Coincident increases in oxytocin receptor expression and EMG responsiveness to oxytocin in the ovine cervix at oestrus

This study has localised oxytocin receptor (OTR) mRNA expression within the cervix of non-pregnant ewes and related this to changes in the sensitivity of the cervical musculature to administered oxytocin (OT) during the oestrous cycle by recording electromyographic (EMG) activity. Cervices were collected from 34 ewes at specified time points throughout the cycle. OTR mRNA expression was localised by in situ hybridisation and results were expressed as optical density measurements from autoradiographs in each of four different cervical regions. EMG recordings were made for up to 8 h per day from four non-pregnant ewes undergoing seasonal oestrous cycles between Days −3 and +3 relative to oestrus (Day 0). The highest concentrations of OTR mRNA were detectable within the luminal epithelium (LE) of the cervix, with values increasing from Day 15 of the cycle, peaking during the follicular phase (P<0.001, compared to the mid-luteal phase) and returning to basal by Day 2. There was a small but significant increase in OTR mRNA hybridisation (above basal/luteal phase values) within the stromal cells (STR) adjacent to the lumen (P<0.05) during the same time period, but no differences from basal values were detectable in the dense collagenous annular ring or in tissue superficial to this. Analysis of pooled EMG activity recorded daily from the cervix indicated that endogenous contractile activity was higher on Day 0 than on the Days +1 (P<0.05), −2, +2 and +3 (P<0.001). The response to bolus intravenous (i.v.) injections of 25 mU OT (25 mU) varied with day of the cycle. This dose produced a measurable and significant response on Days 0 (P<0.001) and +1 (P<0.001), but not on any of the other days, indicating that the sensitivity of the cervical musculature to OT peaked on these days. These data show that the cervix is highly responsive to OT at oestrus. This coincides with an increase in OTR mRNA expression in the luminal epithelial cells, suggesting the likely production of an intermediary messenger between the epithelial and smooth muscle cells.     

The expression of angiogenic growth factors and their receptors in ovarian follicles throughout the estrous cycle in the ewe

Healthy follicles are highly vascularized whereas those undergoing atresia have poor vascularity, suggesting a relationship between follicular vascularization and follicular function. Vascularization is regulated by angiogenic factors, and among them vascular endothelial growth factor (VEGF) and angiopoietin-Tie (Ang-Tie) systems are of central importance. The objectives of this study were to determine if VEGF, VEGF receptor-2 (VEGFR-2), and components of the Ang-Tie system are expressed in ovarian follicles at both the protein and mRNA levels and to explore if their expression is related to the stage of the estrous cycle in the ewe. Ovaries from cyclic ewes were collected during the luteal phase (n = 5) or before (n = 5), during (n = 4), and after (n = 4) the preovulatory luteinizing hormone (LH) surge. After fixation, ovaries were wax-embedded, serially sectioned, and analyzed for both protein and mRNA expression of VEGF, VEGFR-2, angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), Tie-1 (mRNA only), and Tie-2. mRNA was studied by in situ hybridization using digoxigenin-11-UTP-labeled ovine riboprobes. A similar pattern of expression was observed for mRNA and protein for all of the factors. Both mRNA and protein expression of VEGF, VEGFR-2, Ang-1, Ang-2, Tie-1 (mRNA only), and Tie-2 in the granulosa and theca cells of follicles ≥2 mm in diameter was significantly different among the stages of the estrous cycle, with the highest expression detected at the post-LH surge stage. Theca cells expressed significantly greater levels of the six angiogenic factors compared with granulosa cells at all stages of the estrous cycle. Expression levels in granulosa and theca cells were comparable between small (2.0 to 2.5 mm) and medium (2.5 to 4.0 mm) follicles, but large follicles (>4.0 mm) expressed higher mRNA and protein levels (all P < 0.05) for all factors at all stages of the estrous cycle. These data show (i) that VEGF, VEGFR-2, and the Ang-Tie system are present in both granulosa and theca cells of the ovarian follicle, (ii) that thecal cells consistently express greater levels of all of these factors compared with granulosa cells, and (iii) that their levels of expression are related to the stage of the estrous cycle and to follicle size.     

