Circulating concentrations of oxytocin during the estrous cycle and early pregnancy in sheep

Plasma concentrations of oxytocin and progesterone have been measured by radioimmunoassay in jugular venous blood obtained daily from 5 sheep during 2 estrous cycles and in early pregnancy.Concentrations of oxytocin were relatively high (15–30 pg/ml) during the luteal phase of the cycle, but fell at estrus (to 1–17 pg/ml). A fall in oxytocin was also observed on day 15 of pregnancy, when, as expected, progesterone levels remained high. It is suggested that raised basal levels of oxytocin are unlikely to cause the increasing uterine release of prostaglandin F_2α which occurs at the end of the estrous cycle.     

Oviductal progesterone concentration and its spatial distribution in cyclic and early pregnant cows

Changes and local distribution of oviductal progesterone (P(4)) concentration during the estrous cycle and early pregnancy in cows were investigated. Intact reproductive tracts were collected from 16 Holstein cows at an abattoir. Samples were classified in to 4 stages (follicular, postovulatory, luteal and early pregnant,< 20 d) based on visual observation of corpus luteum (CL), uterine characteristics and luteal P(4) concentrations. Oviducts were separated from the uterus at the utero-tubal junction and divided into 4 parts: fimbriae, proximal, medial and distal parts. Luteal tissue samples were also collected. Progesterone levels in oviductal and luteal tissues were determined by radioimmunoassay (RIA). Comparatively higher (P < 0.001) P(4) levels were found in stages with a functioning CL ( luteal phase and early pregnancy) than in those with a regressing CL (follicular phase and post ovulation). The oviduct ipsilateral to the CL bearing ovary during the luteal phase and early pregnancy showed higher ( P < 0.001) P(4) concentrations than the contralateral side. Such a difference was not observed during the follicular phase or post ovulation. The ipsilateral oviduct to the functioning CL at early pregnancy showed higher (P <0.05) P(4) levels than at the luteal phase, while no significant difference in luteal P(4) levels between these 2 stages was observed. Neither were any differences in P(4) concentration within the oviduct observed during any phase of the estrous cycle or during early pregnancy. A positive relationship between luteal and oviductal P(4) concentrations was noted. In conclusion, changes in P(4) levels in the oviduct depend on the location and functional stage of the CL. Localized levels of P(4) in the oviduct may be due to local delivery of P(4) from the CL.     

Expression of vascular endothelial growth factor and its receptors in the porcine corpus luteum during the estrous cycle and early pregnancy

The present study was undertaken to determine the expression of vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/kinase insert domain-containing receptor (Flk-1/KDR), in the porcine corpus luteum (CL) during the estrous cycle and early pregnancy. Immunohistochemical studies localized proteins of VEGF ligand-receptor system in the cytoplasm of luteal cells and in some blood vessels. Western blot analysis revealed significantly higher levels of VEGF protein during early and mid-luteal phase (vs. late luteal phase; P<0.001 and P<0.01, respectively). Quantification of VEGF mRNA in the CL showed increased mRNA levels during entire luteal phase (vs. Days 16-17; P<0.05). Expression of Flt-1 protein remained high during luteal phase (P<0.001), but the mRNA levels tended to increase from the early to the late luteal phase. Elevated protein expression of Flk-1/KDR was found in the mid-luteal phase (vs. Days 16-17; P<0.05). However, induction of Flk-1/KDR mRNA expression occurred earlier, in early luteal phase. The lowest VEGF, Flt-1 and Flk-1/KDR mRNA and protein levels were observed in regressed CL (P<0.001). During pregnancy, VEGF, Flt-1 and Flk-1/KDR mRNA and protein expression was comparable to the mid-luteal phase. In conclusion, the present study has demonstrated dynamic expression of VEGF and its receptors in the porcine CL during the estrous cycle and early pregnancy. These data suggest that the VEGF ligand-receptor system may play an important role in the development and maintenance of the CL in pigs.     

