KLB dysregulation mediates disrupted muscle development in intrauterine growth restriction.

Intrauterine growth restriction (IUGR) is a leading cause of neonatal morbidity and mortality in humans and domestic animals. Developmental adaptations of skeletal muscle in IUGR lead to increased risk of premature muscle loss and metabolic disease in later life. Here, we identified ß-Klotho (KLB), a fibroblast growth factor 21 (FGF21) co-receptor, as a novel regulator of muscle development in IUGR. Using the pig as a naturally-occurring disease model, we performed transcriptome-wide profiling of fetal muscle (day 90 of pregnancy) from IUGR and normal-weight (NW) littermates. We found that, alongside large-scale transcriptional changes comprising multiple developmental, tissue injury and metabolic gene pathways, KLB was increased in IUGR muscle. Moreover, FGF21 concentrations were increased in plasma in IUGR fetuses. Using cultures of fetal muscle progenitor cells (MPCs), we showed reduced myogenic capacity of IUGR compared to NW muscle in vitro, as evidenced by differences in fusion indices and myogenic transcript levels, as well as mechanistic target of rapamycin (mTOR) activity. Moreover, transfection of MPCs with KLB small interfering RNA promoted myogenesis and mTOR activation, whereas treatment with FGF21 had opposite and dose-dependent effects in porcine and also in human fetal MPCs. In conclusion, our results identify KLB as a novel and potentially critical mediator of impaired muscle development in IUGR, through conserved mechanisms in pigs and humans. Our data shed new light onto the pathogenesis of IUGR, a significant cause of lifelong ill-health in humans and animals. KEY POINTS: Intrauterine growth restriction (IUGR) is associated with large-scale transcriptional changes in developmental, tissue injury and metabolic gene pathways in fetal skeletal muscle. Levels of the fibroblast growth factor 21 (FGF21) co-receptor, ß-Klotho (KLB) are increased in IUGR fetal muscle, and FGF21 concentrations are increased in IUGR fetal plasma. KLB mediates a reduction in muscle development through inhibition of mechanistic target of rapamycin signalling. These effects of KLB on muscle cells are conserved in pig and human, suggesting a vital role of this protein in the regulation of muscle development and function in mammals.

Localization and in silico-based functional analysis of miR-202 in bull testis.

Bull fertility is pivotal to the prosperity of the cattle industry worldwide. miR-202 has been shown to be gonad specific and to have key roles in gonad function in different species. To further understand the involvement of miR-202 in bull reproduction, this study aimed to establish its localization in bovine testicular tissue and to identify putative biological functions using bioinformatics approaches. We assessed the miR-202 expression in paraffin-embedded tissue samples collected form an abattoir using in situ hybridization. miR-202 was present in Sertoli cells and in germ cells at different stages of development. Using available databases, a total of 466 predicted gene targets of miR-202 were identified. Functional annotation revealed that miR-202 target genes were mainly associated with protein modification and phosphorylation processes as well as longevity regulating pathway. Moreover, genes in the longevity regulating pathway mapped to PI3K/Akt/mTOR pathway which is involved in promoting proliferation of testicular cells and spermatogenesis. These findings suggest that miR-202 plays important roles in regulating proliferation and viability of testicular cells including somatic and germ cells.

Analyses of bovine luteal fractions obtained by FACS reveals enrichment of miR-183-96-182 cluster miRNAs in endothelial cells.

Our previous studies showed that the miRNA clusters, miR-183-96-182 and miR-212-132, may be critical in promoting luteal cell survival and progesterone production in both bovine and humans. To further understand their involvement in luteal development, this study aimed to establish the expression of these miRNAs in different bovine luteal cell types, namely, endothelial and steroidogenic, isolated using fluorescence-activated cell sorting (FACS). We isolated each of the two cell populations based on the presence of the endothelia surface marker, CD144, and uptake of the lipophilic dye, Nile Red, respectively. Using quantitative Polymerase Chain Reaction (qPCR) in the sorted cell fractions we confirmed that CD144 and the endothelia-specific miRNA, miR-126, were predominantly expressed in endothelial cells (CD144+), whereas HSD3B1 was expressed predominantly in steroidogenic cells (Nile RedHI). Finally, we found that whereas the miR-212-132 cluster was expressed at similar levels in luteal endothelial and steroidogenic cells, miR-183-96-182 was expressed at > 4-fold higher levels in endothelial than in steroidogenic cells (P < 0.05), suggesting that these two miRNA clusters, and particularly miR-183-96-182, may be important in functionally regulating not only steroidogenic cells but also endothelial cells in the corpus luteum (CL).

