Cervicovaginal Epithelium Research Articles

Metabolic Activation of Carbon Tetrachloride by the Cervico‐Vaginal Epithelium in Rodents

The metabolism and binding of 14C-labelled carbon tetrachloride (CCl4) in the genital tract of female adult or juvenile NMRI-mice and Sprague-Dawley rats (mainly in the pro-oestrous/oestrous stage) and an adult New Zealand rabbit were studied. A marked irreversible binding of radioactivity in the squamous cervico-vaginal epithelium of mice given intravenous injections of 14C-CCl4 was revealed by autoradiography of solvent-extracted tissue. The localization of binding in the mouse genital tract incubated with 14C-CCl4 under air was similar to that observed in vivo. Bound radioactivity was also present in the cylindrical epithelium of the rabbit vagina incubated with 14C-CCl4 in vitro. For a comparison, no preferential binding of radiolabelled diethylstilbestrol or ethinylestradiol was observed in the mouse cervico-vaginal epithelium. The level of irreversible binding to PMSG-primed (pregnant mare's serum gonadotrophin) vaginal epithelial 100 x g supernatants of mice and rats incubated with 14C-CCl4 under air was low. Addition of the reducing agent dithionite to the incubations increased the binding in the vaginal epithelium 20-fold. In juvenile mice and rats injected with 14C-CCl4, the levels of metabolites in the epithelium were low, whereas PMSG-primed juvenile rats contained a higher level of metabolites. The results show that the cervico-vaginal epithelium can metabolically activate CCl4 to reactive metabolites and suggest that the metabolism is under endocrine control.

Treatment with different antiestrogens in the neonatal period and effects in the cervicovaginal epithelium and ovaries of adult mice: a comparison to estrogen-induced changes.

Female mice of the NMRI strain were treated for the first 5 days after birth with the following compounds: diethylstilbestrol (DES), MER-25 (ethamoxytriphetol), tamoxifen, ICI 47.699 (the cis-isomer of tamoxifen, an estrogen agonist), clomiphene, nafoxidine or 17 beta-estradiol-3-benzoate (E2). Females were killed at 8 wk or 6 mo and, in the case of tamoxifen also at 12 mo. The cervicovaginal region and the ovaries were prepared for histological studies. MER-25 had no effect on either the cervicovaginal epithelium or ovarian histology. Tamoxifen, clomiphene and nafoxidine resulted in extensive regions with a heterotopic columnar epithelium (HCE) in the cervicovaginal preparations. At 8 wk these regions were more widespread than those observed after treatment with DES and E2. While earlier studies have shown a progressive development of DES-induced HCE, that induced by the antiestrogens regressed with time. All ovaries from adult females treated with DES or E2 lacked corpora lutea. For the antiestrogens there were ovaries with or without corpora lutea, and this treatment was not incompatible with fertile females. It is concluded that in the neonatal period, the cervicovaginal epithelium is more sensitive to antiestrogens than central structures (hypothalamic nuclei), but for DES the opposite is true.

Effects of long-term implantation of vaginal concretions on the cervicovaginal epithelium of mice.

In two experiments, neonatal female BALB/cCrgl or BALB/cfC3HCrgl mice were given subcutaneous injections of 5 micrograms 17 beta-estradiol or sesame oil for the first 3 days of life and were ovariectomized at 60 days of age, at which time vaginal concretions (Experiments I and II) or silica (Experiment II) were implanted intravaginally. Mice were examined at 12 months of age. Three abnormal cervicovaginal epithelial responses were noted: persistent vaginal stratification/cornification (PVS); prominent vaginal squamocolumnar junction (SCJ); epithelial pegs, downgrowths, or lesions (dysplasias). PVS, not present in unimplanted controls, occurs in at least half of the members of the neonatally estrogen-treated groups; implants of concretions or silica did not increase its incidence significantly. Although SCJ was observed in implanted but not in unimplanted controls, its incidence was significantly higher in neonatally estrogen-treated mice than in either control group. The elevated incidence in neonatally estrogen-treated mice was not increased further by implantation of concretions or silica. In neonatally estrogenized mice, the subsequent implantation of a concretion significantly increased the incidence of cervicovaginal abnormalities. Increased PVS and SCJ are teratological consequences of neonatal exposure to a small amount of estrogen; on the other hand, increased dysplasias may, in part, be responses of the estrogenized vaginal epithelium to the concretions.

