Progesterone dependent uptake of uteroglobin by rabbit endometrium

Summary Unlabeled or³H-labeled UGL, isolated from rabbit lungs, was injected intraluminally into the uteri of ovariectomized low dose progesterone substituted and unsubstituted animals as well as into mated animals on the fifth day of pregnancy. It was offered to the endometrium, in this last case, in competition with endogenously synthesized UGL.³H-labeled IgG served as a control. The animals were killed 24 h after the experiment. Immunohistograms and autoradiograms showed that UGL was taken up by the whole endometrium of the ovariectomized animals but only when progesterone was present. In the preimplantation uterus, uptake was limited to clusters of smaller cells accumulated in the lumen adjacent to the epithelium, whereas IgG remained within the uterine lumen, forming a dense layer on the endometrial surface. A preliminary report of parts of this investigation has been presented at the 15th EDBO International Embryological Conference, June 21–25, 1982, in Strasbourg (France) (Abstract 187)     

Uteroglobin and the accumulation of progesterone in the uterine lumen of the rabbit

Summary This study was undertaken to determine whether the influx of progesterone into the uterine lumen of the rabbit, in the preimplantation period, is dependent onuteroglobin (UGL). Rabbits were ovariectomized and, three months later, treated with two defferent doses of progesterone. Purified UGL was injected into one uterine horn and, as a control,immunoglobulin G (IgG) was injected into the other. After four days, the animals were sacrificed their uteri flushed, and the progesterone content of the washes was determined by radioimmunoassay.Animals with the lower serum progesterone level (2.8 ng/ml) had a significantly different uterine horn progesterone content. The hormone accumulation in the horn containing UGL was 2.3 to 7.5 times higher than in the horn containing IgG. Animals with a higher serum progesterone level (7.2 ng/ml) showed no differences. The hormone content was equally high in both horns, presumably due to the synthesis of endogenous UGL being reactivated by the hormone treatment.The validity of these experiments as models for the events during early pregnancy and the physiological role of progesterone available inside the uterus are discussed.     

Varying effects of an IUD on decidualization in mice.

Chronically implanted IUDs consisting of silk suture threads induced decidualization in regions of the uterus remote from the suture site in ovariectomized mice treated with a regimen of progesterone and oestrogen which sensitizes the uterus to a decidual stimulus. In these conditions the IUDs did not inhibit decidualization induced by instilled oil, although they did so in pregnant animals of the same strain. Varying the dose of progesterone and oestrogen did not produce conditions in which IUD's inhibited oil-induced decidualization in ovariectomized mice and progesterone treatment did not prevent IUDs inhibiting decidualization in pregnant animals. However, when ovariectomized mice, sensitized as before, were primed repeatedly with oestrogen to simulate continuing oestrous cycles after IUD insertion, the IUD's inhibited oil-induced decidualization. This involved the premature loss of instilled oil from the uterine lumen and was associated with heavy infiltration of leucocytes into the luminal epithelium. Numbers of leucocytes free in the uterine lumen did not appear to be critical. It appears that contact between the oil and the luminal epithelial surface must be sustained for some length of time to induce a decidual reaction; brief contact is not sufficient to trigger the response.     

Ultrastructural studies of the effect of steroid hormones on pars recta secretions in Bufo arenarum

In amphibians, eggs are released from the ovaries into the coelomic cavity after hormonal stimulation, while related preparatory events occur in the oviducts. We have reported an association between the biological activity of the pars recta (PR), the female reproductive cycle, and ultrastructural modifications of PR epithelium induced by steroid hormones in Bufo arenarum. This article describes (1) the electron microscopic features of PR epithelium and its secretions after ovariectomy; and (2) the effect of either 17β-estradiol, 5α-dihydrotestosterone, progesterone, or a combination of progesterone plus 17β-estradiol on PR epithelium and secretions in ovariectomized animals. Our results indicate that treatment solely with 5α-dihydrotestosterone, 17β-estradiol or progesterone is ineffective in counteracting the effect of ovariectomy, although some effect was noted. The PR secretions of ovariectomized control and ovariectomized animals stimulated with any one of the steroid hormones analyzed, are composed of flocculent material and membranous vesicles. By contrast, treatment of ovariectomized animals with a combination of 17β-estradiol and progesterone, restores the appearance of the pars recta epithelium to that of the nonovariectomized control animals. Progesterone, in the presence of 17β-estradiol, appears to be responsible for triggering the release of secretory granules into the PR lumen. J. Morphol. 231:1–10, 1997. © 1997 Wiley-Liss, Inc.     

