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The agouti (Dasyprocta leporina) and paca (Agouti paca) are among the largest extant rodent species. Although these species have considerable economic potential, there are few reports in the literature concerning male reproductive biology in sexually mature agoutis and pacas. The objectives of the present study were to perform detailed stereologic and histologic analyses of the testis and estimate spermatogenic cycle length in these 2 Neotropical rodent species as well as to compare the data with those from other well-studied rodent members of the Hystricomorpha and Myomorpha suborders. Eight adult agoutis and 6 adult pacas were used. The spermatogenic cycle in agoutis and pacas lasted 9.5 ± 0.03 and 11.5 ± 0.16 days, respectively, whereas the total duration of spermatogenesis for these 2 species was 42.8 ± 0.16 and 51.6 ± 0.7 days, respectively. Most of the parameters investigated were similar to those obtained for the other members of the Hystricomorpha suborder. As a result of the combination of high values of seminiferous tubule volume density, number of Sertoli cells per gram of testis, Sertoli cell efficiency, and a relatively short duration of spermatogenesis, the spermatogenic efficiency found in agoutis (52 ± 2 × 10(6)) and pacas (39 ± 2 × 10(6)) was particularly high when compared with that of previously investigated mammalian species. The data presented in this investigation would be useful for studies related to animal production as well as improvement and conservation programs involving these 2 large Neotropical rodent species.

Although the collared peccary (Tayassu tajacu) is found throughout the Americas, with a high potential for domestication and commercial exploitation, there are few data on the reproductive biology of this mammalian species. The aim of the present study was to investigate testis structure, spermatogenic cycle length, Sertoli cell efficiency, and spermatogenic efficiency. Twelve adult peccaries were used for biometrical, histological, and stereological analyses; 3 of these peccaries received intratesticular injections of (3)H-thymidine for the determination of the duration of spermatogenesis. Testis weight and gonadosomatic index were 23.7 +/- 1.8 g and 0.2% +/- 0.1%, respectively. Seminiferous tubule volume density was 77.4% +/- 1.7%. Leydig cells occupied 12.8% +/- 1.8% of the testis parenchyma and presented a peculiar cytoarchitecture in the periphery of the seminiferous tubule lobes. The premeiotic, meiotic, and postmeiotic stage frequencies were very similar to those found for wild and domestic boars. The spermatogenic cycle and entire spermatogenic process (based on 4.5 cycles) lasted approximately 12.3 +/- 0.2 and 55.1 +/- 0.7 days, respectively. Daily sperm production per gram of testis in the collared peccary was approximately 23.4 +/- 2 x 10(6), which is similar to that of domestic and wild boars. The knowledge generated in the present study could be used in reproduction and animal improvement programs and provides important information that may be used for comparative reproductive biology with previously investigated mammalian species.

In most eutherian mammals, sexually dimorphic masculinization is established by androgen-producing fetal Leydig cells in the embryonic testis. Fetal Leydig cells, which lack expression of the testis-determining gene SRY, arise after the appearance of SRY-expressing Sertoli cells. Therefore, the appearance and differentiation of fetal Leydig cells are probably regulated by factors derived from Sertoli cells. Results from mouse genetic models have revealed that maintenance and differentiation of fetal Leydig cell population depends upon a balance between differentiation-promoting and differentiation-suppressing mechanisms. Although paracrine signaling via Sertoli cell-derived Hedgehog ligands is necessary and sufficient for fetal Leydig cell formation, cell-cell interaction via Notch signaling and intracellular transcription factors such as POD1 are implicated as suppressors of fetal Leydig cell differentiation. This review provides a model that summarizes the recent findings in fetal Leydig cell development.

The objective of this study was to determine whether dietary-induced mild fetal/neonatal hyperthyroidism influenced the initiation of spermatogenesis and the development of the adult-type Leydig cell population. Previously, the effects of neonatally induced hyperthyroidism have been investigated in rats using rather high doses (5 to 10 microg/100 g body weight) of tri-iodothyronine, which not only influenced testicular development, but also negatively affected the general body condition of the animals. To induce hyperthyroidism the diet of the dams was supplemented with 15 mug thyroxine (T(4))/100 g body weight 2 weeks prior to mating and the dams and their offspring were kept on this diet until sacrifice. Pups were killed between days 7 and 64 after birth. At the age of 12 days plasma thyroid-stimulating hormone (TSH) levels tended to be lower in hyperthyroid pups, and from the age of 15 days onwards plasma TSH levels were significantly lower in hyperthyroid animals. Concomitantly, plasma T(4) levels were significantly elevated. From the age of 12 days onwards, plasma follicle-stimulating hormone levels were lower in hyperthyroid animals compared with age-matched control groups. Sertoli cell differentiation did not seem to be influenced by the mild hyperthyroid condition, as no difference in tubule lumen formation was observed between euthyroid and hyperthyroid animals. Nevertheless, a small effect on the progression of spermatogenesis was observed 15 days after birth, as the most advanced type of germ cells in the control testis were pachytene spermatocytes, whereas in the hyperthyroid testis these were leptotene and zygotene spermatocytes. Leydig cell proliferation was decreased in the hyperthyroid pups at the age of 15 days and slightly elevated at later ages, suggesting a possible slower onset of the proliferative activity of these cells than in the euthyroid control animals. Taken together, the present results suggest that even mild dietary-induced hyperthyroidism transiently affects the development of the adult-type Leydig cell population as well as the initial progression of spermatogenesis.

The abundant production of testicular estrogens and the presence of both ESR1 and ESR2 within boar testes are consistent with a role for estrogen in testicular development and/or function in this species. This study was aimed at determining the role of endogenous estrogen in the regulation of testicular development and function, including the effects on testis weight, histology, sperm production (detergent-resistant spermatid numbers), Sertoli cell numbers, and Leydig cell volume in the boar. Twenty-eight littermate pairs of boars were assigned to groups as follows: 1 boar from each pair was assigned to the control group (vehicle) and the other was assigned to treatment and received 0.1 mg/kg body weight of an aromatase enzyme inhibitor (letrozole) orally each week beginning at 1 week of age until castration at 2, 3, 4, 5, 6, 7, or 8 months of age. Testes were weighed and testicular parenchyma was recovered for determination of histology and detergent-resistant spermatid numbers, and for determination of Sertoli cell number and Leydig cell volume by staining for GATA-4 and 17-alpha hydroxylase/17-20 lyase respectively. Testes of aromatase-inhibited boars initially exhibited delayed lumen formation, lower testicular weight, fewer detergent-resistant spermatids, and fewer Sertoli cells, but by 7 to 8 months, these boars had recovered and had larger testes, more detergent-resistant spermatids per testis, and more Sertoli cells. Total Leydig cell volume increased in proportion to testis size. Reducing endogenous estrogen is consistent with a delay in testicular maturation/puberty that allows for a longer window for the proliferation of Sertoli cells and maturation of Leydig cells, resulting in larger testes and higher spermatid production.

Oral administration of sodium tungstate to adult male streptozotocin-diabetic rats for 3 months normalized serum levels of glucose, insulin, luteinizing hormone, and follicle-stimulating hormone. These effects were accompanied by an increase in reproductive performance, which was related to a strong improvement in Leydig cell function markers, such as the recovery of the number of Leydig cells and serum testosterone levels. Moreover, this in vivo recovery was related to a concomitant increase in the cell expression of insulin receptors. Tungstate treatment did not modify Leydig cell function in healthy rats. Furthermore, the addition of tungstate or insulin to the mTLC-1 cell line from Leydig cell origin increased the phosphorylation states of MAP-kinase and glycogen synthase kinase-3. Our results indicate that tungstate treatment in diabetic rats leads to a recovery of reproductive performance by increasing the number of Leydig cells. This increase contributes to the recovery of their functionality, thereby improving the overall function of these cells. We propose that this improvement is caused by the combined effect of the tungstate-induced normalization of insulin glucose and luteinizing hormone serum levels and a direct action of the effector on Leydig cells through modulation of at least MAP-kinase and glycogen synthase kinase-3 activities.

