Date of Award

2006

Level of Access

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Animal Sciences

Advisor

James Weber

Second Committee Member

Robert Taft

Third Committee Member

Carol Kim

Abstract

The laboratory mouse is the leading model for the genetic study of human diseases. With over 3,000 unique strains of mice, The Jackson Laboratory (TJL) in Bar Harbor, ME is the largest repository of mutant mice. New strains are continuously developed worldwide and many are sent to TJL for safekeeping and global distribution to researchers. Maintaining live colonies for all strains is not feasible and poses a challenge. Assisted reproductive technologies (ARTs) such as in vitro fertilization (IVF) and cryopreservation have proven essential in meeting this challenge. Embryos produced during IVF, after being cryopreserved, are stored in liquid nitrogen (-196°C) until they are needed. The performance of each strain when used for ARTs is influenced heavily by genetics. Fortunately there are a small group of standardized strains of laboratory mice, called inbred strains, which provide the genetic background for most mutant strains. The performance of inbred strains was evaluated when used for several ARTs, including: superovulation, in vitro fertilization, embryo cryopreservation, and surgical embryo transfer. Sperm from the same ten strains was studied to recognize strains where the male was the contributing factor to poor IVF results. An inbred strain, SJLJJ, was identified with male subfertility in vitro. The biology and genetics of SJLIJ sperm were examined to identify the etiology of this subfertility. When observed using bright field microscopy, SJLIJ sperm had a hairpin bend in the midpiece-principal piece junction of the tail when calcium was present in the medium. During fertilization, sperm use mechanical and enzymatic forces to penetrate the zona pellucida surrounding the egg. When the zona was removed, SJL/J sperm were able to fertilize normally. The hairpin bend may hinder SJL/J sperm from generating the force needed to penetrate the zona. Similar bending has been reported in sperm with cytoskeletal defects. Transmission electron microscopy revealed no major defects in the morphology of SJL/J sperm flagella. The inability to regulate cell volume has also been reported to cause hairpin bends in sperm. The bend of SJL/J sperm could be reversed using a detergent to disrupt the plasma membrane; however, SJL/J sperm remained bent even when the osmolality of the surrounding environment was changed. From these results, it is concluded that the bending is not the result of a defect in tail structure, cell swelling, or calcium acting directly on the flagella. The bending may be caused by a defect in a plasma membrane calcium channel, but further studies are needed to determine this. Genetic mapping studies revealed a region on Chromosome 17 that is correlated with the SJL/J hairpin sperm phenotype. Several candidate genes in this region have been identified and include: Fert2, a testis specific protein kinase; Efhb, a protein containing calcium binding domains; and Kcnh8, a potassium voltage gated channel. The calcium-induced flagellar bend identified in SJL/J sperm most likely causes the reduced fertility in vitro. Further studies are needed to identify how calcium is involved in sperm bending and to identify the genes involved.

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