Alteration in testicular cell components following transiently induced ischaemia in prepubertal bulls.

The aim of the present study was to evaluate transient testicular ischaemia (induced using elastrator bands) in Jersey calves on testicular morphology and development. Treatments (at 27 +/- 5 days of age) consisted of control (0 h banding) and banding for 2, 4 or 8 h (n = 4 in each group). After castration (at 60 +/- 5 days of age), the right testis was used for calculation of cell components per testis according to the point-counting method. Bodyweight (59.8 +/- 6.2 kg) and scrotal circumference (SC) at banding (9.1 +/- 0.2 cm) did not differ between groups. Fresh testis weight, scrotal temperature immediately before band removal and daily SC growth were decreased in ischaemic (4 and 8 h) testes compared with controls (P < 0.05). In addition, the number of Sertoli and Leydig cells was significantly reduced in the 8 h ischaemic treatment group (P < 0.05). Transiently induced ischaemia significantly decreased the number of germ cells in the 8 h ischaemic treatment group (13 +/- 5 x 10(6) cells) compared with the 0, 2 and 4 h ischaemic treatment groups (38 +/- 6, 32 +/- 6 and 33 +/- 5 x 10(6) cells, respectively; P < 0.05). These results suggest that transiently induced ischaemia for 8 h significantly decreases the number of germ, Sertoli and Leydig cells in prepubertal testis.

Changes in the testis seminiferous tubules and interstitium in prepubertal bull calves immunised against inhibin at the time of gonadotropin administration.

The aim of the present study was to evaluate the effects of gonadotropin administration at initiation of inhibin passive immunisation in Jersey bull calves (age 27 +/- 5 days) on testicular morphology and development. Primary treatments consisted of control (keyhole limpet haemocyanin, KLH; n = 9) or immunisation against inhibin (INH; n = 9). Subsets of calves were randomly assigned within primary treatments (TRT) to receive saline ( n = 3 per TRT), follicle-stimulating hormone (FSH; n = 3 per TRT) or gonadotrophin-releasing hormone (GnRH, n = 3 per TRT). The right testis was removed (age 118 +/- 5 days) to determine volumes of testicular components and cell numbers per testis using stereology. Data were analysed using the MIXED procedure of the SAS program. Antibody titres against inhibin were increased in INH bulls compared with KLH bulls (P < 0.05). In addition, a significant immunisation x hormone treatment interaction was noted for the number of germ cells. Administration of FSH at the time of initial immunisation against inhibin significantly increased the number of germ cells (92.2 +/- 9 x 10(6) cells) compared with INH+saline bulls (54.9 +/- 10 x 10(6) cells), with INH+GnRH bulls having an intermediate number of cells (64.5 +/- 9 x 10(6) cells; P < 0.05). These results suggest that gonadotropin administration at the time of inhibin immunisation increases the number of germ cells in the testis.

Leydig cells, thyroid hormones and steroidogenesis.

Leydig cells are the primary source of androgens in the mammalian testis. It is established that the luteinizing hormone (LH) produced by the anterior pituitary is required to maintain the structure and function of the Leydig cells in the postnatal testis. Until recent years, a role by the thyroid hormones on Leydig cells was not documented. It is evident now that thyroid hormones perform many functions in Leydig cells. For the process of postnatal Leydig cell differentiation, thyroid hormones are crucial. Thyroid hormones acutely stimulate Leydig cell steroidogenesis. Thyroid hormones cause proliferation of the cytoplasmic organelle peroxisome and stimulate the production of steroidogenic acute regulatory protein (StAR) and StAR mRNA in Leydig cells; both peroxisomes and StAR are linked with the transport of cholesterol, the obligatory intermediate in steroid hormone biosynthesis, into mitochondria. The presence of thyroid hormone receptors in Leydig cells and other cell types of the Leydig lineage is an issue that needs to be fully addressed in future studies. As thyroid hormones regulate many functions of Sertoli cells and the Sertoli cells regulate certain functions of Leydig cells, effects of thyroid hormones on Leydig cells mediated via the Sertoli cells are also reviewed in this paper. Additionally, out of all cell types in the testis, the thyrotropin releasing hormone (TRH), TRH mRNA and TRH receptor are present exclusively in Leydig cells. However, whether Leydig cells have a regulatory role on the hypothalamo-pituitary-thyroid axis is currently unknown.

