1 Centro de Estudios en Ginecología y Reproducción, CEGyR, Buenos Aires, 2 Centro de Investigaciones en Reproducción, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina and 3 Departments of Obstetrics and Gynecology, and Animal Science, University of MissouriColumbia, S141 ASRC, 920 E Campus Drive, Columbia, MO 652115300, USA
4 To whom correspondence should be addressed at: Pittsburgh Developmental Center of MageeWomens Research Institute and Departments of ObstetricsGynecologyReproductive Sciences and Cell Biology and Physiology, University of Pittsburgh, 204 Craft Ave, Pittsburgh, PA 15213, USA. e-mail: vrawe@mail.magee.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: fertilization failure/human zygotes/nuclear envelope/nuclear pore complex/pronuclear development
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The assembly of the NE following mammalian fertilization starts with membrane-free chromatin (Szöllosi et al., 1972; Longo, 1973
) represented by two distinct entities: a set of maternal chromosomes and the sperm nucleus. Subsequently, the assembly of the NE takes place in a fashion similar to that seen in somatic cells, regardless of whether the sperm entered the oocytes during IVF or ICSI (Sutovsky et al., 1997
, 1998).
Although the assembly and fusion of membrane vesicles during NE formation have been studied extensively in somatic cells and in a cell-free system (reviewed by Poccia and Collas, 1996), little is known about the pathways leading to the formation of NPC on the zygotic NE, especially in mammalian models. Previous studies in bovine have demonstrated that the fertilizing sperm triggers the assembly of annulate lamellae (AL), in parallel with NPC insertion into NE (Sutovsky et al., 1998
). AL, the cytoplasmic stacks of NPC (reviewed by Kessel, 1992
), cross-react with a nucleoporin-specific antibody mAb 414 in Xenopus egg extracts (Meier et al., 1995
) and in mammalian pronuclear zygotes (Sutovsky et al., 1998
; Ramalho-Santos et al., 2000
).
Despite the continuous improvement of IVF techniques, several levels at which fertilization can fail have been described (Asch et al., 1995; Rawe et al., 2000
). The arrest at two-pronuclei (2PN) stage is one of frequently seen patterns. Although AL have been found in human metaphase II oocytes (Baca and Zamboni, 1967
) and pronuclear zygotes (Hertig and Adams, 1967
; Zamboni et al., 1966
; Van Blerkom et al., 1987
), it is not known how and when NPC are incorporated into NE of developing human pronuclei, or if NPC and/or AL play any role during human fertilization and zygotic development.
In the present study we used immunofluorescence (IF) with an NPC- and AL-specific antibody, mAb 414, to visualize the assembly of NPC and AL in the fertilized and arrested human zygotes. Ultrastructural features of fertilized and arrested human zygotes were characterized by electron microscopy (EM).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nucleoporin and chromatin labelling
Nine prophase I (PI), 11 metaphase I (MI) and 10 metaphase II (MII) human oocytes were screened after the labelling of nucleoporins and chromatin by conventional epifluorescence microscopy. At the same time, a total of 21 supernumerary fertilized zygotes (11 from IVF and 10 from ICSI) and 13 arrested fertilized zygotes (PN formation without further cleavage after 40 h of insemination or injection, six from IVF and seven from ICSI) were screened for nucleoporins and chromatin labelling using a modified protocol of Messinger and Albertini (1991
). For this purpose, zona pellucida was removed by a brief incubation with acid Tyrodes medium, and denuded oocytes/zygotes were fixed and permeabilized for 20 min at 37°C in a microtubule stabilizing buffer (0.1 mol/l PIPES, pH 6.9, 5 mmol/l MgCl2·6H2O, 2.5 mmol/l EGTA containing 2.0% formaldehyde, 0.5% Triton X-100, and 1 µmol/l taxol). Fixed oocytes/zygotes were blocked for 1 h at 37°C with 2% bovine serum albumin (BSA), 2% powdered milk, 2% normal goat serum, 0.1 mol/l glycine and 0.01% Triton X-100 in PBS. If necessary, oocytes/zygotes were stored for up to 3 days at 4°C in this solution. To identify NPC, oocytes were incubated for 40 min at room temperature with 1:250 dilution of antibody mAb414 (BabCo; Berkeley, CA, USA) (Davis and Blobel, 1986
, 1987; Meier et al., 1995
; Sutovsky et al., 1998
) in PBS containing 0.1% BSA and 0.02% sodium azide (PBS + BSA). After a short wash in blocking solution, oocytes were incubated in 1:500 dilution of fluorescein-conjugated goat anti-mouse immunoglobulin G (IgG), for 1 h at room temperature. Finally, treated oocytes were washed three times in PBS + BSA, counterstained with Hoechst 33258 (1 µg/ml) for 30 min at room temperature, washed again in PBS + BSA and mounted between a microscopy slide and a coverslip for examination using Olympus BX-40 epifluorescent microscope. Images were photographed using Kodak Ektachrome film (1600ASA) and processed using Adobe Photoshop 5.0 software (Adobe System Inc.). For control staining, PBS + BSA alone replaced the specific antibody solution. Secondary antibodies and reagents used were purchased from Sigma (St Louis, MO, USA).
