Indian lournal of Experimental Biology Vol. 37,lune 1999, pp. 589-593

Maturation-promoting factor during oocyte maturation of the catfish, Heteropneustes fossilis and the carp, Labeo rohita

K Balamurugan & S Haider·

Department of Zoology, Banaras Hindu University, Varanasi 221 005, India

Received 11 December 1998; revised 19 March 1999

Cytosolic extract (CE) from mature oocytes of the catfish (H. fossilis) and carp (L. rohita) induces 100% genninal vesicle breakdown when microinjected into Clarias immature unstimulated oocytes. For the purpose of micro injection in fish oocytes a dose of - 50 nl is suggested as the optimum dose. SDS-P AGE analysis of CE prepared from immature and 17a, 20/3-DP­induced mature oocyte showed some prominent and faint protein bands in the region of 34, 46 and 48 kDa. Two bands, at 32 and 34 kDa were detected in immature as "Yell as mature oocytes by immunoblotting of SDS-PAGE with anti-PST AIR antibody. Anti-cyelin B I and B2 antibodies recognized 46 and 48 kDa proteins of mature oocytes, respectively. Anti-cyelin A antibody did not recognize any proteins of immature or mature oocytes. These findings indicate that catfish and carp MPF contain p34a1c2 homologs and eyclin B molecules.

Full-grown postvitellogenic oocytesof fish are arrested at the first meiotic prophase. Normally, the resumption of prophase-arrested meiosis (defined as oocyte maturation) is induced by the sequential actions of three major factors, namely, gonadotropin secreted from the pituitary gland, maturation-inducing steroid (MIS) synthesized and secreted from the follicle cells surrounding the oocyte, and maturation­promoting factor (MPF) activated in the oocyte cytoplasm 1.3. The first two factors are species specific whereas MPF acts in. a nonspecies-specific manner across the different phylal ,4.

Numerous studies from our laboratory have suggested that pituitary gonaditropin trigger maturation by stimulating the follicle cells to synthesize 170.., 20J3-dihydroxy-4-pregnen-3-one (170.., 20J3-DP) that acts directly on the oocyte to cause maturation in catfishes and carps5.12. Moreover, we have purified and characterized 170.., 20J3-DP as the naturally occurring and physioio~ically active MIS in the catfish, Clarias batrachusl3

• This steroid has been shown to be one of the most. potent steroids for inducing oocyte maturation in a number of teleost species l ,4,14.16. However, when 170.., 20J3-DP was microinjected into goldfish 1 or catfish (Balamurugan and Haider unpublished) oocytes, it did not induce germinal vesicle breakdown (GVBD)- the first visible event of oocyte maturation. This suggests that some

·Correspondent author

other factor, possibly a cytoplasmic factor (MPF) is produced in the oocytes in response to 170.., 20J3-DP and is the direct cause of oocyte maturation. MPF has been shown to induce maturation when injected into full-grown immature unstimulated oocytes17

•20.

In spite of efforts in several laboratories, progress in MPF identification, characterization and purification in fish had been very slow until recently though it was first purified from Xenopui l and later in the starfish22,23 mature oocytes. The breakthrough in purification was achieved by Yamashita et al. 19

who purified MPF from fish (carp, Cyprinus carpio) mature oocytes. More recently, we demonstrated the presence of MPF in 170.., 20J3-DP stimulated oocytes of Indian catfi.sh, C batrachus and showed that MPF consists of two subunits: the anti-PST AIR reactive 32 kDa, 34 kDa and anti-cyclin B I reactive 46 kDa proteins24. It is now established that MPF is a heterodimer of cdc2 and cyclin B proteins. The former one is known as the catalytic subunit which is a serine/threonine protein kinase homologous to the gene prodyct of the cdc2 gene of fission yeast having protein Qf relative molecular weight 34 kDa, referred to as p34cdC2, while the later regulatory subunit of MPF is cyclin I9,22.31 .

