The roles of progestagen and uterine irritant in the maintenance of cystic endometrial hyperplasia in the canine uterus

Cystic endometrial hyperplasia (CEH) was induced in the left uterine horns of 14 mature ovariectomised greyhound bitches with an intra-luminal silk suture (uterine irritant) and treatment with estradiol benzoate and megestrol acetate (to simulate stages of a normal canine estrous cycle). Right uterine horns served as suture-free controls. From Day 30 of simulated diestrus, bitches received treatments of suture removal (n = 4), progestagen withdrawal (n = 5) or both (n = 5). Necropsies were performed 3 or 9 weeks later. At 3 weeks, severe cystic endometrial hyperplasia was present in all (6/6) left horns and in no (0/6) right horns. At 9 weeks, the left horns in 5/6 of bitches subjected to progestagen-withdrawal had recovered (in varying degrees) from cystic endometrial hyperplasia, whereas no recovery was evident in the left horns of bitches (n = 2) that continued to receive progestagen. This study demonstrated that: (i) cystic endometrial hyperplasia was reversible upon withdrawal of progestagen; (ii) progestagen maintained cystic endometrial hyperplasia in the presence or absence of irritant; and (iii) persistent endometrial irritation in the absence of progestagen may not maintain cystic endometrial hyperplasia.     

Serum estradiol-17 beta, progesterone and respective uterine cytosol receptor concentrations in bitches with spontaneous pyometra

The role of serum estradiol-17 beta (E(2)) and progesterone (P(4)) in relation to uterine estrogen (ER) and progesterone receptors (PR) was investigated in canine cystic endometrial hyperplasia-pyometra (CEH-P). Blood and uterine samples were collected pre- and post-ovariohysterectomy, respectively, from 54 bitches presenting spontaneous CEH-P and 25 healthy control bitches. Competitive enzyme immunoassays (EIA) and enzyme ligand immunoassays (ELIA) were applied to estimate serum hormones and uterine cytosol active receptors, respectively. Animals were classified in the stages of first half of diestrus, second half of diestrus and early anestrus on the basis of reproductive history, clinical signs, uterine and ovarian macro- and microscopic inspection and serum P(4) concentration. Bitches with CEH-P, compared to their respective stage controls, exhibited (a) similar P(4) fluctuations, (b) higher E(2) concentrations, (c) lower PR concentrations during diestrus first and second half and (d) lower ER concentrations during diestrus first half and early anestrus. Negative correlation was detected between P(4) and ER within both CEH-P and control groups. It was concluded that P(4) was the main uterine receptor regulator for both PR and ER during diestrus and early anestrus in healthy and affected uteri. However, in CEH-P bitches, high P(4) levels in diestrus appeared to over-activate uterine PRs, leading to stronger PR self-down regulation and ER suppression. These findings indicate an increased sensitivity of CEH-P uterus to P(4) action. During early anestrus, a complementary role of endogenous E(2) was considered, since reduction of P(4) action appeared to permit uterine ER replenishment and activation by relatively high E(2) levels.     

Apoptosis in canine corpus luteum during spontaneous and prostaglandin-induced luteal regression

