Identification of telocytes in the oviduct of the mare.

Telocytes (TCs), a recently discovered special type of stromal cells, have been identified in many organs of many species, including the female and male reproductive system, with proposed multiple potential bio-functions such as homeostasis, immunomodulation, tissue remodeling and regeneration, embryogenesis, angiogenesis and even tumorigenesis. The aim of this study was to investigate the existence, and characteristics of telocytes in normal equine oviduct. To identify them, we used routine light microscopy, non-conventional light microscopy (NCLM), transmission electron microscopy (TEM), and immunohistochemistry. We found that telocytes of the equine oviduct can be recognized in fixed specimens by light microscopy (methylene blue staining), with more details on Epon semi-thin sections (toluidine blue staining) by NCLM, and that they showed positive immunostaining for CD34. The telocytes, with their typical long and moniliform prolongations, formed networks in the stromal space of the submucosa, muscular and serosa layers, particularly in the lamina propia where they were observed in greater quantity. By TEM we have also confirmed the presence of cells ultrastructurally identifiable as telocytes (cells with telopodes alternating podomers and podoms) in the aforementioned locations. Direct intercellular contacts between epithelial cells and neighboring telocytes were evidenced. EIn conclusion, we demonstrated that telocytes are present in the equine oviduct as previously reported in other species. The potential implication of telocytes in multiple potential functions of physiological and pathological processes deserves further investigation.

Adaptations for matrotrophy in the female reproductive system in the pseudoscorpion Chelifer cancroides (Chelicerata: Pseudoscorpiones, Cheliferidae).

Pseudoscorpiones (pseudoscorpions, false scorpions) is an order of small terrestrial chelicerates. While most chelicerates are lecithotrophic, that is, embryos develop due to nutrients (mostly yolk) deposited in the oocyte cytoplasm, pseudoscorpions are matrotrophic, that is, embryos are nourished by the female. Pseudoscorpion oocytes contain only a small amount of yolk. The embryos develop within a brood sac carried on the abdominal site of the female and absorb nutrients by a pumping organ. It is believed that in pseudoscorpions nutrients for developing embryos are produced in the ovary during a postovulatory (secretory) phase of the ovarian cycle. The goal of our study was to analyze the structure of the female reproductive system during the secretory phase in the pseudoscorpion Chelifer cancroides, a representative of the family Cheliferidae, considered to be one of the most advanced pseudoscorpion taxa. We use diverse microscopic techniques to document that the nutritive fluid is produced not only in the ovaries but also by the epithelial cells in the oviducts. The secretory active epithelial cells are hypertrophic and polyploid and release their content by fragmentation of apical parts. Our observations also indicate that fertilization occurs in the oviducts. Moreover, in contrast to previous findings, we show that secretion of the nutritive material starts when the fertilized oocytes reach the brood sac and thus precedes formation of the pumping organ. Summing up, we show that C. cancroides exhibits traits of advanced adaptations for matrotrophy due to coordinated secretion of the nutritive fluid by the ovarian and oviductal epithelial cells, which substantially increases the efficiency of nutritive fluid formation. Since the secretion of nutrients starts before formation of the pumping organ, we suggest that the embryos are able to absorb the nutritive fluid also in the early embryonic stages.

TRPV4 is involved in levonorgestrel-induced reduction in oviduct ciliary beating.

Previous studies revealed the increasing risk of tubal pregnancy following failure of levonorgestrel (LNG)-induced emergency contraception, which was attributed to the reduced ciliary motility in response to LNG. However, understanding of the mechanism of LNG-induced reduction in the ciliary beat frequency (CBF) is limited. The transient receptor potential vanilloid (TRPV) 4 channel is located widely in the female reproductive tract and generates an influx of Ca2+ following its activation under normal physiological conditions, which regulates the CBF. The present study aimed to explore whether LNG reduced the CBF in the Fallopian tubes by modulating TRPV4 channels, leading to embryo retention in the Fallopian tubes and subsequent tubal pregnancy. The study provided evidence that the expression of TRPV4 was downregulated in the Fallopian tubes among patients with tubal pregnancy and negatively correlated with the serum level of progesterone. LNG downregulated the expression of TRPV4, limiting the calcium influx to reduce the CBF in mouse oviducts. Furthermore, the distribution of ciliated cells and the morphology of cilia did not change following the administration of LNG. LNG-induced reduction in the CBF and embryo retention in the Fallopian tubes and in mouse oviducts were partially reversed by the progesterone receptor antagonist RU486 or the TRPV4 agonist 4α-phorbol 12,13-didecanoate (4α-PDD). The results indicated that LNG could downregulate the expression of TRPV4 to reduce the CBF in both humans and mice, suggesting the possible mechanism of tubal pregnancy. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Sex steroids drive the remodeling of oviductal extracellular matrix in cattle.

