Epidermal growth factor receptor pathway substrate 8 （Eps8） expression in maturing testis

Aim: Although epidermal growth factor receptors are expressed in the testes, whether they signal through epidermal growth factor receptor pathway substrate 8 (EpsS) is unknown. Here we evaluated the expression pattern of Eps8 in the maturing testis. Methods: The expression of Eps8 was analysed by Northern blotting, immunocytochemistry and Western blotting in primary Sertoli cell cultures and in testicular tissue of rodents. Results: Eps8 is specifically expressed in gonocytes, Leydig and Sertoli cells of the neonatal rats and in Leydig and Sertoli cells of the adult rats and mice. Although gonocytes express EpsS, no signal was found in prepubertal or mature spermatogonia and the expression level of Eps8 in Sertoli cells increases with age. No regulation of Eps8 expression in primary immature rat Sertoli cells by Follicle stimulating hormone (FSH) was detected by Western blotting. Conclusion: Eps8 seems to be involved in the growth factor-controlled regulation of cell proliferation and differentiation in the seminiferous epithelium. Eps8 is a possible marker for gonocytes and in Sertoli cells it could be involved in crosstalk with other growth factor pathways. (Asian J Androl 2004 Sep; 6: 195-203)     

Molecular identity, expression and functional analysis of interleukin-lα and its isoforms in rat testis

Interleukin-1α(IL-1α) is a proinflammatory cytokine that has also been found to act as a paracrine mediator involved in the regulation of testicular functions. The present review provides an overview of the role of IL-1α in testicular physiology. Bioactive IL-1α isolated from adult rat testis was found to consist of three distinct immunoreactive protein species with apparent sizes of 45, 24 and 19 kDa. These isoforms showed bioactivity in athymocyte proliferation and steroidogenesis assays with different biopotencies. The background of the molecular heterogeneity and processing, secretion and regulation of the isoforms of testicular IL-1α are discussed. All three isoforms have been found to be secreted into the testis tubular lumen and interstitial space. We have provided evidence that IL- 1α is a paracrine factor that may be of importance in, e.g., the regulation of Leydig cell steroidogenesis.Pathophysiologically, testicular IL-1α may contribute to testicular relapse of acute lymphocytic leukemia in boys.     

Molecular identity, expression and functional analysis of interleukin-1α and its isoforms in rat testis

Interleukin-1α (IL-1α) is a proinflammatory cytokine that has also been found to act as a paracrine mediator involved in the regulation of testicular functions. The present review provides an overview of the role of IL 1α in testicular physiology. Bioactive IL-1α isolated from adult rat testis was found to consist of three distinct immunoreactive protein species with apparent sizes of 45, 24 and 19 kDa. These isoforms showed bioactivity in a thymocyte proliferation and steroidogenesis assays with different biopotencies. The background of the molecular heterogeneity and processing, secretion and regulation of the isoforms of testicular IL-1α are discussed. All three isoforms have been found to be secreted into the testis tubular lumen and interstitial space. We have provided evidence that IL-1α is a paracrine factor that may be of importance in, e.g., the regulation of Leydig cell steroidogenesis. Pathophysiologically, testicular IL-1α may contribute to testicular relapse of acute lymphocytic leuke     

Effects of luteinizing hormone and androgen on the development of rat progenitor Leydig cells in vitro and in vivo

Progenitor Leydig cells are derived from stem cells. The proliferation and differentiation of progenitor Leydig cells significantly contributes to Leydig cell number during puberty. However, the regulation of these processes remains unclear. The objective of the present study was to determine whether luteinizing hormone （LH） or androgen contributes to the proliferation and differentiation of progenitor Leydig cells. Fourteen-day-old male Sprague-Dawley rats were treated for 7 days with NalGlu, which is a gonadotropin- releasing hormone antagonist, to reduce the secretion of LH in the pituitary and thus, androgen in the testis. Rats were co-administered with LH or 7a-methyl-nortestosterone （MENT）, which is an androgen resistant to metabolism by 5a-reductase 1 in progenitor Leydig cells, and the subsequent effects of LH or androgen were measured. 3H-Thymidine was also intravenously injected into rats to study thymidine incorporation in progenitor Leydig cells. Progenitor Leydig cells were examined. NalGlu administration reduced progenitor Leydig cell proliferation by 83%. In addition, LH or MENT treatment restored Leydig cell proliferative capacity to 73% or 50% of control, respectively. The messenger RNA levels of proliferation-related genes were measured using real-time PCR. The expression levels of Igfl, Lifr, Pdgfra, Bcl2, Ccnd3and Pcnawere upregulated by MENT, and those of Pdgfra, Ccnd3and Pcnawere upregulated by LH. Both LH and MENT stimulated the differentiation of progenitor Leydig cells in vitro. We concluded that both LH and MENT were involved in regulating the development of progenitor Leydig cells.     

