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J Kalous

Publications and source records attributed to J Kalous.

12 recordsLinked to original sources
PubMed2001

Resumption of meiosis induced by meiosis-activating sterol has a different signal transduction pathway than spontaneous resumption of meiosis in denuded mouse oocytes cultured in vitro.

The sterol 4,4-dimethyl-5-cholesta-8,14,24-trien-3-ol (follicular fluid meiosis-activating sterol [FF-MAS]) isolated from human follicular fluid induces resumption of meiosis in mouse oocytes cultured in vitro. The purpose of this study was to examine the hypothesis that differential signal transduction mechanisms exist for FF-MAS-induced and spontaneous in vitro resumption of meiosis in mouse oocytes. Mouse oocytes were dissected from ovaries originating from mice primed with FSH 48 h before oocyte collection. Mechanically denuded germinal vesicle (GV) oocytes were in vitro matured in medium supplemented with hypoxanthine and FF-MAS or allowed to mature spontaneously; both groups were exposed to individual compounds known to inhibit specific targets in the cell. After 20-22 h of in vitro maturation, resumption of meiosis was assessed as the frequency of oocytes in GV breakdown (GVBD) stage. Pertussis toxin (2.5 microg/ml) did not influence resumption of meiosis in either group. Dibutyryl cyclic GMP (320 microM) inhibited FF-MAS-induced GVBD, but not spontaneous GVBD, whereas the subtype 5 phosphodiesterase-inhibitor zaprinast (50 microM) inhibited GVBD in both groups. Microinjection of the catalytic subunit of cAMP-dependent protein kinase into oocytes inhibited spontaneous GVBD, but not FF-MAS-induced GVBD. An inhibitor of cytoplasmic polyadenylation, cordycepin (80 microM), inhibited or retarded spontaneous GVBD to a further extent than it did FF-MAS-induced GVBD. Spontaneous GVBD was more sensitive to the histone H1 kinase-inhibitor olomoucine (250 microM) than was FF-MAS-induced GVBD. Addition of the mitogen-activated protein kinase (MAPK)-inhibitor PD 98059 (50 microM), phospholipase C-inhibitor U-73122 (10 microM), p21(ras)-inhibitor lovastatine (250 microM), and the src-like kinase inhibitor PP2 (20 microg/ml) inhibited FF-MAS-induced GVBD, but not spontaneous GVBD. Both MAPKs, extracellular regulated kinase (ERK) 1 and ERK2, were phosphorylated under FF-MAS-induced meiotic resumption, in contrast to spontaneous meiotic resumption, in which ERK1 and ERK2 phosphorylation occurred 2 h after GVBD. In the present study, we show that FF-MAS acts through an MAPK-dependent pathway, and we suggest that src-like kinase, p21(ras), and phosphoinositide signaling lie upstream of MAPK in the FF-MAS-activated signaling pathway. Clearly, striking pathway differences are present between spontaneous versus FF-MAS-induced meiotic resumption.

Animals↗
PubMed1998

Interplay between CDC2 kinase and MAP kinase pathway during maturation of mammalian oocytes.

Two principal kinases, p34cdc2 kinase and MAP kinase play a pivotal role in maturation of mammalian oocytes. In the porcine and bovine oocytes both kinases are activated around the time of germinal vesicle breakdown (GVBD). Butyrolactone I (BL I), a specific inhibitor of cdk kinases, prevents effectively and reversibly resumption of meiosis in the porcine and bovine oocytes. Neither p34cdc2 kinase nor MAP kinase are activated in oocytes inhibited in the GV stage. The bovine oocytes maintained for 48 h in the medium supplemented with BL I, progress subsequently to metaphase II in 91%, their cumuli expand optimally and after in vitro fertilization they possess two pronuclei. When the cdc2 kinase is blocked in the porcine oocytes by BL I, MAP kinase, activated by okadaic acid treatment, is able to substitute cdc2 kinase and induce GVBD. The histone H1 kinase activity sharply decreases in the metaphase II oocytes treated by BL I and one or two female pronuclei are formed. These data indicate that BL I is a useful tool either for the two step in vitro culture of mammalian oocytes or for their activation in nuclear transfer experiments.

4-Butyrolactone↗
PubMed1998

Effects of follicle-stimulating hormone, bovine somatotrophin and okadaic acid on cumulus expansion and nuclear maturation of blue fox (Alopex lagopus) oocytes in vitro.

