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.     

Estrogen- and progesterone-dependent secretory changes in the uterus of the sheep.

This study was undertaken to determine the effects of 17 beta-estradiol (E) and progesterone (P) on polypeptide synthesis and release from the uterus of the sheep. Uterine flushings (UF) and endometrium were obtained from ovariectomized untreated animals, ovariectomized animals treated with E (approximately 5-10 pg/ml) for 6 days (6E) and ovariectomized animals primed with E for 6 days then treated with P (approximately 1.5-3 ng/ml), in the continued presence of E, for an additional 6 days (6EP). Endometrium was cultured (24 h) in the presence of 3H-leucine (3H-leu) or 3H-glucosamine (3H-glcN), and newly synthesized and released proteins were detected in culture media by fluorography of 10% SDS gels. The quantity of proteins in UF and radiolabeled proteins in explant culture media did not change between treatment groups (p < 0.05). Qualitative changes in the synthesis and release of proteins were observed depending on the steroid treatment. An M(r) 57,000 protein was present in UF and 3H-leu-labeled culture media obtained from animals treated only with E and an M(r) > 200,000 was present in 3H-leu-labeled culture media of endometrium obtained from 6E and 6EP animals. An M(r) 44,000 protein was present only in UF from 6EP animals but could not be detected in endometrial culture media from animals undergoing this steroid treatment. These data show that the endometrium of the ovariectomized sheep undergoes alterations in secretory protein patterns which depend on the presence of E and P.     

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.     

The detection and purification of a cat uterine secretory protein that is estrogen dependent (CUPED)

An estrogen-dependent secretory protein (CUPED) was detected and purified from uterine flushings of ovariectomized cats treated with 17 beta-estradiol. The protein was not detected in uterine flushings obtained from untreated ovariectomized animals or estrogen-primed animals treated with progesterone for 4 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of uterine flushings showed the presence of 1 or 2 protein bands with relative mobility values less than reduced and denatured thyroglobulin (Mr = 330,000). The protein was purified by differential centrifugation and gel filtration chromatography. Antiserum was raised against this purified protein in rabbits. The specificity of the antiserum to uterine fluid proteins was assessed by immunoblotting of electrophoretically transferred proteins. The antiserum cross-reacted with electrophoretically separated CUPED protein bands in uterine flushings. This protein may represent the content of the estradiol-induced secretory granules present in endometrial epithelial cells.     

The ultrastructure of mammary explants from virgin ovariectomized goats during hormone-induced lactogenesis

The effect of insulin (I), cortisol (F) and prolactin (P) on the ultrastructural morphology of epithelial cells of cultured mammary explants from virgin ovariectomized (OV-X) goats were studied. The epithelial cells showed little structural organization and were devoid of fat droplets and secretory protein granules at zero time of culture. The cytoplasm contained few profiles of smooth and rough endoplasmic reticulum and the Golgi apparatus was rudimentary. After being cultured in Waymouth's medium without added hormones the epithelial cells were indistinguishable from epithelial cells of uncultured explants. The addition of I induced changes mainly in the appearance of nucleoli. The nucleoli were enlarged and fibrillogranular areas with light spaces were observed. The most obvious cytological changes of epithelial cells of explants cultured in the presence of I and F are translocation of the nucleus into the basal cytoplasm, increase of rough endoplasmic reticulum, an increase in the size of the Golgi apparatus, presence of one or two lipid droplets and in some cells vacuoles with protein granules were present. Mitochondria were more abundant. The epithelial cells of explants cultured in the presence of I, F and P were characterized by the polarization of organelles within the cytoplasm and by the formation and release of protein granules and small and large fat droplets. The cell nucleus was in the basal cytoplasm, the Golgi apparatus was supranuclear. The rough endoplasmic reticulum was extensively developed and formed large sacs. Golgi vacuoles contained protein granules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light and electron microscopic observation on the cervical epithelium of the rabbit. I

