Spindlin 1 is essential for metaphase II stage maintenance and chromosomal stability in porcine oocytes.

Study question: What is the function of Spindlin 1 (Spin1) in metaphase II stage oocytes in pigs? Summary answer: Depletion of Spin1 induces spontaneous oocyte activation and overexpression of Spin1 causes multinuclear formation through induction of DNA damage in porcine oocytes. What is known already: Little is known about the function of Spin1 in oocytes and embryos. In mouse oocytes, Spin1 is specifically expressed during gametogenesis and is essential for meiotic resumption. In somatic cells, Spin1 promotes cancer cell proliferation and activates WNT/T-cell factor signaling. Study design size, duration: After knockdown (KD) or overexpression of Spin1 in porcine MII-stage oocytes, MII maintenance was checked following additional culture for 24 h. Investigated parthenotes were cultured up to the four cell (72 h) or blastocyst (7 days) stages. Participants/materials, setting, methods: Spin1 was knocked down in porcine oocytes and embryos via microinjection of pig Spin1-targeting siRNA. For Spin1 overexpression, porcine Spin1-eGFP cRNA was generated. Additionally, for rescue experiments, cRNA encoding siRNA-resistant mouse Spin1 was added to the pig Spin1-targeting siRNA. For the overexpression and rescue experiments, microinjection and culture were performed using the same methods as the KD experiments. Main results and the role of chance: KD of Spin1 in MII-stage porcine oocytes reduced metaphase-promoting factor and mitogen-activated protein kinase activities, resulting in spontaneous pronuclear formation without calcium activation. However, the DNA damage response was triggered by Spin1 overexpression, generating the checkpoint protein γH2A.X. Furthermore, Spin1 overexpression blocked metaphase-anaphase transition and led to multinucleation in oocytes and embryos. Large scale data: None. Limitations, reasons for caution: This study is based on in vitro investigations with abnormal expression levels of Spin1. This may or may not accurately reflect the situation in vivo. Wider implications of the findings: Spin1 is essential to maintain MII arrest, but a high level of Spin1 induces DNA damage in oocytes and embryos. Thus, a system to accurately regulate Spin1 expression operates in porcine MII-stage oocytes and embryos. Study funding and competing interest(s): This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2015R1D1A1A01057629). The authors declare no competing financial interests.

Role of ataxia-telangiectasia mutated (ATM) in porcine oocyte in vitro maturation.

Ataxia-telangiectasia mutated (ATM) is critical for the DNA damage response, cell cycle checkpoints, and apoptosis. Significant effort has focused on elucidating the relationship between ATM and other nuclear signal transducers; however, little is known about the connection between ATM and oocyte meiotic maturation. We investigated the function of ATM in porcine oocytes. ATM was expressed at all stages of oocyte maturation and localized predominantly in the nucleus. Furthermore, the ATM-specific inhibitor KU-55933 blocked porcine oocyte maturation, reducing the percentages of oocytes that underwent germinal vesicle breakdown (GVBD) and first polar body extrusion. KU-55933 also decreased the expression of DNA damage-related genes (breast cancer 1, budding uninhibited by benzimidazoles 1, and P53) and reduced the mRNA and protein levels of AKT and other cell cycle-regulated genes that are predominantly expressed during G2/M phase, including bone morphogenetic protein 15, growth differentiation factor 9, cell division cycle protein 2, cyclinB1, and AKT. KU-55933 treatment decreased the developmental potential of blastocysts following parthenogenetic activation and increased the level of apoptosis. Together, these data suggested that ATM influenced the meiotic and cytoplasmic maturation of porcine oocytes, potentially by decreasing their sensitivity to DNA strand breaks, stimulating the AKT pathway, and/or altering the expression of other maternal genes.

Effect of anti-PMSG on distribution of estrogen receptor alpha and progesterone receptor in mouse ovary, oviduct and uterus.

It is well established that estrogen and progesterone are critical endogenous hormones that are essential for implantation and pregnancy in females. However, the distribution of estrogen receptor α (ERα) and progesterone receptor (PR) in female reproductive tracts is elusive. Herein, we report that after serial treatments with pregnant mare's serum gonadotrophin (PMSG) with or without anti-PMSG (AP), mice could regulate the distribution of ERα and PR in the murine ovary, oviduct and uterus and the level of estradiol in serum. ERα and PR regulation by PMSG and anti-PMSG was estrous cycle-dependent and critical for promoting the embryo-implantation period. Furthermore, our results suggested that AP-42 h treatment is more effective than the other treatments. In contrast, other treatment groups also affected the distribution of ERα and PR in mouse reproductive tracts. Thus, we found that anti-PMSG has the potential to restore the distribution of ERα and PR, which could effectively reduce the negative impact of residual estrogen caused by the normal superovulation effect of PMSG in mice.

The association of mitochondrial potential and copy number with pig oocyte maturation and developmental potential.

ATP is critical for oocyte maturation, fertilization, and subsequent embryo development. Both mitochondrial membrane potential and copy number expand during oocyte maturation. In order to differentiate the roles of mitochondrial metabolic activity and mtDNA copy number during oocyte maturation, we used two inhibitors, FCCP (carbonyl cyanide p-(tri-fluromethoxy)phenyl-hydrazone) and ddC (2'3-dideoxycytidine), to deplete the mitochondrial membrane potential (Δφm) and mitochondrial copy number, respectively. FCCP (2000 nM) reduced ATP production by affecting mitochondrial Δφm, decreased the mRNA expression of Bmp15 (bone morphogenetic protein 15), and shortened the poly(A) tails of Bmp15, Gdf9 (growth differentiation factor 9), and Cyclin B1 transcripts. FCCP (200 and 2000 nM) also affected p34(cdc2) kinase activity. By contrast, ddC did not alter ATP production. Instead, ddC significantly decreased mtDNA copy number (P < 0.05). FCCP (200 and 2000 nM) also decreased extrusion of the first polar body, whereas ddC at all concentrations did not affect the ability of immature oocytes to reach metaphase II. Both FCCP (200 and 2000 nM) and ddC (200 and 2000 µM) reduced parthenogenetic blastocyst formation compared with untreated oocytes. However, these inhibitors did not affect total cell number and apoptosis. These findings suggest that mitochondrial metabolic activity is critical for oocyte maturation and that both mitochondrial metabolic activity and replication contribute to the developmental competence of porcine oocytes.