Immunization with oral KISS1 DNA vaccine inhibits testicular Leydig cell proliferation mainly via the hypothalamic-pituitary-testicular axis and apoptosis-related genes in goats.

This study aimed to analyze the effect and mechanism of immunization of oral KISS1 DNA vaccine on the proliferation of goat testicular Leydig cells. Ten 8-week-old male goats were randomly divided into KISS1 DNA vaccine and control groups for immunization (five goats each group). These goats were sacrificed at 8 weeks after primary immunization, and the tissue samples of hypothalamus, pituitary, and testis and Leydig cell samples were collected for RT-PCR and CCK8 assay. Immunization with the oral KISS1 DNA vaccine effectively inhibited the proliferation of Leydig cells, the expression of hypothalamus KISS1, GPR54, and GnRH mRNA, pituitary GnRHR and LH mRNA, testicular LHR mRNA, and apoptosis-inhibitory gene Bcl-2 mRNA in Leydig cells. By contrast, the immunization enhanced the mRNA expression of apoptosis-promoting gene Bax and Clusterin in Leydig cells. These findings indicate that immunization with the oral KISS1 DNA vaccine can inhibit the proliferation of goat testicular Leydig cells mainly via the hypothalamic-pituitary-testicular axis and apoptosis-related genes.

T Lymphocytes and Testicular Immunity: A New Insight into Immune Regulation in Testes.

The immune privilege of the testes is necessary to prevent immune attacks to gamete-specific antigens and paternal major histocompatibility complex (MHC) antigens, allowing for normal spermatogenesis. However, infection and inflammation of the male genital tract can break the immune tolerance and represent a significant cause of male infertility. Different T cell subsets have been identified in mammalian testes, which may be involved in the maintenance of immune tolerance and pathogenic immune responses in testicular infection and inflammation. We reviewed the evidence in the published literature on different T subtypes (regulatory T cells, helper T cells, cytotoxic T cells, γδ T cells, and natural killer T cells) in human and animal testes that support their regulatory roles in infertility and the orchitis pathology. While many in vitro studies have indicated the regulation potential of functional T cell subsets and their possible interaction with Sertoli cells, Leydig cells, and spermatogenesis, both under physiological and pathological processes, there have been no in situ studies to date. Nevertheless, the normal distribution and function of T cell subsets are essential for the immune privilege of the testes and intact spermatogenesis, and T cell-mediated immune response drives testicular inflammation. The distinct function of different T cell subsets in testicular homeostasis and the orchitis pathology suggests a considerable potential of targeting specific T cell subsets for therapies targeting chronic orchitis and immune infertility.

High Throughput scRNA-Seq Provides Insights Into Leydig Cell Senescence Induced by Experimental Autoimmune Orchitis: A Prominent Role of Interstitial Fibrosis and Complement Activation.

Leydig cells (Lc), located in the interstitial space of the testis between seminiferous tubules, produce 95% of testosterone in male individuals, which is pivotal for male sexual differentiation, spermatogenesis, and maintenance of the male secondary sex characteristics. Lc are prone to senescence in aging testes, resulting in compromised androgen synthesis capability upon aging. However, little is known about whether Lc undergo senescence in a chronic inflammatory environment. To investigate this question, mouse models of experimental autoimmune orchitis (EAO) were used, and Lc were analyzed by high throughput scRNA-Seq. Data were screened and analyzed by correlating signaling pathways with senescence, apoptosis, androgen synthesis, and cytokine/chemokine signaling pathways. EAO did induce Lc senescence, and Lc senescence in turn antagonized androgen synthesis. Based on the correlation screening of pathways inducing Lc senescence, a plethora of pathways were found to play potential roles in triggering Lc senescence during EAO, among which the Arf6 and angiopoietin receptor pathways were highly correlated with senescence signature. Notably, complement and interstitial fibrosis activated by EAO worsened Lc senescence and strongly antagonized androgen synthesis. Furthermore, most proinflammatory cytokines enhanced both senescence and apoptosis in Lc and spermatogonia (Sg) during EAO, and proinflammatory cytokine antagonism of the glutathione metabolism pathway may be key in inducing cellular senescence during EAO.

