Subcellular localization of nardilysin during mouse oocyte maturation

Archives of Biochemistry and Biophysics 434 (2005) 187–194 www.elsevier.com/locate/yabbi

Subcellular localization of nardilysin during mouse oocyte maturation Zhangliang Maa,b,¤, Xuebing Wanga, Steven Hockmanc, E. Charles Snowa, Louis B. Hershb a

Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky Medical Center, 800 Rose St., Lexington, KY 40536, USA b Department of Molecular and Cellular Biochemistry, University of Kentucky Medical Center, 800 Rose St., Lexington, KY 40536, USA c Pulmonary Critical Care Medicine Branch, National Heart, Lung and Blood Institute, National Institute of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA Received 22 September 2004, and in revised form 18 October 2004 Available online 25 November 2004

Abstract We have previously shown that the peptidase, nardilysin, contains a bipartite nuclear localization signal that permits the enzyme to cycle between the nucleus and cytoplasm. In the present study, we report that nardilysin accumulates in the nucleus of an oocyte as a function of its maturation. Nardilysin is predominantly localized in the cytoplasm of an oocyte when initially placed into culture. The enzyme starts to accumulate in the nucleus within 30 min of in vitro culture. After 3 h, nardilysin is found as a spherical structure surrounded by condensed chromosomal DNA. After 18 h of in vitro culture, it co-localizes with
-tubulin at the spindle apparatus. Cilostamide, a phosphodiesterase 3A inhibitor that inhibits meiosis, blocks accumulation of nuclear nardilysin. This Wnding demonstrates that the nuclear entry of nardilysin is tightly controlled in the oocyte. Taken together, these experiments strongly suggest a role for nardilysin in meiosis through its dynamic translocation from cytosol to nucleus, and then to the spindle apparatus.  2004 Elsevier Inc. All rights reserved. Keywords: Nardilysin; Oocyte; Nuclear localization; Meiosis; Maturation

Nardilysin (E.C. 3.4.24.61) is a peptidase that cleaves a variety of neuropeptides in vitro including dynorphins A and B, -neoendorphin, somatostatin 28 [1,2], and
endorphin [3]. The enzyme was originally identiWed based on its ability to cleave peptides at arginine containing dibasic pairs of amino acids [4]. However, it has been shown that the enzyme can cleave
-endorphin at single basic residue (M-R and/or F-K) [3]. Similarly, its distribution inside a cell is polymorphic. Although nardilysin is primarily localized in the cytosol [1], the enzyme can also be found on the cell surface [5], as well as secreted [2]. Recently, we have reported that the Nterminal region of nardilysin contains a bipartite nuclear

*

Corresponding author. Fax: +1 859 257 8994. E-mail address: [email protected] (Z. Ma).

0003-9861/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2004.10.027

localization signal (NLS1) that permits the enzyme to shuttle between the cytosol and nucleus [6]. At present neither its function nor its in vivo substrates are known. However, the abundance of the enzyme in testis, where its expression is restricted to germ cells, suggests that nardilysin is involved in spermatogenesis [7]. Nardilysin is also found to be a receptor for heparin-binding EGF like growth factor. This receptor activity does not require catalytically active enzyme [8]. It is well known that fully grown mammalian oocytes are arrested at prophase stage of the Wrst meiotic division as indicated by the presence of a large centrally 1 Abbreviations used: NLS, nuclear localization signal; GV, germinal vesicle; GVBD, germinal vesicle breakdown; NPC, nuclear pore complex; MEM, minimum essential medium; PDE, phosphodiesterase; PKA, protein kinase A; CDKs, cyclin-dependent kinases.

