Signal For Maternal Recognition Of Pregnancy Research Articles

Ruminant conceptus-maternal interactions: interferon-tau and beyond.

Embryonic or fetal loss in cattle is associated with problems that occur during oocyte maturation, early embryonic development, conceptus elongation, maternal recognition of pregnancy (MRP), and/or placental attachment and implantation. Many of these problems manifest as inadequate or asynchronous communication between the developing conceptus and endometrium, resulting in pregnancy failure. This review will provide an overview of how various conceptus-endometrial paracrine signaling systems control the fate of early pregnancy in cattle and other ruminants. We begin by summarizing the actions of interferon-tau, the classic MRP signal in ruminates, and then explore how other secretory factors derived from either the conceptus or endometrium influence establishment and maintenance of pregnancy. Insight into how the endometrium responds to male vs. female conceptuses or conceptuses produced by in vitro methods will also be described. Specific focus will be placed on describing how "omic" technologies and other cutting-edge techniques have assisted with identifying novel conceptus and/or endometrial factors and their functions. Recent findings indicate that the endometrial transcriptome and histotroph are altered by conceptus sex, quality, and origin, suggesting that the endometrium is a sensor of conceptus biochemistry. Although the endometrium has a certain level of flexibility in terms of conceptus-maternal interactions, this interplay is not sufficient to retain some pregnancies. However, new information inspires us to learn more and will help develop technologies that mitigate early embryonic loss and reproductive failure in ruminants and other animals.

The timing of the maternal recognition of pregnancy signal is specific to individual mares

Over several breeding seasons, eighteen inseminated mares were examined at 8(±1)h intervals from late oestrus until after ovulation. The time of ovulation was determined as 4h before the first detection of ovulation. Examinations for pregnancy began at 8h intervals between 236 to 260h (late Day 9 to 10) post ovulation. When present an embryo was reduced by compression with a total of 132 reductions on Days 10 (n=15), 11 (n=47), 12 (n=36), 13 (n=27), 14 (n=5) and 15 (n=2). Ten mares were used on multiple occasions, undergoing between 3 and 16 embryo reductions each. Mares were subsequently examined, first to determine non-pregnancy, and then to determine whether luteolysis or luteostasis occurred. Luteostatic mares maintained an ultrasonically identifiable corpus luteum, and between Days 22 and 25 were returned to oestrus by administration of PGF2α. Increasing numbers of mares became luteostatic following embryo reduction as the number of days post ovulation increased. The earliest initiation of luteostasis occurred when an embryo was reduced at 252h (10.5 days). Between the 8h examinations on Days 10 to 12 there was an increase in the proportion of mares undergoing luteostasis. When mares were used on multiple occasions, individual animals had aset time point within a ±8h window post ovulation when they responded to the MRP signal and after which, but not before, embryo reduction resulted in luteostasis. For the eight mares which had embryos reduced ≥6 occasions, the time when luteostasis occurred was consistent in an individual but varied between mares between Day 10 (244–252h) and Day 14 (348h). For example, one mare which did not recognise the presence of an embryo until Day 14 (348h), had 10 reductions undertaken between Days 11 to 13 (268–332h) and all resulted in luteolysis, but all 3 reductions at or after Day 14 (348h) resulted in luteostasis. Despite the late timing of when the MRP signal in this animal was recognised, no spontaneous embryo loss had been seen in three previous pregnancies. These results suggest that the timing of the luteolytic cascade is variable between mares but relatively constant in its timing in an individual animal. This would imply a consistent set of physiological processes that program the endometrium for the luteolytic cascade in any given animal. Further data is needed to investigate whether oestrous cycle length and variation in the oestrous and dioestrous phases of the mare play a role in initiating the mechanisms that enable the uterus to respond to the MRP signal.

