Temporal relationships among testosterone production, steroidogenic acute regulatory protein (StAR), and P450 side-chain cleavage enzyme (P450scc) during Leydig cell aging

Previous studies have shown that the capacity of Leydig cells from aged (21-24-month-old) Brown Norway rats to produce testosterone is reduced from young (4-month-old) levels, and that this is correlated with reductions in steroidogenic acute regulatory protein (StAR), peripheral benzodiazapine receptor (PBR), and the levels and activities of the steroidogenic enzymes. The age(s) at which particular changes in the steroidogenic pathway occur, and the relationship of particular changes to reduced testosterone production, are not known. We examined 3 critical components of the steroidogenic pathway, cyclic adenosine monophosphate (cAMP) production, StAR, and P450 side-chain cleavage enzyme (P450scc) in relationship to age-related decreases in testosterone production. Leydig cells isolated from Brown Norway rats of increasing ages (4, 9, 15, and 20 months) were evaluated. The ability of Leydig cells to produce testosterone was reduced at 9 months, although not significantly. Significant reductions in testosterone production were first seen in cells isolated from rats of 15 months of age, and further reductions occurred thereafter. Reduced testosterone was correlated with reductions in StAR, P450scc mRNA, and protein. Significant decline in luteinizing hormone-stimulated intracellular cAMP levels was seen by 9 months, before significant reductions in testosterone, StAR, and P450scc. Further declines in cAMP levels were seen at 15 and 20 months. These studies suggest that age-related reductions in intracellular cAMP may lead to the reduced testosterone production that characterizes aged Leydig cells. This suggestion is supported by recent studies from our lab demonstrating that long-term (3 days) culture of old Leydig cells with dbcAMP restored testosterone production to levels approximating those of young cells.     

自噬信号因子P70S6K参与Cox7a2调控睾酮生成机制研究

目的:研究Cox7a2对TM3 Leydig细胞睾酮生成的影响以及涉及其中的自噬调控信号的作用。方法:构建Cox7a2荧光表达载体,转染TM3 Leydig细胞。ELISA测定睾酮水平,Western印迹检测Cox7a2对睾酮合成快速调节蛋白StAR表达和自噬调控因子P70S6K磷酸化水平的影响。结果:在TM3 Leydig细胞中,LH刺激能增加促进StAR蛋白表达,增加睾酮合成水平。Cox7a2在TM3小鼠睾丸Leydig细胞中抑制P70S6K磷酸化水平,降低StAR蛋白的表达,进而抑制LH诱导的睾酮合成。结论:Cox7a2通过抑制快速调节蛋白StAR的表达减少LH诱导的睾酮分泌,这至少和Cox7a2抑制自噬调控因子P70S6K有关。     

Cox7a2 mediates steroidogenesis in TM3 mouse Leydig cells

Aim:To investigate the regulatory function of Cox7a2 on steroidogenesis and the mechanism involved in TM3 mouseLeydig cells.Methods:The cDNA of CoxTa2 was cloned from TM3 mouse Leydig cells.It was subcloned to pDsRed-Express-N1 and transfected back into TM3 mouse Leydig cells for Cox7a2 overexpression by transient gene transfection.Steroidogenesis affected by overexpressed Cox7a2 was studied by ELISA.To elicit the mechanism of this effect,expression of steroidogenic acute regulatory(StAR)protein and reactive oxygen species(ROS)were examined byWestern blot and fluorometer,respectively.Results:The cDNA of Cox7a2(249 bp)was cloned from Leydig cells andconfirmed by DNA sequencing.After constructed pDsRed-Express-N1-Cox7a2 was transfected back into TM3 mouseLeydig cells,Cox7a2 inhibited not only luteinizing hormone(LH)-induced secretion of testosterone but also the expres-sion of StAR protein.At the same time,Cox7a2 increased the activity of ROS in TM3 mouse Leydig cells.Conclusion:Cox7a2 inhibited LH-induced StAR protein expression,and consequent testosterone production,at least in part,byincreasing ROS activity in TM3 mouse Leydig cells.(Asian J Androl 2006 Sep;8:589-594)     

