Nongravid Horn Research Articles

Transforming growth factor beta family expression at the bovine feto-maternal interface

BackgroundEndometrial remodelling is necessary for implantation in all mammalian species. The TGF beta super-family plays a crucial role in this event in humans and mice. However, the role of TGF beta super-family members during implantation is still unclear in ruminants. In the present study, the spacio-temporal expression of TGF beta super-family members including activin was explored in bovine trophoblasts and endometrial tissue during the peri-implantation period in order to elucidate whether it is essential for promoting cell proliferation at the implantation site.MethodsGene expression in the fetal membrane and endometrium of the gravid and non-gravid horn around Day 35 of gestation were analyzed with a custom-made oligo-microarray in cattle. The expression of activin and its related genes was also analyzed with quantitative RT-PCR. Activin-like activity in trophoblastic tissue and BT-1 cells was examined using a fibroblast cell proliferation test and Western blotting.ResultsThe expression of various TGF beta super-family related genes including activin was detected in trophoblasts and the endometrium in cattle. The most intensive activin expression was found in the gravid horn endometrium, and rather intense expression was detected in the non-gravid trophoblastic tissue. Extracts from the fetal membrane including trophoblasts and purified activin both stimulated fibroblast proliferation effectively, and activin was immunologically detected in BT-1 cells, which have trophoblastic features.ConclusionsSpecific expression of the activin gene (gene name: inhibin beta A) was found in the gravid horn endometrium during peri-implantation. An activin-like molecule, which was derived from the endometrium and trophoblasts, stimulated the proliferation of fibroblast cells. These results suggested that as in other species, the activity of TGF beta super-family members including activin-like molecules plays a pivotal role in endometrial remodelling, which is an essential process in implantation and placentogenesis during the peri-implantation period in cattle.

The Sphingosine 1-Phosphate (S1P) Signaling Pathway Is Regulated During Pregnancy in Sheep1

Because sphingosine 1-phosphate (S1P) is a potent stimulator of angiogenesis, we hypothesized that the S1P pathway is activated to stimulate endometrial/placental angiogenesis during pregnancy. We initially localized S1P signaling pathway members in the gravid and nongravid uterine horns of unilaterally pregnant ewes. Sphingosine kinase-1 expression was greater in gravid compared to nongravid horns. In situ hybridization revealed elevated expression of sphingosine 1-phosphate phosphatase (SGPP1) in gravid interplacentomal endometrial stroma on Days 20 and 40 compared to the nongravid uterine horn, but expression increased in endometrium of the nongravid uterine horn between Days 40 and 120. SGPP1 expression increased in placentomes late in gestation. Sphingosine 1-phosphate lyase mRNA was modestly expressed at Day 20 and then decreased. In contrast, sphingosine 1-phosphate receptor 1 (S1PR1) mRNA increased in endometrium and caruncular stroma of the gravid uterine horn. Treatment with FTY720 and VPC23019, S1P receptor antagonists, blocked human and ovine endothelial cell invasion using an in vitro model of sprouting angiogenesis. Knockdown of S1PR1 with siRNA reduced invasion responses as well. We previously reported that delta-like 4 (DLL4) and A disintegrin and metalloproteinase with thrombospondin-like repeats 1 (ADAMTS1) participate in endothelial cell invasion stimulated by S1P and growth factors in vitro, and thus investigated whether their expression correlated with areas undergoing angiogenesis in vivo. DLL4 expression was similar to S1PR1, while ADAMTS1 mRNA was expressed by endometria of both nongravid and gravid horns, as well as conceptus and placentomes. These results establish that S1P signaling pathway members and S1P- and growth factor-regulated genes are prominent in uterine and placental tissue and in some cases are correlated with areas undergoing angiogenesis. Thus, S1P signaling may be crucial for proper fetal-placental development.

Local Uteroplacental Influences are Responsible for the Induction of Uterine Artery Myogenic Tone during Rat Pregnancy

Uterine artery constrictor responses to elevation of intraluminal pressure (myogenic tone) are considerably enhanced in late pregnant rats, although the underlying causes remain unknown. A single uterine horn ligation model was used to differentiate local from systemic influences, and to test the hypothesis that factors associated with the site of placentation, rather than systemic hormonal changes, are primarily involved in the induction of this adaptive process. Radial uterine arteries were dissected from the gravid and nongravid uterine horns of late pregnant rats, cannulated, and pressurized. Changes in arterial diameter and smooth muscle [Ca(2+)](i) in response to the elevation of intraluminal pressure were studied using intact and endothelium-denuded arteries loaded with the ratiometric Ca(2+)-sensitive dye fura-2. Elevations of pressure from 10 to 60 and 100 mm Hg resulted in passive arterial distention of arteries from nongravid horns with a minor change in [Ca(2+)](i). In contrast, arteries from gravid horns developed myogenic tone associated with a significant elevation in [Ca(2+)](i). Synchronous oscillations in [Ca(2+)](i) and lumen diameter were frequently observed in vessels from gravid horns. Endothelial denudation augmented tone in the gravid horn but did not uncover myogenic tone in vessels from the nongravid horn. In summary, pregnancy-associated uterine artery myogenic behavior is due to an upregulation of calcium-handling mechanisms, occurs independently of the endothelium, and is induced by local uteroplacental influences.

