GAMETE BIOLOGY AND GAMETOGENESIS - B
Tuesday, August 3, 2004
10:30 AM–12:30 PM
(420) SPHINGOSINE-1-PHOSPHATE ATTENUATES CHEMOTHERAPY-INDUCED DEPLETION OF PRIMORDIAL AND PRIMARY FOLLICLES IN VIVO.
Lee, Ho-Joon1, Perez, Gloria1, Tilly, Jonathan1, 1 Vincent Center for Reproductive Biology, Boston, MA, USA
ABSTRACT- A major side-effect of chemotherapy in women is the damage sustained by their ovaries, characterized by massive germ cell loss leading to infertility and premature ovarian failure. Currently, no therapeutic means exists to block such damage. However, we recently reported that ceramide promotes apoptosis in oocytes, and that, the ceramide metabolite, sphingosine-1-phosphate (S1P), prevents oocyte death caused by radiotherapy in vivo. Although S1P also prevents chemotherapy-induced oocyte apoptosis in vitro, it remains to be determined whether it protects oocytes from chemotherapy-induced death in vivo. Here we show that a single intrabursal injection of S1P (200 micromolar) given 3 h before doxorubicin (DXR) treatment partially protects the primordial and primary follicle pool from chemotherapy-induced depletion. Twenty four hours after DXR treatment (5 mg/Kg BW) the pool of primordial follicles was decreased by 75% and the primary follicles by 50%. In contrast, when S1P was administered before DXR treatment, primordial follicles were decreased by 40% and primary follicles by 21%. To examine the mechanim(s) of S1P action in germ cells, we treated isolated oocytes with DXR (200 nM) in the absence or presence of S1P (10 micromolar) for one hour. Some oocytes were then washed with fresh medium and transferred into new medium (without DXR or S1P) for an additional 2 h of incubation. Oocytes were fixed at 30 min and 3 h after the initial treatment. Because DXR is autofluorescent, we were able to follow its trafficking in oocytes by fluorescence microscopy. The remaining oocytes were incubated for 24 h in the presence of DXR±S1P to assess apoptosis. Treatment of oocytes with S1P inhibited both cellular and DNA fragmentation induced by DXR. This protection was associated with an impaired trafficking of DXR to the DNA (its presumed macromolecular target for apoptosis induction) in oocytes cultured with S1P. We conclude that S1P is protective against both chemotherapy- and radiotherapy-induced ovarian damage, and that S1P alters intracellular trafficking of cytotoxic drugs in oocytes. (Supported by R01-AG12279 to JLT and a Harvard Center of Excellence in Women′s Health Grant to GIP).
KEY WORDS: apoptosis, sphingosine-1-phosphate, oocytes, chemotherapy