Localization of oviductal glycoproteins within the zona pellucida and perivitelline space of ovulated ova and early embryos in baboons (Papio anubis).

The estrogen-dominated baboon oviductal epithelium synthesizes and secretes a family of oviduct-specific glycoproteins. The objective of this study was to determine if these glycoproteins become associated with ova and early embryos. Ovarian and oviductal eggs obtained from superovulated baboons 72 h post-hCG were subjected to an indirect immunofluorescent assay that used a polyclonal antibody prepared toward the baboon oviduct-specific glycoproteins. Oviductal ova as well as 2-cell and 4-cell embryos showed intense, specific fluorescence within their zonae pellucidae. Ovarian ova did not exhibit fluorescence. Oviductal eggs were also fixed and processed for peroxidase-antiperoxidase immunocytochemistry and colloidal gold immunoelectron microscopy to confirm the immunofluorescent data and to determine the subcellular distribution of the antigens. Oviductal ova as well as 2-cell and 3-cell embryos exhibited immunolabeling localized within the zona. Gold particles were distributed uniformly throughout the width of the zona. Occasional groupings of gold particles were observed within the zona. Also, in most eggs, immunoreactivity was observed associated with flocculent material in the perivitelline space as well as the vitelline membrane. Furthermore, immunogold labeling above background level was noted in the cytoplasm of the eggs, particularly in the blastomeres of 3-cell embryos. Collectively, these results indicate that baboon estrogen-dependent oviductal secretory glycoproteins become intimately associated with oviductal ova and with embryos.

Immunohistological distribution of the secretory endometrial protein, 'pregnancy-associated endometrial alpha 2-globulin', a glycosylated beta-lactoglobulin homologue, in the human fetus and adult employing monoclonal antibodies.

We have previously demonstrated that pregnancy-associated endometrial alpha 2-globulin (alpha 2-PEG), the human glycosylated beta-lactoglobulin homologue (HG-BLG), is quantitatively the major secretory soluble protein product of the secretory endometrium during the latter half of the menstrual cycle and decidua spongiosa of the gestational endometrium during early pregnancy, and is principally localized to the glandular epithelium. In the present study employing monoclonal antibodies in immunohistological techniques, the distribution and localization has been examined in normal and pathological tissues of the adult and first-trimester fetus. No significant staining for alpha 2-PEG was detected in any nonreproduction-associated tissue in the normal adult nor any tissue in the fetus. In the adult, most intense staining was associated with the endometrial glandular epithelium in the uterus or in ectopic sites in patients with endometriosis. During the menstrual cycle and pregnancy, appearance of alpha 2-PEG in endometriosis was strongly linked with its appearance in uterine endometrial tissue, suggesting that endometriotic tissue exhibited competence to respond to the same hormonal milieu required to induce synthesis in the uterine endometrium. Localization to the mucosal epithelium of the Fallopian tube was consistent with synthesis of alpha 2-PEG, albeit at low levels, and staining at this site reflected fluctuations of staining within the uterus. Of the pathological specimens examined, staining was only detected in a proportion of ovarian carcinomas. No staining was detected in the mammary gland, a site of beta-lactoglobulin synthesis, whether obtained during pregnancy or lactation.(ABSTRACT TRUNCATED AT 250 WORDS)

Human 'pregnancy-associated endometrial alpha 1-globulin', a 32 kDa insulin-like growth factor-binding protein: immunohistological distribution and localization in the adult and fetus.

We have previously shown that pregnancy-associated alpha 1-globulin, a small molecular weight (32 kDa) insulin-like growth factor-binding protein (IGF-BP), is quantitatively the major secretory protein product of the decidualized endometrium during human pregnancy and is localized principally in the decidual cell. In the present study, employing monoclonal antibodies in immunohistological techniques, the distribution and localization of IGF-BP has been examined in normal and pathological tissues of the adult and first trimester fetus. In the adult, most intense reactivity was associated with endometrial stroma and their derived decidual cells in the uterus or in ectopic sites in patients with endometriosis. During the menstrual cycle, the appearance of IGF-BP in endometriotic tissue was linked with its appearance in uterine endometrial tissue. The only other adult cells where significant staining was detected was in the luteal cells of the corpus luteum. Production of the protein was not a feature of carcinomas. In the fetus, the protein was localized in lymphoid-myeloid progenitor cells and hepatocytes of the liver and at lower levels in testicular Leydig cells and adenocortical cells. These observations suggest highly specific tissue expression of this protein and support a specialized role for this protein in progenitor cells of the lymphomyeloid system, in certain steroid hormone-producing cells and in the decidual cell in pregnancy.