Transfer of Day 11 ruptured embryos can prevent luteolysis in recipient mares

Previous studies transferring Day 11 embryos to Day 11 or 12 recipient mares and then manually crushing the transferred embryo 24 hours later resulted in 83% (5/6) and 100% (6/6) mares, respectively, entering a period of luteostasis. Undertaking manual reduction 12 hours post transfer resulted in 100% (5/5) of Day 11 recipient mares becoming luteostatic. This work suggested that the mare could recognise the presence of an embryo within the uterus after only a short period of time. The present experiment aimed to shorten the window of the presence of an intact embryo still further by evaluating the ability of ruptured or damaged Day 11 embryos transferred to recipient mares to initiate a period of luteostasis. A total of sixteen Day 11 embryos were flushed from the uteri of donor mares. Embryos were moved to a petri dish containing 5mL of Vigro Hold Plus Solution where they were measured and graded. The intact embryos were ruptured using two 100µm diameter acupuncture needles, ensuring the blastocoele fluid was released into the medium. The ruptured embryo and the medium were then transferred to recipient mares at Day 11 (n = 8) or 12 (n = 8) post ovulation using a standard embryo transfer technique. Control Day 11 (n = 8) and 12 (n = 8) recipient mares received 5mL of Vigro Hold Plus. All recipient mares were monitored on alternate days post transfer using a combination of ultrasonography,including assessment of the corpus luteum using colour Doppler, and serial blood serum samples assayed for progesterone in peripheral blood to determine if, and when, they returned to oestrus. Mares with a functional corpus luteum with blood flow and progesterone levels >1ng/mL up to 25 post ovulation were deemed to have entered a period of luteostasis and at this time point luteolysis was achieved by administration of an i.m injection of prostaglandin. Transfer of the ruptured embryo and medium resulted in significantly more Day 12 recipients entering a period of luteostasis compared to Day 11 recipients (75% vs. 12.5%; 6/8 vs. 1/8; P= 0.04, respectively). Day 11 and 12 control recipient mares that received only medium had an incidence of luteostasis of 12.5% (1/8) in each group. Hence the presence of medium containing blastocoele fluid, a ruptured capsule and trophoblast within the uterus can prevent luteolysis around the time of maternal recognition of pregnancy (MRP) in the mare. If all or only some of the components of the ruptured Day 11 embryo are required to initiate luteostasis needs further study. If the mechanisms underlying luteostasis with this model mirror those of the MRP signal in pregnant mares this study also raises questions about the duration of the MRP signal and how long embryo migration within the uterus is needed to prevent luteolysis.

106 Maternal recognition of pregnancy (MRP) and mares: Is the epithelial sodium channel (ENaC) involved?

Maternal recognition of pregnancy (MRP) describes the prevention of luteolysis via the early conceptus. By prolonging the presence of the corpus luteum (CL), the conceptus ensures an environment capable of supporting pregnancy. In mares, MRP is enigmatic; the horse is one of few domesticated species wherein the MRP signal has yet to be discovered. Equine conceptus mobility throughout the uterine horns, termed migration, is known to be essential for pregnancy establishment. However, while the MRP signal in several other species is conceptus-derived, such a signal has yet to be observed from the equine embryo. We hypothesize that the migration of the equine conceptus itself activates MRP, potentially through epithelial sodium channel (ENaC). ENaC has been previously detected in multiple equine organs, observed in the uteri of various mammalian species, and also found to be upregulated during preimplantation in mice. However, neither the presence of ENaC within the mare uterus nor its potential involvement with the equine pregnancy has been researched. The aim of this study was 1) to determine if ENaC was present within the endometrium of the mare uterus and 2) to describe the location of ENaC if observed. Estrus synchronization was completed in mares (n = 14) during peak breeding season. Mares were subsequently blocked by age and breed and then placed into one of 2 groups: control mares (C; n = 7) and treatment mares (T; n = 7). Transrectal ultrasonography (US) was completed in all mares daily when one or more ovarian follicles reached 25 mm. When a mare had a 35 mm or larger follicle present, she received 2,000 international units of human chorionic gonadotropin intramuscularly to induce ovulation (OV) and US continued daily. Additionally, T mares were artificially inseminated (AI) with fresh, extended semen every day until the day after OV and were subsequently checked for pregnancy 12 d post-OV. C mares had the cervix manually manipulated the day after OV to replicate physical stimulation that occurs to the cervix when AI; however, no semen was deposited in C mare reproductive tracts. Endometrial biopsies were collected from all mares before OV, after OV, and during early pregnancy (when applicable) and samples were then processed for ENaC immunohistochemistry analysis. ENaC protein was immunodetected in all C and T mare endometrial samples and was localized to both uterine luminal epithelium and uterine glands. While further analysis will be conducted to quantify endometrial ENaC expression in C versus T (open versus pregnant) mares, the present histological data confirms that ENaC is present within the equine uterus.

