Chronic corticosterone treatment impairs Leydig cell 11beta-hydroxysteroid dehydrogenase activity and LH-stimulated testosterone production.

The effects of excess corticosterone on luteinizing hormone (LH)-stimulated Leydig cell testosterone production and activity of 11beta-HSD was studied. Adult male rats (200-250 g body weight) were treated with corticosterone-21-acetate (2 mg/100 g body weight, i.m., twice daily) for 15 days. Another set of rats was treated with corticosterone (dose as above) plus LH (ovine LH 100 microg/kg body weight, s.c., daily) for 15 days. Corticosterone administration significantly increased serum and testicular interstitial fluid (TIF) corticosterone but decreased testosterone levels. Administration of LH with corticosterone partially prevented the decrease in serum and TIF testosterone. The oxidative activity of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) was significantly decreased in Leydig cells of rats treated with corticosterone alone and in combination with LH. The direct effect of corticosterone on Leydig cell steroidogenic potency was also studied in vitro. Addition of corticosterone to Leydig cell culture showed a dose dependent effect on LH-stimulated testosterone production. Corticosterone at 50 and 100 ng/ml did not alter LH-stimulated testosterone production, but at high doses (200-400 ng/ml), decreased basal and LH-stimulated testosterone production. Basal and LH-stimulated cAMP production was not altered by corticosterone in vitro. It is concluded from the present study that elevated levels of corticosterone decreased the oxidative activity of 11beta-HSD and thus resulting in impaired Leydig cell steroidogenesis and the inhibitory effects of corticosterone on testosterone production appear to be mediated through inhibition of LH signal transduction at post-cAMP level.     

Chronic administration of corticosterone impairs LH signal transduction and steroidogenesis in rat Leydig cells

The mechanism involved in the inhibitory actions of chronic corticosterone treatment on Leydig cell steroidogenesis was studied in adult Wistar rats. Rats were treated with corticosterone-21-acetate (2 mg/100 g body weight, i.m., twice daily) for 15 days and another set of rats was treated with corticosterone plus ovine luteinizing hormone (oLH) (100 microg/kg body weight, s.c., daily) for 15 days. Chronic treatment with corticosterone increased serum corticosterone but decreased serum LH, testosterone, estradiol and testicular interstitial fluid (TIF) testosterone and estradiol concentrations. Administration of LH with corticosterone partially prevented the decrease in serum and TIF testosterone and estradiol. Leydig cell LH receptor number, basal and LH-stimulated cAMP production were diminished by corticosterone treatment which remained at control level in the corticosterone plus LH treated rats. Activities of steroidogenic enzymes, 3beta- and 17beta-hydroxysteroid dehydrogenase (3beta-HSD and 17beta-HSD) were significantly decreased in corticosterone treated rats. LH plus corticosterone treatment did not affect 3beta-HSD activity but decreased 17beta-HSD activity, indicating a direct inhibitory effect of excess corticosterone on Leydig cell testosterone synthesis. The indirect effect of corticosterone, thus, assume to be mediated through lower LH which regulates the activity of 3beta-HSD. Basal, LH and cAMP-stimulated testosterone production by Leydig cells of corticosterone and corticosterone plus LH treated rats were decreased compared to control suggesting the deleterious effect of excess corticosterone on LH signal transduction and thus steroidogenesis.     

Regulation by dexamethasone of the 3 beta-hydroxysteroid dehydrogenase activity in adult rat Leydig cells.

The effect of long-term in vitro treatment with dexamethasone, insulin and/or LH on the 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity and the testosterone level was examined in cultures of Leydig cells from adult rats. A rapid and simple method for measuring the 3 beta-HSD activity has been developed, in which the NADH, generated by 3 beta-HSD, reduced nitroblue tetrazolium to a product with absorption maximum at 560 nm. Km for the reaction was 8.1 microM and Vmax was 12.7 nmol/min x mg protein. Addition of 0.1 or 1 microM dexamethasone for 44 h decreased the 3 beta-HSD activity to 83% and the basal testosterone level to 64% of control value after 22 and 44 h of culture. Addition of 1 nM insulin inhibited the 3 beta-HSD activity to 90% after 44 h of culture, whereas the testosterone level was increased after 3 h. Addition of 0.1 ng/ml LH did not affect the 3 beta-HSD activity in Leydig cells from adult rats. Concomitant treatment of the cells with dexamethasone and insulin inhibited the 3 beta-HSD activity to 74%, indicating an additive effect, whereas no additive effect on the testosterone level was observed. The results demonstrate that the 3 beta-HSD activity can be measured in a rapid and reliable way by measuring the reduction of nitroblue tetrazolium. Furthermore, the results suggest that dexamethasone acts on 3 beta-HSD through a mechanism different from that of insulin, as an additive effect was observed.     

Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure

Depriving rats of luteinizing hormone (LH) causes Leydig cells to lose smooth endoplasmic reticulum and diminishes their P450 C17-hydroxylase/C17,20-lyase activity (Wing et al., 1984). LH administration to hypophysectomized rats prevents these changes in Leydig cell structure and function (Ewing and Zirkin, 1983). We adopted a multistep procedure of rat Leydig cell isolation to study the trophic effects of LH on steroidogenesis in the Leydig cell. Our method employs vascular perfusion, enzymatic dissociation, centrifugal elutriation, and Percoll gradient centrifugation. The purified Leydig cell fraction obtained after Percoll density-gradient centrifugation contains 95% well-preserved 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)-staining cells with ultrastructural characteristics of Leydig cells. These Leydig cells produced 248 and 29 ng of testosterone/10(6) Leydig cells when incubated for 3 h with and without a maximally stimulating concentration of ovine LH. Purified Leydig cells obtained from control rats and rats treated with testosterone-estradiol (T-E) implants for 4 days to inhibit LH production were incubated with a saturating concentration (2 microns) of pregnenolone. Leydig cells from control and T-E-implanted rats produced 537 and 200 ng of testosterone/10(6) Leydig cells X 3 h, respectively, suggesting a defect in the steroidogenic reactions converting pregnenolone to testosterone in Leydig cells from T-E-implanted rats. By using rabbit antibodies to the P450 C17-hydroxylase/C17,20-lyase pig microsomal enzyme, immunoblots of one-dimensional sodium dodecyl sulfate polyacrylamide gels of Leydig cell microsomal protein from control and 4- and 12-day T-E implanted rats revealed a continued loss of enzyme as the period of LH withdrawal continues. These results show that Leydig cells from animals deprived of LH had diminished capacity to convert pregnenolone to testosterone and reduced P450 C17-hydroxylase/C17,20-lyase content.     

Specific destruction of Leydig cells in mature rats after in vivo administration of ethane dimethyl sulfonate

Effects of ethane dimethyl sulfonate (EDS) on Leydig cells have been studied using the following parameters: morphology, histochemistry of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and esterase, quantitative activity of esterase, testosterone concentrations in plasma, and steroid production by isolated interstitial cells in vitro. Degenerating Leydig cells were observed within 16 h after the injection of mature rats with EDS (75 mg/kg body weight). At that time the testosterone concentration in plasma and the specific activity of esterase in testis tissue were decreased to approximately 35% and 60% of the control value, respectively. At 48 h after EDS only a few normal Leydig cells were left and the plasma testosterone concentration was less than 5% of the control value. The specific activity of esterase in total testis tissue was similar to the activity of dissected tubules from untreated rats. At 72 h no Leydig cells could be detected and no 3 beta-HSD and esterase-positive cells were present. At that time macrophages were still present in the interstitium and the appearance of the spermatogenic epithelium was normal, but 1 wk after EDS the elongation of spermatids was disturbed, probably due to a lack of testosterone. In some of the animals the cytotoxic effects of EDS on Leydig cells could be partly inhibited by human chorionic gonadotropin treatment. The basal steroid production by interstitial cells from mature rats 72 h after EDS was not significant and no stimulation by LH was observed, whereas no effect of EDS could be detected on steroid production by interstitial cells isolated from immature rats and mice 72 h after treatment. Other compounds with similar structures, such as butane dimethyl sulfonate (busulfan) and ethane methyl sulfonate (EMS) had no effect on Leydig cells from mature rats. It is concluded that EDS specifically destroys Leydig cells in mature rats.     

