Female Reproductive Tract Epithelium Research Articles

Constitutive expression and distinct properties of IFN-epsilon protect the female reproductive tract from Zika virus infection.

The immunological surveillance factors controlling vulnerability of the female reproductive tract (FRT) to sexually transmitted viral infections are not well understood. Interferon-epsilon (IFNɛ) is a distinct, immunoregulatory type-I IFN that is constitutively expressed by FRT epithelium and is not induced by pathogens like other antiviral IFNs α, β and λ. We show the necessity of IFNɛ for Zika Virus (ZIKV) protection by: increased susceptibility of IFNɛ-/- mice; their "rescue" by intravaginal recombinant IFNɛ treatment and blockade of protective endogenous IFNɛ by neutralising antibody. Complementary studies in human FRT cell lines showed IFNɛ had potent anti-ZIKV activity, associated with transcriptome responses similar to IFNλ but lacking the proinflammatory gene signature of IFNα. IFNɛ activated STAT1/2 pathways similar to IFNα and λ that were inhibited by ZIKV-encoded non-structural (NS) proteins, but not if IFNε exposure preceded infection. This scenario is provided by the constitutive expression of endogenous IFNε. However, the IFNɛ expression was not inhibited by ZIKV NS proteins despite their ability to antagonise the expression of IFNβ or λ. Thus, the constitutive expression of IFNɛ provides cellular resistance to viral strategies of antagonism and maximises the antiviral activity of the FRT. These results show that the unique spatiotemporal properties of IFNε provides an innate immune surveillance network in the FRT that is a significant barrier to viral infection with important implications for prevention and therapy.

Mini-Percoll Technique Induces Similar Capacitation Features in Domestic Ruminant Frozen-Thawed Spermatozoa Regardless of the Presence of Heparin

Background: Sperm capacitation is a process consists of a series of functional, biochemical, and biophysical modifications that render the ejaculated sperm competent for oocyte fertilization. Secreted by the female reproductive tract epithelium, heparin promotes capacitation by binding to and removing seminal plasma proteins, which are adsorbed to the sperm PM and would inhibit capacitation. There is substantial evidence that cryopreservation promotes capacitation-like changes in bull, ram and buck sperm. Our general hypotheses were: (a) cryopreserved ram sperm suffer capacitation more quickly than buck and bull sperm under the same conditions; (b) the capacitation status of ruminant cryopreserved sperm is similar whether or not heparin is present after the mini-Percoll technique; and (c) ruminant frozen-thawed sperm selected by mini-Percoll and incubated within media without heparin supplementation is not impaired in terms of capacitation status and sperm agglutination. This study aimed to compare sperm parameters of ovine, caprine, and bovine frozen-thawed sperm after mini-Percoll processing followed by incubation with or without heparin supplementation.Materials, Methods & Results: Commercial semen of all species were used. Sperm samples were selected by mini-Percoll and supplemented (or not) with heparin within an incubation medium for 18 h. Sperm kinematics (CASA system analyzes), capacitation status (CTC staining) and sperm agglutination were evaluated after thawing, mini-Percoll, 1.5 h, 3 h, 6 h and 18 h. In comparison with post-thawing analysis, ovine species demonstrated a reduction (P < 0.05) in most of the sperm motility parameters after mini-Percoll. Conversely, ovine samples presented the highest (P < 0.05) rate of acrosome-reacted cells after mini-Percoll. Heparin supplementation did not affect most of the parameters evaluated (P > 0.05). In caprine and bovine species, a lower (P < 0.05) rate of sperm agglutination was observed in the presence of heparin at 18 h of incubation. In the absence of heparin, ovine samples showed a higher (P < 0.05) agglutination rate compared to the bovine species after long incubation period.Discussion: The present study compared sperm parameters (sperm kinematics, agglutination rate and capacitation status) of ruminant frozen-thawed sperm after mini-Percoll selection followed by in vitro incubation with or without heparin supplementation. In this study, it was observed the same rate of capacitated cells after the sperm selection (min-Percoll) between ruminant species. This indicate that the capacitation process occurs similarly between ruminant species, refuting the first hypothesis of this study. The presence of heparin did not influence the capacitation status of ruminant frozen-thawed sperm after mini-Percoll selection, it demonstrates that the second hypothesis was supported by this study making more economic and practical the use of ruminant frozen-thawed semen. The absence of heparin in the incubation medium did not harmed the capacitation status and sperm agglutination of ruminant frozen-thawed sperm. This supported the third hypothesis of the current study and indicate that the use of mini-Percoll technique regardless the presence of heparin could be a useful alternative for the preparation of ruminant frozen-thawed sperm. In conclusion, the capacitation status of ruminant frozen-thawed sperm is similar whether or not heparin is present after the mini-Percoll technique.