Mechanical stretch regulates hypertrophic phenotype of the myometrium during pregnancy

The adaptive growth of the uterus is a critical event that involves changes in cellular phenotypes throughout pregnancy. In early pregnancy, uterine growth is due to hyperplasia of uterine smooth muscle cells (SMCs) within the myometrium; however, the major component of myometrial growth occurs after mid-gestation. This study sought to test the hypothesis that increase in myometrial growth seen during late pregnancy is due to SMC hypertrophy caused by mechanical stretch of uterine tissue by a growing fetus(es) by providing direct measurements of individual SMC size. We employed a stereological approach to calculate the average cell volumes of uterine myocytes through diameter measurements using the Stereoinvestigator statistical software. Uterine tissues were collected from nonpregnant Wistar rats, as well as from gravid and nongravid horns of unilaterally pregnant animals on gestational days (d) 8 (early gestation), 14 (mid-gestation), 19 (late gestation), 22 (term), and 4 days post partum. Anti-caveolin-1 immunostaining was used to clearly delineate SMC boundaries. The stereological analysis revealed that the dramatic increase in myometrial growth seen during late gestation (d19-22) is due to a threefold increase in the size of uterine myocytes. A significant increase in SMC volumes was detected in the gravid uterine horn as compared with the corresponding empty horn of unilateral term pregnant animals (day 22, mean cell volume 1114 vs 361 microm(3), P<0.05), indicating the effect of uterine occupancy. The restriction of the hypertrophy to cells within the gravid horn suggests that it may be a response to the biological mechanical stretch of uterine walls by the growing fetus(es) and placenta(s).

The relationship between consecutive pregnancies in Thoroughbred mares. Does the location of one pregnancy affect the location of the next, is this affected by mare age and foal heat to conception interval or related to pregnancy success

Delayed uterine involution is a major cause of early reproductive failure in mares. Involution is affected by mare age, and foaling to covering interval. Involution rates vary between the previously non-gravid horn (PNGH), which recovers the quicker, and the previously gravid horn (PGH). Location of a pregnancy and its likely success may, therefore, be affected by its location relative to the previous pregnancy. This study aimed to determine: (i) the location of concepti in consecutive pregnancies; (ii) whether this varies with mare age or foaling to conception interval; (iii) whether location in relation to the previous pregnancy affects success. 1383 Thoroughbred mares were monitored by ultrasonic scanning during oestrus and early pregnancy. Significantly ( p < 0.01) more pregnancies were located in the PNGH (79.2%) than the PGH (20.8%). The number of pregnancies in PGH significantly increased with mare age ( p < 0.01) and foaling to conception interval ( p < 0.05). Significantly ( p < 0.001) more pregnancies located in the PGH (16.5%) failed, than those in the PNGH (4.6%). It can be concluded that most pregnancies locate in the PNGH where their chances of success are greatest. The larger number of pregnancies locating in the PGH in older mares and those with shorter foaling to conception intervals may in part account for the higher conceptus mortality rates in such mares. Hence breeding older mares on alternate years and maximising foaling to conception interval may improve reproductive success. Alternatively termination of pregnancies located in the PGH followed by timely recovering may be justifiable as might ET in older mares covered close to foaling.

Effect of the conceptus on uterine prostaglandin-F2α and prostaglandin-E2 release and synthesis during the periimplantation period in the pig

The present study was conducted to evaluate the effect of the conceptus on uterine prostaglandin-F2alpha (PGF2alpha) and prostaglandin-E2 (PGE2) release and the expression of prostaglandin synthase enzymes during the periimplantation period in the pig. A surgically generated model with conceptuses developing in only one of the uterine horns was created. The highest concentration of PGF2alpha and PGE2 was found in the gravid uterine horn, compared with the non-gravid horn and the intact horn of cyclic gilts. Endometrial concentration of both PGs in pregnant gilts was elevated regardless of the conceptus in the uterine horn, whereas only myometrial PGE2 concentration increased during pregnancy. Expression of prostaglandin-E2 synthase (mPGES-1) mRNA in the endometrium was upregulated during the oestrous cycle, while protein expression presented a similar pattern to that of PGE2 concentration in the uterine flushings. Prostaglandin-F2alpha synthase (PGFS) mRNA and protein expression in the endometrium did not differ between pregnancy and oestrous cycle but PGFS mRNA in the myometrium increased during pregnancy both in the gravid and the non-gravid uterine horns. We suggest a local effect of the conceptus on PG release pathways but also a more systemic effect within the whole uterus with regard to PG synthesis and accumulation in uterine tissues.