The extracellular matrix (ECM) is a group of molecules that offer structural and biochemical support to cells and interact with them to regulate their function. Also, growth factors (GFs) stored in the ECM can be locally released during ECM remodeling. Here, we hypothesize that the balance between ECM components and remodelers is regulated according to the ovarian steroid milieu to which the oviduct is exposed during the periovulatory period. Follicular growth was manipulated to generate cows that ovulated small follicles (SF-small corpus luteum [SCL]; n = 20) or large follicles (LF-large corpus luteum [LCL]; n = 21) and possess corresponding Estradiol (E2) and Progesterone (P4) plasmatic concentrations. Ampulla and isthmus samples were collected on day 4 (day 0 = ovulation induction) and immediately frozen or fixed. The transcriptional profile (n = 3/group) was evaluated by RNA sequencing. MMP Antibody Array was used to quantify ECM remodelers' protein abundance and immunohistochemistry to quantify type I collagen. Transcriptome analysis revealed the over-representation of ECM organization and remodeling pathways in the LF-LCL group. Transcription of ECM components (collagens), remodelers (ADAMs and MMPs), and related GFs were upregulated in LF-LCL. Protein intensities for MMP3, MMP8, MMP9, MMP13, and TIMP4 were greater for the LF-LCL group. Type I collagen content in the mucosa was greater in SF-SCL group. In conclusion, that the earlier and more intense exposure to E2 and P4 during the periovulatory period in LF-LCL animals stimulates ECM remodeling. We speculate that differential ECM regulation may contribute to oviductal receptivity to the embryo.

Oviductal extracellular vesicles (oviductosomes, OVS) are conserved in humans: murine OVS play a pivotal role in sperm capacitation and fertility.

