Function of RanGAP1 in Mouse Oocyte Fertilization

Hao Wei-jie, Sun Lu-shuang, Hu Xiao-yang, Xu Ying, Ding Cong, Yan Yun-qin, and Cao Yun-kao*

1 Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine, College of Veterinary Medicine, Northeast Agricultural University,Harbin 150030, China

2 College of Life Sciences, Northeast Agricultural University, Harbin 150030, China

Abstract: RanGAP1 is a Ran GTPase-activating protein that plays a pivotal role in the majority of nucleocytoplasmic transport pathways. The protein is limited to somatic cells. In this study, the localization and possible functions of RanGAP1 were examined during mouse oocyte fertilization. Immuno fluorescence analysis showed that after sperm penetration, RanGAP1 was found to diffuse within the cytoplasm, but concentrated in the microtuble of the reversed spindle and the constriction ring between the oocyte and the second polar body; with the expansion of sperm chromatin, RanGAP1 began to move to the region around the expanding sperm and oocyte chromatin, and gradually concentrated around the growing parents pronuclei. After the male and female pronuclei apposed,the membrane of one pronuclei brokefirst, numerous concentrated RanGAP1 dots were observed in the chromosome region. With the chromatin condensing into chromosome, the parents chromosomes mixed together and prepared to start thefirst mitosis, the condensed RanGAP1 was just the shape of the microtuble to assemble thefirst mitosis spindle. These showed that RanGAP1 played an important role in regulating spindle functions, chromosome alignment, PB2 extrusion and pronuclei nuclear envelope assembly/disassembly in mouse oocyte fertilization.

Key words: RanGAP1, immuno fluorescence, nucleus assembly, mouse oocyte, fertilization

Introduction

Mouse oocyte fertilization is the fusion of haploid gametes, oocyte and sperm, to form the diploid zygote.It occurs when sperm and oocyte recognize each other, the sperm head enters into the oocyte, the sperm chromosome depolymerization and the extrusion of the second polar body from the zygote, the formation of male and female pronucleus, the male and female chromatin fuse to form a new, genetically distinct organism. It is a complex and precisely orchestrated process.

RanGTPase signaling pathway controls numerous cellular processes, which include nucleocytoplasmic transport (Macara, 2002), DNA replication (Hughes et al., 1998), the entry into and out of mitosis or meiosis(Görlich and Kutay, 1999), microtubule nucleation and spindle assembly (Dasso, 2002), and nuclear breakdown/reassembly throughout the cell cycle in mitosis and meiosis (Zhang et al., 1998). RanGAP1 is a key factor of the classical RanGTPase signaling pathway, which is localized in the cytoplasmic surface of nuclear pores and catalyzes RanGTP hydrolysis to RanGDP (Matunis et al., 1996; Izaurralde et al.,1997). RanGAP1 is involved in nucleocytoplasmic transport (Mahajan et al., 1997), mitotic spindle assembly (Joseph et al., 2002; Joseph et al., 2004)and nuclear envelope reconstruction process (Zhang and Clarke, 2001). Thus, it is important in a variety of mitotic cellular activities. And the role of RanGAP1 in mouse oocyte meiosis and its molecular mechanism (in press) has been studied, but the roles of RanGAP1 in mouse fertilization are not reported.

In this study, the localization of RanGAP1 was investigated, during mouse oocyte fertilization using confocal laser scanning microscopy.

Materials and Methods

Ethics statement

Mice care and use were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. ICR mice (6–8 weeks old) were housed and bred in a temperature-controlled room.They received a standard murine chow diet, and were kept on a cycle of 12 h light and 12 h darkness, with the darkness starting from 7:00 p.m. The mice were killed by cervical dislocation. All the efforts were made to minimize suffering and the only procedure performed on the dead animals was to obtain ovaries and the collection of oocytes from the ovaries.

Antibodies and reagents

Rabbit polyclonal anti-RanGAP1 antibody (c-5)used in Western blot and immunofluorescence was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz and CA, USA). The secondary horseradish peroxidase (HRP)-labeled goat IgG antibody was purchased from Beijing Biosynthesis Biotechnology Co (Beijing, China). All other reagents were purchased from Sigma-Aldrich, unless otherwise mentioned.

Zygote collection

In vivo fertilized zygotes were collected 16 h after hCG from the oviduct ampullae of superovulated females that had been mated with the same strain of males. After removing cumulus cells with 300 IU • mL-1hyaluronidase in M2 medium, zygotes were cultured in M16 medium (Sigma) until using. Zygotes at different stages of development were collected for confocal microscopy.

lmmuno fluorescence and confocal microscopy

Zygotes werefixed in 4% paraformaldehyde in PBS with 0.5% Triton X-100 for 30 min at room temperature, followed by blocking in 1% bovine serum albumin for 24 h at 4℃. Thereafter, zygotes were incubated with rabbit polyclonal anti-RanGAP1 antibody (1 : 100) and mouse monoclonal anti-αtubulin-FITC antibody (1 : 100) overnight at 4℃.After three washed (5 min each) in washing buffer(0.1% Tween 20 and 0.01% Triton X-100 in PBS),the zygotes were treated with FITC-labeled goat IgG secondary antibody (Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China) to stain RanGAP1 for 1 h at room temperature (this step was omitted for α-tubulin). After three 5-min washed in PBS containing 0.1% Tween 20 and 0.01% Triton X-100, the oocytes were mounted on glass slides with mounting medium containing 4', 6-diamidino-2-phenylindole(DAPI) to stain DNA. Finally, the oocytes were examined by using an FV1000 confocal laser-scanning microscope (Olympus).

