the effects of proteasome inhibitor lactacystin on mouse oocyte meiosis and first cleavage

Science in China Ser. C Life Sciences 2005 Vol.48 No.3 287 294 287

Copyright by Science in China Press 2005

The effects of proteasome inhibitor lactacystin on mouse oocyte meiosis and first cleavage

TAN Xin1,3, PENG An2, WANG Yongchao2 & TANG Zuoqing3

1. School of Life Sciences and Technology, Beijing Institute of Technology, Beijing 100081, China; 2. College of Life Sciences, Beijing Normal University, Beijing 100875, China; 3. College of Basic Medicine, Capital University of Medical Sciences, Beijing 100054, China Correspondence should be addressed to Tan Xin (email: [email protected])

Received March 29, 2004; revised July 23, 2004

Abstract In order to study the effects of ubiquitin-proteasome pathway (UPP) on mouse oo-cyte meiosis and cleavage, oocytes undergoing maturation and parthenogenetic activation and 1-cell embryos were treated with lactacystin, a specific inhibitor of proteasome. The results indi-cated that the rate of GVBD was not influenced by the treatment, but polar body extrusion, par-thenogenesis and first cleavage were inhibited. Immunofluorescent staining using anti -tubulin antibody indicated that the continuous treatment of lactacystin from GV stage disorganized microtubules and spindle assembly. When metaphase stage oocytes were treated with the drugthe already formed spindle structure was not affected , but the oocytes were arrested at meta-phases. The 1-cell embryos were arrested at interphase or metaphase of first mitosis when they were incubated in the drug. Proteasome regulatory subunit PA700 was located in the spindle region, as indicated by immunofluorescence. These results suggest that UPP has effects on the process of oocyte meiosis and early cleavage in many aspects, including normal organization of spindle at prophase and segregation of chromosomes at anaphase for normal meiosis. Keywords: proteasome, oocyte, meiosis, cleavage.

DOI: 10.1360/04yc0047

Many studies have shown that the ubiq-uitin-proteasome pathway (UPP) for the degradation of short-lived proteins plays a key role in regulating cell cycle progression[1 3]. At least two distinct prote-olytic pathways are required for cell cycle process. The first pathway promotes transition from G1 to S phase, and the second initiates the onset of anaphase and exit from mitosis. The inhibition of UPP will re-sult in the blockage of cell cycle process. The knowl-edge of the role of UPP in cell division, however, mainly came from studies on somatic cells. Meiosis differs from mitosis in some aspects: It is physiologi-cally arrested at some special stages; homologous

chromosomes segregate in anaphase I in stead of seg-regation of sister chromatids in the case of mitosis; and the interphase between two meioses is quite dif-ferent from the usual cell cycle. Meiotic division com-prises a set of complicated mechanisms, many of which have been studied, including regulation of dif-ferent protein kinases, interactions between the nu-cleus-cytoplasm, etc.[4,5]. It has been demonstrated that UPP plays important roles in gametogenesis and re-production cycle, including spermatogenesis, sper-miogenesis, degradation of sperm cytochondriome, the regulation in the early stage of pregnancy, the recon-struction of endometrium in menstrual cycle, and the

288 Science in China Ser. C Life Sciences

degradation of steroid hormone receptors[6]. But the data about the regulation of meiosis by UPP are still limited.

Studies on lower-order eukaryotes indicated that proteasome take roles on the regulation of meiosis. The activity of proteasome changed during oocyte maturation[7,8] and some key proteins, such as cyclin B, are degraded by proteasome in those species[9,10]. Josefsberg et al.[11] have also discovered that protea-some is involved in the first meiotic division of rat oocytes, but the general knowledge about the meiotic division of mammalian oocytes remains limited. We are not fully clear about the exact roles of UPP in dif-ferent stages of meiosis.

Lactacystin is an irreversible inhibitor of protea-some[12], which specifically inhibits the trypsin-like and chymotrypsin-like activities of the 20S protea-some. It does not inhibit other proteases[13]. In this study, lactacystin was used to evaluate the effect of proteasome on the maturation, activation of oocytes, and early cleavage.

1 Materials and methods

1.1 Collection of oocytes

(i) Isolation and culture of GV stage oocytes: Kun-ming strain mice of 4 weeks old were injected intrap-eritoneally with 5 i.u. pregnant mare’s serum gonad-otrophin (PMSG). Mice were killed by cervical dislo-cation at 48 h post-PMSG, and GV stage oocytes were collected from the ovaries by puncturing antral folli-cles in the M2 medium (Sigma) with a 4# needle. The oocytes were washed for several times with M2 me-dium to remove cumulus cells and other tissues. 3.5 mm plastic culture vessels were used to incubate the oocytes in M16 medium (Sigma) under paraffin oil at 37 , 5% CO2 in moist air.

