Beta microseminoprotein is not a prostate-specific protein. Its identification in mucous glands and secretions.

Beta microseminoprotein (beta inhibin, PSP94), an unglycosylated protein of 94 amino acids with unknown function, is one of the predominating proteins in the secretion of the human prostate gland. In this work the authors have demonstrated that the expression of beta microseminoprotein is not restricted to the prostate and that the protein has a previously unrecognized widespread occurrence in the human body. According to radioimmunoassay, beta microseminoprotein immunoreactivity is present in many nonprostatic body fluids. The highest concentrations were found in secretions from the respiratory tract; in tracheobronchial fluid sometimes even at concentrations comparable to that in seminal plasma (about 1 g/l). Intermediate concentrations were found in gastric juice and some samples of secretion from the uterine cervix, whereas tears, saliva, pancreatic juice, bile, and mucus from the colon had low concentrations. According to gel chromatography, the molecular size of the beta microseminoprotein immunoreactivity present in tracheal fluid, gastric juice, and secretion from the uterine cervix did not differ from that of beta microseminoprotein in seminal plasma. The beta microseminoprotein immunoreactive component present in gastric juice had the same amino-terminal amino acid sequence as prostatic beta microseminoprotein (14 residues identified in material purified from gastric juice), providing further evidence for chemical identity of a nonprostatic beta microseminoprotein with the prostatic protein. Immunohistochemical staining with affinity-purified antibodies demonstrated the presence of beta microseminoprotein in many tissues, including the goblet cells in the tracheobronchial epithelium, tracheobronchial submucosal glands, certain mucosal cells in the antrum of the stomach, some glands of Brunner in the duodenum, and in parts of the mucosa of the colon. At least in the respiratory tract, the staining was localized to mucus-containing cells. beta microseminoprotein immunoreactivity also was localized to the cilia of the ciliated epithelium in the respiratory tract, the fallopian tubes, and the Gartner ducts of the uterine cervix. The pattern of tissue distribution of beta microseminoprotein found in this work indicates a connection of beta microseminoprotein with mucous secretions.

Immunohistochemical localization of androgen receptors with mono- and polyclonal antibodies to androgen receptor.

Rat, human, and mouse tissues were stained immunohistochemically using mono- and polyclonal androgen receptor antibodies. Monoclonal antibodies were raised in rats and used to stain human and mouse tissues; polyclonal antibodies were raised in rabbits and used to stain rat tissues. Frozen tissue sections were incubated with the appropriate androgen receptor antibody and staining was completed by the indirect avidin-biotin peroxidase method. A comprehensive survey of rat and mouse tissues was performed. Antibody staining was found exclusively in the nucleus of certain specific cell types, suggesting that the androgen receptor is a nuclear protein. All male sexual organs in the rat showed strong positive nuclear staining for androgen receptor. Weaker positive reactions were seen in kidney, liver, adrenal cortex and pituitary gland. Furthermore, positive staining for androgen receptor was exhibited in skeletal, cardiac and smooth muscle cells, and central nervous tissue. Female reproductive organs also contained androgen receptor-positive cells. The spleen was found to be the only organ examined which did not stain for androgen receptor. The monoclonal antibody could also demonstrate androgen receptor-positive cells in a human prostatic cancer and in a prostate with benign hyperplasia. These data demonstrate the use of antibodies in revealing cellular/subcellular distribution of androgen receptor in target tissues.

Progesterone treatment suppresses estrogen receptor in the sex skin of Macaca nemestrina.

We measured tightly bound nuclear estrogen receptors (ER) in sex skin biopsies obtained from pig-tailed macaques (Macaca nemestrina) which were previously ovariectomized and treated with an estradiol-progesterone regimen. Incubation of fresh tissue slices with a saturating concentration of [3H]estradiol (E2) was done to determine the capacity of nuclear acceptor sites to bind activated ER with high affinity. The radiolabeled ER was extracted from nuclei with 0.5 M KCl, complexed with an anti-ER monoclonal antibody, and quantitated by analysis on sucrose gradients. Even though serum E2 levels were unchanged, 7 and 14 days of sequential progesterone (P) treatment decreased ER amounts below those found after 7, 14 and 21-23 days of E2 treatment. ER regulation in sex skin of this species is similar to that found in macaque reproductive tract; P suppresses ER levels even in the presence of continuous E2. The tissue responses of sex skin to the hormone treatments correlated well with the measured fluctuations of tightly bound nuclear ER, which suggests the functional significance of this ER component.