The correlation between serum epidermal growth factor/testicular epidermal growth factor receptor and spermatogenesis in rat

<正>Objective: To investigate the correlation between epidermal growth factor (EGF)/testicular epidermal growth factor receptor (EGF-R) and spermatogenesis in rat. Methods: Forty mature male Spraque-Dauley (SD) rats were randomly assigned to four groups, ten rats in each: sham operation group (SOG), sialoadenectomy group(SG), sialoade-nectomy group with injection of EGF (0. 25 μg·kg-1·d-1, SG-EGF Ⅰ) and sialoadenectomy group with injection of EGF (0. 50 μg·kg-1·d-1 , SG-EGF Ⅱ). The rats were routinely feed, and blood and testes were obtained on the 48th day after the operation. Serum EGF concentrations were determined by radioimmunoassay (RIA) , expression of EGF-R in testes was examined by the immunohistochemical method, and the spermatogenesis was pathologically checked. Results: Serum EGF levels in SG-EGFIand SG decreased significantly when compared with those of SOG (P<0. 05 and P< 0. 01, respectively). The testicular function of spermatogenesis showed a moderate to severe impairment in SG. The expression of EGF-R in Leydig cells decreased in SG(P<0. 05). The two dosage groups of EGF replacement had different effects. There were no significant differences of EGF-R expression in testicular germ cells, Sertoli cells and Leydig cells in SOG, SG-EGFⅠand SG-EGFⅡ(P>0. 05). Conclusion: EGF may play an important role in the regulation of spermatogenesis. Serum EGF concentration and high expression of EGF-R in Leydig cells have a positive correlation with spermatogenic function of the testes.     

Effect of Ureaplasma urealyticum infection on IL-laand IL-6 secretion by rat Sertoli cells

Objective: To observe the effect of Ureaplasma urealyticum (UU) infection on the IL-1α and IL-6 secretion by rat Sertoli cells. Methods: Eight 20-day-old UU-free male SD rats (average weight 40 g) were used. Under sterile condition, the testes were removed and separately digested with collagenase typeⅡand hyaluronidase. High purity Sertoli cells were then isolated and adjusted to a concentration of 8×105/mL with DMEM/Ham's F-12. In the infected group, 1 mL Sertoli cell suspension and 100 mL UU (serotype 8, T960) were introduced into one well of a 24 well culture plate. In the control group, 1 mL Sertoli cell suspension and 100 mL medium were introduced. IL-1αand IL-6 were determined in the culture supernatant with ELISA. Results: The production of IL-1αwas significantly lower and of IL-6 significantly higher in the infected than those in the control groups (P<0.01). Conclusion: UU infection reduces the IL-1αand increases the IL-6 secretion by rat Sertoli cells. UU infection is probably involved in     

Stage-specific localization of transforming growth factor β1 and β3 and their receptors during spermatogenesis in men

Aim: To investigate the stage-specific localization of transforming growth factor (TGF) β1 and β3 during spermatogenesis in adult human testis, Methods: The localization of TGFβ1 and β3 was investigated by immunohistochemical staining method employing specific polyclonal antibodies. Results: Both TGFβ1 and β3 and their receptors were preponderant in the Leydig cells. TGFβ1 could not be detected in the seminiferous tubules. TGFβ3 and TGFβ-Receptor (R) Ⅰ were mainly seen in the elongated spermatids, while TGFβ-RⅡ in the pachytene spermatocytes and weak in the spermatogonia, spermatids and Sertoli cells. Only TGFβ-RⅡ was detected in the Sertoli cells.TGFβ3, TGFβ-RⅠ and TGFβ-RⅡ showed a staining pattern dependent upon the stages of the seminiferous epithelium cycle. Conclusion: TGFβ isoforms and their receptors are present in the somatic and germ cells of the adult humantestis, suggesting their involvement in the regulation of spermatogenesis.     

