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Describes the immunodetection and quantification of benzo[a]pyrene (B[a]P) adducts in ovarian cells of women exposed to cigarette smoke. Presence of a relationship between B[a]P-DNA adduct levels and smoke exposure and dose, both recent and long-term; Immunostaining confined to the nucleus, suggesting adduct formation by covalent binding to DNA.

Pathogenic prion protein scrapie (PrP(sc)) may contaminate soils for decades and remain in water in colloidal suspension, providing infection pathways for animals through the inhalation of ingested dust and soil particles, and drinking water. We used molecular dynamics simulations to understand the strong binding mechanism of this pathogenic peptide with clay mineral surfaces and compared our results to experimental works. We restricted our model to the moiety PrP(92-138), which is a portion of the whole PrP(sc) molecule responsible for infectivity and modeled it using explicit solvating water molecules in contact with a pyrophyllite cleavage plane. Pyrophyllite is taken as a model for common soil clay, but it has no permanent structural charge. However, partial residual negative charges occur on the cleavage plane slab surface due to a slab charge unbalance. The charge is isotropic in 2D and it was balanced with K(+) ions. After partially removing potassium ions, the peptide anchors to the clay surface via up to 10 hydrogen bonds, between protonated lysine or histidine residues and the oxygen atoms of the siloxane cavities. Our results provide insight to the mechanism responsible for the strong association between the PrP(sc) peptide and clay nanoparticles and the associations present in contaminated soil and water which may lead to the infection of animals.

We performed a case-controlled study to study the immunoreactivity patterns and staining intensity of alphavbeta5 and alphavbeta6 integrins in ectopic and eutopic endometrium from women with endometriosis and compared them with those in normal control endometrium. Forty eutopic and ectopic samples from patients with endometriosis were compared with 12 control endometrial samples. Staining was evaluated using a computerized image analysis system by a blinded, independent observer. The immunoreactivity patterns for both alphavbeta5 and alphavbeta6 were very similar. In eutopic endometrium from women with or without endometriosis, the immunostaining intensity score for both integrins was stronger during the secretory than the proliferative phase. Immunoreactivity in endometriosis was greater than in eutopic endometrium. There were no significant menstrual cycle-related staining differences in endometriosis. The percentage of blood vessels immunolabeled was greater in endometriosis than eutopic endometrium from endometriosis patients and greater in eutopic endometrium from these patients compared with control endometrium. Ectopic tissue from patients with stages I and II endometriosis showed significantly higher staining intensity than ectopic tissue from patients with stages III and IV disease. Determination of the specific functional consequences of the differences observed in alphavbeta5 and alphavbeta6 integrin immunostaining may provide an increased understanding regarding the pathogenesis of endometriosis.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The cellular localization of a 24,000 (24K) mol wt protein was evaluated by monoclonal antibody immunocytochemistry in atrophic and persistent proliferative endometrium as well as in various forms of hyperplastic and neoplastic human endometria. 24K was chiefly located in the cytoplasm of ciliated cells and their precursor forms, clear cells. It was absent in atrophic, resting, and regressive forms of cystic glandular hyperplasia. Persistent proliferative endometria with a low degree of proliferation has occasional 24K immunostained cells, whereas those with active proliferation contained intense 24K immunostaining. A similar pattern characterized the active forms of cystic glandular hyperplasia. The immunostaining reaction decreased in adenomatous and atypical adenomatous hyperplasia as well as in well differentiated adenocarcinomas. In poorly differentiated carcinomas, 24K protein was virtually absent. These findings suggest that 24K protein is estrogen and ciliated cell related, and endometrial ciliogenesis and 24K protein are morphological and biochemical markers, respectively, of the estrogenic endometrial response. Their increase and decrease in hyperplastic and neoplastic endometria support the concept that early hyperplasia is highly sensitive to estrogenic stimulation, whereas with increasing architectural and cytologic atypia, the estrogenic response decreases as a reflection of growing cell populations that are independent of estrogenic influence. Immunostaining with 24K is not sensitive enough to discriminate estrogen-independent cells of atypical adenomatous hyperplasia from those of early, well differentiated adenocarcinoma.