Induction of tolerance in vitro by autologous murine testicular cells.

We have investigated the regulation of self tolerance in mice by examining lymphocyte reactivity in vitro against two subpopulations of autologous testicular cells: germ cells that were derived from the seminiferous tubules, and interstitial somatic cells. In the presence of germ cells, lymphocyte proliferation was strongly reduced. In contrast, somatic interstitial cells stimulated lymphocyte proliferation. In both cases, reactive lymphocytes were mostly T cells. Suppressor T cells activated by autologous germ cells were nonspecific and capable of inhibiting lymphocyte proliferation against autologous and allogeneic somatic testicular cells as well as against allogeneic spleen cells. Suppression was abrogated after treatment of the responder lymphocytes with anti-Ly-2.2 serum plus complement. Lymphocyte proliferation by autologous interstitial cells was considerably reduced, but not completely abolished, by complement-dependent lysis with anti-Thy-1.2 serum. This may indicate the participation in proliferation of a lymphoid cell population other than T cells.

Interleukin-18 levels and mouse Leydig cell apoptosis during lipopolysaccharide-induced acute inflammatory conditions.

Interleukin (IL)-18 is an inflammasome-mediated cytokine produced by germ cells, Leydig cells, and resident macrophages that is indispensable in the maintenance of homeostasis in the testis. We previously demonstrated that endogenous IL-18 induces testicular germ cell apoptosis during acute inflammation when plasma IL-18 levels are very high. However, the impact of acute inflammation and IL-18 on Leydig cells remained unclear. TM3 cells, a mouse Leydig cell line, and RAW264.7 cells, a mouse macrophage cell line, were stimulated with lipopolysaccharide (LPS) or recombinant IL-18 (rIL-18). We assessed the expression of inflammatory cytokines, caspase cleavage, and markers of apoptotic pathways. In Leydig cells, caspase 3 cleavage was increased and death-receptor-mediated apoptotic pathways were activated after LPS stimulation. However, LPS stimulation did not increase IL-18 expression in the Leydig cell line. When high-dose rIL-18 was administered to the Leydig cell line to mimic levels seem after inflammation, rIL-18 upregulated Tnf-α mRNA, Fadd mRNA, and Fas protein, promoted cleavage of caspase-8 and caspase-3, and induced apoptosis. Low-dose rIL-18 did not stimulate apoptosis. To determine if the high level of IL-18 seen in the testes after inflammation was derived from immune cells, we examined IL-18 protein expression in a macrophage cell line, RAW264.7. In contrast to the TM3 cells, IL-18 was significantly increased in RAW264.7 cells after LPS stimulation. These results suggest that high-dose IL-18 derived from macrophages is harmful to Leydig cells. Reducing the overexpression of IL-18 could be a new therapeutic approach to prevent Leydig cell apoptosis as a result of acute inflammation.

Biochemical, immunohistochemical and morphometrical investigation of the effect of thymoquinone on the rat testis following exposure to a 900-MHz electromagnetic field.

Long-term use of cell phones emitting electromagnetic fields (EMFs) have raised concerns regarding public health in recent year. We aimed to investigate the possible effects of 900 MHz EMF exposure (60 min/day for 28 days) on the rat testis. Another objective was to determine whether the deleterious effect of EMF radiation would be reduced by the administration of thymoquinone (TQ) (10 mg/kg/day). Twenty-four male adult Wistar albino rats were randomly selected, then assigned into four groups as followControl, EMF, TQ and EMF + TQ. Testicular samples were analyzed using histological, stereological, biochemical and immunohistochemical techniques. Total numbers of primary spermatocytes and spermatids as well as Leydig cells were significantly decreased in the EMF group compared to the Control group (p < 0.05). In the EMF + TQ group, the total number of primary spermatocytes was significantly increased compared to the EMF group (p < 0.05). Superoxide dismutase (SOD) activity was significantly increased in the EMF group compared to the Control group (p < 0.05). Also, serum testosterone levels and wet weight of testes were significantly decreased in the EMF group compared to the Control group (p < 0.05). Our findings suggested that exposure to a 900 MHz EMF had adverse effects on rat testicular tissue and that the administration of TQ partially mitigated testicular oxidative damages caused by EMF radiation.