FOXL2 is a Progesterone Target Gene in the Endometrium of Ruminants.

Forkhead Box L2 (FOXL2) is a member of the FOXL class of transcription factors, which are essential for ovarian differentiation and function. In the endometrium, FOXL2 is also thought to be important in cattle; however, it is not clear how its expression is regulated. The maternal recognition of pregnancy signal in cattle, interferon-Tau, does not regulate FOXL2 expression. Therefore, in the present study, we examined whether the ovarian steroid hormones that orchestrate implantation regulate FOXL2 gene expression in ruminants. In sheep, we confirmed that FOXL2 mRNA and protein was expressed in the endometrium across the oestrous cycle (day 4 to day 15 post-oestrus). Similar to the bovine endometrium, ovine FOXL2 endometrial expression was low during the luteal phase of the oestrous cycle (4 to 12 days post-oestrus) and at implantation (15 days post-oestrus) while mRNA and protein expression significantly increased during the luteolytic phase (day 15 post-oestrus in cycle). In pregnant ewes, inhibition of progesterone production by trilostane during the day 5 to 16 period prevented the rise in progesterone concentrations and led to a significant increase of FOXL2 expression in caruncles compared with the control group (1.4-fold, p < 0.05). Ovariectomized ewes or cows that were supplemented with exogenous progesterone for 12 days or 6 days, respectively, had lower endometrial FOXL2 expression compared with control ovariectomized females (sheep, mRNA, 1.8-fold; protein, 2.4-fold; cattle; mRNA, 2.2-fold; p < 0.05). Exogenous oestradiol treatments for 12 days in sheep or 2 days in cattle did not affect FOXL2 endometrial expression compared with control ovariectomized females, except at the protein level in both endometrial areas in the sheep. Moreover, treating bovine endometrial explants with exogenous progesterone for 48h reduced FOXL2 expression. Using in vitro assays with COS7 cells we also demonstrated that progesterone regulates the FOXL2 promoter activity through the progesterone receptor. Collectively, our findings imply that endometrial FOXL2 is, as a direct target of progesterone, involved in early pregnancy and implantation.

Maternal Recognition of Pregnancy in the Horse: Are MicroRNAs the Secret Messengers?

The signal for maternal recognition of pregnancy (MRP) has still not been identified in the horse. High-throughput molecular biology at the embryo–maternal interface has substantially contributed to the knowledge on pathways affected during MRP, but an integrated study in which proteomics, transcriptomics and miRNA expression can be linked directly is currently lacking. The aim of this study was to provide such analysis. Endometrial biopsies, uterine fluid, embryonic tissues, and yolk sac fluid were collected 13 days after ovulation during pregnant and control cycles from the same mares. Micro-RNA-Sequencing was performed on all collected samples, mRNA-Sequencing on the same tissue samples and mass spectrometry was conducted previously on the same fluid samples. Differential expression of miRNA, mRNA and proteins showed high conformity with literature and confirmed involvement in pregnancy establishment, embryo quality, steroid synthesis and prostaglandin regulation, but the link between differential miRNAs and their targets was limited and did not indicate the identity of an unequivocal signal for MRP in the horse. Differential expression at the embryo–maternal interface was prominent, highlighting a potential role of miRNAs in embryo–maternal communication during early pregnancy in the horse. These data provide a strong basis for future targeted studies.

Maternal recognition of pregnancy signal or endocrine disruptor: The two faces of oestrogen during establishment of pregnancy in the pig

Timing of conceptus growth and attachment to the uterine luminal epithelium is regulated by progesterone secretion from the corpus luteum and by expression of progesterone receptor in the uterine epithelia and stroma. Conceptus growth and uterine attachment are temporally associated with the disappearance of progesterone receptors from uterine epithelia. While the loss of progesterone receptor from the endometrial epithelia on day 10 of the oestrous cycle and pregnancy has been well documented, the factors involved with cell specific down-regulation of progesterone receptor are yet to be established. We propose that several progesterone stimulated factors activate nuclear factor kappa B (NF-kB) within the uterine epithelia, which leads to inhibition of progesterone receptor and concomitant stimulation of endometrial genes expressed during early conceptus development. Although oestrogens secreted by pig conceptuses function to establish pregnancy, timing of endometrial exposure to oestrogen is critical. Early oestrogen administration alters the pattern of gene expression through the NF-kB system desynchronising the uterine environment for conceptus implantation resulting in later embryonic loss.

