Uterine lymphocyte distribution and interleukin expression during early pregnancy in cows

Both the production of cytokines and the distribution of immune cells within the uterus change during early pregnancy. Evidence obtained mainly from mice indicates that these changes are important for implantation and in preventing a maternal immune response to the conceptus. The ruminant embryo also produces interferon tau at this time, the signal for the maternal recognition of pregnancy. The relationship between these events in cows was studied using uteri from three groups of animals on day 16 after observed oestrus: (i) cyclic controls, (ii) pregnant and (iii) inseminated but with no embryo present. Embryo size and the antiviral activity in uterine flushings (indicative of the interferon tau concentration) were measured. Sections of intact uterus were frozen for the localization and quantitation of CD4(+) (T lymphocytes), CD14(+) (macrophages) and CD21(+) (B lymphocytes) uterine cells by immunohistochemistry. The expression of interleukin (IL)-1alpha, IL-2, IL-6 and IL-10 mRNAs in uterine extracts was measured by RT-PCR. Neither embryo size, interferon tau concentration nor pregnancy status influenced the distribution of CD4(+), CD14(+) or CD21(+) cells in the day 16 uterus. Endometrial IL-1alpha mRNA was detected in most cows across the groups, whereas IL-2 mRNA was only present in the non-pregnant uterus. IL-6 and IL-10 mRNAs were not detectable in any uteri. In conclusion, IL-2 mRNA expression is detectable in the non-pregnant but not the pregnant uterus on day 16 and interferon t is unlikely to play a role in the redistribution of immune cells in the uterus during early bovine pregnancy.     

Platelet-activating factor in the rabbit uterus during early pregnancy

Platelet-activating factor (PAF) concentrations were low in the non-pregnant, oestrous uterus (mean +/- s.e.m.: 2.2 +/- 1.2 pmol/g, n = 3). However, uterine PAF increased dramatically during pregnancy to a maximum of 37.8 +/- 4.90 pmol/g (n = 7) on Day 5. By Day 7, PAF concentrations in the uteri of pregnant rabbits had returned to levels similar to those found at oestrus. In contrast, uterine PAF in pseudopregnant rabbits peaked at 30.6 +/- 2.8 pmol/g (n = 8) on Day 4, declined to 20.5 +/- 2.4 pmol/g (n = 8) on Day 5 and then remained at that concentration through Day 7. Uterine PAF co-migrated with synthetic PAF (1-O-hexadecyl-2-acetyl-sn-glycero-phosphocholine) in both thin-layer and normal-phase high-performance liquid chromatography. PAF activity in the uterus during pregnancy and pseudopregnancy was found almost exclusively in the endometrium; little or no PAF was found in myometrium, uterine flushings or blastocysts. While no PAF was detected in blastocysts on Days 5 and 6 of pregnancy, the presence of the embryo appears to modulate biosynthesis and/or degradation of PAF by the uterus, since PAF decreased significantly in uterine tissue apposed to the implanting embryo (but not in similar areas between such attachment sites). Increased concentrations of PAF in the preimplantation rabbit uterus followed by a dramatic decrease on the day of blastocyst attachment suggest that this potent inflammatory autacoid may play a vital role in implantation.     

Estrogen regulates the expression of the small proline-rich 2 gene family in the mouse uterus

Small proline-rich (SPRR) proteins are structural components of the cornified cell envelope (CE), a specialized structure beneath the plasma membrane of stratified squamous epithelia. They are divided into four families, of which SPRR2 is the most complex consisting of 11 members (2a-2k) in the mouse. To assess the possible influence of estrogen on expression of the SPRR2 family in the uterus, we examined the effect of 17b-estradiol (E2) on SPRR2 mRNA levels on ovariectomized (OVX) adult mice. We employed a combination of laser capture microdissection (LCM) and semiquantitative RT-PCR to examine expression in particular uterine cell types - luminal epithelia, and stromal and muscle cells. We also used quantitative real-time PCR to measure levels of the mRNA of several SPRR2 proteins in the mouse uterus over the estrous cycle and during early pregnancy. Expression of SPRR2a, 2b, 2c, 2d, 2e, 2f and 2g mRNA was increased by estrogen treatment. SPRR2a, 2b, 2d and 2e were highly expressed on day 1 and 2 of pregnancy, but decreased markedly by days 3-6. Interestingly, several members of the SPRR2 family were preferentially up-regulated at implantation sites compared to inter-implantation sites around day 4 of pregnancy. They were abundant during proestrus and estrus but declined rapidly during metestrus. These results indicate that estrogen is a key regulator of the expression of the SPRR2 family in the mouse uterus during the estrous cycle and early pregnancy. In addition, they suggest that some members of the family play an important role in uterine processes such as the estrous cycle, early pregnancy and implantation.     

