Gonadotropin Regulation of Retinoic Acid Activity in the Testis.

Initiation of spermatogenesis in primates is triggered at puberty by an increase in gonadotropins; i.e., follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Prior to puberty, testis of the monkey contains only undifferentiated germ cells. However, sermatogonial differentiation and spermatogenesis may be initiated prior to puberty after stimulation with exogenous LH and FSH. Retinoic acid (RA) signaling is considered to be a major component that drives spermatogonial differentiation. We were interested in evaluating the relative role of LH and FSH, either alone or in combination, in regulating the retinoic acid signaling in monkey testis. Sixteen juvenile male rhesus monkeys (Macaca mulatta) were infused with intermittent recombinant single chain human LH (schLH) or recombinant human FSH (rhFSH) or a combination of both for 11 days. We then analyzed the expression of the several putative RA signaling pathway related genes; i.e. RDH10, RDH11, ALDH1A1, ALDH1A2, CYP26B1, CRABP1, CRABP2, STRA6, STRA8 in the testis after 11 days of stimulation with vehicle, LH, FSH and combination LH/FSH using quantitative real-time PCR (qPCR). The qPCR results analysis showed that administration of gonadotropins affected a significant change in expression of some RA signaling related genes in the monkey testis. The gonadotropins, either alone or in combination dramatically increased expression of CRABP2 (p≤0.001), whereas there was a decrease in ALDH1A2 expression (p≤0.001). Moreover, combined gonadotropin treatment led to the significant decrease in CRABP1 expression (p≤0.05). These findings are the first evidence that the activity of retinoic acid signaling in the monkey testis is regulated through gonadotropins (LH/FSH) levels.

The testicular transcriptome associated with spermatogonia differentiation initiated by gonadotrophin stimulation in the juvenile rhesus monkey (Macaca mulatta).

STUDY QUESTION: What is the genetic landscape within the testis of the juvenile rhesus monkey (Macaca mulatta) that underlies the decision of undifferentiated spermatogonia to commit to a pathway of differentiation when puberty is induced prematurely by exogenous LH and FSH stimulation? SUMMARY ANSWER: Forty-eight hours of gonadotrophin stimulation of the juvenile monkey testis resulted in the appearance of differentiating B spermatogonia and the emergence of 1362 up-regulated and 225 down-regulated testicular mRNAs encoding a complex network of proteins ranging from enzymes regulating Leydig cell steroidogenesis to membrane receptors, and from juxtacrine and paracrine factors to transcriptional factors governing spermatogonial stem cell fate. WHAT IS KNOWN ALREADY: Our understanding of the cell and molecular biology underlying the fate of undifferentiated spermatogonia is based largely on studies of rodents, particularly of mice, but in the case of primates very little is known. The present study represents the first attempt to comprehensively address this question in a highly evolved primate. STUDY DESIGN, SIZE, DURATION: Global gene expression in the testis from juvenile rhesus monkeys that had been stimulated with recombinant monkey LH and FSH for 48 h (N = 3) or 96 h (N = 4) was compared to that from vehicle treated animals (N = 3). Testicular cell types and testosterone secretion were also monitored. PARTICIPANTS/MATERIALS, SETTING, METHODS: Precocious testicular puberty was initiated in juvenile rhesus monkeys, 14-24 months of age, using a physiologic mode of intermittent stimulation with i.v. recombinant monkey LH and FSH that within 48 h produced 'adult' levels of circulating LH, FSH and testosterone. Mitotic activity was monitored by immunohistochemical assays of 5-bromo-2'-deoxyuridine and 5-ethynyl-2'-deoxyuridine incorporation. Animals were bilaterally castrated and RNA was extracted from the right testis. Global gene expression was determined using RNA-Seq. Differentially expressed genes (DEGs) were identified and evaluated by pathway analysis. mRNAs of particular interest were also quantitated using quantitative RT-PCR. Fractions of the left testis were used for histochemistry or immunoflouresence. MAIN RESULTS AND THE ROLE OF CHANCE: Differentiating type B spematogonia were observed after both 48 and 96 h of gonadotrophin stimulation. Pathway analysis identified five super categories of over-represented DEGs. Repression of GFRA1 (glial cell line-derived neurotrophic factor family receptor alpha 1) and NANOS2 (nanos C2HC-type zinc finger 2) that favor spermatogonial stem cell renewal was noted after 48 and 96 h of LH and FSH stimulation. Additionally, changes in expression of numerous genes involved in regulating the Notch pathway, cell adhesion, structural plasticity and modulating the immune system were observed. Induction of genes associated with the differentiation of spermatogonia stem cells (SOHLH1(spermatogenesis- and oogenesis-specific basic helix-loop-helix 1), SOHLH2 and KIT (V-Kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog)) was not observed. Expression of the gene encoding STRA8 (stimulated by retinoic acid 8), a protein generally considered to mark activation of retinoic acid signaling, was below our limit of detection. LARGE SCALE DATA: The entire mRNA data set for vehicle and gonadotrophin treated animals (N = 10) has been deposited in the GEO-NCBI repository (GSE97786). LIMITATIONS REASONS FOR CAUTION: The limited number of monkeys per group and the dilution of low abundance germ cell transcripts by mRNAs contributed from somatic cells likely resulted in an underestimation of the number of differentially expressed germ cell genes. WIDER IMPLICATIONS OF THE FINDINGS: The findings that expression of GDNF (a major promoter of spermatogonial stem cell renewal) was not detected in the control juvenile testes, expression of SOHLH1, SOHLH2 and KIT, promoters of spermatogonial differentiation in mice, were not up-regulated in association with the gonadotrophin-induced generation of differentiating spermatogonia, and that robust activation of the retinoic acid signaling pathway was not observed, could not have been predicted. These unexpected results underline the importance of non-human primate models in translating data derived from animal research to the human situation. STUDY FUNDING/COMPETING INTEREST(S): The work described was funded by NIH grant R01 HD072189 to T.M.P. P.A. was supported by an Endocrine Society Summer Research Fellowship Award and CONICET (Argentine Research Council), S.N. by a grant from Vali-e-Asr Reproductive Health Research Center of Tehran University of Medical Sciences (grant #24335-39-92) to Dr Batool Hosseini Rashidi, and M.P.H. by grants from the National Health and Medical Research Council of Australia, and the Victorian State Government's Operational Infrastructure Support Program. The authors have nothing to disclose.

