Estrogen inhibits interleukin-18 mRNA expression in the mouse uterus

Interleukin-18 (IL-18) is a proinflammatory cytokine expressed in female reproductive organs in humans, rats and mice. The physiological roles of uterine IL-18 and the regulatory mechanisms of IL-18 gene expression are unclear. The present study aimed to clarify the effects of estradiol-17beta (E2) and progesterone (P4) on IL-18 mRNA expression in the mouse uterus. Distribution and expression levels of IL-18 mRNA were studied using an RNase protection assay. Expression of IL-18 mRNA was observed in all organs studied, including testes, ovaries and uteri. The uterine IL-18 mRNA level of estrous mice was higher than that of diestrous mice. E2 treatment (1, 5, 25 or 250 ng/mouse) decreased uterine IL-18 mRNA levels in ovariectomized mice dose-dependently. E2 treatment acutely decreased IL-18 mRNA levels 3 h after injection, but these levels returned to the initial level after 48 h. P4 treatment (1 mg/mouse) decreased uterine IL-18 mRNA levels after 12 h, but levels returned to the initial level after 48 h. Both uterine IL-18 and IL-18Ralpha mRNAs were detected in cultured endometrial epithelial and stromal cells. These results suggest that uterine IL-18 expression is reduced by sex steroid hormones and that IL-18 acts on endometrial cells in a paracrine or autocrine manner.     

Expression of interleukin-18 receptor mRNA in the mouse endometrium

Interleukin-18 (IL-18) is a proinflammatory cytokine involved in chronic inflammation, autoimmune diseases, and a variety of cancers, and is expressed in mouse uteri. Our previous study suggested that IL-18 acts as a paracrine factor, regulating endometrial function. To elucidate the physiological roles of IL-18 in the mouse endometrium, the expression of the IL-18 receptor (IL-18R) alpha subunit was analyzed. IL-18Ralpha mRNA was expressed in several mouse organs in addition to the endometrium. In situ hybridization analysis using a biotin-labeled mouse IL-18Ralpha riboprobe demonstrated that IL-18Ralpha mRNA expression was detected in glandular epithelial cells, stromal cells around uterine glands, and myometrial cells in the mouse uterus, suggesting that these cells are targets for IL-18. The uterine IL-18Ralpha mRNA expression level changed with the estrous cycle. The uterine IL-18Ralpha mRNA levels of estrous mice were higher than those of diestrous mice. In addition, the IL-18Ralpha mRNA levels in uteri at 3 and 14 days after ovariectomy were higher than those at diestrus and decreased following treatment with estradiol-17beta or progesterone. These findings suggest that IL-18Ralpha gene expression is regulated by estrogen and progesterone and that the uterine IL-18 system is involved in the regulation of uterine functions in a paracrine manner.     

Immunophenotypical characterization of lymphocyte subpopulations of the uterus of non-pregnant and pregnant goats

The increased susceptibility during pregnancy to certain pathogens that cause abortions may be related to changes in the distribution and phenotype of lymphocyte subpopulations in the uterus. Histological, electron microscopic and immunocytochemical techniques were used in this study to examine whether such variations occur in different stages of the reproductive cycle of goats. The study of non-pregnant goats showed that most uterine lymphocytes were T cells and displayed both an intraepithelial and stromal distribution. CD8+ T lymphocytes were more numerous than CD4+ T lymphocytes. In the endometrial epithelium two lymphocyte subpopulations were observed: non-granulated CD2+ CD8+ T lymphocytes and granulated CD2+ CD8- T lymphocytes. During gestation, no lymphocytes were observed in the placentomal area, while a decreased number of T lymphocyte subpopulations were found in the inter-placentomal area. In the inter-caruncular epithelium, non-granulated CD2+ CD8+ T lymphocytes disappeared, whereas the granulated CD2+ CD8- T lymphocyte subpopulations increased their number and changed their morphology.     