Expression and localization of Smad1, Smad2 and Smad4 proteins in rat testis during postnatal development

Aim：To study the expression and regulation of Smad1,Smad2 and Smad4 proteins(intracellular signaling molecules of transforming growth factor-β family)in rat testis during postnatal development.Methods:The whole testes were collected from SD rats aged 3,7,14,28 and 90(adults)days.The cellular localization and developmental changes were examined by immunohistochemistry ABC method with the glucose oxidase-DAB-nickel enhancement technique.Quantitative analysis of the immunostaining was made by the image analysis system.The Smads proteins coexistence in the adult rat testis was tested by the double immune staining for CD14-Smad4 and Smad2-Smad4.The protein expression of Smad during rat testicular development was examined by means of Western blots.Results: Smadl,Smad2 and Smad4 were present throughout testicular development.The immunostaining of Smad1 and Smad2 were present in spermatogenic cells.A positive immunoreactivity was located at the cytoplasm,but the nucleus was negative,Smadl was immunolocalized at the d14,d28 and adult testes,while Smad2,at the d7,d14,d28 and adult testis.There was positive immunoreaction in the Sertoli cells and Leydig cells as well.The immunolocalization of Smad4 was exclusively at the cytoplasm of Leydig cells and the nuclei were negative throughout the testicular development.No expression was detected in the germ cells.The results of image and statistical analysis showed that generally the expression of Smad1,Smad2 and Smad4 in the testis tended to increase gradually with the growth of the rat.Conclusion;The present data provide direct evidences for the molecular mechanism of TGF-β action in rat testes during postnatal development and spermatogenesis.     

CD99 and CD106 （VCAM—1） in human testis

Aim: The expression of the cytokines IL-2, IL-6, IL-10, IFN-γ and TNF-α and the adhesion proteins CD99 and CD106 was studied in the human testis at the protein level. Methods: The expression of the cytokines and the adhesion proteins was assessed using immunohistochemistry and immunoblotting. Results: None of the cytokines studied was present in the human testis, but CD99 and CD106 (VCAM-1) strongly were expressed in all the testes investigated. CD99 was present in the interstitial tissue of the human testis as well as in the Sertoli cells. The identity of the CD99 interstitial cells is unclear. CD106 (VCAM-1) was present in Leydig cells as well as the basal parts of the Sertoli cells in the seminiferous tubules. In immunoblotting, CD99 was demonstrated at molecular ratios of 46-57 (kD). This is a novel isoform of the molecule. Conclusion: The human testis produces both CD99 and CD106 and as CD106 mediates cell binding to lymphocytes, it is possible that the human Leydig cells adhere to lymphocytes l     

Stroma-epithelium crosstalk in prostate cancer

The critical role played by stroma-epithelium crosstalk in carcinogenesis and progression of prostate cancer has been increasingly recognized. These interactions are mediated by a variety of paracrine factors secreted by cancer cells and/or stromal cells. In human prostate cancer, reactive stroma is characterized by an increase in myofibroblasts and a corresponding amplification of extracellular matrix production and angiogenesis. Permanent genetic mutations have been reported in stromal cells as well as in tumour cells. Transforming growth factor-J3, vascular endothelial growth factor, platelet-derived growth factor and fibroblast growth factor signalling pathways are involved in the process of angiogenesis, whereas hepatocyte growth factor, insulin-like growth factor-l, epidermal growth factor, CXC12 and Interleukin-6 play active roles in the progression, androgen-independent conversion and distal metastasis of prostate cancer. Some soluble factors have reciprocal interactions with androgens and the androgen receptor （AR）, and can even activate AR in the absence of the androgen ligand. In this article, we review the complex interactions between cancer cells and the surrounding microenvironment, and discuss the potential therapeutic targets in the stromal compartment of prostate cancer.     

Paracrine regulation of testicular function

Spermatogenesis and spermiogenesis are controlled by FSH and testosterone but need also the participation of several paracrine and autocrine mechanisms of regulations. The relationships between peritubular, Sertoli and Leydig cells are currently investigated. High intratesticular testosterone levels are maintained by a binding to a protein called ABP which is synthetized by Sertoli cell and regulated by pituitary FSH. Leydig cell testosterone, peritubular cell P-Mod-S (protein modulating Sertoli function) and Sertoli cell FRP (follicle regulatory protein). Accumulation of testosterone results to aromatase activity modulation. Aromatization is stimulated by FSH, activin, alpha-MSH but is inhibited by aromatase inhibitor, inhibin, FSHBI (FSH binding inhibitor). Other molecules, growing factors, mitogenic factors, energetic substrates are synthetized in the testis under the control of germ cells. Understanding of these mechanisms of intratesticular regulation will permit to discover therapies capable of correcting certain fertility dysfunctions.     

