Testicular descent occurs in two consecutive stages: the transabdominal stage and the inguinoscrotal stage. Androgens play a crucial role in the second stage by influencing the development of the gubernaculum, a structure that pulls the testis into the scrotum. However, the mechanisms of androgen actions underlying many of the processes associated with gubernaculum development have not been fully elucidated. To identify the androgen-regulated genes, we conducted large-scale gene expression analyses on the gubernaculum harvested from luteinizing hormone/choriogonadotropin receptor knockout ( Lhcgr KO) mice, an animal model of inguinoscrotal testis maldescent resulting from androgen deficiency. We found that the expression of secreted protein acidic and rich in cysteine (SPARC)-related modular calcium binding 1 ( Smoc1 ) was the most severely suppressed at both the transcript and protein levels, while its expression was the most dramatically induced by testosterone administration in the gubernacula of Lhcgr KO mice. The upregulation of Smoc1 expression by testosterone was curtailed by the addition of an androgen receptor antagonist, flutamide. In addition, in vitro studies demonstrated that SMOC1 modestly but significantly promoted the proliferation of gubernacular cells. In the cultures of myogenic differentiation medium, both testosterone and SMOC1 enhanced the expression of myogenic regulatory factors such as paired box 7 ( Pax7 ) and myogenic factor 5 ( Myf5 ). After short-interfering RNA-mediated knocking down of Smoc1 , the expression of Pax7 and Myf5 diminished, and testosterone alone did not recover, but additional SMOC1 did. These observations indicate that SMOC1 is pivotal in mediating androgen action to regulate gubernaculum development during inguinoscrotal testicular descent.
Animals ; Male ; Mice ; Testis/growth & development* ; Mice, Knockout ; Androgens/pharmacology* ; Testosterone/pharmacology* ; Receptors, LH/metabolism* ; Calcium-Binding Proteins/metabolism*

Animals ; Diethylhexyl Phthalate ; toxicity ; Female ; Ovary ; drug effects ; metabolism ; ultrastructure ; Rats, Sprague-Dawley ; Receptors, LH ; metabolism ; Receptors, LHRH ; metabolism ; Toxicity Tests

At present, there is no reliable in vitro assembled prepubertal testis-like biomimetic organ culture system designed to assess the functional effects of human gonadotropins on Sertoli and Leydig cells. Spermatogenesis is regulated by endocrine, paracrine, and juxtacrine factors (testicular cross-talk), mainly orchestrated by gonadotropins such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH) that play a pivotal role by stimulating Leydig and Sertoli cells, respectively. The aim of our study was to set up an in vitro prepubertal porcine bioengineered construct as a new model for experimental studies on reassembled Sertoli and Leydig cells. We have evaluated Sertoli and Leydig cells obtained from 15- to 20-day-old neonatal pig testes in terms of purity and function. Subsequently, purified Sertoli and enriched Leydig cells were subjected to coincubation to obtain an in vitro prepubertal porcine testis-like culture system. We performed enzyme-linked immunosorbent assay (ELISA) for anti-Müllerian hormone (AMH), inhibin B, and testosterone secretion in the medium, and Real-Time PCR analysis of AMH, inhibin B, FSH-r, aromatase, LHr, and 3β-HSD mRNA expression levels. This in vitro testis-like system was highly responsive to the effects of human gonadotropins and testosterone. AMH mRNA expression and secretion declined, and inhibin-B increased, while FSH-receptor expression was downregulated upon FSH/LH exposure/treatment. Finally, the production of testosterone was increased selectively upon LH treatment. In summary, our proposed model could help to better determine the action of human gonadotropins on Sertoli and Leydig cells. The potential usefulness of the system for shedding light into male infertility-related issues is evident.
3-Hydroxysteroid Dehydrogenases/metabolism* ; Animals ; Animals, Newborn ; Anti-Mullerian Hormone/metabolism* ; Aromatase/metabolism* ; Cell Culture Techniques ; Enzyme-Linked Immunosorbent Assay ; Follicle Stimulating Hormone/pharmacology* ; Hormones/pharmacology* ; In Vitro Techniques ; Inhibins/metabolism* ; Leydig Cells/metabolism* ; Luteinizing Hormone/pharmacology* ; Male ; Models, Biological ; Real-Time Polymerase Chain Reaction ; Receptors, FSH/metabolism* ; Receptors, LH/metabolism* ; Sertoli Cells/metabolism* ; Swine ; Testis/metabolism* ; Testosterone/metabolism*

