The Journal of Reproduction and Development Supplement

The first successful cryopreservation of teleost embryos was reported in the Japanese flounder (Chen and Tian, 2005). To develop optimum protocols for cryopreservation of flounder embryos, we examined the toxicity of cryoprotectants to embryos, their water-and cryoprotectant-permeability, and survival of cryopreserved embryos. To examine the toxicity of the cryoprotectants (DMSO, ethylene glycol (EG), methanol (MeOH) and propylene glycol (PG)), embryos at the blastula, gastrula, tail bud and pre-hatch stages were exposed to cryoprotectant solutions at 15°C and their survival was assessed by their ability to hatch. To determine the permeability, embryos were exposed to solutions with a cryoprotectant or sucrose at 25°C, and relative volume changes were examined. MeOH and PG were less toxic than DMSO and EG to embryos at all the stages. Of the four stages, embryos at the tail bud stage showed the highest hatching rate after exposure to MeOH and PG. When embryos were suspended in cryoprotectant or sucrose solutions, they showed minimal volume change, suggesting low water- and cryoprotectant-permeability. When embryos were cryopreserved with vitrification solutions (FVS1 and FVS3) containing MeOH and PG (13% + 20% and 18% + 27%, respectively), embryos at all stages examined became opaque during cooling, indicating intracellular ice formation during cooling. The low-membrane permeability of Japanese flounder embryos might make them difficult to be cryopreserved. Although Chen and Tian reported successful cryopreservation of flounder embryos using FVS1 and FVS3, our study failed to repeat their results.

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[Objectives] The ng/fg parthenogenetic embryos harboring the 13-kb H19 deletion in ng allele successfully developed to term. However, the majority of the parthenotes exhibit severe growth retardation and die either just before or soon after recovery. The imprinted Igf2 and Dlk1 genes are thought to play important role for the extended development and survival. To explore a reason for the developmental retardation and the lethality in the parthenotes, we focused on hematopoiesis and assessed the expression of Igf2 and Dlk1, known as major regulator for hematopoietic stem cell differention and hematopoisis. [Methods] The ng/fg parthenotes were reconstructed by serial nuclear transfer. Total RNAs were extracted from the liver of E12.5 parthenotes ng(wt) /fg(wt) and ng (H19D13) /fg(wt) and controls. The cDNA was used for the quantitative gene expression analysis by real-time PCR. Liver cells of ng(wt) /fg(wt) and ng(H19D13) /fg(wt) parthenotes and controls at E12.5 were incubated with anti-Ter119 and anti-c-Kit, and followed by using FACS analysis. [Result] The expression of Igf2 in liver was repressed in ng(wt) /fg(wt) parthenotes, and decreased in ng(H19D13) /fg(wt) parthenotes at E15.5. The Dlk1 was repressed in the liver of both types of parthenotes. Further, the flow cytometry analysis of the liver cells at E12.5 showed that the immature erythroid cells were evidently increased in ng(wt) /fg(wt) parthenotes, but the more mature erythroid cells were decreased in ng(wt) /fg(wt) parthenogenetic liver. [Conclusion] The present results show that the retardation of liver development and hematopoietic stem cell differentiation in parthenotes could be involved in parthenogenetic lethality.

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[Introduction] The first mitotic cleavage plane correlates with the sperm entry position fertilization cone, or the second polar body (2pb) (Gardner et al., 1997; Karolina et al., 2002). Recently, Hiiragi et al. (2004) have shown that the first mitotic cleavage plane of the mouse egg is not predetermined but is rather defined by the topology of the two apposing pronuclei. [Methods] Mouse zygotes were produced by ICSI using oocytes and sperm from B6D2F1 mice. First, sperm heads were injected into oocytes at different positions at angles to the line of the metaphase II site and the center of egg (45° to 180°). Then male and female pronuclear position were observed 2 h interval until the early 2-cell embryo. Second, in order to examine the effects of the microtubule network on the migration of male and female pronuclei, eggs 2 h after ICSI were treated with 0.02 μg/ml demecolcine or 5 μg/ml cytochalasin B. Then the positions of male and female pronuclei were examined at 12 or 16 h after ICSI. Finally, the positions of the spindle, male and female chromosomes to the 2pb during the first cleavage were observed. [Results] The female pronucleus was always located in the space side of the 2pb and this tie is caused by a tie-microtubule cluster between the 2pb and female pronucleus. The male pronucleus migrated to the center of zygote and usually made angles 90 to 135° to the line of the female nucleus and the 2pb. Additionally, the final position of the male pronucleus was independent of the sperm injection site. In conclusions: The first mitotic cleavage plane of the mouse zygote is defined by the tie of the female pronucleus and the 2pb through a tie-microtubule cluster.

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We previously demonstrated the specific distribution of a calcium-dependent phospholipids binding protein, annexin 5, in the testis. As we found that annexin 5 was involved in gonadotropin secretion in the pituitary gonadotropes, it is hypothesized to be involved in some testicular function. To examine this possibility, in the present study, we studied first the effect of hemicastration and unilateral cryptorchidism on annexin 5 content and distribution in the testis. Effects of hCG, GnRH and testosterone on annexin 5 expression were also evaluated. Hemicastration increased annexin 5 content of the remaining testis after 24h. It was thought that this increase was induced by enhanced gonadotropin secretion after castration, because hCG (50 IU/head) augmented testicular content of annexin5 within 24h. It seemed that the interstitium was expanded with the augmentation of annexin 5 expression after hCG treatment. On the other hand, unilateral cryptorchidism also increased the annexin 5 expression of the cryptorchid testis during four weeks observation. Annexin 5 in the cryptorchid testis concentrated to intra-seminiferous tubules but not in the interstitium. The increase of testicular annexin 5 content seen after hemicastration and cryptochidism is thought to be induced by diverse mechanisms and in different cell species. Intratesticular administration of GnRH analog and subcutaneous testosterone injection both increased annexin 5 expression. These present data reveal multiple stimuli give changes in annexin 5 content in various cell species of the testis. Pituitary luteinizing hormone is suggested to augment annexin 5 synthesis in the interstitial cells.

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