Vascular cognitive impairment: Advances and trends.

The presence of vascular neurocognitive impairment (whatever the severity) is always associated with a functional impact and increased risk of dependency and institutionalization. However, vascular cognitive impairment remains underdiagnosed, and the mechanisms underlying post-stroke cognitive disorders are still poorly understood. However, the advent of new criteria and a standardized international neuropsychological battery is expected to lead to improved diagnosis and management, and the development of novel techniques (such as brain imaging and amyloid PET) should improve our understanding of the mechanisms underlying vascular cognitive impairment and help to identify potential targets for therapy.

Detection of benzo[a]pyrene diol epoxide-DNA adducts in embryos from smoking couples: evidence for transmission by spermatozoa.

Tobacco smoking is deleterious to reproduction. Benzo[a]pyrene (B[a]P) is a potent carcinogen in cigarette smoke. Its reactive metabolite induces DNA-adducts, which can cause mutations. We investigated whether B[a]P diol epoxide (BPDE) DNA adducts are detectable in preimplantation embryos in relation to parental smoking. A total of 17 couples were classified by their smoking habits: (i) both partners smoke; (ii) wife non-smoker, husband smokes; and (iii) both partners were non-smokers. Their 27 embryos were exposed to an anti-BPDE monoclonal antibody that recognizes BPDE-DNA adducts. Immunostaining was assessed in each embryo and an intensity score was calculated for embryos in each smoking group. The proportion of blastomeres which stained was higher for embryos of smokers than for non-smokers (0.723 versus 0.310). The mean intensity score was also higher for embryos of smokers (1.40+/-0.28) than for non-smokers (0.38+/-0.14; P = 0.015), but was similar for both types of smoking couples. The mean intensity score was positively correlated with the number of cigarettes smoked by fathers (P = 0.02). Increased mean immunostaining in embryos from smokers, relative to non-smokers, indicates a relationship with parental smoking. The similar levels of immunostaining in embryos from both types of smoking couples suggest that transmission of modified DNA is mainly through spermatozoa. We confirmed paternal transmission of modified DNA by detection of DNA adducts in spermatozoa of a smoker father and his embryo.

Immunodetection of benzo[a]pyrene adducts in ovarian cells of women exposed to cigarette smoke.

Benzo[a]-pyrene (B[a]P) is a potent mutagen and carcinogen present in cigarettes. We report here on immunodetection and quantification of B[a]P-DNA adducts in granulosa-lutein cells of patients undergoing in-vitro fertilization (IVF) and embryo transfer, who were exposed to cigarette smoke. Follicular fluids (FF) and granulosa-lutein cells were obtained from the same follicular aspirate from 32 women self-reported as active smokers, passive smokers, or non-smokers. Cells were immunostained with 5D11, an anti-B[a]P diolepoxide monoclonal antibody that recognizes DNA adducts. Cotinine, a reliable marker for recent smoke exposure and dose, was assessed by radioimmunoassay in 32 FF samples. Individual scores of cell immunoreactivity were highly correlated with FF cotinine concentrations. Evaluations of immunostaining intensity in 9770 granulosa-lutein cells from the 32 women revealed higher average scores in active and passive smokers, relative to non-smokers. In passive smokers the average level of cell immunostaining was 63% of that of active smokers. These relationships provide quantitative evidence that B[a]P-DNA adduct levels are related to smoke exposure and dose, both recent and long term. Immunostaining was confined to the nucleus, suggesting adduct formation by covalent binding to DNA. Presence of adducts in granulosa-lutein cells from women exposed to cigarette smoke may increase the risk for DNA damage.

Immunodetection of cotinine protein in granulosa-lutein cells of women exposed to cigarette smoke.

OBJECTIVE: To detect immunoreactivity to cotinine protein, a major metabolite of nicotine, in granulosa-lutein cells from patients exposed to cigarette smoke, as measured by levels of cotinine in follicular fluid (FF) samples. DESIGN: Controlled immunocytochemical study. SETTING: Hospital IVF-ET program treating infertile patients. PATIENT(S): Twenty-eight women classified by self-reported smoking habits: active smokers (n = 17), passive smokers (n = 4), and nonsmokers (n = 7). INTERVENTION(S): Ovarian hyperstimulation. MAIN OUTCOME MEASURE(S): Grades of immunostaining intensity were assessed in granulosa-lutein cells. Patient scores of cell immunostaining were calculated and regressed on levels of FF cotinine. RESULT(S): Cotinine levels in FF were higher in active smokers than in passive smokers or nonsmokers. Cotinine immunostaining was visualized in the nucleus and cytoplasm of granulosa-lutein cells. Mean grades and mean scores of immunostaining intensity were higher in active smokers than in passive smokers or nonsmokers. There was a strong positive correlation between scores of cell immunostaining and FF cotinine levels. CONCLUSION(S): The association between cotinine expression in granulosa-lutein cells and FF cotinine provides reliable evidence for a dose-related effect. This constituent of cigarette smoke appears to interact directly with and incorporate into these ovarian cells. Our approach seems useful for monitoring ovarian exposure to environmental toxins.

