Oviduct-specific Glycoprotein Research Articles

In Silico analysis of molecular mimic between Der p 23 against allergenic sources

To identify through In Silico analysis the possible molecular mimicry between Der p 23 and antigens from allergenic sources. Identity was sought between Der p 23 and proteins from the mite families Pyroglyphidae, Acaridae, Chortoglyphidae and Echimyopodidae, through PSI-BLAST and They used PRALINE and EMBOSS for the alignments. Antigens with resolved experimental structure were obtained from Protein Data Bank and those not reported were generated using Swiss Model server and ALPHAFOLD 2. Epitope prediction was carried out with the Ellipro server and Pymol 2.3 was used to visualize the 3D models. The analysis between Pyroglyphidae allergens and Der p 23 showed identity with the endochitinase-like protein of D. pteronyssinus, and the type 2 chitin binding domain of D. farinae, with identities between 85 and 100%, with coverage of 100%, and 75% respectively. The allergens Der f 23 and Der p 23 of D. farinae and D. pteronyssinus had 100% coverage with identities of 85.42% and 79.59%, respectively. Among the allergens of Tyrophagus putrescentiae, binding to chitin, oviduct-specific glycoprotein and Cda4p were included, which had identity values corresponding to 40%, 42.22% and 34.78%, with coverage values that did not exceed the 55%. No results were found for Chortoglyphidae and Echimyopodidae. There is molecular mimicry and structural homology between Der P 23 and allergens from allergic sources of the Pyroglyphidae and Acaridae families. Potential epitopes were identified in Der p 23, which could present cross-reactivity with the proteins of the allergenic sources studied, which must be demonstrated in In vitro and In vivo studies. In vitro and in vivo work is needed to demonstrate the results obtained in the In Silico analysis.

Long-term culture of feline oviduct epithelial cells on permeable filter supports

Basic knowledge about cellular and molecular mechanisms underlying feline reproduction is required to improve reproductive biotechnologies in endangered felids. Commonly, the domestic cat (Felis catus) is used as a model species, but many of the fine-tuned, dynamic reproductive processes can hardly be observed in vivo. This necessitates the development of in vitro models. The oviduct is a central reproductive organ hosting fertilization in the ampulla and early embryonic development in the isthmus part, which also functions as a sperm reservoir before fertilization. In other species, culturing oviduct epithelial cells in compartmentalized culture systems has proven useful to maintain oviduct epithelium polarization and functionality. Therefore, we made the first attempt to establish a compartmentalized long-term culture system of feline oviduct epithelial cells from both ampulla and isthmus. Cells were isolated from tissue samples (n = 33 animals) after routine gonadectomy, seeded on permeable filter supports and cultured at the liquid–liquid or air–liquid interface. Cultures were harvested after 21 days and microscopically evaluated for epithelial differentiation (monolayer formation with basal–apical polarization) and protein expression of marker genes (oviduct-specific glycoprotein, acetylated tubulin). Due to the heterogeneous and undefined native tissue material available for this study, the applied cell culture approach was only successful in a limited number of cases (five differentiated cultures). Even though the protocol needs optimization, our study showed that the compartmentalized culture approach is suitable for maintaining differentiated epithelial cells from both isthmus and ampulla of the feline oviduct.

Recombinant human OVGP1 increases intracellular calcium and further potentiates the effects of progesterone on human sperm

PurposeTo investigate the effects of recombinant human oviduct–specific glycoprotein (rHuOVGP1) alone and in combination with progesterone (P4) on intracellular Ca2+ concentration [Ca2+]i and to investigate if rHuOVGP1 in combination with P4 can further enhance tyrosine phosphorylation (pY) of sperm proteins during human sperm capacitation.MethodsFluorometric flow cytometry was performed to examine the effects of rHuOVGP1 on [Ca2+]i in human sperm during capacitation. Confocal microscopy was used in conjunction with live cell imaging to analyze the influence of rHuOVGP1 and P4 on [Ca2+]i in the sperm tail and to examine the involvement of CatSper channels in their effect on [Ca2+]i. Western blot analysis was performed to assess the protein levels of p105, a major tyrosine-phosphorylated sperm protein.ResultsrHuOVGP1 increases [Ca2+]i in human sperm at the beginning of capacitation and further increases and sustains the level of [Ca2+]i in the sperm tail following the addition of P4. Inhibition of CatSper channels impedes the effects of rHuOVGP1 on [Ca2+]i in the sperm tail. P4 alone can increase pY of a major human sperm protein, p105, yet yields a further increase when used in combination with rHuOVGP1.ConclusionThe present study revealed that rHuOVGP1 may work with P4 to upregulate [Ca2+]i at the beginning of capacitation in part through CatSper channels which, in turn, leads to the downstream event of pY of sperm proteins and enhancement of sperm capacitation.

