Stage-specific alterations in the apical membrane glycoproteins of endometrial epithelial cells related to implantation in rabbits

The rabbit endometrial epithelium undergoes differentiation prior to the time of blastocyst implantation, including loss of surface negativity and a change in glycocalyx morphology. Nonpregnant (estrous) and pseudopregnant rabbits were used to study specific alterations in proteins and saccharide composition of the luminal epithelial membrane and its glycocalyx related to the acquisition of receptivity to implantation. Pregnant animals were used to study further modification of the luminal surface by implanting blastocysts. The apical surface of luminal epithelial cells was solubilized by a 15-min intraluminal incubation of 1% Triton X-100 containing protease inhibitors. Proteins in extract solutions were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Three new polypeptides (24 kDa, 42 kDa and 58 kDa) were identified in uteri from receptive rabbits. Binding of succinyl Wheat Germ Agglutinin (sWGA) and Ricinus communis Agglutinin (RCA-I) lectins to the 24 kDa and 42 kDa components on Western blots of extracts separated by SDS-PAGE identified them as glycoproteins. Additionally, other polypeptides (26 kDa, 80-86 kDa and 145 kDa) showed changes in affinity for WGA, RCA-I or concanavalin A (Con A), depending on the hormonal state. Correlating with these findings was an increased binding of these lectins to intact nonciliated cells in uteri of receptive rabbits compared to estrous animals; ciliated cells bound Dolichos biflorus Agglutinin (DBA) specifically, regardless of the hormonal condition. Treatment of uteri from estrous animals, or Western blots of proteins from these animals, with neuraminidase prior to lectin exposure suggested the presence of glycoproteins having a sialic acid-D-galactose terminus in nonreceptive rabbits. Reduced binding of lectin to intact cells at implantation sites and to blots of proteins isolated from these sites, compared to nonimplantation sites, was noted. These results provide evidence for stage-specific alterations in protein and saccharide composition of the apical surface of endometrial epithelium prior to implantation, and indicate that implanting blastocysts further modify the luminal surface.     

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.     

Actin binding protein expression is altered in uterine luminal epithelium by clomiphene citrate, a synthetic estrogen receptor modulator

Clomiphene citrate (CC), a synthetic oestrogen, is often prescribed as a superovulator in treating infertility. Although CC works efficiently, pregnancy rates following CC treatment are approximately 10 times lower than "natural" rates. This study investigates how a dose of 1.25 mg CC given to ovariectomized rats before the implantation priming hormones (a single dose of progesterone for 3 days and a dose of estradiol-17beta on d3, P-P-PE), alters the expression and distribution of alpha-actinin, gelsolin and vinculin. Actin binding proteins show a specific distribution within the uterine epithelium during implantation, linking the actin cytoskeleton to integrin expression on the uterine surface and in this way aiding "adhesiveness" for blastocyst apposition to the uterine epithelium. In this study, immunocytochemistry on frozen uterine sections using mouse monoclonal antibodies against alpha-actinin, gelsolin and vinculin and peroxidase-conjugated secondary antibodies, show that CC, administered before the P-P-PE regimen, down-regulates the expression of vinculin, does not alter the expression of gelsolin and up-regulates alpha-actinin on the uterine apical surface, when compared to P-P-PE treated animals. All three proteins are down-regulated on the apical surface of the luminal epithelium and glands in all groups when compared to pregnant controls. Vinculin was only localized in the basolateral compartment of the uterine epithelial cells in the CC treated groups. By down-regulating these proteins on the uterine surface and up-regulating vinculin on the basolateral membrane of the epithelium, CC may impede adhesion and invasion of blastocysts at implantation. These results may aid the exogenous manipulation of uterine tissue to control fertility and improve assisted reproductive out-comes.     

Effect of hormone replacement therapy on the elemental contents of uterine tissue

For the past years, different therapies based on steroid hormone supplementation or modulators of estrogen receptors have been used after menopause to prevent or manage osteoporosis. Although these treatments seem to be beneficial, they have some negative effects in the uterus and breast. The objective of this study was to assess variations for the concentrations of K, Ca, Mn, Fe, Cu, Zn, and Se in uterine tissue of Wistar rats. Ovriectomized rats were subjected to estrogen, progesterone, raloxifene, and tibolone supplementation and compared with nonovariectomized control animals. Elemental contents determined by the particle-induced X-ray emission (PIXE) technique revealed major alterations in Fe, Ca, Mn, and Se in the uterus of ovariectomized rats relative to control animals. After ovariectomy, a significant increase in Ca and Fe and a significant decrease in Mn and Se contents were determined in the uterus. For the ovariectomized groups in which animals, received raloxifene, tibolone, estrogen, and estrogen combined with progesterone supplementation, an overall recovery in Mn, Fe, and Se contents was verified. Elemental concentration in the progesterone-supplemented group did not significantly differ from ovariectomized animals receiving placebo. The alterations found for ovariectomized animals receiving placebo and progesterone suggest tissue impairment and trace element imbalance, contrasting with the remaining supplemented groups where an enhancement of tissue activity might justify similar concentration levels relative to controls, because most of the elemental contents altered after ovariectomy.     