Effect of Folate Deficiency on the Reproductive Organs of Female Rhesus Monkeys: a Cytomorphological and Cytokinetic Study

The effect of folate deficiency was studied on the cytomorphology and the kinetics of proliferation of ovarian granulosa cells, and the epithelial cells lining the uterus, cervix and vagina of six sexually mature female rhesus monkeys. The cellular kinetics were studied by in vivo labeling with [3H]thymidine, followed by autoradiography of the histological sections of tissue biopsies taken at various time intervals and labeled mitosis curves. The folate-deficient monkeys showed an increased number of atresic and cystic ovarian follicles with depletion of granulosa cells. There was also a significant impairment of the proliferation kinetics of granulosa cells as indicated by decreased [3H]thymidine labeling index (L.I.), and marked prolongation of the pre-DNA-synthetic time (G1 phase); the DNA-synthetic (S phase) and postsynthetic (G2 phase) periods were only mildly prolonged. These changes may have impaired the ovarian synthesis of the steroidal hormones, estrogen and progesterone. The cervicovaginal epithelium showed megaloblastosis, multinucleation and impairment of orderly proliferaton and maturation. Less pronounced changes were also seen in the uterus. The cytomorphological and functional disturbances in the uterus, cervix and vagina may be partly secondary to those in the ovaries, and partly due to the depletion of their essential folate coenzymes.folate deficiency cytomorphology cytokinetics [3H]thymidine rhesus monkey

Genital warts and cervical cancer. I. Evidence of an association between subclinical papillomavirus infection and cervical malignancy.

A blind comparative survey was undertaken to study the prevalence of subclinical papillomavirus infection (SPI) in a representative sample of women treated surgically for invasive or preinvasive cervical neoplasia. According to a semiobjective rating system, 73 of 80 women (91%) with cervical neoplasia and ten of 80 matched controls (12.5%) showed histologic evidence of human papillomavirus (HPV) infection. Sixty of the controls (75%), but none of the study group, had normal cervicovaginal epithelium. A highly significant statistical relationship exists between subclinical papillomavirus infection of the lower genital tract and the occurrence of cervical neoplasia (F = 378; P less than 0.001; X2 = 109, P less than 0.001). The prevalence of SPI was seven times greater in the study group than in comparable controls of equivalent disease status. Because both are covariables of promiscuity, statistical association exist between cervical neoplasia and all sexually transmitted diseases. However, the strength, specificity and consistency of this relationship suggest that SPI may be a precursor or cervical malignancy. This contention is given biologic plausibility by a broad fabric of supporting epidemiology, virologic and clinicopathologic evidence.

The effects of intrauterine exposure to stilboestrol on the cervicovaginal epithelia.

THE epithelial disposition of the lower genital tract in 10 females exposed to diethylstilboestrol (DES)in utero was studied. Significant deviations from the normal pattern were found including the development of squamous carcinomain situ in one patient.

Effect of epidermal chalones on induction and growth of tumors of the cervicovaginal epithelium in mice

Interferon is observed in culture medium in interaction of L-target cells (synchronized in the G1 and S phase and unsynchronized) with lymphocytes (intact, immune and actinomycin D-treated). Interferon production is induced after 30 min (20--25 units/ml) of interaction between immune lymphocytes and L cells in the G1 phase, reaches the maximum after 10 hours (70--75 units/ml) and slightly decreases after 48 hours (60--65 units/ml). In interaction of both immune and actinomycin D-treated lymphocytes with L cells in the G1 and S phase, interferon production correlates with the cytotoxic lymphocyte effect on these cells.