Effect of long term deprivation of luteinizing hormone on Leydig cell volume, Leydig cell number, and steroidogenic capacity of the rat testis.

Leydig cells atrophy, losing cytoplasmic volume and the capacity for testosterone secretion, within 1-2 weeks of LH deprivation. We investigated the effects of long term (0-16 weeks) LH deprivation on the volume of an average Leydig cell, the volume of Leydig cells per testis, the number of Leydig cells per testis, and testosterone secretion by in vitro perfused testes. Endogenous LH was suppressed in adult rats by testosterone/estradiol-filled (TE) Silastic implants. The presence of Leydig cells in testes was verified by 1) morphological examination using light and electron microscopy, 2) histochemical localization of 3 beta-hydroxysteroid dehydrogenase activity (3 beta HSD), and 3) conversion of pregnenolone to progesterone by in vitro perfused testes. Marked quantitative differences existed in Leydig cell morphology among control and treated rats. The volume of an average Leydig cell and the total volume of Leydig cells per testis decreased (P less than 0.01) rapidly and progressively after TE implantation. At 16 weeks, the average Leydig cell lost 90% of its cytoplasmic volume and 65% of its nuclear volume. Analysis of variance failed to detect a significant decline in Leydig cell number per testis, despite a 16% reduction from the value in control rats (22.2 +/- 1.5 x 10(6)) in rats treated for 16 weeks (18.7 +/- 1.5 x 10(6)). After TE implantation, LH-stimulated testosterone secretion by in vitro perfused testes diminished (P less than 0.01) rapidly to 5% of the control values at 1 week and less than 0.3% of the control value from 4-16 weeks. In contrast, 25% of 3 beta HSD activity was retained (P less than 0.01 vs. controls) at 16 weeks, based on the rate of pregnenolone conversion to progesterone. Moreover, testes of treated rats secreted progesterone at a rate twice that of controls, when the steroid secretion rates were expressed per volume of Leydig cell cytoplasm. Loss of the testosterone-secreting capacity of testes after LH withdrawal was associated with a loss in the volume, but not a significant loss in the number, of Leydig cells. Thus, LH was required to maintain the differentiated structure and function of Leydig cells, but was not required to maintain the overwhelming majority of Leydig cells in the adult rat testis through 16 weeks. Moreover, at least one steroidogenic enzyme, 3 beta HSD, was retained by Leydig cells after long term LH deprivation.

Leydig cell peroxisomes and sterol carrier protein-2 in luteinizing hormone-deprived rats.

We investigated the effects of 8 days of LH withdrawal on rat Leydig cell peroxisomal volume, total and intraperoxisomal catalase and sterol carrier protein-2 (SCP2) contents, and LH-stimulated testosterone secretion in vitro. Three groups of adult male Sprague-Dawley rats, i.e. control, TE-implanted (testosterone-17 beta-estradiol-filled Silastic implants to suppress LH), and TELH-implanted (TE-implanted and LH replacement via Alzet mini osmotic pumps), were used. After 8 days, Leydig cell organelle volumes (stereology), intraperoxisomal catalase and SCP2 contents (immunocytochemistry), LH-stimulated testosterone secretion by isolated Leydig cells in vitro (determined by RIA), and total catalase and SCP2 contents in equal numbers of Leydig cells (immunoblot analyses) were determined. Results showed that the TELH-implanted rats were identical to controls in every parameter tested. Testis volume and Leydig cell number per testis in control and TE-implanted rats were not significantly different; however, reductions (P < 0.05) were observed in the average volume of a Leydig cell (one third of controls) and the volume of Leydig cells per testis. All Leydig cell organelle volumes tested were significantly lower in TE-implanted rats than in the controls; however, the volumes of smooth endoplasmic reticulum (SER) and peroxisomes were the most reduced (lowered to one sixth of control values). LH-stimulated testosterone secretion per Leydig cell in vitro correlated well with these changes in the volumes of Leydig cell SER and peroxisomes. Intraperoxisomal catalase in Leydig cells was unchanged in TE-implanted rats, although immunoblotting demonstrated a loss of total catalase content (which reflected the reduction in the volume of peroxisomes). SCP2 in Leydig cells of TE-implanted rats was undetectable with immunoblot analysis (explained by the reductions in Leydig cell peroxisome volume and intraperoxisomal SCP2). These results demonstrate that the organelles SER and peroxisomes and the protein SCP2 in Leydig cells are more LH dependent than the other organelles (e.g. mitochondria, lysosomes) and protein catalase, respectively. Moreover, the findings of this study are consistent with the hypothesis that Leydig cell peroxisomes play a significant role in testosterone production.

