Uterine biology in pigs and sheep

There is a dialogue between the developing conceptus (embryo-fetus and associated placental membranes) and maternal uterus which must be established during the peri-implantation period for pregnancy recognition signaling, implantation, regulation of gene expression by uterine epithelial and stromal cells, placentation and exchange of nutrients and gases. The uterus provide a microenvironment in which molecules secreted by uterine epithelia or transported into the uterine lumen represent histotroph required for growth and development of the conceptus and receptivity of the uterus to implantation. Pregnancy recognition signaling mechanisms sustain the functional lifespan of the corpora lutea (CL) which produce progesterone, the hormone of pregnancy essential for uterine functions that support implantation and placentation required for a successful outcome of pregnancy. It is within the peri-implantation period that most embryonic deaths occur due to deficiencies attributed to uterine functions or failure of the conceptus to develop appropriately, signal pregnancy recognition and/or undergo implantation and placentation. With proper placentation, the fetal fluids and fetal membranes each have unique functions to ensure hematotrophic and histotrophic nutrition in support of growth and development of the fetus. The endocrine status of the pregnant female and her nutritional status are critical for successful establishment and maintenance of pregnancy. This review addresses the complexity of key mechanisms that are characteristic of successful reproduction in sheep and pigs and gaps in knowledge that must be the subject of research in order to enhance fertility and reproductive health of livestock species.     

Uterine biology in pigs and sheep

ABSTRACT: There is a dialogue between the developing conceptus (embryo-fetus and associated placental membranes) and maternal uterus which must be established during the peri-implantation period for pregnancy recognition signaling, implantation, regulation of gene expression by uterine epithelial and stromal cells, placentation and exchange of nutrients and gases. The uterus provide a microenvironment in which molecules secreted by uterine epithelia or transported into the uterine lumen represent histotroph required for growth and development of the conceptus and receptivity of the uterus to implantation. Pregnancy recognition signaling mechanisms sustain the functional lifespan of the corpora lutea (CL) which produce progesterone, the hormone of pregnancy essential for uterine functions that support implantation and placentation required for a successful outcome of pregnancy. It is within the peri-implantation period that most embryonic deaths occur due to deficiencies attributed to uterine functions or failure of the conceptus to develop appropriately, signal pregnancy recognition and/or undergo implantation and placentation. With proper placentation, the fetal fluids and fetal membranes each have unique functions to ensure hematotrophic and histotrophic nutrition in support of growth and development of the fetus. The endocrine status of the pregnant female and her nutritional status are critical for successful establishment and maintenance of pregnancy. This review addresses the complexity of key mechanisms that are characteristic of successful reproduction in sheep and pigs and gaps in knowledge that must be the subject of research in order to enhance fertility and reproductive health of livestock species.     

Pregnancy recognition signaling mechanisms in ruminants and pigs

Maternal recognition of pregnancy refers to the requirement for the conceptus (embryo and its associated extra-embryonic membranes) to produce a hormone that acts on the uterus and/or corpus luteum (CL) to ensure maintenance of a functional CL for production of progesterone; the hormone required for pregnancy in most mammals. The pregnancy recognition signal in primates is chorionic gonadotrophin which acts directly on the CL via luteinizing hormone receptors to ensure maintenance of functional CL during pregnancy. In ruminants, interferon tau (IFNT) is the pregnancy recognition signal. IFNT is secreted during the peri-implantation period of pregnancy and acts on uterine epithelia to silence expression of estrogen receptor alpha and oxytocin receptor which abrogates the oxytocin-dependent release of luteolytic pulses of prostaglandin F2-alpha (PGF) by uterine epithelia; therefore, the CL continues to produce progesterone required for pregnancy. Pig conceptuses secrete interferon delta and interferon gamma during the peri-implantation period of pregnancy, but there is no evidence that they are involved in pregnancy recognition signaling. Rather, pig conceptuses secrete abundant amounts of estrogens between Days 11 to 15 of pregnancy required for maternal recognition of pregnancy. Estrogen, likely in concert with prolactin, prevents secretion of PGF into the uterine venous drainage (endocrine secretion), but maintains secretion of PGF into the uterine lumen (exocrine secretion) where it is metabolized to a form that is not luteolytic. Since PGF is sequestered within the uterine lumen and unavailable to induce luteolysis, functional CL are maintained for production of progesterone. In addition to effects of chorionic gonadotrophin, IFNT and estrogens to signal pregnancy recognition, these hormones act on uterine epithelia to enhance expression of genes critical for growth and development of the conceptus.     