Potential roles of gonadotropins to control pulsatile retinoic acid signaling during spermatogenesis.

Spermatogenesis is a highly regulated process that takes place in the seminiferous tubules of testis. This process initiates at puberty with differentiation of spermatogonia and their meiotic entry. The initiation of spermatogenesis depends on gonadotropins secreted by the pituitary gland; i.e., follicle-stimulating hormone (FSH) and luteinizing hormone (LH). In the absence of FSH and LH only premeiotic germ cells are present in the testis. The prepubertal development phase in juvenile testis is characterized by a protracted hypogonadotropic state, which only consists of Sertoli and undifferentiated germ cells in the seminiferous epithelium. All germ cells in the juvenile testis are undifferentiated spermatogonia, which are proliferating in a relatively gonadotropin-independent manner. It has been revealed that vitamin A deficient (VAD) animals are also infertile, and only premeiotic germ cells (undifferentiated spermatogonia) are present in their seminiferous tubules. The developmental block in VAD animal can be removed by administration of retinol and germ cell differentiation reinitiates in a synchronous manner. Recent studies have revealed that the biologically active form of vitamin A, retinoic acid (RA), regulates germ cell differentiation and lead to the generation of the cycle of the seminiferous epithelium and normal spermatogenic wave. Recent study has shown that synchronous spermatogenesis at neonatal mouse, but not after initiation of meiosis, can be induced by treating vitamin A sufficient males with RA. The treatment of neonatal males at different ages with exogenous RA has revealed that although RA is sufficient to induce differentiation of spermatogonial at 4 dpp and earlier, it fails to alter asynchrony and it does not irreversibly cause a spermatogonial differentiation. These observations led us to suggest that gonadotropins trigger differentiation of spermatogonia and spermatogenesis through regulation of RA signaling in the seminiferous epithelium of the adult testis.

Elevated Expression of the Testis-specific Gene WBP2NL in Breast Cancer.