Immunohistochemical localization of Met5-enkephalin-Arg6-Gly7-Leu8 in the female genital organs and in the paracervical ganglion of the pig.

Indirect immunofluorescence method was used to study the localization and distribution of the proenkephalin A-derived octapeptide, Met5-enkephalin-Arg6-Gly7-Leu8 (MEAGL), in the paracervical ganglion and in the female genital organs of the pig. In the paracervical ganglion, a subpopulation of principal neurons and nerve fibers contained MEAGL immunoreactivity. In the vagina, numerous MEAGL-immunoreactive nerve fibers were localized in the muscular membrane, under the serous membrane and in the submucous layer. The uterine cervix contained a great number of immunoreactive nerve fibers in muscular membrane and in submucous and subserous layers. The pattern of distribution of MEAGL-immunoreactive nerve fibers in the uterine horns was similar to that of cervix, but their number in the uterine horns was lower. MEAGL-immunoreactive fibers were also observed through different oviductal layers. In the ovary a low number of immunoreactive fibers were seen in the medullary and cortical parts of the organ. The results of this study indicate that the female genital organs, particularly the uterus and vagina, of the pig receive dense innervation by nerve fibers containing the proenkephalin A-derived octapeptide MEAGL. The presence of MEAGL in principal neurons and fibers of the paracervical ganglion suggests that a large proportion of them originate from neurons of the paracervical ganglion.

Molecular pathology of the lower female genital tract. The papillomavirus model.

Papillomavirus-related genital neoplasms are one area where molecular biology has had an impact at many levels. Studies of cell transformation, gene expression, and genome organization have linked papillomaviruses to neoplasia; they have also provided data suggesting potential pathways by which the papillomaviral genome exerts its effect on cells. Molecular epidemiological studies using clinical material have identified specific HPV types with neoplasia, profiled the populations at risk for these infections, and supported the emerging concept of latent infection. Studies using in situ hybridization have confirmed the close relationship of neoplastic change with certain infections (such as HPV-16), and have detailed the transcription patterns of the papillomavirus genome in warts, precancers, and carcinomas. The technology of in situ hybridization has facilitated the evaluation of archive material; using this material, the close relationship between HPV type 18 and adenocarcinomas and small-cell carcinomas has been described. Methods for expressing HPV proteins in bacteria have produced a spectrum of antisera to specific gene products, which in turn will facilitate mapping their distribution in tissues, determining their biological significance, and clarifying the host immune response to genital papillomavirus infections. Although these multidisciplinary approaches help to promote an understanding of genital HPV infections and their related neoplasms as well as clarifying the role of HPV in the evolution of genital neoplasia, the clinical utility of this information has not yet been established.

Peptide histidine methionine and vasoactive intestinal peptide: occurrence and relaxant effect in the human female reproductive tract.

The occurrence of the neuropeptides peptide histidine methionine (PHM) and vasoactive intestinal peptide (VIP) in the human female genital tract was studied by means of immunochemistry and radioimmunoassay in combination with gel chromatography. In addition, the effect of PHM and VIP on smooth muscle activity was investigated in vitro. The regional distribution of PHM as determined by radioimmunoassay correlated with that of VIP. This finding agreed with the immunohistochemical data, which, in addition, provided evidence for colocalization of the two peptides in nerve fibers. These fibers were most abundant in the vagina and the uterine cervix, where they seemed to innervate blood vessels, smooth muscle, and epithelial cells. The concentrations of immunoreactive PHM and VIP were found to be similar in all areas except in the vagina, where the PHM concentration was fourfold that of VIP. Gel chromatography of vaginal extract revealed a high concentration of a C-terminally extended form of PHM, suggesting differential processing pathways of the VIP precursor. Both PHM and VIP inhibited, in a dose-dependent manner, the smooth muscle activity in strips from the Fallopian tube and the myometrium. Administered in combination, PHM and VIP had an additive effect and displayed the same efficacy as VIP alone, indicating that the peptides act via a common receptor.

Neuropeptide Y in the human female genital tract: localization and biological action.

