Immunolocalization of INSL3 in dog foetal Leydig cells and the LGR8 receptor in the gubernaculum testis

To study the effect of diethylstilbestrol (DES) at different doses on transabdominal testicular descent in rats and the expression of INSL3 in the testis and HOXA10 in the gubernaculum. Fifty E13.5 (embryonic day 13.5) pregnant female SD rats were randomly divided into five groups that received a subcutaneous injection of DMSO, 2.5, 5.0, 10.0 and 20.0 mg/kg DES (group A, B, C, D and E), respectively. Male offspring were killed at E19.5, and then fetal mortality, the degree of transabdominal testicular ascent (DTA) was determined by a stereomicroscope. The mRNA expressions of INSL3 in the testis and HOXA10 in the gubernaculum were determined by RT-PCR. The expression of INSL3 protein was determined by Western blotting. Male fetal mortality in group A, B, C, D, and E were 3.57%, 6.90%, 12.00%, 19.23% and 36.36%, respectively, which showed a dose-effect relationship between DES and the male fatal mortality (r=0.999, P<0.01). DTA in group B, C, D and E were (23.7+/-1.7) U, (38.8+/-1.9) U, (49.3+/-1.8) U and (58.6+/-2.1) U that were significantly larger than that in group A [(8.5+/-1.3) U] (q=46.12, 88.53, 120.44 and 141.37, respectively, P<0.01). There was also a dose-effect relationship between DES and DTA. In group B, C, D, and E, the expression of INSL3 mRNA were 0.9570+/-0.1490, 0.6760+/-0.1380, 0.0170+/-0.0040 and 0.0013+/-0.0003, respectively; the expressions of INSL3 protein were 0.8360+/-0.1520, 0.5310+/-0.1070, 0.0140+/-0.0020 and 0.0011+/-0.0003, respectively, which were significantly larger than the expression of INSL3 mRNA (1.801+/-0.126) and INSL3 protein (1.612+/-0.134) in group A (qmRNA=40.4840, 52.4402, 83.1585 and 82.0582, respectively, and qprotein=38.6151, 52.2747, 77.2756 and 76.1983, respectively, P<0.01). The expression of HOXA10 mRNA in group A, B, C, D, and E were 0.945+/-0.125, 0.940+/-0.119, 0.656+/-0.115, 0.544+/-0.118 and 0.463+/-0.114, respectively. Compared with the expression of HOXA10 mRNA in group A, the expression of group B was not significantly different (q=0.2213, P>0.05), those in other groups were down-regulated significantly (q=12.4304, 17.2477 and 20.2789, respectively, P<0.01). DES inhibited transabdominal testicular descent dose-dependently via down-regulating the expression of INSL3. HOXA10 may play no role in low-dosage DES induced intra-abdominal cryptorchidism, but down-regulated HOXA10 mRNA was involved in high-dosage DES induced ones.

Objective To investigate the expression levels and the clinical significance of INSL3 and LGR8 in patients with unilateral cryptorchidism.Methods The expression levels of INSL3 and LGR8 were evaluated using RTRCR in gubernaculum and perididymis in 42 unilateral cryptorchidism (high position 4 cases; middle 31 cases; low 7 cases).Controls were taken from 40perididymis in 40 hydrocele patients and 40 hernia sacs in female indirect inguinal hernia patients.Results The expression levels of INSL3 and LGR8 in perididymis tissues and gubernaculums of different type of unilateral cryptorchidism showed no significant difference (P＞0.05).The expression levels of INSL3 and LGR8 in gubernaculums were lower than those in perididymis tissues (P＜0.05) ; and the expressions of INSL3 and LGR8 in perididymis tissues in unilateral cryptorchidism were lower than those in hydrocele testis (P＜0.05).The expressions of INSL3 and LGR8 in hemia sac of female indirect inguinal hernia were lower than those in perididymis of hydrocele testis (P＜0.05).Conclusions The expression levels of INSL3 and LGR8 in perididymis tissues and gubernaculums of different type of unilateral cryptorchidism have no significant difference.It would imply that INSL3 and LGR8 might have a role in different stages of testicular descent.Lower levels of INSL3 and LGR8 may be associated with altered gubernacular development and cryptorchidism. Key words: Insulin like factor-3; Receptors,G-protein-coupled; Cryptorchidism

