Equine Placenta Research Articles

Placental homogeneity: Characterizing transcriptional variation among equine chorioallantoic locations

The proper function of the placenta is essential for the health and growth of the fetus and the mother. The placenta relies on dynamic gene expression for its correct and timely development and function. Although numerous studies have identified genes vital for placental functions, equine placental molecular research has primarily focused on single placental locations, in sharp contrast with the broader approach in human studies. Here, we hypothesized that the molecular differences across different regions of the equine placenta are negligible because of its diffuse placental type with a macroscopic homogenous distribution of villi across the placental surface. We compared the transcriptome and stereological findings of the body, pregnant horn, and non-pregnant horn within the equine chorioallantois. Our transcriptomic analysis indicates that the variation between regions of the placenta within individuals is less than the variation observed between individuals. A low number of differentially expressed genes (DEGs) (n = 8) was identified when comparing pregnant and non-pregnant horns within the same placenta, suggesting a remarkable molecular uniformity. A higher number of DEGs was identified when comparing each horn to the body (193 DEGs comparing pregnant horn with body and 207 DEGs comparing non-pregnant horn with body). Genes with a higher expression in the body were associated with processes such as extracellular matrix synthesis and remodeling, which is relevant for placental maturation and placenta-endometrial separation at term and implies asynchrony of these processes across locations. The stereological analysis showed no differences in microcotyledonary density, and width between the locations. However, we observed a greater chorioallantoic thickness in the body and pregnant horn compared to the non-pregnant horn. Overall, our findings reveal a uniform transcriptomic profile across the placental horns, alongside a more distinct gene expression pattern between the uterine body and horns. These regional differences in gene expression suggest a different pace in the placental maturation and detachment among the placental locations.

Quantitative microCT imaging of a whole equine placenta and its blood vessel network

Placental structure is linked to function across morphological scales. In the placenta, changes to gross anatomy, such as surface area, volume, or blood vessel arrangement, are associated with suboptimal physiological outcomes. However, quantifying each of these metrics requires different laborious semi-quantitative methods. Here, we demonstrate how, with minimal sample preparation, whole-organ computed microtomography (microCT) can be used to calculate gross morphometry of the equine placenta and a range of additional metrics, including branching morphometry of placental vasculature, non-destructively from a single dataset. Our approach can be applied to quantify the gross structure of any large mammalian placenta.

Characterization of the equine placental microbial population during nocardioform placentitis

Nocardioform placentitis is a poorly understood disease of equine late gestation. The presence of nocardioform, filamentous branching gram-positive bacteria, has been linked to the disease, with Crossiella equi, Amycolatopsis spp., and Streptomyces spp. being the most frequently identified bacteria. However, these bacteria are not found in all clinical cases in addition to being isolated from healthy, normal postpartum placentas. To better understand this form of placentitis, we analyzed the microbial composition in the equine placenta (chorioallantois) of both healthy postpartum (control; n = 11) and nocardioform-affected samples (n = 22) using 16S rDNA sequencing. We found a lower Shannon index in nocardioform samples, a higher Chao1 index in nocardioform samples, and a difference in beta diversity between control and nocardioform samples (p < 0.05), suggesting the presence of dysbiosis during the disease. In the majority of the NP samples (77 %), one of the following genera—Amycolatopsis, Crossiella, Lentzea, an unidentified member of the Pseudonocardiaceae family, Mycobacterium, or Enterococcus —represented over 70 % of the relative abundance. Overall, the data suggest that a broader spectrum of potential opportunistic pathogens could be involved in nocardioform placentitis, extending beyond the traditionally recognized bacteria, resulting in a similar histomorphological profile.