Gonadotropin-inhibitory hormone (GnIH) and its receptor in the female pig: cDNA cloning, expression in tissues and expression pattern in the reproductive axis during the estrous cycle

Since its discovery, gonadotropin-inhibitory hormone (GnIH) has appeared to act as a key neuropeptide in the control of vertebrate reproduction. GnIH acts via the novel G protein-coupled receptor 147 (GPR147) to inhibit gonadotropin release and synthesis. To determine the physiological functions of GnIH in the pig, a study was conducted to clone and sequence the cDNA of the GnIH precursor and GPR147. Our results demonstrated that the cloned pig GnIH precursor cDNA encoded three LPXRF and that its receptor possessed typical transmembrane features. Subsequently, tissue expression studies revealed that GnIH was mainly expressed in the brain, corresponding largely with the tissue expression patterns of GPR147 in the pig. The expression patterns in the reproductive axis of the female pig across the estrous cycle were also systemically investigated. The hypothalamic levels of both GnIH and its receptor mRNA were lowest in estrus and peaked in the proestrus and diestrus phases. The highest pituitary GnIH mRNA level was detected in the metestrus, and its receptor displayed a somewhat similar pattern of expression to that of the ligand. However, the expression patterns of GnIH and GPR147 were negatively correlated in the ovary. Immunolocalization in the ovary during the estrous cycle revealed that the immunoreactivities of GnIH and GPR147 were mainly localized in the granulosa and theca cells of the antral follicles during proestrus and estrus and in the luteal cells during metestrus and diestrus. Taken together, this research provided molecular and morphological data for further study of GnIH in the pig.     

Effect of the season on some aspects of the estrous cycle in Martina Franca donkey

The Martina Franca (MF) donkey breed, with 48 jackasses and 515 jennies, is considered an endangered breed according to the data from the Monitoring Institute for Rare Breeds and Seeds in Europe. The knowledge of the estrous cycle characteristics has a great impact for assisted reproduction, especially in endangered species. In this study, the estrous cycle characteristics were investigated in 12 MF jennies throughout the year. Estrous cycle, estrous and diestrous lengths, follicular growth and ovulation, and estradiol-17β (E2) and progesterone (P4) plasma concentrations were monitored in MF jennies and compared in different seasons. In all jennies (100%) estrous cycle was detected during the whole year, with no differences in the estrous cycle length among seasons. However, a significant increase of estrous length in spring and summer compared with autumn and winter was found. Diestrus was shorter in summer than in the other seasons. Estrous behavior was always shown and characterized by rhythmic eversion of the vulvar labia (winking) with exhibition of the clitoris, urination, male receptivity and clapping, with sialorrhoea, neck and head extension, and back ears. Estrus was characterized by the ovulation of a larger follicle in spring and summer than in autumn and winter. The pattern of E2 and P4 plasma concentrations during the estrous cycle were similar to that reported for the mare, but without differences among the four seasons, so that a negligible effect of environmental conditions on ovarian E2 and P4 secretion was hypothesized, despite the larger diameter of the ovulating follicle in spring and summer.     

Expression of Smad2 and Smad4, transforming growth factor-beta signal transducers in rat endometrium during the estrous cycle, pre-, and peri-implantation

SMADs are intracellular signaling molecules that transmit signals elicited by members of transforming growth factor-β (TGF-β) superfamily. To decipher the mechanism of TGF-β signaling during the estrous cycle and implantation, we performed in situ hybridization to investigate the expression patterns of mRNAs for Smad2 and Smad4 in rat endometrium during the estrous cycle and on Days 0.5, 1.5, 2.5, 3.5, 4.5, 5.5, and 6.5 of pregnancy. Intense epithelial expression of Smad2 mRNA at diestrus and proestrus was reduced at estrus and metaestrus, while Smad4 maintained its constitutive expression during the estrous cycle. During pre-implantation, both Smads were accumulated in the luminal epithelium and the glandular epithelium. Contrary to the dramatic Smad4 expression, Smad2 was highly down-regulated on Day 2.5 and was increased on Day 3.5. During peri-implantation, both Smads were expressed in the luminal epithelium, subepithelial stroma, and the primary decidual zone. Smad4 was down-modulated on Day 5.5. These results suggest that (a) both Smads are involved in the tissue remodeling of cycling and pregnant rat uteri; (b) TGF-β signaling functions mainly in the epithelium during pre-implantation and Smad2 is involved in the endometrial switch from the neutral phase to the receptive phase; (c) TGF-β signaling is down-regulated at the time when trophoblast invasion begins and both Smads are involved in the formation of the primary decidual zone.     