Failure to detect specific binding sites for prostaglandin F-2 alpha in membrane preparations from rat endometrium

Membrane preparations from endometria of rats in different physiological states (e.g. pseudopregnancy, ovariectomized animals receiving progesterone + oestradiol or oestradiol alone) were studied for [3H]PGF-2 alpha binding by methods which detected PGF-2 alpha binding in ovary preparations and PGE binding in the same endometrial preparations. There was no evidence of high-affinity binding sites for [3H]PGF-2 alpha. Saturable [3H]PGF-2 alpha binding that increased with the onset of uterine sensitivity was detected but this binding does not fulfil all the criteria required for a PGF-2 alpha receptor and is probably due to binding to PG metabolizing enzymes in our preparations, or to binding of [3H]PGF-2 alpha to PGE binding sites. The failure to detect specific PGF-2 alpha binding sites seems to reflect a true absence of these sites in the rat endometrium.     

Cytoplasmic oestradiol-binding sites and their relationship to oestradiol content in the endometrium of cattle.

Twenty-three cows and heifers were killed at known times during the oestrous cycle or during the first 35 days of pregnancy. Duplicate cytosol preparations were made from the endometrium of each uterine horn and both the binding-site concentration and the oestradiol level were determined for each sample. During the cycle, the oestradiol concentration was only 0-2 to 1-7% of the concentration of binding sites which varied considerably between Days 19 and 5 (47,665 +/- 7538 sites/cell, mean +/- S.E.M.) and Days 6 to 18 (7060 +/- 444 sites/cell). The concentration of binding sites remained low in pregnant animals (6689 +/- 492), although the oestradiol concentration was high about 20 days after insemination, resulting in almost 14% of the sites being occupied. Five inseminated animals in which no conceptus was found when they were slaughtered 19 to 22 days later had low concentrations of binding sites but two animals had high levels of oestradiol with 13% and 15%, respectively, of their cytoplasmic sites being occupied. It is suggested that these animals had recently lost their conceptuses. Two ovariectomized cows and one non-cyclic animal contained high concentrations of oestradiol-binding sites in the uterine cytoplasm. No significant difference was found between the uterine horn adjacent to the ovary with the CL and the contralateral horn in early pregnancy or during the luteal phase of the oestrous cycle. An animal killed 1 week after parturition contained fourfold more sites in the involuting horn than in the opposite horn. It is suggested that progesterone plays a major role in regulating oestrogen-induced replacement of cytoplasmic binding sites.     

Prostaglandin E production by uteri of ovariectomized pregnant rats receiving antiprogesterone steroid or in which progesterone has been withdrawn.

Antiprogesterone steroid, ZK98299 (Schering, Germany) or RU38486 (Roussel Uclaf, France), has been administered to ovariectomized early pregnant rats receiving continuous steroid replacement. At 24 h later, uterine explants of rats treated with ZK98299 produced significantly greater amounts of prostaglandin E (PGE) than did controls or animals treated with RU38486. The PGE/PGF2 alpha production ratio for uteri of rats treated with ZK98299 or RU38486 was markedly lowered compared to controls, and a significant decrease occurred in the PGE/6-keto PGF1 alpha production ratio for rats treated with RU38486. For ovariectomized early pregnant rats in which progesterone has been withdrawn, a significant reduction in uterine PGE production occurred when compared to control animals. There was also a marked decrease in PGE/PGF2 alpha production ratio, and the PGE/6-keto PGF1 alpha production ratio tended to be lowered relative to controls. The stimulated production (as by ZK98299) or unchanged production of PGE (as by RU38486) indicates a selective action on uterine PGE synthesis among the antiprogesterone steroids, and these findings cannot be explained simply in terms of a blockage of progesterone receptors.     