МОРФОЛОГИЯ ПРОВИЗОРНЫХ ОРГАНОВ ЛОШАДИ ВО ВЗАИМОСВЯЗИ С ЖИЗНЕСПОСОБНОСТЬЮ НОВОРОЖДЕННЫХ ЖЕРЕБЯТ

The aim of the study is to identify the morphofunctional features of the fetal part of the equine placenta in relation to prenatal growth and development of newborn foals. The fetal parts of the placentas of the most viable foals of the first subtype have a maximum weight of (8.02 ± 0.24) kg, area of (14287.48 ± 311.72) cm2, length of the umbilical cord of (57.68 ± 0.75) cm, thickness of (3.76 ± 0.07) cm. The diameter of the arteries and veins in the umbilical cord is almost the same – 11–12 mm, their lumen is strongly narrowed, the thickness of the wall of the umbilical arteries and vein is also equal to (5–6 mm). The thickness of the chorion reaches (8.00 ± 0.91) mm, the number of villi per 1 cm2 of the chorion is (41.80 ± 1.56) pcs., the thickness of the allantoamnion is (6.20 ± 0.58) mm, and the diameter of the vessels is from 4 to 6 mm, their number on the allantoamnion section of 100 cm2 is 5–10. The weight of the fetal parts of the placentas of less viable foals of the second subtype is (5.93 ± 0.24) kg, the area is (10854.92 ± 407.67) cm2, the length of the umbilical cord is (60.00 ± 0.84) cm, the diameter is (3.62 ± 0.06) cm. The diameter of the vein in the umbilical cord is slightly larger (12 mm) than the arteries (11 and 8 mm). The thickness of the chorion decreases to 4–6 mm (5.20 ± 0.37) mm, the number of villi per 1 cm2 of the chorion is 38.40 ± 1.17, there are areas in which the height and density of the villi are even smaller. The thickness of the allantoamnion is (4.06 ± 0.45) mm, and the diameter of the vessels is from 4 to 6 mm, from 3 to 7 vessels are found in an area of 100 cm2 of the allantoamnion. The weight of the fetal parts of the placentas of the third subtype of the least viable foals is significantly (by 37.37 %) lower than the first, as is the area, the length of the umbilical cord is (57.20 ± 0.66) cm, the diameter is (3.44 ± 0.05) cm. The diameter of the vein in the umbilical cord is slightly smaller (9 mm) than the arteries (12 and 11 mm). The chorion is unevenly covered with small, slightly branching villi, not shiny, its thickness reaches (2.84 ± 0.07) mm, and the number of villi per 1 cm2 is from 20 to 40. The diameter of the walls of the allantoamnion vessels decreases and is no more than 3.5 mm at most, their number is from 3 to 5 per 100 cm2.

Novel Placenta-Derived Liquid Product Suitable for Cosmetic Application Produced by Fermentation and Digestion of Porcine or Equine Placenta Using Lactic Acid Bacterium Enterococcus faecalis PR31

Since ancient times, the placenta has been used to produce cosmetic and health food products, whereas fermentation is a technology that has been used to produce foods and cosmetics. For application in cosmetics, traditional placental extracts produced solely by proteolysis have not had enough moisturizing properties or the ability to stimulate the proliferation of epidermal keratinocytes. We combined these two traditional approaches to produce raw materials without such drawbacks that are suitable for cosmetic applications. Using a unique lactic acid bacterial strain, Enterococcus faecalis PR31, to directly ferment and digest both porcine and equine placentas, we produced the following liquid products: placenta ferment filtrates. The ferment filtrates stimulated the proliferation of not only normal human dermal fibroblasts but also epidermal keratinocytes. The ferments had higher equilibrium water content properties than traditional placental extracts, and the ferment derived from the porcine placenta maintained high stratum corneum water content levels for up to 6 h after its application on the skin. Metabolome analysis revealed various molecules that were increased by fermentation, among which lactic acid was assumed to play a central role in the high moisturizing properties. To conclude, the placenta ferment filtrates developed in this study are beneficial for cosmetic applications.

Microbial Profiling of Amniotic Fluid, Umbilical Blood and Placenta of the Foaling Mare.

