PROLIFERATION AND APOPTOSIS IN LONG TERM DIABETIC MOUSE OVARY: AN ANALYSIS OF INTRINSIC AND EXTRINSIC PATHWAY OF APOPTOSIS

BARRIOS MARCELA; FRAUNHOFFER NICOLAS.; MEILERMAN ABUELAFIA ANALIA.; DRAGO HUGO; ESPOSITO GIULIANA; VITULLO ALFREDO.

New York

Encuentro; 2012 Germ Cells Meeting; 2012

Cold Spring Harbor

Type I diabetes mellitus (T1D) accounts for about 10 ? 15% of all cases of diabetes. It is a multifactorial autoinmune disease for which susceptibility is determined by genetic, environmental and immunological factors. Recently, emerging evidence has shown that oocytes from diabetic mice experience delayed maturation, abnormal cellular metabolism, mitochondrial dysfunction and meiotic defects. These changes will be related with alteration in the follicular structure and an increase of apoptosis frequency in diabetic mice. The main objective of the present study was to analyze the balance between the apoptosis pathways during the follicular loss in diabetic mice. 25 BALB/c female mice (age 30 days; 15 diabetic and 10 controls) were used in this assay. To generate a T1D model, female mice received five injections of streptozotocin at a dose of 60 mg/kg, during 5 consecutive days. Animals were selected as diabetic when glucose level was > 200 mg/dl. Weight and food intake was recorded weekly both in diabetic and control animals. Three diabetic and 2 control animals were sacrificed at day 15, 20, 40, 70 and 80 post-treatment. The ovaries were removed and weighed; one of them was fixed in 4% PFA for immunohistochemistry and immunofluorescence and the other one was frozen at -80°C for Western Blot analysis. The proteins studied were: Proliferating Cell Nuclear Antigen (PCNA), BCL-2 family proteins ? Bax, Bcl-2 and Bid cleaved, Fas/Fas-L system and Caspase family - cleaved caspase 8 and active caspase 3. Bax and Bcl-2 expression was constant at all time-points, with no differences between control and diabetic mice. Extrinsic pathway markers (Fas, Fas-L, cleaved Bid and cleaved caspase 8) showed strong cytoplasmatic immunostaining in oocyte and granulosa cells of diabetic mice with higher levels at day 20, decreasing its expression between the day 40 and 80. Active caspase 3 was positive in granulosa cells of atretic antral follicles with constant levels of expression in all time-points. PCNA was constantly detected in all samples. This study shows that during the earliest stages of streptozotocin - induced T1D, a switch from intrinsic to extrinsic apoptosis pathway takes place in the mouse ovary. At more advanced times, follicular moves again to intrinsic death mechanisms. Clearly, apoptosis activity, both intrinsic and extrinsic, is enhanced in the ovary of diabetic-induced mice affecting follicular survival and compromising fertility.