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 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.     

Detection and quantification of CUPED, an estrogen-dependent uterine protein, in uterine fluid and endometrial tissue of estrous and pregnant cats

Uterine flushings, media from cultured endometrial explants, and endometrial tissue obtained from estrous and pregnant cats were analyzed for the presence of a previously characterized, high-molecular-weight, estrogen-dependent glycoprotein (CUPED) by polyacrylamide-gel electrophoresis, Western blots, radioimmunoassay, and immunocytochemistry. Elevated levels of CUPED were present within the flushings, media, and tissue of estrous and 3-day postcoital animals. High levels of CUPED were also present in the flushings of 5-day postcoital animals; but the ability of endometrial explants to synthesize CUPED during short-term culture was greatly reduced, and only some of the endometrial glands contained CUPED secretory granules. CUPED was essentially nondetectable in the flushings, media, and tissues of animals pregnant for 7 or more days. Thus CUPED is present within the uterine lumen of the cycling cat at the time of sperm migration through the uterus and also for the first day or two that the developing blastocyst is present within the uterine lumen. The disappearance of CUPED from the tissue and flushings was correlated with the luteal production of progesterone.     

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.     

Postnatal development of the Mongolian gerbil uterine glands

The development of gerbil uterine glands has been described. Gland formation starts about day 6 postnatally (p.n.). At day 10 p.n. the glandular lumen contains already some fluid, and the glandular cells are in an activated state. The apical shape of the glandular cells depends mainly on the width of the lumen. Secretory vesicles in early developmental stages are electron-lucent. As sexual maturity is reached, secretory granules become electron-dense. This change in secretory granular structure indicates a change in the quality of uterine gland fluid.     

Immunocytochemical localization and messenger ribonucleic acid levels of a progesterone-dependent endometrial secretory protein (cathepsin L) in the pregnant cat uterus.

The purpose of this study was to localize immunocytochemically a progesterone-dependent protein (PDP) and to determine PDP mRNA levels during the initial stage of the implantation period. Uterine tissue was collected from Day 0-18 postcoital animals. The tissue was processed for immunocytochemical localization of PDP, and the endometrial RNA was isolated and analyzed for PDP gene expression by slot-blot hybridization. PDP was detected immunocytochemically as early as Day 5 postcoitus in the epithelial cells of the deep uterine glands, and the intensity of immunostaining appeared to peak by Day 12 postcoitus. PDP was absent in the endometrium obtained from implantation sites after Day 16 postcoitus, but the synthesis of PDP was maintained in the endometrium obtained from nonimplantation sites. Immunogold electron microscopy demonstrated that PDP was present in electron-dense granules of the glandular epithelial cells. PDP mRNA was detectable in the endometrium at Day 5 postcoitus and peaked around Day 10 postcoitus. PDP mRNA was absent in the endometrium from implantation sites after Day 16 postcoitus, but was maintained in the endometrium from nonimplantation sites. In summary, the results of this study illustrate that PDP is synthesized within the epithelial cells of the deep uterine glands, packaged within membrane-bound secretory granules, and released into the uterine lumen. Also, the process of implantation alters the gene expression in a very localized way since PDP mRNA and PDP-positive granules were absent in the endometrial glands obtained from the implantation site within 1-2 days of the onset of implantation, whereas both PDP mRNA and PDP-positive granules were maintained in the endometrial glands from nonimplantation-site regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

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     

An ultrastructural study of the cysts in chicken ultimobranchial glands,with special reference to C-cells

Summary The ultimobranchial glands of the chicken were examined by electron microscopy and immunocytochemistry using a calcitonin antiserum. Electron microscopy confirmed the presence of C-cells, containing numerous secretory granules storing calcitonin, in the luminal lining of cyst-like structures found in these glands. These cells were furnished with prominent microvillar projections at their luminal surface, and the cytoplasm of the apical region was filled with fibril material. Furthermore, the cells contained prominent junctional complexes and desmosomes at their apico-lateral surfaces. In these C-cells, secretory granules were concentrated near the lumen and some were attached to the apical cell membrane. The luminal content of the cysts had a colloid-like and flocculent appearance, and was frequently seen attached to the cytoplasmic projections or apical cell membrane of the C-cells. Since the cysts progressively increase in volume and number with age, it is suggested that they may partly play a role in the storage of excess or unneeded hormonal products.     

