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Lorraine Richardson, James P Hanrahan, Tharmala Tharmalingam, Stephen Carrington, Pat Lonergan, Alexander C O Evans and S. Fair

The aim of this study was to investigate the properties and to functionally characterise the cervical mucus that modulates sperm transport through the cervix by using ewe breeds with a divergent pregnancy rate (Belclare and Suffolk; high and low, respectively) following cervical insemination using frozen-thawed semen. Sperm number, as well as sialic acid and fucose content in both the channels and in the lumen of different regions of the cervix were quantified in inseminated Belclare and Suffolk ewes. Expression of glycosyltransferase and MUC genes, glycosidase activity and sialic acid speciation in follicular phase cervical tissue and mucus were assessed. More spermatozoa were found in the cervical channels in the region closest to the cervical os in Belclare than Suffolk ewes (P<0.05) and Suffolk ewes had a higher sialic acid content in the cervical channels than Belclare ewes (P<0.05) in all regions of cervix. Suffolk ewes had significantly higher expression of FUT1, ST6GAL1 and MUC5AC than Belclare ewes. There was no difference between the breeds in glycosidase activity (P>0.05). Levels of Neu5Ac were higher in Belclare than Suffolk ewes (P<0.05) and levels of Neu5Gc was higher in Suffolk than Belclare ewes (P<0.05). Competitive sperm penetration assays demonstrated that frozen-thawed sperm progression increased when cervical mucus was incubated with sialyllactose prior to a sperm penetration test (P<0.05). These results suggest that the difference between Belclare and Suffolk ewes in sperm transport with frozen-thawed semen is due to the higher concentration of sialic acid within channels, which binds to spermatozoa and reduces their ability to traverse the cervix.

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Katarzyna Joanna Szymańska, Nerea Ortiz-Escribano, Etienne Van den Abbeel, Ann Van Soom and Luc Leybaert

Vitrification of immature germinal vesicle-stage oocytes is a promising method in assisted reproduction but is associated with reduced developmental potential and low birth rates. Cumulus-oocyte complexes (COCs) express several connexins that form hexameric hemichannels, which interact head to head to create a gap junction or exist as unopposed free hemichannels. The latter are normally closed but open under stress conditions and may exert detrimental effects. We determined whether minimizing hemichannel opening and cell death during vitrification could improve COC quality. Bovine immature COCs underwent vitrification, storage and warming, followed by dye uptake to assess hemichannel opening and TUNEL staining to detect cell death. Based on these scores, we optimized the procedure by tuning the equilibration time, temperature, cryoprotectant concentration and extracellular Ca2+ concentration and assessed its impact on maturation, cleavage and blastocyst formation after parthenogenetic activation. We found that the major stressor resides in the cooling/warming phase of the vitrification procedure and observed that hemichannel opening and cell death in cumulus cells measure different aspects of cell stress. Optimization of the hemichannel and cell death readouts demonstrated that combined minimal hemichannel opening/cell death gave the highest cleavage rates but had no effect on maturation and blastocyst formation. Neither hemichannel nor cell death optimization performed better than the non-optimized protocol, leading to the conclusion that cell stress factors other than those detected by hemichannel dye uptake or TUNEL positivity are involved.

Open access

Jie Mei, Yuan Yan, Shi-Yuan Li, Wen-Jie Zhou, Qun Zhang, Ming-Qing Li and Hai-Xiang Sun

