The ran GTPase regulates mitotic spindle assembly.

Ran is an abundant nuclear GTPase with a clear role in nuclear transport during interphase but with roles in mitotic regulation that are less well understood. The nucleotide-binding state of Ran is regulated by a GTPase activating protein, RanGAP1, and by a guanine nucleotide exchange factor, RCC1. Ran also interacts with a guanine nucleotide dissociation inhibitor, RanBP1. RanBP1 has a high affinity for GTP-bound Ran, and it acts as a cofactor for RanGAP1, increasing the rate of GAP-mediated GTP hydrolysis on Ran approximately tenfold. RanBP1 levels oscillate during the cell cycle [4], and increased concentrations of RanBP1 prolong mitosis in mammalian cells and in Xenopus egg extracts (our unpublished observations). We investigated how increased concentrations of RanBP1 disturb mitosis. We found that spindle assembly is dramatically disrupted when exogenous RanBP1 is added to M phase Xenopus egg extracts. We present evidence that the role of Ran in spindle assembly is independent of nuclear transport and is probably mediated through changes in microtubule dynamics.

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.

[Reliability of pregnancy diagnosis in sows using direct radioimmunoassay of estrone-sulfate]. / Spolehlivost diagnostiky gravidity u prasnic prímou radioimunoanalýzou estron-sulfátu.

The pregnancy diagnosis in sows using direct radioimmunoassay of estrone-sulphate in the blood serum without sample extraction is described. It was found that for the pregnancy diagnosis the period between days 22 and 30 of pregnancy can be used since in this period the estrone-sulphate concentrations in all pregnant sows markedly exceeded those of 64 non-pregnant animals. The estrone-sulphate estimation cannot be used for the pregnancy diagnosis before day 22 and between day 30 and 40 because the estrone-sulphate concentrations in most samples collected in these periods were lower than 4 nmol.l-1.

[The effect of repeated administration of Oestrophan (SPOFA) on the quality of sexual function in breeding stock]. / Stanovení vlivu opakovaného podání Oestrophanu (SPOFA) na kvalitu pohlavních funkcí plemenic.

The objective of the study was to check a contingent negative effect of repeated treatment of heifers by PGF2 alpha--Oestrophan (SPOFA)--on the course and quality of their ovarian activity and external heat signs. Changes in progesterone concentrations were evaluated three times a week. Three heifers were subjected to treatment eight times and two heifers nine times. Of 42 applications of Oestrophan (SPOFA) the response was positive in 95.2% (forty heats). When Oestrophan (SPOFA) was administered on the fourth and sixth day of the sexual cycle, in two cases no heat nor luteolysis of corpus luteum was detected; this was demonstrated by hormonal investigation. Of the forty evaluated heats, 82.5% were classified as strongly and medium expressive, the remaining were weakly expressive. The onset of heat was found to be on the average 72.6 hours after Oestrophan (SPOFA) administration, in 9.1% of the cases the heat started about 48 hours after administration, 12.4% of cows showed the heat signs between 72nd and 96th hours.

[Levels of progesterone, 17 beta-estradiol and 11-hydroxycorticosteroid in the blood in cows in different types of puerperal conditions]. / Koncentrace progesteronu, 17 beta-estradiolu a 11-hydroxykortikosteroidü v krevní plazme krav pri rozdílném prubehu puerperia.

Changes in progesterone (P4), 17 beta-estradiol (E2) and 11-hydroxycorticosteroid (11-OHCS) concentrations in blood plasma were determined in daily intervals from the day of parturition to day 15 post partum (p.p.) in cows with physiological puerperium (n = 8), with puerperal endometritis (n = 6), and with placenta retention (n = 6). Cows with puerperium disorders (endometritis, placenta retention) had significantly higher P4 levels in the period from day 3 to day 7 p.p. than cows with physiological puerperium. E2 concentrations decreased to basal values following the parturition in cows with spontaneous parturitions and subsequent expulsion of the placenta. A delayed decrease in E2 concentrations after parturition and a significant increase on day 5 and day 7 p.p. were recorded in the group of animals with placenta retention. Significantly higher levels of 11-OHCS in blood plasma were detected by day 5 or by day 8 p.p. in cows with placenta retention and puerperal endometritis. Extraovarial sources of sexual steroids resulting in endocrine malfunctions are discussed as well as likely consequences for puerperium.

