Decondensation Of The Sperm Nucleus Effects On The Male Pronucleus And Embryo Development Of Porcine Oocytes After Intracytoplasmic Sperm Injection (ICSI)

Intracytoplasmic, sperm .injection (ICSI) is the new way of solving men's sterility. And as a new technique of embryo engineering of domestic animal, ICSI can solve the problem of procine polyspermy. Since ICSI is injects directly single sperm to the intracytoplasm of oocyte, the physiological and biochemical reactions which were produced when sperm contected and perforated the zona pellucida and plasma membrane of oocyte are omitted, and the process of injecting sperm into oocyte. bring about, the weak activation of oocyte and the decrease of activity of maturation-promoting factor (MPF) ahead of time. If sperms were not been treated by appropriate methods, and the, cytoplasm of oocytes were not maturated completely or oocytes were not been activated fully, decondensation of some sperm nucleus would be hindered, and affected the efficiencies of ICSI. If the methods of promoting decondensation of some sperm nucleus were not appropriate, it would be bring about the increase of the rates of parthenogenetic development. The object of this study was to explore an effective method to promoting decondensation of the sperm nucleus, and improve the efficiencies of ICSI in porcine.The present study investigated the, sperm morphological change and factors affecting the pronuclear formation and embryo development of porcine oocytes after ICSI. Furthermore, we study the effects of different methods to induce the decondensation of the sperm nucleus on the male pronucleus and embryo development of porcine oocytes after ICSI, such as activation of oocytes, sperm pretreatment with dithiothreitol (DTT) or glutathione (GSH), and adding cysteine (Cys) and epidermal growth factor (EGF) to the maturation medium. The results as following:In Exp. 1, in vitro matured oocytes were injected by ICSI using boar sperms and activated by the electrical pulse, then they were transferred to the in vitro culture (IVC) mediums. At 6h, 9h, 12h, 15h and 18h after ICSI, the oocytes were stained with Hoechst33342 to assess the sperm morphological change and the pronuclear formation. At 6h after ICSI, 20.2% of the sperms had a swell head and, only 2.2% was decondensed, in this time, the male or female pronuclear hadn't been found. At 9h after ICSI, 12.2% of the sperms was decondensed and 20.0% of the oocytes had been activated. And at 12h after ICSI, the rate of the decondensed sperms had been increased to 30.0%, 5.4% had been formed male pronucleus. Pronuclear formation was asynchronous, the male pronucleus formed about 3 hours later than female pronucleus, at 9h, 12h 15h, and 18h after ICSI, 20.0%, 46.2%, 63.5% and 70.3% oocytes formed female pronucleus respectively, while 5.4%, 25.5% and 39.1% of oocytes formed the male pronucleus respectively at 12h, 15h and 18h after ICSI.In Exp. 2, sperm injected, sham injection or non-injection oocytes were either activated byCaCl₂ (1.8pl, 30mM), ionomycin (15umol/l, 40min) or electrical pulse (a single dc-pulse of 0.4KV/cm for 90us). The results were that activation rates of sham injected and non-activated oocytes were higher than those of non-injected and non-activated oocytes ((12.8%¹3.3%)vs(8.3 % ⁸.9%)), but obviously lower than those of sham injected and activated oocytes ((12.8% 13.3 %) vs (18.3 % ⁶4.3 % )). This suggested the activate effect of porcine oocytes was poor when they were activated only by injection, in order to improve the activation rates of oocytes, it was necessary to carry out the man-made activation. Comparing the three different protocols of man-made activation, we found that there were no significant difference among those activation rates of the injected oocytes which were either activated by CaCl₂, ionomycin or electrical pulse (69.4% versus 67.3% versus 70.4%, P>0.05). Fertilization rates of sperm injected oocytes activated by CaClr-injection were higher than those stimulated by ionomycin (46.4% versus 31.2%, P<0.05), but no difference with those stimulated by electrical pulse (46.4% versus 38.8%, P>0.05). While parthenogenetic development rates of sham injected and control oocytes activated by CaClr-injection were significantly lower than those activated by ionomycin and electrical pulse (5.9% versus 45.3%, 51.6%; 4.5% versus 38.8%, 46.5%, respectively; P<0.01). After being activated by CaClr-injection, the sperm injected oocytes showed a higher (48.1% versus 17.0%, P<0.01; 30.2 