(ii) To obtain ovulated oocytes, mice were su-perovulated by intraperitoneal injection of 5 i.u. PMSG and 5 i.u. human chorionic gonadotrophin (hCG) with 48 h apart. The animals were killed 16 h after hCG injection by cervical dislocation. Cumu-lus-oocyte complexes (COC) were obtained by cutting

the ampulla of oviduct in M2 medium with a needle. Cumulus masses were dispersed by a brief exposure to hyaluronidase (Sigma, 300 i.u./mL in M2 medium). After washing 3 times with M2 medium, the oocytes were incubated in M16 medium under the condition mentioned about.

(iii) To obtain fertilized oocytes, female mice were intraperitoneally injected with 5 i.u. PMSG followed by 5 i.u. hCG 48 h later (6:00 pm), then they were raised with male mice in the same cage overnight. Female mice with vagina plug were chosen the next morning and were killed 18 h post-hCG. COC were obtained by cutting the ampulla of oviduct with a nee-dle in M2 medium and incubated in M16 medium for 3 h. The cumulus cells around fertilized oocytes were dispersed automatically. The fertilized oocytes were washed for 3 times with M2 medium to remove cu-mulus cells. Then they were again incubated in M16 medium.

1.2 Drug treatments of oocytes

Lactacystin was dissolved in H2O as stock solutions at concentrations of 13.3 mmol/L; Cycloheximide (CHX, Sigma) was dissolved in physiological saline at concentrations of 17.8 mmol/L. Oocytes were treated according to the following designs. (i) GV stage oo-cytes just removed from ovaries were incubated in M16 medium containing different concentrations of lactacystin. Then observation, fixation, and im-muno-fluorescent stain of the oocytes were performed at certain times. (ii) Metaphase II stage oocytes were incubated in M16 medium containing different con-centrations of lactacystin. 0.5 h later, CHX was added to the final concentration of 35.5 mol/L to partheno-genetically activate the oocytes. The observation, fixa-tion, and immuno-fluorescent stain of the oocytes were performed 7 h later. (iii) Oocytes collected from mated mice 18 h after hCG injection were incubated in M16 medium containing 20 mol/L lactacystin. The pronuclear rate was estimated 6 h later. The fertilized oocytes with pronuclei were chosen to be incubated again in M16 medium to 24 h. Then the eggs were observed, fixed, and immuno-fluorescently stained.

Effects of proteasome inhibitor lactacystin on mouse oocyte meiosis & first cleavage 289

1.3 PI staining, immunofluorescence, and confocal microscopy

PI staining and immunofluorescence were per-formed as described previously with some modifica-tions[14]. Oocytes in different stages were stripped of the zona pellucida using acidified Tyrode’s solution (pH 2.5). After being washed with M2 medium, they were fixed in 3.7% paraformaldehyde in 0.01 mol/L PBS (pH7.4) containing 0.005% triton X-100 for 30 min, followed by permeabilization in 0.01 mol/L PBS (pH7.4) containing 0.025% triton X-100 and 0.3% BSA for 30 min. After blocking in 0.01 mol/L PBS containing 150 mmol/L glycine and 0.3% BSA for 30 60 min, the oocytes were incubated with 1:100 mouse anti- tubulin (Santa Cruz Biotechnology) di-luted in 0.01 mol/L PBS containing 0.3% BSA and 0.005% triton X-100, or 1:50 rabbit anti-proteasome activator PA700 subunit 10B (Calbiochem) diluted in 0.01 mol/L PBS containing 1% BSA for 2 h at 37 or overnight at 4 . After four washes of 5 min each in 0.01 mol/L PBS containing 0.005% triton X-100, the oocytes were incubated with fluorescein isothiocy-anate (FITC)-labeled anti mouse or anti rabbit anti-bodies at 1:200 dilution in 0.01 mol/L PBS containing 0.3% BSA and 0.005% triton X-100 for 1 h at 37 in the dark. After being rinsed once with 0.01 mol/L PBS containing 0.005% triton X-100, the oocytes were stained with 10 g/mL propidium iodide (PI, Sigma) in PBS for 5 min. Finally, they were washed twice in PBS containing 0.005% triton X-100. We first dropped one droplet of 0.21mol/L DABCO on a glass slide, then the oocytes were mounted on it. The four corners of the glass slide were coated with a thin layer of Vaseline and a cover-slide was covered. Observation was taken with a fluorescent microscope and a laser scanning confocal microscope.