Oviductal epithelium of the baboon: hormonal control and the immuno-gold localization of oviduct-specific glycoproteins.

Oviducts were obtained from a series of cycling and ovariectomized steroid-treated baboons. The lining epithelium of the ampulla and isthmus was analyzed by light and electron microscopy. Both morphological and cytomorphometric analyses revealed that the morphological and functional state of the oviductal epithelium in the baboon is controlled by the ovarian steroids. Additionally, a clear cephalocaudal steroid-responsive gradient was observed when the data from the ampulla and isthmus of the same animal were compared. Within the ampulla, estradiol induced hypertrophy, hyperplasia, ciliogenesis, and secretory activity, whereas adding progesterone to the treatment regimen (+/- estradiol) resulted in atrophy, deciliation, apoptosis, and loss of the secretory activity. These cyclic processes were less evident in the isthmus. We also used an indirect electron microscopic immunogold technique and a previously characterized polyclonal antibody to determine the localization of oviduct-specific glycoproteins. These glycoproteins were present in every secretory granule observed, regardless of oviduct region, electron density, or size of the secretory granule. In summary, our data show that 1) estradiol induces and maintains the mature epithelium of the baboon oviduct, 2) steroid withdrawal or the administration of progesterone causes regression of the epithelium, and 3) the previously identified estradiol-dependent oviduct-specific glycoproteins are synthesized within and released from the secretory epithelial cells.

Estrogen and progestin receptors in the reproductive tract of male and female primates.

With the aid of monoclonal antibodies specific to the estrogen and progestin receptors, we have examined the cellular localization of these proteins in the reproductive tract of male and female macaques. Two striking findings have resulted from our work with these new reagents. First, these receptors are detectable only in cell nuclei, regardless of hormonal treatment, and second, they are often detectable in stromal, but not epithelial cells when the epithelial cells undergo various estrogen or progestin-dependent events. The latter observation has led us to conclude that stromal cell-epithelial cell interactions may play previously unappreciated roles in the hormonal control of the primate reproductive tract. The lines of evidence that have drawn us to this conclusion will be reviewed.

Sulfated oviductal glycoproteins in the rabbit: quantitation by competitive enzyme-linked immunosorbent assay.

The rabbit oviductal epithelium synthesizes and secretes a family of antigenically related, sulfated oviductal glycoproteins (SOG). Anti-SOG monoclonal antibodies (Mabs) were produced and two (Mab 1 and Mab 2) were selected for further characterization. Periodate oxidation of Western blots of oviductal fluid did not affect the binding of Mab 1 or Mab 2, thus suggesting that these antibodies recognized protein rather than carbohydrate epitopes on SOG. The specificity of Mab 1 was determined by Western blot analysis of tissues obtained from estrous rabbits and from the male rabbit reproductive tract. SOG was identified in tissue extracts of both the oviductal ampulla and isthmus. Cervix was the only non-oviductal tissue with which Mab 1 cross-reacted. Mab 1 was used to isolated SOG from whole oviductal fluid by immuno-affinity chromatography. Affinity-purified SOG and Mab 1 were used to develop a quantitative, SOG-specific, competitive enzyme-linked immunosorbent assay. This assay was used to quantify SOG in rabbit oviductal fluid collected during estrus and pseudopregnancy. SOG secretion during pseudopregnancy was resolved into two transient episodes of increased secretion. Maximum SOG secretion (X = 1039 +/- 199 micrograms/day) occurred within 48 h of the induction of pseudopregnancy. A second period of enhanced SOG secretion (X = 308 +/- 46 micrograms/day) occurred during the fifth and sixth days of pseudopregnancy. Baseline SOG secretion occurred during estrus at approximately 60% of maximum postovulatory secretion.

