Scanning electron microscopy of the surface epithelium of the bovine endometrium.

The surface epithelium of the bovine endometrium comprises at least 2 cell types (ciliated cells and secretory cells with microvilli), but their distribution and morphological changes over the estrous cycle are poorly understood. The objective was to quantify the number of ciliated cells and assess morphological changes in secretory cells on the uterine surface epithelium during the estrous cycle. Caruncular endometrium (CAR) and intercaruncular endometrium (ICAR) samples were collected from the uterine body, the horn ipsilateral to the corpus luteum or dominant follicle (H-CL/DF), and the horn contralateral to the corpus luteum or dominant follicle (H-NCL/NDF) from heifers following slaughter on d 0 (estrus; n = 5) or d 14 (mid-luteal phase; n = 5) of the estrous cycle. Samples were prepared for scanning electron microscopy at 1,000× magnification. Four to 10 fields (256 × 225 µm) for each sample were examined (n = 567 images). The number of ciliated cells was counted and the surface was scored for the morphology of the secretory cells (0 = absence of microvilli on surface; 3 = 100% of surface covered with microvilli). Ciliated cells were present in both the CAR and ICAR regions. The number of ciliated cells per field increased from d 0 to 14 in CAR and decreased from d 0 to14 in ICAR. The scanning electron microscopy revealed a general lack of uniformity in the lawn of microvilli on the surface of the endometrium. Based on the scores, approximately 25% of the fields had a surface that was <50% covered by microvilli. Depletion of microvilli may be explained by a normal process where apical protrusions are formed and either regress back into the cell surface or break to release their contents into the uterine lumen. These studies support the hypothesis that the surface of the luminal epithelium changes during the estrous cycle through a process that involves remodeling of the apical surface. The morphology of the apical surface may have a key role in governing pregnancy establishment.

Global transcriptomic response of bovine endometrium to blastocyst-stage embryos.

The aims of this study were (i) to investigate changes in the global transcriptome of bovine endometrial explants induced by exposure to blastocysts, (ii) to investigate if male and female blastocysts elicit a differential response in the endometrial transcriptome in vitro and (iii) to determine whether bovine endometrium responds to the presence of murine embryos. In Experiment 1, endometrial explants from the same uterus were cultured for 6 h with or without 20 in vitro-produced bovine blastocysts. In Experiment 2, endometrial explants were cultured with male or female bovine blastocysts produced in vitro by IVF either using sex-sorted semen or conventional unsorted semen followed by embryo sexing based on a biopsy. In Experiment 3, endometrial explants were cultured alone or in the presence of bovine blastocysts (n = 25) or murine blastocysts (n = 25). Following culture, explants were snap frozen and stored at -80°C until RNA extraction, qPCR or RNA-Seq. Culture with bovine blastocysts increased endometrial expression of 40 transcripts, all of which were interferon-tau induced. Culture with male or female bovine blastocysts increased transcript abundance of five classic interferon-stimulated genes (MX1, MX2, ISG15, OASY1, RSAD2) in explants; however, there was no difference in abundance of transcripts previously reported to be related to embryonic sex (IFNAR1, IFNAR2, CTGF, ARTN, SLC2A1, SLC2A5). Exposure to murine blastocysts did not elicit any detectable change in transcript abundance. These findings, coupled with our previous data, indicate that very local, interferon-tau-induced changes in endometrial gene expression occur in response to blastocysts; whether such changes play any role in subsequent pregnancy recognition remains to be established.

Genome-wide association analysis and gene set enrichment analysis with SNP data identify genes associated with 305-day milk yield in Holstein dairy cows.