The presence of a microbiome/microbiota in the placenta is hotly debated. In previous studies, the presence of bacteria in equine amniotic fluid and umbilical blood was independent of foal health. The objective of the present study was to determine if the same bacteria are present in the equine placenta as in amniotic fluid and umbilical blood. Samples were obtained from 24 parturient mares and foals. Placental bacterial DNA was extracted, and the microbiome was identified using 16S rRNA sequencing. All amniotic fluid samples contained some polymorphonucleocytes; bacteria were isolated from four samples. Aerobic or anaerobic growth was found in 18 and 3 umbilical blood samples, respectively. Serum amyloid A was <5 mg/L in all 24 samples, total WBC varied between 2900 and 10,700/µL, and fibrinogen varied between 0 and 5.16 g/L. In jugular blood, serum amyloid A was <5 mg/L in all 24 foals, total white blood count was 3200 to 8100/µL, and fibrinogen was 0.44 to 4.42 g/L. The diversity of bacterial microbiota was similar in all placental regions at the phylum level but differed at the genus level; the most abundant phyla were Proteobacteria (42-46.26%) and Actinobacteria (26.91-29.96%). In conclusion, bacteria were found in the fetal compartments and placenta of healthy equine pregnancies; however, we can neither prove nor disprove the hypothesis that the placenta has its own microbiome.

Metagenetic Analysis of the Pregnant Microbiome in Horses

Placentitis is the leading cause of infectious abortion in the horse. Additionally, it can result in weak and/or growth restricted offspring. While the etiology of ascending placentitis is well described in mares, less is known regarding the pathogenesis of other types, such as nocardioform placentitis. This study aims to identify the microbial communities in different body sites of the pregnant mare in early gestation to establish a core microbiome that may be perturbed in pathologic pregnancies such as placentitis. We hypothesize that the equine placenta harbors a distinct resident microbiome in early pregnancy when characterized by metagenetics and that there will be a disparity in bacterial communities from the oral, vaginal, and fecal microbiome. Samples were collected from the oral cavity, vagina, anus, and the allantoic portion of the allantochorion (“placenta”) from five pregnant mares between 96 and 120 days of gestation. The V4 region of the 16S rRNA gene was amplified for Illumina MiSeq sequencing to examine core bacterial communities present in the different body sites. Microbial community composition of the pregnant ponies by body site was significantly different (Bray–Curtis dissimilarity). The placenta was significantly different from the feces, oral cavity, and vagina. Alpha diversity measuring the Shannon diversity matrix was significant, with the body sites being a compounding variable, meaning there was a difference in richness and evenness in the different microbial communities. Feces had the greatest alpha diversity, while the oral cavity and placenta similarly had the least. In conclusion, metagenetics did reveal distinct community differences in the oral, fecal, vaginal, and placenta cavities of the horse. The equine placenta does show similarities in its microbial communities to the oral cavity. Further research needs to be completed to investigate how bacteria may be translocated to the placenta from these other body sites and how they contribute to the development of placentitis.

Epigenetic regulation of collagen in the mare's placenta: putative relationship with maternal age