Characterization and differentiation of CD51+ Stem Leydig cells in adult mouse testes.

It was reported previously that adult mouse stem Leydig cells (SLCs) express CD51 (integrin α-chain V). However, it is still unclear whether all CD51+ cells are SLCs. In the present study, we found that CD51+ cells can be classified into two sub-groups, a weakly-staining group (CD51+) and a strongly-staining group (CD51++). The CD51+ cells expressed common SLC marker genes, including Nestin, Pdgfra and Coup-tf2, while CD51++ cells did not express these genes. Instead, they expressed macrophage markers, such as F4/80, Cd115 and Tnfa. When these cells were induced to differentiate in vitro, the CD51+ cells, but not CD51++ cells, formed Leydig cells. Overall, our results showed that although SLCs expressed CD51, not all CD51-expressing cells are SLCs. The cells that expressed high levels of CD51 are actually macrophages.

Activation of the NLRP3 Inflammasome Pathway by Prokineticin 2 in Testicular Macrophages of Uropathogenic Escherichia coli- Induced Orchitis.

Infections of the reproductive tract are known to contribute to testicular inflammatory impairment, leading to an increase of pro-inflammatory cytokines such as IL-1ß, and a decline in sperm quality. Prokineticin 2 (PK2), a secretory protein, is closely associated with the secretion of pro-inflammatory cytokines in inflamed tissue. It was reported that increased PK2 is related to the upregulation of IL-1ß, but the underlying mechanism remains elusive. Here, we illustrated that PK2 was upregulated in testicular macrophages (TM) in a rat model of uropathogenic Escherichia coli (UPEC) infection, which induced the activation of the NLRP3 inflammasome pathway to boost IL-1ß secretion. Administration of PK2 inhibitor alleviated the inflammatory damage and suppressed IL-1ß secretion. Moreover, PK2 promoted NLRP3 expression and the release of cleaved IL-1ß from TM to the supernatants after the challenge with UPEC in vitro. IL-1ß in the supernatants affected Leydig cells by suppressing the expression of genes encoding for the enzymes P450scc and P450c17, which are involved in testosterone production. Overall, we revealed that increased PK2 levels in TM in UPEC-induced orchitis may impair testosterone synthesis via the activation of the NLRP3 pathway. Our study provides a new insight into the mechanisms underlying inflammation-associated male infertility and suggests an anti-inflammatory therapeutic target for male infertility.

Antiviral responses of human Leydig cells to mumps virus infection or poly I:C stimulation.

BACKGROUND: The immuno-privileged status of the testis is essential to the maintenance of its functions, and innate immunity is likely to play a key role in limiting harmful viral infections, as demonstrated in the rat. In men mumps virus infects Leydig cells and has deleterious effects on testosterone production and spermatogenesis. The aim of this study was to test whether mumps virus infection of isolated human Leydig cells was associated with an inhibition of their innate antiviral defences. METHODS: Leydig cell production of mRNA and protein for interferons (IFNs) and of three antiviral proteins-2'5' oligoadenylate synthetase (2'5'OAS), double-stranded RNA-activated protein kinase (PKR) and MxA-was investigated, in the absence or presence of mumps virus or viral stimuli including poly I:C, a mimetic of RNA viruses replication product. RESULTS: Stimulated or not, human Leydig cells appeared unable to produce routinely detectable IFNs alpha, beta and gamma. Although the level of PKR remained unchanged after stimulation, the expression of 2'5'OAS and MxA was enhanced following either mumps virus or poly I:C exposure (P < 0.05 versus control). CONCLUSIONS: Overall, our results demonstrate that mumps virus replication in human Leydig cells is not associated with a specific inhibition of IFNs or 2'5'OAS, MxA and PKR production and that these cells display relatively weak endogenous antiviral abilities, as opposed to their rat counterparts.