Long-Term Effects of Neonatal Estrogen Treatment on Mitogen Responsiveness of Mouse Spleen Lymphocytes<xref ref-type="fn" rid="FN2">2</xref>

Neonatal female NMRI mice were given injections of olive oil (controls) or daily doses of corticosterone (10 micrograms), 17 beta-estradiol (10 micrograms), or diethylstilbestrol (DES) (0.01, 0.1, 1, or 5 micrograms) for the first 5 days after birth. The 5-micrograms dose of DES resulted in a persistently reduced in vitro mitogen response to concanavalin A or bacterial lipopolysaccharide of spleen lymphocytes from 6-, 10-, and 18-week-old or 17-month-old females. DES injections from day 6 through day 10 did not influence the later mitogen response. Treatment of ovariectomized 10-week-old females with 5 micrograms DES for 5 days resulted in a tendency to a reduced mitogen response (not statistically significant) 24 hours after the last DES injection. Four weeks later, the mitogen response was the same in experimental and control females. Different possible mechanisms for the persistent effect on the mitogen response are discussed. Neonatal DES treatment not only resulted in persistent changes in the cervicovaginal epithelium and in the hypothalamic-pituitary gland control system but also in the spleen lymphocyte mitogen response. The altered mitogen response should be a stimulus for a detailed analysis of the immune system in women exposed to DES during fetal life, some of whom develop later in life clear cell adenocarcinoma of the uterine cervix and vagina.

The Isozyme Pattern of Cyclic Amp‐Dependent Protein Kinase and the Distribution of a Cervicovaginal Antigen in Experimental Carcinoma of the Cervix Uteri of Mice

Tissue-sections from 12 methylcholanthrene-induced carcinomas of the cervix uteri of mice were tested for the presence of an antigen normally confined to the cervicovaginal epithelium. The antigen was detected in 10 of the 12 tumours investigated with indirect immunofluorescence, and in all 7 tumours studied with the more sensitive method of mixed hemagglutination. The concentration of the antigen was generally higher in the well-differentiated areas of the tumours, but it was also found associated with solitary tumour cells, apparently invading the stroma. The presence of CVA in the tumours suggests an origin of the tumour cells from the cervicovaginal epithelium. The cyclic AMP dependent protein kinase (EC 2. 7. 1. 37) in the tumour cytosols was studied by chromatography on agarose and DEAE-cellulose. The enzyme showed the same properties as that from normal vaginal epithelium. The tumour cells thus contain an apparently normal complement of this enzyme, which is believed to be responsible for most of the intracellular actions of cyclic AMP.

ADENYLATE CYCLASE ACTIVITY IN MOUSE VAGINAL EPITHELIUM. AGE RELATED CHANGES IN BASAL ENZYME ACTIVITY AND ESTRADIOL SENSITIVITY.

Using neonatal and immature female NMRI mice, age related variations in histochemically demonstrable adenylate cyclase activity were studied in the vaginal epithelium. Basal enzyme activity increased from day 3 to 6 after birth, only later to decrease. Almost no activity was seen on day 14. An estradiol induced increase in enzyme activity occurred in 3- and 6-day-old animals but not in 14-day-old animals. In spite of this difference both neonatal and 14-day-old animals respond to estradiol with an increased amount of a cellular product with antigenic properties specific for the cervicovaginal epithelium. The results are discussed in relation to estrogen induced irreversible effects in the neonatal cervicovaginal epithelium.

Estradiol-17 ? and cAMP: in vitro studies on the cervicovaginal epithelium of neonatal mice

In organ culture of the cervicovaginal epithelium from neonatal mice, the epithelium synthesizes a material with specific antigenic properties (CVA). CVA was studied with immunofluorescence and the amount estimated semiquantitatively. In line with earlier studies, adenosine 3',5'-cyclic monophosphate (dibutyryl derivative (dcAMP), increased the amount of CVA, but adenosine 2',3'-cyclic monophosphate did not. Addition of histamine to the culture medium moderately increased the amount of CVA, whereas the anti-histamine diphenhydramine (H1-antagonist) slightly reduced the strong increase induced by dcAMP and estradiol in combination. No effect was seen under similar conditions using the H2-antagonist metiamide. Taken together with earlier results it is considered possible that the action of histamine and diphenhydramine is related to effects on the cell membranes. Phentolamine had no effect. The dcAMP effect was inhibited by actinomycin D and cycloheximide.