The effect of inhibition of aromatase enzyme activity on Leydig cell number and ultrastructure in beagles.

We have shown previously that administration of an orally active competitive aromatase inhibitor 4-(5,6,7,8-tetrahydroimidazo [1,5a] pyridin-5-yl) benzonitrile monohydrochloride to adult male beagles increases peripheral blood LH and testosterone concentrations, and that testes from treated dogs produce more testosterone when perfused in vitro than age-matched controls. In the present study we posed the question of whether the increased testosterone secretion by testes from these same aromatase-treated dogs was due to Leydig cell hypertrophy or hyperplasia, and if the latter, whether cytoplasmic organelles are increased. Beagles were treated with the inhibitor at a dosage of 2.5 mg/kg.day for 25 weeks and were euthanized by an overdose of iv sodium pentobarbital; testes were perfusion-fixed, embedded, and sectioned for stereological analysis. There were no significant differences in testis volume and absolute volumes of seminiferous tubules, blood vessels, lymphatic space, macrophage cells, and mesenchymal cells between the control and treated dogs. In contrast absolute interstitium volume and the absolute volume of Leydig cells per testis were significantly increased (P less than 0.05) in treated dogs. This increased Leydig cell volume per testis was due to increased volume of individual Leydig cells rather than to increases in Leydig cell number per testis. Additional studies showed that the surface area per Leydig cell of smooth endoplasmic reticulum, outer and inner mitochondrial membranes, and membranes of lipid droplets per testis were significantly higher (P less than 0.05) in the treated dogs as compared to the controls. In summary, the results of this study lead us to conclude that aromatase inhibition in the mature dog causes Leydig cell hypertrophy rather than hyperplasia and increased surface area per Leydig cell of subcellular organelles that contain enzymes involved in steroid biosynthesis.

Luteinizing hormone causes rapid and transient changes in rat Leydig cell peroxisome volume and intraperoxisomal sterol carrier protein-2 content.

The aim of the present study was to investigate the effects of a single injection of LH on rat Leydig cell peroxisome volume and peroxisomal sterol carrier protein-2 (SCP2) content. Sexually mature Sprague-Dawley rats (n = 5) were injected sc with 500 micrograms LH and euthanized, and trunk blood was collected at 0, 0.5, 1, 2, and 3 h. Additionally, LH-treated rats were whole body perfused-fixed, and their testes were processed for qualitative and quantitative histochemical and immunocytochemical studies at 0, 0.5, 1, and 2 h. Peroxisomes were identified by cytochemical staining for catalase activity with the alkaline 3,3'-diaminobenzidine tetrahydrochloride method. Catalase and SCP2 were immunolocalized in Leydig cell organelles via 10-nm AuroProbe EM protein-A gold particles. Peak plasma testosterone concentrations were observed 1 and 2 h after the single sc LH injection. The average volume of a Leydig cell was unchanged by the LH treatment at all time points tested. Similarly, the absolute volumes of smooth endoplasmic reticulum and mitochondria per Leydig cell were unchanged at all time points tested. By contrast, the absolute volume of peroxisomes per Leydig cell increased 3-fold 0.5 h after LH injection (P less than 0.01) and then returned to control values by 2 h. The absolute volume of negative bodies (single membrane-bound cytoplasmic organelles lacking catalase) per Leydig cell was elevated above the control value 0.5 and 1 h after LH injection. Western blot analysis demonstrated a single protein at 14 and 60 kDa with anti-SCP2 and anticatalase, respectively, for both homogenates obtained from liver and purified Leydig cells. Quantitative immunocytochemical studies demonstrated that the gold particle density representing SCP2 over peroxisomes increased 5-fold 0.5 h after the LH injection (P less than 0.01) and then returned to control values by 2 h. In contrast, the gold particle density representing catalase over peroxisomes was not different in control and LH-injected groups. We conclude that a single sc injection of LH causes a rapid, specific, and transient increase in both the volume of peroxisomes and the peroxisomal content of SCP2 in Leydig cells.

Luteinizing hormone on Leydig cell structure and function.