Milk production traits, such as 305-day milk yield (305MY), have been under direct selection to improve production in dairy cows. Over the past 50 years, the average milk yield has nearly doubled, and over 56% of the increase is attributable to genetic improvement. As such, additional improvements in milk yield are still possible as new loci are identified. The objectives of this study were to detect SNPs and gene sets associated with 305MY in order to identify new candidate genes contributing to variation in milk production. A population of 781 primiparous Holstein cows from six central Washington dairies with records of 305MY and energy corrected milk were used to perform a genome-wide association analysis (GWAA) using the Illumina BovineHD BeadChip (777 962 SNPs) to identify QTL associated with 305MY (P < 1.0 × 10-5 ). A gene set enrichment analysis with SNP data (GSEA-SNP) was performed to identify gene sets (normalized enrichment score > 3.0) and leading edge genes (LEGs) influencing 305MY. The GWAA identified three QTL comprising 34 SNPs and 30 positional candidate genes. In the GSEA-SNP, five gene sets with 58 unique and 24 shared LEGs contributed to 305MY. Identification of QTL and LEGs associated with 305MY can provide additional targets for genomic selection to continue to improve 305MY in dairy cattle.

Insights into conceptus elongation and establishment of pregnancy in ruminants.

This review integrates established and new information on the factors and pathways regulating conceptus-endometrial interactions, conceptus elongation and establishment of pregnancy in sheep and cattle. Establishment of pregnancy in domestic ruminants begins at the conceptus stage (embryo or fetus and associated extra-embryonic membranes) and includes pregnancy recognition signalling, implantation and the onset of placentation. Survival and growth of the preimplantation blastocyst and elongating conceptus require embryotrophic factors (amino acids, carbohydrates, proteins, lipids and other substances) provided by the uterus. The coordinated and interactive actions of ovarian progesterone and conceptus-derived factors (interferon-τ and prostaglandins) regulate expression of elongation- and implantation-related genes in the endometrial epithelia that alter the uterine luminal milieu and affect trophectoderm proliferation, migration, attachment, differentiation and function. A comparison of sheep and cattle finds both conserved and non-conserved embryotrophic factors in the uterus; however, the overall biological pathways governing conceptus elongation and establishment of pregnancy are likely conserved. Given that most pregnancy losses in ruminants occur during the first month of pregnancy, increased knowledge is necessary to understand why and provide a basis for new strategies to improve pregnancy outcome and reproductive efficiency.

Review: Implantation and placentation in ruminants.

Ruminants have a unique placenta in comparison to other mammalian species. Initially, they possess a non-invasive epitheliochorial type of placenta during conceptus elongation. As the conceptus trophectoderm begins to attach to the luminal epithelium (LE) of the endometrium, binucleate cells (BNCs) develop within the trophoblast of the chorion. The BNCs migrate and fuse with the uterine LE to form multinucleate syncytial plaques in sheep and hybrid trinucleate cells in cattle. This area of the ruminant placenta is semi-invasive synepitheliochorial. The BNCs form the foundation of the placental cotyledons and express unique placenta-specific genes including pregnancy-associated glycoproteins and chorionic somatomammotropin hormone 2 or placental lactogen. Attachment and interdigitation of cotyledons into endometrial caruncles form placentomes that are subsequently vascularized to provide essential nutrients for growth of the fetus. This chapter review will discuss historical and current aspects of conceptus implantation and placenta development in ruminant ungulates with a focus on cattle and sheep. Single-cell analysis promises to provide a much more detailed understanding of the different cell populations and insights into pathways mediating trophoblast and placenta. This fundamental is required to understand pregnancy loss and develop strategies to improve pregnancy outcomes in ruminants.

Impact of postpartum metritis on the regeneration of endometrial glands in dairy cows.