Leydig cell aging: steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme

Primary points of control in steroidogenesis are the transport of cholesterol from intracellular stores to the inner mitochondrial membrane, and the subsequent conversion of cholesterol to pregnenolone by the cholesterol side-chain cleavage enzyme (P450scc). Testosterone production has been shown to decline in Brown Norway rat Leydig cells as the rats age. To better understand the mechanism by which aging Leydig cells lose steroidogenic function, we examined the effect of aging on steroidogenic acute regulatory protein (StAR), an important Leydig cell cholesterol transfer protein, and on P450scc. Leydig cells isolated from middle-aged (14 months) and old (24 months) rats produced significantly less testosterone than cells from young (4 months) rats. StAR mRNA (1.7 kilobase [kb]) was significantly reduced in Leydig cells from middle-aged and old rats, by 26% and 52%, respectively. Significant reductions also were seen in the steady-state levels of mRNA for P450scc, of 29% and 50%, respectively. Western blots revealed significant reductions in StAR protein, by 47% and 74%, respectively, and in P450scc protein, by 38% and 54%, respectively. In response to LH stimulation in vitro, testosterone production by Leydig cells in young, middle-aged, and old rats increased by 30-, 40-, and 33-fold, respectively, although the amounts of testosterone produced by the young cells significantly exceeded that produced by the middle-aged and old cells. StAR protein also increased in response to LH by 1.4- , 3-, and 11-fold, respectively, whereas P450scc protein remained unchanged. These results are consistent with the conclusion that compromise of StAR-mediated cholesterol transport may play a key role in age-related reductions in Leydig cell steroidogenesis. However, because P450scc is reduced in old Leydig cells, the reaction catalyzed by this enzyme would be rate-limiting under circumstances in which saturating amounts of cholesterol entered the mitochondria.     

Chronic stress induces ageing-associated degeneration in rat Leydig cells

Several studies have suggested that stress and ageing exert inhibitory effects on rat Leydig cells. In a pattern similar to the normal process of Leydig cell ageing, stress-mediated increases in glucocorticoid levels inhibit steroidogenic enzyme expression that then results in decreased testosterone secretion. We hypothesized that chronic stress accelerates the degenerative changes associated with ageing in Leydig cells. To test this hypothesis, we established a model of chronic stress to evaluate stress-induced morphological and functional alterations in Brown Norway rat Leydig cells; additionally, intracellular lipofuscin levels, reactive oxygen species (ROS) levels and DNA damage were assessed. The results showed that chronic stress accelerated ageing-related changes: ultrastructural alterations associated with ageing, cellular lipofuscin accumulation, increased ROS levels and more extensive DNA damage were observed. Additionally, testosterone levels were decreased. This study sheds new light on the idea that chronic stress contributes to the degenerative changes associated with ageing in rat Leydig cells in vivo.     

低剂量睾酮疗法降低TM3睾丸间质细胞的氧化损伤

睾酮替代疗法对老年男性及性腺机能减退症的治疗是有意义的。然而,外源性睾酮对睾丸间质细胞的作用尚不清楚,需要进一步阐明。本研究表明睾酮补充疗法能降低睾丸间质细胞的氧化损伤。本文用睾丸间质细胞TM3作为体外细胞模型。研究发现睾酮剂量为100nmol L-1治疗时可以产生细胞保护作用,但睾酮补充剂量≥500nmol L-1时就会产生细胞毒作用。在睾酮剂量为100nmol L-1治疗时能够显著降低ROS的产生,脂质过氧化物含量,缺氧诱导因子（HIF）-1α的稳定性和活性。睾酮剂量为500nmol L-1时产生的活性氧比100nmol L-1时增加了1．72倍。与对照组相比,睾酮剂量为50nmol L-1时类固醇激素合成急性调节蛋白（STAR）的表达增加了1．58倍（P〈0．01）．睾酬补充疗法降低了化学性诱导缺氧。低剂量睾酮补充疗法治疗睾丸间质细胞通过降低ROS和脂质过氧化物,增加StAR蛋白表达,减缓缺氧应激即降低HIF-1α测子的稳定性性产生细胞保护作用。研究中发现当睾酮剂量≥500nmol L-1时,氧化损伤增加。为阐明睾酮替代疗法的效果,需进一步阐明睾丸间质细胞中华酮量效关系受哪种机制调控。     

Steroidogenesis decline accompanied with reduced antioxidation and endoplasmic reticulum stress in mice testes during ageing