Localization and regulation of expression of the FAR-17A gene in the hamster flank organs.

A quantitative in situ hybridization study was carried out to determine the precise localization and androgen regulation of the flank organ regulated (FAR-17A) mRNA expression in the different cellular components of the hamster flank organs. Although FAR-17A mRNA was highly expressed in the epithelial cells of the sebaceous glands, it was also found in the outer root sheath of the hair follicles and in melanocytes. The changes in FAR-17A mRNA levels, in the size of the flank organ and sebaceous gland areas as well as in the weight of the seminal vesicles and prostate, were compared following castration and after 5alpha-dihydrotestosterone treatment. FAR-17A mRNA levels were already significantly decreased 1 d after castration, in parallel with a concomitant decrease in the number of labeled cells with the FAR-17A probe. A maximal decrease was found 7 d after castration. The other parameters were significantly reduced later. After 7 d of treatment with dihydrotestosterone, all values returned to those found in intact animals. Similar stimulatory effects on these parameters were observed after treatment with the adrenal sex steroid precursor dehydroepiandrosterone. These data show that all of the components of the flank organs (sebaceous glands, hair follicles, and melanocytes) express the flank organ regulated (17A) type gene (FAR-17A) gene and that its expression is stimulated by treatment with either dihydrotestosterone or dehydroepiandrosterone. Moreover, FAR-17A mRNA levels respond to androgen stimulation more rapidly than the standard morphologic parameters, revealing that the FAR-17A gene could be a more sensitive and cell specific marker to study the mechanisms of androgen action in the skin.

The ontogeny of pit-1 expression in the human fetal pituitary gland.

Pit-1 expression has been reported to be cell-type-specific in the adult human pituitary and in human pituitary adenomas. In contrast, studies of rodent fetal adenohypophysial development as well as mature rodent glands have indicated that the pit-1 mRNA is ubiquitously expressed and the protein is under translational control. To determine the ontogeny and localization of Pit-1 expression in the human fetus, we examined fetal pituitaries (n = 23) at various stages of gestation from 6 weeks to term using in situ hybridization (ISH) for pit-1 mRNA, immunohistochemical localization of Pit-1 protein, and combined ISH for pit-1 mRNA with immunohistochemistry for pituitary hormones. At 6 and 7 weeks of gestation, the cells surrounding both limbs of Rathke's cleft showed a moderate specific signal for pit-1 mRNA. At 7 weeks, only a few cells were immunoreactive for ACTH and there was no definite colocalization of that hormone with pit-1 mRNA. At 8 and 9 weeks of gestation, there was definite preferential expression of pit-1 mRNA in cells containing growth hormone (GH) but not ACTH, as well as in cells with no detectable hormone immunopositivity. At midgestation and after, there was clear correlation between pit-1 mRNA expression and hormone content; cells with GH, prolactin and/or thyrotropin immunoreactivity had abundant pit-1 mRNA, whereas those containing ACTH, FSH or LH were negative for pit-1 mRNA by ISH and its protein by immunohistochemistry. The signal for pit-1 mRNA was stronger in intensity and present in more cells in fetal than in adult adenohypophyses. The ontogeny of pit-1 mRNA expression indicates that it precedes the onset of pituitary hormone detection. Its abundance in the human pituitary early in gestation may reflect its role in cytodifferentiation and cell proliferation. The correlation between pit-1 mRNA detection and Pit-1 protein localization is consistent with a cell-type-specific pretranslational regulatory mechanism for Pit-1 expression in the developing human adenohypophysis.

Local and systemic reduction by topical finasteride or flutamide of hamster flank organ size and enzyme activity.