Spontaneous luteal regression and prostaglandin-induced luteolysis in bitches were evaluated by measuring the apoptotic index for DNA fragmentation and the relative level of Bax gene expression in ovaries removed from nine untreated nonpregnant bitches at selected times during diestrus and in nine pregnant bitches after 1 day of administering abortive doses of a PGF-analog gel formulation given intravaginally at selected times during gestation. Nonpregnant diestrus was divided into three periods (early, mid and late) based on vaginal cytology and plasma progesterone concentration. Pregnant bitches were treated with a PGF-analog gel at corresponding stages of pregnancy (early, mid and late) and evaluated by ultrasound. Another eight pregnant bitches were similarly studied and serum progesterone concentrations were determined after 1, 2, 3 or 4 days of PGF-analog gel. Corpora lutea obtained by ovariohysterectomy were analyzed for apoptotic internucleosomal DNA fragmentation relative to that in a control cell line (U937), using an apoptotic DNA ladder kit and gel electrophoresis and for relative expression of the pro-apoptotic Bax gene by RT-PCR and electrophoresis. In nonpregnant bitches, the DNA fragmentation apoptotic index was greater in late than in early diestrus (P < 0.01). The index after 1 day of PGF-analog gel was higher in early pregnant bitches than in early diestrus bitches (P < 0.05); it was highest in midpregnancy (P < 0.05). The degree of apoptosis was related to the number of times PGF-analog gel was administered. Bax mRNA was detected in the corpus luteum (CL) and Bax expression increased from early to middiestrus in nonpregnant subjects (P < 0.05). Potential elevation in Bax due to PGF-analog gel treatment in pregnancy was only significant in relation to normal diestrus during early pregnancy (P < 0.01). In conclusion, we inferred that the effects of endogenous or exogenous prostaglandin on CL life span in bitches involved increases in apoptotic activity and that increased apoptosis was implicated in normal luteal regression in nonpregnant bitches.     

Epidermal growth factor, transforming growth factor-α, and epidermal growth factor receptor expression and localization in the canine endometrium during the estrous cycle and in bitches with pyometra

Gene expression and immunohistochemical localization of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and epidermal growth factor receptor (EGF-R) were compared between the endometrium of bitches (Canis familiaris) with pyometra accompanied by cystic endometrial hyperplasia (CEH) and that of healthy bitches at similar stages of the estrous cycle. In normal bitches, endometrial TGF-α mRNA levels were highest at proestrus and gradually decreased as the cycle progressed to anestrus. Epidermal growth factor receptor mRNA levels were not significantly affected by the stage of the estrous cycle. Epidermal growth factor mRNA levels were higher at Day 35 of diestrus than at other stages of the estrous cycle (P < 0.05). In bitches with pyometra, endometrial TGF-α and EGF-R mRNA levels did not differ significantly from those at diestrus in normal bitches, but EGF mRNA levels were lower than those at Day 35 of diestrus in normal bitches (P < 0.05). In normal bitches, positive immunohistochemical staining for TGF-α, EGF, and EGF-R was mainly present in the glandular and luminal epithelial cells of the endometrium. In contrast, in bitches with pyometra, immunoreactivity for EGF was clearly present in endometrial stromal cells. Inflammatory cells that had infiltrated the endometrial stroma stained strongly for TGF-α and EGF-R. Luminal and glandular epithelial cells also stained positive for EGF-R. In conclusion, expression of TGF-α by inflammatory cells and a low level of expression and differential localization of EGF may be involved in aberrant growth of endometrial glands and development of CEH.     

Plasma concentrations of 13,14-dihydro-15-keto prostaglandin F2-alpha (PGFM), progesterone and estradiol in pregnant and nonpregnant diestrus cross-bred bitches

The canine corpus luteum (CL) typically sustains elevated plasma progesterone concentrations for 2 months or more, with a peak approximately 15-25 days after ovulation, followed by a slow decline. The processes involved in the slow, protracted regression of the CL over the remaining 1.5-2-month period in nonpregnant bitches and until shortly prepartum in pregnant bitches are not well characterized. The rapid luteolysis that occurs immediately prepartum appears to be a result of a prepartum rise in peripheral PGF. The potential role of PGF in the slow regression process in the several weeks preceding parturition and in nonpregnant bitches after 15-25 days after ovulation is not known. Therefore, plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F2-alpha (PGFM), progesterone (P4) and estradiol (E2) were determined and compared in bitches during nonpregnant diestrus (n = 9) or pregnancy (n = 8). During the gradual decrease in plasma concentrations of progesterone in both groups, the P4 pattern appeared unrelated to changes in either E2 or PGFM concentrations. The PGFM pattern was different between diestrus and pregnant bitches (P > 0.01); there was an apparent progressive but slow increase in PGFM in pregnant bitches from Days 30 to 60, followed by a large increase prior to parturition; concentrations declined immediately postpartum. However, there were no increases in PGFM during the same interval in nonpregnant bitches. Mean estradiol concentrations were sporadically elevated during the last third of pregnancy and less so in nonpregnant diestrus; there was no acute prepartum increase in estradiol associated with the PGFM increase. In summary, although there were no apparent changes in peripheral PGF2alpha concentration involved in regulating the slow protracted phase of luteal regression in nonpregnant bitches, modest increases in PGFM may play a role in ovarian function after mid-gestation in pregnant bitches. Furthermore, the acute prepartum rise in PGFM was not dependent on any concomitant increase in estradiol concentrations.     