Immunocytochemical localization of transforming growth factor α, epidermal growth factor and epidermal growth factor receptor in the cat endometrium and placenta

This study was undertaken to determine the immunocytochemical localization of transforming growth factor α, epidermal growth factor and epidermal growth factor receptor in the endometrium of ovariectomized cats treated with oestradiol-17β and/or progesterone and in the endometrium and placenta of pregnant cats. Specific immunostaining was observed for all three antibodies. Moderate immunostaining for transforming growth factor α was observed in the epithelium of ovariectomized and oestrogen-treated cats. Dark epithelial staining was observed throughout pregnancy. The epithelial cells in progesterone-treated and peri-implantation animals contained dense deposits of reaction product, which were not reduced in intensity when immunoabsorbed antiserum was used. For epidermal growth factor, light--moderate epithelial staining was observed in ovariectomized and steroid-treated animals, and this increased in pregnant cats. Stromal staining for both the transforming and the epidermal growth factors was limited in steroid-treated animals and increased as pregnancy continued. Dark staining for epidermal growth factor receptor was observed in the epithelium and stroma in all the animals studied. The tips of surface epithelial convolutions in the non-implantation sites were always more darkly stained than in other regions of the surface epithelium. Staining in the placental trophoblast was limited to the syncytiotrophoblast for the two growth factors and the cytotrophoblast for the receptor during most of pregnancy and was absent late in pregnancy. The placental maternal giant cells contained specific immunoreactivity for all the immunogens from the middle of pregnancy to term. This study demonstrates that the two growth factors and the epidermal growth factor receptor are present in the endometrium and placenta of cats and suggests that these growth factors may play an autocrine/paracrine role during reproduction     

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.     

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.     

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.     

Effects of ovariectomy and steroid replacement on the genital tract of the viviparous lizard, Xantusia vigilis

Two glandular components are described in the genital tract of Xantusia: tubal glands in the Fallopian tube and goblet cells in the uterine villi. Sperm or seminal receptacles occur between adjacent villi in the uterus. Forty ovariectomized lizards carrying a silk loop in the wall of the left uterus were treated for two weeks with either progesterone, estradiol-17 β, progesterone plus estradiol or vehicle. Uteri with loops serving as a local irritant, did not differ significantly from the contra-lateral uteri in any group, hence a response similar to the deciduomal reaction of mammals is not found in this lizard. The weight of the genital tract is similar in sham-operated and in ovariectomized lizards injected with either progesterone or the vehicle. Maximal increase in weight of the tract is noted with estradiol treatment, while simultaneous administration of both steroids is followed by a moderate increase of oviducal weight. Tubal glands and sperm receptacles in ovariectomized lizards injected with either the vehicle or progesterone are smaller than those of the sham-operated or ovariectomized lizards treated with estradiol or with estradiol plus progesterone. Goblet cells are small and lack secretory granules in ovariectomized lizards injected with either the vehicle, or with estrogen or progesterone alone. Both steroids, given together, restore the size and apparent secretory activity of the goblet cells. It is concluded that in this viviparous species, both estrogen(s) and progestin(s) are essential for the maturation of the genital tract in the preovulatory stage.     

Endometrial surface and secretory alterations associated with embryonic mortality in gilts administered estradiol valerate on days 9 and 10 of gestation

Administration of estrogen to gilts on Days 9 and 10 of pregnancy results in total embryonic loss by Day 18. The present study examined changes in the uterine endometrial surface and secretion during conceptus attachment in control and estrogen-treated (Days 9 and 10) pregnant gilts. Gilts were unilaterally hysterectomized on either Days 12 and 14 or Days 16 and 18 of gestation. Uterine horns were flushed with saline and conceptuses were evaluated. Intact conceptuses were recovered from all control gilts, whereas estrogen-treated gilts contained normal intact conceptuses only on Day 12 of gestation. Antiviral activity, which reflects conceptus viability, was reduced (p less than 0.01) in uterine flushings after Day 14 in estrogen-treated gilts. Culture of endometrial explants with [3H]glucosamine revealed several glycoproteins that are synthesized during the period of conceptus attachment; however, no difference in glycoprotein synthesis between treatment groups was detected by analysis with two-dimensional PAGE and fluorography. Analyses of the uterine epithelium by scanning and transmission electron microscopy demonstrated that estrogen administration caused an alteration in the uterine surface, a thinning of the uterine epithelial glycocalyx, and a reduction of cationic ferritin binding to the microvilli of the uterine epithelium. Results indicate that conceptus mortality after early administration of estrogen is associated with alterations in the uterine endometrial surface during the period of conceptus attachment in the pig.     

