Pituitary and Leydig cell function in boars actively immunized against gonadotrophin-releasing hormone

Crossbred boars were (a) immunized against GnRH conjugated to human serum globulin (200 micrograms GnRH-hSG) in Freund's adjuvant at 12 weeks of age and boosted at weeks 18 and 20 (N = 10), (b) served as controls and received hSG only in adjuvant (N = 10), or castrated at weaning (N = 10). At 24 weeks of age (immediately before slaughter), the boars were challenged with saline or pig LH (1 microgram/10 kg body weight). After slaughter, fresh testicular fragments were incubated with pig LH (0.05 and 0.2 ng/2 ml medium) to assess the effects of immunization on Leydig cell function. Pituitary contents of LH and FSH, and testicular LH receptor content were also measured. The results indicated that plasma LH and testosterone concentrations, pituitary LH content, testicular LH receptor content, testis and sex accessory organ weights were significantly reduced in GnRH-immunized boars compared to hSG-adjuvant controls. However, plasma and pituitary FSH content were not affected by high antibody titres generated against GnRH. The testicular testosterone response to exogenous LH in vivo and in vitro was significantly reduced (P less than 0.05) in GnRH-immunized boars. These results indicate that active immunization against GnRH impairs pituitary and Leydig cell functions in boars.     

Morphofunctional alterations in testicular cells of deslorelin-treated boars: an immunohistochemical study

In this study we thoroughly scrutinized testes morphology and investigated whether treatment of recipient boars with gonadotropin-releasing hormone (GnRH)-agonist deslorelin could alter the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), luteinizing hormone receptors (LHRs), and androgen receptors (ARs) in testicular cells. An implant containing 4.7 mg of the GnRH-agonist deslorelin was subcutaneously inserted into crossbred male pigs at 91 and 147 days of age. Testicular traits, morphology of the testes, the proteins' expression, and testosterone concentration in blood plasma were analyzed in all boars after slaughter at 175 days of age. Histological analysis revealed significant alterations in both the interstitial tissue and seminiferous tubules of experimental animals after 28 and 84 days of deslorelin treatment. The intensity of the AR immunostaining within the testis appeared as a function of the severity of testicular dysgenesis. Time-dependent action of deslorelin on the expression of LHR and 3beta-HSD in Leydig cells was also detected. Staining for LHR and 3beta-HSD was very weak or the Leydig cells were immunonegative. Concomitantly, a significant decrease in plasma testosterone level was found in both groups of deslorelin-treated boars when compared with the control group. This is the first report showing the cellular distribution of AR, LHR, and 3beta-HSD in testicular cells of deslorelin-treated boars. It is concluded that morphological and immunohistochemical studies are important for the evaluation of testicular histoarchitecture and steroidogenic function. Subsequently, the endocrine control of reproduction in the GnRH-agonist deslorelin-treated males will be better understood.     

Effect of testosterone on testicular steroidogenesis in the hypogonadal (hpg) mouse

Previous studies have shown that androgens have direct inhibitory effects on steroidogenesis in active Leydig cells. It is not clear what effect androgens have on inactive Leydig cell either through direct action on the cell itself or indirectly through stimulation of Sertoli cell activity. The hpg mouse has undetectable levels of circulating gonadotrophins and the gonads fail to develop post-natally. The effect of androgen treatment on testicular steroidogenesis and morphology was examined in these animals. Treatment with testosterone propionate for two weeks significantly increased testicular and seminal vesicle weight. Seminiferous tubules showed marked development in androgen-treated animals, indicating increased Sertoli cell activity, but the abnormal Leydig cell morphology of the hpg testis was unchanged. Androgen production per testis in vitro was low in control hpg animals and remained unaffected by treatment with androgen. Similarly, the pattern of [3H]pregnenolone metabolism was not significantly affected by androgen treatment. The androgen content of the testis was higher in androgen-treated animals but this could be accounted for by uptake of administered steroid from the circulation. It is concluded that androgens have no direct trophic effect on Leydig cells and that stimulation of Sertoli cell activity is not, in itself, sufficient to affect Leydig cell function.     

