Mouse Female Reproductive Tract Research Articles

Uncoupling Protein 2, but Not Uncoupling Protein 1, Is Expressed in the Female Mouse Reproductive Tract

Uncoupling proteins (UCPs) are transporters of the inner mitochondrial membrane. Whereas UCP1 is uniquely present in brown adipose tissue where it uncouples respiration from ATP synthesis and activates respiration and heat production, UCP2 is present in numerous tissues, and its exact function remains to be clarified. Two sets of data provided the rationale for this study: (i) the intriguing report that UCP1 is present in uterus of mice (Nibbelink, M., Moulin, K., Arnaud, E., Duval, C., Penicaud, L., and Casteilla, L. (2001) J. Biol. Chem. 276, 47291-47295); and (ii) an observation that Ucp2(-/-) female mice (homozygous matings) have smaller litters compared with Ucp2(+/+) animals (S. Rousset and A.-M. Cassard-Doulcier, unpublished observations). These data prompted us to examine the expression of UCP1 and UCP2 in the reproductive tract of female mice. Using wild type, Ucp1(-/-) mice, and Ucp2(-/-) mice, we were unable to detect UCP1 in uterus of mice with appropriate antibodies, and we conclude that the signal assigned to UCP1 by others was neither UCP1 nor UCP2. Using a polyclonal antibody against UCP2 and tissues from Ucp2(-/-) mice as controls, UCP2 was detected in ovary, oviduct, and uterus. Expression of Ucp2 mRNA was also observed in ovary and uterus using in situ hybridization analysis. Bone marrow transplantation experiments revealed that the UCP2 signal of the ovary was restricted to ovarian cells. UCP2 level in ovary decreased during follicular growth and increased during the pre-ovulatory period, during which aspects of an inflammatory process are known to exist. Because UCP2 down-regulates reactive oxygen species, a role in the regulation of inflammatory events linked to the preparation of ovulation is suggested.

Distribution and regulation of ENaC subunit and CFTR mRNA expression in murine female reproductive tract.

The present study investigated the regional distribution and cyclic changes in the mRNA expression of epithelial Na+ channel (ENaC) subunit and cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel, in adult female mouse reproductive tract. In situ hybridization revealed that in contrast to the abundant expression of CFTR, ENaC (alpha, beta, gamma) mRNA signal was not detected throughout the estrus cycle in the ovary and oviduct. Messenger RNA for all ENaC subunits was abundantly detected in the cervical and vaginal epithelia throughout the estrus cycle but for CFTR, mRNA was found only at proestrus. In the uterine epithelium, alphaENaC mRNA was detected at diestrus but not found at any other stage, while CFTR mRNA was only detected at early estrus but not other stages. Semi-quantitative RT-PCR detected mRNA for all ENaC subunits in the uterus throughout the cycle with maximal expression at diestrus and CFTR mRNA was only found in the early stages of the cycle. The involvement of ENaC and CFTR in Na+ absorption and Cl- secretion was demonstrated in cultured endometrial epithelia using the short-circuit current technique and found to be influenced by ovarian hormones. Taken together, these data indicate a main secretory role of the ovary and oviduct and a predominantly absorptive role of the cervix and vagina. The present results also suggest an ability of the uterus to secrete and absorb at different stages of the estrus cycle. Variations in the fluid profiles may be dictated by the regional and cyclic variations in expression of ENaC and CFTR and are likely to contribute to various reproductive events in different regions of the female reproductive tract.

Roles of p63 in differentiation of Müllerian duct epithelial cells.

