Biosynthesis and immunocytochemical localization of an estrogen-dependent glycoprotein and associated morphological alterations in the sheep ampulla oviduct.

Published reports have shown that an M(r) 90,000-92,000 protein is released into the oviductal lumen of the sheep, during estrus at a time corresponding to ovulation and fertilization, where it associates with the embryo. The objectives of this study were (a) to determine whether estradiol-17 beta (E) alone or in combination with progesterone (P) induces the synthesis of the M(r) 90,000-92,000 protein from the ampulla and/or isthmus oviduct; (b) to monitor structural alterations in oviductal epithelial cells associated with the synthesis of this protein; and (c) to generate a polyclonal antiserum to the protein and use the antiserum to verify its cellular location and tissue specificity. Oviductal flushings and explant culture media were obtained from ovariectomized animals treated with E alone or with E plus P. The M(r) 90,000-92,000 protein was present in 3H-leucine- and 3H-glucosamine-labeled culture media of the ampulla (not isthmus) oviduct in animals treated with E alone or with E plus P. The glycoprotein was detected in gels of oviductal flushings obtained from animals treated only with E. A specific polyclonal antiserum to the protein was made and cross-reacted on Western blots of oviductal flushings from E-treated animals and ampulla (not isthmus) oviduct culture media from animals treated with E alone or with E plus P. The secretory apparatus of the epithelial cells of the ampulla oviduct matured and differentiated in response to E. Light and electron microscopic immunocytochemistry localized the M(r) 90,000-92,000 glycoprotein to secretory granules in the nonciliated cells of the ampulla oviduct. Immunoperoxidase reaction product was absent in tissue sections and Western blots of other reproductive and nonreproductive tract tissues obtained from steroid-treated animals. Therefore, the secretory cells of the ampulla oviduct of the sheep synthesize and release an E-induced, oviduct-derived M(r) 90,000-92,000 glycoprotein.     

Ovarian steroid hormone involvement in endogenous opioid modulation of LH secretion in mature ewes during the breeding and non-breeding seasons

The opioid antagonist WIN-44441-3 (WIN-3, Sterling-Winthrop) caused significant increases in LH secretion in ovariectomized ewes treated with progesterone but not in ovariectomized animals treated with oestradiol-17 beta. In the non-breeding season, plasma LH concentrations in ovariectomized ewes without steroid therapy, given oestradiol-17 beta or oestradiol-17 beta and progesterone together were not affected by treatment with WIN-3 on Day 6 after ovariectomy (there was a significant increase in LH as a result of WIN-3 treatment 13 days after ovariectomy in sheep given no steroid therapy). However, WIN-3 treatment of ovariectomized sheep given progesterone resulted in a significant increase in plasma LH. WIN-3 was ineffective when given to intact ewes treated with progesterone during the non-breeding season. With ovariectomized sheep during the breeding season there was again no response to WIN-3 at 6 days after ovariectomy in sheep given oestradiol-17 beta, but significant LH elevations in animals given no steroid, those given progesterone and those given progesterone + oestradiol-17 beta. The lack of an LH response to WIN-3 in ovariectomized sheep treated with oestradiol-17 beta did not result from a reduced pituitary response to GnRH since such animals responded normally to exogenous GnRH treatment. Overall, these results are consistent with the idea that, irrespective of the time of year, progesterone exerts negative feedback upon LH release at least in part through an opioidergic mechanism, whereas oestradiol-17 beta exerts negative feedback through steps unlikely to involve opioids. Progesterone can override the effect of oestradiol-17 beta during the breeding season only. Further, there appears to be a steroid-independent opioid involvement in LH suppression, operating at both times of year.     