To gain an understanding of the mechanisms by which Leydig cell steroidogenic function degenerates with ageing, we explored steroidogenic gene expression in relation to antioxidation status and endoplasmic reticulum (ER) stress during the ageing of mice. Expression of StAR, P450scc and other steroidogenic enzymes decreased starting at middle age (12‐month‐old) compared to that of the young control (3‐month‐old) mice. The immunohistochemical staining intensity of 3β‐HSD for Leydig cells was significantly weaker in the aged (24‐month‐old) group than that in the young control group. The number of Leydig cells showed no significant difference between the groups. A progressive reduction in antioxidants MnSOD and GPx4 was observed in the testicular tissue with down‐regulated SIRT1 protein level in the middle‐aged and aged (24‐month‐old) mice. The number of testicular macrophages was significantly higher in the aged group than that in the middle‐aged and young mice. Age‐associated up‐regulation of ER stress markers such as GRP78 and Chop was observed. These results suggested that oxidative stress and ER stress might play a role in the deficit of Leydig cell steroidogenic function during ageing.     

老年大鼠睾丸间质细胞结构和功能变化的实验研究

目的:研究老年SD大鼠(PADAM动物模型)睾丸间质细胞形态、分泌功能变化,探讨老年大鼠睾丸间质细胞的功能状态。方法:分别取青年SD大鼠和老年各20只,静脉血测定血清总睾酮和游离睾酮的浓度,并通过组织切片和透射电镜观察两个年龄组大鼠睾丸间质细胞形态学变化;此外,分别用hCG、Forskolin刺激体外培养的两个年龄组大鼠的睾丸间质细胞,比较培养基中睾酮和孕酮的浓度。结果:老年大鼠的血清总睾酮[(3.07±0.75)nmol/L]和游离睾酮[(0.71±0.65)nmol/L]均比青年大鼠[(10.89±6.11)nmol/L和(2.42±1.02)nmol/L]显著降低(P<0.05);细胞形态有较显著差异;体外培养的大鼠睾丸间质细胞分泌能力显著降低(P<0.05)。结论:老年SD大鼠血清睾酮和游离睾酮浓度显著低于青年SD大鼠,原因在于睾酮合成酶系统整体功能衰退。     

Cholesterol transport,peripheral benzodiazepine receptor,and steroidogenesis in aging Leydig cells

The cellular mechanisms responsible for age-related decline in the ability of Leydig cells to produce testosterone are not yet fully understood. The decline in testosterone production could result from a reduction in the Leydig cell enzymatic activities mediating testosterone synthesis, the amount of substrate available for these enzymes, or both. In the present study, we examined the effect of age on a critical early step in the steroidogenic pathway, the transport of cholesterol into mitochondria. Leydig cells were isolated from the testes of young and old Brown Norway rats and incubated with human chorionic gonadotropin (hCG) and the side-chain cleavage cytochrome P450scc inhibitor aminoglutethimide (AMG). Mitochondria were isolated from these cells in the presence of AMG. Upon removal of AMG, the mitochondria from old cells produced 80% less steroid than those from young cells, only a fraction of which could be accounted for by a decrease in P450scc activity. These results suggest that the accumulation of hormonally recruited cholesterol into mitochondria is defective in old Leydig cells. With this in mind, we turned our attention to peripheral benzodiazepine receptor (PBR), a mitochondrial cholesterol-binding protein known to be involved in mediating cholesterol transport. PBR messenger RNA (mRNA) and protein expression were decreased in old cells. Moreover, both the dissociation constant (Kd) and the number of binding sites (Bmax) of the PBR were decreased in the old cells by 50% and 30%, respectively. Taken together, these results suggest that alterations in cholesterol transport and in PBR may play critical roles in age-related decreases in testosterone production in Brown Norway rat Leydig cells.     

In vitro effects of substance P and arginine-vasopressin on testosterone production in Leydig cells of short and long photoperiodic hamsters

Summary. The aim of the present study was to determine the interaction between substance P (SP) and arginine-vasopressin (AVP) on basal and LH-stimulated testosterone production by Leydig cells isolated from hamsters kept under long or short days (LD-hamsters, SD-hamsters, respectively). SP inhibited the testosterone production of Leydig cells, its effect being more pronounced in the case of LH-stimulated steroidogenesis in LD-hamsters. Similarly, the addition of AVP to the culture medium resulted in a diminution of basal, as well as LH-stimulated testosterone secretions. When Leydig cells were co-incubated with SP (10⁻⁷ M) and AVP (10⁻⁷ M), a strong inhibition of the testosterone production by 50–60% was established in LD-animals. However, even within the experimental circumstances in SD-hamsters, the modulation of testosterone production by SP and AVP was evident. The reported results suggest that there is an interference of two regulatory pathways, namely photoperiodic dependence and paracrine control of testicular steroidogenesis in hamsters.     

