Effects of age and altrenogest treatment on conceptus development and secretion of LH, progesterone and eCG in early-pregnant mares

The treatment of early pregnant mares with a history of repeated early embryonic loss with the progestin altrenogest has become routine; however no controlled studies on the efficiency of altrenogest to prevent embryonic losses are available so far. In the present study, we have investigated effects of altrenogest treatment in mares on conceptus development and the secretion of LH, progesterone, and eCG until day 100 of pregnancy. In addition, differences related to age of mares were assessed. Mares were treated with altrenogest (0.044 mg/kg per os once daily) or sunflower oil (10 ml per os once daily) from day 6 to day 100 after ovulation. Blood samples for analysis of LH, progesterone, and eCG were collected. The size of the embryonic vesicle and embryo/fetus was determined by ultrasound. No difference in the per cycle pregnancy rate between altrenogest-treated (75%) and sunflower oil-treated mares (74%) was detected (n.s.). A significant effect of age but not of altrenogest treatment on mean diameter of the embryonic vesicle was found between days 12 and 22 of pregnancy (e.g. day 15: control, 4-8 years: 22.9 ± 1.0 mm, >8 years: 22.0 ± 1.7 mm, altrenogest, 4-8 years: 26.1 ± 2.0 mm, >8 years: 20.4 ± 1.0 mm, P < 0.05). A significant effect of age and treatment on size of the embryo proper between days 30 and 45 was detected (P < 0.05). In the control group but not in the altrenogest group, size of the embryo proper respective fetus was negatively correlated with age of the mares (day 30: r = −0.834, P < 0.05; day 35: r = −0.506, P < 0.05). Plasma concentrations of LH and progesterone were neither effected by age nor by treatment of mares, but significant effects of age and altrenogest treatment on eCG concentrations between days 40 and 130 were detected (P < 0.05). The present study demonstrates for the first time a positive influence of altrenogest-treatment on a retarded development of the embryo respective fetus around the beginning of placentation in mares older than 8 years.     

Effect of conceptus secretions on HOXA10 and PTGS2 gene expression, and PGE2 release in co-cultured luminal epithelial and stromal cells of the porcine endometrium at the time of early implantation

Homeobox A10 (HOXA10) gene expression was demonstrated in the endometrium of adult porcine uteri, however there is little information concerning the role of this gene in the pig. Objectives of the present study were to examine: 1) the expression of HOXA10 in the endometrium of cyclic and early pregnant gilts; 2) the effect of estradiol (E₂) and progesterone (P₄) on HOXA10 expression in porcine luminal epithelial (LE) and stromal (ST) cells in vitro; 3) the effect of E₂ and conceptus-exposed medium (CEM) on HOXA10 and prostaglandin endoperoxide synthase (PTGS2) gene expression and prostaglandin (PG) E₂ secretion from LE and ST cells in a co-culture model. The abundance of HOXA10 mRNA was increased on day 15 of pregnancy in comparison to day 15 of the estrous cycle. Moreover, increased HOXA10 mRNA level was detected in ST cells after E₂ and P₄ treatment. E₂ stimulated the expression of HOXA10 in LE cells cultured on collagen and pre-treated with steroids, but not in LE on plastic surfaces. Addition of CEM to LE cells cultured in collagen-coated inserts of the co-culture system resulted in elevated HOXA10 and PTGS2 gene expression and PGE₂ secretion in these cells, but not in ST cells cultured in basal compartments. ST cells directly treated with E₂ or CEM showed higher levels of HOXA10 and PTGS2 expression. Blocking of estrogen receptors with ICI-182,780 did not influence the stimulatory effect of CEM. We conclude that HOXA10 expression in the porcine endometrium is closely related to the implantation process and stimulated by conceptus products. Moreover, the co-culture system of LE and ST cells is a promising model for the study of endometrial response to conceptus-derived factors.     

Effects of flunixin meglumine, recombinant bovine somatotropin and/or human chorionic gonadotropin on pregnancy rates in Nelore cows