Detection of a progesterone-dependent secretory protein synthesized by cat endometrium

Uterine flushings and culture media from endometrial explants incubated in the presence of radiolabeled amino acids were analyzed using one-(1-D) and two-dimensional (2-D) gel electrophoresis to identify proteins synthesized by the endometrium and subsequently released into the uterine lumen. 1-D and 2-D analyses of uterine flushings and culture media of endometrial explants obtained from 7- to 11-day pregnant cats (pre-implantation) showed a Mr 30,000 protein that appeared on 2-D gels as a family of macromolecules with isoelectric points between 6.5 and 7.0. This family of macromolecules was also present in the culture media of implantation-site tissue obtained from 12- to 16-day pregnant cats and of nonimplantation-site endometrium obtained form 12- to 28-day pregnant cats. The Mr 30,000 protein was absent in uterine flushings and culture media from estrous and 3- to 5-day-pregnant cats. In ovariectomized, steroid-treated animals, the Mr 30,000 protein was only detected in flushings and media from those animals treated with progesterone, regardless of the presence or absence of estradiol-priming and/or simultaneous estradiol treatment. In daily flushings obtained from ovariectomized, steroid-treated cats equipped with an indwelling uterine catheter, the Mr 30,000 protein was absent during the 14 days of estradiol treatment and was first detected 3-4 days after the onset of estradiol plus progesterone treatment. This protein was not detected in serum from estrous, 9-day pregnant, ovariectomized, and ovariectomized, steroid-treated animals. This study shows that 1) a progesterone-dependent protein, with an approximate molecular weight of 30,000 and an isoelectric point of 6.5-7.0, first appears within the uterine lumen soon after the arrival of the blastocyst and continues to be present during implantation; 2) the synthesis and release of the Mr 30,000 protein is dependent on progesterone regardless of the presence or absence of estradiol; and 3) the onset of secretion of the Mr 30,000 protein requires 3-4 days of continuous progesterone treatment in the estradiol-primed cat.     

The effect of estrogen and progesterone on structural changes in the uterine glandular epithelium of the ovariectomized sheep.

The development of epithelial cells of the uterine glands of ovariectomized sheep in response to estradiol-17 beta (E) and progesterone (P) was studied using light and electron microscopy. Animals that had been ovariectomized for six weeks were placed in one of three experimental treatment groups. Group I animals (untreated controls) received no steroid treatment. Group II animals (E alone) received one 4-cm E implant (E approximately 5-10 pg/ml) and their uteri were removed after 2, 4, or 6 days. Group III animals (E-primed, P-treated) received an E implant (E approximately 5-10 pg/ml) for 6 days and then were treated with six 13-cm P implants (P approximately 1.5-3 ng/ml), in the continued presence of E, for 2, 4 or 6 days. Six weeks after ovariectomy the epithelial cells of the uterine glands were low cuboidal and morphologically appeared to be synthetically inactive. Following 2 days of E treatment the epithelial cells had significantly increased in cell height, and protein-synthesizing organelles were well developed. Maturation of the secretory apparatus continued throughout E treatment. The Golgi complex and rough endoplasmic reticulum (RER) were abundant. Lysosomal-like granules and granules of varying electron density were present in the cytoplasm. The chronic administration of P to E-primed animals did not result in any further increase in cell height. Elongated mitochondria, a cup-shaped Golgi apparatus, extensive apical microvilli, and irregularly shaped membranous profiles in the supranuclear cytoplasm characterized these uterine epithelial cells. Lysosomal-like granules, small vesicles, and scattered patches of glycogen were seen in the cytoplasm. These data show that the uterine epithelial cells of the ovariectomized sheep undergo morphological alterations in protein-synthesizing organelles and apical specializations that depend on the presence of E and P.     

Synthesis of uterine endometrial proteins during early diestrus in the cyclic and pregnant dog, and after estrogen and progesterone treatment.