STUDY QUESTIONS: Are extracellular vesicles (EVs) in the murine oviduct (oviductosomes, OVS) conserved in humans and do they play a role in the fertility of Pmca4-/- females? SUMMARY ANSWER: OVS and their fertility-modulating proteins are conserved in humans, arise via the apocrine pathway, and mediate a compensatory upregulation of PMCA1 (plasma membrane Ca2+-ATPase 1) in Pmca4-/- female mice during proestrus/estrus, to account for their fertility. WHAT IS KNOWN ALREADY: Recently murine OVS were identified and shown during proestrus/estrus to express elevated levels of PMCA4 which they can deliver to sperm. PMCA4 is the major Ca2+ efflux pump in murine sperm and Pmca4 deletion leads to loss of sperm motility and male infertility as there is no compensatory upregulation of the remaining Ca2+ pump, PMCA1. Of the four family members of PMCAs (PMCA1-4), PMCA1 and PMCA4 are ubiquitous, and to date there have been no reports of one isoform being upregulated to compensate for another in any organ/tissue. Since Pmca4-/- females are fertile, despite the abundant expression of PMCA4 in wild-type (WT) OVS, we propose that OVS serve a role of packaging and delivering to sperm elevated levels of PMCA1 in Pmca4-/- during proestrus/estrus to compensate for PMCA4's absence. STUDY DESIGN, SIZE, DURATION: Fallopian tubes from pre-menopausal women undergoing hysterectomy were used to study EVs in the luminal fluid. Oviducts from sexually mature WT mice were sectioned after perfusion fixation to detect EVs in situ. Oviducts were recovered from WT and Pmca4-/- after hormonally induced estrus and sectioned for PMCA1 immunofluorescence (IF) (detected with confocal microscopy) and hematoxylin and eosin staining. Reproductive tissues, luminal fluids and EVs were recovered after induced estrus and after natural cycling for western blot analysis of PMCA1 and qRT-PCR of Pmca1 to compare expression levels in WT and Pmca4-/-. OVS, uterosomes, and epididymal luminal fluid were included in the comparisons. WT and Pmca4-/- OVS were analyzed for the presence of known PMCA4 partners in sperm and their ability to interact with PMCA1, via co-immunoprecipitation. In vitro uptake of PMCA1 from OVS was analyzed in capacitated and uncapacitated sperm via quantitative western blot analysis, IF localization and flow cytometry. Caudal sperm were also assayed for uptake of tyrosine-phosphorylated proteins which were shown to be present in OVS. Finally, PMCA1 and PMCA4 in OVS and that delivered to sperm were assayed for enzymatic activity. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human fallopian tubes were flushed to recover luminal fluid which was processed for OVS via ultracentrifugation. Human OVS were negatively stained for transmission electron microscopy (TEM) and subjected to immunogold labeling, to detect PMCA4. Western analysis was used to detect HSC70 (an EV biomarker), PMCA1 and endothelial nitric oxide synthase (eNOS) which is a fertility-modulating protein delivered to human sperm by prostasomes. Oviducts of sexually mature female mice were sectioned after perfusion fixation for TEM tomography to obtain 3D information and to distinguish cross-sections of EVs from those of microvilli and cilia. Murine tissues, luminal fluids and EVs were assayed for PMCA1 (IF and western blot) or qRT-PCR. PMCA1 levels from western blots were quantified, using band densities and compared in WT and Pmca4-/- after induced estrus and in proestrus/estrus and metestrus/diestrus in cycling females. In vitro uptake of PMCA1 and tyrosine-phosphorylated proteins was quantified with flow cytometry and/or quantitative western blot. Ca2+-ATPase activity in OVS and sperm before and after PMCA1 and PMCA4 uptake was assayed, via the enzymatic hydrolysis rate of ATP. MAIN RESULTS AND THE ROLE OF CHANCE: TEM revealed that human oviducts contain EVs (exosomal and microvesicular). These EVs contain PMCA4 (immunolabeling), eNOS and PMCA1 (western blot) in their cargo. TEM tomography showed the murine oviduct with EV-containing blebs which typify the apocrine pathway for EV biogenesis. Western blots revealed that during proestrus/estrus PMCA1 was significantly elevated in the oviductal luminal fluid (OLF) (P = 0.02) and in OVS (P = 0.03) of Pmca4-/-, compared to WT. Further, while PMCA1 levels did not fluctuate in OLF during the cycle in WT, they were significantly (P = 0.02) higher in proestrus/estrus than at metestrus/diestrus in Pmca4-/-. The elevated levels of PMCA1 in proestrus/estrus, which mimics PMCA4 in WT, is OLF/OVS-specific, and is not seen in oviductal tissues, uterosomes or epididymal luminal fluid of Pmca4-/-. However, qRT-PCR revealed significantly elevated levels of Pmca1 transcript in Pmca4-/- oviductal tissues, compared to WT. PMCA1 could be transferred from OVS to sperm and the levels were significantly higher for capacitated vs uncapacitated sperm, as assessed by flow cytometry (P = 0.001) after 3 h co-incubation, quantitative western blot (P < 0.05) and the frequency of immuno-labeled sperm (P < 0.001) after 30 min co-incubation. Tyrosine phosphorylated proteins were discovered in murine OVS and could be delivered to sperm after their co-incubation with OVS, as detected by western, immunofluorescence localization, and flow cytometry. PMCA1 and PMCA4 in OVS were shown to be enzymatically active and this activity increased in sperm after OVS interaction. LARGE SCALE DATA: None. LIMITATIONS REASONS FOR CAUTION: Although oviductal tissues of WT and Pmca4-/- showed no significant difference in PMCA1 levels, Pmca4-/- levels of OVS/OLF during proestrus/estrus were significantly higher than in WT. We have attributed this enrichment or upregulation of PMCA1 in Pmca4-/- partly to selective packaging in OVS to compensate for the lack of PMCA4. However, in the absence of a difference between WT and Pmca4-/- in the PMCA1 levels in oviductal tissues as a whole, we cannot rule out significantly higher PMCA1 expression in the oviductal epithelium that gives rise to the OVS as significantly higher Pmca1 transcripts were detected in Pmca4-/-. WIDER IMPLICATIONS OF THE FINDINGS: Since OVS and fertility-modulating cargo components are conserved in humans, it suggests that murine OVS role in regulating the expression of proteins required for capacitation and fertility is also conserved. Secondly, OVS may explain some of the differences in in vivo and in vitro fertilization for mouse mutants, as seen in mice lacking the gene for FER which is the enzyme required for sperm protein tyrosine phosphorylation. Our observation that murine OVS carry and can modulate sperm protein tyrosine phosphorylation by delivering them to sperm provides an explanation for the in vivo fertility of Fer mutants, not seen in vitro. Finally, our findings have implications for infertility treatment and exosome therapeutics. STUDY FUNDING AND COMPETING INTEREST(S): The work was supported by National Institute of Health (RO3HD073523 and 5P20RR015588) grants to P.A.M.-D. There are no conflicts of interests.

Changes in localization and density of CD63-positive exosome-like substances in the hen oviduct with artificial insemination and their effect on sperm viability.

