maintenance of luteinizing hormone responsiveness and testosterone production.

The aim of this study was to establish the minimum conditions required to maintain adult rat Leydig cell testosterone production and luteinizing hormone (LH) responsiveness in short term culture, at a level similar to that observed in vivo, which could be used to study factors which may have a delayed or chronic effect on Leydig cell function. Percoll gradient-purified adult rat Leydig cells (5.0 x 10(4)/250 microliters) were cultured in Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12) with 0.1% bovine serum albumin at 32 degrees C for up to 3 days, with daily medium changes. A combination of submaximal rat LH (0.1 ng/ml) and a maximal concentration of rat serum lipoproteins (0.5 mg/ml) maintained testosterone production at between 5 and 15 ng/10(6) cells/h; subsequent stimulation of the Leydig cells with a maximum dose of rat LH (8 ng/ml) over 24 h resulted in testosterone production of 75-240 ng/10(6) cells/h on all 3 days of culture. However, the addition of 0.1% fetal calf serum instead of rat lipoproteins could not maintain LH-stimulated testosterone production in the same culture period. In cultures containing submaximal LH and lipoproteins, levels of testosterone production and responses to maximal LH stimulation were constant over the culture period when expressed as either testosterone production per 10(6) cells plated, or testosterone production per microgram DNA recovered at end of incubation. Reduction of the oxygen tension from 19% to 5%, or to 1% did not significantly alter testosterone production by Leydig cells under these established conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leydig cell hypoplasia: a cause of male pseudohermaphroditism.

A patient with male pseudohermaphroditism is presented whose testicular biopsy showed marked paucity of the Leydig cell populations to which is attributed testosterone deficiency and impaired spermatogenesis.     

Destruction of testicular Leydig cells reveals a role of endogenous inhibin in regulating follicle-stimulating hormone secretion in the adult male rat

It has previously been demonstrated that passive immunoneutralization of endogenous inhibin results in a dramatic elevation in follicle-stimulating hormone (FSH) secretion in the adult female rat but not in the adult male. The purpose of the present study was to investigate whether the effects of immunoneutralizing endogenous inhibin on FSH secretion in the adult male rat might be masked by the presence of additional, compensating, FSH-suppressing factors. This was determined by examining the individual and combined effects of removing the testicular influences provided by the Leydig cells using the selective toxicant, ethane dimethane sulfonate (EDS), and passive immunoneutralization of endogenous inhibin. Within 24 h of a single i.p. injection of EDS, plasma testosterone levels were lowered to near assay limits and by 3 days were undetectable. Plasma FSH levels were significantly elevated 3 and 7 days after EDS treatment, but not to the levels observed in rats castrated for similar periods of time. Castration of rats, treated 3 days earlier with EDS, resulted in a further significant increase in FSH secretion as compared with EDS-treated, sham-operated controls, indicating that the testes were providing an additional FSH-suppressing factor(s) other than those originating in the Leydig cells. Injection of anti-inhibin serum, into rats treated 3 or 7 days earlier with EDS, induced a further significant increase in FSH secretion that raised plasma FSH to a level comparable to that observed in male rats castrated for similar periods of time. Plasma LH secretion was also dramatically elevated by EDS treatment to levels that equaled or exceeded those observed in similarly timed castrates. Pituitary sensitivity, as tested by the injection of an exogenous challenge of luteinizing hormone-releasing hormone (LHRH), was significantly increased 3 or 7 days after either EDS treatment or castration in terms of LHRH-stimulated LH release, but not in terms of LHRH-stimulated FSH release. Immunoneutralization of endogenous inhibin induced no further observable changes in pituitary sensitivity to LHRH. These results demonstrate that in the absence of the Leydig cells a secondary role is revealed for endogenous inhibin in suppressing FSH secretion that, in combination with the Leydig cell influence(s), accounts for the postcastration increase in FSH. The need to remove the Leydig cell influence(s) to reveal an effect of endogenous inhibin on FSH secretion in the adult male rat may suggest that the inhibin effect is normally masked by the presence of the comparatively larger suppressive influence(s) derived from the Leydig cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Leydig cell apoptosis after the administration of ethane dimethanesulfonate to the adult male rat is a Fas-mediated process.

Leydig cells undergo apoptosis in response to the cytotoxin ethane dimethanesulfonate (EDS), with numbers declining at 12-18 h and maximal apoptosis at 24 h postinjection. The Bcl-2 family members, Bcl-2, Bcl-xl, and Bax, appear not to be involved in this process. To further investigate this phenomena, a single dose of EDS was administered to adult rats to induce the killing of Leydig cells. The interstitial cells were examined up to 3 days after EDS administration by Western blot analysis for the Bcl-2 family members (Bak and Bcl-w). Western blotting showed that Bak expression in the interstitial cell preparations was unchanged after EDS, and immunohistochemistry showed that it was not up-regulated in Leydig cells in response to EDS. Bcl-w expression in the Leydig cells and interstitial cell preparations was unchanged until 48 h when it became undetectable, suggesting that Leydig cell-associated Bcl-w is not involved in initiating apoptosis. We also investigated the role of the Fas system in Leydig cell apoptosis. Both Fas receptor and Fas ligand protein levels increased after EDS, peaking at 12-18 h and declining thereafter. Fas receptor and ligand were shown by immunohistochemistry to be present in Leydig cells, and after EDS all Leydig cells became strongly positive for both proteins. The intensity of staining increased in the early stages of apoptosis and decreased as the nuclear morphology became more fragmented. These data suggest that Bcl-2 family members are not involved in Leydig cell apoptosis after EDS administration. However, up-regulation of the Fas system does occur, implicating activation of Fas receptor in the induction of Leydig cell apoptosis.     