The objectives of this study were to identify and characterize dog uterine endometrial proteins synthesized de novo in explant culture during early luteal phase, to examine distribution of these proteins prior to the embryo's entering the uterus and during its free-floating period prior to implantation, and to examine regulation of endometrial proteins by estrogen and progesterone (P4) treatments. Uterine endometrium was collected from cyclic and pregnant bitches on diestrus Days 3, 7, and 10 as determined by loss of cornification of vaginal epithelium, and from ovariectomized dogs after treatment with corn oil, estrogen, P4, or estrogen followed by 1 or 2 wk of P4. Tissue was incubated in an explant culture system in the presence of [3H]leucine or [35S]methionine. The rate of incorporation of [3H]leucine into nondialyzable macromolecules indicated no significant change in rates of incorporation by status (pregnant vs. nonpregnant), day, or steroid treatment. Uterine endometrial-conditioned culture medium, analyzed by two-dimensional SDS-PAGE and fluorography, revealed a complex array of at least ten proteins or protein complexes in cyclic and pregnant bitches. No difference in protein pattern was detected by status; however, differences in distribution were apparent by day of cycle or early pregnancy. Two major proteins, cP5 (M(r) 54,686) and cP6 (M(r) 23,010) appeared to be differentially expressed. Expression of cP5, maximal on diestrus Day 3, decreased as the cycle or pregnancy progressed to diestrus Day 10. In contrast, expression of cP6, a minor protein on diestrus Day 3, appeared to be up-regulated for each status to Day 10, with increased intensity and multiple isoelectric and molecular-weight variants. In ovariectomized steroid-treated dogs, two-dimensional SDS-PAGE showed that pattern and distribution of specific proteins were affected by treatment. Acidic protein cP1 (M(r) 87,600), synthesized after corn oil and P4 treatment, was suppressed with estradiol (E2). Proteins cP2 (M(r) 40,000 and M(r) 42,000), present with all treatments, were intensified with P4. A high-M(r) basic protein complex (cP3) and acidic protein cP4 were expressed with E2 and maintained with P4 treatment. Proteins cP5 and cP6, while not induced by E2 or P4 alone, required E2 priming for P4 induction. Protein cP5 was down-regulated while cP6 was up-regulated with P4 for 2 wk. Proteins induced by estrogen followed by 1 or 2 wk of P4 treatments were similar to those released by endometrial explants collected from pregnant and cyclic bitches on Days 3, 7, and 10 of spontaneous diestrus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential regulation of prostaglandin EP and FP receptors in pregnant sheep myometrium and endometrium during spontaneous term labor.

In the present study, we characterized the mRNA abundance of prostaglandin E(2) receptor subtypes (EP1 and EP3, which stimulate excitatory responses; EP2 and EP4, which stimulate inhibitory responses) and the FP receptor in pregnant sheep myometrium and endometrium in relation to parturition. Myometrial and endometrial poly(A) RNA was extracted from control ewes at 143-147 days gestational age (dGA, n = 6) and from ewes in spontaneous term labor at 145-147 dGA (n = 6), and was subjected to Northern blot analysis for FP, EP1, EP2, EP3, and EP4 mRNA. Myometrial EP3, EP4, and FP mRNA abundance increased during labor (P<0.05); EP2 mRNA did not change. EP1 mRNA was not detectable in the myometrium. Endometrial EP2 and EP4 mRNA remained unchanged during labor. EP3 mRNA was expressed at a very low level, and EP1 and FP mRNA were not detected in endometrium in any animals studied. In conclusion, there is differential expression in myometrium and endometrium of EP subtypes and FP receptor in relation to labor. Increases in EP3 and FP, together with increased prostaglandin production from intrauterine tissues, may lead to the switch in the myometrial contraction pattern that occurs during labor. These differences within and between myometrium and endometrium may result from different anatomical location, such as longitudinal or circular layers of myometrium, or vascular location.     

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.     

Progesterone-regulated secretion of the serpin-like proteins of the ovine and bovine uterus.

The uterine milk (UTM) proteins are the major progesterone-regulated proteins secreted by the sheep uterus during pregnancy. Recently, proteins related to the UTM proteins have been identified in uterine secretions of the pregnant cow and sow. The present objective was to determine the time course for induction of the UTM proteins in sheep and cattle. Twelve ovariectomized ewes received subcutaneous injections of either vehicle for 10 days or 100 mg/d of progesterone for 10 days or 30 days. The presence of UTM proteins was examined by Western blotting of uterine flushings and by immunoabsorption of radiolabeled UTM proteins from conditioned medium of endometrial explant cultures performed with [35S]methionine precursor. Uterine milk proteins were present in slight amounts in uterine flushings and endometrial-conditioned culture medium of some ewes in the control group, but amounts of proteins were greatly enhanced by progesterone after 10 or 30 days of treatment. Prolonged exposure to progesterone (30 days versus 10 days) increased amounts of UTM proteins. Immunohistochemical analysis of endometrium indicated that the major site of UTM proteins was the glandular epithelium. In the second experiment, nine ovariectomized cows were treated daily with vehicle for 12 days or 750 mg progesterone for 12 or 30 days. Uterine flushings and conditioned endometrial culture medium were examined for UTM proteins by Western blotting. Uterine milk proteins were present to some degree in cows treated with vehicle, and an enhancement in amounts of UTM proteins was not observed until after 30 days of progesterone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estradiol regulation of secretory component in the uterus of the rat: evidence for involvement of RNA synthesis

