Hepacivirus A Infection in Horses Defines Distinct Envelope Hypervariable Regions and Elucidates Potential Roles of Viral Strain and Adaptive Immune Status in Determining Envelope Diversity and Infection Outcome.

Hepacivirus A (also known as nonprimate hepacivirus and equine hepacivirus) is a hepatotropic virus that can cause both transient and persistent infections in horses. The evolution of intrahost viral populations (quasispecies) has not been studied in detail for hepacivirus A, and its roles in immune evasion and persistence are unknown. To address these knowledge gaps, we first evaluated the envelope gene (E1 and E2) diversity of two different hepacivirus A strains (WSU and CU) in longitudinal blood samples from experimentally infected adult horses, juvenile horses (foals), and foals with severe combined immunodeficiency (SCID). Persistent infection with the WSU strain was associated with significantly greater quasispecies diversity than that observed in horses who spontaneously cleared infection (P = 0.0002) or in SCID foals (P < 0.0001). In contrast, the CU strain was able to persist despite significantly lower (P < 0.0001) and relatively static envelope diversity. These findings indicate that envelope diversity is a poor predictor of hepacivirus A infection outcomes and could be dependent on strain-specific factors. Next, entropy analysis was performed on all E1/E2 genes entered into GenBank. This analysis defined three novel hypervariable regions (HVRs) in E2, at residues 391 to 402 (HVR1), 450 to 461 (HVR2), and 550 to 562 (HVR3). For the experimentally infected horses, entropy analysis focusing on the HVRs demonstrated that these regions were under increased selective pressure during persistent infection. Increased diversity in the HVRs was also temporally associated with seroconversion in some horses, suggesting that these regions may be targets of neutralizing antibody and may play a role in immune evasion.IMPORTANCE Hepacivirus C (hepatitis C virus) is estimated to infect 150 million people worldwide and is a leading cause of cirrhosis and hepatocellular carcinoma. In contrast, its closest relative, hepacivirus A, causes relatively mild disease in horses and is frequently cleared. The relationship between quasispecies evolution and infection outcome has not been explored for hepacivirus A. To address this knowledge gap, we examined envelope gene diversity in horses with resolving and persistent infections. Interestingly, two strain-specific patterns of quasispecies diversity emerged. Persistence of the WSU strain was associated with increased quasispecies diversity and the accumulation of amino acid changes within three novel hypervariable regions following seroconversion. These findings provided evidence that envelope gene mutation is influenced by adaptive immune pressure and may contribute to hepacivirus persistence. However, the CU strain persisted despite relative evolutionary stasis, suggesting that some hepacivirus strains may use alternative mechanisms to persist in the host.

In vitro growth inhibition of Theileria equi by bumped kinase inhibitors.

INTRODUCTION: Theileria equi, an etiologic agent of equine piroplasmosis, is a tick-transmitted hemoprotozoan of the phylum Apicomplexa. Recent outbreaks of piroplasmosis in the United States have renewed interest in safe and effective treatment options. Although imidocarb dipropionate (IMD) is the drug of choice for clearance of T. equi, adverse reactions and recently documented resistance support the need for alternative therapeutic strategies. The recently described bumped kinase inhibitors (BKIs) are a new class of compounds that could potentially be used as safe and effective alternatives to IMD. In an initial effort to evaluate this potential, herein we determined the T. equi growth inhibitory activity of 11 BKIs relative to that of IMD and the previously tested BKI 1294. Because some BKIs have known human ether-à-go-go related gene (hERG) channel activity, we also assessed the hERG activity of each compound with the goal to identify those with the highest potency against T. equi coupled with the lowest potential for cardiotoxicity. RESULTS: Six BKIs inhibited T. equi growth in vitro, including the previously evaluated BKI 1294 which was used as a positive control. All six compounds were significantly less potent (higher 50% effective concentration (EC50)) than IMD. Two of those compounds were more potent than BKI 1294 control but had similar hERG activity. Although the remaining three compounds had similar to lower potency than BKI 1294, hERG EC50 was higher for three of them (BKI 1735, BKI 1369 and BKI 1318). CONCLUSIONS: The BKI compounds evaluated in this study inhibited T. equi in vitro and had diverse hERG activity. Based on these considerations, three compounds would be suitable for further evaluation. While these results provide a foundation for future work, in vivo pharmacokinetic, pharmacodynamics, and safety studies are needed before BKI compounds can be recommended for clinical use in T. equi infected horses.

Experimental transmission of equine hepacivirus in horses as a model for hepatitis C virus.

UNLABELLED: 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.