Response of optic gland pathways to thermal stress in the reproductive phase of female Octopus maya

ABSTRACT The octopus optic glands are the source of multiple signalling molecules that control the transitions through different physiological stages, such as maturation, ageing and death. This study focused on the role of the optic glands during the reproductive phase of Octopus maya, investigating the molecular mechanisms that prevent reproduction at elevated temperatures. RNA sequencing was used to analyse and compare optic gland transcriptomic profiles of female O. maya before, during and after egg-laying under optimal and thermal stress (elevated temperature) conditions. Each stage showed well-defined transcriptomic patterns, and all were strongly affected by thermal stress. At optimal temperature, the optic glands contribute importantly to regulating the energetic balance, as suggested by the expression of genes that modulate the metabolic rate, arterial blood pressure and the management of ATP sources in response to food deprivation. At elevated temperatures, the optic glands of mated females (before egg-laying) showed upregulation of genes encoding neuropeptides (FMRF-amide, APGW-amide, PRQFV-amide, FCAP, buccalin and myomodulin) and the dopamine beta-hydroxylase gene. During egg-laying at elevated temperatures, the neuropeptide receptor capaR and the sex peptide receptor were upregulated whereas homologs of fetuin-B and oviduct-specific glycoprotein genes, crucial for fertilization, were downregulated. These changes in optic gland gene expression in O. maya may be the basis for the high-temperature inhibition of egg-laying, previously observed from this octopus species. Furthermore, after egg-laying, during egg incubation thermal stress affected the expression of genes involved in steroid and thyroid hormone biosynthesis and in inflammatory and apoptotic pathways.

Progestin Significantly Inhibits Carcinogenesis in the Mogp-TAg Transgenic Mouse Model of Fallopian Tube Cancer.

Recent studies suggest that the fallopian tube epithelium (FTE) harbors the precursor for high-grade ovarian cancer, creating opportunities for targeting the FTE for ovarian cancer prevention. Preclinical evidence supports progestins as ovarian cancer preventives, but the effect of progestins on the FTE is not well characterized. The murine oviduct-specific glycoprotein promotor-driven simian virus 40 large T-Antigen (mogp-TAg) transgenic mouse model develops neoplastic lesions in the fallopian tube in a manner similar to that described in human fallopian tube and ovarian cancers. In this study, we investigated the inhibitory effects of the progestin depo-medroxyprogesterone acetate (DMPA) on fallopian tube carcinogenesis following treatment for 3 and 7 weeks in 5-week-old mogp-TAg mice. Overall, compared with vehicle-treated mice, the fallopian tube of DMPA-treated mice was significantly smaller (P < 0.0005), accumulated fewer p53-positive cells, had normal distribution of ciliated cells, less nuclear pleomorphism and epithelial tufting, and had a significantly lower proliferative index (P = 0.001). Accumulation of p53 signatures and serous tubal intraepithelial carcinomas (STIC) in the fallopian tube was significantly reduced in the DMPA (P < 0.0005) treatment group. Moreover, the fallopian tube of the DMPA-treated mice developed significantly less adenocarcinoma compared with vehicle (P < 0.005) at both treatment time points. DMPA treatment significantly induced cleaved caspase-3 (P < 0.0005) in the FTE compared with vehicle suggesting that apoptosis is involved in DMPA-related clearance of abnormal cells from the fallopian tube. These data demonstrate that DMPA targets early events in fallopian tube carcinogenesis by clearing genetically damaged cells, leading to marked reduction in adenocarcinoma, supporting progestins as chemopreventive agents for fallopian tube and ovarian cancers. PREVENTION RELEVANCE: The fallopian tube is thought to harbor the cell of origin for most ovarian cancers. We show in a mouse model of fallopian tube cancer that progestin eradicates the earliest known precancerous lesions and markedly inhibits fallopian tube carcinogenesis, adding to growing preclinical evidence supporting progestins as potent ovarian cancer chemopreventive agents.