CA 125 in normal tissues and carcinomas of the uterine cervix, endometrium and fallopian tube. I. Immunohistochemical detection.

The distribution of cancer antigen 125 (CA 125) has been investigated in normal tissues and carcinomas of the Müllerian duct by immunohistochemical methods using the monoclonal antibody OC 125. Detection of CA 125 was most intense in cryostat sections and decreased in formalin fixed and paraffin embedded tissues according to the duration of fixation. Enzymatic digestion with neuraminidase or alkaline hydrolysis abolished specific staining suggesting the antigen is a sialylsaccharide bound to protein by alkali-labile linkage. Immunohistochemical staining demonstrated the presence of CA 125 in all normal glandular epithelia of the endocervix, endometrium and fallopian tube in different distribution patterns. In normal endometrium the cellular distribution pattern was related to the menstrual cycle. In endocervical, endometrial and tubal adenocarcinomas CA 125 was found in 73% of cases. In glandular structures the antigen was concentrated at the luminal surface of the tumour cells, in solid tumour areas it was spread throughout the cytoplasm or concentrated in large cytoplasmic vacuoles. The expression of CA 125 was considerably lower in solid tumour areas. These data show that CA 125 is not a true "tumour marker", but a product of female genital mucosae and of their cancerous derivates provided their synthesizing ability is not lost in the course of pathologic differentiation.

CA 125 in normal tissues and carcinomas of the uterine cervix, endometrium and Fallopian tube. II. Immunoradiometric determination in secretions, tissue extracts and serum.

The study deals with the occurrence of cancer antigen 125 (CA 125) in the normal and neoplastic uterine cervix, endometrium and fallopian tube and its applicability as a tumour marker. CA 125 concentrations were measured in 52 secretion specimens, in cytosol fractions of 97 tissue biopsies and in serum from 47 women with nonmalignant disorders and from 334 patients with carcinomas. High quantities of CA 125 (780-454860 U/ml) were detected in cervical mucus, intra-uterine and tubal fluid, exceeding those in the corresponding serum samples by factors of up to 2000. CA 125 concentrations were 9-53 fold higher in cytosol fractions of normal and neoplastic glandular epithelia of the endocervix and endometrium than in those of cervical squamous epithelia and the cervical wall. Despite similarly high antigen concentrations in normal glandular epithelia and adenocarcinomas serum levels elevated to above 65 U/ml were only found in patients with malignant tumours. The positivity rates in serum increased with tumour extent and were 0-43% for primary and 63-79% for recurrent cervical, endometrial and tubal adenocarcinomas. During long-term follow-up, CA 125 serum concentrations were concordant with the clinical course in 10 out of 11 patients with progressive carcinomas. According to these results, the release of CA 125 into the peripheral blood is apparently dependent on the infiltrative growth and the mass of the tumour rather than on the local tissue concentrations. The clinical use of CA 125 is limited to the detection of advanced adenocarcinomas of the Müllerian duct.

Immunohistochemical localization of calcitonin gene-related peptide and bombesin/gastrin-releasing peptide in nerve fibers of the rat, guinea pig and pig female genital organs.

The localization and distribution of calcitonin gene-related peptide (CGRP) and bombesin/gastrin-releasing peptide (GRP) immunoreactivity were studied in the rat, guinea pig and pig female genital organs with indirect immunohistochemical technique. In the rat, guinea pig and pig, CGRP and GRP immunoreactivities were localized in nerve fibers of the uterus, ovary and oviduct. Generally, CGRP-immunoreactive nerve fibers were intensely stained, while GRP-immunoreactive nerve fibers exhibited moderate immunoreactivity. The number of GRP-immunoreactive nerve fibers in these organs was lower in comparison with that of CGRP-immunoreactive nerve fibers. The pattern of distribution of these nerve fibers was very similar in different genital organs of all species studied. In the uterus of rat, guinea pig and pig, CGRP- and GRP-immunoreactive nerve fibers and nerve bundles were observed in the muscular membrane and around blood vessels. Some delicate CGRP- and GRP-immunoreactive nerve fibers were also present in the submucous layer of the uterus. In the oviduct, CGRP- and GRP-immunoreactive nerve fibers were seen in the muscular membrane, around blood vessels and in the submucous layer. In the ovary, CGRP- and GRP-immunoreactive nerve fibers were distributed in medullary stroma, in close contact with blood vessels and between follicles of different stages of development.

