Long-term alterations in histology and steroid receptor levels of the genital tract and mammary gland following neonatal exposure of female BALB/cCrgl mice to various doses of diethylstilbestrol.

The relation of the dosage of diethylstilbestrol (DES) administered neonatally to the incidence and severity of genital tract and mammary gland lesions and to the levels of sex hormone receptors was examined using a mouse model for human intrauterine DES exposure. Female BALB/cCrgl mice received various doses of DES (ranging from 5 X 10(-1)-10(-5) micrograms daily for the first 5 days of life) or the sesame oil vehicle alone. In the vagina, at all ages examined (1, 2, 6, and 12 months) cytosolic estrogen receptors are consistently decreased after high doses of neonatal DES (10(-1) and 1 microgram). In contrast, at the same ages, vaginal cytosolic progestin receptors increase after identical doses. In the uterus, the 1-microgram dose of neonatal DES also consistently decreases cytosolic estrogen receptors while increasing cytosolic progestin receptors at 1, 2, and 6 months of age. Histologically, neonatal doses of 5 X 10(-2) micrograms DES result in vaginal lesions at 2 months. With age, this threshold level decreases, implying interaction with an altered hormonal milieu. The uterus shows a sensitivity similar to that of the vagina in regard to the histopathological effects of neonatal DES. The ovary and mammary glands are 10- to 100-fold more sensitive to neonatal DES exposure.

Demonstration and characterization of estrogen receptor in chimpanzee sex skin: correlation between nuclear receptor levels and degree of swelling.

The presence of cytosol and nuclear estrogen receptors has been demonstrated in chimpanzee sex skin. Both receptors sedimented at approximately 4S in sucrose gradients containing 0.6 M KCl. Both had a steroid specificity and high affinity for estrogen that conform to an estrogen receptor. The absence of such receptors in the pigmented skin surrounding the sex skin and in the abdominal skin indicates their discrete localization in the sex skin. While the cytosol receptor remained low (less than 10 fmol/mg cytosol protein), the nuclear estrogen receptor showed a fluctuation during the menstrual cycle, attaining the highest level during the late follicular phase (90.4 +/- 8.4 fmol/mg nuclear extract protein; nearly 3-4 times the level during the early follicular and luteal phases). When ovariectomized animals were treated with mestranol, the concentration of nuclear estrogen receptor increased from below detection to 74 fmol/mg nuclear extract protein. An increase in serum progesterone during the luteal phase, despite concurrently elevated serum estradiol levels, was associated with a reduction of the nuclear estrogen receptor as was the administration of progesterone and mestranol to estrogen-primed ovariectomized animals. Changes in the concentration of the nuclear estrogen receptor were positively correlated with changes in the degree of the sexual swelling. These results strongly suggest that estrogen controls the sexual swelling by acting through specific receptors in the sex skin, and that counteraction by progesterone of estrogen stimulation of the sexual swelling is effected through a reduction in nuclear estrogen receptors.

Immunocytochemical localization of estrogen receptors in the macaque reproductive tract with monoclonal antiestrophilins.

Monoclonal antibodies to the estrogen receptor (anti-ER) were used to develop an immunocytochemical method to detect ERs in frozen sections of the macaque reproductive tract. Specific nuclear, but not cytoplasmic, staining occurred with two different methods: direct, in which an antiestrophilin -horseradish peroxidase conjugate was used as the first antibody, and indirect, in which a mixture of antiestrophilins was used in the first incubation step. Nuclear staining was absent when various control antibodies replaced the anti-ER. In uteri from spayed monkeys treated with estradiol (E2) for 14 days, nuclear staining was always present. In uteri from similar animals treated for an additional 14 days with E2 and progesterone, nuclear staining was almost completely absent. Mean endometrial nuclear ER levels, measured by an exchange assay, were 5-fold greater in the E2-treated than in the E2-plus progesterone-treated group. In addition, when samples of estrogenized uterus and oviduct were incubated for 60 min in vitro with 100 nM E2, the intensity of nuclear staining increased in parallel with an increase in the concentration of nuclear ER. The nuclei of stromal, smooth muscle, and epithelial cells of the estrogenized oviduct, cervix, and vagina as well as smooth muscle cells of the estrogenized myometrium were also receptor positive. Nontarget tissues, such as duodenum, colon, esophagus, and skeletal muscle, contained no cells that showed specific nuclear staining. Some staining of cytoplasmic and extracellular components occurred in all preparations. These latter reactions were nonspecific, because they were present in many nontarget tissues or when control antibodies replaced the anti-ER. With current methods, only nuclear ERs can be reliably localized in frozen sections of monkey tissues with monoclonal antiestrophilins .

