Construction of Conditional Acid Ceramidase Knockout Mice andin vivoEffects on Oocyte Development and Fertility

Abstract

The number of resting follicles in the ovary and their successful maturation during development define the fertile female lifespan. Oocytes, enclosed within follicles, are subject to natural selection, and the majority will undergo apoptosis during prenatal life through adulthood. Our previous studies revealed high levels of the lipid hydrolase, acid ceramidase (AC), in human and mouse oocytes, follicular fluid and cumulus cells. In addition, supplementation of in vitro fertilization media with recombinant AC enhanced the survival of oocytes and preimplantation embryos. Herein we constructed and used a conditional knockout mouse model of AC deficiency (cACKO) to further investigate the role of this enzyme in oocyte survival in vivo. Immunohistochemical staining, activity assays, and western blot analysis revealed that AC expression was high in the ovaries of normal mice, particularly in the theca cells. After induction of the AC gene knockout with tamoxifen (TM), AC levels decreased in ovaries, and ceramide was correspondingly elevated. A novel immunostaining method was developed to visualize follicles at various stages, and together with light microscopic examination, the transition of the follicle from the secondary to antral stage was found to be defective in the absence of AC. Western blot analysis showed elevated BAX and PARP expression in TM-treated cACKO mouse ovaries compared to control animals. In parallel, the levels of BCL-2 and anti-Mullerian hormone, a marker of ovarian reserve, were decreased. In addition to the above, there was a significant decrease in fertility observed in the TM-treated cACKO mice. Together, these data suggest that AC plays an important role in the preservation of fertility by maintaining low ceramide levels and preventing apoptosis of theca cells, thereby promoting survival of the follicle during the transition from the secondary to antral stage.

The emergence of acid ceramidase as a therapeutic target for acute myeloid leukemia

“…Additionally, development of a fluorogenic AC substrate to measure enzyme activity facilitates the identification and characterization of these inhibitors 64, 65 . Importantly, a conditional AC knockout mouse has been developed that may be useful to further validate AC as a target in particular cancer types 66 . This review will focus on new agents since previous reviews detail several of these compounds including ceranibs, derivatives of N- oleoylethanolamide (NOE), and multiple AC-targeting drugs of the LCL series of B13 analogs 67-71 .…”

Inhibitors of Ceramidases

“…aCDase expression has also been found to be essential factor for the survival of newly formed embryos by controlling the conversion of ceramide to S1P (Li et al, 2002;Eliyahu et al, 2007). Moreover, Eliyahu et al (2012) recently showed that aCDase activity plays a crucial role in the ovarian development and oocyte survival by regulating the cellular levels of ceramide, thereby preventing apoptosis of the theca cells.…”

The molecular medicine of acid ceramidase

“…Observations regarding the early lethality of these mice led to the use of AC to enhance survival of oocytes and embryos, and to improve the outcome of in vitro fertilization (see below). Second, a conditional (floxed) AC mouse was constructed and bred to a tamoxifin (TM) inducible Cre mouse (Eliyahu et al, 2012). After TM induction these mice also survived despite lipid storage in some tissues.…”