The present studies were undertaken to characterize the response of uterine secretory component (SC) to estradiol. Administration of estradiol for 3 days to ovariectomized rats before incubation of uterine tissues resulted in a marked accumulation of SC in the incubation media. When uteri from ovariectomized rats treated with progesterone or testosterone were incubated, very little SC accumulated in the media, indicating that the estradiol-stimulated increase is hormone-specific. When uteri from rats that received estradiol for 6 days were compared with uteri from 3-day treated rats, SC release during a 24-hr incubation period was the same. This finding indicates that in the presence of prolonged estradiol exposure, SC production continues. The estradiol-induced accumulation of SC in culture is not due to the release of pre-formed uterine SC. When tissue SC levels were measured after 3 days of estradiol treatment, very little tissue SC was found relative to that released into culture media during 24 hr of incubation. The addition of actinomycin D to the incubation media markedly inhibited SC release by uteri from estradiol-treated rats. The release of SC was also inhibited by alpha-amanitin, a known inhibitor of Type II polymerase. These studies demonstrate that estradiol stimulation of SC is markedly reduced by inhibitors of RNA synthesis, and suggest that estradiol regulation of SC is mediated through uterine mRNA synthesis.     

Ovarian steroid-regulated synthesis and secretion of complement C3 and factor B in mouse endometrium during the natural estrous cycle and pregnancy period.

We demonstrate the presence of complement factor B (Bf) and complement C3 in uterine luminal fluid collected from estrogen-stimulated immature and adult female mice. We examined the synthesis and secretion of these two proteins in mouse endometrium at various stages of the natural estrous cycle and during the pregnancy period. The mRNA levels of these two proteins increased markedly in proestrus and estrus and declined sharply in metestrus to an undetectable level. The Bf mRNA remained undetectable, whereas a readily detectable C3 mRNA level reappeared, in diestrus. Meanwhile, these two proteins were immunolocalized to the apical cytoplasm of glandular and luminal epithelial cells of the endometrium during the estrous cycle. Administration of an estrogenic steroid to immature or ovariectomized adult mice markedly stimulated the expression of Bf, C3, and their RNA messages in the endometrium, whereas injection of progesterone alone to ovariectomized animals did not stimulate their expression. Expression of C3 was remarkably enhanced, whereas that of Bf changed only slightly, after injection of combined estrogen and progesterone to ovariectomized animals. In pregnant mice (Day [D] 1 = day of vaginal plug), Bf mRNA was at a high level on D1 and D2, dropped to an almost undetectable level from D3 to D8, and then increased to a low level thereafter until delivery. The C3 mRNA was at a high level on D1, dropped on D2 to an almost undetectable level from D3 to D9, increased to a very high level from D10 to D18, and then declined sharply before delivery. Immunohistochemical patterns of both proteins in the endometrium during preimplantation were positively correlated with changes in their mRNA levels.     

