Oxytocin induces PGE2 release from bovine cervical mucosa in vivo.

Oxytocin receptor (OTR) concentrations in bovine cervical mucosa rise steeply a few days before estrus to high concentrations and fall rapidly after estrus. To study the physiological role of these OTR, the effect of OT on the release of PGE, from the cervical mucosa of periestrous cows in vivo was determined by inserting bags made of dialysis tubing containing isooncotic saline solution in the endocervix for two 2-h periods, a fresh bag for each period. During the first period no treatment was given, during the second period OT (100 IU) or saline was injected i.m. PGE2 content in the second bag was significantly greater in OT-treated cows than in saline-treated cows. In a second experiment cervical resistance to stretch, achieved by distention of a balloon inside the cervical canal, was measured in periestrous cows before and 10 h after i.m. injection of OT, or endocervical application of 2.5mg PGE1 in a jelly, or the inactive jelly. A significant reduction in the resistance was achieved with both OT and PGE1; in the doses given the effect of PGE1 was longer lasting than that of OT.

Secretion of oxytocin in pregnant and parturient cows: corpus luteum may contribute to plasma oxytocin at term.

Plasma oxytocin (OT) concentrations were determined in 14 late-pregnant and parturient Angus-Hereford cows. Jugular and utero-ovarian veins were cannulated for simultaneous withdrawal of blood samples. Samples were collected at 10-min intervals for 6 h once weekly beginning 60-14 days before the date of expected delivery (group 1), or daily 3-7 days before the due date (group 2). In a third group, samples were collected at 15-min intervals every other day for 12 h beginning 1 wk before calving. Basal levels of OT were low, the overall mean for both veins was 0.46 +/- 0.03 microU/ml until a week before parturition, and then increased to 0.77 +/- 0.1 microU/ml (P < 0.02). Spurts of OT occurred intermittently on all days. Interpeak intervals averaged 71.0 +/- 10.7 min until Day -14, and from Day -14 to Day -1 the intervals were 44.0 +/- 5.3 min (P < 0.05). From Day -60 to Day -25 the amplitudes of OT peaks were low and similar in both veins (mean 1.37 +/- 0.1 microU/ml). From Day -14 to Day -1 the peak amplitudes were 3.6 +/- 0.4 microU/ml on average (P < 0.02). During the last 2 wk the utero-ovarian peak of OT was frequently higher than the peripheral peak. In addition, a number of spurts were observed in the utero-ovarian vein only (solo peaks). On the day of parturition during the first stage of labor, peak amplitudes had increased to 7.3 +/- 2.0 microU/ml, and the interpeak intervals had become shorter than before labor (mean 25.1 +/- 2.6 min). A large surge of OT initiated the expulsive stage of labor. Basal levels rose to 43.1 +/- 16 microU/ml and 38.7 +/- 12.6 microU/ml, and peak levels to 77.4 +/- 19.1 microU/ml and 91.6 +/- 21 microU/ml in the jugular and utero-ovarian veins, respectively. Interpeak intervals had decreased to 17.2 +/- 3.3 min (P < 0.05). Oxytocin levels remained high after delivery of the calf until the placenta was expelled. The posterior pituitary was the source of circulating OT during most of gestation and labor, but the solo peaks observed during late gestation in the utero-ovarian vein were probably of luteal origin or possibly of caruncular origin, because near term, both tissues express OT mRNA. Fetal posterior pituitary is another possible source for these peaks. Our conclusions are that during bovine pregnancy, low amplitude spurts of OT are secreted intermittently; near term, both the frequency and peak amplitude of the spurts increase; and during labor, a dramatic increase in plasma OT precedes the expulsion of the calf. The main source of OT is the posterior pituitary, but near term, a utero-ovarian source secretes additional OT into the systemic circulation.

Word deafness presenting as a sudden hearing loss.

Sudden hearing loss is a condition frequently seen in ENT departments and is usually due to otological pathology. We present a patient who described symptoms of a sudden inability to hear but who, on further questioning and investigations, had 'word deafness' due to bilateral temporoparietal infarcts. We discuss the clinical syndromes associated with these infarcts and the specific management of word deafness.

Functional importance of bovine myometrial and vascular LH receptors and cervical FSH receptors.