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placed germinal vesicle (GV, nucleus) [9]. Meiotic arrest is maintained until the preovulatory gonadotropin surge. With stimulation by hormones in vivo, an oocyte exits from prophase arrest and undergoes germinal vesicle breakdown (GVBD) in which the nuclear membrane is broken and chromosomal DNA is condensed. This is followed by emitting polar bodies and then the oocyte becomes a metaphase II (MII) arrested egg. The series of events that start with GVBD and end at MII leads to the production of a mature oocyte which is ready for fertilization by a sperm [10,11]. When isolated from an ovary and cultured in a suitable medium, meiotically competent oocytes will spontaneously mature as evidenced by germinal vesicle breakdown (GVBD). However, if the medium is supplemented with a chemical that leads to an increase in intracellular cAMP levels (e.g., cAMP phosphodiesterase 3A inhibitor, cilostamide, or a cAMP analogue, db-cAMP) [12,13], the meiotic response is suppressed. The oocytes remain in prophase I arrest, as indicated by the presence of the germinal vesicle (GV), thus they are naturally synchronized cells that are arrested at prophase. In culture, the oocytes will spontaneously pass prophase as indicated by the appearance of the GVBD. Nardilysin is abundant in the germ cells of testis [7], and has been reported to be present in the ovary [14]. We previously reported that low levels of endogenous nardilysin can be found in the nuclei of HEK 293 cells. Similarly, low levels of recombinant nardilysin were found in the nucleus of transfected NIH3T3 cells [6]. Nardilysin appears to shuttle between the nucleus and cytoplasm, with the majority of the enzyme localized to the cytoplasm. We have further demonstrated that nardilysin possesses a functional nuclear localization signal at its N-terminus, which is responsible for its nuclear localization. The amount of nuclear nardilysin can be enhanced though the addition of either spermine or leptomycin B to cells. As nardilysin is a 140 kDa protein, it cannot pass through the nuclear pore complex (NPC) by passive diVusion, it must be actively transported. Deletion or mutation of the N-terminal nuclear localization signal caused nardilysin to remain in the cytoplasm [6]. Taking these results into account, it was of interest to determine whether it is present in oocytes and its subcellular distribution during oocyte maturation. In the present study, we conWrmed that nardilysin is expressed in oocytes and is translocated into the nuclei of oocytes in a cell-cycle regulated fashion.

Materials and methods Materials Monoclonal Cy3-anti-
-tubulin antibody, embryo tested mineral oil, albumin, and DAPI were purchased

from Sigma (St. Louis, MO). FITC-anti-nucleoporin monoclonal antibody was the product of BD Transduction Laboratories (San Diego, CA). Rabbit anti-sera to phosphodiesterase 3A, TRITC-anti-histone H1, and normal rabbit sera were from Santa Cruz Biotech. (Santa Cruz, CA). Alexa Fluor 488 chicken anti-rabbit and Alexa Fluor 594 chicken anti-mouse sera were from Molecular Probes (Eugene, OR). Anti-sera to recombinant nardilysin were produced in rabbits by Bethyl Labs (Montgomery, TX). Minimum essential medium (MEM), 200 mM glutamine, and 4–20% gradient SDS– polyacrylamide gels were obtained from Invitrogen (Carlsbad, CA). Unless indicated, all other materials were from Sigma (St. Louis, MO). Oocyte collection and culture All mice used in this study were maintained in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Ovaries from mice (Black Swiss, 3–5 weeks old) were harvested, placed in maturation medium (MEM with 2 mM L-glutamine, 25 mM Hepes, pH 7.4 100 U/ml penicillin, 100 g/ml streptomycin, and 0.23 mM sodium pyruvate) with 10 M cilostamide [15]. The isolated ovaries were punched with a Wne needle to release the cumulus-oocyte complex from antral follicles. Denuded oocytes were obtained by repeated pipetting cumulus-oocytes through a pulled Pasteur pipette under a stereomicroscope (Zeiss, Germany). After washing at least Wves times in a 50 l droplet of fresh maturation medium under mineral oil, the oocytes were incubated within a drop (50 l) of maturation medium covered by mineral oil in a 5% CO2 atmosphere at 37 °C for the desired time. For blocking tests, 10 M cilostamide was maintained in the maturation medium all the time. Cultured oocytes were Wxed in PBS with 2% of paraformaldehyde (Electron Microscopy Sciences, Washington, PA) at room temperature for 45 min. The Wxed oocytes were then transferred to a droplet of blocking solution (PBS with 0.3% BSA, 100 mM glycine) for »15 min. Neutralized oocytes were washed at least for Wve times with the blocking solution and then they were used readily or kept at 4 °C in blocking solution. Western blot analysis Six hundred denuded oocytes freshly isolated were treated with 15 l of lysis buVer (50 mM Tris–HCl, pH 7.4, containing 10 g/ml aprotinin, 2 g/ml leupeptin, 2 g/ml pepstatin, and 0.2 mM phenylmethylsulfonyl Xuoride) for 10 min, and mixed with an equal volume of 2£ SDS loading buVer (100 mM Tris–HCl, pH 6.8, 200 mM DTT, 4% SDS, 0.2% bromophenol blue, and 20% glycerol) and heated at 100 °C for 5 min. A sample of 0.1 g of puriWed recombinant nardilysin [16] was used as a positive control. Extracts of oocytes were