Reactive oxygen and nitrogen species regulate porcine embryo development during pre-implantation period: A mini-review

Significant porcine embryonic loss occurs during conceptus morphological elongation and attachment from d 10 to 20 of pregnancy, which directly decreases the reproductive efficiency of sows. A successful establishment of pregnancy mainly depends on the endometrium receptivity, embryo quality, and utero-placental microenvironment, which requires complex cross-talk between the conceptus and uterus. The understanding of the molecular mechanism regulating the uterine-conceptus communication during porcine conceptus elongation and attachment has developed in the past decades. Reactive oxygen and nitrogen species, which are intracellular reactive metabolites that regulate cell fate decisions and alter their biological functions, have recently reportedly been involved in porcine conceptus elongation and attachment. This mini-review will mainly focus on the recent researches about the role of reactive oxygen and nitrogen species in regulating porcine embryo development during the pre-implantation period.     

The immunological approach to pregnancy diagnosis: a review

The developing embryo/fetus bears antigens which are foreign to the mother and it could be expected that immune rejection of the conceptus would occur. One of the reasons why the fetus is not rejected is because a depression of the maternal immune response takes place during pregnancy. Serum from pregnant animals of several species has been shown to contain a factor, early pregnancy factor (EPF), which is immunosuppressive. EPF has been detected as early as six hours after mating and its detection could aid diagnosis of early pregnancy in all species.     

Effect of estrogen inhibitors on conceptus estrogen synthesis and development in the gilt

Two estrogen antagonists (keoxifene and clomiphene) and two aromatase inhibitors (LY56110 and 4-hydroxyandrostenedione, 4-OHA) were utilized to determine the role of conceptus estrogen in trophoblastic elongation and maintenance of pregnancy in the pig. Pregnant gilts were unilaterally hysterectomized on day 10.5, and infused via a uterine arterial catheter with 200 mg of keoxifene or vehicle. The remaining uterine horn was removed based on time estimated for conceptus elongation. In a second study, pregnant gilts were injected daily with 200 mg (i.m.) of clomiphene or vehicle during pregnancy (days 10-16) and hysterectomized on day 30. A third study assessed in vitro aromatase inhibition by 4-OHA and LY56110 using trophoblastic microsomes incubated with [1 beta, 2 beta-3H]-androstenedione for 6 hr. In a fourth study, in vivo inhibition of aromatase activity was determined. For this study pregnant gilts, unilaterally hysterectomized on day 10.5, received either 4-OHA, LY56110, or vehicle. Conceptus development and uterine estrogens were quantified. None of the estrogen antagonists and aromatase inhibitors interferred with conceptus elongation. Uterine protein, calcium and acid phosphatase were similar (P greater than .10) between keoxifene- and vehicle-treated gilts. Embryonic survival of clomiphene- and vehicle-treated gilts was similar (91.5 vs 87.4%). In vitro, 4-OHA and LY56110 had 50% inhibitory concentrations of 0.1 microM and 13 nM. Treatment of gilts with 4-OHA reduced total estrogens in uterine flushings by 57% (P less than .02), whereas treatment with LY56110 did not significantly lower total estrogen content in uterine flushings. Estrogen antagonists were not effective in blocking conceptus elongation and maintenance of pregnancy. Although estrogen synthesis can be inhibited in vitro, dosages of aromatase inhibitors used were not totally effective in vivo.     

