Effect of duration of estradiol exposure on embryo survival and endometrial gene expression in anestrous embryo recipient mares.

Previous studies indicate a positive correlation between the duration of estrus prior to ovulation and likelihood of pregnancy in embryo recipient mares. However, the mechanisms by which the duration of estrus before may affect fertility remains unclear. This study aimed to determine the effect of different durations of estradiol exposure, prior to progesterone administration, on embryo viability in anestrous recipient mares, and endometrial expression of genes thought to influence embryo survival. Three groups of anestrous recipient mares treated with different duration of estradiol were used: long (LE), short (SE) and no treatment (NE). Day 8 embryos were transferred into recipient mares four days after long-acting progesterone administration and recovered 48h later to examine embryo growth and viability. The endometrial gene expression profile of selected genes was also investigated. The likelihood of recovering an embryo 48h after transfer was 46.1% (6/13), 62.5% (5/8) and 85.7% (6/7) for recipient mares from the NE, SE and LE groups, respectively (P = .09). Embryos recovered from the different groups of recipients did not, however, differ in size, morphology or the proportion of nuclei undergoing mitosis (P > .05). Abundance of mRNA for uterocalin (P19) and insulin-like growth factor 1 (IGF1) were increased in the LE compared to the NE group, while fibroblast growth factor 2 (FGF2), progesterone receptor (PGR) and insulin-like growth factor 1 receptor (IGF1R) transcript abundances were increased (P < 0.05) in the NE group compared to both SE and LE groups. In conclusion, a longer exposure of the endometrium to estradiol before progesterone tended to improve embryo survival within 48h of transfer. However, the grade, growth rate, and proportion of mitotic cells in surviving embryos did not differ among groups. If embryos are destined to fail in a suboptimal endometrial environment, they die and disappear quickly. Moreover, a more adequately estradiol-primed uterus, before the progesterone rise, seems to create a uterine environment, in terms of P19, IGF1, FGF2 and PGR gene expression, more conducive to embryo survival and further development.

In Vitro-Produced Equine Blastocysts Exhibit Greater Dispersal and Intermingling of Inner Cell Mass Cells than In Vivo Embryos.

In vitro production (IVP) of equine embryos is increasingly popular in clinical practice but suffers from higher incidences of early embryonic loss and monozygotic twin development than transfer of in vivo derived (IVD) embryos. Early embryo development is classically characterized by two cell fate decisions: (1) first, trophectoderm (TE) cells differentiate from inner cell mass (ICM); (2) second, the ICM segregates into epiblast (EPI) and primitive endoderm (PE). This study examined the influence of embryo type (IVD versus IVP), developmental stage or speed, and culture environment (in vitro versus in vivo) on the expression of the cell lineage markers, CDX-2 (TE), SOX-2 (EPI) and GATA-6 (PE). The numbers and distribution of cells expressing the three lineage markers were evaluated in day 7 IVD early blastocysts (n = 3) and blastocysts (n = 3), and in IVP embryos first identified as blastocysts after 7 (fast development, n = 5) or 9 (slow development, n = 9) days. Furthermore, day 7 IVP blastocysts were examined after additional culture for 2 days either in vitro (n = 5) or in vivo (after transfer into recipient mares, n = 3). In IVD early blastocysts, SOX-2 positive cells were encircled by GATA-6 positive cells in the ICM, with SOX-2 co-expression in some presumed PE cells. In IVD blastocysts, SOX-2 expression was exclusive to the compacted presumptive EPI, while GATA-6 and CDX-2 expression were consistent with PE and TE specification, respectively. In IVP blastocysts, SOX-2 and GATA-6 positive cells were intermingled and relatively dispersed, and co-expression of SOX-2 or GATA-6 was evident in some CDX-2 positive TE cells. IVP blastocysts had lower TE and total cell numbers than IVD blastocysts and displayed larger mean inter-EPI cell distances; these features were more pronounced in slower-developing IVP blastocysts. Transferring IVP blastocysts into recipient mares led to the compaction of SOX-2 positive cells into a presumptive EPI, whereas extended in vitro culture did not. In conclusion, IVP equine embryos have a poorly compacted ICM with intermingled EPI and PE cells; features accentuated in slowly developing embryos but remedied by transfer to a recipient mare.