To date, informations about MPF molecule has been confined to carp (C carpio) and catfish (C batrachus). Studies using other fish species may provide further evidence to suggest that the presence and actions of MPF are highly conserved. In this

590 INDIAN J EXP BIOL, JUNE 1999

report we identify the components of the MPF molecule in another catfish, Heteropneustes fossilis and carp, Labeo rohita during oocyte maturation by immunoblotting with mouse anti-PST AIR (p34cdc2

homologs) and rabbit anti-cyclins A, B I and B2.

Materials and Methods Animals and oocytes-Female catfish (H fossilis)

and carp (L. rohita) were purchased ,?ommercially and maintained in laboratory until use. Full-grown immature oocytes were manually isolated from ovarian: fragments. Mature oocytes were obtained by incubating oocytes at 23±2°C in freshly prepared incubation medium5 containing I Ilg/ml 17a,20J3-DP for 18 hr. Maturational processes were assessed by immersing the oocytes in a clearing solution (5% formalin and 4% acetic acid), facilitating microscopic examination of the occurrence ofGVBD.

Extraction of MP F-Immature and mature oocytes were obtained as described previously and briefly washed and homogenized gently with a loose-fitting pestle in a centrifuge tube ~sing an extraction buffer (80 mM J3-glycerophosphate, 50 mM NaF, 20 mM EGT A, 20 mM a-napthylphosphate, IS mM MgCh. I mM ATP, I mM dithiothreitol, 300 IlM p­amidinophenyl methane sulfonyl fluoride (p­APMSF), 3 Ilg/ml leupeptin, 20 mM Hepes, pH 7.5; after Yamashita et al. 19). The homogenates were centrifuged at 25000 g for IS min at 2°C. The turbid supernatant was collected and centrifuged at 100,000 g for I hr at 4°C. The soluble fraction below the yellow fat layer was collected and A TP-y-S and cytochalasin B were added to final concentrations of I mM and 10 Ilg/ml, respectively. These cytosolic extracts (referred to as CE hereafter) were used to assess the MPF activity and sodium dodecyl sulfate­polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

Assay for MPF activity--In order to assess the MPF activity, the CE prepared from the immature and mature oocytes of fl. fossilis and L. rohita were microinjected (for microinjection procedure see Haider and Balamurugan20

) into unstimulated (pre­incubated in the presence of 10 Ilg/ml cycloheximide for 2 hr) immature oocytes of catfish. Approximately 20, 50 and 70 nl of CE (protein concentration 5 mglml) were microinjected into Clarias oocytes through their animal pole only. All the injected oocytes were maintained at 23±2°C in the freshly

prepared incubation medium containing 10 Ilg/ml cycloheximide and the MPF activity was recorded by observing the incidence of GVBD.

Electrophoresis and immunoblotting--Proteins pres.ent in CE were analyzed by 12.5% SDS-PAGE32

along with Sigma molecular weight markers and visualized by silver staining. For immunoblotting, proteins were transferred from gels to nitrocellulose membrane (Hybond, Amersham) in a buffer contain­ing 48 mM Tris, 39 mM glycine, 0.037% sodium dodecyl sulfate and 20% methanol, p H 8.3 for 3.5 hr by semidry eiectroblotting. The membrane was then rinsed in Tris·-buffered saline (TBS: 20 mMTris, 137 mMNaCI, pH 7.6) and blocked with 2% BSA in TBS containing 0.1 % Tween 20 (TTBS) for 3 hr. After washing the membrane thrice (1 5 min each) with TTBS, incubated with primary antibody (I :500 dilution) against mouse-PSTAIR sequence of p34cdc2

or rabbit cyclins A or B I and B2 for 3 hr and washed again three times. The membrane was then incubated with horseradish peroxidase (HRP)-conjugated second antibody (dilution I: 1000; Nil, New Delhi) for 3 hr. Following three washes with TTBS, the peroxidase activity was visualized by treating the membrane with 4-chloro-I-napthol and 3% H20 2 in T~S. All incubations were carried out at room temperature.