Air-liquid interface cell culture: From airway epithelium to the female reproductive tract.

The air-liquid interface (ALI) approach is primarily used to mimic respiratory tract epithelia in vitro. It is also known to support excellent differentiation of 3D multilayered skin models. To establish an ALI culture, epithelial cells are seeded into compartmentalized culture systems on porous filter supports or gel substrata. After an initial propagation period, the culture medium is removed from the apical side of the epithelium, exposing the cells to the surrounding air. Therefore, nutritive supply to the cells is warranted only by the basolateral cell pole. Under these conditions, the epithelial cells differentiate and regain full baso-apical polarity. Some types of epithelia even generate in vivo-like apical fluid or mucus. Interestingly, the ALI culture approach has also been shown to support morphological and functional differentiation of epithelial cells that are not normally exposed to ambient air in vivo. This review aims at giving a brief overview on the characteristics of ALI cultures in general and ALI models of female reproductive tract epithelia in particular. We discuss the applicability of ALI models for the investigation of the early embryonic microenvironment and for its implications in assisted reproductive technologies.

Mechanisms of antigen transport from the female reproductive tract to the gastrointestinal tract

Abstract One of the major functions of the female reproductive tract (FRT) is the establishment and maintenance of pregnancy, resulting in healthy offspring. Although many pathogens (e.g. Chlamydia spp.) infect the FRT, no vaccines against these pathogens have been licensed to date. This lack of success has been in part attributed to the inability of the FRT to efficiently internalize antigens. We showed that FRT epithelium of mice can internalize nanoparticles (NPs) < 40 nm and that protein coated 20 nm NPs prime the intestinal mucosa for secretion of IgA. This finding led us to hypothesize that NPs reach the GI tract following per-vaginal (PV) instillation. Using multiphoton and fluorescence microscopy, we show that PV-instilled antigens (DAPI, Dextran, NPs) reach the stomach antrum and the small intestine within 1–3 h. It is proposed that Chlamydia spp. colonizes the GI tract following PV instillation by entering the circulation. We show that colonization of the GI tract by C. muridarum is dependent on cell transport. While within 7 days of PV infection Chlamydia colonizes the GI tract of controls, while no Chlamydia can be detected in iliac lymph nodes (ILNs) or GI tracts of CCR7−/− mice. Moreover, treatment of mice with FTY720 inhibits colonization of the GI tract, but it does not preclude infection of ILNs by Chlamydia. This finding indicates that Chlamydia transport from the ILNs to the GI tract depends on sphingosine 1-phosphate-mediated cell egress. Elucidation of mechanisms by which C. muridarum disseminates systemically to colonize the lymphoid tissues and the GI tract will be important for understanding both the immunity to and pathogenesis of Chlamydia spp.