Z. Ma et al. / Archives of Biochemistry and Biophysics 434 (2005) 187–194

electrophoresed on a 4–20% of gradient polyacrylamide gel containing 0.1% SDS. The resolved proteins were electrophoretically transferred to a PVDF membrane, probed with rabbit anti-nardilysin and HRP-conjugated goat anti-rabbit IgG (H + L), and visualized using the ECL detection system (Amersham–Pharmacia Biotech). ImmunoXuorescence staining Fifty to sixty Wxed oocytes were permeabilized with blocking buVer containing 0.1% of Triton X-100 at RT for 5 min, and washed Wve times with blocking solution. Then, they were incubated with the appropriate primary antibody (1:50–1:100) at 4 °C overnight. After washing with blocking solution for Wve times, they were incubated with Alexa Fluor 488/594 matched with the appropriate primary antibody at RT for 1 h, followed by Wve times washing with blocking solution, and then staining with DAPI (1.5 g/ml) at RT for »5 min. The stained oocytes were equilibrated in PBS, pH 8.1/glycerol (1/1, vol/vol), then put on a slide framed with Vaseline (Altana, Melville, NY) and mounted with Vectashield Mounting Medium (Burlingame, CA). The samples were covered with a No. 1 micro cover slide (VWR, West Chester, PA), and sealed with nail polish (Electron Microscopy Sciences, Washington, PA). Images were recorded with a Leica TCS SP confocal microscope (Heidelberg, Germany). Unless otherwise noted, all images with a single Wgures were taken using identical excitation and detection voltages. Depletion of anti-sera to nardilysin Recombinant nardilysin was expressed and puriWed as described [16]. PuriWed recombinant nardilysin was added to anti-nardilysin anti-sera, incubated at RT for 2 h, and centrifuged at 10,000g for 5 min, and the supernatant was transferred to a new tube. This depleted antisera and normal rabbit sera are used to stain oocytes as negative controls. All above experiments were repeated independently at least three times.

Results Since nardilysin was found to be involved in spermatogenesis [7] and in small amount in human ovary [14], it was of interest to know whether nardilysin was present in oocytes. Because nardilysin was expressed in the ovary, it did not necessarily preclude that it was expressed in oocytes. In the present study, we examined the presence of nardilysin in mouse oocytes. We found that it was not only being expressed in oocytes, but also exhibiting nuclear localization as well as co-localization with the spindle apparatus during oocyte maturation

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Fig. 1. Western blot analysis of endogenous nardilysin in oocytes. Oocytes crude extracts were prepared as described under Materials and methods and subjected to a 4–20% gradient SDS–PAGE, then transferred to PVDF and probed with anti-sera to recombinant nardilysin, following incubation with HRP conjugated goat anti-rabbit IgG and ECL to detect signal through exposure to X-ray Wlm. Lane 1: 0.1 g of puriWed recombinant nardilysin; lane 2: crude extracts of oocytes.

(see details given later). As shown in Fig. 1, nardilysin is clearly demonstrated to be expressed in the oocytes by Western blot analysis using rabbit anti-sera to nardilysin. This is further supported by later data gained through immunochemistry assay (see details in Fig. 3). To document that the oocytes used in this study undergo normal maturation processes, they were monitored with a Leica stereomicroscope (Heidelberg, Germany) and were found to develop through the normal maturation process. As shown in Fig. 2 and Table 1, at 0 h time point of in vitro culture, 100% of the cells were at the GV stage. After 3 h of in vitro culture, 43% of the cells in medium without the use of the inhibitor of phosphodiesterase 3A (PDE 3A), cilostamide (10 M), were characterized by GVBD and after 18 h, »80% of these cells passed the GV stage, characterized either by GVBD (29%) or by formation of a polar body (PB, 51%). This process could be eVectively blocked with cilostamide. As seen in Table 1, at least 93% of oocytes in the group with 10 M of cilostamide were kept in the GV stage, only 4% of oocytes were in GVBD stage, and no oocyte showed the development of a polar body. These data show that the oocytes as maintained in this study underwent normal maturation and this normal maturation was eVectively blocked by cilostamide as previously reported [11,13,16,17]. Normal maturing and cilostamide blocked oocytes were stained with rabbit anti-sera to recombinant nardilysin. As seen in Fig. 3A, nardilysin is predominantly localized in the cytoplasm when placed into culture. If there is any present in the nucleus of an oocyte, it is below the detectable level of immunochemistry assay. This result agrees with previous reports [1,6,7] that the enzyme is mainly found in the cytoplasm. Within 30 min of in vitro culture, cytoplasmic nardilysin has not dramatically changed relative to that in oocytes at the 0 time point, however, it now can clearly be seen to begin to accumulate in the nuclei of the oocytes (Fig. 3B) compared with that at 0 h time point (Fig. 3A). After 3 h of in vitro culture, chromosomal DNA appears to either surround nardilysin or condensed chromosome DNA is surrounded by nardilysin (Fig. 3C). After 18 h of in vitro culture, nardilysin localizes with condensed chromo-