Avian sperm are stored in the sperm storage tubules (SSTs) of the hen oviduct for a prolonged period. However, the precise mechanisms by which sperm are kept alive in the SSTs are still not fully understood. The aim of this study was to determine whether exosomes are secreted by SST cells and play a role in the survival of sperm. Utero-vaginal junction (UVJ) tissue from approximately 50 wk old White Leghorn hens was collected before (control group) and after intravaginal insemination with seminal plasma (SP group) or semen (AI group). The samples were used to prepare frozen sections and total protein extraction. The localization of the CD63, an exosome marker, was determined by immunohistochemistry and its protein level in the UVJ mucosal tissues was examined by Western blot. Exosomes were isolated from the culture media of UVJ and vaginal mucosa cells by ultracentrifugation and characterized by SDS-PAGE and Western blot. The viability and motility of sperm incubated with exosomes were also examined. CD63 was localized in the apical region of UVJ mucosal epithelium cells and SST cells of control, SP, and AI groups. The CD63 protein decreased in SST cells surrounding resident sperm and tended to appear in the SST lumen in the AI group. The protein level of CD63 in UVJ mucosal tissues was significantly higher in the AI group than control. The CD63 protein (approximately 75 kDa) was detected in ultracentrifugation pellets from the culture medium of UVJ and vagina cells. The viability of sperm incubated with 1 µg/µl vaginal exosomes was significantly decreased but was not affected by UVJ exosomes. These results suggest that exosomes were synthesized by SST cells and may be secreted into SST lumen when sperm were stored in SSTs. The role of SST exosomes in sperm storage needs to be examined further.

Steroid hormones regulate sperm-oviduct interactions in the bovine.

After insemination in the cow, a sperm reservoir is formed within the oviducts, allowing the storage and then progressive release of spermatozoa toward the ovulated oocyte. In order to investigate the hormonal regulation of these events in vitro, the ovarian steroids 17ß-estradiol (E2) and progesterone (P4) were added at various concentrations to monolayers of bovine oviduct epithelial cells (BOEC) before or during co-incubation with spermatozoa. Main findings demonstrate that (1) a 18-h pretreatment of BOEC with 100 pg/mL and 100 ng/mL of E2 decreased by 25% the ability of BOEC to bind spermatozoa after 10 min, and for the highest dose of E2, 60 min of co-incubation; (2) P4 at concentrations of 10, 100 and 1000 ng/mL induced the release within 60 min of 32-47% of bound spermatozoa from BOEC; this sperm-releasing effect was maintained after a 18-h pretreatment of BOEC with 100 pg/mL of E2; (3) E2 in concentrations above 100 pg/mL inhibited the releasing effect of P4 on bound sperm in a dose-dependent manner; (4) spermatozoa bound to BOEC, then released from BOEC by the action of P4-induced higher cleavage and blastocyst rates after in vitro fertilization than the control group. These results support the hypothesis that the dynamic changes in steroid hormones around the time of ovulation regulate the formation of the sperm reservoir and the timed delivery of capacitated spermatozoa to the site of fertilization.

Progesterone Prevents High-Grade Serous Ovarian Cancer by Inducing Necroptosis of p53-Defective Fallopian Tube Epithelial Cells.

High-grade serous ovarian carcinoma (HGSOC) originates mainly from the fallopian tube (FT) epithelium and always carries early TP53 mutations. We previously reported that tumors initiate in the FT fimbria epithelium because of apoptotic failure and the expansion of cells with DNA double-strand breaks (DSB) caused by bathing of the FT epithelial cells in reactive oxygen species (ROSs) and hemoglobin-rich follicular fluid (FF) after ovulation. Because ovulation is frequent and HGSOC is rare, we hypothesized that luteal-phase progesterone (P4) could eliminate p53-defective FT cells. Here we show that P4, via P4 receptors (PRs), induces necroptosis in Trp53-/- mouse oviduct epithelium and in immortalized human p53-defective fimbrial epithelium through the TNF-α/RIPK1/RIPK3/MLKL pathway. Necroptosis occurs specifically at diestrus, recovers at the proestrus phase of the estrus cycle, and can be augmented with P4 supplementation. These results reveal the mechanism of the well-known ability of progesterone to prevent ovarian cancer.

