319 APPROACHES TO IMPROVE INTRACYTOPLASMIC SPERM INJECTION MEDIATED TRANSGENESIS AND MAXIMIZE THE USE OF SEX-SORTED SPERM IN BOVINE

Intracytoplasmic sperm injection (ICSI) mediated transgenesis is an effective tool for transgenic animal production. However, ICSI in cattle remains inefficient. In this work, we assayed approaches to improve egfp expressing blastocysts production by ICSI: the sperm pretreatment with heparin and l-glutathione (Hep-GSH), the use of sex-sorted sperm (SS), the refrozen/thawing of SS sperm, and the combination of these. Quality of ICSI blastocysts was analysed by studying the expression of 4 genes, and the rates of DNA fragmentation. Cumulus-oocyte complexes from slaughtered cow ovaries were in vitro-matured for 21 h. Nonsorted (NS) and sex-sorted (SS) frozen straws were thawed. Some of them were incubated with 80 μM Hep-15 mM GSH for 20 h (Hep-GSH+). The Hep-GSH-control group was not pretreated. Semen samples were co-incubated with 50 ng µL–1 of pCX-EGFP for 5 min before ICSI. Moreover, the SS sperm that are usually discarded after ICSI were cryopreserved and used for ICSI after a second thawing (ICSI SS refrozen). The ICSI NS, sham, and diploid parthenogenetic (Diplo PA) controls were included. Oocytes were activated with 5 µM ionomycin for 4 min, TCM-199 for 3 h (except for diploid PA), and 1.9 mM DMAP for 3 h. Cleavage and blastocyst/egfp expression rates were evaluated on Days 2 and 7 post-ICSI, respectively. Results are shown in Table 1. Relative expression of HMGN1, GLUT5, AQP3, and OCT4 genes from ICSI NS Hep-GSH+ and IVF blastocysts were compared by qPCR. Data were analysed by the pair-wise fixed reallocation randomisation test. None of the 4 genes showed differences between groups. The DNA fragmented nucleus index/blastocyst cell numbers were determined by TUNEL assay, not showing differences between groups (Kruskal–Wallis test, P ≤ 0.05). Means ± s.d. were 29 ± 17/91 ± 27 for ICSI Hep-GSH+; 27 ± 15/63 ± 34 for ICSI Hep-GSH–; 28 ± 17/68 ± 17 for ICSI SS, 28 ± 13/75 ± 24 for ICSI SS refrozen; and 21 ± 13/105 ± 59 for IVF SS control. The Hep-GSH pretreatment can increase blastocyst and transgene expressing blastocysts rates after TM-ICSI, except when SS semen is used. Interestingly, the use of SS sperm for ICSI can be maximized by cryopreservation and reuse of discarded sperm cells. The parameters analysed in this work indicate that the proposed approaches do not affect blastocyst quality. Therefore, Hep-GSH pretreatment of NS sperm and refrozen SS sperm could be applied for TM-ICSI in bovine for the production of transgenic animals. Table 1.In vitro development and egfp expression of ICSI embryos fertilized with nonsorted (NS) and sex-sorted (SS) sperm pretreated with Hep-GSH, refrozen, or both

Analysis of DNA fragmentation in bovine somatic nuclear transfer embryos using TUNEL

The production of cloned animals is an inefficient process because of early or late embryonic losses. This study focused on the DNA fragmentation that occurs during embryonic development. The occurrence of DNA fragmentation was examined in bovine embryos produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (NT) using the terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL). IVF and NT embryos at the two-cell to blastocyst stage were stained by TUNEL for the analysis of DNA-fragmented nuclei and with propidium iodide for determination of the total number of cells. DNA fragmentation was first detected in NT embryos at the four-cell stage, but in IVF embryos at the six- to eight-cell stage. The percentage of embryos with at least one DNA-fragmented nucleus increased with the advance of the developmental stage of embryos in both IVF and NT groups. The DNA-fragmented nucleus index in NT embryos that developed beyond the four-cell stage was significantly higher (P<0.01) than that of IVF embryos at the same stage. In the both IVF and NT groups, TUNEL-labelled cells were detected in almost all blastocysts and were mainly observed in presumptive inner cell mass (ICM) cells of embryos. The DNA-fragmented nucleus index was negatively correlated with the total number of cells in NT blastocysts, but this relationship was not observed in IVF blastocysts. These results suggest that the high occurrence of DNA fragmentation observed in NT embryos may be related to early embryonic loss after transfer.

F-actin disorganization in apoptotic cell death of cultured rat renal proximal tubular cells

The mechanism of nephrotoxin-induced apoptosis was studied in rat renal proximal tubular cells (PTC) exposed to the nephrotoxin S-(1,2-dichlorovinyl)-L-cysteine (DCVC). After a 6-h incubation, DCVC caused a condensation of heterochromatin and a fragmentation of the nucleus in 84 and 16% of the cells, respectively, which is indicative of apoptosis. This was confirmed biochemically by agarose gel electrophoresis demonstrating the formation of DNA fragments with multiples of 200 bp. The antioxidant N,N'-diphenyl-p-phenylenediamine prevented neither the fragmentation of the nucleus nor the formation of DNA fragments, but it did prevent lactate dehydrogenase release and bleb formation by DCVC. Apoptosis induced by DCVC was closely associated with F-actin disorganization: every cell with a fragmented nucleus displayed completely disorganized F-actin, while cells with a normal nucleus still possessed at least some intact F-actin also induced apoptosis in PTC. Similarly, dithiothreitol, which damages F-actin in PTC, caused apoptosis of PTC. These data suggest a causal relationship between F-actin disorganization and apoptosis of PTC.

ETUDE AU MICROSCOPE ELECTRONIQUE DES TRANSFORMATIONS NUCLEAIRES DE E. COLI K12 S ET K12 S (λ26) APRES IRRADIATION AUX RAYONS ULTRAVIOLETS ET AUX RAYONS X

Nuclear transformations induced in E. coli K12S and K12S(λ26) by ultraviolet radiations and x-rays have been studied with ultrathin sections in the electron microscope. The nucleoplasm keeps its normal aspect during "fragmentation" and during "condensation" of the nucleus into the "vesicular" form. Serial sections show that the "fragmented" nucleus consists in reality of only one very tortuous vacuole. No difference either in the shape or in the fine structure of the nucleus could be observed between the lysogenic strain and the non-lysogenic strain. A high concentration of NaCl has a "condensation" effect on the fragmented nuclei and decreases the induction rate.