Interferon-gamma and interleukin-4 mRNA expression by peripheral blood mononuclear cells from pregnant and non-pregnant cattle seropositive for bovine viral diarrhea virus

The acceptance of the fetal allograft by pregnant women and mice seems to be associated with a shift from a Th 1 dominated to a Th 2 dominated immune response to certain infectious agents. The goal of this study was to examine cytokine expression in peripheral blood mononuclear cells (PBMCs) from cattle immune to bovine viral diarrhea virus (BVDV) to determine whether pregnancy also has an influence on the type of immune response in this species. Forty-six heifers and cows between 14 months and 13 years of age were included in this study. Twenty-four were seropositive and 22 seronegative for BVDV. Eleven of the seropositive animals and 11 of the seronegative animals were in the eighth month of gestation, the remaining animals were virgin heifers. PBMC from these animals were analyzed for Interferon (IFN)-gamma and Interleukin (IL)-4 mRNA expression by real-time RT-PCR after stimulation with a non-cytopathic strain of BVDV. Additionally, an ELISA was performed to measure IFN-gamma in the supernatants of stimulated cell cultures. In BVDV seropositive animals, IFN-gamma mRNA levels were significantly higher than in BVDV seronegative animals and there was a significant positive correlation between the changes in IFN-gamma and IL-4 mRNA expression. There was, however, no significant difference in IFN-gamma and IL-4 mRNA levels between pregnant and non-pregnant animals. These results are inconsistent with BVDV inducing a Th1 or Th2 biased immune response. Furthermore, a shift in the cytokine pattern during bovine pregnancy was not evident.     

Serum concentrations of leptin in pregnant and non-pregnant bitches

Leptin regulates body weight and several physiological processes including reproduction. We evaluated the circulating levels of leptin in pregnant and non-pregnant bitches as well as their correlation with body weight, food intake and number of foetuses. Nineteen healthy German shepherd bitches were used and divided in two groups (pregnant n = 12 and non-pregnant n = 7). Blood samples were collected every 15 days starting from ovulation (Day 0) throughout pregnancy (pregnant group, P) or throughout luteal phase (non-pregnant group, NP) In pregnant bitches, leptin concentrations increased from the day of ovulation (1.32 ± 0.06 ng/ml) up to day 45 (1.51 ± 0.06 ng/ml; p < .01) and returned to baseline values from day 60 post-ovulation. In non-pregnant bitches, leptin concentrations remained constant throughout the whole observation period (estimated marginal mean ± SE=1.33 ± 0.38 ng/ml). Pairwise comparisons showed significant differences between P and NP at day 45 post-ovulation (p < .05). Multivariable models indicated that, controlling for time and litter size, there was a positive relationship between leptin concentration and BW (p < .05) although Pearson coefficients showed that the correlation between BW and leptin was only significant in NP animals at day 45 (r = 0.76, p < .05). The multivariable approach also suggested that, holding BW and time constant, leptin concentrations tend to increase as the number of puppies increased (p = .06). Our study supports indirectly the contribution of the feto-placental unit to the circulating maternal leptin concentration.     

Increased Th1/Th2 (IFN-gamma/IL-4) Cytokine mRNA ratio of rat embryos in the pregnant mouse uterus

Somatic cell nuclei can be dedifferentiated in ooplasm from another species, and interspecies cloned embryos can be implanted into the uteri of surrogates. However, no full pregnancies have been achieved through interspecific mammalian cloning. Rat blastocysts transferred into mouse uteri provide a unique model for studying the causes of interspecific pregnancy failure. In this study, intraspecific pregnancy (mouse-mouse) and interspecific pregnancy (rat-mouse) models were established. On Day 9 of pregnancy, the fetoplacental units were separated from the uterine implantation sites and the expression of messenger (m)RNA was quantitated by real-time PCR. We compared the mRNA expression levels of type-1 T helper (Th1) and type-2 T helper (Th2) cytokines, interferon-gamma (IFN-gamma), and interleukin-4 (IL-4) in fetoplacental units between intraspecific and interspecific pregnancy groups. The mRNA expression of IFN-gamma in the fetoplacental units of the interspecific pregnancy group was significantly higher than that of the intraspecific pregnancy group (P<0.05). The mRNA expression of IL-4 in the interspecific pregnancy group was significantly lower than that in the intraspecific pregnancy group (P<0.05). We also analyzed the ratio of IFN-gamma/IL-4 mRNA, and an increased IFN-gamma/IL-4 mRNA ratio was observed in the interspecific pregnancy compared with that in the intraspecific pregnancy group. The IFN-gamma and IL-4 mRNA expressions indicate that there is a Th1/Th2 imbalance in the feto-maternal interface of interspecific pregnancies. Bias of Th1 cytokine dominance may be a barrier to reproductive success between species.     