versus 26.3, P<0.05) rates of cleavage and total cell number of blastocysts compared to control ones. Those results indicated that the efficiencies of ICSI of the porcine oocytes which were activated by CaClr-injection was highest among three different activated protocols.In Exp. 3, after incubation of the sperms with 2mM, 4mM, 6mM and 8mM DTT for lh, phase -contrast microscopy showed that 56.2%, 73.5%, 87.4% and 95.3% of treated sperms displayed altered morphology respectively. The head of these sperm exhibited pronounced bending near the equatorial segment, and the midpiece of the tail appeared to be swollen and bent. As the concentration of DTT increased, the morphology change of treated sperms become more appearent. No such change were seen in untreated sperm. Further incubation of the sperm in mTBM with DTT or not for up to 6h, stained with Hoechst33342 every 2h, those treatments led to progressive time-dependent decondensation of the nucleus. Again, no such decondensation was seen in the untreated sperm. Oocytes injected with 6mM DTT-treated sperms showed a significantly higher 2PN+2Pb formation rates (60.6% versus 48.9%, 52.7%, 51.2%, 46.7%, respectively; P<0.01) and cleavage rates (49.1% vs 43.2%, 43.8%, 40.5%, 44.3%, respectively; P<0.05) compared to oocytes injected with 2mM, 4mM or 8mM DTT-treated sperms and control ones. Decondensed sperm head (DSH) formation rates of 6mM DTT-treated protocols were higher than 2mM or 4mM DTT-treated protocols (80.8% versus 72.6%, 75.1%; P<0.05) and control protocol (80.8% versus 69.2%; P<0.01),but no significantly difference with 8mM DTT-treated protocol (80.8% versus 83.3%, P>0.05). The survival rate was lower in 8mM DTT-treated protocol than 2mM ,4mM or 6mM DTT-treated protocols and control protocol (83.9% versus 88.0%, 87.2%, 87.0%, 88.5%; P<0.05). As the concentration of DTT increased, total cell number of blastocysts decreased. Total cell number of blastocysts from oocytes injected 6mM DTT-treated sperms were higher than 8mM DTT-treated protocol (26.2 versus 23.0, P<0.01), but all lower than control protocol (26.2, 23.0 versus 29.8, P<0.01). This suggested that 6mM DTT-treated protocol was better than other DTT-treated protocols, and at the same time of leading to decondensation of the sperm nucleus, the side effects of DTT affects the development quality of porcine embryos by ICSI.In Exp. 4, motile sperm were pretreated with 0.25mM, 0.50mM, 1.0 mM GSH before ICSI, and non-treated sperm served as controls. As the concentration of GSH increased, the trends of the rates of 2PN+2Pb formation, MPN formation and decondensation of the sperm nucleus were upward. Oocytes injected 0.50mM GSH-treated sperms showed a higher rates of clearage than 0.25mM or l.0mM GSH-treated protocols and controls (56.3% versus 51.1%, 51.6%, 44.9%, respectively; P<0.05) , and the rate of blastocyst formation was higher than controls(7.3% versus 2.8%, P<0.05). No differences were observed in the rates of blastocyst formation among three GSH-treated protocols. This suggested that the efficiencies of pretreated sperms with 0.50mM GSH were better than other GSH-treated protocols. We compared the effects of pretreated sperms with 0.50mM GSH and 6mM DTT on the pronuclear formation of porcine oocytes after ICSI and their subsequent development. Oocytes injected with 0.50mM GSH-treated sperms showed a higher rates of MPN formation (75.3% vs 69.2%, P<0.05) , 2PN+2Pb formation (66.1% vs 60.6%, P<0.01) and clearage (56.3% vs 49.1%, P<0.01) than 6mM DTT-treated protocol, and total cell number of blastocysts (35.8 vs 26.2, P<0.01) were significantly higher in the former than the latter. Those results indicated that the efficiencies of pretreated sperms with 0.50mM GSH were better than 6mM DTT during the course of porcine ICSI.In Exp. 5, we examined the effects of adding 0.6mM Cys and10ng/ml EGF to the maturation medium on pronuclear formation of porcine oocytes after ICSI and their subsequent development. Adding Cys and EGF to the modified TCM199 (protocol I) increased the rate of nuclear maturation compared to the controls (the modified TCM199) ( 78.1% versus 70.2%, P<0.05), but no difference with only adding EGF to he modified TCM199 (protocol II) (78.1% versus 76.4%, P>0.05). The rates of 2PN+2Pb formation (72.7% versus 65.4%, 66.1%), MPN formation(78.9% versus 74.2%, 74.2%), survival of injected oocytes (91.5% versus 88.4%, 87.2%) and blastocyst formation (10.8% versus 7.6%, 7.2%) were significantly higher in protocol I than in protocol Hand the controls...