Microtubule nucleation from the fibrous corona by LIC1-pericentrin promotes chromosome congression.

Error-free chromosome segregation in mitosis and meiosis relies on the assembly of a microtubule-based spindle that interacts with kinetochores to guide chromosomes to the cell equator before segregation in anaphase. Microtubules sprout from nucleation sites such as centrosomes, but kinetochores can also promote microtubule formation. It is unclear, however, how kinetochore-derived microtubules are generated and what their role is in chromosome segregation. Here, we show that the transient outer-kinetochore meshwork known as the fibrous corona serves as an autonomous microtubule nucleation platform. The fibrous corona is essential for the nucleation of kinetochore-derived microtubules, and when dissociated from the core kinetochore, it retains microtubule nucleation capacity. Nucleation relies on a fibrous-corona-bound pool of the LIC1 subunit of the dynein motor complex, which interacts with the γ-tubulin-tethering protein pericentrin (PCNT). PCNT is essential for microtubule nucleation from fibrous coronas, and in centrosome-depleted cells, where nearly all mitotic nucleation occurs at fibrous coronas, chromosome congression is fully dependent on PCNT. We further show that chromosomes in bovine oocytes, which naturally lack centrosomes, have highly expanded fibrous coronas that drive chromosome-derived microtubule nucleation. Preventing fibrous corona expansion in these cells impairs chromosome congression and causes spindle assembly defects. Our results show that fibrous coronas are autonomous microtubule-organizing centers that are important for spindle assembly, which may be especially relevant in acentrosomal cells such as oocytes.

Parental genomes segregate into distinct blastomeres during multipolar zygotic divisions leading to mixoploid and chimeric blastocysts.

BACKGROUND: During normal zygotic division, two haploid parental genomes replicate, unite and segregate into two biparental diploid blastomeres. RESULTS: Contrary to this fundamental biological tenet, we demonstrate here that parental genomes can segregate to distinct blastomeres during the zygotic division resulting in haploid or uniparental diploid and polyploid cells, a phenomenon coined heterogoneic division. By mapping the genomic landscape of 82 blastomeres from 25 bovine zygotes, we show that multipolar zygotic division is a tell-tale of whole-genome segregation errors. Based on the haplotypes and live-imaging of zygotic divisions, we demonstrate that various combinations of androgenetic, gynogenetic, diploid, and polyploid blastomeres arise via distinct parental genome segregation errors including the formation of additional paternal, private parental, or tripolar spindles, or by extrusion of paternal genomes. Hence, we provide evidence that private parental spindles, if failing to congress before anaphase, can lead to whole-genome segregation errors. In addition, anuclear blastomeres are common, indicating that cytokinesis can be uncoupled from karyokinesis. Dissociation of blastocyst-stage embryos further demonstrates that whole-genome segregation errors might lead to mixoploid or chimeric development in both human and cow. Yet, following multipolar zygotic division, fewer embryos reach the blastocyst stage and diploidization occurs frequently indicating that alternatively, blastomeres with genome-wide errors resulting from whole-genome segregation errors can be selected against or contribute to embryonic arrest. CONCLUSIONS: Heterogoneic zygotic division provides an overarching paradigm for the development of mixoploid and chimeric individuals and moles and can be an important cause of embryonic and fetal arrest following natural conception or IVF.

Effect of Overfeeding Shetland Pony Mares on Embryonic Glucose and Lipid Accumulation, and Expression of Imprinted Genes.