Presence of vascular endothelial growth factor (VEGF) is not only limited to cells directly involved in angiogenesis but has also been demonstrated in steroidogenic cells like testicular Leydig cells. Because Leydig cells are subjected to regulation by gonadotropic hormones and produce steroid hormones, we have investigated here the effects of human chorionic gonadotropin (hCG) or steroid hormones on VEGF expression in cultured mouse tumor Leydig cells (MA-10 cells) and have then analyzed the underlying molecular mechanisms. Northern blot analysis and enzyme-linked immunosorbent assays revealed increases in VEGF mRNA and protein levels, respectively, over 3-20 hours in MA-10 cells after stimulation with hCG or 8-Br-cAMP. Although MA-10 cells lack the classical progesterone receptor, progesterone was able to stimulate VEGF expression. Promoter analyses and antibody supershift experiments suggested that the proximal region is able to constitutively bind the transcription factors Sp1 and Sp3. Mutations of 2 potential Sp1 binding sites in the proximal region showed the requirement of these motifs for stimulation of VEGF by hCG and 8-Br-cAMP. The distal cytosine-rich sequence interacts with so far-unidentified faster migrating factors. Following stimulation with hCG or 8-Br-cAMP, the binding of these proteins was increased in the complexes formed in the proximal and distal regions. VEGF expression in Leydig cells is regulated by gonadotropin via a cAMP-dependent mechanism, and the transcription factors Sp1 and Sp3 appear to be involved in the activation of the promoter. Progesterone also appears to play a role in the regulation of VEGF, acting presumably via a nonconventional receptor that remains to be characterized yet.

Procedures for purification of Leydig cells have facilitated studies of their regulatory biology. A multistep procedure that includes a filtration with nylon mesh (100-micron pore size) to separate interstitial cells from the seminiferous tubules, combining centrifugal elutriation and Percoll density gradient sedimentation, has been used to obtain a 95% enrichment of rat Leydig cells. However, the number of recovered Leydig cells by this procedure represents only a small fraction of the 25 million, on average, that exist in the adult rat testis. The objective of this study was to test whether the yield of purified Leydig cells might be enhanced by substitution of unit-gravity sedimentation (S method) for the filter step (F method). We also asked whether a greater number of Leydig cell clusters, macrophages, or both would be recovered by this new method, and if the presence of Leydig cell clusters is associated with increased capacity for testosterone production in vitro. The number of purified Leydig cells was 1.9-fold higher for the S method than for the F method, with no differences in purity assessed by 3beta-hydroxysteroid dehydrogenase histochemical staining. Leydig cell clusters were also found in greater numbers with the S method both after collagenase dispersion and at the end of the purification. No differences were seen in testosterone production or in the number of macrophages present in the Leydig cells that were prepared by the 2 methods. These results indicate that the new method recovers greater numbers of Leydig cells by collecting clustered Leydig cells that are systematically eliminated when a filtration step is used.

The surprising findings that male mice lacking the estrogen receptor (ER) alpha (alphaERKO) have atrophic testis and are infertile proved that ERalpha is involved in normal testicular function. To obtain compatible in vitro model systems for alphaERKO male mice, we immortalized different cell lines from the testis of alphaERKO and wild-type (C57BL/6) mice with the human papilloma virus E6/E7 genes. The established cell lines were characterized for Sertoli, Leydig, and peritubular cell markers by means of messenger RNA expression and functional assays. One wild-type-derived cell line showed Leydig cell-specific marker gene expression and produced testosterone after stimulation with cyclic adenosine monophosphate. Most wild-type cell lines expressed androgen receptor and a functional ERalpha as shown by high estrogenic activity in a luciferase-based transactivation assay. Most notably, the wild-type-derived WL3, and the ES4 cell line derived from alphaERKO mice expressed ERbeta and showed ER-mediated transcriptional activity, but no ERalpha protein expression. These cell lines with and without functional ERalpha or ERbeta enable the analyses of ER subtype-specific responses and their function in testicular cell signaling, morphogenesis, and neoplasia.

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.

We examined the effects of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and epidermal growth factor (EGF) on the expression and kinase activity of cyclin-dependent kinase 5 (Cdk5) in Leydig TM3 and Sertoli TM4 cell lines. Hormonal regulation of the expression and activity of Cdk5 by using normal and hypophysectomized rat testes was also investigated to elucidate its role. Cdk5 levels and kinase activity were significantly elevated in TM3 cells that were grown in the presence of 7.5% serum, EGF, or LH and were associated with an increase in testosterone production compared with controls. These increases were accompanied by an increase in proliferation of TM3 cells after treatment with serum or EGF but not with LH suggest that Cdk5 may be involved in cellular differentiation that is induced with LH treatment. In contrast, the presence of neither serum, EGF, nor FSH had a significant effect on Cdk5 activity levels in the Sertoli TM4 cell line, and there was no correlation with proliferative activity or transferrin levels. A significant decrease in Cdk5 expression and activity were noted in rat testis after hypophysectomy compared with normal rat testis and is associated with a simultaneous decrease in testosterone and transferrin levels. Immunohistochemical analysis revealed that Cdk5 was strongly expressed in the nuclei and cytoplasm of Leydig cells, Sertoli cells, spermatogonia, and peritubular cells of normal adult rat testis. After hypophysectomy, the pattern of Cdk5 staining differed markedly from that in normal rat testis and a profound reduction in staining of Cdk5 was observed in each tubule. Our results suggest that LH and EGF influence and modulate Cdk5 expression and activity in Leydig TM3 cells and may, conceivably, be involved in signal transduction cascades that are initiated by hormones or growth factors. Cdk5 in Sertoli TM4 cells is likely to possess some constitutive functions that are not affected by the cells' proliferation state. Moreover, Cdk5 is probably involved in the constitutive and hormonally stimulated activities of the rat testis, in addition to its involvement in cell proliferation.

Steroidogenesis and spermatogenesis decrease in aging Brown Norway rats. We therefore hypothesized that there must be accompanying morphological changes taking place in the seminiferous tubules of the aging testis. The testes of Brown Norway rats ranging in age from 3 to 24 months were prepared for light and electron microscopy. To assess the integrity of the blood-testis barrier with age, a lanthanum nitrate study was done. The normal seminiferous tubules present in rats at 3 and 12 months of age were largely replaced at 24 months by fully regressed tubules that were virtually devoid of germ cells and contained large intercellular spaces. An electron-microscopic study of these regressed tubules showed a complete loss of cyclical variations of the organelles of the Sertoli cells. The nucleus was more irregularly shaped and was present at various levels in the epithelium. The endoplasmic reticulum was a loose, vesiculated network that was unlike the elaborate, tubular, anastomotic network noted in young animals. The lysosomes were large, oddly-shaped, and contained lipidic inclusions, in contrast to the distinct membrane-bound lysosomes and dense core bodies found in the young animals. Adjacent Sertoli cell processes encompassed large, empty intercellular spaces, possibly occupied previously by germ cells. The typical Sertoli-Sertoli junctions of the blood-testis barrier in the young animal were rarely seen at 24 months and were replaced by focal contact points, usually between three Sertoli cell processes. In the aged animals, lanthanum nitrate permeated the basal and adluminal compartments, extending between Sertoli cell processes and entering the intercellular spaces and lumen. In summary, during aging, there is a breakdown of the blood-testis barrier, and there are striking changes in the appearance of Sertoli cells. These results suggest a possible intrinsic limitation that prevents stem cells from renewing themselves, whether because of a degeneration of immunological origin or because of a lack of Sertoli cell support.

