Dominant expression of porcine Calbindin-D9k in the uterus during a luteal phase

Calbindin-D9k (CaBP-9k) is a member of intracellular calcium binding proteins, which have a high affinity to calcium. CaBP-9k is mainly expressed in the mammalian intestine, uterus and placenta, and is regulated in tissue- and species-specific manners. Previous studies have shown that CaBP-9k expression is mainly controlled by steroid hormones and their receptors. Thus, we further investigated the expression and regulation of CaBP-9k during an estrus cycle in the pig uterus by Northern blot and immunoblot analysis in this study. In addition, serum levels of estrogen (E2) and progesterone (P4) were measured using ELISA. The CaBP-9k mRNA is highly expressed in the porcine uterus during a luteal phase compared to a follicular phase, and its mRNA level in a luteal phase is increased up to 10-fold compared to a follicular phase. In parallel to the level of CaBP-9k mRNA, the CaBP-9k protein is also dominantly expressed in the porcine uterus, and strongly expressed in the epithelium and glands of the porcine uterus during a luteal phase. Although, the localization of the CaBP-9k protein is scarcely detected at follicular phase, it is dominantly expressed in the porcine uterus during a luteal phase. In addition, the serum P4 level was significantly increased during a luteal phase compared to a follicular phase, whereas no difference was observed in E2 levels between follicular and luteal phases, indicating that the ratio of P4/E2 is remarkably increased in porcine uterus during a luteal phase compared to a follicular phase. In conclusion, these results suggest that P4 may play an important role in the up-regulation of CaBP-9k gene in the porcine uterus in a luteal phase, which is unlike the condition in the rat uterus. In addition, the porcine CaBP-9k may be dominantly expressed in the epithelium and glandular structure of pig uterus during a luteal phase. It may also be differentially regulated during this cycle presumably by steroid hormones, especially up-regulated P4 levels in this tissue.     

TRPV6 and Calbindin-D9k-expression and localization in the bovine uterus and placenta during pregnancy

ABSTRACT: BACKGROUND: Transient receptor potential channel type 6 (TRPV6) and Calbindin-D9k (CaBP-9 k) are involved in the active calcium (Ca2+) transport mechanism in many tissues including placenta and uterus, suggesting a role in the establishment and maintenance of pregnancy. Moreover, TRPV6 and CaBP-9 k seem to support the materno-fetal Ca2+ transport that is crucial for fetal Ca2+ homeostasis, bone growth and development. However, it is unknown if these proteins are also involved in the aetiology of pathologies associated with parturition in cows, such as retained fetal membranes (RFM). The aim of the current study was to create an expression profile of uterine and placentomal TRPV6 and CaBP-9 k mRNAs and proteins during pregnancy and postpartum in cows with and without fetal membrane release. METHODS: Uteri and placentomes of 27 cows in different stages of pregnancy and placentomes of cows with and without RFM were collected. Protein and mRNA expression of TRPV6 and CaBP-9 k was investigated by real-time PCR, immunohistochemistry and Western blot. RESULTS: In the uterine endometrium, highest TRPV6 and CaBP-9 k expression was found in the last trimester of pregnancy, with a particular increase of protein in the glandular epithelium. In the placentomes, a gradual increase in TRPV6 mRNA was detectable towards parturition, while protein expression did not change significantly. Placentomal CaBP-9 k expression did not change significantly throughout pregnancy but immunohistochemistry revealed an increase in staining intensity in the maternal crypt epithelium. Immunohistochemical, stronger placental CaBP-9 k signals were seen in animals with RFM compared to animals with an undisturbed fetal membrane release, while protein levels, measured by Western blot analyses did not change significantly. CONCLUSIONS: The results of the present study demonstrate a dynamic expression of TRPV6 and CaBP-9 k during pregnancy in the bovine uterine endometrium and placentomes, suggesting a functional role for these proteins in Ca2+ metabolism during pregnancy. The temporal and spatial expression patterns indicate that TRPV6 and CaBP-9 k may be involved in materno-fetal Ca2+ transport, mainly through an interplacentomal transport, and that both proteins may participate in physiological processes that are crucial for fetal and placental development. However, neither TRPV6 nor CaBP-9 k seem to be causative in the retention of fetal membranes.     

Microsomal lipid peroxidation in human pregnant uterus and placenta

A study was made of the microsomal lipid peroxidation of the pregnant human uterus and placenta. It was found that the lipid peroxidation of the microsomal fraction of the uterus is specific for prostaglandin formation: the lipid peroxidation was enhanced by arachidonic acid, and inhibited by anti-prostaglandins. Accordingly, it is suitable as a screening test for the pharmacological examination of anti-prostaglandin effects. The lipid peroxidation in the placenta is not specific. In both tissues examined the lipid peroxidation is linked to ascorbic acid.     