Maternal overfeeding is associated with disturbances in early embryonic epigenetic reprogramming, leading to altered expression of imprinted genes and nutrient transporters, which can affect both fetal and placental development and have lasting effects on the health of resulting offspring. To examine how maternal overfeeding affects the equine embryo, Shetland pony mares were fed either a high-energy (HE: 200% of net energy requirements) or maintenance (control) diet. Mares from both groups were inseminated, and day-seven embryos were recovered and transferred to recipients from the same or the alternate group. The expression of a panel of imprinted genes, glucose and amino acid transporters, and DNA methyltransferases (DNMTs) were determined in conceptus membranes after recovery on day 28 of gestation (late pre-implantation phase). The expression of nutrient transporters was also assessed in endometrium recovered from recipient mares immediately after conceptus removal. In addition, glucose uptake by day-28 extra-embryonic membranes, and lipid droplet accumulation in day-seven blastocysts were assessed. Maternal overfeeding resulted in elevated expression of imprinted genes (IGF2, IGF2R, H19, GRB10, PEG10 and SNRPN), DNMTs (DNMT1 and DNMT3B), glucose (SLC2A1), fructose (SLC2A5) and amino acid (SLC7A2) transporters following ET from an HE to a control mare. Expression of amino acid transporters (SLC1A5 and SLC7A1) was also elevated in the endometrium after ET from HE to control. Maternal overfeeding did not affect lipid droplet accumulation in blastocysts, or glucose uptake by day-28 membranes. It remains to be seen whether the alterations in gene expression are maintained throughout gestation and into postnatal life.

Dual spindles assemble in bovine zygotes despite the presence of paternal centrosomes.

The first mitosis of the mammalian embryo must partition the parental genomes contained in two pronuclei. In rodent zygotes, sperm centrosomes are degraded, and instead, acentriolar microtubule organizing centers and microtubule self-organization guide the assembly of two separate spindles around the genomes. In nonrodent mammals, including human or bovine, centrosomes are inherited from the sperm and have been widely assumed to be active. Whether nonrodent zygotes assemble a single centrosomal spindle around both genomes or follow the dual spindle self-assembly pathway is unclear. To address this, we investigated spindle assembly in bovine zygotes by systematic immunofluorescence and real-time light-sheet microscopy. We show that two independent spindles form despite the presence of centrosomes, which had little effect on spindle structure and were only loosely connected to the two spindles. We conclude that the dual spindle assembly pathway is conserved in nonrodent mammals. This could explain whole parental genome loss frequently observed in blastomeres of human IVF embryos.

Effect of a long-term high-energy diet on cardiovascular parameters in Shetland pony mares.

BACKGROUND: Changes in cardiovascular parameters, including blood pressure (BP) and cardiac anatomical dimensions, are an inconsistent feature of the equine metabolic syndrome. The order in which these changes arise is unknown. OBJECTIVES: Determine the order in which EMS-associated changes in cardiovascular parameters arise. ANIMALS: Twenty Shetland pony mares. METHODS: High-energy (HE) diet mares were fed 200% of net energy requirements for 1 (n = 3) or 2 (n = 7) consecutive diet-years, with 17 weeks of hay-only between years. Noninvasive BP measurements and echocardiograms were performed during both years. Resting 24-hour ECGs and measurements of autonomic tone (splenic volume and packed cell volume [PCV]) were performed at the end of diet-year 1. Results were compared to control mares receiving a maintenance diet for 1 (n = 7) or 2 (n = 3) consecutive years. RESULTS: In year 1, HE mares had significantly higher values than control mares for mean relative left ventricular wall thickness (P = .001). After 2 diet-years, mean systolic (P = .003), diastolic (P < .001) and mean arterial BP (P = .001), heart rate (HR; P < .001), and mean left ventricular wall thickness (P = .001) also were significantly increased in HE compared to control mares. No pathological arrhythmias or differences in splenic volume or PCV were detected. CONCLUSIONS AND CLINICAL IMPORTANCE: Ingesting a HE diet first induced minor changes in BP, and progressed to left-sided cardiac hypertrophy in Shetland pony mares. These findings are of interest given the increasing incidence of obesity in horses.

Compromised MPS1 Activity Induces Multipolar Spindle Formation in Oocytes From Aged Mares: Establishing the Horse as a Natural Animal Model to Study Age-Induced Oocyte Meiotic Spindle Instability.

