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The female reproductive tract (FRT) undergoes extensive remodeling during reproductive cycling. This recurrent remodeling and how it shapes organ-specific aging remains poorly explored. Using single-cell and spatial transcriptomics, we systematically characterized morphological and gene expression changes occurring in ovary, oviduct, uterus, cervix, and vagina at each phase of the mouse estrous cycle, during decidualization, and into aging. These analyses reveal that fibroblasts play central-and highly organ-specific-roles in FRT remodeling by orchestrating extracellular matrix (ECM) reorganization and inflammation. Our results suggest a model wherein recurrent FRT remodeling over reproductive lifespan drives the gradual, age-related development of fibrosis and chronic inflammation. This hypothesis was directly tested using chemical ablation of cycling, which reduced fibrotic accumulation during aging. Our atlas provides extensive detail into how estrus, pregnancy, and aging shape the organs of the female reproductive tract and reveals the unexpected cost of the recurrent remodeling required for reproduction., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for enumerating the gene expression dynamics at single-cell resolution. Various organs comprising distinct cellular composition and architecture require unique approaches for highly viable single-cell preparation and reliable sequencing results. Here, we describe an optimized protocol for isolating the female reproductive tract (FRT), dissecting different FRT regions, and preparing high-viability single cells from the uterine endocervix and ectocervix to generate a complete molecular cell atlas by scRNA-seq for studying normal physiology and disease. For complete details on the use and execution of this protocol, please refer to Chumduri et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Visualizing precise spatial patterns of an organ-wide gene and protein expression among diverse cell types can provide critical insights into the fundamental processes underlying normal tissue homeostasis and disease development. Here, we describe an optimized protocol for single-molecule RNA in situ hybridization (smRNA-ISH), immunohistochemistry, and cell lineage analysis of the female reproductive tract organs using commercially available smRNA-ISH probes, antibodies, and inducible Cre-mice. The high-resolution multispectral fluorescence imaging is performed using wide-field epifluorescence or confocal microscopy combined with a slide scanner. For complete details on the use and execution of this protocol, please refer to Chumduri et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

The expression of nitric oxide synthase (NOS) in male and female urogenital tissues has been investigated by using conventional light microscopical immunoperoxidase staining. We present an improved immunohistochemical method for the specific and simultaneous detection of endothelial and neuronal NOS (eNOS/nNOS) in vaginal tissue. Specific antibodies have been used in combination with the tyramide signal amplification method. We found a subepithelial meshwork of varicose nerve fibers. A subpopulation of fibers presented immunoreactivity specific for nNOS. Epithelial cells also showed cytoplasmatic labeling for nNOS. Arteries presenting signals for eNOS in their endothelial layer were found in close proximity to nNOS-positive nerve fibers., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Healthy functioning of the female reproductive tract (FRT) depends on balanced and dynamic regulation by hormones during the menstrual cycle, pregnancy and childbirth. The mucosal epithelial lining of different regions of the FRT-ovaries, fallopian tubes, uterus, cervix and vagina-facilitates the selective transport of gametes and successful transfer of the zygote to the uterus where it implants and pregnancy takes place. It also prevents pathogen entry. Recent developments in three-dimensional (3D) organoid systems from the FRT now provide crucial experimental models that recapitulate the cellular heterogeneity and physiological, anatomical and functional properties of the organ in vitro. In this review, we summarise the state of the art on organoids generated from different regions of the FRT. We discuss the potential applications of these powerful in vitro models to study normal physiology, fertility, infections, diseases, drug discovery and personalised medicine.

We conducted a cytological search for amyloid structures in female reproductive cells of Gallus gallus domesticus and Drosophila melanogaster . We have shown that the amyloid-specific dye, Thioflavin S, but not Congo red, stains some cytoplasmic and even nuclear structures in chicken ovaries. In fruit fly eggs both Thioflavin S and Congo red specifically stain eggshell structures such as micropyle, dorsal appendages and pillars. Moreover, these structures, when stained with Congo red, demonstrate birefringence in polarized light, which is a characteristic feature of all classical amyloids. Our data show that female reproductive cells during evolution began to use amyloid fibrils to form various functional structures necessary for development under certain environmental conditions.

This study aimed to detect the presence of glutathione peroxidase 8 (GPx8) in rat during preimplantation period of pregnancy. Females were killed on first (D1), third (D3), and fifth (D5) day of pregnancy. The presence of GPx8 in embryos was detected under the confocal microscope, the presence of GPx8 in genital organs was confirmed immunohistochemically, and the amount of GPx8 was determined using densitometry. We found that GPx8 is dispersed in the cytoplasm of oocytes, while after fertilization, it is concentrated in granules. From 4-cell stage till blastocyst, GPx8 reaction was found in the perinuclear region. In the ovary, GPx8 was seen in granulosa-lutein cells, in plasma of blood vessels, and inside Graafian follicles. In oviduct, GPx8 was detected in the plasma and in the extracellular matrix (ECM). Moreover, epithelial cells of isthmus were positive. In uterus, GPx8 was observed in the uterine glands, in the plasma, and in ECM. On D5, the enzyme disappeared from the uterine glands and appeared in fibroblasts. Densitometry revealed that the highest amount of GPx8 was on D1 and subsequently declined. To our knowledge, this is the first paper describing GPx8 presence in the oocytes, preimplantation embryos, and female genital organs in mammals. Our results improve the understanding of antioxidant enzymes presence during pregnancy in defense against oxidative stress, which is considered to be one of the main causes of infertility.

The functional characterization and regulation of tissue resident and non-resident CD8+ T cells in the human female reproductive tract (FRT) as women age remains a gap in our knowledge. Here we characterized the cytotoxic activity and granular contents of CD8+ T cells from the FRT in pre- and postmenopausal women. We found that under steady-state conditions, CD8+ T cells from endometrium (EM), endocervix and ectocervix displayed direct cytotoxic activity, and that cytotoxicity increased in the EM after menopause. Cytotoxic activity was sensitive to suppression by TGFβ exclusively in the EM, and sensitivity to TGFβ was reduced after menopause. Under steady-state conditions, cytotoxic activity (measured as direct killing activity), cytotoxic potential (measured as content of cytotoxic molecules) and proliferation are enhanced in non-resident CD8+ (CD103-) T cells compared to tissue resident (CD103+) T cells. Upon activation, CD103+ T cells displayed greater degranulation compared to CD103- T cells, however the granular content of perforin, granzyme A (GZA) or granzyme B (GZB) was significantly lower. After menopause, degranulation significantly increased, and granular release switched from predominantly GZB in premenopausal to GZA in postmenopausal women. Postmenopausal changes affected both CD103+ and CD103- subpopulations. Finally, CD103+ T cells displayed reduced proliferation compared to CD103- T cells, but after proliferation, cytotoxic molecules were similar in each population. Our results highlight the complexity of regulation of cytotoxic function in the FRT before and after menopause, and are relevant to the development of protective strategies against genital infections and gynecological cancers as women age., (Copyright © 2020 Rodriguez-Garcia, Shen, Fortier and Wira.)

Female reproductive medicine may not have been entirely overlooked in the history of medical research, but it has never been given the attention that it deserves. There are signs, however, that the spotlight is turning toward the most essential of human processes.