Effects of thyroid and luteinizing hormones on the onset of precursor cell differentiation into leydig progenitor cells in the prepubertal rat testis

Leydig cells in the adult rat testis differentiate during the neonatal-prepubertal period. However, the stimulus for the initiation of their differentiation is still not clear. In the present study our objectives were to test the effects of thyroid hormone and LH on the initiation of precursor cell differentiation into Leydig cells in the prepubertal rat testis. Four groups of Sprague-Dawley rats were used. All treatments began at postnatal Day 1. Rats in groups I, II, and III received daily s.c. injections of saline (200 microl, controls), triiodothyronine (T(3), 50 microg/kg body weight, hyperthyroid), and LH (ovine LH 10 microg/rat/day), respectively. Rats in group IV were made hypothyroid from postnatal Day 1 by adding 0.1% propylthiouracil (PTU) to their mother's drinking water. Testes of rats were collected at 7, 8, 9, 10, 11, 12, 16, and 21 days of age, fixed in Bouin's solution, and embedded in paraffin for immunocytochemical studies. Immunoexpression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and LH receptors (LHR) in testicular interstitial cells (other than the fetal Leydig cells) was observed using the avidin-biotin method. In control rats, out of all spindle-shaped cell types in the testis interstitium, only the peritubular mesenchymal cells showed positive immunolabeling for 3beta-HSD, beginning from the postnatal Day 11. However, positive immunolabeling for LHR was first detected in these cells at Day 12, i.e., after acquiring the steroidogenic enzyme activity. In T(3)-treated rats 3beta-HSD positive spindle-shaped cells were first observed at Day 9 (i.e., 2 days earlier than controls), and LHR-positive cells were first observed on Day 11 (2 days later than obtaining 3beta-HSD immunoactivity); they were exclusively the peritubular mesenchymal cells. The 3beta-HSD- and LHR-positive spindle-shaped cells were absent in the testis interstitium of LH-injected rats from Days 7 through 12 but were present at postnatal Day 16. In addition, more fetal Leydig cell clusters and fetal Leydig cells in mitosis were present in LH-treated rats compared to rats in all other treatment groups. Following their first detection, the number of positive cells for each protein continued to increase at each subsequent age in controls, T(3)-, and LH-injected groups. In PTU rats, 3beta-HSD and LHR-positive spindle-shaped cells were absent throughout the experimental period. From these observations, it is possible to suggest the following regarding the developing rat testis interstitium. 1) The precursor cells for the adult generation of Leydig cells in the postnatal rat testis are the peritubular mesenchymal cells. 2) Luteinizing hormone does not initiate the onset of mesenchymal cell differentiation into Leydig cells, instead it delays this process. However, daily LH treatment causes mitosis in fetal Leydig cells and increase in fetal Leydig cell clusters. 3) Thyroid hormone is critical to initiate the onset of mesenchymal cell differentiation into adult Leydig cells.     

Effects of Etomidate on the Steroidogenesis of Rat Immature Leydig Cells

Background

Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established.

Methodology

Immature Leydig cells isolated from 35 day-old rats were cultured with 30 μM etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidate (0.3–30 μM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of etomidate on CYP11A1 and HSD3B1 activity.

Results and Conclusions

In intact Leydig cells, 30 μM etomidate significantly inhibited androgen synthesis. Further studies showed that etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC₅₀) values of 12.62 and 2.75 μM, respectively. In addition, etomidate inhibited steroidogenesis-related gene expression. At about 0.3 μM, etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 μM), it also reduced the expression levels of Cyp11a1, Hsd17b3 and Srd5a1. In conclusion, etomidate directly inhibits the activities of CYP11A1 and HSD3B1, and the expression levels of Cyp11a1 and Hsd17b3, leading to the lower production of androgen by Leydig cells.     

Activation of GPER-1 Estradiol Receptor Downregulates Production of Testosterone in Isolated Rat Leydig Cells and Adult Human Testis

Purpose

Estradiol (E2) modulates testicular functions including steroidogenesis, but the mechanisms of E2 signaling in human testis are poorly understood. GPER-1 (GPR30), a G protein-coupled membrane receptor, mediates rapid genomic and non-genomic response to estrogens. The aim of this study was to evaluate GPER-1 expression in the testis, and its role in estradiol dependent regulation of steroidogenesis in isolated rat Leydig cells and human testis.

Materials and Methods

Isolated Leydig cells (LC) from adult rats and human testicular tissue were used in this study. Expression and localization studies of GPER-1 were performed with qRT-PCR, immunofluorescence, immunohistochemistry and Western Blot. Luteinizing Hormone (LH) -stimulated, isolated LC were incubated with estradiol, G-1 (GPER-1-selective agonist), and estrogen receptor antagonist ICI 182,780. Testosterone production was measured with radioimmunoassay. LC viability after incubation with G-1 was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay.