Molecular mechanism of cell proliferation in rodent uterus during the estrous cycle

The rodent uterus is a widely studied target tissue for sexual steroid hormone action. The aim of the present study was to assess the molecular mechanism that participates in the initiation of cell proliferation of the rat uterine epithelial cells during the estrus (E)–metestrus (M) transition. Cell proliferation, ERα, c-fos, cyclin D1 and D3, cdk4, and cdk6 proteins were assessed in these animals by immunohistochemistry. Estradiol (E₂) and progesterone (P₄) plasma levels were assessed by RIA. The results indicate that the glandular epithelium starts to proliferate at 21:00 h on estrus day, and initiates at least 3 h before the luminal epithelium does. Fos expression was markedly increased during the afternoon of estrus day, and its increase was in parallel to ERα expression. Interestingly, both, cyclin D1 and D3 were abundantly expressed in the luminal and glandular epithelia, and nuclear immunolabelling of cyclin D1 and D3 precedes BrdU incorporation in the cell. cdk4 and cdk6 were localized in the nuclei in both epithelia throughout the studied time course. In addition, cdk4 was more abundant throughout estrus and metestrus days than cdk6. The overall results indicate that ERα, Fos and cyclins D1 and D3, cdk4 and cdk6 are expressed in both glandular and luminal epithelia of the rat uterus during the E–M transition. In conclusion, there is a good correlation between sequential expression of these proteins and cell cycle progression in the rat uterine epithelial cells during the estrous cycle. However, the differences observed in the cellular localization, time course of expression and the cellular types that express both cyclins between physiological and pharmacological conditions, demonstrated different mechanisms of regulation and should be due to the complex hormonal milieu during the estrous cycle.     

Studies on the relationship between paracoccidioidomycosis in ddY mice and their estrous cycle

The relationship between paracoccidioidomycosis in ddY mouse and its estrous cycle was studied. Adult ddY mice of both sexes were used as experimental animals. Estrous cycle of female mice was examined before inoculation of Paracoccidioides brasiliensis yeast cells and mice were divided into 5 groups such as proestrus, estrus, metestrus-I, metestrus-II and diestrus. Each mouse was inoculated intravenously with 10⁶ P. brasiliensis yeast cell units and sacrificed on day 28 after inoculation. Their internal organs were cultured, and in addition, their histopathologies were studied. As a result, there was no difference in the organ cultures among the male and the female mice of 5 groups. However, histopathologically, the female groups at estrus, metestrus-I and metestrus-II were affected more severely than the male group, and the susceptibility of the female mice to the fungus was closely related to their estrous cycles.Abbreviations BHI-D brain heart infusion agar supplemented with 1.0% of anhydrous dextrose - PAS periodic acid-Schiff techniques - PBS phosphate buffered saline solution - SD standard deviation     

A Mathematical Model for the Actions of Activin,Inhibin, and Follistatin on Pituitary Gonadotrophs

The timed secretion of the luteinizing hormone (LH) and follicle stimulating hormone (FSH) from pituitary gonadotrophs during the estrous cycle is crucial for normal reproductive functioning. The release of LH and FSH is stimulated by gonadotropin releasing hormone (GnRH) secreted by hypothalamic GnRH neurons. It is controlled by the frequency of the GnRH signal that varies during the estrous cycle. Curiously, the secretion of LH and FSH is differentially regulated by the frequency of GnRH pulses. LH secretion increases as the frequency increases within a physiological range, and FSH secretion shows a biphasic response, with a peak at a lower frequency. There is considerable experimental evidence that one key factor in these differential responses is the autocrine/paracrine actions of the pituitary polypeptides activin and follistatin. Based on these data, we develop a mathematical model that incorporates the dynamics of these polypeptides. We show that a model that incorporates the actions of activin and follistatin is sufficient to generate the differential responses of LH and FSH secretion to changes in the frequency of GnRH pulses. In addition, it shows that the actions of these polypeptides, along with the ovarian polypeptide inhibin and the estrogen-mediated variations in the frequency of GnRH pulses, are sufficient to account for the time courses of LH and FSH plasma levels during the rat estrous cycle. That is, a single peak of LH on the afternoon of proestrus and a double peak of FSH on proestrus and early estrus. We also use the model to identify which regulation pathways are indispensable for the differential regulation of LH and FSH and their time courses during the estrous cycle. We conclude that the actions of activin, inhibin, and follistatin are consistent with LH/FSH secretion patterns, and likely complement other factors in the production of the characteristic secretion patterns in female rats.     