2 Results

2.1 The effect of lactacystin on mouse oocyte matura-tion, parthenogenetic activation and the first cleavage

The result showed that GV rate 4 h after in vitro culture was not influenced by the inhibition of the catalytic activity of proteasome under the treatment of

lactacystin, which means the restart of meiosis is not influenced by the effect (Fig. 1), but first polar body extrusion was significantly inhibited by lactacystin (p<0.01). This effect is positively correlated with the dosage of the drug (Fig. 1). Lactacystin also inhibited the rate of parthenogenetic activation of superovulated oocytes by CHX, as confirmed by the rate of pronu-clear formation (Fig. 2) (p<0.01), which is also posi-tively correlated with the dosage of the drug. When oocytes were fertilized by mating and were obtained 18 h post-hCG, they had completed the second polar body extrusion but had not formed pronuclei. Treat-ment of lactacystin to such fertilized oocytes for 6 h did not have significant effect on the rate of pronuclear formation (86.6% for the experimental group com-pared with 90.5% for the controls, p>0.05), but as showed by Fig. 3, the first cleavage was significantly inhibited (p<0.01).

Fig. 1. Effects of lactacystin on GV rate and PB rate of in vitro cul-tured oocytes. The means of three different experiments are presented along with their standard errors. * p<0.01, compared with control; ** p<0.01, compared with control and *.

2.2 The effect of Lactacystin on spindle assembly

Under the treatment of lactacystin, the chromatin condensed normally after GVBD, but the assembly of microtubule was disturbed as showed by im-munofluorescent staining of -tubulin (Figs. 4, 5(a) and 5(b)). When GV stage oocytes were treated with 20 mol/L lactacystin and observed 13 h later, there was only 17.5% of the oocytes with spindle structure, compared with 74.2% in the controls (p<0.01). Of the remaining 82.5%, we found chromosomes scattered in


Fig. 2. Effects of lactacystin on the parthenogenetic activation rate of M II oocytes. M II oocytes were incubated at different concentrations of lactacystin. 35.5 mmol/L CHX was added 0.5 h later. Fractions of oo-cytes with pronucleus were obtained 8 h later. The means of three dif-ferent experiments are presented along with their standard errors. * p<0.01, compared with control; ** p<0.01, compared with control and *.

Fig. 3. Effects of lactacystin on the first cleavage. Fertilized oocytes were incubated in 20 mmol/L lactacystin. The data were obtained 24 h later. The means of two different experiments are presented. * p<0.01, compared with control.

Fig. 4. Effects of lactacystin on microtubule organization during oocyte meiotic maturation. Microtubule organization of mouse oocytes was visual-ized by fluorescent microscopy after 13 h in vitro culture. The means of two different experiments are presented. * p<0.01, compared with control. the central region of oocytes. The microtubules were disorganized, forming radiation, bundle or mass pat-tern near the chromosomes (Fig. 5(a)), or forming multi polar pattern around chromosomes (Fig. 5(b)). Besides, the oocytes treated with lactacystin which had spindle were all in metaphase whereas 27.4% of controls was in anaphase (two mass of chromatin were seen). MII phase oocytes treated with 20 mol/L lac-tacystin for 2 h had the normal spindle structure which was not different from controls (Fig. 5(c)). The results

indicated that the inhibited effect of lactacystin on the activity of proteasome disturbed the spindle assembly when the drug was used before the cell entered meta-phase, resulting in disarranged organization of micro-tubules. But when lactacystin was used at metaphase when spindle had already formed, the spindle kept the normal structure and did not disassemble. The obser-vation of fertilized oocytes which were treated with lactacystin and were stained by -tubulin and PI showed that most of them were arrested at the inter-


phase with two pronuclei which had nucleoli; a few were arrested at the metaphase of the first mitosis (Figs. 6 and 3). This result indicated that the inhibited effect of lactacystin on the first cleavage inferred dif-ferent mechanisms: Inhibiting entering prophase by preventing either pronuclear envelope disassembly or chromatin condensation, or arresting one-cell embryo at the metaphase of first mitosis.

