Influence of progesterone on oocyte quality and embryo development in cows

In cattle, the majority of embryo loss occurs very early during pregnancy (approximately Day 16), around or prior to maternal recognition of pregnancy. The actions of P4 in controlling LH pulsatility and ovarian follicular development may impinge negatively on oocyte quality. A considerable proportion of embryo loss may be attributable to inadequate circulating progesterone (P4) concentrations and the subsequent downstream consequences on endometrial gene expression and histotroph secretion into the uterine lumen. Conceptus growth and development require the action of P4 on the uterus to regulate endometrial function, including conceptus–maternal interactions, pregnancy recognition, and uterine receptivity for implantation. This review summarizes recent data highlighting the role of progesterone in determining oocyte quality and embryo development in cattle.     

Supplemental progesterone during early pregnancy exerts divergent responses on embryonic characteristics in sows and gilts

Progesterone (P4) plays a key role in pregnancy establishment and maintenance; during early pregnancy, P4 stimulates the production and release of uterine secretions necessary for conceptus growth prior to implantation; therefore, exogenous P4 supplementation may improve embryo development. This study evaluated the effects of supplementation during early pregnancy with long-acting injectable progesterone or altrenogest on embryonic characteristics of sows and gilts. Thus, a total of 32 sows and 16 gilts were used. On day 6 of pregnancy sows and gilts were allocated to one of the following groups: non-supplemented; supplemented with 20 mg of altrenogest, orally, from days 6 to 12 of pregnancy; supplemented with 2.15 mg/kg of long-acting injectable progesterone on day 6 of pregnancy. Animals were killed on day 28 of pregnancy, and ovulation rate, embryo survival, embryo weight, crown-to-rump length, uterine glandular epithelium and endometrial vascularization were assessed. Treatments had no effect on pregnancy rate, embryo survival or endometrial vascular density (P > 0.05). Non-supplemented gilts presented larger and heavier embryos compared to gilts from supplemented groups (P < 0.05). Sows in the altrenogest group presented larger and heavier embryos compared to non-supplemented sows and sows supplemented with long-acting injectable progesterone. In conclusion, supplementation of sows and gilts with progestagen from day 6 of pregnancy can be used as a means to improve embryo survival without deleterious effects.     

The effect of undernutrition on the establishment of pregnancy in the ewe

The relationship between nutrition and reproduction in sheep has been the subject of research in several international groups. This review will particularly focus on the effects of undernutrition on the potential causes of reproductive failure including abnormalities of the ovum or the embryo, luteal inadequacy and failure of the supply of progesterone to the uterus, or the mechanisms involved in maternal recognition of pregnancy. The level of nutrition and peripheral progesterone concentrations are inversely related, and increased rates of embryo loss, associated with higher progesterone concentrations in ewes with low levels of nutrition have been reported. Undernutrition may act through changes in the distribution of progesterone in the endometrium. Thus, lower endometrial levels on day 5 of the cycle in ewes fed half of their maintenance requirements have been observed, providing a link between the known role of progesterone in embryo survival by the modulation of uterine function and the higher embryo losses found in undernourished ewes. The evidence of an effect of maternal nutrition on IFNtau secretion from the conceptus and of PGF2alpha production from the uterus is presented. Moreover, undernutrition provokes a reduction in the sensitivity of the endometrium to progesterone that may affect embryo survival. Finally, a state of undernutrition induces changes in the endometrial sensitivity to steroid hormones at early stages of pregnancy that could adversely alter uterine environment to the detriment of embryo survival.     

Composition of the bovine uterine proteome is associated with stage of cycle and concentration of systemic progesterone

Early embryonic loss accounts for over 70% of total embryonic and foetal loss in dairy cattle. Early embryonic development and survival is associated with the concentration of systemic progesterone. To determine if the uterine proteome is influenced by stage of cycle or systemic progesterone concentrations, uterine flushings were collected from the ipsi‐ and contralateral uterine horns of beef heifers on Days 7 (n = 10) and 15 (n = 10) of the oestrous cycle. Animals were separated into low or high progesterone groups based on plasma progesterone concentrations on Day 5 of the cycle. Samples were albumin depleted before iTRAQ® labeling and subsequent strong cation exchange‐LC‐MS/MS analyses. A total of 20 proteins were up to 5.9‐fold higher (p < 0.05) and 20 were up to 2.3‐fold lower on Day 15 compared to Day 7. In addition, the expression of a number of proteins on Day 7 and/or 15 of the cycle was correlated with progesterone concentrations during Days 3–7 or the rate of change in progesterone between Days 3 and 7. This study highlights the dynamic changes occurring in the microenvironment surrounding the embryo during this period. The findings here also support the hypothesis that progesterone supports embryonic development by altering the maternal uterine environment.     

