Submission Number: MIC-4-1-19

Abstract Number: 72

SPHINGOLIPID SIGNAL TRANSDUCTION IN CORPUS LUTEUM APOPTOSIS: SIMILARITIES BETWEEN RAT AND RABBIT.

Michael Abdo* ¹, MA Bogoyevitch ², R Sabbadini ³ and AM Dharmarajan* ¹

Department of Anatomy & Human Biology, University of Western Australia, Perth, Western Australia ¹
Department of Biochemistry, University of Western Australia, Perth, Western Australia ²
Department of Biology, San Diego State University, San Diego, California, USA ³

Abstract:
The apoptotic signal is initiated and mediated through a variety of factors. In the present study, we report the possible involvement of the sphingolipid second messengers, ceramide and sphingosine, in mediating corpus luteum (CL) apoptosis. Furthermore we report the possible involvement of the mitogen-activated protein kinases (MAPK) and the effect of caspase inhibition on this signal transduction pathway. Rat CL (Wistar) were non-enzymatically dissected on Day 16 of pregnancy and cultured in MEM, supplemented with 30% FBS for 6h with and without C2-ceramide (50M), dihydroceramide-an inactive form of ceramide (50M) and sodium aurothiomalate-a general caspase inhibitor (SAM, 10mM). Rabbit CL (New Zealand White) were collected on Day 11 of pseudopregnancy following perfusion for 4h with C2-ceramide (50M), dihydroceramide (50M), sphingosine (50M), and SAM (10mM). DNA was isolated, and DNA integrity assessed by 3-end labelling [Dharmarajan et al (1994) Endocrine Journal 2:295-303]. Sphingosine levels in rabbit CL were assessed on Day 11 and Day 18 of pseudopregnancy, and following hCG treatment, by HPLC analysis [Sabbadini et al (1993) Biochem Biophys Res Comm 193(2):752-758]. The expression of MAPK was assessed by SDS-PAGE using antibodies specific to both total and phosphorylated c-JUN NH₂-terminal protein kinase (JNK), extracellular signal-related kinase (ERK 1 & 2), and p38 MAPK. Rat CL treated with C2-ceramide showed a significant increase (P<0.05) in DNA fragmentation after only 2h incubation. Rabbit CL demonstrated a significant increase in DNA fragmentation (fold changes) when treated with C2-ceramide (2.10.1) and sphingosine (3.70.4). In both animal models cell death was inhibited by treatment with SAM. Sphingosine levels increased with increasing levels of DNA fragmentation, but decreased following hCG treatment. Furthermore, Western blot analyses revealed the presence of activated p38 MAPK following C2-ceramide treatment. These results suggest, for the first time, a potential role for the sphingolipid signal transduction pathway in CL apoptosis within both the rat and rabbit. Moreover, the data suggest an interaction between the sphingolipid and caspase signal transduction pathways. Supported by NHMRC, UWA(UPA), ARC Small & Raine Foundation.

Keywords: Corpus Luteum, Apoptosis, Sphingolipid

This abstract is being presented at: 8:00 AM in session:
Apoptosis