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J Motlík

Publications and source records attributed to J Motlík.

At least 19 recordsLinked to original sources
PubMed1998

Secretion of cumulus expansion-enabling factor (CEEF) in porcine follicles.

The objective of this study was to find out whether porcine cumulus and mural granulosa cells can secrete cumulus expansion-enabling factor (CEEF). Culture drops of M-199 medium were conditioned with denuded porcine oocytes (1 oocyte/microliter), cumulus cells from oocytectomized complexes (1 OOX/microliter), pieces of mural granulosa isolated from preantral to preovulatory follicles (1000 cells/microliter), or oviductal cells (1000 cells/microliter) for 24 hr. The production of CEEF was assessed by the addition of mouse OOX and follicle-stimulating hormone (FSH) (1 microgram/ml) to microdrops of the conditioned medium. After 16-18 hr, expansion of the mouse OOX was scored on a scale of 0 to 4 by morphologic criteria. Mouse OOX did not expand in nonconditioned FSH-supplemented medium. Immature porcine oocytes produced +3 to +4 expansion of the mouse OOX. Granulosa cells isolated from preantral and early antral follicles and cumulus cells isolated from all states of follicle development constitutively secreted CEEF under in vitro conditions. Mural granulosa cells of small, medium, and preovulatory (PMSG) follicles also secreted CEEF in vitro; however, FSH or leutenizing hormone (LH) stimulation was essential for this secretion. Hormonally induced secretion of CEEF was accompanied by expansion of the mural granulosa itself. Granulosa cells isolated from follicles of gilts 20 hr after PMSG and human chorionic gonadotropin (hCG) administration did not produce CEEF and did not expand in response to FSH and LH in vitro. CEEF activity also was found in the follicular fluid of small antral follicles, was reduced in medium follicles, and was not detectable in PMSG-stimulated follicles. However, CEEF activity was reestablished in the follicular fluid of preovulatory follicles by hCG injection, conceivably due to increased production of CEEF by cumulus cells. We conclude that (1) porcine cumulus and mural granulosa cells are capable of CEEF production in vitro and (2) autocrine secretion of CEEF by cumulus cells is involved in regulation of porcine cumulus expansion both in vitro and in vivo.

Animals↗
PubMed1997

Establishment of the block against sperm penetration in parthenogenetically activated bovine oocytes matured in vitro.

The ability of a single electric pulse to mimic a block against sperm penetration in bovine oocytes matured in vitro was investigated. Confocal laser scanning microscopy detected a global loss of spots, presumed to be cortical granules, stained with Lens culinaris agglutinin, in pulsed oocytes. Transmission electron microscopy revealed that cortical granule exocytosis occurred within 1 min of stimulation and the number of remaining cortical granules was significantly reduced in all pulsed oocytes. The ability of pulsed oocytes to undergo fertilization in vitro was also affected, as only 31% of the pulsed oocytes were penetrated compared with 87% in the control group. Since incidences of penetration in pulsed oocytes (31%), and of polyspermy in control oocytes (18%) did not differ and were highly correlated (P = 0.009) among trials (n = 15), the induced block is considered to be comparable with the natural block triggered by a spermatozoon. The increased resistance of the zona pellucida to pronase E observed in pulsed oocytes suggests that the induced block depends, at least partly, on modifications of zona pellucida glycoproteins. Finally, the majority (66%) of pulsed, penetrated oocytes did not form male pronuclei, probably as a consequence of asynchrony between the formation of female pronucleus and sperm penetration. The reduced ability of the cytoplasm to induce the formation of a male pronucleus was accompanied by a fall in histone H1 kinase activity to basal values by 3 h after stimulation. These results demonstrate that a single electric pulse can induce a block against sperm penetration similar to that of the spermatozoon itself.

Acrosome↗
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↗
PubMed1995

Inhibition of protein synthesis affects histone H1 kinase, but not chromosome condensation activity, during the first meiotic division of pig oocytes.