Comprehensive analysis of blood cells and plasma identifies tissue-specific miRNAs as potential novel circulating biomarkers in cattle.

BACKGROUND: The potential of circulating miRNAs as biomarkers of tissue function, both in health and disease, has been extensively demonstrated in humans. In addition, circulating miRNA biomarkers offer significant potential towards improving the productivity of livestock species, however, such potential has been hampered by the absence of information on the nature and source of circulating miRNA populations in these species. In addition, many miRNAs originally proposed as robust biomarkers of a particular tissue or disease in humans have been later shown not to be tissue specific and thus to actually have limited biomarker utility. In this study, we comprehensively analysed miRNA profiles in plasma and cell fractions of blood from cattle with the aim to identify tissue-derived miRNAs which may be useful as biomarkers of tissue function in this important food animal species. RESULTS: Using small RNA sequencing, we identified 92 miRNAs with significantly higher expression in plasma compared to paired blood cell samples (n = 4 cows). Differences in miRNA levels between plasma and cell fractions were validated for eight out of 10 miRNAs using RT-qPCR (n = 10 cows). Among miRNAs found to be enriched in plasma, we confirmed miR-122 (liver), miR-133a (muscle) and miR-215 (intestine) to be tissue-enriched, as reported for other species. Profiling of additional miRNAs across different tissues identified the human homologue, miR-802, as highly enriched specifically in liver. CONCLUSIONS: These results provide novel information on the source of bovine circulating miRNAs and could significantly facilitate the identification of production-relevant tissue biomarkers in livestock. In particular, miR-802, a circulating miRNA not previously identified in cattle, can reportedly regulate insulin sensitivity and lipid metabolism, and thus could potentially provide a specific biomarker of liver function, a key parameter in the context of post-partum negative energy balance in dairy cows.

Pericytes in Veterinary Species: Prospective Isolation, Characterization and Tissue Regeneration Potential.

Although pericytes have long been known for their roles in blood vessel regulation, it was not until a decade ago that their tissue regeneration potential began to be considered, after studies showed that pericytes were the in vivo counterparts of mesenchymal stem/stromal cells (MSCs). The prospective isolation and culture expansion of pericytes brought great excitement as it opened the way to the therapeutic use of well-defined cell populations with known regenerative potential to overcome concerns associated with the use of traditional MSC preparations. Studies first in humans and later in the horse and other domestic species showed that indeed cultured pericytes had key characteristics of MSCs, namely, their immunophenotype and the abilities to grow clonally and to differentiate into mature mesenchymal cells both in vitro and vivo. Several studies with human pericytes, and to a much lesser extent with animal pericytes, have also shown significant promise in tissue repair in different disease models. This review summarizes current knowledge on the tissue regeneration properties of pericytes from domestic animals and outlines future steps necessary for realizing their full potential both in clinical veterinary medicine and in preclinical testing of human therapies using large animal models, including the need for robust approaches for isolation, culture and appropriate in vivo testing of the tissue regenerative properties of pericytes in these species.

Circulating miRNA signatures of early pregnancy in cattle.