Bovine myometrium and cervix contain luteinizing hormone/human chorionic gonadotropin (LH/hCG) binding sites, LH receptor (LH-R) messenger RNA (mRNA), and LH-R protein. Expression of LH-R is dependent on the stage of the cycle. LH-R expression is high during the luteal phase but weak during the follicular phase. In both myometrium and cervix, LH activates both the adenylate cyclase and phospholipase C pathways, and the effect of LH on both pathways at each stage of the cycle is correlated with the amount of LH-R present in the tissue. Because activation of cyclic AMP (cAMP) is associated with myometrial quiescence, we suggest that LH activation of uterine cAMP could serve to keep the uterus quiescent during the luteal phase. On the other hand, in the uterine vein LH-R mRNA and LH-R are maximal during preestrus/estrus as opposed to the luteal phase. In the uterine vein, LH increases the expression of cyclooxygenase and production of both prostaglandin E2 (PGE2) and PGF2 alpha. Because PGF2 alpha is the physiological luteolytic signal in the cow, we suggest that this increase in prostaglandin production by the uterine vein is part of the physiological events leading to luteolysis. In addition to uterine LH-R, the bovine cervix at preestrus/estrus has high levels of follicle-stimulating hormone receptor (FSH-R) and its corresponding mRNA. As with LH-R, activation of FSH-R by FSH is associated with activation of a G protein-coupled receptor family that mediates the cAMP and inositol phosphate signaling pathways. Activation of these signaling pathways is associated with an increase in the expression of cyclooxygenase and production of PGE2. Because expression of the FSH receptor was maximal at the time of the FSH peak in the blood, we suggest a physiological role for FSH in the cervix relaxation and opening at estrus.

Efficacy of an intravaginal progesterone insert and an injection of PGF2alpha for synchronizing estrus and shortening the interval to pregnancy in postpartum beef cows, peripubertal beef heifers, and dairy heifers.

The objective was to test the efficacy of an intravaginal progesterone insert and injection of PGF2alpha for synchronizing estrus and shortening the interval to pregnancy in cattle. Cattle were assigned to one of three treatments before a 31-d breeding period that employed artificial insemination. Control cattle were not treated, and treated cattle were administered PGF2alpha or an intravaginal progesterone-releasing insert (CIDR) for 7 d and treated with PGF2alpha on d 6. The treatments were applied in one of three experiments that involved postpartum beef cows (Exp. 1; n = 851; 56+/-0.6 d postpartum), beef heifers (Exp. 2; n = 724; 442.5+/-2.8 d of age), and dairy heifers (Exp. 3; n = 260; 443.2+/-4.5 d of age). Luteal activity before treatment was determined for individual cattle based on blood progesterone concentrations. In Exp. 1, there was a greater incidence of estrus during the first 3 d of the breeding period in CIDR+PGF2alpha-treated cows compared with PGF2alpha-treated or control cows (15, 33, and 59% for control, PGF2alpha, and CIDR+PGF2alpha, respectively; P < 0.001). The improved estrous response led to an increase in pregnancy rate during the 3-d period (7, 22, and 36% for control, PGF2alpha, and CIDR+PGF2alpha, respectively; P < 0.001) and tended to improve pregnancy rate for the 31-d breeding period for cows treated with CIDR+PGF2alpha, (50, 55, and 58% for control, PGF2alpha, and CIDR+PGF2alpha, respectively, P = 0.10). Improvements in rates of estrus and pregnancy after CIDR+PGF2alpha, were also observed in beef heifers. Presence of luteal activity before the treatment period affected synchronization and pregnancy rates because anestrous cows (Exp. 1) or prepubertal heifers (Exp. 2) had lesser synchronization rates and pregnancy rates during the first 3 d of the breeding period as well as during the entire 31-d breeding period. The PGF2alpha, and CIDR+PGF2alpha but not the control treatments were evaluated in dairy heifers (Exp. 3). The CIDR+PGF2alpha-treated heifers had a greater incidence of estrus (84%) during the first 3 d of the breeding period compared with the PGF2alpha-treated heifers (57%), but pregnancy rates during the first 3 d or during the 31-d breeding period were not improved for CIDR+PGF2alpha compared with PGF2alpha-treated heifers. In summary, the concurrent treatment of CIDR and PGF2alpha improved synchronization rates relative to PGF2alpha alone or control. Improved estrus synchrony led to greater pregnancy rates for beef cows and beef heifers but failed to improve pregnancy rates for dairy heifers.