Homeostatic leukocyte trafficking into and within the female reproductive tract (FRT) contributes to fertility and reproductive health. It is unclear how this process is regulated in the anatomically distinct reproductive tissues, or whether the genes involved are affected by cyclical changes in reproductive hormones. In tissues such as skin and intestine, mouse studies have defined evolutionarily conserved molecular mechanisms for tissue-specific homing, interstitial positioning, and leukocyte egress. Chemokine family members are invariably involved, with the chemokine expression profile of a tissue regulating leukocyte content. Reproductive tissues (ovary, vagina, cervix, uterine horn) of 8 week old virgin female C57BL/6 mice ( n = 20 ) were collected, and expression of mRNA for leukocyte markers and chemokines conducted by qPCR. Lymphocytic and myeloid cell populations within the uterus, cervix, bone marrow and PALN from virgin C57BL/6 mice were determined by flow cytometric analysis. Variation in leukocyte content between reproductive tissues is evident, with the uterus and cervix containing complex mixtures of lymphocytes and myeloid cells. Twenty-six chemokine genes are expressed in the FRT, many by several component tissues, some preferentially by one. Most striking are Xcl1 and Ccl28 , which are restricted to the uterus. Ccl20 and genes encoding CXCR2 ligands are primarily transcribed in cervix and vagina. Ovary shows the lowest expression of most chemokine genes, with the notable exception of Ccl21 and Ccl27 . We also identify eight chemokines in the vagina whose expression fluctuates substantially across the oestrous cycle. These data reveal complex chemokine networks within the FRT, and provide a framework for future studies of homeostatic leukocyte trafficking into and within these tissues. Abbreviations : BM: bone marrow; DC: dendritic cell; DN: double negative; FRT: female reproductive tract; FSC: forward scatter; NK: natural killer; PALN: para-aortic lymph node; SSC: side scatter; Tregs: regulatory T cells.

The objective of this study was (1) to describe the US appearance and obtain reference values for the uterus and ovaries in nongravid and gravid queens with histologically confirmed reproductive tracts without disorders, (2) to provide US measurements of the reproductive tract compared to gross macroscopic and water-bath post-OVH US measurements in nongravid queens, and (3) to describe the sonographic appearance of the female reproductive tract during the different histopathologic phases of the reproductive cycle in nongravid and gravid queens. Ninety-three queens from a "trap, neuter, return" program were included in this study. Sonographic evaluation of the reproductive tract was performed in all queens, and measurements of the corpus uteri, uterine horns, and ovaries were recorded. Following OVH, macroscopic measurements were obtained, and a water-bath US evaluation of these tissues and measurements was recorded. Samples from the corpus uteri and both the uterine horns and ovaries were collected for histopathologic examination after all measurements had been recorded. Seventy-two reproductive tracts met the inclusion criteria by having a histopathologically confirmed normal reproductive tract. Sixty-three queens were nonpregnant and 9 were pregnant. The ovaries and uterus were sonographically visible in all queens regardless of reproductive status. The ovaries were ovoid in shape, and the uterus appeared as a tubular structure with distinct wall layers (serosa and indistinct myometrium and myometrium, or serosa, myometrium, and endometrium), with variable echogenicity of the inner layers. The layering of the uterine wall, observed during the second half of pregnancy, was described. Ovarian follicles were visible in 66/72 (92%) cats. However, the CL was only visible in 40/72 (55%) cats. The reference values of the left ovarian length, right ovarian length, uterine horn diameter, and uterine body are 7.1-13.9, 7.3-13.6, 1-5.8, and 1.5-5.3 mm, respectively, in a nongravid uterus. The uterine wall thickness during pregnancy varied from 2.4 to 6.8 mm. There was a significant positive correlation between US measurements obtained in vivo and those obtained macroscopically and in a water bath post-OVH. The body weight, follicular size, sonographic visibility of the uterine wall layering, the histopathologic luteal phase, and the active/inactive status on histopathology had a significant effect on the uterine measurements (p < 0.05). It was not possible to describe the exact US features of the reproductive tract during the different histopathologic phases. In conclusion, ultrasonographic reference values for the normal female reproductive tract in cats were determined. The results of this study indicated that the ovaries and uterus were visible in cats regardless of reproductive status., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Herpes Simplex Virus Type 2 (HSV-2) is one of the most prevalent sexually transmitted viruses and is a known risk factor for HIV acquisition in the Female Genital Tract (FGT). Previously, we found that curcumin can block HSV-2 infection and abrogate the production of inflammatory cytokines and chemokines by genital epithelial cells in vitro. In this study, we investigated whether curcumin, encapsulated in nanoparticles and delivered by various in vivo routes, could minimize inflammation and prevent or reduce HSV-2 infection in the FGT. Female mice were pre-treated with curcumin nanoparticles through oral, intraperitoneal and intravaginal routes, and then exposed intravaginally to the tissue inflammation stimulant CpG-oligodeoxynucleotide (ODN). Local intravaginal delivery of curcumin nanoparticles, but not intraperitoneal or oral delivery, reduced CpG-mediated inflammatory histopathology and decreased production of pro-inflammatory cytokines Interleukin (IL)-6, Tumor Necrosis Factor Alpha (TNF-α) and Monocyte Chemoattractant Protein-1 (MCP-1) in the FGT. However, curcumin nanoparticles did not demonstrate anti-viral activity nor reduce tissue pathology when administered prior to intravaginal HSV-2 infection. In an alternative approach, intravaginal pre-treatment with crude curcumin or solid dispersion formulations of curcumin demonstrated increased survival and delayed pathology following HSV-2 infection. Our results suggest that curcumin nanoparticle delivery in the vaginal tract could reduce local tissue inflammation. The anti-inflammatory properties of curcumin delivered to the vaginal tract could potentially reduce the severity of HSV-2 infection and decrease the risk of HIV acquisition in the FGT of women.

Bunocotyle progenetica is a hemiuroid digenean whose sexual adults become fully developed and lay their eggs inside the rediae in the molluscan host. In this study, the fine structure of the germinal mass, brood cavity and birth canal in the B. progenetica rediae was examined using transmission electron and confocal microscopy. The large germinal mass attached to the body wall has a cellular composition typical for this organ. The characteristic traits of this germinal mass are weakly developed supporting tissue and the presence of deep lacunae opening into the brood cavity. These lacunae presumably participate in feeding the deeply lying embryos and facilitate their release into the brood cavity. The germinal mass is also characterized by intensive degeneration of cellular elements, which may represent a mechanism controlling the offspring number, limited in this species by the size of the redial brood cavity. The brood-cavity lining consists of flattened cells bearing lamellar projections and is connected anteriorly with the epithelium of the birth canal. The brood-cavity musculature, which is well developed in other hemiuroid digeneans, is significantly reduced in B. progenetica, most likely because their cystophorous cercariae remain inside the rediae, removing the need for muscle contractions pushing them through the brood cavity. The birth canal comprises three regions distinguished by the structure of the lining and muscle arrangement. The comparison of rediae of B. progenetica with parthenitae of other digeneans has shown that the organization of the redial reproductive apparatus in this species may have been influenced by life-cycle modification.

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., (© 2019 Blackwell Verlag GmbH.)

