Fluctuation of Aquaporin 1, 5, and 9 Expression in the Pig Oviduct during the Estrous Cycle and Early Pregnancy

Thirteen mammalian aquaporin (AQPs) isoforms with a unique tissue-specific pattern of expression have been identified. To date, 11 isoforms of AQP have been reported to be expressed in female and male reproductive systems. The purpose of our study was to determine the localization and quantitative changes in the expression of AQP1, 5 and 9 within the pig oviduct during different stages of the estrous cycle and early pregnancy. The results demonstrated that AQP1, 5, and 9 were clearly detected in all studied stages of the estrous cycle and pregnancy. AQP1 was localized within oviductal blood vessels. In cyclic gilts, the expression of AQP1 protein did not change significantly between days 10–12 and 14–16 but increased on days 2–4 and 18–20. AQP5 was localized in smooth muscle cells and oviductal epithelial cells. The expression of AQP5 protein did not change significantly between days 10–12 and 14–16 of the estrous cycle but increased on days 2–4 and 18–20. The anti-AQP9 antibody labeled epithelial cells of the oviduct. The expression of AQP9 did not change significantly between days 10–12 and 14–16 of the estrous cycle but increased on days 2–4 and 18–20. In pregnant gilts, expression of AQP1, 5, and 9 did not change significantly in comparison with the estrous cycle. Therefore, a functional and distinctive collaboration seems to exist among diverse AQPs in water handling during the different oviductal phases in the estrous cycle and early pregnancy.     

Estradiol induces and progesterone inhibits the preovulatory surges of luteinizing hormone and follicle-stimulating hormone in heifers

Objectives were to determine: 1) whether estradiol, given via implants in amounts to stimulate a proestrus increase, induces preovulatory-like luteinizing hormone (LH) and follicle-stimulating hormone (FSH) surges; and 2) whether progesterone, given via infusion in amounts to simulate concentrations found in blood during the luteal phase of the estrous cycle, inhibits gonadotropin surges. All heifers were in the luteal phase of an estrous cycle when ovariectomized. Replacement therapy with estradiol and progesterone was started immediately after ovariectomy to mimic luteal phase concentrations of these steroids. Average estradiol (pg/ml) and progesterone (ng/ml) resulting from this replacement were 2.5 and 6.2 respectively; these values were similar (P greater than 0.05) to those on the day before ovariectomy (2.3 and 7.2, respectively). Nevertheless, basal concentrations of LH and FSH increased from 0.7 and 43 ng/ml before ovariectomy to 2.6 and 96 ng/ml, respectively, 24 h after ovariectomy. This may indicate that other ovarian factors are required to maintain low baselines of LH and FSH. Beginning 24 h after ovariectomy, replacement of steroids were adjusted as follows: 1) progesterone infusion was terminated and 2 additional estradiol implants were given every 12 h for 36 h (n = 5); 2) progesterone infusion was maintained and 2 additional estradiol implants were given every 12 h for 36 h (n = 3); or 3) progesterone infusion was terminated and 2 additional empty implants were given every 12 h for 36 h (n = 6). When estradiol implants were given every 12 h for 36 h, estradiol levels increased in plasma to 5 to 7 pg/ml, which resembles the increase in estradiol that occurs at proestrus. After ending progesterone infusion, levels of progesterone in plasma decreased to less than 1 ng/ml by 8 h. Preovulatory-like LH and FSH surges were induced only when progesterone infusion was stopped and additional estradiol implants were given. These surges were synchronous, occurring 61.8 +/- 0.4 h (mean +/- SE) after ending infusion of progesterone. We conclude that estradiol, at concentrations which simulate those found during proestrus, induces preovulatory-like LH and FSH surges in heifers and that progesterone, at concentrations found during the luteal phase of the estrous cycle, inhibits estradiol-induced gonadotropin surges. Furthermore, ovarian factors other than estradiol and progesterone may be required to maintain basal concentrations of LH and FSH in heifers.     