The female reproductive system of Zearaja chilensis (Quichenot, 1848) (Chondrichthyes, Rajidae): gametogenesis and microscopic validation of maturity criteria / El sistema reproductor femenino de Zearaja chilensis (Quichenot, 1848) (Chondrichthyes, Rajidae): gametogénesis y validación microscópica de los criterios de madurez

Chondrichthyans are extremely susceptible to overfishing due, among other things, to their reproductive adaptations. Basic knowledge of reproductive parameters is essential, both of ecological and for economic points of view. Zearaja chilensis is a valuable economic resource in South America. This work analyzes the microanatomy of female reproductive system, as well as the size of the onset of vitellogenesis. The material was fixed in Bouin and processed using routine histological techniques. Both ovaries are equally functional. Folliculogenesis depicts the same pattern of other Chondrichthyans. Follicles with different degrees of maturation coexist in mature animals, with the exception of oogonia, which were only found in immature individuals. Likewise, atretic follicles were recorded in all stages of maturation. The size of yolk input, microscopically recorded, is lower than the detected at naked eye. Oviductal glands and uterus show similarity with those reported in other lecitotrophic cartilaginous fish. This work reports, for the first time, the morfofunctional microanatomy of the species, and puts to the test the accuracy of the commonly employed criteria for the determination of sexual maturity, a critical data when determining management policies.

Los condrictios son extremadamente susceptibles a la explotación pesquera debido, entre otras cosas, a sus adaptaciones reproductivas. Los conocimientos básicos de sus parámetros reproductivos son esenciales, ya sea desde el punto de vista ecológico como económico. Zearaja chilensis constituye un importante recurso económico en América del Sur. En este trabajo se analiza la anatomía microscópica del sistema reproductor femenino y la talla de inicio de la vitelogénesis. El material se fijó en Bouin y se procesó mediante técnicas histológicas de rutina. Ambos ovarios son igualmente funcionales. La foliculogénesis presenta el mismo patrón de otros condrictios. Los folículos con diferentes grados de maduración coexisten en animales maduros, con la excepción de las oogonias, que sólo se observaron en los individuos inmaduros. Los folículos atrésicos se registraron en todas las etapas de la maduración. La talla de inicio de la vitelogénesis, microscópicamente registrada, es inferior a la detectada en el ojo desnudo. La glándula oviductal y el útero muestran similitud con lo reportado en otros peces cartilaginosos lecitotróficos. Este trabajo describe por primera vez, la microanatomía morfofuncional de la especie y pone a prueba la precisión de los criterios comúnmente empleados para la determinación de la madurez sexual, una información crítica para la determinación de las políticas de gestión.

Celsr1 is required for the generation of polarity at multiple levels of the mouse oviduct.

The oviduct is an important organ in reproduction where fertilization occurs, and through which the fertilized eggs are carried to the uterus in mammals. This organ is highly polarized, where the epithelium forms longitudinal folds along the ovary-uterus axis, and the epithelial multicilia beat towards the uterus to transport the ovulated ova. Here, we analyzed the postnatal development of mouse oviduct and report that multilevel polarities of the oviduct are regulated by a planar cell polarity (PCP) gene, Celsr1. In the epithelium, Celsr1 is concentrated in the specific cellular boundaries perpendicular to the ovary-uterus axis from postnatal day 2. We found a new feature of cellular polarity in the oviduct - the apical surface of epithelial cells is elongated along the ovary-uterus axis. In Celsr1-deficient mice, the ciliary motion is not orchestrated along the ovary-uterus axis and the transport ability of beating cilia is impaired. Epithelial cells show less elongation and randomized orientation, and epithelial folds show randomized directionality and ectopic branches in the mutant. Our mosaic analysis suggests that the geometry of epithelial cells is primarily regulated by Celsr1 and as a consequence the epithelial folds are aligned. Taken together, we reveal the characteristics of the multilevel polarity formation processes in the mouse oviduct epithelium and suggest a novel function of the PCP pathway for proper tissue morphogenesis.

Expression and secretion of plasma membrane Ca2+-ATPase 4a (PMCA4a) during murine estrus: association with oviductal exosomes and uptake in sperm.

PMCA4, a membrane protein, is the major Ca(2+) efflux pump in murine sperm where its deletion leads to a severe loss of hyperactivated motility and to male infertility. We have previously shown that the PMCA4b splice variant interacts with CASK (Ca(2+/)CaM-dependent serine kinase) in regulating sperm Ca(2+). More recently we detected that PMCA4a isoform, in addition to its presence in testis, is secreted in the epididymal luminal fluid and transferred to sperm. Here we show that Pmca4 mRNA is expressed in both the 4a and 4b variants in the vagina, uterus, and oviduct. Immunofluorescence reveals that PMCA4a is similarly expressed and is elevated during estrus, appearing in the glandular and luminal epithelia. Western analysis detected PMCA4a in all tissues and in the luminal fluids (LF) of the vagina (VLF), uterus (ULF), and the oviduct (OLF) collected during estrus. It was ~9- and 4-fold higher in OLF than in VLF and ULF, and only marginally present in LF collected at metestrus/diestrus. Fractionation of the LF collected at estrus, via ultracentrifugation, revealed that 100% of the PMCA4a resides in the vesicular fraction of the ULF and OLF. Transmission electron microscopy (TEM) revealed that OLF vesicles have an exosomal orientation (with the cytoplasmic-side inward), a size range of 25-100 nm, with the characteristic CD9 biomarker. Thus, we dubbed these vesicles "oviductosomes", to which PMCA4a was immunolocalized. Incubation of caudal sperm in the combined LF or exosomes resulted in up to a ~3-fold increase of sperm PMCA4a, as detected by flow cytometry, indicating in vitro uptake. Our results are consistent with the increased requirement of Ca(2+) efflux in the oviduct. They show for the first time the presence of oviductal exosomes and highlight their role, along with uterosomes and vaginal exosomes, in post-testicular sperm acquisition of PMCA4a which is essential for hyperactivated motility and fertility.

