76 DEVELOPMENTAL CAPABILITY OF TRANSGENIC NUCLEAR-TRANSFERRED PIG EMBRYOS PRODUCED USING THE NOVEL METHOD OF PSEUDOPHYSIOLOGICAL ACTIVATION

2010 ◽  
Vol 22 (1) ◽  
pp. 196 ◽  
Author(s):  
M. Samiec ◽  
M. Skrzyszowska ◽  
R. Slomski
Keyword(s):  
Somatic Cell ◽  
Donor Cell ◽  
Fibroblast Cell ◽  
Original Method ◽  
Calcium Oscillations ◽  
Cell Nuclei ◽  
Novel Method ◽  
Transgenic Embryos

The physicochemical stimuli, which are commonly used for artificial activation of porcine nuclear-transferred (NT) oocytes, can affect detrimentally or cytotoxically the clonal cybrids and thereby inhibit the development or decrease the quality of cloned embryos. Therefore, we have recently developed a novel method of pseudophysiological transcomplementary (transcytoplasmic) activation to stimulate the developmental program of porcine oocytes reconstructed by somatic cell nuclear transfer. The mechanism underlying this original technique of activation is transcytoplasmic influx of sperm-derived proteins triggering intracellular calcium oscillations, which is mediated via heterologous (rabbit) zygote-descended cytoplasts. The purpose of our study was to estimate the in vitro developmental competences of porcine transgenic cloned embryos following pseudophysiological activation of oocytes receiving pWAPhGH-GFPBsd gene construct-nucleofected fetal fibroblast cell nuclei. In the cloning procedure, IVM pig oocytes were used as recipient cells for cell nuclei of positively selected transgenic fibroblast cells. The reconstruction of enucleated oocytes was performed by intracytoplasmic injection of either the somatic cell-derived karyoplast or whole tiny nuclear donor cell. The activation of porcine NT oocytes was achieved by electrofusion of them with the xenogeneic cytoplasts isolated from in vivo-derived rabbit zygotes (i.e. with the so-called zygoplasts), which led to the formation of triple xenocytoplasmic hybrids (xenocybrids). The rabbit zygotes had been flushed postmortem from the separated oviducts of superovulated postpubertal female donors 18 to 20 hafter administration of hCG and copulation. Single rabbit zygote-descended cytoplasts were inserted into the perivitelline space of previously reconstructed pig oocytes. The resulting zygoplast-NT oocyte couplets underwent fusion, which was induced by generation of 2 successive DC pulses of 1.2 kV cm-1 for 60 μs. The electrofusion medium consisted of 0.3 M Ca2+-deprived mannitol supplemented with 0.1 mM MgSO4 and 0.2 mg mL-1 fatty-acid-free BSA. The transcytoplasmically activated xenocybrids were cultured in vitro for 6 to 7 days up to morula/blastocyst stages. A total of 183/207 (88.4%) oocytes reconstructed with nucleofected fibroblast cell nuclei were successfully fused with zygoplasts. Out of 183 cultured NT embryos, 138 (75.4%) were cleaved. The rates of transgenic NT embryos that reached the morula and blastocyst stages yielded 106/183 (57.9%) and 65/183 (35.5%), respectively. In conclusion, the original method of pseudophysiological activation of porcine NT oocytes turned out to be relatively efficient, which has been confirmed by the high percentages of pWAPhGH-GFPBsd transgenic embryos developing in vitro to morula and blastocyst stages.

49 INTRA-GENUS CLONED EMBRYOS PRODUCTION AND PREGNANCIES DERIVED FROM LEOPARD CAT NUCLEI AND DOMESTIC CAT ENUCLEATED EGGS

2006 ◽  
Vol 18 (2) ◽  
pp. 133 ◽  
Author(s):  
I. K. Kong ◽  
H. S. Lee ◽  
N. H. Kim ◽  
L. H. Kim ◽  
H. D. Shin ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Nuclear Genome ◽  
Fibroblast Cell ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Cell Nuclei ◽  
Reconstructed Embryos ◽  
Significant Difference ◽  
Leopard Cat