Reproduction in domestic ruminants during the past 50 yr: discovery to application.

The study of reproductive physiology in domestic ruminants has progressed from the whole animal to the molecular level in an amazingly short period of time. The volume of information on this subject is enormous; therefore, we have focused on domestic ruminants, with an emphasis on cattle. To date, artificial insemination (AI) is perhaps the most powerful technique that reproductive physiologists and geneticists have provided the livestock industry for genetic improvement. Early efforts to establish AI as a tool were initiated in Russia around 1899 and since that time major advances in methods of semen collection, evaluation of male fertility, cryopreservation of sperm, sex-sorted semen, and estrous cycle control have occurred. The preceding advances not only led to the widespread use of AI, but also contributed to our fundamental understanding of ovulation control, timing of insemination, gamete biology, and cryopreservation. In regards to anestrus, our understanding of the concept of neuroendocrine control of the pituitary gland and the role of steroid feedback led to the Gonadostat Theory, which proposes that onset of puberty is due to a decrease in the negative feedback of gonadal steroids over time. Subsequent studies in prepuberal and postpartum sheep and cattle established that a short luteal phase frequently precedes the first normal length cycle that is accompanied by estrous expression. This observation led to the common practice of treating prepuberal heifers and anestrous postpartum cows with a short-term progestin treatment (e.g., Controlled Internal Drug Release) to induce normal estrous cycles. In domestic ruminants, fertilization rate is high (85% to 95%); however, significant embryonic mortality before or around the time of maternal recognition of pregnancy (MRP) reduces the pregnancy rate to a single breeding. Significant effort has been directed at determining the time of MRP, the signal for MRP, as well as elucidating the physiological, cellular, and molecular dialogue between the conceptus and uterine environment. Advancements have now led us to the ability to edit the genome to alleviate disease and possibly improve production traits. In summary, major advancements in our understanding of reproductive biology have stemmed from efforts to establish the AI and embryo transfer technique and reduce the negative impact of anestrus and embryonic mortality in domestic ruminants.

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 48hours. 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 (24h) and 97 (48h) 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.

Differential expression of ISG 15 mRNA in peripheral blood mononuclear cells of nulliparous and multiparous pregnant versus non-pregnant Bos indicus cattle.

Embryonic mortality is found to be the main source of reproductive wastage in domestic ruminants. Many genes are involved in the growth and development of the embryo, and the interferon-stimulated gene 15 (ISG 15) is one of the major gene stimulated by interferon tau, the maternal recognition of pregnancy signal in ruminants. In this study, both genomic and cDNA sequences of ISG 15 from Bos indicus (Deoni breed) were amplified and characterized. The genomic sequence of Deoni ISG 15 exhibited 99% identity with Bos taurus and 97% identity with that of Bos mutus and Bubalus bubalis. Moreover qRT-PCR analysis revealed constitutive expression of the ISG 15 mRNA in peripheral blood mononuclear cells of Deoni heifers and multiparous cows during early pregnancy. Fourteen Deoni heifers and fifteen multiparous Deoni cows were synchronized for timed AI by CIDR-Ovsynch protocol, and six animals were kept as cyclic control in each group. Blood samples were collected on days 7, 14, 16, 18, 21, 30 and 45 from the day of AI. Pregnancy was confirmed by plasma progesterone level through ELISA. A significantly higher expression of ISG 15 mRNA was found on day 16 (p<.05) and day 18 (p<.05) of pregnancy in nulliparous heifers. Although in multiparous Deoni cows ISG 15 expression was greater in pregnant cows, difference was statistically non-significant. The result of this study indicates that ISG 15 gene expression is upregulated during 16-18days of pregnancy and could be used as an early pregnancy marker in dairy cows especially in heifers.