Two pharyngeal hypophyses from patients with endocrine disorder were examined light microscopically and immunocytochemically. The pharyngeal hypophysis from a patient with primary hypothyroidism was hypertrophic, with TSH cell hyperplasia; while that from a patient treated with metoclopramide, a dopamine-receptor-blocking drug, showed PRL cell hyperplasia. These findings strongly suggest that under certain circumstances the pharyngeal hypophysis is able to respond with specific changes to variations in the endocrine feedback.

Fifteen pharyngeal and sellar pituitary glands, obtained at autopsy from unselected adult patients, were compared in an attempt to elucidate the functional significance of the pharyngeal pituitary. The study was carried out using light microscopy and an indirect peroxidase technique to detect the presence of prolactin, GH, ACTH, LH, FSH, TSH and lipotrophin (LPH) immunoreactive cells. A quantitative analysis of these cell types in each gland was performed. Neither the pharyngeal nor the sellar pituitaries were abnormal in six cases. In this group the average percentage of immunoreactive cells in the sellar vs the pharyngeal pituitary was 11.3 vs 7.4 for FSH cells, 13.3 vs 4.4 for LH cells, 6.4 vs 5.2 for TSH cells, 14 vs 1.5 for ACTH cells, 13.1 vs 6 for LPH cells, 29.4 vs 5.2 for GH cells and 21.2 vs 8.5 for prolactin cells. A comparative statistical evaluation of the seven hormone-producing cell types indicated that, in most cases, the percentage of immunoreactive cells was significantly higher in the sellar pituitary. Examination of serial sections revealed hyperplasia, with or without microadenomas, in nine sellar pituitaries. In these abnormal cases most of the pharyngeal pituitary glands showed hyperplasia of the same cell type as was found hyperplastic in the sellar adenohypophysis. However, hyperplasia restricted to the sellar pituitary was also seen. There were two cases in which the pharyngeal pituitary was almost lacking in immunoreactive cells; in one of them the sellar pituitary had GH and prolactin cell hyperplasia. The results obtained confirm that under normal conditions the pharyngeal pituitary is not an important source of adenohypophyseal hormones.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Eight adult human pharyngeal pituitary glands taken at autopsy were studied by immunocytochemistry to reveal the presence of ACTH-, lipotropin-, FSH-, LH-, TSH-, PRL-, and GH-immunoreactive cells. All of these cell types were found and quantitated in pharyngeal hypophyses from patients with no evidence of endocrine disorder. The percentage of the seven hormone-producing cell types varied from gland to gland from 1-30%; there were no marked histological differences between sexes or attributable to age. The cellular composition of the pharyngeal hypophysis shows that this gland has the capacity to produce at least seven hormones.

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.

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).

The follicular structures present in the human pituitary gland were studied, at the light-microscopic level, using histochemical and immunocytochemical techniques. The antisera applied in the peroxidase-antiperoxidase procedure were anti-hFSH beta, anti-hLH beta, anti-hPRL, anti-hGH, anti-hTSH beta, anti-hLPH beta, anti-pACTH, and anti-hACTH. In the 10 normal pituitaries examined, follicles were always found in the three areas of the adenohypophysis. The wall of the pars distalis follicles showed the seven immunoreactive cell types studied, while follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells were the only ones present in the wall of the pars tuberalis follicles. Most of the cell types studied were also present in the wall of the intermediate area follicles, but these follicles had characteristics not found in the other two areas. They were very large, with frequent interconnections forming a three-dimensional network of anastomotic cavities, and the colloid had different histochemical affinity. None of the hormones studied could be detected by immunocytochemistry within the follicular colloid. Three of the ten pituitary adenomas examined showed numerous follicular structures. Some of the follicles in the adenomatous pituitaries were similar to those found in the normal adenohypophysis, but there were also follicles filled with only traces of colloid and numerous blood cells in the cavity, and follicles filled with neoformed connective tissue. In one of these cases, FSH/LH immunoreactive adenoma cells were seen in the wall of the follicles. The results obtained suggest that the finding of pituitary adenomas with follicular structures is not uncommon and that the follicles originate from the tumor cells. In addition, the follicles seem to have several functional stages, explaining the finding of different types of follicular formation.