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 immunocytochemical localization of a cat uterine protein that is estrogen dependent (CUPED)

An estrogen-dependent polypeptide (CUPED), which was purified from uterine flushings of estrogen-treated cats, was localized in endometrial epithelial cells of cats using the peroxidase-antiperoxidase immunocytochemical staining procedure. Epithelial cells from animals treated with estradiol for 4, 7, or 14 days and estrogen-primed animals treated with progesterone for 2 days showed positive immunostaining. Staining was absent in untreated ovariectomized animals and in estrogen-primed animals treated with progesterone for 4 days. Specific cytoplasmic staining was confined to apical secretory granules in nonciliated cells of deep uterine glands. Staining was also commonly observed in the lumen of deep glands. Immunostaining was absent in the cells of the surface epithelium, stroma, and myometrium. In addition, other organs such as the oviduct, kidney, liver, pancreas, and lung showed no evidence of specific immunocytochemical staining. Therefore, the estrogen-dependent polypeptide obtained from uterine flushings of estrogen-treated ovariectomized cats is a uterine-specific secretory product that is packaged in apical cytoplasmic granules of uterine epithelial gland cells before being released into the uterine lumen.     

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.     

Effect of progesterone on tachykinin concentrations in the hypothalamus and anterior pituitary of female siberian hamsters.

The effect of progesterone on SP- and NKA-like immunoreactive substances in the hypothalamus and anterior pituitary was studied in ovariectomized and in ovariectomized, estrogen treated Siberian hamsters. Neither ovariectomy nor progesterone or estradiol treatment resulted in apparent changes in the tachykinin concentration in the hypothalamus. No effect of the treatments was seen on the release of tachykinins by hypothalami incubated in vitro in presence of high KCl concentrations. Ovariectomy resulted in a significant increase in the concentrations of both tachykinins in the anterior pituitary, as compared with intact animals. Progesterone (5 mg/animal) significantly reduced tachykinin concentrations in the anterior pituitary, as compared with the values found in ovariectomized animals. Estradiol completely suppressed the post-ovariectomy increase in anterior pituitary tachykinins, and progesterone did not significantly modify the response to estradiol. Lower doses of progesterone (250 microg or 1 mg/animal) significantly reduced NKA concentrations in the anterior pituitary of ovariectomized Siberian hamsters, but SP concentrations, although showing a similar tendency, were not significantly different in progesterone-treated as compared with ovariectomized, control animals. These results suggest that progesterone may modulate tachykinin stores in the anterior pituitary gland of Siberian hamsters.     

Secretion and reabsorption of uterine luminal fluid in rats

Treatment of ovariectomized rats with oestradiol-17beta and progesterone demonstrated that oestradiol-17beta causes secretion of sodium, potassium and water into the lumen of the uterine horn and that progesterone causes reabsorption of these substances.     

Effect of steroids and the estrous cycle on uterine neuromedin U receptor expression

We investigated the role of estrogen and progesterone on rat uterine NmU receptor expression in both intact and ovariectomized animals and examined receptor expression through the estrous cycle. Chronic administration of beta-estradiol 3-benzoate (E2) or progesterone in intact animals was devoid of any effect. RU486 caused a 2-fold up-regulation in NmU receptor density. Ovariectomy caused a 60% decrease in receptor density, but chronic E2 administration to ovariectomized rats significantly increased NmU receptor density. The estrous cycle had no significant effect on NmU receptor density. These results suggest that NmU receptor expression is estrogen-dependent, whereas progesterone or a progestin-induced factor is involved in the modulation of this expression.     