[The effect of the light schedule on biochemical indicators in the blood, serum and urine of heifers during their estrus cycle]. / Vliv svetelného rezimu na biochemické ukazatele krve, séra a moce jalovic v prubehu ríjového cyklu.

The dynamics of 38 biochemical parameters of blood, serum and urine was studied in ten heifers during their oestrous cycle in a light house (80-340 lux) and a dark small house (10-40 lux). In the light stable a significant influence (P less than 0.05) was found to be exerted on five parameters and in the dark stable nine parameters, four being influenced in the same way in both houses: haematocrit value and haemoglobin in blood and cholesterol and progesterone in serum; the relationship of progesterone content in serum to the days of oestrous cycle was particularly high in the light house. The comparison of the parameters between the groups demonstrated a significant (P less than 0.05) influence of light regime, exerted on some days of the oestrous cycle upon the levels of inorganic phosphorus, total protein and aspartate aminotransferase activity (AST) in serum and upon haematocrit, haemoglobin and ketone bodies in the blood of heifers. However, the light regime did not influence the levels of progesterone which were somewhat higher in the light house but with no statistically significant difference from the dark house. A significant difference between the groups was obtained in the levels of sodium and phosphorus in urine (P less than 0.05) between the first and tenth days of the oestrous cycle. No differences in the oestrous cycle between the heifers in the light and dark stables were recorded. Neither was the duration of heat influenced significantly; it was only less manifest in the dark stable.

Phenotyping of pig alpha 1B-glycoprotein (PO2) and haemopexin by 1D polyacrylamide gel electrophoresis and immunoblotting.

Described is an alternative procedure for the phenotyping of pig alpha 1B-glycoprotein (PO2) and haemopexin. The procedure is based on the separation of serum samples by horizontal polyacrylamide gel electrophoresis, passive blotting onto a nitrocellulose (NC) sheet, and immunochemical detection using a mixture of a primary antibody (rabbit anti-pig alpha 1B or anti-pig haemopexin) and a peroxidase-labelled secondary antibody. Several NC copies can be obtained from a single gel and these can be developed with different monospecific antisera.

Fertilisation competence of bovine normally matured or aged oocytes derived from different antral follicles: morphology, protein synthesis, H1 and MBP kinase activity.

We have investigated the fertilisation competence, protein synthesis, histone H1 kinase and myelin basic protein (MBP) kinase activities in three categories of bovine oocytes (derived from three size categories of follicles: M--medium, 2.5-5.0 mm; S--small, 1.5-2.5 mm; T--tiny, 1.0-1.5 mm). In contrast to more or less normal meiotic maturation (85.6%) and fertilisation (70.8%) of M oocytes cultured for 24 h, the fertilisation of M oocytes cultured for 40 h was associated with increased rates of retarded male pronuclear development and retention of the second polar body. The S and T oocytes cultured for 24 h or 40 h were mostly arrested at defective late diakinesis-metaphase I (77.5-100%) stage. After fertilisation of S and T oocytes cultured for 24 h no polar body was extruded and formation of one, three or four female pronuclei, together with mostly normal male pronuclei, was observed. The fertilisation of S and T oocytes after 40 h culture resulted in a higher number of female and a decreased number of male pronuclei. A major change in the pattern of protein synthesis was associated with the resumption of meiosis. There were no significant differences in the profile of protein synthesis between oocyte categories in all groups either matured or fertilised. The H1 kinase activity reached comparable increased levels in oocytes of all categories matured for 24 h and decreased during the 40 h culture, most significantly in M oocytes. The MBP kinase activity was at approximately the same high level in all categories of oocytes after 24 h of culture and remained stable until 40 h. The fertilisation after 24 h of culture resulted, in M oocytes, in low levels of both H1 and MBP kinase activities; in S oocytes, only H1 kinase was completely inactivated while MBP kinase activity decreased to some extent; in T oocytes, both H1 and MBP kinase activity decreased. Fertilisation of all oocyte categories after 40 h culture resulted in complete inactivation of both these kinases to their basal levels.

Genetic polymorphism of serum vitamin D-binding protein (GC) in sheep and mouflon.