Transgenic mice overexpressing the LH receptor in the female reproductive system spontaneously develop endometrial tumour masses

The receptor for the luteinizing hormone (LH-R) is aberrantly over expressed in cancers of the reproductive system. To uncover whether LH-R over expression has a causative role in cancer, we generated a transgenic (TG) mouse which overexpresses the human LH-R (hLH-R) in the female reproductive tract, under the control of the oviduct-specific glycoprotein (OGP) mouse promoter (mogp-1). The transgene was highly expressed in the uterus, ovary and liver, but only in the uterus morphological and molecular alterations (increased proliferation and trans-differentiation in the endometrial layer) were detected. A transcriptomic analysis on the uteri of young TG mice showed an up regulation of genes involved in cell cycle control and a down regulation of genes related to the immune system and the metabolism of xenobiotics. Aged TG females developed tumor masses in the uteri, which resembled an Endometrial Cancer (EC). Microarray and immunohistochemistry data indicated the deregulation of signaling pathways which are known to be altered in human ECs. The analysis of a cohort of 126 human ECs showed that LH-R overexpression is associated with early-stage tumors. Overall, our data led support to conclude that LH-R overexpression may directly contribute to trigger the neoplastic transformation of the endometrium.

Oviduct fluid during IVF moderately modulates polyspermy in in vitro-produced goat embryos during the non-breeding season

The present study determined i) the presence of proteins (oviduct-specific glycoprotein, OVGP1; heat shock protein-70A, HSPA1A; heat shock protein-A8, HSPA8; annexin A1, ANXA1; annexin A5, ANXA5; and myosin-9, MYH9) known to be involved in early reproduction in the oviduct fluid (OF) of anestrous goats; and ii) the functional effect of during IVF on polyspermy modulation and embryonic development. In vitro-matured oocytes were co-cultured with spermatozoa (1.0, 2.0, or 4.0 x 106 cells/mL) for 18 h in SOF medium supplemented with 5 μg/mL of heparin, 4 μg/mL gentamicin, and 10% estrus sheep serum (CTRL1, CTRL2, and CTRL4 groups) or the same medium plus 10% OF (OF1, OF2, and OF4 groups) obtained from anestrus goats. The analysis of OF by western blotting confirmed the presence of the six proteins tested for. The increase in sperm concentration had no effect (P > 0.05) on the penetration rate in any group; however, monospermy rate decreased as sperm concentration was increased in both OF and CTRL. Regardless of the concentration used, when data were pooled, OF supplementation improved (P < 0.05) monospermy and tended (P = 0.057) to enhance IVF efficiency. Additionally, IVF efficiency was higher (P < 0.05) in OF1 than in OF4 [60 ± 13 vs 37 ± 5%). The development capacity was not affected (P > 0.05) by the sperm concentration and OF treatment, and the average values were cleavage (72 ± 2.6%), blastocyst (37 ± 3.0%), blastocyst in relation to the cleaved (51 ± 4.8%), hatched (62 ± 1.2%), and number of cells per blastocyst (174 ± 1.8%). In conclusion, the six proteins analyzed are present in the OF of anestrous goats, and the supplementation of this OF during IVF may modulate the polyspermy incidence and enhance IVF efficiency, especially when 1x106 sperm per mL is used.

The Combined Effects of Recombinant Human Oviductin (rHuOVGP1) and Progesterone on Tyrosine Phosphorylation of Sperm Proteins