Effects of ovarian hormones on cell membranes in the rat uterus

Freeze-fracture techniques have been used to study tight junctions on the lateral plasma membrane of cells of the luminal epithelium of the rat uterus under various hormonal regimes. Tight junctions from ovariectomized control rats extended some 0.5 μm down the lateral membrane and the junctional strands often formed a network of closely packed, circular compartments. Following treatment of rats with estrogen for 3 days the tight junctional regional still extended 0.5 μm down the lateral membrane, but the strands ran more parallel to the apical surface. They did not enclose circular compartments. After treatment with progesterone, either alone or with estrogen in such a way as to condition the ovariectomized uterus for implantation, a third pattern of junctional organization emerged. In these animals the junctional region extended 1.1 μm down the lateral membrane and the strands frequently crosslinked, enclosing compartments of varying and irregular size and shape. Our observations suggest that ovarian hormones could regulate the contents of the uterine lumen by altering the structure extent of the tight junctions which connect the epithelial cells enclosing the lumen.     

In vivo response of secretory component in the rat uterus to antigen, IFN-gamma, and estradiol

Intrauterine immunization of ovariectomized rats with SRBC is known to elicit pronounced IgA and IgG antibody responses in uterine secretions of immunized uteri. To determine whether secretory component (SC), the receptor for transporting polymeric IgA from tissues to mucosal surfaces, was also influenced by Ag, ovariectomized rats were immunized and boosted by placing SRBC into the lumena of individual uterine horns. In response to Ag, the levels of polymeric IgA, as well as free SC and SC bound to polymeric IgA, increased in uterine secretions. When ovariectomized animals were treated with estradiol, a fivefold increase in SC levels was observed in the immunized horns, indicating that a hormone response is superimposed on the Ag-induced stimulation of uterine SC. To determine whether IFN-gamma influences the presence of SC in uterine secretions, IFN-gamma was placed in the uterine lumena of ovariectomized nonimmunized rats. When uterine secretions were analyzed, significantly higher levels of SC were found in IFN-gamma-exposed uteri than were present in saline treated control animals. In contrast, intrauterine instillation of IFN-gamma had no effect on the levels of IgA in uterine secretions. This response was specific for IFN-gamma in that IFN-alpha/beta had no effect on uterine SC or IgA levels. These results indicate that intrauterine instillation of Ag, in addition to evoking pronounced antibody responses, stimulates the production of SC, which may be responsible for the transport of polymeric IgA from tissue to uterine secretions. Furthermore, they indicate that IFN-gamma placed in the uterine lumen stimulates SC production and suggest that the uterine SC response to Ag may be mediated by the action of IFN-gamma on uterine epithelial cells.     

Role played by the ovary and adrenals in early receptivity during 4-day cycle in the female rat

The mechanisms involved in the control of precocious sexual receptivity were studied in 4-day cyclic female Wistar rats injected with 10 μg estradiol benzoate (EB) and caged with a male during the night from diestrus II to proestrus. Early mating frequencies were compared in intact females, in animals ovariectomized on the morning of diestrus I, in adrenalectomized and in adrenalectomized-ovariectomized females. No change in early sexual receptivity occurred either in ovariectomized, or in adrenalectomized animals. On the contrary, a significant decrease of precocious mating frequencies was noted in adrenalectomized-ovariectomized females. The role played by the ovary in the control of precocious receptivity was supposed to be due to the secretion of progesterone which has been evidenced on the late afternoon of diestrus II in estrogen treated females.Concerning the mechanisms by which the adrenals may compensate for the ovaries in the control of early sexual receptivity in estrogen-primed females it was observed that notwithstanding an inhibitory action exerted by EB on the adrenal progesterone secretion, a low rate of progesterone was maintained in the peripheral plasma which was compatible with early mating in ovariectomized animals.     