2.3 Localization of the proteasome in mouse oocytes during meiotic division

By immunofluorescent staining with antibody against proteasome activator PA700 subunit 10B and PI staining, we found that proteasome existed in the

Fig. 5. Microtubule organization of mouse oocytes treated with 20

mol/L lactacystin. (a) and (b) GV stage oocytes were treated with 20 mol/L lactacystin and were collected 13 h after maturation culture. -tubulin and PI staining were performed. (a) The cytoplasmic micro-

tubules radiating from chromosomes; (b) the cytoplasmic microtubules forming multi polar pattern around chromosomes; (c) Superovulated oocytes were treated with 20 mol/L lactacystin for 2 h, then they were stained with -tubulin and PI. The oocytes were at metaphase II, the structure of spindle was normal. Green, -tubulin; red, DNA.

Fig. 6. Microtubule organization of 1-cell embryos treated with 20 mol/L lactacystin. Fertilized eggs were treated with 20 mol/L lacta-

cystin and were collected after 24 h culture. -tubulin and PI staining were performed. (a) Most 1-cell embryos were in the pronuclear stage with two pronuclei. (b) Some 1-cell embryos were at metaphase of mitosis I. Left: -tubulin; middle: DNA; right: merged.

whole oocyte. But in GV stage, it mainly localized in GV and periphery of GV. After GVBD, proteasome was distributed evenly in the oocyte. When spindle formed, it localized to the region of spindle (Fig. 7(a) and (b)), but lactacystin inhibited the accumulation by disturbing spindle formation (Fig. 7(c)).

Fig. 7. The localization of proteasome subunit PA700 at different stages of oocyte meiosis. (a) GV stage oocytes were collected. PA700 and PI staining were performed. Left, PA700; middle, DNA; right, merged. (b) Metaphase oocytes. Staining with anti-PA700 showed that PA700 concentrated to the region of spindle. Green, PA700; red, DNA. (c) GV stage oocytes were treated with 20 mol/L lactacystin for 13 h in vitro and were stained with anti-PA700 and PI. Green, PA700; red, DNA.

3 Discussion In this research, we studied the effect of degradation

of proteins through ubiquitin-proteasome pathway in mouse oocyte on the meiosis and early cleavage. Our results indicated that inhibition of the catalytic activity of the proteasome arrested the oocytes or fertilized oocytes at different stages.

Our study demonstrated that the inhibition of the catalytic activity of the proteasome did not affect GVBD of in vitro mouse oocytes. This is in contrast to the cases in some low-order eukaryotes[15,16], but is consistent with the research on rats[11]. Josefsherg et al. considered such difference between low-order eu-karyotes and mammals resulted from the different mechanisms of reinitiating meiosis. In low-order eu-karyotes, the oocytes meiosis reinitiates upon external stimulation, such as sperm or hormones, whereas


mammal oocytes resume meiosis spontaneously upon their separation from the follicle, which may involve a different signal pathway. It has been proposed that the mechanisms are different for mammals between in vitro and in vivo oocyte meiotic reinitiation[17], so why proteasome accumulates in and around GV and if pro-teasome has some roles in in vivo oocyte meiotic re-initiates deserve further investigation.

Lactacystin inhibited spindle assembly during the oocyte maturation. Lactacystin is a highly specific inhibitor of proteasome, so it is confirmed for the first time that proteasome is needed for the organization of microtubules and spindle assembly. It has been known that some kinases which are important for spindle as-sembly, such as Aurora-A kinase, Nek2A that is highly enriched at the centrosome, and cyclin A, are degraded by proteasome at the prophase of somatic cells. The over activities of those kinases interfere with the func-tion of spindle and centrosome. Aurora-A associated with centrosomes and spindle. Overexpression of Aurora-A would lead to mitotic spindle aberration[18]. But if there is the same mechanism in meiosis remains to be investigated. Our result also showed that the microtubules often formed radiate figure from con-densed chromosomes or formed multi polar pattern in the oocytes treated with lactacystin and whose spindle was not found, indicating the abnormality of polar location for microtubules. Under the normal condition, there may exist many microtubule organizing centers (MTOC) in oocytes, and the assembly of microtubules mainly begins at two poles of spindle. The process relies on the correct localization of the poles. Some important molecules, such as Plk (polo-like kinase), MAPK, and -tubulin have been found to be located at the poles of spindle[19]. The relation between those molecules and the abnormalities will be investigated in future. To take more consideration, which proteins are involved in the fail of spindle assembly when the ac-tivity of proteasome is inhibited and if any other factor takes action deserve future study.

We also confirmed that though lactacystin inhibited the normal assembly of spindle or led to malformation of spindle, it could not disassemble already formed spindle, indicating that the molecular events and regu-

lations in the early stage of oocyte maturing division are different from those in the M II arrested stage, so the target molecules degraded by proteasome are dif-ferent between the two stages.

