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.
The treatment of mice with WIN 18,446 suppresses retinoic acid-mediated differentiation of spermatogonia and synchronizes spermatogenesis.
contraception; retinoids; spermatogenesis; testis; WIN 18,446
Meiosis is essential for generation of healthy gametes in both sexes and involves recombination and segregation of homologous chromosomes to produce haploid gametes. The initiation of meiosis in both sexes relies upon retinoic acid (RA) (Griswold MD, Hogarth CA, Bowles J, Koopman P. Initiating Meiosis: The Case for Retinoic Acid. Biol Reprod 2012; 86(35):1–7). Previous studies have demonstrated that the stimulated by retinoic acid gene 8 (Stra8) was required for meiotic progression in both the mouse ovary and postnatal testis. To identify additional candidates that may play a role during meiosis, we used microarray databases to generate lists of transcripts with expression profiles similar to that of Stra8 in the embryonic ovary and postnatal testis. One such gene, establishment of cohesion 1 homolog 2 (Saccharomyces cerevisiae) (Esco2), has been described as a regulator of sister chromatid cohesion during mitosis. This study describes the first in-depth analysis of ESCO2 localization and regulation during meiosis in both males and females. ESCO2 colocalized with the gamma H2A histone family member X (H2AFX) in pachytene spermatocytes, indicating that ESCO2 is a component of the XY body. In pachytene cells of the embryonic ovary, ESCO2 colocalized with H2AFX, which is consistent with the presence of ESCO2 in areas of double-stranded breaks. In addition, the expression of Esco2 was found to be regulated by RA in the postnatal testis. These data indicate that ESCO2 may play a vital role in meiosis in both males and females.
Establishment of cohesion 1 homolog 2 (ESCO) co-localizes with histone H2AFX is consistent with ESCO2 being present in areas of double-stranded breaks.
germ cells; meiosis; ovary; testis
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.
Down-regulation by retinoic acid of Mir-17-92 (Mirc1) cluster and its paralog Mir-106b-25 (Mirc3) cluster miRNAs contributes to retinoic acid-induced spermatogonial differentiation.
microRNA; Mir-106b-25 cluster; Mir-17-92 cluster; retinoic acid; spermatogonial differentiation
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.
Upregulation of Mirlet7 family miRNAs by retinoic acid potentially contributes to retinoic acid-induced spermatogonial differentiation.
Lin28; microRNAs; Mirlet7 family; retinoic acid; spermatogonial differentiation
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.
The bis-(dichloroacetyl)-diamine, WIN 18,446, inhibits the enzymatic conversion of retinol to retinoic acid, and suppresses the retinoic acid-mediated expression of Stra8 in the neonatal and adult mouse testis and the embryonic gonad.
Bis-(dichloroacetyl)-diamines; retinoic acid; spermatogenesis; Stra8; testis
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.
Three genes, Esco2, Setdb2, and Uba6 show a Stra8-like microarray expression profile, and their proteins localize specifically to germ cells during the mitotic-to-meiotic transition.
gamete biology; germ cells; meiosis; spermatogenesis; testis
Spermatogenesis is an intricately regulated process of cellular differentiation transforming spermatogonial stem cells to spermatozoa. Elimination of the transcription factor EGR4 generates subfertile male mice yet the expression and function of EGR4 in the mammalian testis has yet to be fully investigated. We performed in situ hybridization and immunofluorescence to identify Egr4 transcript and protein localization in the developing murine testis. EGR4 was detected in both germ and somatic cells in the neonatal testis but was specific to germ cells inside the seminiferous epithelium from juvenile development onward. EGR4 also displayed distinct intracellular localization patterns within specific cell populations of the testis. In addition, Egr4-deficient testis tubules regress from relatively normal to Sertoli cell and undifferentiated spermatogonia only over time. Taken together, these data suggest that Egr4 may regulate spermatogenesis at multiple steps, with roles in the dividing Sertoli cells, peritubular myoid cells, and the meiotic and elongating haploid germ cell populations.
spermatogenesis; EGR4; testis; germ cells
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.
differentiation; gonocytes; in vitro; retinoic acid; spermatogenesis; spermatogonia; spermatogonial differentiation; Stra8
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.
in vivo; preleptotene spermatocytes; retinoic acid; spermatogonia; STRA8