Worldwide, around 15% of couples who seek to conceive suffer from infertility. Assisted reproductive technology (ART) would have never been possible without research endeavors. However, to further improve the fertilization rates of ART procedures, there is still more to be done. The mammalian oviductal cells synthesize and secrete a major glycoprotein know as oviductin, or oviduct-specific glycoprotein (OVGP1). This protein has been implicated in enhancing sperm capacitation, sperm motility, sperm penetration and fertilization. Our lab has successfully produced recombinant human oviductin (rHuOVGP1), which has been shown to enhance tyrosine phosphorylation of sperm proteins, a biochemical hallmark of capacitation that takes place in the sperm tail. Additionally, the sex hormone progesterone (P4) has been implicated in increasing the influx of calcium ions through the CatSper channel which is similarly located in the sperm tail and is an important aspect of capacitation. Here we performed a study in the hope of gaining a better understanding of the mechanism of sperm capacitation by examining if rHuOVGP1 works synergistically with P4 to regulate tyrosine phosphorylation of sperm proteins during capacitation. Fresh sperm samples were processed and capacitated at different time points in the absence or presence of rHuOVGP1 (50 mg/mL) with or without P4. The results obtained indicate that both rHuOVGP1 and P4 can enhance tyrosine phosphorylation of sperm proteins, however, the best result was obtained when rHuOVGP1 was used in combination with P4. The addition of rHuOVGP1 with P4 to the capacitation medium may improve the fertilization rates of ART procedures.

Structural and functional characterization of buffalo oviduct-specific glycoprotein (OVGP1) expressed during estrous cycle.

Oviduct-specific glycoprotein (OVGP1) is a high molecular weight chitinase-like protein belonging to GH18 family. It is secreted by non-ciliated epithelial cells of oviduct during estrous cycle providing an essential milieu for fertilization and embryo development. The present study reports the characterization of buffalo OVGP1 through structural modeling, carbohydrate-binding properties and evolutionary analysis. Structural model displayed the typical fold of GH18 family members till the boundary of chitinase-like domain further consisting of a large (β/α)8 TIM barrel sub-domain and a small (α+β) sub-domain. Two critical catalytic residues were found substituted in the catalytic centre (Asp to Phe118, Glu to Leu120) compared with the active chitinase. The carbohydrate-binding groove in TIM barrel was lined with various conserved aromatic residues. Molecular docking with different sugars revealed the involvement of various residues in hydrogen-bonding and non-bonded contacts. Most of the substrate-binding residues were conserved except for a few replacements (Ser13, Lys48, Asp49, Pro50, Asp167, Glu199, Gln272 and Phe275) in comparison with other GH18 members. The residues Trp10, Trp79, Asn80, Gln272, Phe275 and Trp334 were involved in recognition of all six ligands. The α+β sub-domain participated in sugar-binding through Thr270, Gln272, Tyr242 and Phe275. The binding assays revealed significant sugar-binding with purified native and recombinant OVGP1. Phylogenetic analysis revealed that OVGP1 was closely related to AMCases followed by other CLPs and evolution of OVGP1 occurred through several gene duplications. This is the first study describing the structural characteristics of OVGP1 that will further help to understand its interaction with gametes to perform crucial reproductive functions.

Equine chorionic gonadotropin increases estradiol levels in the bovine oviduct and drives the transcription of genes related to fertilization in superstimulated cows.

In the bovine oviduct, estradiol (E2) stimulates secretion and cell proliferation, whereas progesterone (P4) suppresses them. In this study, we have evaluated the effect of two superstimulatory protocols (follicle-stimulating hormone [FSH] or FSH combined with equine chorionic gonadotropin [eCG]) on the oviductal levels of E2 and P4 and its outcome on oviductal cells. Compared with the control group (a single pre-ovulatory follicle), we have observed that the cows submitted to FSH/eCG treatment showed a higher concentration of E2 in the oviduct tissue, together with a higher abundance of messenger RNA encoding steroid receptors (ESR1 and progesterone receptor), and genes linked to gamete interactions and regulation of polyspermy (oviduct-specific glycoprotein 1, heat-shock protein family A member 5, α-l-fucosidase 1 [FUCA1], and FUCA2) in the infundibulum and ampulla segments of the oviduct. However, we did not observe any modulation of gene expression in the isthmus segment. Even though the FSH protocol upregulated some of the genes analyzed, we may infer that the steady effect of FSH combined with eCG on oviduct regulation might benefit fertilization and may potentially increase pregnancy rates.