The light and electron microscopy of the cervical epithelium of ovulatory, estrous, and long-term ovariectomized rabbits have been studied to determine what structural changes occur under different hormonal conditions. The percentage of nonciliated secretory cells is 49.6 in ovulatory, 43.6 in estrous, and 23.7 in long-term ovariectomized rabbits, and of ciliated cells is 50.2 in ovulatory, 56.2 in estrous, and 76.3 in long-term ovariectomized animals. The values for the ovulatory and estrous rabbits are significantly different at the P less than 0.05 level from those of the ovariectomized animals. In all 3 groups the general ultrastructure of the normal ciliated cells is similar. Interestingly, the Golgi complex is very prominent in all. Glycogen bodies occur frequently only in ciliated cells of ovariectomized and occasionally of estrous animals. Abnormalities in ciliation are quite common in the ovariectomized rabbits. The structure of the nonciliated secretory cells varies appreciably within and between the 3 groups. In these cells from well-developed epithelia of certain ovulatory and estrous animals, the apical cytoplasm contains secretory granules of at least three types. In addition, very irregularly shaped, dense, perinuclear granules occur, which may be another type of secretory granule or lysosomes. As compared to ciliated cells, the secretory cells have less prominent Golgi complexes, more abundant bundles of intermediate filaments, a more extensive glycocalyx on their apical surface, and more heterochromatic nuclei. In comparison to the cells of well-developed epithelia, the nonciliated cells of some other ovulatory and estrous rabbits are less well differentiated with fewer or no secretory granules and less well developed organelles. In the nonciliated cells of the long-term ovariectomized rabbits, there are no secretory or dense perinuclear granules. There is a decrease in the number of organelles that are involved in secretion, in the size of the cells, and in the amount of nuclear euchromatin.     

Uterotropic action of indomethacin on the rat uterus

Administration of indomethacin (10 mg/kg body weight, twice daily for 6 days) resulted in a significant (P less than 0.01) increase in the weight, and cross-sectional area of uteri of ovariectomized rats, whereas no such effects were observed following indomethacin administration to normal cycling rats. Prostaglandin F2 alpha (PGF2 alpha) content (ng/uterus) and concentration (ng/g wet weight) in the uterus of indomethacin-treated animals were reduced 40.4% and 60.8%. Simultaneous administration of either estradiol-17 beta (E2), progesterone testosterone with indomethacin to ovariectomized rats failed to reduce the uterine weight increase. On the contrary, concomitant administration of E2 (25 or 100 ng/day) and indomethacin resulted in uterine weight increases which were greater than those associated with indomethacin alone. Uterine E2 content was significantly higher in animals treated with indomethacin plus E2 as compared to those given estradiol alone. Uterine uptake of 2,4,6,7-[3H]E2 following i.v. administration was greater in animals pretreated with indomethacin (5 mg/kg, twice daily) for 3 days than in ovariectomized controls. These results suggest that prostaglandins may be involved in the regulation of uterine growth.     

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.     

Estradiol regulation of secretory component: Expression by rat uterine epithelial cells

Sex hormones are known to play an important role in the regulation of mucosal immunity in the female reproductive tract. The purpose of this study was to examine the effect of estradiol (E₂) on secretory component (SC) expression by epithelial cells in the rat uterus and to determine whether SC mRNA is present in uterine tissues and is under hormonal control. When ovariectomized rats treated with E₂ for 3 days and sacrificed 12 h after the last injection, expression of SC on luminal and glandular epithelial cells, as determined by immunohistochemistry, was elevated when compared to control animals. To determine whether E₂ regulation of SC involves mRNA synthesis, uterine RNA was extracted and analyzed by Northern blot. These experiments demonstrated that SC RNA is present in uteri from intact rats and markedly increased when ovariectomized animals are treated with E₂. In other studies, uterine epithelial cells from adult rats were isolated and grown on permeable membranes for 5 to 10 days. Under these conditions, isolated epithelial cells grow to confluence, form tight junctions, and preferentially secrete SC into the apical medium. These studies identify epithelial cells as a key target cell in the uterus for the regulation of mucosal immunity by E₂, which we postulate will play an important role in studies to prevent and/or control the spread of sexually transmitted diseases.     