Effects of d-propranolol and estradiol on the cervicovaginal epithelium.

The cervicovaginal epithelium of neonatal mice produces a material with specific antigenic properties (CVA) and this material is produced in increased amounts after estradiol treatment. Using a cytochemical method, estradiol treatment was shown to result in an increase of adenylate cyclase activity in the same epithelium. When d-propranolol is injected together with estradiol, the increase in CVA is inhibited, while the hormone-induced proliferation of epithelial cells is not influenced. When adenylate cyclase activity is studied under identical conditions, the estradiol-promoted increase in enzyme activity is largely counteracted by d-propranolol. These findings would suggest that Adenosine 3"5"-cyclic monophosphate (cAMP) has a role in some, but not all, estradiol-mediated effects in the neonatal cervicovaginal epithelium.

Studies on the differentiation pattern and hormonal sensitivity of an antigenic material specific for the cervicovaginal epithelium in fetal and neonatal mice

By use of an indirect mixed haemagglutination method for tissue sections, the Müllerian cervicovaginal epithelium of fetal and neonatal mice has been shown to contain a material (CVA) with antigenic properties specific for this epithelium. The method is highly sensitive and permits a semiquantitative estimation of CVA in the epithelium. The studies showed that the cervicovaginal epithelium undergoes a multiphasic differentiation pattern. An estradiol injection 1 day prior to killing the animals strongly increased the amount of demonstrable CVA. The quantitative response to estradiol varied with the age of the animals. Notably, estradiol given as early as the day of birth stimulated CVA accumulation in the vaginal epithelium. Differences between the cervical epithelium and the vaginal epithelium regarding the response to estradiol are described.

Estrogen and antagonist-induced structural changes in the cervico-vaginal epithelium of immature rats.

Estrogen enhances proliferation and keratin formation in the cervico-vaginal epithelium of immature rats, whereas the estrogen antagonist, CI-628 (Parke-Davis), represses mitosis and induces mucinogenesis. Combinations of antagonist and estrogen allow differentiation of an epithelium composed of both keratin-forming and mucous cells. Both estrogen and antagonist induce synthesis of a cervico-vaginal endogenous peroxidase in the intermediate and superficial layers of the epithelium. The secreted vaginal mucus of antagonist-treated animals stains intensely for exogenous peroxidase.

Oral contraceptive hormones, folate metabolism, and the cervical epithelium

The currently available evidence concerning disorders of folate metabolism in women taking oral contraceptives has been reviewed. A disturbance in folate balance serious enough to cause symptoms (i.e., megaloblastic anemia) occurs very rarely. In some series, but not in others, serum and/or red cell folate concentrations have been reduced in oral contraceptive users. It is doubtful whether sex steroids affect polyglutamate folate absorption. About 20 percent of women taking contraceptive hormones manifest mild megaloblastic changes on Papanicolaou smears of the cervicovaginal epithelium which disappear after folic acid therapy. The current evidence, however, would not indicate that any significant benefit would ensue from routine folate supplementation in women on oral contraceptives.

Adenylate cyclase in an estradiol sensitive tissue: a cytochemical study.

Estradiol was found to stimulate the activity of adenylate cyclase in the cervicovaginal epithelium of neonatal mice, demonstrated with adenylyl-imidodiphosphate and also with adenosine triphosphate (ATP) as substrate. Measures were taken to exclude interference by other ATP-degrading enzymes. The deposits of the reaction product, indicating enzyme activity, were localized on the plasma membrane of all sides of the epithelial cells. A similar distribution of enzyme activity was recorded in both controls and estradiol-stimulated animals with either of the two substrates; however, the deposits appeared most prominent after estradiol-treatment. In view of a previous report on increased activity of the cyclic AMP-degrading enzyme phosphodiesterase after estradiol-treatment, it was concluded that the parallel elevation in activity of both adenylate cyclase and cyclic AMP-phosphodiesterase indicates a role of cyclic AMP in the action mechanism of estradiol.