Expression of prostaglandin synthesis pathway enzymes in the porcine corpus luteum during the oestrous cycle and early pregnancy

Prostaglandins (PGs) of luteal origin may have paracrine and/or autocrine actions on the functions of the corpus luteum (CL). Previously, we have shown that enzymes of PG synthesis pathway such as prostaglandin E synthase (mPGES-1), prostaglandin F synthase (PGFS) and prostaglandin 9-ketoreductase (CBR1) are important in regulation of PG production in the conceptuses and endometrium of cyclic and pregnant pigs. Therefore, localization and expression patterns of these enzymes were determinated in porcine CL. The PGFS protein content was lower in metestrus and higher around luteolysis, and then decreased in late regressing CL. PGFS protein levels were lower on days 5-8 of pregnancy and did not differ between days 10 and 25. Elevated expression of mPGES-1 mRNA was found in early luteal phase. The mPGES-1 protein content, similarly to PGFS, was higher during luteolysis. mPGES-1 mRNA and protein levels were constant between days 5 and 25 of pregnancy. PGFS and mPGES-1 expression was down-regulated on days 16-17 of the oestrous cycle when compared to the corresponding days of pregnancy. Enhanced mPGES-1/PGFS ratio occurred during early luteal phase and days 5-8 of pregnancy. Expression of CBR1 mRNA and protein was constant during the cycle and pregnancy. Our studies revealed higher mPGES-1/PGFS ratios in the CL during early luteal phase and corresponding days of pregnancy that could favor PGE(2) synthesis and may be important in the control of luteal development. However, PG synthesis in the endometrium/conceptus rather than in the CL could be involved in luteolysis and maternal recognition of pregnancy in pigs.     

Endocrine profiles during the estrous cycle and pregnancy in the Baird's tapir (Tapirus bairdii)

Serum samples were collected 1–3 times weekly from two Baird's tapirs (Tapirus bairdii) for 6 months in 1987–1988, and for more than 3 consecutive years beginning in 1989 to characterize hormone patterns during the estrous cycle and pregnancy. Based on serum progesterone concentrations, mean (±SEM) duration of the estrous cycle (n = 20) was 30.8 ± 2.6 days (range, 25–38 days) with a luteal phase length of 18.1 ± 0.4 days (range, 15–20 days). Mean peak serum progesterone concentrations during the luteal phase were 1.35 ± 0.16 ng/ml, and nadir concentrations were 0.19 ± 0.03 ng/ml during the interluteal period. Distinct surges of estradiol preceded luteal phase progesterone increases in most (14/20) cycles. Gestation length was 392 ± 4 days for three complete pregnancies. Mean serum progesterone concentrations increased throughout gestation and were 1.83 ± 0.13, 2.73 ± 0.13, and 4.30 ± 0.16 ng/ml during early, mid- and late gestation, respectively. Serum estradiol concentrations began to rise during mid-gestation, increasing dramatically during the last week of pregnancy. Patterns of serum estriol and estrone secretion during pregnancy were similar to that observed for estradiol. In contrast to progesterone and estrogens, serum cortisol concentrations were unchanged during pregnancy or parturition. Females resumed cycling 16.2 ± 2.0 days after parturition (n = 4) and, on two occasions, females became pregnant during the first postpartum estrus. These data suggest that the tapir cycles at approximately monthly intervals and that increases in serum progesterone are indicative of luteal activity. The interluteal period is relatively long, comprising approximately 40% of the estrous cycle. During gestation, progesterone concentrations are increased above luteal phase levels, and there is evidence of increased estrogen production during late gestation. The absence of increased cortisol secretion at the end of gestation suggests that this steroid does not play a major role in initiating parturition in this species. © 1994 Wiley-Liss, Inc.     