Natural fluctuation and gonadal hormone regulation of astrocyte immunoreactivity in dentate gyrus

The number and the surface density of cells immunoreactive for the specific astrocytic marker glial fibrillary acidic protein (GFAP), were evaluated in both the hilus of the dentate gyrus and the granular layer of the vermis of the cerebellar cortex of adult female rats during the different phases of the estrous cycle, after ovariectomy and after the pharmacological administration of estradiol and/or progesterone to overiectomized rats. Although no significant differences were detected in the number of immunoreactive cells among the different experimental groups studied, their surface density showed significant changes in the hilus of the dentate gyrus. The surface density of immunoreactive cells was increased in the afternoon of proestrus and on the morning of estrus compared to the morning of proestrus, diestrus, and metestrus, was decreased after ovariectomy, and showed a dose-dependent increase in ovariectomized rats injected with 17β estradiol (1, 10, or 300 μg/rat), alone or in combination with progesterone (500 μg/rat). In contrast, it was not affected by the administration of 17β estradiol (300 μg/rat). The surface density of immunoreactive cells was significantly increased over control values by 5 h after the injection of 17β estradiol (300 μg/rat) and as early as 1 h after the administration of progesterone. The separate injection of either 17β estradiol or progesterone had smaller effects on the surface density of immunoreactive cells than did the administration of both hormones together. The surface density of GFAP-immunoreactive cells reached maximal values by 24 h after the administration of 17β estradiol and/or progesterone and returned to control levels by 48 h after the combined injection of progesterone and 17β estradiol, while in the rats that were injected with only one of the two hormones, the surface density of immunoreactive cells remained over control values for at least 9 days. No such hormonal effects on GFAP-immunoreactive cells were observed in the cerebellar cortex. © 1993 John Wiley & Sons, Inc.     

Influence of Ovarian Hormones on Cortical Spreading Depression and Its Suppression by L-kynurenine in Rat

Migraine is sexually dimorphic and associated in 20–30% of patients with an aura most likely caused by cortical spreading depression (CSD). We have previously shown that systemic L-kynurenine (L-KYN), the precursor of kynurenic acid, suppresses CSD and that this effect depends on the stage of the estrous cycle in female rats. The objectives here are to determine the influence of ovarian hormones on KCl-induced CSD and its suppression after L-KYN by directly modulating estradiol or progesterone levels in ovariectomized rats. Adult female rats were ovariectomized and subcutaneously implanted with silastic capsules filled with progesterone or 17β-estradiol mixed with cholesterol, with cholesterol only or left empty. Two weeks after the ovariectomy/capsule implantation, the animals received an i.p. injection of L-KYN (300 mg/kg) or NaCl as control. Thirty minutes later CSDs were elicited by applying KCl over the occipital cortex and recorded by DC electrocorticogram for 1 hour. The results show that both estradiol and progesterone increase CSD frequency after ovariectomy. The suppressive effect of L-KYN on CSD frequency, previously reported in normal cycling females, is not found anymore after ovariectomy, but reappears after progesterone replacement therapy. Taken together, these results emphasize the complex role of sex hormones on cortical excitability. The CSD increase by estradiol and, more surprisingly, progesterone may explain why clinically migraine with aura appears or worsens during pregnancy or with combined hormonal treatments.     

Steroid feedback inhibition of pulsatile secretion of gonadotropin-releasing hormone in the ewe

The long-term negative feedback effects of sustained elevations in circulating estradiol and progesterone on the pulsatile secretion of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) were evaluated in the ewe following ovariectomy during the mid-late anestrous and early breeding seasons. GnRH secretion was monitored in serial samples of hypophyseal portal blood. Steroids were administered from the time of ovariectomy by s.c. Silastic implants, which maintained plasma concentrations of estradiol and progesterone at levels resembling those that circulate during the mid-luteal phase of the estrous cycle; control ewes did not receive steroidal replacement. Analysis of hormonal pulse patterns in serial samples during 6-h periods on Days 8-10 after ovariectomy disclosed discrete, concurrent pulses of GnRH in hypothalamo-hypophyseal portal blood and LH in peripheral blood of untreated ovariectomized ewes. These pulses occurred every 97 min on the average. Treatment with either estradiol or progesterone greatly diminished or abolished detectable pulsatile secretion of GnRH and LH, infrequent pulses being evident in only 3 of 19 steroid-treated ewes. No major seasonal difference was observed in GnRH or LH pulse patterns in any group of ewes. Our findings in the ovariectomized ewe provide direct support for the conclusion that the negative-feedback effects of estradiol and progesterone on gonadotropin secretion in the ewe include an action on the brain and a consequent inhibition of pulsatile GnRH secretion.     

