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RESEARCH |
1 Department of Animal Sciences, University of Missouri, S-141 ASRC, 920 E Campus Drive, Columbia, Missouri 65211, USA, 2 Julius-Maximilians-University, Würzburg, Germany, 3 Research Institute of Animal Production, Constantine the Philosopher University, Nitra, Slovak Republic, 4 Tumor Biology Program, Mayo Clinic, Rochester, Minnesota, USA, 5 Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri, USA and 6 Obstetrics and Gynecology, University of Missouri, Columbia, Missouri, USA
Correspondence should be addressed to G Manandhar; Email: manandharg{at}missouri.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Introduction |
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Among various resident molecules of centrosomes, centrins are 20 kDa, Ca+2-binding contractile proteins occurring in undifferentiated cells of phylogenetically diverse eukaryotic organisms (Salisbury 1995, Schiebel & Bornens 1995) and in differentiated cells in which centrioles are conserved (Wolfrum 1991, 1992, 1995). The pericentriolar proteins (e.g., 
-tubulin) do not always accompany canonical centrosomes, as they can exist outside the centrosome in some cells (Novakova et al. 1996, Wilson et al. 1997) or be present in the spindle poles/astral foci lacking an organized centrosome (Gueth-Hallonet et al. 1993, Palacios et al. 1993, Callaini et al. 1999). In contrast, centrin is intimately associated with the microtubular triplets (Wolfrum 1995, Manandhar et al. 1999) or the distal lumen of centrioles (Levy et al. 1996, Paoletti et al. 1996). During mouse and primate spermiogenesis, centriole degeneration is correlated with the loss of their centrin labeling (Manandhar et al. 1999, Manandhar & Schatten 2000). Taken together, these studies suggest that punctate centrin localization can be used as a marker of centrioles.
Mammals have several isoforms of centrin (Paoletti et al. 1996). Three have been well defined and expressed in different types of cells, specializing in different functions. Centrin-2 (cen2p) is ubiquitously expressed in somatic cells (Wolfrum & Salisbury 1998, Hart et al. 1999) and involved in centriole replication (Salisbury et al. 2002). Human CEN2 is localized in the chromosome Xq28 (Tanaka et al. 1994, Chatterjee et al. 1995). Human Centrin-1 gene (HsCEN1) is intron-less retroposon derived from CEN2 and localized in the chromosome 18A2 (Errabolu et al. 1994, Hart et al. 1999). It is expressed in developing spermatids when the expression of CEN2 is shut down (Hart et al. 1999). Besides spermatids, CEN1 expression has been found in differentiating ciliated cells of the retina (Wolfrum & Salisbury 1998) and nasal epithelium (Laoukili et al. 2000). Hence, it is likely to be involved in the formation of axonemes of the motile organelles. The Centrin-3 gene (CEN3) is more closely related to yeast CDC31 than to CEN1 or CEN2. Yeast cdc31p is localized in the bridge of the spindle pole body (SPB) playing an important role in the SPB duplication (Baum et al. 1986). Human cen3p is expressed less abundantly in somatic epithelial cells (LeDizet et al. 1998, Laoukili et al. 2000) and mainly enriched in centrosomes. Therefore, it may be important for ciliogenesis or centriole replication. Cen3p migrates slower in SDS-PAGE (~23 kDa) than cen1p/2p (Middendorp et al. 1997).
Centrin follows unique mode of degeneration during spermatogenesis in different animal species. Mouse spermatozoa lose centrin completely along with the centioles (Manandhar et al. 1999). In primates, it is partially degenerated correlating with the partial degradation of distal centrioles (Manandhar & Schatten 2000). Hence, the spermatozoa are likely to contribute centrin to the embryonic centrosomes in higher mammals. The present work has shown that boar spermatozoa possess centrin similar to primate spermatozoa. But the oocytes do not retain the gametic centrin contributed by spermatozoa after in vitro fertilization (IVF) or the somatic centrin contributed by pig embryonic fibroblast (PEF) after somatic cell nuclear transfer (SCNT). Despite the presence of residual CEN1, CEN2 mRNAs in the oocytes and embryos, the embryonic centrin is regenerated only during late blastocyst stage.