In the mouse female reproductive tract, p63, a homologue of the p53 tumor suppressor gene, is highly expressed in basal cells of the vaginal and cervical epithelium, but not in the uterine epithelium. P63 is undetectable in the undifferentiated epithelium of the embryonic Müllerian duct. The Mullerian vaginal epithelium becomes p63 positive and stratified during the first week of postnatal development. P63 expression in the Müllerian vaginal epithelium is induced by vaginal mesenchyme. When vaginal mesenchyme was combined with uterine epithelium from newborn mouse, the uterine epithelium was induced to undergo vaginal differentiation and to express p63. Conversely, when the vaginal epithelium from the newborn mouse was recombined with uterine mesenchyme, it underwent uterine differentiation and failed to express p63. After the uterine epithelium or vaginal epithelium differentiates, the expression status of p63 in uterine (negative) and vaginal (positive) epithelia is not altered by heterotypic mesenchyme. Studies with p63-null mice demonstrate that p63 is essential for vaginal epithelial differentiation, because p63-null Müllerian vaginal epithelium developed as uterine epithelium. Thus, p63 determines whether Müllerian duct epithelial cells become uterine or vaginal. Misexpression of p63 in uterine and vaginal epithelial lesions induced by neonatal diethylstilbestrol (DES) exposure induces pathological changes. Irregularities in p63 expression (and thus epithelial differentiation) are observed in the uterine and vaginal epithelia of neonatally DES-exposed mice during the first week of postnatal development. Thus, neonatal DES exposure abnormally transforms uterine and vaginal epithelial differentiation by perturbing epithelial expression of p63 during development.

Wnt-7a maintains appropriate uterine patterning during the development of the mouse female reproductive tract

The murine female reproductive tract differentiates along the anteroposterior axis during postnatal development. This process is marked by the emergence of distinct cell types in the oviduct, uterus, cervix and vagina and is dependent upon specific mesenchymal-epithelial interactions as demonstrated by earlier heterografting experiments. Members of the Wnt family of signaling molecules have been recently identified in this system and an early functional role in reproductive tract development has been demonstrated. Mice were generated using ES-mediated homologous recombination for the Wnt-7a gene (Parr, B. A. and McMahon, A. P. (1995) Nature 374, 350-353). Since Wnt-7a is expressed in the female reproductive tract, we examined the developmental consequences of lack of Wnt-7a in the female reproductive tract. We observe that the oviduct lacks a clear demarcation from the anterior uterus, and acquires several cellular and molecular characteristics of the uterine horn. The uterus acquires cellular and molecular characteristics that represent an intermediate state between normal uterus and vagina. Normal vaginas have stratified epithelium and normal uteri have simple columnar epithelium, however, mutant uteri have stratified epithelium. Additionally, Wnt-7a mutant uteri do not form glands. The changes observed in the oviduct and uterus are accompanied by a postnatal loss of hoxa-10 and hoxa-11 expression, revealing that Wnt-7a is not required for early hoxa gene expression, but is required for maintenance of expression. These clustered hox genes have been shown to play a role in anteroposterior patterning in the female reproductive tract. In addition to this global posterior shift in the female reproductive tract, we note that the uterine smooth muscle is disorganized, indicating development along the radial axis is affected. Changes in the boundaries and levels of other Wnt genes are detectable at birth, prior to changes in morphologies. These results suggest that a mechanism whereby Wnt-7a signaling from the epithelium maintains the molecular and morphological boundaries of distinct cellular populations along the anteroposterior and radial axes of the female reproductive tract.

Smooth Muscle Cell Fate Determination and Pattern Formation in the Mouse Female Reproductive Tract • 307

Smooth Muscle Cell Fate Determination and Pattern Formation in the Mouse Female Reproductive Tract • 307

Expression of Hox a Genes in the Mouse and Human Female Reproductive Tract

Expression of Hox a Genes in the Mouse and Human Female Reproductive Tract

O-176 Expression of hox a genes in the mouse and human female reproductive tract

O-176 Expression of hox a genes in the mouse and human female reproductive tract

Potentiation of diethylstilbestrol‐induced alterations in the female mouse reproductive tract by transforming growth factor‐α transgene expression