The influence of steroids on vascular tension of isolated superficial veins of the nose and face during the estrous cycle of gilts

The arrangement of the superficial facial veins enables blood flow from the nasal cavity into the peripheral circulation by two pathways: through the frontal vein into the cavernous sinus and through the facial vein into the external jugular vein. The current study was designed to determine whether estradiol and progesterone affect the vascular tone of the superficial veins of the nose and face in cycling gilts (Sus scrofa f. domestica) and to analyze the immunolocalization of progesterone receptors and estradiol receptors in these veins. The influence of hormones on vascular tension differed depending on the type of vessel and the phase of the estrous cycle. Estradiol decreased vascular tension in the nasal vein during the follicular phase (P < 0.05) and increased tension in the frontal vein during the luteal phase (P < 0.05). Progesterone increased the vascular tension of the frontal vein (P < 0.05) and decreased the tension of the other veins (P < 0.05) in both phases of the cycle. Expression of estradiol receptor β but not of progesterone receptor was observed in the superficial veins of the nose and face. In conclusion, the effect of ovarian steroid hormones on the vascular tension of the superficial veins of the nose and face in female pigs as well as the reactivity of these veins to steroid boar pheromones can affect the blood supply from the nasal cavity to the venous cavernous sinus. We propose that the ovarian steroid hormones that modulate the vascular tension of the nasal and facial veins may also influence the action of boar pheromones absorbed into the nasal mucosa in gilts and may reach the brain via local destination transfer.     

Effect of a postnatal high-fat diet exposure on puberty onset,estrous cycle regularity,and kisspeptin expression in female rats

Kisspeptin, encoded by Kiss1, plays a key role in pubertal maturation and reproduction as a positive upstream regulator of the hypothalamic-pituitary-gonadal (HPG) axis. To examine the role of high-fat diet (HFD) on puberty onset, estrous cycle regularity, and kisspeptin expression, female rats were exposed to HFD in distinct postnatal periods. Three groups of rats were exposed to HFD containing 60% energy from fat during the pre-weaning period (postnatal day (PND) 1–16, HFD PND 1–16), post-weaning period (HFD PND 21–34), or during both periods (HFD PND 1–34). Puberty onset, evaluated by vaginal opening, was monitored on days 30–34. Leptin, estradiol (E₂), Kiss1 mRNA levels, and number of kisspeptin-immunoreactive cells in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) were measured at day 34. Body weight increased only in rats exposed to HFD during post-weaning period, whereas the timing of vaginal opening was unaffected in all three groups. Leptin, Kiss1 mRNA levels, and number of kisspeptin-immunoreactive cells at day 34 were not affected by HFD. Additionally, the estrous cycle regularity was monitored in rats exposed to HFD for 40 days from weaning. Leptin, E₂, and Kiss1 mRNA levels in the AVPV and ARC were measured after the HFD exposure. Thirty-three percent of rats exposed to HFD exhibited irregular estrous cycles and a two-fold increase in leptin. By contrast, E₂ level and Kiss1 mRNA levels were not affected by the treatment. These data show that postnatal HFD exposure induced irregular estrous cycles, but had no effect on puberty onset or kisspeptin.     

Changes in the distribution of ZO-1, occludin,and claudins in the rat uterine epithelium during the estrous cycle

During the estrous cycle, the endometrium epithelium experiences marked cellular structural changes. For fertilization to proceed, maintenance of an adequate uterine environment by ovarian hormones is essential. Epithelial cells lining the uterine lumen are associated with each other by tight junctions (TJs), which regulate the passage of ions and molecules through the paracellular pathway. The aim of the present study was to assess by confocal immunofluorescence the distribution pattern of the TJ proteins ZO-1, occludin, and claudins 1–7 in the rat uterus during the estrous cycle. Our results reveal that on proestrus, the day when mating takes place, ZO-1, occludin, and claudins 1 and 5 are located in the TJs, while claudins 3 and 7 display a basolateral distribution. In contrast, on metestrus day, when no sexual mating occurs and the uterine lumen is devoid of secretions, none of these proteins were detected in the TJ region, and only a diffuse cytosolic staining was observed for some of the proteins. On estrus and diestrus days, an intermediate situation was encountered, since ZO-1 localized in the TJs, whereas occludin was no longer detectable in the TJs. The distribution of claudins during these stages varied from the lowermost portion of the basolateral membrane to its apex. In conclusion, the results show that the protein composition of TJs present in the luminal epithelial cells of the uterus changes during the different days of the estrous cycle, and suggest that the expression of TJ proteins participates in providing an adequate environment for a successful fertilization.This work was supported by grants PAPIIT (IN210902, IX228504) and PAIP (6190-08) from the National Autonomous University of Mexico (UNAM), and by grants G34511-M and 37846-N from the Mexican National Council on Science and Technology (CONACYT).     