Human OVGP1 enhances tyrosine phosphorylation of proteins in the fibrous sheath involving AKAP3 and increases sperm-zona binding

PurposeTo investigate if the recombinant human oviduct-specific glycoprotein (rHuOVGP1)–enhanced tyrosine-phosphorylated (pY) proteins are components of specific structure(s) of the sperm tail and if rHuOVGP1 binds to the oocyte and enhances sperm-egg binding.MethodsImmunofluorescent staining and confocal microscopy were performed to examine the localization of pY proteins, outer dense fiber (ODF), and A-Kinase Associated Protein 3 (AKAP3) in human sperm during capacitation. Western blot and immunoprecipitation were employed to analyze protein levels of pY proteins and AKAP3. Immunofluorescent staining was performed to examine the binding of rHuOVGP1 to human oocytes. The effect of rHuOVGP1 on enhancing sperm-zona binding was examined using hemizona assay.ResultspY proteins were detected mainly in the fibrous sheath (FS) surrounding the ODF with a relatively weak immunoreaction in the neck and mid-piece. Western blot analysis revealed co-migration of the pY 105 kDa protein with AKAP3, which was further confirmed by immunoprecipitation correlating immunofluorescent results of co-localization of pY proteins with AKAP3 in the sperm tail. rHuOVGP1 binds specifically to the zona pellucida (ZP) of human oocytes. Prior incubation of sperm and/or ZP with rHuOVGP1 increased sperm-egg binding.ConclusionsThe present study revealed that one of the major rHuOVGP1-enhanced pY proteins could be AKAP3 of the FS and that rHuOVGP1 is capable of binding to human ZP and its presence in the medium results in an increase in sperm-zona binding. Supplement of rHuOVGP1 in in vitro fertilization media could be beneficial for enhancement of the fertilizing ability of human sperm.

Oviduct proteins in fertilization and early embryo development

The oviduct controls the environment in which the gametes are transported and fuse, and in which embryonic development begins. The ultrastructural topography of the ampulla and isthmus is similar, consisting of ciliated and secretory cells, but a different array of proteins is secreted by each segment along with various serum components. Amino acids are selectively secreted by the oviduct; these amino acids probably interact with the gametes or embryo to facilitate the processes of fertilization and development. An oviduct-specific glycoprotein is synthesized by the ampulla of sheep and cattle in response to oestrogen and secreted mainly from day-1 to day 3 of the ovarian cycle. This oestrus-associated glycoprotein (EGP) has a variable molecular mass of 80-97 kDa and a pI value ranging from 4.7 to 5.5. The bovine (b) and ovine (o) EGP genes are 95.5% identical and consist of 1560 base pairs encoding 519 amino acids containing one N-linked and several O-linked glycosylation sites. The terminal glycosides are N-acetylglucosamine and galactose-N-acetylgalactosamine for bEGP, and fucose, galactose and sialic acid residues are also identified for oEGP. EGP binds to zona pellucida and blastomere membranes, but evidence for EGP binding to sperm membranes is equivocal. After in vitro fertilizaton the proportion of sheep oocytes cleaving was increased in the presence of oEGP, but when single-cell embryos were cultured with oEGP, these cleavage rates were reduced. In addition, consistent positive effects of oEGP were observed on blastocyst formation. Elaboration of the mechanism of synthesis of EGP, its action and its role in fertilization and embryo development is important for our understanding of the events of early pregnancy.

Oviduct specific glycoprotein expression in goats (Capra aegagrus) testes and ovaries

The OVGP have important rule in male reproduction to improve physiological and membrane fluidity and helps increase calcium ions into membranes that stimulate membrane bonding with intracellular cAMP thus enhancing the integrity of intact membranes of spermatozoa. In female reproduction, OVGP have important rule to support fertilization. In pig and cow, there have been found where was the expression of OVGP in ovarium and it have been used in vitro fertilization. This study was conducted to determine the expression of OVGP in local goat (Capra aegragus) testis and ovarium. The testes and ovaries that used in this study was taken form slaughterhouse whith maximum time three hours from organs harvesting. Observation of OVGP expression in the testis and ovarium was done by immunohistochemical method. The results showed that OVGP expression in goat testes was seen in the base cells of seminiferous tubules of spermatogonium, spermatocyte cells and in interstitial cells or leydig cells, on the other hand OVGP expression showed in follicle tertier and corpus luteum of ovarium. Based on these results it can be concluded that there is an expression of OVGP in local goat testis and ovarium that have important rules of male and female reproduction.

Influence of elevated temperature on bovine oviduct epithelial cells (BOECs).