Luteolysis in early diestrous beagle bitches

To induce luteolysis early in diestrus, 15 mated beagles were treated with 250mug/kg prostaglandin F(2)alpha (PG), administered subcutaneously twice daily for 4 d, from Day 5 to Day 8 after the onset of cytological diestrus. They were divided into three groups of five bitches each: Group 1 received PG only. Group 2 received PG and 20 mug/kg/day bromocryptine orally. Group 3 received PG and 0.1mg/kg/day dexamethasone intramuscularly. Fifteen untreated beagles served as controls. In each of the three treatment groups, four of the five bitches (80%) underwent complete luteolysis and did not whelp. The three bitches which did not undergo luteolysis whelped normally. All control bitches whelped normally. It was concluded that luteolysis is possible in bitches early in diestrus and the inclusion of bromocryptine or dexamethasone did not appear to influence luteolysis.     

Degeneration and apoptosis of endometrial cells in the bitch

The relationships between changes in plasma progesterone concentrations, degeneration of the luminal epithelium, the occurrence of apoptosis of endometrial cells and endometrial leucocyte populations in the bitch were determined. Mature bitches (n = 15) were euthanized and necropsied when in diestrus (Days 7-75, n = 12) or in anestrus (Days 10, 32 and 53). Degeneration of the luminal epithelium was observed in bitches in late diestrus (Days 38-75, n = 5) when plasma progesterone concentrations were decreasing and in anestrus (Days 10 and 32, n = 2) when plasma progesterone concentrations were < 0.5 ng/mL. Endometrial leucocyte populations increased after degeneration of the luminal epithelium (around Day 42 of diestrus). Apoptosis was mainly observed in the basal glandular epithelial cells and endothelial cells of blood capillaries in all except anestrous bitches. Very few apoptotic cells were found in the superficial glandular epithelial cells and stromal cells. Higher apoptotic indices were detected in the basal glandular epithelium on Days 12-42 of diestrus than at other stages. Therefore, apoptosis of glandular basal epithelial cells occurred mainly in early diestrus, degeneration of cells of the luminal epithelium occurred from mid-diestrus to early anestrus, and the increase in leucocyte numbers may have been a consequence and not a cause of luminal epithelial degeneration.     

In vitro PGF2alpha production by endometrium and corpus luteum explants from pregnant and nonpregnant diestrus bitches and placental explants from pregnant bitches