Quantification of an estrogen-dependent cat uterine protein (CUPED) in uterine flushings of estrogen- and progesterone-treated ovariectomized cats by radioimmunoassay

We previously reported the purification of an estrogen-dependent cat uterine protein (CUPED) and the preparation of a specific anti-CUPED serum in rabbits. Here, we describe a specific radioimmunoassay for CUPED using the purified CUPED and anti-CUPED serum that was utilized to quantify CUPED in daily uterine flushings obtained from steroid-treated ovariectomized cats. The radioimmunoassay was sufficiently sensitive to measure 0.1-100 ng CUPED. CUPED levels were low in untreated ovariectomized cats, increased within one day after the onset of treatment with estradiol, and remained elevated as long as estradiol was unopposed by progesterone. The levels of CUPED decreased when progesterone was added to the treatment regimen either 7, 14, or 28 days after the initiation of estradiol treatment. The data indicate that the presence of CUPED in the uterine flushings is dependent on the presence of estradiol and the absence of progesterone, that CUPED appears in the uterine lumen within one day after the onset of treatment with estradiol, and that the levels of CUPED are sharply reduced within one day of administration of progesterone and become nondetectable after three days.     

An immunohistochemical study of the distribution of ABH antigens in human submandibular glands at the light and electron microscopic levels.

The distribution of blood group antigens ABH in submandibular glands was studied at light and electron microscopy levels by applying ImmunoGold Silver Staining (IGSS) and post-embedding ImmunoGold (IGS) methods, respectively. In IGSS treated samples, a cytoplasmic and a surface form of antigen localization were discernible in the glandular parenchyma. The former was restricted to most mucous cells and to scattered serous cells: A and B antigens were demonstrated in mucous cells of A and B type glands, while H antigen appeared in most mucous and occasional serous elements regardless of the blood type of donors. The latter appeared as a strong H reactivity on cell surfaces of serous acini and ducts regardless of the patient blood type. The IGS method was applied both on non-osmicated samples embedded in LR White resin and on osmicated, Epon embedded samples. In non-osmicated tissues, antigen labelling was revealed in secretory granules and cell surfaces. Positive secretory granules were found in most mucous cells and occasional serous, intercalated, and striated duct cells. A and B antigens weakly reacted in mucous cells of A and B type glands, respectively, while strong H reactivity was seen in mucous, serous, intercalated and striated duct cells of glands of all types. Surfaces labelled with H antigen were found on both lumenal and basolateral membranes of striated ducts in glands of all types. IGS method applied on osmicated, Epon embedded samples, selectively revealed blood group antigens in secretory granules of serous cells but not in the apical vesicles of striated ductal cells. Cell surfaces were completely unreactive.     

Receptors for luteinizing hormone-releasing hormone.

Antisera to luteinizing hormone-releasing hormone (LH-RH) confer on Araldite sections of occasional rat pituitaries moderate immunocytochemical staining to the large secretory granules of gonadotrophs. Treatment of the sections with LH-RH before anti-LH-RH yields strong staining in all animals, irrespective of presence or absence of staining without pretreatment. This enhancement of staining is specific for LH-RH and is a high affinity, saturable reaction. Staining with or without LH-RH pretreatment is absent when anti-LH-RH absorbed with insolubilized LH-RH is used. Staining is inhibited by carboxyterminally-deficient LH-RH, unaffected by aminoterminally deficient LH-RH.     

An immunocytochemical study of TSH beta storage in rat thyroidectomy cells with and without D or L thyroxine treatment.

