Experimental evidence showing that no mitotically active female germline progenitors exist in postnatal mouse ovaries

Abstract

It has been generally accepted for more than half a century that, in most mammalian species, oocytes cannot renew themselves in postnatal or adult life, and that the number of oocytes is already fixed in fetal or neonatal ovaries. This assumption, however, has been challenged over the past decade. In this study, we have taken an endogenous genetic approach to this question and generated a multiple fluorescent Rosa26(rbw/+);Ddx4-Cre germline reporter mouse model for in vivo and in vitro tracing of the development of female germline cell lineage. Through live cell imaging and de novo folliculogenesis experiments, we show that the Ddx4-expressing cells from postnatal mouse ovaries did not enter mitosis, nor did they contribute to oocytes during de novo folliculogenesis. Our results provide evidence that supports the traditional view that no postnatal follicular renewal occurs in mammals, and no mitotically active Ddx4-expressing female germline progenitors exist in postnatal mouse ovaries.

Fig. 1.

Adult mouse ovaries can support, but do not contribute oocytes to, de novo folliculogenesis by transplanted fetal ovarian cells. (A–C) EGFP-expressing ovarian cells from 12.5-dpc Rosa26^rbw/+ fetuses were collected and dispersed through a 40-μm-pore strainer to avoid cell aggregation. Approximately 2 × 10⁵ cells were injected into each ovary of adult WT (C57BL/6) mice at three different sites. Four weeks after the transplantation, 53 newly formed, EGFP-positive follicles were observed in the ovaries of six recipient females. Representative primary (A), secondary (B), and early antral (C) follicle stages are shown. All EGFP-positive follicles were composed of EGFP-positive oocytes (red arrowheads) and EGFP-positive granulosa cells (red arrows). This shows that both structures have developed from the transplanted fetal ovarian cells. Some EGFP-positive cells were seen in the theca-interstitial layer and stromal area of several EGFP-negative follicles (white arrows), but no chimeric follicles with EGFP-positive granulosa cells and EGFP-negative oocytes, and no EGFP-negative granulosa cells and EGFP-positive oocytes, were found. This suggests that adult mouse ovaries do not contribute oocytes during de novo folliculogenesis initiated by transplanted fetal ovarian cells. (D–F) Two-month-old WT females were sterilized by i.p. injection of busulfan (30 mg/kg bodyweight) and cyclophosphamide (120 mg/kg bodyweight). Two weeks later, EGFP-expressing ovarian cells from 12.5-dpc Rosa26^rbw/+ fetuses were injected into the sterilized ovaries. (D) Four weeks after the transplantation, 32 EGFP-positive follicles were observed in the ovaries of 10 recipient females. All follicles were made up of EGFP-positive oocytes and EGFP-positive granulosa cells of fetal ovarian origin, and no EGFP-negative oocytes were found. This indicates that depletion of endogenous follicles by chemotherapy drugs does not stimulate the adult mouse ovaries to contribute oocytes to de novo folliculogenesis. (E) Hematoxylin staining corresponding to D. (F) Magnified image of the framed area in D. EGFP-positive oocytes (red arrowheads) and EGFP-positive granulosa cells (red arrows) are indicated. O, oocyte.

Fig. 2.

Ddx4-positive cells from postnatal mouse ovaries are mitotically inactive. The gonadal cells from PD 8 or adult Rosa26^rbw/+;Ddx4-Cre male and female mice were dissociated into single cells, filtered through a 40-μm-pore cell strainer to remove large oocytes and cell aggregates, and then cultured on STO feeder cells for 12 h. The cells were then subjected to live cell imaging with an EM-CCD camera for 72 h. Photos were taken from the RFP channel every 2 h. (A) Representative images showing that RFP-expressing (Ddx4-positive) spermatogonial stem cells (SSCs) from postnatal testes (n = 264, 10–15 μm in size) divided one to three times (enclosed in circles and squares). (B) Representative images showing that none of the observed RFP-positive cells (n = 1517, 10–15 μm) from postnatal ovaries proliferated (arrow and arrowhead). Images shown are individual frames from Movies S1 and S2 (more detailed observation throughout the 72-h period is available in Movies S1 and S2).

Fig. 3.

Clonal cells found in long-term ovarian cell cultures are Ddx4-negative and are not germline progenitors. The gonadal cells from PD 8 or adult Rosa26^rbw/+;Ddx4-Cre male and female mice were cultured on STO feeder cells for extended periods of time. (A) Representative colony formed by RFP-expressing (Ddx4-positive) postnatal testicular cells after an 8-d in vitro culture (arrow). (B) Representative colony formed by EGFP-expressing (Ddx4-negative) postnatal ovarian cells in passage 5 (arrowhead). (C) EGFP-positive clonal cells found in B did not express pluripotent stem cell marker Sox2 or germline markers Oct4, Stella, or Ddx4, as determined by semiquantitative RT-PCR. Gapdh was used as a control. Primers used are listed in Table S1. (D) Larger oocyte-like cells were observed among Rosa26^rbw/+;Ddx4-Cre postnatal ovarian cells that were cultured for 3 weeks without being passed. These larger cells are, however, EGFP positive (arrow), indicating that they are Ddx4 negative and thus are not of germline lineage. All experiments were repeated at least three times.

Fig. 4.

Ddx4-negative ovarian clonal cells from Rosa26^rbw/+;Ddx4-Cre females cannot differentiate into oocytes or granulosa cells in vivo. (A) EGFP-expressing (Ddx4-negative) clonal cells from ovaries of adult Rosa26^rbw/+;Ddx4-Cre females (as described in Fig. 3B) were cultured for five passages and then injected into adult WT (C56BL/6) ovaries (as described in Fig. 1). Four weeks later, no fluorescent oocytes or granulosa cells were found, and EGFP-expressing cells were seen only in the theca-interstitial area of several nonfluorescent growing follicles (arrows). (B) In a follicular reconstitution system, the above-mentioned EGFP-expressing (Ddx4-negative) clonal cells were mixed with 14.5 dpc WT fetal ovarian cells, and the cell pellets were transplanted under the kidney capsules of bilaterally ovariectomized WT recipient females. Four weeks later, no fluorescent oocytes or granulosa cells were found in the reconstituted ovarian tissues. EGFP-positive cells were dispersed only in the theca-interstitial area of some nonfluorescent growing follicles (arrows). All experiments were repeated at least three times. O, oocyte.