The meiotic competence and meiosis resumption of Blue fox (Alopex lagopus) oocytes from anoestrous animals were followed. Oocyte-cumulus complexes (OCC) were cultured in modified TC 199 medium with or without FSH, recombinant bovine somatotrophin (bST) and okadaic acid (OA). The results showed that oocytes less than 100 microns in diameter did not achieve germinal vesicle breakdown (GFBD) by 72 h of culture, which indicates their meiotic incompetence. Oocytes larger than 100 microns in diameter underwent GVBD after 48 h of culture (27%) and reached metaphase II (MII) after 72 and 96 h (20% and 27%) in control medium. Both bST and OA accelerated resumption of meiosis (bST: 55% GVBD and 42% MII after 48 h; OA: 66% GVBD after 18 h). In contrast, FSH significantly reduced meiosis resumption (only 3% GVBD and MII after 72 h) and induced changes in the shape of cumulus granulosa (CG) cells and F-actin assembly typical for cumulus expansion. However, the innermost layers of CG cells (corona radiata) remained connected with the oocyte via gap junctions until the end of culture. Cumuli of oocytes cultured in control, bST-supplemented or OA-supplemented medium did not expand (changes in cell shape and F-actin redistribution did not occur). Moreover, especially in media with bST and OA an increased detachment and rapid disconnection of their gap junctions with the oocyte were observed. These results suggest that under in vitro conditions FSH stimulates expansion of the CG cells and the attached membrana granulosa cells but in contrast it secures heterologous gap junctions between cytoplasmic processes of the corona radiata cells and oolemma during 3 days of culture. Thus, in agreement with the in vivo situation in which Canidae oocytes are ovulated in the GV stage, the cumulus, mainly corona radiata cells, controls resumption of meiosis in Blue fox oocytes under in vitro conditions also.

Animals↗
PubMed1996

Co-culture with pig membrana granulosa cells modulates the activity of cdc2 and MAP kinase in maturing cattle oocytes.

Bovine cumulus-enclosed oocytes, initially cultured up to diakinesis (8 h of initial culture) or metaphase I (12 h of initial culture), were subsequently co-cultured for 6 h in contact with pig membrana granulosa (PMG) cells and then assayed for histone H1 and MAP kinase activities. In addition, the phosphorylation state of ERK 1,2 proteins was determined by Western blotting. The alterations in nuclear envelope breakdown, meiotic spindle formation and the patterns of chromosome condensation were analysed by immunofluorescence and transmission electron microscopy. The diakinesis-stage oocytes (initially cultured for 8 h) already possessed high histone H1 kinase and MAP kinase activities that were correlated with condensed and partially individualised chromosomes. The ERK 1 and most ERK 2 proteins were partly phosphorylated. Following the 6 h co-culture of these oocytes with PMG a rapid decrease in MAP kinase activity and a slower decrease in histone H1 kinase occurred, as well as ERK 1 and ERK 2 dephosphorylation. Both kinase activities and ERK 1,2 phosphorylation were fully restored following the release of the oocytes from co-culture and a subsequent culture in the absence of PMG. Moreover, the clumped bivalents were reindividualised and 56% of these oocytes reached metaphase II after 20 h of culture without PMG. The metaphase I oocytes, initially cultured for 12 h, displayed a fusiform meiotic spindle and a metaphase array of chromosomal bivalents, accompanied by high levels of both histone H1 and MAP kinase activity. Co-culture of MI oocytes with PMG abolished the activity of both kinases and caused the dephosphorylation of ERK 1 and ERK 2. Furthermore, the spindle microtubules were depolymerised and the chromosomal bivalents clumped into a single mass. Neither of the protein kinase activities nor the meiotic spindle were restored following subsequent culture in the absence of PMG for up to 20 h. These observations indicate that under in vitro conditions membrana granulosa cells can cause a prompt decrease in histone H1 and MAP kinase activities, and metaphase I oocytes. While these events are fully reversible in late diakinesis oocytes, metaphase I oocytes did not complete maturation after release from co-culture.

Animals↗
PubMed1993

Pig membrana granulosa cells prevent resumption of meiosis in cattle oocytes.