Light and electron microscopic observations on the cervical epithelium of the rabbit: II

The endocervical epithelium of long-term ovariectomized rabbits treated for 1-10 days with 5 micrograms of estradiol benzoate every 12 hr has been studied by light and electron microscopy. In addition, morphometric data on ciliated and nonciliated cells of rabbits treated for 2, 6, and 10 days are compared to those on untreated ovariectomized, estrous, and ovulatory rabbits. The percentage of ciliated cells increases after ovariectomy to 76.3% and that of secretory cells decreases to 23.7% as compared to estrous controls. Treatment of ovariectomized rabbits with estradiol results in a gradual increase in ciliated and secretory cell area, height, and nuclear area. After 10 days of treatment, cell areas are significantly larger than those in the ovulatory or estrous controls; cell height and nuclear areas have returned to preovariectomized levels; and the percentages of ciliated and secretory cells have reached those of estrous levels. Estradiol stimulates mitotic division of secretory cells but affects ciliogenesis minimally. In ciliated cells, estradiol treatment results in a modest increase in polysomes and granular endoplasmic reticulum and in striking increases in the size of the Golgi complex and in the number of lipofuscin bodies as compared to those in the ovariectomized controls. In secretory cells, estradiol treatment brings about an increase in the numbers of polysomes, Golgi complexes, and cisternae of the granular endoplasmic reticulum, in the sizes of the nucleoli, and in the amount of euchromatin. Secretory granules appear in some cells after 2 days of estradiol stimulation and increase in number through 10 days of treatment. Perinuclear granules are more pleomorphic and heterogeneous in structure and more numerous in the 6- to 10-day-estradiol-treated than in ovulatory animals, and they may function as lysosomes degrading excess secretory product. Deep apical concavities of the secretory cells occur most often after 2 and 6 days of treatment.     

Immunogenic properties of modified antigen E. III. Effect of repeated injections of modified antigen on immunocompetent cells specific for native antigen.

It has been shown that ragweed antigen E loses its major antigenic determinants after denaturation in 8 M urea, but urea-denatured (UD) antigen and an alpha-polypeptide chain isolated from the denatured molecules are capable of priming mouse T cells specific for native antigen. Weekly injections of 10mug UD antigen or alpha-chain into antigen E-primed animals depressed the ongoing IgE antibody response, whereas injections of the same dose of antigen E failed to depress the antibody response. It was found by adoptive transfer experiments that helper activity of antigen E-primed splenic T cells was depressed by the treatment of the donors with either modified antigen or native antigen E. The same treatment of antigen E-primed animals depressed the DNA synthetic response of their splenic T cells to antigen E. The treatment of antigen E-primed animals with UD antigen resulted in a decrease of antigen E-specific IgE-B cells and IgG-B cells in their spleen, whereas the treatment with native antigen expanded the B cell populations. In view of the results obtained in the mouse, cellular basis for the immunologic effects of hyposensitization treatment is discussed.     

Effect of substance P on thyrotropin secretion from the pituitary gland in the rat

The role of substance P (SP) on thyrotropin (TSH) secretion was investigated in ovariectomized (OVX) female, estrogen-primed OVX, and normal male rats. Third ventricular administration of SP induced a significant increase in plasma TSH levels when compared to control animals in E-primed OVX rats (p less than 0.001). The plasma TSH levels increased in a dose-related manner and reached maximum levels at 10 min after injection. In contrast, intraventricularly injected SP failed to alter plasma TSH levels in both OVX rats and normal male rats. Intravenous administration of SP dramatically stimulated TSH release in E-primed OVX rats (p less than 0.001), whereas SP had no effect on the release of TSH when injected in OVX rats and normal male rats. To investigate any direct action of SP on TSH release from the anterior pituitary gland, synthetic SP was incubated with dispersed anterior pituitary cells harvested from E-primed OVX rats and normal male rats. SP, in the dose range between 10(-8) M and 10(-6) M, failed to alter the release of TSH into the culture medium in vitro. These findings indicate that SP has a stimulatory role in the control of TSH release by an action on the hypothalamus but only in estrogen-primed rats.     

