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Global methylation levels differ in in vitro- and in vivo-developed embryos. Follicular fluid (FF) contains extracellular vesicles (EVs) containing miRNAs that affect embryonic development. Here, we examined our hypothesis that components in FF affect global DNA methylation and embryonic development. Oocytes and FF were collected from bovine ovaries. Treatment of zygotes with a low concentration of FF induced global DNA demethylation, improved embryonic development, and reduced DNMT1/3A levels. We show that embryos take up EVs containing labeled miRNA secreted from granulosa cells and the treatment of zygotes with EVs derived from FF reduces global DNA methylation in embryos. Furthermore, the methylation levels of in vitro-developed blastocysts were higher than those of in their vivo counterparts. Based on small RNA-sequencing and in silico analysis, we predicted miR-29b, -199a-3p, and -148a to target DNMTs and to induce DNA demethylation, thereby improving embryonic development. Moreover, among FF from 30 cows, FF with a high content of these miRNAs demethylated more DNA in the embryos than FF with a lower miRNA content. Thus, miRNAs in FF play a role in early embryonic development. [ABSTRACT FROM AUTHOR]

The present study examined the effect of polysaccharides gels made of xanthan gum and locust bean gum (gel culture system) on oocyte maturation and explored the molecules causing the beneficial effect of the gel culture system. Oocytes and cumulus cells complexes were collected from slaughterhouse-derived ovaries and cultured on a plastic plate or gel. The gel culture system improved the rate of development to the blastocyst stage. The oocytes that matured on the gel contained high lipid contents and F-actin formation, and the resultant 8-cell stage embryos had low DNA methylation levels compared to their plate counterparts. RNA sequencing of the oocytes and embryos revealed the differentially expressed genes between the gel and plate culture systems, and upstream regulator analysis revealed estradiol and TGFB1 as top activated upstream molecules. The medium of the gel culture system contained higher concentrations of estradiol and TGFB1 than that of the plate cultures system. Supplementation of the maturation medium with either estradiol or TGFB1 resulted in high lipid content in oocytes. In addition, TGFB1 improved the developmental ability of the oocytes and increased F-actin content while reducing DNA methylation levels in the 8-cell stage embryos. In conclusion, the gel culture system is useful for embryo production, potentially through the upregulation of TGFB1. [ABSTRACT FROM AUTHOR]

Problem: Systemic inflammation induced by infection, which is associated with testicular inflammation, predisposes males to subfertility. Recently, the nucleotide‐binding oligomerization domain, leucine‐rich repeat‐, and pyrin domain‐containing 3 (NLRP3) inflammasome was identified as a key mediator of inflammation, and excessive activation of the NLRP3 inflammasome was shown to contribute to the pathogenesis of a wide variety of diseases. However, the mechanisms underlying infectious inflammation in the testis remain unclear. We investigated the effect of lipopolysaccharide (LPS)‐induced systemic inflammation on the role of the NLRP3 inflammasome in murine testes. Method of study: We performed in vivo and in vitro studies using an LPS‐induced model of NLRP3 inflammasome activation and testicular inflammation. Results: Intraperitoneal administration of LPS significantly impaired sperm motility in the epididymis of wild type (WT) and NLRP3‐knockout (KO) mice. LPS administration stimulated interleukin (IL)‐1β production and secretion in the testes of WT mice, and these adverse effects were improved in the testes of NLRP3‐KO mice. LPS administration also stimulated neutrophil infiltration as well as its chemoattractant C‐C motif chemokine ligand 2 (CCL2) in WT testes, which were suppressed in NLRP3‐KO testes. In in vitro cell culture, treatment with LPS and NLRP3 inflammasome activation significantly induced IL‐1β and CCL2 secretion from WT but not NLRP3‐KO testicular cells. Conclusions: Taken together, our results suggest that testicular cells have the potential to secrete IL‐1β and CCL2 in an NLRP3 inflammasome‐dependent manner and that these cytokines from the testis may further exacerbate testicular function, resulting in subfertility during infectious diseases. [ABSTRACT FROM AUTHOR]