To better understand the process of slow luteal regression of the nonpregnant cycle in dogs and the acute luteolysis that occurs prepartum, the present study investigated in vitro PGF2alpha production by the endometrium, corpus luteum and placental explants obtained at known times of the cycle from pregnant bitches (days 63, 64 and immediately postpartum; day 0 = estimated day of the ovulatory LH surge) and from nonpregnant diestrus bitches (approximately days 65, 75 and 85). Both basal PGF2alpha production and its production in the presence of the protein kinase C (PKC) stimulator 12,13-phorbol dibutyrate (PDBu) were determined. For PDBu-supplemented incubations, mean PGF2alpha production (pg/mL/mg/6 h) by endometrium explants of the nonpregnant bitches in late diestrus was highest on day 65 (205 +/- 87) and reduced to low levels (38 +/- 17 and 11 +/- 11) on days 75 and 85, respectively. The production by corpus luteum explants from these bitches was significantly less on day 65 (46 +/- 14) than that of the day 65 endometrium explants, and was slightly increased on day 85 (103 +/- 52). The corresponding mean PGF2alpha production by the endometrium explants of pregnant bitches was on average much greater (i.e., two to three-fold) compared to nonpregnant bitches (P < 0.01) and involved high concentrations at day 64 (1523 +/- 467) and postpartum, compared to somewhat lower levels on day 63 (830+/-65); luteal PGF production (165 +/- 4) was also higher than in nonpregnant bitches around day 65. For pregnant bitches, PGF production per gram of tissue in the endometrium explants was greater than for the CL or placenta explants (180 +/- 37). Therefore, the endometrium of the pregnant bitch has an increased capability to produce PGF2alpha immediately prepartum, which on a tissue weight basis, exceeds that of either corpora lutea or the placenta. However, assuming a larger mass of placental tissue in vivo, we inferred that the placenta may contribute substantially to peripheral PGF concentrations.     

Luteal function in the bitch: changes during diestrus in pituitary concentration of and the number of luteal receptors for luteinizing hormone and prolactin

The concentration of unoccupied luteal receptors for luteinizing hormone (LH) and prolactin, and the concentration of these two hormones in the pituitary was determined in 11 groups of bitches (n = 3 or 4/group) representing stages from proestrus through Day 80 of diestrus. Despite dramatic changes in serum concentrations of progesterone, the concentration of luteal receptors for LH and prolactin was quite constant throughout the entire luteal phase. In association with the ovulatory surge of LH, pituitary concentration of LH decreased abruptly from proestrus to Day 2 of diestrus, and was then gradually replenished during the remainder of diestrus. The concentration of prolactin in the pituitary did not vary significantly from proestrus through late diestrus.     

Changes in ovine conceptus and endometrial function following asynchronous embryo transfer or administration of progesterone

The secretory protein profile from conceptuses collected from naturally mated ewes on Days 10, 12, 14, and 16 was characterized by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. The presence of the anti-luteolysin ovine trophoblast protein-1 (oTP-1) in culture medium from Day 10 conceptuses was confirmed by fluorography, Western blotting, and radioimmunoassay (RIA). On each of the days studied, oTP-1 was the dominant secretory protein, and was secreted in increasing quantities as pregnancy progressed. In a second experiment, Day 6 embryos were transferred to either Day 6 (SR) or Day 4 (AR) recipients. Three mated ewes (P) received daily injections of 50 mg progesterone on Days 4-9. Controls consisted of 2 groups of pregnant ewes (D8 and D10). Conceptuses and ipsilateral endometrium were collected 4 days following transfer of conceptuses to SR and AR ewes, on Day 10 in P and D10 ewes, and on Day 8 in D8 ewes. Conceptus volume was estimated upon recovery from the uterus. Tissues were cultured with 35S-methionine, and the medium was analyzed for total and trichloroacetic acid-precipitable radiolabeled proteins. Levels of specific endometrial secretory proteins were determined after protein separation by 2D-PAGE and estimation of the radioactivity associated with discrete radiolabeled proteins on fluorographs. The concentration of oTP-1 in conceptus culture medium was estimated by RIA. Thirty endometrial proteins were investigated. All 30 proteins were present in endometrial cultures from SR, AR, D10, and P ewes, but 13 proteins were absent from D8 ewes. Levels of three proteins were higher in AR compared to D8 (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