Inducible nitric oxide synthase in the rat testis: evidence for potential roles in both normal function and inflammation-mediated infertility

In vitro data have indicated that nitric oxide (NO) inhibits Leydig cell testosterone production, suggesting that NO may play a role in the suppression of steroidogenesis and spermatogenic function during inflammation. Consequently, we investigated expression of the inflammation-inducible isoform of NO synthase (iNOS) in the inflamed adult rat testis and the ability of a broad-spectrum inhibitor of NO production, L-nitro-L-arginine methyl ester, to prevent Leydig cell dysfunction during inflammation. Unexpectedly, immunohistochemical and mRNA data established that iNOS is expressed constitutively in Leydig cells and in a stage-specific manner in Sertoli, peritubular, and spermatogenic cells in the normal testis. Expression was increased in a dose-dependent manner in all these cell types during lipopolysaccharide (LPS)-induced inflammation. In noninflamed testes, treatment with the NO synthase inhibitor reduced testicular interstitial fluid formation and testosterone production without any effect on serum LH levels. Administration of the inhibitor did not prevent the suppression of testicular interstitial fluid and testosterone production that occurs within 6 h after LPS treatment. Collectively, these data indicate a novel role for iNOS in autocrine or paracrine regulation of the testicular vasculature, Leydig cell steroidogenesis, and spermatogenesis in the normal testis. The data suggest that increased NO is not the major cause of acute Leydig cell dysfunction in the LPS-treated inflammation model, although a role for NO in this process cannot be excluded, particularly at other time points. Moreover, up-regulation of iNOS may contribute to the seminiferous epithelium damage caused by LPS-induced inflammation.     

Effects of anabolic steroid (19-nortestosterone) on the secretion of testicular hormones in the stallion.

The aim of this study was to clarify the effect of anabolic steroids on the testicular endocrine function of mature stallions. Mature thoroughbred stallions were treated with 800 mg nandrolone decanoate every 3 weeks for 3 months. After the first treatment, plasma concentrations of LH, immunoreactive inhibin and testosterone decreased rapidly to the nadir. These hormones were maintained at significantly lower concentrations compared with concentrations in intact stallions. Histology of the testicular tissue indicated the arrest of advanced spermatogenesis in the seminiferous tubules and a severe depletion of the number of Leydig cells in the interstitial compartment as a result of treatment. Most of the immunopositive cells for the inhibin alpha-subunit and steroidogenesis enzymes in the interstitial compartment decreased below detectable amounts, whereas immunopositive reactions of inhibin alpha-subunit in the seminiferous tubules were clearly observed. In conclusion, the treatment of mature stallions with nandrolone decanoate caused a decrease in the secretion of ir-inhibin and testosterone from the testis, the depletion of the number of Leydig cells and a decrease below detectable amounts of inhibin alpha-subunit and steroidogenesis enzymes. The concentration of ir-inhibin in the peripheral blood may be a useful marker for the examination of testicular activity in stallions being treated with anabolic steroids.     

Limitations imposed by testicular blood flow on the function of Leydig cells in rats in vivo

Testis blood flow per testis closely follows testis weight in rats made aspermatogenic by a single exposure of the testis to 43 degrees C for 30 min or 500 rad (5 Gy) of irradiation from a caesium source, or following ligation of the efferent ducts. Aspermatogenesis following these treatments was associated with only minor changes in the concentrations of testosterone in peripheral blood before stimulation with human chorionic gonadotrophin (hCG), and a reduced responsiveness to hCG when testis weight had fallen after heating. The concentrations of testosterone in testicular venous blood was normal or above normal during aspermatogenesis resulting from heat or irradiation, and only slightly reduced following efferent duct ligation. Consequently testosterone production (defined as the product of plasma flow and the veno-arterial concentration difference for testosterone) was markedly reduced during aspermatogenesis, both before and after stimulation with hCG. It appears that the reduced blood flow limits the amount of testosterone leaving the testis, and while the Leydig cells are capable under some circumstances of compensating partially for this fall by increasing the concentration of testosterone in the testicular venous blood, this compensation is not complete when there are severe reductions in blood flow. Therefore one can conclude that the mass of the tubules is the main determinant of testis blood flow and the Leydig cells must manage with what the tubules require.     

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.     