BACKGROUND: Low fertility remains a leading cause of poor productivity in dairy cattle. In this context, there is significant interest in developing novel tools for accurate early diagnosis of pregnancy. MicroRNAs (miRNAs) are short RNA molecules which are critically involved in regulating gene expression during both health and disease. MiRNAs have been shown to regulate ovarian function, uterine receptivity, embryonic development and placental function. Circulating miRNAs can provide useful biomarkers of tissue function and disease; importantly, differential miRNA profiles have been linked to pregnancy and preeclampsia in humans. This study sought to establish the potential of circulating miRNAs as biomarkers of early pregnancy in cattle. RESULTS: We applied Illumina small-RNA sequencing to profile miRNAs in plasma samples collected from eight non-pregnant heifers on Days 0, 8 and 16 of the oestrous cycle and 11 heifers on Days 16 and 24 of pregnancy. We sequenced a total of 46 samples and generated 9.2 million miRNA reads per sample. There were no differences in miRNA read abundance between any of the pregnant and non-pregnant time-points (FDR > 0.1). As a complementary approach, we analysed sample pools (3-4 samples/pool) corresponding to Days 0, 8 and 16 of the oestrous cycle and Day 24 of pregnancy (n = 3 pools/group) using Qiagen PCR arrays. A total of 16 miRNAs were differentially expressed (FDR < 0.1) in plasma between pregnant and non-pregnant animals. RT-qPCR validation using the same plasma samples confirmed that miR-26a was differentially upregulated on Day 16 pregnant relative to non-pregnant heifers (1.7-fold; P = 0.043), whereas miR-1249 tended to be upregulated in Day 16 pregnant heifers (1.6-fold; P = 0.081). Further validation in an independent group of heifers confirmed an increase in plasma miR-26a levels during early pregnancy, which was significant only on Day 24 (2.0-fold; P = 0.027). CONCLUSIONS: Through genome-wide analyses we have successfully profiled plasma miRNA populations associated with early pregnancy in cattle. We have identified miR-26a as a potential circulating biomarker of early pregnancy.

Transcriptome profiling of granulosa and theca cells during dominant follicle development in the horse.

Several aspects of equine ovarian physiology are unique among domestic species. Moreover, follicular growth patterns are very similar between horses and humans. This study aimed to characterize, for the first time, global gene expression profiles associated with growth and preovulatory (PO) maturation of equine dominant follicles. Granulosa cells (GCs) and theca interna cells (TCs) were harvested from follicles (n = 5) at different stages of an ovulatory wave in mares corresponding to early dominance (ED; diameter ≥22 mm), late dominance (LD; ≥33 mm) and PO stage (34 h after administration of crude equine gonadotropins at LD stage), and separately analyzed on a horse gene expression microarray, followed by validation using quantitative PCR and immunoblotting/immunohistochemistry. Numbers of differentially expressed transcripts (DETs; ≥2-fold; P < 0.05) during the ED-LD and LD-PO transitions were 546 and 2419 in GCs and 5 and 582 in TCs. The most prominent change in GCs was the down-regulation of transcripts associated with cell division during both ED-LD and LD-PO. In addition, DET sets during LD-PO in GCs were enriched for genes involved in cell communication/adhesion, antioxidation/detoxification, immunity/inflammation, and cholesterol biosynthesis. In contrast, the largest change in TCs during the LD-PO transition was an up-regulation of genes involved in immune activation, with other DET sets mapping to GPCR/cAMP signaling, lipid/amino acid metabolism, and cell proliferation/survival and differentiation. In conclusion, distinct expression profiles were identified between growing and PO follicles and, particularly, between GCs and TCs within each stage. Several DETs were identified that have not been associated with follicle development in other species.

Characterization of microRNAs differentially expressed during bovine follicle development.

