Fetal Leydig cells: What we know and what we don't.

During male fetal development, testosterone plays an essential role in the differentiation and maturation of the male reproductive system. Deficient fetal testosterone production can result in variations of sex differentiation that may cause infertility and even increased tumor incidence later in life. Fetal Leydig cells in the fetal testis are the major androgen source in mammals. Although fetal and adult Leydig cells are similar in their functions, they are two distinct cell types, and therefore, the knowledge of adult Leydig cells cannot be directly applied to understanding fetal Leydig cells. This review summarizes our current knowledge of fetal Leydig cells regarding their cell biology, developmental biology, and androgen production regulation in rodents and human. Fetal Leydig cells are present in basement membrane-enclosed clusters in between testis cords. They originate from the mesonephros mesenchyme and the coelomic epithelium and start to differentiate upon receiving a Desert Hedgehog signal from Sertoli cells or being released from a NOTCH signal from endothelial cells. Mature fetal Leydig cells produce androgens. Human fetal Leydig cell steroidogenesis is LHCGR (Luteinizing Hormone Chronic Gonadotropin Receptor) dependent, while rodents are not, although other Gαs -protein coupled receptors might be involved in rodent steroidogenesis regulation. Fetal steroidogenesis ceases after sex differentiation is completed, and some fetal Leydig cells dedifferentiate to serve as stem cells for adult testicular cell types. Significant gaps are acknowledged: (1) Why are adult and fetal Leydig cells different? (2) What are bona fide progenitor and fetal Leydig cell markers? (3) Which signaling pathways and transcription factors regulate fetal Leydig cell steroidogenesis? It is critical to discover answers to these questions so that we can understand vulnerable targets in fetal Leydig cells and the mechanisms for androgen production that when disrupted, leads to variations in sex differentiation that range from subtle to complete sex reversal.

GLI3 resides at the intersection of hedgehog and androgen action to promote male sex differentiation.

Urogenital tract abnormalities are among the most common congenital defects in humans. Male urogenital development requires Hedgehog-GLI signaling and testicular hormones, but how these pathways interact is unclear. We found that Gli3XtJ mutant mice exhibit cryptorchidism and hypospadias due to local effects of GLI3 loss and systemic effects of testicular hormone deficiency. Fetal Leydig cells, the sole source of these hormones in developing testis, were reduced in numbers in Gli3XtJ testes, and their functional identity diminished over time. Androgen supplementation partially rescued testicular descent but not hypospadias in Gli3XtJ mutants, decoupling local effects of GLI3 loss from systemic effects of androgen insufficiency. Reintroduction of GLI3 activator (GLI3A) into Gli3XtJ testes restored expression of Hedgehog pathway and steroidogenic genes. Together, our results show a novel function for the activated form of GLI3 that translates Hedgehog signals to reinforce fetal Leydig cell identity and stimulate timely INSL3 and testosterone synthesis in the developing testis. In turn, exquisite timing and concentrations of testosterone are required to work alongside local GLI3 activity to control development of a functionally integrated male urogenital tract.

Injection molded open microfluidic well plate inserts for user-friendly coculture and microscopy.

Open microfluidic cell culture systems are powerful tools for interrogating biological mechanisms. We have previously presented a microscale cell culture system, based on spontaneous capillary flow of biocompatible hydrogels, that is integrated into a standard cell culture well plate, with flexible cell compartment geometries and easy pipet access. Here, we present two new injection molded open microfluidic devices that also easily insert into standard cell culture well plates and standard culture workflows, allowing seamless adoption by biomedical researchers. These platforms allow culture and study of soluble factor communication among multiple cell types, and the microscale dimensions are well-suited for rare primary cells. Unique advances include optimized evaporation control within the well, manufacture with reproducible and cost-effective rapid injection molding, and compatibility with sample preparation workflows for high resolution microscopy (following well-established coverslip mounting procedures). In this work, we present several use cases that highlight the usability and widespread utility of our platform including culture of limited primary testis cells from surgical patients, microscopy readouts including immunocytochemistry and single molecule fluorescence in situ hybridization (smFISH), and coculture to study interactions between adipocytes and prostate cancer cells.

Steroidogenic factor 1 promotes aggressive growth of castration-resistant prostate cancer cells by stimulating steroid synthesis and cell proliferation.

The dependence of prostate cancer on androgens provides a targeted means of treating advanced disease. Unfortunately, androgen deprivation therapies eventually become ineffective, leading to deadly castration-resistant prostate cancer (CRPC). One of many factors implicated in the transition to CRPC is the onset of de novo steroidogenesis. Although reactivation of steroid receptors likely plays a pivotal role in aggressive CRPC, little is understood regarding the mechanisms whereby prostate cancer cells initiate and maintain steroidogenesis. We hypothesize that steroidogenic factor 1 (SF1, NR5A1, AD4BP), a key regulator of steroidogenesis in normal endocrine tissues, is expressed in CRPC where it stimulates aberrant steroidogenesis and fuels aggressive growth. Notably, SF1 is not expressed in normal prostate tissue. Our results indicated that SF1 was absent in benign cells but present in aggressive prostate cancer cell lines. Introduction of ectopic SF1 expression in benign human prostate epithelial cells (BPH-1) stimulated increased steroidogenic enzyme expression, steroid synthesis, and cell proliferation. In contrast, data from an aggressive human prostate cancer cell line (BCaPT10) demonstrated that SF1 was required for steroid-mediated cell growth because BCaPT10 cell growth was diminished by abiraterone treatment and short hairpin RNA-mediated knockdown of SF1 (shSF1). SF1-depleted cells also exhibited defective centrosome homeostasis. Finally, whereas xenograft experiments in castrated hosts with BCaPT10 control transplants grew large, invasive tumors, BCaPT10-shSF1 knockdown transplants failed to grow. Therefore, we conclude that SF1 stimulates steroid accumulation and controls centrosome homeostasis to mediate aggressive prostate cancer cell growth within a castrate environment. These findings present a new molecular mechanism and therapeutic target for deadly CRPC.