The aim of this study was to evaluate the influence of elevated temperature on bovine oviduct epithelial cells (BOECs), based on the expression and localization of both heat shock protein 70 (HSP70), responsible for the cellular defence mechanism, and oviduct specific glycoprotein 1 (OVGP1) which is the most important embryotrophic protein. BOECs were cultured alone and co-cultured with cattle embryos at control (38.5°C) and elevated temperature (41°C) for 168 h. The elevated temperature had no effect on the viability of BOECs but exerted a negative effect on embryo development. The elevated temperature increased the expression of HSP70 and decreased the expression of OVGP1 at both mRNA and protein levels in BOECs cultured alone and those co-cultured with embryos. However, the presence of embryos limited the decrease in OVGP1 expression in BOECs at elevated temperature but did not alter the expression of HSP70. These results demonstrate for the first time the influence of elevated temperature on BOECs, consequently providing insights into the interactions between the embryo and the oviduct at elevated temperature.

ISOLATION AND PROFILLING OVIDUCT SPECIFICT GLYCOPROTEIN IN OVIDUCTAL FLUID OF KACANG GOAT (Capra aegagrus hircus)

Frozen semen supplemented by Oviduct Specific Glycoprotein (OVGP) is a new way to solve the decrease of in vitro fertility on goats caused by polyspermy and to increase the efficiency of artificial insemination on goats. Oviduct Specific Glycoprotein has associated with zona pellucida and localized in perivitelline space. The existence of Oviduct Specific Glycoprotein in perivitelline space may be linked to a mechanism that blocks polyspermic through vitelin block. Oviduct Specific Glycoprotein can be found in the fluid that was secreted by the oviduct (oviductal fluid). This study was aimed to determine Oviduct Specific Glycoprotein profile in the oviductal fluid of Kacang goats Malang district, West Java Province, Indonesia. Oviduct Specific Glycoproteins were collected from an oviductal liquid of kacang goats and samples were loaded to 12% SDS-PAGE to determine the weight of the molecule of Oviduct Specific Glycoprotein, further confirmed by western blot. The result of this study showed that molecule weight of Oviduct Specific Glycoprotein been collected from oviductal liquid was approximately 55-65 kDa. After being confirmed by western blotting the molecule weight of Oviduct Specific Glycoprotein was 65 kDa.

Semen modulated secretory activity of oviductal epithelial cells is linked to cellular proteostasis network remodeling: Proteomic insights into the early phase of interaction in the oviduct in vivo

The oviductal epithelium is crucial for the integrity of the female organ. Previously we got evidence that the surface proteome of oviductal epithelial cells (Oecs) is promptly altered in response to insemination and thus suggested that this early phase plays a notable regulatory role in maintaining cellular function. This study further aimed to assess the effect of semen on the cellular and molecular mechanisms in rabbit Oecs. A quantitative gel-based proteomic approach was applied to analyze changes at three time points (0h, 1h, 2h) after intrauterine insemination (IUI) compared to time matched controls. Within two hours the abundance of 22 protein species was evidently altered in the intracellular fraction. Functional analysis revealed that the proteins were primarily involved in proteostasis as well as metabolic processes. The analysis of phosphoproteins specified a role of mitogen-activated protein kinase (MAPK) signaling molecules. Concurrently, semen increased oviduct-specific glycoprotein (OVGP1) secretion. A correlation between OVGP1 abundance and microtubule-associated proteins 1A/1B–light chain 3 lipidation was observed. The localization and changes in abundance of selected proteins were corroborated by antibody-based methods. These results clearly show that the early phase of interaction acts as a trigger for cellular adaptation to meet an altered demand in the female organ. SignificanceThe oviductal epithelium and its secreted proteins exert a pivotal role in reproductive processes, including the final maturation of male gametes. Thereby, the regulation and subsequently the functionality of the oviductal epithelial cell layer are important factors for the establishment of the appropriate milieu in the female reproductive tract. Notably, male gametes themselves have been shown to be an extrinsic modulatory factor of the oviductal epithelium. Accordingly a comprehensive knowledge about the underlying cellular and molecular mechanisms in the epithelial cells is of interest, also with regard to in vitro purposes. So far, the role of the early phase of interaction in the female organ has not been considered in detail. To get a further insight into the underlying cellular and molecular mechanisms, herein we analyzed the effect of semen on oviductal epithelial cells (Oecs) on the intracellular proteome level within the first two hours after insemination. The present study revealed a directed response of Oecs in vivo and disclosed intracellular pathways that are affected by the interplay between semen and the female reproductive tract. The prompt adaptation of the secretory activity and remodeling of the oviductal epithelium was accompanied by the concerted alterations of protein species that are primarily involved in the maintenance of cellular homeostasis. Besides emphasizing the importance of the early interaction phase for subsequent reproductive processes, the gained knowledge might further be implemented for in vitro applications as well.