Increase in Leydig cell responsiveness in the unilaterally cryptorchid rat testis and its relationship to the intratesticular levels of testosterone

Adult rats were made unilaterally cryptorchid (UCD) and 6-7 weeks later Leydig cells were isolated from the scrotal and abdominal testes and their capacity to secrete testosterone in vitro was compared. Basal testosterone production by Leydig cells from the abdominal testes of UCD rats was lowered, compared with cells from the contralateral scrotal testes, whilst their responsiveness to both human chorionic gonadotrophin and an LH releasing hormone agonist was enhanced two- to threefold (P less than 0.001) compared both with cells from the contralateral scrotal testes and with cells isolated from untreated rats of the same age. In the UCD rats, concentrations of testosterone in testicular interstitial fluid (IF) were reduced (P less than 0.001) by 70-90% in abdominal, compared with scrotal, testes. A similar reduction was evident in the levels of testosterone in spermatic venous blood, and both this decrease and that in IF levels of testosterone varied according to the degree of testicular involution. The ontogeny of the above changes was investigated. After induction of unilateral cryptorchidism, the weight of the abdominal compared with the scrotal testis declined slowly, such that by day 5 there was only a 25% reduction in weight compared with a 70% reduction by day 40. In contrast, the levels of testosterone in IF from abdominal testes declined rapidly, such that by day 5 an 80% reduction was attained, compared with scrotal testes, with little further change by day 40.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leydig cell hyperplasia and the maintenance of bone volume: bone histomorphometry and testicular histopathology in 29 male leprosy autopsy cases

This study was conducted to determine if osteoporosis in male leprosy patients is caused by testicular atrophy. Bone volume (BV/TV), trabecular number (TbN), trabecular thickness (TbTh), and trabecular separation (TbSp) were measured in two areas in decalcified paraffin sections of lumbar bones from 29 male leprosy and 6 male nonleprosy autopsy cases. We found significant differences in the average BV/TV measurements among the 7 patients with nodular Leydig cell hyperplasia (BV/TV 12.24%) and the 22 patients without hyperplasia (BV/TV 7.35%) and 6 patients without leprosy (BV/TV 12.98%). Bone volume was maintained in patients with nodular Leydig cell hyperplasia, and we determined no clinical factor other than the Leydig cell hyperplasia that reflected the bone volume. The osteoporosis of male leprosy patients was attributed to secondary gonadal dysfunction due to testicular atrophy, and Leydig cell hyperplasia appears to preserve bone volume.     

Growth hormone-secretory patterns in androgen-resistant (testicular feminized) rats

To investigate the role of androgen receptors in the expression of the male GH-secretory pattern in adult rats, the GH-secretory patterns in androgen-resistant (testicular feminized) rats were compared with their normal male and female littermates. All animals were prepared with intraatrial Silastic catheters and bled every 15 min for 8 h (0800-1600 h). Normal male littermates displayed a characteristic low frequency, high amplitude pattern of GH secretion with bursts of GH occurring every 2.5-3 h and separated by prolonged trough periods where GH values remained low or undetectable (less than 5 ng/ml) for 45-90 min. Normal female littermates showed a characteristic high frequency, low amplitude pattern of GH secretion with pulses of GH occurring every hour. Compared to normal male littermates, females had lower individual GH peak amplitudes and shortened GH-trough periods which contain higher GH levels. GH-secretory profiles displayed by testicular-feminized animals qualitatively and quantitatively resembled those of the normal female littermates. These data suggest that androgen receptors are necessary for the expression of masculine GH-secretory patterns.     

A Leydig cell stimulatory factor produced by human testicular tubules

It is demonstrated that tubular fragments derived from human testes and cultured in vitro produce a factor that stimulates the production of testosterone by human interstitial cells and by Percoll-purified Leydig cells from rat and mouse origin. The active principle in the conditioned media is a thermo-labile and trypsin-sensitive protein with an MW greater than 10,000. The factor is active in the presence as well as in the absence of maximally effective concentrations of LH and its activity is not accompanied by measurable changes in cAMP production. There are several points of analogy between this factor and a Leydig cell stimulatory protein produced by rat Sertoli cells. Molecular weight fractionation of spent media from human testicular tubules using an Amicon ultrafiltration system results in a 38- to 102-fold increase in Leydig cell stimulatory activity in a fraction corresponding to a molecular weight of 10,000 up to 30,000. These figures are comparable to those observed after molecular weight fractionation of spent media from rat Sertoli cells. Dose-response curves with partially purified preparations from human and rat origin yield parallel dose-response curves. In rat Sertoli cells as well as in human testicular tubules, the production of the active principle is stimulated by FSH and dibutyryl cAMP. Finally, maximally effective concentrations of the active principles of human and rat origin display no additive effects whereas additive effects are clearly evident with other Leydig cell stimulatory factors such as LHRHa and EGF. The hypothesis is advanced that the Leydig cell stimulatory factors from tubular origin may act as paracrine regulatory molecules responsible for the effects of FSH on Leydig cell function.