Hormonal regulation of apoptosis in the endometrium of common marmosets (Callithrix jacchus)

Phase-dependent apoptotic changes in the human endometrium during an ovarian cycle imply a potential role of steroids in the regulation of apoptosis. The present study was undertaken to determine the direct role of hormones in endometrial apoptosis in marmosets (Callithrix jacchus), a primate species which shows similarity to humans in terms of the cycle length and pattern. Endometrial apoptosis was detected by 3'-end labeling (TUNEL) in various phases of ovarian cycle in naturally cycling healthy marmosets (n=14) and also in ovariectomized marmosets (n=13) treated with either estradiol alone (E) or progesterone alone (P) or estradiol followed by progesterone (E+P). Expressions of apoptosis associated genes such as Bcl-2 family members (Bax and Bcl-2), proliferating cell nuclear antigen (PCNA)--a proliferation marker and steroid receptors, ERalpha and PR A were analysed by immunohistochemical methods. Apoptosis was intense in the glandular epithelial cells of endometrium during the mid-luteal phase as compared to other phases in naturally cycling animals; in the E+P group as compared to other groups of ovariectomized animals (P<0.05). Pronounced apoptosis in the mid-luteal phase was accompanied by the increased expression of Bax in glandular epithelial cells; while Bcl-2 immunoreactivity remained unchanged. PCNA expression was higher in the naturally cycling animals in the follicular phase and in the E group of the ovariectomized animals as compared those in the other groups. Immunoreactive ERalpha and PR A in glandular epithelial cells were most abundant during early follicular phase in naturally cycling animals and in both E and E+P groups among the ovariectomized animals. The present study highlights the importance of apoptosis in endometrial remodeling during the ovarian cycle and secondly, the role of both estradiol and progesterone in the regulation of apoptosis.     

Maintenance of gonadotrophs in pituitary autografts under the kidney capsules of female rats given sex hormones or LRH

Female Sprague-Dawley rats were hypophysectomized and the anterior pituitary gland was immediately placed under the kidney capsule. For 1 week after surgery, groups of pituitary autograft-bearing animals were treated with twice-daily injections of estradiol 17 beta (E), progesterone (P), estradiol 17 beta and progesterone (EP), or luteinizing hormone-releasing hormone (LRH). Within 2--4 hours following the last injection, the pituitary grafts were removed and placed into organ culture. They were maintained in culture with or without added LRH (10(-7) M) for 1 hour at 37 degrees C. The culture media were then frozen for later radioimmunoassay of FSH and LH. The tissues were kept in culture for an additional 24 hours, at which time they were fixed and prepared for immunocytochemistry or electron microscopy. Results showed that treatment of the animals with E, EP, or LRH enhanced the release of FSH and LH into the culture media, and that the release of these hormones was increased further by acute incubation with LRH. The ultrastructure of the gonadotrophs was well maintained by treating the animals with E or the combination of E and P or with LRH. Graft tissue from animals treated with LRH, which was incubated subsequently for 24 hours with LRH, showed the best maintenance of gonadotroph morphology. This experimental procedure should be useful for obtaining gonadotrophs for use in establishing gonadotroph cell lines.     

Estradiol stabilizes estrogen receptor messenger ribonucleic acid in sheep endometrium via discrete sequence elements in its 3'-untranslated region

The preovulatory surge of estrogen up-regulates estrogen receptor-alpha (ER) gene expression in the uterus during the estrous/menstrual cycles of female mammals. Previously, we demonstrated that the 5-fold increase in ER mRNA levels in endometrium of ovariectomized ewes treated with a physiological dose of estradiol (E2) is entirely due to an increase in ER mRNA stability. Our current work confirms that the E2 effect is specific to ER mRNA. The sequence of ER mRNA, cloned from sheep endometrium, shows a high degree of conservation with those of other species, even in the 5'- and the very long 3'-untranslated regions. In a cell-free assay, ER mRNA demonstrates greater stability with endometrial extracts from E2-treated ewes compared with those from untreated ovariectomized ewes. The E2-enhanced stability of ER mRNA was ablated by prior treatment of the extracts with proteinase K, 70 C heat, and oxidizing and alkylating reagents, indicating that a protein is responsible for stabilization of the message. The 3'-untranslated region of ER mRNA contains discrete sequences required for E2-enhanced stability, four of which were identified by extensive deletion mutant analyses. Transfer of two of the four minimal E2-modulated stability sequences conferred E2-enhanced stability to a heterologous RNA. These minimal E2-modulated stability sequences contain a common 10-base, uridine-rich sequence that is predicted to reside in a loop structure. Throughout our studies, estrogen stabilization of ER mRNA in sheep endometrium resembled that of vitellogenin mRNA in frog liver, indicating conservation of this ancient mechanism for enhancing gene expression in response to estrogen.     