Expression of functional luteinizing hormone (LH) receptor and its messenger ribonucleic acid in bovine endometrium: LH augmentation of cAMP and inositol phosphate in vitro and human chorionic gonadotropin (hCG) augmentation of peripheral prostaglandin in vivo.

Bovine endometrium contains LH/hCG binding sites and LH increases endometrial production of prostaglandin H synthase-2 (PGHS-2) and prostaglandin synthesis. This study showed that uterine endometrium contained both LH receptor mRNA transcript and a 93-kDa immunoreactive protein that bound to anti-rat LH receptor antibody. LH receptor and its mRNA were expressed maximally in the endometrium of cows from the luteal phase compared to the follicular phase of the estrous cycle. Furthermore, there was a response shown when incubation of endometrial minces from both pre-estrus/estrus and luteal phase (but not post-ovulatory phase) with LH or oxytocin (20 ng/ml) that resulted in a significant (p<0.02) increase in cAMP and total inositol phosphates. When Day 15 cows were injected i.v. with 3000 units hCG, the increase in peripheral 13,14-dihydro-15-keto PGF(2alpha) was 2.5-fold higher than saline controls or oxytocin. We conclude that LH stimulates endometrial cAMP and total inositol phosphates which results in increased formation of uterine PGHS-2 similar to LH effect on ovarian PGHS-2. The increased 13,14-dihydro-15-keto PGF(2alpha) production induced in vivo by injections of hCG indicates that LH may have a reinforcing role in luteolysis.

A biomechanical, radiologic, and clinical comparison of outcome after multilevel cervical laminectomy or laminoplasty in the rabbit.

STUDY DESIGN: A rabbit model was used to compare clinical outcome, radiographic changes, and biomechanical flexibility after cervical laminectomy and open-door laminoplasty. OBJECTIVE: This study tested the hypothesis that radiographic changes and biomechanical flexibility could explain the differences in clinical outcome after cervical laminectomy and laminoplasty. SUMMARY OF BACKGROUND DATA: Although multilevel cervical laminoplasty is thought to have advantages over cervical laminectomy, clinical outcome studies have been contradictory, and no experimental study has examined the possible mechanisms for the differences after healing. METHODS: Twenty-four New Zealand White rabbits were randomized into four groups: normal, sham, C3-C6 wide laminectomy, and C3-C6 open-door laminoplasty. Clinical, radiographic, and biomechanical data were collected and compared up to 3 months after surgery. RESULTS: Laminectomy had a statistically significant poorer clinical outcome when compared with laminoplasty after 3 months of healing. Radiologic analysis showed statistically significant angular deformity in the laminectomy group compared with laminoplasty and control groups at 3 months. In contrast, biomechanical measures of flexibility, neutral zone, and range of motion showed only small differences between any of the groups at any time. CONCLUSIONS: The presence of deformity, and not a change in flexibility, is responsible for the differences in clinical outcome observed after laminectomy compared with laminoplasty in this model.

Clinical outcome scales for use in a rabbit model of cervical myelopathy.

This study determined the ability of an upper extremity Tarlov scale, a lower extremity Tarlov scale, and the Durham scale to predict the development of myelopathy and the likelihood of survival in a rabbit model of surgical treatments for cervical spondylotic myelopathy. Forty-eight rabbits were evaluated using the scales after cervical spinal surgery. Logistic regression analysis revealed that all three scales could predict the occurrence of myelopathy. However, only the Durham and lower extremity Tarlov scales also predicted the likelihood of survival. The Durham scale is offered as a useful predictor of myelopathy and survival in an animal model of surgical treatments for cervical spondylotic myelopathy. The lower extremity Tarlov scale is also a useful predictor of outcome; however, the upper extremity Tarlov scale is not recommended.

B-chain sequence and in situ hybridization of the rabbit placental relaxin-like gene product.

