A Potential Autocrine Role for Interferon Tau in Ovine Trophectoderm

Interferon tau (IFNT), a type I IFN produced by the conceptus trophectoderm, is the signal for maternal pregnancy recognition in ruminants. The purpose of this study was to investigate whether IFNT effected on the proliferation of ovine trophectoderm cells in an autocrine manner. Elongated ovine conceptuses (Days 15, Day 0 = day of mating) were collected for isolation of mononuclear ovine trophectoderm (oTr‐1) cells, and conceptuses (Days 15 and 20, n = 4 and 3, respectively) were collected for RNA extraction. We demonstrated that the IFNT receptor, IFNAR1, was expressed in trophectoderm of day 15 and 20 conceptuses. Interestingly, the ovine trophectoderm cell line oTr‐1 cultured in the presence of recombinant bovine IFNT (rbIFNT) displayed increased expressions of IFN‐stimulated genes (ISGs), such as IFN‐stimulated gene 15 (ISG15), 2‐5‐oligoadenylate synthetase 1 (OAS1) and bone marrow stromal cell antigen 2 (BST2). Meanwhile, the presence of rbIFNT in the culture media could promote the cell proliferation in a dose‐dependent manner. Furthermore, the connective tissue growth factor, which has diverse functions in cell proliferation and is involved in conceptus elongation, was upregulated in oTr‐1 cell by rbIFNT treatment in vitro. These data indicated that IFNT could act as an autocrine factor to regulate trophectoderm cell proliferation.     

Effects of Exogenous Insulin on Luteolysis and Reproductive Cyclicity in the Mare

Insulin is a pancreatic hormone that classically regulates carbohydrate and fat metabolism, but also appears to play a role in various reproductive processes. A preliminary study suggested insulin production by day 10 to 18 equine conceptuses. The aim of the present study was to examine the hypothesis that insulin is the conceptus signal responsible for maternal recognition of pregnancy (MRP) in the mare, or otherwise influences reproductive cyclicity during the MRP period. Six Warmblood mares were treated daily during days 7 to 17 after ovulation of two successive oestrous cycles with either (short and intermediate acting) insulin or control saline. Mares were assigned randomly to treatment, and crossed over during the subsequent cycle. Time of ovulation and corpus luteum surface area were determined by serial transrectal ultrasonographic examination of the mares' ovaries, and daily jugular vein blood samples were analysed for progesterone and luteinizing hormone (LH) concentrations. On day 14 of dioestrus, the luteolytic drive was examined by measuring systemic 15-ketodihydroprostaglandin F_{2 α} (PG-metabolite) release in response to oxytocin challenge. In addition, yolk sac fluid recovered from 32 day 10 to 14 equine conceptuses was analysed for insulin concentrations. Insulin administration did not affect luteal size, dioestrus length, the interovulatory interval, or circulating LH concentrations. Insulin administration also failed to suppress oxytocin-induced PGF_{2 α} release, and tended to depress systemic progesterone concentrations. Finally, insulin could not be detected in the yolk sac fluid of day 10 to 14 equine conceptuses by radio-immunoassay. It is concluded that insulin administered daily during days 7 to 17 of dioestrus has little or no effect on reproductive cyclicity in the mare, and is unlikely to be the MRP signal.     

Expression Pattern of CD34 at the Maternal–Foetal Interface During Pregnancy in Pigs

The pig exhibits a non‐invasive, epitheliochorial placentation. Adhesion molecules are indispensable for successful implantation and establishment of placentation. CD34 is an adhesion molecule belonging to the immunoglobulin superfamily (IgSF). To take the first step to investigate the role of CD34 in placentation, we examined the expression pattern of CD34 at the maternal–foetal interface in Yorkshire gilts on days 15, 26, 50 or 95 and in Meishan gilts on days 26, 50 or 95 of pregnancy (n = 3 gilts/breed/day of pregnancy) by immunohistochemical technique. The CD34‐positive signals were detected in uterine luminal epithelium and trophectoderm in Yorkshire pigs; the staining for CD34 was located in trophectoderm but barely detectable at the uterine luminal epithelium on day 15 of pregnancy. Then, the expression of CD34 increased dramatically in both the uterine luminal epithelium and trophectoderm by day 26, and weak staining intensity was observed at the maternal–foetal interface on days 50 and 95 of pregnancy. The expression pattern of CD34 in Meishan pigs is similar to that in Yorkshire pigs except that only a few positive signals were observed at the luminal epithelium on day 26 of pregnancy. These results suggest that CD34 may be involved in mediating the cell‐to‐cell adhesion between trophectoderm and the luminal epithelial cells during early pregnancy in pigs.     

Progesterone concentration in defatted milk of dairy cows in early pregnancy.