Endometrial protein secretion during early pregnancy in entire and ovariectomized ewes

The secretion and synthesis of protein in vitro by explants of endometrium were examined in entire ewes during the first 10 days of the oestrous cycle and during an equivalent interval in ovariectomized ewes which received injections of oestradiol and progesterone. The schedule of steroid injections given was designed to simulate endogenous ovarian secretion of progesterone during the luteal phase before oestrus, of oestradiol around oestrus and of progesterone during the luteal phase after oestrus. The rate of protein synthesis and tissue RNA:DNA and protein:DNA ratios in intercaruncular and caruncular endometrium were generally higher in entire than in ovariectomized ewes. In ovariectomized ewes oestradiol increased these activities at 2-4 days after oestrus, whereas progesterone preceding oestradiol caused increases at oestrus, but not thereafter. In entire ewes and in ovariectomized ewes receiving the full steroid treatment regimen, protein secretion was high at oestrus and declined markedly during the next 4-6 days. In ovariectomized ewes not receiving progesterone before oestradiol, secretion increased between 4 and 6 days after oestrus, or during the equivalent stage of treatment in ewes which did not show oestrus. The omission of this progesterone did not modify secretion by caruncular endometrium. Oestradiol increased protein secretion by both tissues. The data suggest that progesterone given before oestradiol (or its equivalent in entire ewes) inhibits the secretion, at about 4-7 days after oestrus, of uterine proteins which may impair embryo development in ovariectomized ewes which do not receive this progesterone.     

Ethanol alters vasoactive intestinal peptide-induced steroid release from immature rat ovaries in vitro

The present study was conducted to examine the acute effects of ethanol (ETOH) on basal and VIP-induced release of testosterone (T) and estradiol (E2) from immature ovaries in vitro. Ovaries were collected from anestrus (A) and both naturally occurring and pregnant mare's serum gonadotropin (PMSG)-induced early proestrus (EP) animals. The ovaries were incubated in either media alone, media plus 1 microM VIP, media plus ETOH in doses ranging from 25 to 100 mM, or media plus each dose of ETOH containing VIP. The present results demonstrate that ETOH did not affect either basal or VIP-induced steroid release from ovaries collected from A animals. Likewise, the ETOH did not alter basal steroid secretion from EP animals; however, the drug significantly reduced the VIP-stimulated release of both T and E2 from EP ovaries. Thus, these data demonstrate for the first time that ETOH is capable of altering prepubertal ovarian responsiveness to VIP, a peptide known to be involved in the developmental regulation of ovarian function.     

In vitro synthesis of oviductal secretory proteins by estrogen-treated ovariectomized gilts.

The objective of this study was to identify, characterize, and examine oviductal secretory proteins (OSP) synthesized de novo by whole oviduct (WO), ampulla (A), and isthmic (I) tissue from ovariectomized (OVX), corn oil (CO)-, estrogen (E)-, progesterone (P)-, and E + P-treated gilts. Oviducts were collected from OVX gilts after CO, E, P, or E + P treatment for 11 consecutive days and tissue was incubated with 3H-leucine (3H-leu). Rates of 3H-leu incorporation into nondialyzable macromolecules by WO explants were greater (P less than 0.01) with E- compared to CO-, P-, or E + P-treated gilts and greater (P less than 0.05) by A explants with E- compared to CO-, P-, or E + P-treated gilts. An effect of location was noted, with A having a greater (P less than 0.01) rate of incorporation than WO or I. Conditioned culture medium was analyzed by one (1D)- and two-dimensional (2D) sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and fluorography. Analyses by 1D-SDS-PAGE revealed three major E-dependent bands (335,000, 100,000, and 80,000 M(r)) in WO and A, and one (335,000 M(r)) in the I. A 20,000 M(r) band found in A was inhibited by E, while a 60,000 M(r) band found in the A was induced by P. Analyses by 2D-SDS-PAGE resolved major E-dependent bands 2 (100,000 M(r)) and 3 (80,000 M(r)) into basic and acidic 100,000 M(r) proteins and a 75,000-85,000 M(r) protein (pI less than 4), respectively, found in WO and A, but not in I. A basic 20,000 M(r) protein and an acidic 45,000 M(r) complex, both found in A, were inhibited by E. Gel filtration of culture medium revealed a high M(r) fraction (greater than 2 x 10(6)) that was induced by E and was 6.8-fold greater in medium from A than from I. This study clearly demonstrates that 1) WO and A tissue from E-treated gilts de novo synthesize and secrete three major proteins (basic 100,000, acidic 100,000, and 75,000-85,000 M(r)); 2) these E-dependent proteins are not found in I or with other treatment; 3) several protein complexes synthesized by A are inhibited by E treatment; and 4) a high M(r) fraction, produced primarily in the A, is induced or amplified by E.     