Purpose: The present study investigated the effects of docosahexaenoic acid (DHA) on the growth of bovine oocytes. Methods: Oocytes and granulosa cell complexes (OGCs) were collected from early antral follicles (0.4‐0.7 mm) on the surface of ovaries harvested from a slaughterhouse. The OGCs were cultured with 0, 1, and 10 μmol/L docosahexanoic acid (DHA) for 16 days. Results: Antrum formation of the OGCs and the number of granulosa cells (GCs) surrounding the oocytes were comparable among groups, whereas supplementation of 0.1 μmol/L of DHA significantly improved oocyte growth. Oocytes grown with DHA had a higher fertilization rate, acetylation levels of H4K12, and ATP contents, as well as a lower lipid content compared with those grown without DHA. In addition, GCs surrounding OGCs grown with DHA had low lipid content compared with vehicle counterparts. Furthermore, when GCs were cultured in vitro, DHA increased ATP production, mitochondrial membrane potential, and reduced lipid content and levels of reactive oxygen species. RNA‐seq of GCs revealed that DHA increased CPT1A expression levels and affect genes associated with focal adhesion, oxidative phosphorylation, and PI3K‐AKT etc Conclusion: The results suggest that DHA supplementation affects granulosa cell characteristics and supports oocyte growth in vitro. Docosahexaenoic acid improves growth of oocytes derived from early antral follicles. [ABSTRACT FROM AUTHOR]

Problem: The oviduct is essential for reproduction. We previously showed that oviduct epithelial cells (OECs) isolated from aged cows expressed higher levels of inflammatory cytokines, including interleukin (IL) 1A and IL1B. In addition, aging is associated with tissue dysfunction and cellular senescence via a senescence‐associated secretory phenotype (SASP) and immune cell accumulation. We investigated whether IL1A or IL1B causes SASP production, cellular senescence, and inflammatory responses in bovine OECs. Method of Study: The OECs were isolated from bovine oviducts from young (mean 50.3 months) and aged cows (mean 157.0 months) and cultured. Results: Treatment with IL1A or IL1B induced SASP production (IL8, IL6, TNFA, and CCL2) and mRNA expression of cell adhesion molecules in bovine OECs, but both IL1s did not induce cellular senescence in OECs and migration of polymorphonuclear neutrophils (PMNs). Cultured medium of OECs treated with IL1s, especially IL1B, dramatically induced PMN migration. Treatment with the CCL2 inhibitor, but not IL8 or its receptor CXCR2 inhibitors, significantly reduced immune cell migration in IL1B‐treated OEC‐cultured medium. Treatment with IL1B increased PMN adhesion to OECs, resulting in further SASP production in OECs due to a PMN‐OEC interaction. Conclusion: We suggest that senescence‐associated IL1s cause SASP production in bovine OECs and CCL2 induced by IL1B is essential for the migration of immune cells to OECs. Specifically, IL1B regulates PMN migration and adhesion to bovine OECs, and PMNs accelerate inflammatory cytokine production from bovine OECs via a direct interaction. These phenomena may contribute to chronic oviductal inflammation, resulting in subfertility. [ABSTRACT FROM AUTHOR]

Abstract Background After ovulation in the cow, the corpus luteum (CL) rapidly develops within a few days with angiogenesis and progesterone production. CL formation resembles an inflammatory response due to the influx of immune cells. Neutrophils play a role in host defense and inflammation, and secrete chemoattractants to stimulate angiogenesis. We therefore hypothesized that neutrophils infiltrate in the developing CL from just after ovulation and may play a role in angiogenesis of the CL. Methods and Results Polymorphonuclear neutrophils (PMN) were detected in CL tissue by Pas-staining, and interleukin-8 (IL-8, a neutrophil-specific chemoattractant) was measured in supernatant of the CL tissue culture: considerable amounts of PMNs and the high level of IL-8 were observed during the early luteal phase (days 1-4 of the estrous cycle). PMNs and IL-8 were low levels in the mid and late luteal phases, but IL-8 was increased during luteal regression. The PMN migration in vitro was stimulated by the supernatant from the early CL but not from the mid CL, and this activity was inhibited by neutralizing with an anti-IL-8 antibody, indicating the major role of IL-8 in inducing active PMN migration in the early CL. Moreover, IL-8 stimulated proliferation of CL-derived endothelial cells (LECs), and both the supernatant of activated PMNs and IL-8 stimulated formation of capillary-like structures of LECs. Conclusion PMNs migrate into the early CL partially due to its major chemoattractant IL-8 produced at high levels in the CL, and PMNs is a potential regulator of angiogenesis together with IL-8 in developing CL in the cow.

Purpose: This retrospective observational study investigated relationships between the abundance of cell‐free mitochondrial DNA (cf‐mtDNA) in spent culture medium (SCM) of human‐expanded blastocysts and their morphokinetics to address the question of whether the abundance of cf‐mtDNA in SCM could predict the quality of blastocysts. Methods: Embryos (n = 53) were individually cultured in a time‐lapse incubator until they reached the expanded blastocyst stage (5 or 6 days), following which copy numbers of cf‐mtDNA in SCM (20 μL) of expanded blastocysts were determined using real‐time PCR. Results: The duration between start of blastulation to expanded blastocyst (tEB–tSB) and between that of the blastocyst stage to expanded blastocyst (tEB–tB) significantly and positively correlated with the abundance of cf‐mtDNA in the SCM (tEB–tSB: r =.46; P <.01; tEB–tB: r =.47; P <.01). The abundance of cf‐mtDNA in the SCM was significantly greater in blastocysts with blastocyst collapse (BC), than without BC, and significantly and positively correlated with the number of BC. Conclusions: The abundance of cf‐mtDNA in the SCM was associated with expansion duration and BC. Thus, cf‐mtDNA abundance in the SCM serves as a marker to predict the quality of expanded blastocysts. [ABSTRACT FROM AUTHOR]