The hamster flank organ is a widely used model of the control of sebaceous gland activity by androgens and anti-androgens. Finasteride, a 5 alpha-reductase inhibitor, was administered locally on the surface of the right flank organ and right ear twice daily for 4 weeks. The treatment caused similar 12% to 30% reductions in the size of the sebaceous glands in both flank organs. Moreover, relative mRNA levels of the androgen-regulated FAR-17a gene measured by in situ hybridization as well as [3H]-thymidine incorporation and 5 alpha-reductase activity were similarly decreased in the two flank organs after topical application. The pure anti-androgen flutamide, at the same doses, exerted a more potent effect on all the same parameters, and the effect was also comparable on both the treated and untreated sides of flank organs. Finasteride and flutamide significantly decreased ventral and dorsal prostatic weights after topical application. The present data show that the topical administration of finasteride, in analogy with flutamide, causes local inhibition of sebaceous gland growth in both the costovertebral organs and ears. However, as demonstrated by the similar inhibitory effect in the contralateral untreated side and the reduced weight of the dorsal and ventral lobes of the prostate and seminal vesicles, finasteride and flutamide both exert significant systemic effects.

Immunocytochemical detection of androgen receptor in human temporal cortex characterization and application of polyclonal androgen receptor antibodies in frozen and paraffin-embedded tissues.

Immunocytochemical and biochemical studies have demonstrated the presence of androgen receptor protein in various regions of the rodent and non-human primate cortex. Localization of androgen receptor in the human brain has, however, not been studied as extensively, because of difficulties in obtaining suitable tissue samples. In the present study, we have localized androgen receptors in both frozen and paraffin-embedded temporal cortex from epileptic patients undergoing resection. Polyclonal antibodies were raised against fusion proteins containing fragments of the human androgen receptor protein. The antibodies were affinity-purified against the corresponding fusion protein. Immunoprecipitation and Western blotting using extracts from human cell lines demonstrated the specificity of the antibodies for the human androgen receptor and lack of cross-reactivity with other steroid hormone receptors. Immunocytochemistry was performed on frozen and paraffin sections of human temporal cortex and in paraffin-embedded benign hyperplastic prostates (BPH), as well as prostate and breast carcinomas, by the streptavidin-biotin-peroxidase method. Antigen-retrieval was performed in paraffin-embedded sections using microwave irradiation. Specific nuclear and cytoplasmic immunoreactivity for androgen receptor was detected in neurons, astrocytes, oligodendrocytes, and microglia cells of the temporal cortex. In contrast, only nuclear staining was observed in BPH, prostate and breast carcinomas. Immunoprecipitation of human temporal cortex lysate and subsequent Western blot analysis demonstrated the expression of a 98 kDa immunoreactive protein, slightly smaller than the reported molecular weight of the wild-type androgen receptor. These results provide further evidence for the expression of androgen receptor in the human temporal cortex. The use of these immunocytochemical techniques should enable the retrospective determination of possible changes in androgen receptor expression in a variety of archival paraffin-embedded tissues, including samples of the human central nervous system.

Expression of pit-1 messenger ribonucleic acid and protein in the human placenta.

It is well established that the human placenta produces a wide range of hormones similar to those secreted by the pituitary and hypothalamus. However, the physiological role and regulation of placental hormone synthesis and release are still largely unknown. GH (GH-N) is expressed in the pituitary, where it requires the tissue-specific transcription factor Pit-1. Chorionic somatomammotropin A (CS-A) and CS-B as well as the placental GH variant (GH-V), which also belong to the GH gene family and are located in the same chromosomal cluster, are expressed in the placental syncytiotrophoblast. The presence of Pit-1-binding sites in the CS-A and GH-V promoter regions predicts that Pit-1 may be expressed in the placenta. However, this has not yet been demonstrated. To examine possible similarities in the regulation of these genes in the pituitary and placenta, we studied the expression of pit-1 messenger ribonucleic acid (mRNA) in the human placenta, transformed human placental cells, and the JEG-3 choriocarcinoma cell line. Polymerase chain reaction (PCR) products of the expected size were amplified from first and third trimester placentas, transformed placental cells, and JEG-3 complementary DNA by reverse transcription-PCR. The pit-1-specific sequence was confirmed by restriction endonuclease digestion, Southern hybridization, and DNA sequencing. Human pituitary tissue was used as a positive control; no PCR product was obtained from hippocampus (negative control). In situ hybridization of placental tissue sections revealed the presence of pit-1 mRNA in first and third trimester syncytiotrophoblast. Pit-1 protein was localized by immunohistochemistry with the same tissue distribution and a nuclear localization pattern. These data demonstrate expression of pit-1 mRNA and Pit-1 protein in the human placenta, thus questioning its role as a pituitary-specific regulator of GH-N gene transcription. The expression of Pit-1 in the placenta, together with its previously demonstrated capability to bind to and activate the CS-A and the GH-V promoters, suggests that it may play a role in the regulation of hormones belonging to the GH gene family in both pituitary and placenta.