Distribution of CD68-, CD8-, MHCI- and MHCII-positive cells in the bull and ram testis and epididymis.

The mammalian testis possesses a special immunological environment because of its properties of remarkable immune privilege and effective local innate immunity. The testicular immune privilege protects immunogenic germ cells from systemic immune attack, and local innate immunity is important in preventing testicular microbial infections. Thus, this study aimed to immunohistochemically demonstrate the distribution and localization of CD68-, CD8-, MHCI- and MHCII-positive immune cells in the testes and epididymes. Negative immunoreactivity was detected in the seminiferous tubule epithelium and peritubular myoid cells of the testes upon staining in CD68, CD8 and MHC Class I. Positive CD68 immunoreaction was determined in the Sertoli cells and some Leydig cells. The detection of positive cells for CD8 clearly indicated the presence of lymphocytes. Furthermore, the staining with MHCI intensity was ascertained to vary from weak to moderate in the Sertoli and Leydig cells and connective tissue cells. MHCII-positive immunoreactivity was determined in myoid cells and Leydig cells in the interstitial area. The epithelium of the epididymis showed positive staining for CD68 and CD8, but the stroma displayed a rather weak staining. In the ram epididymis, neither intraepithelial nor interstitial positive reaction was observed for MHCI. In the epididymis, the basal cells displayed a stronger staining for MHCII. In conclusion, these cells not only contribute to local immunity through their direct effects on the quality of fertility in males, but also contribute either directly or indirectly to immune privilege by minimizing the development of both autoimmune reactions and potentially harmful risks.

Differential effects of histamine on Leydig cell and testicular macrophage activities in wall lizards: precise role of H1/H2 receptor subtypes.

The present study in the wall lizard, Hemidactylus flaviviridis, was aimed to understand the role of histamine (HA) in the regulation of Leydig cell and testicular macrophage activities, for the first time, in ectothermic vertebrates. Although HA did not affect the testosterone production from unstimulated Leydig cells, it had dual concentration-related effects, stimulatory at a low concentration of 10(-10) M while inhibitory at a high concentration of 10(-5) M, on FSH-induced testosterone production. This suggests that HA did not influence the basal Leydig cell steroidogenesis, but modulated the FSH-stimulated testosterone production in a biphasic manner depending upon its concentration. However, HA failed to affect the FSH-stimulated Leydig cell proliferation, indicating that HA modulated the testosterone production from Leydig cells without influencing their proliferation in wall lizards. HA, apart from Leydig cells, differentially regulated the testicular macrophage immune responses. It inhibited phagocytosis and superoxide production at high concentration (10(-5) M), while stimulated superoxide production and could not affect phagocytosis at low concentration (10(-10) M). Using selective H1 and H2 antagonists, pyrilamine and famotidine respectively, H1 receptor subtype was seen responsible for mediating the inhibitory effect of HA on Leydig cell steroidogenesis and testicular macrophage immune responses at high concentration, while H2 receptors were involved for the stimulation at low concentration.

Mumps virus-induced innate immune responses in mouse Sertoli and Leydig cells.

Mumps virus (MuV) infection frequently causes orchitis and impairs male fertility. However, the mechanisms underlying the innate immune responses to MuV infection in the testis have yet to be investigated. This study showed that MuV induced innate immune responses in mouse Sertoli and Leydig cells through TLR2 and retinoic acid-inducible gene I (RIG-I) signaling, which result in the production of proinflammatory cytokines and chemokines, including TNF-α, IL-6, MCP-1, CXCL10, and type 1 interferons (IFN-α and IFN-ß). By contrast, MuV did not induce the cytokine production in male germ cells. In response to MuV infection, Sertoli cells produced higher levels of proinflammatory cytokines and chemokines but lower levels of type 1 IFNs than Leydig cells did. The MuV-induced cytokine production by Sertoli and Leydig cells was significantly reduced by the knockout of TLR2 or the knockdown of RIG-I signaling. The local injection of MuV into the testis triggered the testicular innate immune responses in vivo. Moreover, MuV infection suppressed testosterone synthesis by Leydig cells. This is the first study examining the innate immune responses to MuV infection in testicular cells. The results provide novel insights into the mechanisms underlying the MuV-induced innate immune responses in the testis.

