Conditional deletion of the retinoblastoma (Rb) gene in ovarian granulosa cells leads to premature ovarian failure

Abstract

The retinoblastoma protein (RB) regulates cell proliferation and survival by binding to the E2F family of transcription factors. Recent studies suggest that RB also regulates differentiation in a variety of cell types, including myocytes, neurons, adipocytes, and chondrocytes. Rb mutations have been found in ovarian cancer; however, the role of RB in normal and abnormal ovarian function remains unclear. To test the hypothesis that loss of Rb induces ovarian tumorigenesis, we generated an ovarian granulosa cell conditional knockout of Rb (Rb cKO) using the Cre/lox recombination system. Rb cKO females showed 100% survival and no ovarian tumor formation through 9 months of age, but they developed progressive infertility. Prepubertal Rb cKO females showed increased ovulation rates compared with controls, correlating with increased follicle recruitment, higher Fshr and Kitl mRNA levels, and lower anti-Müllerian hormone levels. In contrast, the ovulation rate of 6-wk-old females was similar to that of controls. Morphometric analysis of Rb cKO ovaries from 6-wk-old and older females showed increased follicular atresia and apoptosis. Rb cKO ovaries and preantral follicles had abnormal levels of known direct and indirect target genes of RB, including Rbl2/p130, E2f1, Ccne2, Myc, Fos, and Tgfb2. In addition, preantral follicles showed increased expression of the granulosa cell differentiation marker Inha, decreased levels of Foxl2 and Cyp19a1 aromatase, and abnormal expression of the nuclear receptors Nr5a1, Nr5a2, and Nr0b1. Taken together, our results suggest that RB is required for the temporal-specific pattern of expression of key genes involved in follicular development.

Figure 1

Generation of the Rb cKO Mice and Fertility Studies A, Breeding scheme used to generate Rb cKO mice. B, Efficiency of recombination of the Rb floxed allele by the Amhr2Cre allele in granulosa cells. PCR analysis of genomic DNA from granulosa cells derived from two Rb^flox/−Amhr2Cre⁻ (Rb ^flox/−) and three Rb^flox/−Amhr2Cre⁺ (Rb cKO) mice. Note that the recombined band (Rec) is present only in Amhr2Cre⁺ granulosa cells. C, Quantitative RT- PCR analysis of exon 19 (floxed) deletion in granulosa cells. D and E, Fertility studies of Rb cKO females. Ten Rb^flox/− and Rb cKO females were bred to stud males for 6 months, and the number of pups per litter and number of litters per month were recorded. D, Average number of pups per litter. Rb cKO females had a significantly lower number of pups per litter. E, Total number of pups. The cumulative number of pups produced by 10 Rb cKO females was also lower than that of controls. **, P < 0.01; a, P < 0.05.

Figure 2

Histological Analysis and Follicle Counts of the Ovaries of Prepubertal Control and Rb cKO Mice A, Ovaries from 3-wk-old WT and Rb^flox/−Amhr2Cre⁺ (Rb cKO) mice. Note the increased amount of growing follicles present in the Rb cKO ovary. B and C, Follicle counts of 2-wk-old (B) and 3-wk-old (C) WT and Rb cKO mice. Follicles were counted in five histological sections derived from four to five ovarian samples and the number of follicles per square millimeter was calculated as described in Materials and Methods. Two-week-old Rb cKO ovaries show a significantly higher number of primary and preantral (multilayer) follicles compared with WT ovaries, whereas 3-wk-old Rb cKO ovaries show a significantly higher number of secondary and preantral follicles compared with WT. Only follicular stages that displayed significant differences are shown. For counts in all follicle stages, please refer to supplemental Fig. 1, A and B. Magnification, ×50. *, P < 0.05.

Figure 3

Histological Analysis and Follicle Counts of the Ovaries of Adult Control and Rb cKO Mice Ovaries from 6-wk-old (A) and 12-wk-old (B) WT and Rb cKO mice. Note the increased amount of oocyte remnants (yellow arrows) present in the Rb cKO ovaries at both 6 wk (C) and 12 wk (D) of age. Follicle counts of 6-wk-old (E) and 12-wk-old (F) Rb cKO mice show a significant increase in the number of atretic follicles and oocyte (zona pellucida) remnants. Only follicular stages that displayed significant differences are shown. For counts in all follicle stages, please refer to supplemental Fig. 2, A and B. Magnification, ×50 (A and B) and ×100 (C and D). *, P < 0.05.