The study investigated the gross and microscopic anatomy of the genital organs of 20 agoutis at different stages of the estrous cycle and four in the final trimester of pregnancy. Specimens were euthanized and their reproductive organs were fixed in a 4% paraformaldehyde or 2.5% glutaraldehyde solution and submitted to routine histological techniques for light and scanning electron microscopy. In the ovary, during the proestrus phase, we observed developing follicles and corpus luteum (CL) in regression; during estrus, there were Graafian follicles; during metestrus, there was a hemorrhagic corpus, whereas in diestrus, there was a mature CL. The uterus was partially double because the cervix was cranially septate but caudally, the septum disappeared, forming a single ostium that opened into the vagina. Changes occurred along the estrous cycle in the uterine and vaginal epithelia, that is, an increase in the uterine epithelium height accompanied by an increase of thickness of the vaginal epithelium during the follicular phase and a decrease of thickness of both epithelia during the luteal phase. The endometrial lining was composed of a simple cuboidal epithelium to simple columnar epithelium with basal nuclei. The vaginal mucosa consisted of epithelium that varied from nonkeratinized stratified squamous (luteal phase) to keratinized stratified squamous (follicular phase). The clitoris was external to the vagina. It presented two protruding lateral keratinized spicules and a centralized urethra, with no common parts between the urinary and genital tracts. Anatomical and histological changes were observed mainly in the cervix, vagina and spicules of the clitoris during the EC., (© 2019 Wiley Periodicals, Inc.)

CD8 + T cells provide a critical defence from pathogens at mucosal epithelia including the female reproductive tract (FRT). Mucosal immunisation is considered essential to initiate this response, however this is difficult to reconcile with evidence that antigen delivered to skin can recruit protective CD8 + T cells to mucosal tissues. Here we dissect the underlying mechanism. We show that adenovirus serotype 5 (Ad5) bio-distributes at very low level to non-lymphoid tissues after skin immunisation. This drives the expansion and activation of CD3 - NK1.1 + group 1 innate lymphoid cells (ILC1) within the FRT, essential for recruitment of CD8 + T-cell effectors. Interferon gamma produced by activated ILC1 is critical to licence CD11b + Ly6C + monocyte production of CXCL9, a chemokine required to recruit skin primed CXCR3 + CD8 + T-cells to the FRT. Our findings reveal a novel role for ILC1 to recruit effector CD8 + T-cells to prevent virus spread and establish immune surveillance at barrier tissues.

The broad-snouted caiman (Caiman latirostris) is a crocodilian species that inhabits South American wetlands. As in all other crocodilians, the egg incubation temperature during a critical thermo-sensitive window (TSW) determines the sex of the hatchlings, a phenomenon known as temperature-dependent sex determination (TSD). In C. latirostris, we have shown that administration of 17-β-estradiol (E 2 ) during the TSW overrides the effect of the male-producing temperature, producing phenotypic females (E 2 SD-females). Moreover, the administration of E 2 during TSW has been proposed as an alternative way to improve the recovery of endangered reptile species, by skewing the population sex ratio to one that favors females. However, the ovaries of E 2 SD-female caimans differ from those of TSD-females. In crocodilians, the external genitalia (i.e. clitero-penis structure or phallus) are sexually dimorphic and hormone-sensitive. Despite some morphological descriptions aimed to facilitate sexing, we found no available data on the C. latirostris phallus histoarchitecture or hormone dependence. Thus, the aims of this study were: (1) to establish the temporal growth pattern of the phallus in male and female caimans; (2) to evaluate histo-morphological features and the expression of estrogen receptor alpha (ERα) and androgen receptor (AR) in the phallus of male and female pre-pubertal juvenile caimans; and (3) to determine whether the phallus of TSD-females differs from the phallus of E 2 SD-females. Our results demonstrated sexually dimorphic differences in the size and growth dynamics of the caiman external genitalia, similarities in the shape and spatial distribution of general histo-morphological compartments, and sexually dimorphic differences in innervation, smooth muscle fiber distribution, collagen organization, and ERα and AR expressions. The external genitalia of E 2 SD-females differed from that of TSD-females in many histological features and in the expression of ERα and AR, resembling patterns described in males. Our results alert on the effects of estrogen agonist exposure during TSW and suggest that caution must be taken regarding the use of E 2 SD as a procedure for wildlife population management., (Copyright © 2018. Published by Elsevier Inc.)

The aim of this study was to evaluate the effectiveness of antiretroviral treatment (ART) on the proportion and functions of Th17 and Treg cells in peripheral blood and female genital tract (FGT) respectively. To this aim, samples from 41 HIV-neg, 33 HIV+ ART-naïve and 32 HIV+ ART+ subjects were obtained. In peripheral blood, altered Th17 and Th17/Treg proportions were normalized in HIV+ ART+, but certain abnormal Treg and activated T-cell proportions were still observed. In FGT, abnormal patterns of secretion for Th17-related cytokines were observed in cervical mononuclear cells (CMCs) from HIV+ women, even in those from HIV+ ART+, compared to the HIV-neg group. Moreover, these altered patterns of secretion were associated with diminished levels of CXCL5 and CXCL1 chemokines and with an immunoregulatory skew in the CCL17/CCL20 ratio in ectocervix samples of these women. Finally, ART did not restore proportions of Th17-precursor cells with gut-homing potential in PBMCs, and positive correlations between these cells and the levels of IL-17F and IL-21 production by CMCs may suggest that a better homing of these cells to the intestine could also imply a better restoration of these cells in the female genital tract. These results indicate that antiretroviral treatment did not restore Th17-related immune functions completely at the female mucosal level.

Tenofovir (TFV) treatment of female reproductive tract (FRT) cells results in differential accumulation of intracellular Tenofovir diphosphate (TFV-DP) in different cell types, with greater concentrations in epithelial cells (100-fold) and fibroblasts (10-fold) than in CD4+ T cells. The possibility that TFV-DP accumulation and retention in epithelial cells and fibroblasts may alter TFV availability and protection of CD4+ T cells against HIV infection, prompted us to evaluate TFV and/or Tenofovir alafenamide (TAF) release from FRT cells. Endometrial, endocervical and ectocervical polarized epithelial cells and fibroblasts were pre-loaded with TFV or TAF, and secretions tested for their ability to inhibit HIV infection of activated blood CD4+ T cells. Epithelial cell basolateral secretions (1, 2 and 3 days post-loading), but not apical secretions, suppressed HIV infection of CD4+ T cells, as did secretions from pre-loaded fibroblasts from each site. Intracellular TFV-DP levels in epithelial cells following preloading with TFV or TAF correlated directly with ARV protection of CD4+ T cells from HIV infection. When added apically to epithelial cells, TFV/TAF was released basolaterally, in part through Multidrug Resistant Protein transporters, taken up by fibroblasts and released into secretions to partially protect CD4+ T cells. These findings demonstrate that epithelial cells and fibroblasts release TFV/TAF for use by CD4+ T cells and suggest that the tissue environment plays a major role in the sustained protection against HIV infection.