Non-suture technique for rabbit oviduct anastomosis with 2-octyl cyanoacrylate: a histopathologic and biomechanical analysis.

AIM: The aim of this study was to investigate histological and biomechanical properties of oviduct anastomosis with 2-octyl cyanoacrylate (OCA) in the rabbit. MATERIAL AND METHODS: Sixty female rabbits were randomly divided equally into three groups: A (control), B (traditional catgut suture), and C (non-suture technique using OCA). After suture or OCA anastomosis, gross examination (adhesion formation) and histopathology (hematoxylin-eosin), ultrastructure (transmission electron microscopy), and biomechanics (bursting pressure) on para-anastomotic site were investigated on oviduct taken at 1 (A1, B1, C1) and 4 (A2, B2, C2) weeks, respectively. RESULTS: Adhesion score in group B was more severe than that in groups A and C at 1 and 4 weeks. Histopathology showed that acute endosalpingitis in group B was the most intense at 1 week, followed by significantly more tissue stimulation induced by catgut and foreign-body giant cells in group B than in group C at 4 weeks. Ultrastructural damage of ciliated cells was reversed partly (B2) and completely (C2) at 4 weeks. Bursting pressure in C1 was weaker than that in B1, followed by no significant difference at 4 weeks. CONCLUSION: Non-suture using OCA for oviduct anastomosis can be accepted as a new-perspective technique.

Alginate hydrogels for three-dimensional organ culture of ovaries and oviducts.

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States(1). The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae(2,3). Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease. Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation(4). Alginate hydrogels can be used to support the growth of many types of tissues(5). Alginate is a linear polysaccharide composed of repeating units of ß-D-mannuronic acid and α-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues(6,7). Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling(5). The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro. A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Ultrastructural and functional aspects of the spermatheca in the american harlequin bug, Murgantia histrionica (Hemiptera: Pentatomidae)

The spermatheca of Murgantia histrionica (Hahn) was investigated using fluorescence, scanning and transmission electron microscopy. The aim of the study was to elucidate the structure of this organ, pointing out differences between mated and unmated females. Results have shown an elaborated cuticular structure associated with muscular and glandular tissues. The spermatheca is joined with the common oviduct by the spermathecal duct, forming a thin saccular dilation through two consecutive invaginations. The distal part of the organ is formed by a series of two communicating cuticular chambers. The first cylindrical-shaped chamber, corresponding to the coiled region, is wrapped by longitudinal muscular fibers suspended between two cuticular flanges. The contractions of these fibers compress a deformable zone of the cylinder, pumping the sperm toward the spermathecal duct. Without contractions the cylinder results to be isolated from the proximal part of the spermatheca by means of a valve. The second chamber, corresponding to the spermatheca, is made of two parts: a truncated-conical sub chamber, with a constant cuticular thickness, bearing on itself the distal flange, where muscular fibers are attached. The second part is a bulb-like structure wrapped in a glandular epithelium. The secretory units are composed by two cells: a secretory cell and an associated duct cell. Every evacuating duct shows a little reservoir just after the terminal apparatus, and converge inside the distal bulb after a tortuous path. The functional implications of this structure in the reproductive biology of M. histrionica are discussed.

Modifications in the oviducts of workers and queens of Melipona quadrifasciata anthidioides (Hymenoptera: Apidae) with different ages.

