Mouse Genital Tract Research Articles

Genital antibody responses in mice after intranasal infection with an attenuated candidate vector strain of Bordetella pertussis.

Intranasal administration of live attenuated Bordetella pertussis, from which the pertussis toxin gene has been deleted, has previously been shown to give rise to high levels of serum immunoglobulin G (IgG) antibodies against both the protective antigen filamentous hemagglutinin (FHA) and heterologous antigens genetically fused to FHA. Here, we extend these results by demonstrating that anti-FHA IgA and IgG antibodies are also produced in the genital tract of mice, both in the vagina and in the uterus, after a single intranasal administration of B. pertussis. By comparing the immune responses induced after infection with wild-type virulent B. pertussis with that induced by infection with an attenuated pertussis toxin-deficient strain, we conclude that pertussis toxin produced by the virulent bacteria does not modify antibody production to FHA in the genital tract of B. pertussis-infected mice. The intranasal infection with either the attenuated or the virulent B. pertussis strain also led to the development of immunologic memory that could be efficiently boosted with purified FHA administered either intranasally or intravaginally to give rise to a significant increase in the levels of specific IgA and IgG produced locally in the genital tract, as well as of specific antibodies in the serum. These observations suggest that attenuated B. pertussis could be a promising vector for intranasal administration to induce antibody responses against antigens from sexually transmitted pathogens fused to FHA.

Subclinical chlamydial infection of the female mouse genital tract generates a potent protective immune response: implications for development of live attenuated chlamydial vaccine strains.

Chlamydia trachomatis is a major cause of sexually transmitted disease (STD) for which a vaccine is needed. CD4(+) T-helper type 1 (Th1) cell-mediated immunity is an important component of protective immunity against murine chlamydial genital infection. Conventional vaccine approaches have not proven effective in eliciting chlamydial-specific CD4 Th1 immunity at the genital mucosa. Thus, it is possible that the development of a highly efficacious vaccine against genital infection will depend on the generation of a live attenuated C. trachomatis vaccine. Attenuated strains of C. trachomatis do not exist, so their potential utility as vaccines cannot be tested in animal models of infection. We have developed a surrogate model to study the effect of chlamydial attenuation on infection and immunity of the female genital tract by treating mice with a subchlamydiacidal concentration of oxytetracycline following vaginal infection. Compared to untreated control mice, antibiotic-treated mice shed significantly fewer infectious organisms (3 log(10)) from the cervico-vagina, produced a minimal inflammatory response in urogenital tissue, and did not experience infection-related sequelae. Antibiotic-treated mice generated levels of chlamydia-specific antibody and cell-mediated immunity equivalent to those of control mice. Importantly, antibiotic-treated mice were found to be as immune as control untreated mice when rechallenged vaginally. These findings demonstrate that subclinical chlamydial infection of the murine female genital tract is sufficient to stimulate a potent protective immune response. They also present indirect evidence supporting the possible use of live attenuated chlamydial organisms in the development of vaccines against chlamydial STDs.

Does Chlamydia trachomatisMoPn enter a microbiologically-inapparent state during experimental infection of the mouse genital tract?

Microbiologically-inapparent chlamydial infection may contribute towards the immunopathogenesis of these diseases. Although morphologically and physiologically aberrant non-cultivable chlamydiae can be induced reversibly in cell culture, evidence for these forms in infections of animals and humans is indirect. A mouse model of salpingitis caused by the mouse pneumonitis biovar of Chlamydia trachomatis(MoPn) was used to determine the existence of non-cultivable organisms in vivo. Following intravaginal inoculation, mice yielded high chlamydial counts for 7–14 days, with a decline in culture-positivity by 21–28 days. A significant elevation of IFN γproduction in infected tissues was measured for 21 days and, from 28–70 days, all mice were culture-negative and developed characteristic hydrosalpinges. MoPn was detected by PCR in vaginal swabs of 80% and 69% respectively of culture-negative animals at 21 and 28 days. In a second study, 100%, 63% and 50% of culture-negative genital tissue homogenates were PCR-positive at 21, 28 and 42 days. Immunosuppression with either cyclophosphamide or hydrocortisone failed to regenerate cultivable chlamydiae. Tissues were disrupted by homogenization and inoculated intranasally to MF1 mice which are extremely susceptible to MoPn, but all culture-negative specimens were non-infectious. The significance of the PCR-positive culture-negative specimens requires further investigation, since these may represent a non-cultivable state in the deeper tissues of the mouse genital tract which may be beyond the reach of reactivating triggers.