Several tissue clearing methods have been developed for three-dimensional imaging of thick specimens. Here, we applied CUBIC and ScaleS approaches to whole-mounted vaginal wall to reveal spatial distribution of γδ T lymphocytes, the key cells engaged in the epithelial homeostasis control and immune surveillance. Both methods rendered the tissue transparent and enabled detection of the green fluorescent protein (GFP)-expressing γδ T cells in vaginal samples of Tcrd-H2BeGFP transgenic mice. Upon additional immunolabeling, however, only CUBIC preserved the GFP signal and allowed for cell localization assessment during the estrous cycle. Using a combination of single- and two-photon microscopy, we found that during the diestrus phase the number of γδ T cells in the vaginal wall increased compared to estrus, while the proportion of cells residing in epithelium and stroma remained constant, irrespective of the cycle phase, and was close to 3:1, respectively. Moreover, the distance from epithelial γδ T cells to laminin-positive basal membrane and collagen-rich stroma also increased in diestrus in spite of thinning of epithelium upon shedding cornified cells. Our data indicate that γδ T cells sense sex hormone fluxes which influence their number and position them closer to the vaginal lumen in the diestrus phase., (© 2018 Australasian Society for Immunology Inc.)

Background: Estrogens are known to orchestrate reproductive events and to regulate the immune system during infections and following tissue damage. Recent findings suggest that, in the absence of any danger signal, estrogens trigger the physiological expansion and functional specialization of macrophages, which are immune cells that populate the female reproductive tract (FRT) and are increasingly being recognized to participate in tissue homeostasis beyond their immune activity against infections. Although estrogens are the only female gonadal hormones that directly target macrophages, a comprehensive view of this endocrine-immune communication and its involvement in the FRT is still missing., Objective and Rationale: Recent accomplishments encourage a revision of the literature on the ability of macrophages to respond to estrogens and induce tissue-specific functions required for reproductive events, with the aim to envision macrophages as key players in FRT homeostasis and mediators of the regenerative and trophic actions of estrogens., Search Methods: We conducted a systematic search using PubMed and Ovid for human, animal (rodents) and cellular studies published until 2018 on estrogen action in macrophages and the activity of these cells in the FRT., Outcomes: Our search identified the remarkable ability of macrophages to activate biochemical processes in response to estrogens in cell culture experiments. The distribution at specific locations, interaction with selected cells and acquisition of distinct phenotypes of macrophages in the FRT, as well as the cyclic renewal of these properties at each ovarian cycle, demonstrate the involvement of these cells in the homeostasis of reproductive events. Moreover, current evidence suggests an association between estrogen-macrophage signaling and the generation of a tolerant and regenerative environment in the FRT, although a causative link is still missing., Wider Implications: Dysregulation of the functions and estrogen responsiveness of FRT macrophages may be involved in infertility and estrogen- and macrophage-dependent gynecological diseases, such as ovarian cancer and endometriosis. Thus, more research is needed on the physiology and pharmacological control of this endocrine-immune interplay.

Introduction: At its basic level, HIV infection requires a replication-competent virus and a susceptible target cell. Elevated levels of vaginal inflammation has been associated with the increased risk of HIV infection as it brings highly activated HIV target cells (CCR5+CD4+ T cells; CCR5+CD4+CD161+ Th17 T cells) to the female genital tract (FGT) where they interact with HIV. Decreased HIV risk has been associated with a phenotype of decreased immune activation, called immune quiescence, described among Kenyan female sex workers who were intensely exposed to HIV yet remain uninfected. Current prevention approaches focus on limiting viral access. We took the novel HIV prevention approach of trying to limit the number of HIV target cells in the genital tract by reducing inflammation using safe, affordable and globally accessible anti-inflammatory drugs., Methods: We hypothesized that the daily administration of low doses of acetylsalicylic acid (ASA 81 mg) or hydroxychloroquine (HCQ 200 mg) would reduce inflammation thereby decreasing HIV target cells at the FGT. Low-risk HIV seronegative women from Nairobi, Kenya were randomized for six weeks therapy of ASA (n = 37) or HCQ (n = 39) and tested to determine the impact on their systemic and mucosal immune environment., Results: The results showed that HCQ use was associated with a significant reduction in the proportion of systemic T cells that were CCR5+CD4+ (p = 0.01) and Th17 (p = 0.01). In the ASA arm, there was a 35% and 28% decrease in the proportion of genital T cells that were CD4+CCR5+ (p = 0.017) and Th17 (p = 0.04) respectively. Proteomic analyses of the cervical lavage showed ASA use was associated with significantly reduced amount of proteins involved in the inflammatory response and cell recruitment at the mucosa, although none of the individual proteins passed multiple comparison correction. These changes were more apparent in women with Lactobacillus dominant microbiomes., Conclusion: Together, these data indicate that taking low-dose ASA daily was associated with significant reduction in HIV target cells at the FGT. This study provides proof-of-concept for a novel HIV-prevention approach that reducing inflammation using safe, affordable and globally accessible non-steroidal anti-inflammatory agents is associated with significant reduction in the proportion of HIV-target cells at the FGT., (© 2018 The Authors. Journal of the International AIDS Society published by John Wiley & sons Ltd on behalf of the International AIDS Society.)

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

The mucosal tissues of the gut and female reproductive tract (FRT) are susceptible to pathogen infections including bacteria, viruses, and parasites, and are also the targets for immune disorders such as Crohn's disease, inflammatory bowel disease (IBD), and many types of cancers. However, the role of the mucosal immune cells to control these diseases is largely unknown. The limited availability of human mucosal biopsy tissue and the low number of cells that can be isolated from these tissues hampers the characterization of the phenotype and function of human mucosal immune cell subsets. Therefore, human-immune-system humanized mice are surrogate models to investigate the human mucosal immune cell responses during the course of the disease. The current protocols used to harvest the immune cells from the mucosal tissues, however, result in low recovery of cells with poor viability. We have established a novel protocol, which results in a high yield of human lymphocytes with high viability to overcome this issue. The immune cells obtained from a single DRAG mouse by our protocol were sufficient for conducting functional assays and for flow cytometry analyses including phenotypic, exhaustion, and functional panels., (Published by Elsevier B.V.)

Several growth factor families have been shown to be involved in the function of the female reproductive tract. One subfamily of the fibroblast growth factor (FGF) superfamily, namely the FGF8 subfamily (including FGF17 and FGF18), has become important as Fgf8 has been described as an oocyte-derived factor essential for glycolysis in mouse cumulus cells and aberrant expression of FGF18 has been described in ovarian and endometrial cancers. In this review, we describe the pattern of expression of these factors in normal ovaries and uteri in rodents, ruminants and humans, as well as the expression of their receptors and intracellular negative feedback regulators. Expression of these molecules in gynaecological cancers is also reviewed. The role of FGF8 and FGF18 in ovarian and uterine function is described, and potential differences between rodents and ruminants have been highlighted especially with respect to FGF18 signalling within the ovarian follicle. Finally, we identify major questions about the reproductive biology of FGFs that remain to be answered, including (1) the physiological concentrations within the ovary and uterus, (2) which cell types within the endometrial stroma and theca layer express FGFs and (3) which receptors are activated by FGF8 subfamily members in reproductive tissues., (© 2018 Society for Reproduction and Fertility.)

For over three decades, HIV infection has had a tremendous impact on the lives of individuals and public health. Microbicides and vaccines studies have shown that immune activation at the genital tract is a risk factor for HIV infection. Furthermore, lower level of immune activation, or what we call immune quiescence, has been associated with a lower risk of HIV acquisition. This unique phenotype is observed in highly-exposed seronegative individuals from different populations including female sex workers from the Pumwani cohort in Nairobi, Kenya. Here, we review the link between immune activation and susceptibility to HIV infection. We also describe a new concept in prevention where, instead of targeting the virus, we modulate the host immune system to resist HIV infection. Mimicking the immune quiescence phenotype might become a new strategy in the toolbox of biomedical methods to prevent HIV infection. Clinical trial registration on clinicaltrial.gov: #NCT02079077.