Effect of ACTH (tetracosactide) on steroid hormone levels in the mare. Part B: effect in ovariectomized mares (including estrous behavior)

The mare is the only non-primate species known to display estrous signs after ovariectomy and adrenal hormones have been implicated as a possible cause. Moreover, in several species, estradiol seems to have a stimulatory effect on the hypothalamic-pituitary-adrenal axis. The aim of the present study was to compare the effect of ACTH (tetracosactide) on pertinent hormones [cortisol, progesterone, androstenedione, testosterone (intact and ovariectomized mares) and estradiol (ovariectomized mares only)] in intact mares in estrus with the same mares after ovariectomy (n=5). Blood samples were collected hourly from 12:00 until 14:00 h the following day (half-hourly between 14:00 and 17:00 h) on two occasions, with saline or ACTH treatment at 14:00 h (saline treatment day or ACTH treatment day). The mares, both when intact and after ovariectomy, showed a significant increase in all measured hormones, except estradiol (not measured in intact mares), after ACTH treatment, lasting at least 3h post-treatment (P<0.001). On the saline treatment day, cortisol levels in ovariectomized mares were lower than in intact mares in the evening (18:00-23:00 h), but higher at night (24:00-05:00 h). No differences in cortisol response between mares, when intact and after ovariectomy, were found after ACTH treatment (P=0.3). Androstenedione levels were lower (P<0.001) and increased less after ACTH treatment in ovariectomized mares, as compared to when intact (P<0.05). Progesterone concentrations were lower in the ovariectomized mares at night (24:00-05:00 h) on the saline treatment day and at all times on the ACTH treatment day (P<0.05). Testosterone concentrations were lower in ovariectomized mares on both treatment days, as compared to when intact (P<0.001). It was concluded that ovariectomy affected basal cortisol pattern. Ovarian androstenedione and testosterone contributed to the basal circulating levels and, in the case of androstenedione, was stimulated by ACTH. Endogenous estradiol did not act stimulatory on adrenal gland hormone production in the mare.     

Sufficient progesterone-priming prior to estradiol stimulation is required for optimal induction of the cervical prostaglandin system in pregnant sheep at 0.7 gestations

The purposes of this study were to determine the separate and interactive functions of progesterone and estradiol in regulating the cervical prostaglandin (PG) system in pregnant sheep at 0.7 gestations. At 106-108 days of gestational age (dGA), ewes were treated with vehicle for 14 days (n = 5) or vehicle for 12 days followed by estradiol 5 mg twice a day, intramuscularly for 2 days (n = 5) or progesterone 100 mg, twice a day, intramuscularly for 14 days (n = 5) or progesterone 100 mg twice a day, intramuscularly for 10 days and then 2 days vehicle followed by estradiol 5 mg twice a day intramuscularly for 2 days (n = 5). At 121-123 dGA, cervical tissues were obtained under halothane anesthesia. Cervical RNA and protein were extracted and analyzed for prostaglandin-endoperoxide synthase 2 (COX2), two PGE(2) receptors, PTGER2 and PTGER4, and estrogen receptor alpha (ESR1) by Northern and Western blot analysis. Immunocytochemistry and in situ hybridization were applied to localize cellular distribution of COX2, PTGER2, and PTGER4 in the cervix. Data were analyzed by ANOVA. COX2 and PTGER4 mRNAs and proteins were increased (P < 0.05) in ewes treated with combined estradiol and progesterone but not in ewes treated with estradiol or progesterone alone compared with controls. ESR1 mRNA was increased in ewes treated with progesterone and estradiol plus progesterone. In contrast, PTGER2 mRNA and protein remained the same after all treatments. COX2 mRNA and protein were localized only in cervical glandular epithelial cells, whereas PTGER2 and PTGER4 were localized in both cervical glandular epithelial and smooth muscle cells. In conclusion, these data suggest that additional progesterone priming at 0.7 gestations synergizes with estradiol to induce cervical COX2, PTGER4, and ESR1 and support our hypothesis that stimulation of the cervical PG system by estradiol is optimized by sufficient progesterone priming in the pregnant sheep cervix.     