The objective was to compare pharmacological strategies aiming to inhibit prostaglandin F2 alpha (PGF_2α) synthesis (flunixin meglumine; FM), stimulate growth of the conceptus (recombinant bovine somatotropin; bST) and progesterone (P₄) synthesis (human chorionic gonadotropin; hCG), as well as their combinations, regarding their ability to improve pregnancy rates in beef cattle. Lactating Nelore cows (N = 975), 35 to 70 days postpartum, were synchronized and inseminated by timed artificial insemination (TAI) on Day 0. On Day 7, cattle were allocated into eight groups and received one of the following treatments: saline (S) on Days 7 and 16 (Group Control); S on Day 7 and FM on Day 16 (Group FM); bST on Day 7 and S on Day 16 (Group bST); bST on Day 7 and FM on Day 16 (Group bST + FM); hCG on Day 7 and S on Day 16 (Group hCG); hCG on Day 7 and FM on Day 16 (Group hCG + FM); bST and hCG on Day 7 and S on Day 16 (Group bST + hCG), or bST and hCG on Day 7 and FM on Day 16 (Group bST + hCG + FM). The aforementioned treatments were administered at the following doses: 2.2 mg/kg FM (Banamine^®; Intervet Schering-Plough, Cotia, SP, Brazil), 500 mg bST (Boostin^®; Intervet Schering-Plough), and 2500 IU hCG (Chorulon^®; Intervet Schering-Plough). Pregnancy diagnosis was performed 40 days after TAI by transrectal ultrasonography. Pregnancy rates were not significantly different among treatments. However, there was a main effect of hCG treatment to increase pregnancy rates (63.0 vs. 55.4%; P = 0.001). Concentrations of P₄ did not differ significantly among groups on Day 7 or on Day 16. However, consistent with the higher pregnancy rates, hCG increased P₄ concentrations on Day 16 (10.6 vs. 9.6 ng/mL, respectively; P = 0.05). We concluded that hCG treatment 7 days after TAI improved pregnancy rates of lactating Nelore cows, possibly via a mechanism leading to induction of higher P₄ concentrations, or by reducing the luteolytic stimulus during maternal recognition of pregnancy.     

Gastrulation and the establishment of the three germ layers in the early horse conceptus

Experimental studies and field surveys suggest that embryonic loss during the first 6 weeks of gestation is a common occurrence in the mare. During the first 2 weeks of development, a number of important cell differentiation events must occur to yield a viable embryo proper containing all three major germ layers (ectoderm, mesoderm, and endoderm). Because formation of the mesoderm and primitive streak are critical to the development of the embryo proper, but have not been described extensively in the horse, we examined tissue development and differentiation in early horse conceptuses using a combination of stereomicroscopy, light microscopy, and immunohistochemistry. Ingression of epiblast cells to form the mesoderm was first observed on day 12 after ovulation; by Day 18 the conceptus had completed a series of differentiation events and morphologic changes that yielded an embryo proper with a functional circulation. While mesoderm precursor cells were present from Day 12 after ovulation, vimentin expression was not detectable until Day 14, suggesting that initial differentiation of mesoderm from the epiblast in the horse is independent of this intermediate filament protein, a situation that contrasts with other domestic species. Development of the other major embryonic germ layers was similar to other species. For example, ectodermal cells expressed cytokeratins, and there was a clear demarcation in staining intensity between embryonic ectoderm and trophectoderm. Hypoblast showed clear α1-fetoprotein expression from as early as Day 10 after ovulation, and seemed to be the only source of α1-fetoprotein in the early conceptus.     

Expression profile and role of prostacyclin receptor (PTGIR) in peri-implantation porcine conceptuses

Prostacyclin (prostaglandin I₂ [PGI₂]) signaling system not only plays a pivotal role in vascular function in many species but is also important during early pregnancy in rodents and ruminants. Recently, abundant concentrations of PGI₂ were found in the endometrium and uterine lumen of gilts at the time of implantation. In the present study, conceptuses collected on Days 10, 12, 14, 16, and 18 of pregnancy were examined for the expression of PGI₂ receptors, PTGIR. Moreover, the effect of iloprost (a PGI₂ analogue) on attachment, proliferation, and apoptosis in conceptus trophoblast (Tr) cells was investigated in vitro. Increased PTGIR mRNA expression was observed in Day 16 trophoblasts compared with Days 10, 12, and 14 conceptuses (P < 0.001) and Day 18 trophoblast tissue (P < 0.01). Embryos from Day 18 of gestation revealed greater PTGIR mRNA expression compared with Day 16 embryos (P < 0.01). In contrast to mRNA, PTGIR protein level in conceptus and trophoblast tissue was high on Days 12 and 14, followed by a decrease observed on Day 16. On Day 18 of pregnancy, PTGIR protein was detected in both trophoblast and embryonic tissue. Iloprost stimulated attachment and proliferation of Tr cells, but this effect was abolished by the addition of the PTGIR-specific antagonist, CAY10441, into culture medium. Addition of iloprost neither did affect the ratio of BAX/BCL-2 gene expression in cultured Tr cells nor did protect these cells from staurosporine-induced apoptosis. In summary, PTGIR is expressed in porcine conceptuses, and PGI₂ acting through this receptor may promote the attachment and proliferation of Tr cells, thereby facilitating conceptus implantation.     