The study of morphological features of highly eusocial bees has helped to answer a series of questions concerning their biology. The labor division places the females into two castes, queen with reproductive function and worker with a wide variety of tasks. However, in different species and under different conditions, workers can develop ovaries and lay trophic eggs that are eaten by the queen or used to originate males. In this work, the development of the oviducts was monitored in workers and virgin queens of Melipona quadrifasciata anthidioides specimens of different ages to verify whether there is permanent sterility of these queens and workers due to aging. Lateral and common oviducts of virgin queens aged <7, 10, 15, 20, and 25 days old, physogastric queens, nurse, and forager workers were analyzed for histology and ultrastructure. Although the structural organization of the lateral and common oviducts were very similar, differences in width of the cuticle and the degree of chromatin condensation were observed, indicating differences in the development of this organ of the reproductive system between the castes. It was also demonstrated that electron-lucent vesicles appear to be related to the formation of the cuticle that lines the oviducts. Because no cellular death characteristics were found, it can be inferred that the absence of mating of the queens as old as of 25 days of age does not cause permanent sterility.

Morphological study of the oviductal gland in the smallnose fanskate Sympterygia bonapartii (Müller and Henle, 1841) (Chondrichthyes, Rajidae) / Estudo morfológico da glândula oviductal no smallnose fanskate Sympterygia bonapartii (Müller and henle, 1841) (Chondrichthyes, Rajidae)

The oviductal gland is an exclusive structure of cartilaginous fishes that produces the egg jelly, forms the tertiary egg envelopes and stores sperm. The biological importance of this structure is related to the special features of the reproductive strategy of the group and to its phylogeny, considering that egg-laying is the ancestral condition in this fish (Dulvy and Reynolds, 1997). This gland of the smallnose fanskate shows four morphofunctional zones. The lining epithelium along the gland is columnar with secretory and ciliated cells. Secretions are mucous and/or proteic according to the zone, and to their specific functions. This is the first report about the microanatomy of the female reproductive tract of S. bonapartii with evidence of sperm storage in the genus.

A glândula oviductal é uma estrutura exclusiva das elasmobrânquias que produz a geléia do ovo, o envoltório terciário do ovo e armazena espermatozóides. Sua importância está relacionada com as características especiais da estratégia reprodutiva do grupo e com a filogenia destes peixes em que oviparidade é a condição ancestral (Dulvy and Reynolds, 1997). A glândula oviductal do smallnose fanskate mostra quatro zonas morfofuncionais. O revestimento em toda a glândula é epitélio colunar ciliado com células glandulares e ciliadas. As secreções são diferentes entre as zonas e relacionada com as suas funções específicas. A informação aqui exposta constitui o primeiro relato sobre a micro-anatomia do trato reprodutivo feminino de S. bonapartii com evidencias de armazenamento do esperma neste gênero.

Effect of progesterone administration on the distribution of oviductal carbohydrates in Rana tigrina.

Our aim has been to determine whether carbohydrate distribution in the oviducts of progesterone-treated animals is comparable with that of seasonal breeders in Rana tigrina. Like many other anurans, R. tigrina oviduct exhibits a short straight portion (pars recta, pr) at the beginning followed by a long, highly coiled portion (pars convoluta, pc). Histologically, the oviduct of this species revealed some unique features, one of which was intense toluidine blue staining, specifically in the upper mucosal glands of pc4. Based on lectin reactivities in the epithelial cells and mucosal glands, patterns of lectin staining in the seasonal breeders were classified into seven types: R1-R3 (for pr) and C1-C4 (for pc). Typically, some lectins reacted selectively either with ciliated cells (concanavalin A) or non-cialiated cells (Ricinus communis agglutinin I and wheatgerm agglutinin); however, Bandeiraea simplicifolia agglutinin I reacted with both cell types. These staining patterns were different in the progesterone-treated animals. Differences in glycan distribution in the oviductal secretions were revealed by lectin blotting. Compared with the seasonal breeders, an enhanced staining of some lectins was noted in the hormone-treated animals: either an increased staining intensity of existing protein bands or additional staining of new protein bands. Inversely, the staining of wheatgerm agglutinin was markedly diminished in the hormone-treated animals, suggesting the inhibitory effect of progesterone on oviductal glycan distribution. Whether alteration in glycan distribution upon progesterone treatment affects the physiological properties of the released jelly substances remains to be addressed.

Tissue remodeling: a mating-induced differentiation program for the Drosophila oviduct.