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Materials and Methods |
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Sperm-head preparation
Boar spermatozoa were washed by suspending in cold (4 °C) PBS and centrifuged. The pellets were resuspended in cold PBS containing 0.1% Triton X-100 and sonicated for 1 min on ice at 30% intensity with Branson Sonifier (Branson Ultrasonic Corp, Danbury, CT, USA). The suspensions were layered on 70% sucrose cushions and centrifuged at 100 000 g for 1 h at 4 °C. They were examined under light microscope to check the purity of the sperm-head preparations, and further sonicated and centrifuged until the purity of the sperm-head fraction was more than 99%.
Oocyte maturation, in vitro fertilization and embryo culture
Porcine ovaries were collected from a local abattoir. Cumulus–oocyte complexes (COCs) were aspirated from antral follicles (3–6 mm size) and washed three times in HEPES-buffered Tyrode lactate medium containing 0.1% (w/v) polyvinyl alcohol (TL-HEPES-PVA). For maturation to MII stage, they were transferred to 500 µl drop of maturation medium consisting of tissue culture medium (TCM) 199 (Gibco) supplemented with 0.1% PVA, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 µg/ml luteinizing hormone (LH), 0.5 µg/ml follicle-stimulating hormone (FSH), 10 ng/ml epidermal growth factor, 10% porcine follicular fluid, 75 µg/ml penicillin G, and 50 µg/ml streptomycin. The medium drops were overlaid with mineral oil in four-well dishes (Nunc, Roskilde, Denmark) and incubated at 38.5 °C, 5% CO2 in air. After 22 h of culture, the oocytes were transferred to fresh maturation medium without LH and FSH, and cultured for another 22 h.
For in vitro fertilization, the cumulus cells of the matured COCs were removed by vortexing with 0.1% hyaluronidase in TL-HEPES-PVA medium and washed with Tris-buffered medium (mTBM) (Abeydeera et al. 1998) containing 0.2% (w/v) BSA. About 25–30 oocytes were placed in 50 µl drops of mTBM, covered with mineral oil, and incubated for about 30 min until spermatozoa were added. A semen pellet was thawed in PBS containing 0.1% PVA (PBS–PVA) and centrifuged through two-layered Percoll gradients (60 and 40%) at 2500 r.p.m. for 10 min. The spermatozoa were resuspended, washed twice in PBS–PVA, and resuspended in mTBM. The sperm suspension was added to the 50 µl oocytes-containing medium to give a final sperm concentration of 106/ml. Oocytes were co-incubated with spermatozoa for 6 h, and transferred to 500 µl drops of NCSU-23 medium (Petters & Wells 1993) containing 0.4% BSA for further culture.
Somatic cell nuclear transfer
The PEF cells isolation and nuclear transfer were done according to the previously described method (Park et al. 2001). PEF cells were isolated from a day 35 crossbred porcine fetus. The tissue was cut into small pieces and incubated for 30 min at 37 °C in PBS containing 0.05% trypsin and 0.02 mM EDTA. The cell suspension was centrifuged, pellet washed, resuspended, and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM L-glutamine, 0.1 mM Na-pyruvate, 75 µg/ml penicillin G, 50 µg/ml streptomycin, and 15% (v:v) fetal calf serum (FCS). The cells were cultured for 10–13 days, through two passages before being used as nuclear donors. In vitro matured oocyte freed from cumulus cells were prepared as described above. They were enucleated by aspirating the first-polar body and adjacent cytoplasm in the enucleation medium (TCM 199 supplemented with HEPES, 0.3% BSA, and 7.5 µg/ ml cytochalasin B) with beveled glass pipette, 30 µm diameter. The fibroblast cells were injected into the perivitelline space of the oocytes. The oocyte–fibroblast pairs were placed between 0.2 mm diameter platinum electrodes kept 1 mm apart in fusion/activation medium. Fusion/activation was induced with two DC pulses (1 s interval) of 1.2 kV/cm for 30 µs using BTX Electro-Cell Manipulator 200 (BTX, San Diego, CA, USA). The medium used for injection was the same as for enucleation but without cytochalasin B (CB). The medium used for fusion and activation consisted of 0.3 M m annitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.5 mM HEPES. The nuclear transfer embryos were cultured in NCSU-23 medium for various periods to obtain different stage embryos.