Electromyographic activity and noradrenaline content of the rabbit oviduct under different hormonal states.

Spontaneous electromyographic (EMG) activity of the oviduct recorded in vivo in untreated, estrogen-treated, and progesterone-treated castrated rabbits was found to exhibit two main patterns: short spike bursts lasting 1-10 s and long trains of action potentials lasting several minutes, which constituted the major component of EMG activity. After estrogen treatment, both wet weight and noradrenaline (NA) content of the castrated rabbit oviduct were enhanced mainly at the ampullary-isthmic junction; long trains of discharges were significantly shorter (2.0-2.7 min vs 3.6-4.6 min) and appeared at more frequent intervals (9.8-12.2 min vs 14.2-22.6 min). After progesterone treatment, spontaneous EMG activity was not significantly different from that in untreated castrated rabbits (as was the NA content) except at the ampullary-isthmic junction. NA injection elicited a stimulatory response of the oviduct lasting 1-7 min in the three hormonal states. Phentolamine strongly depressed spontaneous EMG activity but the inhibition was more transient in castrated rabbits than in estrogen-treated and progesterone-treated animals. Propranolol had no effect on spontaneous EMG activity. These data and the high NA concentrations found in all parts of the isthmus support the hypothesis that adrenergic innervation plays a role in the organization of oviductal motility in the rabbit.

Estrogen and progesterone receptors in the oviduct during egg transport in cyclic and pregnant rats.

We investigated the temporal relationships between ovum transport and changes in the concentration of nuclear steroid receptors in the oviduct of cyclic and pregnant rats. A lack of parallelism between estrogen and progesterone fluctuations in plasma and their respective nuclear receptor concentrations in the oviduct predominated during egg transport. In pregnant animals, oviductal egg transport took 24 h longer than in nonpregnant animals. In both conditions, transport was initiated while the action of estrogen and progesterone on the oviduct--measured as nuclear receptor accumulation--was decreasing. Three or four days later, depending on whether the animal was pregnant, the eggs entered the uterus shortly after an increase in the nuclear receptor accumulation of both hormones. Treatment with RU486, a progesterone receptor-blocking agent known to cause premature arrival of eggs in the uterus, advanced estrogen receptor accumulation in the oviduct of pregnant rats. These data suggest that the arrival of eggs in the uterus is timed by a transitory increase in nuclear estrogen receptor in the oviduct that does not necessarily reflect a similar change of circulating estradiol. Moreover, in pregnant rats, the onset of this estrogenic action is delayed by a progesterone receptor-mediated effect that hinders nuclear estrogen receptor accumulation.

Endocytosis in the epithelium of the mouse oviduct.

We studied the pathway of serum protein transport into the lumen of the mouse oviduct by localizing several tracer proteins in the oviduct after intravenous injection on days 1, 5, and 11 of pregnancy. Fluorescent proteins were observed in the lamina propria and in vesicles in the lumenal epithelial cells mainly in the preampulla segment on days 5 and 11 of pregnancy. In the isthmus, there was much less fluorescence in the lamina propria and no fluorescent vesicles in lumenal epithelial cells. This is similar to previous observations on day 1 and indicates that the uptake of serum proteins into lumenal epithelial cells in the preampulla is not limited to the time when embryos are present in the oviductal lumen. Horseradish peroxidase (HRP) was present in the lamina propria of the preampulla on days 1 and 5, but direct tracer movement into the oviductal lumen was blocked by the epithelial junctional complexes. Within the epithelial cells, HRP was localized in endocytic vesicles along the basolateral membrane, multivesicular bodies (mvb), elongated dense bodies below the nucleus (bdb), and many small vesicles near the apical surface of the cells. Ferritin was also used as a tracer and was observed in the same locations as HRP. Acid phosphatase in the epithelial cells of the preampulla on day 1 was localized in mvb and bdb, indicating that these structures are lysosomes. It appeared that HRP and ferritin followed two pathways after basolateral endocytosis by the epithelial cells in the preampulla: 1) they were transported to apical vesicles that may release their contents into the oviductal lumen, or 2) they were transported to lysosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of progesterone receptor with monoclonal antibodies to the human progestin receptor.

