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Vitrification has been widely used for oocyte cryopreservation, but there is still a need for optimization to improve clinical outcomes. In this study, we compared the routine droplet merge protocol with modified multi-gradient equilibration vitrification for cryopreservation of mouse oocytes at metaphase II. Subsequently, the oocytes were thawed and subjected to intracytoplasmic sperm injection (ICSI). Oocyte survival and spindle status were evaluated by morphology and immunofluorescence staining. Moreover, the fertilization rates and blastocyst development were examined in vitro. The results showed that multi-gradient equilibration vitrification outperformed droplet merge vitrification in terms of oocyte survival, spindle morphology, blastocyst formation, and embryo quality. In contrast, droplet merge vitrification exhibited decreasing survival rates, a reduced proportion of oocytes with normal spindle morphology, and lower blastocyst rates as the number of loaded oocytes increased. Notably, when more than six oocytes were loaded, reduced oocyte survival rates, abnormal oocyte spindle morphology, and poor embryo quality were observed. These findings highlight that the vitrification of mouse metaphase II oocytes by the modified multi-gradient equilibration vitrification has the advantage of maintaining oocyte survival, spindle morphology, and subsequent embryonic development.
This study attempted to investigate and validate whether epididymis cold storage could be a suitable alternative for short-term preservation of spermatozoa. Mouse cauda epididymides and spermatozoa were preserved at 4–8°C from 1 day to 6 weeks. From days 1 to 10, motility and fertility were daily examined when motility loss occurred. From week 1, spermatozoa were used for intracytoplasmic sperm injection (ICSI) at weekly intervals to test their fertility, and spermatozoa DNA integrity was determined by comet assay. We found that motility and progressive motility scores gradually decreased with storage time. In nearly all spermatozoa, DNA integrity was maintained from days 1 to 10, but the percentage of spermatozoa with damaged DNA significantly increased from week 2 to week 6. Spermatozoa retained fertility until day 6, although fertility gradually decreased after day 3. From week 1 to week 5, fertilization rates by ICSI were more than 82.69% but decreased gradually after week 3. We found that spermatozoa preserved in the epididymis at 4–8°C had progressively lower motility, fertility and proportion of undamaged DNA, but could still fertilize oocytes. However, all the parameters of cold-preserved spermatozoa were completely inferior to that from cold-preserved cauda epididymides. The results imply that cold storage of cauda epididymides could be conducive to short-term preservation of spermatozoa, and the cold-stored spermatozoa can resist DNA denaturation, which is necessary for maintaining reproductive ability.
Round spermatid injection (ROSI) into mammalian oocytes can result in the development of viable embryos and offspring. One current limitation to this technique is the identification of suitable round spermatids. In the current paper, round spermatids were selected from testicular cells with phase contrast microscopy (PCM) and fluorescence-activated cell sorting (FACS), and ROSI was performed in two strains of mice. The rates of fertilization, embryonic development and offspring achieved were the same in all strains. Significantly, round spermatids selected by PCM and FACS were effectively used to rescue the infertile Pten-null mouse. The current results indicate that FACS selection of round spermatids can not only provide high-purity and viable round spermatids for use in ROSI, but also has no harmful effects on the developmental capacity of subsequently fertilized embryos. It was concluded that round spermatids selected by FACS are useful for mouse strain rederivation and rescue of infertile males; ROSI should be considered as a powerful addition to the armamentarium of assisted reproduction techniques applicable in the mouse.
The goal of this project was to determine whether the originating strain of mouse embryonic stem (ES) cells affects the maintenance of their pluripotency under uniform culture conditions. ES cells from two strains of mice, E14 and C2J, were tested. Both ES cell lines were cultured in KOSR + 2i medium and then injected into C57BL/6J blastocysts. Our results demonstrate that this medium could support both E14 and C2J ES cells to keep their pluripotency, though E14 ES cells were found to have a higher chimeric rate than C2J ES cells. However, analysis by backcrossing revealed that C2J and E14 ES cells have the same ability for germline transmission. Our results demonstrate that ES cells derived from E14 and C2J cells have the same capacity for germline transmission when injected into C57BL/6J blastocysts; however, due to the limitation of mixed genetic background between E14 cells and host C57BL/6J embryos, C2J ES cells are preferable to E14 ES cells for use in gene-targeting and should become the cell line of choice for the generation of genetically engineered mutant mouse lines.
The maintenance and preservation of strains of mice used in biomedical research presents a unique challenge to individual investigators and research institutions. The goal of this study was to assess a comprehensive system for mouse strain conservation through a combination of natural mating, sperm cryopreservation and assisted reproductive technology. Our strategy was based on the collection and cryopreservation of fresh epididymal sperm from male mice by semi-vasectomy; these mice were then naturally mated for breeding purposes. If no satisfactory results were obtained from natural breeding, then the cryopreserved sperm were used for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI); resultant embryos were then transferred into pseudopregnant-recipient female mice. Our results show that some semi-vasectomized mouse strains can be conserved by natural breeding, and that sterile males can be compensated for through the use of IVF and ICSI technology. As such, we believe this system is suitable for the purpose of strain conservation, allowing the continuation of natural breeding with the safeguard of assisted reproduction available.
