Caspase activity inhibition delays programmed spermatogenic cell death in vitro.
Programmed cell death or apoptosis was analyzed in rat Sertoli-spermatogonial cell cocultures prepared from 2-9 day old rats using time-lapse video microscopy, a cell viability fluorescence microscopy assay, immunocytochemical markers, and cell-permeable caspase inhibitory peptides with reversible and irreversible effects. We show that apoptosis can initially affect a single member of a spermatogonial cell cohort and that single non-viable spermatogonial cells can remain conjoined to viable spermatogonial cells. The integrity of the cytoskeletal F-actin network and the presence on Bcl-2 immunoreactivity are valuable markers of spermatogonial cell viability. Apoptotic bodies released into the culture medium are generally eliminated after culture medium replenishment; however, spermatogonial apoptotic cell remnants can be taken up by Sertoli cells, which are known to represent a phagocytic somatic population within the seminiferous epithelium. Cell permeable caspase-1 and caspase-4 inhibitory peptides with reversible and irreversible action were supplemented to a serum-free hormone-growth factor-supplemented medium. In the absence of the caspase inhibitory peptide, the viability of spermatogonial cells decreases gradually with time in coculture. However, the addition of caspase inhibitory peptides causes a significant accumulation of spermatogenic cells per unit surface area. Although inhibition of caspases, the executors of spermatogonial cell death, results in a substantial increase of spermatogonial cells in the cocultures, it remains to be determined what the differentiation potential of caspase-inhibited spermatogonial cell cohorts is.
Tres
Laura L
LL
Department of Cell Biology and Anatomical Sciences, The Sophie Davis School of Biomedical Education/The City University of New York Medical School, New York, NY 10031, USA. tres@med.cuny.edu
Rosselot
Carolina
C
Kierszenbaum
Abraham L
AL
D000199:Actins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D061945:Caspase Inhibitors; D002470:Cell Survival; D002478:Cells, Cultured; D018920:Coculture Techniques; D016895:Culture Media, Serum-Free; D003599:Cytoskeleton / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D013006:Growth Hormone / Q000378:metabolism; D007150:Immunohistochemistry; D008297:Male; D018715:Microscopy, Video; D010587:Phagocytosis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D012708:Sertoli Cells / Q000378:metabolism* / Q000502:physiology; D013094:Spermatozoa / Q000378:metabolism* / Q000502:physiology*; D013997:Time Factors
D000199:Actins; D015415:Biomarkers; D061945:Caspase Inhibitors; D016895:Culture Media, Serum-Free; D019253:Proto-Oncogene Proteins c-bcl-2; D013006:Growth Hormone