RNA isolation
Frozen–thawed spermatozoa were centrifuged through a discontinuous two-layer Percoll gradient (45/95%). For each batch of RNA isolation, 20 oocytes, 20 pre-blastocyst, or 5 blastocyst stage embryos were used. Cumulus cells, zona pellucida, and attached spermatozoa were cleaned from oocytes and embryos according to the methods described earlier. Oocytes and embryos were washed thrice with PBS prepared with diethyl pyrocarbonate-treated (0.1% DEPC) and -autoclaved distilled water. Samples were transferred to RNase-free Eppendorf tubes, to which 0.6 ml of RLT buffer (Qiagen) and 1% ß-mercaptoethanol (Sigma) were added. The lysates were processed for RNA isolation or stored at –80 °C for future use. Total RNA was isolated with RNeasy Mini or Micro Kit with carrier RNA (Qiagen). Possible contaminating genomic DNA was removed on the column by RNase-free DNase Set (Qiagen). The eluents were supplemented with 20U SUPERase-In RNase inhibitor (Ambion, Austin, TX, USA).
Reverse transcriptase-PCR (RT-PCR) and nested PCR
CEN2 and CEN3 mRNAs were detected by RT-PCR and nested PCR methods. The RT-PCR was done in two steps. In the first step, cDNAs were synthesized using Qiagen Sensiscript RT-PCR kit. Total RNA extracts were preheated at 65 °C for 5 min to denature secondary structure and chilled rapidly on ice for 10 min. RT buffer, dNTP, RNase inhibitor (SUPERase-In RNase inhibitor, Ambion, Austin, TX, USA) and Oligo-dT primer (Promega), and total RNA were added according to the manufacturer’s specification to make the 20 µl RT mixture. The RT reactions were run for 180 min at 37 °C and the resulting cDNAs were stored at –20 °C until used. CEN2 and CEN3 primers were designed according to the published sequence of Homo sapiens centrin genes retrieved from the GenBank (Table 1
). PCR was performed in an Eppendorf Mastercycler PCR machine using a HotMaster hot start PCR kit (Eppendorf, Hamburg, Germany). In all experiments, an internal positive control using boar ß-actin primers in the samples and negative controls was performed by substituting the test sample with water. The PCR settings were 94 °C 2 min, 50 cycles of 94 °C 20 s, 57 °C 10 s, 68 °C 25 s, and 68 °C 1 min for final extension. The PCR products were electrophoresed on 1.2% agarose gels, stained with 0.5 µg/ml ethidium bromide, and photographed with u.v. illumination. Nested PCR reactions were performed on the first-round PCR products that were analyzed by electrophoresis. The PCR products were sequenced by Applied Biosystems 3730 DNA Analyzer using Applied Biosystems Prism BigDye Terminator cycle sequencing chemistry, at the DNA core facility of the University of Missouri – Columbia. The resultant sequences were edited and analyzed by Genetool Lite (BioTools Inc., Edmonton, Alberta, Canada).
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Electrophoresis was done on 4–20% gradient gels and the resolved proteins were transferred to PVDF membrane using wet transfer system (Towbin et al. 1979). Wash and incubation were done with Tris-buffered saline containing 0.25% Tween-20 (TBS-TW) and 1% non-fat milk. The membranes were blocked with 10% non-fat milk in TBS-TW. They were incubated either with E7 antibody or with 20H5 overnight at 4 °C. After washing, the membranes were incubated with HRP-conjugated goat anti-mouse IgG (10 000x dilution), processed for chemiluminiscence using a commercial kit (SuperSignal, Pierce, Rockford, IL, USA), and visualized by exposing to X-ray film. For simultaneous detection of ß-tubulin and centrin, the PVDF membranes were first incubated with E7 antibody for 1 h at room temperature, transferred to mAb 20H5 without washing, and incubated overnight at 4 °C. They were further processed as described above.
Immunofluorescence labeling
Diluted spermatozoa were applied to poly-L-lysine-coated coverslips, fixed, and permeabilized with cold (–20 °C) methanol. The PEF cells were grown on coverslips and fixed in a similar way. The cells were sequentially incubated with 5% normal goat serum (blocking), mAb 20H5, TRITC-conjugated anti-mouse IgG antibody (80x dilution), Ab E7, FITC-conjugated anti-mouse IgG antibody (80 x dilution), 4, 6-diamino-2-phenylindole (DAPI) (5 µg/ml), and mounted with VectaShield (Vector Lab Inc., Burlingame, CA, USA). Control labelings were done by following similar protocol, but without incubating with mAb 20H5.