The postpartum uterus involutes to its pre-pregnant and fully functional state within approximately 60 d after calving. Uterine glands are essential for fertility but little is known about their regeneration postpartum. Likewise, the effect of uterine disease (metritis) on gland regeneration is unknown. We hypothesized that uterine glands would be regenerated early postpartum and that metritis would be associated with slower gland regeneration to affect their numbers later postpartum during the breeding period. Postpartum dairy cows were diagnosed as healthy (n = 17 and 9 for experiment [Exp.] 1 and 2) or metritis (n = 17 and 10 for Exp. 1 and 2, respectively) at 7 to 10 d postpartum. Cows were slaughtered at approximately 1 mo (Exp. 1) or approximately 80 or 165 d (Exp. 2) postpartum for the collection of the uterus. Uterine tissue was sectioned and the number of glandular cross-sections per unit area was counted and cross-sectional area measured. Cellular proliferation within the luminal epithelium (LE) and glandular epithelium (GE) was quantified by MKI67 (marker of cellular proliferation) immunohistochemistry. In early postpartum cows (Exp. 1), the greatest amount of MKI67 staining was found in the deep endometrium (cells closest to the myometrium). Cows with purulent material in the uterine lumen at d 30 slaughter (Exp. 1) had fewer endometrial glands per unit area in the deep and middle endometrium when compared with nonpurulent cows. The MKI67 staining was less in the deep endometrial GE and LE for purulent compared with nonpurulent cows. Estrus cyclicity was associated with a greater number of gland cross-sections in the deep and middle endometrium. Later postpartum (80 and 165 d; Exp. 2), there was greater glandular development compared with Exp. 1 and a tendency for a lesser number of gland cross-sections per unit area in diseased cows without an effect on MKI67 staining in the GE or LE. We conclude that uterine disease slows the development of uterine glands early postpartum (by 1 mo) through a mechanism that involves cellular proliferation within the GE. The impact of the early postpartum disease on glandular development later postpartum (Exp. 2) appeared to be less. Additional time, therefore, may allow recovery of the GE in later postpartum cows.

Influence of sire fertility status on conceptus-induced transcriptomic response of the bovine endometrium.

The aim was to examine the effect of sire fertility status on conceptus-induced changes in the bovine endometrial transcriptome. To generate elongated conceptuses, Day 7 blastocysts produced in vitro using frozen-thawed sperm from Holstein Friesian bulls (3 High fertility, HF and 3 Low fertility, LF) were transferred in groups of 5-10 into synchronized heifers (n = 7 heifers per bull) and recovered following slaughter on Day 15. Day 15 endometrial explants recovered from the uterine horn ipsilateral to the corpus luteum were recovered from synchronized cyclic heifers (n = 4). Explants from each heifer were co-cultured for 6 h in RPMI medium alone (Control) or with 100 ng/ml ovine recombinant interferon tau (IFNT) or with a single conceptus from each HF or LF bull. After 6 h, explants were snap frozen and stored at -80°C. Extracted mRNA was subjected to RNA-seq and the resulting data were analyzed with R software. The numbers of differentially expressed genes (DEG; FDR<0.05) were: HF vs. Control: 956; LF vs. Control: 1021; IFNT vs. Control: 1301; HF vs. LF: 2. Unsurprisingly, the majority of DEG (658) were common to all comparisons and were related to IFNT-induced changes in the endometrium. Prior to applying the adjusted p-value, there were 700 DEG between HF and LF, with 191 and 509 genes more expressed in HF or LF, respectively (p < 0.05). Overrepresentation analysis of KEGG pathways (FDR<0.05), revealed that DEG with higher expression in LF were involved in cell cycle and proteolysis, while those upregulated DEG by HF conceptuses were strongly associated with immune process pathways, such as TNF, NF-kappa B, cytokine-cytokine receptor interaction, and TLR signaling. These pathways were also enriched by DEG upregulated by IFNT compared to the Control. Furthermore, only the HF, and not the LF group, affected the expression of most genes in these pathways (p < 0.05) according to a negative binomial regression model. Finally, a weighted gene co-expression network analysis revealed two clusters of co-expressed genes associated with the HF conceptuses (p < 0.05), which were also enriched for the aforementioned pathways. In conclusion, HF conceptuses, similar to IFNT treatment, stimulated multiple pathways involved in immune response, which were apparently not affected by LF conceptuses.

