Microtubule and chromatin organization during the first cell-cycle following intracytoplasmic injection of round spermatid into porcine oocytes

Lee, Jang-Won; Kim, Nam‐Hyung; Lee, Hoon Taek; Chung, Kyuha

doi:10.1002/(sici)1098-2795(199806)50:2<221::aid-mrd13>3.0.co;2-9

Cited by 29 publications

(8 citation statements)

References 37 publications

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“…Electrical stimulation was handled according to the method of Lee et al . () with slight modifications. Selected oocytes were rinsed three times in activation medium (0.25 mol/L Mannitol, 0.1 mmol/L CaCl 2 , 0.1 mmol/L MgCl 2 , 0.5 mmol/L Hepes, 0.1% bovine serum albumin (BSA)), then pre‐incubated in this medium for 5 min at 39°C.…”

Section: Methodsmentioning

confidence: 99%

Effect of FH535 on in vitro maturation of porcine oocytes by inhibiting WNT signaling pathway

Shi

Cheng

et al. 2017

Animal Science Journal

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Wingless-int (WNT) signaling pathway is vital to modulate life processes, including cell fate determination, cell differentiation, cell proliferation, cell apoptosis and embryogenesis. To demonstrate the uncertain effect of the canonical WNT signaling pathway on oocyte maturation, immature porcine oocytes were collected and cultured in vitro with the WNT/β-catenin inhibitor FH535. The concentrations of FH535 were selected as 0.00, 0.01, 0.10, 1.00 and 10.00 μmol/L. The results showed that the optimum concentration of FH535 on oocyte maturation was 1.00 μmol/L. In this concentration, the proportion of MII oocytes increased (P < 0.05). The rate of cleavage was the same with the control (P > 0.05), while the rate of blastocysts in the 1.00 μmol/L FH535 treated group was higher than that of control (P < 0.01). Additionally, the average number of nuclei in blastocysts raised significantly (P < 0.05). The inhibition of WNT could regulate expression of maturation-related genes, including Cdc-2, Bmp-15, Gdf-9 and Mos. In the 1.00 μmol/L FH535 treated group, the messenger RNA level of β-catenin showed no significant change compared to the control (P > 0.05), but the protein abundance was decreased (P < 0.05). This study revealed that the inhibition of FH535 on the WNT signaling pathway could promote the maturation of porcine oocytes and altered gene expressions in vitro.

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Section: Methodsmentioning

confidence: 99%

Effect of FH535 on in vitro maturation of porcine oocytes by inhibiting WNT signaling pathway

Shi

Cheng

et al. 2017

Animal Science Journal

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“…Electrical stimulation was as described by Lee et al [18] with slight modifications. Briefly, reconstructed embryos were washed and preincubated 20 sec in activation medium (0.25 M mannitol solution supplemented with 0.01% polyvinyl alcohol, 0.5 mM Hepes, 0.1 mM CaCl 2 ·H 2 O, and 0.1 mM MgCl 2 ·6H 2 O with pH 7.2) at room temperature.…”

Section: Activation Of Oocytesmentioning

confidence: 99%

Production of Cloned Pigs by Whole-Cell Intracytoplasmic Microinjection1

Lee

Tian

et al. 2003

Biology of Reproduction

103

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Cloning by somatic cell nuclear transfer has been successfully achieved by both fusing of a donor cell with and injecting an isolated donor cell nucleus into an enucleated oocyte. However, each of the above methods involves extended manipulation of either the oocytes (fusion) or the donor cells (nucleus isolation). Additionally, cloning efficiency can be reduced by low fusion rate of the cell fusion method, and specialized micromanipulation equipment and exacting nucleus isolation techniques are required for the nucleus injection method. Here we report a whole-cell injection technique for nuclear transfer in pigs and the production of cloned piglets with comparable, if not higher, efficiency than the other two nuclear transfer procedures. First, we tested the feasibility of this technique with three types of frequently used donor cells (cumulus, mural granulosa, and fibroblasts) and obtained the optimal nuclear reprogramming conditions for these cells. We further improved our protocol by avoiding ultraviolet exposure during enucleation and achieved a 37% blastocyst rate. We then conducted whole-cell injection using skin fibroblasts from the ear of a sow transgenic for two genes, the porcine lactoferrin and the human factor IX, and produced four live-born cloned transgenic piglets from three recipients. The present study demonstrated the applicability of producing normal, cloned piglets by the simple and less labor-intensive whole-cell intracytoplasmic injection.

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“…Following ROSI, the maternally synthesized MTOCs, instead of the sperm-derived MTOCs, drive the behavior of microtubules in fertilized oocytes and embryos (Lee et al, 1998). However, in rabbits, the maternal MTOCs often function improperly and fail to align the chromosomes or nuclei in the parthnogenetically activated rabbit oocytes, including those reconstructed with blastomeres or somatic cells (Pinto-Correia et al, 1993;Yin et al, 2002).…”

Section: Is the Sperm Centrosome Necessary For Fertilization?mentioning

confidence: 99%

Functional assessment of centrosomes of spermatozoa and spermatids microinjected into rabbit oocytes

Tachibana¹,

Ogonuki²,

Ugajin³

et al. 2008

Molecular Reproduction Devel

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Although intracytoplasmic sperm injection (ICSI) is a widely used assisted reproductive technique, the fertilization rates and pregnancy rates of immature spermatids especially in round spermatid injection (ROSI) remain very low. During mammalian fertilization, the sperm typically introduces its own centrosome which then acts as a microtubule organizing center (MTOC) and is essential for the male and female genome union. In order to evaluate the function of immature germ cell centrosomes, we used the rabbit gamete model because rabbit fertilization follows paternal pattern of centrosome inheritance. First, rabbit spermatids and spermatozoa were injected into oocytes using a piezo-micromanipulator. Next, the centrosomal function to form a sperm aster was determined. Furthermore, two functional centrosome proteins (gamma-tubulin and centrin) of the rabbit spermatogenic cells were examined. Our results show that the oocyte activation rates by spermatozoa, elongated spermatids, and round spermatids were 86% (30/35), 30% (11/36), and 5% (1/22), respectively. Sperm aster formation rates after spermatozoa, elongated spermatids, and round spermatids injections were 47% (14/30), 27% (3/11), and 0% (0/1), respectively. The aster formation rate of the injected elongating/elongated spermatids was significantly lower than that of the mature spermatozoa (P = 0.0242). Moreover, sperm asters were not observed in round spermatid injection even after artificial activation. These data suggest that poor centrosomal function, as measured by diminished aster formation rates, is related to the poor fertilization rates when immature spermatogenic cells are injected.

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Microtubule and chromatin organization during the first cell-cycle following intracytoplasmic injection of round spermatid into porcine oocytes

Cited by 29 publications

References 37 publications

Effect of FH535 on in vitro maturation of porcine oocytes by inhibiting WNT signaling pathway

Effect of FH535 on in vitro maturation of porcine oocytes by inhibiting WNT signaling pathway

Production of Cloned Pigs by Whole-Cell Intracytoplasmic Microinjection1

Functional assessment of centrosomes of spermatozoa and spermatids microinjected into rabbit oocytes

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