Several different miRNAs have been proposed to regulate ovarian follicle function; however, very limited information exists on the spatiotemporal patterns of miRNA expression during follicle development. The objective of this study was to identify, using microarray, miRNA profiles associated with growth and regression of dominant-size follicles in the bovine monovular ovary and to characterize their spatiotemporal distribution during development. The follicles were collected from abattoir ovaries and classified as small (4-8  mm) or large (12-17  mm); the latter were further classified as healthy or atretic based on estradiol and CYP19A1 levels. Six pools of small follicles and individual large healthy (n=6) and large atretic (n=5) follicles were analyzed using Exiqon's miRCURY LNA microRNA Array 6th gen, followed by qPCR validation. A total of 17 and 57 sequences were differentially expressed (greater than or equal to twofold; P<0.05) between large healthy and each of small and large atretic follicles respectively. Bovine miRNAs confirmed to be upregulated in large healthy follicles relative to small follicles (bta-miR-144, bta-miR-202, bta-miR-451, bta-miR-652, and bta-miR-873) were further characterized. Three of these miRNAs (bta-miR-144, bta-miR-202, and bta-miR-873) were also downregulated in large atretic follicles relative to large healthy follicles. Within the follicle, these miRNAs were predominantly expressed in mural granulosa cells. Further, body-wide screening revealed that bta-miR-202, but not other miRNAs, was expressed exclusively in the gonads. Finally, a total of 1359 predicted targets of the five miRNAs enriched in large healthy follicles were identified, which mapped to signaling pathways involved in follicular cell proliferation, steroidogenesis, prevention of premature luteinization, and oocyte maturation.

Equine Induced Pluripotent Stem Cell Culture.

Groundbreaking work by Takahashi and Yamanaka in 2006 demonstrated that non-embryonic cells can be reprogrammed into pluripotent stem cells (PSCs) by forcing the expression of a defined set of transcription factors in culture, thus overcoming ethical concerns linked to embryonic stem cells. Induced PSCs have since revolutionized biomedical research, holding tremendous potential also in other areas such as livestock production and wildlife conservation. iPSCs exhibit broad accessibility, having been derived from a multitude of cell types and species. Apart from humans, iPSCs hold particular medical promise in the horse. The potential of iPSCs has been shown in a variety of biomedical contexts in the horse. However, progress in generating therapeutically useful equine iPSCs has lagged behind that reported in humans, with the generation of footprint-free iPSCs using non-integrative reprogramming approaches having proven particularly challenging. A greater understanding of the underlying molecular pathways and essential factors required for the generation and maintenance of equine iPSCs and their differentiation into relevant lineages will be critical for realizing their significant potential in veterinary regenerative medicine. This article outlines up-to-date protocols for the successful culture of equine iPSC, including colony selection, expansion, and adaptation to feeder-free conditions.

Derivation and long-term maintenance of porcine skeletal muscle progenitor cells.

Culture of muscle cells from livestock species has typically involved laborious enzyme-based approaches that yield heterogeneous populations with limited proliferative and myogenic differentiation capacity, thus limiting their use in physiologically-meaningful studies. This study reports the use of a simple explant culture technique to derive progenitor cell populations from porcine muscle that could be maintained and differentiated long-term in culture. Fragments of semitendinosus muscle from 4 to 8 week-old piglets (n = 4) were seeded on matrigel coated culture dishes to stimulate migration of muscle-derived progenitor cells (MDPCs). Cell outgrowths appeared within a few days and were serially passaged and characterised using RT-qPCR, immunostaining and flow cytometry. MDPCs had an initial mean doubling time of 1.4 days which increased to 2.5 days by passage 14. MDPC populations displayed steady levels of the lineage-specific markers, PAX7 and MYOD, up until at least passage 2 (positive immunostaining in about 40% cells for each gene), after which the expression of myogenic markers decreased gradually. Remarkably, MDPCs were able to readily generate myotubes in culture up until passage 8. Moreover, a decrease in myogenic capacity during serial passaging was concomitant with a gradual increase in the expression of the pre-adipocyte markers, CD105 and PDGFRA, and an increase in the ability of MDPCs to differentiate into adipocytes. In conclusion, explant culture provided a simple and efficient method to harvest enriched myogenic progenitors from pig skeletal muscle which could be maintained long-term and differentiated in vitro, thus providing a suitable system for studies on porcine muscle biology and applications in the expanding field of cultured meat.

Association between blood miR-26a levels following artificial insemination, and pregnancy outcome in dairy cattle.