Bovine endometrial legumain and TIMP‐2 regulation in response to presence of a conceptus

The precise mechanism of placentation in the bovine species where a restricted trophoblast invasion occurs to form the synepitheliochorial placenta is not fully understood. This study initially investigated the conceptus-maternal interactions in the peri-attachment period by comparing the proteins present at Days 16 and 18 in uterine luminal fluid (ULF) of pregnant with nonpregnant cows using 2-D gel electrophoresis. Nine protein spots were identified that were present in greater amounts in pregnant compared to nonpregnant ULF: carbonic anhydrase, ezrin, heat shock protein 70, isocitrate dehydrogenase, nucleoside diphosphate kinase, peroxiredoxin 1, purine nucleoside phosphorylase, thioredoxin and triosephosphate isomerase and four proteins that were less abundant in ULF from the gravid compared to the nongravid horns or nonpregnant uteri: cystatin E/M, legumain, retinol-binding protein (RBP) and tissue inhibitor of matrix metalloproteinase 2 (TIMP-2). Successful placentation requires the remodelling of the endometrial surface therefore uterine mRNA and protein expression of legumain, a protease activator, and TIMP-2, a protease inhibitor, was examined in detail during the oestrous cycle and from Days 13 to 31 of pregnancy. Both mRNAs were up-regulated in the endometrium during the luteal phase of the oestrous cycle and during early pregnancy. Although legumain and TIMP-2 mRNA expression levels were similar between uterine horns at the same day of pregnancy, the amount of protein differed between gravid and nongravid horns possibly modulated by interferon-tau or by other factors produced by the conceptus. These events at the conceptus-maternal interface may provide localised control of protease activity necessary for controlling trophoblast invasion of the endometrium.

Endometrial expression of the insulin-like growth factor system during uterine involution in the postpartum dairy cow

Rapid uterine involution in the postpartum period of dairy cows is important to achieve a short interval to conception. Expression patterns for members of the insulin-like growth factor (IGF) family were determined by in situ hybridisation at day 14±0.4 postpartum (n=12 cows) to investigate a potential role for IGFs in modulating uterine involution. Expression in each uterine tissue region was measured as optical density units and data were analysed according to region and horn. IGF-I mRNA was localized to the sub-epithelial stroma (SES) of inter-caruncular and caruncular endometrium. Both IGF-II and IGF-1R expression was detected in the deep endometrial stroma (DES), the caruncular stroma and myometrium. IGFBP-2, IGFBP-4 and IGFBP-6 mRNAs were all localised to the SES of inter-caruncular and caruncular uterine tissue, and in the DES and caruncular stroma, with IGFBP-4 mRNA additionally expressed in myometrium. IGFBP-3 mRNA was only detectable in luminal epithelium. IGFBP-5 mRNA was found in myometrium, inter-caruncular and caruncular SES and caruncular stroma. These data support a role for IGF-I and IGF-II in the extensive tissue remodelling and repair which the postpartum uterus undergoes to return to its non-pregnant state. The differential expression of binding proteins between tissues (IGFBP-3 in epithelium, IGFBP-2, -4, -5 and -6 in stroma and IGFBP-4 and -5 in myometrium) suggest tight control of IGF activity within each compartment. Differential expression of many members of the IGF family between the significantly larger previously gravid horn and the previously non-gravid horn may relate to differences in their rate of tissue remodelling.

Endometrial gland surface density and hyperaemia of the endometrium during early pregnancy in the mare

The morphology and surface density of endometrial glands responsible for the nutrition of the placenta requires further study employing computer assisted morphometric analysis. To analyse changes in the morphology and surface density of endometrial glands situated adjacent to, and remote from, the conceptus during the first 30 days of gestation. Endometrial biopsies were recovered, under videoendoscopic control, from both nonpregnant and pregnant mares 12 days after ovulation, as well as from pregnant mares on Days 16, 20, 25 and 30 of gestation. One biopsy was taken at the base of the nongravid uterine horn and the other from beneath the conceptus at the base of the gravid horn. The samples were evaluated by computer assisted morphometric analysis to determine the surface area per unit volume (Sv) of endometrial glands in the stratum spongiosum. No differences in morphology or density of the glands were discernable between pregnant and nonpregnant mares on Day 12 day after ovulation. On Day 16, however, the glands beneath the conceptus appeared to be at a significantly lower density than the clearly more tortuous glands at the base of the nongravid horn. Between Days 20 and 30 of gestation the endometrium in direct contact with the conceptus was visibly more reddened and hyperaemic than elsewhere in the uterus. However, the Sv of glands beneath the conceptus had now increased again so they appeared similar to those in the nongravid horn, due mainly to a reduction in the level of stromal oedema in the former. The embryo-maternal interactions that are vital during early pregnancy to suppress the cyclical luteolytic cascade and support nidation of the conceptus, were characterised here with local endometrial hyperaemia, histotroph production and a decrease in endometrial gland Sv at the commencement of the stationary phase. Both the components secreted by the endometrial glands and the factors liberated by the conceptus during early pregnancy need further investigation.