Inhibitory mechanisms of lead on steroidogenesis in MA-10 mouse Leydig tumor cells

Lead can directly influence Leydig cell steroidogenesis, which results in reduction of testosterone and causes low sperm counts in human beings and animals. This study investigated the effect of 6 h incubation time of lead on steroidogenesis in MA-10 mouse Leydig tumor cells. Lead acetate, ranging from 10(-8) to 10(-5) M, caused profounder inhibitory effects on human chorionic gonadotropin (hCG)- and dibutyryl cAMP (dbcAMP)-stimulated progesterone production for 6 h in MA-10 mouse Leydig tumor cells. Lead acetate significantly inhibited hCG- and dbcAMP-stimulated progesterone production from 20 to 35% in MA-10 cells at 6 h. Lead suppressed the expression of steroidogenesis acute regulatory (StAR) protein from 30 to 55%. Moreover, the activities P450 side-chain cleavage (P450scc) enzyme and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) were reduced by lead from 15 to 25%. Thus, after 6 h exposure to lead caused profounder inhibitory effects on StAR protein expression and steroidogenic enzymes and then progesterone production compared to 2- or 3-h lead treatments in MA-10 mouse Leydig tumor cells.     

The effects of Tremella aurantia on testosterone and corticosterone productions in normal and diabetic rats

Tremella aurantia (TA) has been traditionally used as food and crude medicine in Chinese society. The polysaccharide isolated from the fruiting bodies of TA exhibits significant hypoglycemic activity in diabetic mouse models of insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM). Diabetes will cause sexual dysfunction in patients. In the present study, we examined if the treatment of TA on IDDM and NIDDM rats will restore steroidogenesis and then the reproductive function. The fruiting bodies (FB), mycelium (TM) and polysaccharide (GX) of TA were fed to the IDDM and NIDDM rats, and testosterone and corticosterone levels in plasma, the weight of steroidogenic organs, and the expression of steroidogenic acute regulatory (StAR) protein and P450scc enzyme were determined. Plasma testosterone productions were significantly suppressed with the feeding of FB or TM in normal rat (p < 0.05). Testosterone productions were also significantly suppressed in IDDM diabetes rats (p <0.05), and FB or TM could not restore the inhibitory effects (p > 0.05). There was no significant difference of the testosterone production between normal and NIDDM rats (p > 0.05). In plasma corticosterone production, there were no differences among control, FB- or TM-fed normal rats (p > 0.05). Corticosterone levels were reduced in IDDM rats compared to control, and FB or TM could restore its level. Corticosterone levels were induced in NIDDM rats compared to control (p <0.05), but FB, TM or GX significantly brought the corticosterone back (p < 0.05) to the control levels. Considering steroidogenic organs, IDDM rats with or without TA treatments had heavier testis and adrenal glands, but not epididymis, than normal rats with or without TA treatments. There were no effects of TA on the weight of steroidogenic organs among normal and NIDDM rats. However, GX feeding in NIDDM rat had lesser testis weight compared to NIDDM rats. The expression of StAR protein and P450scc enzyme were not different among groups in IDDM and NIDDM rats. Plasma testosterone productions were suppressed in normal rats with the feeding of TA (FB and TM). IDDM rats did have lower testosterone, but not in NIDDM, and FB or TM could not restore the inhibitory effects. The induction of IDDM or NIDDM rats did affect steroidogenesis and steroidogenic organ weights, and the feeding of TA had different effects on steroidogenesis in different types of diabetic rats.     

Effects of varicocele on testosterone, apoptosis and expression of StAR mRNA in rat Leydig cells

The aim of this study was to explore the effects of varicocele on the morphology and function of Leydig cells in the rat testis. Forty male Sprague-Dawley rats were divided into two groups: the experimental group underwent surgery to create a left varicocele (VC), and the control group underwent a sham operation. Serum testosterone and intratesticular testosterone levels were measured using a radioimmunoassay after 4 and 8 weeks of operation. Leydig cells were studied for apoptosis and expression of steroidogenetic acute regulatory (StAR) protein mRNA levels. Serum testosterone levels declined after 4 and 8 weeks of operation but were not significant (P>0.05). However, the intratesticular testosterone levels after 8 weeks were significantly decreased compared with the control group (P<0.01). The mean apoptosis index of Leydig cells in the experimental group was significantly higher than that in the control group after 4 or 8 weeks (P<0.01). StAR mRNA levels in the Leydig cells of the experimental group were significantly lower compared to those of the control group (P<0.01). Our data show that varicocele did impair Leydig cell function by increasing apoptosis and suppressing the expression of the StAR protein.     