Decidualization renders the endometrium transiently receptive to an implanting blastocyst although the underlying mechanisms remain incompletely understood. The aim of this study was to determine the role of chemokine CXCL16 and its receptor CXCR6 in the decidualization during pregnancy. Here, the expression of CXCL16 was investigated in endometrial tissues, decidua, and placenta in this study. Compared with endometrial tissue, protein expression of CXCL16 was significantly higher in tissues from the fertile control samples, especially in villus. Meanwhile, the primary trophoblast cells and decidual stromal cells (DSCs) secreted more CXCL16 and expressed higher CXCR6 compared to endometrial stromal cells (ESCs) in vitro. Stimulation with the inducer of decidualization (8-bromoadenosine 3′,5′-cyclic with medroxyprogesterone acetate, 8-Br-cAMP plus MPA) significantly upregulated the expression of CXCL16 and CXCR6 in ESCs in vitro. After treatment with exogenous recombinant human CXCL16 (rhCXCL16) or trophoblast-secreted CXLC16, decidualised ESCs showed a significant decidual response, mainly characterised by increased prolactin (PRL) secretion. Simultaneously, PI3K/PDK1/AKT/Cyclin D1 pathway in decidualised ESCs were activated by rhCXCL16, and AKT inhibitor GS 690693 abolished the PRL secretion of ESCs that was triggered by rhCXCL16. Finally, the impaired CXCL16/CXCR6 expression could be observed at the maternal-foetal interface from patients who have experienced spontaneous abortion. This study suggests that the CXCL16/CXCR6 axis contributes to the progression of ESC decidualization by activating PI3K/PDK1/AKT/Cyclin D1 pathway. It unveils a new paradigm at the maternal-foetal interface in which CXCL16 is an initiator for the molecular crosstalk that enhances decidualization of ESCs.

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M. Luján Scalise, María Marta Amaral, Julieta Reppetti, Alicia E Damiano, Cristina Ibarra and Flavia Sacerdoti

Shiga toxin (Stx2) producing Escherichia coli infections during early gestation may impair placentation through a Stx2 damage of extravillous trophoblast (EVT) cells. We have previously demonstrated that Stx2 injected in rats in the early stage of pregnancy causes spontaneous abortion by a direct cytotoxic effect in the highly perfused feto-uteroplacental unit. The main aim was to evaluate the effects of Stx2 on EVT in order to understand the possible adverse effects that the toxin may cause on trophoblast cells during early pregnancy. Swan 71 and HTR-8 cell lines were used as human EVT models. The presence of Stx2 receptor, globotriaosylceramide (Gb3), on Swan 71 and HTR-8 cells was evaluated by thin layer chromatography. The effects of Stx2 on cell viability were evaluated by neutral red uptake, migration by wound healing assay and invasion was determined by the “transwell chamber” assay. Metalloproteinase activity (MMP-2) was evaluated by zymography and tubulogenesis was analyzed by counting the total tube length and the number of branches formation. We have demonstrated that Swan 71 expresses high levels of Gb3 compared to HTR-8 cells. Stx2 decreased significantly Swan 71 viability in a dose dependent manner after 72 h of toxin exposure. Furthermore, Stx2 impaired migration, invasion and tube-like formation of Swan 71 cells and decreased the MMP-2 activity. These cytotoxic effects were partially prevented by aminoguanidine, an inducible nitric oxide synthase inhibitor. These studies demonstrate that the function and viability of EVT cells may be altered by Stx2 and suggest that NO overexpression may be involved in the detrimental effects.

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Yu Chen, Hongshi Yu, Andrew J Pask, Asao Fujiyama, Yutaka Suzuki, Sumio Sugano, Geoff Shaw and Marilyn B Renfree

The development of the mammalian phallus involves hormone-dependent mesenchymal–epithelial signalling mechanisms that contribute to urethral closure and regulation of phallus elongation and growth. In marsupials, most differentiation and growth of the phallus occurs post-natally, making them amenable to direct hormone treatment. Expression of IGFs, FGFs, EFNB2, MAFB, DLX5 and AP-1 mRNAs in the phallus at day 50 post-partum (pp) were altered after treatment of tammar wallaby young from day 20 to 40 pp with androgen, oestrogen or after castration at day 25 pp. However, the most interesting changes occurred in the IGF pathway genes. Androgen treatment upregulated IGF1 in female phalluses and oestrogen treatment upregulated IGF1 in male phalluses, but it was downregulated by castration. IGFBP3 was higher in female phalluses and downregulated by androgen. IGF1 expression was higher in all untreated male than in female phalluses from day 50 to 150 pp, but IGFBP3 had the reverse pattern. At day 90 pp, when urethral closure in males is progressing and male phallus growth is accelerating. IGF1 and PCNA protein were only detected in the male urorectal septum, suggesting for the first time that closure and elongation may involve IGF1 activation of cell proliferation specifically in male phalluses. These effects of sex steroids on gene expression and on the IGF1 signalling pathway in particular, suggest that the developing phallus may be especially susceptible to perturbation by exogenous hormones.