Aneuploidy originating during meiosis in oocytes is the major cause of reduced fertility, implantation failure and miscarriage in women beyond their mid-thirties. Loss of chromosome cohesion, and defective microtubule dynamics and spindle assembly are, in turn, the major contributors to the error-prone nature of chromosome segregation in the oocytes of older women. However, the underlying molecular defects are not well understood. Altered function of MPS1 and AURKC have been shown to induce multipolar spindle phenotypes in murine oocytes and cancer cells, however, their role in reproductive aging associated oocyte aneuploidy is not known. Although age-related gamete and embryonic aneuploidy has been studied in female rodents, the horse may be a more appropriate animal model. Similar to women, aged mares suffer from reduced fertility and an increased incidence of oocyte aneuploidy. Moreover, mares show a long interval (decades) to reproductive senescence and, unlike rodents but similar to women, horse oocytes assemble the meiotic spindle in a slow and unstable manner, independent of microtubule organizing centers. In this study we found that oocytes from aged mares have lower expression of mRNA for Mps1, Spc25 and AurkC than oocytes from young mares while gene expression for other meiosis regulators did not differ. To assess the ability of horse oocytes to correctly form a bipolar spindle, in vitro matured MII oocytes were allowed to re-form their spindle after nocodazole-induced microtubule depolymerization. To investigate the importance of MPS1 and AURKC function in spindle (re)assembly, various concentrations of a MPS1 inhibitor (MPS1i, Compound 5) or an AURK inhibitor (AURKi, ZM447439) were included after nocodazole washout. MII oocytes from aged mares showed a higher incidence of spindle abnormalities after exposure to MPS1i. In contrast, Aurora kinase inhibition severely impaired microtubule organization and spindle formation in all oocytes, irrespective of mare age. In conclusion, gene expression for the kinases Mps1, Spc25, and AurkC is reduced in oocytes from aged mares. Moreover, spindle (re)assembly in aged mares' oocytes is more unstable when Mps1 is inhibited. Overall, this suggests that compromised Mps1 activity predisposes to meiotic spindle instability in aged mare oocytes. This spindle instability could predispose to chromosome segregation errors.

Overfeeding Extends the Period of Annual Cyclicity but Increases the Risk of Early Embryonic Death in Shetland Pony Mares.

Obesity has been associated with altered reproductive activity in mares, and may negatively affect fertility. To examine the influence of long-term high-energy (HE) feeding on fertility, Shetland pony mares were fed a diet containing 200% of net energy (NE) requirements during a three-year study. The incidence of hemorrhagic anovulatory follicles (HAF) and annual duration of cyclicity were compared to those in control mares receiving a maintenance diet. Day-7 embryos were flushed and transferred between donor and recipient mares from both groups; the resulting conceptuses were collected 21 days after transfer to assess conceptus development. HE mares became obese, and embryos recovered from HE mares were more likely to succumb to early embryonic death. The period of annual cyclicity was extended in HE compared to control mares in all years. The incidence of HAFs did not consistently differ between HE and control mares. No differences in embryo morphometric parameters were apparent. In conclusion, consuming a HE diet extended the duration of cyclicity, and appeared to increase the likelihood of embryos undergoing early embryonic death following embryo transfer.

The horse as a natural model to study reproductive aging-induced aneuploidy and weakened centromeric cohesion in oocytes.

Aneuploidy of meiotic origin is a major contributor to age-related subfertility and an increased risk of miscarriage in women. Although age-related aneuploidy has been studied in rodents, the mare may be a more appropriate animal model to study reproductive aging. Similar to women, aged mares show reduced fertility and an increased incidence of early pregnancy loss; however, it is not known whether aging predisposes to aneuploidy in equine oocytes. We evaluated the effect of advanced mare age on (1) gene expression for cohesin components, (2) incidence of aneuploidy and (3) chromosome centromere cohesion (measured as the distance between sister kinetochores) in oocytes matured in vitro. Oocytes from aged mares showed reduced gene expression for the centromere cohesion stabilizing protein, Shugoshin 1. Moreover, in vitro matured oocytes from aged mares showed a higher incidence of aneuploidy and premature sister chromatid separation, and weakened centromeric cohesion. We therefore propose the mare as a valid model for studying effects of aging on centromeric cohesion; cohesion loss predisposes to disintegration of bivalents and premature separation of sister chromatids during the first meiotic division, leading to embryonic aneuploidy; this probably contributes to the reduced fertility and increased incidence of pregnancy loss observed in aged mares.