AQP1 gene expression is upregulated by arginine vasopressin and cyclic AMP agonists in trophoblast cells

Aquaporins (AQPs) are water channels that regulate water flow in many tissues. As AQP1 is a candidate to regulate placental fluid exchange, we sought to investigate the effect of arginine vasopressin (AVP) and cAMP agonists on AQP1 gene expression in first trimester-derived extravillous cytotrophoblasts (HTR-8/Svneo) and two highly proliferative carcinoma trophoblast-like cell lines but with a number of functional features of the syncytiotrophoblast namely; JAR and JEG-3 cells. Our data demonstrated that AVP (0.1 nM) significantly increased the expression of AQP1 mRNA at 10 h in HTR-8/SVneo and JEG-3 cells (P<0.05). Both SP-cAMP, a membrane-permeable and phosphodiesterase resistant cAMP, and forskolin, an adenylate cyclase stimulator significantly increased AQP1 mRNA expression in all cell lines after 2 h in a dose-dependent manner (P<0.05) with a parallel increase in protein expression. In the time course study, 5 microM of either SP-cAMP or forskolin significantly stimulated AQP1 mRNA expression after 2 h in HTR-8/SVneo cells and after 10 h in JAR and JEG-3 cells. AQP1 protein expression was highest after 20 h in both HTR-8/SVneo and JEG-3 cells (P<0.05). AVP-stimulated cAMP elevation was blocked in the presence of 9-(tetrahydro-2'-furyl) adenine (SQ22536) (100 microM), a cell-permeable adenylate cyclase inhibitor (P<0.05). These results indicate that in trophoblasts-like cells AQP1 gene expression is upregulated by both AVP and cAMP agonists. Furthermore, our data demonstrate that a cAMP-dependent pathway is responsible for the AVP effect on AQP1. Thus, modulation of AQP1 expression by maternal hormones may regulate invasion and fetal-placental-amnion water homeostasis during gestation.     

Progesterone reduces the effect of the serotonin 1B/1D receptor antagonist, GR 127935, on lordosis behavior

Ovariectomized rats were hormonally primed with 10 μg estradiol benzoate or with estradiol benzoate plus 500 μg progesterone. Rats received a bilateral infusion with 200 ng of the 5-HT_1B/1D receptor antagonist, N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)-1-1′-biphenyl-4-carboxamide hydrochloride (GR 127935), into the ventromedial nucleus of the hypothalamus (VMN), followed by a 5 min restraint or home cage experience. In estrogen-primed females that had experienced minimal handling between ovariectomy and use in the experiment, infusion with the water vehicle transiently inhibited lordosis behavior, and the 5-HT_1B/1D receptor antagonist amplified this inhibition. There were no effects in rats hormonally primed with estrogen and progesterone. Handling for two days before the experiment reduced the effects of the infusions in estrogen-primed rats. However, when a 5 min restraint experience followed infusion with GR 127935, there was a significant decline in lordosis behavior that persisted for 10 to 15 min after the experience. Regardless of the prior experience or type of infusion, the addition of progesterone to the hormonal priming completely prevented the lordosis inhibition. These findings are consistent with prior evidence that progesterone protects against the inhibitory effects of a 5 min restraint experience on lordosis behavior. Moreover, these are the first experiments to demonstrate an inhibitory effect of a selective 5-HT_1B/1D receptor antagonist in the VMN on lordosis behavior of estrogen primed, but not estrogen and progesterone primed, ovariectomized rats.     

Hormonal regulation of sialic acid-binding (SAS) protein synthesis of rat uterus

Sialic acid binding proteins (SAS) of rat uteri have been found in all three stages of the estrous cycle. To study the control of synthesis of these proteins two different animal models were used I-immature female rats (25 d) where the hormones estradiol (E₂) and progesterone (P₄) were given separately and together, and II-adult female rats where hormone treatment commenced 14 days after ovariectomy. The data indicated that E₂ initiated the synthesis of SAS proteins in the immature animals, while P₄ could inhibit SAS synthesis, either given alone or together with E₂. However, prior priming of the rat with E₂ and subsequent administration of P₄ stimulated SAS protein synthesis.     