Breast cancer is one of the most common causes of cancer death in women; therefore, the study of molecular aspects of breast cancer for finding new biomarkers is important. Recent studies have shown that WW domain-binding protein 2 (WBP2) is important for the oncogenic property of breast cancer. WWP2 N-terminal-like (WBP2NL) is a testis-specific signaling protein that induces meiotic resumption and oocyte activation events. Our previous study revealed that WBP2NL gene expression is elevated in actively dividing cells and it might be associated with cellular proliferation and tumorigenic process. However, the clinical relevance and importance of WBP2NL gene in cancer has not been understood yet. Therefore, we were interested in analyzing the expression of WBP2NL gene in human breast cancer tissues and breast cancer cell lines, for the first time. We used reverse transcription-polymerase chain reaction (RT-PCR) and semi-nested RT-PCR to evaluate the expression of WBP2NL in malignant breast cancer and adjacent noncancerous tissue (ANCT) samples, as well as MCF-7 and MDA-MB-231 cell lines. The WBP2NL gene was expressed in 45 out of 50 (90%) breast cancer tissues and overexpressed in the MDA-MB-231 cell line. We suggest that WBP2NL may play roles in breast cancer activation maybe through binding to a group I WW domain protein. The elevated expression of WBP2NL gene in breast cancer and MDA-MB-231 cell line leads us to suggest that WBP2NL might be considered as a novel prognostic factor for early diagnosis of breast cancer.

Application of three-dimensional culture systems to study mammalian spermatogenesis, with an emphasis on the rhesus monkey (Macaca mulatta).

In vitro culture of spermatogonial stem cells (SSCs) has generally been performed using two-dimensional (2D) culture systems; however, such cultures have not led to the development of complete spermatogenesis. It seems that 2D systems do not replicate optimal conditions of the seminiferous tubules (including those generated by the SSC niche) and necessary for spermatogenesis. Recently, one of our laboratories has been able to induce proliferation and differentiation of mouse testicular germ cells to meiotic and postmeiotic stages including generation of sperm in a 3D soft agar culture system (SACS) and a 3D methylcellulose culture system (MCS). It was suggested that SACS and MCS form a special 3D microenvironment that mimics germ cell niche formation in the seminiferous tubules, and thus permits mouse spermatogenesis in vitro. In this review, we (1) provide a brief overview of the differences in spermatogenesis in rodents and primates, (2) summarize data related to attempts to generate sperm in vitro, (3) report for the first time formation of colonies/clusters of cells and differentiation of meiotic (expression of CREM-1) and postmeiotic (expression of acrosin) germ cells from undifferentiated spermatogonia isolated from the testis of prepubertal rhesus monkeys and cultured in SACS and MCS, and (4) indicate research needed to optimize 3D systems for in vitro primate spermatogenesis and for possible future application to man.

The Evaluation of WBP2NL-Related Genes Expression in Breast Cancer.

Breast cancer is the most frequent cause of mortality in women all around the world; therefore, study on molecular aspects of breast cancer is necessary for finding new biomarkers. Recent studies have shown that WW Binding Protein 2 (WBP2) is an important protein for the oncogenic property of cancer. We have previously evaluated the WW Binding Protein 2 N-Terminal Like (WBP2NL) gene expression in cancerous cell line and breast tumor tissues, and reported changes in expression, which could increase tumorigenic cell growth. However, the molecular mechanisms of WBP2NL and its clinical relevance have not been investigated. In this study, the expression of WBP2NL-related genes in the invasive breast carcinoma and normal breast tissues was evaluated for the first time. Analysis of WBP2NL-related genes expression was performed with reverse transcription-PCR and real time-PCR detection method. The target genes studied were as follow: WW domain containing E3 ubiquitin protein ligase 1(WWP1), membrane associated guanylatekinase containing WW and PDZ domain-1 (MAGI1), neural precursor cell expressed developmentally down-regulated 4 (NEDD4), formin binding protein-4 (FNBP4), BCL2-associated athanogene-3 (BAG3), WW domain-containing oxidoreductase (WWOX), yes-associated protein-1 (YAP1), WW domain containing transcription regulator (WWTR1), member RAS oncogene family (RAB2A), and small G protein signaling modulator 3 (SGSM3). The expression of WWP1, BAG3, and WWTR1 was significantly increased in breast cancer. In contrast, the expression of WWOX, YAP1, RAB2A, and SGSM3 was significantly decreased. The MAGI1 and NEDD4 expression was increased, while the expression of FNBP4 was unchanged. These findings lead us to suggest that WBP2NL might play roles as an anti-apoptotic factor or co-activator to promote breast cancer cell survival and proliferation.

A vector-based system for the differentiation of mouse embryonic stem cells toward germ-line cells.

