Mitochondrial function and intracellular distribution is severely affected in in vitro cultured mouse embryos.

Studies of mitochondrial dynamics have identified an intriguing link between energy supply balance and mitochondrial architecture. This suggests that inappropriate culture conditions might inhibit mitochondrial functions, and affect embryonic development. Therefore, this study was conducted to determine whether in vitro culture (IVC) might affect mitochondrial function, distribution, organization (by Mitotracker Green), gene expression on RNA level (by qPCR), and protein expression and localization (by western blot and immunostaining) involved in regulation of mitochondrial functions. Mitochondria in 2-cell IVC embryos were less numerous compare to IN VIVO while the localization and distribution do not differ between the groups. Mitochondria of in vivo blastocysts formed elongated network along the cells, while in IVC were fragmented, rounded, and aggregated mainly in the perinuclear region. Additionally, mitochondria of IN VIVO embryos moved back and forth along their long axis on radial tracks, while in IVC blastocysts were much less active. mtDNA copy number in IVC blastocysts (92,336.65 ± 5860.04) was significantly lower than that of IN VIVO (169,103.92 ± 16,322.41; P < 0.02) as well as lower protein expressions responsible for mitochondrial fusion was observed in IVC blastocysts. Results indicate that in vitro culture affect on perturbations in mitochondrial number and function, which is associated with decreased developmental competence of in vitro produced mouse embryos.

Establishment of a Wisent (Bison bonasus) Germplasm Bank.

The wisent, or European bison (Bison bonasus), belongs to the same family (Bovidae) as the American bison and domestic cattle. The wisent is the largest mammal in Europe, and is called the "Forest Emperor". The wisent is listed as "Vulnerable" on the IUCN Red List, and is protected by international law. Achievements in reproductive biotechnology have opened new possibilities for the cryoconservation of the wisent germplasm. Therefore, this research aimed to improve a strategy for the protection and preservation of the European bison through the creation of a wisent germplasm bank, based on the following procedures: isolation and in vitro maturation (IVM) of oocytes, in vitro fertilization (IVF) of matured oocytes, in vitro embryo culture (IVC), and embryo cryopreservation. Wisent ovaries were isolated from females outside the reproductive season, and eliminated from breeding for reasons other than infertility. Cumulus-oocyte complexes (COCs) were isolated from follicles greater than 2 mm in diameter and matured for 24 h and 30 h. After IVM, COCs were fertilized in vitro with wisent sperm. The obtained wisent zygotes, based on oocytes matured for 24 h and 30 h, were cultured for 216 h. Embryos at the morula and early blastocyst stages were vitrified and then warmed and transferred to interspecies recipients (Bos taurus). USG and biochemical tests were used to monitor pregnancies. This study obtained embryos in the morula and early blastocyst stages only after oocytes were fertilized and matured for 30 h. On average, per oocyte donor, 12.33 ± 0.5 COCs were isolated, and only 9.33 ± 0.61 COCs were qualified for in vitro maturation (75.68%), while 9.16 ± 0.48 COCs were matured (84.32%). On average, per donor, 5.5 ± 0.34 embryos were cleaved (59.96%) after 48 h post-fertilization (hpf), and 3.33 ± 0.21 achieved the eight-cell stage (36.52%) after 96 hpf, while 1 ± 0.21 morula and early blastocyst stages (10.71%) were achieved after 216 hpf. A total of six embryos (one morula and five early blastocysts) were obtained and vitrified; after warming, five of them were interspecies transferred to cattle (Bos taurus). On day 41 after fertilization, 3 out of 5 pregnancies were detected based on USG, P4, and PAG tests. However, no pregnancy was observed on day 86 after fertilization, indicating embryo resorption. This study shows that obtaining wisent embryos in vitro, and subsequent cryopreservation to create a wisent embryo bank, can be applied and implemented for the wisent protection program.

Influence of embryo transfer on embryo preimplantation development.

OBJECTIVE: To investigate the impact of injection speeds of the transferred load on embryo development. DESIGN: A laboratory model for in vitro simulation of ET was developed to investigate the impact of varying injection speeds of the transferred load on embryo development. SETTING: Academic research institutes of reproduction biotechnology and private centers of reproductive medicine. ANIMAL(S): Mouse hybrid F(1) females (C57bl/10 J × CBA-H; N = 15) aged 2-3 months. INTERVENTION(S): In vitro exposure of mouse embryos with either the fast ET (ejection speed, >1 m/s) or slow ET (ejection speed, <0.1 m/s) and consecutive culture for 36 hours. MAIN OUTCOME MEASURE(S): Development rate, morphology and apoptotic index of embryos. RESULT(S): The development rate was the slowest in embryos exposed to the fast ET. Morphological changes in response to ET were observed only among embryos exposed to the fast ET. The mean apoptotic index was 17.6% in the group exposed to the fast ET, 5.6% in the group exposed to the slow ET, and 2.58% in the control group. CONCLUSION(S): A reduction of the ejection speed of the transferred load allows avoidance of a developmental delay and diminishes injury of the embryos. Therefore, it is reasonable to suggest transferring the embryos at the lowest possible ejection speed.

