Roles of MAPK and Spindle Assembly Checkpoint in Spontaneous Activation and MIII Arrest of Rat Oocytes

Rat oocytes are well known to undergo spontaneous activation (SA) after leaving the oviduct, but the SA is abortive with oocytes being arrested in metaphase III (MIII) instead of forming pronuclei. This study was designed to investigate the mechanism causing SA and MIII arrest. Whereas few oocytes collected from SD rats at 13 h after hCG injection that showed 100% of mitogen-activated protein kinase (MAPK) activities activated spontaneously, all oocytes recovered 19 h post hCG with MAPK decreased to below 75% underwent SA during in vitro culture. During SA, MAPK first declined to below 45% and then increased again to 80%; the maturation-promoting factor (MPF) activity fluctuated similarly but always began to change ahead of the MAPK activity. In SA oocytes with 75% of MAPK activities, microtubules were disturbed with irregularly pulled chromosomes dispersed over the spindle and the spindle assembly checkpoint (SAC) was activated. When MAPK decreased to 45%, the spindle disintegrated and chromosomes surrounded by microtubules were scattered in the ooplasm. SA oocytes entered MIII and formed several spindle-like structures by 6 h of culture when the MAPK activity re-increased to above 80%. While SA oocytes showed one Ca2+ rise, Sr2+-activated oocytes showed several. Together, the results suggested that SA stimuli triggered SA in rat oocytes by inducing a premature MAPK inactivation, which led to disturbance of spindle microtubules. The microtubule disturbance impaired pulling of chromosomes to the spindle poles, caused spindle disintegration and activated SAC. The increased SAC activity reactivated MPF and thus MAPK, leading to MIII arrest

Evaluation of in vitro cultured rat oocytes, from different strains, by spindle morphology and maturation-promoting-factor activity combined with nuclear-transfer experiments.

Although successful nuclear transfer (NT) has been reported in the rat 6 years ago, somatic cell nuclear transfer (SCNT) in the rat could not be repeated. Our experiments with rat SCNT reveal the difficulties related to rat cloning. We first focussed on the most appropriate rat strain that could be used as an oocyte donor. Then we describe how rat oocytes can be kept in a nonactivated state during in vitro culture, because the latter undergo spontaneous partial activation through rapid extrusion of the second polar body after isolation from the oviduct. In the SCNT experiments performed with the one-step manipulation technique it was possible to produce rat embryos, which developed in vivo up to the blastocyst stage. In addition, we identified the implantation sites of SCNT rat embryos reconstructed with Sprague-Dawley (SD) oocytes. Furthermore, different rat strains were used as oocyte donors and their oocytes were cultured under different conditions to establish a stable nonactivating oocyte culture system. The ratio of activated to nonactivated oocytes was measured by spindle-stability and maturation promoting factor (MPF) activity. These measurements indicated that a substrain of the SD rat strain, the so-called OFA-SD strain, is the one providing the most stable oocytes, when their oocytes are cultured in the presence of the proteasome inhibitor MG132. However, it was not possible to obtain any implantation sites with reconstructed oocytes derived from the OFA-SD strain transferred to foster mothers. This goal was not achieved, even when the trichostatin A (TSA) treatment was used, which is known to enhance the cloning efficiency of reconstructed mouse, porcine, bovine, and rabbit oocytes both in vitro and in vivo by enhancing the reprogramming efficiency of the recipient nucleus

First successful pregnancy in Switzerland after prospective sex determination of the embryo through the separation of X-chromosome bearing spermatozoa

QUESTION UNDER STUDY: The feasibility and the potential advantages of separating X-chromosome bearing spermatozoa for the prevention of a severe X-chromosome linked disorder with the use of intracytoplasmic sperm injection are presented. METHOD: A carrier of muscular dystrophy type Becker was treated with intracytoplasmic sperm injection, using spermatozoa previously stained with the Hoechst dye 33342 and sorted with flow cytometry. RESULTS: After transfer of one single blastocyst, an intrauterine pregnancy arose. In the ninth week of gestation, the female sex of the embryo was confirmed with proof of absence of the SRY gene of the Y-chromosome. After normal pregnancy, the patient delivered a healthy daughter. CONCLUSIONS: The staining of spermatozoa with specific markers and sorting with flow cytometry provides a means of preventing significant disease in the offspring and may help in reducing the number of surplus embryos needed for preimplantation genetic diagnosis