Immunohistochemical studies on the expression and estrogen dependency of EGF and its receptor and C‐fos proto‐oncogene in the uterus and vagina of normal and neonatally estrogen‐treated mice

Background The final target cell response to estrogen is dependent not only on the estrogen receptor, but also on autocrine/paracrine interactions with growth factors (e.g., EGF) and proto-oncogenes (e.g., c-fos). Because neonatal estrogen treatment results in permanent changes in the female mouse genital tract (permanent vaginal cornification, cervical adenosis and tumors, changed growth control mechanisms in uterus), it was of interest to study possible acute and permanent effects of such treatment on distribution and levels of EGF, its receptor (EGF-r), and c-fos and to relate such changes to morphological development and appearance of epithelial abnormalities. Methods Immunohistochemical techniques using frozen sections from the uterus and vagina of neonatal and adult (ovariectomized, estradiol-treated) females, treated with olive oil or diethylstilbestrol in neonatal life. Results A difference in stromal-epithelial distribution of EGF was demonstrated with respect to region studied (uterus, vagina) and age (neonatal, adult). EGF was localized mainly in the uterine stroma but in both vaginal epithelium and stroma (with a different pattern compared to uterus). In neonatal females, EGF occurred in both tissue components in both regions, and the distribution pattern was quite different from that in adult females. The EGF level was increased by estrogen in adult but not in neonatal females. EGF-r and c-fos occurred in both uterine epithelium and stroma and in the vaginal epithelium; levels and distribution pattern were affected by estrogen. Neonatal estrogen treatment increased the levels of uterine EGF and c-fos in adult life. Conclusions There are distinct developmental changes in the distribution and estrogen sensitivity of EGF. Only further studies can prove or disprove the association between the earlier reported disturbed growth control mechanisms in the uterus of adult but neonatally estrogen-treated females and the increased levels of uterine EGF and c-fos. The present results do not seem to explain mechanisms involved in the origin of neonatally estrogen-induced cervicovaginal epithelial abnormalities, nor do they explain the earlier described difference in estrogen-induced proliferative response between the uterine cervix and uterus proper. © 1996 Wiley-Liss, Inc.

Immunohistochemical studies on the expression and estrogen dependency of EGF and its receptor and C-fos proto-oncogene in the uterus and vagina of normal and neonatally estrogen-treated mice.

The final target cell response to estrogen is dependent not only on the estrogen receptor, but also on autocrine/paracrine interactions with growth factors (e.g., EGF) and proto-oncogenes (e.g., c-fos). Because neonatal estrogen treatment results in permanent changes in the female mouse genital tract (permanent vaginal cornification, cervical adenosis and tumors, changed growth control mechanisms in uterus), it was of interest to study possible acute and permanent effects of such treatment on distribution and levels of EGF, its receptor (EGF-r), and c-fos and to relate such changes to morphological development and appearance of epithelial abnormalities. Immunohistochemical techniques using frozen sections from the uterus and vagina of neonatal and adult (ovariectomized, estradiol-treated) females, treated with olive oil or diethylstilbestrol in neonatal life. A difference in stromal-epithelial distribution of EGF was demonstrated with respect to region studied (uterus, vagina) and age (neonatal, adult). EGF was localized mainly in the uterine stroma but in both vaginal epithelium and stroma (with a different pattern compared to uterus). In neonatal females, EGF occurred in both tissue components in both regions, and the distribution pattern was quite different from that in adult females. The EGF level was increased by estrogen in adult but not in neonatal females. EGF-r and c-fos occurred in both uterine epithelium and stroma and in the vaginal epithelium; levels and distribution pattern were affected by estrogen. Neonatal estrogen treatment increased the levels of uterine EGF and c-fos in adult life. There are distinct developmental changes in the distribution and estrogen sensitivity of EGF. Only further studies can prove or disprove the association between the earlier reported disturbed growth control mechanisms in the uterus of adult but neonatally estrogen-treated females and the increased levels of uterine EGF and c-fos. The present results do not seem to explain mechanisms involved in the origin of neonatally estrogen-induced cervicovaginal epithelial abnormalities, nor do they explain the earlier described difference in estrogen-induced proliferative response between the uterine cervix and uterus proper.

CD4+ T cells play a significant role in adoptive immunity to Chlamydia trachomatis infection of the mouse genital tract.