Control of gonadotrophin release in Scottish Blackface and Finnish Landrace ewes during seasonal anoestrus

The patterns of LH and FSH secretion were measured in 4 experimental groups of Finnish Landrace and Scottish Blackface ewes: long-term (18 months) ovariectomized ewes (Group 1), long-term ovariectomized ewes with an oestradiol implant, which has been shown to produce peripheral levels of approximately 5 pg/ml (Group 2), long-term ovariectomized ewes with an oestradiol implant for 18 months which was subsequently removed (surgery on Day 0) (Group 3) and short-term ovariectomized ewes (surgery on Day 0) (Group 4). LH and FSH concentrations were monitored in all groups at approximately weekly intervals, before and after Day 0. Finnish Landrace ewes in Groups 1, 2 and 3 had significantly higher mean FSH concentrations than did Scottish Blackface ewes (P less than 0.01). FSH and LH concentrations increased significantly in Groups 3 and 4, but values in Group 4 were significantly lower (P less than 0.01) than those in Group 1 ewes even up to 30 days after ovariectomy. In Group 3, LH concentrations increased to levels similar to those in Group 1. The pattern of LH release was, however, significantly different, with a lower LH pulse frequency (P less than 0.05), but higher pulse amplitude (P less than 0.05). This difference was maintained at least until 28 days after implant removal. We suggest that removal of negative feedback by ovariectomy demonstrates an underlying breed difference in the pattern of FSH secretion and that ovarian factors other than oestradiol are also involved in the negative-feedback control of hypothalamic/pituitary gland function. Furthermore, negative-feedback effects can be maintained for long periods, at least 28 days, after ovariectomy or oestradiol implant removal.     

Combinatorial effects of the phytoestrogen genistein and of estradiol in uterus and liver of female Wistar rats

The knowledge about safety of phytoestrogens on proliferative endpoints in the endometrium is rather limited, particularly when low amounts of estrogens are present like in postmenopausal women. Therefore, we now studied how genistein (GEN) exposure affects proliferative endpoints in the endometrium in estrogenized animals. We investigated the effects of GEN (10 mg/(kg day) BW) on uterine proliferation and on general uterine response markers in intact female rats and ovariectomized (OVX) female rats co-treated with different doses of estradiol (E2; 1 or 4 μg/(kg day) BW). In parallel we investigated generalized hepatic effects of GEN in this co-stimulatory protocol. In agreement to our previous results, GEN treatment of OVX animals for 3 days results in a faint stimulation of the uterine wet weight. In intact animals and in OVX animals co-treated with E2 no effects of GEN on uterine wet weight were detectable. GEN treatment did not affect the uterine epithelial height in intact animals but resulted in a decrease of the protein and mRNA expression of the proliferation marker PCNA. In OVX animals co-treated with E2, GEN antagonized the E2 stimulated increase of the uterine epithelial height and epithelial PCNA expression. Besides PCNA, GEN effects on the uterine mRNA expression of IGF-1, IGF-1R, Complement C3, estrogen receptor- (ER) and -β (ERβ), as well as progesterone receptor were investigated in intact and OVX co-treated animals. Overall there was a tendency in all combinatorial groups that GEN counteracts E2 function in uterine tissue. Surprisingly, while investigating estrogenic response markers in liver, we observed very strong effects of GEN on hepatic marker gene expression. GEN significantly down-regulated CaBP9K and IGFBP1 mRNA levels in intact animals. In OVX animals hepatic CABP9K and IGFBP1 mRNA levels were not affected by E2 treatment. GEN treatment, even in combination with E2, decreased the hepatic CaBP9K expression below the levels observed in untreated animals. Interestingly co-treatment of OVX rats with low dose E2 and GEN resulted in a significant increase of IGFBP1 mRNA expression. Summarising our results we conclude that (1) GEN treatment in the presence of E2 is safe regarding proliferative responses in the endometrium of adult animals; (2) the observation of differences of the GEN activity in intact and OVX/E2 substituted animals can be taken as a hint that GEN may interact mechanistically with progestins which has to be proven in detail in future investigations and (3) the detection of strong effects of the phytoestrogen GEN on hepatic gene expression may point to the need of future investigations to rule out the possibility of adverse responses in this organ.     