A series of rat and mouse monoclonal antibodies to the human progesterone receptor (PR) has recently been produced. These antibodies were used for the immunocytochemical identification of PR in several mammalian species including humans. The specificity of the monoclonal antibodies for PR was confirmed by using competition studies with purified PR and by comparison of the immunostained tissues, known from steroid binding assays to be receptor rich, with immunostained tissues known to be receptor-poor. Immunoreactive PR was found in the nuclei of uterine epithelial, stromal, and smooth muscle cells; benign ductal and lobular epithelial cells of the breast; ovarian surface epithelium; ovarian stroma and luteal cells; pulmonary parenchymal cells; and selected pituitary parenchymal cells. A proportion of the following selected human tumors expressed PR: breast carcinomas, endometrial carcinomas, ovarian carcinomas, and meningiomas. PR was localized to the nuclei of all progesterone target tissues even under conditions where the vast majority of the receptor is unoccupied by steroid, suggesting that the unoccupied as well as the steroid-occupied form of PR are predominantly nuclear proteins, as observed previously for estrogen receptor and rabbit PR.

Immunocytochemistry of ion transport mediators in the genital tract of female rodents.

With immunocytochemistry, we have determined distribution of sodium, potassium-adenosine triphosphatase (Na+, K+-ATPase) and of three isoenzymes of carbonic anhydrase (CA) and have shown absence of the chloride channel, Band 3 protein, in the genital tract of female rodents. Staining for Na+,K+-ATPase was strongest in the ampullary oviduct and uterine glands in the mouse. In the mouse and rat ovary, immunostaining evidenced CA I, II, and III in theca interna cells where the enzyme could affect the pH of follicular fluid. The zona pellucida of the ovary and cytoplasmic foci in follicular granulosa cells stained for content of only CA I in mouse ovary, suggesting synthesis of a zona pellucida component by granulosa cells. CA II in mouse oviductal epithelium increased from the negative infundibulum to the variably positive ampulla and isthmus to the uniformly positive interstitial segment. The content of CA III varied inversely to that of CA II. The prevalence of CA II-positive cells apparently corresponded with that of nonciliated cells, whereas abundance of CA III-positive cells concurred with that of ciliated cells in regions of the mouse oviduct. The rat oviduct lacked CA II but, like that of the mouse, showed CA III in the proximal region. The staining for CA II in surface epithelium exceeded the reactivity in glandular epithelium in the mouse uterus, except during estrus. In contrast, rat uterus evidenced CA II in glandular but not surface epithelium. These results testify to possible significance of various ion transport mechanisms for biologic activities of diverse cells in the female genital tract.

Plasma and tissue concentrations of ceforanide and cefazolin in women undergoing hysterectomy.

Thirty-four women who underwent vaginal hysterectomy received either ceforanide or cefazolin as perioperative antimicrobial prophylaxis. Samples of plasma, myometrium, endometrium and fallopian tubes were obtained at various intervals after injection and were assayed for cephalosporin concentration. Following intramuscular injection approximately 1 h prior to surgery, both drugs provided adequate tissue levels at the time of the procedure. Although both antimicrobials achieved similar tissue concentrations, all tissue samples for ceforanide exceeded the MIC90 for Escherichia coli while in the cefazolin group 9/18 myometrial samples and 10/15 endometrial samples fell below the MIC90 for this organism.

Cytoplasmic and nuclear progesterone receptors in human fallopian tube and their relationship to plasma steroids during the menstrual cycle.

Progesterone (P) binding sites in the ampullary and isthmic regions of the fallopian tube and corpus myometrium were measured during the follicular, ovulatory, and luteal phases. Both cytosolic and nuclear fractions were used for P receptor determination. The concentrations of P receptors were similar in the isthmus and myometrium in all three phases. Although the absolute concentration of nuclear P receptor was always lower than that of the cytosolic receptor, the overall pattern of receptor concentration in the different regions was the same, being highest in the ampulla. Both cytosolic and nuclear receptor concentrations in all tissues were lowest in the luteal phase and highest in the ovulatory phase. In all tissues examined in the luteal phase the cytosolic P receptor but not the nuclear receptor was inversely related to plasma P concentration. The data are compatible with a dual control of P receptors by estrogen and P.