We reported that the nucleotide sequence of a cDNA generated from rabbit placental poly(A)(+) RNA using porcine preprorelaxin primers was identical to SQ10, a product of squamous differentiated tracheal epithelial cells. However, these results did not confirm that SQ10 was the biologically active rabbit relaxin that had been isolated previously yet not sequenced. In this study, a 7-kDa protein isolated from rabbit placentas exhibited relaxin bioactivity and cross-reacted with a porcine relaxin antiserum. A partial amino acid sequence of this protein revealed a sequence identical to that of SQ10. Although the amino acid sequence of the putative relaxin receptor-binding domain found in the B chain of relaxin was modified in SQ10 from CGRDYVR to CRNDFVR, the placental protein was bioactive. These results suggest that SQ10 is the rabbit relaxin. In situ hybridization, using an SQ10 riboprobe, indicated radiolabeling in the syncytiotrophoblast cells of the rabbit placenta. The pattern of labeling corresponded with the immunohistochemical staining for relaxin observed with use of a porcine relaxin antiserum. These results indicate that the syncytiotrophoblast cells are a site of synthesis for SQ10 and that the immunostaining is not solely the result of sequestering SQ10 through receptor-mediated endocytosis. A potential role for relaxin in implantation is discussed.

Accumulation of cyclooxygenase-2 gene transcripts in uterine tissues of pregnant and parturient cows: stimulation by oxytocin.

Cyclooxygenase 1 and 2 (COX-1 and COX-2) mRNA were measured by ribonuclease protection assays in total RNA extracted from intercaruncular and caruncular endometrium, myometrium, cotyledons, and cervical mucosa of pregnant cows. Tissues were obtained at gestational ages of 150 days and 275 days and at term not in labor, at term in labor, and 6-12 h postpartum. Additionally, the effect of oxytocin (OT) on COX-2 expression was determined in intercaruncular endometrium of six third-trimester cows (between 230 and 270 days of pregnancy), three of which were injected with OT (200 IU) and three with saline 2 h before tissues were harvested. Prostaglandin F2alpha (PGF2alpha) metabolite was measured in plasma samples taken at 15-min intervals before and after the injections. Results showed that COX-2 mRNA was expressed in every type of tissue examined, although in different concentrations and beginning at different stages. Other than in seminal vesicular and prostate glands used as positive controls, low concentrations of COX-1 mRNA were detected only in myometrium and caruncles. Cotyledons had the highest concentration of COX-2 transcripts at all stages studied. Caruncles had about half the concentration of COX-2 transcripts that was seen in cotyledons, and on Day 150 even less. COX-2 mRNA expression in both tissues increased with advancing gestation, but there was no difference between samples from term-no-labor and term-in-labor cows. COX-2 mRNA concentrations in endometrium and myometrium were low; they varied randomly during pregnancy with no significant increase until postpartum, when COX-2 transcripts in endometrium had increased severalfold whereas those in myometrium were similar to values before parturition. Cervical mucosa expressed COX-2 mRNA weakly until term but had increased markedly at parturition. Injection of 200 IU of OT induced a substantial increase in endometrial COX-2 mRNA concentration within 2 h; this was associated with linearly increasing plasma concentrations of 13, 14-hydroxy-15-keto-prostaglandin F2alpha, which were still rising at termination of the experiment. The results suggest that endogenous OT is a major factor in induction of COX-2 expression and PGF2alpha release at term and during parturition in cows.

Ontogeny of oxytocin receptors and oxytocin-induced stimulation of prostaglandin synthesis in prepubertal heifers.

Developmental aspects of oxytocin (OT) receptors (OTR) in uterine tissues before puberty are not known. Bovine ovaries secrete some estradiol, but no progesterone, before puberty; the circulating levels of estradiol are between 1 and 3 pg/ml until puberty. Cross-bred Angus-Brahman heifers, in which puberty occurs around 12 months of age, were used to determine the concentrations of OTR from the late fetal stage to adulthood. PGF2alpha release in response to OT was determined in 3-, 6-, and 9-month-old heifers (n = 4 each). Myometrium, endometrium, and cervical mucosa were obtained from 3-week-old, 3-month-old, 6-month-old, and 9-month-old heifers and from adult cows at estrus. Whole uterus and cervix were taken from third trimester fetuses and at birth. [3H]OT binding and specificity, localization of immunoreactive (ir) OTR, OTR messenger RNA, and OT-induced release of PGF2alpha were determined. The uterus from fetuses and the neonate expressed OTR messenger RNA and bound [3H]OT. At 3 weeks of age, OTR concentrations per mg protein were very low, but at 3 months of age they had increased markedly in all three tissues. At 6 and 9 months of age, levels of OTR had risen further and were similar to those in adult cows at estrus. Prepubertal uterus also possessed separate vasopressin VP1 subtype receptors. The ir-OTR was localized in luminal epithelial cells of endometrium and cervical mucosa, most of which were ir positive, whereas in myometrium, clusters of ir-OTR-positive cells were found among large numbers of ir-OTR-negative cells. The PGF2alpha response to OT was insignificant in heifers of all age groups, in contrast to that in cows at estrus. Endometrial cells from 4- to 5-month-old heifers did not respond to OT with PG release in the absence or presence of added arachidonic acid. Tumor promoters, lipopolysaccharide, and interleukin-2 also failed to elicit PG release in vitro, although they induced PG release in similar cell cultures from cyclic cows. In summary, uterine tissues of prepubertal heifers have high levels of OTR, which appear to be developmentally regulated. These receptors are not coupled to PG synthase, or alternatively, the PG synthase gene is not expressed before puberty, possibly because the tissues have had no previous exposure to progesterone.