Neonatal estrogen exposure causes numerous abnormalities in the female reproductive tract, including carcinogenesis. One mechanism by which neonatal estrogen elicits teratogenic and carcinogenic effects is epigenetic and involves the modulation of a number of estrogen-regulated genes including epidermal growth factor (EGF). Because of the evidence that there is an integral relationship between the EGF family, estrogen action, and the regulation of the growth and differentiation of the reproductive tract, we used transforming growth factor-α (TGFα) transgenic mice to investigate the interaction of constitutive TGFα expression with the potent estrogen diethylstilbestrol (DES) in the induction of reproductive-tract alterations. Our study was designed to determine whether TGFα expression could modulate DES-induced carcinogenesis of the female mouse reproductive tract. The animals were homozygous TGFα transgenic female mice from the MT42 line and the parental CD-1 outbred mice. The presence of the TGFα transgene significantly increased the incidence of DES-induced vaginal adenosis, uterine endometrial hyperplasia, uterine polyps, hypospadia, benign ovarian cysts, and pituitary adenomas. However, constitutive TGFα expression did not promote reproductive-tract neoplasia. This study demonstrates that TGFα participates in the regulation of developmental and morphogenic events in the Müllerian duct and urogenital sinus, suggesting a role for TGFα in the pathogenesis of reproductive-tract diseases. Furthermore, we showed that although constitutive expression of the TGFα transgene did have an effect on the reproductive tract, TGFα overexpression alone could not substitute for DES as a reproductive-tract carcinogen or as a promoter of uterine neoplasia, indicating that DES-induced carcinogenesis requires events in addition to the overexpression of this single peptide growth factor. © 1996 Wiley-Liss, Inc. 1 This article is a US Government work and, as such, is in the public domain in the United States of America.

Potentiation of diethylstilbestrol-induced alterations in the female mouse reproductive tract by transforming growth factor-alpha transgene expression.

Neonatal estrogen exposure causes numerous abnormalities in the female reproductive tract, including carcinogenesis. One mechanism by which neonatal estrogen elicits teratogenic and carcinogenic effects is epigenetic and involves the modulation of a number of estrogen-regulated genes including epidermal growth factor (EGF). Because of the evidence that there is an integral relationship between the EGF family, estrogen action, and the regulation of the growth and differentiation of the reproductive tract, we used transforming growth factor-alpha (TGF alpha) transgenic mice to investigate the interaction of constitutive TGF alpha expression with the potent estrogen diethylstilbestrol (DES) in the induction of reproductive-tract alterations. Our study was designed to determine whether TGF alpha expression could modulate DES-induced carcinogenesis of the female mouse reproductive tract. The animals were homozygous TGF alpha transgenic female mice from the MT42 line and the parental CD-1 outbred mice. The presence of the TGF alpha transgene significantly increased the incidence of DES-induced vaginal adenosis, uterine endometrial hyperplasia, uterine polyps, hypospadia, benign ovarian cysts, and pituitary adenomas. However, constitutive TGF alpha expression did not promote reproductive-tract neoplasia. This study demonstrates that TGF alpha participates in the regulation of developmental and morphogenic events in the Müllerian duct and urogenital sinus, suggesting a role for TGF alpha in the pathogenesis of reproductive-tract diseases. Furthermore, we showed that although constitutive expression of the TGF alpha transgene did have an effect on the reproductive tract, TGF alpha overexpression alone could not substitute for DES as a reproductive-tract carcinogen or as a promoter of uterine neoplasia, indicating that DES-induced carcinogenesis requires events in addition to the overexpression of this single peptide growth factor.