Sertoli cells in the boar testis: changes during development and compensatory hypertrophy after hemicastration at different ages

Changes in Sertoli cell numbers and testicular structure during normal development and compensatory hypertrophy were assessed in crossbred Meishan x White Composite males. Boars were assigned at birth to unilateral castration at 1, 10, 56, or 112 days or to remain as intact controls through 220 days. The first testes removed were compared to assess testicular development. At 220 days, testicular structure was evaluated in boars representing the 25% with the largest (Lg) testis and the 25% with the smallest (Sm) testis in each treatment group. The number of Sertoli cells per testis reached a maximum by Day 56 in Sm testis but not until Day 112 in Lg testis boars, indicating a longer duration of Sertoli cell proliferation in Lg testis boars. Unilateral castration of Lg testis boars on Days 1, 10, 56, and 112 caused the weight of the remaining testis to hypertrophy by 149%, 135%, 119%, and 120%, respectively, and total sperm production to increase to 127%, 128%, 97%, and 106%, respectively. However, Sertoli cell numbers changed little in hemicastrate boars. In Lg testis boars, compensatory hypertrophy primarily involved proliferation of Leydig cells and expansion of existing Sertoli cells with little increase in Sertoli cell numbers, but in Sm testis boars, it involved expansion of existing Leydig and Sertoli cells without increase in cell numbers. These results indicate that Lg and Sm testis boars display intriguing differences during both development and compensatory hypertrophy, and they identify a unique animal model for further studies of factors that program and control Sertoli cell proliferation.     

Immunolocalization of androgen receptors in testicular cells of prepubertal and pubertal pigs

In the testis, androgen receptors are known to mediate autocrine and paracrine effects of androgens on Leydig cell function and spermatogenesis. The pig presents some unusual features with regard to the synthesis of testosterone and estrogens in the male gonads. In testes from prepubertal males, testosterone level was lower than in testes from adult boars, while estrogen secretion was relatively high and comparable to that of mature porcine gonad. Immunolocalization of androgen receptors and intensity of immunohistochemical staining was age-dependent. In testis sections from adult boars, androgen receptors were found in nuclei of all somatic cells such as Leydig cells, Sertoli cells, and peritubular-myoid cells, whereas in sections from immature pigs only in the Leydig cell cytoplasm showed positive immunoreaction for androgen receptors. In control tissue sections incubated with omission of the primary antibody, no positive staining was observed. Detection of the androgen receptors in testicular cells of the pig is important for understanding of their central role in mediating androgen action.     

Early sexual maturity in local boars of Northeastern India: age-related changes in testicular growth, epididymal sperm characteristics and peripheral testosterone levels

The present study reports the age related changes in the peripheral testosterone levels, testicular and epididymal growth and development and cauda epididymal spermiogram in local pigs of Northeastern India, which attain sexual maturity around 3 months of age. Local boars (n = 20) were castrated at monthly intervals from 2 to 6 months of age (4 boars per month) to study the testicular growth and development and the epididymal spermiogram. Blood samples, collected from local boars (n = 6) at monthly intervals from 2 to 6 months of age, were analyzed for testosterone levels by radioimmunoassay. Compared to Hampshire boars, significantly (P < 0.05) high testosterone levels were observed in the local boars as early as 2 months of age. The mean (± SEM) level of testosterone in the local boars at 2, 3 and 4 months of age was 11.89 ± 1.52, 20.45 ± 1.33 and 20.38 ± 2.0 ngml⁻¹, respectively. Though there was consistently significant (P < 0.05) difference in the body weight between Hampshire and local pigs, the same was not observed in case of testicular weight except at 3 and 6 months of age. In line with the above observation, the testis:body weight ratio (gram testis per kg body weight) was significantly (P < 0.05) higher in the local boars compared to the Hampshire boars at any time of observation, which ranged from 0.8 to 1.0 in case of Hampshire and from 2.3 to 3.0 in local boars. The sperm concentration in the cauda epididymal fluid of local boars at 2, 3 and 6 months of age was 2255 ± 186.6, 3685 ± 103.8 and 4325 ± 146.2 million/ml, respectively and the sperm motility, viability and total abnormality was 73.3, 75.2 and 6.2%, respectively at 3 months of age. Taken together, the testosterone level, testicular growth and development and epididymal spermiogram indicate the trait of early sexual maturity in the local pigs as compared to Hampshire.     

Effect of surgery and efferent duct ligation on testicular blood flow and testicular steroidogenesis in the rat

A moderate reduction in testicular blood flow was observed in both testes 24 h after unilateral efferent duct ligation without any corresponding change in testosterone secretion as indicated in the peripheral blood, in testicular venous blood, or in testicular tissue fluid. At 21 days a pronounced unilateral decrease in blood flow was associated with the extensive degeneration of tubules in the testis on the ligated side. These changes were also associated with decreased testosterone secretion by the testis on the ligated side, although Leydig cell function was not abolished since testosterone in the tissue increased rather than decreased. It is therefore concluded that testicular blood flow may play an important role in the changes of testosterone secretion that follow unilateral efferent duct ligation.     

