| dc.description.abstract | One of the most fascinating aspects of biology is the conception, growth, and birth of the young. Central to these processes are gamete maturation, fertilization, and early embryonic development. An understanding of these processes is important not only to increase our knowledge in reproductive science but also in medically assisted reproductive technology (MART) to treat infertility.
Chapter 1 of this thesis (General Introduction) provides comprehensive information on the biology of sperm maturation, fertilization, and preimplantation embryo development in mammals relevant to the content of the thesis. It also describes the need for using rodents as animal models to study early mammalian development, some aspects of which are studied in the present investigation (Chapters 2–5).
Studies are necessary on male gamete (sperm) maturation since one of the significant causes of infertility is subfertile male status with poor-quality spermatozoa. Various sperm motility stimulants are used to accomplish assisted reproduction using such spermatozoa. Pentoxifylline (PF), a methylxanthine derivative, is one such compound used to enhance sperm motility and is applied in human in vitro fertilization (IVF) programs. However, the mechanism by which PF elicits the motility-stimulating effect on spermatozoa is not well understood. Moreover, the possible adverse effects of PF on fertilization and embryonic development remain to be established using a suitable rodent model.
The second chapter of this thesis describes PF-induced capacitation (CAP) and acrosome reaction (AR) in spermatozoa using a hamster model. This chapter also deals with the mechanism of action of PF during sperm maturation. Pentoxifylline induced early onset of sperm maturation, and this early onset was associated with an early cAMP rise and intracellular mobilization of [Ca²]i but not due to a rise in cGMP or uptake of Ca². The activity of protein kinase A, but not that of protein tyrosine kinase or protein kinase C (PKC), was necessary for PF-induced CAP, while activities of PKC and phospholipase A2 and their signaling pathways were necessary for PF-induced AR. Incidentally, this is the first report to document a detailed study on the possible biochemical mechanism of action of PF in mammalian spermatozoa.
The third chapter deals with the influence of PF on fertilization and pre-/post-implantation embryo development in hamsters. An improvement in the IVF rate was achieved using 0.45 mM PF-treated, unwashed spermatozoa. Continuous exposure of a low concentration (23 M, exposed to oocytes during IVF using 0.45 mM PF-treated spermatozoa) of PF to hamster 8-cell embryos was not detrimental to blastocyst development in vitro and improved their development (72.5 ± 4.8 vs. 57.5 ± 2.7), producing live births. Moreover, higher concentrations (>0.45 mM) of PF were inhibitory to embryo development, and this inhibition was exhibited in a concentration-dependent manner.
Since mammalian preimplantation embryos develop in a protected environment in the female reproductive tract, information on embryo development must therefore be derived from experiments on cultured embryos. Our ability to sustain normal embryo development in vitro is far from satisfactory. Clearly, the major problem is the unsuitability of the presently available embryo culture media. Hence, achieving normal development of embryos in vitro by optimizing media components is a major challenge to mammalian embryologists. As observed in Chapter 3, the percentage of blastocysts developed from 8-cell embryos has been modest, and there is a need to optimize embryo culture media that better support blastocyst development.
The fourth chapter of this thesis therefore focuses on the influence of energy substrates on the development of hamster 8-cell embryos. Supplementation of optimized concentrations of succinate (0.5 mM) and malate (0.01 mM) to a modified hamster embryo culture medium-2 (HECM-2m) supported 100% development of embryos to high-quality (as judged by the mean cell number per blastocyst) viable blastocysts (confirmed by embryo transfer). It was shown that non-glucose oxidizable energy substrates were the most preferred media components for hamster embryo development. It was also demonstrated that one of the causes of glucose inhibition of embryo development was reduced ATP production.
Poor preimplantation embryo development occurs not only in vitro but also in vivo. A substantial number of preimplantation embryos developing in vivo are either retarded, deformed, or degenerated and are believed to be lost. However, the extent and causes of such embryonic loss are not clear in most mammals studied. Hence, there is a need to understand the extent of developmental retardation and abnormality of in vivo-produced embryos and the possible causes of their loss.
The fifth chapter of this thesis describes the results regarding embryonic loss in vivo and the plausible causes of such loss using a rat model. A qualitative and quantitative assessment was made on the in vivo development of rat preimplantation embryos and their timing of migration in the reproductive tract. In qualitative terms, there was a substantial degree of developmental retardation and abnormality in embryos recovered during the preimplantation period, i.e., days 1–5 of pregnancy. In quantitative terms, it was estimated that 44% of recovered embryos belonged to asynchronously cleaving, deformed/degenerate embryos, and about 56% of recovered preimplantation embryos were of the expected developmental age.
Towards the plausible causes of embryonic loss, an assessment was made to investigate bacterial infection and the levels of endotoxin in the female reproductive tract and their correlation with the developmental status of preimplantation embryos. It was observed that one or more aerobic bacteria were present in the vagina, but the upper reproductive tract was sterile in all animals. The occurrence of common vaginal bacterial flora or vaginal endotoxin was not associated with the incidence of impaired development of preimplantation embryos. However, the presence of pathogenic bacteria in the vagina of day 4 pregnant animals or the presence of endotoxin in the upper reproductive tract was associated with developmental abnormality of embryos. | |