Regulatory cytokine expression and interstitial fluid formation in the normal and inflamed rat testis are under leydig cell control.

Leydig cells have been implicated in several inflammation-related responses of the testis. Specifically, these cells produce the proinflammatory cytokines interleukin-1 (IL-1) and IL-6, stimulate macrophage recruitment, and promote interstitial fluid formation. In addition, the immunoregulatory cytokines macrophage migration inhibitory factor (MIF), transforming growth factor-beta1 (TGFbeta1), and interferon-gamma (IFNgamma) are constitutively expressed by testicular cells, including the Leydig cells. In the present study, the contribution of the Leydig cell to testicular inflammatory responses was examined in adult male rats treated with the Leydig cell-specific toxin, ethane dimethane sulfonate (EDS). Intratesticular testosterone levels were modulated by subcutaneous testosterone implants. After 10 days, animals received an injection of lipopolysaccharide (LPS) to induce an inflammatory response, or saline alone, and were killed 3 hours later. Both depletion of Leydig cells by EDS and LPS treatment caused a decrease in collected testicular interstitial fluid to about 35% of control levels, but the effects were not additive. Maintenance of intratesticular testosterone reversed the interstitial fluid decline following EDS treatment and partially prevented the LPS-induced effect. MIF, TGFbeta1, and IFNgamma were expressed in both the normal and inflamed testis at similar levels. In contrast, EDS treatment caused a significant decline in expression of all 3 cytokines, which was prevented by the testosterone implants. These data indicate that 1) expression of TGFbeta1, MIF, and IFNgamma in the testis is not dependent on the presence of intact Leydig cells but is under direct testosterone control and 2) the decline in testicular interstitial fluid during inflammation involves the Leydig cells, acting via both androgens and nonandrogenic secretions. These data provide further support for a significant role for the Leydig cell in modulating the testicular response to inflammation.

High androgen receptor immunoexpression in human "Sertoli cell only" testis.

Our purpose was to evaluate cellular androgen receptor (AR) distribution and intensity of immunostaining in the human azoospermic testis. Thirty six biopsy specimens from azoospermic men were immunostained, using a monoclonal antibody of human AR. The localization and the intensity of AR immunostaining was evaluated in Sertoli Cell Only (SCO) testis (G1, n = 21), in spermatogenesis arrest testis (G2, n = 11) and in histologically normal testis (G3, n = 4). We found an AR immunostaining in Sertoli, peritubular myoid and Leydig cells, but not in germ cells. The intensity of the immunostaining varied substantially between biopsy specimens of different patients. Sertoli and Leydig cells AR immunostaining (score and intensity) in SCO group was higher than in the other groups. For Sertoli cells, the score means of AR immunoreactivity were 20 +/- 2.36, 10.18 +/- 1.0 and 1 +/- 1, for G1, G2 and G3 groups, respectively. For Leydig cells, the score means were 10.24 +/- 1.37, 6 +/- 0.71 and 0, for G1, G2 and G3 groups, respectively. We found significant differences between G1 and G2 (p = 0.0008), between G1 and G3 (p = 1.54 10-7) and G2 and G3 (p = 0.00032). These results suggest that in the testis AR is located exclusively in somatic cells and its expression is higher in SCO syndrome than in normal and in arrest spermatogenesis testes.

The implication of pro-inflammatory cytokines in the impaired production of gonadal androgens by patients with pulmonary tuberculosis.