The influence of protein synthesis on the regulation of the first meiotic division was studied in pig oocytes. We show that histone H1 kinase activity gradually increases during in vitro culture of pig oocytes, reaching maximum in metaphase I stage after 24 hr of culture. However, in the presence of the protein synthesis inhibitor cycloheximide, histone H1 kinase is not activated during the whole culture period, and after 24 hr it is approximately at the same level as in prophase-stage oocytes. The gradual increase in phosphorylation of six proteins of molecular weights 39, 48, 53, 66, 96, and 120 kDa, observed during the first 24 hr of culture, was not detected when cycloheximide was added to the culture medium. Similarly, the decrease in phosphorylation of a 90-kDa protein was not seen in cycloheximide-treated oocytes. On the other hand, the levels of both MPF components, p34cdc2 and cyclin B, which were found to be nearly constant during the first meiotic division, were not influenced by cycloheximide treatment as revealed by Western blotting. The process of germinal vesicle breakdown (GVBD) was totally blocked by cycloheximide. The condensation of chromatin, however, was not influenced, suggesting that GVBD and chromosome condensation could be regulated independently. The different degrees of MPF activation involved in these processes, as well as the nature of the protein(s) which must be synthesized for triggering GVBD, are discussed.

Animals↗
PubMed1994

Chromatin condensation and histone H1 kinase activity during growth and maturation of rabbit oocytes.

Fully grown rabbit oocytes, isolated from preovulatory follicles, exhibit highly condensed bivalents within an intact germinal vesicle while a very low level of histone H1 kinase activity could be detected in their extracts. Chromatin condensation started in growing oocytes isolated from antral follicles presenting a diameter of 0.5 mm. This event was accompanied by a transient rise in histone H1 kinase activity which culminated in large antral follicles measuring 0.75 to 1 mm in diameter. However, the extent of histone H1 kinase activity observed in these growing oocytes remained far less important than that recorded in extracts prepared from in vitro cultured metaphase I and metaphase II oocytes. Moreover, this activity was insufficient to induce germinal vesicle breakdown which will only occur with an increasing efficiency, following in vitro culture of medium, large, and fully grown antral follicles.

Animals↗
PubMed1993

Ultrastructural cytochemistry of the nucleus and nucleolus in growing rabbit oocytes.

Ultrastructural changes of the germinal vesicle during the growth of rabbit oocytes were studied by means of light and electron microscopy, 3H-uridine autoradiography, Ag-NOR staining and E-PTA staining. Particular interest was paid to the nucleologenesis and condensation of chromatin. In contrast to other mammalian species, chromosome condensation in rabbit oocytes occurred concomitantly with rRNA synthesis-dependent nucleolar compaction and preceded nuclear envelope breakdown and resumption of meiosis.

Animals↗
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↗
PubMed1992

Development of pronuclei in pig oocytes activated by a single electric pulse.

Pig oocytes were matured in vitro in a modified M-199 medium for 44 h, subjected to electrical stimulation and scored for activation 6 h later. Sham pulsed oocytes, exposed to electroporation medium and an a.c. field, did not develop the female pronucleus any more frequently than occurs spontaneously (8.3% within 50 h of culture). However, a single d.c. pulse proved extremely efficient in activating pig oocytes. Pulses of 0.75-1.65 kV cm-1 lasting 30 or 100 microseconds activated at least 90% of matured oocytes. The developmental pathway taken by the activated oocytes depended on the parameters of the pulse. The lowest effective stimulation (0.45 and 0.60 kV cm-1 for 30 microseconds) frequently produced oocytes that remained in pre-pronuclear stages of activation (29.4 and 42.3%, respectively). Extrusion of the second polar body and creation of one pronucleus was the most frequent type of activation (in up to 88.2% among the activated oocytes). The strongest stimulations used (1.05-1.65 kV cm-1 for 100 microseconds) often yielded oocytes that failed to extrude the second polar body and formed two or more pronuclei (up to 56.3%). Under optimal stimulation (0.75 kV cm-1), the activated oocytes proceed synchronously to interphase of the first mitotic division. Anaphase II is reached within 30 min and telophase Ii at 1 h after application of the pulse. The second polar body is extruded about 2 h after activation. Well-defined swelling pronuclei were found in oocytes 5-6 h after activation. The relationship between the stage of oocyte maturation and susceptibility to activation was investigated. The period of culture in which the oocytes develop the activation competence (32-36 h of culture) overlapped with the period in which the oocytes complete meiosis (28-38 h). This suggests that ageing in meiotic arrest is not essential for pig oocytes to become activated by electric pulses. Activation of pig oocytes was accompanied by release of cortical granules. In sections of control (metaphase II) oocytes, an average of 7.3 intact cortical granules per 10 microns of overlying cytoplasmic membrane was found. This number dropped to 1.5 in 10 microns within 30 min after the pulse.

