Although the mechanisms of in vitro postovulatory aging

Although the mechanisms of in vitro postovulatory aging and in vivo reproductive aging differ, there are some remarkable cytological similarities relevant to human reproduction. It has been reported that aging of human and mouse oocytes can result in disorganization of the spindle and defects in chromosome alignment (Battaglia et al., 1996), including increased kinetochore distance (Merriman et al., 2012). Moreover, the frequent fragmentation seen in our postovulatory aged oocytes is reminiscent of the frequent fragmentation observed in human preimplantation embryos. Fragmentation is a common feature of human embryos (Antczak and Van Blerkom, 1999; Hardy, 1999), and is reported to correlate with low implantation rate (Alikani et al., 1999) and low live-birth rate (Luke et al., 2014; Racowsky et al., 2011), as well as with chromosomal abnormalities (Chavez et al., 2012) and apoptosis (Hardy, 1999). The molecular mechanisms leading to fragmentation are unclear; our data show that ectopic localization of pi3k inhibitors B1 to cytoplasmic clusters is likely responsible for the frequent fragmentation of postovulatory aged oocytes. Thus genome transfer may be useful to restore developmental potential of oocytes for patients with high embryo fragmentation. Additional experiments are required to address these questions in human oocytes.
Experimental Procedures
Author Contributions
Acknowledgments This research was supported the NYSCF-Robertson award to D.E., and laboratory startup funds to D.E. from Columbia University. M.Y. is supported by the New York Stem Cell Foundation and in part by the Uehara Memorial Foundation and the Takeda Science Foundation, Japan. D.E. is an NYSCF-Robertson Investigator. The authors would like to thank Chuang-Chen Lin for carrying out embryo transfer, and Lauren Bauer Vensand, Carmen Dusenberry, Travis Kroeker, and Brandon Pearl for expert laboratory support.
Introduction Pancreatic agenesis is a rare congenital disease caused by a mutation in PDX1 (Stoffers et al., 1997), GATA4 (Shaw-Smith et al., 2014), or most commonly GATA6 (Chao et al., 2015; De Franco et al., 2013; Lango Allen et al., 2012; Stanescu et al., 2014). The majority of GATA6 mutations leading to pancreatic agenesis are de novo heterozygous mutations. Some GATA6 mutations have incomplete penetrance as determined by patients having identical mutations to pancreatic agenesis patients, but displaying either adult-onset diabetes or an absence of pancreatic abnormalities (Bonnefond et al., 2012; De Franco et al., 2013). The majority of pancreatic agenesis patients also display a combination of other defects including congenital heart defects, gut abnormalities, and intrauterine growth retardation (Chao et al., 2015). GATA6 belongs to a six-member family of transcription factors that bind to the consensus sequence (A/T)GATA(A/G). GATA1, GATA2, and GATA3 are mainly expressed in hematopoietic cell lineages, while GATA4, GATA5, and GATA6 are predominantly expressed in the heart, gonads, and endodermal-derived tissues (Viger et al., 2008). GATA6 is known to regulate endodermal gene expression and development of endoderm-derived organs (Molkentin, 2000). In mice, GATA6 is expressed in the primitive streak, heart, lung, intestine, gonads, adrenal, and pancreatic tissues (Koutsourakis et al., 1999; Liu et al., 2002). Within the adult pancreatic tissue, GATA6 is expressed in both the exocrine tissue and the islets of Langerhans (Sartori et al., 2014). In contrast to the severe disease phenotype found in humans with GATA6 heterozygous mutations, GATA6 heterozygous mice are fertile and phenotypically normal. Homozygous GATA6 null mice are embryonic lethal (Morrisey et al., 1998). Using tetraploid complementation, GATA6 has been shown to be essential for extra-embryonic endoderm development explaining the embryonic lethality (Koutsourakis et al., 1999; Zhao et al., 2005); however, GATA6 null cells can contribute to the definitive endoderm. Analysis of a loss of GATA6 in pancreas progenitors or adult β cells has demonstrated minimal impact on endocrine function, with normal numbers of β cells and no overt signs of diabetes despite a mild impact on endoplasmic reticulum stress (Carrasco et al., 2012; Martinelli et al., 2013; Sartori et al., 2014; Xuan et al., 2012).