The uterus and early pregnancy failure in the mare

The environment the equine conceptus finds itself in when it arrives in the uterus some 6 days after ovulation will determine if it will thrive or die. The uterus will be its home for approximately the next 11 months and as such it needs to both coordinate the growth of the placenta and ensure there is enough nourishment passing to this organ for the fetus to develop normally. The trophoblast, endometrium, maternal ovaries and, later in pregnancy, the fetal gonads, all play roles in the hormonal changes that orchestrate these events. Although failures of these processes later in pregnancy can have catastrophic effects for the fetus, it is in early gestation that the foundations for a successful pregnancy are laid. This paper therefore concentrates on some of the noninfectious influences the uterus may have on survival of the young conceptus.     

滋养层细胞囊泡与奶牛低品质胚胎共移植对受胎率的影响

将奶牛低品质胚胎与囊胚滋养层细胞囊泡（trophoblastic vesicles,TRV）共移植,TRV来源于体外受精培养 7 d、体内培养7 d囊胚的囊胚滋养层细胞。胚胎移植受体牛超声波妊娠诊断共进行2次,即发情的第26～43天和第38～73天。第1次妊娠诊断时试验组（与TRV共移植组）妊娠率（66.7%,16/24）显著高于对照组（34.5%,10/29）（P＜0.05）。在第1次和第2次妊娠诊断期间试验组有3头牛的胚胎死亡。试验组和对照组的分娩率分别为41.7%（10/24）和27.6%（8/29）,两者间无显著差异。两组间受体牛的妊娠期和后代出生重相似。     

The immunology of early pregnancy in farm animals

The mammalian conceptus undergoes development in the face of a functional immune system. This characteristic of viviparity creates opportunities and perils for the conceptus. In the period up to hatching from the zona pellucida, the conceptus appears immunologically inert with low expression of major histocompatibility complex (MHC) genes and little evidence for alterations in the function of maternal immune cells. The conceptus may benefit from cytokines produced by leucocytes resident in the reproductive tract or by other cells of the reproductive tract. One cytokine in particular, colony-stimulating factor 2, can promote preimplantation development and cause changes in conceptus function that increase the likelihood that the conceptus develops to term. It is not clear whether activation of specific types of immune responses in early pregnancy can enhance the likelihood of pregnancy success. Semen deposition causes inflammation in the reproductive tract, but there is little evidence that this process is beneficial to pregnancy unless the uterus was exposed to a prior inflammatory event. Around the time of placentation, the period of immunological inertness of the conceptus is replaced by a period of immune activation caused by the expression of interferon genes in ruminants and pigs and MHC class I genes in invasive trophoblast of the horse. The large-scale changes in the endometrium that occur as a result are likely to play an important role to ensure continued development of the conceptus. In contrast, other immune responses generated as a result of disease in the reproductive tract or other tissues (mastitis) can lead to death of the conceptus. Because of this hazard, as well as the possibility that cell-mediated immune responses against the conceptus could occur, the immune system is regulated by both the mother and conceptus to reduce immune responsiveness during pregnancy.     

An overview of molecular and cellular mechanisms associated with porcine pregnancy success or failure

Prenatal mortality remains one of the major constraints for the commercial pig industry in North America. Twenty to thirty per cent of the conceptuses are lost early in gestation and an additional 10-15% is lost by mid-to-late gestation. Research over the last two decades has provided critical insights into how uterine capacity, placental efficiency, genetics, environment, nutrition and immune mechanisms impact successful conceptus growth; however, the exact cause and effect relationship in the context of foetal loss has yet to be determined. Similar to other mammalian species such as the human, mouse, rat, and primates, immune cell enrichment occurs at the porcine maternal-foetal interface during the window of conceptus attachment. However, unlike other species, immune cells are solely recruited by conceptus-derived signals. As pigs have epitheliochorial placentae where maternal and foetal tissue layers are separate, it provides an ideal model to study immune cell interactions with foetal trophoblasts. Our research is focused on the immune-angiogenesis axis during porcine pregnancy. It is well established that immune cells are recruited to the maternal-foetal interface, but their pregnancy specific functions and how the local milieu affects angiogenesis and inflammation at the site of foetal arrest remain unknown. Through a better understanding of how immune cells modulate crosstalk between the conceptus and the mother, it might be possible to therapeutically target immune cells and/or their products to reduce foetal loss. In this review, we provide evidence from the literature and from our own work into the immunological factors associated with porcine foetal loss.     