One-dimensional polyacrylamide gel electrophoresis (1D PAGE) followed by immunoblotting revealed genetic polymorphism of GC protein in sheep (variants F, S, V) and mouflon (variants F and S, apparently identical to F and S of sheep). The frequency of Gcs allele ranged from 0.84 to 1.0 in the 12 breeds of sheep studied. GcV allele was observed only in Tsigai breed with a frequency of 0.017.

Evidence for the presence of alpha 1B-glycoprotein in mammalian sera: immunoblotting studies.

1. A monospecific antiserum to pig alpha 1B-glycoprotein (PO2) was produced in rabbits and was used to search for homologues of alpha 1B in sera of 41 mammalian species belonging to seven orders. 2. Specific reactions were detected in the sera of representatives of Insectivora, Primates, Carnivora, Proboscidea, Perissodactyla and Artiodactyla. No cross-reactions were observed in the sera of two species of Rodentia (mouse, rat). 3. Cross-reactions in the sera of Erinaceus europaeus, Homo sapiens and Macaca mulatta were rather weak; this indicates a greater structural difference between the alpha 1 B of Insectivora and Primates and that of the other mammalian orders. 4. Electrophoretic patterns of alpha 1 B were, in most cases, heterogeneous, the most heterogeneous being in ruminants. 5. Evidence was obtained that the alpha 1 B of sheep is identical with the earlier described (Juneja and Gahne (1980) Anim. Blood Grps Biochem. Genet. 11, 81-92.) polymorphic post-transferrin (Ptf).

Radioimmunoassay of progesterone in cow milk.

Fertilization checks in dairy cattle performed by estimation of progesterone in milk require a simple technique, the use of a sample taken independently of milking procedure and a simple management of the results. The laboratory equipped with a sample processor and pipetting device, high throughput counter for 125iodine and a tabletop computer can analyse about 1000 samples of fat-free milk in one day. The results are used for computer management of reproduction and veterinary service in farms with 600 to 2000 cows.

Modifications of the mouse zona pellucida during oocyte maturation and egg activation: effects of newborn calf serum and fetuin.

A precocious but limited loss of cortical granules (CG) occurs during mouse oocyte maturation both in vivo and in vitro. Although CG loss during maturation in vivo is not associated with changes in the zona pellucida (ZP), a maturation-associated conversion of ZP2 to ZP2f occurs during oocyte maturation in vitro in serum-free medium. We now demonstrate that a maturation-associated change of ZP3 to ZP3f, as assessed by a reduction in sperm binding, also occurs during maturation in vitro in serum-free medium, and that both newborn calf serum (NCS) and fetuin, each of which inhibits the ZP2 conversion, also inhibit the ZP3 conversion. The concentration-dependence of the NCS- and fetuin-mediated inhibition of the ZP2 conversion, coupled with the concentration of fetuin present in NCS, is consistent with fetuin being the component present in NCS that is primarily responsible for this inhibition. Although NCS can inhibit the ZP modifications that occur during oocyte maturation in vitro, ionophore treatment of eggs, which results in an extensive release of CGs over a short period of time, overcomes the inhibitory effect of NCS on the ZP2 conversion. Results of these studies suggest a potential regulatory function of serum-derived components in the formation of a fertilizable egg.

p95, the major phosphotyrosine-containing protein in mouse spermatozoa, is a hexokinase with unique properties.

Mouse sperm contain a major phosphotyrosine-containing protein of M(r) 95,000 (nonreducing conditions) which has been implicated as a sperm membrane receptor for the egg zona pellucida glycoprotein, ZP3 (Leyton, L., and Saling, P. (1989) Cell 57, 1123-1130; Leyton, L., LeGuen, P., Bunch, D., and Saling, P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11692-11695). This protein was purified and subjected to limited tryptic digestion and subsequent amino acid analysis. Three sequenced peptides revealed 100% amino acid identity to a mouse hepatoma hexokinase (Arora, K. K., Fanciulli, M., and Pederson, P. L. (1990) J. Biol. Chem. 265, 6481-6488). The purified protein, which migrated at M(r) 116,000 under reducing conditions (p95/116), reacted with an antiserum to the purified rat brain hexokinase, type 1, and comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the purified rat brain enzyme under both nonreducing and reducing conditions. Unlike p95/116, the rat brain enzyme was not a phosphotyrosine-containing protein. The p95/116 protein could be immunoprecipitated with the hexokinase antiserum or an O-phosphotyrosine antibody. Limited tryptic digestion of the purified p95/116 and the rat brain enzyme generated subsets of identical peptides which reacted with the hexokinase antiserum. However, p95/116 also contained phosphotyrosine-containing peptides that were not present in the rat brain hexokinase. When different mouse tissues were probed with the hexokinase antiserum all tissues, with the exception of liver, contained immunoreactive protein. In contrast, only sperm and testis possessed a phosphotyrosine-containing form of hexokinase. These data suggest that the germ cell component of the testis possesses a unique tyrosine-phosphorylated form of hexokinase.