Expression and localization of progesterone receptor membrane component 1 and 2 and serpine mRNA binding protein 1 in the bovine corpus luteum during the estrous cycle and the first trimester of pregnancy

The aim of this study was to evaluate the mRNA and protein expression and the localization of progesterone receptor membrane component 1 (PGRMC1), PGRMC2, and the PGRMC1 partner serpine mRNA binding protein 1 (SERBP1) in the bovine CL on Days 2 to 5, 6 to 10, 11 to 16, and 17 to 20 of the estrous cycle as well as during Weeks 3 to 5, 6 to 8, and 9 to 12 of pregnancy (n = 5–6 per each period). The highest levels of PGRMC1 and PGRMC2 mRNA expression were found on Days 6 to 16 (P < 0.05) and 11 to 16, respectively, of the estrous cycle and during pregnancy (P < 0.001). The level of PGRMC1 protein was the highest (P < 0.05) on Days 11 to 16 of the estrous cycle compared with the other stages of the estrous cycle and pregnancy, whereas PGRMC2 protein expression (P < 0.001) was the highest on Days 17 to 20 and also during pregnancy. The mRNA expression of SERBP1 was increased (P < 0.05) on Days 11 to 16, whereas the level of its protein product was decreased (P < 0.05) on Days 6 to 10 of the estrous cycle and was at its lowest (P < 0.001) on Days 17 to 20. In pregnant cows, the patterns of SERBP1 mRNA and protein expression remained constant and were comparable with those observed during the estrous cycle. Progesterone receptor membrane component 1 and PGRMC2 localized to both large and small luteal cells, whereas SERBP1 was observed mainly in small luteal cells and much less frequently in large luteal cells. All proteins were also localized in the endothelial cells of blood vessels. The data obtained indicate the variable expression of PGRMC1, PGRMC2, and SERBP1 mRNA and protein in the bovine CL and suggest that progesterone may regulate CL function via its membrane receptors during both the estrous cycle and pregnancy.     

Luteoprotective roles of luteinizing hormone are mediated by not only progesterone production but also glucocorticoid conversion in bovine corpus luteum

Luteinizing hormone (LH) is known as a key regulator of corpus luteum (CL) function, but the luteoprotective mechanisms of LH in the maintenance of bovine CL function are not well understood. The current study investigated if LH increases cell viability and induces cortisol conversion, and if the luteoprotective action of LH is mediated by stimulating the local production and action of progesterone (P4) and/or cortisol. Cultured bovine luteal cells obtained at the mid‐luteal stage (Days 8–12 of the estrous cycle) were treated for 24 hr with LH (10 ng/ml) with/without onapristone (OP, a specific P4 receptor antagonist; 100 µM), cortisone (1 µM), and aminoglutethimide (AGT, a specific inhibitor of cytochrome P450 side‐chain cleavage; 100 µM). LH with and without OP significantly increased the mRNA and protein expressions of 11β‐hydroxysteroid dehydrogenase (HSD11B) 1, but did not affect the mRNA or protein expression of HSD11B2. These treatments also significantly increased HSD11B1 activity. Cell viability was significantly increased by LH alone or by LH in combination with cortisone and OP. LH in combination with OP or AGT significantly decreased cell viability as compared to LH alone. The overall results suggest that LH stimulates not only P4 production but also HSD11B1 expression, thereby increasing the cortisol concentration in the bovine CL, and that LH prevents cell death through these survival pathways. LH may consequently support CL function during the luteal phase in cattle. Mol. Reprod. Dev. 80: 204–211, 2013. © 2013 Wiley Periodicals, Inc.     