Old,new and the newest concepts of inhibition of luteolysis during early pregnancy in pig

In 1977 Bazer and Thatcher proposed that maternal recognition of pregnancy in the pig involves the secretion of PGF(2alpha) towards the uterine lumen (exocrine) rather than towards the uterine venous drainage (endocrine) as occurs in the non-pregnant pig during the mid to late stages of the estrous cycle. The retrograde transfer of PGF(2alpha) from the venous blood and uterine lymph into the uterus and the ability of the uterine vein and artery wall to accumulate PGF(2alpha) could constitute a part of putative mechanism of corpus luteum protection during early pregnancy. A luteotropic/anti-luteolytic effect of PGE(2) in the pig also has been frequently demonstrated and it seems that the most effective agent in changing PGE(2):PGF(2alpha) secretion is estradiol. The role for oxytocin during luteolysis and early pregnancy is controversial. It appears, however, that the main function of this hormone is autocrine and/or paracrine stimulation of PGF(2alpha) secretion. Pig trophoblastic interferons, unlike those of ruminants, do not themselves exert an anti-luteolytic effect in pigs. It is likely, that cytokines and angiogenic growth factors are involved in the initiation of luteolysis and/or maintenance of corpora lutea (CL).A discovery of functional LH receptors in porcine endometrium opened a new possibility for this hormone in luteolysis and perhaps in recognition of pregnancy in pigs. The endogenous LH pulses can provoke prostaglandin secretion from endometrium in pigs. On the other hand prolongation of up-regulation of LH receptors in endometrium of early pregnant gilts can additionally increase angiogenic factor production before the process of implantation is completed. Finally new integrated concepts of luteolysis and inhibition of luteolysis in pigs based on selectively reviewed information are presented.     

T-cell tolerance to the developing equine conceptus

One of the most intriguing and dramatic examples of immunological tolerance is displayed by the mammalian foetal-placental unit, which thrives as a semi-allograft in the mother's uterus during pregnancy. The success of the so-called foetal allograft stands in stark contrast to the failure of most tissue and organ grafts to survive without genetic matching of donor and recipient or drastic immunosuppression of the recipient's immune system. Experiments conducted over the past 60 years have revealed multiple mechanisms that enable the conceptus to avoid immunological detection or destruction. Many of these mechanisms are directed towards evading immune-mediated damage by maternal T lymphocytes, and they can be grouped into three classes: (i) downregulation of major histocompatibility complex (MHC) gene expression in placental trophoblast cells; (ii) local and systemic alterations in maternal immune reactivity; and (iii) innate defence mechanisms of the trophoblast cells that comprise the barrier between foetal and maternal tissues. The redundancy in these protective mechanisms helps ensure the transmission of life from generation to generation and provides a rich field of study of ways in which functional immunological tolerance can be manifest. The variation in placental forms and function among mammalian species present opportunities to discover and understand novel tolerogenic mechanisms that may have broad application in biology, medicine and animal husbandry. This review focuses on the evidence obtained from studies of pregnancy in the mare that support the case for selective T-cell tolerance to the mammalian conceptus.     