Effects of neonatal treatment with phytoestrogens, genistein and daidzein, on sex difference in female rat brain function: estrous cycle and lordosis

It is well known that neonatal exposure to estrogen induces masculinization or defeminization of the brain. In this study, the effects of neonatal treatment with two kinds of soybean isoflavone aglycone, genistein (GS) and daidzein (DZ), on the estrous cycle and lordosis behavior were investigated. Female rats were injected subcutaneously with 1 mg GS, 1 mg DZ, 100 microg estradiol (E2), or oil daily for 5 days from birth. As a result, vaginal opening was advanced in GS- or E2-treated females. A vaginal smear check indicated that oil- or DZ-treated females showed a constant 4- or 5-day estrous cycle, whereas GS- or E2-treated rats showed a persistent or prolonged estrus. Ovariectomy was performed in all females at 60 days of age. The ovaries in the GS- or E2-treated groups were smaller than those in the oil- and DZ-treated groups and contained no corpora lutea. In the DZ group, although corpora lutea were seen, ovaries were smaller than that of control females. Behavioral tests were carried out after implantation of E2-tubes. All of the oil- or DZ-treated females showed lordosis with a high lordosis quotient (LQ). On the other hand, as male rats, LQs were extremely low in the E2-treated group, when compared to the oil-treated group. In the GS-treated group, the mean LQ was lower than that in the oil-treated group, but higher than those in the E2-treated female or male groups. These results suggest that genistein acts as an estrogen in the sexual differentiation of the brain and causes defeminization of the brain in regulating lordosis and the estrous cycle in rats. In addition, neonatal daidzein also has some influence on ovarian function.     

QRFP in female rats: effects on high fat food intake and hypothalamic gene expression across the estrous cycle

Pyroglutamylated arginine-phenylalanineamide peptide (QRFP) is a neuropeptide involved in feeding behavior. Central administration of QRFP selectively increases the intake of a high fat diet in male rats. QRFP administration also stimulates the hypothalamic-pituitary-gonadal axis via gonadotrophin-releasing hormone in male and female rats. Prepro-QRFP mRNA is expressed in localized regions of the mediobasal hypothalamus which are abundant in neurotransmitters, neuropeptides and receptor systems important for food intake regulation and reproductive behaviors. The current experiments were conducted to investigate the effects of centrally administered QRFP-26 on the intake of a high fat diet (HFD, 60% kcal from fat) in female rats and to investigate alterations in hypothalamic prepro-QRFP and its receptors, GPR130a and GPR103b, mRNA levels over the estrous cycle. In Experiment 1, female rats were administered QRFP-26 (intracerebroventricular; 0.3 nmol, 0.5 nmol, 1.0 nmol) in rats consuming either a HFD or a low fat diet. All doses of QRFP-26 selectively increased the intake of the HFD in female rats. These data suggest that QRFP-26 regulates the intake of energy dense foods in female rats, which is similar to previous findings in male rats. In Experiment 2, hypothalamic levels of prepro-QRFP mRNA and its receptors were assessed during diestrus, proestrus, or estrus. The level of prepro-QRFP mRNA in the ventromedial/arcuate nucleus (VMH/ARC) of the hypothalamus was increased during proestrus, which suggests that endogenous estrogen levels regulate QRFP expression in the VMH/ARC. These data suggest that QRFP may play a role in coordinating feeding behaviors with reproductive function when energy demand is increased.     