Animals↗
PubMed1991

Follicle and oocyte growth in early postnatal calves: cytochemical, autoradiographical and electron microscopical studies.

The initiation of oocyte and follicle growth was studied in 1- and 3-d-old calf ovaries using cytochemical, autoradiographical and electron microscopical approaches. Attention was only paid to unilaminar ovarian follicles that were classified into 3 categories: unilaminar flattened (UF), unilaminar flatto-cuboidal (UFC) and unilaminar cuboidal (UC) ovarian follicles when the oocyte was surrounded by 1 layer of flattened, a mixture of flattened and cuboidal and entirely cuboidal follicle cells, respectively. Our findings suggested that oocytes within each of these follicle categories were in different developmental stages. Furthermore, electron microscopic observations revealed that early after birth, oocyte nuclei characteristic of diplotene configuration (aggregation of the nuclear chromatin into moderately electron-dense small patches and fibrillo-granular texture of the nucleolus) were encountered in 41% of the UF follicles. The rest of the UF as well as all of the UFC and UC follicles were found to contain dictyate oocytes in which the chromatin was highly decondensed and the nucleolus differentiated into fibrillar, fibrillo-granular and granular components. The present results also indicated that the complete transition of the surrounding follicle cells from flattened to cuboidal shape and the morphological changes of the oocyte endoplasmic reticulum and mitochondria were 2 complementary events essential for initiation of oocyte growth.

Animals↗
PubMed1991

Lack of effect of oocytectomy on expansion of the porcine cumulus.

Oocyte-cumulus cell complexes (OCC) and complexes with an attached piece of membrana granulosa (C + P), isolated from prepubertal or cyclic gilts stimulated with pregnant mares' serum gonadotrophin, were cultured in media supplemented with follicle-stimulating hormone (FSH; 0.01-1.0 micrograms/ml) or forskolin (50-100 mumol/l) for 24 and 32 h. FSH and forskolin each induced dose-dependent cumulus and membrana granulosa expansion. After 2 h of culture, FSH (0.1 microgram/ml) or forskolin (100 mumol/l) increased the contents of intracellular adenosine 3',5'-phosphate (cAMP) in OCC from prepubertal gilts to almost 10 times that in unstimulated complexes. After 24 h of culture in media supplemented with FSH (0.1 microgram/ml) or forskolin (100 mumol/l), the oocytectomized OCC and C + P showed similar expansion to that of the control groups. The intracellular cAMP contents in intact and oocytectomized OCCs were similar in all groups except those treated with FSH, in which the intact OCCs had significantly higher contents than their oocytectomized counterparts (P less than 0.01). After hyaluronidase treatment, cumulus and membrana granulosa cells of intact and oocytectomized OCC and C + P were suspended, except for those of the innermost layers of the corona radiata. The results suggest that increases in cAMP contents and synthesis of an extracellular, hyaluronidase-sensitive mucus by pig OCC and C + P induced by FSH or forskolin are not dependent on the oocyte.

Animals↗
PubMed1990

Combined effects of protein synthesis and phosphorylation inhibitors on maturation of mouse oocytes in vitro.