Estrogen Induces Retinoid Receptor Expression in Mouse Cervical Epithelia

Steroid hormones and retinoids are powerful regulators of normal epithelial differentiation and function in the mouse female reproductive tract, where their actions are mediated by nuclear receptors. The expression pattern, heterodimeric interactions, and availability of ligand of these transcription factors are thought to contribute to the biological response. Estrogen (E2) induces proliferation and squamous differentiation of the cervical and vaginal epithelium during the mouse estrous cycle, while progesterone and retinoids maintain the simple columnar epithelium of the endocervix and uterine horns. We wanted to investigate whether retinoid receptors are responsive to estrogen status during cervical epithelial differentiation induced by a single dose of estrogen in ovariectomized adult mice. Northern blot analysis demonstrated a prolonged induction of RXRα and RARγ gene expression by E2in the mouse cervix and vagina. When the induction of RXRα and RARγ was compared to genes known to be responsive to E2, including estrogen receptor (ER) and c-fos, RXRα was induced within 0.5 h of hormone treatment, while RARγ induction was evident by 4 h. The induction of these retinoid receptors suggests that they may be implicated in epithelial growth and differentiation in response to E2. Moreover, potential heterodimeric interactions among these receptors indicate that normal, cyclical epithelial differentiation results from the interplay of these molecules. Usingin situhybridization analysis, RXRα transcripts were localized preferentially in the basal cells, while ER mRNA was expressed throughout the epithelium of the ectocervix and vagina. Furthermore, ER transcripts were highly expressed in the stratified squamous foci induced by mild vitamin A depletion in the columnar epithelium of the endocervix and uterine horns. Therefore, the induction of RXRα and RARγ by E2and their expression pattern in relation to ER suggest that they are needed to coordinate specific genetic programs that result in cervical epithelial growth and differentiation.

Expression of collagen alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains in the pregnant mouse uterus.

The alpha 1, alpha 2, and alpha 3 chains of collagen VI and mRNAs for these chains were localized in the female mouse reproductive tract by immunofluorescence and in situ hybridization. High levels of collagen VI protein and mRNAs were present in the endometrium and myometrium of the uterus up to Day 4.5 of pregnancy. After embryo implantation, reduction in collagen VI protein within the decidualizing endometrium correlated with significantly reduced steady-state levels of alpha 1(VI), alpha 2(VI), and alpha 3(VI) mRNAs, indicating either transcriptional down-regulation of collagen VI gene expression or decreased stability of transcripts. High levels of alpha 1(V1) and alpha 2(VI) mRNAs, but not alpha 3(VI) mRNA, in cells surrounding the uterine epithelium in the mesometrial region did not correlate with deposition of collagen VI protein in this region. These data are consistent with an important role of alpha 3(VI) in assembly of collagen VI heterotrimers. However, distinct immunostaining with antiserum to alpha 2(VI) chains in the extracellular matrix immediately beneath the uterine epithelium may indicate that alpha 2(VI) chains are deposited without the alpha 1(VI) or alpha 3(VI) collagen chains. No collagen VI protein or mRNAs were detected in any tissue layers of the embryo on Days 5.5 or 6.5 of gestation.

Activation of apoptosis in early mouse embryos by 2'-deoxyadenosine exposure.

Adenosine deaminase (ADA) catalyzes the irreversible hydrolytic deamination of adenosine and deoxyadenosine to nontoxic derivatives. The importance of this reaction in the female reproductive tract of mice is suggested by pronounced utero-placental expression of ADA, and by embryolethality of the potent ADA-inhibitor deoxycoformycin (dCF) on day 7-8 of gestation. The present study investigated the effects of dCF, adenosine, and deoxyadenosine on the mouse neurula. Morphological cell death was monitored by the acridine orange reaction (AOR), and biochemical cell death by internucleosomal DNA cleavage (IDC). A strong AOR appeared in day 7-8 embryos between 3 and 4.5 hr post-exposure to dCF in utero; there was no apparent effect on day 6 or day 9 embryos. Most embryonic tissues were responsive, although the heart and extraembryonic membranes were resistant. Up to 75% of the embryonic chromatin was degraded in a regular pattern in concert with the AOR. Immediate activation of "whole-body" apoptosis was reproduced in short-term whole embryo culture with 0.1 mM deoxyadenosine in the presence of 0.01 mM dCF. This was not activated by exposure to dCF alone nor to adenosine; however, high concentrations of adenosine completely blocked the response to deoxyadenosine, whereas niacinamide inhibited the AOR without changing IDC. The cytotoxic effect of deoxyadenosine was correlated with an expansion of embryonic dATP pools determined by high-performance liquid chromatography analysis. The results suggest that deoxyadenosine is the embryotoxic metabolite which accumulates in the antimesometrium of pregnant mice treated with dCF. Exposure to this metabolic toxin activates apoptosis in day 7-8 embryos through an adenosine-sensitive, NAD-dependent mechanism.