Conceptus-induced changes in the endometrial transcriptome: how soon does the cow know she is pregnant?

This study sought to determine the earliest response of the bovine uterine endometrium to the presence of the conceptus at key developmental stages of early pregnancy. There were no detectable differences in gene expression in endometria from pregnant and cyclic heifers on Days 5, 7, and 13 postestrus, but the expression of 764 genes was altered due to the presence of the conceptus at maternal recognition of pregnancy (Day 16). Of these 514 genes, MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD9, EIF4E, and IFIT2 increased to the greatest extent in pregnant endometria (>8-fold log2 fold change increase). The expression of OXTR, Bt.643 (unofficial symbol), and KCNMA1 was reduced the most, but short-term treatment with recombinant ovine interferon tau (IFNT) in vitro or in vivo did not alter their expression. In vivo intrauterine infusion of IFNT induced the expression of EIF4E, IFIT2, IFI44, ISG20, MX2, RSAD2, SAMD9, and USP18. These results revealed for the first time that changes that occur in the endometrial transcriptome are independent of the presence of a conceptus until pregnancy recognition. The differentially expressed genes (including MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD, and EIF4E) are a consequence of IFNT production by the conceptus. The identified genes represent known and novel early markers of conceptus development and/or return to cyclicity and may be useful to identify the earliest stage at which the endometrial response to the conceptus is detectable.

Effect of pregnancy and progesterone concentration on expression of genes encoding for transporters or secreted proteins in the bovine endometrium.

The objective of this study was to determine the temporal and spatial expression patterns of genes encoding transporters, as well as selected secreted proteins that may be regulated by progesterone (P4) and/or the presence of the conceptus in the bovine endometrium. Estrus-synchronized beef heifers were randomly assigned to either: 1) pregnant, high P4; 2) pregnant, normal P4; 3) cyclic, high P4; or 4) cyclic, normal P4. Uteri were collected on days 5, 7, 13, and 16 of the estrous cycle or pregnancy. Localization of mRNAs for ANPEP, CTGF, LPL, LTF, and SLC5A1 in the uteri was determined by radioactive in situ hybridization, and expression quantified in the endometria by quantitative real-time PCR. ANPEP localized to luminal (LE) and superficial glandular (sGE) epithelia of all heifers on days 5 and 7 only. SLC5A1 mRNA was detected in the LE and sGE on days 13 and 16 in all heifers, and expression increased on day 16 in pregnant groups. CTGF localized weakly to the LE and GE on days 5 and 7 but increased on days 13 and 16 with an increase (P < 0.05) in CTGF expression in high P4 (day 7) and pregnant heifers (day 16). Both LPL and LTF localized to the GE only on days 5 and 7. In conclusion we have characterized the temporal expression pattern of these genes and modulation of their transcript abundance by P4 (CTGF, LPL) and/or the conceptus (CTGF, SLC5A1) likely modifies the uterine microenvironment, enhancing histotroph composition and contributing to advanced conceptus elongation.

Loci associated with conception rate in crossbred beef heifers.

The inability of beef cattle to maintain full term pregnancies has become an economic concern for the beef industry. Herd management and nutritional improvements have alleviated environmental impacts on embryonic and fetal loss, yet additional gains can be made through genomic selection. The objectives of this study were to identify loci and gene-sets in crossbred beef heifers associated with the number of services required to become pregnant (TBRD) and heifer conception rate at first service (HCR1). Heifers (n = 709) from a commercial beef operation underwent one round of artificial insemination, before exposure to bulls for natural service for 50 days. Pregnancy and time of conception was determined by ultrasound 35 days after the breeding season. Heifers were genotyped using the GeneSeek (Lincoln, NE) Bovine GGP50K BeadChip prior to genome-wide association analyses (GWAA) conducted using an EIGENSTRAT-like model to identify loci associated (P < 1 × 10-5) with TBRD and HCR1. One locus was associated (P = 8.97 × 10-6) with TBRD on BTA19 and included the positional candidate gene ASIC2, which is differentially expressed in the endometrium of fertility classified heifers, and the positional candidate gene, SPACA3. Gene-set enrichment analyses using SNP (GSEA-SNP) data, was performed and identified one gene-set, oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen as enriched (NES = 3.15) with TBRD and contained nine leading edge genes that contributed to the enrichment of the gene set. The enriched gene-set is involved in catalyzing oxidation-reduction reactions, which have been associated with oxidative stressors impacting pregnancy success. No loci were associated nor gene-sets enriched with HCR1. Identification of loci, positional candidate genes, gene-sets and leading edge genes enriched for fertility facilitate genomic selection that allows producers to select for reproductively superior cattle, reduce costs associated with infertility, and increase percent calf crop.