Early pregnancy diagnosis is key to maximise productivity of dairy herds. We previously showed that an increase in the levels of miR-26 could be detected as soon as day 8 of pregnancy in heifers. The aims of this study were to determine whether 1) plasma miR-26 levels would be distinctly elevated, retrospectively, early after artificial insemination in lactating cows with successful compared to failed pregnancies, 2) the early increase in miRNA levels in cows with successful pregnancy could be accounted for by changes in miRNA expression in white blood cells (WBCs), presumably induced by the effects of embryo-derived interferon tau (IFNt), and 3) plasma miRNA levels may provide a reliable early predictor of pregnancy that could be used at a herd level. Blood samples were taken from a total of 34 dairy cows (lactation number 1 to 4) before (D0) and 9 and 18 days after artificial insemination at oestrus, followed by confirmation of pregnancy status by ultrasound on D32. In addition, WBCs collected from non-pregnant cows (n = 4) were stimulated in vitro with recombinant ovine IFNt (0-100 pg/ml). Levels of miRNAs and ISG15, a known IFNt-induced gene, were quantified by qPCR. Relative to D0, a larger increase in plasma miR-26a (P = 0.04) occurred on D9 in cows later confirmed to be pregnant (n = 12) than in cows with a failed pregnancy (n = 22). Expression of miR-26a in WBCs was not affected (P>0.1) by pregnancy status or IFNt stimulation in vitro, in contrast to ISG15 expression which increased markedly (P<0.0001) both in WBC samples collected on D18 from animals later confirmed to be pregnant, and in WBCs after stimulation with IFNt in vitro. Finally, ROC analyses revealed that miR-26a on D9 or D18 could predict pregnancy outcome with much lower accuracy than WBC ISG15 on D18 (Likelihood ratio, 2.3 vs 15.4). In summary, a modest increase in plasma miR-26a levels occurs during early pregnancy in mature dairy cows which may not accounted for by changes in miRNA levels in WBCs or the effects of IFNt. Moreover, compared to ISG15, changes in miR-26a levels may not provide an accurate test for early diagnosis of pregnancy in cows.

Associations between testicular development and fetal size in the pig.

BACKGROUND: Impaired reproductive performance is the largest contributing factor for the removal of boars from commercial systems. Intrauterine growth restricted piglets represent 25% of the total number of piglets born and have impaired reproductive performance. This study aimed to improve the understanding of temporal changes in testicular gene expression during testes development in fetuses of different size. The lightest and closest to mean litter weight (CTMLW) male Large White × Landrace littermates were collected at gestational days (GD) 45, 60 and 90 (n = 5-6 litters/GD). RESULTS: Testes weight and testes weight as a percentage of fetal weight were not associated with fetal size at GD60 or 90. Fetal plasma testosterone was not associated with fetal size at GD90. There was no association between fetal size and seminiferous tubule area and number, number of germ or Sertoli cells per tubule. The lightest fetuses tended to have wider seminiferous tubules compared to the CTMLW fetuses at GD90 (P = 0.077). The testicular expression of KI67 (P ≤ 0.01) and BAX:BCL2 ratio (P = 0.058) mRNAs decreased as gestation progressed. Greater SPP1 mRNA expression was observed at GD60 when compared with GD45 and 90 (P ≤ 0.05). Lower expression of DMRT1 and SPP1 (P < 0.01) mRNAs was observed in testes associated with the lightest fetuses compared to the CTMLW fetuses at GD90. CONCLUSIONS: These findings provide novel insights into the expression profiles of genes associated with testicular development and function. Further, these data suggest that programming of reproductive potential in IUGR boars occurs late in gestation, providing a platform for further mechanistic investigation.

Associations between foetal size and ovarian development in the pig.

