Cytoplasmic changes in relation to nuclear maturation and early embryo developmental potential of porcine oocytes: Effects of gonadotropins, cumulus cells, follicular size, and protein synthesis inhibition*

Abstract

Morphological and biochemical changes indicative of cytoplasmic maturation in relation to nuclear maturation progression and early embryo developmental potential was studied. Fluorescently labeled microfilaments and cortical granules were visualized by using laser scanning confocal microscopy. The mitogen-activated protein (MAP) kinase phosphorylation and cyclin B1 levels were revealed by Western blot. With the maturation of oocytes, cortical granules and microfilaments were localized at the cell cortex. A cortical granule-free domain (CGFD) and an actin-thickening area were observed over both the MII spindle of a mature oocyte and chromosomes of a nocodazole-treated oocyte, suggesting that chromosomes, but not the spindle, determined the localization of CGFD and actin-thickening area. In oocytes that are incompetent to resume meiosis, as indicated by the failure of germinal vesicle breakdown (GVBD), peripheral localization of cortical granules and microfilaments, phosphorylation of MAP kinase and synthesis of cyclin B1 did not occur after 44 hr in vitro. These cytoplasmic changes were also blocked when GVBD of meiotically competent oocytes was inhibited by cycloheximide. Culture of oocytes in a chemically defined medium showed that biological factors such as gonadotropins, cumulus cells and follicle size affected both nuclear and cytoplasmic maturation as well as embryo developmental potential. Absence of gonadotropins or removal of cumulus cells alone did not significantly influence GVBD or cyclin B1 levels, but decreased the final maturation and developmental ability of oocytes. A combination of gonadotropin absence and cumulus removal decreased GVBD, MAP kinase phosphorylation and embryo development. A high proportion of oocytes derived from small follicles were able to resume meiosis, synthesize cyclin B(1), phosphorylate MAP kinase and translocate CGs, but their maturation and embryo developmental ability were limited. Removal of cumulus cells from small follicle-derived oocytes severely affected their ability to undergo cytoplasmic and nuclear maturation.

Intracytoplasmic Sperm Injection – Factors Affecting Fertilization

“…It should be noted that the immature oocytes collected in stimulated cycles have already been under stimulation with high doses of gonadotropins and are exposed to hCG before retrieval. The nuclear maturation, cytoplasmic maturation and ensuing developmental capacity of these oocytes may very well be different in comparison with immature oocytes collected from small antral follicles of unstimulated ovaries in the typical IVM procedure [151].…”

Effect of sperm preparation method on in vitro fertilization in pigs

“…Several procedures have been designed to remove diluent media, cryoprotectants or decapacitating factors from the seminal plasma. Among these methods are swim-up (Berger et al, 1985;Parrish and Foote, 1987), the Percoll discontinuous gradient (Grant et al,1994;Jeong and Yang, 2001), Sephadex gel filtration (Steeno et al, 1985), glass wool filtration (Pereira et al, 2000), glass bead columns (Daya et al, 1987) and albumin gradients (Han et al, 1999;Iwasaki et al, 1999;Sun et al, 2001). However, some research teams prefer not to treat spermatozoa before IVF, but concentrate them by centrifugation before dilution in the fertilization medium (Martinez et al, 1996;Gadea and Matas, 2000).…”

Identification and Characterization of an Oocyte Factor Required for Porcine Nuclear Reprogramming

“…Accompanying the nuclear maturation process, many cytoplasmic changes, termed cytoplasmic maturation, occur (20,21). Some proteins, regarded as reprogramming factors, are largely synthesized from stored mRNAs during the process of cytoplasmic maturation (9).…”