Effects of oestradiol-17 beta and progesterone on the number of plasma cells in uteri of ovariectomized mice

Immunoglobulins A and G were localized by immunoperoxidase labelling in uteri of ovariectomized mice treated with oestradiol-17 beta and progesterone. The administration of oestradiol or progesterone alone to ovariectomized mice for 3 days increased the number of IgA plasma cells from about 1 to 14 per histological section. When the two hormones were administered simultaneously for 3 days the number of plasma cells per section was equal to or greater than with either hormone alone. Treatment with oestradiol followed by progesterone in a sequence that prepares the uterus for implantation resulted in about 31 IgA plasma cells per section. Counts of IgG plasma cells showed similar trends but the numbers were smaller. The results indicate that progesterone increases rather than decreases the number of plasma cells in the mouse uterus. This is consistent with observations on intact mice during oestrus and pregnancy and suggests that the marked increase in endometrial plasma cells at the time of implantation in mice is a response to progesterone acting on an oestrogen-primed uterus.     

Oviduct response to prostaglandins: Influence of estradiol and progesterone

Oviductal motility was measured in the isthmus of ovariectomized New Zealand rabbits. The effects of estradiol and progesterone on spontaneous motility and on the response of the oviduct to exogenously administered prostaglandin E₁ (PGE₁) and PGF_2α were determined. Estradiol treatment significantly increased both the amplitude (P<0.05) and frequency (P<0.01) of spontaneous contractions. The amplitude of spontaneous activity was less following progesterone treatment than following estradiol treatment (P<0.05). Progesterone treatment increased the duration of oviduct response to PGE₁ (P<0.05). Estradiol treatment had no effect on the response to PGE₁. Increased oviductal activity caused by PGF_2α lasted significantly (P<0.01) longer in ovariectomized, untreated animals than in ovariectomized animals treated with estradiol or progesterone. Progesterone was more effective than estradiol in decreasing the duration of the response to PGF_2α. These effects of steroid hormones on the responsiveness of the oviduct to PGE₁ and PGF_2α could contribute to the physiological control of egg transport. The nadir of ovarian hormone influence, as in the recently ovariectomized animals and as occurs immediately after ovulation, is associated with a high responsiveness of the oviduct to PGF_2α. This could effectively increase isthmic occlusion and prevent the eggs from passing through the oviduct prematurely. The gradual increase in ovarian estradiol and progesterone secretion during the 3 days following coitus could result in decreased responsiveness to PGF_2α and increased responsiveness to PGE₁. These changes might cause relaxation of isthmic tone and allow movement of eggs through the isthmus into the uterus.     

Prolactin as a factor in the uterine response to progesterone in rabbits

The direct effect of prolactin on uteroglobin production and on uterine endometrial oestrogen and progesterone receptor concentrations was tested by using ovariectomized rabbits (at least 12 weeks) treated with prolactin; prolactin + progesterone; prolactin + oestradiol + progesterone; oestradiol + progesterone; or progesterone alone. Prolactin treatment produced a significant (P less than 0.05) increase in the concentration of cytosolic oestrogen and progesterone receptors, restoring the concentrations to values found at oestrus. However, the concentration of nuclear receptors remained low. In the remaining treatment categories there was no significant (P greater than 0.05) increase in the concentration of oestrogen and progesterone receptors compared with those in ovariectomized controls. However, the sequential treatment of ovariectomized animals with prolactin + progesterone stimulated uteroglobin production to a concentration equal to that found in intact rabbits on the 5th day of pregnancy. This was not achieved by prolactin or progesterone alone or with oestradiol. These results suggest that prolactin acts as an essential factor in the rabbit uterine response to progesterone, perhaps by the modulation of progesterone receptor activity.     

A bioassay for the evaluation of antiproliferative potencies of progesterone antagonists.