Aquaporin 4 regulates the effects of ovarian hormones on monoamine neurotransmission

Aquaporin 4 (AQP4) is the predominant water channels in the brain of mammals. Our previous study has reported that AQP4 knockout induced sex-specific alterations in neurotransmission, indicating that AQP4 might regulate the interaction between sex hormones and neurotransmission. In the present study, we found that AQP4 knockout decreased the concentrations of estrogen and progestogen. Further study showed that exogenous estrogen decreased DA and 5-HT in cortex, reduced DA and 5-HT in striatum, but increased 5-HT in hippocampus in AQP4+/+ male mice. However, in AQP4-/- male mice, exogenous estrogen almost did not alter the levels of neurotransmitters except for decreasing DA in cortex. In female mice, ovariectomy decreased DA in the striatum of AQP4+/+ mice, but did not alter the levels of DA in AQP4-/- mice. These findings reveal for the first time that AQP4 regulates not only water and ion homeostasis but also the functions of ovarian hormone and neurotransmitter.     

The influence of ovarian hormones on the rat oviductal and uterine concentration of noradrenaline and 5-hydroxytryptamine

This paper describes the effects of estradiol and progesterone on the concenirations of noradrenaline and 5-hydroxytryptamine in the Wistar rat oviduct and uterus. The levels of noradrenaline and 5-hydroxytryptamine are higher in the oviduct than in the uterus whereas p-tyrosine and tryptophan are similar in both tissues. Estradiol treatment reduced the oviductal concentration of noradrenaline but not 5-hydroxytryptamine in oviduct, while the concentrations of both noradrenaline and 5-hydroxytryptamine were reduced in uterine horn. The levels of noradrenaline in the oviduct and uterus in rats in estrus were lower than those of diestrous rats. Bilateral ovariectomy produced an increase in uterine noradrenaline and 5-hydroxytryptamine levels. These changes were reversed in the presence of ovarian hormones as indicated by experiments where unilateral ovariectomy was performed. Reserpine administration reduced noradrenaline concentration in both the oviduct and the uterus but did not change oviductal or uterine 5-hydroxytryptamine.These results indicate the existence of noradrenaline within postganglionic sympathetic nerve terminals and suggest that estrogens increase the utilization and the synthesis of noradrenaline in both the oviducts and the uterine horns. With respect to 5-hydroxytryptamine the data support the concept that it is mainly associated with mast cells.     

The acute effects of bilateral ovariectomy or adrenalectomy on progesterone, testosterone and estradiol serum levels depend on the surgical approach and the day of the estrous cycle when they are performed

Bilateral ovariectomy or adrenalectomy are experimental tools used to understand the mechanisms regulating the hypothalamus-pituitary-ovarian and the hypothalamus-pituitary-adrenal axis. There is evidence that acute unilateral perforation of the dorsal peritoneum in rats results in significant changes in progesterone, testosterone and estradiol serum concentrations. Because different surgical approaches for unilateral or bilateral ovariectomy or adrenalectomy, sectioning the superior ovarian nerve or the vagus nerve are used, we compare the acute effects on hormone serum concentrations resulting from the unilateral or bilateral dorsal approach to performing bilateral ovariectomy or adrenalectomy with those obtained when an unilateral incision is performed in the ventral abdomen. In general, the progesterone, testosterone and estradiol serum concentrations were higher in animals with ventral approach than in those with dorsal surgery, the effects varying depending on the day of the estrous cycle when surgery was performed. The results suggest that the neural signals arising from different zones of the peritoneum and/or the abdominal wall play different roles in the mechanisms regulating steroid hormones concentrations.     