Human chorionic gonadotrophin in early gestation induces growth of estrogenic ovarian follicles and improves primiparous sow fertility during summer

Reduced summer farrowing rates may be due to inadequate corpora luteal (CL) support. Porcine CL become dependent on LH from 12 d of pregnancy and the embryonic estrogen signal for maternal recognition of pregnancy (MRP) is initiated at about 11–12 d after insemination. We hypothesised that injection of the LH analogue human chorionic gonadotropin (hCG) would induce growth of estrogenic follicles and, by mimicking the signal for MRP and stimulating progesterone secretion, increase primiparous sow fertility. In Experiment 1, during a 28 d lactation 53 mixed parity sows were full-fed either throughout lactation (n=16) or until 18 d and then feed restricted during the last 10 d of lactation (n=36). At 12 d after mating restrict-fed sows were injected with 1000IU hCG (n=17) or were not injected (n=19); the full-fed sows acted as non-treated positive controls. Transrectal ovarian ultrasound exams were performed on days 12, 16, 20, 24, and 28; blood samples were obtained on days 12, 14, and 15 for estradiol and progesterone assay. For Experiment 2, during the summer months primiparous sows received 1000IU hCG 12 d after mating (n=28) or were non-injected controls (n=27). Pregnancy status was determined at 28 d and sows allowed to go to term to determine farrowing rates and litter sizes. In Experiment 1, injection of hCG increased (P<0.001) follicle diameter and serum concentrations of estradiol (P<0.01) and progesterone (P<0.05). There were no effects of lactation feeding level on wean-estrus interval, farrowing rate or subsequent litter size. In Experiment 2, hCG injection was associated with a higher pregnancy rate (P<0.05) and farrowing rate (P<0.08). There was no effect on litter size. These data confirm that hCG stimulates growth of estrogenic follicles and CL function, and improves primiparous sow fertility during the summer months.

Conceptus elongation in ruminants: roles of progesterone, prostaglandin, interferon tau and cortisol.

The majority of pregnancy loss in ruminants occurs during the first three weeks after conception, particularly during the period of conceptus elongation that occurs prior to pregnancy recognition and implantation. This review integrates established and new information on the biological role of ovarian progesterone (P4), prostaglandins (PGs), interferon tau (IFNT) and cortisol in endometrial function and conceptus elongation. Progesterone is secreted by the ovarian corpus luteum (CL) and is the unequivocal hormone of pregnancy. Prostaglandins (PGs) and cortisol are produced by both the epithelial cells of the endometrium and the trophectoderm of the elongating conceptus. In contrast, IFNT is produced solely by the conceptus trophectoderm and is the maternal recognition of pregnancy signal that inhibits production of luteolytic pulses of PGF2α by the endometrium to maintain the CL and thus production of P4. Available results in sheep support the idea that the individual, interactive, and coordinated actions of P4, PGs, IFNT and cortisol regulate conceptus elongation and implantation by controlling expression of genes in the endometrium and/or trophectoderm. An increased knowledge of conceptus-endometrial interactions during early pregnancy in ruminants is necessary to understand and elucidate the causes of infertility and recurrent early pregnancy loss and provide new strategies to improve fertility and thus reproductive efficiency.

The Buserelin Enigma; How Does Treatment with this GnRH Analogue Decrease Embryo Mortality?

The use of the GnRH analogue buserelin administered in mid dioestrus has been shown to increase early pregnancy rate, reduce embryo loss and increase litter size in several species. The mechanism by which this works is unclear and may differ between species. In cattle it is believed that buserelin, by reducing oestrogen concentrations, causes the development of endometrial oxytocin receptors to be delayed. Luteal oxytocin binds with these receptors to promote the formation and secretion of prostaglandin. Any delay in the release of PGF2α would allow an underdeveloped embryo extra time to produce and release the maternal recognition of pregnancy signal, which might otherwise be lost following luteolysis. This may also be the mechanism in sheep and pigs although an increase in progesterone concentrations could also be important. However in the mare, following buserelin administration at 9 or 10 days after the detection of ovulation, pregnancy rates are already increased by 12-13 days compared with untreated controls, i.e. before the time of luteolysis, without any change in progesterone concentrations. In addition to an increased pregnancy rate, the twin embryo rate is also increased and subsequent embryo loss rate reduced. The possible mechanisms by which this may result in the mare are discussed.

PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM: Biological role of interferon tau in endometrial function and conceptus elongation12

This review integrates established and new information on the biological role of ovarian progesterone (P4) and interferon tau as well as conceptus- and endometrial-derived factors, PG and cortisol, in endometrial function and conceptus elongation during the periimplantation period of pregnancy in ruminants. Interferon tau is the maternal recognition of pregnancy signal that inhibits production of luteolytic pulses of PGF2α by the endometrium to maintain corpora lutea and their production of P4, the unequivocal hormone of pregnancy. Conceptus-endometrial interactions in ruminants are complex and involve carefully orchestrated temporal and spatial alterations in endometrial gene expression during pregnancy. Available results from studies in sheep support the idea that the individual, interactive, and coordinated actions of P4, interferon tau, PG, and cortisol regulate expression of elongation- and implantation-related genes in the endometrial epithelia and that P4 and PG are essential regulators of conceptus elongation. The outcome of these gene expression changes is alterations in endometrial secretions that govern conceptus elongation via effects on trophectoderm proliferation, migration, attachment, and adhesion. An increased knowledge of conceptus-endometrial interactions during early pregnancy in ruminants is necessary to understand and elucidate the causes of recurrent pregnancy loss and to provide a basis for new strategies to improve pregnancy outcome and reproductive efficiency.

Intrauterine administration of plant oils inhibits luteolysis in the mare

The maternal recognition of pregnancy (MRP) signal in the mare has not been determined, although oestrogens have been proposed as a potential candidate. To determine effects of intrauterine administration of oestrogen and various oils on cyclic luteolysis in the mare. Intrauterine oestradiol or fatty acids may suppress luteolysis in the cycling mare when administered during late dioestrus. A single 1 ml dose of slow-release oestradiol (10 mg/ml) in fractionated coconut oil was infused into the uterine lumen of cycling mares on Days 6, 8, 10, 12 or 14 post ovulation (n=12 in each group). Four further groups, each of 12 mares, received an intrauterine infusion of either 1 ml of fractionated coconut oil, peanut oil, mineral oil or a slow-release preparation of oestradiol (10 mg/ml) in mineral oil on Day 10 post ovulation. Serial blood samples were assayed for progesterone concentrations to monitor luteal function. Intrauterine administration of oestradiol in fractionated coconut oil showed peak efficacy at Day 10 when luteolysis was delayed in 11/12 (92%) mares. The ability of the treatment to delay luteolysis was not significantly different when administered on Days 8 (9/12; 75%), 12 (10/12; 83%) or 14 (6/12; 50%) of dioestrus, but declined significantly when given on Day 6 (3/12; 25%). Oestradiol was not needed to initiate luteostasis since fractionated coconut oil alone or peanut oil administered at Day 10 induced the same high rate of luteal persistence (11/12; 92% for both oils). In contrast, mineral oil did not prolong luteal lifespan, either when administered alone (2/12; 17%) or combined with oestradiol (3/12; 25%). These results do not unequivocally rule out a possible involvement of embryonic oestrogens in MRP in the mare but suggest it is unlikely. The results demonstrate that plant oils can postpone luteolysis, suggesting they may modulate synthesis or release of prostaglandins from the mare's endometrium. Administration of fractionated coconut or peanut oil on Day 10 post ovulation provides an effective and practical method of prolonging luteal function ('pseudopregnancy') thereby suppressing unwanted oestrous behaviour. Further studies to elucidate the mechanism by which this is achieved may increase understanding of both luteostasis and MRP signal in the mare.

Interferon regulatory factor 6 (IRF6) is expressed in the ovine uterus and functions as a transcriptional activator

Interferon tau (IFNT), the maternal recognition of pregnancy signal in sheep and other ruminants, is secreted by the conceptus and regulates the expression of a number of genes in a cell-specific manner within the uterus. The response of different endometrial cell types to IFNT appears to be specified by IFN regulatory factors (IRFs). IRF2, a potent repressor of gene transcription, is expressed only by luminal (LE) and superficial glandular epithelia (sGE), whereas IRF1 and IRF9, activators of gene transcription, are expressed only in GE and stromal cells of the uterus during early pregnancy. In the present study, IRF6 was found to be expressed in LE/sGE and middle GE of the ovine uterine endometrium as well as conceptus trophectoderm. IRF family members can regulate transcription via IFN-stimulated response elements (ISREs). Transient transfection analyses found that IRF6 enhanced basal activity of ISRE-containing promoters, but did not enhance IFNT stimulation of ISRE-containing promoters in variety of different cell types. Further, IRF6 did not cooperate with IRF1 or reduce IRF2 repression of ISRE-containing promoter activity. These results establish that IRF6 is a transcriptional activator that is preferentially expressed in the endometrial epithelia and conceptus trophectoderm. IRF6 is hypothesized to play critical roles in endometrial gene expression as well as in conceptus trophectoderm growth and differentiation.

