Anaerobic microflora of the female reproductive tract

The microflora of the female reproductive tract is very diverse and plays an important role in both normal and pathological states. The data on the mechanisms of colonization resistance which involve the vaginal microbios (the production of H2O2, organic acids, bacteriocin-like substances, competition for adhesion sites) are presented. The data on the role of individual antagonistically active substances of anaerobic bacteria in suppressing gonococci, fungi, microorganisms, associated with bacterial vaginosis, etc. are given. The leading role of anaerobic microorganisms in the appearance of microecological disturbances, including bacterial vaginosis, is emphasized. The role of the pathogenic properties of anaerobic bacteria for the development of different pathological processes, such as premature birth, postnatal and postoperative purulent septic diseases, inflammation of pelvic organs, cancer of the neck of uterus, is discussed.     

Generation of human female reproductive tract epithelium from human embryonic stem cells

Background

Recent studies have identified stem/progenitor cells in human and mouse uterine epithelium, which are postulated to be responsible for tissue regeneration and proliferative disorders of human endometrium. These progenitor cells are thought to be derived from Müllerian duct (MD), the primordial female reproductive tract (FRT).

Methodology/Principal Findings

We have developed a model of human reproductive tract development in which inductive neonatal mouse uterine mesenchyme (nMUM) is recombined with green fluorescent protein (GFP)-tagged human embryonic stem cells (hESCs); GFP-hESC (ENVY). We demonstrate for the first time that hESCs can be differentiated into cells with a human FRT epithelial cell phenotype. hESC derived FRT epithelial cells emerged from cultures containing MIXL1⁺ mesendodermal precursors, paralleling events occurring during normal organogenesis. Following transplantation, nMUM treated embryoid bodies (EBs) generated epithelial structures with a typical MD phenotype that expressed the MD markers PAX2, HOXA10. Functionally, the hESCs derived FRT epithelium responded to exogenous estrogen by proliferating and secreting uterine-specific glycodelin A (GdA).

Conclusions/Significance

These data show nMUM can induce differentiation of hESC to form the FRT epithelium. This may provide a model to study early developmental events of the human FRT.     

Development of the mammalian female reproductive tract

The female reproductive tract (FRT), which includes the oviduct, uterus, cervix and vagina, is critical for mammalian reproduction. Recent research using knockout mice has contributed substantially to our understanding of the molecular mechanisms governing FRT development. Aside from satisfying our curiosities about the origin of life, these studies have provided us with a better understanding of FRT disorders and ways to improve female fertility. Here we review genes that are involved in various stages of sexual duct formation and development in mammals. In addition, the effect of exogenous estrogen such as DES on FRT development is also discussed.     

Assessment of human sperm function after recovery from the female reproductive tract

The physiology and fertile life of human spermatozoa in the female reproductive tract have received little previous attention. A technique was developed for recovering spermatozoa from human cervical mucus at various intervals after artificial insemination. The functions of these cells as measured by penetration of the human zona pellucida and fusion with the zona-free hamster oocytes were examined. Penetration into the zona pellucida was consistently observed when sperm were recovered from 1 to 80 h after insemination. Penetration through the zona into the perivitelline space (PVS) was seen from 1 to 72 h after insemination. Fusion of human sperm with zona-free hamster oocytes was observed from 1 to 48 h after insemination. Motile sperm were recovered 112 and 120 h after insemination with swimming speeds comparable to freshly capacitated spermatozoa. Concentrations of recovered sperm at these longer intervals from insemination were insufficient for sperm-oocyte assays. These studies demonstrate that human spermatozoa aged in vivo may be recovered from cervical mucus for physiologic study, and suggest that the fertile life of human sperm may be 80 h or more.     

Developmental genetics of the female reproductive tract in mammals

The female reproductive tract receives the oocytes for fertilization, supports the development of the fetus and provides the passage for birth. Although abnormalities of this organ system can result in infertility and even death, until recently relatively little was known about the genetic processes that underlie its development. By drawing primarily on mouse mutagenesis studies and the analysis of human mutations we review the emerging genetic pathways that regulate female reproductive-tract formation in mammals and that are implicated in congenital abnormalities of this organ system. We also show that these pathways might be conserved between invertebrates and mammals.     

