Debra Mitchell

Expression of Stimulated by Retinoic Acid Gene 8 (Stra8) and Maturation of Murine Gonocytes and Spermatogonia Induced by Retinoic Acid In Vitro

Abstract Vitamin A deficiency in the mouse results in an arrest in the progression of undifferentiated spermatogonia to differentiating spermatogonia. The supplement of retinol to vitamin-A-deficient mice reinitiates spermatogenesis in a synchronous manner throughout the testes. It is unclear whether the effects of retinoids are the result of a direct action on germ cells or are indirectly mediated through Sertoli cells. The expression of Stimulated by retinoic acid gene 8 (Stra8), which is required for spermatogenesis, is directly related to the availability of retinoic acid (RA). Analysis of gene expression by microarrays revealed moderate levels of Stra8 transcript in gonocytes and high levels in A and B spermatogonia. Stra8 mRNA levels were greatly reduced or absent in germ cells once they entered meiosis. This study examined the effect of retinoic acid on cultured neonatal testes and isolated gonocytes/spermatogonia in vitro. THY1+ and KIT+ germ cells were isolated by magnetic-activated cell sorting from the testes of mice of different ages. Isolated germ cells were cultured and treated with either vehicle (ethanol) or RA without feeder cells. We found that 1) Stra8 is predominantly expressed in premeiotic germ cells, 2) RA stimulates gonocyte DNA replication and differentiation in cultured neonatal testes, 3) in the absence of feeder cells, RA directly induces the transition of undifferentiated spermatogonia to differentiating spermatogonia by stimulating Stra8 and Kit gene expression, 4) RA dramatically stimulates Stra8 expression in undifferentiated spermatogonia but has a lesser impact in differentiating spermatogonia, 5) endogenous Stra8 gene expression is higher in differentiating spermatogonia than in undifferentiated spermatogonia and could mediate the RA effects on spermatogonial maturation, and 6) RA stimulates a group of genes involved in the metabolism, storage, transport, and signaling of retinoids.

Expression of Stimulated by Retinoic Acid Gene 8 (Stra8) in Spermatogenic Cells Induced by Retinoic Acid: An In Vivo Study in Vitamin A-Sufficient Postnatal Murine Testes

Abstract Vitamin A is required for male fertility and normal spermatogenesis. Retinoic acid (RA), an active metabolite of vitamin A, is necessary for spermatogonial maturation and proper entry of germ cells into meiotic prophase in the postnatal testes. The expression of Stra8, which is essential for successful meiosis in both male and female gonads and normal spermatogenesis, is directly related to the availability of RA. This study examined the developmental expression pattern of Stra8 transcript in both male and female gonads, provided specific cellular localization of STRA8 protein in the postnatal and adult testis, and investigated RA actions in adult germ cells in a vitamin A-sufficient condition. The peak of Stra8 mRNA expression coincided with the onset of meiosis in postnatal testes. STRA8 protein was detected in gonocytes as early as 5 days postpartum. The expression of STRA8 protein in the neonatal testes was not uniform among spermatogonia, perhaps heralding the asynchronous beginning of spermatogenesis. In adult testes, the highest level of Stra8 mRNA and protein was found in seminiferous epithelial stages VI–VIII. STRA8 protein was localized to some type A and B spermatogonia, preleptotene spermatocytes, and early leptotene spermatocytes. In the vitamin A-sufficient adult testes, RA but not retinol acetate stimulated Stra8 mRNA expression. STRA8 protein expression in adult spermatogonia was induced by RA stimulation, suggesting its role in spermatogonial differentiation. Retinoic acid also increased the number of preleptotene spermatocytes exhibiting 5-bromo-2-deoxyuridine incorporation, indicating a more synchronized premeiotic DNA replication.

Two miRNA Clusters, Mir-17-92 (Mirc1) and Mir-106b-25 (Mirc3), Are Involved in the Regulation of Spermatogonial Differentiation in Mice

ABSTRACT Increasing evidence indicates that microRNAs (miRNAs) may be critical players in spermatogenesis. The miRNA expression profiles of THY1+-enriched undifferentiated spermatogonia were characterized, and members of Mir-17-92 (Mirc1) and its paralog Mir-106b-25 (Mirc3) clusters are significantly downregulated during retinoic acid-induced spermatogonial differentiation, both in vitro and in vivo. The repression of microRNA clusters Mir-17-92 (Mirc1) and Mir-106b-25 (Mirc3) by retinoic acid in turn potentially upregulates the expression of Bim, Kit, Socs3, and Stat3. The male germ cell-specific Mir-17-92 (Mirc1) knockout mice exhibit small testes, a lower number of epididymal sperm, and mild defect in spermatogenesis. Absence of Mir-17-92 (Mirc1) in male germ cells dramatically increases expression of Mir-106b-25 (Mirc3) cluster miRNAs in the germ cells. These results suggest that Mir-17-92 (Mirc1) cluster and Mir-106b-25 (Mirc3) cluster miRNAs possibly functionally cooperate in regulating spermatogonial development.