Membrana granulosa was isolated from healthy large antral follicles of prepubertal or cyclic gilts stimulated with PMSG or PMSG and hCG. Ultrastructural observations revealed that pieces of pig membrana granulosa were associated with the basement membrane. The cattle cumulus-enclosed oocytes (COC) were placed in the rolled pieces of the pig membrana granulosa (PMG). After 8 and 24 hr of coculture with PMG from prepubertal gilts, only 16% and 21% of oocytes underwent GVBD, respectively. PMG from PMSG-stimulated cyclic gilts blocked the resumption of meiosis in all COC. The inhibitory effect of heterologous granulosa cells was fully reversible. When COC were initially incubated for 2 and 4 hr, subsequent culture in PMG prevented GVBD in 100% and 36% of oocytes, respectively. This suggests that functional contact between COC and PMG was established during the first 2 hr of coculture. To follow metabolic cooperation between PMG and COC, PMG was prelabeled with 3H-uridine and cocultured with COC. Autoradiography on semithin sections revealed the intensive passage of 3H-uridine from PMG into the cumulus layer and an oocyte. COC placed in PMG after GVBD (8 and 12 hr of an initial incubation) did not extrude the first polar body. PMG isolated from cyclic gilts after PMSG and hCG stimulation also inhibited GVBD of COC. Since nearly all COC placed in PMG isolated 10 and 12 hr after hCG remained in the GV stage after 24 hr of coculture, the hCG stimulation did not substantially diminish the meiosis inhibiting activity of PMG.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Okadaic acid accelerates germinal vesicle breakdown and overcomes cycloheximide- and 6-dimethylaminopurine block in cattle and pig oocytes.

Pig and cattle oocytes, when released from the follicle, spontaneously resume first meiotic division within 20 or 8 hr, respectively. In oocytes of both species, the activity of histone H1 kinase increases during maturation, exhibiting a maximum in metaphase I. Treatment of these oocytes with okadaic acid results in acceleration of germinal vesicle breakdown (GVBD) and of histone H1 kinase activation. This effect is more important in pig oocytes, in which the acceleration rises for 6 hr, as compared to 2 hr in cattle. Moreover, under these conditions, H1 kinase activity measured after 12 hr of culture appears higher than that observed in control metaphase I oocytes. When added to prophase oocytes, both cycloheximide and 6-DMAP (6-dimethylaminopurine) block GVBD and histone H1 kinase activation. Okadaic acid, at a concentration of 2.5 microM, is able to release the inhibitory effect exerted by cycloheximide on histone H1 kinase activity; however, GVBD occurred only in two-thirds of pig and one-quarter of cattle oocytes after 20 hr of culture. In addition, okadaic acid fully reverses the effect of 6-DMAP on H1 kinase activity and on GVBD in both species. The opposite effects of 6-DMAP and okadaic acid on MPF activation are discussed, as well as the nature of the protein, which has to be synthesized during the first meiotic division and may be involved in the MPF activation cascade.

Adenine↗
PubMed1993

Is cAMP decrease essential for resumption of meiosis in mouse oocytes?

Resumption of meiosis was inhibited in mouse oocyte cumulus complexes (OCC) co-cultured with pig membrane granulosa (PMG). After 3 and 6 h of co-culture these oocytes possessed an intact nuclear envelope and their nucleolar surface was associated with granules approximately 80 nm in diameter. Preincubation of OCCs for 30, 45, 60 or 90 min followed by co-culture with PMG for 2 h of either OCCs or denuded oocytes resulted in germinal vesicle breakdown (GVBD) in approximately 0, 30, 70 and 100% mouse OCCs and in approximately 30, 60, 80 and 100% denuded oocytes, respectively. It seems that the inhibitory contact between mouse oocytes and PMG was established during the first h of co-culture. After isolation from antral follicles the oocytes contained 2.75 fmol cyclic adenosine 3', 5'-monophosphate (cAMP). When OCCs were co-cultured for 1, 2 or 3 h with PMG, the amount of cAMP per oocyte was 1.34, 1.33 and 1.51 fmol, respectively. After culture of OCCs in control medium the amount of cAMP was 1.21, 1.39 and 2.16 fmol, respectively. The present results suggest that the inhibitory activity of PMG is not species-specific. Moreover, PMG prevented resumption of meiosis in mouse oocytes in spite of the cAMP drop in oocyte cytoplasm characteristic of the resumption of meiosis.

Animals↗
PubMed1989

[The physician and the computer. 30. Evaluation of time, different aspects of the same problem].

The authors mention different ways in which a give problem can be described within the framework of the theory of sets, logic, mathematical analysis, statistic and cybernetics. As an example of the confrontation of the mentioned aspects they discuss different approaches to the evaluation of time as a variable--temporal logic, Markov chains, theory of catastrophes which play a part in medicine, in particular in prognostic considerations. These different aspects make possible solutions which may differ and their application depends in particular on the purpose for which they are intended.

Logic↗