Biomedical subjects

F P Daen

Publications and source records attributed to F P Daen.

5 recordsLinked to original sources

PubMed1995

Histone H1 kinase activity during in vitro fertilization of pig follicular oocytes matured in vitro.

The present study was conducted to clarify the relationship between histone H1 kinase (H1K) activity and events associated with in vitro fertilization of pig follicular oocytes matured in vitro. Histone H1 kinase has been shown to be homologous with a maturation promoting factor (MPF). Cumulus-oocyte complexes obtained from prepubertal gilts were cultured for 46 h in a modified Waymouth's MB752/1 medium and were then inseminated in vitro with frozen-thawed and preincubated epididymal boar spermatozoa. At 4, 6, 8 and 10 h post insemination, the oocytes were stained with 10 microg/ml Hoechst-33342 and examined under a fluorescent microscope for the stage of fertilization, according to morphological changes of oocyte nuclear chromatin and the extent of sperm penetration. Sperm penetration was observed to occur within 4 h post insemination (20.5%), and the percentage of fertilized oocytes increased (P < 0.01) to 72.9% at 8 h post insemination. Pronuclear formation was observed from 6 h post insemination (3.3%) and the percentage increased (P < 0.01) to 46.8% at 10 h post insemination. In each examination period, H1K activities in unfertilized oocytes at metaphase-II remained unchanged (112.0 fmol/h/oocyte) and were higher (P < 0.01) than those in fertilized oocytes (30.1 fmol/h/oocyte). The H1K activity in fertilized oocytes such as oocytes emitting a second polar body, oocytes with an enlarging sperm head(s) and oocytes with multiple pronuclei did not differ significantly. These results suggest that MPF in pig oocytes is inactivated shortly after sperm penetration and is maintained at the basal level throughout pronuclear formation.

Journal Article↗

PubMed1995

Serum factor(s) stimulating cumulus expansion in porcine oocyte-cumulus complexes matured and fertilized in vitro.

We have investigated the factor(s) present in serum that stimulate cumulus expansion (accumulation of hyaluronic acid in the intercellular space of cumulus cells) and male pronucleus (mPN) formation in porcine oocytes matured and fertilized in vitro. After 24 hr of culture, oocyte-cumulus complexes that matured in the serum of pig, cow, rabbit and rat showed an expansion of the surrounding cumulus cell matrices. The cumulus expansion stimulating effect is not species-specific. Porcine serum was fractionated by ultracentrifugation at 220,000 x g at 10 degrees C for 48 hr, resulting in four fractions. The oocyte-cumulus complexes that matured in the 2nd fraction exhibited a marked expansion of the cumulus cell layers, including the corona radiata. Complexes cultured in the remaining fractions, however, showed very little or no expansion. There was no expansion of the oocytectomized complexes matured in the 2nd fraction. Serum was not effective in the formation of the male pronucleus. The active factor(s) was heat stable at 100 degrees C for 30 min and showed a relatively strong activity at a molecular weight of > 1.0 and < 6.5 kDa. These results indicate that serum contains a low molecular weight, heat stable factor(s) that acts on the oocyte to stimulate cumulus expansion.

Animals↗

PubMed1995

Decrease of histone H1 kinase activity in relation to parthenogenetic activation of pig follicular oocytes matured and aged in vitro.

Changes of histone H1 kinase activity before and after electrical stimulation were connected with the ability of cytoplasm of pig oocytes to be activated parthenogenetically when matured and aged in vitro. Cumulus-oocyte complexes were collected from prepubertal gilts and cultured in a modified Waymouth's MB752/1 medium. The first mature oocytes were observed after 30 h of culture. After 36 h of culture, about 65% of oocytes had matured (reached metaphase II stage with the first polar body). When oocytes matured after 36 h of culture were stimulated with an electric pulse and subsequently cultured for 10 h, only 7% became parthenogenetically activated (formation of a female pronucleus). When oocytes matured for 60 h and 72 h underwent the same treatment, significantly more became activated parthenogenetically (46% and 57%, respectively). Oocytes matured for 72 h but not stimulated electrically also exhibited high spontaneous parthenogenetic activation (24%). Activation of oocytes resulted either in the formation of a female pronucleus(ei) or in fragmentation of oocytes. Fragmentation in stimulated and nonstimulated oocytes increased significantly after 72 h of culture (37% and 18%, respectively). Histone H1 kinase activity in immature oocytes at the germinal vesicle stage was low (17.2 fmol h-1 per oocyte). However, when oocytes were cultured for 36 and 48 h, histone H1 kinase activity increased significantly (168.2 and 138.5 fmol h-1 per oocyte, respectively). Prolonged culture (60 h and 72 h) resulted in a significant decrease in histone H1 kinase activity (94.3 and 49.3 fmol h-1 per oocyte, respectively). When oocytes cultured for up to 72 h were electrically stimulated, histone H1 kinase activity in activated oocytes (oocytes that formed a female pronucleus and fragmented oocytes) was significantly lower (24.7 mol h-1 per oocyte) than that in nonactivated oocytes (99.9 mol h-1 per oocyte). The present data clearly indicate that the gradual decrease of histone H1 kinase activity is correlated with ageing of oocytes matured in vitro and with their ability to be parthenogenetically activated.

Animals↗

PubMed1994

The effect of pig follicular fluid fractions on cumulus expansion and male pronucleus formation in porcine oocytes matured and fertilized in vitro.

The aim of this study was to analyse the substance(s) present in porcine follicular fluid that promotes cumulus expansion and male pronucleus formation in pig oocytes matured and fertilized in vitro. The follicular fluid was separated into four fractions by ultracentrifugation at 220,000 g at 10 degrees C for 48 h. Oocyte-cumulus-cell complexes obtained from prepubertal gilts were cultured in vitro for 48 h in each of the fractions after reconstitution with a modified Krebs-Ringer bicarbonate solution. Each fraction containing oocyte-cumulus complexes was then fertilized in vitro with frozen-thawed and preincubated epididymal boar spermatozoa. After 24 h of culture, oocytes that matured in fraction 1 showed a marked expansion of the surrounding cumulus, as did those matured in pig follicular fluid. However, complexes cultured in the remaining fractions exhibited very little or no expansion. No difference was observed in the degree of expansion caused by follicular fluid collected from small, medium or large follicles. Further analysis of fraction 1 by HPLC gave several subfractions, one of which (22) induced a significantly greater expansion of the cumulus than did others (P < 0.01). Fraction 1 also induced higher rates of male pronuclei formation (62%) than did fractions 2, 3 and 4 (22%, 35% and 30%, respectively). The rate of male pronuclei formation in pig follicular fluid from large follicles was significantly lower (33%) than in fluid from small or medium follicles (89% and 78%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Comparison of histone H1 kinase activity during meiotic maturation between two types of porcine oocytes matured in different media in vitro.

Histone H1 kinase (H1K) activity was assayed during meiotic maturation in porcine oocytes matured in a modified Krebs-Ringer bicarbonate solution (KRB) or in porcine follicular fluid (pFF) in vitro. Oocytes matured in KRB displayed lower male pronucleus formation ability, delayed first polar body emission, and a higher spontaneous activation rate than oocytes matured in pFF. In oocytes matured in pFF, H1K activity was low at the germinal vesicle stage and increased about 8-fold at first and second metaphases, with a transient depression at first anaphase and telophase. The H1K activity at second metaphase in oocytes matured in KRB was significantly lower than that in oocytes matured in pFF. These results suggest that the maturation medium used influences the fluctuation pattern of H1K activity and the biological characteristics of porcine oocytes cultured in vitro.

Animals↗