In denuded mouse oocytes, neither 3 nor 5 hours of preincubation in dbcAMP (1 mM) and cycloheximide (10 micrograms/ml), followed by further 3 hours in cycloheximide only, lowered the rate of GVBD (93% and 92%, respectively). It means that 3 and 5 hours preincubation in cycloheximide did not impair the ability of mouse oocytes to resume meiosis in medium with the protein synthesis inhibitor. To test the combined effects of inhibition of protein phosphorylation and protein synthesis, oocytes were cultured for 3, 4, or 5 hours in 2 mM of 6-DMAP and subsequently for 3 hours in 10 micrograms/ml cycloheximide. The incubation in 6-DAMP for 4 or 5 hours diminished (63% or 35% of GVBD, respectively) the ability of mouse oocytes to resume meiosis when subsequent protein synthesis was blocked by cycloheximide. However, the highly condensed bivalents were always visible in GVs. Thus the above treatment did not prevent chromatin condensation although GVBD was blocked.

Adenine↗
PubMed1990

Cell-cycle aspects of growth and maturation of mammalian oocytes.

In this review, recent data concerning growth and maturation of nonmammalian and mammalian female germ cells are compiled with regard to the increased understanding of somatic cells mitotic cycles, from yeast to human tissues. These data allow us to conclude that growing oocytes of nonvertebrates, lower vertebrates, and mammals resemble somatic cells in the G1 phase of the mitotic cycle in their metabolic and cell cycle behavior. Transcriptional and translational activity of growing oocytes and G1 somatic cells is not compatible with the activation of maturation promoting factor (MPF), with chromatin condensation or with nuclear membrane disintegration. Growing oocytes, even when they are in the dictyate stage of the first meiotic division, promptly inactivate MPF introduced into their cytoplasm by fusion or microinjection, just as do somatic interphase cells. In mammals, the LH surge induces "de novo" RNA and protein synthesis in granulosa cells. This metabolic change in granulosa cells abolishes their inhibitory activity, and meiosis in fully grown oocytes in preovulatory follicles is then resumed. Resumption of meiosis requires an activation of pre MPF molecules within oocytes. This can be achieved either without (mouse, rat, and rabbit) or with (pig, sheep, and cow) an active protein synthesis by the oocytes. The species specificity is probably dependent on the presence or absence of cyclin-like and/or mos-like molecules in fully grown oocytes. Both major events during GVBD, chromatin condensation, and nuclear envelope disintegration require protein phosphorylation. Experimentally, these two phosphorylation activities can be separated one from another. The active MPF molecules are amplified autocatalytically in amphibian and starfish oocytes. However, an increase of MPF activity in mouse and pig oocytes, similarly as in Rana pipiens and sturgeon oocytes, requires an active protein synthesis.

Animals↗
PubMed1990

Two sensitivity levels of cattle oocytes to puromycin.

Germinal vesicle breakdown (GVBD) in cumulus-enclosed and denuded cattle oocytes was sensitive to puromycin at concentrations at or above 50 micrograms/ml. Media supplemented with 5-25 micrograms/ml of puromycin did not significantly reduce either rate or sequence of GVBD after 8 h of culture (82-96% GVBD). In concentrations of 50, 75, and 100 micrograms/ml, GVBD occurred in 15, 4, and 2% of oocytes, respectively. However, 50 micrograms puromycin/ml did postpone the time sequence of GVBD, since all treated oocytes underwent GVBD after 20 h of culture. Oocytes arrested in the germinal vesicle (GV) stage possessed GV filled with highly condensed bivalents. The puromycin block (100 micrograms/ml) was fully reversible, and the time sequence of GVBD was two times faster than in control medium. Proteins important for GVBD were synthesized during the first 4 h of culture, and 81% of oocytes underwent GVBD when puromycin (100 micrograms/ml) was added after 4 h of preincubation in control medium. The first polar body (I PB) expulsion was more sensitive to inhibition of protein synthesis, as shown by the observation that 2.5 and 5 micrograms puromycin/ml significantly (69 and 61%) reduced the incidence of Metaphase II, and 10 micrograms/ml highly significantly (31%) reduced it. The I PB expulsion in concentrations of 25 and 37 micrograms puromycin/ml was less than 5%. The subsequent culture in puromycin (8 h) and 6-dimethylaminopurine (8 h) proved that nuclear membrane breakdown is less sensitive to inhibition of protein phosphorylation than the process of chromatin condensation.