Characterization, expression, and immunohistochemical localization of 5 alpha-reductase in human skin.

Human skin has been shown to contain a high level of 5 alpha-reductase activity, the enzyme that catalyses the conversion of the weak androgen testosterone into dihydrotestosterone, the most potent androgen. Because two types of 5 alpha-reductase genes have been characterized in humans, we have cloned 5 alpha-reductase cDNAs from adult human keratinocyte and skin fibroblast cDNA libraries to identify and gain better knowledge of the 5 alpha-reductase expressed in normal human skin. Nucleotide sequence analysis shows that the clones obtained correspond to the type I 5 alpha-reductase. RNase protection analysis using (poly A)+ RNA obtained from human skin and prostate also confirms that type I 5 alpha-reductase is the predominant type expressed in normal skin, whereas type II 5 alpha-reductase is the major form found in the prostate. Following polymerase chain reaction amplification of human keratinocyte and skin fibroblast cDNA, a low level of type II 5 alpha-reductase cDNA has been detected. Using antipeptide antibodies raised in rabbits against the peptide sequence covering amino acids 227 -240 to perform immunohistochemical localization of 5 alpha-reductase, we have found that 5 alpha-reductase is distributed in sweat and sebaceous glands, as well as in the epidermal cell layers, thus providing the basis for the important role of androgens in human skin and its appendages.

Immunohistochemical localization of the oestrogen-responsive 110 kDa and 74 kDa polypeptides and complement component C3 in the rat genital tract after oestrogen treatment and during the oestrous cycle.

In previous studies we showed that two oestrogen-responsive rat uterine secretory polypeptides of molecular mass 110 and 74 kDa share sequence similarities with the alpha and beta chains of rat complement component C3. Using polyclonal antibodies specific to the 110 kDa polypeptide, the 74 kDa polypeptide and rat serum C3, we studied the localization of these proteins in the rat genital tract using an indirect immunoperoxidase technique. The results showed that in oestradiol-treated rats immunostaining for the three antigens was heterogeneously distributed in the epithelia of the endometrium, cervix and vagina and that the intensity of staining was greater after oestrogen treatment than in controls. In oestradiol-treated immature rats, the immunoreactivity for the 110 kDa and 74 kDa polypeptides was greater in the endometrial and vaginal epithelia than in the cervical epithelium, whereas immunoreactivity for C3 was greatest in the vaginal epithelium. In cyclic rats, staining by all three antibodies was seen only during pro-oestrus and oestrus, during which stages the immunostaining for the 110 kDa and 74 kDa polypeptides was detected only in the endometrial epithelium, whereas immunostaining for C3 was also found in the epithelia of the cervix and vagina. In general, consecutive sections of these tissues revealed a close correlation between the immunostaining for the 110 kDa and 74 kDa polypeptides and C3. However, there were some sections that showed clear differences in staining, suggesting that more than one C3-related protein species was detected in the female rat reproductive tract.

Cell type-specific expression of the pituitary transcription activator pit-1 in the human pituitary and pituitary adenomas.

Pit-1 is a transcription factor that has been shown to be critical for pituitary-specific activation of the GH and PRL genes. In rodents and humans, differentiation and/or maintenance of somatotroph, lactotroph, and thyrotroph phenotypes are dependent on expression of a functional pit-1 gene. In rodents, Pit-1 protein is detectable in only these three cell types; however, pit-1 mRNA transcripts appear to be present at comparable levels in all adenohypophysial cell types, suggesting that translational controls may dictate the pattern of Pit-1 expression. We examined the distribution of pit-1 transcripts in the human pituitary and pituitary adenomas. All tumors were characterized by immunocytochemistry, electron microscopy, and tissue culture for accurate classification. Northern blot analysis demonstrated abundant levels of pit-1 mRNA in somatotroph, mammosomatotroph, and lactotroph adenomas. Two clinically silent adenomas that expressed TSH as well as gonadotropins contained detectable levels of pit-1 mRNA. No pit-1 expression was otherwise detected in corticotroph, gonadotroph, null cell, or oncocytic adenomas. In situ hybridization localized pit-1 mRNA transcripts in adenomas that contained GH, PRL, or TSH, but not in adenomas composed of other cell types. Pit-1 mRNA was also localized to selected subpopulations of the human nontumorous adenohypophysis that contained immunoreactivity for GH, PRL, and/or TSH. Pit-1 protein immunoreactivity was detected in the nuclei of adenomas that expressed pit-1 mRNA, but not in those that were negative for pit-1 mRNA; it was also localized only in cells containing GH, PRL, or TSH beta in the nontumorous adenohypophysis. These data demonstrate selective expression of the human pit-1 gene in adenohypophysial cell types responsible for GH, PRL, and/or TSH synthesis and are consistent with a predominantly pretranslational regulatory mechanism for Pit-1 expression in the human.