The effects of luteinizing hormone (LH) and human chorionic gonadotrophic hormone (hCG) on Leydig cell structure and function are reviewed in this paper under two main headings; responses to LH and hCG stimulation and responses to LH deprivation. With acute LH stimulation, up to 2 hours following the LH injection, there was no change in the volume of a Leydig cell. However, Leydig cell peroxisomal volume and intraperoxisomal SCP2 content showed a rapid and transient change. These changes can be considered to be specific because: i) no other Leydig cell organelle including smooth endoplasmic reticulum (SER) showed such a change, and ii) only the intraperoxisomal SCP2 but not catalase (a marker enzyme for peroxisomes) showed such a change within 30 minutes of LH stimulation. As these changes occurred prior to the peak testosterone levels following this treatment, it is suggested that SCP2 and peroxisomes may have an association with testosterone biosynthesis prior to cholesterol transport into mitochondria. With LH or hCG stimulation for longer periods, i.e. one day or more, the same morphological changes are produced in Leydig cells irrespective of the age of the species, dosage of LH or hCG, and with single or multiple doses. These changes include, Leydig cells hypertrophy and/or hyperplasia, increase in the cellular organelle content (mostly SER and mitochondria) and depletion of lipid droplets. In addition, a recent study showed that Leydig cell peroxisomal volume, SCP2 content, the amount of intraperoxisomal SCP2 and testosterone secretory capacity were also significantly increased in response to chronic LH treatment. The effects of LH deprivation by whatever means (e.g. hypophysectomy, with testosterone and 17 beta-estradiol silastic implants, LH antisera) on Leydig cell structure and function is generally described as opposite to those observed following LH or hCG stimulation. These include Leydig cell hypotrophy and hypoplasia, reductions in the cytoplasmic organelle content in general and specific reductions in SER and peroxisomal volumes, reductions in total catalase and SCP2 in Leydig cells together with reductions in the intraperoxisomal SCP2 content in Leydig cells and their testosterone secretory capacity.

Effects of thyroid hormones on Leydig cells in the postnatal testis.

Thyroid hormones (TH) stimulate oxidative metabolism in many tissues in the body, but testis is not one of them. Therefore, in this sense, testis is not considered as a target organ for TH. However, recent findings clearly show that TH have significant functions on the testis in general, and Leydig cells in particular; this begins from the onset of their differentiation through aging. Some of these functions include triggering the Leydig stem cells to differentiate, producing increased numbers of Leydig cells during differentiation by causing proliferation of Leydig stem cells and progenitors, stimulation of the Leydig cell steroidogenic function and cellular maintenance. The mechanism of action of TH on Leydig cell differentiation is still not clear and needs to be determined in future studies. However, some information on the mechanisms of TH action on Leydig cell steroidogenesis is available. TH acutely stimulate testosterone production by the Leydig cells in vitro via stimulating the production of steroidogenic acute regulatory protein (StAR) and StAR mRNA in Leydig cells; StAR is associated with intracellular trafficking of cholesterol into the mitochondria during steroid hormone synthesis. However, the presence and/or the types of TH receptors in Leydig cells and other cell types of the Leydig cell lineage is still to be resolved. Additionally, it has been shown that thyrotropin-releasing hormone (TRH), TRH receptor and TRH mRNA in the testis in many mammalian species are seen exclusively in Leydig cells. Although the significance of the latter observations are yet to be determined, these findings prompt whether hypothalamo-pituitary-thyroid axis and hypothalamo-pituitary-testis axis are short-looped through Leydig cells.

Cell-cell interactions in the testis of teleosts and elasmobranchs.

