Date of Award

Summer 2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Program/Concentration

Biomedical Sciences

Committee Director

Andrei Zalensky

Committee Member

Sergio Oehninger

Committee Member

Silvina Bocca

Committee Member

Jacob Meyer

Committee Member

Peter Sutovsky

Abstract

Chromosome organization is regarded as an important factor in regulating gene expression. In addition to the histone code, it is a part of the epigenetic mechanisms participating in fertilization and early embryonic development. Recent studies have demonstrated that chromosomes of human spermatozoa have well-defined spatial organization that includes preferred intranuclear positioning. The hypothesis of this dissertation is that sperm chromosome architecture participates in controlled transformation and activation of the paternal genome following fertilization.

Adequate model systems are necessary to study early nuclear events characteristic of human fertilization. This research focused on introduction and application of heterologous intracytoplasmic sperm injection (ICSI) to study sperm chromosomes remodeling in ooplasm. ICSI of human sperm into bovine and hamster oocytes was explored in detail and the latter pair has been chosen for further studies as robust and apparently faithful models of the paternal gamete development.

In this work, it was established that after ICSI: 1) sperm cells with partially digested membranes and depleted of acrosome decondense more rapidly and to a greater extent than intact cells; and 2) marked sperm-to-sperm variability in degree of chromatin swelling exists during all stages of pronuclear development as detected by DNA staining in zygotes. The latter characteristic is proposed to reflect the cryptic nuclear heterogeneity in population of morphologically, mature normal sperm.

Chromatin transformation in pronuclei was established by immunolocalizaton of protamines in zygotes. Protamine withdrawal is rapid (completed within I hr) and correlates with pronuclear area rather than with specific time post ICSI. We propose that such behavior is again a consequence of hidden variability in sperm population.

The final part involved the post fertilization study of two human sperm chromosomes — Homo Sapiens (HSA) chromosomes 18 and 19. Findings demonstrated that in sperm: 1) both chromosomes are packed into compact territories; and 2) similar to somatic cells, gene-rich HSA19 is positioned internally while gene-poor HSA18 peripherally. The unwinding of HSA I8/19 in developing pronuclei was described. To visualize structure remodeling of sperm chromosomes in zygotes, novel FISH procedures were developed.

These findings suggest that remodeling of human sperm in heterologous ICSI faithfully mimics early steps of natural human fertilization and for the first time demonstrated several novel aspects of nuclear and chromosome reorganization during male pronuclei development.

DOI

10.25777/m83v-tk02

ISBN

9781124294537

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