Immunolocalization of the hyaluronan receptor CD44 in the reproductive tract of the mare

Hyaluronan (HA), a glycosaminoglycan, is a major component of the pericellular matrix which envelopes mammalian cells. Binding of hyaluronan to one of its specific receptors, CD44, modulates transduction of intracellular signals which direct a variety of processes, including embryogenesis, wound healing, inflammation, and neoplasia. Since regulation of these processes is critical to equine reproductive success, localization of constitutive CD44 expression was evaluated by immunohistochemical methods in ovarian, oviductal, and uterine tissues from healthy mares. Ovarian stroma contained thecal cells with varying CD44 immunopositivity. Follicular and granulosa cells of some antral and atretic follicles were positive for CD44. In the oviduct, the luminal epithelium was variably positive for CD44, with overall decreasing intensity of immunostaining from the infundibulum to the isthmus. The CD44 molecule was expressed strongly by surface epithelial cells of the uterine endometrium, but was present only rarely among cells of uterine glands. In addition, CD44 was expressed by smooth muscle cells of vascular walls, oviduct, and uterus. Since CD44 is known to modulate cell movement and differentiation, and was present at multiple sites in the reproductive tract of normal mares, we inferred there may be an important role for the HA-CD44 signaling pathway in reproductive function and inflammation.     

Immunobiology of the reproductive tract in a female baboon

The aim of this study was to assess the suitability of various antihuman antibodies directed against immunocomponent cells to identify components involved in cellular and humoral immune responses in the immune organs of a female baboon, and to use these reagents to analyze the immunobiology of its reproductive tract. A female baboon of reproductive age was euthanized in the luteal phase of the menstrual cycle, and samples of spleen, intestines, tonsil, lymph nodes, Fallopian tube, uterus, cervix, and vagina were removed. Tissues were either fixed in 10% unbuffered formaldehyde, Bouin's fluid, or 95% ethanol containing 5% glacial acetic acid, and embedded in paraffin, or frozen unfixed. Frozen sections were then fixed in 100% acetone. Subsequently, tissue sections were reacted with the following antihuman antibodies directed against CD3, CD45RA, CD45RO, CD4, CD8, CD20, CD68, HLA-DR, CD57, CD103, CD15, and TIA-1: IgA, IgG, IgM, J-chain, secretory component, and neutrophil elastase, using routine immunohistology techniques. Human tissues (spleen, small intestine, lymph node, and tonsil) were used as positive controls. All antihuman antibodies crossreacted with baboon tissues, except neutrophil elastase, CD15, CD45RO, CD57, and CD1A. The distribution of immune cells in the reproductive tract of the female baboon was comparable to that in the human and offers the potential for this primate to be used as a model for the study of human reproductive immunology.     

Localization and quantity of hyaluronan in urogenital organs of male and female rats

The histochemical distribution of hyaluronan was analysed in various urogenital organs of male and female (non-pregnant and pregnant) rats by use of a hyaluronan-binding protein and avidin biotin/peroxidase staining. Microwave-aided fixation was used to preserve the extracellular location of hyaluronan. The concentrations of hyaluronan in the different tissues were measured with a highly sensitive radio-assay. Hyaluronan accumulated predominantly in the connective tissue around smooth muscle fibres and in the subepithelial lamina propria. Abundant hyaluronan also occurred in perivascular and perineural connective tissue. In the female urogenital organs, hyaluronan content was high in the vagina and urinary bladder, and highest in the vagina during pregnancy. In the uterus, the surface epithelium of the endometrium stained intensely. In the ovary, the zona pellucida of the oocyte and the theca interna cell layer of the follicles and the follicular fluid of mature follicles exhibited prominent staining. The corpus luteum was devoid of hyaluronan, whereas enlarged corpora lutea of pregnancy exhibited weak, patchy staining. In male urogenital organs, staining for hyaluronan was absent from the testis and epididymis, whereas the erectile connective tissue of the penis stained intensely. The hyaluronan concentrations were high in penile tissue and urinary bladder, while testis, epididymis and the ductus deferens contained only little hyaluronan.     