Concentrations of luteinising hormone and progesterone in pregnant and non-pregnant heifers

An experiment was conducted to determine if concentrations of luteinising hormone or progesterone were different in pregnant or non-pregnant heifers for seven days before and 20 days after a successful or non-successful insemination. Heifers with an oestrous cycle length of 18 to 24 days only were used and they were bled at 08.00, 16.00 and 24.00 each day for seven days before and for 20 days after insemination with thawed semen (treatment 1) or semen diluent (treatment 2). Animals allocated to treatment 3 had the embryo nonsurgically flushed from the uterus at days 10 to 12 while animals allocated to treatment 4 were inseminated with semen diluent and then had a viable embryo transferred to the uterus between days 10 and 12. All animals were slaughtered between 19 and 21 days after insemination and pregnancy rate determined. There were no differences in basal luteinising hormone levels between treatments. Blood concentrations of progesterone were not different before insemination and for 16 days after insemination for pregnant (11 out of 15) and non-pregnant heifers (14) allocated to treatments 1 and 2. Between days 17 and 20, progesterone concentrations declined in non-pregnant heifers. Transfer of an embryo to non-pregnant heifers on day 10 to 12, did not affect progesterone concentrations, but non-surgical flushing of the embryo caused a decline in blood concentrations of progesterone. It was concluded that basal blood concentrations of luteinising hormone and progesterone, in samples taken three times daily were not different in pregnant or non-pregnant heifers before and for 16 days after insemination.     

Progesterone receptor mRNA levels during pregnancy, labor, lactation and the estrous cycle in rat uterus

Progesterone plays important roles in the regulation of female reproduction. In this study, progesterone receptor (PR) mRNA levels in rat uterus during pregnancy, labor, lactation and the estrous cycle were examined by competitive RT-PCR. During pregnancy and lactation, PR mRNA levels had decreased on day 20 of pregnancy (P20) and P21 compared with P15 but increased during labor. After a decline on day 1 of lactation (L1), PR mRNA levels had increased again on L3 and L14 compared with P15, P18, P20, P21 and P21pm (at 2200-2300 h on P21). There was no significant change in the PR mRNA level during the estrous cycle. The PR mRNA level did not change during 1 week of progesterone treatment or afterwards. Injection of 17beta-estradiol did not affect PR mRNA levels in rats treated with progesterone or those without any injections. In rats on P18, 17beta-estradiol injection did not change PR mRNA levels after sham-operation but induced an increase in PR mRNA levels of rats ovariectomized 6 h before the treatment. These results suggest that uterine PR mRNA levels are differently regulated during late pregnancy, labor and lactation, and during labor estrogen is one of the essential factors for the increase in PR mRNA levels.     

Plasma progesterone concentrations in pregnant and non-pregnant llamas (Lama glama)

Female llamas ovulate in response to copulation, and progesterone secretion by the corpus luteum indicates recent ovulation (mating) and, or, pregnancy. The plasma progesterone concentration was 0.9 to 1.4 ng/ml in five non-pregnant llamas and 7.4 to 9.2 ng/ml in three llamas in the last month of pregnancy. After ovulation had been induced in nine of 10 llamas by a single intramuscular injection of 500 or 750 iu of human chorionic gonadotrophin, the plasma progesterone concentration increased after two days from 0.5 to 1.2 ng/ml to 4.6 to 10.3 ng/ml after six to nine days and returned to basal values after 10 to 13 days, reflecting the life-span of a corpus luteum in the absence of conception. After a male llama had been introduced into a group of 13 females, 10 matings which resulted in eight conceptions occurred in the first 11 days, and 11 of the llamas became pregnant. The llamas' progesterone concentrations increased after mating and remained high if conception had occurred: 6 to 12 ng/ml in months one to four, and 5 to 9 ng/ml in months five to nine of the 11-month gestation. Two of the 13 llamas had high concentrations of progesterone although they did not become pregnant.     

Comparative analysis between endometrial proteomes of pregnant and non-pregnant ewes during the peri-implantation period

Background:Early pregnancy failure has a profound impact on both human reproductive health and animal production.2/3 pregnancy failures occur during the peri-implantation period;however,the underlying mechanism(s)remains unclear.Well-organized modification of the endometrium to a receptive state is critical to establish pregnancy.Aberrant endometrial modification during implantation is thought to be largely responsible for early pregnancy loss.Result:In this study,using well-managed recipient ewes that received embryo transfer as model,we compared the endometrial proteome between pregnant and non-pregnant ewes during implantation period.After embryo transfer,recipients were assigned as pregnant or non-pregnant ewes according to the presence or absence of an elongated conceptus at Day 17 of pregnancy.By comparing the endometrial proteomic profiles between pregnant and non-pregnant ewes,we identified 94 and 257 differentially expressed proteins(DEPs) in the endometrial caruncular and intercaruncular areas,respectively.Functional analysis showed that the DEPs were mainly associated with immune response,nutrient transport and utilization,as well as proteasome-mediated proteolysis.Conclusion:These analysis imply that dysfunction of these biological processes or pathways of DEP in the endometrium is highly associated with early pregnancy loss.In addition,many proteins that are essential for the establishment of pregnancy showed dysregulation in the endometrium of non-pregnant ewes.These proteins,as potential candidates,may contribute to early pregnancy loss.     