The oocytes and embryos, except blastocysts, were vortexed with 0.5% hyaluronidase to remove the cumulus cells. They were briefly (5–7 s) treated with 0.5% protease and pipetted repeatedly in PBS–0.5%BSA to remove the distended zona pellucida. The oocytes were fixed with cold methanol. The blastocysts were fixed without removing the residual zona pellucida. The oocytes and embryos were triple labeled with mAb 20H5, E7, and DAPI as described above.
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Results |
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). Porcine centrin genes are phylogenetically closer to human and canine than rodent counterparts. CEN2 and CEN3 cDNA sequences of porcine MII oocyte have been submitted to the GenBank (accession nos. DQ011859
[GenBank]
and DQ066552
[GenBank]
).
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). However, enhanced amplification by nested PCR revealed the presence of CEN2 mRNA in all stages of pre-implantation embryos (Fig. 1B). CEN2 mRNA was not detectable in the ejaculated boar spermatozoa by RT-PCR or nested PCR. CEN3 mRNA was observed in oocytes and pre-implantation embryos of all stages by RT-PCR method, but not in spermatozoa (Fig.1C). Enhanced amplification by nested PCR revealed CEN3 mRNA in GV and MII oocytes, but not in boar spermatozoa or water control (Fig. 1D).
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). The centrin band of the PEF cells migrated slightly faster in the gel than that of the spermatozoa. The PEF and sperm centrins are likely to be cen2p and cen1p respectively, because cycling somatic cells express chiefly CEN2, while CEN1 expression is restricted to retina and testicular cells (Middendorp et al. 1997, Hart et al. 1999). The expression of CEN2 gene is shut down in germ cells during the early spermiogenesis stage and compensated by CEN1 expression (Hart et al. 1999). mAb E7 reacted with the 55 kDa ß-tubulin band in both the extracts (Fig. 2B).
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). The upper and lower bands correspond to, cent1p and cen2p respectively (Wiech et al. 1996, Middendorp et al. 1997, Hart et al. 1999). The immunoreactive protein of boar spermatozoa migrated exactly as the upper band of testicular cell extracts (Fig. 2C). The lower band of the testicular cell extracts coincided precisely with the immunoreactive bands of PEF cell extracts. Centrin was detected in the GV-stage oocytes. It migrated similar to PEF cells or the lower band of the testicular cell extracts, and hence likely to be cen2p. Centrin was not detectable in the extracts of purified sperm heads, MII oocytes, one- or two-cell stage embryos by western blotting (Fig. 2C). The PVDF membranes were probed with 20H5 and E7 antibodies to simultaneously detect centrin and ß-tubulin in the same blots, the latter serving as an intrinsic positive control. The GV oocytes, MII oocytes, and one- and two-cell embryo extracts displayed approximately similar sized 55 kDa bands, but the centrin band was visible only in the GV oocyte extracts.
Boar sperm and fibroblast centrin becomes undetectable after fertilization or SCNT, and embryonic centrin first, appears at blastocyst stage
The PEF cells possess cen2p. Anti-centrin antibody mAb 20H5 labeled the putative centrioles of PEF cells (Fig. 3A). In some cells, the centrioles labeled by the anti-centrin antibody were visible as two orthogonally oriented cylindrical structures (Fig. 3A; inset) similar to the centriolar duplex. Immunofluorescence of boar spermatozoa labeled with mAb 20H5 revealed two spots in the connecting piece region of the sperm flagellum corresponding to two residual centrioles (Fig. 3B and C). In the separated sperm head-tails, two centrin spots were observed associated with the proximal part of the tail.
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). In germinal vesicle breakdown (GVBD) stage, the centrin appeared as dispersed fine particles in the nuclear area (Fig. 3E). The GV oocytes labeled with goat anti-mouse antibody as control preparations did not show any recognizable structure (Fig. 3F). Centrin was not detectable in the in vitro matured MII oocytes except for a diffuse background (Fig. 2G). The spindle polar regions were unfocussed, broad, and lacked cognate centrosomes or centrin-labeled structures.
Punctate centrin labeling was not observed in the in vitro fertilized zygotes or in the pre-blastocyst embryos by immunofluorescence (Fig. 3H–I). Apparently, the centrin contributed by sperm centrosome was lost after fertilization in the zygotes. Centrin reappeared in the completely expanded blastocysts. The putative centrosomes at the spindle poles of mitotic blastomeres were labeled by mAb 20H5 (Fig. 3K). The interphase cells exhibited various patterns of centrin labeling. In pre-hatched blastocysts, some of the blastomeres showed fine speckles of centrin in the nuclear area (Fig. 3J) as seen in GV oocytes (Fig. 3D). Most of the pre-hatched blastocyst cells lacked a defined labeling except for a diffuse cytoplasmic background. Hatched blastocysts possessing twice as many nuclei as pre-hatched blastocysts revealed putative centrosomes labeled as distinct centrin spots. Most of the cells displayed one or two punctuated spots in the juxtanuclear area (Fig. 3L). Some cells possessed multiple fine particles, but were confined to smaller areas. Blastocysts labeled with TRITC-conjugated secondary antibody but omitting mAb 20H5 did not reveal any recognizable structure (data not shown).