The ability of CD4+ and CD8+ T cells to adoptively immunize mice against Chlamydia trachomatis infection of the mouse genital tract was studied. Adoptive transfer experiments were performed with splenic CD4+ or CD8+ T cells obtained from mice following resolution of a primary genital tract infection and after a secondary chlamydial challenge. The results show that donor CD4+ T cells, but not CD8+ T cells, obtained from mice following resolution of a primary infection or after secondary challenge were effective in transferring significant antichlamydial immunity to the genital tracts of naive animals. The lymphokine profiles in the culture supernatants of proliferating Chlamydia-specific CD4+ T cells obtained from mice following resolution of a primary infection and after secondary challenge were assayed by an enzyme-linked immunoadsorbent assay. Protective CD4+ T cells restimulated in vitro secreted interleukin 2, gamma interferon, and interleukin 6, lymphokine profiles characteristic of both Th1- and Th2-like responses. Resting CD4+ T cells obtained from mice 4 months following resolution of a primary infection were also capable of conferring significant levels of adoptive protective immunity to naive mice. These findings support an important role for CD4+ T cells in acquired immunity to chlamydial infection of the genital tract and indicate that protective CD4+ immune responses in this model are relatively long lived.

Changes in the hormone dependency of epithelial cell proliferation in the genital tract of mice following neonatal oestrogen treatment

The genital tract epithelium of the female laboratory mouse has been widely studied as a model of oestrogen-dependent growth and proliferation. Perturbation of the hormonal imprinting of these tissues during neonatal development has also been used to study the development of pathological abnormalities, particularly in the cervical epithelium. This study demonstrates that mice treated neonatally from days 1–5 with supraphysiological concentrations of oestrogen are able to maintain high levels of proliferation following the removal of the ovaries later in adult life. This high level of proliferation was shown to be independent of the ovarian oestrogens and of oestrogens produced peripherally by aromatisation. These results suggest conversion of the genital tract in these mice to a fully hormonal “independent” state. However, neonatal treatment with oestrogen was not found to produce a uniform change to hormonal independence. Further challenge of the adult ovariectomised mice with oestrogen, demonstrated that a population of cells still retained the ability to respond to the mitogenic influence of this hormone.

Roxithromycin treatment of mouse chlamydial salpingitis and protective effect on fertility

We used a mouse model of acute chlamydial salpingitis to evaluate the efficacy of roxithromycin in preventing irreversible inflammatory damage leading to tubal infertility. Female C3H/He mice were genitally inoculated with a human strain of Chlamydia trachomatis and then treated with roxithromycin glutamate subcutaneously. Treatment was initiated either 7 or 10 days postinfection (p.i.) and continued for 7 days at a dosage of 50 or 100 mg/kg of body weight per 24 h. The course of the disease was monitored serologically, bacteriologically, and histologically. At the end of the treatment, the mice were encaged with males and their reproductive capacity was recorded over a 19-week period. The protective effect of roxithromycin was assessed in terms of fertility parameters in comparison with values for noninfected control mice. When treatment was initiated on day 7 p.i. and given in twice-daily 25-mg/kg doses, all the mice remained fertile and the total number of offspring was similar to that of sham-infected mice (17.3 +/- 3.3 versus 17.2 +/- 2.3). When treatment was initiated on day 10 p.i. and given in a single daily dose of 50 or 100 mg/kg, 90 and 70% of the mice, respectively, remained fertile; however, in terms of total offspring, fertility was lower in the group treated with the lower dose (5.6 +/- 1.4 versus 13.0 +/- 3.8). Roxithromycin was found to be effective against C. trachomatis in the mouse genital tract, but fertility was only partially preserved when the time between infection and treatment was prolonged.

Anti-masculinizing action of estradiol and cyproterone acetate: Regulation of a protein fraction with phospholipase-A 2 stimulatory and masculinizing activities

Recently we have identified a protein fraction (55–63 K) from male and testosterone-exposed female mouse genital tract, which stimulates phospholipase A 2 (PLA 2) and induces masculine differentiation in an undifferentiated mouse genital explant, suggesting a role of this protein in the action of testosterone. In the current study we have further investigated the role of this protein by determining whether anti-masculinizing agents, namely, estradiol and cyproterone acetate, have any effect on the production of this protein. The results described here indicate that a protein fraction containing PLA 2 stimulatory activity was present in both control male and estradiol- or cyproterone acetate-exposed male fetal genital tract. However the specific activity of the PLA 2-stimulatory protein was significantly higher in the control males than in the experimental males. We did not find any major difference in the behavior of this protein fraction in various chromatographic steps except that in CM-sepharose column; the PLA 2-stimulatory activity from the male preparation was eluted in two overlapping peaks with 0.3 and 0.25 M NaCl and that from the treated males was eluted only with 0.25 M NaCl. The SDS-gel analysis of this protein fraction revealed a doublet band (55 and 63 K) in control samples and primarily a 63 K band in experimental samples. The protein fraction from all these sources showed a significant difference in their biological activity. The control male preparation induced Wolffian duct whereas the estradiol sample was completely ineffective and the cyproterone acetate sample was partially effective in inducing Wolffian duct. Thus, it appears that the protein fraction has a role in the masculinizing action of testosterone.