Relationship between peripheral estrogen concentrations at insemination and subsequent fetal loss in cattle

In a survey on pregnancy rate and embryonic losses in dairy cattle on 6 Israeli farms, cows (n = 78) were divided into 3 groups on the basis of ultrasonography at 21 d post insemination; pregnancy diagnosis at 40 to 50 d post insemination and blood progesterone (P4) levels at 21 d. The groups were either pregnant (P4 level > 1.0 ng/ mL); not pregnant (P4 < 0.5 ng/mL), or showed early embryo loss (P4 > 1.0 ng/mL and the presence of an embryonic vesicle on D 21 but later returned to estrus or were found not pregnant on D 40 to 50). On the day of insemination, peripheral estrogen was significantly higher (P < 0.05) in the early embryo loss group (15.3 +/- 1.1 pg/mL, n = 27) than in pregnant (9.4 +/- 0.6 pg/mL, n = 26) or not pregnant (9.6 +/- 0.7 pg/mL, n = 25) group. The cows on 3 farms which were fed 1 to 2 kg/d of vetch (Vicia sativa), an estrogenic legume, had higher estrogen concentrations on the day of insemination than cows (2 farms) fed other legumes (13.7 +/- 0.64, n = 58 vs 10.7 +/- 0.8 pg/mL, n = 42; P < 0.01). On one of the 3 farms, vetch was replaced with alfalfa after the first year. Following the cessation of vetch feeding the estrogen concentrations in the blood decreased from 32 +/- 5 pg/mL to 14 +/- 2 pg/mL (n = 9). These data suggest that high peripheral estrogen on the day of insemination is associated with early embryonic loss. These data also indicate that estrogen concentrations on the day of insemination can be influenced by diet.     

Inhibiting uterine PC6 blocks embryo implantation: an obligatory role for a proprotein convertase in fertility

Successful embryo implantation involves complex interactions between the embryo and the uterus and is critical in establishing pregnancy. Proprotein convertase (PC) 6 (PC6) is one of the PC endoproteases regulating protein function through posttranslational activation of precursor proteins, including growth and differentiation factors. Here we show that PC6 protein is induced in the uterine stromal cells specifically at the site of embryo attachment during early pregnancy in mice. In vivo blocking of uterine production of PC6 protein using morpholino antisense oligonucleotides in mice resulted in total inhibition of implantation, revealing a vital role for PC6 in modulating the uterus for embryo implantation. Studies in primates (rhesus monkey and human) showed a dramatic upregulation of endometrial PC6 during the phase of uterine receptivity and at implantation, particularly during a critical uterine cell differentiation process termed decidualization. Thus, the current studies have demonstrated that PC6 is an essential molecule in modulating uterine function to support the establishment of embryo implantation. Interestingly, PC6 is one of the PCs identified to be important in processing the coat protein of HIV; inhibition of PCs has been suggested to be an effective approach to reduce HIV transmission. We therefore propose the novel concept that PC6 could be a potential nonhormonal target in the female reproductive tract for dual protection for women, both in preventing pregnancy and reducing HIV infection.     

Relationship of plasma estradiol-17beta, total estrogen, and progesterone to estrus behavior in mithun (Bos frontalis) cows

The objectives of this study were (1) to establish the characteristics of estrus behavior in mithun cows (n = 12) and (2) to determine the relationships between this behavior and the plasma concentrations of estradiol-17beta (E2), total estrogen, and progesterone. Estrus was detected by visual observations of estrus signs, per recta examination of genitalia and bull parading thrice a day for three consecutive cycles. Among the behavioral signs of estrus, the cow to be mounted by bull (100%) was the best indicator of estrus followed by standing to be mounted (92%). Per rectum examination of genital organs revealed relaxed and open os externa of cervix, turgid uterus, and ovaries having palpable follicles in all animals. The mean (+/-SEM) length of estrus cycle and duration of estrus were recorded to be 21.8 +/- 0.69 days and 12.6 +/- 1.34 h, respectively. Endocrine profiles during the peri-estrus period showed that the mean highest peak concentrations of E2 (27.29 +/- 0.79 pg/ml) and total estrogen (45.69 +/- 2.32 pg/ml) occurred at -3.90 +/- 2.27 and -3.89 +/- 2.26 h prior to the onset of estrus, respectively. Plasma progesterone concentration was basal (0.14 +/- 0.001 ng/ml) during the peri-estrus period. Plasma E2 and total estrogen were found to increase from 6 days before estrus to reach a peak level on the day of estrus and decline thereafter to basal level on day 3 of the cycle. The plasma progesterone concentration was the lowest on the day of estrus showing gradual increase to register a peak level on day 15 of the cycle. Estrus behavior was found to be positively correlated with the maximum peak concentration of E2 (r = 0.89; P < 0.0001) and total estrogen (r = 0.66; P = 0.019) during the peri-estrus period. The mean total estrogen concentration during the peri-estrus period was significantly correlated with estrus behavior (r = 0.60; P = 0.04). The correlations between the estrus behavior and E2:progesterone ratios at 6 days before the onset of estrus (r = 0.92) and on the day of estrus (r = 0.95) was significant. The total estrogen:progesterone ratios at 6 days before the onset of estrus and on the day of estrus were also positively correlated with the estrus behavior (r = 0.86 and 0.88). In conclusion, our results suggest that the maximum peak concentration of E2 and total estrogen and mean level of total estrogen during the peri-estrus period and the E2:progesterone and total estrogen:progesterone ratios on 6 days before the onset of estrus and on the day of estrus are the important factors contributing the behavioral manifestation of estrus in mithun cows.     