OESTRADIOL-17 : ITS INFLUENCE ON CYCLIC 3'5'-ADENOSINE MONOPHOSPHATE PHOSPHODIESTERASE AND 5'-NUCLEOTIDASE IN THE CERVICOVAGINAL EPITHELIUM OF NEONATAL MICE

Summary. This study concerns the histochemical demonstration of cyclic 3′,5′-adenosine monophosphate phosphodiesterase (cAMP-PD) and 5′-nucleotidase in the cervicovaginal epithelium of neonatal mice. Newborn animals showed no demonstrable activity of either of the enzymes; some cAMP-PD activity was present in 3-day-old animals, but no 5′-nucleotidase activity was seen at this age. Oestradiol-17β promoted an increased activity of both enzymes in 3-day-old animals. The localization of both enzymes was probably adjacent to the plasma membrane, with an increased activity at the luminal surface. This investigation has shown that cAMP-degrading enzymes are present in this tissue and that the `action mechanism' of oestradiol involves an effect on the cAMP-level regulating system.

Adequate staining of Trichomonas vaginalis by McManus' periodic acid-schiff stain.

A histochemical study of the cervicovaginal epithelium was undertaken; it was found that Trichomonas vaginalis show an intense affinity for periodic acid-Schiff stain. In order to confirm this observation, 16 proven cases of vaginal trichomoniasis were studied. Two routine stains, two histochemical stains for glycogen, and five histochemical stains for mucosubstances were performed on a specimen from each case. Reactivity of T. vaginalis to Schiff’s reagent was found to be due to an important glycogen and acidic sulfated mucopolysaccharide content not heretofore recognized.The morphologic characteristics of T. vaginalis stained with the McManus periodic acid-Schiff stain are described, as well as some bases for differential diagnosis. The undulating membrane was demonstrated for the first time in stained smears. The use of periodic acid-Schiff stain is recommended for a simple and accurate laboratory diagnosis of vaginal trichomoniasis.

Cervicovaginal epithelium: Its origin and development

Studies on the derivation and differentiation of the cervicovaginal epithelium are reviewed. Several experimental systems point to a dual origin of the vaginal epithelium in most species studied: Anteriorly there is Müllerian epithelium, and posteriorly there is sinus epithelium. The relative contribution varies. Man is an exception as no Müllerian epithelium is included in the vaginal epithelium. The development of the Müllerian epithelium, both the appearance of a specific antigen and the morphologic transformation into a stratified epithelium, has been analyzed with the neonatal mouse used as a test system. The high proliferative activity in the Müllerian epithelium seems to be a prerequisite for the differentiation process. By inhibiting the proliferation, it is possible to change the prospective fate of the epithelium irreversibly. A pathogenetic model for diethylstilbestrol-induced vaginal adenocarcinomas in young girls is given. Finally, the epidermization process in the uterine cervix is discussed. The mouse Müllerian epithelium has proved to be very valuable for studying many basic aspects of cellular differentiation problems; it may also contribute to elucidation of epithelial changes in the human cervix uteri.

Epithelial ultrastructure and the distribution of an estradiol sensitive antigen in the vagina of adult mice

The distribution of an antigenic material specific for the cervicovaginal epithelium (CVA) was studied in the vaginal epithelium of the adult mouse with immunofluorescence and immunoferritin techniques. The antigen localization has been correlated to the fine structure of the vaginal epithelium in various states of functional activity. The antigen distribution in adult ovariectomized mice and in ovariectomized mice treated with estradiol was compared with that in normal cycling mice. CVA was found to be associated with the exterior of the cell membrane of vaginal epithelium cells, being part of the glycocalyx.