Expression of VEGF-receptor system in conceptus during peri-implantation period and endometrial and luteal expression of soluble VEGFR-1 in the pig

In view of the importance of vascular events observed during gestation, it was hypothesized that the VEGF-receptor system plays a critical role during early pregnancy and maternal recognition of pregnancy in pigs. This hypothesis was tested by examining the expression of the VEGF-receptor system in the porcine conceptus. Additionally, the endometrium, corpus luteum (CL) and embryos were studied for the expression of soluble VEGF receptor 1 (sVEGFR-1), the strong endogenous antagonist of VEGF. The expression patterns show that VEGF164 mRNA levels increase gradually in line with conceptus development, whereas VEGF120 and VEGFR-2 remain unchanged during the peri-implantation period. Interestingly, elevated VEGFR-1 expression was observed in conceptuses on days 15-16 of gestation (P < 0.05). Comparison of the endometrial sVEGFR-1 mRNA expression revealed up-regulation on days 12 and 15-16 of pregnancy (P < 0.01 and P < 0.05, respectively). Furthermore, increased sVEGFR-1 levels were observed on day 12 of the estrous cycle in the CL (P < 0.05). Concluding, it seems that conceptus-derived VEGF164 plays crucial role in peri-implantation vascular events in pigs. These results support a potential role of VEGFR-1 in the proper growth and development of porcine conceptus during pregnancy. Moreover, expression patterns of sVEGFR-1 in the endometrium of pregnant pigs suggest that it may participate in vascular remodeling important for successful implantation. Finally, luteal sVEGFR-1 may be involved in the maintenance of CL function whenever pregnancy occurs in pigs.     

Post-implantation mouse conceptuses produce paracrine signals that regulate the uterine endometrium undergoing decidualization

The uterus undergoes a series of dramatic changes in response to an implanting conceptus that, in some mammalian species, includes differentiation of the endometrial stroma into decidual tissue. This process, called decidualization, can be induced artificially in rodents indicating that the conceptus may not be essential for a proper maternal response in early pregnancy. In order to test this hypothesis, we determined if and how the conceptus affects uterine gene expression. We identified 5 genes (Angpt1, Angpt2, Dtprp, G1p2 and Prlpa) whose steady-state levels in the uterus undergoing decidualization depends on the presence of a conceptus. In situ hybridization revealed region-specific effects which suggested that various components of the conceptus and more than one signal from the conceptus are likely responsible for altering decidual cell function. Using cell culture models we found that trophoblast giant cells secrete a type I interferon-like molecule which can induce G1p2 expression in endometrial stromal cells. Finally, decidual Prlpa expression was reduced in the uterus adjacent to Hand1- and Ets2-deficient embryos, suggesting that normal trophoblast giant cells in the placenta are required for the conceptus-dependent effects on Prlpa expression in the mesometrial decidua. Overall, these results provide support for the hypothesis that molecular signals from the mouse conceptus have local effects on uterine gene expression during decidualization.     

Regulation of expression and role of leukemia inhibitory factor and interleukin-6 in the uterus of early pregnant pigs

Cytokines, which are generally involved in the process of inflammation, may also play a critical role in conceptus implantation. We examined: (1) the expression profiles of leukemia inhibitory factor (LIF) and interleukin (IL)-6 mRNA and their protein content in the endometrium of cyclic and pregnant gilts on Days 10 to 18 after estrus; (2) the effect of conceptus-exposed medium on LIF and IL-6 synthesis in the endometrium; (3) the profiles of IL6R and LIFR mRNA expression in pig conceptuses collected on Days 10 to 18 of pregnancy; and (4) the effect of LIF and IL-6 on the attachment and proliferation of porcine trophoblast cells. The expression of LIF mRNA in the endometrium increased between Days 10 and 12 in both cyclic and pregnant gilts, and tended to be higher in Day 12 pregnant animals compared with nonpregnant ones. The LIF protein content in the uterine lumen peaked on Day 12 of pregnancy, and was higher than on Day 12 of the estrous cycle. Endometrial IL-6 mRNA expression was upregulated on Day 12 in pregnant gilts compared with nonpregnant animals. Moreover, a higher content of IL-6 protein was observed in pregnant than in cyclic gilts. The addition of conceptus-exposed medium resulted in up-regulation of LIF and IL6 mRNA expression, and increased IL-6 content in endometrial slices. In conceptuses, increased mRNA expression was detected on Days 10 to 14 for IL6R and on Day 14 for LIFR, when compared with other days studied. LIF and IL-6 stimulated the attachment and proliferation of trophoblast cells in vitro. In summary, LIF and IL-6 are important components of embryo-uterine interactions during early pregnancy in the pig, and may contribute to successful conceptus implantation.