In this paper we present the state of knowledge on cell-cell interactions in the testis of two groups of anamniote vertebrates--teleosts and elasmobranchs--which include most fish. In these fish, the structural organization of the testis differs fundamentally from that which characterizes amniotes in which the germinal tissue is located in tubules open at both ends and consists of a permanent population of Sertoli cells associated with successive stages of germ cell development. In fish, the spermatogenic unit of testis is the spermatocyst, which corresponds to one germ cell or to a clone of isogenetic germ cells, enclosed by one or several Sertoli cells, which form the wall of the cyst. In fish testis, the Sertoli cells do not represent a permanent population of cells. Although both are of the cystic type, the teleost and elasmobranch testes are differently organized. In elasmobranchs, primary spermatogonia and Sertoli cells lie initially free within the interstitial tissue, before becoming sequestered by a basement membrane; the testis is then composed of a mass of spermatocysts which contain many Sertoli cells, each being associated with a clone of germ cells. In contrast, in teleosts, the cysts are confined to large elongated structures limited by a basement membrane. These structures are either lobules originating under the albuginea or tubules which, in contrast to those of mammals, are anastomosed. In the lobules, the spermatocysts start to develop at the blind end of the lobules and migrate towards the efferent system, whereas in the tubules, the spermatocysts are located against the basement membrane, all along the tubules and do not migrate. In elasmobranchs, unlike teleosts, Leydig cells are either absent from the interstitial tissue or rare and undifferentiated and their role in steroid production is at best marginal. While many studies have focused on topographical and functional interactions between the diverse cell types present in mammalian testis, only a few studies have brought particular attention to these aspects in fish. In fish, like in mammals, testicular cell-cell interactions are based on structural elements and chemical factors. Occasionally, various adhering junctions have been observed, essentially in teleosts, between Sertoli cells, between Sertoli cells and germ cells, between germ cells themselves, and interstitial cells. Furthermore, in some teleost species, using horseradish peroxidase or lanthanum salts, the presence of tight junctions between Sertoli cells has been correlated to the occurrence of a Sertoli barrier. In these species, the barrier develops after meiosis so that only haploid germ cells are shielded from the vascular system. In fish, recent development of techniques which enable the preparation and in vitro culture of enriched populations of testicular cells and of spermatocysts, has allowed investigations on functional aspects of cell-cell interactions. In particular, data have been obtained, in the trout, on the control of spermatogonia proliferation by Sertoli cell-conditioned media and, in the dogfish, on the steroidogenic activity of Sertoli cells, in relation to the differentiation stage of the associated germ cells. Furthermore information exists, in the trout, showing that intratubular macrophages may participate in the re-initiation of spermatogonial proliferation. In conclusion, the cytoarchitecture of fish testis, as compared to that of mammals, presents original features which provide unique opportunities to develop fruitful studies for a better understanding of the complex control mechanisms underlying testicular function in vertebrates.

Mitosis in normal adult guinea pig Leydig cells.

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.

Heterogeneity of adult mouse Leydig cells with different buoyant densities.

The authors investigated the morphologic characteristics and human chorionic gonadotrophin (hCG)-stimulated testosterone production of adult mouse Leydig cells in vitro, which have different buoyant densities. Leydig cells from five testes of Swiss outbred male mice (15 weeks old) were isolated and purified by mechanical dispersion followed by density gradient centrifugation using Percoll. Two groups of Leydig cells were obtained with different buoyant densities: group 1 had densities of 1.0667 to 1.0515 g/cm3 and group 2 had densities of 1.0514 to 1.0366 g/cm3. In vitro testosterone production of these Leydig cells, in response to different doses of hCG (0, 5, 25, 125, 625, and 3125 pg/mL), was determined by radioimmunoassay. Leydig cells were fixed and processed for electron microscopic stereology to quantify the organelles by volumes and surface area. In Leydig cells of Group 1, testosterone production per cell in vitro in response to 0 and 5 pg/mL hCG was not significantly different (P greater than 0.05). Increases in the dose up to 25 pg/mL produced a significant increase (P less than 0.05) in testosterone production, although hCG doses of 125 and 625 pg/mL did not produce further increases in testosterone levels. However, 3125 pg/mL hCG further elevated the testosterone production by those Leydig cells with high buoyant density. In Leydig cells in group 2, the patterns of testosterone production in response to hCG doses of 0, 5, and 25 pg/mL were similar to those of Leydig cells in group 1. Those Leydig cells with low buoyant density, however, were unable to stimulate further testosterone production by an hCG dose of 3125 pg/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental cryptorchidism in the adult mouse. III. Qualitative and quantitative electron microscopic morphology of Leydig cells.