Immunohistochemical demonstration of relaxin in the genital tract of pregnant and nonpregnant women.

The biotin-avidin immunoperoxidase staining method and antisera against highly purified porcine relaxin were used to localize relaxin in the genital tract of pregnant and nonpregnant women. Formalin-fixed tissue specimens from normal placenta, decidua, myometrium, vagina, corpus luteum, and Fallopian tubes were studied. In pregnant women, relaxin was found in the placental syncytiotrophoblast, decidua, and corpus luteum. In nonpregnant women, relaxin was identified in the corpus luteum and endometrium in the secretory, but not in the proliferative, phase. Myometrium, cervix, vagina, and Fallopian tubes were negative for relaxin. This is the first report describing relaxin in the nonpregnant corpus luteum, and we also confirm results of an early disputed study claiming that endometrium in the secretory phase contains relaxin. The origin and biological role of human endometrial relaxin remain to be studied.

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.

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.

Substance P in obstetrics and gynecology.

The occurrence and distribution of the neurohormone substance P in the urogenital tract suggest a role for substance P in regulation of blood flow, smooth-muscle activity, and sensation. The distribution and pharmacologic effects of substance P in the brain suggest an involvement of substance P in regulating the release of gonadotropins and prolactin from the pituitary and in the gonadal-hypothalamic feedback. As substance P is also present in endocrine-like cells of the diffuse endocrine system in the urogenital tract, it may serve as a tumor marker for carcinoid and related tumors, particularly in the ovary and the uterine cervix. This review is limited to the distribution and effects of substance P in the female genital tract and its relevance in female reproduction.

Calcitonin gene-related peptide (CGRP) in the female rat urogenital tract.

CGRP-immunoreactivity was found throughout the female rat urogenital tract by specific radioimmunoassay, and shown to be present in nerve fibres by immunocytochemistry. The highest concentrations of CGRP-like immunoreactivity were found in the urinary tract, with lower levels in regions of the genitalia. Chromatographic analysis of bladder and vaginal extracts on Sephadex G-50 columns and HPLC revealed at least three CGRP-immunoreactive peaks. The major peak emerged in the same position as synthetic rat CGRP. CGRP nerve fibres were associated mainly with blood vessels, non-vascular smooth muscle, squamous epithelium and uterine and cervical glands, and were particularly abundant in the ureter and bladder. CGRP-immunoreactivity was depleted by neonatal treatment with capsaicin and after surgical section of pelvic and/or hypogastric nerves. Immunocytochemistry demonstrated that depletion occurred predominantly in the mucosal layer of the urogenital tract. These findings indicate a sensory function for most of the CGRP-immunoreactive nerves in the rat urogenital tract.

Distribution of galanin immunoreactivity in the genitourinary tract of man and rat.

Galanin has been shown to be present in substantial quantities in the human and rat genitourinary tract by radioimmunoassay and immunocytochemistry. The highest concentrations measured by radioimmunoassay were found in the human vas deferens, corpus cavernosum and spongiosum and in the vagina and cervix. In man gel chromatographic analysis showed two molecular forms. The earlier eluting peak was different from porcine galanin standard. There was only one molecular form in the rat which emerged in an earlier position than the porcine standard. Galanin immunoreactive nerve fibres demonstrated in the genitourinary tract were found both in man and rat. They were found within smooth muscle and in close relationship to blood vessels. The presence and distribution of galanin in the genitourinary system suggest the possibility that this neuropeptide could play a role in the regulation of smooth muscle tone, blood flow and motility.

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.

Gonadotropin- and estrogen-induced increase of peripheral-type benzodiazepine binding sites in the hypophyseal-genital axis of rats.