Results

GPER-1 mRNA is abundantly expressed in rat LC and human testis. Co-localization experiments showed high expression levels of GPER-1 protein in LC. E2-dependent activation of GPER-1 lowers testosterone production in isolated rats LCs and in human testis, with statistically and clinically significant drops in testosterone production by 20–30% as compared to estradiol-naïve LC. The exposure to G-1 does not affect viability of isolated LCs.

Conclusions

Our results indicate that activation of GPER-1 lowers testosterone levels in the rat and human testis. The expression of GPER-1 in human testis, which lack ERα, makes it an exciting target for developing new agents affecting testosterone production in men.     

The aging Leydig cell: 2. Two distinct populations of Leydig cells and the possible site of defective steroidogenesis

Using metrizamide gradient centrifugation two populations of Leydig cells were found in both 60-90 day-old and 24 month-old rats. Cells from both Band 2 (B2) and Band 3 (B3) responded to LH stimulation with increased cyclic AMP formation; however, only B3 cells produced significant amounts of testosterone. Cells from both B2 and B3 of the old rats synthesized less cyclic AMP and testosterone than cells from their younger counterparts. In response to LH stimulation, 0.01 - 1.0 mIU/ml, no appreciable difference of cyclic AMP formation could be detected between young and old Leydig cells. Maximal testosterone production occurred when 1 mIU/ml LH was used. Only when LH concentration was increased to 10 and 100 mIU/ml, did young Leydig cells produce significantly more cyclic AMP than old Leydig cells. After addition of 5X10(-7)M of pregnenolone or progesterone to the incubation medium, both young and old Leydig cells produced comparable amounts of testosterone. These results demonstrate no impairment of old rat Leydig cells to synthesize testosterone from pregnenolone and progesterone.     

Exposure to Perfluorooctane Sulfonate In Utero Reduces Testosterone Production in Rat Fetal Leydig Cells

Background

Perfluorooctane sulfonate (PFOS) is a synthetic material that has been widely used in industrial applications for decades. Exposure to PFOS has been associated with decreased adult testosterone level, and Leydig cell impairment during the time of adulthood. However, little is known about PFOS effects in utero on fetal Leydig cells (FLC).

Methods and Results

The present study investigated effects of PFOS on FLC function. Pregnant Sprague Dawley female rats received vehicle (0.05% Tween20) or PFOS (5, 20 mg/kg) by oral gavage from gestational day (GD) 11–19. At GD20, testosterone (T) production, FLC numbers and ultrastructure, testicular gene and protein expression levels were examined. The results indicate that exposures to PFOS have affected FLC function as evidenced by decreased T production, impaired FLC, reduced FLC number, and decreased steroidogenic capacity and cholesterol level in utero.

Conclusion

The present study shows that PFOS is an endocrine disruptor of male reproductive system as it causes reduction of T production and impairment of rat fetal Leydig cells.     

Intercellular communication between Sertoli cells and Leydig cells in the absence of follicle-stimulating hormone-receptor signaling

Effective interactions among the various compartments of the testis are necessary to sustain efficiency of the spermatogenic process. To study the intercellular communication between the Sertoli and Leydig cells in the complete absence of FSH receptor signaling, we have examined several indices of Leydig cell function in FSH receptor knockout (FORKO) mice. The serum testosterone levels were reduced in the 3- to 4-mo-old adult FORKO males compared to wild-type mice despite no significant alteration in circulating LH levels. Treatment with ovine LH resulted in a dose-dependent increase in serum testosterone levels in all three genotypes (+/+, +/-, and -/-). However, the response in FORKO males was significantly reduced. Similarly, the total intratesticular testosterone per testis was also lower, but the intratesticular testosterone per milligram of testis was significantly elevated in the FORKO males. Western blot analysis revealed an apparent higher expression of the enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD) as well as LH-receptor density in the testis of FORKO males. Immunohistochemistry also showed an increase in the intensity of 3beta-HSD staining in the testicular sections of FORKO males. Although LH receptor binding increased per unit weight in FORKO mice, the total LH binding remained the same in all genotypes. Taken together, the results of the present study suggest that, in the absence of FSH receptor signaling, the testicular milieu is altered to affect Leydig cell response to LH such that circulating testosterone is reduced in the adult mutant. Studies are currently under way to understand the mechanisms underlying this phenomenon.     