Nuclear localization of androgen receptor in heterogeneous samples of normal, hyperplastic and neoplastic human prostate.

To facilitate an understanding of how androgens participate in the genesis of human benign hyperplasia and carcinoma we assayed androgen receptor in the epithelium and stroma of human prostatic tissue from 57 patients. Immunohistochemical staining of human androgen receptor was performed on 106 sections of normal prostate, benign prostatic hyperplasia (BPH) and prostate cancer. To determine variability of androgen receptor staining sections taken from different portions of the gland were studied. Frozen tissue sections were incubated with monoclonal antiandrogen receptor antibodies and staining was completed by the indirect avidin-biotin peroxidase method. Antibody staining was found mainly in the nucleus of prostatic epithelial cells, although some stromal cells also showed positive staining. Unlike normal prostate, there was a heterogeneous distribution of androgen receptor in BPH and prostate cancer. The androgen receptor content in well differentiated adenocarcinoma epithelium was significantly higher compared to moderately (p less than 0.05) and poorly (p less than 0.05) differentiated adenocarcinoma. Regardless of the origin of stromal tissue, some staining was observed. In each specimen studied the androgen receptor staining was consistent qualitatively and quantitatively for each pathological component throughout the specimen. These data confirm that androgen receptor is a nuclear receptor protein. Furthermore, they show the ability of monoclonal antibodies to reveal cellular/subcellular distribution of androgen receptor, and demonstrate a correlation between the degree of tumor differentiation and androgen receptor content in epithelial but not in stromal cells. These observations may have important implications for understanding the variable tumor response to hormone therapy.

Corticotropin-releasing hormone, luteinizing hormone-releasing hormone, growth hormone-releasing hormone, and somatostatin-like immunoreactivities in biopsies from breast cancer patients.

The presence of immunoreactive adrenocorticotropin-releasing hormone (CRH), luteinizing hormone-releasing hormone (LHRH), growth hormone-releasing hormone (GHRH), and somatostatin has been investigated by immunohistochemistry in forty biopsies from breast cancer patients. All of these hypothalamic hormones were found in about 30% of the samples, seen in the cytoplasm or in the nuclei of the tumor cells. Positive immunostaining for the hypothalamic hormones was present in colloid, lobular, and infiltrating ductal carcinomas. There was not a clear relationship between occurrence of staining for the hypothalamic hormones and the histologic grade of tumors or the clinical stage of the disease. Immunoreactive LHRH was more frequently found in breast tumors with estrogen and progesterone receptors. On the other hand, preneoplastic breast lesions expressed mainly somatostatin, while immunoreactivity was absent in normal mammary tissue.

Analysis of a 24-kilodalton (KD) protein in the human uterine cervix during abnormal growth.

The authors have previously studied the presence and distribution of a 24-kilodalton (KD) estrogen-regulated protein in the human normal cervix (Am J Obstet Gynecol 1986; 155:1090-1096). This protein has recently been identified as a heat-shock protein, and in order to continue its study the authors have now examined its expression in preneoplastic to neoplastic cervical samples. The study involved 53 patients, the presence of 24-KD protein together with keratin and carcinoembryonic antigen (CEA) was investigated by immunohistochemical analysis. Cytosol samples from 15 patients with squamous cervical carcinomas were also studied by the Western blot technique, and the presence of estrogen receptors was analyzed biochemically. The 24-KD protein was observed in cervical intraepithelial neoplasias (CIN), but it was not useful to identify the different degrees of CIN examined. The 24-KD protein, keratin, and CEA were predominantly expressed in well and moderately differentiated squamous carcinomas in the more differentiated areas, and the protein was also found in cervical adenocarcinomas. The presence of 24-KD protein did not correlate with that of estrogen receptors in squamous cervical carcinomas. The Western blot and the immunohistochemical studies revealed that the antibody to 24-KD protein does not cross-react with epitopes of CEA and keratins.