SECRETED PHOSPHOPROTEIN 1 ISOLATED FROM UTERINE SECRETIONS PROMOTES INTEGRIN-MEDIATED ADHESION OF PORCINE TROPHECTODERM CELLS

In domestic pigs, over two-thirds of reproductive losses occur during the first 25 days of gestation, a period during which rapid conceptus development, trophoblast adhesion and implantation are initiated. Successful conceptus implantation requires pregnancy-specific alterations in extracellular matrix (ECM) at the conceptus-maternal interface. The pig luminal epithelium (LE) immediately adjacent to the implanting conceptus secretes SPP1 (also known as osteopontin), an ECM molecule involved in cell-cell and cell-ECM communication that is believed to mediate conceptus-uterus interactions. We hypothesized that SPP1 expressed by the uterine LE bridges to integrin receptors on conceptus trophectoderm to stimulate adhesion at the conceptus-maternal interface. The objective of this study was to determine if SPP1 isolated from uterine secretions (uterine milk) supports trophectoderm cell adhesion in vitro. To accomplish this, we employed a unilaterally pregnant sheep model, where the ovary ipsilateral to the right uterine horn was removed and a double ligature placed at the base of the right uterine horn at the bifurcation. At the following estrus, sheep were mated to generate gravid and nongravid uterine horns, hysterectomized on day 120 and the uterine milk within nongravid horns collected. SPP1 was purified from uterine milk using a DEAE-Sephacel column (ion exchange chromatography) followed by dual phenyl-Sepharose columns (hydrophobic chromatography). A 45 kDa fragment, previously identified at the apical surface of the sheep LE during pregnancy, and a 25 kDa fragment were isolated. The spontaneously immortalized pig trophectoderm cell line pTr2 was allowed to attach to serial dilutions (20 μg/ml to 40 ng/ml) of the following proteins: bovine milk SPP1 (bSPP1; purified in the manner described above), ovine uterine milk SPP1 (oSPP1), recombinant rat SPP1 containing the RGD cellbinding sequence (rSPP1-RGD), recombinant rat SPP1 containing an RAD sequence in place of the RGD sequence (rSPP1-RAD), full-length human fibronectin (FN; positive control) and BSA (negative control). Dose-dependent cell adhesion was observed for bSPP1, oSPP1 and rSPP1- RGD, all of which supported higher levels of adhesion than FN at concentrations ranging from 0.15 and 1.25 μg/ml. Only at concentrations of 2.5 μg/ml and higher did FN support greater pTr2 adhesion than the various SPP1 proteins. Cell adhesion was not supported by rSPP1-RAD over BSA controls at any concentration, strongly suggesting that adhesion to the various SPP1 proteins and FN occurred through RGD-binding integrin receptors. Integrins require the presence of divalent cations to allow ligand binding. This property was used to further confirm that pTr2 cell adhesion to SPP1 was integrin-mediated. Cells were allowed to attach in the presence of varying levels of divalent cations: 1) no cations; 2) 1 mM Ca2+ + 2 mM Mg2+; 3) 2 mM Ca2+ + 1 mM Mg2+; or 4) 0.1 mM Mn2+. No attachment was observed in the absence of divalent cations, confirming that porcine trophectoderm attachment to SPP1 is integrin-mediated. This study is the first to demonstrate that a native form of uterine SPP1, secreted by the uterine glandular epithelium and present at the conceptus-maternal interface, can promote dose- and cation-dependent integrin-mediated adhesion of pig trophectoderm cells. (Supported by NRI USDA Grant 2006-35203-17199). (platform)

Uterine artery vasoconstriction during exercise in gravid and nongravid uterine horns in rabbit pregnancy

In rabbits, the uterine artery vasoconstrictor response to exercise is attenuated in late gestation. We asked whether this alteration in vascular control is related to systemic circulating factors associated with pregnancy. Uterine artery blood flow (UtBF; Transonic Systems) was measured in, and uterine artery conductance (UtC) was calculated for, gravid (G) and nongravid (NG; due to oviduct ligation) uterine horns in 4 rabbits during treadmill exercise (2 min at 10 m/min, 7% grade). Rabbits were studied in the nonpregnant state (NP) and during pregnancy (P) on days 10 (earlyP), 20 (midP) and 28 (termP) of a 31 d gestation. Values are mean(SD). At rest, the increases in UtBF and UtC during gestation were greater in the G vs. NG horn (UtBF at termP, G: 20(2) ml/min; NG: 8(4) ml/min). During exercise when NP, the vasoconstrictor response (% change in UtC) was similar in the two horns [G: −34(14)% NG: −35(7)%]. In the G horn, the %change in UtC was unaffected in earlyP [−35(13)%] but was attenuated (P<0.05) at midP[−23(13)%] and termP[0(2)%]. In contrast, in the NG horn the % change in UtC was enhanced in earlyP [−72(14)%], and unaffected at midP [−49(23)%] and termP [−54(16)%]. There was a significant interaction (P<0.01) between uterine horn status and gestation time, which indicated that the G and NG horns responded differently to exercise in earlyP and termP. The preservation of the uterine vasoconstrictor response to exercise in a nongravid horn in a termP rabbit suggests that systemic circulating factors are not primarily responsible for the attenuated uterine artery vasoconstrictor response to exercise in pregnancy. Supported by R15 HL77128 and MWU intramural award.