Alteration in expression of estrogen receptor isoforms alpha and beta, and aromatase in the testis and its relation with changes in nitric oxide during aging in mice

The aim of present study was to investigate the changes in the testicular expression of aromatase, ER alpha, ER beta and iNOS protein and correlate these with serum testosterone and nitric oxide levels, to elucidate the role of estrogen and nitric oxide in the testis during aging. This study showed localization of aromatase and ER alpha mainly in the Leydig cell and showed close correlation of testicular aromatase level with circulating testosterone level suggesting that estrogen may be modulating testicular steroidogenesis. Localization ER alpha mainly in the mitotically active germ cell suggest possible role of estrogen in germ cell proliferation. This study showed basal level of nitric oxide during reproductively active period, whereas increased serum nitric oxide coincides with decreased testicular activity in old age. This study showed inverse correlation between aromatase and NO level. Treatment with either SNP or L-NAME on testicular steroidogenic factor (3-beta HSD/ StAR) or germ cell survival factor (Bcl2) showed that increased NO causes decreased steroidogenesis and increased germ cell apoptosis. In conclusion this study suggest that estrogen modulate steroidogenesis and germ cell survival in reproductively active period whereas in old age decreased estrogen concentration causes increased nitric oxide which in turn decreases testicular steroidogenesis and germ cell apoptosis.     

Testicular lipids in mice with testicular feminization

Morphological, histochemical and biochemical studies of the testis of mice with testicular feminization (tfm/y) reveal a large accumulation of lipids in Leydig cells and in Sertoli cells. In Leydig cells of tfm/y mice, lipid droplets do not exhibit the special relationship with smooth endoplasmic reticulum that exists in normal adult Leydig cells. Compared to the surgically-cryptorchid control, the tfm/y testis contains more lipid in Leydig cells but less in Sertoli cells. There are also quantitative differences in testicular lipids in tmf/y and normal testes but no significant differences were noted between tfm/y and surgically-cryptorchid testes. The testes of both the genetically defective and surgically-cryptochid animals contain increased amounts of total lipids and phospholipids, and of free and esterified cholesterols. Exogenous testosterone has no effect on lipids or other characteristics of these cells. The present results suggest that the increased lipids in tfm/y mice result from a genetic disorder that asserts itself (1) in Leydig cells where it is associated with, and is probably a result of, impaired lipid metabolism and steroidogenesis, and (2) in Sertoli cells where it is perhaps attributable to arrested spermatogenesis and impaired steroidogenesis.     

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.     

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.     

Expression of FGF2 and TGFalpha and testis morphology during testicular hypertrophy subsequent to hemicastration in the neonatal boar

The objective was to ascertain fibroblast growth factor-2 (FGF2), epidermal growth factor (EGF), and transforming growth factor-alpha (TGFalpha) mRNA expression and testis morphology during accelerated testicular growth after hemicastration in the neonatal boar. On Day 10 after birth (Day 0), boars were assigned to control (n = 28), no treatment; hemicastrated (n = 28), left testis removed. The right testis in both groups (n = 7) was removed on Days 5, 10, 15, and 20. Expression of mRNA for FGF2, EGF, and TGFalpha was determined by qRT-PCR using TaqMan. Testicular morphology was determined on Day 15. On Day 10, hemicastrated boars had a greater (P = 0.01) testis weight (6.2 +/- 0.8 g; mean +/- SEM) than controls (4.3 +/- 0.4 g) and on Day 15 testis weight in hemicastrated boars (8.8 +/- 0.8 g) was twice (P < 0.01) that of control boars (4.2 +/- 0.3 g). Seminiferous tubule volume was approximately doubled in hemicastrated boars (P < 0.01) and was associated with an increase (P < 0.01) in Sertoli cell number. Interstitial compartment volume was greater (P < 0.01) in hemicastrated boars. Leydig cell numbers were similar (P = 0.14) but volume was greater (P < 0.01) for hemicastrates. There were no differences (P > 0.05) between control and hemicastrated boars in TGFalpha or FGF2 expression on Day 5 or Day 10, and EGF was not detected. It was concluded that upregulation of TGFalpha or FGF2 expression is not a pre-requisite for enhanced testicular growth and increased Sertoli cell proliferation that occurs subsequent to hemicastration in the neonatal boar.     