We studied the functional morphology of the female reproductive system of the purple stone crab Danielethus crenulatus. The most remarkable feature is the relative storage capacity and extensibility of the seminal receptacles. These receptacles are a pair of simple sacs that lack internal structures dividing the internal lumen. Differences in seminal receptacle size and contents are accompanied by conspicuous changes in receptacle lining at a tissue level. Full seminal receptacles contain discrete sperm masses formed by hardened fluid and densely packed spermatophores. Different sperm masses are likely from different mates and their stratified disposition within the seminal receptacles is compatible with rival sperm displacement and last sperm precedence. Additionally, the anatomical structure of the vulva and vagina suggest active female control over copula. We discuss our results in the general context of sperm storage in brachyurans and the implications for the mating system of this species., (© 2017 Wiley Periodicals, Inc.)

Because of the poor knowledge of the morphology of the female reproductive organs of most brachyuran crabs, this study investigated two Atlantic representatives of the family Leucosiidae, Ilia nucleus (Linnaeus, 1758) and Persephona mediterranea (Herbst, 1794), using histological methods and magnetic resonance imaging (MRI). While the vagina conforms to the concave type, the arrangement of the two chambers of the seminal receptacle differs strongly from that of other eubrachyuran sperm storage organs. Both chambers are oriented laterally within the crab's body. This is in contrast to the dorso-ventral orientation described in most other known brachyuran crabs. The lateral chamber is covered by cuticle, whereas the medial chamber is covered by a holocrine glandular epithelium. The oviduct connection is located ventrally, posterior to the vagina. The oviduct orifice is characterized by a transition from the epithelium lining the oviduct to the seminal receptacle's holocrine glandular epithelium. Moreover, muscle fibres are attached to the oviduct orifice and to the sternal cuticle. This musculature can be interpreted as an important feature in the fertilization and egg-laying process by supporting and controlling the inflow of eggs into the seminal receptacle lumen. The results of this study are compared to the morphology of the seminal receptacle of another leucosiid crab, Ebalia tumefacta (Montagu, 1808), and to those of other known eubrachyuran crabs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The aim of this study was to monitor the oestrous cycle using vaginal cytology, ultrasound and measurement of hormone levels associated with the modification of external genitalia in female Euphractus sexcinctus. Five adult female six-banded armadillos were used for the study. Every three days, we chemically restrained the animals with a combined dose of ketamine and xylazine for 90 days. On each occasion, we conducted vaginal cytology and monitored the alterations in the vulval appearance. In addition, we obtained blood samples for serum estradiol and progesterone analysis and evaluated the ovaries by ultrasonography (8 MHz). As results, at least two entire cycles were monitored per female as based on external oestrous signs. We determined that six-banded armadillos' oestrous cycle lasts 23.5 ± 3.12 days, comprising 8.8 ± 1.4 days for oestrogen phase, in which we verified vaginal bloody discharge, vulvar oedema, presence of mucus and ease of introduction of the swab. During oestrus, females presented an oestrogen peak of 240.66 ± 12.69 pg ml(-1) , on average, with a positive visualization of ovary follicles by ultrasound. The progesterone phase lasts 15.62 ± 2.1 days, characterized by the absence of bloody secretion and difficulty in introducing the swab; there was verification of a progesterone plateau of 10.83 ± 1.86 ng ml(-1) , on average, with identification of corpora lutea in 60% of the ovaries. This is apparently the first description of the six-banded armadillos' oestrous cycle, which proves the efficiency of a multiparametric analysis to monitor it., (© 2016 Blackwell Verlag GmbH.)

In order to understand branchiuran reproductive biology, it is imperative to know the sites of oogenesis and oocyte maturation, locate the accessory reproductive glands, and identify the fertilization site with the present knowledge of the sperm transfer mechanism of the genus Argulus. With these objectives, we attempted to describe the female reproductive system of Argulus bengalensis using serial histological sections through the ovaries and associated ducts in the transverse, longitudinal, and sagittal planes. The reproductive organs include a median ovary, one pair of ovarian lumina, a median oviduct, and a pair of collateral accessory glands. A duct from each of the collateral accessory glands leads into the proximal part of the median oviduct, which opens to the exterior through a genital opening at the distal end. The glandular secretion presumably contributes to the jelly coat of the egg. The ovary is bound with a tunica propria which extends further diametrically inside the ovary forming the paired lumina. The lumina are confluent into the median oviduct. Two distinct areas, the germarium and differentiating zones, are clearly distinguishable within the ovary. The tunica propria itself houses the oogonia within a matrix, serving as the germarium. Transmission electron micrograph reveals that the matrix is made of collagen. The collagen matrix confers elasticity to the tunica propria to accommodate the postvitellogenic oocytes within the ovarian lumen. The differentiating zone is situated in between the germarium: dorsally it is covered with a chromatophore layer. The ovary is ensheathed by a circum ovarian striated muscle. The presence of spermatophores in the ovarian lumen indicates the fertilization site. J. Morphol. 277:707-716, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The majority of new Human Immunodeficiency Virus (HIV)-1 infections are acquired via sexual transmission at mucosal surfaces. Partial efficacy (31.2%) of the Thai RV144 HIV-1 vaccine trial has been correlated with Antibody-dependent Cellular Cytotoxicity (ADCC) mediated by non-neutralizing antibodies targeting the V1V2 region of the HIV-1 envelope. This has led to speculation that ADCC and other antibody-dependent cellular effector functions might provide an important defense against mucosal acquisition of HIV-1 infection. However, the ability of antibody-dependent cellular effector mechanisms to impact on early mucosal transmission events will depend on a variety of parameters including effector cell type, frequency, the class of Fc-Receptor (FcR) expressed, the number of FcR per cell and the glycoslyation pattern of the induced antibodies. In this study, we characterize and compare the frequency and phenotype of IgG (CD16 [FcγRIII], CD32 [FcγRII] and CD64 [FcγRI]) and IgA (CD89 [FcαR]) receptor expression on effector cells within male and female genital mucosal tissue, colorectal tissue and red blood cell-lysed whole blood. The frequency of FcR expression on CD14+ monocytic cells, myeloid dendritic cells and natural killer cells were similar across the three mucosal tissue compartments, but significantly lower when compared to the FcR expression profile of effector cells isolated from whole blood, with many cells negative for all FcRs. Of the three tissues tested, penile tissue had the highest percentage of FcR positive effector cells. Immunofluorescent staining was used to determine the location of CD14+, CD11c+ and CD56+ cells within the three mucosal tissues. We show that the majority of effector cells across the different mucosal locations reside within the subepithelial lamina propria. The potential implication of the observed FcR expression patterns on the effectiveness of FcR-dependent cellular effector functions to impact on the initial events in mucosal transmission and dissemination warrants further mechanistic studies.