Modelling oviduct fluid formation in vitro.

Oviduct fluid is the microenvironment that supports early reproductive processes including fertilisation, embryo cleavage, and genome activation. However, the composition and regulation of this critical environment remains rather poorly defined. This study uses an in vitro preparation of the bovine oviduct epithelium, to investigate the formation and composition of in vitro derived oviduct fluid (ivDOF) within a controlled environment. We confirm the presence of oviduct specific glycoprotein 1 in ivDOF and show that the amino acid and carbohydrate content resembles that of previously reported in vivo data. In parallel, using a different culture system, a panel of oviduct epithelial solute carrier genes, and the corresponding flux of amino acids within ivDOF in response to steroid hormones were investigated. We next incorporated fibroblasts directly beneath the epithelium. This dual culture arrangement represents more faithfully the in vivo environment and impacts on ivDOF composition. Lastly, physiological and pathophysiological endocrine states were modelled and their impact on the in vitro oviduct preparation evaluated. These experiments help clarify the dynamic function of the oviduct in vitro and suggest a number of future research avenues, such as investigating epithelial-fibroblast interactions, probing the molecular aetiologies of subfertility, and optimising embryo culture media.

Human fallopian tube epithelium co-culture with murine ovarian follicles reveals crosstalk in the reproductive cycle.

Do interactions between human fallopian tube epithelium and murine follicles occur during an artificial reproductive cycle in a co-culture system in vitro? In a co-culture system, human fallopian tissues responded to the menstrual cycle mimetic by changes in morphology and levels of secreted factors, and increasing murine corpus luteum progesterone secretion. The entire fallopian tube epithelium, including ciliated and secretory cells, can be regulated in the reproductive cycle. Currently, there are no in vitro culture models that can monitor fallopian tissues in real time in response to factors produced by the ovary. In addition, there are no reports on the impact of fallopian tissue on ovarian function during the menstrual cycle. Human fallopian tissue (n=24) was obtained by routine hysterectomies from women (aged 26-50 years, mean age=43.6) who had not undergone exogenous hormonal treatment for at least 3 months prior to surgery. CD1 female mice were used for ovarian follicle isolation. The human fallopian epithelium layers were either co-cultured with five murine multilayer secondary follicles (150-180 μm follicles, encapsulated in one alginate gel bead) for 15 days or received stepwise steroid hormone additions for 13 days. The fallopian tissue morphology and cilia beating rate, as measured by an Andor Spinning Disk Confocal, were investigated. Oviduct-specific glycoprotein 1 (OVGP1), human insulin-like growth factor 1 (hIGF1), vascular endothelial growth factor A (VEGF-A) and interleukin 8 (IL8) as biological functional markers were measured either by ELISA or western blot to indicate dynamic changes in the fallopian epithelium during the reproductive cycle generated by mouse follicles or by stepwise steroid hormone induction. Three or four patients in each experiment were recruited for replicates. Data were presented as mean±SD and further analyzed using one-way ANOVA followed by Tukey's multiple comparisons test. The cultured fallopian tube epithelium responded to exogenous steroid hormone stimulation, as demonstrated by enhanced cilia beating rate (~25% increase, P=0.04) and an increase in OVGP1 secretion (P=0.02) in response to 1 nM estradiol (E2) treatment when compared with 0.1 nM E2. Conversely, 10 nM progesterone plus 1 nM E2 suppressed cilia beating rate by ~30% (P=0.008), while OVGP1 secretion was suppressed by 0.1 nM E2 plus 50 nM progesterone (P=0.002 versus 1 nM E2 alone). Human fallopian tube epithelium was co-cultured with murine secondary follicles to mimic the human menstrual cycle. OVGP1 and VEGF-A secretion from fallopian tissue was similar with stepwise hormone treatment and when cultured with murine follicles. However, the secretion patterns of hIGF1 and IL8 differed in the luteal phase when comparing steroid treatment with follicle co-culture. In co-culture, hIGF1 secretion was suppressed in the luteal versus follicular phase (P=0.005) but stepwise hormone treatment had no effect on hIGF1. In co-culture, IL8 secretion was also suppressed on luteal phase day 15 (P=0.013) versus follicular phase day 7, but IL8 secretion increased continuously under high E2/progesterone treatment (P=0.003 for D13 versus D3). In the co-culture system, the corpus luteum continuously produced progesterone in the presence of fallopian tube tissue until Day 18 while, without fallopian tissue, progesterone started to drop from Day 13. One limitation of this study is that murine follicles were used to mimic the human menstrual cycle. However, although secretion patterns of peptide hormones such as inhibins and activins differ in mice and humans, the co-culture system used here did reveal interactions between the tissues that govern reproductive function. In vitro co-culture models of fallopian reproductive tissues with ovarian follicles can provide an important tool for understanding fertility and for uncovering the mechanisms responsible for reduced fertility. In addition, the role of oviductal secretions and how they influence ovarian function, such as the production of progesterone during the menstrual cycle, can be uncovered using this model. None. This work was funded by grants from the NIH (UH3TR001207), the American Cancer Society (RSG-12-230-01-TBG) and NIH (R01EB014806). The authors declare no competing financial interest.