A collaboration of aquaporins handles water transport in relation to the estrous cycle in the bitch uterus

Fluid movement through uterine cell membranes is crucial, as it can modulate the tissue imbibition pattern in the different phases of the estrous cycle. To gain insight into the mechanisms underlying steroid-controlled water handling, the presence and distribution of aquaporins (AQPs), integral membrane channel proteins permitting rapid passive water movement, was explored in bitch uterine tissues. Immunohistochemistry and Western immunoblot analysis were used to study the presence of AQP1, AQP2, and AQP5 in the layers of the bitch uterine wall during the different estrous phases. Presence of endothelial nitric oxide-generating enzyme NO synthase (NOS3) was also investigated, as it is known that the vasodilator NOS3 might be involved in the development of uterine edema. The results demonstrated the following: (1) AQP1, AQP2, and AQP5 were present in the uterus of cycling bitches. (2) AQP1 was localized within uterine mesometrial, myometrial, and endometrial blood vessels and in the circular and longitudinal layers of myometrium. AQP1 localization and expression were unaffected by the estrous cycle. (3) The estrogenic milieu was probably at the basis of AQP2 expression in the glandular and luminal epithelium of the endometrium. (4) AQP5 water channels were present in the apical plasma membrane of uterine epithelial cells in coincidence with plasma progesterone increase. (5) NOS3 was localized in the myometrial and epithelial tissues as well as in blood vessels indicating a contribution of this vasoactive peptide to the uterine imbibition processes. Thus, we can hypothesize that a functional and distinctive collaboration exists among diverse AQPs in water handling during the different functional uterine phases.     

The regulation of precopulatory behavior by ovarian hormones in the female Mongolian gerbil

The hormonal regulation of precopulatory behavior in the female Mongolian gerbil was studied using two groups (N = 6) of sexually experienced females. A novel testing procedure was used which involved females living continuously with test males for several days. The test males showed either full sexual behavior (copulating males, C) or only precopulatory behavior (noncopulating males, NC). Experiment 1 investigated changes during the estrous cycle and following ovariectomy in females. Experiment 2 studied the effects of hormonal treatment of these ovariectomized females with 6 micrograms estradiol benzoate (EB) followed by 0.4 mg progesterone (P) or by 0.04 ml arachis oil. When tested with NC males, females displayed a greater range of precopulatory behavior. The patterns could be classified into three groups according to the manner of response to ovariectomy and hormone treatment. Group I patterns (approach, leave, and olfactory investigation of the male's head) were affected by neither ovariectomy nor EB treatment relative to Day 3 levels (Day 3, day preceding estrus; Day 4, estrus), but they were increased to estrous levels by EB and P. Group II patterns (darting, foot-stomping, and the present and piloerection postures) appeared only during estrus, did not appear after ovariectomy, and reappeared only after sequential EB and P treatment. Group III patterns (investigation of the male's anogenital area, allogrooming, ventral gland marking, and sand-rolling) were reduced relative to both estrus and Day 3 levels by ovariectomy and increased above Day 3 levels by EB alone; EB and P treatment further increased Group III patterns to the level of estrus. It is suggested that female precopulatory behavior patterns differ in their responsiveness to ovarian hormones. Estrogen appears to affect those patterns associated with the earliest stages of estrus (Group III).     

Oviductal plasminogen activator inhibitor-1 (PAI-1): mRNA, protein, and hormonal regulation during the estrous cycle and early pregnancy in the pig

