Two classes of ovarian primordial follicles exhibit distinct developmental dynamics and physiological functions

Abstract

In the mammalian ovary, progressive activation of primordial follicles serves as the source of fertilizable ova, and disorders in the development of primordial follicles lead to various ovarian diseases. However, very little is known about the developmental dynamics of primordial follicles under physiological conditions, and the fates of distinct populations of primordial follicles also remain unclear. In this study, by generating the Foxl2-CreER(T2) and Sohlh1-CreER(T2) inducible mouse models, we have specifically labeled and traced the in vivo development of two classes of primordial follicles, the first wave of simultaneously activated follicles after birth and the primordial follicles that are gradually activated in adulthood. Our results show that the first wave of follicles exists in the ovaries for ∼3 months and contributes to the onset of puberty and to early fertility. The primordial follicles at the ovarian cortex gradually replace the first wave of follicles and dominate the ovary after 3 months of age, providing fertility until the end of reproductive life. Moreover, by tracing the time periods needed for primordial follicles to reach various advanced stages in vivo, we were able to determine the exact developmental dynamics of the two classes of primordial follicles. We have now revealed the lifelong developmental dynamics of ovarian primordial follicles under physiological conditions and have clearly shown that two classes of primordial follicles follow distinct, age-dependent developmental paths and play different roles in the mammalian reproductive lifespan.

Figure 1.

Labeling the first wave of activated follicles in Foxl2-CreER^T2;mT/mG mouse ovaries. (A) Illustration of the tamoxifen-induced labeling of pregranulosa and granulosa cells of the first wave of activated follicles in postnatal Foxl2-CreER^T2;mT/mG mice. In Foxl2-expressing pregranulosa and granulosa cells, the CreER^T2 recombinase is not active and the cells express membrane-targeted Tomato (mT), a red fluorescent protein. Upon tamoxifen induction, the activated CreER^T2 recombinase mediates the deletion of the mT region and switches on the expression of membrane-targeted green fluorescent protein (mG) resulting in follicles that are labeled with green pregranulosa and granulosa cells. (B and C) Efficient labeling of the first wave of activated follicles by administration of tamoxifen to pregnant females. Pregnant females were given a single intraperitoneal injection of tamoxifen at E16.5, and the ovaries of their pups at PD3 (B) and PD13 (C) were analyzed. Note that the first wave of activated follicles in the ovarian medulla was efficiently labeled (arrows) whereas primordial follicles in the ovarian cortex were not labeled (arrowheads). Although not all pregranulosa and granulosa cells were labeled by the low dosage of tamoxifen used, a follicle was considered successfully labeled as long as one somatic cell turned green. (D) Labeling of all follicles by tamoxifen injection at PD5. The female mice were given a single intraperitoneal injection of tamoxifen at PD5 and were analyzed at PD13. This protocol of tamoxifen administration led to the labeling of all follicles, including the first wave of follicles (arrows) and the cortical primordial follicles (arrowheads).

Figure 2.

Tracing the development of the first wave of activated follicles in Foxl2-CreER^T2;mT/mG mouse ovaries. Tamoxifen was administered to pregnant females at E16.5. The Foxl2-CreER^T2;mT/mG pups born to these females were sacrificed at different ages to analyze their ovaries. (A and B) Most of the growing follicles in ovaries collected from PD23 (A) and PD35 (B) mice show labeled green granulosa cells (arrows), indicating that they have developed from the first wave of activated follicles. The presence of labeled antral follicles (AF) indicates that follicles from the first wave contribute to the onset of puberty and to fertility during young adulthood. A few unlabeled red growing follicles (arrowheads) can also be seen, indicating that they developed from the cortical primordial follicles. (C) At PD45, both labeled green (arrows) and unlabeled red (arrowheads) growing follicles are observed, and some unlabeled follicles have reached the antral stage (AF). (D) At PD90, the majority of the growing follicles, including antral follicles (AF), are unlabeled (arrowheads). The number of labeled follicles (arrow) has decreased, and most of these are at advanced stages or have differentiated into corpora lutea (CL). (E) At PD105, only green debris can be observed in the ovary (arrow). (F) At 4 months, no green fluorescent cells can be observed. (G) Quantification of labeled and unlabeled growing follicles at different ages. At PD105, the first wave of activated follicles becomes depleted, and the follicles grown from cortical primordial follicles become the only source of the growing follicle pool. (H) Quantification of labeled and unlabeled antral follicles and CL as a marker for their contributions to fertility. At PD105, no antral follicles or CL are from the first wave of follicles. In G and H, n = 4 for PD13, n = 4 for PD23, n = 5 for PD35, n = 5 for PD45, n = 5 for PD60, n = 5 for PD90, n = 4 for PD105 and n = 4 for 4 months. Values are means ± SD.

Figure 3.

Labeling of primordial follicles in adult mouse ovaries using the Sohlh1-CreER^T2;R26R mouse model. (A) Illustration of the tamoxifen-induced labeling of adult primordial follicles in the Sohlh1-CreER^T2;R26R mice. In the oocytes of primordial follicles, the CreER^T2 mediated by the Sohlh1 promoter is not active and no β-galactosidase is expressed before tamoxifen is given. Upon tamoxifen administration, the CreER^T2 recombinase becomes activated and mediates the removal of the STOP sequence that is in front of the lacZ (the gene encoding β-galactosidase) cDNA. This results in the expression of lacZ in the oocytes and the production of blue-colored β-galactosidase staining. (B–D) Labeling of only primordial follicles in adult mouse ovaries. Three-month-old Sohlh1-CreER^T2;R26R females were given intraperitoneal injections of tamoxifen for 3 consecutive days. (B) The labeled primordial follicles (shown by the blue dots in the oocytes) can be seen 5 days after the injection. Arrows indicate the unlabeled growing follicles. (C) Labeled primordial follicles are still seen in ovaries 7 weeks after tamoxifen injection. (D) Labeled primordial follicles are still seen in the ovaries 14 months after tamoxifen injection.

Figure 4.

Tracing the development of primordial follicles in adult Sohlh1-CreER^T2;R26R mice. Sohlh1-CreER^T2;R26R adult females were injected with tamoxifen and sacrificed at different time points. The Y-axis is the percentage of mice showing labeled follicles. The numbers of mice used for each time point are shown above the bars. Representative labeled follicles are shown in the right panels. (A) A minimum of 7 days is needed for labeled primordial follicles to reach the primary stage. Labeled primary follicles can be found in all mice sacrificed from 9 to 57 days after tamoxifen injection. (B) A minimum of 23 days is needed for labeled primordial follicles to develop into secondary follicles. (C) A minimum of 37 days is needed for labeled primordial follicles to develop into the early antral stage. (D) A minimum of 47 days is needed for labeled primordial follicles to develop into the antral stage.

Figure 5.

Labeled primordial follicles persisted to the end of the reproductive lifespan in Sohlh1-CreER^T2;R26R mouse ovaries. Sohlh1-CreER^T2;R26R adult females were given tamoxifen and sacrificed 10 months later. (A) A representative labeled primary follicle with a labeled primordial follicle in the same field. (B) A representative labeled secondary follicle with a labeled primordial follicle in the same field. (C) A representative labeled early antral follicle. (D) A representative labeled antral follicle.