A model for cystic endometrial hyperplasia/pyometra complex in the bitch

The objective of this study was to develop a reliable model for the study of the cystic endometrial hyperplasia and pyometra complex (CEH/P) in the bitch. Greyhound bitches (n = 15) were ovariectomised and allocated into three groups (Group 1, n = 5; Group 2, n = 5; Group 3, n = 10, including 5 used from Group 1). Simulated proestrus, estrus and diestrus were induced by treatment with estradiol benzoate and megestrol acetate. The duration of cervical opening during estrus was determined by the intra-vaginal infusion of radio-opaque medium and subsequent radiography of the uterus (Group 1). One milliliter of a culture of Escherichia coli (with five uro-pathogenic virulence factors as identified by PCR: pap, sfa, hlyA, cnf1 and fim) was inoculated intra-vaginally daily throughout the simulated estrus (Group 2). One milliliter of the culture (n = 6) or sterile Luria-Bertani broth (n = 4) was introduced directly into the uterus on simulated diestrus Days 8 or 12 (Group 3). Necropsies were performed 12 and 7-14 days after the inoculation (Groups 2 and 3). The cervix remained open throughout the duration of simulated estrus (5-6 days) in four out of five bitches, and for a shorter duration (3 days of a 6-day estrus period) in one bitch (Group 1). CEH/P was induced by inoculation of bacteria into the uterus (10/10 bitches) but not into the vagina (0/5 bitches), (P = 0.003). A model for the study of CEH/P has been validated.     

Canine corpus luteum regression: apoptosis and caspase-3 activity

The present study evaluated the occurrence of apoptosis and caspase-3 activity in the canine corpus luteum during the period of luteal regression in eight pregnant and nine nonpregnant diestrus bitches. Intact luteal cells were obtained from corpora lutea in both peripartum pregnant bitches and nonpregnant diestrus bitches at approximately 65 d (range 63-68) after estrus, but not at days 75 and 85 in nonpregnant bitches. In all bitches, apoptotic cells were rarely detected and when present, those cells were more easily detected using the hematoxylin and eosin technique than using the critical electrolyte concentration technique. The luteal structures at 75 and 85 d of diestrus had histological characteristics similar to a corpus albicans. Caspase-3 activity was detected in morphologically normal corpora lutea from both pregnant and diestrus bitches around day 65, and also in the later structures considered corpus albicans tissue. These results suggested that apoptosis may not be the major mechanism involved in canine functional luteal regression, and that caspase-3 participated in both functional and morphological luteolysis and in the tissue reorganization involved in corpus albicans formation.     

Expression of 3beta-hydroxysteroid dehydrogenase, cytochrome p450 17alpha-hydroxylase/17,20-lyase and cytochrome p450 aromatase enzymes in corpora lutea of diestrous and early pregnant mares

In the pregnant mare, luteal estrogen production increases at the onset of equine chorionic gonadotropin (eCG) secretion by endometrial cups. In previous studies, we have demonstrated that eCG stimulates luteal androgen and estrogen production in pregnant mares. To further elucidate the regulation of steroidogenesis within the equine corpus luteum (CL) of pregnancy, we examined the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/17,20 lyase (P450(17alpha)) and cytochrome P450 aromatase (P450(arom)) in luteal tissue samples collected during diestrus (Days 7 to 10) and pregnancy before (Days 29 to 35) and after (Days 42 to 45) the onset of eCG secretion. Immunoblot analyses revealed a single protein per enzyme with molecular weights of 48 kDa (3beta-HSD), 58 kDa (P450(17alpha)) and 56 kDa (P450(arom)). Steady-state levels of 3beta-HSD were lower in luteal tissue of diestrus than pregnancy, but expression did not change during pregnancy. Steady-state expression of P450(17alpha) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, P450(17alpha) expression was significantly higher after the onset of eCG secretion. Steady-state expression of P450(arom) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, luteal expression of P450(arom) was significantly lower after the onset of eCG secretion. These data support the hypotheses that eCG has a differential effect on the expression of luteal steroidogenic enzymes, that the eCG-induced increase in luteal estrogen production is the result of an increase in available aromatizable androgen due to an increase in P450(17alpha) expression and activity, and that increased luteal estrogen production is not due to an increase in aromatase expression.     