The leopard cat (Prionailurus bengalensis), a member of the felidae family, is currently listed as threatened by the Ministry of Environment in South Korea. In exotic or endangered species, the lack of oocytes and recipients precludes the use of traditional somatic cell nuclear transfer (NT), and an approach such as intragenus NT may be the only alternative for producing embryos and offspring. In the present study, we used the leopard cat (LC) as a somatic cell donor to evaluate the in vivo developmental competence, after transfer into domestic cat recipients, of cloned embryos produced by the fusion of LC fibroblast cell nuclei with domestic cat cytoplasts. A total of 412 enucleated domestic cat oocytes were reconstructed with either male (Treatment A) or female (Treatment B) adult LC fibroblasts. There was no significant difference in fusion rate (60.4 vs. 56.9%) between Treatment A and B. Of the fused couplets, the cleavage and blastocyst developmental rate in Treatment A were greater than those in Treatment B (69.5 vs. 60.9%; 8.3 vs. 7.8%; P < 0.05). In treatment A, in vivo developmental studies at 30-45 days postimplantation demonstrated 4.8% (21/435) of reconstructed embryos (n = 435) had entered into the uterine lining of recipients, but only 1.4% (6/435) formed fetuses. However, all of the reconstructed embryos failed to develop to term (65 days). Microsatellite analyses confirmed that the nuclear genome of the cloned fetuses were LC in origin.

47 DIFFERENTIAL DEVELOPMENTAL ABILITY OF EMBRYOS CLONED FROM TISSUE-SPECIFIC STEM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 142
Author(s):  
K. Inoue ◽  
N. Ogonuki ◽  
H. Miki ◽  
S. Noda ◽  
S. Inoue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Donor Cell ◽  
Fibroblast Cell ◽  
High Rate ◽  
Full Potential ◽  
Cell Nuclei ◽  
Cell Stage

Although cloning animals by somatic cell nuclear transfer is generally an inefficient process, use of appropriate donor cell types may improve the cloning outcome significantly. Among the donor cells tested so far, mouse embryonic stem cells have given the best efficiency in terms of the development of reconstructed embryos into offspring. In this study, we examined whether 2 in vitro-produced pluripotent stem cells—neural stem cells (NSCs) and mesenchymal stem cells (MSCs)—could be better nuclear donors than other differentiated cells. Embryos were reconstructed by transfer of nuclei from NSCs or MSCs with full potential for differentiation in vitro. Most (76%) of the 2-cell NCS embryos developed to the 4-cell stage; 43% implanted and 1.6% developed to term after transfer to pseudopregnant recipients. These rates were very similar to those of embryos cloned from fibroblast cell nuclei. Interestingly, in the patterns of zygotic gene expression, NSC embryos were more similar to in vitro-fertilized embryos than fibroblast cloned embryos. By contrast, embryos reconstructed using MSC nuclei showed lower developmental ability and no implantation was obtained after embryo transfer. Chromosomal analysis of the donor MSCs revealed very high frequencies of monosomy and trisomy, which might have caused the very poor post-implantation development of embryos following nuclear transfer. Thus, in vitro-produced pluripotent cells can serve as donors of nuclei for cloning mice, but may be prone to chromosomal aberrations leading to a high rate of cloned embryo death.

79 PSEUDOPHYSIOLOGICAL TRANSCOMPLEMENTARY ACTIVATION OF GOAT OOCYTES RECEIVING ADULT EAR CUTANEOUS FIBROBLAST CELL NUCLEI

2010 ◽  
Vol 22 (1) ◽  
pp. 198
Author(s):  
M. Skrzyszowska ◽  
M. Samiec
Keyword(s):  
Somatic Cell ◽  
Plasma Membranes ◽  
Dermal Fibroblast ◽  
Fibroblast Cell ◽  
Vero Cells ◽  
Atmospheric Conditions ◽  
Cell Nuclei ◽  
Developmental Potential ◽  
Water Saturated