Comparative analysis between endometrial proteomes of pregnant and non-pregnant ewes during the peri-implantation period

Background

Early pregnancy failure has a profound impact on both human reproductive health and animal production. 2/3 pregnancy failures occur during the peri-implantation period; however, the underlying mechanism(s) remains unclear. Well-organized modification of the endometrium to a receptive state is critical to establish pregnancy. Aberrant endometrial modification during implantation is thought to be largely responsible for early pregnancy loss.

Result

In this study, using well-managed recipient ewes that received embryo transfer as model, we compared the endometrial proteome between pregnant and non-pregnant ewes during implantation period. After embryo transfer, recipients were assigned as pregnant or non-pregnant ewes according to the presence or absence of an elongated conceptus at Day 17 of pregnancy. By comparing the endometrial proteomic profiles between pregnant and non-pregnant ewes, we identified 94 and 257 differentially expressed proteins (DEPs) in the endometrial caruncular and intercaruncular areas, respectively. Functional analysis showed that the DEPs were mainly associated with immune response, nutrient transport and utilization, as well as proteasome-mediated proteolysis.

Conclusion

These analysis imply that dysfunction of these biological processes or pathways of DEP in the endometrium is highly associated with early pregnancy loss. In addition, many proteins that are essential for the establishment of pregnancy showed dysregulation in the endometrium of non-pregnant ewes. These proteins, as potential candidates, may contribute to early pregnancy loss.

Electronic supplementary material

The online version of this article (doi:10.1186/s40104-015-0017-0) contains supplementary material, which is available to authorized users.     

Serum progesterone, oestrone and oestradiol in pregnant and non-pregnant red deer hinds.

Changes in serum progesterone (P), oestrone (E1) and oestradiol (E2) concentrations were monitored in 34 female red deer (Cervus elaphus) shortly after the end of the breeding season and at mid-gestation. Pregnancy could be detected on the basis of serum P, but there were no significant differences between the pregnant and non-pregnant animals farmed animals in E1 and E2 concentrations. Twenty-five pregnant hinds captured in winter showed serum P levels similar to those found in farmed deer during the gestation period.     

Concentrations of isoflavones and their metabolites in the blood of pregnant and non-pregnant heifers fed soy bean

The present study compared the changes in isoflavones (daidzein and genistein) and their metabolite (equol and para-ethyl-phenol) concentrations in the blood plasma of cyclic and pregnant heifers after feeding with soy bean. Twelve healthy heifers were divided into three groups: cyclic heifers (days 8-12 of the estrous cycle; control group; n=4), an early pregnancy group (2 months pregnant; n=4) and a late pregnancy group (8 months pregnant; n=4). All heifers were fed a single dose of 2.5 kg of soy bean and then blood samples were taken from the jugular vein for 8 h at predetermined intervals. The concentrations of soy bean-derived isoflavones and their active metabolites were measured in the blood plasma on an HPLC system. In the blood plasma of the early- and late-pregnant heifers, we found lower concentrations and time-dependent decreases in daidzein and genistein in comparison to cyclic heifers (P<0.05). Moreover, we noticed significant increases of equol and para-ethyl-phenol in the blood plasma of the early-pregnant heifers (P<0.05). In contrast, in the blood plasma of the late-pregnant heifers, we did not find an increase in the isoflavone metabolite concentrations compared with the early-pregnant heifers (P>0.05). In conclusion, physiological status (cyclicity or pregnancy) of the females influenced the concentrations of isoflavone metabolites in the blood plasma of the heifers. The stage of pregnancy affects isoflavone absorption, biotransformation and metabolism differently and results in higher concentrations of active metabolites of isoflavones during early pregnancy in comparison to their lower concentrations during late pregnancy. Therefore, we surmise that cows are more sensitive to active isoflavone metabolite actions during early pregnancy than cyclic heifers and heifers in late pregnancy.