Effect of long-term overfeeding of a high-energy diet on glucose tolerance in Shetland pony mares.

BACKGROUND: Overfeeding is associated with obesity and insulin dysregulation (ID), which are both risk factors for equine metabolic syndrome. How chronic overfeeding affects development of these factors is poorly understood. OBJECTIVES: To examine the influence of long-term high-energy diet provision on body condition and ID. ANIMALS: Eleven Shetland pony mares. METHODS: In a 3-phase study, the high-energy group (n = 7) was fed 200% of net energy (NE) requirements (hay; concentrate: 36% sugar and starch, 13% fat) for 24 weeks, followed by 17 weeks hay-only feeding before resuming the high-energy diet (n = 4) for an additional 29 weeks. Mares were weighed weekly. Oral glucose tolerance tests were performed 3 to 4 times per dietary period. Results were compared with those of a control group (phase 1, n = 4; phases 2 and 3, n = 6) that received 100% NE requirements, using a general linear mixed model with post hoc Bonferroni testing. RESULTS: The mean body weight of the high-energy group increased by 27% per high-energy feeding period. During both feeding periods, area under the curve (AUC) for plasma glucose concentration decreased (P < .01), whereas AUC for plasma insulin concentration increased. Mean basal plasma glucose concentration and peak plasma insulin concentrations were higher (P < .05) in the high-energy group than in the control group. CONCLUSION AND CLINICAL IMPORTANCE: Feeding a high-energy diet to healthy nonobese Shetland pony mares led to more efficient glucose metabolism within 5 weeks, followed by significant hyperinsulinemia and obesity. Hyperinsulinemic status was reversed during 17 weeks of hay-only feeding, regardless of body condition, but returned rapidly after restarting the high-energy diet.

Asynchronous Embryo Transfer Followed by Comparative Transcriptomic Analysis of Conceptus Membranes and Endometrium Identifies Processes Important to the Establishment of Equine Pregnancy.

Preimplantation horse conceptuses require nutrients and signals from histotroph, the composition of which is regulated by luteal progesterone and conceptus-secreted factors. To distinguish progesterone and conceptus effects we shortened the period of endometrial progesterone-priming by asynchronous embryo transfer. Day 8 embryos were transferred to synchronous (day 8) or asynchronous (day 3) recipients, and RNA sequencing was performed on endometrium and conceptuses recovered 6 and 11 days later (embryo days 14 and 19). Asynchrony resulted in many more differentially expressed genes (DEGs) in conceptus membranes (3473) than endometrium (715). Gene ontology analysis identified upregulation in biological processes related to organogenesis and preventing apoptosis in synchronous conceptuses on day 14, and in cell adhesion and migration on day 19. Asynchrony also resulted in large numbers of DEGs related to 'extracellular exosome'. In endometrium, genes involved in immunity, the inflammatory response, and apoptosis regulation were upregulated during synchronous pregnancy and, again, many genes related to extracellular exosome were differentially expressed. Interestingly, only 14 genes were differentially expressed in endometrium recovered 6 days after synchronous versus 11 days after asynchronous transfer (day 14 recipient in both). Among these, KNG1 and IGFBP3 were consistently upregulated in synchronous endometrium. Furthermore bradykinin, an active peptide cleaved from KNG1, stimulated prostaglandin release by cultured trophectoderm cells. The horse conceptus thus responds to a negatively asynchronous uterus by extensively adjusting its transcriptome, whereas the endometrial transcriptome is modified only subtly by a more advanced conceptus.

In vitro production of horse embryos predisposes to micronucleus formation, whereas time to blastocyst formation affects likelihood of pregnancy.