The morphology of Leydig cells of control and 28-day-old cryptorchid mice was studied by electron microscopy and stereologic techniques. Leydig cell profiles of control mice were larger in section when compared to cryptorchid mice, but no differences were observed in the distribution of organelles in Leydig cells in the two groups. Quantitatively, the absolute volumes of smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), mitochondria, lysosomes, multivesicular bodies, peroxisomes, cytoplasmic matrix, nucleus, lipid droplets, membrane whorls, ribosomal aggregates, and annulate lamellae per Leydig cell were reduced significantly after 28 days of cryptorchidism. However, the absolute volumes of these organelles per testis were not significantly different between control and cryptorchid mice, due to the increase in Leydig cell number per testis in the cryptorchid testis, compared to the controls, except that the absolute volume of Golgi per Leydig cell was not significantly different between control and cryptorchid rats, but the absolute volume of Leydig cell Golgi was significantly lower in control rats. Based on these results, we conclude that, morphologically, a 28-day cryptorchid mouse Leydig cell clearly approximates a "half unit" of a control Leydig cell.

Experimental cryptorchidism in the adult mouse: I. Qualitative and quantitative light microscopic morphology.

Morphologic changes in the testes of adult mice after experimentally induced cryptorchidism were studied by light microscopy and stereology. Increasing duration of cryptorchidism resulted in a gradual decrease in the volume of seminiferous tubules per testis, and this was associated with germ cell degeneration. The volumes of Sertoli cell lipid droplets increased, and dilations of the intercellular space between the Sertoli cell junctions was observed in the cryptorchid testis. The luminal volume of the seminiferous tubule was reduced by 50% after 28 days of cryptorchidism. However, the volumes of intertubular tissue and Leydig cells in control and cryptorchid testes were not significantly different. Leydig cell number per testis increased, and the average volume of a Leydig cell decreased gradually with the progression of the cryptorchid state. The volume of the connective tissue cells in the intertubular area increased, but no significant volume change was observed in the volume of intertubular macrophages. After 28 days, the cryptorchid testis contained a significantly increased volume of blood vessels and a reduced volume of lymphatic space per testis. These observations clearly demonstrate that, although the mouse is a species closely related to the rat, the morphologic changes that occur in the Leydig cell population after induction of experimental cryptorchidism in this species is different.

Experimental cryptorchidism in the adult mouse: II. A hormonal study.

Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone levels and secretory capacity of the in vitro stimulated testis were determined in control and bilateral cryptorchid mice after 7, 14, 21, and 28 days. In a separate study, serum FSH, LH, and testosterone levels were measured in unilateral and bilateral cryptorchid, hemicastrate, and bilaterally castrate adult mice after 28 days of treatment. Serum FSH levels were significantly increased in bilaterally cryptorchid mice compared to controls (0 days) at 7, 14, 21, and 28 days, but serum LH and testosterone levels did not change. At 28 days, the elevated serum FSH levels in the unilateral and bilateral cryptorchid mice were not different than those in hemicastrate mice. However, the FSH levels in bilaterally castrate mice after 28 days were significantly higher than all other groups. Serum LH and testosterone levels were significantly different only in the bilaterally castrate group, compared to control levels. Both the normal and cryptorchid testes of all ages studied were capable of producing equal levels of testosterone in vitro, both basally and with a human chorionic gonadotropin (hCG) dose of 700 mIU/ml (maximum stimulatory dose for both the normal and the 28 day cryptorchid testes). Changes that occur in mice with experimentally induced cryptorchidism are not identical to those seen in the rat. Serum FSH levels increase, but no changes occur in serum LH and testosterone levels. Additionally, a cryptorchid mouse testis is not hyperresponsive to hCG stimulation in vitro.

Studies on peroxisomes of the adult rat Leydig cell.

The aims of this study were to differentially identify peroxisomes and lysosomes in Leydig cells of the sexually mature rat using cytochemical techniques, to describe the size and shape of peroxisomal profiles, and to localize catalase and sterol carrier protein-2 (SCP2) in Leydig cell peroxisomes. Peroxisome profiles, identified by cytochemical staining for catalase activity using 3,3'-diaminobenzidine tetrahydrochloride (DAB) were categorized according to their longest diameter as small (less than 0.18 microns), intermediate (0.18-0.45 microns), and large (more than 0.45 microns); and according to their shape, which were designated as circular, oval, and dumbbell. Together these peroxisomal profiles occupied 11.2 microns 3/Leydig cell. Lysosomes, identified in the same tissue sections as acid phosphatase positive organelles, occupied 12.9 microns 3/Leydig cell. Negative bodies with morphology identical to cytochemically unstained peroxisomes also were detected. These organelles occupied 14.5 microns 3/Leydig cell. Catalase was immunolocalized exclusively in Leydig cell peroxisomes using AuroProbe EM protein A G10 (ie, 10 nm gold particles). Sterol carrier protein-2 was immunolocalized in Leydig cell peroxisomes by AuroProbe EM protein A G15 (ie, 15 nm gold particles). Immunolocalization of catalase and SCP2 using 10 nm and 15 nm gold particles in the same peroxisomes confirmed that Leydig cell peroxisomes contain SCP2. Taken together, these results show conclusively that adult rat Leydig cell peroxisomal profiles occur in different shapes and sizes, which suggests the existence of a network of peroxisomes, rather than peroxisomes occurring as separate isolated organelles. More importantly, the present study demonstrates for the first time that Leydig cell peroxisomes contain SCP2.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of insulin-like growth factor-I on androgen production by highly purified pubertal and adult rat Leydig cells.

