Functional activity of rat testicular cells in culture

Testicular cells from adult hypophysectomized rats were cultured for 10 or 12 days, and the effect of treatment with hCG (10 ng/ml) on testosterone and progesterone production and the activity of the Leydig cell enzyme, 3 beta-hydroxysteroid dehydrogenase, were studied. Regardless of hormone treatment, on 4th day in culture a decline in the steroidogenic activity of cultured cells could be observed. Treatment with hCG resulted in stimulation of steroidogenesis on days 6 to 10 in culture, as measured by testosterone and progesterone production. Hormone treatment stimulated or inhibited the enzyme activity depending on the presence or absence in the culture medium of 10(-6) M spironolactone, an inhibitor of 17 alpha-hydroxylase, or an anti-androgen, cyproterone acetate.     

Temporal relationship of the prolactin-dependent LH-induced LH receptor to the LH stimulus

The time course for LH induction of luteinizing hormone (LH) receptors as reflected in binding of ¹²⁵l-labeled hCG was investigated in hypophysecto-mized adult male rats. A low dose of oLH (10 μg) was administered to hypophysectomized adult male rats following pretreatments with prolactin, follicle-stimulating hormone (FSH), growth hormone (GH), or saline. Testicular binding of hCG was determined at different times following the LH injection using Leydig cell membrane preparations from a testicular homogenate. Seven days after hypophysectomy, hCG binding was at a nadir of 19 ± 7% (mean ± SD) of control values. Pretreatment with prolactin (100 μg/day) for 7 days was associated with a nonsignificantly different hCG binding that was 30 ± 5% of control values. Prolactin pretreatment plus a single 10 μg LH i.p. injection increased ¹²⁵l hCG binding up to 56 ± 10% of control values within 30 minutes of the LH injection. Luteinizing hormone-induced hCG binding persisted at a high level (51 ± 4% of control values) for 2 hours but returned to hypophysectomized control levels 6 hours after the i.p. LH injection. Seven days pretreatment with FSH or GH at 100 μg/day plus 10-μg LH injections was also tested. Neither FSH nor GH had a statistically significant effect on hCG binding nor could they mimic the ability of prolactin to allow for LH induction of hCG binding in the hypophysectomized adult male rats. These studies suggest that the induction or “up-regulation” of Leydig cell hCG binding by ovine LH is rapid and specifically dependent upon pre-exposure to prolactin.     

Regulation of 5 alpha-reductase activity in cultured immature leydig cells by human chorionic gonadotropin

The present studies examined the hormonal regulation of 5 alpha-reductase activity in cultured immature rat Leydig cells. Within the testis 5 alpha-reductase was concentrated in the interstitial cell compartment, and among interstitial cells, the enzyme was localized primarily in Band 3 of Percoll density gradients, which contains the majority of Leydig cells. Among various factors reported previously to stimulate testicular 5 alpha-reductase activity when administered in vivo to immature rats (LH/hCG, FSH, luteinizing hormone releasing hormone or prolactin), only LH/hCG directly stimulated 5 alpha-reductase activity of cultured immature Band 3 cells. Neither growth hormone which was reported previously to stimulate hepatic 5 alpha-reductase activity, nor insulin, insulin-like growth factor-I, or epidermal growth factor, which have been reported to modulate Leydig cell function, had any effect on 5 alpha-reductase activity of Band 3 cells. These studies suggest that the major factor directly stimulating 5 alpha-reductase activity in Leydig cells during early maturation is LH. However, it is possible that other factors acting indirectly may modulate the maturational rise in 5 alpha-reductase activity.     

Testicular steroidogenesis after human chorionic gonadotropin desensitization in rats.

When a single injection of 500 I.U. of human chorionic gonadotropin (hCG) is given to rats there is an initial acute rise of plasma testosterone and of testicular content for both cyclic AMP and testosterone. This response correlates with an increase in both lyase and 17 alpha-hydroxylase activities. Thereafter both plasma and testicular testosterone decline and do not increase after a second injection of hCG. During this period of desensitization, isolated Leydig cells were insensitive to the steroidogenic stimulatory effect of both hCG and dibutyryl cyclic AMP. The post-cyclic AMP block is not due to an alteration of the cyclic AMP-dependent protein kinase but it is correlated with a decrease in both lyase and 17 alpha-hydroxylase activities of the Leydig cell's microsomes. This decrease is not caused by the absence of the recently described cytosol activator of this enzyme because its addition did not restore the enzymatic activity. Within 60 to 96 h after hCG injection there was a spontaneous increase of both plasma and testicular testosterone and this parallels the recovery of lyase and 17 alpha-hydroxylase activities. These results suggest that both enzymatic activities are regulated, directly or indirectly, by hCG, and that this is partly responsible for the hCG-induced steroidogenic refractoriness of Leydig cells.     

