Differential expression of miR-374b through late gestation.

<p>Expression of circulating miRNAs was analyzed using quantitative PCR through late gestation (8, 9 or 10 months) and post-partum (PP). One miRNA, miR-374b, was found to be differentially regulated. Data shown represent the ΔCt value (dCt), calculated as ΔCt = Ct_(target)−Ct_{(mean of sample)}, with each dot representative of a separate sample. A lower ΔCt value indicates a higher expression level; for each 1-point reduction in ΔCt, an approximately 2-fold increase in expression can be assumed. Significantly different samples are indicated by varying superscripts.</p

Evaluation of circulating miRNAs during late pregnancy in the mare

<div><p>MicroRNAs (miRNAs) are small, non-coding RNAs which are produced throughout the body. Individual tissues tend to have a specific expression profile and excrete many of these miRNAs into circulation. These circulating miRNAs may be diagnostically valuable biomarkers for assessing the presence of disease while minimizing invasive testing. In women, numerous circulating miRNAs have been identified which change significantly during pregnancy-related complications (e.g. chorioamnionitis, eclampsia, recurrent pregnancy loss); however, no prior work has been done in this area in the horse. To identify pregnancy-specific miRNAs, we collected serial whole blood samples in pregnant mares at 8, 9, 10 m of gestation and post-partum, as well as from non-pregnant (diestrous) mares. In total, we evaluated a panel of 178 miRNAs using qPCR, eventually identifying five miRNAs of interest. One miRNA (miR-374b) was differentially regulated through late gestation and four miRNAs (miR-454, miR-133b, miR-486-5p and miR-204b) were differentially regulated between the pregnant and non-pregnant samples. We were able to identify putative targets for the differentially regulated miRNAs using two separate target prediction programs, miRDB and Ingenuity Pathway Analysis. The targets for the miRNAs differentially regulated during pregnancy were predicted to be involved in signaling pathways such as the STAT3 pathway and PI3/AKT signaling pathway, as well as more endocrine-based pathways, including the GnRH, prolactin and insulin signaling pathways. In summary, this study provides novel information about the changes occurring in circulating miRNAs during normal pregnancy, as well as attempting to predict the biological effects induced by these miRNAs.</p></div

Differential expression of circulating miRNAs in pregnant mares.

<p>Expression of circulating miRNAs was analyzed using quantitative PCR to identify miRNAs with significantly different expression during pregnancy. Samples were grouped into pregnant (8, 9, 10 m gestation, postpartum (PP)) or non-pregnant (diestrus) samples and analyzed by one-way ANOVA corrected for false discovery rate (Benjamini-Hochberg; P < 0.05), with post-hoc analysis performed by student’s t-test (P < 0.1). Data were normalized by ΔCt = Ct_(target)−Ct_{(mean of the sample)}, with each point representing the ΔCt for an individual mare. A lower ΔCt value indicates a higher expression level; for each 1-point reduction in ΔCt, an approximately 2-fold increase in expression can be assumed. Significantly different samples are indicated by varying superscripts.</p

Reimmunization increases contraceptive effectiveness of gonadotropin-releasing hormone vaccine (GonaCon-Equine) in free-ranging horses (<i>Equus caballus</i>): Limitations and side effects

<div><p>Wildlife and humans are increasingly competing for resources worldwide, and a diverse, innovative, and effective set of management tools is needed. Controlling abundance of wildlife species that are simultaneously protected, abundant, competitive for resources, and in conflict with some stakeholders but beloved by others, is a daunting challenge. Free-ranging horses (<i>Equus caballus</i>) present such a conundrum and managers struggle for effective tools for regulating their abundance. Controlling reproduction of female horses presents a potential alternative. During 2009–2017, we determined the long-term effectiveness of GnRH vaccine (GonaCon-Equine) both as a single immunization and subsequent reimmunization on reproduction and side effects in free-ranging horses. At a scheduled management roundup in 2009, we randomly assigned 57 adult mares to either a GonaCon-Equine treatment group (<i>n</i> = 29) or a saline control group (<i>n</i> = 28). In a second roundup in 2013, we administered a booster vaccination to these same mares. We used annual ground observations to estimate foaling proportions, social behaviors, body condition, and injection site reactions. We found this vaccine to be safe for pregnant females and neonates, with no overt deleterious behavioral side effects during the breeding season. The proportion of treated mares that foaled following a single vaccination was lower than that for control mares for the second (<i>P =</i> 0.03) and third (<i>P</i> = 0.08) post-treatment foaling seasons but was similar (<i>P</i> = 0.67) to untreated mares for the fourth season, demonstrating reversibility of the primary vaccine treatment. After two vaccinations, however, the proportion of females giving birth was lower (<i>P</i> <0.001) than that for control mares for three consecutive years and ranged from 0.0–0.16. The only detectable adverse side effect of vaccination was intramuscular swelling at the vaccination site. Regardless of vaccine treatment (primary/secondary), approximately 62% (34/55) of immunized mares revealed a visible reaction at the vaccine injection site. However, none of these mares displayed any evidence of lameness, altered gait or abnormal range of movement throughout the 8 years they were observed in this study. Our research suggests that practical application of this vaccine in feral horses will require an initial inoculation that may provide only modest suppression of fertility followed by reimmunization that together could result in greater reduction in population growth rates over time.</p></div