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Fig. 2. Oocytes maturation process. Oocytes were isolated and cultured as described under Materials and methods. At the indicated time, cultured oocytes in the presence or absence of 10 M cilostamide were observed and their images were taken using a Leica stereomicroscope. Upper panel, normal maturation process: (A) fresh GV stage, (B) late GV stage, (C) GVBD stage, (D) polar body stage. Lower panel, blocked maturation process: (E)–(H) all in GV stage (100£ magniWcations).

somal DNA in a spindle shape (Fig. 3D). Co-staining of 18-h-cultured oocytes (Fig. 4) with anti-nardilysin and Cy3-monoclonal anti-
-tubulin reveals that nardilysin co-localizes with
-tubulin at the spindle apparatus. This Wnding strongly suggests that nardilysin may be involved in the process of meiosis for separation of chromosomal DNA. As can be seen in Figs. 3E–H, when the maturation process was blocked by cilostamide, nardilysin did not enter into the nucleus even after 18 h of culture. This experiment implies that its translocation into the nucleus of an oocyte is tightly dependent on the maturation process. During our observations, nardilysin is closely related to the change of chromosomal DNA from 30 min to 3 h in vitro culture, however, it never co-localizes with histone H1 or chromosomal DNA (Figs. 3C and D, Fig. 4). To conWrm the accumulation of nuclear nardilysin in the oocyte, a FITC conjugated antibody to the nuclear envelope protein, nucleoporin [18], was used to co-stain with anti-sera to nardilysin. As seen in Fig. 5, nardilysin is mainly found in the cytoplasm with no detectable nuclear nardilysin at 0 h time point (Fig. 5A). This agrees with the results from Fig. 3A, as well as a previous report [6]. After 3 and 18 h of in vitro culture with the inhibition of 10 M cilostamide, there was no nuclear nardilysin observed within nucleoporin (Figs. 3C and D) at detectable levels. This experiment indicates that entry of nuclear nardilysin is inhibited by cilostamide. However, in the absence of cilostamide in the medium, nardilysin appears in the nucleus within 30 min of in vitro culture (Fig. 5B). This also matches with the result of in Fig. 3B. In a previous study, we found that only up to 10% of transfected cells showed nuclear nardilysin. The reason for this small amount of nuclear localization [6], may be caused by the cells being unsynchronized and at diVerent stages of the cell cycle, and only those transfected cells at late G2 stage (equals to the prior stage of GVBD of an oocyte) may

show nuclear localization. Although data are not shown here, we found it very diYcult to distinguish the nucleus from the cytoplasm by FITC-anti-nucleoporin in oocytes cultured for either 3 h or 18 h without cilostamide. We think this is because the nuclear envelope of the oocyte was broken at that time, and nucleoporin cannot be used as a control marker any more from this time point. To exclude the possibility that all cytosolic proteins may transfer into the nucleus of an oocyte during its maturation, oocytes at diVerent stages of development were probed with rabbit anti-sera to phosphodiesterase (PDE) 3A, a cytoplasmic protein [19] as a marker. As can be seen in Fig. 6, no nuclear PDE 3A is found at early stages nor is it found at the spindle apparatus in the polar body stage. This result implies that nuclear nardilysin is not an artifact of the system. Furthermore, the speciWcity of the anti-nardilysin anti-sera was established in experiments where oocytes were stained either with non-immune normal rabbit sera or with nardilysin depleted anti-sera, Fig. 7.