Elongation of Müllerian ducts and connection to urogenital sinus determine the borderline of uterine and vaginal development

In female mice, proximal, middle and caudal Müllerian ducts (MDs) differentiate into oviduct, uterus and vagina, respectively. The fates of female reproductive tract epithelia are determined by the mesenchyme. However, the mesenchymal fate determination system is still unclear. It is reported that presence or absence of retinoic acid (RA) signaling in MD mesenchyme induced uterine or vaginal mesenchyme, respectively. To analyze determination of the borderline, RA signal switching factors were found to play critical roles. Expression of a RA metabolizing enzyme, CYP26A1, was high in the epithelium of caudal MD and urogenital sinus, indicating that the enzyme causes the absence of RA signaling in the region. mRNA expression of some transcription factors regulating Aldh1a2, RA synthesis enzyme expressed in MDs, in other tissues was detected in MDs. When the transcription factor genes were overexpressed in a uterine mesenchymal cell line, C/ebpδ overexpression stimulated Aldh1a2 expression. Furthermore, C/EBPδ protein was strongly expressed in the proximal and middle regions of the MDs and bound to the Aldh1a2 promoter in vivo. Since C/ebpδ mRNA expression was maintained at the same level in proximal, middle and caudal MDs, we hypothesize that a high frequency of mitosis induces a low level protein expression in MD mesenchyme. In fact, the mitotic activity was significantly high in caudal mesenchyme, and a mathematical model showed that a gradient of protein was induced by cell proliferation. Therefore, morphogenesis of MDs controls the fate of mesenchyme via RA degradation in urogenital sinus and a gradient of proteins involved in RA synthesis.

Modeling early embryo-maternal interactions in vitro

Environmental conditions experienced during early embryonic development influence growth, metabolism, and gene expression of the embryo as well as the epigenetic profile of the offspring. The environment of the early embryo consists of the luminal fluid within the oviduct and uterus and the epithelial cells composing this fluid. Whether the embryo is able to shape its own microenvironment by interacting with the epithelial lining of the oviduct/uterus and which factors potentially interfere with or regulate these interactions remains to be elucidated. As early embryonic signals and the respective maternal responses are subtle and local events, it is challenging to study them in vivo. Therefore, adequate in vitro-models optimally mimicking the contact zone between the maternal reproductive tract and the early embryo are needed to a) elucidate basic mechanisms involved in early embryonic development and b) reduce the number of experimental animals used for such studies. Functional epithelial cells are generally defined by a polarized distribution of organelles and proteins. Proper polarization is tightly connected with physiological cell behavior and in vivo-like reactivity of the epithelium. Therefore, this review summarizes current strategies for in vitro preservation of epithelial cell polarity. It presents recent advances in 3D culture of female reproductive tract epithelia and embryo-epithelial co-cultures. A special emphasis is set on compartmentalized culture systems, powerful tools for studying early embryo-maternal interactions in vitro. In such systems, cultured epithelial cells are manipulable from their basolateral as well as their apical cell pole, allowing concomitant application of embryonic as well as maternal effectors from the appropriate cellular compartment.

Mucosal Humoral Immune Response to SIVmac239∆nef Vaccination and Vaginal Challenge.

Live attenuated vaccines such as SIV with a deleted nef gene have provided the most robust protection against subsequent vaginal challenge with wild-type (WT) SIV in the SIV-rhesus macaque model of HIV-1 transmission to women. Hence, identifying correlates of this protection could enable design of an effective HIV-1 vaccine. One such prechallenge correlate of protection from vaginal challenge has recently been identified as a system with three components: 1) IgG Abs reacting with the viral envelope glycoprotein trimeric gp41; 2) produced by plasma cells in the submucosa and ectopic tertiary lymphoid follicles in the ectocervix and vagina; and 3) concentrated on the path of virus entry by the neonatal FcR in the overlying epithelium. We now examine the mucosal production of the Ab component of this system after vaginal challenge. We show that vaginal challenge immediately elicits striking increases in plasma cells not only in the female reproductive tract but also at other mucosal sites, and that these increases correlate with low but persistent replication at mucosal sites. We describe vaginal ectopic follicles that are structurally and functionally organized similar to follicles in secondary lymphoid organs, and we provide inferential evidence for a key role of the female reproductive tract epithelium in facilitating Ab production, affinity maturation, and class switch recombination. Vaccination thus accesses an epithelial-immune system axis in the female reproductive tract to respond to exposure to mucosal pathogens. Designing strategies to mimic this system could advance development of an effective HIV-1 vaccine.