Top twenty pathways predicted for the miRNA differentially regulated through late gestation (miR-374b).

<p>Top twenty pathways predicted for the miRNA differentially regulated through late gestation (miR-374b).</p

Top twenty pathways predicted for miRNAs differentially regulated during pregnancy (miR-133b, miR-204b, miR-454, miR-486-5p).

<p>Top twenty pathways predicted for miRNAs differentially regulated during pregnancy (miR-133b, miR-204b, miR-454, miR-486-5p).</p

Comparative relative risk reduction (RRR), 95% confidence intervals, and p-values associated with differences in foaling proportions between GonaCon-treated and control mares during 2009–2017.

<p>Comparative relative risk reduction (RRR), 95% confidence intervals, and p-values associated with differences in foaling proportions between GonaCon-treated and control mares during 2009–2017.</p

Comparative probability of foaling and pregnancy for treatment and control groups of free-ranging feral horses (<i>Equus caballus)</i> mares selected for this experiment.

<p>Mares were treated with a primary vaccination of GonaCon-Equine in October 2009 and then reimmunized with the same vaccine in September 2013 at scheduled gathers at Theodore Roosevelt National Park, North Dakota, USA. GonaCon vaccinations occurred at the time points represented by the red arrows. Symbols correspond to observed p-values for relative risk comparisons between treatment groups within years (p-value between 0.05 and 0.1 = +, for < 0.05 = x, and for < 1x10^-05 = *).</p

Placentome morphology and fetal weight.

<p><b>A</b>) Testosterone propionate treatment (TP) decreased the number of type A placentomes, and increased type C and type D placentomes collected at gestational day 90. <b>B</b>) TP treatment did not affect placental weight while <b>C</b>) female fetuses from TP ewes had significantly reduced body weight at gestational day 90 compared to female fetuses from controls. * Indicates P≤0.06</p

Androgen Receptor and Histone Lysine Demethylases in Ovine Placenta

<div><p>Sex steroid hormones regulate developmental programming in many tissues, including programming gene expression during prenatal development. While estradiol is known to regulate placentation, little is known about the role of testosterone and androgen signaling in placental development despite the fact that testosterone rises in maternal circulation during pregnancy and in placenta-induced pregnancy disorders. We investigated the role of testosterone in placental gene expression, and focused on androgen receptor (AR). Prenatal androgenization decreased global DNA methylation in gestational day 90 placentomes, and increased placental expression of AR as well as genes involved in epigenetic regulation, angiogenesis, and growth. As AR complexes with histone lysine demethylases (KDMs) to regulate AR target genes in human cancers, we also investigated if the same mechanism is present in the ovine placenta. AR co-immunoprecipitated with KDM1A and KDM4D in sheep placentomes, and AR-KDM1A complexes were recruited to a half-site for androgen response element (ARE) in the promoter region of <i>VEGFA</i>. Androgenized ewes also had increased cotyledonary VEGFA. Finally, in human first trimester placental samples KDM1A and KDM4D immunolocalized to the syncytiotrophoblast, with nuclear KDM1A and KDM4D immunostaining also present in the villous stroma. In conclusion, placental androgen signaling, possibly through AR-KDM complex recruitment to AREs, regulates placental VEGFA expression. AR and KDMs are also present in first trimester human placenta. Androgens appear to be an important regulator of trophoblast differentiation and placental development, and aberrant androgen signaling may contribute to the development of placental disorders.</p></div