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.

Peroxisomes and intracellular cholesterol trafficking in adult rat Leydig cells following Luteinizing hormone stimulation.

The present study was designed to explore the intracellular cholesterol trafficking in Leydig cells of adult rats following Luteinizing hormone (LH) injection. Histochemical techniques were used to demonstrate distribution of free cholesterol in Leydig cells of control and LH-injected rats. Two groups of sexually mature male Sprague Dawley rats (n=4/group) were used. Fifteen min following an injection of 200 microl of either saline (control) or luteinizing hormone (LH, 500 microg in saline) testes of rats were fixed by whole body perfusion using 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer for 20 min. Fixed testes were cut into 3 mm3 and kept immersed in the fixative for further 15 min. Tissue cubes were then incubated at 37 degrees C in a medium containing cholesterol oxidase, 3,3'-diaminobenzidine tetrahydrochloride, horseradish peroxidase and dimethyl sulfoxide to histochemically localize free cholesterol in Leydig cells and processed for electron microscopy. Thin sections of these tissues were stained with aqueous uranyl acetate and lead citrate and examined with a Philips 201C electron microscope. In Leydig cells of control rats, free cholesterol was detected primarily in lipid droplets and plasma membrane. In the majority of Leydig cells, peroxisomes were unstained for free cholesterol, but occasionally few stained ones were present. Staining was not detected in mitochondria and smooth endoplasmic reticulum (SER) in Leydig cells of control rats. In LH-injected rats, lipid droplets, many peroxisomes, inner and outer mitochondrial membranes and some cisternae of SER in Leydig cells showed staining for free cholesterol. Fusion of Leydig cell peroxisomes with lipid droplets and mitochondria was also observed in the LH treated rats. These findings suggested that peroxisomes in adult rat Leydig cells participate in the intracellular cholesterol trafficking and delivery into mitochondria during LH stimulated steroidogenesis. Lipid droplets are used as one source for cholesterol for this process.

Signs of aging are apparent in the testis interstitium of Sprague Dawley rats at 6 months of age.