BMP-regulated exosomes from Drosophila male reproductive glands reprogram female behavior.

Male reproductive glands secrete signals into seminal fluid to facilitate reproductive success. In Drosophila melanogaster, these signals are generated by a variety of seminal peptides, many produced by the accessory glands (AGs). One epithelial cell type in the adult male AGs, the secondary cell (SC), grows selectively in response to bone morphogenetic protein (BMP) signaling. This signaling is involved in blocking the rapid remating of mated females, which contributes to the reproductive advantage of the first male to mate. In this paper, we show that SCs secrete exosomes, membrane-bound vesicles generated inside late endosomal multivesicular bodies (MVBs). After mating, exosomes fuse with sperm (as also seen in vitro for human prostate-derived exosomes and sperm) and interact with female reproductive tract epithelia. Exosome release was required to inhibit female remating behavior, suggesting that exosomes are downstream effectors of BMP signaling. Indeed, when BMP signaling was reduced in SCs, vesicles were still formed in MVBs but not secreted as exosomes. These results demonstrate a new function for the MVB-exosome pathway in the reproductive tract that appears to be conserved across evolution.

Manifestations of immune tolerance in the human female reproductive tract

Like other mucosal surfaces (e.g., the gastrointestinal tract, the respiratory tract), the human female reproductive tract acts as an initial barrier to foreign antigens. In this role, the epithelial surface and subepithelial immune cells must balance protection against pathogenic insults against harmful inflammatory reactions and acceptance of particular foreign antigens. Two common examples of these acceptable foreign antigens are the fetal allograft and human semen/sperm. Both are purposely deposited into the female genital tract and appropriate immunologic response to these non-self antigens is essential to the survival of the species. In light of the weight of this task, it is not surprising that multiple, redundant and overlapping mechanisms are involved. For instance, cells at the immunologic interface between self (female reproductive tract epithelium) and non-self (placental trophoblast cells or human sperm) express glycosylation patterns that mimic those on many metastatic cancer cells and successful pathogens. The cytokine/chemokine milieu at this interface is altered through endocrine and immunologic mechanisms to favor tolerance of non-self. The “foreign” cells themselves also play an integral role in their own immunologic acceptance, since sperm and placental trophoblast cells are unusual and unique in their antigen presenting molecule expression patterns. Here, we will discuss these and other mechanisms that allow the human female reproductive tract to perform this delicate and indispensible balancing act.

Generation of Human Female Reproductive Tract Epithelium from Human Embryonic Stem Cells

BackgroundRecent 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 FindingsWe 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/SignificanceThese 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.

Mǔllerian cyst of the mesentery: A case report of an unusual location

Mǔllerian cysts or paramesonephric cysts arise from the fused embryonic ducts, which typically regress in the uterus. These cysts are usually located paravertebrally. We present an unusual case of a Mǔllerian cyst developing within the mesentery of the ileocecum that was successfully resected. The patient presented to our surgical unit with abdominal pain and swelling. She underwent all the necessary tests to rule out other pathologies before she underwent right hemicolectomy. The patient was discharged without complications. Histopathology confirmed the presence of female reproductive tract epithelium, which was conclusive of a Mǔllerian cyst or paramesonephric cyst. Mǔllerian cysts are rarely malignant, and they are usually treated surgically. The incidence of Mǔllerian cysts is one in 105,000, with almost equal sex distribution. Their unusual intraperitoneal location further demonstrates their uncommon presentation.