Hormonal control of the expression of antibody-defined lactosaminoglycans in the mouse uterus

The uterus undergoes a number of hormone-induced changes during estrus and early pregnancy. Changes in the uterine glycoprotein population have been investigated by using the monoclonal antibodies SSEA-1, IIC 3, A5, and C6. These antibodies detect specific terminal or side-chain modifications of lactosaminoglycan molecules. In ovariectomized female mice treated with estrogen, SSEA-1 was the only antigen expressed at the uterine epithelium. Progestational stimuli for 2 days induced SSEA-1 expression in the uterine glands and the sialylated form of A5 at the uterine epithelium. Three days of progesterone treatment induced IIC3 expression within the uterine glands. An additional day of progesterone treatment resulted in a uterine epithelial expression of IIC3. The expression of these antigens in the progestational phase were confirmed in naturally mated females. In these preparations, expression of the sialylated form of C6 was also observed, beginning on Day 2.0 of gestation. These observations suggest subtle modification of lactosaminoglycan chains during the hormonally induced preparative and receptive phases of the mouse uterus.     

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.     

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.     

Physiological aspects of blastocyst uterine interaction

An interaction between the blastocyst and the uterus is essential for establishment of pregnancy. Because maternal estrogen is not an absolute requirement, estrogen of embryonic origin has been implicated in this process in the pig and the rabbit. Furthermore, estrogen forming capacity has been documented in the blastocyst of these species. However, while the complete machinery for steroid synthesis in the pig balstocyst has been demonstrated, the issue is still unresolved for the rabbit blastocyst. In the present communication we have shown that 17α-hydroxylase and C17–20-lyase, enzymes involved in the formation of androgens (C¹⁹-steroids) from C²¹-steroids (progestins), are present in day-6 rabbit blastocysts. C17–20-lyase activity was undetectable to low in day-5 and increased in day-6 balstocysts. The activity was further increased in day-6 blastocysts cultured for 24 h. Because prostaglandins have been implicated in uterine vascular changes at about the time of implantation and pregnancy establishment, and because catechol estrogens are more potent than phenolic estrogens in stimulating prostaglandin synthesis in the blastocyst and the uterus, we determined catechol estrogen forming capacity in the rabbit and pig blastocyst. Catechol estrogen forming capacity (estrogen-2/4-hydroxylase) in the pig blastocyst appears on day 10 of pregnancy, peaks on day 12 and then declines. Our preliminary experiments also indicate that day-6 rabbit blastocysts have catechol estrogen forming capacity. On the basis of our present findings and of others, we propose that catechol estrogens of embryonic origin mediate the stimulatory effect of estrogens on prostaglandin synthesis in the embryo and/or the uterus and thus participate in the process of establishment of pregnancy.     

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.     

GABA consumption during early pregnancy impairs endometrial receptivity and embryo development in mice

γ‐Aminobutyrate (GABA) is commonly used as a food supplement and a health care product by young females, due to its positive roles in relieving stress, alleviating anxiety, and improving sleep. However, its recommended daily dose in different products varies widely. Besides, it is unknown whether, and how, GABA consumption during early pregnancy influences pregnancy establishment. In this study, we found that when pregnant mice were treated with a high (12.5 mg/g) dose of GABA (orally) during preimplantation, there was a reduction in the number of implantation sites on day 5 of pregnancy. Also, among these unimplanted embryos, most exhibited morphological degeneration and developmental retardation, and only a few of them developed into blastocysts but could not implant into the uterus. Moreover, the expression of uterine receptivity–related factors—LIF, E‐cadherin, and HOXA10—were all downregulated, while the number of uterine glands was reduced in the high GABA dose group. Finally, in vitro results demonstrated that GABA (ranging from 10 to 50 μg/μL) markedly inhibited preimplantation embryo development in a dose‐response manner. However, this inhibitory effect was not observed when the embryos were pretreated with 40 μΜ 2‐hydroxysaclofen, a GABA_B antagonist, indicating that GABA exerts its inhibitory effects via its B‐type receptor. Our results suggest that exposure to certain GABA concentrations, during early pregnancy, can impair preimplantation embryo development via its B‐type receptor, and endometrial receptivity, which greatly disturbs early embryo implantation in mice. These findings could raise concerns about GABA consumption during the early stages of pregnancy.     