Statistical analyses

All the results came from at least three repetitions.

Results

Subcellular localization of RanGAP1 in mouse fertilized oocytes

As shown in Fig. 1, with the sperm penetrating,the oocyte was released from MⅡ oocyte arrest and exhausted the second polar body, RanGAP1 was found to diffuse within the cytoplasm, but concentrated in the microtuble of the reversed spindle and the constriction ring between the oocyte and the second polar body (Fig. 1b). With the expansion of sperm chromatin, RanGAP1 began to move to the region around the expanding sperm and oocyte chromatin, it was concentrated in the second polar body (Fig. 1c).

Fig. 1 Immuno fluorescence localization of RanGAP1 during mouse oocyte fertilization

With the formation of the male and female pronuclei, the concentrated RanGAP1 formed a ball around the pronuclei membrane, in other places of the cytoplasm, RanGAP1 was uniformly distributed(Fig. 1d). With the growth of pronuclei, they came closer to each other, the green fluorescent staining of RanGAP1 became increasingly stronger, the concentration of RanGAP1 seemed even higher than the cytoplasm except the concentrated ball (Fig. 1e-h).

After the male and female pronuclei apposed, the membrane of one pronuclei brokefirst, numerous concentrated RanGAP1 dots were observed in the chromosome region (Fig. 1i-j). With the chromatin condensing into chromosome, the parent chromosomes mixed together and prepared to start thefirst mitosis, the condensed RanGAP1 was just the shape of the microtuble to assemble thefirst mitosis spindle(Fig. 1k-l).

Discussion

It was demonstrated that RanGAP1 played important roles in nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope reconstruction process in mitosis, its role in meiotic spindle assembly was also be tested, subsequent chromosome alignment, and polar body extrusion. In this study, the localization of RanGAP1 was investigated during mouse oocyte fertilization by immunofluorescence and confocal microscopy, in order to inspect if RanGAP1 had any roles in mouse oocyte fertilization.

It was found that RanGAP1 was mainly localized in the cytoplasm with a higher concentration in the microtuble of the reversed spindle and the constriction ring between the oocyte and the second polar body,because the position of the cleavage furrow depended on the spindle. It demonstrated that RanGAP1 was required for asymmetric meiotic divisions in oocytes.In mitosis, SUMO-1 conjugated targets RanGAP1 to kinetochores and mitotic spindles in HeLa cells (Joseph et al., 2002). The localization of RanGAP1 was analogous to Ran (Cao et al., 2005). RanGAP1 was the key factor of classical RanGTPase signaling pathway,which was required for proper spindle formation in oocyte meiosis (Sazer and Dasso, 2000; Dasso, 2001).RanGAP1 was involved in spindle and kinetochores in mitosis by altering the concentrations of Ran-GTP available to mediate MT stabilization (Joseph et al.,2002). On the other hand, the position of the cleavage furrow was defined by the spindle (Dumont et al.,2007). Depletion of Cdc42 disrupted the progression of the cleavage furrow. Oocytes were reported to extrude much larger polar bodies than the controls in the presence of RanQ69L (Hutten et al., 2008),similar to the described effects of the knockdown of RanGAP1 on PB1 extrusion in oocyte maturation.So, RanGAP1 appeared to be involved in the second polar body extruded by microtubules in fertilization.Further study was necessary to determine exactly how RanGAP1 regulating mouse oocyte fertilization.

After the second polar body was extruded, with the expansion of parent chromosomes, RanGAP1 began to move to the region around the expanding sperm and oocyte chromatin, it showed that RanGAP1 might have some roles in chromosome depolymerizing into chromatin and do some prepares for the pronucleus formation. With the formation of the male and female pronuclei, the concentrated RanGAP1 formed a ball around the pronuclei membrane, and was much lower in the nucleolus. RanGTPase had been implicated in cell cycle control, including the transition from mitosis (or meiosis) to interphase, when the compartmentalization of the nucleus was established.In an experiment using a cell-free system of Xenopus egg extracted to examine the function of Ran during the assembly of pronuclei from demembranated Xenopus sperm heads, it was found that increased concentration of RanGTP produced a large, stable microtubule aster nucleated from the sperm centrosome and arrested nuclear assembly, blocking chromatin decondensation. In contrast to RanGTP,excessed RanGDP had no inhibitory effects on aster formation and nuclear assembly. RanGDP/GTP switch might play a role in co-ordinating changes in the structure of microtubules and the reassembly of the nucleus associated with the transition from mitosis(or meiosis) to interphase (Zhang et al., 1999). Ran's concentration in the interphase nuclei was either required directly to allow nuclear envelope assembly/disassembly to occur, or needed to modify chromatin.Ran and its cofactors RanBP2, RanGAP and RCC1 were all essential for reformation of the nuclear envelope after cell division. Reducing the expression of any components of Ran GTPase cycle by RNAi led to strong extranuclear clustering of integral nuclear envelope proteins and nucleoporins (Askjaer et al., 2002).

In mammalian cells, it had been demonstrated that RanGAP1 with its C-terminal SUMOs was located at the nuclear membrane through interaction with the nuclear pore protein Nup358/ RanBP2 in interphase,where it maintained RanGTP gradient across the nuclear membrane for normal nuclear transport (Hutten et al., 2008; Saitoh et al., 2006). So, Ran GAP1 should play an important function in the control of nucleocytoplasmic transport in fertilized oocyte.

Conclusions

In conclusion, thefindings showed that RanGAP1 played an important role in regulating spindle assembly, chromosome alignment, PB2 extrusion and pronuclei nuclear envelope assembly/disassembly in mouse oocyte fertilization.