Effect of prostaglandins on hormonal function of cultured rat Leydig cells

The effect of PGF2 alpha and its analogues on androgen production and activity of delta 5,3 beta-hydroxysteroid dehydrogenase in rat Leydig cells in vitro was investigated. Prostaglandin of the F type inhibit the enzyme activity and hormone secretion by cultured Leydig cells. This effect was considerably stronger in Leydig cells isolated from mature rats, than by Leydig cells from immature animals.     

Rapid Leydig cell proliferation and luteinizing hormone receptor replenishment in the neonatal rat testis after a single injection of human chorionic gonadotropin

Two-day-old rats were stimulated with a single dose of human chorionic gonadotropin (hCG). Changes in the Leydig cell number, mitotic activity, cell size, and number of luteinizing hormone (LH) receptors were studied. The Leydig cell number of the hCG-treated animals was 1.8 times that of the control on Day 1 and remained elevated for the rest of the 5-day experiment (p less than 0.0001). On Day 1 the number of Leydig cell mitoses in the hCG group was greater (p less than 0.05) than in the controls. The Leydig cell size increased transiently to two times that of the control (p less than 0.01) within the first day after the treatment and returned to control size by Day 5. The number of LH receptors per testis decreased 81% in 1 day (p less than 0.01), but returned to control level by Day 3. Since Leydig cell numbers were constant after Day 1, the rapid receptor recovery was obviously due to restoration of the binding sites rather than increased cell number. The present results demonstrate a rapid proliferative response and rapid LH receptor replenishment in the fetal-neonatal Leydig cells after gonadotropic stimulation. These responses of fetal-type Leydig cells are in clear contrast to those observed in adult testes after a similar stimulation.     

Evidence that Leydig precursors localize in immature band two cells isolated on Percoll gradients

The present studies examined responses to hCG and/or insulin of 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase activity (3 beta-HSD) in cultured Band 2 and Band 3 cells from 25- to 40-day-old rats isolated on Percoll gradients. In Band 2 cells, from 25-day-old rats enzyme activity increased about 3- and 2.5-fold, after 6 days of exposure to hCG or insulin, respectively. However, hCG did not stimulate enzyme activity in Band 2 cells from 30-, 35- and 40-day-old animals, and responses to insulin alone or insulin plus hCG declined with age. In Band 3 cells only insulin increased enzyme activity at each age. Neither hCG or insulin altered DNA levels in Band 2 or Band 3 cells, suggesting that increased activity in Band 2 cells from 25-day-old rats was not due to cellular replication. However, hCG increased the number of cells staining positive for 3 beta-HSD about 4-fold in Band 2 cells from 25-day-old rats. Insulin did not increase the number of positive staining cells in Band 2 and Band 3 cells from 25-day-old rats, suggesting that its major effect was to increase enzyme activity in existing cells. These results suggest that during a limited period of maturation precursor cells in Band 2, which are undetected by histochemical staining for 3 beta-HSD, can be converted to Leydig cells in culture by hCG.     

Regulatory mechanism of Toona sinensis on mouse leydig cell steroidogenesis

Toona sinensis (TS), a kind of arbor, widely distributes nowadays in Asia. The leaves of TS have been used as an effective nutritious food in Chinese society for a long time. It was reported that Toona sinensis can induce apoptosis of cancer cells, reduce plasma glucose in diabetic rats, and improve lipolysis of differentiated 3T3-L1 adipocyte and its uptake of glucose. It has also been shown that TS may increase dynamic activity of human sperm. Thus, we are interested to investigate whether Toona sinensis has any effect on mouse Leydig cell testosterone production, which correlates to sperm activity. Primary mouse Leydig cells were purified to conduct the in vitro experiments. Different concentrations of crude Toona sinensis were added to primary mouse Leydig cells and the testosterone production was determined. The results showed that crude TS significantly inhibited both basal and human chorionic gonadotropin (hCG)-stimulated testosterone productions in dose dependent manner, respectively (P<0.05). Crude TS also reduced the forskolin- and dibutyryl-cAMP (dbcAMP)-stimulated testosterone production (P<0.05), which indicated that crude TS might affect protein kinase A (PKA) signal transduction pathway at the site after the formation of cyclic AMP. Moreover, TS inhibited Leydig cell steroidogenesis by suppressing the activity of steroidogenic enzymes including P450 side chain cleavage enzyme, 3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, 20 alpha-hydroxylase and 17 beta-hydroxysteroid dehydrogenase (P<0.05). In summary, these results suggested that TS inhibited steroidogenesis by suppressing the cAMP-PKA signaling pathway and the activities of steroidogenic enzymes in normal mouse Leydig cells.     