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K Herzog, L Debertolis, J P Kastelic, M Schmicke, S E Ulbrich and H Bollwein

The objective was to characterize effects of Escherichia coli LPS (given i.v.) on corpus luteum (CL) and embryonic viability in early pregnant cattle. Eight non-lactating German Holstein cows were given 0.5 µg/kg LPS on 35 ± 3 day (mean ± s.e.m.) of pregnancy, whereas seven heifers, 41 ± 6 day pregnant, were given 10 mL saline (control group). Transrectal B-mode examinations of the CL were done at −1, 3, 6, 12, 24, 48, 72 and 96 h relative to treatment. Blood samples were collected at −1, 0.5, 1, 2, 3, 4, 6, 9, 12, 24, 48, 72 and 96 h. At 12 and 48 h, the CL was biopsied. None of the cows still in the experiment 10 day after LPS (n = 7) had embryonic loss. In LPS-treated cows, luteal area decreased (from 4.1 to 3.1 cm2; P ≤ 0.05) within 6 h and until 48 h. Luteal blood flow decreased by 39% (P ≤ 0.05) within the first 6 h after LPS, but returned to pre-treatment values by 48 h. Plasma P4 decreased by 62% (P ≤ 0.05), reached a nadir (2.7 ± 0.6 ng/mL) at 12 h after LPS and was not restored to pre-treatment (P ≤ 0.05). In luteal tissue, mRNAs for STAR and for FGF1 were lower (P ≤ 0.05) in LPS than in saline-treated cattle at 12 h, with no difference between groups at 48 h. Levels of mRNAs for CASP3 and FGF2 were not different between groups (P > 0.05) at 12 or 48 h after treatment. In conclusion, LPS transiently suppressed CL function, but did not induce embryonic mortality.

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Xiang Xiao, Yue Yang, Baiping Mao, C. Yan Cheng and Ya Ni

SRC family kinases (SFKs) are known regulators of multiple cellular events, including cell movement, differentiation, proliferation, survival, and apoptosis. SFKs are expressed virtually by all mammalian cells. They are non-receptor protein kinases that phosphorylate a variety of cellular proteins on tyrosine, leading to activation of protein targets in response to environmental stimuli. Among SFKs, SRC, YES, and FYN are the ubiquitously expressed and best studied members. In fact, SRC, the prototypical SFK, was the first tyrosine kinase identified in mammalian cells. Studies have shown that SFKs are regulators of cell junctions, and function in endocytosis and membrane trafficking to regulate junction restructuring events. Herein, we briefly summarize recent findings in the field regarding the role of SFKs in the testis in regulating spermatogenesis, particularly in Sertoli-Sertoli and Sertoli-germ cell adhesion. While it is almost 50 years since the identification of the oncogene v-Src encoded by Rous sarcoma transforming virus, the understanding of SFK involvement during spermatogenesis in the testis remains far behind that in other epithelia and tissues. The goal of this review aims to bridge this gap.

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Xiaoqing Yang, Meivita Devianti, Yuan H Yang, Yih Rue Ong, Ker Sin Tan, Shanti Gurung, Jean L Tan, Dandan Zhu, Rebecca Lim, Caroline E Gargett and James A Deane