Uterine epithelial morphology and progesterone receptors in a mifepristone-treated viviparous lizard Pseudemoia entrecasteauxii (Squamata: Scincidae) during gestation

Structural and functional changes to the uterus associated with maintenance of pregnancy are controlled primarily by steroid hormones such as progesterone. We tested the hypothesis that progesterone regulates uterine structural changes during pregnancy in the viviparous skink, Pseudemoia entrecasteauxii, by treating pregnant females with the progesterone receptor antagonist mifepristone at different stages of pregnancy. Expression and distribution of progesterone receptor was determined using Western blot and immunohistochemistry. During early pregnancy, mifepristone treatment resulted in altered uterine epithelial cell surface morphology and high embryo mortality, but did not affect females at mid and late stages of pregnancy. Females treated with mifepristone in early pregnancy exhibited abnormal uterine epithelial cell morphology such as lateral blebbing and presence of wide gaps between cells indicating loss of intercellular attachment. Chorioallantoic membranes of the embryo were not affected by mifepristone treatment. Two isoforms (55 kDa and 100 kDa) of progesterone receptor were identified using immunoblots and both isoforms were localized to the nucleus of uterine epithelial cells. The 55 kDa isoform was expressed throughout pregnancy, whereas the 100 kDa isoform was expressed during mid and especially late pregnancy. In P. entrecasteauxii, mifepristone may prevent successful embryo attachment in early pregnancy through its effects on uterine epithelial cells but may have little effect on pregnancy once the maternal-embryo structural relationship is established.     

In vivo analysis of progesterone receptor action in the uterus during embryo implantation

In order for a successful pregnancy to occur, the embryo must attach to the luminal epithelial cells and invade into the stroma. Then, the surrounding stromal cells need to undergo decidualization in order to establish the vasculature necessary for survival of the embryo. These events in early pregnancy are tightly regulated by the steroid hormones, estrogen (E2) and progesterone (P4), through their cognate receptors, the estrogen receptor (ER) and the progesterone receptor (PR), respectively. Using a mouse model in which the PR has been ablated, it was demonstrated that the PR is necessary for embryo implantation and decidualization. Therefore, understanding the mechanism of PR action in the adult uterus is necessary in order to understand the events of early pregnancy. Insights from both mouse models and human samples have been integral in elucidating uterine PR action. These studies have shown that not only PR target genes, but also mediators of PR action are important for correct PR action in early pregnancy. Many of the genes involved in PR action in early pregnancy have also been shown to have roles in uterine diseases such as endometriosis and endometrial cancer. Therefore, the integration of mouse and human studies on PR action in the uterus will be important for the future understanding of uterine diseases and in the development of treatment for these diseases.     

In vivo analysis of progesterone receptor action in the uterus during embryo implantation

In order for a successful pregnancy to occur, the embryo must attach to the luminal epithelial cells and invade into the stroma. Then, the surrounding stromal cells need to undergo decidualization in order to establish the vasculature necessary for survival of the embryo. These events in early pregnancy are tightly regulated by the steroid hormones, estrogen (E2) and progesterone (P4), through their cognate receptors, the estrogen receptor (ER) and the progesterone receptor (PR), respectively. Using a mouse model in which the PR has been ablated, it was demonstrated that the PR is necessary for embryo implantation and decidualization. Therefore, understanding the mechanism of PR action in the adult uterus is necessary in order to understand the events of early pregnancy. Insights from both mouse models and human samples have been integral in elucidating uterine PR action. These studies have shown that not only PR target genes, but also mediators of PR action are important for correct PR action in early pregnancy. Many of the genes involved in PR action in early pregnancy have also been shown to have roles in uterine diseases such as endometriosis and endometrial cancer. Therefore, the integration of mouse and human studies on PR action in the uterus will be important for the future understanding of uterine diseases and in the development of treatment for these diseases.     