Adenine↗
PubMed1989

Activity of maturation promoting factor in pig oocytes after microinjection and serial transfer of maturing cytoplasm.

Microinjection of 120 pl homologous or heterologous (mouse) maturing cytoplasm into immature pig oocytes resulted in germinal vesicle breakdown (GVBD) and chromosome condensation (CC) after 8 h of culture. In contrast, nearly all control oocytes remained at the germinal vesicle stage. Immature cytoplasm did not induce nuclear maturation of recipient oocytes when injected. The cytoplasm of the oocytes injected with maturing cytoplasm preserved the capacity to cause GVBD in the second recipients. These results suggest that the level of injected maturation promoting factor (MPF) must rise before GVBD can occur. When the pig oocytes injected with 120 pl of mouse maturing cytoplasm were cultured in cycloheximide for 8 h, the presence of GVBD was dramatically reduced. Thus, in pig oocytes the increase in active MPF up to the effective concentration requires protein synthesis.

Animals↗
PubMed1989

RNA and protein synthesis requirements for the resumption of meiosis in rabbit oocytes: the role of cumulus cells.

In vitro maturation of rabbit cumulus-enclosed oocytes was fully inhibited in alpha-amanitin- (100 micrograms/ml) and cycloheximide- (5 micrograms/ml) supplemented media. The inhibition was reversible and substantially reduced by delaying the addition of alpha-amanitin (2h) or cycloheximide (3 h). In contrast, both drugs did not inhibit germinal vesicle breakdown in denuded oocytes. Co-culture of granulosa cells (1 x 10(6)/ml) with denuded oocytes did not substitute for an intact cumulus. The data presented here suggest that the resumption of meiosis in rabbit cumulus-enclosed oocytes is dependent upon early transcriptional and translational events which probably occur within the cumulus cells.

Amanitins↗
PubMed1989

In vitro techniques of bovine oocyte maturation, fertilization and embryo culture resulting in the birth of a calf.

Oocyte cumulus complexes were aspirated from 3 to 5 mm follicles of cows prestimulated with 2.000 IU PMSG 24 h before slaughter. Oocytes matured in culture were fertilized in vitro by heparinized freshly ejaculated or epididymal spermatozoa. The cultivation procedure for fertilized eggs was the same as that used for cultivation of oocytes. From 163 matured oocytes, 109 cleaved to the 2-cell stage 24 h after fertilization and after 6 days of cultivation, 18 developed to the late morula and 18 to the blastocyst stages. Eleven blastocyts and 1 late morula were transferred surgically to the uteri of 7 recipient heifers. Two heifers became pregnant: one delivered a bull-calf at term, while the other pregnancy resulted in abortion at the 3rd month. The examination of some embryos by transmission electron microscopy showed an almost normal morphology for most cells. The degenerated cells contained mostly electron-dense residual bodies of unknown origin.

Animals↗
PubMed1988

Does autocatalytic amplification of maturation-promoting factor (MPF) exist in mammalian oocytes?

The method of polyethylene-glycol-induced fusion of mammalian oocytes was applied to study maturation-promoting factor (MPF) activity. After homologous fusions of one maturing--late diakinesis (LD), metaphase I (MI)--pig or mouse oocyte to one, two, or three immature-germinal vesicle (GV)--oocytes, giant cells were cultured in control or cycloheximide supplemented medium for 3 hours. The occurrence of germinal vesicle breakdown (GVBD) and premature chromosome condensation (PCC) served as a control of MPF activity. In giant cells composed of one maturing and one, two or three immature oocytes, GVBD and PCC were observed in all cases after cultivation in the control medium. In the presence of cycloheximide, the completion of GVBD and PCC remained high when one maturing and one immature oocyte were fused (83.7% and 95.7% of GVBD in pig and mouse, respectively). However, in giant cells composed of one maturing and up to three immature oocytes, all GVs were broken down only occasionally (4.8% and 11.7% in pig and mouse, respectively). These results suggest that in pig and mouse oocytes MPF does not amplify autocatalytically, but requires active protein synthesis for its production.

Animals↗