Invitro embryo production is an increasingly popular means of breeding horses. However, success is limited by a high incidence of early embryo loss. Although there are various possible causes of pregnancy failure, chromosomal abnormalities, including aneuploidy, are important potential contributors. This study evaluated the frequency of micronucleus formation as a proxy for aneuploidy in invitro-produced (IVP) and invivo-derived horse blastocysts. Associations between IVP embryo morphology, frequency of nuclear abnormalities and the likelihood of pregnancy were investigated. IVP blastocysts exhibited a higher frequency of cells with micronuclei than invivo-derived embryos (10% vs 1% respectively; P=0.05). This indication of chromosomal instability may explain the higher incidence of pregnancy failure after transfer of IVP embryos. However, the frequency of micronuclei was not correlated with brightfield microscopic morphological characteristics. Nevertheless, IVP embryos reaching the blastocyst stage after Day 9 of invitro culture were less likely to yield a pregnancy than embryos that developed to blastocysts before Day 9 (27% vs 69%), and embryos that had expanded before transfer were more likely to undergo embryonic death than those that had not expanded (44% vs 10%). These findings indicate that current embryo culture conditions are suboptimal and that the speed of embryo development is correlated with pregnancy survival.

Vitrifying immature equine oocytes impairs their ability to correctly align the chromosomes on the MII spindle.

Vitrified-warmed immature equine oocytes are able to complete the first meiotic division, but their subsequent developmental competence is compromised. Therefore, the present study investigated the effects of vitrifying immature horse oocytes on the chromosome and spindle configuration after IVM. Cumulus-oocytes complexes (COCs) were collected and divided into two groups based on mare age (young ≤14 years; old ≥16 years). COCs were then either directly matured invitro or vitrified and warmed before IVM. Spindle morphology and chromosome alignment within MII stage oocytes were assessed using immunofluorescent staining, confocal microscopy and three-dimensional image analysis. Vitrification reduced the ability of oocytes to reach MII and resulted in ultrastructural changes to the meiotic spindle, including shortening of its long axis, and an increased incidence of chromosomes failing to align properly at the metaphase plate. We hypothesise that aberrant chromosome alignment is an important contributor to the reduced developmental competence of vitrified equine oocytes. Contrary to expectation, oocytes from young mares were more severely affected than oocytes from older mares; we propose that the reduced effect of vitrification on oocytes from older mares is related to pre-existing compromise of spindle assembly checkpoint control mechanisms in these mares.

Effect of the duration of estradiol priming prior to progesterone administration on endometrial gene expression in anestrous mares.

Field data indicate that a longer period of estrus prior to ovulation correlates positively with fertility. To test the hypothesis that the duration of exposure to estrogens prior to progesterone dominance influences endometrial function, we used anestrous mares to simulate varying durations of estrus (3 groups of 5 mares): long (LE), short (SE), and no estrus (NE), as determined by the duration of estradiol priming prior to progesterone treatment: 7, 2 and 0 days for the LE, SE and NE, respectively. Endometrial biopsies were recovered 4 days after progesterone administration in all groups for real time quantitative reverse transcription PCR (RT-qPCR) and immunohistochemical analyses. A total of 17 genes believed to contribute to a "receptive endometrium" for embryo development and viability were evaluated by RT-qPCR. Of the genes evaluated, the expression of FGF-2 (fibroblast growth factor-2) decreased with increased length of preceding estrus, whereas P19 (uterocalin) expression was higher in the LE than in the SE or NE groups. In conclusion, a lower abundance of FGF-2 and higher abundance of uterocalin, a lipocalin protein known to play an important role in providing lipids to the embryo, could contribute to a more receptive endometrium in mares following a long estrus.

Expression of glucose transporters in the endometrium and early conceptus membranes of the horse.