Results Presence of MPF activity in mature oocytes of

H fossilis and L. rohita-CE prepared from immature and 17a, 20J3-DP-induced (mature) oocytes of H. fossilis and L. rohita were assayed for MPF activity. When microinjected into isolated unstimulated (cycloheximide treated) immature oocytes of Clarias the CE from immature oocytes of both the fishes failed to i':lduce maturation whereas the CE of mature oocytes induced oocyte maturation but the induction of GVBD varied according to the amount injected. Oocytes injected with -70 nl of CE could not retain this volume as it burst out of the oocytes .. When the oocytes were injected with -20 nl , less than 20% of oocytes underwent GVBD even after 5 hr whereas 100% GVBD was recorded with a dose of -50 nl after 4 hr of injection.

Protein migration pattern by kIDS-PAGE and immunoblotting--The CE obtained from immature and mature oocytes of H fossilis and L. rohita were analyzed on 112.5% SDS-PAGE along with Sigma

BALAMURUGAN & HAIDER : MA TURA nON-PROMOTING FACTOR IN OOCYTES 591

molecular weight markers and visualized by silver staining. Protein migration pattern did not show any marked difference between the immature and mature CE obtained either from H fossilis or from L. rohita (Fig.). Apart from some prominent and faint bands, protein bands in the region of 34, 46 and 48 kDa were also observed in immature and mature lanes. For characterization of MPF molecules, a few immuno­blotting experiments were conducted using antibodies against PST AIR sequence of p34cdc2

, and cyclins A, B) and B2. Anti-PST AIR (p34Cdc2 homo logs ) antibody cross-reacted with two bands corresponding to 32 and 34 kDa proteins of immature (not shown) and mature (Fig. 2, lanes ) and 2) oocyte CE from both the fishes . However, anti-cyclins B I and B2 antibodies recognized 46 and 48 kDa proteins, respectively of mature CE only (Fig. 2, lanes 4 and 5) but did not cross react with any proteins present in immature CE (not shown) either of H fossilis or of L. rohita oocytes. Anti-cyclin A antibody did not show any cross reactivity with anyone of the proteins from mature (Fig. 2, lane 3) or immature (not shown) oocyte CE of both the fishes .

Discussion MPF has been found to be ubiquitous and is not a

species specific cytop lasmic factor. Its transfer from

,kOa I

48 :::. 46 --34

• L

Mr .... 97

1

H

M kOa

48 ~·46

34

Fig. l--SDS-PAGE analysis of cytosolic extracts from Labeo rohita (L) and Heleropneusles fossilis (H) oocytes. Samples were prepared (for details see Materials and methods ) from 100 oocytes. Five ~I of aliquotes were loaded from immature (I) and mature (M) oocyte extracts with Sigma molecular weight markers (Mr) and protein bands were visualized by silver staining.

kOo 1 2

34" -~' .-32"" --~ -.

L H

3 4

L

5 kOo

H

s:48 . 46

Fig. 2-lmmunoblotting with anti-PSTAIR (lanes I and 2), anti­cyeli n A (lane 3) and anti-cyelins B I and B2 (lanes 4 and 5) antibodies. Anti-eST AIR reactive 32 and 34 kDa proteins are present in immature (not shown) as well as in mature oocyte extracts of Labeo rohita (L) and Heteropneustes foss ilis (H). Anti­cyclins B I and B2 antibodies cross-reacted with 46 and 48 kDa proteins, respectively, of mature oocyte extracts only. Anti-cyelin A antibody did not show any cross reactivity with any proteins from mature (lane 3) or from immature (not shown) oocyte extracts of both the fi shes.

oocytes of a given species into immature oocytes of other species causes maturation in the rec ipient oocytes. Dettlaff et al. 33 demonstrated that cytoplasm taken from mature oocytes could induce maturation when injected into immature oocytes and reported MPF-like activity in the sturgeon. However, this activity was not detected in conditions of inhibited protein synthesis . Yamashita et a/19 suggested that inactivity of sturgeon MPF when injected into immature sturgeon oocyte under protein synthesis inhibition, might be a problem of the characteristics of the recipient oocytes, rather than of sturgeon MPF itself. We also demonstrate that the CE prepared from steroid-stimulated oocytes of H fossilis and L. rohita induced maturation when injected into immature, unstimulated Clarias oocytes denoting the presence of true, post-translational MPF activity in mature catfish and carp oocytes.