Thyroid hormones: their role in testicular steroidogenesis

Thyroid hormones are important for growth and development of many tissues. Altered thyroid hormone status causes testicular abnormalities. For instance, juvenile hypothyroidism/neonatal transient hypothyroidism induces macroorchidism, increases testicular cell number (Sertoli, Leydig, and germ cells) and daily sperm production. Triiodothyronine (T3) receptors have been identified in sperm, developing germ cells, Sertoli, Leydig, and peritubular cells. T3 stimulates Sertoli cell lactate secretion as well as mRNA expression of inhibin-alpha, androgen receptor, IGF-I, and IGFBP-4. It also inhibits Sertoli cell mRNA expression of Müllerian inhibiting substance (MIS), aromatase, estradiol receptor, and androgen binding protein (ABP) and ABP secretion. T3 directly increases Leydig cell LH receptor numbers and mRNA levels of steroidogenic enzymes and steroidogenic acute regulatory protein. It stimulates basal and LH-induced secretion of progesterone, testosterone, and estradiol by Leydig cells. Steroidogenic factor-1 acts as a mediator for T3-induced Leydig cell steroidogenesis. Although the role of T3 on sperm, germ, and peritubular cells has not yet been completely studied, it is clear that T3 directly regulates Sertoli and Leydig cell functions. Further studies are required to elucidate the direct effect of T3 on sperm, germ, and peritubular cells.     

Paracrine Factors in Adult Rat Testis Gonadotrophin Control of Opioids and LHRH Like Peptide

A paracrine regulation involves agents which are produced by one cell type and act on an other one within an organ. In rodent testis, local control mechanisms modulate the actions of the gonadotrophins according to local requirements. Two groups of peptides-opioids and testicular LHRH are defined as paracrine factors and in vivo they are both modified by HCG. In vitro, after HCG exposure, we first localized an opioid like material in Sertoli cells cytoplasma by immunohistochemistry. This material is detected in freeze dried homologous culture media using a dot immunobinding technique. With a longer HCG exposure, an LHRH like material is then visualized in the basal compartment of the Sertoli cells and it is detected in freeze dried homologous culture media by the same technical procedure than for opioid material. By adding synthetic enkephalins to culture medium, we obtain the same results as with the endogenous opioid material, excreted after HCG addition. If naloxone a potent opiate antagonist, is added to the culture medium previously to HCG or enkephalins, the Sertoli cells cytoplasma are no more immunoreactives with the anti-enkephalin serum and no LHRH material is neither visualized by immunohistochemical technique neither detected in culture media. We conclude that testicular opioids, synthetized by the Leydig cells and which have specific Sertoli cells receptors are one Leydig-Sertoli paracrine communication factor. One way of response to their receptor fixation is the synthesis and excretion by Sertoli cells of testicular LHRH. This one is known to act on Leydig cells via specific receptors and it is one Sertoli-Leydig cells paracrine communication factor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of neuropeptide — Y and its C-terminal flanking peptide immuno-reactivity in the seminiferous tubules of human testis

The presence of neuropeptide Y (NPY) and its C-terminal flanking peptide (C-PON) was described by immunohistochemistry in human testes. The immunopositive material was visualized in the spermatogenic elements of the seminiferous tubules. More NPY occurred in the younger testis and more C-PON in the older ones. NPY positive material was present mainly in the spermatogonia, and in the primary spermatocytes, where C-PON also occurred. The megalospermatocytes, present in aged testis, showed C-PON immunoreactivity. Both NPY and C-PON were present in granular form in the perinuclear zone of the cells. No positive material was detected in the Sertoli cells or in the Leydig cells. It is possible that NPY and its precursor are synthetized within the testis and might play a role in the paracrine and/or autocrine regulation of spermatogenesis.     

Macrophage migration inhibitory factor (MIF) as a paracrine mediator in the interaction of testicular somatic cells

Originally the macrophage migration inhibitory factor (MIF) was described as a classical T-cell cytokine. Recently, a much broader tissue distribution for MIF has been revealed. We demonstrated that MIF protein and mRNA are present in the Leydig cells of the normal adult rat testis. Addition of recombinant MIF to cultures of rat seminiferous tubules resulted in decreased secretion of inhibin, whereas follistatin and activin levels remained unchanged, suggesting a paracrine role for MIF in Sertoli cell regulation. Furthermore, MIF showed unique compensatory production in the rat testis. Depletion of the original MIF source, the Leydig cells, by the specific toxin EDS prompted MIF expression by the previously negative Sertoli cells. Leydig cell re-population of the interstitial tissue by precursor cells resulted in a switch back to production by Leydig cells. Therefore, testicular MIF appears to be under very tight paracrine control. MIF has thus been identified as a new mediator in the cross-talk between Leydig cells and the somatic cells of the seminiferous tubules of the rat testis.     