The aim of the study was to determine the in vitro developmental potential of caprine cloned embryos following pseudophysiological (transcytoplasmic) transcomplementary activation of oocytes reconstructed with ear skin-derived fibroblast cell nuclei. The source of nuclear recipient cells were IVM doe oocytes. The reconstruction of the previously enucleated oocytes (i.e. ooplasts) was performed by microinjection of either the somatic cell-derived karyoplasts or intact whole tiny nuclear donor cells directly into the cytoplasm. The reconstructed oocytes were incubated in Upgraded B2 INRA medium for 30 min to 1 h before their pseudophysiological activation. The activation was achieved by electrofusion of clonal cybrids with the allogeneic cytoplasts isolated from caprine IVF-created zygotes, which led to the formation of triple allocytoplasmic hybrids (allocybrids). These originate from 3 sources: (1) homologous whole nuclear donor fibroblast cells or their karyoplasts; (2) enucleated oocytes (ooplasts), and (3) zygote-derived cytoplasts. Single zygote-descended cytoplasts (the so-called zygoplasts) were inserted into the perivitelline space of previously reconstituted oocytes. The resulting zygoplast-clonal cybrid couplets were subsequently subjected to electrofusion, which was induced by application of a single DC pulse of 2.4 kV cm-1 for 15 μs. The electrofusion of zygoplast and reconstructed oocyte plasma membranes occurred in an isotonic dielectric solution deprived of Ca2+ ions. The transcytoplasmically activated clonal cybrids were cultured in vitro in Upgraded B2 INRA medium for 48 h at 38.5°C in a 100% water-saturated atmosphere of 5% CO2 and 95% air. Afterward, cleaved embryos were co-cultured with Vero cells in medium supplemented with 10% fetal bovine serum for an additional 96 to 144 h up to morula and blastocyst stages under the same thermal and atmospheric conditions. A total of 53/78 (67.9%) oocytes reconstructed with fibroblast cell nuclei were successfully fused with zygoplasts. From among 53 cultured cloned embryos, 34 (64.2%) cleaved. The rates of embryos that reached the morula and blastocyst stages were 21/53 (39.6%) and 11/53 (20.8%), respectively. In conclusion, the relatively high percentages of morulae and blastocysts were noticed among in vitro-cultured caprine cloned embryos produced by the strategy of pseudophysiological transcytoplasmic activation of oocytes reconstructed with adult dermal fibroblast cell nuclei. Therefore, the use of cytoplasmic components originating from zygotes as the stimuli for activation of nuclear-transferred oocytes appeared to be an effective procedure in the generation of goat blastocysts by somatic cell cloning.

No differences in sheep somatic cell nuclear transfer outcomes using serum-starved or actively growing donor granulosa cells

2003 ◽  
Vol 15 (3) ◽  
pp. 157 ◽  
Author(s):  
T. T. Peura ◽  
K. M. Hartwich ◽  
H. M. Hamilton ◽  
S. K. Walker
Keyword(s):  
Somatic Cell ◽  
Granulosa Cells ◽  
Nuclear Transfer ◽  
Survival Rates ◽  
Fetal Loss ◽  
Donor Cell ◽  
Perinatal Period ◽  
Offspring Mortality

The aim of this study was to compare serum-starved and non-starved donor cells in sheep nuclear transfer with a special emphasis on cloning outcomes. Sheep oocytes, derived either in vivo or in vitro, were fused with cultured serum-starved or actively growing adult granulosa cells. Resulting blastocysts were transferred to recipients fresh or after vitrification, and subsequent pregnancies followed to term. Donor cell treatment did not significantly affect preimplantation development, pregnancy rates, fetal loss or neonate survival rates. Of 22 lambs born, ten survived the immediate perinatal period but all succumbed at various timepoints within the first few weeks of life. The results of the study suggest that the use of serum-starved cells offers no advantages or disadvantages to cloning outcomes. Neither were significant differences in outcomes observed when using either in vivo- or in vitro-derived oocytes or embryos transferred fresh or after vitrification. Yet, these results continue to highlight problems associated with somatic cell cloning as indicated by offspring mortality. It remains unclear whether the high offspring mortality in the current study was related to species, associated with the cell lines used or the result of other causes.

scholarly journals 38BIRTH OF AFRICAN WILD CAT CLONED KITTENS

2004 ◽  
Vol 16 (2) ◽  
pp. 141 ◽  
Author(s):  
M.C. Gomez ◽  
C.E. Pope ◽  
A.M. Giraldo ◽  
L. Lyons ◽  
R.F. Harris ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
Pcr Amplification ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Felis Silvestris ◽  
Cell Nuclei ◽  
Oocyte Aspiration