RiboTag analysis of actively translated mRNAs in Sertoli and Leydig cells in vivo.

Male spermatogenesis is a complex biological process that is regulated by hormonal signals from the hypothalamus (GnRH), the pituitary gonadotropins (LH and FSH) and the testis (androgens, inhibin). The two key somatic cell types of the testis, Leydig and Sertoli cells, respond to gonadotropins and androgens and regulate the development and maturation of fertilization competent spermatozoa. Although progress has been made in the identification of specific transcripts that are translated in Sertoli and Leydig cells and their response to hormones, efforts to expand these studies have been restricted by technical hurdles. In order to address this problem we have applied an in vivo ribosome tagging strategy (RiboTag) that allows a detailed and physiologically relevant characterization of the "translatome" (polysome-associated mRNAs) of Leydig or Sertoli cells in vivo. Our analysis identified all previously characterized Leydig and Sertoli cell-specific markers and identified in a comprehensive manner novel markers of Leydig and Sertoli cells; the translational response of these two cell types to gonadotropins or testosterone was also investigated. Modulation of a small subset of Sertoli cell genes occurred after FSH and testosterone stimulation. However, Leydig cells responded robustly to gonadotropin deprivation and LH restoration with acute changes in polysome-associated mRNAs. These studies identified the transcription factors that are induced by LH stimulation, uncovered novel potential regulators of LH signaling and steroidogenesis, and demonstrate the effects of LH on the translational machinery in vivo in the Leydig cell.

Turning a spermatogenic wave into a tsunami: synchronizing murine spermatogenesis using WIN 18,446.

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.

Localization and regulation of murine Esco2 during male and female meiosis.

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.

The RNase III enzyme DROSHA is essential for microRNA production and spermatogenesis.

DROSHA is a nuclear RNase III enzyme responsible for cleaving primary microRNAs (miRNAs) into precursor miRNAs and thus is essential for the biogenesis of canonical miRNAs. DICER is a cytoplasmic RNase III enzyme that not only cleaves precursor miRNAs to produce mature miRNAs but also dissects naturally formed/synthetic double-stranded RNAs to generate small interfering RNAs (siRNAs). To investigate the role of canonical miRNA and/or endogenous siRNA production in spermatogenesis, we generated Drosha or Dicer conditional knock-out (cKO) mouse lines by inactivating Drosha or Dicer exclusively in spermatogenic cells in postnatal testes using the Cre-loxp strategy. Both Drosha and Dicer cKO males were infertile due to disrupted spermatogenesis characterized by depletion of spermatocytes and spermatids leading to oligoteratozoospermia or azoospermia. The developmental course of spermatogenic disruptions was similar at morphological levels between Drosha and Dicer cKO males, but Drosha cKO testes appeared to be more severe in spermatogenic disruptions than Dicer cKO testes. Microarray analyses revealed transcriptomic differences between Drosha- and Dicer-null pachytene spermatocytes or round spermatids. Although levels of sex-linked mRNAs were mildly elevated, meiotic sex chromosome inactivation appeared to have occurred normally. Our data demonstrate that unlike DICER, which is required for the biogenesis of several small RNA species, DROSHA is essential mainly for the canonical miRNA production, and DROSHA-mediated miRNA production is essential for normal spermatogenesis and male fertility.

Two miRNA clusters, Mir-17-92 (Mirc1) and Mir-106b-25 (Mirc3), are involved in the regulation of spermatogonial differentiation in mice.

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.

Altered testicular gene expression patterns in mice lacking the polyubiquitin gene Ubb.

Ubiquitin (Ub) is an essential protein found in all eukaryotic cells and plays important roles in a variety of cellular functions including germ cell development. We have previously reported that targeted disruption of the polyubiquitin gene Ubb results in male and female infertility in Ubb(-/-) mice, with germ cells arrested at meiotic prophase I. Although reduced Ub levels in germ cells are believed to be responsible for the fertility defect in Ubb(-/-) mice, it is still unclear how reduced Ub levels result in sterility. Here we describe the results of a microarray analysis of the murine testicular transcriptome, which demonstrates dramatically altered gene expression patterns in Ubb(-/-) mice, possibly related to reduced levels of histone 2A (H2A) ubiquitylation. We find that large numbers of genes related to fertility, metabolism, transcription, and the ubiquitin-proteasome system (UPS) are misregulated in Ubb(-/-) mice. Such wide-ranging alterations in gene expression suggest that loss of the Ubb gene does not mimic a single-gene defect phenotype, but instead may affect gene expression more globally. These dramatic changes in gene expression could, at least in part, contribute to the complex fertility and metabolic phenotypes seen in these mice.