Transmission electron microscopy
Six arrested zygotes (2PN stage) and five control zygotes were fixed for 1 h in a fixative composed of 2.5% glutaraldehyde and 0.6% paraformaldehyde in 0.25 mol/l cacodylate buffer (pH 7.2), then washed in 0.1 mol/l cacodylate buffer containing 0.2 mol/l sucrose and post-fixed for 1 h in 1% osmium tetroxide. Following dehydration by ascending ethanol series (30100%), oocytes were infiltrated by a series of washes in a mixture of propylene oxide and Polybed 812 (Polyscience, Warrington, PA, USA) EM resin and embedded in PolyBed 812. Ultrathin sections were cut on a PorterBlum Sorvall MT2B ultramicrotome and stained in two steps with uranyl acetate and lead citrate. Thin sections were examined and photographed in a Zeiss EM 9A electron microscope (Zeiss, Oberkochen, Germany). Negatives were scanned and printed using Adobe Photoshop 5.0 software.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Transmission electron microscopy of control and arrested zygotes
To provide more support for the observations of fertilized oocytes using IF, we explored the distribution of NPC and AL by EM. At the ultrastructural level (Figure 3), the presumed normally fertilized zygotes displayed small stacks of AL distributed through the cytoplasm and adjacent to the NE (Figure 3A). In contrast, large assemblies of AL were predominantly found in the vicinity of the male and female pronuclei in the arrested zygotes (Figure 3B, C). An interesting feature of arrested zygotes was the presence of numerous mitochondria with accumulation of the electron-dense material in mitochondrial matrix (Figure 3C). On serial ultrathin sections, such mitochondria were found to be distributed evenly throughout the zygotic cytoplasm, but were not detected in the control zygotes. In the supernumerary fertilized zygotes (controls), the EM study revealed an apparently normal pronuclear development (Figure 3D). While the NE of such zygotes contained numerous NPC (Figure 3E), the number of NPC was greatly diminished in the arrested zygotes (Figure 3F, Table II). Internalization of AL into both pronuclei, described previously (Zamboni et al., 1966; Van Blerkom and Motta, 1989
) in presumably normal zygotes, and the presence of NPC-free stretches of NE were recorded in the arrested zygotes (Figure 3F), but not in the control ones.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
While the arrested zygotes showed 2PN in light microscopy, and their overall appearance was similar to control 2PN zygotes (Figure 2E, F), epifluorescence revealed DNA condensation and fragmentation in the arrested zygotes. It is possible that DNA within the pronuclei of arrested zygotes became fragmented and condensed while the NE remained intact, thus maintaining normal appearance in light microscopy. This is supported by the observation that all arrested zygotes screened by immunofluorescence showed 2PN configurations indistinguishable from normal zygotes when examined by light microscopy. DNA fragmentation and disappearance of NPC from NE of the arrested zygotes could be signs of abortive apoptotic process.
Previous studies suggested that NPC are involved in selective nucleocytoplasmic transport in the pronuclear bovine zygotes (Sutovsky et al., 1998). The anomalies of AL assembly were shown to accompany fertilization arrests after ICSI in rhesus monkey (Ramalho-Santos et al., 2000
). NPC are removed from the sperm NE during spermatid elongation in the testis (Sutovsky et al., 1999
) and the NE of mature sperm is removed completely during fertilization in order to make the sperm chromatin accessible to oocyte cytoplasmic factors necessary for the remodelling of the sperm nucleus (Perreault et al., 1984
; Poccia and Collas, 1996
; Sutovsky et al., 1996, 1997; Sutovsky and Schatten, 1997, 2000; Usui et al., 1997
; Wright, 1999
).