BACKGROUND: In both vertebrates and invertebrates, the oviduct is an epithelial tube surrounded by visceral muscles that serves as a conduit for gamete transport between the ovary and uterus. While Drosophila is a model system for tubular organ development, few studies have addressed the development of the fly's oviduct. Recent studies in Drosophila have identified mating-responsive genes and proteins whose levels in the oviduct are altered by mating. Since many of these molecules (e.g. Muscle LIM protein 84B, Coracle, Neuroglian) have known roles in the differentiation of muscle and epithelia of other organs, mating may trigger similar differentiation events in the oviduct. This led us to hypothesize that mating mediates the last stages of oviduct differentiation in which organ-specific specializations arise. RESULTS: Using electron- and confocal-microscopy we identified tissue-wide post-mating changes in the oviduct including differentiation of cellular junctions, remodeling of extracellular matrix, increased myofibril formation, and increased innervation. Analysis of once- and twice-mated females reveals that some mating-responsive proteins respond only to the first mating, while others respond to both matings. CONCLUSION: We uncovered ultrastructural changes in the mated oviduct that are consistent with the roles that mating-responsive proteins play in muscle and epithelial differentiation elsewhere. This suggests that mating triggers the late differentiation of the oviduct. Furthermore, we suggest that mating-responsive proteins that respond only to the first mating are involved in the final maturation of the oviduct while proteins that remain responsive to later matings are also involved in maintenance and ongoing function of the oviduct. Taken together, our results establish the oviduct as an attractive system to address mechanisms that regulate the late stages of differentiation and maintenance of a tubular organ.

Ultrastructure of anterior uterus of the oviduct and the stored sperm in female soft-shelled turtle, Trionyx sinensis.

Ultrastructure of sperm storage in female soft-shelled turtle, Trionyx sinensis was examined under light and electron microscopes. Sperm storage tubules are restricted to the anterior of the uterus. These tubules developed either by folding or fusion of the oviductal mucosal folds and are lined by both ciliated and secretory cells. Ciliated cells are characterized by a few microvilli and prominent cilia in the apical membranes. A prominent feature of the secretory cell is the presence of secretory granules in the supranuclear region. The size, shape, and electron density of these granules vary markedly. The secretory product is released mainly by exocytosis into the oviductal lumen, where it appears as flocculent material. The unique structure in the base of the epithelium, the basal border of the cell -- the basal lamina -- and a blood vessel layer, is presumed to be a important barrier, by which the nourishment exchange and the microenvironment maintenance are ensured. The gland cell is presented with numerous, round, membrane-bound secretory granules of moderate to high electron densities. We divide these granules into three types according to their appearance: (1) membrane bounded granules with high-homogeneous electron density, (2) membrane bounded granules with moderate-homogeneous electron density, (3) membrane bounded, electron dense granules with concentric structures. These granules are presented as different stages of the secretions in the gland cell. The junction complexes are markedly distributed between cells, which are important in keeping stability and the microenvironment maintenance of the sperm storage tubules. Sperm stored in the tubules are heterogeneous in cytology. In addition to the mature sperm in the lumen, sperm with large chromatic granules are found, which are presumed to be immature sperm and are being in the process of nuclear condensation. Several spermatozoa in the tubules are exhibited with definitive indications of degeneration of the nuclei. The nuclear volume increases. The electron density of the central cores in mitochondria declines, combined with the deterioration of concentric membrane structure. Those changes are possibly due to the long time storage of the sperm in sperm storage tubules, and the leakage of reactive oxygen species is suggested to be a major cause. We conclude that the ultrastructure character of sperm storage in the oviduct of Trionyx sinensis is unique, in addition to having a basal function in secretion and the cilia swing, the tubules also provide an available microenvironment for the sperm to long time stored. The degenerative sperm in the tubules might be related to paternity-specific reproductive adaptations, and the sperm competition might occur during long time storage.

Reproductive tissue regression: involvement of caspases, inducible nitric oxide synthase and nitric oxide during moulting in White Leghorn hens.

Moulting is a natural physiological process where the reproductive system of birds undergoes complete remodeling in preparation for the next laying cycle. In domestic chickens, moulting is artificially induced by feed withdrawal to recycle the old laying flock for best profit margins. This has received severe criticism from animal welfare organizations, forcing several countries to stop this practice. Several alternative methods to feed withdrawal methods were developed but were found to produce inconsistent results. Understanding the actual mechanism of moulting would help in designing a new animal welfare friendly method. The present investigation attempted to study the molecular mechanism of moulting in White Leghorn hens. Eighty-four layers (75 weeks) were divided into two groups. The birds in the first group were subjected to moulting by feed withdrawal (FW) while the other group received high dietary Zn (ZnF) treatment for 10 days. Six birds from each group were sacrificed on 0, 1-4, 6 and 10 days of moulting and mRNA expression of caspases-1, -2 and iNOS, along with the apoptotic ladder pattern and nitric oxide (NO) in the ovary and oviduct, was investigated. The mRNA expression of iNOS was upregulated with a corresponding increase in NO levels. Caspases-1 and -2 were differentially upregulated in the ovary and oviduct of moulted birds. A constant decline in serum estradiol and progesterone levels was also observed. It can be concluded that the pattern of reproductive regression during moulting by the two methods is different, as the expression of genes studied in the present investigation is different.