A bioassay which allows quantification of the antiproliferative potency of progesterone antagonists on the mammary gland was developed. For this purpose, ovariectomized rats were substituted with oestrone and progesterone and a further group simultaneously treated with the progesterone antagonists Mifepristone (= RU 38.468), Onapristone (= ZK 98.299), or ZK 112.993 (Schering AG, Berlin). A morphometric analysis of the tubulo-alveolar buds in the inguinal mammary glands revealed a dramatic antiproliferative effect of the progesterone antagonists after as little as 3 days of treatment. Several less specific mammary gland growth parameters (weight, DNA- and RNA-content) proved to be less sensitive. This bioassay measures the potency of progesterone antagonists to competitively antagonize the specific effects of progesterone on the target organ mammary gland. Further advantages of this bioassay are the use of a hormonally standardized biological system, the quantitative results, the small amount of test compound necessary, as well as the substitution with progesterone and oestrone since the antiproliferative potency of progesterone antagonists on experimental hormone dependent mammary carcinomas is most potently displayed in ovariectomized animals substituted with both sex hormones.     

Ovarian hormones and the duration of sexual receptivity in the female golden hamster

The induction of sexual receptivity and its maintenance after copulation in ovariectomized female golden hamsters (Mesocricetus auratus) was found to be a function of the levels of ovarian hormones administered. Various combinations of estradiol benzoate (between 0.6 and 666 μg) and progesterone (between 0.05 and 5.0 mg) were administered in two experiments. Although some animals responded at 0.6 μg, higher levels of estradiol benzoate (1–6 μg or more) were more effective in inducing levels of lordosis equivalent to those seen in intact females in natural estrus. After mating, a depression in lordosis was observed in both ovariectomized and intact females. However, in ovariectomized females (excluding animals that did not respond initially) the duration of postcopulatory receptivity was a function of the level of progesterone administered. High levels of progesterone tended to prolong slightly the duration of postcopulatory receptivity.     

Effect of estradiol and progesterone on the rate of incorporation of radiolabeled arachidonate into phospholipids and triglycerides of the rat uterus

To obtain information on the effect of estradiol (E) and progesterone (P) on the overall rate of the acylation and deacylation reactions with [3H]arachidonate ([3H]AA) in the rat uterus, we investigated the effect of chronic treatment of ovariectomized rats with 2-200 micrograms/day E-dipropionate (EPP) and with 2 mg P alone or with the combination of these steroids on the extent and the time course of the in vitro incorporation of [3H]AA into various phospholipids (PLs) and the triacylglycerol fraction (TG). The results demonstrate that physiologic doses of E leads to a rapid equilibrium of the deacylation-acylation cycle only in the case of phosphatidylinositol (PI) whereas at high (200 micrograms/day) dose level it exerts the same effect on phosphatidylcholine (PC), phosphatidylethanolamine (PE) as well as on TG. P alone has no remarkable effects in the ovariectomized rat, but it decreases markedly the incorporation of [3H]AA into PI and TG in intact animals. Furthermore, P decreases the incorporation of [3H]AA into PI in the ovariectomized rat treated with 2 micrograms/day EPP as well as attenuating the enhanced labeling of PC, PE and PI caused by higher doses. Time-course studies provide evidence that all of these effects of progesterone can be accounted for by its ability to decrease the rate of deacylation and, therefore, to prolong the time needed to attain equilibrium in the rates of acylation and deacylation reactions of the various lipids with arachidonate. These data offer a novel outlook on the regulatory role of progesterone and estrogens in uterine function.     

Estrogen-dependent synthesis and release of a protein (CUPED) by cultured uterine explants from the domestic cat

Media from cultured cat endometrial explants were analyzed for the presence of a previously characterized high molecular weight estrogen-dependent secretory protein (CUPED) by polyacrylamide gel electrophoresis and radioimmunoassay (RIA). Both L-[(3)H]-leucine and D-[(3)H]-glucosamine were incorporated into newly synthesized CUPED during the culture of endometrial explants obtained from estradiol-treated ovariectomized cats, but not during the culture of tissue obtained from untreated ovariectomized animals or estradiol-primed ovariectomized animals treated with progesterone. The addition of tunicamycin to the culture medium inhibited the synthesis and release of the glycosylated form of CUPED from endometrial explants obtained from estradiol-treated cats. These data demonstrate that CUPED is synthesized and released in vitro from endometrial explants obtained from estradiol-treated cats as a glycoprotein possessing N-linked oligosaccharide chains.