After fertilization arrest at 1-cell stage, the AL- and NPC-specific antibody mAb 414 (Meier et al., 1995; Sutovsky et al., 1998
) revealed the accumulation of AL in the cytoplasm of arrested zygotes. AL formed large patches assembled near the fragmented pronuclei, as previously observed in some rhesus monkey ICSI oocytes (Sutovsky et al., 1996
; Ramalho-Santos et al., 2000
). It is possible that the pronuclear development and nucleocytoplasmic communication were impaired in these zygotes. Consequently, the pronuclear development was not completed and the mitotic division had not taken place. This is consistent with the observation that the microinjection of an NPC antagonist, wheat germ agglutinin, prevented embryo development in bovine (Sutovsky et al., 1998
). Similarly, the S-phase entry was blocked in rhesus monkey embryos with deviant PN and AL appearance after ICSI (Ramalho-Santos et al., 2000
). Studies of Xenopus oocytes suggest that one of the NPC components, RanBP2 (also called Nup358), is a key factor in the interphase nucleocytoplasmic transport, but also in the control of mitotic events, including spindle assembly during metaphase and the reformation of the NE during telophase (Greber and Carafoli, 2002
).
Alternatively, the abnormal biogenesis or function of the zygotic centrosome, documented previously in some cases of human fertilization failure (Asch et al., 1995; Van Blerkom, 1996
), could affect the recruitment of NPC and cytoplasmic trafficking of AL in some of the arrested, fertilized zygotes, mainly in those not showing pronuclear apposition. The nocodazole-induced disruption of the microtubules within fertilized bovine oocytes was indeed shown to derail the pronuclear recruitment of NPC, PN development and cytoplasmic movement of AL in fertilized bovine oocytes (Sutovsky et al., 1998
).
EM studies confirmed the pathological reorganization of NPC and AL in arrested zygotes, previously observed by IF. Large assemblies of AL have been found in the cytoplasm of arrested 2PN zygotes, while smaller stacks of AL were seen in the control ones. A similar pattern of AL assembly was observed by IF in normally fertilized zygotes that did not cleave after cryopreservation/thaw. Cytoskeletal damage caused by cryopreservation (Sathananthan et al., 1988; Van der Elst et al., 1992
; Park et al., 1997
; Wei-Hua et al., 2001
; Boiso et al., 2002
) may affect the pattern of AL distribution. The association of AL with microtubules has been demonstrated previously (reviewed by Kessel, 1992
; Sutovsky et al., 1998
). The electron-dense deposits observed in the mitochondria throughout the cytoplasm of arrested zygotes were reminiscent of calcium phosphate deposits in preimplantation embryos of mouse t-mutant (Hillman and Tasca, 1973
). In our studies, such mitochondria were distributed randomly throughout the cytoplasm of arrested zygotes, and could be suggestive of apoptotic process, known to start with alterations of the mitochondrial membrane and matrix. Furthermore, it might be interesting to speculate that the release of activated caspases by these abnormal mitochondria could lead to the digestion of many cellular proteins responsible for cell cycle regulation, DNA damage recognition and repair and regulation of the cellular structure (for review see Nalepa and Zukowska-Szczechowska, 2002
).
The results presented in this study document that the patterns of NPC and AL distribution in human oocytes that failed to fertilize do not mimic those seen in normal zygotes. Such observations suggest a differential distribution of NPC and AL in the normal and arrested pronuclear zygotes. These findings have implications for understanding the aetiology and cellular basis of early developmental failure in humans.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Asch, R., Simerly, C. S., Ord, T., Ord, V. A. and Schatten, G. (1995) The stages at which human fertilization arrests: microtubule and chromosome configurations in inseminated oocytes which fail to complete fertilization and development in humans. Hum. Reprod., 10, 18971906.[Abstract]
Baca, M. and Zamboni, L. (1967) The fine structure of human follicular oocytes. J. Ultrastruct. Res., 19, 354381.[ISI][Medline]
Benavente, R., Dabauvalle, M-C., Scheer, U. and Chaly, N. (1989) Functional role of newly formed pore complexes in postmitotic nuclear reorganization. Chromosoma, 98, 233241.[CrossRef][ISI][Medline]
Boiso, I., Marti, M., Santalo, J., Ponsa, M., Barri, P.N. and Veiga, A. (2002) A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage. Hum. Reprod., 17, 18851891.