Transforming growth factor-alpha is a potential mediator of estrogen action in the mouse uterus.

To better understand the role of peptide growth factors in sex steroid hormone-mediated growth of the female reproductive tract, the effect of estrogen on the expression of transforming growth factor-alpha (TGF alpha) in mouse uterus was investigated. Our results show that estrogen induces the expression of TGF alpha mRNA in the mouse uterus in a dose- and time-dependent manner. The up-regulation of TGF alpha transcripts occurs predominantly in uterine epithelial cells. RIA and Western blot analysis demonstrate that immunoreactive TGF alpha protein is secreted at high levels into mouse uterine luminal fluid after estrogen treatment. The induction of uterine TGF alpha mRNA is specific to estrogen; nonestrogenic steroids did not induce expression. Antibody specific to TGF alpha significantly reduces estrogen-mediated uterine growth, which supports the concept that TGF alpha is a mitogen for the reproductive tract. Analysis of TGF alpha/EGF receptors by binding, affinity labeling, and phosphorylation studies indicates that functional receptors are present in the mouse uterus after estrogen exposure. Thus, our data support a physiological role for TGF alpha and its receptor pathway in the female mouse reproductive tract.

Transforming growth factor-alpha is a potential mediator of estrogen action in the mouse uterus

To better understand the role of peptide growth factors in sex steroid hormone-mediated growth of the female reproductive tract, the effect of estrogen on the expression of transforming growth factor-alpha (TGF alpha) in mouse uterus was investigated. Our results show that estrogen induces the expression of TGF alpha mRNA in the mouse uterus in a dose- and time-dependent manner. The up-regulation of TGF alpha transcripts occurs predominantly in uterine epithelial cells. RIA and Western blot analysis demonstrate that immunoreactive TGF alpha protein is secreted at high levels into mouse uterine luminal fluid after estrogen treatment. The induction of uterine TGF alpha mRNA is specific to estrogen; nonestrogenic steroids did not induce expression. Antibody specific to TGF alpha significantly reduces estrogen-mediated uterine growth, which supports the concept that TGF alpha is a mitogen for the reproductive tract. Analysis of TGF alpha/EGF receptors by binding, affinity labeling, and phosphorylation studies indicates that functional receptors are present in the mouse uterus after estrogen exposure. Thus, our data support a physiological role for TGF alpha and its receptor pathway in the female mouse reproductive tract.

Secretory immunoglobulin binding to bacteria in the mouse uterus after mating

Bacteria of several species were present in the mouse uterus on the morning after mating, as demonstrated by bacteriol cultures and microscopic examination of Gram-stained uterine luminal contents. The similarity between bacteria cultured from the vagina before mating and from the uterus after mating suggested that bacteria were introduced into the uterus from the vagina, possibly by coitus. The bacteria were cleared from the uterus about two days after mating. Immunohistochemical labeling of smears of the luminal contents on the morning after mating demonstrated IgA, IgG, and possibly IgM bound to many of the bacteria. The bacteria were often agglutinated, and there was a correlation between the intensity of immunoglobulin labeling on bacteria and the extent of agglutination. The amount of antibody bound to bacteria in multiparous mice was about the same as in mice that had not been mated previously. We observed both IgG and IgA on bacteria when organisms from vaginal cultures were incubated for 60 min in the uteri of estrogen-primed, virgin, female mice. This indicated that the uterus was the source of at least part of the immunoglobulins bound to bacteria. We did not demonstrate that the immunoglobulins bound to bacteria were specific anti-bacterial antibodies, but the binding persisted through three washing steps and there wasno immunoglobulin binding to sperm in the same preparations. Neutrophils in the uterine lumen on the day after mating contained phagocytosed bacteria. These results suggest that the secretory immune system in the female mouse reproductive tract may play a role in returning the uterus to an aseptic state after mating by at least three mechanisms: direct blocking of attachment sites involved in bacterial binding to mucosal epithelium, agglutination of bacteria and thus reduction in the number of organisms available for binding to the epithelium, and opsonization of bacteria for phagocytosis by neutrophils.