Expression of functional luteinizing hormone (LH) receptor and its messenger ribonucleic acid in bovine uterine veins: LH induction of cyclooxygenase and augmentation of prostaglandin production in bovine uterine veins.

We have previously reported that bovine endometrium contains LH/human CG binding receptors and LH induces cyclooxygenase and prostaglandin production in the bovine endometrium. The present study investigated 1) whether bovine uterine vein and artery contain LH receptor messenger RNA (mRNA) and receptor protein and 2) whether LH can regulate the formation of vasoactive eicosanoids by the uterine vein. The uterine vein endothelium, but not the uterine artery, contained LH receptor mRNA transcript essentially identical to that found in the bovine corpus luteum. The uterine vein endothelium also contained a 95-kDa immunoreactive receptor protein that bound to rat anti-LH receptor antibody in Western blots. The LH receptor mRNA and LH receptor were maximally expressed in the uterine vein from cows in proestrus/estrus compared with cows in luteal or postovulatory phases. Incubation of endothelial minces of uterine vein with LH resulted in a 2-fold increase in cyclooxygenase concentration as determined by Western blot using an antibody to ram seminal vesicle cyclooxygenase. The increase in cyclooxygenase was maximal in cows in proestrus/estrus compared with postovulatory and luteal phase cows. Incubation of proestrous/estrous uterine vein or artery minces with LH or mellitin (a phospholipase A2 stimulator) caused increased production of eicosanoids. In the uterine vein, LH caused a significant increase in both PGF2alpha (basal 4.1 +/- 0.4 vs. 5.7 +/- 0.4 ng/100 mg x 6 h, P < 0.01; N = 9 cows) and PGE2 (basal 5.7 +/- 0.3 vs. 7.7 +/- 0.8 ng/100 mg x 6 h, P < 0.01; N = 6 cows) but had no effect on prostaglandin production by the artery. Mellitin increased PGF2alpha production by both uterine vein and artery minces but had no effect on PGE2 production in either tissue. Addition of steroids (progesterone, estradiol) or cytokines (tumor necrosis factor-alpha, IL-6) to the uterine vascular tissues had essentially no effect on prostanoid production. In summary, bovine uterine vein from proestrous/estrous cows expressed the LH receptor and its mRNA. Expression of the receptor may have physiological significance as LH induces cyclooxygenase and increases prostaglandin release in the uterine vein. The maximal stimulation of the receptor and its mRNA at proestrus/ estrus may serve to increase the amounts of prostanoids reaching the regressing corpus luteum either directly by increasing prostanoid production or indirectly by increasing the blood flow to the ovary.

Oxytocin antagonist [1-D(CH2)5,Tyr(ME)2,Thr4,Tyr-NH2(9)]ornithine vasotocin inhibits oxytocin-induced prostaglandin F2alpha release in late-pregnant cows.

