Inversely, only weak labeling of CFTR was associated with granulosa cells and oocytes in atretic follicles

Inversely, only weak labeling of CFTR was associated with granulosa cells and oocytes in atretic follicles. to discovery of non-invasive biomarkers of oocyte quality in assisted human reproduction and in large animal embryo transfer programs. that are competent to produce normal offspring is a goal that would facilitate a variety of technological advances in assisted human and animal reproduction, such as fertilization (IVF), intracytoplasmic sperm injection (ICSI), IVF with gender sorted spermatozoa, creation of transgenic embryos, somatic cell nuclear transfer in animals, stem cell technology, cryopreservation and development of embryos followed by non-surgical embryo transfer. Unfortunately, it is often difficult to assess the developmental and reprogramming potential of oocytes or embryos without inflicting irreparable damage to them. Various morphological, biochemical, genomic and proteomic approaches to discover and then characterize novel markers that would permit non-invasive assessments PU-WS13 of oocyte and embryo quality have been pursued with modest success. Pig oocytes, obtainable in PU-WS13 much larger quantities than rodent or non-human primate oocytes, are an excellent biomedical model as they mimic some of the temporal events of oocyte maturation and early development observed in humans. For example, pig is the only known animal species besides the rat that makes the major transition from maternal control of development to zygotic control of development during the four-cell stage as in the human [1]. It is relatively easy to create model AXUD1 batches of good- and poor-quality pig oocytes by using an maturation (IVM) system with and without gonadotropins. Before the preovulatory gonadotropin surge, PU-WS13 mammalian oocytes are arrested at the dictyate stage of meiosis I. The gonadotropin discharge induces a cascade of events including chromatin condensation, germinal vesicle breakdown, re-initiation of the oocyte cell cycle, expansion of the oocyte-surrounding cumulus mass, first polar body extrusion and second meiotic arrest at metaphase-II [2]. Pincus and Enzmann [3] first observed that mammalian oocytes released from large ovarian follicles spontaneously resume meiosis and many of them are able to reach metaphase of second meiosis when cultured =538) and 59.0% without gonadotropins (=114); blastocyst formation rate of 38.9% in ova fertilized after maturation with gonadotropins (=228) and 25.4% in ova matured without goandotropins (=68). After maturation, the oocytes and the spent culture media were stored at ?80C until ExacTag? quantitative proteomic analysis was performed. For oocyte storage, PU-WS13 zonae were carefully removed by pronase treatment and the zona-free oocytes were deposited in batches of 100 in a minimal volume of protein-free TLH medium in 0.6 L Eppendorf tubes. Oocytes in tubes were gently spun for 5 s in a benchtop microcentrifuge at 6000 rpm and the remaining medium was carefully removed by aspiration from tube under a stereomicroscope. Cellular debris was removed from the culture media by centrifugation for 10 min at 2000 prior to storage in a final volume of ~500 L/tube. 2.2 ExacTag? quantitative proteomic analysis Quantitative proteomic analysis was performed by using the ExacTag? (Perkin Elmer) labeling kit according to the manufacturers directions. This kit is designed for high sensitivity, which makes it optimal for work with low concentration protein samples. In control experiments using different concentrations of bovine serum albumin with ExacTag kit, we were able to detect protein at concentrations as PU-WS13 low as 0.001 pmol. The preparation of oocytes was dissolved in 1 mL of 2-D gel loading buffer (2D-Xtract; G Biosciences) made from dried powder and used immediately. The solution also contained 10 mM DTT and a protease cocktail (Protearrest). The solution was incubated at room temperature for 30 min prior to precipitation with six volumes of chilled acetone..