The goal was to increase pseudopregnant mice production by estrous cycle staging by visual examination before pairing and to determine the effect of such pseudopregnant recipients on embryo transfer. To compare methods of estrous cycle staging over 14 days, groups consisted of 10 females in proestrus-estrus and 10 vasectomized males; group 1: only daily visual observation; group 2: daily visual observation and cytological examination on day 1; group 3: daily visual observation and daily cytological examination. The average time to first vaginal plug was 1.8 days in group 1, 2.7 days in group 2, and 3.2 days in group 3, whereas the average time between consecutive vaginal plugs was 9.2 days (group 1), 10 days (group 2), and 9.25 days (group 3). The average time between consecutive estrous cycles was 9.7 days (group 1), 11.8 days (group 2), and 9.4 days (group 3). The congruence between visual and cytological examination in determining proestrus-estrus in group 2 was 100% and that for the four stages in group 3 was 79% with a range of 44% to 100%. From 162 plug-positive females originally selected in proestrus-estrus, 49%, 30%, 19%, and 2% were plug-positive on Day 1, Day 2, Day 3, and Day 4, respectively, showing that pseudopregnant mice production was significantly increased on the first 2 days. From 192 plug-positive females originally selected randomly, these values were 31%, 21%, 35%, 10%, and 3% on d1, d2, d3, d4, and d5, respectively. No significant differences were observed between groups with respect to embryo transfers with fresh or cryopreserved embryos although the number of pups born per litter was higher in group A with fresh (7.57 vs. 6.39) and cryopreserved-thawed embryos (5.0 vs. 4.38). Furthermore, the sex ratio and the genotype of the pups were not significantly affected., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Routine infertility investigations in the male and female include imaging techniques such as ultrasonography and endoscopy (fertiloscopy). However, these techniques lack the resolution to localize vital sperm or to reveal detailed morphological analysis of the oviduct which is often the cause of infertility in females. Therefore we set out to evaluate the efficiency of optical coherence tomography (OCT) as a diagnostic imaging tool for micron-scale visualization of the male and female genital tract. Using the bovine as a model, the optical features of the Telesto(TM) , Ganymede(TM) (both Thorlabs) and Niris(TM) (Imalux) OCT imaging systems were compared., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Elevated inflammatory cytokines (EMCs) at mucosal surfaces have been associated with HIV susceptibility, but the underlying mechanisms remain unclear. We characterized the soluble mucosal proteome associated with elevated cytokine expression in the female reproductive tract. A scoring system was devised based on the elevation (upper quartile) of at least three of seven inflammatory cytokines in cervicovaginal lavage. Using this score, HIV-uninfected Kenyan women were classified as either having EMC (n=28) or not (n=68). Of 455 proteins quantified in proteomic analyses, 53 were associated with EMC (5% false discovery rate threshold). EMCs were associated with proteases, cell motility, and actin cytoskeletal pathways, whereas protease inhibitor, epidermal cell differentiation, and cornified envelope pathways were decreased. Multivariate analysis identified an optimal signature of 16 proteins that distinguished the EMC group with 88% accuracy. Three proteins in this signature were neutrophil-associated proteases that correlated with many cytokines, especially GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-1β (interleukin-1β), MIP-3α (macrophage inflammatory protein-3α), IL-17, and IL-8. Gene set enrichment analyses implicated activated immune cells; we verified experimentally that EMC women had an increased frequency of endocervical CD4(+) T cells. These data reveal strong linkages between mucosal cytokines, barrier function, proteases, and immune cell movement, and propose these as potential mechanisms that increase risk of HIV acquisition.

The Drosophila Kelch protein is required to organize the ovarian ring canal cytoskeleton. Kelch binds and cross-links F-actin in vitro, and it also functions with Cullin 3 (Cul3) as a component of a ubiquitin E3 ligase. How these two activities contribute to cytoskeletal remodeling in vivo is not known. We used targeted mutagenesis to investigate the mechanism of Kelch function. We tested a model in which Cul3-dependent degradation of Kelch is required for its function, but we found no evidence to support this hypothesis. However, we found that mutant Kelch deficient in its ability to interact with Cul3 failed to rescue the kelch cytoskeletal defects, suggesting that ubiquitin ligase activity is the principal activity required in vivo. We also determined that the proteasome is required with Kelch to promote the ordered growth of the ring canal cytoskeleton. These results indicate that Kelch organizes the cytoskeleton in vivo by targeting a protein substrate for degradation by the proteasome., (Copyright © 2015 by the Genetics Society of America.)

Relaxin hormone secreted into the circulation during pregnancy was discovered through its effects on pubic symphysis relaxation and parturition. Genetic inactivation of the relaxin gene or its cognate relaxin family peptide receptor 1 (RXFP1) in mice caused failure of parturition and mammary nipple enlargement, as well as increased collagen fiber density in the cervix and vagina. However, the relaxin effect on discrete cells and tissues has yet to be determined. Using transgenic mice with a knockin LacZ reporter in the Rxfp1 allele, we showed strong expression of this gene in vaginal and cervical stromal cells, as well as pubic ligament cells. We produced a floxed Rxfp1 allele that was used in combination with the Tagln-cre transgene to generate mice with a smooth muscle-specific gene knockout. In pregnant females, the ROSA26 reporter activated by Tagln-cre was detected in smooth muscle cells of the cervix, vagina, uterine artery, and in cells of the pubic symphysis. In late pregnant females with conditional gene ablation, the length of pubic symphysis was significantly reduced compared with wild-type or heterozygous Rxfp1(+/-) females. Denser collagen content was revealed by Masson trichrome staining in reproductive tract organs, uterine artery, and pubic symphysis. The cervical and vaginal epithelium was less developed than in heterozygous or wild-type females, although nipple size was normal and the dams were able to nurse their pups. In summary, our data indicate that relaxin/RXFP1 signaling in smooth muscle cells is important for normal collagen turnover and relaxation of the pubic symphysis during pregnancy., (© 2015 by the Society for the Study of Reproduction, Inc.)

Growing evidence suggests important roles for specialized platelet-derived growth factor receptor alpha-positive (PDGFRalpha(+)) cells in regulating the behaviors of visceral smooth muscle organs. Examination of the female reproductive tracts of mice and monkeys showed that PDGFRalpha(+) cells form extensive networks in ovary, oviduct, and uterus. PDGFRalpha(+) cells were located in discrete locations within these organs, and their distribution and density were similar in rodents and primates. PDGFRalpha(+) cells were distinct from smooth muscle cells and interstitial cells of Cajal (ICC). This was demonstrated with immunohistochemical techniques and by performing molecular expression studies on PDGFRalpha(+) cells from mice with enhanced green fluorescent protein driven off of the endogenous promoter for Pdgfralpha. Significant differences in gene expression were found in PDGFRalpha(+) cells from ovary, oviduct, and uterus. Differences in gene expression were also detected in cells from different tissue regions within the same organ (e.g., uterine myometrium vs. endometrium). PDGFRalpha(+) cells are unlikely to provide pacemaker activity because they lack significant expression of key pacemaker genes found in ICC (Kit and Ano1). Gja1 encoding connexin 43 was expressed at relatively high levels in PDGFRalpha(+) cells (except in the ovary), suggesting these cells can form gap junctions to one another and neighboring smooth muscle cells. PDGFRalpha(+) cells also expressed the early response transcription factor and proto-oncogene Fos, particularly in the ovary. These data demonstrate extensive distribution of PDGFRalpha(+) cells throughout the female reproductive tract. These cells are a heterogeneous population of cells that are likely to contribute to different aspects of physiological regulation in the various anatomical niches they occupy., (© 2015 by the Society for the Study of Reproduction, Inc.)