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

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

Long-term alterations on the male mouse genital tract associated with neonatal exposure to cyproterone acetate biochemical data

The effects of neonatal administration of cyproterone acetate on the growth, hormone responsiveness, DNA and protein concentrations, protein profiles, protein synthetic patterns and nuclear androgen binding sites of epididymis, vas deferons and seminal vesicles were investigated in adult mice. The weight of epididymis and seminal vesicle was significantly depressed and the reductions observed were secondary to cellular hypoplasia in epididymis and to cellular hypotrophy in seminal vesicle. The 3 organs studied showed a limited response to exogenous androgens at adulthood. When assessed by the number of cells the response of the 3 organs was similar to that of controls but it was significantly reduced from 25 to 35% when measured in term of cellular concentration of proteins. The protein profiles from homogenates of whole organs and the protein synthetic patterns after [ 35S]methionine incorporation, analyzed by polyacrylamide gel electrophoresis, showed reproducible persistent alterations. The total number of nuclear androgen binding sites was significantly reduced in seminal vesicles.

Influenza specific antibodies in the female genital tract of mice after oral administration of live influenza vaccine.

In order to investigate the possible stimulation of antibodies in the genital tract by immunization female NMRI-mice were given orally a live influenza vaccine (A/PR/8/34, H1N1) on two occasions which were 10 days apart. Subsequently, virus specific IgA antibodies measured by an enzyme immunoassay in homogenates of urinary bladder, uterus and vagina and also in uterine washings. Specific IgA antibodies were not detectable in the sera of immunized mice. The high IgA titer in uterine washings, and in the homogenates suggests enhancement by vaccine of IgA antibody production in the genital tract.

Enhancement of colonization of Ureaplasma urealyticum in the mouse genital tract by estrogen treatment

Experimental colonization by Ureaplasma urealyticum in the vagina of Balbc mice was greatly enhanced by administration of estrogen, whereas it was not affected by treatment with progesterone. Ureaplasma was recovered from almost half of the estrogen-treated animals 7 days after inoculation, and the organism persisted in all of these animals until 21 days after inoculation; on the other hand, the organism was isolated in less than 20% of the untreated, progesterone-treated, or danazol-treated mice 7 days after inoculation and was no longer isolated in any of these groups 21 days after inoculation. At least a hundredfold more organisms were recovered from the estrogen-treated mice as compared to the untreated mice 14 days after inoculation and thereafter. U. urealyticum was also isolated from the uterine cavity and salpinx in approximately half of the mice in which the organism was isolated from the vagina.

Embryonic cholinesterase activity during morphogenesis of the mouse genital tract. Light- and electron-microscopic observations.

In the genital tract of male and female mouse embryos cholinesterase activity is described that is independent from innervation. The enzyme activity is localized in the mesenchyme at the junction of Wolffian and Müllerian ducts with the urogenital sinus. During male development prostate buds and vesicular glands grow out into the cholinesterase-active mesenchyme. During female development the active mesenchyme participates in the downgrowth of the vaginal anlage. Ultrastructurally the cholinesterase activity is localized in the perinuclear cisterna and in smooth endoplasmic reticulum of the mesenchymal cells. The enzyme activity disappears with definitive differentiation of the tissue. The embryonic cholinesterase is a component of a primitive muscarinic system. Its relation to the morphogenetic action of testosterone and its possible general functions are discussed.

The interplay of host and organism factors in infection of the mouse genital tract by Mycoplasma pulmonis.

Mice of strain TO, in groups of ten, were inoculated intravaginally with Mycoplasma pulmonis organisms. Seven mice became infected after inoculation of organisms with strong haemadsorptive capacity, four after inoculation of organisms with diminished adsorptive capacity following ten passes in medium, and none after inoculation of apparently non-adsorbing organisms which had been passed 50 times. There appeared to be a correlation, therefore, between the ability to infect and the cytadsorptive capacity of the organisms. There was only a minimal vaginal polymorphonuclear leucocyte (PMNL) response in the infected mice and most of them had ceased to be infected by 35-42 days. In contrast, mice treated with progesterone had enhanced infections; all those given strongly haemadsorbing organisms, and organisms passed ten times, became infected and remained so for at least 42 days. Furthermore, at least ten fold more organisms were recovered from progesterone-treated than from untreated mice, and the PMNL response was much greater. Most of the progesterone-treated mice given organisms passed 50 times did not become infected, but some did, and the organisms recovered from them were fully cytadsorptive. It is postulated that a few cytadsorbing organisms in this inoculum were induced to infect under the enhancing effect of progesterone.