Segmental distribution and gestational changes of GABA-transaminase activity in the rat oviduct.

The occurrence and the localization of 4-aminobutyrate:2-oxoglutarate transaminase (GABA-transaminase) in the non-pregnant and pregnant rat oviduct were examined using biochemical and enzyme histochemical techniques. Specific GABA-transaminase activity was detected in the ampullary and isthmic portions of the oviduct as well as in the utero-tubal junction. The enzymic activity was lower in the ampullary than in the isthmic or intramural segments of the oviduct. Pregnancy induced a significant increase of GABA-transaminase activity in each portion of the oviduct. Enzyme histochemistry showed the highest GABA-transaminase reactivity at the level of the epithelial cells of the oviduct irrespective of the portion of the tube examined. A faint specific activity was demonstrated in the smooth muscle of the oviduct while the serosa did not show specific staining. Our findings indicate that: the observed increase of GABA-transaminase activity in the oviduct of the pregnant rat may be responsible for the reduced GABA levels in the oviduct during gestation; and the extraneuronal localization of GABA-transaminase activity does not seem to support the suggestion of a possible GABAergic innervation of the oviduct.

The regional distribution of NPY-, PHM-, and VIP-containing nerves in the human female genital tract.

The regional distributions of neuropeptide Y (NPY), peptide histidine-methionine (PHM), and vasoactive intestinal polypeptide (VIP) immunoreactivities in the human female genital tract have been estimated by specific radioimmunoassays, and their molecular forms determined by chromatography. The localisation and distribution of these three peptides was carried out by immunocytochemistry. The vagina and cervix contain high concentrations of NPY- and VIP-immunoreactive nerves, mainly localised around the vascular and nonvascular smooth muscle. VIP-containing nerves were, in addition, seen beneath the cervical and, in particular, the vaginal epithelium. A comparatively high level of immunoreactive NPY is found in the fallopian tube, mainly around the circular muscle coat. There is evidence that VIP is a neurotransmitter in the female genital tract, and these results suggest a similar role for NPY and PHM.

Gamma-aminobutyric acid in the genital tract of the rat during the oestrous cycle.

The highest values of gamma-aminobutyric acid (GABA) in the genital tract of the rat at different stages of the oestrous cycle were found in the oviduct (3.5-7 micrograms/mg protein) and the lowest in the ovary (50-100 ng/mg protein). The values for uterus and vagina ranged between 80 and 150 ng/mg protein. GABA (10-30 ng/microliter) was also found in fluid in the ovarian bursa. At 11:00 h, on the day of oestrus, GABA content increased in the ovaries but values in the oviducts were maximal at 11:00 h on the day of pro-oestrus. Variations in GABA content of the vagina were also found. Uterine cervix or uterine horn showed no changes during the oestrous cycle. The GABA content was not uniform throughout the oviduct: the highest values were found in the portion next to the ovary. At 10 days after removal of the right oviduct, GABA values in the ovary and ovarian bursa fluid decreased on the operated side. At 1 month after surgery, the values in ovary were normal but the values in ovarian bursa fluid were still low, suggesting that the source of ovarian GABA was not the oviduct. The variations observed in the present paper suggest an involvement of GABA in reproductive physiology.

Human fallopian tube contains placental protein 14.

Radioimmunoassay and immunoperoxidase staining were used to study the tissue content and localization of an endometrial protein, placental protein 14, in the human fallopian tube. Placental protein 14 immunoreactivity was found in saline extracts from all fallopian tubes tested (n = 14). In the fimbrial part the placental protein 14 content was higher in the secretory than in the proliferative phase (p less than 0.01). No difference was found in the placental protein 14 content between the isthmic, ampullar, and fimbrial parts of the tube. Immunoperoxidase staining localized placental protein 14 to the ciliated and secretory epithelial cells of the mucosa in all parts of the tube regardless of the phase of menstrual cycle. The occurrence of the same protein in the endometrium and fallopian tube is compatible with the common embryonic origin from the müllerian duct of these tissues.