Discussion Oocyte meiosis resumption is a very complex process in which many proteins and/or enzymes such as protein kinase A (PKA), cyclin-dependent kinases (CDKs), cyclins, MAPK, PDE 3A, and cyclases are thought to be involved [20–23]. Nardilysin has been reported as being enriched in the male germ cell during sperm maturation [7], as well as being expressed in the human ovary [14]. To explore a possible function for the previously described nardilysin in ovary, its presence and subcellular distribution in an oocyte are examined in the present research. Here, we directly provide evidence that nardilysin is expressed in oocytes by Western blotting analysis and immunochemistry.

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Fig. 3. ImmunoXuorescent subcellular localization of nardilysin by confocal microscopy in maturing oocytes. Oocytes were collected and cultured in the presence or absence of cilostamide at the indicated time as described in Fig. 2, and then they were Wxed by 2% paraformaldehyde and immunostained as described under Materials and methods with anti-nardilysin (green); anti-histone H1 (red); and DAPI (blue), following incubation with appreciated Alexa Fluor 488/594 conjugated secondary antibodies, respectively. Then, the oocytes were put into a slide as described under Materials and methods, and observed through a Leica confocal TCS SP microscope. Images were taken through an image linked system controlled by Leica confocal software, LCS lite. Upper panel and lower panel are the same as those described in Fig. 2 (1000£ magniWcations). Table 1 EVect of cilostamide on spontaneous resumption of meiosis by mice oocytes in vitro

Maturation medium Maturation medium + 10 M of cilostamide

No. of tested

0 h (%) GV

GVBD

PB

3 h (%) GV

GVBD

PB

18 h (%) GV

GVBD

PB

100 100

100 100

0 0

0 0

57 100

43 0

0 0

13 93

29 4

51a 0b

Note. PB, polar body. a Seven oocytes in fresh medium group and bthree oocytes in medium plus 10 M cilostamide were dead.

Fig. 4. ImmunoXuorescent co-localization of nardilysin and
-tubulin by confocal microscopy in polar body stage during maturing oocytes. Oocytes were collected and cultured for 18 h in the absence of cilostamide as described in Fig. 2, then Wxed and immune-stained as in Fig. 3 with anti-nardilysin (green), Cy3-anti-
-tubulin (red) and DAPI (blue) respectively. Images were taken in the manner as in Fig. 3 (1000£ magniWcations).

In dynamic studies of oocytes maturation, nardilysin was found to be predominantly localized in the cytoplasm at 0 h time point. This result concurs with most reports regarding the localization of the enzyme [1,6,7]. However, nardilysin is seen to accumulate in the nucleus of an oocyte within 30 min of development, a time at which nuclear-envelop-breakdown begins to occur. This experiment demonstrates that nardilysin can enter into the nucleus of a cell as previously proposed [6] based on its presence in the nuclei of HEK 293 cells and NIH 3T3 cells. We assume that the mechanism of its nuclear localization in an oocyte may be in the same manner as that

we previously conWrmed through its N-terminal bipartite NLS [6]. After 3 h of in vitro culture, nardilysin formed a sphere surrounded by condensed chromosomal DNA, prior to the spindle-forming step. At the polar body stage, nardilysin was co-localized with
-tubulin in the spindle apparatus. This dynamic change of the localization of nardilysin suggests this enzyme may play role (s) in separating condensed chromosomal DNA. One possible function for nuclear nardilysin is that it might degrade peptides or hormones which are involved in the contraction of motor proteins during meiosis. Alternatively, as nardilysin possesses a functional acidic domain

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Fig. 5. ImmunoXuorescent nuclear accumulation of nardilysin determined by confocal microscopy in the early stage of maturing oocytes. Oocytes were collected and cultured at indicated time in the presence or absence of cilostamide as in Fig. 2. They were Wxed and immune-stained as in Fig. 3 with rabbit anti-nardilysin (red) and FITC-anti-nucleoporin (green), following incubation with Alexa Fluor 594 conjugated anti-rabbit antibodies. Images were taken in the manner as in Fig. 3. (A) A freshly isolated oocyte. (B) A 30 min activated oocyte in the absence of cilostamide. (C) and (D) 3 and 18 h activated oocytes in the presence of cilostamide, respectively. (1000£ magniWcations).