The African wild cat (AWC, Felis silvestris lybica; 2n=38) is one of the smallest wildcats, and it’s future is threatened by hybridization with domestic cats (Felis silvestris catus; 2n=38). Nuclear transfer (NT) is a potentially valuable tool for retaining genetic variability, and could assist in the continuation of species with few remaining individuals. Inter-species nuclear transfer into domestic cat (DSH) supports development of somatic cell nuclei from AWC (Gomez et al., 2003, Biol Reprod 69, 1032–1041). Therefore, the purpose of the present study was to evaluate the in vivo developmental competence of nuclear transfer embryos derived by fusion of African wildcat fibroblasts with domestic cat cytoplasts, after transfer into domestic cat recipients. In vivo- and in vitro-matured domestic cat oocytes were mechanically enucleated in modified Tyrodes salt solution supplemented with 20μgmL−1 of cytochalasin B (CCB) and 2mgmL−1 of sucrose, and reconstructed with AWC fibroblast cells derived from an adult male; cultured and passaged 1 to 3 times before serum-starved with DMEM +0.5% FBS and cultured for 5 additional days before use. Fusion took place in fusion medium (0.3M mannitol and 0.1mMMg+2), and membrane fusion was induced by applying a 3s AC pre-pulse of 20V, 1MHz; followed by two 30μs DC pulses of 240V/mm at intervals of 0.5s. Fused couplets were activated 2–3h after fusion by placing the couplets between two electrodes in a fusion chamber containing 3mL of fusion medium and exposing them to two 60μs DC pulses of 120V/mm. Then, couplets were incubated in 30μL drops of Tyrodes solution containing 1% MEM nonessential amino acids, 3mgmL−1 BSA (IVC-1 medium), and supplemented with 10μgmL−1 cycloheximide and 5μgmL−1 CCB at 38°C in 5% CO2 for 4h. After activation, cloned embryos were cultured in 500μL of IVC-1 medium until the day of the transfer. Derived AWC NT embryos were transferred into the oviducts (Day 1) or uteri (Days 5, 6, 7) of 36 gonadotrophin-treated DSH recipients on Day 1 after ovulation or on Days 5, 6, or 7 after oocyte aspiration, respectively. Pregnancy was assessed by ultrasonography on Days 21 to 23. One domestic cat was still pregnant and ongoing on Day 60. Kittens were delivered by Cesarean section in each of the seven pregnant recipients on days 61 to 67 of gestation. The kittens weighed an average of 86.2g (50.0 to 103g) and died within 36h after delivery. The post-mortem pathology reports revealed that most of them had an immature respiratory system. The clonal status of the kittens was assessed by multiplex PCR amplification of 20 microsatellite markers, including seven markers that are known to be on the X chromosome. Results from these assays confirmed that the AWC kittens had originated from the AWC donor somatic cell line and were not related to the DSH recipient cats. In summary, these results indicate that AWC cloned kittens can be produced by ET of embryos derived from AWC cells into DSH cytoplasts. Research was funded partially by the John &amp; Shirley Davies Foundation. Table 1

scholarly journals Influences of somatic donor cell sex on in vitro and in vivo embryo development following somatic cell nuclear transfer in pigs

2016 ◽  
Vol 30 (4) ◽  
pp. 585-592 ◽  
Author(s):  
Jae-Gyu Yoo ◽  
Byeong-Woo Kim ◽  
Mi-Rung Park ◽  
Deug-Nam Kwon ◽  
Yun-Jung Choi ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Donor Cell

24 EFFECT OF DONOR CELL TYPE ON IN VITRO AND IN VIVO DEVELOPMENTAL COMPETENCE OF CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 142
Author(s):  
N. L. Selokar ◽  
P. Sharma ◽  
D. Kumar ◽  
R. K. Sharma ◽  
P. S. Yadav
Keyword(s):  
Somatic Cell ◽  
Seminal Plasma ◽  
Donor Cell ◽  
Cell Types ◽  
Developmental Competence ◽  
Cell Type ◽  
Donor Cells ◽  
Donor Cell Type