Post fetal death development of endometrial cups in a Jenny donkey (Equus asinus)

Serial ultrasound examinations of the gravid uterus of a Jenny donkey (Equus asinus) carrying a transferred extraspecies horse (Equus caballus) conceptus between 11 and 33 days of gestation demonstrated that the embryo died before Day 33 even though the conceptus persisted in the uterus and the horse chorionic girdle invaded the maternal endometrium to form a circle of typically large horse endometrial cups in the right uterine horn. The high concentrations of horse CG secreted by these cups with its higher than usual ratio of FSH-like to LH-like biological activities of 1.4 compared to the 0.1 ratio of normal donkey CG hyper-stimulated the donkey’s ovaries to develop multiple large accessory follicles and resulting secondary corpora lutea which secreted high concentrations of progestagens that persisted in her bloodstream for the next 90 days. This case illustrated that, in all likelihood, girdle invasion can happen after the death of the embryo indicating autonomy of the chorionic girdle.     

Aspects of the antiluteolytic activity of the conceptus during early pregnancy in ewes

Experiments were undertaken to determine whether the conceptus renders a corpus luteum resistant to the luteolytic action of prostaglandin F2 alpha (PGF2 alpha), and modulates release of this prostaglandin by the uterus of early pregnant ewes. Prostaglandin F2 alpha was luteolytic when administered to indomethacin-treated ewes on d 10 and 11 of the estrous cycle. The same PGF2 alpha treatment was not luteolytic when applied on d 19 and 20 of pregnancy in ewes treated with indomethacin. Pulsatile release of PGF2 alpha (measured by 15-keto-13,14-dihydro PGF2 alpha-PGF2 alpha plasma level, PGFM) was observed between d 14 and 16 of the cycle but not during the same period of pregnancy. Ablation of the conceptus on d 17 resulted in progressive restoration of PGFM surges and subsequent luteolysis. Estradiol-17 beta (E2-17 beta) administration on d 12 of the cycle induced earlier PGFM surges and luteal regression. The same E2-17 beta treatment administered on d 14, 19 and 33 of pregnancy failed to induced PGFM pulses and luteolysis. In the absence of the conceptus (surgical ablation), E2-17 beta treatment was luteolytic (PGFM surges) on d 17 but not on d 33. We conclude that the conceptus controls the amount and pattern of PGF2 alpha released by the uterus, as well as the sensitivity of the uterus to E2-17 beta as early as d 14 of pregnancy. Simultaneously, an embryonic protective effect takes place at the luteal level.     

Monoclonal antibodies to equine interferon-alpha (IFN-alpha): new tools to neutralize IFN-activity and to detect secreted IFN-alpha

Interferon-alpha (IFN-alpha) is a type I interferon that is secreted during the early stages of the innate immune response and is often induced upon infection with viral pathogens. IFN-alpha production affects multiple downstream events influencing both innate and adaptive immune responses. Here, we describe the expression of an equine rIFN-alpha/IgG4 fusion protein in mammalian cells. The anti-viral activity of rIFN-alpha/IgG4 was found to be 70-fold higher than that of a previously described IFN-gamma/IgG1 as tested by bioassay. The purified rIFN-alpha was subsequently used for the generation of six monoclonal antibodies (mAbs) to equine IFN-alpha. Four of these mAbs inhibited the protective anti-viral effect of equine leukocyte IFN in bioassays. One mAb (clone 240-2) showed a high-neutralizing capacity. An ELISA was established using two anti-equine IFN-alpha mAbs (clones 29B and 240-2) and its analytical sensitivity for was found to be around 800 pg/ml and 3 U/ml for rIFN-alpha and equine leukocyte IFN, respectively. When analyzing samples with a likely dominance of IFN-alpha among type I IFNs, such as supernatants from equine peripheral blood mononuclear cells stimulated with CpG-oligodeoxyribonucleotides, the results obtained by ELISA and IFN bioassay showed a high agreement (r(2)(sp)=0.98). When analyzing samples likely containing a mixture of type I IFNs, such as serum and nasal secretions from virally infected horses, the ELISA only detected some of the IFN-activity recorded in the bioassay. Overall, the data showed that the new anti-equine IFN-alpha mAbs are valuable tools to detect native IFN-alpha for further characterization of the early innate immune response and anti-viral immunity in horses.