Uterine blood supply as a main factor involved in the regulation of the estrous cycle--a new theory

The paper presents a new theory on the physiological mechanism of initiation of luteolysis, function of endometrial cells and protection of corpus luteum. This theory is based on previous studies published by the authors and their coworkers on the retrograde transfer of PGF2alpha in the uterine broad ligament vasculature during the estrous cycle, early pregnancy and pseudopregnancy. The studies were focused on cyclic changes in uterine blood supply and the apoptosis of endometrial cells. Moreover, the results of many other authors are cited. The statements of the theory are as follows: 1. The initiation of luteolysis is a consequence of regressive changes in the endometrium which are due to the reduction of the uterine blood supply below the level necessary to provide for the extended needs of active endometrium. 2. During the luteal phase, both a considerable increase in uterine weight and a decrease in blood flow through the uterine artery, resulting from increasing progesterone concentration, reduce the uterine blood supply. In comparison to the volume of blood flowing to the porcine uterus during the estrus period, only 30-40% of the blood volume is determined on day 12 of the estrous cycle. The uterine weight at that time is 40-60% larger than that in the early luteal phase. Thus, due to the considerable constriction of uterine blood vessels, there is a discrepancy between the requirement for oxygen and other factors transported by blood and the possibility of supplying the uterus with these substances. After reaching the threshold of uterine blood supply level, which in pigs takes place around day 12 of the estrous cycle, regressive changes and PGF2alpha release from endometrial cells occurs. 3. Estrogens and progesterone are the major factors affecting blood flow in vessels supplying the uterus. The factors that modulate, complement and support vasodilation and vasoconstriction are: PGE2, LH, oxytocin, cytokines, neurotransmitters and other local blood flow regulators. In some animal species these modulators, especially those of embryonic origin, may be crucial for the status of uterine vasculature. 4. During early pregnancy, the action of embryo signals (estrogens, cytokines), endometrial PGE2 as well as LH results in the relaxation of the uterine artery (pigs: day 12) and, consequently, in an increase in uterine blood supply. This reaction of the maternal recognition of pregnancy effectively prevents regressive changes in well developed endometrial cells to occur. 5. Local uptake and retrograde transfer of PGF2alpha into the uterine lumen during early pregnancy protects corpus luteum from PGF2alpha luteolytic action. 6. During the period of regressive changes resulting from the limited uterine blood supply, endometrial cells restrain PGF2alpha synthesis. They are, however, still capable of releasing prostaglandin when uterine blood supply is improved after the embryo appears in the uterus. This potential capability for PGF2alpha synthesis was demonstrated in in vitro studies when endometrial cells collected during its regressive phase were incubated in medium and stimulated by LH and oxytocin. 7. Prostaglandin F2alpha pulses in venous blood flowing from the uterus do not confirm pulsatile secretion of PGF2alpha. The pulses may result from the pulsatile excretion of PGF2alpha with venous blood according to the rhythmic uterine contractions associated with oxytocin secretion. 8. The results supporting this concept are presented and discussed in due course. The critique of Bazer and Thatcher's theory on exocrine versus endocrine secretion of prostaglandin F2alpha during the estrous cycle is also depicted.     

Interaction between uterine PGE and PGF2α production and the nitridergic system during embryonic implantation in the rat

Embryonic implantation is a complex process in which both maternal andembryonic signals are involved. In the present study, we evaluated changes in uterine prostaglandins production and nitric oxide synthase (NOS) activity during the course of early pregnancy and their interaction during implantation in rats. Uterine phospholipase A₂ (PLA₂) activity is increased on days 5 (day of ovoimplantation) and 6, compared to preimplantation days (3 and 4). This enhanced activity might be responsible for the observed increase in uterine PGE and PGF_2α production observed on day 5 of pregnancy, which induces endometrial vascular permeability and decidualization. When embryo access to the uterus is impaired, the increase of PG production is suppressed. During postimplantation, PGE levels return to preimplantation values, while PGF_2α decreased with respect to preimplantation values. Uterine NOS activity is also increased on day 4 and reaches a maximum on day 5, with a profile similar to PGE and PGF_2α Dexamethasone administered in vivo decreased uterine NOS activity on day 4 of pregnancy but not on day 5, suggesting the presence of at least two types of NOS enzymes in the early days of pregnancy. A competitive inhibitor of NOS, L-NAME (600 and 1000 μM) induced a decrease in PGE and PGF_2α production in uterine tissue on day 5 of pregnancy. These results suggest the existence of a physiologically relevant nitridergic system which modulates prostaglandin production in the rat uterus during embryonic implantation.