Placental plasticity influences fetal development, pregnancy success, and foal growth. Older fertile mares deliver heavier foals and placentas, with increased collagen deposition. In humans, epigenetics regulates placental development and function. Epigenetics, through DNA methylation, also modulates endometrial fibrosis. As such, we hypothesized that as mares get older, collagen deposition in the placenta might be epigenetically modulated. This study assessed if transcription of collagen genes in the equine placenta was epigenetically modulated through DNA methyltransferases (DNMTs), and affected by aging. Immediately after foaling, samples from different chorioallantoic areas (gravid horn, non-gravid horn, and body) were collected from young (n=10; 4-6 years) and older mares (n=10; 12-18 years). Gene transcription of collagen type 1 (COL1A1), collagen type 3 (COL3A1), and DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) was determined (qRT-PCR). Unpaired T-test and Mann-Whitney test were used with statistical significance set a p <0.05. In older mares, COL1A1 mRNA levels were higher in the gravid than in the non-gravid horn (p<0.05), and also upregulated in comparison to young mares’ placentas (p<0.05). In young mares, these transcripts were increased in the placenta body in comparison to the gravid (p<0.05) and non-gravid horns (p<0.01). In older mares, COL3A1 transcriptionwas higher in gravid and non-gravid horns, with respect to the same placenta regions in young mares (p<0.05). In older mares, DNMT1 mRNA expression in the body was lower than in younger mares (p<0.05). For all placentas, DNMT1 transcripts in the non-gravid horn were lowest, compared to the gravid horn (p<0.01) and body (p<0.001). In young mares, DNMT1 mRNA expression in the body was also higher than in the gravid horn (p<0.05). With mares aging, there was a downregulation of DNMT3A transcripts in gravid and non-gravid placenta horns, compared to young mares’ placentas (p< 0.05). In older mares, its levels were lowest in the gravid horn, compared to the non-gravid horn (p<0.05) and body (p<0.01). Similarly, in older mares’ placentas, DNMT3B transcripts were downregulated in the gravid horn (p<0.05) and placenta body (p<0.001), compared to young mares. DNMT3B mRNA levels were lowest in the non-gravid horn, both in young mares, in comparison to placenta body (p<0.01) and in older mares, with respect to the gravid horn and body (p<0.01). Considering mares aging, hypomethylation indicated by decreased DNMTs transcripts, in parallel with upregulation of collagen gene expression in gravid and non-gravid horns, may suggest epigenetic involvement on placental plasticity. However, further research is needed to unravel the intricacies of epigenetic modulation of collagen in equine placentas.

An in vitro model for equine placental research

A fundamental understanding of placental development and its associated pathologies is crucial to mitigating the losses associated with placental disorders. Organoids are widely used to investigate the developmental biology of organs and in disease modeling. In humans, placental organoids have been successfully developed using trophoblast cells. In this study, we aimed to 1) develop placental organoids using pre-term and term equine placentae, 2) evaluate the organoid structures, 3) compare the organoid transcriptome with their respective chorioallantois (CA), and 4) evaluate organoids based on their starting material (pre-term vs term). The CA was collected from healthy placentae following elective abortion (80-100d of gestation) and following spontaneous term parturition. CA samples were processed for culture, fixed for histology, and stored in RNAlater. For culture, each CA sample was enzymatically digested to a single cell suspension and was seeded into 50µL Matrigel Matrix droplets surrounded by medium. Passage-3 organoids (P3) were used for histology (H&E), immunohistochemistry (IHC), and transcriptomic analysis. Steroid production and conversion were measured by mass spectrometry (MS) of culture media. After optimization of culture conditions, we produced 3D-organoids (Pre-Term, n=6 and Term, n=3). Organoids were passaged up to 12 times over ±3 months and were successfully frozen and re-cultured post-thaw. Total extracted RNA from initial CA samples and organoids was sequenced (150PE; ∼43 million reads/sample). The genes expressed in the organoidsclosely resemble those of the tissue of origin, 19.4% of the expressed genes were differentially expressed (DEGs) between the initial CA and P3 (n=3,949 genes; FDR<0.001). H&E staining of both CA and the organoids indicates the epithelial origin of the cells within the organoids. The enriched expression of trophoblast-specific genes PLAC8B, GATA3, KRT7, VGLL1, and EGFR, and the presence of the trophoblast-specific genes TFAP2A and TFAP2C, and GATA2 in the organoids confirm their trophoblast identity. The presence of SOX2 and OCT4, as demonstrated by IHC, confirms the pluripotency of the organoid cells. The SRD5A1 and SRD5A3 genes were expressed in all organoids and our preliminary MS analysis demonstrated the conversion of progesterone to 5α-DHP, further suggesting functional integrity. Comparison between Pre-Term and Term organoids revealed 552 DEGs (FDR<0.001), with downregulation of immune/inflammatory/defense responses and blood vessel development, and upregulation of molecule transport, development, and adhesion in the Pre-Term group. To conclude, this is the first report of placental organoids in domestic animals. The organoids show structural, transcriptomic, and functional similarities to the equine placenta in vivo and consequently could be used as an in vitro model to study placental development and associated pathologies. Funded by The Foundation for The Horse, GJCRF, UGent BOF, BAEF, L'Oréal-UNESCO FWO, and the Center for Equine Health at UC Davis.