Effects of supplementation with impuberal or adult testicular protein extracts on genital tract and testicular histology as well as hormonal levels in adult busulfan treated rats

24 adult Wistar rats received one SC injection of busulfan (10 mg/kg BW) on day 0. They were injected daily from day 1 to day 10 either with BSA (1 and 2 mg/rat, n = 5 respectively), with acetonic extract of adult ram testicular cytosol (2 mg/rat, n = 10) or with acetonic extract of impuberal calf testicular cytosol (2 mg/rat, n = 4). 10 animals served as control. Animals were killed on day 17. Busulfan treatment induced decrease of total volumes of intertubular tissue and of Leydig cells per testis, of accessory glands, and of germ cells from type A spermatogonia to zygotene spermatocytes. Supplementation with adult testicular extract did not modify accessory glands but increased Leydig cell total volume, individual cellular and nuclear areas and Sertoli cell nuclear areas. It decreased further the germ cells from A spermatogonia to zygotene primary spermatocytes. Supplementation with impuberal testicular extract did not modify accessory glands, Leydig cell total volume, individual cellular and nuclear areas or Sertoli cell nuclear area. It increased the germ cells from A spermatogonia to zygotene primary spermatocytes to values intermediate between those of control and busulfan treated rats.     

Tumor necrosis factor and interleukin-1 stimulate testosterone secretion in adult male rat Leydig cells in vitro

The actions of two cytokines, tumor necrosis factor (TNF) and interleukin-1 (IL-1), on testosterone production by dispersed adult testis cells and purified Leydig cells in culture were studied. In one set of experiments, testis cells from adult (90-day-old) rats were enzymatically dispersed. In another set of experiments, the dispersed testis cells were placed on a Percoll density gradient and were centrifuged to yield purified (greater than 85%) Leydig cells. Both whole testis cells and purified Leydig cells were cultured in the presence of varying doses of TNF or IL-1 with or without maximally stimulating doses of human chorionic gonadotropin (hCG). Both TNF and IL-1 stimulated basal secretion of testosterone in whole testis cells, as well as purified Leydig cells. Additionally, both TNF and IL-1 augmented maximally hCG stimulated testosterone secretion. Both cytokines stimulated testosterone secretion by dispersed testis cells as early as 4 hours, and the effect continued for up to 72 hours. The cytokines slightly, but significantly, stimulated testosterone production in purified Leydig cells after 24 hours, and continued for up to 72 hours. We have concluded from this data that TNF and IL-1 stimulate the testosterone secretion by adult rat Leydig cells. While this effect might be mediated through the action of the cytokines on testicular macrophages, there might also be a direct effect on the Leydig cell since augmentation of secretion occurred in purified Leydig cells, as well as whole testis cells. Therefore, TNF and IL-1 may serve as local regulators of Leydig cell function.     

Evaluation of the effect of peritubular cell secretions and the testicular paracrine factor P-Mod-S on Leydig cell steroidogenesis and immunoactive inhibin production

Testicular peritubular cells have been shown to produce a paracrine factor, termed P-Mod-S, under androgen control that has dramatic effects on Sertoli cell function and may provide an important mode of androgen action in the testis. Therefore, the current study was designed to investigate the possibility that peritubular cell secretory products could feedback and regulate Leydig cell function. The Leydig cell functional parameters that were examined included testosterone production and inhibin secretion. Purified forms of P-Mod-S (P-Mod-S(A) and P-Mod-S(B) shown to be biologically active on Sertoli cells) had no effect on basal or gonadotrophin-stimulated production of testosterone or inhibin by Leydig cells. A preparation of peritubular cell-secreted proteins (PSP) with molecular weights greater than 3 kDa did not influence testosterone production by Leydig cells. PSP, however, did influence cultured Leydig cell morphology and improved cell viability. PSP also had no effect on the ability of LH to stimulate Leydig cell testosterone production. Whilst determining the effect of PSP on Leydig cell inhibin production, PSP was found to contain endogenous levels of inhibin apparently due to 2% contamination of the peritubular cell cultures with Sertoli cells. When this endogenous inhibin level was considered, PSP was found to have no influence on basal or hormone-stimulated production of inhibin by Leydig cells. Results of the current study indicate that peritubular cell secretory products, including the paracrine factor P-Mod-S, do not appear to play a major role in the regulation of Leydig cell function. Therefore, the regulation of Leydig cell function by the seminiferous tubule will primarily be due to Sertoli cell secretory products.