Regulation of protein tyrosine phosphorylation in boar sperm through a cAMP-dependent pathway.

Changes of protein tyrosine phosphorylation in ejaculated boar sperm incubated in vitro were examined with the use of antiphosphotyrosine antibodies and immunoblotting. The intracellular levels of cAMP were modulated by treatment with various combinations of caffeine, 3-isobutyl-1-methylxanthine (IBMX), and dibutyryl cyclic AMP (dbcAMP), and acrosome reactions (ARs) were induced via treatment with divalent cation ionophore A23187. Proteins of Mr 34, 38, 40, and 44 (p34 ... p44) were strongly phosphorylated on tyrosine residues in freshly prepared sperm samples and at the same level during all subsequent treatments. Incubation of sperm in vitro for various periods of time induced an increase of tyrosine phosphorylation of p20, p93, and p175. The tyrosine phosphorylation of p93, p175, and several other sperm proteins was up-regulated in a concentration-dependent manner following treatment of the sperm with dbcAMP, caffeine, or IBMX alone, or with combinations of caffeine and IBMX, respectively, with dbcAMP; the tyrosine phosphorylation of p20 was not correlated with treatment of sperm with cAMP-elevating reagents. The percentage of sperm cells undergoing spontaneous ARs was not affected by the manipulation of cAMP levels and was not correlated with protein tyrosine phosphorylation. In contrast, the addition of calcium to the incubation media decreased protein tyrosine phosphorylation and elevated percentage of spontaneous ARs. The induction of ARs with A23187 caused a significant decrease of tyrosine phosphorylation of p93, p175, and p220/230, indicating that dephosphorylation on protein tyrosine residues might be associated with calcium influx during physiological ARs as well. Proteins p93 and p175 were effectively solubilized in greater than 9M urea/1% triton and in SDS sample buffer, but to only a small extent in triton, while p20 was virtually completely extractable with triton. In conjunction with the previously reported isolation of active tyrosine kinase sp42 from triton extracts of noncapacitated boar sperm cells (Berruti and Porzio, 1992: Biochim Biophys Acta 1118: 149-154), our results suggest that a cAMP-dependent event is required for tyrosine phosphorylation of triton-insoluble proteins such as p93 and p175. On the other hand, the tyrosine phosphorylation of p20 (and potentially other triton-soluble substrates) might not strictly require such cAMP up-regulation. We discuss the differences in the regulation of cAMP-dependent tyrosine phosphorylation in mouse, human, and boar sperm, and suggest that sensitivity to calcium and distinct basal levels of cyclic nucleotide PDE might correspond to species-specific reproduction strategies in mammals.

Modifications of the mouse zona pellucida during oocyte maturation: inhibitory effects of follicular fluid, fetuin, and alpha 2HS-glycoprotein.

Mouse oocyte maturation in vivo or in vitro is accompanied by a precocious release of cortical granules. When oocytes are matured in vitro in the absence of serum, cortical granule exocytosis results in modifications of the zona pellucida (ZP) that are known to constitute the zona pellucida block to polyspermy. Fetuin, a glycoprotein found in newborn calf serum, can inhibit these maturation-associated changes in the zona pellucida. In this report we demonstrate that the human fetuin homologue, alpha 2-HS-glycoprotein, is present in human follicular fluid. Human follicular fluid, or purified human alpha 2-HS-glycoprotein, inhibits the maturation-associated conversion of mouse ZP2 to ZP2f in a concentration-dependent manner. Although the concentration of alpha 2HS-glycoprotein in human follicular fluid is high enough to account for the inhibitory effect of human follicular fluid on this maturation-associated conversion, human follicular fluid immunodepleted of alpha 2HS-glycoprotein retains its full inhibitory activity. Thus, other inhibitory molecules are likely to be present in follicular fluid and contribute to the inhibition of the maturation-associated conversion of ZP2.