Boman, A.L., Delannoy, M.R. and Wilson, K.L. (1992) GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro. J. Cell Biol., 116, 281294.[Abstract]
Burke, B. and Gerace, L. (1986) A cell free system to study reassembly of the nuclear envelope at the end of mitosis. Cell, 44, 639652.[ISI][Medline]
Collas, P., Courvalin, J.-C. and Poccia, D. (1996) Targeting of membranes to sea urchin sperm chromatin is mediated by a lamin B receptor-like integral membrane protein. J. Cell Biol., 135, 17151725.[Abstract]
Davis, L.I. and Blobel, G. (1986) Identification and characterization of a nuclear pore complex protein. Cell, 45, 699709.[ISI][Medline]
Davis, L.I. and Blobel, G. (1987) Nuclear pore complex contains a family of glycoproteins that includes p62: glycosylation through a previously unidentified cellular pathway. Proc. Natl Acad. Sci. USA, 84, 75527556.[Abstract]
Görlich, D. and Mattaj, I.W. (1996) Nucleocytoplasmic transport. Science, 271, 15131518.[Abstract]
Greber, U.F. and Carafoli, E. (2002) Signalling takes control of nucleo-cytoplasmic trafficking. EMBO Rep., 3, 410414.
Hanover, J.A., Cohen, C.K., Willingham, M.C. and Park, M.K. (1987) O-linked N-acetylglucosamine is attached to proteins of the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycoproteins. J. Biol. Chem., 262, 98879894.
Hertig, A.T. and Adams, E.C. (1967) Studies on the human oocyte and its follicle. I. Ultrastructural and histochemical observations on the primordial follicle stage. J. Cell Biol., 34, 647675.
Hillman, N. and Tasca, R.J. (1973) Synthesis of RNA in t 12 -t 12 mouse embryos. J. Reprod. Fertil., 3, 501506.
Kessel, R.G. (1992) Annulate lamellae: Last frontier in cellular organelles. Int. Rev. Cytol., 133, 43120.[ISI][Medline]
Longo, F.J. (1973) Fertilization. A comparative ultrastructural review. Biol. Reprod., 9, 149215.[ISI][Medline]
Macaulay, C. and Forbes, D.J. (1996) Assembly of the nuclear pore: Biochemically distinct steps revealed with NEM, GTPgS and BAPTA. J. Cell Biol., 132, 520.[Abstract]
Meier, E., Miller, B.R. and Forbes, D.J. (1995) Nuclear pore complex assembly studied with a biochemical assay for annulate lamellae formation. J. Cell Biol., 129, 14591472.[Abstract]
Messinger, S.M. and Albertini, D.I. (1991) Centrosome and microtubule dynamics during meiotic progression in the mouse oocyte. J. Cell Sci., 100, 289298.[Abstract]
Nalepa, G. and Zukowska-Szczechowska, E. (2002) Caspases and apoptosis: die and let live. Wiad Lek., 55, 100106.[Medline]
Newport, J.W. and Spann, T. (1987) Disassembly of the nucleus in mitotic extracts: membrane vesicularization, lamin disassembly and chromosome condensation are independent processes. Cell, 48, 219230.[ISI][Medline]
Newport, J.W., Wilson, K.L. and Dunphy, W.G. (1990) A lamin-independent pathway for nuclear envelope assembly. J. Cell Biol., 111, 22472259.[Abstract]
Panté, N. and Aebi, U. (1993) The nuclear pore complex. J. Cell Biol., 122, 977984.[ISI][Medline]
Park, S.E., Son, W.Y., Lee, S.H., Lee, K.A., Ko, J.J. and Cha, K.Y. (1997) Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage. Fertil. Steril., 5, 920926.[CrossRef]
Perreault, S.D., Wolf, R.A. and Zirkin, B.R. (1984) The role of disulfide bond reduction during mammalian sperm nuclear decondensation in vivo. Dev. Biol., 101, 160167.[ISI][Medline]
Pfaller, R., Smythe, C. and Newport, J.W. (1991) Assembly/disassembly of the nuclear envelope membrane: cell cycle-dependent binding of nuclear membrane vesicles to chromatin in vitro. Cell, 65, 209217.[CrossRef][ISI][Medline]
Poccia, D. and Collas, P. (1996) Transforming sperm nuclei into male pronuclei in vivo and in vitro. Curr. Top. Dev. Biol., 34, 2588.[ISI][Medline]
Ramalho-Santos, J., Sutovsky, P., Simerly, C., Oko, R., Wessel, G., Hewitson, L. and Schatten, G. (2000) ICSI choreography: fate of sperm structures after monospermic rhesus ICSI and first cell cycle implications. Hum. Reprod., 15, 26102620.