Human type 2 17beta-hydroxysteroid dehydrogenase mRNA and protein distribution in placental villi at mid and term pregnancy

Background  

During human pregnancy, the placental villi produces high amounts of estradiol. This steroid is secreted by the syncytium, which is directly in contact with maternal blood. Estradiol has to cross placental foetal vessels to reach foetal circulation. The enzyme 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD2) was detected in placental endothelial cells of foetal vessels inside the villi. This enzyme catalyzes the conversion of estradiol to estrone, and of testosterone to androstenedione. It was proposed that estradiol level into foetal circulation could be regulated by 17beta-HSD2.     

Localization of immunoreactive testosterone and 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase in cynomolgus monkey (Macaca fascicularis) testes during postnatal development.

The age-related expression of testosterone and 3beta-HSD in the testes of cynomolgus monkeys was detected using light-microscopic immunocytochemistry. Intense deposits of immunoreactive testosterone were labeled in parts of Leydig cells in neonatal, late infantile, pubertal, and adult testes, and only a few Leydig cells in early infantile testes. The immunoreactive 3beta-HSD was labeled in parts of Leydig cells and in all Sertoli cells in neonatal, late infantile, pubertal, and adult testes, whereas only a few Leydig cells, but no Sertoli cells, were labeled in early infantile testes. The fluctuations of testosterone and 3beta-HSD expression in testes correlated well with those already observed plasma testosterone levels during postnatal development in cynomolgus monkeys.     

Environmental stress alters the developmental pattern of delta 5-3 beta-hydroxysteroid dehydrogenase activity in Leydig cells of fetal rats: a quantitative cytochemical study

Quantitative cytochemistry was used to determine the effect of subjecting pregnant rats to environmental stress on the activity of delta 5-3 beta hydroxysteroid dehydrogenase (3 beta-HSD) in Leydig cells of their fetuses. Enzyme activity was measured by microspectrophotometry in individual Leydig cells in cryostat sections of fetal testes on Days 16-21 postconception. Fetuses of stressed mothers lacked the peak of enzyme activity on Days 18 and 19 of gestation that is characteristic of Leydig cells of normal fetuses at this time. In addition, both before and after these 2 days, 3 beta-HSD activity in Leydig cells of stressed fetuses was significantly higher than normal. The altered developmental pattern of 3 beta-HSD activity in the stressed fetuses largely corresponds to the changes in plasma testosterone found previously in male fetuses of mothers exposed to the same regimen of stress. Thus, in the fetal Leydig cell, the activity of 3 beta-HSD, a key steroidogenic enzyme, can be modified by environmental stress, and provides an index of steroidogenic activity of the fetal testes and of the titers of circulating testosterone.     

Differences between the regulation of cholesterol side-chain cleavage in Leydig cells from mice and rats

A Leydig cell culture system has been used to study the in vitro modulation by luteinizing hormone (LH) of steroidogenesis in Leydig cells isolated from mice and immature rats. Mouse Leydig cells precultured for 24 h in the presence of increasing concentrations of LH (1 ng-1 microgram/ml) showed a dose-dependent decrease of the maximal LH-stimulated testosterone production. After pretreatment with 1 microgram LH/ml, maximal LH-stimulated testosterone production. After production in the presence of excess 20 alpha-hydroxycholesterol (a cholesterol side-chain cleavage substrate) were reduced to approx. 50% of control values. The possible site of action of LH is probably prior to pregnenolone, because testosterone production in the presence of excess pregnenolone was not affected by the LH pretreatment. Immature rat Leydig cells showed no decrease of maximal steroid production after 24 h culture in the presence of 1 microgram LH/ml. These results indicate that the regulation of the cholesterol side-chain cleavage activity during long-term LH action is different in mouse and rat Leydig cells. The properties of the cholesterol side-chain cleavage enzyme in mouse and rat Leydig cells were further investigated with different hydroxylated cholesterol derivatives as substrates. Steroid production by mouse Leydig cells in the presence of (22R)-22 hydroxycholesterol was similar as in the presence of LH. In contrast, steroidogenesis in rat Leydig cells in the presence of (22R)-22 hydroxycholesterol was at least 10-fold higher than in the presence of LH. It is concluded that the cholesterol side-chain cleaving enzyme in the mouse Leydig cell operates at its maximal capacity during short-term LH stimulation and can be inhibited after long-term LH action, whereas in the rat Leydig cell only a fraction of the potential activity is used during short-term LH stimulation, which is not affected during long-term LH action.     