Expression of Mirlet7 Family MicroRNAs in Response to Retinoic Acid-Induced Spermatogonial Differentiation in Mice

Spermatogonial differentiation is orchestrated by the precise control of gene expression involving retinoic acid signaling. MicroRNAs have emerged as important regulators of spermatogenesis, and here we show that the Mirlet7 family miRNAs are expressed in mouse spermatogonia and spermatocytes. Retinoic acid significantly leads to the induction of Mirlet7 miRNAs through suppression of Lin28. We further confirmed both in vitro and in vivo that expressions of Mycn, Ccnd1, and Col1a2, which are targets of Mirlet7, were downregulated during spermatogonial differentiation. These results suggest that Mirlet7 family miRNAs play a role in retinoic acid-induced spermatogonial differentiation.

Turning a Spermatogenic Wave into a Tsunami: Synchronizing Murine Spermatogenesis Using WIN 18,446

ABSTRACT The BDADs (bis-[dichloroacetyl]-diamines) are compounds that can inhibit spermatogenesis via blocking the metabolism of vitamin A. We utilized one specific BDAD, WIN 18,446, to manipulate the endogenous production of retinoic acid (RA) in the testis to further investigate the action of this compound on mammalian sperm production. Transient treatment of adult male mice with WIN 18,446 blocked spermatogonial differentiation and induced significant changes in the cycle of the seminiferous epithelium. WIN 18,446 treatment of neonatal mice also blocked spermatogonial differentiation and, followed by injection of RA, induced synchronous spermatogenesis in adulthood. The net result was pulsatile, rather than normal continuous, release of sperm from the seminiferous epithelium. This study describes a novel technique that can enrich for specific germ cell populations within the testis, representing a valuable new tool for studying spermatogenesis.

Processive Pulses of Retinoic Acid Propel Asynchronous and Continuous Murine Sperm Production

ABSTRACT The asynchronous cyclic nature of spermatogenesis is essential for continual sperm production and is one of the hallmarks of mammalian male fertility. While various mRNA and protein localization studies have indirectly implicated changing retinoid levels along testis tubules, no quantitative evidence for these changes across the cycle of the seminiferous epithelium currently exists. This study utilized a unique mouse model of induced synchronous spermatogenesis, localization of the retinoid-signaling marker STRA8, and sensitive quantification of retinoic acid concentrations to determine whether there are fluctuations in retinoid levels at each of the individual stages of germ cell differentiation and maturation to sperm. These data show that processive pulses of retinoic acid are generated during spermatogonial differentiation and are the likely trigger for cyclic spermatogenesis and allow us, for the first time, to understand how the cycle of the seminiferous epithelium is generated and maintained. In addition, this study represents the first direct quantification of a retinoid gradient controlling cellular differentiation in a postnatal tissue.

Suppression of Stra8 Expression in the Mouse Gonad by WIN 18,446

Bis-(dichloroacetyl)-diamines (BDADs) are compounds that inhibit spermatogenesis and function as male contraceptives in many species; however, their mechanism of action has yet to be fully investigated. It has been proposed that BDADs may function via inhibition of testicular retinoic acid (RA) biosynthesis. We employed an organ culture technique and the expression of a marker for RA activity, Stra8 (stimulated by retinoic acid gene 8), to investigate if the BDAD WIN 18,446 inhibited the biosynthesis of RA from retinol (ROL) in neonatal and adult murine testis and in the embryonic murine gonad. After culturing either whole testes or germ cells isolated from mice at 2 days postpartum (dpp) with WIN 18,446 or with WIN 18,446 plus ROL, Stra8 expression was suppressed, demonstrating that WIN 18,446 inhibited the conversion of ROL to RA in both systems. We also utilized a transgenic mouse containing an RA-responsive LacZ reporter gene to demonstrate limited RA induction of LacZ expression in 2-dpp testes cultured with WIN 18,446 plus ROL. The expression of Stra8 was downregulated in adult mouse testis tubules cultured with WIN 18,446 when compared to tubules cultured with the vehicle control. WIN 18,446 also inhibited the conversion of ROL to RA in embryonic ovaries and testes cultured for 48 h. These murine results provide critical insights regarding how the BDADs can inhibit spermatogenesis by blocking the ability of vitamin A to drive germ cell development. In addition, these techniques will be useful for screening novel inhibitors of RA biosynthesis as potential male contraceptives.