Progesterone-regulated changes in endometrial gene expression contribute to advanced conceptus development in cattle.

The postovulatory rise in circulating progesterone (P4) concentrations is associated with increased pregnancy success in beef and dairy cattle. Our study objective was to determine how elevated P4 alters endometrial gene expression to advance conceptus development. Synchronized heifers were inseminated (Day 0) and randomly assigned to pregnant high P4 or to pregnant normal P4. All high P4 groups received a P4-release intravaginal device on Day 3 after insemination that increased P4 concentrations up to Day 7 (P < 0.05). Tissue was collected on Day 5, 7, 13, or 16 of pregnancy, and endometrial gene expression was analyzed using the bovine Affymetrix (Santa Clara, CA) microarrays. Microarray analyses demonstrated that the largest number of P4-regulated genes coincided with the day when the P4 profiles were different for the longest period. Genes with the largest fold change increase (such as DGAT2 and MSTN [also known as GDF8]) were associated with triglyceride synthesis and glucose transport, which can be utilized as an energy source for the developing embryo. Temporal changes occurred at different stages of early pregnancy, with the greatest difference occurring between well-separated stages of conceptus development. Validation of a number of genes by quantitative real-time PCR indicated that P4 supplementation advances endometrial gene expression by altering the time (FABP, DGAT2, and MSTN) or duration (CRYGS) of expression pattern for genes that contribute to the composition of histotroph.

Coevolution of endogenous betaretroviruses of sheep and their host.

Sheep betaretroviruses offer a unique model system to study the complex interaction between retroviruses and their host. Jaagsiekte sheep retrovirus (JSRV) is a pathogenic exogenous retrovirus and the causative agent of ovine pulmonary adenocarcinoma. The sheep genome contains at least 27 copies of endogenous retroviruses (enJSRVs) highly related to JSRV. enJSRVs have played several roles in the evolution of the domestic sheep as they are able to block the JSRV replication cycle and play a critical role in sheep conceptus development and placental morphogenesis. Available data strongly suggest that some dominant negative enJSRV proviruses (i.e. able to block JSRV replication) have been positively selected during evolution. Interestingly, viruses escaping the transdominant enJSRV loci have recently emerged (less than 200 years ago). Thus, endogenization of these retroviruses may still be occurring today. Therefore, sheep provide an exciting and unique system to study retrovirus-host coevolution. (Part of a multi-author review).

Pregnancy associated glycoproteins (PAGs) and pregnancy loss in high vs sub fertility heifers.