The present study investigated the effects of aging in the testis interstitium in Sprague Dawley rats. Rats of 3, 6 and 24 months of age were used. Testes of rats (n = 5) were fixed by whole body perfusion using a fixative containing 2.5% glutaraldehyde in cacodylate buffer, processed and embedded in eponaraldite. Using 1 microns sections stained with methylene blue, qualitative and quantitative morphological studies were performed. Purified Leydig cell preparations, obtained by collagenase digestion followed by elutriation and density gradient centrifugation, were used to determine luteinizing hormone (LH; 100 ng/ml) stimulated testosterone secretory capacity per Leydig cell in vitro. Testosterone levels in the incubation medium, and testosterone and luteinizing hormone levels in serum of these three groups of rats were determined via radioimmunoassay. Morphological studies revealed that Leydig cells were more abundant in the testis interstitium at 6 and 24 months when compared to 3 months. Moreover, collagen fiber bundles were more frequently observed in the testis interstitium at older ages. Blood vessels of the testis interstitium in 24-month-old rats frequently showed partial and complete occlusion of their lumen and thickening of vessel walls. This feature was also present at 6 months, but less frequently. The results of the stereological studies revealed that the volumes of seminiferous tubules, interstitium and Leydig cells per testis was significantly higher (P < 0.05), at 6 and 24 months of age than those at 3 months. Moreover, volume of macrophages per testis was observed to be significantly higher (P < 0.05) at 6 months when compared to 3 and 24 months, and volume of connective tissue cells per testis was observed to be significantly lower (P < 0.05) at 6 and 24 months when compared to 3 months of age. No significant difference (P > 0.05) was observed for the volume of lymphatic space per testis in the three age groups studied. Volume of interstitial blood vessels per testis was not significantly different at 3 and 6 months of age, but a significantly greater (P < 0.05) volume was observed at 24 months. However, at 6 and 24 months, only 71% and 31% of the total blood vessel volumes respectively had completely open lumen in them; the rest of the blood vessels were either partially (12.5% at 6 months and 17% at 24 months) or completely (16.5% at 6 months and 52% at 24 months) occluded. The number of Leydig cells per testis was doubled at 6 and 24 months of age compared to 3 months. The average volume of a Leydig cell was not significantly different between 3 and 6 months of age, however, at 24 months a significantly lower (P < 0.05) value was observed. LH stimulated testosterone secretory capacity per Leydig cell in vitro was reduced by 50% at 6 months of age compared to 3 months; a further significant (P < 0.05) reduction was observed at 24 months. Serum testosterone and LH levels were not significantly different between 3 and 6 months of age but at 24 months a significantly lower (P < 0.05) value was observed for both of these hormones. In summary, the present study demonstrated many changes in the components of the testis interstitium in the aged Sprague Dawley rat. Modifications in the blood vessels and the occurrence of abundant collagen fibers in the interstitial space could possibly contribute to the reduced testosterone secretory capacity per Leydig cell with advancing in age. The observed Leydig cell hyperplasia could be suggested as a compensatory effort to maintain the normal androgen status of the aged rat, which is rather successful at 6 months but unsuccessful at 24 months. This investigation further revealed that these characteristic changes in the aged testis interstitium at 24 months are also present to some extent at 6 months of age in Sprague Dawley rats, suggesting that aging of the testis in this strain of rats commences early in life.

Effects of continuous and intermittent exposure of lactating mothers to aroclor 1242 on testicular steroidogenic function in the adult male offspring.

Polychlorinated biphenyls (PCBs) are worldwide pollutants and have caused hazardous effects on many animal species including humans. They have been detected in human milk and therefore exposure of newborns to PCBs is unavoidable if they are breast-fed. We present our findings on two experiments performed to test the effects of intermittent and continuous exposure of lactating rats to two different doses (80 microg and 8 microg) of Aroclor 1242 (a PCB congener) on testicular steroidogenic function of their adult male offspring. In experiment I, three groups of lactating dams received daily subcutaneous (SC) injections of either corn oil, 80 microg of Aroclor 1242 and 8 microg of Aroclor 1242 in corn oil, respectively. In experiment II, three groups of lactating dams received two SC injections per week of either corn oil or Aroclor 1242 (80 microg and 8 microg) in corn oil, respectively. Pups in all groups (n=8 per group) were weaned at day 21 and were raised on a normal diet until sacrificed at 90 days. Experiment I: Leydig cell number per testis was significantly (P<0.05) increased and the average volume of a Leydig cell was significantly (P<0.05) reduced in both groups of Aroclor-exposed rats compared to corn oil controls. Both doses of Aroclor resulted in reduced (P<0.05) serum testosterone levels compared to corn oil-treated controls. LH-stimulated testosterone production per testis and per Leydig cell was lower in Aroclor-exposed rats compared to controls. Experiment II: No changes were observed in Leydig cell size and number per testis among the three groups. Serum LH, testosterone and LH-stimulated testicular testosterone production in offspring rats of Aroclor-treated dams were not significantly different (P>.05) from the offspring of corn oil-treated dams. However, these parameters were lower in value in the offspring of dams treated with Aroclor 80 microg compared to the other two groups. LH-stimulated testosterone secretory capacity per Leydig cell was significantly lower in offspring of dams treated with Aroclor compared to controls. Serum T4 and T3 levels were not significantly different among the Aroclor-exposed and control rats in both experiments. These results demonstrate that continuous exposure of lactating mothers to 8 and 80 microg of Aroclor 1242 causes hypotrophy and malfunctioning of Leydig cells in the adult male offspring resulting in a hypoandrogenic status. Intermittent treatment of lactating mothers with 80 microg of Aroclor (but not with 8 microg of Aroclor) also produced malfunctioning of Leydig cells and a hypoandrogenic status in the absence of Leydig cell hypotrophy. However, the Aroclor 8 microg dose was ineffective to produce the above effects.