Interestingly, in some hatched blastocysts, the anti-centrin antibody labeled eccentrically located curved plate-like structures (Fig. 4A–C). In some blastocysts, the centrin plates occurred in a group of adjacent cells. The nuclei of such cells were relatively more condensed than those of the surrounding cells. However, they are not likely to be the cytokinetic structures since they were not observed in the cells possessing residual mid-zone spindles (data not shown). When co-labeled with anti-ß-tubulin antibody, microtubules seemed to emanate centripetally from the centrin plates (Fig. 4D). Possibly, the centrin plates are the junctional complexes that develop between some blastomeres and associate with actin and microtubule bundles (Talbot & Garrett 2001). Further investigation on the centrin plates will be part of a future research project.
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). To investigate the fate of somatic centrin in zygotes, fibroblast cells were fused with enucleated oocytes. The punctate centrin spots of the PEF cells were detectable during the early pronuclear stage (Fig. 4E). With further in vitro incubation of the embryos, the centrin spots seem to breakdown into smaller particles and disperse around the nuclear area (Fig. 4F). In the late pronuclear stage nuclear transfer embryos, the punctate centrin spots were not detectable (Fig. 4G). The early cleavage stage nuclear transfer embryos lacked detectable centrin, similar to in vitro fertilized embryos (data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The present study has shown that centrin disperses as fine particles during GVBD and finally becomes undetectable in MII oocytes. Dispersal of the centrin particles in the perinuclear area of GVBD oocytes is likely to be a consequence of centriole degeneration. Centrioles degenerate in developing oocytes (Szollosi et al. 1972) resulting in dispersal of the centrosomal material (Manandhar et al. 2005). Ultrastructurally, the dispersal of the centrosomal material or centrin particles might be reflected by the fragmentation of -tubulin containing multivesicular aggregates in mouse oocytes (Calarco 2000). The dispersed centrin particles are possibly degenerated by proteolysis in the oocyte cytoplasm, because they were not detectable in MII oocytes or early cleavage-stage embryos by western blotting. The molecular pathway of centrin degeneration in oocytes is largely unknown. The centrosomes harbor various molecular species of proteolytic ubiquitin–proteasome pathway (Wigley et al. 1999), possibly more concentrated in the spermatid centrosomes (Berruti & Martegani 2005). In the mean time, oocytes and early embryos have highly active proteasomal degradation machinery (Sutovsky et al. 2003). Taken together, it is presumed that the endogenous oocyte centrins and centrin incorporated into oocytes by spermatozoa are mainly degraded through the ubiquitin–proteasome pathway.
Boar spermatozoa have two centrin spots. But the zygotes do not retain centrin contributed by spermatozoa. Loss of sperm-borne centrin has also been noted in human zygotes (Simerly et al. 1999). The oocytes are unable to retain even the somatic cell centrin introduced into them by fusing with embryonic fibroblasts (see also, Manandhar et al. 2004, Zhong et al. 2005). These observations suggest that the zygotic centrosomal apparatus can function without centrin. Interestingly, the lack of centrin in porcine zygotic and early embryonic cells is similar to those of mice (Hiraoka et al. 1989), which do not possess centrioles during these stages of pre-implantation embryonic development (Szollosi et al. 1972).
Among the various isoforms of centrin, the somatic variant cen2p has been shown to be important for centriolar duplication (Salisbury et al. 2002). Centrin is incorporated into the daughter centriolar cylinders during the formation process (D’Assoro et al. 2001). Considering these observations along with ultrastructural localization, it is most likely that centrin is an integral component of centrioles. The present study has shown that porcine zygote and early blastomeres lack detectable amounts of centrin, although the zygotes do possess at least one centriole derived from the sperm-borne, paternal centrosome (Sutovsky et al. 2003). Deficiency of cen2p could be a limiting factor for centriole replication in the early embryonic cells. Electron microscopic studies by random sectioning have shown the presence of one or two centrioles in the first cleavage spindle poles of human (Sathananthan et al. 1996) and sheep embryos (Crozet 1990, Crozet et al. 2000). Nevertheless, the embryonic cells might inherit some residual cen2p from GV-stage oocytes or synthesize it in a regulated manner from the stored mRNAs. Though undetectable by conventional biochemical approaches, this centrin pool could support some rounds of embryonic centriole replication.