Figure 4

Confocal Microscopic Analysis of Apoptosis in Six-Week-Old WT and Rb cKO Ovaries Representative micrographs showing TUNEL staining from WT (A) and Rb cKO (B) 6-wk-old ovaries. TUNEL-positive cells are shown in green; cell nuclei are shown in red (propidium iodine). C, Total and TUNEL-positive follicles and cell numbers were quantified in 10 HPFs (×40) per section, and three individual sections were quantified per specimen (three specimens). D, The number of follicles showing four or more TUNEL-positive cells was also recorded. The significant increase in the number of TUNEL-positive cells and follicles observed in Rb cKO ovaries is limited to the preantral stage. Results are presented as the average of the percent of positive cells per total cell number per HPF (apoptotic index) ± sem and the average number of TUNEL-positive follicles per section ± sem. Magnification, ×250. *, P < 0.05.

Figure 5

Histological Analysis of Nine-Month-Old Control and Rb cKO Ovaries A and B, Representative micrographs of WT (A) and Rb cKO (B) mice. Magnification, ×50. C and D, High magnification of ovary from left inset (C) and right inset (D) in B. Note the large amount of periodic acid Schiff-positive zona pellucida remnants (oocyte remnants) (yellow arrowheads). Magnification, ×400.

Figure 6

Estrous Cycle Profiles of 8-Week-Old and 12-Week-Old Rb cKO Females and Serum FSH Levels at Estrus in Eight-Week-Old Rb cKO Females Estrous cycle profiles of 8-wk-old (A) and 12-wk-old (C) Rb cKO females. Note that mutant females have significantly longer diestrus even at the earliest time point (n = 5). B, Serum FSH levels at estrus in 8-wk-old females (n = 4). Note the significant decrease in Rb cKO females. Serum levels at random stages of the estrous cycle are presented in Table 2. *, P < 0.05.

Figure 7

Superovulation and Timed-Mating Experiments in Control and Rb cKO Mice Four to five 3- or 6-wk-old WT and Rb^flox/−Amhr2Cre⁺ (Rb cKO) were superovulated, and the number of ovulated oocytes per female was recorded. Results are presented as average number of oocytes ± sem (n = 4 and 5). B, Confocal analysis of meiosis II oocytes. Oocytes retrieved from superovulated 3-wk-old WT and Rb cKO females were collected, fixed, and immunostained with β-tubulin to visualize the meiotic spindle (green). Chromatin was stained with propidium Iodide (red). Arrowheads show misaligned chromosomes or unattached spindle fibers. Magnification, ×800. D, Stained oocytes were analyzed by confocal microscopy, and the percentage of oocytes showing abnormal spindles and/or chromosome alignment was recorded (n = 3). Results are shown as the average percentage of normal and abnormal oocytes ± sem. *, P < 0.05.

Figure 8

FSHR and FSH Target Gene Expression Changes in Prepubertal Ovaries and Preantral Follicles from Control and Rb cKO Females Real-time PCR analysis of mRNA from 2- and 3-wk-old control (WT and Rb^flox/−) and Rb cKO (Rb^flox/−, Amhr2Cre⁺) ovaries and preantral follicles isolated from 12-d-old females. Ovaries and preantral follicles were collected from three to four independent control and Rb cKO females. Average and sem are shown. A significant increase was seen in the relative quantity (RQ) of Fshr (A and B), Ccnd2 (D and E), and Inha (E and F) in Rb cKO ovaries and preantral follicles. The value of one of the control samples was set to equal 1. *, P < 0.05.

Figure 9

KITL and AMH Changes in Prepubertal Ovaries and Preantral Follicles from Control and Rb cKO Females QPCR of mRNA from 2- and 3-wk-old control (WT and Rb^flox/−) and Rb cKO (Rb^flox/−, Amhr2Cre⁺) ovaries and preantral follicles isolated from 12-d-old females. Ovaries and preantral follicles were collected from three to four independent control and Rb cKO females. Average and sem are shown. A significant increase was seen in the relative quantity (RQ) of Kitl in Rb cKO ovaries (A) and preantral follicles (B). Although no changes were observed in 3-wk-old ovaries (C), a significant decrease was seen in the relative quantity (RQ) of Amh was seen in preantral follicles (D). Western blot analysis of preantral follicles confirmed a significant decrease in AMH levels (E). The value of one of the control samples was set to equal 1. *, P < 0.05.