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.

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.

Exposure to retinoic acid in the neonatal but not adult mouse results in synchronous spermatogenesis.

Retinoic acid (RA) is required for germ cell differentiation, the regulation of which gives rise to a constant production of mature sperm. In testes from 3-day postpartum (dpp) RARE-hsplacZ mice, periodic regions positive for beta-galactosidase activity were observed along the length of the seminiferous tubules. Periodicity was abolished by treatment of neonates with exogenous RA at 2 dpp. To assess the consequences, 2-dpp mice were treated with RA, and the long- and short-term effects were assessed. Long-term effects of neonatal RA exposure included a delay in the appearance of advanced germ cells and the absence of a spermatogenic wave (synchronous spermatogenesis) in the adult. In contrast, RA exposure in vitamin A-sufficient adults did not result in synchronous spermatogenesis but rather induced apoptosis in a subset of spermatogonia. Shortly after (24 h) neonates were exposed, altered expression of known germ cell differentiation and the (Stra8, Kit, Sycp3, and Rec8) meiosis markers and an increase in the number of STRA8 and SYCP3 immunopositive cells were observed relative to those of vehicle controls. However, 48 and 72 h after exposure, a significant reduction in the number of STRA8 and SYCP3 immunopositive cells occurred. Immunohistochemical analysis of a marker for apoptosis demonstrated neonatal exposure resulted in increased germ cell apoptosis, as observed in the adult. Additionally, RA exposure resulted in increased Cyp26a1 expression of the RA-degrading enzyme. Thus, while RA treatment of neonatal and adult mice resulted in apoptosis of spermatogonia, synchronous spermatogenesis occurred only after neonatal RA exposure.

Suppression of spermatogenesis by bisdichloroacetyldiamines is mediated by inhibition of testicular retinoic acid biosynthesis.

The bisdichloroacetyldiamine WIN 18,446 reversibly inhibits spermatogenesis in many species, including humans; however, the mechanism by which WIN 18,446 functions is unknown. As retinoic acid is essential for spermatogenesis, we hypothesized that WIN 18,446 might inhibit retinoic acid biosynthesis from retinol (vitamin A) within the testes by inhibiting the enzyme aldehyde dehydrogenase 1a2 (ALDH1a2). We studied the effect of WIN 18,446 on ALDH1a2 enzyme activity in vitro, and on spermatogenesis and fertility in vivo, in mature male rabbits for 16 weeks. WIN 18,446 markedly inhibited ALDH1a2 enzyme activity in vitro with an IC(50) of 0.3 µM. In vivo, the oral administration of 200 mg/kg WIN 18,446 to male rabbits for 16 weeks significantly reduced intratesticular concentrations of retinoic acid, severely impaired spermatogenesis, and caused infertility. Reduced concentrations of intratesticular retinoic acid were apparent after only 4 weeks of treatment and preceded the decrease in sperm counts and the loss of mature germ cells in tissue samples. Sperm counts and fertility recovered after treatment was discontinued. These findings demonstrate that bisdichloroacetyldiamines such as WIN 18,446 reversibly suppress spermatogenesis via inhibition of testicular retinoic acid biosynthesis by ALDH1a2. These findings suggest that ALDH1a2 is a promising target for the development of a reversible, nonhormonal male contraceptive.

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.

Gene expression in the efferent ducts, epididymis, and vas deferens during embryonic development of the mouse.

The tissues of the male reproductive tract are characterized by distinct morphologies, from highly coiled to un-coiled. Global gene expression profiles of efferent ducts, epididymis, and vas deferens were generated from embryonic day 14.5 to postnatal day 1 as tissue-specific morphologies emerge. Expression of homeobox genes, potential mediators of tissue-specific morphological development, was assessed. Twenty homeobox genes were identified as either tissue-enriched, developmentally regulated, or both. Additionally, ontology analysis demonstrated cell adhesion to be highly regulated along the length of the reproductive tract. Regulators of cell adhesion with variable expression between the three tissues were identified including Alcam, various cadherins, and multiple integrins. Immunofluorescence localization of the cell adhesion regulators POSTN and CDH2 demonstrated cell adhesion in the epithelium and mesenchyme of the epididymis may change throughout development. These results suggest cell adhesion may be modulated in a tissue-specific manner, playing an important role in establishing each tissue's final morphology.

Expression of stimulated by retinoic acid gene 8 (Stra8) and maturation of murine gonocytes and spermatogonia induced by retinoic acid in vitro.

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.