Ezrin immunoexpression in trophoblast cells of the equine placenta

Ezrin expression in microcotyledons and trophoblastic cells plays an important role in vascularization and maternal-fetal exchange. Ezrin is a membrane cytoskeletal crosslinker protein that helps regulate several cytoskeletal-related functions, including cell adhesion (Souza.et.al. Research in Veterinary Science.2023. 154: 84-88). Here, we describe the histopathological findings and expression as detected by an ezrin-specific antibody in pregnant and non-pregnant uterine horns of normal equine placentas. Eighteen placentas from embryo recipient mares of the Mangalarga Machador breed were collected at delivery (n=18; age 4-8 years). All placentas were evaluated for macroscopic characteristics. As part of the histopathological evaluation, pregnant and non-pregnant horn samples underwent hematoxylin-eosin staining to grade and compare the arrangement and vascularity of microcotyledons and allantochorions. Silanized slides were immunostained with an ezrin-specific antibody (clone: H-276; rabbit polyclonal-IgG; Santa Cruz; dilution: 1:500μl; LSAB+ Dako, Carpenteria, California, USA). A sample of human breast ductal adenocarcinoma was used as the positive control. For the negative control, the primaryantibody was replaced with a primary antibody isotype. In the microscopic analysis, the microcotyledonary arrangement in the pregnant horn was more intense than in the non-pregnant horn, except for one case. In terms of vascularization, the horns did not differ in the number or intensity of vessels in the microcotyledons and allantochorion. Ezrin antibody expression in placental horns was more intense in the proximal region of the microcotyledons compared with the basal lamina of the allantochorion and was diffused throughout the microcotyledonary network. The pregnant horn showed greater ezrin expression in trophoblasts compared to the non-pregnant horn. The two horn types showed similar color intensity in most samples however, ezrin was expressed mildly in the pregnant horn, while the non-pregnant horn from the same placenta showed moderate to intense ezrin expression. These observations may reflect a way of modulating maternal–fetal exchange, because trophoblast cells have this function. In summary, ezrin was expressed in trophoblast cells located in the microcotyledons of equine placentas, suggesting that this protein may play a role at the maternal–fetal interface.

Characterization of the equine placental microbial population in healthy pregnancies

In spite of controversy, recent studies present evidence that a microbiome is present in the human placenta. However, there is limited information about a potential equine placental microbiome. In the present study, we characterized the microbial population in the equine placenta (chorioallantois) of healthy prepartum (280 days of gestation, n = 6) and postpartum (immediately after foaling, 351 days of gestation, n = 11) mares, using 16S rDNA sequencing (rDNA-seq). In both groups, the majority of bacteria belonged to the phyla Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidota. The five most abundant genera were Bradyrhizobium, an unclassified Pseudonocardiaceae, Acinetobacter, Pantoea, and an unclassified Microbacteriaceae. Alpha diversity (p < 0.05) and beta diversity (p < 0.01) were significantly different between pre- and postpartum samples. Additionally, the abundance of 7 phyla and 55 genera was significantly different between pre- and postpartum samples. These differences suggest an effect of the caudal reproductive tract microbiome on the postpartum placental microbial DNA composition, since the passage of the placenta through the cervix and vagina during normal parturition had a significant influence on the composition of the bacteria found in the placenta when using 16S rDNA-seq. These data support the hypothesis that bacterial DNA is present in healthy equine placentas and opens the possibility for further exploration of the impact of the placental microbiome on fetal development and pregnancy outcome.