Effects of amphetamine on steroidogenesis in MA-10 mouse Leydig tumor cells

Amphetamine influences plasma and testicular testosterone levels. However, there is no evidence that amphetamine can directly influence Leydig cell functions. In the present study, a MA-10 mouse Leydig tumor cell line was used to determine whether and how amphetamine affected Leydig cell steroidogenesis. MA-10 cells were treated with different concentrations of amphetamine without or with human chorionic gonadotropin (hCG) and/or enzyme precursors over different time durations. Steroid production, enzyme activities and StAR protein expression were determined. Amphetamine alone had no any effect on MA-10 cell steroidogenesis. However, amphetamine (10(-11)M and 10(-10)M) significantly enhanced hCG-treated progesterone production at 3 hr in MA-10 cells (p < 0.05). Furthermore, amphetamine significantly induced more progesterone production upon treatment with 22R-hydroxycholesterol (p < 0.05), a precursor of P450 side-chain cleavage enzyme (P450scc). However, amphetamine did not induce more progesterone production when treated with pregnenolone (p > 0.05), a precursor of 3beta-hydroxysteroid dehydrogenase. In addition, the expressions of StAR protein and P450scc enzyme were not significantly different between hCG alone and hCG plus amphetamine treatment in MA-10 cells (p > 0.05). These results suggested that amphetamine enhanced hCG-induced progesterone production in MA-10 cells by increasing P450scc activity without influencing StAR protein and P450scc enzyme expression or 3beta-HSD enzyme activity.     

Steroid metabolism by purified adult rat leydig cells in primary culture

To characterize Leydig cell steroidogensis, we examined the metabolism of (³H)pregnenolone (3β-hydroxy-5-pregnen-20-one) to androgens in the presence and absence of human chorionic gonadotropin (hCG) as a function of culture duration. Approximately 20–30% of the (³H)pregnenolone was converted to testosterone (17_β-hydroxy-4-androsten-3-one) by purified Leydig cells at 0, 3 and 5 days (d) of culture. Androstenedione (4-androstene-3,17-dione) and dihydrotestosterone (17_β-hydroxy-5_α-androstan-3-one) were also produced while on day 5 of culture, significant amounts of progesterone (4-pregnene-3, 20-dione) were isolated. The Δ⁵ intermediates, 17-hydroxypregnenolone (3β, 17-dihydroxy-5-pregnen-20-one) and dehydroepiandrosterone (3β-hydroxy-5-androsten-17-one), accounted for less than 1% of substrate conversion, indicating a clear preference for Leydig cells to metabolize (³H)pregnenolone via the Δ⁴ pathway. On day 0 of culture, unidentified metabolites consisted of predominately polar steroids while on day 5 of culture, the unidentified metabolites consisted of predominately nonpolar steroids. In the presence of hCG, (³H)pregnenolone metabolism did not differ from basal on day 0 or 3 of culture. HCG increased the conversion of pregnenolone to progesterone and 17-hydroxyprogesterone (17-hydroxy-4-pregnene-3, 20-dione) on 5d. This suggests that Leydig cells cultured for 5d have decreased C_17–20 desmolase activity or that hCG acutely stimulates 3β-hydroxysteroid dehydrogenase and Δ⁴-Δ⁵ isomerase activities.     

LH contamination may explain FSH effects on rat Leydig cells

Treatment of immature, hypophysectomized male rats with 50 micrograms ovine FSH (NIH-FSH-S12) twice a day for 5 days stimulated the maximum quantity of 17 beta-hydroxyandrogen produced by isolated Leydig cells in response to hCG. Pretreatment of the FSH preparation with an LH antiserum in one study markedly reduced and in another study completely abolished this stimulatory effect of FSH, but only slightly impaired the capacity of the hormone to stimulate the Sertoli cell in vivo (epididymal androgen-binding protein). Administration of another highly potent FSH preparation (LER-1881) had no discernible effects on the dose-response characteristics of the Leydig cells but was superior to the NIH-FSH-S12 in its capacity for stimulating the Sertoli cell. When all hormone preparations were tested for their ability to stimulate steroid secretion from normal Leydig cells in vitro, a close correlation was obtained between their Leydig cell-stimulating activity (a measure of LH contamination) and their capacity to alter Leydig cell responsiveness after in-vivo treatment. FSH treatment had no effects on specific LH binding per 10(6) Leydig cells. It is concluded that the stimulatory influence of FSH on rat Leydig cells may to some extent be a result of the LH contaminating the hormone preparation.     