Ontogeny and cellular origin of a rat seminiferous tubule factor(s) that inhibits LH-dependent testosterone production by interstitial cells in vitro

Previous studies have shown the presence of a peptide in spent media from incubated seminiferous tubules (SMST), which inhibits LH stimulation of testosterone production by rat Leydig cells in vitro. The present study has investigated whether the secretion of this inhibitor changes during development in the rat. Seminiferous tubules obtained from rats aged 10, 20, 25, 30, 35, 40, 42, 50 or 60 days were incubated at 32 degrees C for 24 h. Spent media from these incubations were then added to interstitial cells isolated from the testes of rats aged 60 days. Spent media from rats aged 10-30 days had no effect on basal or oLH-stimulated testosterone production by interstitial cells during 3-h incubation. Significant inhibition of LH-stimulated testosterone production was, however, observed with SMST from rats aged 35-60 days. Spent media prepared using tubules from normal, prenatally irradiated (Sertoli cell-enriched) or seminiferous tubules, depleted of peritubular cells, had no effect on basal, but inhibited LH-stimulated, testosterone production. Spent media from peritubular cell cultures had no effect on basal or LH-stimulated testosterone production by interstitial cells. The inhibitory effect of SMST was also dependent on the age of the rats providing the target cells. Interstitial cells from rats aged 10, 20, 50 or 60 days were responsive to the inhibitor while cells from rats aged 30 and 40 days were not. The results of the present study demonstrate that the seminiferous tubule factor(s), which inhibits LH action on interstitial cells, is first secreted at 35 days, a time when the most mature germ cells present are in the early maturation phase. Moreover, interstitial cells are responsive to this factor in both immature (10-20 day-old) and mature (50-60 day-old) rats, but not at ages in between these times. It is suggested that the adult Sertoli cell is the major source of the interstitial cell inhibitor.     

Prenatal exposure to dexamethasone alters Leydig cell steroidogenic capacity in immature and adult rats.

This study examines the effects of prenatal exposure to dexamethasone (DEX) on postnatal testosterone production in male rats. Pregnant female rats were treated on gestation days 14-19 with DEX (100 microg/kg body weight per day; n = 9) or vehicle (n = 9). Results show that 35-day-old male offspring from DEX-treated pregnant females (n = 42) had decreased levels of serum testosterone (45.6% lower, P < .05) compared with control offspring (n = 43), although serum luteinizing hormone (LH) levels were not significantly altered. These findings suggest that a direct programming of developing gonadal cells occurs in response to high levels of maternal glucocorticoid. Indeed, testosterone production was significantly reduced in Leydig cells isolated from immature offspring of DEX-treated pregnant females compared with controls (48.3%, P < .001), and LH stimulation of these cells did not compensate for the lowered steroidogenic capacity. The hypothalamic-pituitary-adrenal axis was also affected, because significant reductions in both serum adrenocorticotropic hormone (ACTH; 26.2%, P < .001) and corticosterone (CORT; 32.3%, P < .001) were measured in DEX-exposed immature male offspring. In contrast, adult male offspring from DEX-treated dams had significantly higher levels of serum ACTH (39.2%, P <. 001) and CORT (37.8%, P < .001). These same animals had higher serum testosterone (31.6%, P < or = .05) and a significant reduction in serum LH (30.8%, P < .001). Moreover, Leydig cells isolated from these adult offspring exhibited an increased capacity for testosterone biosynthesis under basal (38.6%, P < .001) and LH-stimulated conditions (33.5%, P < .001). In summary, sustained changes in steroidogenic capacity were observed in male rats exposed to high levels of glucocorticoid during prenatal development. More specifically, DEX exposure in utero perturbed Leydig cell testosterone production in both pubertal and adult rats.     