Previous studies have shown that reductions in Leydig cell testosterone production occur with aging in the Brown Norway rat. The recent observation that changes in luteinizing hormone (LH) pulse interval and amplitude also occur with aging suggests the possibility that age-related reduced Leydig cell steroidogenesis might be related to changes in LH. We reasoned that, if this is the case, exogenously administered LH should restore testosterone production by aged rat Leydig cells to the higher levels produced by Leydig cells of young rats. To test this hypothesis, young (4-month-old) and aged (21-month-old) rats received testosterone- and estradiol-containing Silastic implants designed to suppress LH and, thus, endogenous Leydig cell testosterone production. At the same time, the rats received miniosmotic pumps programmed to deliver pulsatile ovine LH at a predetermined daily dose. In some experiments, treatment effects were determined by measuring testosterone production by testes perfused in vitro with maximally stimulating ovine LH. In others, Leydig cells were isolated by centrifugal elutriation and Percoll density gradient centrifugation, and their in vitro ability to produce testosterone in response to maximally stimulating LH was determined. Testes or isolated Leydig cells from untreated young rats produced about twice as much testosterone as that produced by Leydig cells from aged rats. The administration of testosterone- and estradiol-filled implants for 5 days reduced testosterone production significantly at both ages. In young rats administered 24 microg LH/day for 5 days, along with the implants, testosterone production was maintained at the high level of the young controls. Comparable treatment of aged rats resulted in testosterone production only at the low level of the aged controls. Indeed, even with higher LH doses (36 microg/day), testosterone production by the aged rat Leydig cells did not rise above the aged-control level. The inability of exogenously administered LH to increase testosterone production by testes and Leydig cells of aged rats suggests that Leydig cell steroidogenic deficits in the aged Brown Norway rat are unlikely to be the result of age-related changes in LH.

Spermatogenesis in Asian men appears to be more susceptible to suppression by steroidal contraceptives administered in clinical trials than spermatogenesis in Caucasian men. The objective of this study was to determine whether ethnic differences exist in testicular structure and spermatogenic potential that might predispose Asians to a high sensitivity to steroidal contraceptives. Testes from 12 Chinese men were compared to those from 8 Hispanic men and 12 non-Hispanic Caucasian men of ages 29+/-3, 30+/-2, and 29+/-3 years, respectively. Testes were fixed by vascular perfusion with glutaraldehyde, further fixed in osmium, embedded in Epon, and evaluated by stereology using 0.5-microm sections stained with toluidine blue. Homogenates of fixed testes were evaluated for the number of Sertoli cells and the daily sperm production based on pachytene primary spermatocytes (PDSP) or spermatids with spherical nuclei (DSP). Paired parenchymal weight was less (P < 0.05) in Chinese men than in Hispanic or Caucasian men. The PDSP per gram of parenchyma was lower (P < 0.05) and the DSP per gram tended to be lower in Chinese men than in other groups. The histologic appearance, volume density, and length per man of seminiferous tubules were the same among the ethnic groups; however, the diameter of seminiferous tubules was less (P < 0.05) in Chinese than in Hispanic or Caucasian men. The PDSP per man and the DSP per man were lower (P < 0.05) in Chinese than in Hispanic or Caucasian men. The number of Sertoli cells per gram was higher (P < 0.05) in Chinese or Caucasian men than in Hispanic men, but the number of Sertoli cells per man was lower (P < 0.05) in Chinese men than in Hispanic or Caucasian men. Sertoli cell function, measured as the number of germ cells accommodated by a single Sertoli cell, was lower (P < 0.05) in Chinese men than in Caucasian men. The volume density of Leydig cell cytoplasm was greatest (P < 0.05) in Chinese men, but the number of Leydig cells was similar among the ethnic groups. Hence, smaller testes coupled with reduced Sertoli cell number and function and reduced daily sperm production could predispose Asian men to have a heightened negative response of testes to steroidal contraceptives, as compared to Caucasian men. Dampening (by exogenous androgens) of any physiological benefit to spermatogenesis that a high volume density of Leydig cell cytoplasm may bestow on the human testis (that Asian men may have evolved to require) would exacerbate ethnic differences in the spermatogenic response to hormonal contraceptives.

The objective of this study was to investigate the role of androgens and nonandrogenic Leydig cell products in maintaining Leydig cell and macrophage numbers in the testis of the adult rat. Adult male Sprague-Dawley rats received Silastic implants containing testosterone and estradiol (T-E) in order to suppress endogenous luteinizing hormone (LH) for 9 weeks. After T-E treatment, Leydig cell and macrophage numbers, quantified using the optical disector approach, were reduced by 40 and 60%, respectively, compared with controls. Administration of human chorionic gonadotropin (hCG) for a period of 10 days restored Leydig cell numbers to control levels, and macrophage numbers were partially restored. Administration of the antiandrogen, flutamide, in combination with hCG treatment in T-E implanted animals prevented the restoration of Leydig cell numbers but did not prevent the recovery of macrophage numbers. In the T-E-implanted animals, there was a decrease in testicular macrophage nuclear size, which was not restored by either hCG or hCG plus flutamide treatment. The results of this study support the hypothesis that LH is the main pituitary regulator of both Leydig cell and macrophage number in the adult rat testis and further indicate that androgens are responsible for maintaining Leydig cell numbers and/or differentiation, but nonandrogenic Leydig cell factors are primarily responsible for controlling macrophage numbers. Testicular macrophage function, as indicated by nuclear size, does not appear to be influenced by LH or testosterone in the adult rat.

We previously reported a strain-related difference in basal 3beta-hydroxysteroid dehydrogenase-isomerase (3betaHSD) activity in response to testosterone in cultured Leydig cells. The data suggested that the response to testosterone was androgen receptor mediated and that testosterone was acting via a trans-acting factor distal to the androgen receptor to regulate Leydig cell basal 3betaHSD activity. This study was designed to determine whether the previous reported strain-related difference in basal 3betaHSD activity in response to testosterone was due to a difference at the 3betaHSD protein and/or at the mRNA level. In C57BL/6J Leydig cells, 2.0 microM testosterone significantly decreased basal 3betaHSD immunoreactive mass by day 6 in culture. Treatment with 2.0 microM testosterone and 2.0 microM hydroxyflutamide, an androgen receptor antagonist, negated the inhibitory effect of testosterone on C57BL/6J 3betaHSD immunoreactive mass. Treatment with 2.0 microM testosterone also significantly decreased 3betaHSD mRNA content in C57BL/6J Leydig cells, which was detectable on day 3 in culture. In contrast to Leydig cells from C57BL/6J mice, Leydig cells from C3H/HeJ mice were not susceptible to the inhibitory effect of testosterone on 3betaHSD. Treatment with 2.0 microM testosterone had no detectable effect on C3H/HeJ 3betaHSD immunoreactive mass or mRNA content at any time point in culture. These data indicate that the testosterone-induced loss of basal 3betaHSD activity in C57BL/6J Leydig cells can be accounted for by the loss of 3betaHSD immunoreactive mass, which is preceded by the loss of 3betaHSD mRNA, and that the strain-related difference in the regulation of 3betaHSD is present at all three levels. Thus, the putative trans-acting factor involved in the mechanism whereby testosterone decreases basal 3betaHSD is likely to regulate the amount of 3betaHSD mRNA.

