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.

Background: miRNA biogenesis requires two RNase III enzymes, DROSHA and DICER. Results: Lack of DROSHA in the male germ line leads to deficiency in miRNA production and male infertility. Conclusion: DROSHA and DICER have both common and unique functions in male germ cell development. Significance: This study reveals an essential role of DROSHA, DICER, and DROSHA-/DICER-dependent small noncoding RNAs spermatogenesis.

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.

Equine hepacivirus (EHCV; nonprimate hepacivirus) is a hepatotropic member of the Flaviviridae family that infects horses. Although EHCV is the closest known relative to hepatitis C virus (HCV), its complete replication kinetics in vivo have not been described, and direct evidence that it causes hepatitis has been lacking. In this study, we detected EHCV in 2 horses that developed post-transfusion hepatitis. Plasma and serum from these horses were used to experimentally transmit EHCV to 4 young adult Arabian horses, two 1-month-old foals (1 Arabian and 1 Arabian-pony cross), and 2 foals (1 Arabian and 1 Arabian-pony cross) with severe combined immunodeficiency (SCID). Our results demonstrated that EHCV had infection kinetics similar to HCV and that infection was associated with acute and chronic liver disease as measured by elevations of liver-specific enzymes and/or by histopathology. Although most of these animals were coinfected with equine pegivirus (EPgV), also a flavivirus, EPgV viral loads were much lower and often undetectable in both liver and blood. Three additional young adult Arabian-pony crosses and 1 SCID foal were then inoculated with plasma containing only EHCV, and evidence of mild hepatocellular damage was observed. The different levels of liver-specific enzyme elevation, hepatic inflammation, and duration of viremia observed during EHCV infection suggested that the magnitude and course of liver disease was mediated by the virus inoculum and/or by host factors, including breed, age, and adaptive immune status. Conclusion: This work documents the complete infection kinetics and liver pathology associated with acute and chronic EHCV infection in horses and further justifies it as a large animal model for HCV. (HEPATOLOGY 2015;61:1533-1546 H epatitis C virus (HCV), a member of the Hepacivirus genus in the family Flaviviridae, is estimated to persistently infect 150 million people throughout the world, and is a leading cause of cirrhosis and hepatocellular carcinoma. Development of a vaccine that prevents persistent infection is an important goal, but significant challenges to prophylaxis include viral immune escape, antigenic diversity of different viral strains, and the limitations of current mouse models for studies of HCV infection, progression, and vaccine development.1,2 Although humans are the only natural hosts for HCV, chimpanzees can be infected with HCV, develop similar clinical disease as humans, and currently represent the best model for vaccine studies.1 However, owing to the proposed listing of all chimpanzees as endangered and the resulting phase out of the use of chimpanzees in research, 3 alternative animal models are needed. Small animal models in which to dissect correlates of vaccine-mediated protection against HCV would have distinct advantages, but in their absence, alternative large animal models deserve consideration. Recent work has identified unique hepaciviruses and viruses of the closely related genus Pegivirus in rodents, bats, and

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.