Oxygen levels affect oviduct epithelium functions in air-liquid interface culture.

Key reproductive events such as fertilization and early embryonic development occur in the lumen of the oviduct. Since investigating these processes in vivo is both technically challenging and ethically sensitive, cell culture models have been established to reproduce the oviductal microenvironment. Compartmentalized culture systems, particularly air-liquid interface cultures (ALI; cells access the culture medium only from the basolateral cell side), result in highly differentiated oviduct epithelial cell cultures. The oxygen (O2) tension within the oviduct is 4-10% across species, and its reduced O2 content is presumed to be important for early reproductive processes. However, cell culture models of the oviduct are typically cultivated without O2 regulation and therefore at about 18% O2. To investigate the impact of O2 levels on oviduct epithelium functions in vitro, we cultured porcine oviduct epithelial cells (POEC) at the ALI using both physiological (5%) and supraphysiological (18%) O2 levels and two different media regimes. Epithelium architecture, barrier function, secretion of oviduct fluid surrogate (OFS), and marker gene expression were comparatively assessed. Under all culture conditions, ALI-POEC formed polarized, ciliated monolayers with appropriate barrier function. Exposure to 18% O2 accelerated epithelial differentiation and significantly increased the apical OFS volume and total protein content. Expression of oviduct genes and the abundance of OVGP1 (oviduct-specific glycoprotein 1) in the OFS were influenced by both O2 tension and medium choice. In conclusion, oviduct epithelial cells can adapt to a supraphysiological O2 environment. This adaptation, however, may alter their capability to replicate in vivo tissue characteristics.

Knockdown of ribosomal protein S3 causes preimplantation developmental arrest in mice.

Ribosomal protein S3 (RpS3), a member of the ribosome 40S subunit, has conventional ribosomal function and additional extraribosomal functions. The aim of the present study was to analyze the expression and localization of RpS3 and its function in early embryogenesis in mice. RpS3 mRNA and protein were expressed in multiple mouse tissues. In the ovary, RpS3 protein was ubiquitously and highly expressed in oocytes and granulosa cells. After ovulation and fertilization, RpS3 mRNA and protein were detected in oocytes and preimplantation embryos. Furthermore, RpS3 protein was localized in the cytoplasm of oocytes and preimplantation embryos. Moreover, knockdown of RpS3 in zygotes led to a significantly decreased rate of blastocyst formation. These results provide the first evidence for a novel function of RpS3 in regulating early embryonic development in mice.

Gene expression and promoter methylation of porcine uncoupling protein 3 gene.

OBJECTIVE: Uncoupling protein 3 gene (UCP3) is a candidate gene associated with the meat quality of pigs. The aim of this study was to explore the regulation mechanism of UCP3 expression and provide a theoretical basis for the research of the function of porcine UCP3 gene in meat quality. METHODS: Bisulfite sequencing polymerase chain reaction (PCR) and quantitative real-time PCR (Q-PCR) were used to analyze the methylation of UCP3 5'-flanking region and UCP3 mRNA expression in the adipose tissue or skeletal muscle of three pig breeds at different ages (1, 90, 210-day-old Putian Black pig; 90-day-old Duroc; and 90-day-old Dupu). RESULTS: Results showed that two cytosine-guanine dinucleotide (CpG) islands are present in the promoter region of porcine UCP3 gene. The second CpG island located in the core promoter region contained 9 CpG sites. The methylation level of CpG island 2 was lower in the adipose tissue and skeletal muscle of 90-day-old Putian Black pigs compared with 1-day-old and 210-day-old Putian Black pigs, and the difference also existed in the skeletal muscle among the three 90-day-old pig breeds. Furthermore, the obvious changing difference of UCP3 mRNA expression was observed in the skeletal muscle of different groups. However, the difference of methylation status and expression level of UCP3 gene was not significant in the adipose tissue. CONCLUSION: Our data indicate that UCP3 mRNA expression level was associated with the methylation status of UCP3 promoter in the skeletal muscle of pigs.

Valosin-containing protein is associated with maintenance of meiotic arrest in mouse oocytes†.

Valosin-containing protein (VCP) is a member of the highly conserved AAA (ATPase associated with a variety of cellular activities) superfamily. A previous study has shown that targeted deletion of Vcp in mice results in early embryonic lethality. The aim of the present study was to analyze the expression and localization of VCP and its function in meiotic arrest of mouse oocytes. Vcp mRNA and protein were expressed in multiple mouse tissues. In the ovary, VCP protein was mainly expressed in oocytes and granulosa cells. After ovulation and fertilization, Vcp mRNA and protein were detected in oocytes and preimplantation embryos. Furthermore, VCP protein was localized in both the cytoplasm and nucleus of germinal vesicle (GV)-stage oocytes and preimplantation embryos. Moreover, knockdown of Vcp in GV-stage oocytes led to a significantly increased rate of germinal vesicle breakdown (GVBD). In addition, inhibition of VCP protein improved the GVBD rate in mouse GV-stage oocytes. When VCP inhibition was reversed, the final GVBD rate returned to normal. These results provide the first evidence for a novel function of VCP in meiotic arrest of mouse oocytes.

Fas-associated protein factor 1 is involved in meiotic resumption in mouse oocytes.

Fas-associated protein factor 1 (FAF1) is a Fas-associated protein that functions in multiple cellular processes. Previous research showed that mutations in Faf1 led to the lethality of cleavage stage embryos in a mouse model. The aim of the present study was to analyze the expression pattern, localization, and function of FAF1 in meiotic resumption of mouse oocytes. FAF1 was exclusively expressed in oocytes at various follicular stages within the ovary and was predominantly localized in the cytoplasm of growing oocytes. Furthermore, Faf1 mRNA and protein were persistently present during oocyte maturation and Faf1 mRNA levels were similar in the germinal vesicle (GV), GV breakdown (GVBD), and metaphase II (MII) stages of oocytes. Moreover, knockdown of Faf1 in GV-stage oocytes led to a significantly decreased rate of GVBD. To our knowledge, these results provide the first evidence regarding a novel function of FAF1 in meiotic resumption in mouse oocytes.

NLRP2 and FAF1 deficiency blocks early embryogenesis in the mouse.

Nlrp2 is a maternal effect gene specifically expressed by mouse ovaries; deletion of this gene from zygotes is known to result in early embryonic arrest. In the present study, we identified FAF1 protein as a specific binding partner of the NLRP2 protein in both mouse oocytes and preimplantation embryos. In addition to early embryos, both Faf1 mRNA and protein were detected in multiple tissues. NLRP2 and FAF1 proteins were co-localized to both the cytoplasm and nucleus during the development of oocytes and preimplantation embryos. Co-immunoprecipitation assays were used to confirm the specific interaction between NLRP2 and FAF1 proteins. Knockdown of the Nlrp2 or Faf1 gene in zygotes interfered with the formation of a NLRP2-FAF1 complex and led to developmental arrest during early embryogenesis. We therefore conclude that NLRP2 interacts with FAF1 under normal physiological conditions and that this interaction is probably essential for the successful development of cleavage-stage mouse embryos. Our data therefore indicated a potential role for NLRP2 in regulating early embryo development in the mouse.