It is estimated that intra-uterine growth restricted piglets represent 25 % of the total number of piglets born. Growth restricted female pigs have impaired reproductive performance postnatally. HHowever, when during gestation this phenotype arises is not known. With this study, the aim was to improve the understanding of foetal ovarian development in normal and small foetuses throughout gestation. Female Large White X Landrace foetuses were obtained at gestational day (GD) 45, 60 and 90 (n = 5-6 litters/GD). Histological analysis of GATA4 stained foetal ovaries at GD60 and 90 indicated there were fewer primary follicles (P ≤ 0.05) in the foetuses weighing the least compared to those with a weight similar to the mean for the litter (CTMLW) at GD90. Plasma oestradiol concentrations were less in the foetuses with lesser weights compared with greater weight foetuses at GD90 (P ≤ 0.05). The RNA was extracted from ovaries of the lesser weight and CTMLW foetuses at GD45, 60 and 90 and qPCR was performed to quantify relative abundance of 12 candidate mRNAs for which encoded proteins that modulate ovarian function and development. Gestational changes in relative abundances of CD31, PTGFR, SPP1 and VEGFA mRNA transcripts were observed. Relative abundance of KI67 (P = 0.066) and P53 (P ≤ 0.05) was less in ovaries of the lesser weight compared to CTMLW foetuses at GD60. There was a lesser relative abundance of PTGFR mRNA transcript in ovaries from the foetuses with lesser weight compared to CTMLW foetuses at GD45 and 60 (P ≤ 0.05). These findings indicate that postnatal differences in the reproductive potential of growth restricted females are programmed early in gestation. It is hoped that further investigation will improve the understanding of the relationship between prenatal reproductive development and postnatal reproductive performance.

Bovine Granulosa Cell Culture.

Culture of granulosa cells has for long provided a useful tool to understand the molecular processes underlying ovarian follicle development. Among all species investigated, cattle have become an excellent model for in vitro studies on follicular biology, both because of their resemblance with humans in terms of follicular biology and the importance of reproductive failure as a cause of lost productivity in the dairy industry. In this chapter, we describe up-to-date methods for the harvesting of granulosa cells from bovine ovaries collected post-mortem, as well as procedures for both culturing granulosa cells in an undifferentiated state and inducing their luteinization in vitro, and for the efficient transfection of granulosa cells with oligonucleotide sequences for the purpose of investigating the function of specific genes in vitro.

Profiling of MicroRNAs in the Biofluids of Livestock Species.

The value of circulating microRNAs (miRNAs) as noninvasive biomarkers of human disease has been extensively demonstrated. Significant potential also exists in other species, particularly in relation to control of veterinary diseases and selection/monitoring of production traits in livestock. Although robust protocols have been developed for miRNA profiling of human biofluids, significant optimization may be required before these can be applied to other species. In this chapter, we describe protocols for small-RNA sequencing and RT-qPCR analyses of plasma samples from livestock species. In addition, we provide brief data analysis protocols for small-RNA sequencing and RT-qPCR data. Finally, we highlight important considerations for these protocols such as low RNA yield, platform-specific biases, and optimal normalization approaches.

Association of plasma microRNA expression with age, genetic background and functional traits in dairy cattle.

A number of blood circulating microRNAs (miRNAs) are proven disease biomarkers and have been associated with ageing and longevity in multiple species. However, the role of circulating miRNAs in livestock species has not been fully studied. We hypothesise that plasma miRNA expression profiles are affected by age and genetic background, and associated with health and production traits in dairy cattle. Using PCR arrays, we assessed 306 plasma miRNAs for effects of age (calves vs mature cows) and genetic background (control vs select lines) in 18 animals. We identified miRNAs which were significantly affected by age (26 miRNAs) and genetic line (5 miRNAs). Using RT-qPCR in a larger cow population (n = 73) we successfully validated array data for 12 age-related miRNAs, one genetic line-related miRNA, and utilised expression data to associate their levels in circulation with functional traits in these animals. Plasma miRNA levels were associated with telomere length (ageing/longevity indicator), milk production and composition, milk somatic cell count (mastitis indicator), fertility, lameness, and blood metabolites linked with body energy balance and metabolic stress. In conclusion, circulating miRNAs could provide useful selection markers for dairy cows to help improve health, welfare and production performance.

The Fate of Autologous Endometrial Mesenchymal Stromal Cells After Application in the Healthy Equine Uterus.