Study of estrogen receptor, progesterone receptor, and the estrogen-regulated Mr 24,000 protein in patients with carcinomas of the endometrium and cervix.

The presence of an estrogen-regulated protein with 24,000 molecular weight has been studied in 47 patients with endometrial carcinomas and in 29 patients with cervical carcinomas in order to correlate its presence with that of estrogen receptors (ERs) and progesterone receptors (PgRs). In the cytosol tumor samples the Mr 24,000 protein was detected by the Western blot technique using a monoclonal antibody (C11), while the presence of ER and PgR was studied by the one-point dextran-coated charcoal assay. In the tumor tissue sections immunohistochemistry was applied to detect Mr 24,000 protein, ER, and PgR; in these cases monoclonal antireceptor antibodies (H222 and mPRI) were used to localize the receptor proteins. In endometrial and endocervical adenocarcinomas the presence of Mr 24,000 protein correlated significantly with that of ER (P less than or equal to 0.05) in the cytosol samples; when the evaluation was performed in the tumor sections, the presence of Mr 24,000 protein correlated with that of ER (P less than or equal to 0.005) and PgR (P less than or equal to 0.05) as well. The study also showed that almost 70% of the well-differentiated adenocarcinomas had ER, PgR, and Mr 24,000 protein. In 25% of the endometrial adenocarcinomas examined the tumors were associated with normal, proliferative, and hyperplastic endometrium; in these cases the presence of ER, PgR, and Mr 24,000 protein was evaluated by immunohistochemistry in the malignant and nonmalignant endometrium. On the other hand, there was a lack of correlation between Mr 24,000 protein, ER, and PgR in the squamous carcinomas of the uterine cervix and in the endometrial adenocarcinomas with squamous cells. In most of these cases the tumors lacked ER and PgR although 80% of them contained the Mr 24,000 protein to a variable degree. It is suggested that Mr 24,000 protein is involved in growth and differentiation (the Mr 24,000 protein is a heat shock protein) and that the gene coding of this protein is under hormonal control only in those tissues where growth and differentiation are strongly hormonally controlled (breast and endometrium).

Ultrastructural and immunoelectron-microscopic study of the human pharyngeal hypophysis.

The ultrastructural characterization of seven cell types in the pharyngeal hypophysis from adult subjects is described. By immunoelectron microscopy, two of the granular cell types were identified as growth-hormone- and prolactin-producing cells. The vascular supply of this gland was mainly composed of capillaries without fenestrations. Review of the literature allows a comparison with the ultrastructure of the sellar adenohypophysis and with the pharyngeal hypophysis of children.

Localization of an estrogen-responsive protein in the human cervix during menstrual cycle, pregnancy, and menopause and in abnormal cervical epithelia without atypia.

The presence and distribution of an estrogen-responsive protein with a molecular weight of 28,000 were investigated in the human cervix with use of a monoclonal antibody. This biochemical marker protein was localized with a light microscope and by immunocytochemical studies of the different cell types and cell layers of the cervix. The study involved 60 patients, 48 of whom were sexually active, eight pregnant, and four in menopause; strips of endometrial tissue were analyzed in 22 patients. In cycling women the estrogen-responsive protein was identified in the subcolumnar cells of the endocervix, whereas in the ectocervix the protein was detected mainly in the parabasal and intermediate cell layers but alternating with unstained areas. There were no significant variations in the presence of the protein in the ectocervical and endocervical epithelium during the different phases of the menstrual cycle. During pregnancy a more intense and homogeneous immunostaining of the protein was seen in the ectocervix, endocervical areas with squamous metaplasia showed strong estrogen-responsive immunostaining, and predecidual and decidual cells were positive for the protein. There were no prominent changes in the presence and distribution of the protein in the abnormal ectocervical samples without atypia. However, in the endocervix the protein detection was useful to follow the evolution of the subcolumnar cells to simple squamous metaplasia. These samples displayed intense estrogen-responsive immunostaining. No immunoreaction was observed in the cervix of menopausal women. The results of the present study have shown that the response of the normal uterine cervix to estrogenic influence is heterogeneous in different cervical cell types and in different sites within the same cell layer; during the normal menstrual cycle the capability of response of the cervical cells to variations of estrogen levels is limited when compared with the endometrium; and during pregnancy and in the process of indirect squamous metaplasia some of the cervical cells seem to be very reactive to estrogenic stimulation. This study defines the normal baseline for further analysis of the estrogen-regulated protein in the uterine cervix during abnormal growth.