The injection of spermatozoa into mouse, human and rabbit oocytes at specific times and positions can result in different rates of viable embryo development. However, it is not clear how the timing and position of round spermatid injection (ROSI) affect pronucleus (PN) formation and blastocyst development of mice. First, we determined the changes in relative position of the first polar body and the spindle, carried out ROSI from 11.5 to 13 h post-hCG administration, then activated by Sr2+, and finally compared the development of ROSI zygotes, including the formation of pronuclei and development of blastocyst. Between 11.5 and 13 h post-hCG administration, the rate of 2PN formation by ROSI at 3 o'clock was the highest among all treated oocytes. Moreover, the blastocyst rate of zygotes with two pronuclei (2PN) was up to 27.41%. These results suggest that the time and position of ROSI can significantly influence the formation of 2PN, that the rates of 2PN formation are closely correlated with blastocyst formation and that the formation of 2PN is necessary for later embryo development.
G2/M somatic nuclei were introduced into enucleated meiotically competent oocytes and subsequently cultured in TCM199 plus 10% fetal calf serum (FCS). Pseudo-first polar bodies could be extruded, but the chromosomes failed to arrange normally. Kinetochores were traced with immunofluorescent microscopy using autoimmune sera from patients with CREST (Calcinosis, Raynaud's phenomenon, Esophageal dysmotility, Sclerodactyly, Telangiectasia) scleroderma. In vitro matured oocytes arrested at second meiotic metaphase and kinetochores were detectable as paired structures aligned at the spindle equator. At meiotic anaphase, present or past the kinetochores separated and remained aligned at the distal sides of the chromosomes until telophase, when their alignment perpendicular to the spindle axis was lost. Kinetochores failed to arrange normally after transferring somatic nuclei into oocytes. Our results suggest that somatic cell nuclei are unable to proceed normally through meiosis when introduced into oocyte meiotic cytoplasm.
The developmental ability of reconstructed blastocysts from C57BL/6 strain mouse inner cell masses (ICMs) and Kunming strain mouse trophoblasts was assessed. The procedure of ICM replacement was as follows: C57BL/6 ICMs were separated from the blastocysts using immunosurgery. A slit was made in the zona pellucida of a Kunming blastocyst to allow its ICM to extrude. The C57BL/6 ICM was injected into the Kunming blastocoele, and the extruded Kunming ICM was cut off. The reconstructed blastocysts were able to re-expand (77%) and hatch (27.3%) in vitro. A total of 64 reconstructed blastocysts and 124 Kunming blastocysts were co-transferred into the uteri of 11 pseudopregnant Kunming mice, and an ICM replacement offspring was born. The results indicate that reconstructed embryos obtained by inter-strain ICM replacement have the ability to develop to term. This technique may provide a method to solve the pregnancy failure in interspecific cloning.
Conventional methods of somatic cell nuclear transfer either by electrofusion or direct nucleus injection have very low efficiency in animal cloning, especially interspecies cloning. To increase the efficiency of interspecies somatic cell nuclear transfer, in the present study we introduced a method of whole cell intracytoplasmic injection (WCICI) combined with chemical enucleation into panda–rabbit nuclear transfer and assessed the effects of this method on the enucleation rate of rabbit oocytes and the in vitro development and spindle structures of giant panda–rabbit reconstructed embryos. Our results demonstrated that chemical enucleation can be used in rabbit oocytes and the optimal enucleation result can be obtained. When we compared the rates of cleavage and blastocyst formation of subzonal injection (SUZI) and WCICI using chemically enucleated rabbit oocytes as cytoplasm recipients, the rates in the WCICI group were higher than those in the SUZI group, but there was no statistically siginificant difference (p>0.05) between the two methods. The microtubule structures of rabbit oocytes enucleated by chemicals and giant panda–rabbit embryos reconstructed by WCICI combined with chemical enucleation were normal. Therefore the present study suggests that WCICI combined with chemical enucleation can provide an efficient and less labor-intensive protocol of interspecies somatic cell nuclear transfer for producing giant panda cloned embryos.
This study assessed the effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection (ICSI) rabbit embryos. Oocytes were recovered from female rabbits superovulated with PMSG and hCG, and epididymal sperm were collected from a fertile male rabbit. The oocyte was positioned with the first polar body at 12 o'clock position, and a microinjection needle containing a sperm was inserted into the oocyte at 3 o'clock. Oolemma breakage was achieved by aspirating ooplasm, and the aspirated ooplasm and sperm were re-injected into the oocyte. The injected oocytes were cultured in M199 medium containing 10% fetal calf serum at 38 °C with 5% CO2 in air. The results showed that oocytes injected at 1 h post-collection produced a higher (p<0.05) fertilization rate than those injected at 4 or 7 h post-collection. Blastocyst rate in the 1 h group was higher (p<0.05) than in the 7 h group. Denuded oocytes (group A) and oocytes with cumulus cells (group B) were injected, respectively. Rates of fertilization and development of ICSI embryos were not significantly different (p<0.05) between the two groups. Four ICSI methods were applied in this experiment. In methods 1 and 2, the needle tip was pushed across half the diameter of the oocyte, and oolemma breakage was achieved by either a single aspiration (method 1) or repeated aspiration and expulsion (method 2) of ooplasm. In methods 3 and 4, the needle tip was pushed to the oocyte periphery opposite the puncture site, and oolemma breakage was achieved by either a single aspiration (method 3) or repeated aspiration and expulsion (method 4) of ooplasm. Fertilization rate in method 2 was significantly higher (p<0.05) than in methods 1 and 3. Blastocyst rates were not significantly different (p<0.05) among methods 1, 3 and 4, but method 2 produced a higher (p<0.05) blastocyst rate than method 3.
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