Construction of conditional acid ceramidase knockout mice and in vivo effects 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.

Fig. 1

Generation of the cACKO mouse model. (A) Schematic depiction of the mouse AC gene modification, showing the Floxed allele before and after TM-induced Cre recombinase-mediated exision. The location of the primers used to detect the Floxed allele (P1/P2 and P3/P4), as well as the primers used to detect the gene rearrangement (P1/P4) also are shown. (B) Representative genotype analysis of a Floxed and cACKO mouse after TM treatment. The 250 bp band derived from within intron 13 (see Fig. 1A) is diagnostic of the Floxed allele, while the 310 bp band is derived from the gene rearrangement. Note that in the TM-treated Floxed mouse, no gene rearrangement occurred in the tail (due to the lack of Cre recombinase expression), while in the tail of the TM-treated cACKO mouse there was 100% conversion. In contrast, in the ovary of this same TM-treated cACKO mouse the gene rearrangement was partial.

Fig. 2

Decreased AC expression in the ovaries of TM-treated cACKO mice. (A) qPCR was used to quantify the levels of the AC mRNA in the ovaries of wild-type (WT), heterozygous complete AC knockout (Het), Floxed mice treated with TM (Floxed+TM), and cACKO mice treated with TM (cACKO+TM). The AC mRNA levels were normalized to SRP18 rRNA as an internal control. Bar heights represent the mean values (fold difference comparing AC to SRP18 expression) from 3 independent experiments. The results showed that the AC mRNA levels were significantly decreased compared to any of the three control groups (t test, P=4.02E-6 for WT mice***, 2E-2 for Het mice**, and 1.7E-4 for Floxed+TM mice*). (B) Western blot analysis showing the levels of AC precursor protein relative to beta actin. Only the cACKO mice treated with TM exhibited a reduction in AC protein levels. Blot is representative of three independent experiments. (C) Significantly decreased AC activity was observed in the ovaries of TM-treated ACKO mice, but not TM-treated Floxed mice (*=t test, P=3E-4 TM-treated cACKO compared to compared to WT mice***, 1E-3 compared to Het mice**, and 6E-3 compared to Floxed mice*).

Fig. 3

Decreased fertility in TM-treated cACKO can be explained by decreased ovarian reserve and apoptosis in the ovary. (A) Western blot analysis revealed a decreased in AMH levels, a marker of ovarian reserve, and its transcription factor Sox-9 in the ovaries of TM-treated cACKO, but not TM-treated Floxed mice. Beta actin was used as a loading control. (B) An increase in the apoptosis markers, PARP and Bax, and a decrease in the anti-apoptosis marker, Bcl-2, also was observed in the ovaries of TM-treated cACKO, but not Floxed mice. Blots are representative of three independent experiments.

Fig. 4

Depiction of follicles in the mouse ovary. (A) A representative H & E stained ovary section from a 3-month-old wild-type female mouse. Scale bar = 50 microns. (B) A higher magnification depicting a representative follicle at each stage. A schematic presentation of the different stage follicles also is provided below (adapted from http://amh-tes,com/).

Fig. 5

Number of follicles at different stages of maturation in TM-treated Floxed and cACKO mice. (A,B) Sample images showing immunostaining using anti-PARD6a and anti-AMH antibodies. PARD6a antibodies assist in the visualization of early stage (primary, early secondary) follicles, while AMH antibodies label secondary and early antral stages. Some labeling of primary follicles also was observed. Localization of the primary antibodies was visualized using a fluorescent second antibody (Cy-3/2) and laser scanning confocal microscopy. Scale bar = 50 microns. (C) Combined number of follicles per ovary in TM-treated cACKO and Floxed mice, assessed as described in the Materials and Methods by a combination of light microscopy and PARD6a and AMH immunostaining. Sterology was performed using the Stero Investigator software. A significant (*t-test, P=0.0002) difference was observed among the antral follicles. NS= non-significant.

Fig. 6

Reduced AC and elevated ceramide in the ovaries of TM-treated cACKO compared to Floxed mice. (A) High AC expression was observed by immunostaining in the ovaries of TM-treated Floxed mice. Arrowheads indicate the theca cells, where AC expression was most prominent. In contrast, AC expression levels were markedly reduced in the ovaries of TM-treated cACKO mice. AC was visualized using a fluorescent second antibody Cy-3 and laser-scanning confocal microscopy. Scale bars indicate 50 microns. Images are representative of more than three independent immunostaining experiments. (B,C) Confocal analysis of ceramide in TM-treated Floxed and cACKO mice. Ceramide was highly elevated in the primary and secondary follicles of TM-treated cACKO (C) compared to Floxed animals (B). Scale bars = 10 microns. Images are representative of three independent immuno-staining experiments. Representative follicles of each stage are shown. Light microscopic images are provided below the immunostained images.