Physical characteristics of the uterus during the bovine estrous cycle and early pregnancy

Uterine tone, uterine contractility and endometrial echotexture were monitored daily in heifers during the estrous cycle (n = 6; Days 0 to 21; ovulation = Day 0) and during early pregnancy (n = 7; Days 0 to 26). Uterine tone was assessed by transrectal palpation and scored from 1 (flaccid) to 5 (turgid) by an operator who had no knowledge of reproductive status, day, or group. The main effect of day was significant, but the group effect and the group-by-day interaction were not. Uterine tone scores were high during the periovulatory period (Days--1, 0, 1), decreased (P < 0.05) to low levels on Days 3 and 4, and then increased (P < 0.05) from Days 4 to 10. The increase in tone during early diestrus was confirmed (P < 0.05) in a second experiment. Uterine contractility was assessed by transrectal ultrasonography during a five-minute scan of the caudal portions of the uterine horns and scored from 1 (minimal contractility) to 4 (maximal contractility). The main effects of day and the group-by-day interaction were significant. Contractility scores in both groups were highest just before or on the day of ovulation (Days--1,0) and then decreased (P < 0.05) until Day 11. After Day 16, the scores increased (P < 0.05) in the nonbred heifers and remained low in the pregnant heifers. Endometrial echotexture scores were different among days (P < 0.0001), between the 2 groups (P < 0.02), and for the group-by-day interaction (P < 0.0001). Echotexture scores in both groups peaked just before ovulation (Day--1) and then decreased (P < 0.05) until Day 4. After Day 16, the scores increased in the nonbred group but remained low in pregnant heifers. In summary, uterine contractility and endometrial scores had similar profiles, being high during the periovulatory period and low thereafter; the levels rose in association with the end of the interovulatory interval in nonbred heifers, but remained at low levels in pregnant heifers. Uterine tone scores were also high during the periovulatory period and decreased to low levels several days postovulation, but then, in contrast with the other end points, began to increase in both the nonbred and pregnant heifers.     

Ovine osteopontin: II. Osteopontin and alpha(v)beta(3) integrin expression in the uterus and conceptus during the periimplantation period.

Osteopontin (OPN) is an acidic 70-kDa glycoprotein that is cleaved by proteases to yield 45-kDa and 24-kDa fragments. The 70-kDa and 45-kDa proteins contain a Gly-Arg-Gly-Asp-Ser (GRGDS) sequence that binds to cell surface integrins (primarily alpha(v)beta(3) heterodimer) to promote cell-cell attachment and cell spreading. A 70-kDa acidic protein was previously detected by two-dimensional (2D) PAGE in Day 17 pregnant endometrial cytosolic extracts using Stainsall and identified as immunoreactive OPN using Western blotting. Three forms of immunoreactive OPN proteins (70, 45, and 24 kDa) were detected by 1D PAGE and Western blot analysis of endometrial extracts. OPN protein in endometrial extracts did not differ between cyclic and pregnant ewes. However, the amount of 45-kDa OPN increased in uterine flushings from pregnant ewes between Days 11 and 17. Immunoreactive OPN was localized to luminal and glandular epithelia of both cyclic and pregnant ewes, and to trophectoderm of Day 19 conceptuses. The alpha(v) and beta(3) integrins were detected on Day 19 endometrium and conceptuses by immunofluorescence. It was reported that OPN mRNA increases in the uterine glands of pregnant ewes and secretion of OPN protein into the uterine lumen increases during early pregnancy. The present results demonstrate accumulation of OPN protein on endometrial LE and conceptus trophectoderm. Therefore, it is hypothesized that progesterone and/or interferon-tau induce expression, secretion and/or proteolytic cleavage of OPN by uterine epithelium. Secreted OPN is then available as ligand for alpha(v)beta(3) integrin heterodimer on trophectoderm and uterus to 1) stimulate changes in morphology of conceptus trophectoderm and 2) induce adhesion between luminal epithelium and trophectoderm essential for implantation and placentation.     