Reproductive inefficiency and infertility are major financial burdens to domestic livestock. Variables associated with these reproductive losses during early gestation include contributions from the oocyte, uterus, sperm, embryo and placenta. Bovine pregnancy associated glycoproteins (PAG) are produced by the binucleate cells of the ruminant placenta and can be used to diagnose pregnancy. Increased circulating concentrations of PAG early in gestation have been correlated with pregnancy success and decreased concentrations are predictive of impending embryonic mortality in both beef and dairy cattle. The objectives of the current study were to determine whether: 1) heifer fertility status is associated with circulating concentrations of PAG and pregnancy loss; and 2) PAG concentrations within the same animal are repeatable across multiple pregnancies. We hypothesized maternal PAG concentrations would be increased in high fertility compared to subfertile heifers but not repeatable across subsequent pregnancies in the same heifer. Serial embryo transfer (ET; n = 4 rounds) was used to classify predominately Angus heifers (n = 92) as highly fertile (HF = 30; 100% pregnancy success) or subfertile (SF = 62; average = 33%; range = 25-75% pregnancy success) based on day 28 ultrasound diagnosis. Blood samples were collected at both day 28 and 44 for quantification of circulating PAG concentrations by an in house PAG ELISA with antibodies raised against early secreted PAGs. Pregnancy was terminated at day 44 of gestation and heifers were allowed 30 days recovery before synchronization for the next ET. Only heifers that were diagnosed pregnant by ultrasound were used in this study (HF: n = 30, SF: n = 62). Serum concentrations of PAGs were not different between HF (5.90 ±â€¯0.27 ng/mL) and SF (5.56 ±â€¯0.31 ng/mL; P = 0.16) heifers at day 28 of gestation nor was there a difference at day 44 of gestation (P = 0.32). Subfertile heifers had increased pregnancy loss between days 28 and 44 of gestation. Based on odds ratio analysis, SF heifers had a 2.41 times chance to undergo pregnancy loss between day 28-44 compared to HF heifers (P < 0.05). There was no correlation (P > 0.05) in maternal circulating concentrations of PAG between pregnancies on day 28 or 44 of gestation in samples obtained from HF heifers. In summary, circulating concentrations of PAG are not different between HF and SF heifers; however, HF classified heifers have decreased pregnancy loss between days 28 and 44 of gestation.

Molecular cloning and characterization of prostaglandin (PG) transporter in ovine endometrium: role for multiple cell signaling pathways in transport of PGF2alpha.

In ruminants, endometrial prostaglandin F(2alpha) (PGF(2alpha)) is the luteolytic hormone. Cellular transport of PGF(2alpha) in the uterine endometrium is critical for regulation of the estrous cycle. Molecular mechanisms responsible for control of PGF(2alpha) transport in endometrium during luteolysis are largely unknown. In the present study, we characterized the prostaglandin transporter (PGT) in ovine endometrium. Ovine PGT cDNA consists of 1935 nucleotides that encode 644 amino acids. In ovine endometria, PGT is highly expressed during the period of luteolysis, between d 14 and 16 of the estrous cycle, in luminal and glandular epithelia. Pharmacological and genomic inhibition of PGT indicates that it is responsible for influx and efflux of PGF(2alpha) in ovine endometrial epithelial cells. Inhibition of PGT during the period of luteolysis prevents the release of oxytocin-induced PGF(2alpha) pulses, and maintains functional corpus luteum and its secretion of progesterone. In ovine endometrial epithelial cells, protein kinase A and protein kinase C pathways are involved in regulating the influx of PGF(2alpha), whereas epidermal growth factor receptor pathways are implicated in regulation of influx and efflux of PGF(2alpha.) The ERK1/2 pathway is associated with efflux of PGF(2alpha), whereas Jun-amino-terminal kinase/stress-activated protein kinase pathways are involved in both efflux and influx of PGF(2alpha.) Phosphatidylinositol 3-kinase pathways are not involved in either influx or efflux of PGF(2alpha) in ovine endometrial epithelial cells. These are the first results to demonstrate a functional role for PGT in regulation of PGF(2alpha) efflux and influx in ovine endometrial cells that influence luteolytic mechanisms in ruminants.

Proline metabolism in the conceptus: implications for fetal growth and development.