Progesterone concentrations have been measured in defatted milk of British Friesian cows of four herds during the oestrus cycles (other than short cycles) immediately before artificial insemination (AI) at oestrus and immediately after AI (in non-pregnant cows), and during early pregnancy. Differences in mean progesterone concentrations between herds were significant (P less than 0.05) on all days within the day 10-18 period after AI, both in pregnant and in non-pregnant, inseminated cows but were not significant between pregnant and non-pregnant cows within herds until day 17 or 18. It is concluded that up to this time (that of luteolysis in non-pregnant cows) undefined factors, variable among herds, can have a much greater influence on the rate of progesterone secretion by corpora lutea and consequent progesterone concentration in plasma and milk than does the presence of conceptuses. Maximum mean progesterone concentration reached during early pregnancy in two herds did not differ significantly; it was reached in the 11-15-day period in one herd but not until 46-50 days in the second. Mean progesterone concentration declined after day 90.     

αv β3 Integrin may participate in conceptus attachment by regulating morphologic changes in the endometrium during peri-implantation in ovine

The objective of this study was to determine expression and potential functions of α(v) and β(3) integrin subunits in ovine endometrium during the peri-implantation period (days 8-17 after fertilization). The morphologic changes in the endometrium were observed histochemically following haematoxylin/eosin (HE) staining, whereas the expressions of α(v) and β(3) integrin subunits were analysed by RT-PCR, immunohistochemistry and Western blot. The filamentous conceptus attached to the luminal epithelium (LE) on day 17 of pregnancy, with no differences in endometrial morphology between days 8-12 of pregnancy. However, endometrial glands in the endometrial stroma (S) underwent extensive hyperplasia from day 14 to day 17, increased reductus of the LE with an obvious proliferation of caruncles, and an increased number and diameter of blood vessels (V) in the endometrium. The relative expression levels of α(v) and β(3) integrin subunits mRNA gradually increased until day 16, but sharply declined on day 17. Western blot analysis revealed that the expression pattern of α(v) and β(3) integrin subunit proteins paralleled that of the corresponding mRNA. In addition, immunohistochemical localization of α(v) and β(3) integrin subunits confirmed their presence in the glandular epithelium (GE), LE and endometrial stroma. Immunostaining on LE and stroma varied with the increasing days of pregnancy, with the strongest immunostaining on days 16 and 17. In conclusion, expression of α(V) and β(3) integrin subunits was closely related to the early progression of pregnancy and conceptus attachment; therefore, we inferred that α(v) β(3) integrin may participate in conceptus attachment by the regulation of endometrial morphology during peri-implantation in ovine.     

Production of calcium maintenance factor Stanniocalcin-1 (STC1) by the equine endometrium during the early pregnant period

A factor responsible for progression to pregnancy establishment in the mare has not been definitively characterized. To identify factors possibly involved in the establishment of equine pregnancy, the endometrium was collected from day 13 (day 0=day of ovulation) cyclic and day 13, 19 and 25 pregnant animals. From initial subtractive hybridization studies, a calcium regulating factor, Stanniocalcin-1 (STC1) mRNA, was found as a candidate molecule expressed uniquely in the pregnant endometrium. Endometrial expression of STC1 mRNA was noted on day 19 and was markedly increased in the day 25 gravid endometrium. STC1 protein was found in the extracts of day 25 gravid endometrium and immunochemically localized in the uterine glands. In addition, STC1 protein was detected in uterine flushing media collected from day 25 pregnant mares. High concentrations of estradiol-17 β (E(2)) were detected in day 25 conceptuses. E(2) levels were much higher in the gravid endometrium than in other regions, whereas progesterone levels did not differ among the samples from different endometrial regions. Expression of STC1 mRNA, however, was not significantly upregulated in cultured endometrial explants treated with various concentrations of E(2) (0.01-100 ng/ml) with or without 10 ng/ml progesterone. These results indicate that an increase in STC1 expression appears to coincide with capsule disappearance in the conceptus, and suggest that STC1 from the uterine glands likely plays a role in conceptus development during the pregnancy establishment period in the mare.     