Rawe, V.Y., Brugo Olmedo, S., Nodar, F.N., Doncel, G.F., Acosta, A.A. and Vitullo, A.D. (2000) Cytoskeletal organisation defects and abortive activation in human oocytes after IVF and ICSI failure. Mol. Hum. Reprod., 6, 510516.
Sathananthan, A., Trounson, A., Freemann, L. and Brady, T. (1988) The effects of cooling human oocytes. Hum. Reprod., 3, 968977.[Abstract]
Snow, C. M., Senior, A. and Gerace, L. (1987) Monoclonal antibodies identify a group of nuclear pore complex glycoproteins. J. Cell Biol., 104, 11431156.[Abstract]
Sutovsky, P. and Schatten, G. (1997) Depletion of glutathione during oocyte maturation reversibly blocks the decondensation of the male pronucleus and pronuclear apposition during fertilization. Biol. Reprod., 56, 15031512.[Abstract]
Sutovsky, P. and Schatten, G. (2000) Paternal contributions to the mammalian zygote: fertilization after spermegg fusion. Int. Rev. Cytol. 195, 165.[ISI][Medline]
Sutovsky, P., Hewitson, L.C., Simerly, C.R., Tengowski, M.W., Navara, C.S., Haavisto, A.J. and Schatten, G. (1996) Intracytoplasmic sperm injection (ICSI) for rhesus monkey fertilization results in unusual chromatin, cytoskeletal and membrane events, but eventually leads to pronuclear development and sperm aster assembly. Hum. Reprod., 11, 17031712.[Abstract]
Sutovsky, P., Oko, R., Hewitson, L. and Schatten, G. (1997) The removal of the sperm perinuclear theca and its association with the bovine oocytes surface during fertilization. Dev. Biol., 188, 7584.[CrossRef][ISI][Medline]
Sutovsky, P., Simerly, C., Hewiston, L. and Schatten, G. (1998) Assembly of nuclear pore complexes and annulate lamellae promotes normal pronuclear development in fertilised mammalian oocytes. J. Cell Sci., 111, 28412854.
Sutovsky, P., Ramalho-Santos, J., Moreno, R.D., Oko, R., Hewitson, L. and Schatten, G. (1999) On-stage selection of single round spermatids using a vital, mitochondrion-specific fluorescent probe MitoTrackerTM and high resolution differential interference contrast (DIC) microscopy. Hum. Reprod., 14, 23012312.
Szöllosi, D., Calarco, P. and Donahue, R.P. (1972) The nuclear envelope: its breakdown and fate in mammalian oogonia and oocytes. Anat. Rec., 174, 325340.[ISI][Medline]
Usui, N., Ogura, A., Kimura, Y. and Yanagimachi, R. (1997) Sperm nuclear envelope: breakdown of intrinsic envelope and de novo formation in hamster oocytes or eggs. Zygote, 5, 3546.[ISI][Medline]
Van Blerkom, J. (1996) Sperm centrosome dysfunction: a possible new class of male factor infertility in the human. Mol. Hum. Reprod., 2, 349354.[Abstract]
Van Blerkom, J. and Motta, P. (1989) Ultrastructure of Human Gametogenesis and Early Embryogenesis. Kluwer, Boston.
Van Blerkom, J., Bell, H. and Henry, G. (1987) The occurrence, recognition and developmental fate of pseudo-multipronuclear eggs after in-vitro fertilization of human oocytes. Hum. Reprod., 2, 217225.[Abstract]
Van der Elst, J., Nerinckx, S. and Van Steirteghem, A.C. (1992) In vitro maturation of mouse germinal vesicle-stage oocytes following cooling, exposure to cryoprotectants and ultrarapid freezing: limited effect on the morphology of the second meiotic spindle. Hum. Reprod., 7, 14401446.[Abstract]
Wei-Hua, W., Li Meng, R., Hackett, J., Odenbourg, R. and Keefe, D.L. (2001) Limited recovery of meiotic spindles in living human oocytes after coolingrewarming observed using polarized light microscopy. Hum. Reprod., 16, 23742378.
Wright, S.J. (1999) Sperm nuclear activation during fertilization. Curr. Top. Dev. Biol., 46, 133178.[ISI][Medline]
Zamboni, L., Mishell, D.R. Jr, Bell, J.H. and Baca, M. (1966) Fine structure of the human ovum in the pronuclear stage. J. Cell Biol., 3, 579600.[CrossRef]
Submitted on May 17, 2002; resubmitted on August 30, 2002; accepted on November 19, 2002.