An early steroidogenic defect in hormone-induced Leydig cell desensitization

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

Developmental pattern of testosterone production by the rat testis

We studied the steroidogenic activity of isolated Leydig ceils derived from rats on fetal day 19 (F₁₉) and postnatal (N) days 1, 12, 24, 34, 45 and adults. Leydig cells, isolated at all ages by the collagenase method, increased in number throughout development with a doubling time of 8 days. Testicular content and serum concentrations of testosterone showed parallel changes during development. Moderate values were found at the early stages (F₁₉ and N₁), with a nadir on day 12, followed by a progressive increment to reach maximal values in adulthood. A reduction in steroidogenic activity of the testis during neonatal life was confirmed by urlar|_vitro studies with isolated Leydig cells. Maximal activity was found in group F₉; testosterone production diminished after birth to reach a minimum in group N₃₄ and rose thereafter to adulthood. Leydig cells were responsive to hCG stimulation at all ages in the following order: N₁>N₃₄>N₁₂>F₁₉>N₂₄>N₄₅>adult. The present study demonstrates the existence of an active and hCG-responsive population of Leydig cells in the rat testis from fetal life to adulthood.     

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

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

Oxytocin in Leydig cells: an immunocytochemical study of Percoll-purified cells from rat testes

Summary Testicular interstitial cells, from rats aged 35 days, were dispersed with collagenase and separated through Percoll into 5 fractions (I–V); fraction I being the least dense. Measurement of basal testosterone production, histo-enzymological staining for 3-hydroxysteroid dehydrogenase activity and electron microscopy indicated that the majority of Ley dig cells were found in fraction IV (corresponding to a density of 1.076–1.097 g/ml). In addition, cells from this fraction responded to hCG treatment in a dose-dependent manner on day 0 and remained responsive after being cultured for 1 day. Immunostaining for oxytocin indicated that this fraction also contained the majority of the oxytocin-immunoreactive cells. On day 1 of culture, 56% of the cell population from fraction IV were positively stained for the steroidogenic enzyme and 75% immunoreactive for oxytocin. This overlap indicates that the Leydig cells were also the oxytocin immunoreactive cells.     

Regulation of pig Leydig cell aromatase activity by gonadotropins and Sertoli cells

The present work was done to investigate the cell localization of testicular aromatase activity and its regulation in immature pig testis using an in vitro model. Leydig cells and Sertoli cells were isolated from immature pig testes and cultured alone or together in the absence or presence of human chorionic gonadotropin (hCG) or porcine follicle-stimulating hormone (pFSH) for 2 days. At the end of incubation, the amounts of testosterone (T), estrone sulfate (E1S) and estradiol (E2) were measured. Then the cells were incubated for 4 h in the presence of saturating concentrations of delta 4-androstenedione (3 microM) and the amounts of E1S and E2 were measured again (aromatase activity). The ability of Sertoli cells to produce estrogens was very low and neither hCG nor pFSH had any significant effect. hCG stimulated, in a dose-dependent manner, the secretion of T and E1S by Leydig cells cultured alone as well as the aromatase activity of these cells. The main estrogen produced by Leydig cells was E1S. pFSH also stimulated the above parameters of Leydig cell function; this may have been due to the contamination of this hormone with luteinizing hormone (LH). Coculture of Leydig cells with Sertoli cells without gonadotropins had very small effects on T and E1S production and on aromatase activity. However, treatment of coculture with increasing concentrations of hCG had a dramatic effect on Leydig cell functions. For each hCG concentration, the amounts of T and E1S secreted, as well as the aromatase activity of the coculture, were 2- to 3-fold higher than those of Leydig cells cultured alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the protective role of vitamin C and E against polychlorinated biphenyl (Aroclor 1254)—induced oxidative damage in Leydig cells