OBJECTIVES: To culture the in vitro mouse embryonic stem cells (mESCs) and to direct their differentiation to germ-line cells; in present study we used a vector backbone containing the fusion construct Stra8-EGFP to select differentiated ES cells that entered meiosis. Retinoic acid was used to differentiate embryonic stem cells to germ cells. MATERIALS AND METHODS: A fragment of Stra8 gene promoter (-1400 to +7) was inserted in ScaI/HindIII multiple cloning site of pEGFP-1 vector. The electroporation was done on embryonic stem cells and positive colonies were selected as puromycin-resistant after three weeks of treatment with puromycin. All-trans retinoic acid (RA) was used for differentiation of mESCs at final concentration of 10(-)5M. The expression of protamine 1 (Prm1) gene was checked as post meiotic marker in differentiated mESCs after 5, 10, 15, 21 and 30 days after RA induction. RESULTS: The PCR amplification by specific primers for Stra8-EGFP fusion gene was detected in DNA sample from mESCs after electroporation and puromycin treatment. GFP-positive mESC colonies were observed after 72 hr RA induction. The protamine 1 gene was expressed after 21 days of RA induction. CONCLUSION: In this study, we demonstrated the in vitro generation of mouse embryonic stem cells to germ cells by using a backbone vector containing the fusion gene Stra8-EGFP. The Stra8 gene is a retinoic acid-responsive protein and is able to regulate meiotic initiation.

Expression analysis of PAWP during mouse embryonic stem cell-based spermatogenesis in vitro.

Postacrosomal sheath WW domain-binding protein (PAWP) is a novel sperm protein identified as a candidate sperm-borne, oocyte-activating factor (SOAF). However, regulation of PAWP gene expression is poorly understood. Therefore, we examined the PAWP gene expression across different stages of mouse embryonic stem cell (ESC)-based spermatogenesis in vitro and compared this expression at different stages of mouse testis development in vivo. Expression of PAWP was also examined in mouse embryonic fibroblasts (MEF), Sertoli cell, and the NIH3T3 cancerous cell line. We used a transgenic mouse ESC line C57BL/6J expressing Stra8-EGFP that was plated in murine ESC medium. To induce differentiation, cells were cultured on gelatin-coated medium with Retinoic Acid (RA) treatment. We applied reverse transcription-PCR and real-time PCR to analyze the differential expression of PAWP mRNA during different stages of mouse ESC differentiation in vitro parallel with mouse testis development in vivo and in cell lines. We found that expression of PAWP is increased during testis development in vivo with greatest expression at postmeiotic phase. It is also highly expressed in mouse ESC-derived germ-like cells after 30 d of RA induction in vitro. PAWP is remarkably expressed in mouse ESC and NIH3T3 cell line. These results indicate that PAWP plays a role in spermatogenesis and germ cell development. Moreover, we suggest PAWP as one of the markers that could be looked in ESC studies as a confirmed testis-specific gene. We also suggest an additional possible role for PAWP in proliferation of cancerous cell in general.

Identification of Spata-19 new variant with expression beyond meiotic phase of mouse testis development.

BACKGROUND: The study of specific genes expressed in the testis is important to understanding testis development and function. Spermatogenesis is an attractive model for the study of gene expression during germ cell differentiation. Spermatogenesis associated-19 (Spata-19) is a recently-identified important spermatogenesis-related gene specifically expressed in testis. Its protein product is involved in sperm cell development and reproduction. In this report we examined the expression of Spata-19 mRNA in mouse testis, fetus, and cell lines. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR), nested PCR, and PCR-restriction fragment length polymorphism (PCR-RFLP) were used to analyze Spata-19 mRNA expression in different stages of mouse testis development, mouse fetus, mouse embryonic fibroblasts (MEF), mouse embryonic stem cells (mESC), Sertoli cells, and NIH/3T3 cells. RESULTS: We identified a novel splice variant of Spata-19 in the mouse genome that it is expressed in the fetus and after the meiotic phase of spermatogenesis, and over-expressed in the post-meiotic stage of mouse spermatogenesis. This novel splice variant was absent in five days old mice testis, mESC, MEF, Sertoli, and NIH/3T3 cell lines. CONCLUSION: The Spata-19 has a large novel splice variant in mouse testis that is expressed beyond meiotic phase of testis development. We suggest that this new Spata-19 mRNA variant might be involved in mitochondrial maintenance in sperm cells, and might be correlated with androgen secretion and male fertility.