Characterization of two labor-induced genes, DSCR1 and TCTE1L, in the pregnant ovine myometrium.

In the present study we characterized two labor-induced genes, DSCR1 (Down syndrome candidate region 1) and TCTE1L (murine t-complex like), which were identified by suppression subtractive hybridization in the pregnant ovine myometrium. DSCR1 and TCTE1L cDNA sequences were retrieved from a custom-made labor-myometrial cDNA library by hybridization screening. The characterized cDNA sequences include 5'-untranslated region (UTR), coding region and 3'-UTR, which are 12 bp, 351 bp and 1716 bp for TCTE1L, and 64 bp, 594 bp and 1539 bp for DSCR1 respectively. The two cDNA sequences encode proteins of 116 and 197 amino acids for TCTE1L and DSCR1 respectively. Northern analysis further confirmed the significant increases of myometrial DSCR1 and TCTE1L mRNA associated with spontaneous term labor (n=6) compared with gestation-matched controls not in labor (n=6). The abundance of DSCR1 and TCTE1L mRNA was attenuated when myometrial contraction was inhibited by Nimesulide (n=6), a specific prostaglandin H synthase 2 inhibitor. Fetal occupancy greatly upregulated DSCR1 and TCTE1L mRNA in the gravid horn during betamethasone-induced premature labor (n=6) compared with the non-gravid horn not in labor (n=3). Estradiol upregulated TCTE1L mRNA, but had no effect on DSCR1 mRNA expression in the non-pregnant sheep myometrium. Progesterone alone had no effect on both DSCR1 and TCTE1L mRNA expression, however progesterone antagonized estradiol's stimulating effect on myometrial TCTE1L mRNA expression in ovariectomized non-pregnant sheep. Upregulation of DSCR1 and TCTE1L in both betamethasone-induced premature labor and spontaneous term labor and inhibition of their expression by Nimesulide suggest a functional role of these two genes in myometrial activation associated with onset of labor. Mechanical stretch, labor and steroids differentially regulated DSCR1 and TCTE1L mRNA in the pregnant and non-pregnant sheep myometrium.

Full induction of rat myometrial 11beta-hydroxysteroid dehydrogenase type 1 in late pregnancy is dependent on intrauterine occupancy.

The 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD-1) enzyme catalyses the conversion of the biologically inert glucocorticoid 11-dehydrocorticosterone to active corticosterone (11-oxoreductase activity) in vivo, and it is dramatically up-regulated in uterine myometrium in the days leading up to parturition. 11beta-HSD-1 is likely to enhance local concentrations of glucocorticoid within the myometrium and thus facilitate uterine contractility, but the stimulus for the increase in myometrial 11beta-HSD-1 is unknown. The objective of the present study was to test whether the induction of myometrial 11beta-HSD-1 is dependent on uterine occupancy or systemic hormonal signals of late pregnancy. This involved use of a unilateral pregnancy (ULP) model in which the gravid and nongravid uterine horns are both exposed to the normal systemic hormonal milieu of pregnancy. Western blot analysis showed that the 11beta-HSD-1 signal was only partially induced in the nongravid horn of ULP rats on Day 22 of pregnancy (term: Day 23). Moreover, artificial distension of this nongravid horn had no effect on myometrial 11beta-HSD-1 immunoreactivity or bioactivity at either Day 16 or Day 22 of pregnancy. Removal of fetuses and placentas on Day 18 reduced myometrial 11beta-HSD-1 bioactivity 4 days later, and this effect was not overcome by artificial maintenance of uterine distension. In contrast, after fetectomy at Day 18 (i.e., removal of the fetus but not placenta), myometrial 11beta-HSD-1 bioactivity was largely maintained on Day 22, indicative of placental support for myometrial 11beta-HSD-1 over this period. In conclusion, our data show that full induction of myometrial 11beta-HSD-1 expression and associated 11-oxoreductase bioactivity late in rat pregnancy is dependent upon intrauterine occupancy. Although the hormonal milieu of late pregnancy appears to stimulate myometrial 11beta-HSD-1 marginally, full induction clearly requires an additional stimulus. Manipulations involving fetectomy and artificial uterine distension indicate that the placenta provides at least part of this stimulus, but uterine stretch does not appear to play a role.