Free radical production and lipid peroxidation are potentially important mediators in testicular physiology and toxicology. Polychlorinated biphenyls (PCBs) are global environmental contaminants that cause disruption of the endocrine system in human and animals. The present study was conducted to elucidate the protective role of vitamin C and E against Aroclor 1254-induced changes in Leydig cell steroidogenesis and antioxidant system. Adult male rats were dosed for 30 days with daily intraperitoneal (ip) injection of 2 mg/kg Aroclor or vehicle (corn oil). One group of rats was treated with vitamin C (100 mg/kg bw/day) while the other group was treated with vitamin E (50 mg/kg bw/day) orally, simultaneously with Aroclor 1254 for 30 days. One day after the last treatment, animals were euthanized and blood was collected for the assay of serum hormones such as luteinizing hormone (LH), thyroid stimulating hormone (TSH), prolactin (PRL), triiodothyronine (T₃), thyroxine (T₄), testosterone and estradiol. Testes were quickly removed and Leydig cells were isolated in aseptic condition. Purity of Leydig cells was determined by 3β-hydroxysteroid dehydrogenase (3β-HSD) staining method. Purified Leydig cells were used for quantification of cell surface LH receptors and steroidogenic enzymes such as cytochrome P₄₅₀ side chain cleavage enzyme (P₄₅₀scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β- HSD). Leydig cellular enzymatic antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), γ-glutamyl transpeptidase (γ-GT), glutathione-S-transferase (GST) and non-enzymatic antioxidants such as vitamin C and E were assayed. Lipid peroxidation (LPO) and reactive oxygen species (ROS) were also estimated in Leydig cells. Aroclor 1254 treatment significantly reduced the serum LH, TSH, PRL, T₃, T₄, testosterone and estradiol. In addition to this, Leydig cell surface LH receptors, activities of the steroidogenic enzymes such as cytochrome P₄₅₀scc, 3β-HSD, 17β-HSD, antioxidant enzymes SOD, CAT, GPX, GR, γ-GT, GST and non-enzymatic antioxidants such as vitamin C and E were significantly diminished whereas, LPO and ROS were markedly elevated. However, the simultaneous administration of vitamin C and E in Aroclor 1254 exposed rats resulted a significant restoration of all the above-mentioned parameters to the control level. These observations suggest that vitamin C and E have ameliorative role against adverse effects of PCB on Leydig cell steroidogenesis.     

Immunocytochemical localization of receptor human chorionic gonadotropin complexes in rat leydig cells

Localization of receptor-bound human chorionic gonadotropin (hCG) in rat testis was studied by the peroxidase-antiperoxidase (PAP) complex method. The rats were injected with a single intravenous dose (1000 IU) of hCG. Three, 6, 12, and 24 hr after injection the testes were removed for localization of the hormone. The hormone localized to the periphery of the Leydig cells at all observation points. The intensity of the staining varied between the cells, suggesting that the number of receptors or the accessibility of the receptors to the circulating hormone varies from one cell to another. The staining surrounded the Leydig cells unevenly, but no progressive patching or capping was found. This observation suggests that hCG binds preferentially to the cell surface areas directed toward the capillaries. Compatible results were obtained with anti-hCG serum and with antisera against the hCG subunits. These results are consistent with previous observations that the luteinizing hormone (hCG) receptors accessible to the circulating hormone are located at the surface of the Leydig cells.     

Effects of catechin,epicatechin and epigallocatechin gallate on testosterone production in rat leydig cells

Catechins have been reported to have many pharmacological properties such as the effects of anti‐oxidative, anti‐inflammatory, anti‐carcinogenic, anti‐ultraviolet, and reduction of blood pressure as well as glucose and cholesterol levels. However, the effect of catechins on the reproductive mechanism is still unknown. In the present study, the effects of catechins on testosterone secretion in rat testicular Leydig cells (LCs) were explored. Both in vivo and in vitro investigations were performed. Purified LCs were incubated with or without catechin (CCN), epicatechin (EC), epigallocatechin gallate (EGCG, 10^?10–10^?8 M) under challenge with human chorionic gonadotropin (hCG, 0.01 IU/ml), forskolin, SQ22536 (an adenylyl cyclase inhibitor), 8‐bromo‐adenosine 3′:5′‐cyclic monophosphate (8‐Br‐cAMP), A23187 (a calcium ionophore), and nifedipine (10^?5 M), respectively. To study the effects of catechins on steroidogenesis, steroidogenic precursors‐stimulated testosterone release was examined. The functions of the steroidogenic enzymes including protein expression of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein were investigated and expressed by Western blotting. Catechins increased plasma testosterone in vivo in male rats. In vitro, low‐dose concentration of catechins increased gonadotropin releasing hormone (GnRH)‐stimulated luteinizing hormone (LH) release by anterior pituitary gland and hCG‐stimulated testosterone release by LCs of male rats. These results suggested that catechins stimulated testosterone production by acting on rat LCs via the mechanism of increasing the action of cAMP, but not P450scc, StAR protein or the activity of intracellular calcium. EC, one of the catechins increased the testosterone secretion by rat LCs via the enzyme activities of 17β‐hydroxysteroid dehydrogenase (17β‐HSD). J. Cell. Biochem. 110: 333–342, 2010. © 2010 Wiley‐Liss, Inc.     

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.