Selection of the donor cell type for somatic cell NT is very important based on its capability to be reprogrammed by the oocyte cytoplasm. A very wide variety of donor cells of different origin have been used for somatic cell NT, having differences in the overall efficiency. The aim of this study was to compare the cloning efficiency of donor cells derived from the ventral side of origin of tail skin and seminal plasma of a buffalo bull (age: 3 years old). Somatic cells from skin and seminal plasma were isolated and cultured as described by Selokar et al. (2014 PLOS ONE 9(3), e90755). Cultured seminal plasma cells had classic epithelial morphology, grew in clusters, were hexagonal in outline shape, and were positive for immunocytochemical detection of keratin marker, indicating that they were of epithelial origin, whereas tail-derived cells were spindle in shape and found positive for vimentin expression, indicating the fibroblast origin. To determine their reprogramming potential, these cells between passages 5 to 8 were used for the production of buffalo cloned embryos by handmade cloning as per the method described by Selokar et al. (2012 Theriogenology 78, 930–936). In brief, oocytes were isolated from slaughter-house ovaries and matured in vitro. After 21 h of maturation, cumulus cell mass and zona pellucida were removed by enzymatic treatment, hyaluronidase and pronase, respectively. Zona-free buffalo oocytes were enucleated on the basis of protrusion cone. A single somatic cell was attached to an enucleated oocyte with addition of phytohemagglutinin, followed by sandwich type of electrofusion between the somatic cell-bearing oocyte and enucleated oocyte using BTX electrofusion machine. Fused oocytes were activated by 4 μM calcium ionophore for 5 min and incubated in 2 mM 6-DMAP for 4 h and were cultured in K-RVCL-50® medium for 7 days on a flat surface in a 4-well dish in an incubator (5% CO2 and 38.5°C temperature). The total numbers of embryos reconstructed from tail-derived cells and semen-derived cells were 132 and 158, respectively. Cleavage and blastocyst rate were calculated from total embryos cultured, and data were analysed by Student’s t-test. We found no significant effect on both cleavage (89.30 ± 2.1 v. 94.1 ± 0.6) and blastocyst rate (40.7 ± 4.0 v. 43.1 ± 9.6) for the embryos produced from cells derived from tail and seminal plasma. To study the in vivo developmental competence of embryos derived from the 2 donor cell types, one embryo of each cell type was transferred into 6 recipient animals. Pregnancies were confirmed by ultrasonography at 30 to 35 days after transfer and monitored regularly at 15-day intervals up to 90 days. Three pregnancies were found for tail-derived cells, whereas no pregnancy was obtained for semen-derived cells. Out of 3 pregnancies obtained, 1 embryonic death was observed before 45 days, and 2 are continuing at advance stage. In conclusion, tail-derived cells are the better donor cell choice for buffalo somatic cell NT research. Currently, our focus is on epigenetic reprogramming behaviour of these 2 different cell types to elucidate the possible reprogramming mechanism.

scholarly journals Developmental ability of cloned embryos from neural stem cells

Reproduction ◽  
2006 ◽  
Vol 132 (6) ◽  
pp. 849-857 ◽  
Author(s):  
Eiji Mizutani ◽  
Hiroshi Ohta ◽  
Satoshi Kishigami ◽  
Nguyen Van Thuan ◽  
Takafusa Hikichi ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Success Rate ◽  
Sertoli Cells ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Cloning Efficiency ◽  
Cell Nuclei ◽  
Developmental Potential ◽  
Undifferentiated State

The success rate is generally higher when cloning mice from embryonic stem (ES) cell nuclei than from somatic cell nuclei, suggesting that the embryonic nature or the undifferentiated state of the donor cell increases cloning efficiency. We assessed the developmental ability of cloned embryos derived from cultured neural stem cell (NSC) nuclei and compared the success rate with that of embryos cloned from other donor cells such as differentiated NSCs, cumulus cells, Sertoli cells and ES cells in the mouse. The transfer of two-cell cloned embryos derived from cultured NSC nuclei into surrogate mothers produced five live cloned mice. However, the success rate (0.5%) was higher in embryos cloned from cultured NSC nuclei than from differentiated NSCs (0%), but lower than that obtained by cloning mice from other cell nuclei (2.2–3.5%). Although thein vitrodevelopmental potential to the two-cell stage of the cloned embryos derived from NSC nuclei (73%) was similar to that of the cloned embryos derived from other somatic cell nuclei (e.g., 85% in Sertoli cells and 75% in cumulus cells), the developmental rate to the morula–blastocyst stage was only 7%. This rate is remarkably lower than that produced from other somatic cells (e.g., 50% in Sertoli cells and 54% in cumulus cells). These results indicate that the undifferentiated state of neural cells does not enhance the cloning efficiency in mice and that the arrest point forin vitrodevelopment of cloned embryos depends on the donor cell type.