The affinity and specificity of an antagonist of oxytocin, [1-D(CH2)5,Tyr(ME)2,Thr4,Tyr-NH2(9)]ornithine vasotocin (OTA), to oxytocin receptors (OTR) in bovine gestational endometrium was determined in displacement experiments with oxytocin (OT) and vasopressin (AVP) analogues and compared to myometrial OTR. OTA had the highest affinity in both tissues. The effect of OTA on OT-induced increase in plasma concentration of 13,14-dihydro-15-keto-prostaglandin F2alpha metabolite (PGFM) was studied in 24 late-pregnant cows. Treatments consisted of i.v. saline; OT (50 IU); OTA (1200 microg); and OTA (400, 1200, or 4000 microg) injected i.v. 5 min before OT (50 IU) (n = 4 each). Samples were collected from jugular vein at 15-min intervals for 30 min before and 3 h after the injection of OT. Progesterone was measured in once-daily samples taken for 7 days after the experiment. OT caused a twofold increase in plasma PGFM within about 60 min (p < 0.005), with levels returning to baseline at 150-180 min; OTA (1200 microg) caused a gradual lowering of basal plasma PGFM over 180 min (p < 0.05). The 400-microg or 1200-microg dose of OTA did not alter OT-induced PGFM response, whereas the 4000-microg dose inhibited it almost completely (p < 0.005). Plasma progesterone declined after the experiment in all cows, with no differences among groups. Because OTA inhibits OT-induced release of endometrial prostaglandin F2alpha it may be a good tocolytic agent.

Synthesis and messenger ribonucleic acid expression of apolipoproteins E and A-I by the bovine corpus luteum during the estrous cycle and pregnancy.

In an attempt to characterize proteins secreted by the corpus luteum, explant cultures of luteal slices from cows taken on Days 3, 7, 11, 14, 17, and 19 of the estrous cycle, and Days 17, 88, 180, and > 240 of pregnancy were incubated with H-leucine for 24 h. Proteins in luteal-conditioned medium were separated by two-dimensional PAGE, transferred to polyvinylidene fluoride membrane, and subjected to N-terminal amino acid microsequencing. Microsequence analysis revealed that the bovine corpus luteum synthesized and released de novo synthesized apolipoproteins (Apo) E and A-I in culture during the estrous cycle and pregnancy. Release of Apo E was observed only on Day 3 of the estrous cycle. Release of Apo A-I was observed on Days 11, 14, 17, and 19 of the estrous cycle, and on all days of pregnancy examined. To demonstrate the presence of the appropriate mRNA and characterize the temporal relationship for these identified proteins, total RNA was isolated from corpora lutea on Days 2, 3, 7, 16, 17, and 20 of the estrous cycle, and on Days 17, 90, 170, 180, and 272 of pregnancy, and submitted to Northern and dot blot analysis. Apo E mRNA was expressed only on Days 2-3 of the estrous cycle and was not expressed on the other days of the cycle or during pregnancy. A single Apo E mRNA transcript about 1.0 kilobase (kb) in size was observed. Expression of Apo A-I mRNA was detected on all days of the estrous cycle and pregnancy examined. Apo A-I cDNA hybridized with a single mRNA transcript about 1.0 kb in size. Apo A-I mRNA levels did not differ among days of the estrous cycle, although higher levels of Apo A-I mRNA were observed during later stages of pregnancy. Serum concentrations of Apo A-I and progesterone were correlated across the estrous cycle but not during the prepartum period or after parturition. This study demonstrates for the first time that the corpus luteum synthesizes Apo E and Apo A-I and expresses their respective mRNAs. The pattern of expression of Apo E and Apo A-I mRNAs paralleled that of de novo synthesis of their respective proteins after incubation of luteal tissue with [H]leucine. The role of luteal apolipoproteins may involve an autocrine/paracrine function influencing luteal development, tissue remodeling, and steroidogenesis.

Relaxin: an ovarian hormone in an avian species (Gallus domesticus).

The objective of this study was to characterize the biochemical, immunological, and biological activity of avian relaxin and to immunolocalize relaxin-like peptides in the ovary of the hen (Gallus domesticus). A relaxin-like peptide was partially purified from ovaries of actively laying hens by size-exclusion chromatography and further purified by ion-exchange chromatography on CM-cellulose. Those fractions containing relaxin immunoreactivity were identified with the use of a homologous porcine relaxin radioimmunoassay on selected column effluent and pooled, and a sample was subjected to SDS-gel electrophoresis. The SDS-gel-separated proteins were electrotransferred onto a nitrocellulose membrane and immunostained with an antiserum to porcine relaxin which showed the presence of a single band of approximately 6000 daltons. The dose-response curve generated by avian relaxin-like peptide in the homologous porcine relaxin radioimmunoassay was parallel to that produced by the porcine relaxin standard. Like porcine relaxin, avian relaxin-like peptide eluted from the Sephadex G-50 in an elution volume for a molecule of approximately 6000 daltons, was retained on CM-cellulose, and was bioactive in in vitro inhibition of spontaneous contractions of estrogen-primed mouse uterus (a relaxin bioassay). Using an antiserum specific to porcine relaxin, avian relaxin-like peptide was immunolocalized to the granulosa cells of postovulatory follicle from ovary of a hen less than 24 hr postoviposition. No immunostaining was detected in the cells of the largest preovulatory follicles or when the antiserum was preabsorbed with porcine relaxin prior to staining. The finding of this study indicates that the avian postovulatory follicle, like the corpus luteum of other vertebrate species (sharks and mammals), contains a relaxin-like peptide.

