Studies On Activation And Development Of Goat Oocytes Following Intracytoplasmic Injection Of Round Spermatids

Intracytoplasmic sperm injection(ICSI) have been widely applied as a research tool for studying fundamental aspects of gamete interaction during fertilization and to clinical treatment of male infertility because of serious Oligoasthenospermia in human. And large numbers of test-tube babies fertilized by ICSI have been born. However, a part of men with non-obstructive azoospermia(NOA) failed to produce mature spermatozoa, but round spermatid (ROS) could be retrieved from ejaculate or from testis biopsy specimens. It has been proved that ROS as the earliest haploid male germ cell was injected into oocyte could form male pronucleus(MPN) and participate in syngamy. Therefore, round spermatid injection(ROSI) as a new method to clinical treatment of male infertility has created many expectations for azoospermic men who want to become genetic fathers. At present, although test-tube babies fertilized by ROSI have been born, many problems in ROSI like low fertilized rate and low rate of baby birth still remain unresolved. Probably, it was the main reasons that oocytes could not be activated after ROSI, and that the centrosome in ROS failed to perform function as microtubule organization center in oocytes, and that round spermatid nuclear remodeling in oocytes was abnormal. Therefore, the issues should be research to improve the rate of fertilization by ROSI, and serves human assisted reproduction better. Additionally, studies on oocytes-activating capacity of ROS, on microtubule-organizing capacity of centrosome of ROS in oocytes, and on round spermatid nuclear remodeling in oocytes, will enrich the content of oocytes-activating mechanism by fertilization, elucidate the effect of centrosome and sperm nuclear compaction during spermiogenesis on fertilization.At present study, techniques including ROSI, immunofluorescence, laser confocal microscopy, protein kinase assay et al, and ovarian oocytes matured in vitro and ROS obtained from goat testis were used. Developmental capacity of goat oocytes after ROSI was studied, and the reasons why goat oocytes after ROSI could not develop to blastocysts were analyzed from several aspects as follows: (1) activation of goat oocytes after ROSI, (2) dynamics of MPF and MAPK activity in goat oocytes after ROSI, (3) calcium oscillation in goat oocytes after ROSI, (4) activation and calcium oscillation of mouse oocytes stimulated by goat ROS, (5) activation and calcium oscillation of goat oocytes injected with increasing goat ROS. Besides, at present study nuclear remodling of spermatozoa and round spermatid nucleus in goat oocytes were compared, microtubule-organizing capacity of ROS centrosome in oocytes was observed, and a protocol to activated goat oocytes injected with ROS was optimized. The main results are as follows:1. Cleavage rate of goat oocytes after ROSI was just 9.9%, and no embryo developed to blastocyst. Cleavage rate and blastocyst rate of goat oocytes after ICSI were 66.1% and 28.6% respectively. The results indicated that goat oocytes failed to developed to blastocysts after round spermatid injection.2. 70.6% of goat oocytes after ICSI were activated, however, only 14.8% of goat oocytes after ROSI were activated. Activation rates of oocytes after ROSI were significantly lower than after ICSI. The results indicated that the reasons why goat oocytes after ROSI failed to develop to blastocyst were the oocytes could not be activated by goat ROS.3. Activation of goat oocytes was improved when oocytes after ROSI were treated with ionomycin. Male pronucleus formed when goat ROSs were injected into goat oocytes followed by treatment with ionomycin.4. The second meiotic progression of goat oocytes after ICSI were as follows: M II stage lasted from 0 to 0.47h, Ana II was 0.47-2.0h, Tel II was 2.0-2.9h, and PN formed since 2.9h.5. The activity of MPF and MAPK in oocytes decreased significantly after ICSI and after ROSI followed by Ionomycin treatment within 6h, however, the MPF and MAPK activity almost unchanged in oocytes until 6h. This indicated that no decreasing in MPF and MAPK activity was the main reason why goat oocytes after ROSI could not be activated6. Calcium oscillation occurred in goat oocytes after ICSI. Increasing in intracellular concentration of free calcium ion[Ca2+]i in goat oocytes after ROSI could not happen. [Ca2+]i rised just at when oocytes were treatment with ionomycin after ROSI. This indicated that goat ROS could not trigger calcium oscillation in goat oocytes was the main reason why MPF and MAPK activity could not be inactivated, which result in oocytes could not be activated.7. Activation of mouse oocytes and calcium oscillation occurred when mouse oocytes were injected with goat ROS. This indicated that oocyte-activating factor had been expressed in goat ROS.8. With increaseing in number of goat injected-ROS, activation rate of goat oocytes was increased significantly, calcium oscillation could be induced by four goat ROSs that were injected into goat oocytes. This indicated that the content of oocyte-activating factor in goat ROS was under the threshold, above which oocytes could be activated.9. Spermatozoa decondensed firstly, and then formed male pronucleus when sperm were injected into oocytes. NEBD of round spermatid nucleus occurred, and then PCC formed when ROS were injected into goat oocytes. NEBD of round spermatid nucleus didn't occur in oocytes until male pronucleus formation when oocytes were treated with ionomycin after ROSI.10. Spermatozoal centrosome organized sperm aster in oocytes , and function as microtubule-organizing center. The centrosome in ROS could not organize sperm aster in oocytes after ROSI followed by treatment with ionomycin. The pattern of microtubule-organizing in ROSI oocytes was different from ICSI and ROSI plus ionomycin, microtubule was self-organized by mediation of chromatin, not by centrosome.11. When protocol to activate the oocytes after ROSI was optimized. Activation rate and cleavage rate were not different when oocytes were activated once or twice before or after ROSI. But the rates of 2PN and blastocyst were higher significantly when oocytes were activated before ROSI than after ROSI. The rate of blastocyst was highest when oocytes were activated before and after ROSI.12. Extrusion of the second pole body and PN formation did not be affected if goat oocytes or ROSI oocytes were treated firstly with ionomycin and then incubated in 6-DMAP 1.5 hours later.13. Although the cleavage rates of oocytes treated twice with ionomycin before and after ROSI followed by incubation in 6-DMAP 1.5 hours later were not affected, the blastocyst rates were increased significantly.In summary, from present study we could arrive at four conclusions. (1) Oocyte- activating factor had been expressed in goat ROS, however, the content of oocytes-activating factor in each goat ROS was lower than threshold that calcium oscillation could be triggered, so [Ca2+]i in oocytes could not be increased, and then MPF and MAPK activity could not be inactivated, thus oocytes could not be activated, therefore, goat oocytes injected with ROS failed to develop to blastocyst. (2) The pattern of nuclear remodeling of ROS was different from spermatozoa. (3) The pattern of microtubule-organizing of round spermatid was also different from spermatozoa. (4) The optimal protocol to activate goat oocytes injected with ROS was that goat oocytes were treated with 10μM ionomycin for 2min, 1h later, oocytes were underwent round spermatid injection, then oocytes were treated with 5μM ionomycin for 2min again, oocytes were incubated in 2mM 6-DMAP for 2h from time that oocytes were firstly treated with ionomycin 1.5h later.