BACKGROUND: The chronic nature of tuberculosis and the protracted immuno-inflammatory reactions are implied in a series of metabolic and immune-endocrine changes accompanying the disease. We explored components from the hypothalamous-pituitary-gonadal axis and their relationship with cytokines involved in disease immunopathology, in male TB patients. METHODS: Plasma samples from 36 active untreated pulmonary TB male patients were used to determine TNF-α, IFN-γ, TGF-ß, IL-6, cortisol, dehydroepiandrosterone, testosterone, progesterone, estradiol, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by ELISA. Healthy controls corresponded to 21 volunteers without contact with TB patients and similar age (40 ± 16,8 years). Testicular histological samples from necropsies of patients dying from TB were immune-stained for IL-1ß, TNF-α, IL-6 and IFN-γ. The TM3 mouse Leydig cell line was incubated with recombinants TNF-α, IFN-γ and TGF-ß, supernatants were collected and used to measure testosterone by ELISA. RESULTS: Patients showed decreased levels of testosterone in presence of high amounts of LH, together with augmented IFN-γ, IL-6 and TGF-ß levels. Testicular histological sections showed abundant presence of IL-1ß, TNF-α, IL-6 and IFN-γ in interstitial macrophages, Sertoli cells and some spermatogonia. In vitro treatment of Leydig cells with these cytokines led to a remarkable reduction of testosterone production.

Influence of Testosterone on Inflammatory Response in Testicular Cells and Expression of Transcription Factor Foxp3 in T Cells.

PROBLEM: Previous studies demonstrated a strong association between low androgen levels and reduced capacity to mount an inflammatory response. However, the mechanisms underlying these observations are largely not understood. METHODS OF STUDY: Generation of CD4+CD25+Foxp3+ regulatory T cells in Leydig cell-conditioned media was determined by flow cytometry and ELISA. Influence of testosterone on cytokine response was measured in LPS-stimulated testicular macrophages, Sertoli and peritubular cells. RESULTS: Leydig cell-conditioned media dose-dependently stimulated expression of transcription factor Foxp3 and secretion of IL-10 in splenic CD4+ T cells, an effect abolished by addition of the anti-androgen flutamide. In isolated Sertoli and peritubular cells, testosterone pre-treatment suppressed the LPS-induced inflammatory response on TNF-α mRNA expression, while no effect was evident in testicular macrophages (TM). CONCLUSIONS: Androgens can influence the immune system under normal conditions by the generation and functional differentiation of regulatory T cells and in testicular inflammation by direct effect on Sertoli and peritubular cells.

Inhibition of NOS-NO System Prevents Autoimmune Orchitis Development in Rats: Relevance of NO Released by Testicular Macrophages in Germ Cell Apoptosis and Testosterone Secretion.

BACKGROUND: Although the testis is considered an immunoprivileged organ it can orchestrate immune responses against pathological insults such as infection and trauma. Experimental autoimmune orchitis (EAO) is a model of chronic inflammation whose main histopathological features it shares with human orchitis. In EAO an increased number of macrophages infiltrate the interstitium concomitantly with progressive germ cell degeneration and impaired steroidogenesis. Up-regulation of nitric oxide (NO)-NO synthase (NOS) system occurs, macrophages being the main producers of NO. OBJECTIVE: The aim of our study was to evaluate the role of NO-NOS system in orchitis development and determine the involvement of NO released by testicular macrophages on germ cell apoptosis and testosterone secretion. METHOD AND RESULTS: EAO was induced in rats by immunization with testicular homogenate and adjuvants (E group) and a group of untreated normal rats (N) was also studied. Blockage of NOS by i.p. injection of E rats with a competitive inhibitor of NOS, L-NAME (8mg/kg), significantly reduced the incidence and severity of orchitis and lowered testicular nitrite content. L-NAME reduced germ cell apoptosis and restored intratesticular testosterone levels, without variations in serum LH. Co-culture of N testicular fragments with testicular macrophages obtained from EAO rats significantly increased germ cell apoptosis and testosterone secretion, whereas addition of L-NAME lowered both effects and reduced nitrite content. Incubation of testicular fragments from N rats with a NO donor DETA-NOnoate (DETA-NO) induced germ cell apoptosis through external and internal apoptotic pathways, an effect prevented by N-acetyl-L-cysteine (NAC). DETA-NO inhibited testosterone released from Leydig cells, whereas NAC (from 2.5 to 15 mM) did not prevent this effect. CONCLUSIONS: We demonstrated that NO-NOS system is involved in the impairment of testicular function in orchitis. NO secreted mainly by testicular macrophages could promote oxidative stress inducing ST damage and interfering in Leydig cell function.