Regulation of bovine cervical secretion of prostaglandins and synthesis of cyclooxygenase by oxytocin.

Prostaglandin E2 (PGE2) can cause softening of the bovine cervix at oestrus when receptors for oxytocin (OT) are maximally present, indicating a relationship between OT and PGE2 production. It was therefore determined whether OT can stimulate prostaglandin synthesis or induce cyclooxygenase expression in cervical external os segments obtained from pre-oestrous-oestrous cows. Tissues were minced and incubated (50-100 mg mL[-1] 6 h[-1]) in the presence of OT (10 ng mL[-1]), progesterone (P4) (5 ng mL[-1]) and/or indomethacin (5 microg mL[-1]). It was found that OT stimulated basal PGE2 (7.79+/-1.22 ng 100 mg[-1], mean+/-s.e.m.; n = 6) in external os segments from pre-oestrous-oestrous cows (P < 0.03), whereas P4 and indomethacin inhibited basal and OT-stimulated PGE2 production (P < 0.05). Basal prostaglandin F2alpha (PGF2alpha) production was minimal (<1 ng 100 mg[-1]) and OT had no effect on its production. Expression of cyclooxygenase was measured by Western blot analysis following incubation of the tissue (100 mg 1.5 mL[-1] 3 h[-1]) in the presence of OT (10 ng mL[-1]) and in the presence of P4 (5 ng mL[-1]). It was found that OT stimulated the induction of cyclooxygenase II (79+/-10%; n = 7, P < 0.05). In contrast, P4 inhibited the basal expression of this enzyme (-40+/-5%, n = 7, P < 0.05) in the presence or absence of OT. It is concluded that, in vitro, OT stimulates PGE2 synthesis by the bovine cervix at oestrus and that this effect is mediated by cyclooxygenase.

Oxytocin receptors in bovine cervix during pregnancy and parturition: gene expression and cellular localization.

OBJECTIVES: Our purpose was to determine the expression of transcripts encoding the oxytocin receptor protein in bovine cervix during pregnancy and parturition, the cellular localization of immunoreactive oxytocin receptors, and oxytocin receptors concentrations in the same tissues. STUDY DESIGN: Ribonuclease protection assay for oxytocin receptor messenger ribonucleic acid was used to determine gene expression in bovine cervical tissues obtained from 20 cows throughout pregnancy and parturition, cellular localization of oxytocin receptors was determined by immunohistochemistry, and tritiated oxytocin binding was measured in each tissue. RESULTS: Oxytocin receptor gene expression and tritiated oxytocin binding were well correlated in each instance. During pregnancy the level of oxytocin receptor messenger ribonucleic acid was very low; it was increased at term with a further, marked increase at parturition. Tritiated oxytocin binding also increased dramatically at parturition and was most abundant in the mucosal layer. Strong oxytocin receptor immunoreactivity was present in mucosal epithelial cells, and scattered muscle cells in the muscular part showed the signal. CONCLUSIONS: Our results, together with the previous finding that oxytocin stimulates prostaglandin E2 release from cervical tissue in vitro, indicate that cervical mucosal epithelial cells are targets for oxytocin at parturition and may mediate release of prostaglandin E2.

A cellular and endocrine characterization of the original and induced corpus luteum after administration of a gonadotropin-releasing hormone agonist or human chorionic gonadotropin on day five of the estrous cycle.