Recent identification of plasminogen activator inhibitor-1 (PAI-1) in the pig oviduct has prompted an evaluation of its mRNA, protein synthesis, and hormonal regulation during the estrous cycle and early pregnancy, defined as time prior to and after maternal recognition of pregnancy. To examine PAI-1 protein synthesis, oviductal tissue was collected from European Large White and Chinese Meishan gilts on days 0, 2, and 5 of early pregnancy, divided into three functional segments, and cultured. Culture media was collected and de novo synthesized PAI-1 analyzed by 2D-SDS-PAGE, fluorography, and densitometry. To determine hormonal regulation of PAI-1 synthesis and secretion, four groups of ovariectomized (OVX) cross-bred gilts were each treated with one of four steroid regimens (corn oil, estrogen, progesterone, or estrogen + progesterone) and tissue collected for RNA or cultured. Steady-state mRNA levels of PAI-1 were evaluated throughout the estrous cycle in cross-bred gilts. To compare steady-state PAI-1 mRNA levels between cyclic and pregnant cross-bred gilts, tissue was collected on days 0, 2, and 12. Quantitative analysis of steady-state levels of PAI-1 mRNA were analyzed by dot-blot hybridization and densitometry. A greater (P < 0.01) synthesis and secretion of PAI-1 protein was found in the isthmus portion of the oviduct relative to either the ampulla or infundibulum regardless of day of pregnancy or breed. No difference could be detected for PAI-1 protein between breeds. The Large White had a greater (P < 0.05) secretion of PAI-1 on day 2 of early pregnancy relative to other days examined. Whole oviductal tissue from cross-bred gilts was found to have a significantly greater amount of PAI-1 mRNA on days 1 and 2 compared to other days examined, while the isthmus had significantly greater levels of mRNA on days 2 and 12. A significant effect of day and segment was detected for levels of PAI-1 mRNA from cyclic and early pregnant cross-bred gilts. PAI-1 mRNA was found to be significantly greater in the isthmus than other segments, regardless of day of the estrous cycle or pregnancy. An interaction was detected for estrogen and progesterone on PAI-1 mRNA (P < 0.05) and protein (P = 0.09). Estrogen was found to inhibit PAI-1 protein synthesis and also inhibited progesterone-mediated stimulation of PAI-1 mRNA. Our results demonstrate expression of PAI-1 mRNA and protein are highest on day 2 of early pregnancy, which is consistent with its proposed function of protecting the oocyte/embryo from enzymatic degradation and/or extracellular matrix remodeling of both oviduct and early cleavage-stage embryo.     

Coexpression of adrenomedullin and its receptors in the reproductive system of the rat: effects on steroid secretion in rat ovary

The present study demonstrates the expression of adrenomedullin (ADM) in the reproductive system of the female rat and its effect on the secretion of estradiol and progesterone. Ovarian ADM and Adm mRNA levels were decreased at estrus, whereas oviductal Adm mRNA levels were low at proestrus. Both tissues were shown to coexpress mRNAs encoding the calcitonin receptor-like receptor and receptor activity-modifying protein 1 (Ramp1), Ramp2, and Ramp3. Gel filtration chromatography of ovarian extracts showed two peaks, with the predominant one eluting at the position of authentic rat ADM (1-50) at estrus and at the position of ADM precursor at diestrus. Positive ADM immunostaining was localized in the granulosa and theca cells of the follicle and corpora lutea of the ovary. Adrenomedullin inhibited FSH-induced estradiol secretion in 2-day-old follicles and also suppressed eCG-stimulated progesterone release in corpora lutea. The inhibitory effect of ADM on the follicles and the corpora lutea was abolished by calcitonin gene-related peptide (8-37) and ADM (22-52), respectively. The presence of ADM and the gene expression of ADM and its receptor components in the female reproductive system suggest a paracrine effect of ADM on ovarian steroidogenesis.     

Steroid hormones acutely regulate expression of a Nudix protein-encoding gene in the endometrial epithelium of sheep

Steroid hormones regulate endometrial gene expression to meet the needs of developing embryos. Our hypothesis is that steroid hormones transiently induce expression of genes in the endometrial epithelium to make the uterine environment different between the earliest days of pregnancy. We identified one such gene product using differential display-polymerase chain reactions. The gene product that was strongly induced in ewes between day 3 and 6 of the estrous cycle was cloned and sequenced to identify it as encoding a member of the Nudix family of hydrolase enzymes. Northern blot analyses indicated that NUDT16 mRNA concentrations were elevated 10-fold in the endometrium of sheep from day 5 to 9 of the estrous cycle and returned to basal levels by day 11. In assays of RNA samples from 15 different tissues from an adult ewe, the concentrations of NUDT16 mRNA were greatest in endometrium. In situ hybridization localized NUDT16 mRNA exclusively to the endometrial epithelial cells of the glands and uterine lumen. In ovariectomized ewes, NUDT16 mRNA was induced by a regimen of alternating estrogen and progesterone therapy designed to mimic the hormonal experiences of a ewe at day 6 of the estrous cycle. The final estrogen treatment in the regimen was critical to the expression of NUDT16 as well as progesterone receptor and estrogen receptor-beta genes. Characterization of the NUDT16 gene identified putative steroid hormone response elements, which can now be investigated to understand its unique pattern of regulation in the earliest days of pregnancy.