Endometrial receptivity defects and impaired implantation in diabetic NOD mice

Implantation failure is a major hurdle to a successful pregnancy. The high rate of postimplantation fetal loss in nonobese diabetic (NOD) mice is believed to be related to an abnormal decidual production of interferon (IFN)gamma. To address whether diabetes alters the natural events associated with successful implantation, certain morphological and molecular features of uterine receptivity in diabetic NOD (dNOD) mice were examined in normally mated pregnancy and in concanavalin A (ConA)-induced pseudopregnancy. As opposed to normoglycemic NOD (cNOD) mice, dNOD mice expressed retarded maturation of their uterine pinopodes and overexpressed MUC1 mucin at implantation sites (P < 0.001). Uterine production of leukemia inhibitory factor (LIF) and phosphorylation of uterine NFkappaBp65 and STAT3-Ty705 were found to be low (P < 0.01) during Day 4.5 postcoitum, whereas IFNgamma was aberrantly overexpressed. Loss of temporal regulation of progesterone receptor A (PR A) and PR B, together with aberrantly increased expression of the protein inhibitor of activated STAT-y (PIASy) (P < 0.01) and reduced recruitment (P < 0.01) of the latter to nuclear progesterone receptor sites were prominent features of decidualization failure occurring at peri-implantation in dNOD mice. In conclusion, the aberrant expression of endometrial IFNgamma in dNOD mice is associated with a nonreceptive endometrial milieu contributing to peri-implantation embryo loss in type 1 diabetes.     

The role of progesterone elevation in IVF

Elevation of progesterone during the late follicular phase of stimulated in-vitro fertilization cycles is a frequent event, which negatively impacts the outcome. Over the years evidence has demonstrated a direct relationship between late-follicular elevated progesterone and endometrial receptivity. In this regard, elective cryopreservation of all good quality embryos and transfer in a subsequent frozen/thawed cycle is the most common strategy adopted by clinicians in case of elevated progesterone. Nonetheless, recent evidence suggests that elective cryopreservation might not entirely resolve the reduced pregnancy outcomes associated with the elevation of progesterone, considering that the increase may affect not only implantation, but also embryo quality.     

Culture of bovine embryos in intermediate host oviducts with emphasis on the isolated mouse oviduct

The oviduct provides the optimal environment for the transport of sperm and oocyte at the earliest stages of mammalian embryo development. During the early postfertilization period, several major developmental events occur in the embryo including (i) the first cleavage division, (ii) activation of the embryonic genome, (iii) compaction of the morula, and (iv) formation of the blastocyst. Most of these events are initiated in the oviduct. The absence of assistance from the oviduct may compromise the developmental ability of the cattle embryo under in vitro culture conditions. The oviducts of several mammalian species, including rabbits, cow, sheep (in situ), and mice (organ culture), can sustain early bovine embryos and yield blastocysts of better quality compared with those of culture conditions in vitro, leading to normal pregnancy rates in recipient animals. This review focuses on the use of oviducts in vitro or in vivo as intermediate hosts for postfertilization culture environment of bovine in vitro-produced zygotes with emphasis on the mouse model.     

Progesterone regulation of preimplantation conceptus growth and galectin 15 (LGALS15) in the ovine uterus

Peri-implantation conceptus (embryo/fetus and associated extraembryonic membranes) growth and development are primarily regulated by secretions from the uterus. This study investigated the effects of progesterone on preimplantation conceptus development and endometrial galectin 15 (LGALS15). Ewes received daily injections of either corn oil (CO) vehicle or 25 mg progesterone (P4) from 36 h postmating to hysterectomy. Treatment with P4 increased blastocyst diameter by 220% on Day 9 and advanced time of elongation of blastocysts to a filamentous conceptus on Day 12. Effects of P4 treatment on blastocyst development were blocked by administration of RU486, a progesterone receptor antagonist. Consistent with early elongation of blastocysts, interferon tau (IFNT) protein was about 50-fold greater in uterine flushes from Day 12 in ewes receiving P4 compared with those receiving CO. Expression of cathepsin L (CTSL) and radical S-adenosyl methionine domain containing 2 (RSAD2), both IFNT-stimulated genes, was increased in endometria of Day 12 P4-treated ewes. LGALS15 mRNA, expressed only in the endometrial luminal epithelium and superficial glands, was detected between Days 9 and 12 and was more abundant in ewes receiving P4 than in those receiving CO on both Days 9 and 12. RU486 treatment ablated P4 induction of LGALS15 mRNA in the endometrial epithelia. LGALS15 protein in uterine flushings was not different on Day 9 but tended to be greater in P4-treated ewes than in those receiving CO on Day 12. The advanced development of blastocysts in P4-treated ewes is hypothesized to involve early induction of specific genes in the endometrial epithelia, such as LGALS15, and undoubtedly components of uterine histotroph.     