To determine whether injection of hCG or GnRH-agonist on d 5 after estrus (d 0) has a differential functional effect on an induced and the original corpus luteum (CL), two experiments were conducted. In Exp. 1, nonlactating Holstein cows were injected on d 5 with saline (n = 4; T1), a GnRH-agonist (Buserelin, 8 micrograms i.m.; n = 4; T2), or hCG (1,000 i.u., i.v., and 2,000 i.u., i.m.; n = 4; T3). Induced CL were removed on d 13 and weights were different (GnRH-agonist < hCG). In vitro production of progesterone by CL tissue (microgram/g; microgram/CL) was affected by treatment (GnRH-agonist < hCG) and dose of LH (ng.mL) in culture media. Experiment 2 was a replicate of Exp. 1, except that the original CL was removed on d 17 for in vitro culture. Day-17 CL weights and in vitro production of progesterone by original CL were not affected by treatment. The daily rate of increase of plasma progesterone from d 6 to d 13 differed: saline < GnRH-agonist < hCG (P < .01). From d 14 to 17, the rate of plasma progesterone decrease was not different between treatments. Electron micrographic study of the original and induced CL indicates that LH-like exposure delays involution of steroidogenic luteal cells. In summary, the higher levels of progesterone from d 6 to d 13 of the estrous cycle following an injection of hCG vs GnRH-agonist on d 5 is due to a greater response of hCG-induced CL.

Morphological characteristics of the bovine corpus luteum during the estrous cycle and pregnancy.

The corpus luteum, one of the biological clocks of the estrous cycle and pregnancy, is known foremost for its production of progesterone that blocks the pituitary release of gonadotropins and prepares the uterus for a pregnancy. The cellular sources of this progesterone are the steroidogenic small and large luteal cells. Other luteal cells that are not steroidogenic, but are believed to have an important role in the function of this gland are the fibroblast, macrophages and endothelial cells. The most prominent luteal cell is the large steroidogenic cell characterized by an abundance of smooth endoplasmic reticulum and densely packed spherical mitochondria that are indicative of its contribution to most of the circulating progesterone believed to be constitutively secreted and not under the control of LH. Other distinguishing features of the large luteal cell are the presence of rough endoplasmic reticulum, prominent Golgi, and secretory granules that are indicative of endocrine cells. This cell undergoes dynamic changes across the estrous cycle and pregnancy, believed to reflect a change in progesterone and protein secretion that will eventually influence a successful pregnancy or another ovulation if pregnancy fails. The morphological characteristics of the bovine luteal cells are the focus of this review.

Oxytocin induces prostaglandin F2 alpha release in pregnant cows: influence of gestational age and oxytocin receptor concentrations.

Brahman cows with known breeding dates received i.v. injections of either 10 or 100 IU oxytocin (OT) on Days 50, 150, 250, or 280 of gestation (n = 6 for each stage). Concentrations of the prostaglandin (PG) F2 alpha metabolite, 13,14-dihydro-15-keto-prostaglandin (PGFM), and OT were measured in samples of peripheral plasma collected at 15-min intervals for 1 h before and 1 h after treatment and then at 30-min intervals for 3 h. Plasma progesterone was measured daily for 14 days after OT injections on Days 50 and 250 of gestation. The increase in plasma OT after injection was dose-dependent (p = 0.001) but not affected by stage of gestation. Plasma PGFM increased after OT in a dose- and stage-dependent manner (p = 0.0001). At Day 280, the increase in plasma PGFM after 100 IU OT was sevenfold greater than at Day 50. Plasma progesterone declined significantly during the 7th to 12th days postinjection and returned to normal pregnancy values by the 14th day (4.4 +/- 0.3 ng/ml) except in two cows treated on Day 50 of gestation that later aborted. In these, plasma progesterone was significantly lower, 2.6 +/- 0.1 ng/ml. In a second experiment, the concentration of OT receptors was determined in endometrium collected from purebred Angus or Hereford cows slaughtered on Days 50, 150, 250, and 280 of gestation (n = 3 or 4 at each stage). Endometrial concentrations of OT receptor changed as a function of gestational age, increasing sixfold from Day 50 to Day 280, which was parallel to the increase by OT of plasma PGFM. Thus, endometrial OT receptors are functionally coupled to PGF2 alpha release during pregnancy, and their concentration determines the magnitude of OT-induced PGF2 alpha release during gestation. Consequently, endogenous OT is a factor in the regulation of PGF2 alpha release from the bovine uterus during pregnancy and parturition.