Zygote development after delayed fertilization : a cytological and genetical analysis of embryos of the mouse

Chapter I gives (1) a brief review of morphological and molecular changes in mammalian oocytes initiated by fertilization, (2) a summary of published data on several aspects of post-ovulatory ageing in relation to embryonic development and (3) a description of methods to time ovulation and fertilization.Precise timing of ovulation and fertilization was a prerequisite for the study of the consequences of delayed fertilization for embryonic development.Chapter II describes the methods of ovulation induction and artificial insemination. We induced ovulation with injections of luteinizing hormone releasing hormone (LHRH) at pro-oestrus of the oestrus cycle. The number of oocytes that ovulated after LHRH administration was similar to that after spontaneous ovulation. Although LHRH was administered 8-12 hrs before the expected endogenous luteinizing hormone (LH) surge, we found no significant effect of LHRH induced ovulation on embryonic mortality. This method of ovulation induction, supplemented with artificial insemination to achieve in vivo fertilization, provided a tool to study aspects of early embryonic development after delayed fertilization.Investigations were carried out to determine in detail the timing of the distinct morphological changes triggered by fertilization of unaged oocytes and oocytes aged post-ovulation for 12 hrs. Sperm penetration was shown to be accelerated by 1 hr 30 min after delayed insemination compared with sperm penetration in unaged oocytes. The rate of progression to the first cleavage division was also influenced by the post-ovulation age of mouse oocytes prior to fertilization: penetrated aged oocytes needed less time (1 hr 30 min) to reach the 2-cell stage than zygotes from unaged oocytes. This observation was confirmed by experiments carried out later: the percentages of 3- and 4-cell embryos from aged and unaged oocytes, collected 36 hrs after insemination, were 14% and 7% respectively (Chapter IV).Fertilization activates, among other things, a cascade reaction of protein synthetic alterations.Chapter III deals with protein synthetic activity in unaged and aged LHRH induced oocytes, in unaged superovulated oocytes and in zygotes derived from these types of oocytes. Polypeptides with a relative molecular weight of 35 kDa were predominantly synthesized by LHRH induced and superovulated secondary oocytes and zygotes from these oocytes. This study of patterns of 35 kDa proteins synthesized by zygotes from aged and unaged LHRH induced oocytes revealed that fertilization dependent protein synthetic changes of the 35 kDa protein complex were advanced in zygotes from aged oocytes with reference to pronuclear development.A fraction (16.7%) of morphologically normal zygotes from unaged superovulated oocytes did not synthesize the 35 kDa protein complex at all.It was of interest to learn more about the in vitro developmental capacity of embryos from aged oocytes.In Chapter IV the results of these investigations are arranged and discussed. One-cell and late 2-cell embryos from aged and unaged oocytes were cultured in the presence and absence of DNA damage or DNA-damaging agents for different periods of time. On the one hand we studied the progression to metaphase of the first cleavage division in zygotes from aged and unaged oocytes fertilized with X-irradiated sperm. On the other hand the in vitro developmental capacity of late 2-cell embryos was evaluated in presence and absence of the thymidine analogue 5-Bromode-oxyuridine (BrdU).Post-ovulatory ageing had an effect on the morphology of male as well as female pronuclear chromosomes of the first cleavage metaphase. We also found a detrimental effect of fertilization with X-irradiated spermatozoa on the morphology of male and female pronuclear chromosomes. This effect was particularly observed in male pronuclear chromosomes of zygotes from aged oocytes. Furthermore, fertilization with X-irradiated spermatozoa led to an arrest at interphase in 27% and 7% of zygotes from aged and unaged oocytes respectively. This arrest was not shown after fertilization with sperm not irradiated with X-rays.This experimental setup also enabled us to compare the amount of radiation induced chromosome damage in zygotes from aged and unaged oocytes. Zygotes from aged oocytes did not contain more chromosome damage than zygotes from unaged oocytes, when the visible chromosome mutations originating from the X-irradiated spermatozoa were analyzed at metaphase of the first cleavage division.In Chapter IV we also have shown that zygotes from aged oocytes and unaged oocytes develop in vitro at similar rates from the late 2-cell stage, collected at 36 hrs after insemination, to the 8-cell stage (24 hrs cultures). However, in vitro development of 2-cell embryos from aged oocytes collected 30 hrs after insemination and cultured for 66 hrs is impaired.To determine the number of sister-chromatid exchanges, we cultured 2-cell embryos from aged and unaged oocytes, collected 36 hrs after insemination, in the presence of BrdU for 24 hrs. SCE levels were not significantly different between embryos from aged and unaged oocytes.Cell proliferation of late 2-cell embryos from aged oocytes, collected 36 hrs post-insemination, from aged oocytes was clearly retarded and asynchronous during the 24 hrs culture period in the presence of 10-6 M BrdU.In Chapter V a cytochemical method is described to determine in individual oocytes the distribution of the activity of SDH (succinate dehydrogenase), an enzyme which is located on the inner membrane of mitochondria. We showed that treatment of oocytes with the drug caffeine prior to cytochemical staining resulted in an intens staining of the cells by a formazan precipitate. We applied the cytochemical staining procedure to preovulatory oocytes of mice. In a maturation experiment in vitro we found that the location of formazan correlated well with the location of mitochondria in subsequent stages of maturation. Unfortunately, this cytochemical staining procedure could not be applied to ovulated and fertilized oocytes, since these cells acquired a poor morphology during the staining procedure and displayed high levels of non -dehydrogenase formazan production.