Normal and abnormal epithelial differentiation in the female reproductive tract

In mammals, the female reproductive tract (FRT) develops from a pair of paramesonephric or Müllerian ducts (MDs), which arise from coelomic epithelial cells of mesodermal origin. During development, the MDs undergo a dynamic morphogenetic transformation from simple tubes consisting of homogeneous epithelium and surrounding mesenchyme into several distinct organs namely the oviduct, uterus, cervix and vagina. Following the formation of anatomically distinctive organs, the uniform MD epithelium (MDE) differentiates into diverse epithelial cell types with unique morphology and functions in each organ. Classic tissue recombination studies, in which the epithelium and mesenchyme isolated from the newborn mouse FRT were recombined, have established that the organ specific epithelial cell fate of MDE is dictated by the underlying mesenchyme. The tissue recombination studies have also demonstrated that there is a narrow developmental window for the epithelial cell fate determination in MD-derived organs. Accordingly, the developmental plasticity of epithelial cells is mostly lost in mature FRT. If the signaling that controls epithelial differentiation is disrupted at the critical developmental stage, the cell fate of MD-derived epithelial tissues will be permanently altered and can result in epithelial lesions in adult life. A disruption of signaling that maintains epithelial cell fate can also cause epithelial lesions in the FRT. In this review, the pathogenesis of cervical/vaginal adenoses and uterine squamous metaplasia is discussed as examples of such incidences.     

Cholesteryl sulfate and sterol sulfatase in the human reproductive tract.

Cholesteryl sulfate is a component of human seminal plasma (avg. 445 mug%) and spermatozoa (15 mug/10 (9) cells) and represents more than 85% of the sterol sulfate fraction. This conjugate is avidly bound by spermatozoa when compared to other steroids or steroid sulfates. Autoradiographic localization of CS associated with the spermatozoa revealed a greater accumulation of the radioactivity in the acrosomal region in many, but not all, of those cells examined. Semen is not a site of metabolism of the sterol sulfate but the enzyme, sterol sulfatase, is present in the human female reproductive tract. This cleavage enzyme was detected in Graafian follicles and the activity in the endometrium was ten-fold that found in the Fallopian tube. These findings lead to the proposal that cholesteryl sulfate, an amphipathic molecule ideally suited for interaction with membrane components and implicated in erythrocyte membrane stabilization, may be involved in membrane modifications of the spermatozoa during the process of fertilization.     

Nutrient concentrations in murine follicular fluid and the female reproductive tract

The culture of murine oocytes and preimplantation embryos in vitro has been used successfully for many years. However, this practice can result in cellular stress and reduced viability. Since this phenomenon is partly attributable to differences in nutrient composition between culture media and maternal tract fluids, we determined the concentrations of glucose, pyruvate, lactate and 19 amino acids in murine preovulatory follicles and oestrous oviductal and uterine fluids. Follicular fluids were aspirated from hyperstimulated ovaries, whereas oviductal fluids (with/without oocyte-cumulus complexes) and uterine fluids were collected from naturally cycling animals. Glucose, pyruvate and lactate concentrations were analysed using ultramicrofluorometric methods, whilst amino acid profiles were determined by reverse-phase high performance liquid chromatography. Mean glucose concentrations in follicular, oviduct (with/without cumulus cells) and uterine fluids were 0.46, 1.09/1.65 and 0.61 mmol l(-1), respectively. Pyruvate concentrations were 0.38, 0.37/0.17 and 0.25 mmol l(-1), respectively, and lactate concentrations were 17.34, 10.92/11.68 and 9.41 mmol l(-1), respectively. Oviductal pyruvate concentration was significantly higher, and glucose significantly lower, in the presence of cumulus cells. Taurine, glycine, alanine, glutamine and glutamate were the major amino acids detected. Concentrations of amino acids differed among fluids, with highest levels being found in the oviduct. The follicular fluid and tract nutrient profiles differed from those of murine maturation, fertilisation and embryo culture media. These data extend our understanding of cellular metabolism and of nutritional environments of the oocyte and early embryo as they progress along the reproductive tract in vivo. These results may also contribute to the formulation of nutritionally more physiological media for mouse oocyte maturation and embryo culture.     