Pressure induced nucleus DNA fragmentation.

PURPOSE: The present study was designed to investigate the impact of pressure on nuclear DNA integrity in viable cells of mouse blastocysts. METHODS: The blastocysts of hybrid F1 females [(C57Bl/10 J × CBA-H);N = 15] aged 2-3 months were exposed into the pressure impulse lasting ~0.021 s and characterized by a positive pressure peak of ~76 mmHg. The nuclear DNA fragmentation index of mouse blastocysts was assessed by TUNEL assay within 60 s after exposure to pressure impulse. RESULTS: The mean nuclear DNA fragmentation index was significantly higher in the experimental group (83%) than in the control group (19.7%); p < 0.001. CONCLUSION(S): A low magnitude pressure impulse can induce nuclear DNA fragmentation in mouse blastocysts. The compression and decompression forces appearing during pressure fluctuations are responsible for the observed DNA shearing.

Influence of embryo transfer on blastocyst viability.

OBJECTIVE: To investigate the impact of injection speeds of the transferred load on embryo viability. DESIGN: Laboratory model for in vitro simulation of embryo transfer (ET). SETTING: Academic research institutes of reproduction biotechnology and private centers of reproductive medicine. ANIMAL(S): Mouse hybrid F1 females, C57bl/10J × CBA-H (N = 15), aged 2 to 3 months. INTERVENTION(S): In vitro exposure of mouse blastocysts to either fast ET with an ejection speed of the transferred load of >1 m/s or slow ET with an ejection speed of <0.1 m/s. MAIN OUTCOME MEASURE(S): Morphologic changes and apoptotic index of blastocysts. RESULT(S): Morphologic changes in response to ET were most prevalent in blastocysts exposed to fast ET. The mean apoptotic index was 52% in the group exposed to fast ET, 25% in the group exposed to slow ET, and 12.8% in control group. CONCLUSION(S): Fast ejection of the transferred load can trigger both morphologic changes and apoptosis in mouse blastocysts. A reduction of the ejection speed of the transferred load minimizes injury to the embryos. Therefore, embryos should be transferred at the lowest possible speed.

Enucleated GV oocytes as recipients of embryonic nuclei in the G1, S, or G2 stages of the cell cycle.

Universal recipients in the G2 phase of mitotic cell cycle (preactivated oocytes, zygotes, blastomeres) accept embryonic nuclei in all the stages of their cell cycle. To test if recipients in the G2 of meiotic cycle (immature oocytes) are universal recipients, mouse germinal vesicle (GV) oocytes were enucleated and reconstructed with blastomere nuclei in the G1, S, or G2 stages. Analysis of their maturation has shown that about 30% of the G1 nuclei and 60% of G2 nuclei allow for normal metaphase II (MII), both in the oocytes with and without the first polar body (1st PB). Among oocytes reconstructed with the S phase nuclei, only 8% or less have normal MII, although 75% of them extrude 1st PB. No phase of donor cell cycle prevented the abnormal acceleration of 1st PB extrusion, found in reconstructed GV oocytes. In conclusion, enucleated GV oocytes are not universal recipients of embryonic nuclei, because they do not accept the S donors. However, both the G1 and G2 donor nuclei can be reprogrammed in the GV oocyte cytoplasm.

Mouse zygotes as recipients in embryo cloning.

Zygotes have not been recognized as nuclear recipients since enucleated zygotes receiving nuclei from beyond two-cell stage embryos are not able to form blastocysts. In the present study, a new technique of zygote enucleation is presented, which consists in selectively removing the nuclear membrane with genetic material of pronuclei, but leaving other pronuclear components in the cytoplasm. With selective enucleation it is possible - after transfer of eight-cell stage nuclei - to obtain 70.5 and 7.8% of preimplantation and full-term development respectively. Origin of cloned mice from introduced nuclei was confirmed by the coat colour and glucose phosphate isomerase (GPI) isozyme of the donor. We suggest that some pronuclear factors - taken away from the zygotes in the karyoplasts upon classical enucleation - are needed to reprogram the introduced nuclei.