Ever since Lohka et al. 21 purified the active MPF in Xenopus eggs as a -200 kDa complex containing -32 and -45 kDa proteins and suggested that MPF is associated with protein kinase activity, several studies have been made on the identification, purification and

characterization of MPF molecules in Xenopui S-27

,

starfish22,23,29 and certain fish I9

,24 . Nurse34 in his

592 INDIAN J EXP BIOL, JUNE 1999

review suggested that one component of purified MPF which appears as 34 kDa is a homolog of cdc2 gene product of fission yeast (p34Cdc2) which serves as a protein kinase for entry into M-phase. Further studies demonstrated that 32 and 34 kDa proteins of purified MPF are recognized by an antiserum against 16 aminoacid conserved peptide of cdc2 gene product (anti-PSTAIR antibody) of fission yeast26. This antibody has been proved to be useful to detect p34cdc2 homologues in cell extracts of several species34. The regulatory subunit of p34cdc2 is referred to as cyclins which has been identified in many organisms including fish 19,20,22.27,35,36.

It has previously been reported that cdc2-like genes are present in Drosophila37, Xenopui 8 and goldfish39 . Cell division kinase or cyclin-dependent kinase, referred as cdk2 is a cdc2-like protein found in Xenopus40

, is highly homologous to p34cdc2 and contains the PST AIR sequence but dot:s not form a complex with cyclin B, In vertebrates, however, cdc2 and (;dk2 apparently play different roles. Experiments in goldfish system have suggested that anti-PST AIR reactive proteins did not change remarkably during oocyte maturation30, despite the increased kinase activity. In our present study, by using anti-PST AIR antibody, we found that p34cdc2 -proteins were present in both immature and mature oocytes of H fossilis and L. rohita.

Although both cyclin A and cyclin B can induce oocyte maturation when injected into immature oocytes41 -44, only eyclin B is a component of MPF that triggers oocyte maturation in vivo I 9,22,27. To date, cyclins A and B have been cloned . from several animal sources45 but goldfish eDNA clones of 'eyclin box' described by Hirai et al31 resembled cyclin B more closely than cyclin A. In the present investigation also anti-cyclin A antibody failed to cross-react with any proteins from immature or mature oocyte extracts of both the fishes suggesting its absence during the maturational period.

In our previous studies2v,24, we demonstrated the incorporation of labelled e5S] methionine in the region of 46 kDa protein band, indicating de novo synthesis of this protein during 17u, 20p-DP-induc­tion. Moreover, anti-cyclin B I antibody recognized 46 kDa protein of mature oocytes only. In Xenopus, two species of cyclin B (B I and B2) are involved in the MPF molecule27 and show 68% identity at the

nucleotide level46, and polyclonal antibody against

cyclin B I and B2 had no cross reactivity27. Yamashita et. al. 19 hav(~ identified two ~pecies of cyclin B from purified MPF of mature carp as the 46 and 48 kDa proteins. While p34cdc2 is found in both immature and mature oocyte extracts of goldfish and did not change during maturation, cyclin B was detected in mature oocyte extracts onl/I. In our present study, antibody against the PST AIR sequence of p34cdc2 recognized 32 and 34 kDa proteins of immature as well as mature oocytes while, 46 and 48 kDa proteins of mature oocytes were recognized by anti-cyclin B I and B2 antibodies, respectively. These four proteins were also detected from purified carp MPFI9 and it was suggested that one molecule of :;dc2 kinase and one molecule of cyclin B form a complex in the native condition. Although the same conclusion can be drawn from the present study as well, it is premature to conclude that (i) the cdc2 kinase and cyclin B form a complex, and also that (ii) the complex is sufficient to induce catfish and carp oocyte maturation. Further studies are nequired to understand the precise role of cdc2 kinase and cyclin B. We are now in the process of purifYing the MPF and identifYing the phosphorylated site(s) of cdc2 kinase of active fish MPF.

Acknowledgement We are grateful to Drs P Nurse, T Hunt and J

Gannon, ICRF Clare Hall Laboratory, UK for providing anti-PST AIR, anti-cyClins A, B I and B2 antibodies. This work was supported by a research grant [37(905)/96/EMR-II] awarded to SH from CSIR, New Delhi.

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