Transrectal combined thickness of the uterus and placenta in normal pregnant Egyptian buffalo-cows

The combined thickness of the uterus and placenta (CTUP) is one of the characteristics that can be used to assess fetal development and/or placental function in bovine. The current study was designed to establish reference values for the CTUP throughout pregnancy in normal pregnant buffalo-cows. The CTUP at the intracotyledonary space was measured monthly from the second month until full term using electronic calipers of the ultrasound machine. The CTUP increased monthly from 2.5 mm at the second month to 12 mm at the full term. During the last trimester, the monthly increase in the CTUP was higher than that recorded during the first and second trimesters. The result of the current study can be used as normal values for future studies of CTUP in pathologically pregnant buffalo-cows.     

Specialized patterns of vascular differentiation antigens in the pregnant mouse uterus and the placenta

The success of pregnancy depends on the ability of trophoblast cells to infiltrate the maternal decidua and breach uterine vessels. To ask whether the antigenic phenotype of maternal endothelial cells (EC) in the vascular zone and central decidua basalis may reflect a specialized programming of these vessels for interaction with the trophoblast, we did a survey of several mouse EC differentiation antigens, including MECA-32, MECA-99, and endoglin. Our results revealed striking differences in the phenotype of endothelial lining of vessels in the distinct compartments of the pregnant uterus during Day 9 of pregnancy and at midgestation. Vessels in the central decidua basalis and the vascular zone showed strong expression of MECA-99 but only weak expression of MECA-32, contrasting with the MECA-99(lo), MECA-32(hi) vessels in the capsularis. The vascular zone in addition stained brightly with anti-endoglin. Importantly, invading trophoblast as well as trophoblast cells lining maternal blood spaces were MECA-99(+), MECA-32(-), and endoglin(-), suggesting that the expression of MECA-99 may reflect a specialized co-programming of these trophoblast and EC for future interaction, but also that trophoblast cells may mimic selected antigenic characteristics of endothelium in association with their role in lining maternal blood spaces. In the term pregnant uterus the expression of all differentiation antigens decreased dramatically, suggesting that trophoblast cells as well as maternal EC lose their selected antigenic characteristics when the process of placentation is complete.     

Biology and physiology of Calbindin-D9k in female reproductive tissues: Involvement of steroids and endocrine disruptors

Although Calbindin-D9k (CaBP-9k), a cytosolic calcium binding protein which has calcium binding sites, is expressed in various tissues, i.e., intestine, uterus, and placenta, potential roles of this gene and its protein are not clearly understood. Uterine CaBP-9k may be involved in controlling myometrial activity related with intracellular calcium level and is not under the control of vitamin D despite the presence of vitamin D receptors. But, it is under the control of the sex steroid hormones, estrogen (E2) and progesterone (P4), in female reproductive systems including the uterus and placenta. Thus, in this review, we summarize recent research literature in regards to the expression and regulation of CaBP-9k in mammals and introduce the research data of recent studies by us and others.     

Regulation and localization of insulin-like growth factor binding protein-5 gene expression in the uterus and placenta of the cyclic and early pregnant ewe

The insulin-like growth factor (IGF) system plays an important role in the regulation of uterine function and placental growth. However, there is little information regarding the localization and regulation of IGF binding protein-5 (IGFBP-5) in the reproductive tract. The distribution of this IGFBP was therefore investigated using in situ hybridization in sections of utero-placental tissue obtained throughout the estrous cycle, up to Day 55 of gestation, and on Days 16-17 from both horns of ewes with unilateral pregnancies that followed uterine transection. In nonpregnant ewes, IGFBP-5 mRNA was present at high concentrations in the maternal caruncles and luminal epithelium, and at moderate levels in myometrium. In these regions IGFBP-5 mRNA showed cyclic variations, with concentrations peaking around ovulation, whereas low expression in the endometrial stroma remained constant. During pregnancy, there was additional localization to the endometrial glands; and in all regions, with the exception of the caruncles, concentrations increased significantly with gestational age. In transected uteri, concentrations in the luminal epithelium of the pregnant horn were significantly higher than those in the nonpregnant horn. In the caruncles, IGFBP-5 mRNA formed an intense band just below the tips of the invading fetal villi. Below this band, IGFBP-5 mRNA localized to form a series of rings, which could create a route to allow the fetal villi access into the caruncular stroma for nutrient exchange. In conclusion, IGFBP-5 is abundantly expressed in the ovine reproductive tract, with both the concentration and localization differentially regulated during the cycle and pregnancy.     