Although there are published studies of proline biochemistry and nutrition in cultured cells and postnatal animals, little is known about proline metabolism and function in the conceptus (embryo/fetus, associated placental membranes, and fetal fluids). Because of the invasive nature of biochemical research on placental and fetal growth, animal models are often used to test hypotheses of biological importance. Recent evidence from studies with pigs and sheep shows that proline is a major substrate for polyamine synthesis via proline oxidase, ornithine aminotransferase, and ornithine decarboxylase in placentae. Both porcine and ovine placentae have a high capacity for proline catabolism and polyamine production. In addition, allantoic and amniotic fluids contain enzymes to convert proline into ornithine, which is delivered through the circulation to placental tissues. There is exquisite metabolic coordination among integrated pathways that support highest rates of polyamine synthesis and concentrations in placentae during early gestation when placental growth is most rapid. Interestingly, reduced placental and fetal growth are associated with reductions in placental proline transport, proline oxidase activity, and concentrations of polyamines in gestating dams with either naturally occurring or malnutrition-induced growth retardation. Conversely, increasing proline availability in maternal plasma through nutritional or pharmacological modulation in pigs and sheep enhances concentrations of proline and polyamines in placentae and fetal fluids, as well as fetal growth. These novel findings suggest an important role for proline in conceptus metabolism, growth and development, as well as a potential treatment for intrauterine growth restriction, which is a significant problem in both human medicine and animal agriculture.

WNTs in the ovine uterus: potential regulation of periimplantation ovine conceptus development.

WNTs (Wingless-type MMTV integration site family member) are involved in critical developmental and growth processes in animals. These studies investigated WNT pathways in the ovine uterus and conceptus during the periimplantation period of pregnancy. WNT2 and WNT2B mRNAs were detected in endometrial stroma. WNT5A and WNT5B mRNAs were most abundant in the stroma and less so in the luminal epithelium, whereas WNT11 mRNA was detected primarily in the glands. WNT7A mRNA was present in the luminal epithelium on d 10, absent on d 12 and 14, and increased between d 16 and 20. Only WNT2, WNT2B, and WNT4 were detected in conceptus trophectoderm. FZD6/8 (frizzled receptor) and GSK3B (glycogen synthase kinase 3beta) mRNAs were detected primarily in endometrial epithelia and conceptus trophectoderm, whereas the LRP5/6 (low-density lipoprotein receptor-related proteins 5 and 6) coreceptor was present in all endometrial cells and the trophectoderm. DKK1 (Dickkopf), a WNT signaling inhibitor, increased in the endometrium from d 16-20. CTNNB1 [catenin (cadherin associated protein) beta1] and CDH1 (E-cadherin) mRNAs were most abundant in the endometrial epithelia and trophectoderm. LEF1 (lymphoid enhancer-binding factor 1) mRNA was expressed primarily in uterine epithelia, whereas TCF7L2 [(transcription factor 7-like 2 (T-cell specific, HMG-box)] was primarily in the conceptus. CTNNB1 and TCF7L2 proteins were both abundant in the nuclei of trophoblast giant binucleate cells. WNT7A stimulated a TCF/LEF-luciferase reporter activity in ovine trophectoderm cells that was inhibited by dominant-negative TCF and Sfrp2 (secreted FZD-related protein 2). WNT7A increased trophectoderm cell proliferation as well as MSX2 (msh homeobox 2) and MYC (myelocytomatosis oncogene) mRNA levels. Wnt5a increased trophectoderm cell migration in a Rho kinase-dependent manner. These results support the hypotheses that canonical and noncanonical WNT signaling pathways are conserved regulators of conceptus-endometrial interactions in mammals and regulate periimplantation ovine conceptus development.

Differential expression of cathepsins and cystatin C in ovine uteroplacental tissues.