25 GENOME EDITING OF SOMATIC CELL NUCLEAR TRANSFER DERIVED ZYGOTES BY CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9 GUIDE RNA INJECTION

2016 ◽  
Vol 28 (2) ◽  
pp. 142
Author(s):  
K. M. Whitworth ◽  
S. L. Murphy ◽  
J. A. Benne ◽  
L. D. Spate ◽  
E. Walters ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Line ◽  
Genome Editing ◽  
Genetically Modified ◽  
Donor Cell ◽  
T7 Promoter ◽  
Guide Rna ◽  
Rag2 Gene

Recent applications of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system have greatly improved the efficiency of genome editing in pigs. However, in some cases, genetically modified pig models need an additional modification to improve their application. The objective of this experiment was to determine whether a combination of somatic cell NT (SCNT) by using a previously modified donor cell line and subsequent zygote injection with CRISPR/Cas9 guide RNA to target a second gene would result in embryos and offspring successfully containing both modifications. Fibroblast cell lines were collected from fumarylacetoacetate hydrolase deficient (FAH–/–) fetuses and used as the donor cell line. Somatic cell NT was performed by standard technique. A CRISPR guide RNA specific for recombination activating gene 2 (RAG2) was designed and in vitro transcribed from a synthesised gBlock (IDT) containing a T7 promoter sequence, the CRISPR Guide RNA (20 bp), and 85 bp of tracer RNA. The gBlock was PCR amplified with Q5 polymerase (NEB, Ipswich, MA, USA) and in vitro transcribed with the MEGAshortscript™ T7 Transcription Kit (Life Technologies, Grand Island, NY, USA). Guide RNA (20 ng μL–1) and polyadenylated Cas9 (20 ng μL–1, Sigma, St. Louis, MO, USA) were co-injected into the cytoplasm of SCNT zygotes at 14 to 16 h after fusion and activation. Injected SCNT were then cultured in vitro in PZM3 + 1.69 mM arginine medium (MU1) to Day 5. Three embryo transfers were performed surgically into recipient gilts on Day 4 or 5 of oestrus (50, 62, or 70 embryos per pig) to evaluate in vivo development. The remaining embryos were cultured in MU1 to Day 7 and analysed for the presence of modifications to the RAG2 gene. Embryos were classified as modified if they contained an INDEL as measured by both gel electrophoresis and DNA sequencing of PCR amplicons spanning the targeted exon. The RAG2 modification rate was 83.3% (n = 6), of which 50% (n = 3) of the embryos contained biallelic modifications. All control embryos contained a wild-type RAG2 gene (n = 5). Embryo transfer resulted in a 33.3% pregnancy rate (1/3). The combination of SCNT and CRISPR/Cas9 zygote injection can be a highly efficient tool to successfully create pig embryos with an additional modification. This additional technique further improves the usefulness of already created genetically modified pig models. This study was funded by the National Institutes of Health via U42 OD011140.

Identification of Perilipin-2 as a lipid droplet protein regulated in oocytes during maturation

2010 ◽  
Vol 22 (8) ◽  
pp. 1262 ◽  
Author(s):  
Xing Yang ◽  
Kylie R. Dunning ◽  
Linda L.-Y. Wu ◽  
Theresa E. Hickey ◽  
Robert J. Norman ◽  
...  
Keyword(s):  
Lipid Droplet ◽  
Lipid Content ◽  
Lipid Droplets ◽  
Intracellular Lipids ◽  
Epidermal Growth ◽  
Novel Method ◽  
Perilipin 2 ◽  
Lipid Droplet Protein

Lipid droplet proteins regulate the storage and utilisation of intracellular lipids. Evidence is emerging that oocyte lipid utilisation impacts embryo development, but lipid droplet proteins have not been studied in oocytes. The aim of the present study was to characterise the size and localisation of lipid droplets in mouse oocytes during the periovulatory period and to identify lipid droplet proteins as potential biomarkers of oocyte lipid content. Oocyte lipid droplets, visualised using a novel method of staining cumulus–oocyte complexes (COCs) with BODIPY 493/503, were small and diffuse in oocytes of preovulatory COCs, but larger and more centrally located after maturation in response to ovulatory human chorionic gonadotrophin (hCG) in vivo, or FSH + epidermal growth factor in vitro. Lipid droplet proteins Perilipin, Perilipin-2, cell death-inducing DNA fragmentation factor 45-like effector (CIDE)-A and CIDE-B were detected in the mouse ovary by immunohistochemistry, but only Perilipin-2 was associated with lipid droplets in the oocyte. In COCs, Perilipin-2 mRNA and protein increased in response to ovulatory hCG. IVM failed to induce Perilipin-2 mRNA, yet oocyte lipid content was increased in this context, indicating that Perilipin-2 is not necessarily reflective of relative oocyte lipid content. Thus, Perilipin-2 is a lipid droplet protein in oocytes and its induction in the COC concurrent with dynamic reorganisation of lipid droplets suggests marked changes in lipid utilisation during oocyte maturation.

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