Expression and regulation of the messenger ribonucleic acid encoding the prostaglandin F 2α receptor in the rat myometrium during pregnancy and labor

Objective: We examined expression of messenger ribonucleic acid encoding the prostaglandin F 2α receptor in the rat myometrium throughout late gestation and its regulation by progesterone and mechanical stretch. Study Design: Normal pregnant rats were killed on gestational day 15, 22, or 23 (during labor) or 1 day post partum. The effects of progesterone on prostaglandin F 2α receptor messenger ribonucleic acid levels were investigated by daily injections of progesterone (4 mg) from day 20 of normal gestation or from day 17 in rats bilaterally ovariectomized on day 17. To investigate the effects of myometrial stretch, unilaterally pregnant rats underwent either sham surgery or placement of a polyvinyl tube 3 mm in diameter in the nongravid uterine horn on day 15 or 18 and were killed 5 days later. Prostaglandin F 2α receptor messenger ribonucleic acid levels in the myometrium were determined by a semiquantitative reverse transcription–polymerase chain reaction method. Results: Myometrial prostaglandin F 2α receptor messenger ribonucleic acid levels significantly increased during both term and ovariectomy-induced preterm labor. This increase was blocked by progesterone. In rats with unilateral pregnancies prostaglandin F 2α receptor messenger ribonucleic acid levels in the nongravid horns were similar to those in the contralateral gravid horns on day 20 and during labor regardless of whether they were stretched by a 3-mm tube. Conclusion: Increased myometrial expression of prostaglandin F 2α receptor messenger ribonucleic acid during term and preterm labor is temporally associated with progesterone withdrawal but is not dependent on mechanical stretch of the myometrium. (Am J Obstet Gynecol 2000;182:919-25.)

Differential expression of myometrial oxytocin receptor and prostaglandin H synthase 2, but not estrogen receptor alpha and heat shock protein 90 messenger ribonucleic acid in the gravid horn and nongravid horn in sheep during betamethasone-induced labor.

In the present study, we characterized four myometrial contraction-associated proteins (mCAPs): oxytocin receptor (OTR), prostaglandin H synthase 2 (PGHS2), estrogen receptor alpha (ERalpha), and heat shock protein 90 (Hsp90) messenger RNA (mRNA) expression in the nongravid horn of pregnant sheep and compared them with their expression in the gravid horn that is exposed to a greater degree of stretch. We also examined the regulatory effects of estrogen and progesterone on OTR mRNA expression in ovariectomized nonpregnant sheep. In addition, we determined the ontogeny of mCAP expression in the gravid horn throughout late pregnancy and during spontaneous term labor. Gravid horn and nongravid horn myometria were removed under general anesthesia from control ewes not in labor at 130-140 days gestational age (dGA; n = 3) and during betamethasone-induced labor (n = 6) at the same gestational age. Gravid horn myometrium was also collected from ewes not in labor at 95 dGA (n = 3), 101-110 dGA (n = 3), 111-120 dGA (n = 3), 121-130 dGA (n = 3), 131-140 dGA (n = 3), and 141-145 dGA (n = 4) and from ewes in spontaneous term labor (n = 4). All ewes were carrying single fetuses. Myometrium was also collected from ovariectomized nonpregnant ewes treated with saline (n = 5), estradiol (50 microg/day; n = 5), progesterone (0.3 g, intravaginally; n = 5), and estradiol plus progesterone (n = 5). Myometrial RNA was extracted and analyzed by Northern blot for OTR, PGHS2, ERalpha, and Hsp90 mRNA, normalized for 18S ribosomal RNA or beta-actin. ERalpha, Hsp90, OTR, and PGHS2 mRNA were all significantly up-regulated during betamethasone-induced labor (P < 0.01) in gravid and nongravid horn myometrium. The level of gravid horn OTR mRNA during labor was 3 times the level of nongravid horn OTR mRNA (P < 0.0001). Gravid horn PGHS2 mRNA was also higher than nongravid horn PGHS2 (P < 0.02). In contrast, in spontaneous term labor nongravid horn, ERalpha and Hsp90 mRNA were similar to gravid horn. Myometrial ERalpha and Hsp90 mRNA remained unchanged throughout late pregnancy and increased at spontaneous term labor (P < 0.05). In contrast, myometrial OTR increased around 130 dGA (P < 0.01) and further increased at spontaneous term labor (P < 0.02). Progesterone significantly inhibited myometrial OTR mRNA expression in nonpregnant sheep and estradiol antagonized progesterone's inhibitory effect. Mechanical stretch differentially regulated mCAP mRNA expression in the ovine gravid horn and nongravid horn. Mechanical stretch appears largely responsible for increased OTR mRNA and to a lesser degree PGHS2 mRNA. In addition, endocrine factors may be required for full activation of OTR and PGHS2 mRNA associated with labor. ERalpha and Hsp90 mRNA are not under the control of uterine stretch in keeping with our previous results, indicating that systemic hormones such as estradiol, are prime regulators for these two mCAP mRNA expression during labor.