Dynamics of the Equine Placental DNA Methylome and Transcriptome from Mid- to Late Gestation.

The placenta is a temporary organ that is essential for the survival of the fetus, with a lifelong effect on the health of both the offspring and the dam. The functions of the placenta are controlled by its dynamic gene expression during gestation. In this study, we aimed to investigate the equine placental DNA methylome as one of the fundamental mechanisms that controls the gene expression dynamic. Chorioallantois samples from four (4M), six (6M), and ten (10M) months of gestation were used to map the methylation pattern of the placenta. Globally, methylation levels increased toward the end of gestation. We identified 921 differentially methylated regions (DMRs) between 4M and 6M, 1225 DMRs between 4M and 10M, and 1026 DMRs between 6M and 10M. A total of 817 genes carried DMRs comparing 4M and 6M, 978 comparing 4M and 10M, and 804 comparing 6M and 10M. We compared the transcriptomes between the samples and found 1381 differentially expressed genes (DEGs) when comparing 4M and 6M, 1428 DEGs between 4M and 10M, and 741 DEGs between 6M and 10M. Finally, we overlapped the DEGs and genes carrying DMRs (DMRs-DEGs). Genes exhibiting (a) higher expression, low methylation and (b) low expression, high methylation at different time points were identified. The majority of these DMRs-DEGs were located in introns (48.4%), promoters (25.8%), and exons (17.7%) and were involved in changes in the extracellular matrix; regulation of epithelial cell migration; vascularization; and regulation of minerals, glucose, and metabolites, among other factors. Overall, this is the first report highlighting the dynamics in the equine placenta methylome during normal pregnancy. The findings presented serve as a foundation for future studies on the impact of abnormal methylation on the outcomes of equine pregnancies.

Markers of equine placental differentiation: insights from gene expression studies.

Development and the subsequent function of the fetal membranes of the equine placenta require both complex and precise regulation of gene expression. Advancements in recent years in bioinformatic techniques have allowed more extensive analyses into gene expression than ever before. This review starts by combining publically available transcriptomic data sets obtained from a range of embryonic, placental and maternal tissues, with previous knowledge of equine placental development and physiology, to gain insights into key gene families relevant to placentation in the horse. Covering the whole of pregnancy, the review covers trophectoderm, yolk sac, chorionic girdle cells, allantoamnion and allantochorion. In particular, 182 predicted 'early high impact' genes were identified (>100 transcripts per million (TPM) and >100 fold-change) that distinguish between progenitor trophectoderm, chorionic girdle tissue and allantochorion. Furthermore, 71 genes were identified as enriched in placental tissues (placental TPM > 10, with minimal expression in 12 non-placental TPM < 1), including excellent candidates for functional studies such as IGF1, apolipoproteins, VGLL1, GCM1, CDX2 and FABP4. It is pertinent that future studies should focus on single-cell transcriptomic approaches in order to determine how these changes in gene expression relate to tissue composition and start to better define trophoblast subpopulations in the equine placenta. Future functional characterisation of these genes and pathways will also be key not only to understanding normal placental development and fetal health but also their potential role in pathologies of pregnancy.

Effects of oral administration of equine placental extract supplement on the facial skin of healthy adult women: A randomized, double-blind, placebo-controlled study.