Spatio-temporal expression and regulation of dermatopontin in the early pregnant mouse uterus

During endometrial differentiation the extracellular matrix (ECM) changes dramatically to prepare for implantation of the embryo. However, the genes regulating the ECM build-up in the uterine endometrium during early pregnancy are not well known. Using the PCR-select cDNA subtraction method, dermatopontin was identified in the uterus of a pregnant mouse on day 4 of gestation. Dermatopontin mRNA increased dramatically on day 3, and was at its highest level at the time of implantation. Administration of RU 486 significantly inhibited mRNA expression by day 4 of gestation, but ICI 182,780 did not. Progesterone markedly induced dermatopontin expression in ovariectomized uteri within 4 h of administration, whereas estrogen had little effect. In silico analysis revealed progesterone receptor binding sites in the dermatopontin promoter region. Decidualization did not induce expression of dermatopontin; instead dermatopontin mRNA became strongly localized at the interimplantation site. In situ hybridization revealed that expression gradually decreased in the luminal epithelial cells as pregnancy progressed, whereas it increased in the stromal cells. The pattern of localization and the changes of intensity of dermatopontin mRNA coincided with those of collagen. Collectively, these results strongly suggest that dermatopontin expression is steroid-dependent. They also suggest that, at the time of implantation, dermatopontin expression is primarily regulated spatio-temporally by progesterone via progesterone receptors, and is modulated by the decidual response during implantation. Dermatopontin may be one of the regulators used to remodel the uterine ECM for pregnancy.     

Characterization and expression of the mouse pregnant specific uterus protein gene and its rat homologue in the intestine and uterus

The pregnant specific uterus protein gene (Psup) is a novel mouse gene expressed in pregnant uterus. This paper describes the identification and expression of the rat homologue of Psup. The gene is highly expressed in the duodenum. Expression decreases in a proximal-distal gradient in the small intestine and was not detected in the cecum and colon. The pattern of expression in the mouse was similar. Expression of Psup in the mouse was localized to the epithelial cells in the intestine and pregnant uterus by in situ hybridization. The data show tissue-specific expression of Psup.     

Acetylesterase isoenzymes in rat uterus and placenta

The occurrence of acetylesterase activity in the uterus and placenta of the rat has been investigated using a general histochemical simultaneous coupling technique after separation on polyacrylamide gradient gels. apart from a complex band associated with serum esterases which was demonstrated in all the tissues studied, several other isoenzyme bands were demonstrable in differing degrees in the yolk sac and the virgin uterus. Two of these bands were evident in metrial gland up to day 16 of pregnancy, and a third became present by day 17. Unlike the other two bands, this new band did not seem to be associated with the large granules of the granulated metrial gland cells. None of these bands were detected in trophoblast. The metrial gland isoenzymes reacted as well at acid pH as at neutral pH. The yolk sac isoenzymes reacted either as well or slightly better at acid pH, and one extra band was demonstrable under acid conditions.     

Influence of cortisol on prostaglandin synthesis by fetal membranes, placenta, and uterus of pregnant rabbits

Two experiments were designed to assess the effects of cortisol on prostaglandin formation in amniotic fluid and the prostaglandin-forming cyclooxygenase in 4 gestational tissues of rabbits. Cortisol treatment (12 mg/kg body wt/h) was initiated on Day 21 of pregnancy and continued for a 24-h period. Each experiment included 5 treated and 5 vehicle-injected controls, killed at 48 (Experiment 1) or 62 h (Experiment 2) after initial injection. In both experiments, amniotic fluid was collected; cortisol, prostaglandin F (PGF), and prostaglandin E2 (PGE2) were quantified by radioimmunoassay. Microsomes prepared from amnion, yolk sac splanchnopleure, uterus, and placenta were analyzed for prostaglandin-forming cyclooxygenase activity. In Experiment 2, blood drawn at 12-h intervals was quantified for PGF, PGE2, and progesterone. In cortisol-treated rabbits, plasma progesterone decreased (p less than 0.01) from 7.2 +/- 0.8 ng/ml on Day 21 (pre-treatment) to 1.6 +/- 0.2 ng/ml on Day 23, 48 h after the initiation of cortisol treatment. By 62 h, PGF, PGE2, and cortisol concentrations were all significantly higher (p less than 0.05) in the amniotic fluid of treated animals. However, prostaglandin-forming cyclooxygenase activity had not increased in most fetal or maternal tissues at either 48 or 62 h. Therefore, even though increased prostaglandin production may be responsible for the cortisol-induced abortion, increased cyclooxygenase activity in the fetal membranes, placenta, or uterus probably is not the primary stimulus for the increased prostaglandin synthesis.