Changes in calcium levels in the endometrium throughout pregnancy and the role of calcium on endometrial gene expression at the time of conceptus implantation in pigs

Calcium plays an essential role in regulating many cellular functions, including proliferation, differentiation, and apoptosis. In spite of its importance in the establishment and maintenance of pregnancy, changes in calcium levels at the maternal–conceptus interface during pregnancy and its action on endometrial gene expression are not well understood. Thus, we examined changes in calcium levels in the endometrium during pregnancy, calcium deposition at the maternal–conceptus interface during pregnancy, and the role of calcium on the expression of endometrial genes related to conceptus implantation during early pregnancy in pigs. The amounts of endometrial calcium increased during mid‐ to late pregnancy, and calcium deposition was mainly localized to endometrial and chorionic epithelial cells at the maternal–conceptus interface during pregnancy and conceptus tissues during early pregnancy. The amounts of total recoverable calcium in uterine flushings were greater on Day 12 of pregnancy than Day 12 of the estrous cycle, and estrogen increased absorption of calcium ions by endometrial tissues. Increasing endometrial calcium levels by treatment with A23187, a calcium ionophore, decreased the expression of the estrogen‐responsive endometrial genes AKR1B1, ESR1, FGF7, IL1RAP, LPAR3, S100G, SPP1, and STC1 and increased the expression of genes related to prostaglandin synthesis and transport, namely PTGES, PTGS2, and SLCO5A1. These data suggest that calcium ions at the maternal–conceptus interface play a critical role in the establishment and maintenance of pregnancy in pigs by regulating the expression of endometrial genes involved in conceptus implantation, as well as the attachment of endometrial epithelial and conceptus trophectoderm/chorionic epithelial cells during pregnancy.     

Urinary steroid evaluations to monitor ovarian function in exotic ungulates: III. Estrone sulfate and pregnanediol-3-glucuronide excretion in the Indian rhinoceros (Rhinoceros unicornis)

A study was undertaken to identify urinary estrogen and progesterone metabolites in the female Indian rhinoceros (Rhinoceros unicornis). Measurements of these metabolites were then used to monitor ovarian function and establish normal levels and patterns of steroid excretion during the estrous cycle and pregnancy. Urine samples were analyzed for estrone sulfate and pregnanediol-3-glucuronide (PDG) by direct radioimmunoassays. Both hormones produced discrete profiles reflecting ovarian activity in nonconceptive cycles. The estrous cycle was observed to be 48 days (range 39–64) with a mean follicular phase of 14.8 days (range 13–19), followed by a mean luteal phase of 19 days (range 17–21). Of the single gestation monitored, PDG levels rose above luteal phase levels by the third month after breeding and remained elevated throughout gestation. The combined estrogen and progesterone metabolite profiles present a complete evaluation of ovarian steriod production in the mature female Indian rhinoceros.     

Total ascorbate and dehydroascorbate concentrations in porcine ovarian stroma, follicles and corpora lutea throughout the estrous cycle and pregnancy

Ovarian tissues are thought to require ascorbate as an antioxidant and enzymatic cofactor for the processes of steroid and collagen synthesis. We measured the concentrations of total ascorbate and oxidized ascorbate (dehydroascorbate, DHA) in ovarian stroma, follicles and corpora lutea (CL) throughout the estrous cycle and pregnancy of the sow. Both total ascorbate and DHA concentrations were greatest in luteal tissue and lowest in ovarian stroma across all stages examined. Within the CL, total ascorbate levels were lowest during the early, early-mid, and late luteal phase and were elevated during the mid-luteal phase. Luteal total ascorbate concentrations were further elevated during early pregnancy and were comparable to mid-luteal phase concentrations during the remainder of gestation. Luteal DHA concentrations decreased from mid to late luteal phase, and were elevated throughout pregnancy. As the CL aged during the cycle, the DHA/total ascorbate ratio decreased and remained low throughout pregnancy. Total ascorbate concentrations in follicular tissue increased during the follicular phase and were lowest during the early luteal phase. The DHA concentrations and DHA/total ascorbate ratios in follicular tissue did not differ with stage. Total ascorbate and DHA concentrations in ovarian stroma were low and did not vary with stage. We conclude that periods of maximal luteal and follicular function are associated with increased concentrations of total ascorbate within the tissue. Furthermore, luteolysis appears to be associated with depletion of luteal ascorbate species.     