One of the major difference between the in vivo and in vitro embryonic environments is the stiffness of the culture substrate. Xanthan gum (XG) and locust bean gum (LBG) are natural materials that are safe, inexpensive and easy to handle. In this study, we investigated the effects of using a polysaccharide culture substrate made from 1% XG and 1% LBG (XG‐LBG gel) on bovine embryonic development. Oocytes collected from bovine ovaries were subjected to maturation, and fertilization to generate embryos at an early developmental stage (>4 cell stage). Cleaved embryos were further cultured in a well of 96‐well cell culture plate coated with or without XG‐LBG gel for 5 days. While the developmental rate up to the blastocyst stage did not differ between the two culture systems (control, 38.0 vs. gel, 38.6%), blastocysts developed on the XG‐LBG gel produced significantly high cell numbers and ATP content. Embryos cultured on XG‐LBG gels for 24 hr had high expression levels of F‐actin and a highly even distribution of E‐cadherin. In addition, embryos developed on XG‐LBG gel demonstrated increased translocation of YAP to the nucleus and increased connective tissue growth factor (CTGF) protein levels (downstream of Hippo signalling). These findings suggest that soft culture substrates improve embryonic development by enhancing mechanotransduction, including YAP‐CTGF signalling. [ABSTRACT FROM AUTHOR]

Reproduction involves tightly regulated series of events and the immune system is involved in an array of reproductive processes. Disruption of well-controlled immune functions leads to infertility, placental inflammation, and numerous pregnancy complications, including preeclampsia (PE). Inflammasomes are involved in the process of pathogen clearance and sterile inflammation. They are large multi-protein complexes that are located in the cytosol and play key roles in the production of the pivotal inflammatory cytokines, interleukin (IL)-1β and IL-18, and pyroptosis. The nucleotide-binding oligomerization domain, leucine-rich repeat-, and pyrin domain-containing 3 (NLRP3) inflammasome is a key mediator of sterile inflammation induced by various types of damage-associated molecular patterns (DAMPs). Recent evidence indicates that the NLRP3 inflammasome is involved in pregnancy dysfunction, including PE. Many DAMPs (uric acid, palmitic acid, high-mobility group box 1, advanced glycation end products, extracellular vesicles, cell-free DNA, and free fatty acids) are increased and associated with pregnancy complications, especially PE. This review focuses on the role of the NLRP3 inflammasome in the pathophysiology of PE. [ABSTRACT FROM AUTHOR]

The phenomenon of aging arises from multiple, complex interactions causing dysfunction in cells and organs. In particular, fertility drastically decreases with age. Previously, we have demonstrated that the functional characteristics of the bovine oviduct and uterus change with the age-dependent upregulation of inflammation and noted that S100A9 triggers inflammatory responses in oviduct epithelial cells. In the present study, we investigated the hypothesis that S100A9 affects reproductive events to aspect such as sperm function, fertilization, and the development of the embryo in cows. To investigate the effect of S100A9 on bovine sperm, we incubated sperms in vitro with S100A9 for 5 h and observed significantly decreased sperm motility and viability. During in vitro fertilization, S100A9 treatment for 5 h did not affect the rate of fertilization, time of first division of embryos, or embryo development to blastocyst stage. Treatment of 2-cell stage embryos with S100A9 for 5 h significantly reduced the proportion of cells undergoing normal division (4–8 cell embryos) and embryo development to the blastocyst stage. In experiment involving 24 h treatment of 2-cell embryos, the development of all embryos stopped at the 2-cell stage in the S100A9-treated group. In blastocyst-stage embryos, S100A9 treatment significantly stimulated the expression of endoplasmic reticulum (ER) and the mRNA expression of ER stress markers, and activated caspase-3 with subsequent nuclear fragmentation. Pre-treatment with an ER stress inhibitor significantly suppressed caspase-3 activation by the S100A9 treatment, suggesting that S100A9 induces blastocyst dysfunction by apoptosis (via caspase-3 activation) depending on ER stress. These results indicate that direct exposure to S100A9 exerted adverse effects on sperm function and embryo development. These findings suggest that excessive dose of S100A9 may have an adverse effect to the reproductive machinery by inducing inflammation and tissue dysfunction. [ABSTRACT FROM AUTHOR]