Cathepsins (CTSs) are peptidases that have biological roles in degrading extracellular matrix, catabolism of intracellular proteins, and processing of pro-hormones. Cystatin C (CST3) is a secreted inhibitor of lysosomal cysteine proteases cathepsin B (CTSB) and CTSL. Our working hypothesis is that cathepsins and cystatins play important roles in implantation and placentation in sheep. Expression of CTSB, CTSD, CTSH, CTSK, CTSL, CTSS, CTSZ, and CST3 mRNAs was detected in ovine uteroplacental tissues with distinct temporal and/or spatial expression patterns between Days 40 and 120 of pregnancy. Of particular note, CTSB, CTSD, and CTSZ mRNAs were predominantly detected in the chorion of the placenta and were more abundant in the placentomes than the intercaruncular endometria. CTSL and CST3 mRNAs were abundant in the endometrial epithelia and chorion, whereas CTSK, CTSS and CTSH mRNAs were most abundant in the stratum compactum stroma of the intercaruncular endometrium. Consistent with localisation of mRNAs, immunoreactive CTSL and CST3 proteins were mainly observed in the intercaruncular endometrial glands and intercotyledonary placenta during later pregnancy. These results support the working hypothesis that CTS and CST3 in uteroplacental tissues are involved in endometrial remodelling and placentation in sheep.

Ovine endogenous betaretroviruses (enJSRVs) and placental morphogenesis.

Endogenous retroviruses (ERVs) account for a substantial portion of the genetic pool of every animal species (e.g. approximately 8% of the human genome). Despite their overwhelming abundance in nature, many questions on the basic biology of ERVs are unanswered. The most important question derives from the observations in many animal species, including humans, of abundant ERVs expressed in the female genital tract. Sheep harbor approximately 20 copies of endogenous betaretroviruses (enJSRVs), which are related to an exogenous oncogenic virus, Jaagsiekte sheep retrovirus (JSRV). enJSRVs are abundantly expressed in the ovine placenta and uterine endometrium throughout gestation. Hyaluronidase 2 (HYAL2), which can serve as a cellular receptor for JSRV and enJSRVs envelope (Env), is expressed by the trophoblast giant binucleate cells and multinucleated syncytia of the placenta. Little is known about the cellular and molecular mechanisms that regulate trophoblast differentiation and syncytia formation during synepitheliochorial placentation in sheep. The temporal and spatial alterations in enJSRVs expression in the ovine uterus and placenta support the hypothesis that trophoblast growth and differentiation into binucleate cells and formation of multinucleated syncytiotrophoblast involves enJSRVs Env and possibly their cellular receptor, HYAL2.

Double ovulation and occurrence of twinning in alpacas (Vicugna pacos).

Twin births are rare in alpacas despite the high incidence of double ovulation and are undesirable because they contribute to early and late pregnancy loss, abortion, and birth of nonviable neonates. The objective of the present study was to determine the incidence and outcome of twin pregnancy in double-ovulating alpacas by retrospective and prospective analysis. Data from double-ovulating females (N = 41) presented for pregnancy diagnosis were analyzed to determine pregnancy status at three stages after mating (14-16, 25-30, and 45-53 days). In a prospective study, adult reproductively sound alpacas (n = 21) were examined by ultrasonography to determine the incidence of multiple ovulations. A subset of those alpacas (n = 10) were euthanized either at 9 days (n = 5) or 14 days (n = 5) after a single mating and administration of GnRH to determine presence and number of embryos. A total of 31 cycles were included in the study to determine the incidence of multiple ovulations. In the retrospective study, twin pregnancies were identified between 25 and 30 days in 47.1% of double-ovulating females. There were more twins from bilateral ovulations (62.5%) than from unilateral ovulations (37.5%). Twin pregnancies were either reduced to a singleton (62.5%) or lost completely (37.5%). One set of twins remained viable until Day 52. In the prospective study, double ovulation occurred in 18.8% of the cycles. Two embryos were collected from two of the four double-ovulating females. In conclusion, twin conception is not rare in alpacas. A high rate of spontaneous reduction of twin pregnancies occurs before Day 45. However, a significant proportion of twin pregnancy may result in loss of both embryos. Determination of the number of ovulations (CL) at the time of early pregnancy diagnosis (14-16 days) should be an integral part of any pregnancy evaluation.