Seminal plasma and male factor signalling in the female reproductive tract

In mammals, insemination results in the transmission of seminal factors that act, in the female reproductive tract, to promote sperm survival, to “condition” the female immune response to tolerate the conceptus and to organise molecular and cellular changes in the endometrium to facilitate embryo development and implantation. These events are initiated when signalling agents, including transforming growth factor-β and other cytokines and prostaglandins secreted by seminal vesicle and prostate glands, interact with epithelial cells in the cervix and uterus to activate cytokine synthesis and to induce cellular and molecular changes resembling a classical inflammatory cascade. The consequences are the recruitment and activation of macrophages, granulocytes and dendritic cells, which have immune-regulatory and tissue-remodelling roles that culminate in improved endometrial receptivity to the implanting embryo. Cytokines elicited by seminal activation have embryotrophic properties and also contribute directly to the optimal development of the early embryo. This review summarises our current understanding of the physiology of responses to seminal plasma in the female reproductive tract and considers the evolutionary significance of seminal plasma in influencing female tissues to promote the success of pregnancy.The author acknowledges the support of the NHMRC of Australia Fellowship and Program Grant schemes.     

Regulation of angiogenic growth factors in the female reproductive tract by estrogens and progestins.

Proper regulation of angiogenesis and vascular permeability is essential for the physiological functioning of the female reproductive tract, and major health problems in women, such as dysfunctional uterine bleeding, endometriosis, and uterine cancer, involve a vascular component. There is a large body of literature that describes the effects of sex steroids on the vasculature of the reproductive tract, but far less is known about the molecular mechanisms that regulate these important actions. We hope that this minireview will help emphasize the need for mechanistic studies in this area to improve treatment and prevention of these major health problems in women. Specifically, we believe it will be important to 1) define the exact roles of FGF, VEGF, and other factors in physiological and pathological events in the reproductive tract and the cell types and receptors involved; 2) identify estrogen and progesterone receptor subtypes, the DNA elements, nuclear protein factors, and signaling pathways that mediate regulation of these genes by sex steroids; 3) elucidate any mechanisms of cross-talk between sex steroids and other regulatory factors in the overall regulation of FGF, VEGF, and other angiogenic/permeability factors; and 4) eventually understand how genetic polymorphisms of key regulatory elements affect angiogenesis and the regulation of vascular function in the female reproductive tract.     

Identification of quiescent, stem-like cells in the distal female reproductive tract

In fertile women, the endometrium undergoes regular cycles of tissue build-up and regression. It is likely that uterine stem cells are involved in this remarkable turn over. The main goal of our current investigations was to identify slow-cycling (quiescent) endometrial stem cells by means of a pulse-chase approach to selectively earmark, prospectively isolate, and characterize label-retaining cells (LRCs). To this aim, transgenic mice expressing histone2B-GFP (H2B-GFP) in a Tet-inducible fashion were administered doxycycline (pulse) which was thereafter withdrawn from the drinking water (chase). Over time, dividing cells progressively loose GFP signal whereas infrequently dividing cells retain H2B-GFP expression. We evaluated H2B-GFP retaining cells at different chase time points and identified long-term (LT; >12 weeks) LRCs. The LT-LRCs are negative for estrogen receptor-α and express low levels of progesterone receptors. LRCs sorted by FACS are able to form spheroids capable of self-renewal and differentiation. Upon serum stimulation spheroid cells are induced to differentiate and form glandular structures which express markers of mature Műllerian epithelial cells. Overall, the results indicate that quiescent cells located in the distal oviduct have stem-like properties and can differentiate into distinct cell lineages specific of endometrium, proximal and distal oviduct. Future lineage-tracing studies will elucidate the role played by these cells in homeostasis, tissue injury and cancer of the female reproductive tract in the mouse and eventually in man.