IntroductionPlacenta extract is used as an ingredient in ointments for treating dermatological diseases, skin dryness, and for skin beautification. However, the clinical effects of the equine placenta on humans and the underlying mechanism of action are unclear. This randomized, controlled, double‐blind study aimed to clinically evaluate the effect of oral intake of equine placental extract on human skin quality.MethodsHealthy women volunteers between the ages of 30 and 59 years (n = 29) were randomly assigned to receive 220 mg of equine placental extract–placebo orally, once daily for 4 weeks. Skin quality parameters such as skin hydration, skin barrier function (transepidermal water loss [TEWL]), and melanin index were assessed at baseline and after 4 weeks of administration.ResultsThe melanin index was significantly increased in the placebo group, whereas it remained unchanged in the equine placenta group. The pattern of melanin index change was significantly different due to intake or no intake of equine placenta supplements over 4 weeks. No significant difference was found in skin hydration and TEWL between the two groups at 4 weeks of postadministration. It was shown that the intake of the equine placenta was more effective in protecting the skin condition against the change of ultraviolet (UV) sensitively than the change in temperature and humidity.ConclusionsEffect of equine placental extract intake was evident on the cheek skin of the equine placenta group where participants were protected from UV‐induced pigmentation. Equine placental extract is useful for decreasing melanin synthesis and melanin content in the human skin and can be used as an effective food supplement to maintain human skin quality.

Estrogens Regulate Placental Angiogenesis in Horses.

A sufficient vascular network within the feto-maternal interface is necessary for placental function. Several pregnancy abnormalities have been associated with abnormal vascular formations in the placenta. We hypothesized that growth and expansion of the placental vascular network in the equine (Equus caballus) placenta is regulated by estrogens (estrogen family hormones), a hormone with a high circulating concentration during equine gestation. Administration of letrozole, a potent and specific inhibitor of aromatase, during the first trimester (D30 to D118), decreased circulatory estrone sulfate concentrations, increased circulatory testosterone and androstenedione concentrations, and tended to reduce the weight of the fetus (p < 0.1). Moreover, the gene expression of CYP17A1 was increased, and the expression of androgen receptor was decreased in the D120 chorioallantois (CA) of letrozole-treated mares in comparison to that of the control mares. We also found that at D120, the number of vessels tended to decrease in the CAs with letrozole treatment (p = 0.07). In addition, expression of a subset of angiogenic genes, such as ANGPT1, VEGF, and NOS2, were altered in the CAs of letrozole-treated mares. We further demonstrated that 17β-estradiol increases the expression of ANGPT1 and VEGF and increases the angiogenic activity of equine endothelial cells in vitro. Our results from the estrogen-suppressed group demonstrated an impaired placental vascular network, suggesting an estrogen-dependent vasculogenesis in the equine CA during the first trimester.

Differential Expression Pattern of Retroviral Envelope Gene in the Equine Placenta.

Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses, which have coevolved with vertebrate genomes for millions of years. The conservation of ERV genes throughout evolution suggests their beneficial effects on their hosts' survival. An example of such positive selection is demonstrated by the syncytin gene, which encodes a protein with affinity for various mammalian placentas that is involved in the formation of syncytiotrophoblasts. Although the horse has an epitheliochorial placenta, in which the fetal trophoblasts are simply apposed to the intact uterine epithelium, we have previously demonstrated that the equine ERV (EqERV) env RNA is unexpectedly expressed in placental tissue. In the present study, we investigated the mRNA expression pattern of the EqERV env gene in different parts of the equine placenta, to gain more insight into its putative role in the fetal–maternal relationship. To this end, we used reverse transcription–quantitative PCR (RT–qPCR) and in situ hybridization assays to analyze different target areas of the equine placenta. The retroviral env gene is expressed in the equine placenta, even though there is no syncytium or erosion of the uterine endometrium. The gene is also expressed in all the sampled areas, although with some quantitative differences. We suggest that these differences are attributable to variations in the density, height, and degree of morphological complexity of the chorionic villi forming the microcotyledons. The involvement of the EqERV env gene in different functional pathways affecting the fetus–mother relationship can be hypothesized.