The objective of this study was to investigate the role of the gonadotropins and, in particular follicle-stimulating hormone (FSH) in maintaining the Leydig cell and macrophage populations of the adult rat testis. Adult male Sprague-Dawley rats received a gonadotropin-releasing hormone (GnRH) immunogen for a period of 12 weeks in order to induce a selective deficiency in luteinizing hormone (LH) and FSH. Recombinant human FSH was then administered for 7, 14 and 21 days and macrophages and Leydig cells per testis quantified using the "optical disector" method. After GnRH immunization, Leydig cell and macrophage numbers were reduced by 18% and 68%, respectively, compared with normal controls, resulting in an increase in the ratio of Leydig cells to macrophages from 4:1 to 9:1. Leydig cells regressed morphologically following GnRH immunization, and macrophage mean nuclear diameter was significantly reduced. Administration of FSH did not restore the numbers of either cell type; however, FSH did increase macrophage nuclear size. Eosinophils and mast cells were also found sparsely scattered throughout the interstitium after GnRH immunization and persisted in the FSH-treated animals. The results of this study indicate that in the adult rat: 1) both Leydig cell and macrophage numbers are reduced in the gonadotropin-deficient testis; 2) FSH has no effect on the number of either cell type in the absence of LH; and 3) testicular macrophage activity, as indicated by nuclear size, is stimulated by FSH, either directly or indirectly.

This study provides quantitative information on the early (up to 3 months) effects of vasectomy on apoptosis in the hamster testis. Groups of five adult male golden hamsters were either bilaterally vasectomized or sham-operated and sacrificed at intervals of 3, 6, and 12 weeks after surgery. In all three postvasectomy groups, testis weight and testicular and plasma testosterone (T) levels were not different from controls. Spermatogenic alterations, ranging from tubules with mild intraepithelial vacuoles to almost completely atrophied tubules, were detected in samples of 1 of 5 testes both at 3 and 12 weeks after vasectomy. Histometric analysis of testicular tissues at 3, 6, and 12 weeks in the postvasectomy groups showed no discernible effect of vasectomy on the absolute volumes of seminiferous tubules, tubular lumen, and total Leydig cells when compared to respective controls. In situ analysis of germ-cell apoptosis, characterized by 3'-end-labeling immunocytochemistry, revealed a significant increase (2.5-fold) in germ-cell apoptosis at stages XIII-I, involving primarily the dividing spermatocytes after 3 weeks of vasectomy. Apoptotic index was not changed from sham-operated animals at 6 and 12 weeks postvasectomy. Interestingly, a very high incidence of macrophage apoptosis was detected in the samples of three out of five testes in the 12 weeks postvasectomy group (39.3%) compared to that of controls (0.8%). These results demonstrate that vasectomy has little or no detrimental effect on the morphologic characteristics of the spermatogenesis or intratesticular concentrations of testosterone in the majority of the animals studied up to 12 weeks postsurgery, although vasectomy transiently (3 weeks postsurgery) activated germ-cell apoptosis, involving dividing spermatocytes at stages XIII-I.

The objective of this study was to determine the effects of pituitary hormones (luteinizing hormone [LH], follicle-stimulating hormone [FSH], growth hormone [GH], and prolactin [PRL]) on interstitial cell proliferation and differentiation in the testis of immature hypophysectomized rats. Macrophages, Leydig cells, precursor mesenchymal cells, endothelial lymphatic cells, and myoid cells were studied. Our experimental approach was aimed at determining whether changes in a cellular subpopulation observed after pituitary hormone treatments were the result of division of existing cells in the population, of differentiation of interstitial precursor cells, or both. In this context, it must be stressed that our data reflected the effects of hormones to prevent the decline of cells due to hypophysectomy rather than their recovery. Macrophage proliferation was taken into account because macrophages closely resemble Leydig cells and are known to proliferate after hormonal treatment. A double-labeling procedure (acid phosphatase and anti-bromodeoxyuridine [anti-BUdR]) revealed that LH, FSH, and PRL increased the number of testicular macrophages 105-, 104-, and 103-fold, respectively, in hypophysectomized rats compared to hypophysectomized control animals. BUdR incorporation in testicular macrophages was greater after PRL treatment than after LH and FSH supplementation. In contrast, we were unable to demonstrate any effect of rat GH on the macrophage population. Light microscopic analysis of plastic embedded sections of treated rat testis revealed that LH increased the numbers of Leydig, precursor mesenchymal, and myoid cells 6-, 4-, and 1.3-fold, respectively. LH also stimulated BUdR incorporation into all interstitial cell types. PRL administration increased both the number of Leydig and precursor mesenchymal cells (each 3-fold) but decreased the number of endothelial lymphatic cells (1.5-fold) when compared to the control animals. In contrast, FSH did not increase the number and proliferation of Leydig cells but exerted a slight proliferative effect on the other interstitial cell populations. In GH-treated rats, the number of precursor mesenchymal cells increased two fold above the control rats. GH also exerted slight proliferative effects on both precursor mesenchymal and myoid cells. Immunohistochemical studies of steroidogenic enzymes in the testicular interstitium of treated rats demonstrated the presence of steroidogenic enzymes, not only in Leydig and precursor mesenchymal cells, but also in some (1%-2%) endothelial lymphatic cells and myoid cells. This may indicate that both of these cell types are also constitutively equipped to perform steroidogenesis or that they are precursor cells undergoing differentiation. Taken together, changes in the number of Leydig cells in our animal model appeared more likely to be dependent on the transformation of precursor cells than on division of preexisting mature Leydig cells.

Treatment of male rat pups with the reversible goitrogen 6-n-propyl-2-thiouracil (PTU), administered by adding 0.1% PTU to the mother's drinking water from birth through weaning, increases adult testis size, number of Sertoli cells, and daily sperm production. Adult Leydig cell numbers are also increased by 70%, despite permanent suppression of serum luteinizing hormone (LH). The present study was designed to test whether this effect results from an increase in proliferation of Leydig cells or the mesenchymal precursors. The labeling indices (LI) of six interstitial cell types were measured by tritiated thymidine autoradiography. With the exception of Leydig cells, LI declined after birth for all interstitial cell types in both control and PTU-treated rats, but mesenchymal cell proliferation was not significantly different in control and treated rats. In contrast, the LI of Leydig cells from PTU-treated rats rose by day 10, remained elevated through day 45 (highest on day 35 at 3.6-fold higher than control, P < 0.05), and declined to control values by day 50. The LI of Leydig cells in control rats was unchanged during the experiment. Proliferating Leydig cells were immature, as shown by their cytoplasmic lipid droplets. Adult Leydig cells, which lack lipid droplets, did not proliferate. Mesenchymal and immature Leydig cells contained thyroid hormone receptor mRNA; levels in adult Leydig cells were reduced but detectable. In conclusion, proliferation of Leydig cells, rather than increased proliferation of their mesenchymal precursors, is the principal mechanism responsible for the increase in Leydig cell number after neonatal hypothyroidism. Increased Leydig cell proliferation could result from direct effects of hypothyroidism on these cells. Alternatively, the increased Sertoli cell population that results from PTU treatment could also stimulate increased Leydig cell proliferation, or both of these mechanisms could contribute to the Leydig cell increase.