INTRODUCTION: Glucose is the primary energy substrate for early conceptus development and, for the first 40 days of gestation, the equine conceptus depends solely on glucose available in the histotroph; thereafter, histotrophic glucose provision continues to support transport across the definitive placenta. METHODS: To investigate glucose provision routes during early equine pregnancy we examined expression of glucose transporters in conceptus membranes and endometrium recovered on days 7, 14, 21 and 28 after ovulation. To further differentiate the contributions of maternal progesterone priming and conceptus-endometrium crosstalk in regulating glucose transporter expression, day 8 embryos were transferred to recipient mares on day 8 (synchronous) or day 3 (asynchronous) after ovulation; conceptuses and endometrium were recovered 6 or 11 days later. RESULTS: The glucose transporters SLC2A1, 2A3, 2A4, 2A8, 2A10 and 5A1 were expressed in equine endometrium. In conceptus membranes, expression of SLC2A1-3, 2A5, 2A8, 2A10, 5A1 and 5A11 increased from day 14, and SLC2A1 protein was highly abundant on the apical trophectodermal membrane and in the endoderm. Asynchronous embryo transfer (ET) resulted in reduced SLC2A1 expression in both the endometrium and conceptus membranes. DISCUSSION: A wide range of glucose transporters are expressed in the pre-implantation equine conceptus and endometrium, presumably to ensure adequate glucose provision to the developing embryo. Endometrial expression of SLC2A1 appears to be regulated by a combination of progesterone-priming and conceptus signalling, and its delayed upregulation after asynchronous ET may contribute to the observed delay in conceptus development.

Amino acid transporter expression in the endometrium and conceptus membranes during early equine pregnancy.

Maternally derived amino acids (AA) are essential for early conceptus development, and specific transporters enhance histotrophic AA content during early ruminant pregnancy. In the present study we investigated AA transporter expression in early equine conceptuses and endometrium, during normal pregnancy and after induction of embryo-uterus asynchrony. 'Normal' conceptuses and endometrium were recovered on Days 7, 14, 21 and 28 after ovulation. To investigate asynchrony, Day 8 embryos were transferred to recipient mares on Day 8 or Day 3, and conceptuses were recovered 6 or 11 days later. Endometrial expression of AA transporters solute carrier family 38 member 2 (SLC38A2), solute carrier family 1 members 4 and 5 (SLC1A4 and SLC1A5) increased during early pregnancy, whereas solute carrier family 7 member 8 (SLC7A8), solute carrier family 43 member 2 (SLC43A2) and solute carrier family 7 member 1 (SLC7A1) SLC7A8, SLC43A2 and SLC7A1 expression decreased and the expression of solute carrier family 1 member 1(SLC1A1) and solute carrier family 7 member 2 (SLC7A2) was unaffected. In conceptus membranes, most transporters studied were upregulated, either after Day 14 (solute carrier family 7 member 5 - SLC7A5, SLC38A2, SLC1A4, SLC1A5 and SLC7A1) or Day 21 (SLC43A2 and SLC7A2). Asynchronous ET indicated that endometrial SLC1A5, SLC1A1 and SLC7A8 are primarily regulated by conceptus factors and/or longer exposure to progesterone. In conclusion, AA transporters are expressed in early equine conceptus membranes and endometrium in specific spatiotemporal patterns. Because conceptuses express a wider range of transporters than the endometrium, we speculate that the equine yolk sac has recruited AA transporters to ensure adequate nutrient provision during an unusually long preimplantation period.

Negative uterine asynchrony retards early equine conceptus development and upregulation of placental imprinted genes.

INTRODUCTION: Placental imprinted genes appear to be sensitive indicators of an inappropriate pre-implantation environment. This study examined the effects of negative uterine asynchrony after embryo transfer (ET) on early horse embryo development, and yolk-sac membrane expression of DNA methyltransferases (DNMTs) and equine specific placental imprinted genes. METHODS: Day 8 embryos were transferred to recipient mares on day 8 (synchronous) or day 3 (asynchronous) after ovulation, and conceptuses were recovered 6 or 11 days later (day 14 or 19 of development). RESULTS: Day 14 conceptuses recovered from an asynchronous uterus had a smaller embryonic disc, in which primitive streak development was visibly retarded compared to conceptuses from a synchronous uterus. Similarly, length, somite number and organogenesis were retarded in day 19 embryos after asynchronous ET. Maternal (GRB10, H19, IGF2R, PHLDA2) and paternal (IGF2, INSR, PEG3, PEG10, DIO3, NDN, SNRPN) imprinted genes and DNMTs (DNMT1, 3A and 3B) were all up-regulated between day 14 and 19 of pregnancy and, for most, mRNA expression was higher in synchronous than asynchronous day 19 yolk-sac membrane. Expression of the paternally imprinted gene HAT1 increased between day 14 and 19 of pregnancy, but was not affected by the asynchrony. DISCUSSION: Conceptus development and upregulation of DNMTs and imprinted genes were delayed rather than dysregulated after transfer into a negatively asynchronous uterus. We propose that this ability to 'reset' conceptus development to uterine stage is an adaptation that explains why horse embryos are unusually tolerant of asynchrony after ET.