Figure 10

Gene Expression Changes of RB Target Genes in Preantral Follicles from Control and Rb cKO Females Real-time PCR analysis of mRNA from WT and Rb cKO (Rb^flox/–, Amhr2Cre⁺) preantral follicles isolated from 12-d-old females. Preantral follicles were collected from three independent control and Rb cKO females. Average and SEM are shown. A significant increase was seen in the relative quantity (RQ) of E2f1 (A), Ccne2 (B), Rbl2/ p130 (D), Tgfb2 (E) and Myc (F) in Rb cKO preantral follicles. No significant changes were observed in Rbl1/p107 levels (C).Other gene expression changes are shown in Fig. 11 and Table 3. The value of one the control samples was set to equal 1. *, P < 0.05.

Figure 11

Other Gene Expression Changes in Preantral Follicles from Control and Rb cKO Females Real-time PCR analysis of mRNA from WT and Rb cKO (Rb^flox/−, Amhr2Cre⁺) preantral follicles isolated from 12- d-old females. Preantral follicles were collected from three independent control and Rb cKO females. Average and sem are shown. A significant decrease was seen in the relative quantity (RQ) of Foxl2 (A), Cyp19a1 (B), and Nr5a1/Sf1(C), whereas a significant increase was observed in the levels of Nr5a2/Lrh-1 (D), Nr0b1/Dax-1 (E), and Fos (F) in Rb cKO preantral follicles. The value of one of the control samples was set to equal 1. *, P < 0.05.

Figure 12

Summary of the Rb cKO Studies and RB Function in Granulosa Cells A, Summary of gene expression changes in Rb cKO ovaries. RB can regulate transcription in a direct or indirect manner, through its interaction with other transcription factors. Direct targets of RB and E2F1 (left) include Tgfb2, E2f1, Myc and Fos, cyclin E2, Rbl2/p130, and E2f5. E2f1, Rbl2/p130, Myc, and E2f5 are known to cause cell cycle arrest and apoptosis, whereas increases in Fos and Ccne2 (cyclin E2) lead to increased cell proliferation. RB indirect transcriptional effects (right) in the ovary include the regulation of Nr5a1/Sf1, possibly via interaction with SP1, and of Kitl via unidentified mechanisms. Amh expression is also negatively affected, likely due to the decrease in Nr5a1/Sf1. The decrease in Amh and increase in Fos, in turn, appear to impact Fshr expression, which mRNA is also stabilized by TGFβ2. The combined effect of increased Fshr and Kitl expression as well as the decrease in Amh expression, leads to increased follicular recruitment. FSHR target genes Inha and Ccnd2 (cyclin D2) are also up-regulated in Rb cKO ovaries. It is unclear whether the observed decrease in Foxl2 is a direct or indirect effect of Rb deficiency; regardless of this, the decrease is likely to affect the expression of Cyp19a1 (decrease) and Nr0b1/Dax-1 (increase). The increase in Nr0b1 will counteract the increase in Nr5ab2/Lrh-1, which in combination with the decrease in Foxl2 and Nr5a1/Sf1 may help explain the lower levels of Cyp19a1 in mutant follicles. B, Conditional deletion of Rb leads to POF. In WT mice, primordial follicles are recruited into the growing pool. Multilayer preantral follicles are responsive to FSH, but early follicular development is under the control of other factors, which either directly or indirectly promote (e.g. KITL) or inhibit (e.g. AMH) follicle growth. Growing follicles are rescued from atresia by a rise in FSH levels and continue to grow until the preovulatory stage, when a surge of LH induces the ovulation of fertilizable gametes. In the Rb cKO model, an increase in Kitl and decrease in Amh levels increases the number of growing follicles. Other molecular changes may turn the ovarian follicles more susceptible to cell death, which in combination with lower FSH levels at estrus, results in increased follicular atresia, lower number of viable gametes and births, early depletion of the follicle pool, and POF. Increased follicular atresia in this model may arise from increased E2f1 levels, which are known to induce apoptosis, and possibly E2f5 and Rbl2/p130. Based on our results, we propose that RB in granulosa cells is necessary for the correct temporal expression of genes required for follicular development and to prevent apoptosis, whereas the cell cycle functions of RB can be compensated by other RB family members.