In vitro effects of Celiptium and MR 14504 on mature rat Leydig cell testosterone production

Percoll-purified mature rat Leydig cells have been used to evaluate the testicular toxicity of two highly potent intercalating agents (Celiptium and MR 14505). Testosterone secretion in the absence and in the presence of human chorionic gonadotropin (hCG) was measured to assess Leydig cell function. Celiptium and MR 14504 induce time- and dose-related inhibitory effects on the production of testosterone by Leydig cells, both in the presence and in the absence of hCG, whatever the concentration of hCG used. We have observed that MR 14504 is about 5 times more potent as an inhibitor of rat Leydig cell steroidogenesis than Celiptium without inducing any cell toxicity. The present study indicates that the Leydig cell is an additional potential site for the primary toxic effects of these drugs in the adult rat testis.     

Androgen action via testicular arteriole smooth muscle cells is important for Leydig cell function, vasomotion and testicular fluid dynamics

Regulation of blood flow through the testicular microvasculature by vasomotion is thought to be important for normal testis function as it regulates interstitial fluid (IF) dynamics which is an important intra-testicular transport medium. Androgens control vasomotion, but how they exert these effects remains unclear. One possibility is by signalling via androgen receptors (AR) expressed in testicular arteriole smooth muscle cells. To investigate this and determine the overall importance of this mechanism in testis function, we generated a blood vessel smooth muscle cell-specific AR knockout mouse (SMARKO). Gross reproductive development was normal in SMARKO mice but testis weight was reduced in adulthood compared to control littermates; this reduction was not due to any changes in germ cell volume or to deficits in testosterone, LH or FSH concentrations and did not cause infertility. However, seminiferous tubule lumen volume was reduced in adult SMARKO males while interstitial volume was increased, perhaps indicating altered fluid dynamics; this was associated with compensated Leydig cell failure. Vasomotion was impaired in adult SMARKO males, though overall testis blood flow was normal and there was an increase in the overall blood vessel volume per testis in adult SMARKOs. In conclusion, these results indicate that ablating arteriole smooth muscle AR does not grossly alter spermatogenesis or affect male fertility but does subtly impair Leydig cell function and testicular fluid exchange, possibly by locally regulating microvascular blood flow within the testis.     

Intratesticular injection of [D-Met2-Pro5]enkephalinamide suppresses testosterone secretion of the testis of immature rat

The possible role of enkephalin in the local control of testicular function was studied in neonatal rats. 5- and 10-day old hemicastrated rats were treated intratesticularly with an enkephalin analog [D-Met2-Pro5]enkephalinamide. In 5-day-old rats local injection of different doses (0.1-0.3 micrograms/testis) of the peptide suppressed basal testosterone secretion in vitro in a dose-dependent manner 2 h posttreatment. Intratesticular administration of naloxone prior to enkephalin treatment prevented the decrease in basal testosterone production induced by the opioid agonist. In 10-day-old animals intratesticular injection of 1.0 and 3.0 micrograms/testis of enkephalinamide reduced serum testosterone concentration and basal testosterone secretion in vitro. Systemic injection of the peptide produced no change in steroidogenesis. These results suggest that enkephalins might be among the intratesticular factors regulating Leydig cell functions.     

Involvement of tumor necrosis factor-alpha in the pathogenesis of autoimmune orchitis in rats

We studied the testicular macrophages of rats with experimental autoimmune orchitis (EAO) and analyzed whether the tumor necrosis factor-alpha (TNFalpha) is involved in germ cell apoptosis and in Leydig cell steroidogenesis. The EAO was induced in adult male Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. In the experimental group, a severe orchitis was observed 80 days after the first immunization. ED1- and ED2-positive macrophages were quantified by immunohistochemistry. The TNFalpha concentration of conditioned media from testicular macrophages (TMCM) was determined by ELISA. The number of apoptotic TNF receptor 1 (TNFR1)-positive germ cells was identified by combining in situ end labeling of apoptotic DNA and immunohistochemical techniques. The effect of TNFalpha on Leydig cell testosterone production was determined by RIA. In rats with EAO, we observed a significant increase in the number of TNFalpha-positive testicular macrophages, the TNFalpha concentration in TMCM, and the number of TNFR1-positive germ cells. Sixty percent of TNFR1-positive germ cells were apoptotic. These results suggest that TNFalpha could be involved in the pathogenesis of EAO. Acting together with other local factors such as Fas-FasL, TNFalpha could trigger germ cell apoptosis. We also demonstrated that TNFalpha inhibited in vitro testosterone production in basal and hCG-stimulated Leydig cells from rats with orchitis.