Parental bias in expression and interaction of genes in the equine placenta

Most autosomal genes in the placenta show a biallelic expression pattern. However, some genes exhibit allele-specific transcription depending on the parental origin of the chromosomes on which the copy of the gene resides. Parentally expressed genes are involved in the reciprocal interaction between maternal and paternal genes, coordinating the allocation of resources between fetus and mother. One of the main challenges of studying parental-specific allelic expression (allele-specific expression [ASE]) in the placenta is the maternal cellular remnant at the fetomaternal interface. Horses (Equus caballus) have an epitheliochorial placenta in which both the endometrial epithelium and the epithelium of the chorionic villi are juxtaposed with minimal extension into the uterine mucosa, yet there is no information available on the allelic gene expression of equine chorioallantois (CA). In the current study, we present a dataset of 1,336 genes showing ASE in the equine CA (https://pouya-dini.github.io/equine-gene-db/) along with a workflow for analyzing ASE genes. We further identified 254 potentially imprinted genes among the parentally expressed genes in the equine CA and evaluated the expression pattern of these genes throughout gestation. Our gene ontology analysis implies that maternally expressed genes tend to decrease the length of gestation, while paternally expressed genes extend the length of gestation. This study provides fundamental information regarding parental gene expression during equine pregnancy, a species with a negligible amount of maternal cellular remnant in its placenta. This information will provide the basis for a better understanding of the role of parental gene expression in the placenta during gestation.

Characterization of Nuclear Progesterone Receptor Isoforms in the Term Equine Placenta.

In equine parturition, the role of progestins along with the nuclear progesterone receptor (nPR) signaling pathway in the placenta is not completely clarified. The progestins play an integral role in maintaining myometrial quiescence during the late stage of pregnancy via acting on nPR isoforms (PRA and PRB; PRB is more active than PRA). The current study aimed to determine the PRA and PRB expressions in the term equine placenta at the gene and protein levels. Six term equine placentas were used in this study. Reverse transcription polymerase chain reaction (RT-PCR) was used to quantify the mRNA expression for PRA and PRB. The protein expression was detected using the Western Blot technique. The results revealed that the mRNA and protein expressions for PRA were significantly higher (P < 0.0001) in the term equine placental tissue compared to the mRNA and protein expressions of PRB. These results demonstrated that nPRs are detectable in the term placenta of mares and PRA is the dominant isoform expressed. The present findings raised the possibility that the PRA plays an important role in the parturition process and expulsion of the placenta in mares.

Placentation in Equids.

This chapter focuses on the early stages of placental development in horses and their relatives in the genus Equus and highlights unique features of equid reproductive biology. The equine placenta is classified as a noninvasive, epitheliochorial type. However, equids have evolved a minor component of invasive trophoblast, the chorionic girdle and endometrial cups, which links the equine placenta with the highly invasive hemochorial placentae of rodents and, particularly, with the primate placenta. Two types of fetus-to-mother signaling in equine pregnancy are mediated by the invasive equine trophoblast cells. First, endocrinological signaling mediated by equine chorionic gonadotrophin (eCG) drives maternal progesterone production to support the equine conceptus between days 40 and 100 of gestation. Only in primates and equids does the placenta produce a gonadotrophin, but the evolutionary paths taken by these two groups of mammals to produce this placental signal were very different. Second, florid expression of paternal major histocompatibility complex (MHC) class I molecules by invading chorionic girdle cells stimulates strong maternal anti-fetal antibody responses that may play a role in the development of immunological tolerance that protects the conceptus from destruction by the maternal immune system. In humans, invasive extravillous trophoblasts also express MHC class I molecules, but the loci involved, and their likely function, are different from those of the horse. Comparison of the cellular and molecular events in these disparate species provides outstanding examples of convergent evolution and co-option in mammalian pregnancy and highlights how studies of the equine placenta have produced new insights into reproductive strategies.

Genomics, transcriptomics and proteomics of the normal and abnormal equine placenta: A better understanding of late gestational function and dysfunction

Genomics, transcriptomics and proteomics of the normal and abnormal equine placenta: A better understanding of late gestational function and dysfunction