Mechanisms involved in calcitonin gene-related Peptide-induced relaxation in pregnant rat uterine artery

Human and rodent studies have demonstrated that calcitonin gene-related peptide (CGRP), a potent vasodilator, relaxes uterine tissue during pregnancy but not during labor. The vascular sensitivity to CGRP is enhanced during pregnancy, compared to nonpregnant human uterine arteries. In the present study, we hypothesized that uterine artery relaxation effects of CGRP are enhanced in pregnant rats compared to nonpregnant diestrus rats (NP-DE) and that several secondary messenger systems are involved in this process. We also hypothesized that the expression of CGRP-A receptor components, calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein (RAMP1), and CGRP-B receptors are greater in pregnant rats. For vascular relaxation studies, uterine arteries from either NP-DE or Day 18 pregnant rats were isolated, and responsiveness of the vessels to CGRP was examined with a small vessel myograph. CGRP-A and CGRP-B receptor expressions were assessed by RT-PCR and Western immunoblotting, respectively. CGRP (10(-10)--10(-7) M) produced a concentration-dependent relaxation of norepinephrine-induced contractions in both NP-DE and Day 18 pregnant rat uterine arteries. Pregnancy increased the vasodilator sensitivity to CGRP significantly (P < 0.05) compared to NP-DE rats. CGRP receptor antagonist, CGRP8-37, inhibited CGRP-induced relaxation of pregnant uterine arteries. The CGRP-induced relaxation was not affected by NG-nitro-l-arginine methyl ester (L-NAME) (nitric oxide inhibitor, 10(-4) M) but was significantly (P < 0.05) attenuated by inhibitors of guanylate cyclase (ODQ, 10(-5) M) and adenylate cyclase (SQ 22536, 10(-5) M). CGRP-induced vasorelaxation was significantly (P < 0.05) attenuated by potassium channel blockers KATP (glybenclamide, 10(-5) M) and K(CA) (tetraethylammonium, 10(-3) M). The expression of CRLR and RAMP1 was significantly (P < 0.05) elevated during pregnancy compared to nonpregnant diestrus state (NP-DE). However, CGRP-B receptor proteins in uterine arteries were not altered with pregnancy compared to those of NP-DE. These studies suggest that CGRP-induced increases in uterine artery relaxation may play a role in regulating blood flow to the uterus during pregnancy and, therefore, in fetal growth and survival.     

Regulation of synthesis of uterine secretory proteins: evidence for differential induction of porcine uteroferrin and antileukoproteinase gene expression

Mechanisms regulating the expression of two pregnancy-associated proteins of the porcine uterus, namely the iron-transport protein uteroferrin (UF) and the lysosomal serine proteinase-inhibitor antileukoproteinase (ALP), were investigated by comparing the effects of estrogen (E), progesterone (P4), and conceptuses on the steady-state levels of their mRNAs. For UF, the expression of mRNA with production and secretion of the corresponding protein was also investigated. Progesterone increased the levels of endometrial UF mRNA and of secreted UF, but did not affect the levels of ALP mRNA in ovariectomized gilts that had received P4 treatment for 8 days. Estrogen inhibited the accumulation of endometrial UF mRNA, increased UF secretion in these gilts, but had no effect on levels of ALP mRNA. Administration of E to gilts on Day 11 of the cycle slightly diminished UF mRNA levels at 1 h post-E; had no effect at 6, 12, and 24 h post-E; and increased levels of secreted UF in uterine luminal fluids 24 h post-E. The presence of conceptuses increased levels of endometrial ALP mRNA and decreased UF protein in uterine luminal fluids, but did not affect levels of endometrial UF mRNA. Myometrium, endometrium, and placenta from Day 75 and Day 105 pregnant gilts were also evaluated for ALP and UF mRNA expression to determine regional expression of these steroid-regulated genes. Myometrium and endometrium expressed comparable levels of UF and ALP mRNAs within Days 75 or 105, but placenta did not express detectable levels of mRNA for either protein. Within the myometrium, UF protein is immunolocalized mostly to the inner circular and to a lesser extent to the outer longitudinal layer of smooth muscle. These results indicate that E, P4, and presence of conceptuses differentially affect endometrial expression of ALP and UF mRNAs and secretion of UF.