Within the human female reproductive tract (FRT), the challenge of protection against sexually transmitted infections (STIs) is coupled with the need to enable successful reproduction. Oestradiol and progesterone, which are secreted during the menstrual cycle, affect epithelial cells, fibroblasts and immune cells in the FRT to modify their functions and hence the individual's susceptibility to STIs in ways that are unique to specific sites in the FRT. The innate and adaptive immune systems are under hormonal control, and immune protection in the FRT varies with the phase of the menstrual cycle. Immune protection is dampened during the secretory phase of the cycle to optimize conditions for fertilization and pregnancy, which creates a 'window of vulnerability' during which potential pathogens can enter and infect the FRT.

Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging. Specimens must be harvested with minimal time after landing to minimize postflight readaptation alterations in protein expression/translation, posttranslational modifications, and expression, as well as changes in gene expression and tissue histological degradation after euthanasia. We report the development of a widely applicable strategy for determining the window of optimal species-specific and tissue-specific posteuthanasia harvest that can be utilized to integrate into multi-investigator Biospecimen Sharing Programs. We also determined methods for ISS-compatible long-term tissue storage (10 months at -80°C) that yield recovery of high quality mRNA and protein for western analysis after sample return. Our focus was reproductive tissues. The time following euthanasia where tissues could be collected and histological integrity was maintained varied with tissue and species ranging between 1 and 3 hours. RNA quality was preserved in key reproductive tissues fixed in RNAlater up to 40 min after euthanasia. Postfixation processing was also standardized for safe shipment back to our laboratory. Our strategy can be adapted for other tissues under NASA's Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities.

Topical microbicides are a leading strategy for prevention of HIV mucosal infection to women; however, numerous pharmacokinetic limitations associated with coitally related dosing strategy have contributed to their limited success. Here we test the hypothesis that adeno-associated virus (AAV) mediated delivery of the b12 human anti-HIV-1 gp120 minibody gene to the lower genital tract of female rhesus macaques (Rh) can provide prolonged expression of b12 minibodies in the cervical-vaginal secretions. Gene transfer studies demonstrated that, of various green fluorescent protein (GFP)-expressing AAV serotypes, AAV-6 most efficiently transduced freshly immortalized and primary genital epithelial cells (PGECs) of female Rh in vitro. In addition, AAV-6-b12 minibody transduction of Rh PGECs led to inhibition of SHIV162p4 transmigration and virus infectivity in vitro. AAV-6-GFP could also successfully transduce vaginal epithelial cells of Rh when applied intravaginally, including p63+ epithelial stem cells. Moreover, intravaginal application of AAV-6-b12 to female Rh resulted in prolonged minibody detection in their vaginal secretions throughout the 79-day study period. These data provide proof of principle that AAV-6-mediated delivery of anti-HIV broadly neutralizing antibody (BnAb) genes to the lower genital tract of female Rh results in persistent minibody detection for several months. This strategy offers promise that an anti-HIV-1 genetic microbicide strategy may be possible in which topical application of AAV vector, with periodic reapplication as needed, may provide sustained local BnAb expression and protection.

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., (© 2014 Corrigan et al.)

The male and female reproductive apparatus of Zorotypus magnicaudelli (Malaysia), Zorotypus huxleyi (Ecuador) and Zorotypus weidneri (Brazil) were examined and documented in detail. The genital apparatus and sperm of the three species show only minor differences. The testes are larger in Z. magnicaudelli. Z. huxleyi lacks the helical appendage in the accessory glands. A long cuticular flagellum is present in Z. magnicaudelli and in the previously studied Zorotypus caudelli like in several other species, whereas it is absent in Z. weidneri, Z. huxleyi, Zorotypus hubbardi, Zorotypus impolitus and Zorotypus guineensis. Characteristic features of the very similar sperm are the presence of: a) two dense arches above the axoneme; b) a 9 + 9+2 axoneme with detached subtubules A and B of doublets 1 and 6; c) the axonemal end degenerating with enlarging accessory tubules; d) accessory tubules with 17 protofilaments; e) three accessory bodies beneath the axoneme; and f) two mitochondrial derivatives of equal shape. The first characteristic (a) is unknown outside of Zoraptera and possibly autapomorphic. The sperm structure differs distinctly in Z. impolitus and Z. hubbardi, which produce giant sperm and possess a huge spermatheca. The presence of the same sperm type in species either provided with a sclerotized coiled flagellum in males or lacking this structure indicates that a different organization of the genital apparatus does not necessarily affect the sperm structure. The flagellum and its pouch has probably evolved within Zoraptera, but it cannot be excluded that it is a groundplan feature and was reduced several times. The fossil evidence and our findings suggest that distinct modifications in the genital apparatus occurred before the fragmentation of the Gondwanan landmass in the middle Cretaceous., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Women constitute almost half of HIV-infected population globally, and the female genital tract (FGT) accounts for approximately 40% of all new HIV infections worldwide. The FGT is composed of upper and lower parts, distinct in their morphological and functional characteristics. Co-factors in the genital microenvironment, such as presence of hormones, semen, and other sexually transmitted infections, can facilitate or deter HIV infection and play a critical role in determining susceptibility to HIV. In this review, we examine some of these co-factors and their potential influence. Presence of physical and chemical barriers such as epithelial tight junctions, mucus, and anti-microbial peptides can actively block and inhibit viral replication, presenting a significant deterrent to HIV. Upon exposure, HIV and other pathogens first encounter the genital epithelium: cells that express a wide repertoire of pattern recognition receptors that can recognize and directly initiate innate immune responses. These and other interactions in the genital tract can lead to direct and indirect inflammation and enhance the number of local target cells, immune activation, and microbial translocation, all of which promote HIV infection and replication. Better understanding of the dynamics of HIV transmission in the female genital tract would be invaluable for improving the design of prophylactic strategies against HIV., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Phosphoinositides regulate myriad cellular processes, acting as potent signaling molecules in conserved signaling pathways and as organelle gatekeepers that recruit effector proteins to membranes. Phosphoinositide-generating enzymes have been studied extensively in yeast and cultured cells, yet their roles in animal development are not well understood. Here, we analyze Drosophila melanogaster phosphatidylinositol 4-kinase IIIα (PI4KIIIα) during oogenesis. We demonstrate that PI4KIIIα is required for production of plasma membrane PtdIns4P and PtdIns(4,5)P2 and is crucial for actin organization, membrane trafficking and cell polarity. Female germ cells mutant for PI4KIIIα exhibit defects in cortical integrity associated with failure to recruit the cytoskeletal-membrane crosslinker Moesin and the exocyst subunit Sec5. These effects reflect a unique requirement for PI4KIIIα, as egg chambers from flies mutant for either of the other Drosophila PI4Ks, fwd or PI4KII, show Golgi but not plasma membrane phenotypes. Thus, PI4KIIIα is a vital regulator of a functionally distinct pool of PtdIns4P that is essential for PtdIns(4,5)P2-dependent processes in Drosophila development.