The fused toes locus is essential for somatic-germ cell interactions that foster germ cell maturation in developing gonads in mice.

Ovarian development absolutely depends on communication between somatic and germ cell components. In contrast, it is not until after birth that interactions between somatic and germ cells play an important role in testicular maturation and spermatogenesis. Previously, we discovered that Irx3 expression was localized specifically to female gonads during embryonic development; therefore, we sought to determine the function of this genetic locus in developing gonads of both sexes. The fused toes (Ft) mutant mouse is missing 1.6 Mb of chromosome 8, which includes the entire IrxB cluster (Irx3, Irx5, Irx6), Ftm, Fts, and Fto genes. Homozygote Ft mutant embryos die around embryonic day 13.5 (E13.5); therefore, to assess later development, we harvested gonads at E11.5 and transplanted them into nude mouse hosts. Our results show defects in somatic and germ cell maturation in developing gonads of both sexes. Testis development was normal initially; however, by 3-wk posttransplantation, expression of Sertoli and peritubular myoid cell markers were decreased. In many cases, gonocytes failed to migrate to structurally impaired basement membranes of seminiferous cords. Developmental abnormalities of the ovary appeared earlier and were more severe. Over time, the Ft mutant ovary formed very few primordial or primary follicles, which contained oocytes that failed to grow and were surrounded by scarce granulosa cells that expressed low levels of FOXL2. By 3 wk after transplantation, it was difficult to identify ovarian tissue in Ft mutant ovary transplants. In summary, we conclude that the Ft locus contains genes essential for somatic-germ cell interactions, without which the germ cell niche fails to mature in both sexes.

Irx3 is differentially up-regulated in female gonads during sex determination.

Irx3 is a member of the Iroquois homeobox gene family that encodes a protein known for its essential role in spinal cord development. Transcript screening of male and female gonads during the critical period of sex determination (E12-13.5) revealed a sexually dimorphic expression pattern for Irx3 with female gonads exhibiting a sixfold increase in expression over time. Whole mount in situ hybridization confirmed the sexually dimorphic nature of Irx3 expression and immunohistochemical analysis of gonads at E13.5 determined that IRX3 and GATA4 proteins co-localized to somatic cells of XX gonads. The Irx3 signal persisted in germ cell-depleted XX gonads resulting from Busulfan treatment suggesting that its expression was independent of germ cell regulation. Quantitative real-time PCR analysis over an extended time course determined that Irx3 message was low initially and then increased in XX gonads until E13.5, remained elevated until birth, diminished shortly after birth, and remained low in the adult ovary. In contrast, Irx3 message was 50% lower in male compared to female gonads at the initial time point, and continued to decrease over time. Further analysis of adult ovaries suggested that IRX3 expression is not present in any subpopulations of cells of the differentiated ovary. Together, these results suggest that the Irx3 signal is restricted to the somatic cell component of XX gonads and is present at a discreet period of ovarian development that ends abruptly at birth. This timing coincides with the transition of female primordial germ cells from mitotic proliferation to meiotic division, and the organization of germ cell cysts prior to primordial follicle development at birth.

The androgen receptor represses transforming growth factor-beta signaling through interaction with Smad3.

In the prostate, androgens negatively regulate the expression of transforming growth factor-beta (TGF-beta) ligands and receptors and Smad activation through unknown mechanisms. We show that androgens (dihydrotestosterone and R1881) down-regulate TGF-beta1-induced expression of TGF-beta1, c-Fos, and Egr-1 in the human prostate adenocarcinoma cell line, LNCaP. Moreover, 5alpha-dihydrotestosterone (DHT) inhibits TGF-beta1 activation of three TGF-beta1-responsive promoter constructs, 3TP-luciferase, AP-1-luciferase, and SBE4(BV)-luciferase, in LNCaP cells either with or without enforced expression of TGF-beta receptors (TbetaRI and TbetaRII). Similarly, DHT inhibits the activation of Smad-binding element (SBE)4(BV)-luciferase by either constitutively activated TbetaRI (T204D) or constitutively activated Smad3 (S3*). Activation of SBE4(BV)-luciferase by S3* in the NRP-154 prostatic cell line, which is androgen receptor (AR)-negative but highly responsive to TGF-beta1, is blocked by co-transfection with either full-length AR or AR missing the DNA binding domain. Immunoprecipitation and GST pull-down assays show that AR directly associates with Smad3 but not Smad2 or Smad4. Electrophoretic mobility shift assays indicate that the AR ligand binding domain directly inhibits the association of Smad3 to the Smad-binding element. In conclusion, our data demonstrate for the first time that ligand-bound AR inhibits TGF-beta transcriptional responses through selectively repressing the binding of Smad3 to SBE.