PARENT SESSION
TESTICULAR SPERMATOGENESIS, MORPHOGENESIS, AND GENE EXPRESSION
Kent
7:30 AM-10:00 AM

(497) CLONING AND TESTICULAR EXPRESSION OF RAT Bcl2 MODIFYING FACTOR.

Show, Matthew¹, Anway, Matthew¹, Zirkin, Barry¹, ¹ Department of Biochemistry and Molecular Biology, Baltimore, MD

ABSTRACT- Bcl-2 Modifying Factor (BMF) is a recently discovered pro-apoptosis protein that contains both a myosin binding domain and a BH3 domain that can interact with the anti-apoptosis proteins of the Bcl-2 family. At steady state, BMF is normally associated with the actin cytoskeleton via its myosin binding domain. When the actin cytoskeleton is perturbed, BMF can lose its attachment to myosin and interact with Bcl-2 family members, thus initiating an apoptotic response. Using a combination of 3′ RACE and a degenerative RT-PCR based strategy, we cloned the full length rat coding sequence of the BMF gene. Sequencing revealed that rat BMF shares a 94% nucleotide sequence identity to the mouse cDNA and an 86% identity to the human cDNA. The predicted protein sequence of rat BMF indicates a 97% and a 92% identity to the mouse and human amino acid sequences, respectively. In the rat, BMF mRNA was found to be expressed robustly in the testis and ovary, and to lesser extents in the kidney, liver, and pancreas. Semi-quantitative RT-PCR expression analyses in the testis revealed that BMF is expressed in freshly isolated Sertoli and Leydig cells, as well as in STAPUT-isolated populations of pachytene spermatocytes, round spermatids, and elongated spermatids. The suppression of intratesticular testosterone (T) has been shown to initiate the apoptotic death of pachytene spermatocytes and anoikis (sloughing) of round spermatids. Populations of isolated pachytene spermatocytes, round spermatids, and elongated spermatids from rats implanted with LH-suppressive T-containing Silastic capsules were analyzed for BMF mRNA expression by RT-PCR. Relative to controls, all populations of germ cells from T-treated rats exhibited a dramatic increase in BMF mRNA. This suggests that BMF may play a role in the loss of germ cells that occurs after intratesticular testosterone concentration is reduced. (Supported by HD36209)

KEY WORDS: testis, bmf, apoptosis, anoikis