As previously shown, Leydig cells in culture dramatically up-regulate the expression of the neural cell adhesion molecule (NCAM) gene and express alternatively spliced forms. Because the family of NCAM adhesion molecules is known to be involved in cell migration and differentiation, we examined the potential involvement of NCAMs in Leydig cell differentiation in the developing testis. We detected NCAM-immunoreactive cells in the rat and mouse at embryonic day (ED) 13-14 in epithelia of mesonephric tubules and cell clusters between the mesonephros and testis. At about ED 17-18, strongly NCAM-immunoreactive cells were demonstrated extending from the mesonephros/mesonephric tubules into the region occupied by the forming rete testis and spreading into the testis itself. Within the testis, interstitial fetal Leydig cells, identified with an antiserum directed against P450-side-chain cleavage enzyme, were also NCAM immunoreactive, although to a lesser degree, and in part expressed NCAM-associated polysialic acid. In situ hybridization histochemistry demonstrated the presence of NCAM mRNA, mainly in the cell population corresponding to the strongly immunoreactive cells. NCAM forms with molecular weights of 140 and 180 kDa, the latter polysialylated, predominate in immunoblots of fetal testes. Because testicular development occurs along the mesonephros, from which precursor cells are thought to migrate into the developing gonad and then differentiate into the various testicular cell types, our results may suggest that the expression of NCAMs and NCAM modification are associated with cells that appear to migrate into the developing testis. Although it remains to be proven whether these cells could be precursor cells of Leydig cells, this assumption is supported by the fact that within the developing testis, NCAMs and NCAM modifications are expressed during differentiation of testicular P450-side-chain cleavage enzyme-positive Leydig cells. Thus, the adhesion molecule NCAM and its variants and modifications are expressed in the developing testis and may be of functional, "morphogenic" importance. Because Leydig cells in vitro and during fetal development express NCAMs, these molecules may prove to be a suitable specific marker for the study of Leydig cell development and differentiation.

The relative capacities for testicular macrophages and resident peritoneal macrophages to secrete the pro-inflammatory cytokine, interleukin 1 (LL-1), in response to stimulation by bacterial lipopolysaccharide (LPS), were compared in vitro. Macrophages were isolated from adult male rat testicular interstitial cells or peritoneal lavage by adherence to glass coverslips or plastic culture dishes. The macrophages were immediately cultured, with or without a maximal dose of LPS (1 mu g/ml), over 24 hours at 32 degrees Celsius. Bioactive LL-1 production was measured by a sensitive thymocyte proliferation bioassay, employing recombinant human LL-1 beta as the reference standard. In comparison with the peritoneal macrophages, testicular macrophages displayed only a very small response to LPS, producing 2.8% of the amount of LL-1 per cell secreted by peritoneal macrophages cultured under identical conditions. Production of authentic LL-1 was confirmed by inhibition of the bioassay response in the presence of human recombinant LL-1 receptor antagonist. A small molecular mass (<10 kDa based on ultrafiltration) inhibitor of LL-1 bioactivity was also present in the medium collected from both cultures, but this inhibitory activity did not account for the differences in activity observed. In cultures of total peritoneal cells under similar conditions, addition of testosterone (10-1,000 ng/ml) did not affect LL-1 production in response to LPS. These data indicate that testicular macrophages have a reduced ability to secrete bioactive IL-1, and they provide further evidence for an altered capacity for immune responses within the testis.

This study examined the potential role of testicular opioids, a pertussis toxin (PT)-sensitive G-protein, and phosphodiesterase in mediating the inhibitory effect of immobilization stress on testicular steroidogenesis in adult rats. The experiments were initiated with enriched preparations of Leydig cells, but the stress effect was not sustained in vitro either as a result of the disruption of the morphology of the testis and/or the time required for Leydig cell isolation. Consequently, testicular fragments from control and stressed (3-hour immobilization) rats were used in these experiments. When fragments from stressed rats were incubated for 2 hours in the absence and presence of human chorionic gonadotropin (hCG) (0.1,1, or 10 mlU), testosterone (T) production in response to 1 and 10 mlU hCG was lower (P < 0.05 and 0.01, respectively) than that from control fragments. Basal T secretion did not differ between stressed and control fragments. Naloxone (1, 10, or 100 mu M), did not alter basal or hCG-stimulated T secretion from control fragments, but it normalized the T response to hCG from stressed fragments. Control fragments also showed a reduced T response (P < 0.05) to hCG in the presence of beta-endorphin (beta-E; 36 nM). Incubation of control fragments with PT (30 ng) did not alter basal or hCG-stimulated T production. However, PT normalized (P < 0.01) hCG-stimulated T secretion from stressed fragments. Methylisobutylxanthine (MIX; 0.125 mM) elevated (P < 0.01) hCG-stimulated T production from control fragments, but hCG-stimulated T secretion from stressed fragments remained subnormal in the presence of the phosphodiesterase inhibitor. The data suggest that acute immobilization stress inhibits gonadotropin-induced T production in adult male rats via a mechanism involving testicular opioids and a PT sensitive G-protein. We found no evidence to suggest that a stress induced increase in the activity of phosphodiesterase was involved in this mechanism.

To test whether the gap junction protein connexin 43 (Cx43) is associated with germ cell differentiation and with the Sertoli cell junctional blood barrier, we recorded the temporal changes in its distribution before birth, through the neonatal period, puberty, and adulthood in guinea pig, and throughout the annual seasonal reproductive cycle in the mink. We used the immunoperoxidase labeling technique on Bouin's perfused-fixed testes and with site-specific polyclonal affinity-purified antibodies against Cx43. Cx43 was localized between Leydig cells in fetal guinea pig testis. In this species, after birth, the appearance of Cx43 concurred with the onset of spermatogenesis. In the seminiferous epithelium, the distribution of Cx43 coincided with the gap junctions of the Sertoli cell junctional blood barrier. In guinea pig and mink, the distribution of the protein in the tubules changed in accordance with the germ cell differentiation in a stage-dependent manner and with the modulation, i.e., the assembly and disassembly of the junctional barrier accompanying the translocation of spermatocytes into the lumenal compartment. In the mink, the reaction product persisted during testicular regression but showed a similar distribution from one tubule to the next. In this paper, we documented the existence of a temporal correlation between the appearance of the gap junction protein Cx43 and both the germ cell differentiation and the modulation in the junctional barrier between Sertoli cells. The paper also discusses the possibility that cell-to-cell communications, generally attributed to gap junctions, may help changes in the barrier to take place in coordination with spermatogenesis.

Previous studies have demonstrated that Leydig cell testosterone production diminishes with age in Brown Norway rats. The objective of the studies presented herein was to test the following possible explanations for age-related decline in steroidogenesis: (1) decline in Leydig cell number; (2) understimulation by luteinizing hormone (LH); (3) reduced ability of individual Leydig cells to produce testosterone; and (4) influence of loss of germ cells. Leydig cells isolated from the testes of young and aged rats by centrifugal elutriation and Percoll density gradient centrifugation were examined for their ability to produce testosterone when stimulated maximally with LH or with dibutyryl cyclic AMP (dbcAMP). Leydig cell number and volume were examined in situ using stereological methods. Serum LH levels were measured using a highly sensitive immunofluorometric assay. Average Leydig cell volume decreased with age, and consistent with this observation, individual Leydig cells isolated from aging rats produced significantly less testosterone than those from young rats whether the cells were cultured in vitro with maximally stimulating LH or with dbcAMP. The age-associated diminished testosterone production could not be explained by changes in Leydig cell number, serum LH levels, Leydig cell responsiveness to LH, or testicular germ cell content. These results, taken together, suggest that the reduced testosterone production seen in aged rats is related to defects in the steroidogenic pathway beyond the LH receptor-cAMP cascade. The nature of the initial age-related changes that cause reduced steroidogenesis is not known, and therefore it is not known whether such changes are intrinsic or extrinsic to the Leydig cells.