Identification and Expression of Potential Regulators of the Mammalian Mitotic-to-Meiotic Transition

Meiosis is unique to germ cells and occurs in a sex-specific manner. The genes regulating meiotic initiation in either sex are yet to be fully elucidated. Recent studies have revealed the importance of retinoic acid and one of its target genes, Stra8, in meiotic initiation in both sexes. Microarray analysis of whole murine embryonic ovary and postnatal testis time course data revealed a single peak of Stra8 expression in each organ at the onset of meiosis; at Embryonic Day 14.5 in the ovary and 10 days postpartum in the testis. In order to identify other genes involved in the initiation of meiosis in mammals, murine testis and ovary microarray data were examined more closely for transcripts with expression profiles similar to Stra8. Three such candidates include establishment of cohesion 1 homolog 2 (Esco2), encoding a protein essential for sister chromatid cohesion; SET domain, bifurcated 2 (Setdb2), the mouse ortholog of Eggless, which is essential for oogenesis in Drosophila; and ubiquitin-activating enzyme 6 (Uba6), a gene with fivefold higher expression in human and mouse testes than any other organ. In situ hybridization and immunohistochemistry or immunofluorescence were performed to localize Esco2, Setbd2, and Uba6 expression in the developing testis. The cellular expression pattern localized all three of these transcripts and their respective proteins to germ cells transitioning from mitosis to meiosis, hence supporting the hypothesis of their involvement in the initiation of meiosis. Future research will be directed at determining a specific role for these three proteins in germ cell differentiation.

Riding the Spermatogenic Wave: Profiling Gene Expression Within Neonatal Germ and Sertoli Cells During a Synchronized Initial Wave of Spermatogenesis in Mice

ABSTRACT Continual sperm production relies on germ cells undergoing spermatogenesis asynchronously. As a result, the testis always contains a mixed population of germ cells at different stages of their differentiation process. The heterogeneous nature of the testis makes profiling gene expression within Sertoli cells or specific populations of germ cells impossible when a wild-type testis is assessed. We recently reported a unique method for synchronizing spermatogenesis without affecting fertility by manipulating RA levels within the neonatal testis. Using this protocol, combined with the RiboTag transgenic mouse line, we have mapped the Sertoli and germ cell translatome during the initial synchronized wave of spermatogenesis. Using microarray analysis, we identified 392 and 194 germ cell and Sertoli cells transcripts, respectively, that dynamically change during spermatogonial differentiation, division, and the onset of meiosis. Functional annotation clustering revealed that transcripts enriched in germ cells were mostly associated with meiosis (21 transcripts), chromatin organization (12 transcripts), and cell cycle (3 transcripts). In addition, glycoproteins (65 transcripts), cell adhesion (15 transcripts), and cell junction (13 transcripts) transcripts were overrepresented in the Sertoli cell-enriched list. These datasets represent the first transcriptional analysis of spermatogonial differentiation, division, and meiotic onset. These data suggest that several of the genes encoding meiotic proteins are expressed and are actively being translated well before germ cells enter meiosis. In addition, this study provides novel candidate genes, Asf1b and Esyt3, that may be involved in the regulation of spermatogonial chromatin reorganization, germ-Sertoli cell interactions, and/or blood-testis barrier formation.

Immunolocalization and Regulation of Cystatin 12 in Mouse Testis and Epididymis

Abstract In previous studies, we identified a new member of the male reproductive tract subgroup within family 2 cystatins, termed cystatin 12 (Cst12, previously known as Cst TE-1 or Cres3). The mouse Cst12 mRNA was primarily localized to the Sertoli cells in the testis and to the epithelial cells of the proximal caput region of the epididymis. In this report, studies were carried out to characterize the cystatin 12 (CST12) protein in mouse testis and epididymis. A recombinant His-CST12 fusion protein was expressed in E. coli and purified to generate an anti-CST12 polyclonal antibody. Western blot analysis showed little or no cross-reaction between the anti-CST12 antibody and several other known male reproductive tract cystatins. Immunohistochemistry revealed that CST12 protein was predominantly localized to the cytoplasm of Sertoli cells in the seminiferous epithelium in a stage-dependent manner. All stages showed high levels of expression except stages VII and VIII, in which very limited expression of CST12 was observed. In the epididymis, CST12 was highly expressed in the cytoplasm of the epithelial cells in the proximal caput and secreted into the lumen. The mouse CST12 protein was also detected in other regions of the epididymis; however, the localization varied greatly along the epididymal tubules. Indirect immunofluorescence showed that CST12 protein was localized to the cytoplasmic droplets in both testicular and epididymal spermatozoa. These observations suggest that CST12 protein may play a specialized role during spermatogenesis and sperm maturation. Northern blot analyses demonstrated that Cst12 transcript levels in the epididymis decreased after castration, and testosterone propionate (T) treatment further repressed the expression of this gene. However, 17-beta estradiol (E) administration maintained the expression of Cst12 mRNA after castration, whereas treatment with both T and E failed to maintain Cst12 mRNA levels in epididymis. These results suggest that androgen and estrogen, probably with other testicular factors, are involved in the regulation of this gene.