Differential regulation of neutrophil chemotaxis to IL‐8 and fMLP by GM‐CSF: lack of direct effect of oestradiol

Neutrophils are a normal constituent of the female reproductive tract and their numbers increase in the late secretory phase of the menstrual cycle prior to menses. Several cytokines are produced in female reproductive tract tissue. In particular granulocyte-macrophage colony-stimulating factor (GM-CSF), a potent activator of neutrophils, is secreted in high concentrations by female reproductive tract epithelia. We previously observed that GM-CSF synergizes strongly with interleukin-8 (IL-8) in enhancing chemotaxis of neutrophils. Thus we investigated whether pretreatment of neutrophils with GM-CSF would prime subsequent chemotaxis to IL-8 in the absence of GM-CSF. Surprisingly, a 3-hr pulse of GM-CSF severely diminished chemotaxis to IL-8, whereas N-formyl-methyl-leucyl-phenylalanine (fMLP)-mediated chemotaxis was retained. Conversely, when cells were incubated without GM-CSF they retained IL-8-mediated migration but lost fMLP chemotaxis. These changes in chemotaxis did not correlate with expression of CXCR1, CXCR2 or formyl peptide receptor. However, IL-8-mediated phosphorylation of p44/42 mitogen-activated protein kinase was greatly reduced in neutrophils that no longer migrated to IL-8, and was diminished in cells that no longer migrated to fMLP. Oestradiol, which is reported by some to exert an anti-inflammatory effect on neutrophils, did not change the effects of GM-CSF. These data suggest that neutrophil function may be altered by cytokines such as GM-CSF through modulation of signalling and independently of surface receptor expression.

Disruption of the upper female reproductive tract epithelium by nonoxynol-9

Background Spermicides containing nonoxynol-9 (N-9) may increase HIV transmission. In women, intravaginal N-9 is found in the uterus shortly after its insertion. Exposure of the female upper reproductive tract to N-9 may alter epithelial integrity, thereby increasing HIV transmission risk. Our goal was to characterize the histological effects of N-9 on uterine epithelium in an animal model. Methods Female mice were exposed to intravaginal or intrauterine Advantage-S™ (N-9), Replens™, K-Y® jelly or water. After various exposure times, mice were sacrificed and stained uterine tissue sections were analyzed by a pathologist blinded to treatment. Results Intravaginal and intrauterine N-9 administration resulted in disruption of uterine epithelium compared to Replens, K-Y jelly or water. N-9 caused rapid (within 10 min), focal, uterine epithelial sloughing and complete epithelial loss within 24 h. Epithelial regeneration began 48 h after exposure N-9 and was completely restored within 72 h; the new epithelial layer, however, was composed of cuboidal cells instead of normally present columnar cells. In addition, hemorrhage and necrosis were present are all time points examined. Conclusions Our results demonstrate for the first time that N-9 has a deleterious effect on uterine epithelium. Although these results were observed in a mouse model, similar disruption of the upper reproductive tract epithelium in women by N-9 may facilitate HIV infection and serve as an unrecognized portal of human HIV transmission.

In-vivo transfection of the female reproductive tract epithelium

Mouse female genital tract was transfected in vivo using the ss-galactosidase reporter gene. To transfect the female tract, DNA/liposome complexes were injected through the infundibulum of the oviducts of adult, immature, and pseudopregnant females. Females which were in different stages of the ovarian cycle were also employed. Transfection was analysed using histochemical, immunological and molecular (Southern blotting, polymerase chain reaction and gene sequencing) procedures. The lower region of the uterine glands and the oviduct epithelium in the isthmus and juncture regions were the most conspicuous transfected areas. The greatest numbers of transfected cells were 6% in the oviduct and 9% in the uterus, meanwhile the duration of expression reached a maximum of 7 days in the oviduct and 14 days in the uterus. The hormonal stage of the genital tract epithelium directly affected transfection, as the highest number of successful transfections occurred during the meta-oestrus and pseudopregnancy stages.

Unique aspects of intracellular signal transduction and tyrosine phosphorylation by the C-FMS encoded macrophage colony stimulating factor-1 (CSF-1R) receptor in benign and neoplastic cells derived from mammary and female reproductive tract epithelia.

Unique aspects of intracellular signal transduction and tyrosine phosphorylation by the C-FMS encoded macrophage colony stimulating factor-1 (CSF-1R) receptor in benign and neoplastic cells derived from mammary and female reproductive tract epithelia.