Insulin-like 3 (INSL3) is a hormone produced by fetal and adult Leydig cells of the testis and by theca and luteal cells of the adult ovary. In males, INSL3 regulates testicular descent during fetal life, whereas in adults, it acts as a germ cell survival factor. In the ovary, INSL3 regulates oocyte maturation. Despite its importance for male sex differentiation and reproductive function in both sexes, very little is known regarding the molecular mechanisms that regulate Insl3 expression. So far, the nuclear receptor NR5A1 is the only transcription factor known to regulate the mouse Insl3 promoter in Leydig cells. NR5A1 by itself, however, cannot explain the spatiotemporal expression pattern of the Insl3 gene. In the present study, we have identified the orphan nuclear receptor NR4A1 as a novel regulator of INSL3 transcription in Leydig cells. Using RT-PCR, we found that Nr4a1 is coexpressed with Insl3 in purified Leydig cells and in several Leydig cell lines. Through detailed analyses of the mouse and human INSL3 promoter in Leydig cells, we have mapped a novel regulatory element located at -100 bp that is essential and sufficient to confer NR4A1 responsiveness. Consistent with a role for NR4A1 in Insl3 transcription, chromatin immunoprecipitation assays revealed that endogenous NR4A1 binds to the proximal Insl3 promoter in vivo. Finally, we found that NR4A1 is also implicated in cAMP-induced Insl3 transcription in Leydig cells. Taken together, our identification of NR4A1 as an important regulator of mouse and human INSL3 promoter activity helps us to better define the tissue-specific regulation of the INSL3 gene in gonadal cells.

Insulin-like 3 (INSL3) is a small peptide produced by testicular Leydig cells throughout embryonic and postnatal life and by theca and luteal cells of the adult ovary. During fetal life, INSL3 regulates testicular descent in males, whereas in adults, it acts as an antiapoptotic factor for germ cells in males and as a follicle selection and survival factor in females. Despite its considerable roles in the reproductive system, the mechanisms that regulate Insl3 expression remain poorly understood. There is accumulating evidence suggesting that androgens might regulate Insl3 expression in Leydig cells, but transcriptional data are still lacking. We now report that testosterone does increase Insl3 mRNA levels in a Leydig cell line and primary Leydig cells. We also show that testosterone activates the activity of the Insl3 promoter from different species. In addition, the testosterone-stimulating effects on Insl3 mRNA levels and promoter activity require the androgen receptor. We have mapped the testosterone-responsive element to the proximal Insl3 promoter region. This region, however, lacks a consensus androgen response element, suggesting an indirect mechanism of action. Finally we show that mono-(2-ethylhexyl) phthalate, a widely distributed endocrine disruptor with antiandrogenic activity previously shown to inhibit Insl3 expression in vivo, represses Insl3 transcription, at least in part, by antagonizing testosterone/androgen receptor action. All together our data provide important new insights into the regulation of Insl3 transcription in Leydig cells and the mode of action of phthalates.

Insulin-like 3 (INSL3) is a hormone produced by Leydig cells (LCs) and leads to physiological testicular descent during embryonic development. We investigated the expression of INSL3 by immunohistochemistry in normal LCs, in Leydig cell tumor (LCT) (n=17 including 15 testes and 2 ovaries) and in Leydig cell hyperplasia (LCH) (n=10). Normally distributed LCs showed strong immunostaining in the cytoplasm in all cases. All 10 cases (100%) of LCH were strongly and diffusely positive in the intertubular areas. Six cases of LCH had nodules raging in size from 0.2 to 0.9 cm with variable INSL3 staining. Fifteen of 17 (88.2%) LCTs showed marked decrease INSL3 staining, 10/17 (58.8%) were completely negative, and 5/17 (29.4%) were only focally positive. Two cases with multifocal LCTs showed strong and diffuse cytoplasmic staining of LCs around seminiferous tubules while the LCTs were negative. Two cases diagnosed as LCT were strongly positive for INSL3. Other sex cord stromal tumors tested were consistently negative including Sertoli-cell tumor (n=4), granulosa cell tumor (n=2), and fibrothecoma (n=1). In conclusion, our results contrast with those of previously published studies, and show that the great majority of LCTs are negative or have decreased expression of INSL3 while its expression is retained in LCH. INSL3 negative nodules within LCH may represent early LCTs. INSL3 immunostaining could be helpful to highlight LCs in cases where it is difficult to identify them (ie, small testicular biopsies performed for infertility workup) and in the differential diagnosis between florid LCH and LCT.