Testicular macrophages were selectively depleted in the right testes of adult rats by an intratesticular injection of dichloromethylene diphosphonate-containing liposomes (Cl2MDP-lp), whereas the left testes were injected with 0.9% NaCl and served as control. Before or after Leydig cell destruction with ethylene dimethane sulfonate (EDS), treatment with Cl2MDP-lp/NaCl was given at different times to study the requirements of macrophages in the different stages of Leydig cell regeneration. On day 30 after EDS treatment, new Leydig cells were abundant in the left, macrophage-containing testes. However, in the right, macrophage-depleted testes, the number of Leydig cells was related to the time elapsed between EDS treatment and macrophage depletion. When macrophages were depleted on day 10 before or on days 4 or 10 after EDS treatment, new Leydig cells were nearly absent at 30 days. However, when macrophages were depleted on days 16 or 22 after EDS treatment, Leydig cells were found at 30 days, but their numbers were equivalent to the number of Leydig cells that were already present in EDS-treated animals at the time the macrophages were depleted. These results indicate that macrophages are needed for the differentiation of Leydig cells from mesenchymal precursors, as well as for the proliferative activity of the newly formed Leydig cells, possibly through the secretion of essential growth factors.

Glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of glutathione (GSH), a tripeptide found in all mammalian cells; this function plays a protective role, as the addition of GSH to an electrophile generally forms a less toxic product. The pi class of GSTs contains homodimers of the Yf subunit, also known as Yp or rat subunit 7; this subunit is found in high concentrations in the testis and epididymis. The objective of the present study was to localize immunocytochemically the Yf subunit in the testis and in the various regions of the epididymis using light, electron, and confocal microscopy. In the testis, immunoperoxidase staining was localized exclusively to Sertoli and Leydig cells. The low cuboidal epithelial cells of the rete testis and the sparse ciliated cells of the ductuli efferents were also immunoreactive. A distinct pattern of immunostaining for the Yf subunit was observed in the different regions of the epididymis. The proximal area of the initial segment showed intense reactivity localized to epithelial basal cells. Basal cells in the middle area of the initial segment were also reactive, as were a second unidentified population of cells located in the apical region of the epithelium. The epithelium, including both principal and basal cells, in the distal initial segment, intermediate zone, and proximal caput epididymidis showed a weak, moderate, or strong degree of reactivity, respectively. In the distal caput epididymidis, however, principal cells showed a checkerboard-like pattern of immunoreactivity, with some cells being intensely stained or faintly stained, whereas others were unreactive. Strikingly, in the corpus and proximal cauda epididymidis, intense immunostaining was localized exclusively over the epithelial basal cells. As viewed in the light and confocal microscope, the intensely stained basal cells showed extensive processes that covered most of the base of the epididymal tubule. Upon quantitation of the immunogold labeling density (the number of gold particles/microns2) in principal and basal cells of the different regions of the epididymis, we observed a sharp decline in immunogold labeling of principal cells coupled with a dramatic increase in labeling of basal cells as we progressed along the tissue, particularly in the transition from the caput to the corpus epididymidis. This study constitutes the first demonstration of a protein that is selectively expressed in epithelial basal cells of the corpus and proximal cauda epididymidis.(ABSTRACT TRUNCATED AT 400 WORDS)

Using in vitro bioassays and radioimmunoassays, the authors examined the interactions between purified 32kD ovine inhibin, testosterone, and dihydrotestosterone on basal and GnRH-stimulated secretion of gonadotropins in primary rat pituitary cell cultures. This study demonstrates that inhibin and androgens: 1) differentially regulate gonadotropin levels, 2) cause changes in the amounts of bioactive and immunoactive FSH and LH released. In conclusion, differences in bio- and immuno-FSH and LH secretion suggest that the endocrine milieu results in not only quantitative but qualitative changes in the secreted gonadotropin isoforms.

The proliferation and differentiation of mast cells and Leydig cells were studied in adult sham operated or hypophysectomized rats after the administration of ethylene dimethane sulphonate (EDS) and in prepubertal rats after neonatal treatment with a gonadotropin-releasing hormone (GnRH) antagonist (Organon 30276; Oss, The Netherlands). After treatment with EDS, two proliferative waves were found. On day 3, several interstitial cell types proliferated, whereas mitotic cells corresponded to differentiating Leydig cells and mast cells around day 20. Differentiating Leydig cells showed a higher mitotic index than that of differentiating mast cells. Hypophysectomized animals showed high mitotic activity 3 days after treatment, but 21 days after treatment differentiating Leydig cells were absent and proliferative activity was reduced. The number of mast cells increased from day 15 to day 30 in EDS-treated rats and from day 15 to day 50 in hypophysectomized, EDS-treated rats. GnRH antagonist-treated rats showed poorly differentiated Leydig cells and abundant mitotic figures on day 23. Proliferation and differentiation of Leydig cells occurred concomitantly with the proliferation and differentiation of mast cells between 23 and 30 days of age. These results suggest that Leydig cells and mast cells in the rat testis share some common regulatory factors.

Leydig cells were isolated and purified from adult and midpubertal rats to study the effects of insulin-like growth factor-I (IGF-I) on steroidogenesis. Androgen production, as measured in Leydig cell conditioned culture media, from four different treatment groups (1 = no hormone; 2 = 70 ng/ml IGF-I; 3 = 0.1 ng/ml LH; 4 = 70 ng/ml IGF-I + 0.1 ng/ml LH) were compared daily. After 3 days in culture, the cells were treated with a maximally stimulating dose of luteinizing hormone (LH) (100 ng/ml) for 3 hours. Androgen production was highest in the cells treated with both IGF-I and low concentrations of LH. In the presence of IGF-I, regardless of LH, cells derived from pubertal animals had a greater increase in steroidogenesis during the culture period than did cells from adult animals. Pretreatment with IGF-I prior to maximal LH stimulation induced a greater increase in androgen production in cells from pubertal rats than in cells from adult animals. It is concluded that IGF-I has a direct effect on Leydig cells and may act synergistically with LH to promote androgen synthesis. The greater response in pubertal cells raises the possibility that IGF-I is important in the maturing process of the testis.