Role of exosomes in the reproductive tract Oviductosomes mediate interactions of oviductal secretion with gametes/early embryo.

The oviductal epithelial membrane releases into the luminal environment extracellular vesicles (EVs) which are pleomorphic in nature and fall into two categories: exosomes and microvesicles. Both of these membrane vesicles are referred to as Oviductosomes (OVS), and to date have been identified in the murine and bovine species. Bovine EVs derived in vivo and from in vitro culture show differences in their protein cargo which includes CD9 and HSC70 biochemical markers and fertility-modulating proteins such as oviduct-specific glycoprotein (OVGP) and Plasma Membrane Ca(2+) ATPase 4 (PMCA4). PMCA4, an essential multifunctional sperm protein, is hormonally-regulated with elevated levels seen in proestrus/estrus. OVS deliver PMCA4 to sperm via a fusogenic mechanism involving the interaction between CD9 and integrins which are present on their surfaces. Studies of OVS are needed to determine the components of their cargoes and their interaction with oocytes and the very early embryo. Based on our present knowledge of their interaction with sperm, they are expected to play pivotal roles in regulating fertility and promise to inform the current IVF practice.

Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding.

Studies carried out in several mammalian species suggest that oviductin, also known as oviduct-specific glycoprotein or OVGP1, plays a key role in sperm capacitation, fertilization, and development of early embryos. In the present study, we used recombinant DNA technology to produce, for the first time, recombinant hamster OVGP1 (rHamOVGP1) in human embryonic kidney 293 (HEK293) cells. rHamOVGP1 secreted in the culture medium was purified by affinity chromatography. The resulting protein migrated as a poly-dispersed band of 160-350 kDa on SDS-PAGE corresponding to the molecular mass of the native HamOVGP1. Subsequent mass spectrometric analysis of the purified rHamOVGP1 confirmed its identity as HamOVGP1. Immunocytochemistry demonstrated binding of rHamOVGP1 to the mid-piece and head of hamster sperm and to the zona pellucida (ZP) of ovarian oocytes. In vitro functional experiments showed that addition of rHamOVGP1 in the capacitation medium further enhanced tyrosine phosphorylation of two sperm proteins of approximately 75 kDa and 83 kDa in a time-dependent manner. After 3 hours of incubation in the presence of rHamOVGP1, a significant increase in acrosome reaction was measured. Pretreatment of either sperm or oocyte with 20 μg/ml of rHamOVGP1 prior to sperm-egg binding assay significantly increased the number of sperm bound to the ZP. Addition of rHamOVGP1 in the medium during sperm-egg binding with either oocyte or sperm pretreated with rHamOVGP1 also saw an increase in the number of sperm bound to ZP. In all experimental conditions, the effect of rHamOVGP1 on sperm-oocyte binding was negated by the addition of monoclonal anti-HamOVGP1 antibody. The successful production and purification of a biologically active rHamOVGP1 will allow further exploration of the function of this glycoprotein in reproductive function.