Morphofunctional analysis of the chief (peptic) glandulocytes in the stomach of hibernating rodents

Ultrastructure of the chief gastric gland cells has been studied in the ground squirrel (Cytellus erythrogenys Brandt) at various seasons of the year. When the ground squirrel is in the active state, the cells studied do not differ from those in other animals. During winter hibernation, their activity is inhibited. During first days of awakening, the activity of the chief cells increases; the protein-synthesizing apparatus, the Golgi complex become activated, secreating granules accumulate. At the same time, the greatest number of the destroing cellular elements and discharge of the residual bodies out of the activizing cells are observed. Restoration of the normal structure in the chief cells is completed by the end of the second week. No signs demonstrating transformation of additional cells into the chief ones are revealed. Non-differentiated cells in the ground squirrel fundal glands are widely distributed along the whole length of the glandular cervix and occur even in the upper parts of its body.     

Paracrine regulation of epithelial progesterone receptor by estradiol in the mouse female reproductive tract

Regulation of progesterone receptor (PR) by estradiol-17beta (E(2)) in mouse uterine and vaginal epithelia was studied. In ovariectomized mice, PR expression was low in both vaginal stroma and epithelium, but high in uterine epithelium. E(2) induced PR in vaginal epithelium and stroma, but down-regulated PR in uterine epithelium. Analysis of estrogen receptor alpha (ERalpha) knockout (ERKO) mice showed that ERalpha is essential for E(2)-induced PR expression in both vaginal epithelium and stroma, and for E(2)-induced down-regulation, but not constitutive expression of PR in uterine epithelium. Regulation of PR by E(2) was studied in vaginal and uterine tissue recombinants made with epithelium and stroma from wild-type and ERKO mice. In the vaginal tissue recombinants, PR was induced by E(2) only in wild-type epithelium and/or stroma. Hence, in vagina, E(2) induces PR directly via ERalpha within the tissue. Conversely, E(2) down-regulated epithelial PR only in uterine tissue recombinants constructed with wild-type stroma. Therefore, down-regulation of uterine epithelial PR by E(2) requires stromal, but not epithelial, ERalpha. In vitro, isolated uterine epithelial cells retained a high PR level with or without E(2), which is consistent with an indirect regulation of uterine epithelial PR in vivo. Thus, E(2) down-regulates PR in uterine epithelium through paracrine mechanisms mediated by stromal ERalpha.     

Ultrastructure of the uterus in an ovariectomized gecko (Hemidactylus turcicus) after administration of exogenous estradiol

The uterus of an oviparous gecko, Hemidactylus turcicus, was analysed after ovariectomized females underwent a period of treatment (up to 14 days) with exogenous estradiol. Analysis focused on the uterine mucosa, which is made up of an epithelial layer and an underlying lamina propria containing the shell glands. These tissues are thought to be responsible for secretion of the eggshell components and were thus chosen for analysis using transmission electron microscopy. In ovariectomized females, the epithelial layer was low and cuboidal with minimal/no differentiation or secretory activity. Treatment with exogenous estradiol resulted in a significant increase in cell height associated with gradual differentiation of the epithelium. Development of non-ciliated cells included production of secretory granules (low electron density) at the apical cell surface. The shell glands showed less obvious changes over the course of treatment. Shell glands contained two cell types: dark cells with darkly staining nuclei and organelles, and light cells with very indistinct nuclei and organelles, except for prominent rough endoplasmic reticulum and free ribosomes. This study provides results consistent with published light microscopy studies for other reptiles and additionally provides ultrastructural details of reptilian uterine development not previously available.