Perivascular mesenchymal stem/stromal cells can be isolated from the human endometrium using the surface marker SUSD2 and are being investigated for use in tissue repair. Mesenchymal stem/stromal cells from other tissues modulate T cell responses via mechanisms including interleukin-10, prostaglandin E2, TGF-β1 and regulatory T cells. Animal studies demonstrate that endometrial mesenchymal stem/stromal cells can also modify immune responses to implanted mesh, but the mechanism/s they employ have not been explored. We examined the immunomodulatory properties of human endometrial mesenchymal stem/stromal cells on lymphocyte proliferation using mouse splenocyte cultures. Endometrial mesenchymal stem/stromal cells inhibited mitogen-induced lymphocyte proliferation in vitro in a dose-dependent manner. Inhibition of lymphocyte proliferation was not affected by blocking the mouse interleukin-10 receptor or inhibiting prostaglandin production. Endometrial mesenchymal stem/stromal cells continued to restrain lymphocyte proliferation in the presence of an inhibitor of TGF-β receptors, despite a reduction in regulatory T cells. Thus, the in vitro inhibition of mitogen-induced lymphocyte proliferation by endometrial mesenchymal stem/stromal cells occurs by a mechanism distinct from the interleukin-10, prostaglandin E2, TGF-β1 and regulatory T cell-mediated mechanisms employed by MSC from other tissues. eMSCs were shown to produce interleukin-17A and Dickkopf-1 which may contribute to their immunomodulatory properties. In contrast to MSC from other sources, systemic administration of endometrial mesenchymal stem/stromal cells did not inhibit swelling in a T cell-mediated model of skin inflammation. We conclude that, while endometrial mesenchymal stem/stromal cells can modify immune responses, their immunomodulatory repertoire may not be sufficient to restrain some T cell-mediated inflammatory events.

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Takayuki Takahashi, Akane Hagiwara and Katsueki Ogiwara

Ovulation, which is induced by the ovulatory luteinizing hormone (LH) surge, is a dynamic process that results in a discharge of one or more fertilizable oocytes from the ovarian follicle into the ovarian cavity or into the abdominal cavity. Follicle rupture is a core event of the ovulatory process and has been the subject of intensive investigation. Many studies have been performed in various vertebrate animals that focused on proteolysis during ovulation. Despite much effort, the proteases responsible for follicle rupture in ovulation have not yet been identified for mammalian species. However, studies conducted using the teleost medaka have recently provided valuable information about the follicle rupture process. Follicle rupture during medaka ovulation is accomplished by a two-step extracellular matrix (ECM) hydrolysis mechanism involving two distinct protease systems, the urokinase-type plasminogen activator-1 /plasmin and the matrix metalloproteinase system. In the 24-h spawning cycle of the fish, the former protease system is activated first, and the latter subsequently becomes active. Proteolytic activities of these systems are regulated by their intrinsic inhibitors. The endocrine regulation of the rupture was examined by investigating the expression of matrix metalloproteinase 15 (Mmp15), which is the only LH-inducible protease among those involved in the rupture process. At least two transcription factors, classical nuclear progestin receptor and CCAAT/enhancer-binding protein β, play critical roles in the expression of the protease transcript. This review also summarizes studies addressing follicle rupture during ovulation conducted using other teleost models to understand the current status of teleost ovulation studies.

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Peter Smith, Jennifer Juengel, Paul Maclean, Christy Rand and Jo-Ann L Stanton

A number of studies have demonstrated effects of gestational undernutrition on fetal ovarian development and postnatal female fertility. However, the mechanism underlying these effects remains elusive. Using a cohort of animals in which altered gestational nutrition affected indicators of postnatal fertility, this study applies RNAseq to fetal ovaries to identify affected genes and pathways that may underlie the relationship between gestational plane of nutrition and postnatal fertility. Pregnant ewes were exposed to either a maintenance diet or 0.6 of maintenance for the first 55 days of gestation followed by an ad libitum diet. Complementary DNA libraries were constructed from 5 to 6 fetal ovaries from each nutritional group at both days 55 and 75 of gestation and sequenced using Ion Proton. Of approximately 16,000 transcripts, 69 genes were differentially expressed at day 55 and 145 genes differentially expressed at day 75. At both gestational ages, genes expressed preferentially in germ cells were common among the differentially expressed genes. Enriched gene ontology terms included ion transport, nucleic acid binding, protease inhibitor activity and carrier proteins of the albumin family. Affected pathways identified by IPA analysis included LXR/RXR activation, FXR/RXR activation, pathways associated with nitric oxide production and citrullination (by NOS1), vitamin C transport and metabolism and REDOX reactions. The data offer some insights into potential mechanisms underlying the relationship between gestational plane of nutrition and postnatal fertility observed in these animals. In particular, the roles of nitric oxide and protease inhibitors in germ cell development are highlighted and warrant further study.