Characterization of polo-like kinase 1 during meiotic maturation of the mouse oocyte.

We have characterized plk1 in mouse oocytes during meiotic maturation and after parthenogenetic activation until entry into the first mitotic division. Plk1 protein expression remains unchanged during maturation. However, two different isoforms can be identified by SDS-PAGE. A fast migrating form, present in the germinal vesicle, seems characteristic of interphase. A slower form appears as early as 30 min before germinal vesicle breakdown (GVBD), is maximal at GVBD, and is maintained throughout meiotic maturation. This form gradually disappears after exit from meiosis. The slow form corresponds to a phosphorylation since it disappears after alkaline phosphatase treatment. Plk1 activation, therefore, takes place before GVBD and MAPK activation since plk1 kinase activity correlates with its slow migrating phosphorylated form. However, plk1 phosphorylation is inhibited after treatment with two specific p34(cdc2) inhibitors, roscovitine and butyrolactone, suggesting plk1 involvement in the MPF autoamplification loop. During meiosis plk1 undergoes a cellular redistribution consistent with its putative targets. At the germinal vesicle stage, plk1 is found diffusely distributed in the cytoplasm and enriched in the nucleus and during prometaphase is localized to the spindle poles. At anaphase it relocates to the equatorial plate and is restricted to the postmitotic bridge at telophase. After parthenogenetic activation, plk1 becomes dephosphorylated and its activity drops progressively. Upon entry into the first mitotic M-phase at nuclear envelope breakdown plk1 is phosphorylated and there is an increase in its kinase activity. At the two-cell stage, the fast migrating form with weak kinase activity is present. In this work we show that plk1 is present in mouse oocytes during meiotic maturation and the first mitotic division. The variation of plk1 activity and subcellular localization during this period suggest its implication in the organization and progression of M-phase.

Rapid, nonradioactive, and quantitative method to analyze zona pellucida modifications in single mouse eggs.

A rapid, nonradioactive method to monitor the ZP2 to ZP2f conversion in the zona pellucida of single mouse eggs has been developed. This assay is based on the chemiluminescent detection of biotinylated ZP2 and ZP2f following electrophoresis under reducing conditions and electrophoretic transfer to Immobilon P. This method is about 10 times faster and detects similar extents of ZP2 to ZP2f conversion following A23187-induced egg activation, when compared to the commonly used radioiodination procedures.

Damaged chromatin does not prevent the exit from metaphase I in fused mouse oocytes.

The presence of checkpoint mechanisms which are able to recognize damaged chromatin and thereafter to prevent exit from metaphase I has been investigated in giant mouse oocytes produced by fusion of a normal metaphase I oocyte with an equivalent oocyte with damaged chromatin. The presence of damaged chromatin did not prevent the onset of anaphase I in both sets of chromatin in the fused cells. Interestingly, fused or unfused cells containing only damaged chromatin failed to enter anaphase and persisted instead in a metaphase-like state. These results demonstrate the fragility of checkpoint controls in mammalian female germ cells.

The hCSE1/CAS protein is phosphorylated by HeLa extracts and MEK-1: MEK-1 phosphorylation may modulate the intracellular localization of CAS.

hCSE1/CAS (CAS), the human homologue of the yeast chromosome segregation gene CSE1, is a nuclear transport factor that plays a role in proliferation and apoptosis. A MEK-1 phosphorylation sequence in CAS raises the possibility that MEK-phosphorylation regulates the function of CAS. CAS protein from cell extracts shows covalent charge modifications; one of these charge variants contains phosphotyrosine. CAS protein can be captured from cell extracts by immobilized anti-phosphotyrosine antibodies. We have produced recombinant protein fragments containing the N-terminal or central portion of CAS and found that the N-terminal fragment, which contains a putative MEK phosphorylation site, is phosphorylated by the HeLa extracts and MEK-1. Treatment of cells with an inhibitor of MEK-1 phosphorylation in vivo changes the intracellular localization of CAS from predominantly cytoplasmic to nuclear. This suggests that a function of CAS in nuclear transport may be regulated by phosphorylation.