The oocytes with the normal spindle were arrested at metaphase of meiosis under the treatment of lacta-cystin, which is coincident with the experimental re-sult from rat[11]. Furthermore, we discovered that lac-tacystin significantly inhibited the re-initiation of sec-ond meiotic division after parthenogenetic activation induced by CHX, leading to lowered pronuclear rate. All these may result from the inhibition of degradation of cyclin B, though many other factors should also be taken into serious consideration. It is interesting to notice that lactacystin did not inhibit the formation of pronuclei when meiosis had finished after fertilization, inferring that the time of inhibited effect of lactacystin on parthenogenetic activation was before the second polar body extrusion. Once the second polar body ex-trusion finished, the formation of pronucleus was not affected by lactacystin. In budding yeast, the transi-tions of metaphase to anaphase and anaphase to the end of mitosis are two independent processes. Both of them need a multi-subunit E3 enzyme: anaphase pro-moting complex (APC), which mediates selective degradation of CUT2 and PDS1 by proteasome, re-sulting in chromosome segregation and the cells leav-ing metaphase; then it mediates degrading cyclin B by proteasome to make cells enter interphase. Normal anaphase spindle movements are triggered by the APC-dependent degradation of at least two classes of proteins: one class that is involved in holding sister chromatids together, and a second class that directly influences the behavior of the mitotic spindle[1]. Dur-ing the first meiotic division, the segregation of ho-mologous chromosomes takes place in stead of segre-gation of sister chromatids as in mitosis, so a different class of proteins which play roles in the anaphase check point and are degraded by protesome may exist, which is still to be discovered.

In conclusion, the inhibited effect of lactacystin on polar body extrusion is involved with many different mechanisms and two of which are clear: inhibition of spindle assembly so the oocytes were not able to enter


7. starfish oocyte maturation,

8.

in the toad, Bufo japonicus, Mol.

9. me upon egg activation,

10. e

11. meiosis

12.

ystin beta-lactone, J. Biol. Chem.,

metaphase; arrest oocytes at metaphase. Both the mechanisms are relevant with spindle. The localizing of proteasome in oocytes in this research supports the assumption. There are two kinds of proteasomes: 20S proteasome that is catalytic particle (CP) and 26S proteasome that is the combination of 20S proteasome with a 19S regulatory particle (RP) named PA700. 26S proteasome has the ability to degradate the specific proteins in ATP-dependent manner, whereas 20S pro-teasome has no such ability. In this study, the im-munofluorescent staining with anti-PA700 antibody was performed to localize the location of proteasomes with the catalytic ability. We found that proteasome dispersed evenly in oocytes before the spindle ap-peared, but gathered to the spindle region after spindle had assembled, implying the functions of proteasome are related with spindle. So proteasome is needed for the normal assembly of spindle from tubulins; on the other hand, proteasome may serve as a spindle assem-bly checkpoint effector which perceives the signal of completion of spindle assembly to degrade cyclin B and other anaphase inhibitors at the correct time and promotes oocytes to transmit into anaphase and inter-phase.

The process from fertilization to first cleavage is quite complicated which comprises the formation of male and female pronuclei, reorganization of nucleo-proteins, substitution of protamines by histones, the approach of male and female pronuclei, breakage of pronuclear membrane and entering of mitosis, etc. We found that lactacystin did not inhibit the formation of pronuclei in fertilized eggs when the drug was used after the completion of the second meiotic division, but inhibited the first cleavage division. PI staining showed that most of the 1-cell embryos were arrested at the stage of pronucleus, but the reason was not clear. The appearance of nucleolus in pronucleus indicated that the synthesis of rRNA began which seems not to be affected by the inhibition of proteasome. A few 1-cell embryos were arrested at metaphase of the first mitosis under the treatment of lactacystin. This result is about the same as the experiment with rat em-bryos[20] and the mechanism may also be involved in the fail of degrading cyclin B by proteasome. We de-

duce that the activity of proteasome is necessary in the process for the regulation of cell cycle factors. The inhibited effect of lactacystin on the first cleavage di-vision, as in the case of meiosis, is the combined re-sults of multi-functions.

Acknowledgements This work was supported by the National Natu-ral Science Foundation of China (Grant No. 30270661), Beijing Natural Science Foundation 5032004 and the Special Funds for Major State Basic Research Project (973) of China (No. G1999053901).

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the effects of proteasome inhibitor lactacystin on mouse oocyte meiosis and first cleavage

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