Differential Expression of Myometrial Oxytocin Receptor and Prostaglandin H Synthase 2, But Not Estrogen Receptor and Heat Shock Protein 90 Messenger Ribonucleic Acid in the Gravid Horn and Nongravid Horn in Sheep during Betamethasone-Induced Labor

In the present study, we characterized four myometrial contraction-associated proteins (mCAPs): oxytocin receptor (OTR), prostaglandin H synthase 2 (PGHS2), estrogen receptor α (ERα), and heat shock protein 90 (Hsp90) messenger RNA (mRNA) expression in the nongravid horn of pregnant sheep and compared them with their expression in the gravid horn that is exposed to a greater degree of stretch. We also examined the regulatory effects of estrogen and progesterone on OTR mRNA expression in ovariectomized nonpregnant sheep. In addition, we determined the ontogeny of mCAP expression in the gravid horn throughout late pregnancy and during spontaneous term labor. Gravid horn and nongravid horn myometria were removed under general anesthesia from control ewes not in labor at 130–140 days gestational age (dGA; n = 3) and during betamethasone-induced labor (n = 6) at the same gestational age. Gravid horn myometrium was also collected from ewes not in labor at 95 dGA (n = 3), 101–110 dGA (n = 3), 111–120 dGA (n = 3), 121–130 dGA (n = 3), 131–140 dGA (n = 3), and 141–145 dGA (n = 4) and from ewes in spontaneous term labor (n = 4). All ewes were carrying single fetuses. Myometrium was also collected from ovariectomized nonpregnant ewes treated with saline (n = 5), estradiol (50 μg/day; n = 5), progesterone (0.3 g, intravaginally; n = 5), and estradiol plus progesterone (n = 5). Myometrial RNA was extracted and analyzed by Northern blot for OTR, PGHS2, ERα, and Hsp90 mRNA, normalized for 18S ribosomal RNA orβ -actin. ERα, Hsp90, OTR, and PGHS2 mRNA were all significantly up-regulated during betamethasone-induced labor (P< 0.01) in gravid and nongravid horn myometrium. The level of gravid horn OTR mRNA during labor was 3 times the level of nongravid horn OTR mRNA (P < 0.0001). Gravid horn PGHS2 mRNA was also higher than nongravid horn PGHS2 (P < 0.02). In contrast, in spontaneous term labor nongravid horn, ERα and Hsp90 mRNA were similar to gravid horn. Myometrial ERα and Hsp90 mRNA remained unchanged throughout late pregnancy and increased at spontaneous term labor (P < 0.05). In contrast, myometrial OTR increased around 130 dGA (P < 0.01) and further increased at spontaneous term labor (P< 0.02). Progesterone significantly inhibited myometrial OTR mRNA expression in nonpregnant sheep and estradiol antagonized progesterone’s inhibitory effect. Mechanical stretch differentially regulated mCAP mRNA expression in the ovine gravid horn and nongravid horn. Mechanical stretch appears largely responsible for increased OTR mRNA and to a lesser degree PGHS2 mRNA. In addition, endocrine factors may be required for full activation of OTR and PGHS2 mRNA associated with labor. ERα and Hsp90 mRNA are not under the control of uterine stretch in keeping with our previous results, indicating that systemic hormones such as estradiol, are prime regulators for these two mCAP mRNA expression during labor.

Increased expression of the rat myometrial oxytocin receptor messenger ribonucleic acid during labor requires both mechanical and hormonal signals.

We investigated the expression of the mRNA encoding the oxytocin receptor (OTR) in rat myometrium throughout gestation and its regulation by progesterone and mechanical stretch. Using a semiquantitative reverse transcription-polymerase chain reaction approach, OTR mRNA was found to increase abruptly at the onset of spontaneous labor at term. Progesterone (4 mg/day) starting on Day 20 of gestation blocked this increase. Ovariectomy on Day 17 induced preterm labor 96 h after surgery and a significant increase in myometrial OTR mRNA levels 48 and 96 h after surgery. Both preterm labor and the rise in myometrial OTR expression were blocked by progesterone. To investigate the effects of stretch on myometrial OTR mRNA expression, unilaterally pregnant rats underwent either sham operation or placement of a tube in the nongravid uterine horn to distend the myometrium. On Day 20, stretch had no effect on OTR expression in the nongravid horns. During labor, OTR mRNA was highly expressed in the gravid horns as well as the nongravid stretched horns. In contrast, the level remained low in the nongravid unstretched horns. These results indicate that expression of rat myometrial OTR mRNA during pregnancy and labor is regulated by coordinated interactions between mechanical and endocrine signals.

Expression of connexin-43 and connexin-26 in the rat myometrium during pregnancy and labor is differentially regulated by mechanical and hormonal signals.