Effects of Exogenous Insulin on Luteolysis and Reproductive Cyclicity in the Mare

Insulin is a pancreatic hormone that classically regulates carbohydrate and fat metabolism, but also appears to play a role in various reproductive processes. A preliminary study suggested insulin production by day 10 to 18 equine conceptuses. The aim of the present study was to examine the hypothesis that insulin is the conceptus signal responsible for maternal recognition of pregnancy (MRP) in the mare, or otherwise influences reproductive cyclicity during the MRP period. Six Warmblood mares were treated daily during days 7 to 17 after ovulation of two successive oestrous cycles with either (short and intermediate acting) insulin or control saline. Mares were assigned randomly to treatment, and crossed over during the subsequent cycle. Time of ovulation and corpus luteum surface area were determined by serial transrectal ultrasonographic examination of the mares' ovaries, and daily jugular vein blood samples were analysed for progesterone and luteinizing hormone (LH) concentrations. On day 14 of dioestrus, the luteolytic drive was examined by measuring systemic 15-ketodihydroprostaglandin F(2 alpha) (PG-metabolite) release in response to oxytocin challenge. In addition, yolk sac fluid recovered from 32 day 10 to 14 equine conceptuses was analysed for insulin concentrations. Insulin administration did not affect luteal size, dioestrus length, the interovulatory interval, or circulating LH concentrations. Insulin administration also failed to suppress oxytocin-induced PGF(2 alpha) release, and tended to depress systemic progesterone concentrations. Finally, insulin could not be detected in the yolk sac fluid of day 10 to 14 equine conceptuses by radio-immunoassay. It is concluded that insulin administered daily during days 7 to 17 of dioestrus has little or no effect on reproductive cyclicity in the mare, and is unlikely to be the MRP signal.

O-189: Elevated stress through SAPK/JNK and AMPK mediate cues at implantation for generating the maternal recognition of pregnancy signal

Stress causes homeostatic responses such as slower cell cycle, cell cycle arrest, and apoptosis in preimplantation mouse embryos and in trophoblast stem (TS) cells. These responses are largely mediated by the stress-signaling enzyme, SAPK/JNK. Under culture conditions modeling post-implantation events, stress enzymes regulate developmental responses, also. Production of sufficient maternal recognition of pregnancy (MRP) proteins requires that TS cells first proliferate and then differentiate a subpopulation of trophoblast giant (TG) cells to secrete sufficient MRP proteins.

Elevated Stress and SAPK/JNK Phospho Mediate Cues at Implantation for Generating the Maternal Recognition of Pregnancy Signal

To test whether stress and stress activated protein kinase/Jun kinase (SAPK/JNK) can mediate the maternal recognition of pregnancy signaling induced during implantation of the early mammalian embryo into the uterus.

Fetomaternal interactions and influences during equine pregnancy

The equine embryo takes 6 days to traverse the oviduct and, when it finally enters the uterus, it remains spherical in shape and moves continually throughout the uterine lumen until day 17 after ovulation to deliver its maternal recognition of pregnancy signal to the entire endometrium. Between day 25 and day 35 after ovulation, the trophoblast cells of a discrete annulate portion of the chorion multiply rapidly and acquire an invasive phenotype and, between day 36 and day 38, migrate deeply into the maternal endometrium to form the equine-unique endometrial protuberances known as endometrial cups. These cups secrete large quantities of a gonadotrophic hormone (eCG) into the maternal circulation which, in conjunction with pituitary FSH, stimulates the development of accessory luteal structures in the maternal ovaries to supplement the supply of progesterone to maintain the pregnancy until the placenta can assume this role at about day 100. The non-invasive allantochorion extends slowly to fill the uterus by days 80-85 and its microcotyledonary architecture, which provides both haemotrophic and histotrophic nutrition for the growing fetus, is not fully established until days 120-140. The fetoplacental unit synthesizes large quantities of steroid hormones during the second half of pregnancy, using fetal C-19 precursors secreted by the enlarged fetal gonads for the production of oestrogens and maternal C-21 precursors for the synthesis of progesterone and large quantities of 5alpha-reduced progestagens. Near term, additional pregnenelone is secreted by the fetal adrenal glands so that the mare exhibits the unusual phenomenon of foaling while maternal serum progestagen concentrations are increasing and oestrogen concentrations are decreasing.