The precise role of cen1p in germ cells is not known except for a presumption that it may compensate for the lack of cen2p when the CEN2 gene expression is shut down during spermiogenesis (Hart et al. 1999). It might be important for the generation of the sperm flagellum, similar to the generation of axonemes of the motile cell organelles (Laoukili et al. 2000). Oocytes do not possess cen1p before fertilization and do not retain the sperm-derived cen1p contributed by spermatozoa. These observations indicate that cen1p may not have a significant role in fertilization or embryo development. Besides cen1p and cen2p, somatic cells may contain another well-defined variant, cen3p, and several other uncharacterized isoforms (Wolfrum & Salisbury 1998). Mouse and human CEN3 genes are homologous to yeast CDC31 gene that regulates spindle pole duplication (Baum et al. 1986, Spang et al. 1993). Human cen3p expression is upregulated during ciliogenesis in nasal epithelial cells and localized in the distal lumen of the basal bodies (Laoukili et al. 2000). Antibody 20H5 does not recognize cen3p (Middendorp et al. 1997), thus we do not know whether the cen3p protein is present in the spermatozoa or embryonic cells.
Despite the presence of centrioles, the early embryonic spindle poles of non-rodent mammals including that of pig do not seem to contain fully functional centrosomes. The spindles observed during first embryonic mitosis are barrel-shaped; their spindle poles are broad and anastral in rhesus monkeys (Wu et al. 1996), cows (Navara et al. 1994), and pigs (Kim et al. 1996, present study). These spindles are remarkably different from astral spindles of sea urchins (Schatten et al. 1986, 1987), but surprisingly similar to the acentriolar embryonic cleavage spindles of mice (Calarco-Gillam et al. 1983, Schatten et al. 1985). Centrin is absent in both non-murine and murine pre-implantation embryonic spindle poles. Centrin may not be directly involved in microtubule nucleation, but when it was knocked down in HeLa cells, the centrosomes could not form tightly focused spindle poles (Salisbury et al. 2002). The centrosomes of early embryonic cells apparently could not anchor and tether the polar ends of spindle microtubules into tight foci, possibly due to the lack of centrin. During the pronuclear stage, the zygotic centrosome acts as a dominant MTOC and organizes the sperm aster in non-rodent mammals. However, after nuclear envelope breakdown at the onset of first embryonic mitosis, the condensing chromosomes seem to dominate MTOC activity and nucleate microtubules around them (Kim et al. 1996, Wu et al. 1996). From these points of view, there are no fundamental differences between non-murine and murine embryonic spindles regardless of the presence or absence of centrioles in their spindle poles. Nevertheless, the polar regions of unfocused anastral spindles might differ from canonical centrosomes due to the absence of important centrosomal proteins (Debec et al. 1995) other than centriolar proteins. The oocytes and embryonic cells are capable of organizing functional spindles without canonical centrosome via an acentrosomal pathway (Manandhar et al. 2005).
Interestingly, centrin reappearance in the blastocyst cells follows a dynamically opposite sequence of events as that of its disappearance during GVBD oocytes. Centrin accumulation in porcine blastocysts cells was first detected as fine particles around the interphase nuclei, similar to a pattern seen in the GVBD oocytes. Subsequently, the blastomere centrosomes resume somatic cell-like centrin labeling.
In summary, the present study shows that boar spermatozoa possess centrin but lack centrin mRNAs. During fertilization, the sperm-derived centrin is not retained by the ovum, a pattern mimicked by the disappearance of donor fibroblast cell centrin after somatic cell nuclear transfer. In contrast, mature oocytes and early-stage embryos possess centrin mRNAs, which are not translated into proteins. Despite inheritance of centrin and centrioles from the spermatozoa, the early pre-implantation cleavages show broad and anastral spindle poles. While early embryonic centrosomes can function without centrin, its reappearance at the blastocyst stage of porcine embryonic development might be related to the onset of the differentiation of pluripotent embryonic cells.
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Acknowledgements |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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