[1,8,16,24] it may interact with other enzymes or proteins to aVect their functions in meiosis. Nardilysin can interact in a non-catalytic manner with heparin-binding EGF like growth factor [8] as well as other proteins (Chow and Hersh, unpublished data) through its acidic domain. Thus, co-localization with
-tubulin at spindle apparatus may be another case of non-catalytic capacity as well. Chesneau et al. [7] reported that nardilysin was found at two fundamental microtubular structures in late spermatides: the manchette and the axoneme. However, in that study nardilysin localization at the spindle apparatus was not found. This may have occurred because the cells were not in meiosis at the time observed. The bottom line of these two independent studies both found nardilysin localized at motor proteins.

The data presented here not only conWrm our previous proposal that nardilysin could enter into the nucleus [6], but also show one of the reasons why it goes into nucleus—to cooperate with
-tubulin during mitosis or meiosis. Of course, we will explore the true reason(s) why it localizes in the spindle apparatus in the near future. Although it has been suggested that the second ATG of nardilysin is the start of translation [25], the present study as well as previous data [6] clearly indicate that nardilysin can translocate into the nuclei of cells. The nuclear localization signal of nardilysin is located between the Wrst and second methionine. If the second methionine were used exclusively, its nuclear localization signal would be absent from the enzyme and it would stay in the cytoplasm absolutely. But previous data as well as the present study are not in this case. In the previ-

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Fig. 6. ImmunoXuorescence of phosphodiesterase (PDE) 3A by confocal microscopy in maturing oocytes. Oocytes were collected, cultured at the indicated time in the absence of cilostamide as in Fig. 2, and then they were Wxed and immune-stained as in Fig. 3 with anti-rabbit the regulatory domain of PDE 3A (green) and TRITC-anti-histone H1 (red), respectively. Images were taken in the manner in Fig. 3 (1000£ magniWcations).

Fig. 7. ImmunoXuorescent of nardilysin by confocal microscopy at polar body stage in the maturing oocytes. Oocytes were collected, cultured for 18 h in the absence of cilostamide as in Fig. 2, and following Wxation and immune-staining as in Fig. 3, and probed with normal rabbit sera (upper panel, green) and depleted anti-sera to nardilysin (lower panel, green), as well as anti-
-tubulin (red), and DAPI (blue) respectively. Images were taken in the manner of Fig. 3 (1000£ magniWcations).

ous study [6], we found that nardilysin could rapidly shuttle in and out of the nucleus, with the equilibrium strongly favoring cytoplasmic enzyme. In the present study, we also found that nardilysin could accumulate in the nuclei of oocytes with the majority of cytoplasm. These two independent experiments concur so well, this means that nuclear nardilysin has to be translated starting from the Wrst ATG cordon. We also noted when cilostamide blocked meiosis of oocytes, nardilysin did not enter into the nucleus either. This suggests that nardilysin’s entry into the nucleus is coupled with the maturation process and its nuclear entry is tightly regulated. There are two regulatory mechanisms that come to mind. First, during oocyte maturation nardilysin translation may switch from the second methionine to the Wrst, producing a form with the nuclear localization signal. Alternatively, a factor required for nardilysin nuclear localization is produced in conjunction with oocyte maturation. It had been shown that there was an increase in nuclear envelope permeability before GVBD during starWsh oocyte maturation [26,27]. However, the authors noted that entry of a 70 kDa protein from the cytoplasm to the nucleus was the limited size of cutting-oV for

nuclear pores even though their passive permeability had been increased [27]. We do not think nuclear nardilysin is a result of leakage when the permeability of the nuclear envelope is increased before GVBD processing, since its molecular size is »140 kDa. This is further supported by the Wnding that PDE 3A, which has a similar molecular size of nardilysin, is exclusively distributed in the cytoplasm and never co-localized with
-tubulin at spindle apparatus during the late stage of meiosis. In summary, nardilysin being tightly regulated enters into the nucleus of an oocyte during the early stage of development. Condensed chromosomal DNA is wrapped by nardilysin or it is surrounded by condensed chromosomal DNA at the GVBD stage. Nardilysin is co-localized with
-tubulin at the spindle apparatus during the polar body stage.

Acknowledgments We thank Dr. Hongtao Xu’s (Department of Pathology and Microbiology, University of Prince Edward Island, Charlottetown, PE C1A 4P3, Canada) and Ms. Kathie Nichol’s (Department of Microbiology, Immu-

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