This study was designed to determine the effects of 17 beta-estradiol (E2) on Leydig cell development in the rat. Mature (60 to 65 days old) male rats received a single intraperitoneal injection of ethane dimethylsulfonate (EDS, 100 mg/kg body weight); untreated rats served as controls. In one series of experiments, groups of EDS-treated rats also received daily injections of either E2 (25 micrograms/100 g body weight), human chorionic gonadotropin (hCG, 20 IU/day), a combination of the two, or vehicle only (EDS controls). Animals were killed on days 2, 4, 10, 16, 24, 30, and 36 after EDS treatment. In another series of experiments, groups of EDS-treated rats received daily injections of hCG and E2 during days 0 through 5, 5 through 30, or 16 through 30 after EDS treatment, and were killed on day 30. In both series of experiments, the steroidogenic capacity and hCG binding capacity of the Leydig cells were examined in short-term in vitro incubations using collagenase-dispersed interstitial cells. Testes were also prepared and examined histologically by light and electron microscopy. E2 treatment of animals during the initial 5 days after EDS administration had no effect on the regeneration of interstitial cells and Leydig cells. Treatment with E2 during days 5 to 30 post-EDS blocked the regeneration of Leydig cells and thereby significantly reduced the increase in interstitial cell numbers. Finally, when E2 treatment was delayed until 16 days post-EDS, there was no significant reduction in the regeneration of interstitial or Leydig cells. These data suggest that an important developmental process that is necessary for Leydig cell regeneration occurs between days 5 and 16 post-EDS.(ABSTRACT TRUNCATED AT 250 WORDS)

In this study, Leydig cells in mitosis in adult guinea pigs were quantified. Testes of adult control guinea pigs (n = 10) were fixed by whole body perfusion with 2.5% glutaraldehyde in cacodylate buffer, postfixed in a mixture of osmium tetroxide-potassium ferrocyanide, and embedded in Epon Araldite for qualitative and quantitative microscopy. Using stereologic techniques, the total number of Leydig cells per testis and the number of dividing Leydig cells per testis were quantified. Light microscopic studies revealed the presence of dividing Leydig cells. Ultrastructural studies on a few of these Leydig cells showed that they contained abundant smooth endoplasmic reticulum and mitochondria, which further supported this identity. The total number of Leydig cells per testis and the number of dividing Leydig cells per testis were determined as 14.1 x 10(6) (standard error [SE] = 0.33) and 8 x 10(3) (SE = 5), respectively. These results indicate that Leydig cells undergo mitosis at a rate of 1 per 1.75 x 10(4) in adult guinea pigs.

The authors examined the possibility that ethane 1,2-dimethanesulphonate (EDS) has a cytotoxic effect on spermatogenesis that is not secondary to androgen withdrawal resulting from the well known cytotoxic effect of EDS on Leydig cells. Adult male rats were implanted with polydimethylsiloxane (PDS) capsules containing testosterone (T) and estradiol (E), and were simultaneously injected with EDS. The PDS-TE implants, by inhibiting luteinizing hormone (LH) production, prevented Leydig cells from repopulating the testis and clamped testosterone within the seminiferous tubules at increasing concentrations relative to implant size. In rats that received EDS alone, the number of advanced spermatids per testis was significantly reduced by 2 weeks, but within 8 weeks returned to the numbers maintained in vehicle-injected control rats or in vehicle-injected rats that received testosterone- and estradiol-filled capsules of 24 cm and 0.1 cm, respectively (PDS-24TE). Surprisingly, in rats that received an EDS injection plus PDS-24TE implants, the number of advanced spermatids per testis was significantly reduced at 8 weeks and severe seminiferous tubule atrophy occurred despite the fact that the testosterone concentration was sufficient to quantitatively maintain spermatogenesis in vehicle-injected rats. In rats injected with EDS and implanted with 24 cm testosterone but not estradiol-filled capsules (PDS-24T), the advanced spermatid number per testis was significantly higher than that in the EDS plus PDS-24TE rats, but significantly lower than that in control rats. These results suggest that EDS may have a cytotoxic effect on the seminiferous epithelium that is independent of the elimination of Leydig cells, and the EDS and estradiol act synergistically to exert a profound toxic effect on spermatogenesis.

Two human chorionic gonadotropin (hCG) responsive cells from rat testicular interstitium were previously isolated on a discontinuous gradient of Percoll. The light cells were non-steroidogenic and bound 125I-labeled hCG with high affinity (Kd 3.0 x 10(-10) mol/L), whereas the steroidogenic heavier cells (Leydig cells) produced cyclic adenosine monophosphate (cAMP) and testosterone in response to hCG stimulation with very little hCG binding. In that study, the heavier cell fraction was contaminated with germ cells, red blood cells, and other cells. These cells have now been further purified on a continuous gradient of Percoll (20 to 60%, v/v), and have resolved into three visible bands. The cells in subfraction I, predominantly damaged Leydig cells, germ cells, and/or residual light cells, bind 125I-labeled hCG with high affinity (Kd 4.09 x 10(-10) mol/L) without producing cAMP and testosterone in response to hCG. Subfraction III consists mainly of red blood cells. The cells in subfraction II, identified as typical Leydig cells by electron microscopy, produce cAMP and testosterone in response to hCG but, again, bind only a small amount of hCG (4.5 +/- 0.3 fmol/2 x 10(6) cells/250 microliters/per hour at 37 degrees C). Thus, further purification of the heavier cell fraction from a discontinuous gradient of Percoll on a continuous gradient of Percoll yields Leydig cells, free of contaminating germ cells and red blood cells, which actively produce cAMP and testosterone with a very low level of hCG binding, the affinity of which is undetectable by current binding techniques.

The two major objectives of this study were to determine (i) whether the pituitary is required to maintain Leydig cell number per testis, and (ii) whether alterations in spermatogenic function in the absence of LH can affect Leydig cell volume, number, and 3H-thymidine incorporation in vivo. Four experimental treatments tested the combinations of two factors: (i) the intact pituitary vs hypophysectomy (Hypox); and (ii) arrested vs active spermatogenesis. Subdermal Silastic capsules were used to deliver a low dosage of estradiol in addition to a low dosage of testosterone (TE), which arrested spermatogenesis, or a high dosage of testosterone (HTE) which maintained active spermatogenesis. All four treatments (TE, HTE, Hypox and Hypox-HTE) inhibited LH secretion for 16 weeks. Control rats were sham hypophysectomized. Leydig cell volume per testis and the volume of an average Leydig cell decreased 70-85% (P less than 0.01 vs controls) in all treated rats, whether deprived of LH (TE) or of all pituitary secretions (Hypox), and whether spermatogenesis was arrested (TE, Hypox) or maintained by exogenous testosterone (HTE, Hypox-HTE). This result suggested that LH was the only factor required to maintain Leydig cell volume, since the absence of other pituitary factors or alterations in spermatogenic function could not override or modify the effect of LH deprivation. No significant differences were found in Leydig cell number per testis or the proportion of Leydig cells labeled with 3H-thymidine among control and experimentally treated rats. In contrast to Leydig cell volume, which depended on LH, Leydig cell number and Leydig cell division were maintained at control values in the absence of pituitary factors and spermatogenic function for 16 weeks.

The proliferation and differentiation of possible Leydig cell precursors in adult rats were studied after destruction of the existing Leydig cells with EDS or after daily treatment with hCG. After 2 days with either treatment, a 12- to 16-fold increase in the number of [3H]thymidine-incorporating interstitial cells was found. In the case of hCG treatment, this was probably due to the high plasma hCG levels. However, after EDS treatment, LH levels start to rise between days 1 and 3, suggesting a paracrine stimulation of the proliferation of interstitial cells. After hCG treatment, a substantial increase in the numbers of Leydig cells was already found at day 2. It was concluded that hCG induced a rapid differentiation, without cell division, of existing precursor cells into recognizable Leydig cells. In rats treated with both EDS and hCG, new Leydig cells were not formed during the first 10 days. This indicates that EDS destroys not only mature Leydig cells but also those Leydig cell precursors that are able to differentiate rapidly into recognizable Leydig cells.