The effect of high concentrations of testosterone on ovarian follicle development was investigated. Primary follicles and granulosa cells were cultured in vitro in media supplemented with a testosterone concentration gradient. The combined effects of testosterone and follicle-stimulating hormone (FSH) on follicular growth and granulosa cell gonadotropin receptor mRNA expression were also investigated. Follicle growth in the presence of high testosterone concentrations was promoted at early stages (days 1-7), but inhibited at later stage (days 7-14) of in vitro culture. Interestingly, testosterone-induced follicle development arrest was rescued by treatment with high concentrations of FSH (400 mIU/mL). In addition, in cultured granulosa cells, high testosterone concentrations induced cell proliferation, and increased the mRNA expression level of FSH receptor (FSHR), and luteinized hormone/choriogonadotropin receptor. It was concluded that high concentrations of testosterone inhibited follicle development, most likely through regulation of the FSH signaling pathway, although independently from FSHR downregulation. These findings are an important step in further understanding the pathogenesis of polycystic ovary syndrome.
Androgens ; pharmacology ; Animals ; Cell Proliferation ; drug effects ; Female ; Follicle Stimulating Hormone ; genetics ; metabolism ; pharmacology ; Gene Expression Regulation, Developmental ; Granulosa Cells ; cytology ; drug effects ; metabolism ; Mice ; Ovarian Follicle ; cytology ; drug effects ; growth & development ; metabolism ; Primary Cell Culture ; RNA, Messenger ; genetics ; metabolism ; Receptors, FSH ; genetics ; metabolism ; Receptors, Gonadotropin ; genetics ; metabolism ; Receptors, LH ; genetics ; metabolism ; Signal Transduction ; drug effects ; genetics ; Testosterone ; antagonists & inhibitors ; pharmacology

To investigate the possibility of in vivo transplantation of Leydig cells as a new biologic androgen replacement therapy, the Leydig cells procured from 6 week-old male Sprague-Dawley rats were autotransplanted, and the level of testosterone secretion and histostructural changes were observed. The renal subcapsular and intraperitoneal transplant showed higher levels of testosterone compared to subcutaneous or scrotal counterparts, and the number of transplanted cells was correlated with the level of measured testosterone. Furthermore, if the Leydig cells were transplanted intraperitoneally after the uptake on synthetic collagen, testosterone levels were higher than the ones simply transplanted without synthetic collagen uptake, resulting in 27 fold increase at 3 months. The activity of 125I-hCG decreased 20 to 40% at each month after transplantation compared to the normal levels, but no statistical significance was noted among different periods. The histologic examination revealed neovascularized capillaries and well demarcated sheet-like group of eosinophilic Leydig cells were observed at 4 weeks. But the evidence of destructive changes such as a focal inflammation with central dystropic ossification could be noted after 3 month. On electron microscopy, the marked indentation of nucleus and presence of lipochrome pigment were seen, and the number and size of smooth endoplasmic reticulum and mitochondria were reduced after 3 month. In conclusion, testosterone output could be increased to the physiologic range by increasing the number of transplant cells or utilizing collagen uptake but further effort is necessary on delaying or preventing the structural and functional decrement of Leydig cells.
Animal ; Cell Count ; Leydig Cells/cytology/metabolism/*transplantation ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, LH/metabolism ; Support, Non-U.S. Gov't ; Testosterone/*biosynthesis ; Transplantation, Autologous