Immunohistochemical localization of immunoglobulins A, G and M in the mouse female genital tract.

The localization of immunoglobulins A, G and M (IgA, IgG, IgM) in the mouse genital tract was studied by immunoperoxidase techniques at oestrus, on the day of mating and at the time of implantation. In the horns and body of the uterus, IgA and IgG were located in plasma cells in the endometrium surrounding uterine glands and in the gland lumina. The numbers of these plasma cells increased markedly between Day 1 and Days 4 and 5 of pregnancy and the ratio of plasma cells containing IgA and IgG was about 3 or 4 to 1 at all stages. Area measurements indicated that the increased number of plasmacytes was not due to an increase in the amount of endometrial, myometrial or glandular tissues. Plasma cells were not detected in the cervix and vaginal fornix at oestrus and Day 1, but a few were present on Day 5. In the oviduct, plasma cells containing IgA and IgG were present only in the preampulla and both immunoglobulins were present in the extracellular space of the lamina propria only in this region. No IgM was detected in any part of the reproductive tract at any of the times studied. Uteri on Day 1 of pregnancy contained bacteria of several kinds, some of which were aggregated and coated with IgA. This suggests that the uterine lumen at this time may contain specific anti-bacterial IgA antibodies. Our observations indicate that the horns and body of the uterus and the preampulla of the oviduct are major sites of a local immune system in the female mouse genital tract.

Localization of diethylstilbestrol metabolites in the mouse genital tract

Localization of diethylstilbestrol metabolites in the mouse genital tract

The distribution and effect ofChlamydia trachomatis in CBA mice inoculated genitally, intra-articularly or intravenously

A "fast" egg-killing human strain of Chlamydia trachomatis was inoculated into normal CBA and congenic CBA/nu mice, which have an impairment of T-cell function and do not produce anti-chlamydial antibodies. The mice were inoculated by the intra-uterine, intra-articular, or intravenous routes. Some of the mice were first treated with progesterone, which allows successful chlamydial infection of the mouse genital tract when the organisms are introduced genitally. Mice were sacrificed up to 27 days after inoculation. Homogenates of joints, genital tract, spleen, liver, kidneys, eyes and lungs were prepared and tested for chlamydiae in cycloheximide-treated McCoy cell cultures. Chlamydiae were detected in the genital tracts and spleens, but not in the joints, of mice inoculated via the intra-uterine route. They were found in the joints and spleens of mice inoculated intra-articularly, and were detected also in spleens and, from the 4th to 6th day after inoculation, in joints of mice given the organisms intravenously. These results were obtained irrespective of whether or not the mice had received progesterone. The numbers of chlamydiae in the spleens and joints of the nude mice were larger and they persisted longer than in the corresponding immunocompetent animals, although this was not true for chlamydiae in the genital tract of mice inoculated via the intra-uterine route. Compartmentalisation of chlamydiae was evident although the spleen was infected consistently irrespective of the route of inoculation and, as mentioned, chlamydiae were found transiently in the joints following intravenous inoculation. This suggests that chlamydiae might also enter the human joint.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of experimental Mycoplasma pulmonis infection of the mouse genital tract by progesterone treatment.

Experimental infection of the vagina of TO and CBA mice with Mycoplasma pulmonis was enchanced greatly by progesterone treatment. Almost all treated animals became infected, whereas only two-thirds of the untreated TO mice and less than half of the untreated CBA mice did so. Almost 1000-fold more organisms were recovered from treated than from untreated mice and the duration of infection was more than doubled. The enhanced infection in the hormone-treated animals was accompanied by a more severe vaginal polymorphonuclear leucocyte response which reached a maximum two weeks after inoculation of M. pulmonis. In the TO mice the eventual decline in cellular response coincided with the gradual disappearance of the mycoplasmal infection. The implications of these findings for genital infections of other animal species are discussed.

Autoradiographic investigation of sperm transit through the male mouse genital tract after tritiated thymidine incorporation.

The transit of spermatozoa in the genital tract of the male mouse was investigated by quantitative light microscopic autoradiography after intraperitoneal injection of tritiated thymidine. Transit duration in the caput and the corpus of the epididymis was shown to be 3 days; the total duration of transit in the genital tract was 5 days. These findings indicate that the time required for the transit of spermatozoa in the epididymal caput and corpus was comparable to that calculated in other mammals studied. However, the duration of sperm storage in the epididymal cauda appeared to be shorter than that previously reported for rodents.