Autophagosomes were observed frequently in electron microscope photographs of Leydig cells from normal adult rat testis. Their formation, evolution and fate were analyzed morphologically in preparations treated to show cytidine monophosphatase (CMPase) and glucose-6-phosphatase (G-6-Pase) activities and in animals sacrificed at various time intervals ranging from 5 min to 6 hrs after a single intratesticular injection of cationic ferritin. Analysis of the morphologic data led to the following interpretation and model. Preautophagosomal structures appeared as flattened, elongated membranous profiles. These expanded, took on a C-shape and fused at their edges to demarcate a small cytoplasmic territory containing normal-looking smooth endoplasmic reticulum (ER) and mitochondria. Such early autophagosomes were thus delimited by two membranes separated by a narrow lumen. Following fusion of these elements with secondary lysosomes, the space between the two membranes increased in size, the inner membrane disintegrated and the enclosed organelles no longer could be identified. The late autophagosomes then reached the cell surface and appeared to exocytose their residual content. In contrast to secondary lysosomes and trans-Golgi elements, which were CMPase-positive, the preautophagosomal flattened membranous elements and early autophagosomes were CMPase-negative. The late autophagosomes on the contrary were CMPase-positive. While ER cisternae were G-6-Pase-positive, the pre-, early and late autophagosomal structures were unreactive for this enzyme. Cationic ferritin tracer experiments showed that only late autophagosomes became labeled with cationic ferritin following their fusion with secondary lysosomes into which the tracer had accumulated following its endocytosis from the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Active immunization of Ile-de-France rams against estradiol (E2) resulted in the production of E2-neutralizing antibodies and an elevation in the plasma concentrations of FSH, LH, and testosterone. The presence of E2 antibodies did not affect the testosterone metabolic clearance rate, indicating that the immunization-mediated 10-fold increase in plasma testosterone was the result of a 10-fold increase in testicular testosterone production. Testis weights, as well as nuclear and cytoplasmic volumes of individual peritubular and perivascular Leydig cells, were greater in E2-immunized rams than in albumin-immunized controls. Leydig cell numbers were not affected by treatment. The E2 antibodies were capable not only of neutralizing the inhibitory effects of endogenous E2 on gonadotropin levels in intact rams, but were able to block the effects of exogenously administered E2 on their FSH and LH secretory response to castration. It is concluded that circulating E2 in the ram is involved in pituitary-testicular endocrine homeostasis and that E2 immunoneutralization can be employed to enhance testosterone secretion in this species.

In hypophysectomized rats, 2 days after the administration of the cytotoxic drug ethane dimethyl sulphonate (EDS), the proliferative activity of Leydig cell precursors increased six-fold. Thus, factors other than LH act locally to stimulate the proliferation of precursor cells after EDS. Twenty-six days after EDS administration, neither cells with the morphological characteristics of Leydig cells nor histochemical enzyme activities, such as 3 beta-HSD and alpha-naphtyl esterase, could be detected in testis tissue. In hypophysectomized rats treated daily with hCG (100 iu) for 7 days, starting at 26 days after EDS, the number of Leydig cells was increased to 48 +/- 11 cells (per 1000 Sertoli cells), which is approximately 4.5% of the intact control level. 3 beta-HSD and alpha-naphtyl esterase activity could be detected, and plasma testosterone levels had increased 15-fold compared with the hypophysectomized controls. These results show that proliferation and some differentiation of precursor cells along the Leydig cell lineage can occur independent of LH, but the final stages of the differentiation process require hCG stimulation.

The endocytic activity of Leydig cells was examined by electron microscopy following the injection, into the interstitial space, of tracers used to examine fluid-phase endocytosis, ie, native ferritin and horseradish peroxidase-colloidal gold (horseradish peroxidase-gold), and adsorptive endocytosis, ie, cationic ferritin. At 5 minutes after injection, native ferritin or horseradish peroxidase-gold was present in the interstitial space and free in the lumen of large endocytic vesicles forming at the cell surface. At 15 minutes, these tracers appeared in the matrix of pale multivesicular bodies, while at 30 minutes and 1 hour, the matrix of dense multivesicular bodies became labeled. Beginning at 1 hour, dense membrane-delimited bodies identified cytochemically as lysosomes were labeled. In the case of cationic ferritin, two distinct pathways were taken. In one pathway, cationic ferritin was observed 5 minutes after injection bound to the plasma membrane of Leydig cells and to the membrane of small and large endocytic vesicles. At subsequent time intervals, cationic ferritin appeared consecutively in pale, dense multivesicular bodies and lysosomes. In a second pathway, cationic ferritin was observed at 5, 15, and 30 minutes bound to the membrane of vesicles of intermediate size seen near the cell surface. At 1, 1 1/2 and 2 hours, cationic ferritin-containing intermediate vesicles appeared in increasing number in the Golgi region. However, cationic ferritin was never observed in the Golgi saccules themselves. At later time intervals (3-6 hours), intermediate vesicles labeled with cationic ferritin progressively disappeared from the Golgi region and the cell. Thus in Leydig cells, while fluid-phase tracers reached lysosomes exclusively, cationic ferritin, a tracer of adsorptive endocytosis, not only reached the lysosomes, but was also carried by the intermediate vesicles to the Golgi region of the cell.

Newborn rat testis was transplanted under the kidney capsule of adult castrated and uncastrated male rats to develop and characterize a model system for studies on Leydig cell development. Two weeks after transplantation, the number of Leydig cells and the size of their nuclei in the transplants had increased. Secretion of testosterone was indicated by increased seminal vesicle weights and decreased pituitary LH in the castrated host animals. Pituitary FSH content increased significantly in the uncastrated animals with transplants, which suggested production of an FSH-stimulating factor. Cells with the morphologic features characteristic of fetal- and adult-type Leydig cells were observed in the transplants. The seminiferous tubules with spermatocytes, incipient lumina, and significantly larger average diameters showed more advanced development than those in the normal 2-week-old testis. By the present morphologic and functional criteria, the kidney subcapsular transplantation technique provides a suitable model for studies of fetal and adult Leydig cell development.

The effects of aging on cyclic AMP (cAMP) dependent protein kinase activity of purified Leydig cells were evaluated. Purified Leydig cells from 20-day-, 40-day-, 60-day- and 30-month-old rats were incubated with or without hCG for 1 hour. At the end of incubation, tubes were centrifuged and supernatants were saved for cAMP and testosterone determinations, and the pellets were prepared for protein kinase assay. Basal cAMP levels of Leydig cells from 20-day- and 40-day-old rats were lower than those of 60-day- and 30-month-old rats. In response to hCG stimulation, Leydig cells of 40-day-old rats produced the highest amounts of cAMP, while 30-month-old had the lowest levels. Testosterone responses of 30-month-old rates were also significantly lower than those of 60-day-old mature rats. When cAMP-dependent protein kinase activities were measured, 30-month-old rats had the lowest total kinase activity. These results suggest that reduced protein kinase activity might contribute to the decreased testosterone synthesis of aged rats.

The reciprocal influence between Leydig and Sertoli cells prepared from pig testis were studied by coculture of both types of cells in either plastic dishes or dishes coated with basement membrane matrix. After 2-3 days in plastic dishes, Sertoli cells produced an increase in the steroidogenic response of Leydig cells to hCG. Pretreatment of the coculture with pFSH enhanced the steroidogenic capacity of Leydig cells and increased the number of hCG receptors. Similarly, the number of FSH binding sites and the FSH-induced plasminogen activator activity secretion of Sertoli cells cocultured with Leydig cells were increased. Pretreatment of the coculture with hCG further enhanced both parameters. The positive reciprocal tropic effects between Leydig cells and Sertoli cells were significantly enhanced when the coculture was carried out on the top of extracellular matrix. In addition, when cells were cocultured under these conditions, but not on plastic dishes, they were organized in cell clusters or island structures, with most of the Leydig cells located in the outer area, whereas Sertoli cells were located inside the islands.