Peripheral-type benzodiazepine binding sites (PBS) were demonstrated in the cell membranes of various organs (ovary, uterus, oviduct, pituitary and kidney) of mature and immature female rats by using the PBS-specific ligand [3H]PK 11195. The equilibrium dissociation constants of [3H]PK 11195 for PBS in mature rats ranged from 3 to 4 nM. The specific binding of [3H]PK 11195 (2 nM) in the hypophyseal-genital axis of immature (19-27 days old) female rats was found to be significantly increased in the ovary and uterus, concurrently with the increase in age. Administration of pregnant mare serum gonadotropin or diethylstilbestrol to immature rats increased the density of PBS in the ovary and uterus 2- to 3-fold but no change was found in the kidney. The affinity of [3H]PK 11195 to these tissues did not change following hormonal treatment. These results suggest that gonadotropin and estrogen are involved in the induction of PBS in the organs of the hypophyseal-genital axis in female rats.

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.

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.

On talc translocation from the vagina to the oviducts and beyond.

The objective of this study was to investigate whether multiple vaginal depositions of neutron-activated talc in the cynomolgus monkey result in the translocation of this material to the uterus and beyond. Within a 45-day period, six monkeys received 30 applications of 125 mg neutron-activated talc, suspended in 0.3 ml physiological saline solution containing 1% carboxymethyl cellulose as a suspending agent. The suspension was deposited in the posterior vaginal fornix of the sedated monkeys. Two days after the final talc application, the animals were anaesthetized. Abdominal lavage was performed and the lavage fluid collected for gamma-ray analysis. Also collected for gamma-ray analysis were the following tissues/organs: ovaries, oviducts, uterus, and vagina with cervix. Six untreated control monkeys underwent the same procedures. The radioisotopes 46Sc, 60Co, 59Fe and 51Cr in the activated talc served as tracers. Only the samples containing vagina and cervix from the dosed monkeys contained varying quantities of talc. This demonstrates that no measurable quantities of talc, deposited by multiple applications in the vaginal fornix of the cynomolgus monkey, translocated to the uterus or beyond.

Copper and zinc concentrations, and superoxide dismutase activities in malignant and nonmalignant tissues of female reproductive organs.

The copper and zinc concentrations in 44 malignant and 48 nonmalignant women tissue samples of reproductive organs in women were measured. In malignant samples, the mean copper concentrations were 110%, 76%, and 38% higher for cervix, endometrium and ovary than the nonmalignant ones. The zinc concentrations in the analysed malignant tissues were lower than that in the corresponding nonmalignant tissues. The results of superoxide dismutase activities determinations demonstrate a considerable lowering of the enzymatic capacities to remove free oxygen radical in malignant tissues. A hypothesis for possible mechanism involving elevated copper concentrations, and decreased zinc concentrations, which may be responsible for malignant processes, are presented.

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.

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.

[A new Acanthocephala Breizacanthus ligur sp. n. (Palaeacanthocephala: Arhythmacanthidae Yamatugi, 1935) parasite of some species of benthic fishes from the Ligurian Sea]. / Su di un nuovo acantocefalo Breizacanthus ligur sp.n. (Palaeacanthocephala: Arhythmacanthidae Yamaguti, 1935) parassita di alcune specie di pesci bentonici del Mar Ligure

Breizacanthus ligur sp.n. is described and figured from several benthic fishes from the Ligurian Sea. The host species are: Argentina sphyraena, Chlorophthalmus agassizi, Gadiculus argenteus, Phycis blennoides, Coelorhynchus coelorhynchus, Capros aper, Callionymus phaeton, Helicolenus dactylopterus. The parasites were considered as belonging to the family Arhythmacanthidae Yamaguti, 1935 and to the genus Breizancanthus Golvan, 1969. Breizancanthus ligur differs from the only two known species of the genus B. irenae and B. chabaudi for the number of longitudinal rows of hooks and-or numbers of hooks in each longitudinal row, arrangment of cement glands, length of body and lenght of male genital apparatus ratio, lenght of female genital apparatus and shape of embroyophore.