The distribution, localization, and smooth muscle effects of neuropeptide Y (NPY) were studied in the human female genital tract. High concentrations of NPY immunoreactivity were demonstrated in the uterine artery, the ovary, the fallopian tube, cervix, and the vagina. The NPY immunoreactivity was confined to nerve fibers. The highest density of nerve fibers was observed in relation to blood vessels, although some NPY-immunoreactive nerves were also seen close to nonvascular smooth muscle. The NPY-immunoreactive material throughout the genital tract was identical to synthetic amidated human NPY with regard to size, hydrophobicity, and charge as evaluated by gel filtration, high-performance liquid chromatography, and isoelectric focusing. NPY (10(-10) to 10(-6) M) exerted a direct vasoconstrictory effect on small arteries dissected from the cervix and an additive effect of NPY and norepinephrine responses was observed. Exogenous NPY did not have a direct effect on nonvascular smooth muscle specimens from the fallopian tube or the myometrium. The close relation between NPY-immunoreactive nerves and blood vessels, the presence of NPY-immunoreactive material identical to amidated synthetic human NPY, and the vasoconstrictory effects of NPY indicate that NPY is involved in the regulation of the blood flow in the human female genital tract.

Met5-enkephalin- and Met5-enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibers in the pig female reproductive system.

The localization of Met5-enkephalin (ME) immunoreactivity in the female genital organs of the rat, guinea pig and pig was studied by indirect immunofluorescence method. In the rat and guinea pig, no ME immunoreactivity was observed in the uterus, fallopian tube or ovary. In the pig uterus and fallopian tube ME-immunoreactive nerve fibers were observed in muscular and submucose layers as well as around the blood vessels. In the pig ovary, ME immunoreactivity was localized in nerve fibers in medullary and cortical parts of the organ. Met5-enkephalin-Arg6-Gly7-Leu8 (MEAGL) immunoreactivity was also studied in the pig uterus, where its distribution was similar to that of ME. The present results suggest that the pig genital organs receive innervation by nerve fibers containing proenkephalin A-derived peptides, which may have a role in modulation of neurotransmission in these organs.

Adverse effects of methylene blue on human sperm motility, components of human reproductive tract fluids, and mouse embryo cleavage.

Because methylene blue exhibits germicidal, oxidation, and reduction properties, the authors asked whether this agent causes adverse effects on gametes, embryos, and/or secretions of the reproductive tract. Time- and dose-dependent inhibition of human sperm motility by methylene blue was observed, as was growth inhibition of 2-cell mouse embryos. Furthermore, the presence of methylene blue in uterine, fallopian tube, and peritoneal fluids altered protein mobility in polyacrylamide gels, and yielded apparent values of follicle-stimulating hormone and estradiol up to 260% of actual values (P less than 0.05). These data suggest that the presence of methylene blue in reproductive tract fluids may provide a false impression of their biochemical and biophysical compositions, and that the use of methylene blue as a chromopertubation agent be conducted with appropriate awareness.

Two fetal antigens (FA-1 and FA-2) and endometrial proteins (PP12 and PP14) isolated from amniotic fluid: localisation in the fetus and adult female genital tract.

Monospecific antisera against two fetal antigens (FA-1 and FA-2), alphafetoprotein (AFP) and two endometrial proteins (PP12 and PP14) were used to examine the distribution of these proteins and antigens in human trophoblast and gestational endometrium in first and third trimesters of pregnancy, normal human ovary and fetal tissues by indirect immunoperoxidase histochemical localisation techniques. Fetal liver stained exclusively for FA-1 and AFP which was used as a reference protein. Staining for FA-2 was seen in fetal connective tissue, in particular the basement membrane. FA-1 and FA-2 did not stain positively in decidua, trophoblast or ovarian tissue. Gestational endometrium stained positively for PP14 exclusively in the glandular epithelium, whilst staining for PP12 was seen only in the stromal cells. Trophoblast, both early and late, and ovarian tissue did not stain positively for any of the four substances tested.

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.

Detection of human papillomavirus deoxyribonucleic acid in the female genital tract.

A total of 336 biopsies, scrapes and exfoliated cells from the cervix and from the lower genital tract were screened for human papilloma (HP) viral sequences of types 6, 11, 16 and 18 by Southern blot, dot blot and filter in situ (FISH) hybridizations with cloned 32P-radiolabeled HPV DNA probes. The specimens included cervical intraepithelial neoplasias (CIN I-III), carcinoma in situ and invasive carcinoma of the cervix and vagina, adenocarcinomas, vulvar and vaginal condylomata acuminata and healthy epithelial samples. The oncogenic HPV 16 was found in 46% of the cervical carcinomas. Most of the type 16 occurrences (75%) represented the third stage of inoperable cases. Similarly, HPV 18 was also most frequently present in this stage as well as in carcinoma in situ and in CIN III (25%, 18%). At the same time, in condylomata acuminata, types 6 and 11 were detectable in 88.7% of cares. In all, 13.5% of the normal samples harboured HPV DNA.