In response to growing concerns that environmental chemicals may have adverse effects on human health by altering the endocrine system, the Endocrine Disruptor Screening Program (EDSP), under the auspices of the United States Environmental Protection Agency (U.S. EPA), recently instituted a Tier I battery of tests including a female pubertal assay. This assay requires dosing of female rats from postnatal day (PND) 22 through PND 42 (or 43), the period of pubertal development in the rat, to identify test articles that may have estrogenic or antiestrogenic effects, or may alter hormones or neurotransmitters. While certain landmarks in female rat reproductive development are published, little is published on the microscopic appearance of the female reproductive tract during prepubertal and pubertal development. In this study, reproductive tissues from three female Sprague-Dawley rats were collected each day from PND 20 through PND 50, such that tissues from a total of 93 rats were collected throughout the prepubertal and pubertal period. Tissues were formalin-fixed, trimmed, paraffin-embedded, sectioned at 5-µm thickness, and examined microscopically. The major histologic features of the female reproductive tract throughout this critical period were described in detail. This information will help pathologists interpret findings observed in female pubertal assays.

The Müllerian duct (MD) forms the female reproductive tract (FRT) consisting of the oviducts, uterus, cervix, and upper vagina. FRT function is vital to fertility, providing the site of fertilization, embryo implantation and fetal development. Developmental defects in the formation and diseases of the FRT, including cancer and endometriosis, are prevalent in humans and can result in infertility and death. Furthermore, because the MDs are initially formed regardless of genotypic sex, mesenchymal to epithelial signaling is required in males to mediate MD regression and prevents the development of MD-derived organs. In males, defects in MD regression result in the retention of FRT organs and have been described in several human syndromes. Although to date not reported in humans, ectopic activation of MD regression signaling components in females can result in aplasia of the FRT. Clearly, MD development is important to human health; however, the molecular mechanisms remain largely undetermined. Molecular genetics studies of human diseases and mouse models have provided new insights into molecular signaling during MD development, regression and differentiation. This review will provide an overview of MD development and important genes and signaling mechanisms involved.

Resolution of Chlamydia genital tract infection is delayed in the absence of MyD88. In these studies, we first used bone marrow chimeras to demonstrate a requirement for MyD88 expression by hematopoietic cells in the presence of a wild-type epithelium. Using mixed bone marrow chimeras we then determined that MyD88 expression was specifically required in the adaptive immune compartment. Furthermore, adoptive transfer experiments revealed that CD4(+) T cell expression of MyD88 was necessary for normal resolution of genital tract infection. This requirement was associated with a reduced ability of MyD88(-/-)CD4(+) T cells to accumulate in the draining lymph nodes and genital tract when exposed to the same inflammatory milieu as wild-type CD4(+) T cells. We also demonstrated that the impaired infection control we observed in the absence of MyD88 could not be recapitulated by deficiencies in TLR or IL-1R signaling. In vitro, we detected an increased frequency of apoptotic MyD88(-/-)CD4(+) T cells upon activation in the absence of exogenous ligands for receptors upstream of MyD88. These data reveal an intrinsic requirement for MyD88 in CD4(+) T cells during Chlamydia infection and indicate that the importance of MyD88 extends beyond innate immune responses by directly influencing adaptive immunity.

Sex differences are well known to be determinants of development, health and disease. Epigenetic mechanisms are also known to differ between men and women through X-inactivation in females. We hypothesized that epigenetic sex differences may also result from sex hormone functions, in particular from long-lasting androgen programming. We aimed at investigating whether inactivation of the androgen receptor, the key regulator of normal male sex development, is associated with differences of the patterns of DNA methylation marks in genital tissues. To this end, we performed large scale array-based analysis of gene methylation profiles on genomic DNA from labioscrotal skin fibroblasts of 8 males and 26 individuals with androgen insensitivity syndrome (AIS) due to inactivating androgen receptor gene mutations. By this approach we identified differential methylation of 167 CpG loci representing 162 unique human genes. These were significantly enriched for androgen target genes and low CpG content promoter genes. Additional 75 genes showed a significant increase of heterogeneity of methylation in AIS compared to a high homogeneity in normal male controls. Our data show that normal and aberrant androgen receptor function is associated with distinct patterns of DNA-methylation marks in genital tissues. These findings support the concept that transcription factor binding to the DNA has an impact on the shape of the DNA methylome. These data which derived from a rare human model suggest that androgen programming of methylation marks contributes to sexual dimorphism in the human which might have considerable impact on the manifestation of sex-associated phenotypes and diseases.

The uterus is an extremely plastic organ that undergoes cyclical remodeling including endometrial regeneration during the menstrual cycle. Endometrial remodeling and regeneration also occur during pregnancy and following parturition, particularly in hemochorial implanting species. The mechanisms of endometrial regeneration are not well understood. Endometrial stem/progenitor cells are proposed to contribute to endometrial regeneration in both humans and mice. BrdU label retention has been used to identify potential stem/progenitor cells in mouse endometrium. However, methods are not available to isolate BrdU label-retaining cells (LRC) for functional analyses. Therefore, we employed a transgenic mouse model to identify H2B-GFP LRCs throughout the female reproductive tract with particular interest on the endometrium. We hypothesized that the female reproductive tract contains a population of long-term LRCs that persist even following pregnancy and endometrial regeneration. Endometrial cells were labeled (pulsed) either transplacentally/translactationally or peripubertally. When mice were pulsed transplacentally/translactationally, the label was not retained in the uterus. However, LRCs were concentrated to the distal oviduct and endocervical transition zone (TZ) following natural (i.e., pregnancy/parturition induced) and mechanically induced endometrial regeneration. LRCs in the distal oviduct and endocervical TZ expressed stem cell markers and did not express ERα or PGR, implying the undifferentiated phenotype of these cells. Oviduct and endocervical TZ LRCs did not proliferate during endometrial re-epithelialization, suggesting that they do not contribute to the endometrium in a stem/progenitor cell capacity. In contrast, when mice were pulsed peripubertally long-term LRCs were identified in the endometrial glandular compartment in mice as far out as 9 months post-pulse. These findings suggest that epithelial tissue of the female reproductive tract contains 3 distinct populations of epithelial cells that exhibit stem/progenitor cell qualities. Distinct stem/progenitor-like cells localize to the oviduct, endometrium, and cervix.

Problem: It is unknown whether viral replication or viral components that subvert innate responses in other cells, specifically the virion host shutoff (VHS) protein, play a role in determining primary genital epithelial cell (GEC) innate antiviral responses., Method of Study: Cultures of primary female GECs were exposed to wildtype (WT), VHS-deleted (vhsB), or UV-inactivated HSV-2. Antiviral pathway induction was evaluated by measuring nuclear factor-κB (NFκB) translocation by immunofluorescent microscopy. Proinflammatory cytokines, chemokines, and interferon (IFN) were measured by Luminex or ELISA. Biological activity of IFN-β was evaluated via VSV-GFP bioassay, by blocking secreted IFN-β with neutralizing antibodies and by measuring interferon-stimulated genes by RT-PCR., Results: Proinflammatory cytokines and chemokines were upregulated in primary GECs in response to replication-competent HSV-2, but suppressed in the presence of the VHS protein. In contrast, upregulation of IFN-β depended on viral replication, but was not affected by VHS. However, the IFN-β produced was biologically active and reduced the viral burden., Conclusion: Viral factors such as replication and the presence of the VHS protein play important roles in regulating innate antiviral responses against HSV-2 from primary GECs., (© 2013 John Wiley & Sons Ltd.)