Binding sites to the beta chain of thyroid stimulating hormone (TSH) were localized in pituitaries of thyroidectomized rats. Immunocytochemical staining was observed in hypertrophied TSH cells ("thyroidectomy cells") and primarily located in dilated rough endoplasmic reticulum. Staining was also found on the few secretory granules and on some of the intracisternal granules. Some of the thyroidectomy cells stained intensely, while others exhibited very little staining. When thyroidectomized rats were treated with thyroxine 4 days before death, the TSH cells contained more secretory granules, and the intracisternal granules were larger and more numerous. L-thyroxine was 10 times as potent as D-thyroxine in promoting the build-up of granules. Both types of granules stained intensely.     

ULTRASTRUCTURAL LOCALIZATION OF CALCITONIN IN THE PARAFOLLICULAR CELLS OF PIG THYROID GLAND WITH CYTOCHROME c-LABELED ANTIBODY FRAGMENTS

Parafollicular cells in mammalian thyroid glands are thought to be responsible for the secretion of calcitonin. In this study, calcitonin was localized in pig thyroid gland by an indirect immunocytochemical technique using rabbit antiserum directed against synthetic porcine calcitonin for the first step, and sheep Fab fragments prepared against rabbit Fab and coupled to cytochrome c for the second step. The antigenic determinants of calcitonin were present only in the parafollicular cells, whose secretory granules were heavily labeled. Labeling of the cytoplasmic matrix is thought to indicate a possible leakage of the polypeptide from the granules. A striking observation was the complete absence of labeling in the cisternae of the rough-surfaced endoplasmic reticulum and of the Golgi apparatus. It is concluded that the secretory granules of parafollicular cells contain calcitonin; the mechanism of synthesis of this peptide is not clearly understood.     

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.     

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.     

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.     

A light microscopic and ultrastructural study on the presence and location of oxytocin in the equine endometrium

It has been reported that oxytocin is produced not only in the hypothalamus and posterior pituitary but also in outside the classical hypothalamo-neurohypophyseal axis such as the ovary, testis, placenta and in some nonreproductive sites. In the mare, oxytocin-mRNA has been identified in the endometrium, and oxytocin and its neurophysin have been identified in the uterus. In the present study, oxytocin was localised in the endometrium of the mare at the light microscopic and ultrastructural level by immunostaining and immunogold labelling of endometrial biopsy specimens collected during estrus.Strong positive immunostaining for oxytocin was found in the secretory vesicles of the secretory (nonciliated) epithelial cells of the uterine lumen and of the superficial glands. Using immunogold labelling, oxytocin was detected in the secretory vesicles of secretory epithelial cells. The vesicles containing immunoreactive oxytocin were present on the luminal surface suggesting that oxytocin is secreted into the uterine lumen by apical exocytosis. There was no positive immunostaining in ciliated epithelial cells of the uterine lumen and endometrial glands, in the stromal cells, or in the basal endometrial glands. To our knowledge, this is the first report of the location of oxytocin in specific secretory cells in the endometrium of any domestic species. This locally synthesised uterine oxytocin may have an important role in the autocrine/paracrine control of uterine contractility and luteolysis in the mare.     

Postnatal development of the interstitial cells (palisade cells) of the pars intermedia in the cat pituitary gland. An immunocytochemical and ultrastructural study.

The postnatal development of the interstitial agranulated cells (so-called palisade cells) of the pars intermedia in the cat was investigated immunocytochemically and at the ultrastructural level. Since the first postnatal days, a strong vimentin immunoreactivity and a weaker S-100 protein immunoreactivity were detected in the marginal cells lining the pituitary cleft and in the interstitial bipolar cells located within the pars intermedia. No glial fibrillary acidic protein cells have been found in the pars intermedia of any of the animals studied. This immunocytochemical pattern was maintained throughout the postnatal development. Ultrastructurally these cells showed a vast number of cytoplasmic filaments and well-developed junctional complexes. Secretory granules were never seen. In older animals they lined microcavities and microchannels where they project microvilli and present pinocytotic vesicles on their apical surface. No transitional forms between these cells and granulated secretory cells were found. There is a large number of axons and synaptic endings in contact with the granulated secretory cells. From our findings we guess that palisade cells are not a glial derivative, but they may share a common origin with secretory granulated cells.     

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.     

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.