Correlation of histology with human papillomavirus DNA detection in the female genital tract.

Genital condyloma and intraepithelial neoplasia secondary to Human Papillomavirus (HPV) infection are characterized by perinuclear halos and marked nuclear atypia (koilocytotic atypia) on cytologic and histologic examination. However, at times the histologic findings, including the degree of nuclear atypia, may be suggestive but not absolutely diagnostic of an HPV related neoplasm. HPV DNA sequences were detected in 63 and 56% of colposcopically visible vaginal and cervical lesions, respectively, that were diagnosed as condyloma or intraepithelial neoplasia. HPV DNA sequences were detected in 14 and 47% of vaginal and cervical lesions, respectively, that did not fulfill the histologic criteria of condyloma or intraepithelial neoplasia (i.e., "nondiagnostic"). When examining cervices from patients with no visible lesion and no recent history of an abnormal pap smear, 5.5% had detectable HPV DNA sequences. The histologic findings in this group were equivalent to the virus-negative cases and similar to the "nondiagnostic" cervical lesions. These findings suggest that the detection rate of HPV DNA in "nondiagnostic" tissues is dependent on the site and presence or absence of a visible lesion. The rate is similar in cervical lesions regardless of the histologic findings whereas it is less in vaginal lesions when the histologic criteria of condyloma or intraepithelial neoplasia are not detected.

Interaction of NPY and VIP in regulation of myometrial blood flow and mechanical activity.

The occurrence, molecular characteristics and biological function of neuropeptide Y (NPY) has been studied in the female genital tract of non-pregnant rabbits. NPY immunoreactivity was demonstrated throughout the genital tract. Maximum concentrations were found in the salpinx (fallopian tube), 570 pmol/g (median) lower within the uterine body (1.5 pmol/g), cervix (2.8 pmol/g) and vagina (3.6 pmol/g). In vitro, NPY had a dose-dependent stimulatory effect on non-vascular smooth muscle (ED50 10(-9) mol/l) as studied by myometrial tension recordings. In vivo, NPY (50 pmol/min.kg) induced a dose-related, non-adrenergic and non-cholinergic decrease in myometrial blood flow. Small C-terminal (NPY31-36) or N-terminal (NPY1-16) fragments of NPY had no effect on myometrial blood flow. NPY was found to interact with the smooth muscle effect of VIP; the presence of VIP (10(-8) mol/l) counteracted the contraction elicited by NPY (10(-8) mol/l) returning the response to control value. VIP and NPY displayed a similar physiological antagonism on myometrial blood flow. There was a clear difference in the response to VIP and NPY as the effect of NPY on myometrial blood flow first appeared after a lag period of 2 minutes whereas the effect of VIP was almost instantaneous. It is concluded that NPY and VIP may interact in the local nervous control of genital functions.

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.

Sensitive detection and typing of human papillomavirus DNA in gynecological cell scrapings by slot-blot hybridization.

A rapid and sensitive method for detecting and typing human papillomaviruses (HPVs) in cell scrapings is presented. DNA from scrapings is extracted and bound to nitrocellulose filters (Slot-Blot). By DNA-DNA hybridization with specific 32P-labelled HPV-probes (types 6/11 or 16/18) the patient's DNA is then analyzed for the presence of, and for the type of, HPV DNA sequences. A parallel hybridization with a human repetitive element (Alu sequence) allows quantitation of the different hybridization results. Experiments with HeLa cell DNA show that as little as 10(4) HPV sequences can be detected and typed specifically with this test. Evaluation of this test is completed within 6 to 7 days after cell collection. This Slot-Blot method was used to analyse 1330 specimens taken at the Bernese Dysplasia Outpatient Clinic. The results reveal a very high percentage (90%) of HPV-positive cases in the patient group examined.

Pharmacokinetic profile of cefotetan in different clinical conditions.

The pharmacokinetic profile of cefotetan was studied in a group of hospitalized patients. The absorption of the molecule (after a single dose of 2 g/i.m.) was good and the drug was found to diffuse satisfactorily in the lungs, prostatic tissue, kidney and in the female genitalia.