Luteinizing hormone triggers a molecular association between its receptor and the major histocompatibility complex class I antigen to produce cell activation.

We studied the involvement of major histocompatibility (MHC) class I antigens on the mechanism of LH/hCG receptor activation. For this purpose we investigated the effects of anti-MHC class I antibodies on hormone-receptor interaction, signal transduction, and MHC class I antigen-receptor interaction. Monoclonal antibodies against MHC class I antigen were able to stimulate testosterone production in mouse Leydig cells with the same potency as LH. This biological effect depends on the concentration of antibody used and could be abolished by a LH antagonist. There is a perfect parallelism, for each monoclonal antibody, between the specificity for a particular haplotype and the response of the target cells from the strains carrying such a haplotype. The same antibodies were able to precipitate the soluble LH/hCG receptors, as both a hormone-receptor complex and a free receptor. The results suggest that bound hormone triggers an association of the MHC class I antigen with the LH/hCG receptor, resulting in activation of the target cell.

Localization of immunoreactive androgen in testicular tissue.

Immunohistochemical localization of androgen in the testes of both rat and squirrel monkey has been performed using an antiserum specific to testosterone and certain related androgens. The bound antibodies were visualized by coupling with antigammaglobulin labeled either with fluorescein isothiocyanate or with a horseradish peroxidase-diaminobenzidine technique. The highest concentration of androgen was found in the interstitial tissue but a significant amount was also present in the seminiferous tubules in the layer adjacent to the tubular wall. Very small amounts of androgen were localized in the tubular wall. This restricted localization in the tubules might indicate either de novo synthesis or active uptake of steroids intermediate in the biosynthesis of testosterone in the seminiferous tubules.

The heterogeneity of isolated adult rat Leydig cells separated on Percoll density gradients: an immunological, cytochemical, and functional analysis.

Intertubular cells, isolated from adult rat testes by collagenase dispersal under conditions designed to minimize cell damage, were fractionated on Percoll density gradients. In the gradient fractions, there was a close cellular correlation between the presence of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), determined by cytochemistry, and other Leydig cell markers (nonspecific esterase, autofluorescence, and an antigen defined by monoclonal antibody LC-1C6). As the reagents for 3 beta HSD cytochemistry are excluded by intact membranes, Leydig cells with damaged plasma membranes were identified by 3 beta HSD reactivity in suspended cell preparations, and the total number of 3 beta HSD-positive (3 beta HSD+) cells in the same preparations was determined after lysis of the cell membrane. Whole cells were differentiated from cytoplasmic fragments by counterstaining with the nuclear dye propidium iodide, and the number of intact Leydig cells in each preparation was determined subsequently by subtracting the number of damaged nucleated 3 beta HSD+ cells from the total number of nucleated 3 beta HSD+ cells. The majority of intact isolated Leydig cells were found in gradient fractions of 1.054-1.096 g/ml density. Acute (3-H) basal and hCG-stimulated testosterone production per intact Leydig cell were dependent upon the concentration of Leydig cells per assay well, indicating that there is cooperativity among Leydig cells in vitro. There was no difference in steroidogenic function among intact Leydig cells from different fractions of the above density gradient range at assay concentrations greater than 10,000 Leydig cells/well. At lower cell concentrations, Leydig cells from gradient fractions of lower density (1.054-1.064 g/ml) produced slightly less testosterone in response to hCG stimulation than Leydig cells from more dense fractions (1.070-1.096 g/ml). Prolonging the exposure of isolated cells to the dispersal conditions caused declines in the apparent buoyant density and basal testosterone and hCG-stimulated cAMP and testosterone production of all Leydig cells, without detectable changes in cell integrity. The data indicate that both the absolute steroidogenic function and the functional heterogeneity of isolated intact Leydig cells are, at least in part, dependent upon the procedures used for their isolation.