Expression of Connexin 43 in the Porcine Foetal Gonads During Development

This study was designed to reveal connexin 43 (Cx43) mRNA and protein expression in porcine foetal gonads using RT‐PCR, immunohistochemistry and Western blot analysis. Expression of Cx43 was investigated in porcine foetal ovaries and testes on days 50, 70 and 90 post coitum (p.c.). RT‐PCR results indicated that Cx43 mRNA was expressed in both foetal ovaries and testes at all gestational ages examined. Cx43 protein was found in the foetal ovary but its distribution varied across ovarian compartments and changed during development. In foetal ovaries, Cx43 was localized between the interstitial cells surrounding egg nests on all investigated days of prenatal period. Moreover, Cx43 expression was observed between germ cells on day 50 p.c. as well as between pre‐granulosa and granulosa cells of primordial and primary follicles on days 70 and 90 p.c. In the foetal testes, Cx43 protein was detected between neighbouring Leydig cells on all examined days of prenatal period and between adjacent Sertoli cells exclusively on day 90 p.c. The presence of Cx43 protein in all investigated foetal gonads was confirmed by Western blot analysis. Cx43 protein detection between pre‐granulosa cells of primordial follicles suggests its role in regulation of the initial stages of follicle development. The Cx43 immunoexpression between neighbouring Leydig and between Sertoli cells indicates its involvement in controlling their functions. We propose that Cx43‐mediated gap junctional communication is involved in the regulation of porcine foetal gonadal development.     

Regulation of endometrial granulocyte macrophage-colony stimulating factor (GM-CSF) in the ewe

Granulocyte macrophage-colony stimulating factor (GM-CSF) increases ovine interferon-tau (oIFNtau) secretion by ovine conceptuses, but endometrial production of GM-CSF has not been characterized. Endometrial GM-CSF expression was evaluated in ovariectomized ewes implanted with estradiol-17beta (E(2)) and/or progesterone (P(4)) for 14 days, in day 14 cyclic and day 14 pregnant ewes. Relative levels of endometrial GM-CSF mRNA were 3-fold higher in E(2)- and E(2)/P(4)-treated ewes than that of control or P(4)-treated ovariectomized ewes. Levels of endometrial GM-CSF mRNA for cyclic ewes were similar to E(2)- and E(2)/P(4)-treated ewes, but amounts of GM-CSF mRNA in pregnant ewes were 2-fold higher. GM-CSF concentrations in endometrial culture media, determined by GM-CSF bioassay, for cyclic and E(2)/P(4)-treated ovariectomized ewes were 3-fold higher than those of control, E(2)- and P(4)-treated ovariectomized ewes; however, amounts of GM-CSF in pregnant ewes were 2-fold higher. Immunoreactive GM-CSF, examined by western blot, was detected in the culture medium from E(2)/P(4)-treated ovariectomized, cyclic and pregnant ewes. Luminal and glandular epithelia and stromal regions were determined to be sites of GM-CSF expression by immunohistochemistry and in situ hybridization techniques. Data indicate that combined E(2) and P(4) treatment of ovariectomized ewes is sufficient to restore GM-CSF expression to the level found in cyclic ewes; however, GM-CSF mRNA and protein in pregnant ewes is 2-fold greater than in ovariectomized or cyclic ewes. These data suggest that the conceptus, in addition to steroids, may play a role in the regulation of endometrial production of GM-CSF.     

The histological observations on the large intestine of the goose (Anser anser) during the pre- and post-hatching periods

The development of the cecum and colon in the goose was investigated during the period from the 15th to 28th day of the incubation and from 1 to 30 days of age after hatching by light microscopy. By day 15 of the incubation, in the cecum and colon, the lumen was surrounded by pseudostratified epithelium. The previllous ridges appeared at 15th and 17th days of the incubation in the colon and ceca, respectively. At the base of previllous ridges, the epithelium changed into a simple prismathic epithelium at 15th and 17th days of the incubation in the colon and cecum, respectively. The villi appeared at the 21st days of the incubation. The crypts and goblet cells appeared on the first day after hatching. In the pre-hatching period, the lamina muscularis mucosa was present only in the colon. The submucosa consisted of loosely aggregated connective tissue in the pre-hatching period. In the post-hatching period, it consisted of a very thin layer of connective tissue. Its presence was only obvious where the cells of the submucosal nerve plexus or occasional large blood vessels considerably increased its thickness. The nerve plexus corresponding to the Auerbach's plexus of the mammalian intestine and submucosal nerve plexus appeared by 15th days of the incubation. From the 15th to 28th day of incubation, the tunica muscularis consisted of circular smooth muscle cells in the ceca. On the 28th day of the incubation a thinner longitudinal muscle layer added to the circular muscle layer. In the colon there was an outer longitudinal and a thicker circular muscle layer.     

Oestradiol-17 beta in the milk of cows from 6 days before to 14 days after their insemination.

In artificially inseminated cows (AI on day 1) peak concentrations of oestradiol-17 beta in defatted milk occurred at median times of day 0 during the pre-ovulatory period, days -5 to 2, and day 6 during the post-ovulatory period, days 2-15. Median peak concentrations during these periods were approximately 5 pg/ml and 3 pg/ml respectively. There were no significant differences in the timing or magnitude of oestradiol-17 beta concentrations between cows that became pregnant to the AI and those that entered normal length oestrus cycles immediately after AI.