The transport of morphologically abnormal sperm in the female reproductive tract of mice

AbstractMice of the PL/J strain exhibit a high percentage of morphologically abnormal sperm and provide a model for studying the function of abnormal sperm. The ability of such sperm to reach the site of fertilization within the female reproductive tract has been investigated. We have found a decrease in the percentage of structurally abnormal sperm within the population that reaches the oviduct. This observation suggests either that there is an active selection against abnormal sperm or that they are physiologically disadvantaged in reaching the site of fertilization.

Immunofluorescent localization of a seminal vesicle proteinase inhibitor in the female reproductive tract of naturally inseminated mice.

The indirect immunofluorescent technique was used to localize a low molecular weight, acid-stable proteinase inhibitor of seminal vesicle origin in the female reproductive tract of mice. In recently inseminated animals (0, 2, and 4 hr postcoitus) the inhibitor was localized in the copulatory plug, on the epithelia of the vaginal fornix and cervix, in the uterine lumen, and on the apical surface of the uterine epithelium. Ten hours postcoitus the inhibitor was found in localized areas on the uterine epithelium, in a sperm-leucocyte mass in the uterine lumen, and in the copulatory plug. The inhibitor was not found in females 24 hr postcoitus. The inhibitor could not be localized in the oviducts of any of the animals tested. The data are interpreted to mean that the inhibitor, transported to the female at ejaculation, coats the surface of the female reproductive tract protecting it from acrosomal enzymes or from invasion by spermatozoa or pathogens.

Oxidative metabolism of spermatozoa from inbred and random bred mice.

Epididymal spermatozoa from the random-bred CFW and from the inbred C57 BL/6 strains of mice were treated either hypotonically or with the antibiotic filipin in order to study the mitochondrial oxidative activities of the two strains in the absence of permeability barriers imposed by the plasma membrane. The percentage of motile spermatozoa from C57BL/6 mice was consistently higher than that of CFW mice, but sperm from the latter fertilized a higher percentage of eggs in vitro. In vivo, there was no apparent difference in fertilizing capacity in vivo: no significant difference between strains was observed. There is a strategy of oxidative metabolism in mouse spermatozoa which is common to the two genetic strains of the species tested, but which differs from that of rabbit and bull spermatozoa. The mitochondria of mouse spermatozoa oxidize L--3-glycerolphosphate but not glutamate in the presence of malate; both activities are present in bull spermatozoa but neither are present in rabbit spermatozoa. In common wit those of the mammalian species thus far studied, the mitochondria of mouse spermatozoa readily oxidize lactate and pyruvate in the presence of malate. They also oxidize acetyl CoA, acetyl carnitine, and long-chain acyl CoA esters directly, without the intermediacy of the carnitine esters. Mouse spermatozoa, therefore, have access to endogenous acyl CoA esters as a source of metabolic energy, which is consistent with their ability to maintain motility for 4-6 hours in the absence of added energy sources. Mouse spermatozoa are self-sufficient with regard to oxidative metabolism, which suggests that energy sources are not readily available to them in the mouse female reproductive tract.

Transitional changes in the surface epithelium of the cycling mouse vagina, cervix and uterus: scanning electron microscopic studies.

During the estrous cycle, the mouse reproductive tract undergoes a number of specific morphological changes. The alterations which occur in the luminal surface of the vagina, cervix and uterus were studied by scanning electron microscopy (SEM). The stage of estrus was identified by vaginal observation, cytology and histology. The lumen of the reproductive tract was observed with the SEM at low powers of magnification as well as at the subcellular level. Surface epithelial alterations associated with mucification and keratinization of vaginal epithelium were identified by SEM; changes in the squamocolumnar junction were studied as were the cyclic variations in the mouse endometrium. This study was done to characterize the morphological appearance of the normal female mouse reproductive tract and thereby establish a normal baseline for the evaluation of samples from subsequent studies of corresponding pathologic tissue. A comparison of the similarity of the epithelial characteristics observed in normal cervix and vagina at diestrus to those in cells described by other investigators in reportedly dysplastic tissue demonstrates the need for this type of systematic approach in SEM studies.