Unfolding protein response signaling is involved in development,maintenance, and regression of the corpus luteum during the bovine estrous cycle

The corpus luteum (CL) is a transient endocrine organ. Development, maintenance, and regression of CL are effectively controlled by dynamic changes in gene expression. However, it is unknown what types of gene are affected during the CL life span of the estrous cycle in bovine. Here, we determined whether unfolded protein response (UPR) signaling via eIF2α/ATF4/GADD34, p90ATF6/p50ATF6, and IRE1/XBP1, which is a cellular stress response associated with the endoplasmic reticulum (ER), is involved in the bovine CL life span. Our results indicated that expression of Grp78/Bip, the master UPR regulator, was increased during the maintenance stage and rapidly decreased at the regression stage. Additionally, UPR signaling pathways genes were found to be involved in luteal phase progression during the estrous cycle. Our findings suggested that Grp78/Bip, ATF6, and XBP1 act as ER chaperones for initiating CL development and maintaining the CL. In addition, we investigated whether ER stress-mediated apoptosis is occurred through three UPR signaling pathways in CL regression stage. Interestingly, pIRE1 and CHOP were found to be involved in both the adaptive response and ER stress-mediated apoptosis. During the CL regression stage, increased expression of pJNK and CHOP, two components of ER stress-mediated apoptotic cascades, occurred before increased level of cleaved caspase 3 were observed. The present investigation was performed to identify a functional link between UPR signaling and CL life span during the bovine estrous cycle. Taken together, results from this study demonstrated that UPR protein/gene expression levels were different at various stages of the bovine CL life span. Variations in the expression of these protein/genes may play important roles in luteal stage progression during the estrous cycle.     

Paradoxial changes in the expression of estrogen receptor alpha in breast cancer multicellular spheroids

Multicellular spheroids are excellent models for the analysis of cancer behavior. Just like small avascular tumors, they present a marked zonal heterogeneity which influences gene expression and thus, growth and response to chemotherapy. In the present paper, we sought to analyze the effects of three-dimensional culture in the expression and distribution of estrogen receptor alpha. Using MCF-7 breast cancer cells, we found that multicellular spheroids in estrogen-containing medium presented a paradoxical regulation of estrogen receptor alpha, with a decrease in protein expression and a marked increase in mRNA steady-state levels. Immunohistochemistry showed that only sparse cells in the periphery of the spheroid expressed estrogen receptor, in sharp contrast with progresterone receptor, which was more extensively expressed and HIF-alpha, which was expressed in the central core of the spheroid. This could mean that both hypoxia and ERA activation by estrogen participate in the expression heterogeneity of this hormone receptor in breast cancer These results are important to considerate in the analysis and interpretation of immunohistochemistry of ERA and downstream targets in samples of solid tumors.     

Uterine secretion of prostaglandin F2 alpha in response to oxytocin in ewes: changes during the estrous cycle and early pregnancy.

Experiment 1 was conducted to determine when the ovine uterus develops the ability to secrete prostaglandin F2 alpha (PGF2 alpha) in response to oxytocin and how development is affected by pregnancy. Pregnant and nonpregnant ewes received an injection of oxytocin (10 IU, i.v.) on Day 10, 13, or 16 postestrus. Jugular venous blood samples were collected for 2 h after injection for quantification of 13,14-dihydro-15-keto-PGF2 alpha (PGFM). In nonpregnant ewes, concentrations of PGFM increased following oxytocin on Day 16 but not on Day 10 or 13. Concentrations of PGFM did not increase following treatment on Day 10, 13, or 16 in pregnant ewes. Therefore, the ability of oxytocin to induce uterine secretion of PGF2 alpha develops after Day 13 in nonpregnant but not in pregnant ewes. Experiment 2 was conducted to precisely define when uterine secretory responsiveness to oxytocin develops. Pregnant and nonpregnant ewes received oxytocin on Day 12, 13, 14, or 15. In nonpregnant ewes, concentrations of PGFM increased following treatment on Days 14 and 15, but not earlier. Peripheral concentrations of progesterone showed that uterine secretory responsiveness to oxytocin developed prior to the onset of luteal regression. As in experiment 1, the increase in concentrations of PGFM following administration of oxytocin was much lower in pregnant than in nonpregnant ewes; however, some pregnant ewes did respond to oxytocin with an increase in PGFM. In experiment 3, pregnant ewes received an injection of oxytocin on Day 18, 24, or 30 postmating. Concentrations of PGFM increased following oxytocin on Days 18 and 24. The conceptus appears to delay and attenuate the development of uterine secretory responsiveness to oxytocin.