Meiotic Maturation Of Porcine Oocytes Research Articles

Microtubule and microfilament dynamics in porcine oocytes during meiotic maturation

Microtubule and microfilament organization in porcine oocytes during maturation in vivo and in vitro was imaged by immunocytochemistry and laser scanning confocal microscopy. At the germinal vesicle stage, microtubules were not detected in the oocyte. After germinal vesicle breakdown, a small microtubule aster was observed near the condensed chromatin. During the prometaphase stage, microtubule asters were found in association with each chromatin mass. The asters then elongated and encompassed the chromatin at the metaphase-I stage. At anaphase-I and telophase-I microtubules were detected in the meiotic spindle. Microtubules were observed only in the second meiotic spindle at the metaphase-II stage. The meiotic spindle was a symmetric, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Taxol, a microtubule-stabilizing agent, did not induce microtubules in oocytes at the germinal vesicle stage. After germinal vesicle breakdown, numerous cytoplasmic foci of microtubules were formed in the entire oocyte when oocytes were incubated in the presence of taxol. Microfilaments were observed as a relatively thick uniform area around the cell cortex and were also found throughout the cytoplasm of oocytes at the germinal vesicle stage. After germinal vesicle breakdown, the microfilaments were concentrated close to the female chromatin. During prometaphase, microfilaments were chromatin moved to the peripheral position. At metaphase-I, two domains, a thick and a thin microfilament area, existed in the egg cortex. Chromosomes were located in the thick microfilament domain of the cortex. In summary, these results suggest that both micro-tubules and microfilaments are closely involved with chromosomal dynamics after germinal vesicle breakdown and during meiotic maturation in porcine oocytes. © 1996 Wiley-Liss, Inc.

Microtubule and microfilament dynamics in porcine oocytes during meiotic maturation.

Microtubule and microfilament organization in porcine oocytes during maturation in vivo and in vitro was imaged by immunocytochemistry and laser scanning confocal microscopy. At the germinal vesicle stage, microtubules were not detected in the oocyte. After germinal vesicle breakdown, a small microtubule aster was observed near the condensed chromatin. During the prometaphase stage, microtubule asters were found in association with each chromatin mass. The asters then elongated and encompassed the chromatin at the metaphase-I stage. At anaphase-I and telophase-I microtubules were detected in the meiotic spindle. Microtubules were observed only in the second meiotic spindle at the metaphase-II stage. The meiotic spindle was a symmetric, barrel-shaped structure containing anastral broad poles, located peripherally and radially oriented. Taxol, a microtubule-stabilizing agent, did not induce microtubules in oocytes at the germinal vesicle stage. After germinal vesicle breakdown, numerous cytoplasmic foci of microtubules were formed in the entire oocyte when oocytes were incubated in the presence of taxol. Microfilaments were observed as a relatively thick uniform area around the cell cortex and were also found throughout the cytoplasm of oocytes at the germinal vesicle stage. After germinal vesicle breakdown, the microfilaments were concentrated close to the female chromatin. During prometaphase, microfilaments were chromatin moved to the peripheral position. At metaphase-I, two domains, a thick and a thin microfilament area, existed in the egg cortex. Chromosomes were located in the thick microfilament domain of the cortex. In summary, these results suggest that both microtubules and microfilaments are closely involved with chromosomal dynamics after germinal vesicle breakdown and during meiotic maturation in porcine oocytes.

Activation of mitogen-activated protein kinase during meiotic maturation in porcine oocytes.

To investigate the involvement of mitogen-activated protein kinase (MAP kinase) in meiotic maturation of porcine oocytes, we assayed MAP kinase activity using myelin basic protein (MBP) as a substrate. MAP kinase activity was low during the germinal vesicle stage, 0-20 h of culture. An abrupt increase was observed at metaphase I (30 h of culture), and activity remained significantly higher than that at 0 h until 50 h of culture, with a transient slight decrease at the time of first polar body extrusion (40 h). Detection of the kinase activity by an in-gel phosphorylation assay confirmed that the 42 and 44 kDa MAP kinases were significantly activated in 45 h cultured oocytes but not in 0 h oocytes, and just slightly in 20 h oocytes. In immunoblotting, however, the 42 and 44 kDa bands were detected in 0, 20 and 45 h cultured oocytes. Furthermore, the signal strength of the two bands did not change during the period of culture, but shifted up to 45 h, indicating that the activation of MAP kinase depended not on the synthesis but on the phosphorylation of this enzyme. These results suggest that the activation of MAP kinase is involved in the regulation of meiotic maturation of porcine oocytes, and especially in the regulation after germinal vesicle breakdown.