Aspects of in vivo oocyte production,blastocyst development,and embryo transfer in the cat

A brief overview of the progress made during the past approximately 40 years on the development of methods for in vitro production of cat embryos and intra- and interspecies embryo transfer is described. The presentation is focused primarily on research done over the past 30 years at the Cincinnati Zoo (1980–1995) and at the Audubon Nature Institute, New Orleans (1996–present) beginning with original studies on determining optimal doses of porcine FSH for ovarian stimulation and uterine embryo recovery, cryopreservation, and transfer. A key early finding was the ability of cats to respond to multiple gonadotropin (porcine FSH) treatments by repeated stimulation of follicular development. With a ≥6-month interval between FSH treatments, over the past 15 years (1998–2013), we have done 1603 laparoscopic oocyte retrievals on 337 cats and recovered >38,000 mature oocytes (mean = 24.1 per laparoscopic oocyte retrieval). The limited information available on in vivo blastocyst development in the cat during the latter portion of the preimplantation period (approximately Days 8 to 12 after coitum or approximately Days 7 to 11 after ovulation) was assembled for the purpose of comparing and contrasting it with the growth, expansion, and zona functioning of in vitro-derived blastocysts. Also, results of transferring morulae and/or blastocysts into synchronous recipients are described to emphasize evidence that appears to allude to an essential role for an intact zona pellucida in successful implantation and subsequent development in the cat. Until 2003, our in vitro-derived embryos were transferred into the uterine horns of recipients to determine the feasibility of producing offspring from such primary methods as IVF, intracytoplasmic sperm injection, SCNT, and embryo cryopreservation. With the exception of SCNT embryos, pregnancy rates were satisfactory, but embryo survival rates were not. Subsequently, after finding that SCNT embryo survival rate could be improved using laparoscopic transfer of early cleavage stage embryos into the oviduct, we applied the technique to embryos derived using IVF with sex-sorted sperm, oocyte vitrification, and embryo cryopreservation. Overall, a pregnancy rate of 67% (14/21) has resulted. Most recently, with the oviductal embryo transfer technique, two litters of Black-Footed cat kittens have been born from intra- and interspecies transfer of cryopreserved embryos.     

Gap junction communication between uterine stromal cells plays a critical role in pregnancy-associated neovascularization and embryo survival

In the uterus, the formation of new maternal blood vessels in the stromal compartment at the time of embryonic implantation is critical for the establishment and maintenance of pregnancy. Although uterine angiogenesis is known to be influenced by the steroid hormones estrogen (E) and progesterone (P), the underlying molecular pathways remain poorly understood. Here, we report that the expression of connexin 43 (Cx43), a major gap junction protein, is markedly enhanced in response to E in uterine stromal cells surrounding the implanted embryo during the early phases of pregnancy. Conditional deletion of the Cx43 gene in these stromal cells and the consequent disruption of their gap junctions led to a striking impairment in the development of new blood vessels within the stromal compartment, resulting in the arrest of embryo growth and early pregnancy loss. Further analysis of this phenotypical defect revealed that loss of Cx43 expression resulted in aberrant differentiation of uterine stromal cells and impaired production of several key angiogenic factors, including the vascular endothelial growth factor (Vegf). Ablation of CX43 expression in human endometrial stromal cells in vitro led to similar findings. Collectively, these results uncovered a unique link between steroid hormone-regulated cell-cell communication within the pregnant uterus and the development of an elaborate vascular network that supports embryonic growth. Our study presents the first evidence that Cx43-type gap junctions play a critical and conserved role in modulating stromal differentiation, and regulate the consequent production of crucial paracrine signals that control uterine neovascularization during implantation.     

The hamster as a model for embryo implantation: insights into a multifaceted process

Defects in preimplantation embryonic development, uterine receptivity, and implantation are the leading cause of infertility, pregnancy problems and birth defects. Significant progress has been made in our basic understanding of these processes using the mouse model, where implantation is ovarian estrogen-dependent in the presence of progesterone. However, an animal model where implantation is progesterone-dependent must also be studied to gain a full understanding of the embryo and uterine events that are required for implantation. In this regard, the hamster is a useful model and this review summarizes the information currently available regarding mechanisms involved in synchronous preimplantation embryo and uterine development for implantation in this species.     

Effects of clomiphene citrate on early pregnancy in guinea-pigs

Clomiphene citrate (2 mg/kg body wt) given on the day of mating can block or interrupt pregnancy in guinea-pigs. Corpus luteum function, uterine histology, implantation and embryo development were studied in clomiphene-treated and control animals on Days 5, 9 and 20 of pregnancy. Following treatment, only 25% of the females were regularly pregnant, presenting large and healthy foetuses. The other females examined showed either pregnancy with embryos undergoing resorption or no sign of pregnancy. In these females, corpus luteum size was reduced, progesterone concentrations were very low and the endometrial glands and the epithelium were often altered. It is concluded that clomiphene causes a reduction in fertility by altering the uterus and, by directly or indirectly inducing luteolysis, causes later pregnancy loss.