Because of their distinct differentiation, immunomodulatory, and migratory capacities, endometrial mesenchymal stromal cells (MSCs) may provide an optimum source of therapeutic cells not only in relation to the uterus but also for regeneration of other tissues. This study reports the fate of endometrial MSCs following intrauterine application in mares. Stromal cell fractions were isolated from endometrial biopsies taken from seven reproductively healthy mares, expanded, and fluorescence labeled in culture. Phosphate-buffered saline (PBS) or MSCs (15 × 106) were autologously infused into each uterine horn during early diestrus and subsequently tracked by fluorescence microscopy and flow cytometry of endometrial biopsies and blood samples taken periodically after infusion. The inflammatory response to cell infusion was monitored in endometrial cytology samples. MSCs were detected in endometrial sections at 6, 12, and 24 h, but not later (7 or 14 days), after cell infusion. Cells were in all cases located in the uterine lumen, never within the endometrial tissue. No fluorescence signal was detected in blood samples at any time point after infusion. Cytology analyses showed an increase in % of polymorphonuclear neutrophils between 1 and 3 h after uterine infusion with either MSCs or PBS and a further increase by 6 h only in mares infused with PBS. In summary, endometrial MSCs were detected in the uterine lumen for up to 24 h after infusion, but did not migrate into the healthy endometrium. Moreover, MSCs effectively attenuated the inflammatory response to uterine infusion. We conclude that endometrial MSCs obtained from routine uterine biopsies could provide a safe and effective cell source for treatment of inflammatory conditions of the uterus and potentially other tissues.

Reproductive stage and sex steroid hormone levels influence the expression of mesenchymal stromal cell (MSC) markers in the equine endometrium.

Mesenchymal stem or stromal cells (MSCs) play key roles in tissue homeostasis. In the cyclic equine endometrium, this may be regulated by changes in serum concentrations of sex steroid hormones. This study was designed to investigate the changes in endometrial expression of MSC markers during reproductive cycles in mares and the influence of sex steroid hormones on endometrial MSC proliferation in vitro. Endometrial biopsies were collected from pony mares at different reproductive stages (estrus; day 5 and 13 after ovulation; seasonal anestrus; 20 h and 7days post-partum; n = 5 per stage) and were analyzed by RT-qPCR. MSC (CD29, CD44, CD73, CD90, CD105) and perivascular (CD146, NG2) markers were present in all samples irrespective of reproductive stage. Transcript levels of most markers were present at lowest levels on day 5 after ovulation and at 20 h post-partum. MSCs isolated from endometrial tissue (n = 6 mares) were cultured in the presence of progesterone (0.01-100 µM) and estradiol 17ß (0.1-1 µM), and cell proliferation was analyzed using alamarBlue® assay. Relative to cells incubated in steroid-depleted media, both progesterone and estradiol 17ß moderately increased cell proliferation (1.1- and 1.2-fold, respectively) independently of the concentration used. In conclusion, our results suggest that levels of MSC markers in equine endometrium dynamically change across reproductive cycles and that MSC populations are in part regulated by sex steroids.

Generation of Functional Myocytes from Equine Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) have revolutionized human biomedicine through their use in disease modeling and therapy. In comparison, little progress has been made toward the application of iPSCs in veterinary species. In that regard, skeletal myocytes from iPSCs would have great potential for understanding muscle function and disease in the equine athlete. In this study, we generated skeletal myotubes by transducing equine iPSC-derived mesenchymal derivatives with an inducible lentiviral vector coding for the human sequence of the myogenic factor, MyoD. Myosin heavy chain-positive myotubes generated from two different iPSC lines were compared to myotubes from adult equine skeletal muscle progenitor cells (MPCs). iPSC myotubes had a smaller mean area than MPC myotubes (≤2-fold). In addition, quantitative polymerase chain reaction analyses showed that iPSC myotubes expressed MYH2 and MYH3 isoforms (at similar or lower levels than MPC myotubes), but they did not express the mature muscle isoform, MYH1. Compared to MPC myotubes, iPSC myotubes expressed reduced levels of the myogenic factors, MYOD1 and MYF6, but did not express MYF5. Finally, iPSC myotubes responded to KCl-induced membrane depolarization by releasing calcium and did so in a manner similar to MPC myotubes. In conclusion, this is the first study to report the generation of functional myocytes from equine iPSCs.