We investigated the effects of uterine stretch on the levels of messenger RNA (mRNA) encoding the gap junction proteins connexin-43 (Cx-43) and connexin-26 (Cx-26) as well as the presence of gap junction plaques formed by Cx-43 within the myometrium. In nonpregnant ovariectomized rats, stretch of one uterine horn with a polyvinyl tube induced a significant increase in myometrial Cx-43 mRNA levels, an effect that was blocked by progesterone; no expression of Cx-26 was detected in the presence or absence of stretch. To investigate whether pregnancy and parturition modified the response to stretch, unilaterally pregnant rats underwent either sham operation or placement of a tube in the nongravid uterine horns. On day 20 of pregnancy, expression of Cx-43 mRNA in gravid horns was low, and stretch did not increase this level. Cx-26 mRNA expression was elevated at this time, but only in the gravid horns. Cx-43 mRNA was highly expressed in the myometrium of gravid horns during labor, but Cx-43 expression in sham-operated, nongravid horns remained low. In contrast, nongravid horns stretched with tubes expressed Cx-43 mRNA at levels similar to those in gravid horns. Levels of Cx-26 mRNA in gravid horns fell between days 20 and 23, and this was not altered by stretch. Punctate Cx-43 immunofluorescence (indicative of gap junction formation) also increased in the myometrium after uterine stretch and in gravid horns during labor. Our data demonstrate that differential mechanisms regulate the expression of Cx-43 and Cx-26 in the pregnant myometrium. Cx-43 expression during labor is dependent upon myometrial stretch under conditions of low progesterone. In contrast, Cx-26 expression during late pregnancy, although requiring the presence of the fetal/placental unit, does not require stretch of the myometrium.

Embryonic survival in the uterus of ewes inseminated at the uterotubal junction on Day 32 post partum

Experiments were conducted to determine 1) if pregnancy initiated on Day 32 post partum would be maintained until lambing, 2) if there is a difference in the ability of the previously gravid or nongravid uterine horn to maintain pregnancy, and 3) if season has an effect on embryo loss. Estrus was induced in ewes on Day 32 post partum. At estrus, ewes were inseminated surgically at the uterotubal junction and assigned to the following groups: 1) inseminated at estrus and laparotomized on Day 3 to collect embryos for determination of fertilization rate (C), 2) inseminated in the previously gravid uterine horn (PG), 3) inseminated in the previously nongravid uterine horn (NG), and 4) inseminated when both horns were previously gravid (BG). Ewes pregnant in the PG, NG and BG groups were allowed to lamb. Conception rate in Group C at embryo collection was 70%. Embryo loss, based on concentrations of progesterone at Day 18 post insemination, was 43, 19 and 18% in the BG, NG and PG group, respectively. The high embryo loss in Group BG occurred only during the breeding season. Only 24% of the ewes that had been inseminated lambed. This was due to the prepartum loss of embryos and fetuses (47, 48 and 33% in Group BG, NG and PG, respectively. In conclusion, the detrimental effects of the uterus on embryo survival was evident within 18 d post insemination in Group BG (breeding season), and embryo loss prior to lambing was high in all the treatment groups (both seasons).

Location of equine oviductal embryos on day 5 postovulation and oviductal transport time of day-5 embryos autotransferred to the contralateral oviduct

In horses, limited data is found regarding the vascular events during uterine involution at the puerperal period. Thus, we aimed to evaluate the morphological aspects (size of uterus and intrauterine fluid content) and the hemodynamics (endometrial and mesometrial vascular perfusion) of the uterus during its postpartum involution process. Ten mares were daily scanned by transrectal color Doppler ultrasonography from the first day postpartum (d1) to the 16th day after first postpartum ovulation (D0 = ovulation). The formerly gravid horn (GH) and formerly nongravid horn (NH) were individually evaluated. A reduction (P < .05) in the uterine diameter was observed during the first 7 days postpartum, and the rate of uterine involution decreased after this period. The involution was completed on d21 and d24 for the NH and GH, respectively. Presence of intrauterine fluid was present in large amounts between d1 and d2 postpartum, followed by a decrease (P < .05) between d4 and d7. No fluid was observed after d16 postpartum or after the third day postovulation (D3). During the early postpartum period, an increase (P < .05) in the endometrial and mesometrial vascularization was detected, respectively, between d1 and d4, and between d1 and d2. The vascular perfusion did not differ after d4 for endometrial tissue, whereas was reduced (P < .05) between d2 and d10 for mesometrium. After the first postpartum ovulation, an increase (P < .05) in vascular perfusion was observed from D0 to D5, followed by a decrease (P < .05) between D5 and D11 and an increase (P < .05) between D11 and D14. The novel vascular perfusion profile here described in the endometrium and mesometrium after ovulation is similar to the uterine vascular profile observed during estrous cycles and early pregnancy, indicating a fast return of the mare's uterus to cycling postpartum conditions.