Progesterone-dependent cathepsin L proteolytic activity in cat uterine flushings

Sequence analysis of a cDNA clone for the progesterone-dependent protein (PDP) of the cat uterus revealed that PDP may be cathepsin L. This study was undertaken to directly measure the cathepsin L activity in uterine flushings from pregnant and ovariectomized steroid-treated animals in order to confirm that PDP is cathepsin L. Optimum activity toward the substrate Z-Phe-Arg-NMec was observed at a pH of 5-6. Z-Phe-Phe-CHN2, a specific inhibitor of cathepsin L, significantly inhibited the proteolytic activity present in uterine flushings. Immunoabsorption of PDP from uterine flushings obtained from progesterone (P)-treated cats reduced cathepsin L proteolytic activity to levels observed in ovariectomized and estradiol (E2)-treated animals. In E2-primed and E2 + P-treated animals, proteolytic activity in uterine flushings was detectable after 7 days and peaked after 11-13 days of E2 + P treatment. This proteolytic activity was also dramatically increased before implantation (10-12 days after coitus) in pregnant cats. Thus, our data indicate that changes in cathepsin L activity in uterine flushings are correlated with changes in PDP, the uterine protein synthesized and released from the epithelial cells of the deep uterine glands. PDP, via its cathepsin L proteolytic activity, may play a role in the implantation process.     

Effects of progesterone on the binding of estradiol-receptor to rabbit uterine chromatin

Three day progesterone treatment of ovariectomized rabbits increased $\text{in}$$\text{vitro}$ uterine estrogen-receptor binding to uterine chromatin. The increased binding was traced to changes in chromatin but not the cytosol. Both the number of chromatin acceptor sites and the binding affinity were higher in treated animals. Furthermore, chromatin acidic protein to DNA ratios from treated rabbits were higher by approximately the same factor as for binding sites. A mechanism of synergistic interaction is suggested.     

A comparison of developmental changes in surface charge in mouse blastocysts and uterine epithelium using DEAE beads and dextran sulfate in vitro

The ability of 3.5-day mouse blastocysts and vesicles prepared from maternal uterine epithelium to adhere to surfaces by charge interactions was compared by observing their adhesion to DEAE-Sephadex beads in the presence of increasing concentrations of dextran sulfate. The adhesion frequency for both the blastocysts and epithelium declined in a manner suggesting that predominantly ionic sites were being titrated, but differences between the two tissues in characteristics of the titration curve and susceptibility to neuraminidase digestion indicated that nonionic interactions were relatively more important for blastocysts. Because the threshold concentration of dextran sulfate required to initiate displacement of uterine epithelium from the DEAE beads was at least 4× that required to initiate the displacement of blastocysts, we argue that the uterine epithelium had at least 4× more interactive charged groups on its surface than the blastocysts. These differences were even more pronounced 4.5 days after mating, a time when attachment to the uterine epithelium is normally first seen in vivo. Blastocysts isolated at this time showed a marked increase in resistance to polyanion competition, but the epithelium showed a nearly 50% decline in surface negative charge that was not compensated by nonionic mechanisms. These observations support the conclusion that the initial adhesion of blastocysts in vivo is accompanied by a reduction in negativity of the uterine epithelial surface and by the formation of new trophoblast cell surfaces that adhere by nonelectrostatic interactions.     

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.     

Decidualization in rats given 5alpha-dihydrotestosterone or its propionate neonatally.

In Experiment 1, female rats were given a single subcutaneous injection of 1.25 mg 5alpha-dihydrotestosterone (DHT) or its propionate (DHTP) on day 5 of postnatal life. All of them showed regular estrous cycles as adults like untreated control animals. At about 60 days of age, the rats were ovariectomized and given 7 daily injections of 2 mg progesterone (P) plus 0.2 mug estradiol-17beta (ED). Uterine trauma applied on the 4th day of P-ED injections resulted in well developed deciduomata in all animals by the day after the last injection. This made a sharp contrast to the failure of female rats receiving testosterone propionate (TP) neonatally to give a positive response under similar experimental conditions (Takewaki and Ohta, 1974). The mean weight of traumatized horns was significantly larger in DHTP-treated rats (but not in DHT-treated rats) than in controls. In Experiment 2, rats were ovariectomized on day 4 and given a dose of 1.25 mg DHT or DHTP on day 5. Controls were ovariectomized on day 4 but not given any steroid on the next day. A series of 7 daily injections of 2 mg P plus 0.2 mug ED was started at about 60 days of age, after the animals had received 3 daily injections of 0.2 mug ED or 30 daily injections of 0.1 mug ED. Incidence of deciduomata following uterine traumatization was markedly lowered only in animals treated with DHTP neonatally and given 0.1 mug ED for 30 days as adults, no significant differences being found in both incidence and size of deciduomata among the other groups. It was suggested that the effects of neonatal steroid administration on uterine responsiveness in adulthood are specific to the steroid. The previous conclusion that persistent estrus in androgen-sterilized rats plays a part in the reduction of uterine responsiveness was confirmed. An exposure of rats to estrogen for a prolonged postpuberal period was without effect, unless the animals had received enough androgen neonatally.     

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.