From Peptide Masses to Pregnancy Maintenance: A Comprehensive Proteomic Analysis of The Early Equine Embryo Secretome, Blastocoel Fluid, and Capsule.

Early pregnancy in the mare is a poorly understood, high risk period during which the embryo communicates its presence to the maternal endometrium. Remarkably, the maternal recognition of pregnancy signal is unknown in the horse. This study aimed to profile the proteins secreted by equine blastocysts into their immediate environment, along with proteins contained in the blastocoel and within the acellular embryo capsule. Embryos were recovered on day 8 after ovulation and cultured for 48 hours. Secretomes of day 9 and day 10 embryos were analyzed by LC-MS/MS and supported by analysis of blastocoel fluid and embryo capsule. Analyses revealed 72 (24 h) and 97 (48 h) unique protein IDs in the embryo secretome, 732 protein IDs in blastocoel fluid, and 11 proteins IDs in the embryo capsule. Novel findings of interest include secretion of a pregnancy specific proteinase (PAG) by the equine embryo at day 10, along with detection of a prostaglandin receptor inhibiting protein (PTGFRN) and a progesterone potentiating factor (FKBP4) in blastocoel fluid. This is the first comprehensive proteomic analysis of the equine embryo secretome, and provides new insights into the unique physiology of early pregnancy in this species.

Fibroblast growth factor-2 expression in the preimplantation equine conceptus and endometrium of pregnant and cyclic mares.

Uterine-derived growth factors and cytokines play essential roles in regulating preimplantation conceptus development. In several species, fibroblast growth factor-2 (FGF2) promotes embryogenesis, trophoblast cell migration, and adhesion. This study investigated mRNA expression for FGF2, its receptors (FGFR1-4), the activating factor FGF binding protein (FGF-BP) in equine endometrium and trophectoderm during early pregnancy and the estrous cycle, and localized FGF2 protein in both endometrium and conceptus tissues. FGF2, FGFRs1-4, and FGFBP mRNAs were expressed in endometrium throughout the estrous cycle and early pregnancy, and in days 14 to 28 conceptus membranes. FGF2 transcription was higher during estrus than on days 7 or 14 of diestrus, suggesting estrogen dependency. Endometrial expression of FGF2 mRNA and protein increased as pregnancy progressed from days 21 and day 28; FGF2 protein was localized predominantly in the luminal and glandular epithelium. FGF2 mRNA was detectable in trophectoderm from as early as day 14, and transcription and translation increased in day 21 and 28 allantochorion. FGF2 protein was localized mainly in the trophectoderm up to day 21 but was present in both trophectoderm and endoderm of day 28 allantochorion. FGFR1 mRNA was down-regulated in the endometrium at day 7 of diestrus but increased again by day 14. Gene expression for all of the FGFR2 splice variants, including FGFR2IIIc, was up-regulated during estrus. During early pregnancy, endometrial FGFR1 expression decreased, whereas FGFR2IIIc expression did not change. Conceptus mRNA expression for all FGFRs increased as pregnancy progressed. FGFBP expression remained unchanged in endometrium, but increased in the conceptus between days 14 and 28, suggesting a role in regulating FGF2 activity in the developing conceptus. We conclude that during weeks 3 and 4 of pregnancy, the equine endometrial epithelium produces FGF2, which may play a role in trophoblast development and adhesion.