Effect of unilateral cryptorchidism on the intertubular tissue of the adult rat testis: evidence for intracellular changes within the Leydig cells

Adult male rats were made unilaterally cryptorchid for 1, 2 or 4 weeks, and the morphological response of the Leydig cells was then studied using morphometric assessment of total Leydig cell volume and number per testis in abdominal and scrotal testes. Serum hormone levels were measured and the steroidogenic properties of isolated Leydig cells were evaluated by in-vitro stimulation with hCG and interstitial fluid (IF) obtained from normal rat testes. Total Leydig cell volume and number per testis were not altered in abdominal vs scrotal testes, although the volume of the abdominal testis was 46, 29 and 21%, respectively, of the volume of the contralateral scrotal testis after 1, 2 and 4 weeks. This reduction was accompanied by significant (P less than 0.05) elevation of the serum levels of FSH and LH, although serum testosterone levels were unchanged from the normal range. Despite the lack of quantitative alterations in Leydig cell morphology, hCG- and IF-stimulated testosterone production was significantly (P less than 0.01) greater by abdominal Leydig cells when compared with scrotal Leydig cells derived from the same animals. Ultrastructural examination of Leydig cells in situ suggested an increase in volumetric density of mitochondria in abdominal Leydig cells. Together with the enhanced steroidogenic responses of these cells, these findings suggest that disruption of spermatogenesis in the cryptorchid testis is accompanied by intracellular activation of Leydig cells. Since these effects were not exhibited by Leydig cells from the scrotal testis it is concluded that local factors within the cryptorchid testis are responsible, at least in part, for regulation of Leydig cell activity.     

环境污染物三丁基氯化锡和氯化三苯锡对大鼠睾丸Leydig细胞的影响

目的:探讨环境污染物三丁基氯化锡(TBT)和氯化三苯锡(TPT)对大鼠睾丸Leyd ig细胞的影响。方法:①用0~80 nmol/L浓度的TBT和TPT处理大鼠睾丸Leyd ig(LC-540)细胞24~96 h,用四唑蓝(MTT)法确定细胞的存活率;②用DNA片段法确定是否存在细胞凋亡;③观察细胞内Ca2+螯合剂氨基苯乙烷四乙酸(BAPTA)和蛋白激酶A(PKA)抑制剂H-89、蛋白激酶C(PKC)抑制剂GF109203X、酪氨酸蛋白激酶(TPK)抑制剂Gen iste in是否可以阻断TBT所致的细胞凋亡;④检测TBT处理的原代大鼠睾丸Leyd ig细胞分泌睾酮的变化。结果:①TBT和TPT影响Leyd ig细胞存活率的强度基本相同,在20~80 nmol/L浓度时细胞存活率呈剂量和时间依赖性降低;②DNA片段法研究证实TBT和TPT可引起细胞凋亡;③BAPTA可阻断20 nmol/L的TBT所致的细胞凋亡,而PKA、PKC和TPK抑制剂对细胞存活率没有影响;④TBT可减少Leyd ig细胞分泌睾酮,并使其对人绒毛膜促性腺激素(hCG)刺激的反应性降低。结论:环境污染物TBT和TPT能直接导致睾丸Leyd ig细胞凋亡,并抑制睾酮分泌,这种致凋亡作用可能与细胞内Ca2+浓度增加有关。     

Increased testosterone production in vitro by Leydig cells from rats with severe autoimmune orchitis

We have previously observed (M. O. Suescun et al. , 1994, Journal of Andrology , 15 , 442–448) that rats with autoimmune orchitis (EAO) exhibit increased testosterone production in vitro by isolated testes. The aim of the present study was to determine whether the increase in testosterone production correlated with an enhanced number of Leydig cells and/or enhanced steroidogenic capacity per Leydig cell. For this purpose, EAO was induced in adult Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. At 80 days after the primary immunization, 60% of rats presented with severe testicular damage characterized by sloughing of the seminiferous epithelium, seminiferous tubule atrophy and interstitial mononuclear cell infiltration. At 160 days after the first immunization, testicular lesions were more severe. A morphometric study, by light microscopy, showed an increase in the number of Leydig cells in rats with EAO (45% increase at 80 days and 50% at 160 days). By electronmicroscopy, testicular sections of rats with EAO revealed the presence of numerous Leydig cells closely associated with macrophages. Most Leydig cells exhibited ultrastructural features of active steroid secreting cells.
The steroidogenic capacity of Percoll-purified Leydig cells from testes of rats with EAO, killed at 80 and 160 days, was evaluated. Leydig cells from rats with EAO exhibited an enhanced steroidogenic response to hCG in vitro at 80 days (38%) and an increase in basal (77%) and post-hCG testosterone production (115%) at 160 days compared to controls. However, these cells were less sensitive to hCG. In conclusion, the results indicate that the enhancement of in-vitro testosterone production observed in rats with EAO is accounted for both by the increased number of Leydig cells and by the increased testosterone production of each Leydig cell.