Association of p34cdc2 and Cyclin B1 during Meiotic Maturation in Porcine Oocytes

The relative levels and association of p34cdc2 and cyclin B1 have been determined in pig oocytes during meiotic progression from G2 to metaphase II (MII). Fully grown G2-arrested porcine oocytes contained large amounts of free p34cdc2 and extremely small amounts of p34cdc2-cyclin B1 complex which did not increase in amount during the GV stage. Cyclin B1 is not synthesized in measurable quantities until 23 hr after the start of maturation. During the first metaphase (MI) the amount of both cyclin B1 and the p34cdc2-cyclin B1 complex increased sharply until 35 hr. Thereafter the amount of the p34cdc2-cyclin B1 complex increased again to 45 hr, resulting in higher levels of the complex in MII than in MI. Moreover, three distinct migration forms of p34cdc2 were detected in pig oocytes by immunoblotting with anti-PSTAIRE antibody, and most of the cyclin B1-associated p34cdc2 was detected in the lower band in MI oocytes but migrated in the second band during MII. These results suggest that cyclin B1 levels in porcine oocytes differ from those in starfish, clam, and Xenopus oocytes in which considerably higher concentrations of cyclin B1 appear to be present during the GV stage and that the nature of the association between p34cdc2 and cyclin B1 changes between the first and the second metaphase.

Effects of Vanadate on Meiotic Maturation of Porcine Oocytes in Vitro.

Oocyte maturation is triggered by the activation of maturation promoting factor (MPF). In activation of MPF during meiotic maturation of porcine oocytes, a possible involvement of phosphorylation and subsequent dephosphorylation of a inhibitory tyrosine residue in p34cdc2 was examined using vanadate, a potent protein tyrosine phosphatase inhibitor. Porcine oocytes were cultured in porcine follicular fluid supplemented with pregnant mare's serum gonadotrophin with or without vanadate for 48 h. Vanadate blocked germinal vesicle breakdown (GVBD) almost completely at the concentration of 250 and 500 μM. The addition of an effective dose of vanadate into the maturation medium at 24 or 30 h after the start of maturation culture revealed that the porcine oocytes before GVBD were sensitive to vanadate and had their GVBD blocked, but those that had undergone GVBD were not-sensitive to vanadate and reached to the second metaphase normally. When vanadate was removed after 24 h treatment, GVBD occurred in 64% of oocytes but most of them were arrested at the first metaphase with dispersed chromosomes and abnormal spindles. These results suggest that tyrosine dephosphorylation in p34cdc2 takes place during the first meiosis but not in the second meiosis and that a prolonged exposure to vanadate might deteriorate microtubules function of the oocytes.

Protein Synthesis in Porcine Follicular Oocytes during In Vitro Meiotic Maturation

To investigate protein synthesis associating with in vitro meiotic maturation in porcine oocytes, follicular oocytes were treated with cycloheximide (10 μg/ml) at 6 h intervals in the culture up to 48 h. Germinal vesicle breakdown(GVBD) but not chromatin condensation was blocked in the oocytes when they were treated at either 0, 6 or 12 h after culture. Also, the transition from metaphase-1(M1) to metaphase-2(M2) was prevented. Pronuclear formation was observed in the oocytes when treated at 24 to 30 h after culture. Using [35S]methionine, the profile of protein synthesis revealed that the proteins of 27, 47, 49 and 70kDa were detected only at 0 to 18 h and that new proteins of 25 and 63kDa were appeared from 24 to 48 h with presence of proteins of 39 and 92kDa throughout culture. These results suggest that in porcine oocytes GVBD and the transition from M1 to M2 require protein synthesis and that the remodelling of protein synthesis occurrs at 24 h after in vitro culture.

Fluctuation of histone H1 kinase activity during meiotic maturation in porcine oocytes

Porcine oocytes cultured in follicular fluid for various periods of up to 48 h were stained with Hoechst-33342 and classified according to maturation before assaying. Histone H1 kinase activity at metaphase I was approximately 10 times that at the germinal vesicle stage. An abrupt reduction in activity was observed in oocytes emitting the first polar body; then the activity increased again to the same level as at metaphase I. This pattern is similar to those reported in non-mammalian species and supports the concepts that histone H1 kinase is ubiquitous in eukaryotes and controls the meiotic cell cycle in mammals.

Further Studies Of Inhibitors Of In Vitro Mammalian Oocyte Maturation

Studies of luteinizing hormone (LH)-mediated serum inhibition and of follicular fluid and granulosa cell inhibition of in vitro porcine, ewe, and cow oocyte meiotic maturation have been carried out. A porcine serum inhibitor or inhibitors with a molecular weight above 15,000, similar to the ewe and cow serum inhibitor(s), have been demonstrated. An LH-inactivated inhibitor or inhibitors in fluid from medium-sized ewe and cow follicles have been shown and a minimally effective porcine follicular fluid inhibitor has been confirmed. The estimated molecular weight of these inhibitors is below 1,000. Granulosa cells from the three species were not found to be dramatically inhibitory. It is postulated that the follicular fluid inhibitor(s) may be derived from the larger serum molecule(s) by an action of LH within an intermediate cell type.