Induction of porcine uterine estrogen sulfotransferase activity by progesterone

Studies have been carried out which were designed to examine the hormonal requirement for the appearance of estrogen sulfotransferase activity in porcine uteri. Mature, ovariectomized (OVX) gilts were housed for 3 weeks before being treated with various regimens of estradiol-17 beta (E2) and progesterone (P). Uteri were then removed, minced, incubated for 2 h with [3H] E2 (10(-8) M) and Na2 35SO4 (10(-4) M) and the labeled metabolic products were extracted and analyzed. Endometrial samples were also taken for the determination of E2 and P cytoplasmic and nuclear receptors (R). It was found that 4 daily injections of 250 micrograms of E2 was sufficient to bring plasma E2 concentrations to that representative of a normal estrous cycle (approx. 30 pg/ml) and to induce cytoplasmic PR to high levels (7000--19000 fmol/mg DNA). Estrogen sulfotransferase activity, which was negligible in OVX and E2-treated pigs, increased to near normal secretory levels (4 pmol product/h per 0.4 g tissue) only in pigs primed with E2 and subsequently treated with E2 and P (25--250 mg/day, 3 days). This treatment also brought about the translocation of PR to the nuclear compartment. The steroid alcohol sulfotransferase activity in these tissues decreased upon ovariectomy and remained unaffected by the hormone treatments. Endometria from treated and untreated pigs were cultured for a period up to 7 days. During this time E2 (10(-8) M) induced and/or maintained PR and P (10(-6) M) was shown to stimulate estrogen sulfurylation concomitant with the translocation of PR to the nucleus. These studies have demonstrated that, in OVX pigs and endometrial cultures, P stimulated uterine estrogen sulfotransferase activity to a level normally found in secretory uteri. In order for P to bring about elevated levels of estrogen sulfurylation it was necessary that the endometrium contain adequate concentrations of cytoplasmic PR (which required E2 priming of the system) and the P receptor complex must display nuclear translocation.     

Uterine production of prostaglandins F2 alpha and 6-keto-F1 alpha by ovariectomized pregnant rats receiving antiprogesterone steroid or in which progesterone has been withdrawn.

Ovariectomized early pregnant rats given continuous steroid replacement therapy have been treated with antiprogesterone steroid, ZK98299 or RU38486. At 24 h following treatment, uterine explants in culture were found to produce significantly greater amounts of PGF2 alpha, but not of 6-keto-PGF1 alpha, when compared to controls. ZK98299 and RU38486 gave almost identical levels of uterine PG production. The 6-keto-PGF1 alpha/PGF2 alpha production ratio for uteri of treated rats was decreased by 45% relative to controls. Similar changes in uterine PGF2 alpha production and 6-keto-PGF1 alpha/PGF2 alpha ratio have been shown for ovariectomized early pregnant rats in which progesterone has been withdrawn when compared to control animals. It has been suggested that inhibiting or withdrawing progesterone in rat uteri exposed to estradiol and progesterone may lead to a stimulation of endoperoxide F-reductase and/or E2 9-ketoreductase activities. The presence of luminal fluid in the uteri was observed for animals treated with antiprogesterone steroid or in which progesterone had been withdrawn. This was associated with a decrease in % dry weight for the uteri of these animals.