The peptide hormone insulin-like 3 (INSL3) is produced almost exclusively by Leydig cells of the male gonad. INSL3 has several functions such as fetal testis descent and bone metabolism in adults. Insl3 gene expression in Leydig cells is not hormonally regulated but rather is constitutively expressed. The regulatory region of the Insl3 gene has been described in various species; moreover, functional studies have revealed that the Insl3 promoter is regulated by various transcription factors that include the nuclear receptors AR, NUR77, COUP-TFII, LRH1, and SF1, as well as the Krüppel-like factor KLF6. However, these transcription factors are also found in several tissues that do not express Insl3, indicating that other, yet unidentified factors, must be involved to drive Insl3 expression specifically in Leydig cells. Through a fine functional promoter analysis, we have identified a 35-bp region that is responsible for conferring 70% of the activity of the mouse Insl3 promoter in Leydig cells. All tri- and dinucleotide mutations introduced dramatically reduced Insl3 promoter activity, indicating that the entire 35-bp sequence is required. Nuclear proteins from MA-10 Leydig cells bound specifically to the 35-bp region. The 35-bp sequence contains GC- and GA-rich motifs as well as potential binding elements for members of the CREB, C/EBP, AP1, AP2, and NF-κB families. The Insl3 promoter was indeed activated 2-fold by NF-κB p50 but not by other transcription factors tested. These results help to further define the regulation of Insl3 gene transcription in Leydig cells.

It has been well known that testosterone is the only connection between testis and bone as it plays an important role in skeletal growth and bone mass accrual. In the past few years, studies showed that factors other than testosterone could also be responsible for bone health. The Leydig cells in the testis produce insulin-like 3 and express cytochrome P450 2R1(CYP2R1)gene which encodes the major enzyme involved in 25-hydroxylation of vitamin D. The understanding of the crosstalk between testis and bone could contribute to defining an improved clinical approach to the biochemical diagnosis and therapeutic management of hypogonadism and male osteoporosis. (Chin J Endocrinol Metab, 2015, 31: 372-374) Key words: Testosterone; Insulin-like 3; Cytochrome P450 2R1; Bone metabolism

100 orchiopexies were performed on 82 boys for cryptorchism from Jun.1983 to Apr.1984.The operative lengthening of the spermatic cord was measured after each step of the procedure.The 4 steps involved in the mobilization procedure are:division of the cremaster muscIe and the gubernaculum testis;(2) separation of the cord from the processus vaginalis;(3) retroperitoneal mobilization of the cord,and (4) replacing the cord behind the inferior epigastric vessels.The average lengthening of the cord achieved was 5.54 cm.Retroperitoneal mobilization alone could make the cord to get an extra length by 2.02 cm.72 testes were available for follow-up study,with all the testes descended into the scrotum,89% in a satisfactory position.

目的 研究环境雌激素(EEs)的经典代表己烯雌酚(DES)对雄性胎鼠睾丸引带肌动蛋白(ACTIN)和增殖细胞核抗原(PCNA)表达的影响.方法怀孕雌性昆明小鼠60只,随机分成6组,实验组于孕9～17 d分别给予DES 0、25、50、100、200 μg·kg-1·d-1,二甲基亚砜(DMSO)作为溶剂,对照组给予等体积NS.孕17 d处死母鼠取出胎鼠,取下腹部,常规固定、制片,应用免疫组织化学方法检测睾丸引带ACTIN和PCNA的表达.结果实验组雄性胎鼠睾丸引带发育较差,ACTIN、PCNA表达明显降低(P＜0.05),均有显著的剂量效应关系.结论 DES 降低雄性胎鼠睾丸引带ACTIN和PCNA的表达,从而影响睾丸引带的收缩及增殖活性,这可能是EEs影响睾丸引带发育及导致隐睾的机制。

目的 研究外源性雌激素(EEs)对胚胎小鼠睾丸引带组织肌动蛋白(Actin)和增殖细胞核抗原(PCNA)的表达以探讨EEs对睾丸引带收缩和增殖活性的影响.方法昆明雌性小鼠180只,随机分成18组,于孕9～17 d每天分别给予DES、17α-E2、17β-E2和E3,每种药物又分别给予不同剂量25、50、100、200μg·kg-1·d-1和等体积的DMSO、生理盐水作空白和正常对照.孕19 d处死母鼠,取出活胎,取其下腹部,常规固定、制片.应用免疫组织化学方法检测睾丸引带组织中肌动蛋白(Actin)和增殖细胞核抗原(PCNA)的表达.结果实验组小鼠睾丸引带组织发育较差,其中Actin含量降低,药物剂量越大,Actin含量越低(P＜0.05);较大剂量(如200和100μg·kg-1·d-1药物组)的雌激素处理组胎鼠睾丸引带中PCNA含量降低明显(P＜0.05).两指标均有显著的剂量效应关系.结论EEs可随药物剂量的不同而不同程度地降低睾丸引带中Actin和PCNA的含量.表明EEs影响胚胎期睾丸引带的收缩和增殖活性,并可能是EEs影响睾丸引带甚或睾丸正常发育(下降)的机制。

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

Estradiol represses Insulin-like 3 expression and promoter activity in MA-10 Leydig cells

