The Research Of Rabbit Somatic Nuclear Transfer With In Vitro Matured Oocyte

Viable animals having been cloned by nuclear transfer from somatic cells (SCNT) in several species improved NT technologies and theories, which helped scientists to understand the essence of developmental biology much better. Embryo gene expression and epigenetic development would be studied easily in nuclear transfer model and production of transgenic amimals or wildlife to be extinct could be another utilized contribution of SCNT.Matured oocyte is necessary for clone as recipient to reprogramme the somatic cell nucleus. Oocytes matured in vivo have been used for a long period, but the high cost and low efficency limit its utilization in SCNT. The cheaper and convenient oocytes matured in vitro from slaughter house abandoned ovaries are usually used for SCNT these years, and viable cloned offspring have been produced in quite a few species. But as our knowledge there is no rabbit offspring being cloned from IVM oocytes.The research work on rabbit cloning from embryo cells started since 1975 and then the cloned blastocysts had been obtained. But after that there was no cloned offspring was born until 1988. In that year Stice and his colleagers had succeeded in production of six cloned rabbits, in which eight-cell embryo nuclei were used as nuclear donors. In 2002, a Franch research group successfully cloned rabbits from cumulus cells. Although obstaining viable offsprings, the recipient oocytes they used were collected from in vivo by superovulation. One major objective of our research is to identify the possibility to use oocytes matured in vitro as cytoplasmic recipient to pruduce cloned rabbits.At first we tried to establish the rabbit oocyte in vitro maturation system. Ovaries from slaughter house were not homogeneous in quality, and in this experiment we compared the diameter and developmental ability of oocytes collceted from different folliciles. We divided the follicles into four groups according their size: (1) <0.5mm; (2) 0.7-1.0mm; (3) 1.0-1.5mm; (4) >1.5mm. As the result we found that oocyte diameter was increased with follicular size, and when the follicles grew to more than 1 mm the oocytes got their maximal diameter (133.1±0.7μm). Oocytes from the first group can not resume meiosis, but the other three groups can resume and finish meiotic maturation and get to MII stage. Although having the similar maturation ability the oocytes from the three larger follicle groups had different maturation speed and embryo developmental potential. Oocytes from 0.7-1.0mm follicles matured slower(12h) and had a relative poor blastocyst development(14.2%) comparing to oocytes in 1.0-1.5mm group(10h and 62.8%) and > 1.5mm group(10h and 50.1%). There was no significant difference between oocytes from the two larger follicle groups in morphs and development.Secondly we attempted to optimize rabbit oocyte chemical activation (CA) method. In this experiment, different doses and treating durations of Ionomysin combinated with 6-DMAP and/or cycloheximide (CHX) were used to activate rabbit oocytes. Then the best CA programme was obstained: Oocytes were treated with 2.5μg/ml ionomycine for five minutes and then were cultued for another one hours in TCM199 supplied with 2mM 6-DMAP and5μg/ml CHX. Under this condition we got 81.9% of cleavage rate and 55.1% of blastocyst rate after cultured for six days.Then we designed the experiments to study the effects of chemical assisted enucleation with demecolcine treatment on rabbit SCNT. Our results indicated that being treated with 0.6μg/ml demecolcine for one hour can induce oocyte to form a cytoplasmic protrusion containing chromatin in rabbit, but the efficiency of which was affected by the age of oocyte. As described in results, yonger oocytes were hardly induced to protrude, only 32.6% of oocytes can form cytoplasmic protrisions when they were treated at the 12th hours of in vitro maturation. But if the treatment was delayed to 14th hours the protrusion formation rate would increase to 86.7%, and then the protrusion formation rate would get to 90.3% in oocytes matured in vitro for 18 hours. Then we tested the spindle position changing in oocytes with different IVM duration. We found that the distince between spindle and first polar body (FPB) increased with the IVM duration. In oocytes IVM for 14 hours there were more than 80% of oocytes could keep the angle between spindle and FPB within 30°, but when the IVM time was prolonged to 18 hours only about 65% of oocytes would be in this state. As our experience enucleation will be difficult when the angle between spindle and FPB is more than 30°(data not shown).Donor somatic cells type and cycle can affect the efficiency of reprogramming after nuclei were transferred into oocytes cytoplasm in SCNT experiments. We used three types of somatic cells including cumuluc cells (CC), skin flbroblast cells (SFC) from ear and fetal flbroblast cells (FFC) as nuclear donor cells. The blastocyst development was not different between SFC and FFC (about 35%), but significant higher than CC group (19.7%). Then we compared the effects of two treatments, serum starvation and contact inhibition, on cell cycle synchronization in rabbit cumulus cells. The results indicated that contact inhibition or serum starvition for 3-5 days would synchronize more than 80% of cells at G0/G1 stage. There was no difference between the two treatments. Recipient oocytes age is also a factor can affect successful rate of SCNT. Our data indicated that all oocytes matured in vitro for 12 to 18 hours could be used as cytoplamic recipients in rabbit nuclear transfer. In these experimnts the cleavage rate can get to about 80%, and more than 30% of oocytes could develop to blastocyst stage. MG132, which was identified to incerease MPF in mouse and goat oocytes, was also used in our experiment to improve the nuclear remodeling and develomental ability of reconstracted enbryos. But what is interesting was that MG132 decreased the blastocyst development rate from 26.6% to 9.23. These results suggested that rabbit oocytes matured in vitro were seldem affected by aging in our SCNT experiment, and MG132 treatment was not helpful.Finally we tried to transfer the 2-4 cell SCNT embryos into recipients. As indicated in results we designed three transferring time programme and 643 embryos were transferred into 23 recipients but unfortunately there was no any progency had been observed at last.