Changes in activities of superoxide dismutase, nitric oxide synthase, glutathione-dependent enzymes and the incidence of apoptosis in sheep corpus luteum during the estrous cycle

Anti-oxidative enzymes play a role in protecting cells from oxidative stress-induced cell death. The present study was conducted to evaluate whether the anti-oxidant and pro-oxidant enzymatic capacities of the sheep corpus luteum (CL) are correlated with steroidogenic and structural status of the gland during the estrous cycle. Steroidogenic activity, apoptosis and superoxide dismutase (SOD1 and SOD2), nitric oxide synthase (NOS), glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferase (GST) activities were determined in the CL at specific developmental stages of the luteal phase. The intensity of apoptotic DNA fragmentation, characteristic of physiological cell death, was much greater in CL at late luteal phase than at early and mid-luteal phase, concomitantly with the diminution in the plasma progesterone concentrations from mid-to late luteal phase. SOD1 and GPX activities increased from early to mid-luteal phase, and increased further at late luteal phase. SOD2 and GST activities were not different between early and mid-luteal phase, but increased at late luteal phase. GSR activity was not different between any luteal phase examined. NOS activity decreased from early to mid- and late luteal phase. These results show that the activities of SOD1, SOD2, NOS, GPX, GSR and GST in the sheep CL are subject to major changes during the estrous cycle, and that the anti-oxidant and pro-oxidant enzymatic capacities of luteal cells are not correlated with cell steroidogenic status and integrity during the late luteal phase.     

Expression profiles of interferon‐stimulated gene 15 and prostaglandin synthases in the ovine lymph nodes during early pregnancy

Lymph nodes are distributed all over the body and are part of the lymphatic system. The interferon‐stimulated gene 15 kDa protein (ISG15) and prostaglandins (PGs) are involved in the establishment of pregnancy and are expressed in the uterus during early pregnancy in sheep. In this study, the ovine lymph nodes were obtained on Day 16 of the estrous cycle, and Days 13, 16, and 25 of pregnancy, and the expression of ISG15 and PG synthases, including cyclooxygenase 1 (COX‐1), COX‐2, prostaglandin E (PGE) synthase (PTGES), and a PGF synthase (aldo‐keto reductase family 1, member B1, AKR1B1) were detected by quantitative real‐time polymerase chain reaction, western blot analysis, and immunohistochemistry analysis. Our results showed that there were peaks in the expression of mRNAs and the proteins of ISG15, COX‐1, COX‐2, PTGES, and AKR1B1 in the lymph nodes during early pregnancy and that the COX‐2 and AKR1B1 proteins were limited to the subcapsular sinus and lymph sinuses. In conclusion, the ISG15, COX‐1, COX‐2, PTGES, and AKR1B1 were upregulated in the maternal lymph nodes, which may be beneficial for the development of conceptus, maternal systemic immunoregulation, and anti‐luteolysis during early pregnancy in sheep.     

Reduction by human chorionic gonadotropin of the luteolytic effect of prostaglandin F2 in ewes

The ability of human chorionic gonadotropin (HCG) to reduce the luteolytic effect of prostaglandin (PGF_2^α) was demonstrated in cycling ewes. As expected, treatment with 10 mg of PGF_2^α alone on Day 10 of the estrous cycle exerted a potent negative effect on the function and structure of corpus luteum (CL) as indicated by reduced plasma progesterone, CL progesterone, and CL weight. However, the identical PGF_2^α treatment failed to significantly reduce either luteal function or luteal weight when administered to ewes that were also treated with HCG on Days 9 and 10 of the estrous cycle. Treatment with HCG alone had a positive effect on CL as indicated by increased plasma progesterone, CL progesterone, and CL weight. Treatment with HCG did not render the CL totally insensitive to the negative effects of PGF_2^α because plasma progesterone was reduced when the dose of PGF_2^α was doubled. Whether CL regressed or continued to function after treatment with both HCG and PGF_2^α appeared to depend upon a balance between the positive and negative effects of the two hormones.