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.     

Detection of platelet-derived growth factor-alpha (PDGF-A) protein in cells of Leydig lineage in the postnatal rat testis

Platelet-derived growth factor-A (PDGF-A) is a locally produced growth factor in the rat testis secreted by both Sertoli cells and Leydig cells. It has been suggested that PDGF-A may be involved in modulation of testosterone production and may be essential to Leydig cell differentiation, however it is not known at what stage of differentiation PDGF-A begins to be expressed in the cells of Leydig lineage in the postnatal rat testis. Therefore, the objectives of this research were to determine at what postnatal age and in which cell type is PDGF-A first expressed in cells of the adult Leydig cell lineage, and does PDGF-A expression coincide with expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), an indicator of steroid hormone synthesis. Male Sprague Dawley rats of postnatal day 1, 7, 9-14, 21, 28, 40, 60, and 90 were used (n=6). Animals were euthanized and their testicles removed, fixed in Bouin's solution, embedded in paraffin, and 5 micrometers sections were prepared. Immunolocalization of PDGF-A and 3beta-HSD was carried out using a peroxidase-streptavidin-biotin method. PDGF-A was first detected in cells of the Leydig cell lineage at postnatal day 10 in progenitor cells, which were surrounding the seminiferous tubules (peritubular). These cells were confirmed to be the progenitor cells and not the mesenchymal or any other spindle-shaped cells in the testis interstitium by immunolocalization of 3beta-HSD and PDGF-A in the cells in adjacent sections of testis tissue from rats of postnatal days 10-14. After postnatal day 10, PDGF-A was continued to be expressed in subsequent cells of the Leydig lineage through day 90 (adult), however, was not present in peritubular mesenchymal precursor cells of the Leydig cell lineage or any other spindle-shaped cells in the testis interstitium at any tested age. These results revealed that PDGF-A first appears in Leydig progenitor cells in the postnatal rat testis at the onset of mesenchymal cell differentiation into progenitor cells at postnatal day 10 and suggest that a functional role(s) of PDGF-A in postnatally differentiated Leydig cells in the rat testis is established at the time of the onset of postnatal Leydig stem cell differentiation. It is suggested that the significance of the first expression of PDGF-A in the Leydig progenitor cells may be associated with inducing cell proliferation and migration of this cell away from the peritubular region during Leydig cell differentiation.     

Luteal expression of cytochrome P450 side-chain cleavage, steroidogenic acute regulatory protein, 3beta-hydroxysteroid dehydrogenase, and 20alpha-hydroxysteroid dehydrogenase genes in late pregnant rats: effect of luteinizing hormone and RU486

A decrease in serum progesterone at the end of pregnancy is essential for the induction of parturition in rats. We have previously demonstrated that LH participates in this process through: 1) inhibiting 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity and 2) stimulating progesterone catabolism by inducing 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity. The objective of this investigation was to determine the effect of LH and progesterone on the luteal expression of the steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450(scc)), 3beta-HSD, and 20alpha-HSD genes. Gene expression was analyzed by Northern blot analysis 24 and 48 h after administration of LH or vehicle on Day 19 of pregnancy. StAR and 3beta-HSD mRNA levels were lower in LH-treated rats than in rats administered with vehicle at both time points studied. P450(scc) mRNA levels were unaffected by LH. The 20alpha-HSD mRNA levels were not different between LH and control rats 24 h after treatment; however, greater expression of 20alpha-HSD, with respect to controls, was observed in LH-treated rats 48 h after treatment. Luteal progesterone content dropped in LH-treated rats at both time points studied, whereas serum progesterone decreased after 48 h only. In a second set of experiments, the anti-progesterone RU486 was injected intrabursally on Day 20 of pregnancy. RU486 had no effect on 3beta-HSD or P450(scc) expression but increased 20alpha-HSD mRNA levels after 8 h treatment. In conclusion, the luteolytic effect of LH is mediated by a drop in StAR and 3beta-HSD expression without effect on P450(scc) expression. We also provide the first in vivo evidence indicating that a decrease in luteal progesterone content may be an essential step toward the induction of 20alpha-HSD expression at the end of pregnancy in rats.