The insulin-like 3 (INSL3) hormone produced by Leydig cells is essential for proper male sex differentiation, but the regulation of Insl3 expression remains poorly understood. This study describes a new physical and functional cooperation between the nuclear receptors SF1 and COUP-TFII in Insl3 expression. INSL3, a hormone abundantly produced by Leydig cells, is essential for testis descent during fetal life and bone metabolism in adults. The mechanisms regulating Insl3 expression in Leydig cells have been studied in several species but remain poorly understood. To date, only a handful of transcription factors are known to activate the Insl3 promoter and include the nuclear receptors AR, NUR77, COUP-TFII, and SF1, as well as the Krüppel-like factor KLF6. Some of these transcription factors are known to transcriptionally cooperate on the Insl3 promoter, but the mechanisms at play remain unknown. Here, we report that COUP-TFII and SF1 functionally cooperate on the Insl3 promoter from various species but not on the Inha, Akr1c14, Cyp17a1, Hsd3b1, Star, Gsta3, and Amhr2 promoters that are known to be regulated by COUP-TFII and/or SF1. The Insl3 promoter contains species-conserved binding sites for COUP-TFII (-91 bp) and SF1 (-134 bp). Mutation of either the COUP-TFII or the SF1 sequence had no impact on the COUP-TFII/SF1 cooperation, but the mutation of both binding sites abolished the cooperation. In agreement with this, we found that COUP-TFII and SF1 physically interact in Leydig cells. Finally, we report that the transcriptional cooperation is not limited to COUP-TFII and SF1 as it also occurred between all NR2F and NR5A family members. Our data provide new mechanistic insights into the cooperation between the orphan nuclear receptors COUP-TFII and SF1 in the regulation of Insl3 gene expression in Leydig cells.

Men are men because of testosterone, and the effects of this hormone are evident in virtually every organ or tissue in a normal adult man. Because testosterone is a classical endocrine hormone with effects throughout the body, many endocrinologists will have failed to realize that, in terms of its effects on fertility, the primary role of testosterone is not endocrine but paracrine, as it is the local action of testosterone on spermatogenesis which determines fertility. When looked at from this perspective it is logical to question the traditional endocrine teaching which states that testosterone secretion is controlled solely by pituitary luteinizing hormone (LH). Certainly, the levels of testosterone and LH in peripheral blood show a close relationship, but it is not the level of testosterone in peripheral blood which determines fertility. If the primary role of testosterone is within the testis, then it is not unreasonable to expect that local mechanisms may exist to regulate the intratesticular levels of this steroid. The object of this review is to examinecritically the evidence for such mechanisms, their possible nature and their physiological and clinical significance (if any). Before reviewing the data on intratesticular control of steroidogenesis it is essential to place it in perspective. To do this, three principal questions need to be answered: (I) Why might intratesticular mechanisms for local control of testosterone production be required? (2) What is the role (or roles) of testosterone within the testis? A proper understanding of how testosterone controls spermatogenesis is essential if the local regulation of testosterone production is to be evaluated from a physiological perspective. (3) What are the constraints or problems relating to studies on the intratesticular control of steroidogenesis? It is essential to understand the potential limitations of studies on this subject so that a reasoned interpretation of the results can be made. Most relevant data is derived from studies in uitro, and the relationship of such data to the situation in uivo is often difficult to establish.

Testicular Leydig cells (LC) regulate the proper development of male individuals, both during fetal life (fetal LC) and puberty (adult LC). In the ovaries of adult women, there are cells that are very similar to Leydig cells, the ovarian hilus cells (OHC), which also produce testosterone. The origin of these cells, in both sexes, remains unknown and is still a matter of debate. We have studied the location, characteristics and relationships of the OHC in 90 patients. The indications for oophorectomy were: metrorrhagia (n=9), prolapse (n=8), endometrial hyperplasia (n=14), cancer (endometrial, myometrial, or cervical) (n=35), uterine leiomyomata (n=14), and various ovarian tumors (cysts and benign tumors, borderline and malignant) (n=10). In addition to the hilus, occasionally the nodules, nests and clusters of OHC were located in the mesovarium, the mesosalpinx, and in the medullar and cortical regions of the ovaries. The morphological (including crystalloids of Reinke) and immunohistochemical (positivity for calretinin and alpha-inhibin) findings were similar to those described for testicular LC. Therefore, OHC can be considered ovarian Leydig cells (OLC). LC are usually found in small numbers in the ovaries, but if one looks for them intentionally, one always finds them. Close relationships were observed between the OLC with nerves and vessels. Moreover, an intraneural location of the OLC was demonstrated in all cases, and these intraneural cells showed similar characteristics to extraneural OLC, suggesting that they derive from endoneural cells which are present in the vegetative nerves of the ovaries.