Intact fetal ovarian cord formation promotes mouse oocyte survival and development

Abstract

Background: Female reproductive potential, or the ability to propagate life, is limited in mammals with the majority of oocytes lost before birth. In mice, surviving perinatal oocytes are enclosed in ovarian follicles for subsequent oocyte development and function in the adult. Before birth, fetal germ cells of both sexes develop in clusters, or germline cysts, in the undifferentiated gonad. Upon sex determination of the fetal gonad, germ cell cysts become organized into testicular or ovarian cord-like structures and begin to interact with gonadal somatic cells. Although germline cysts and testicular cords are required for spermatogenesis, the role of cyst and ovarian cord formation in mammalian oocyte development and female fertility has not been determined.

Results: Here, we examine whether intact fetal ovarian germ and somatic cell cord structures are required for oocyte development using mouse gonad re-aggregation and transplantation to disrupt gonadal organization. We observed that germ cells from disrupted female gonad prior to embryonic day e13.5 completed prophase I of meiosis but did not survive following transplantation. Furthermore, re-aggregated ovaries from e13.5 to e15.5 developed with a reduced number of oocytes. Oocyte loss occurred before follicle formation and was associated with an absence of ovarian cord structure and ovary disorganization. However, disrupted ovaries from e16.5 or later were resistant to the re-aggregation impairment and supported robust oocyte survival and development in follicles.

Conclusions: Thus, we demonstrate a critical window of oocyte development from e13.5 to e16.5 in the intact fetal mouse ovary, corresponding to the establishment of ovarian cord structure, which promotes oocyte interaction with neighboring ovarian somatic granulosa cells before birth and imparts oocytes with competence to survive and develop in follicles. Because germline cyst and ovarian cord structures are conserved in the human fetal ovary, the identification of genetic components and molecular mechanisms of pre-follicle stage germ and somatic cell structures may be important for understanding human female infertility. In addition, this work provides a foundation for development of a robust fetal ovarian niche and transplantation based system to direct stem cell-derived oocyte differentiation as a potential therapeutic strategy for the treatment of infertility.

Figure 1

Female gonadal re-aggregation prior to ovary maturation impaired oocyte development. (A) Female mouse genital ridges and ovaries from e11.5 (embryonic day) to P2 (post-natal day) were disrupted by re-aggregation, transplanted under the kidney capsule for three weeks, and sections were examined by H&E stain for oocyte development in follicles in comparison to intact (B) gonadal transplant controls. (C) For comparison, in vivo control endogenous time points are shown to illustrate e12.5 genital ridge, pre-follicle formation e14.5 differentiating ovary, and post-follicle formation adult ovary. (D) P2 ovaries were also FACS sorted prior to re-aggregation to ensure a single cell suspension and an equivalent dissociation method. 100× magnification.

Figure 2

Quantification of re-aggregation induced impairment of oocyte development. (A) The average number of oocytes in follicles per graft, normalized to the number of sections counted, was determined following three weeks transplantation of intact or re-aggregated samples. No oocytes were detected in re-aggregated samples prior to e13.5. Oocyte numbers increased in re-aggregated samples after e13.5 in parallel to the increasing age of ovary donors, however, oocyte numbers were still significantly reduced from e13.5 to e15.5 compared to intact control samples. (B) Average weight per graft following transplantation of re-aggregated early and late gonadal stages demonstrating equivalent weights. Error bars represent s.d. (n = 3, 150 sections/graft). * = p < 0.05 between e16.5 and e15.5 re-aggregated. **/*** = p < 0.05 between e12.5/e14.5 intact and re-aggregated, respectively.

Figure 3

Genital ridge re-aggregation did not affect oocyte meiotic entry or progression but disrupted oocyte survival and development in follicles. (A) Intact or re-aggregated (re-agg) e12.5 female genital ridges were transplanted for three days, and germ cells were subsequently analyzed for entry and progression through stages of meiosis prophase I by SYCP3 and SYCP1 immunostaining of chromosome synapsis. Blue is DAPI. 630× magnification. (B) Intact or re-aggregated e12.5 samples on day three of transplantation were quantified for the percentage of cells in meiotic prophase I as determined by SYCP chromosomal alignment localization by immunofluorescence. Error bars represent s.d. (n = 3, 100 cells/graft). No statistically significant difference was detected. (C) By 12 days of transplantation, many primordial and primary follicles were observed from the intact e12.5 genital ridge but none were detected from the re-aggregated genital ridge by H&E stain. 200× magnification.

Figure 4

Re-aggregation obstructed intact ovarian cord structure and induced Caspase-independent oocyte loss before follicle formation. (A) The numbers of surviving oocytes per graft were determined by TRA98 immunofluorescence following five, seven, and 12 days of intact or re-aggregated (re-agg) e12.5 female genital ridge transplantation revealing oocyte loss by d7 before follicle formation on d12. Error bars represent s.d. (n = 3) */** = p < 0.05 between d7/d12 re-aggregated and intact, respectively. (B) Re-aggregation induced oocyte loss was Caspase-independent. The percentage of TRA98+ oocytes (100 cells/graft) undergoing apoptosis was assayed by TUNEL and active Caspase2 or Caspase3 co-immunofluorescence following five to seven days of transplantation. Intact = solid bars. Re-aggregated = striped bars. TUNEL+ (red) apoptotic oocytes co-stained for TRA98 (green) were detected in re-aggregated samples following 5 (B') and 7 (B'') days of transplantation (C) Ovarian cord structure was examined by TRA98 germ cell and FOXL2 granulosa somatic cell immunofluorescence over 12 days of e12.5 female genital ridge transplantation. Oocytes and granulosa cells clustered together in defined ovarian cord-like structures (dashed lines) following five to seven days of intact sample transplantation, and ovarian follicles were detected by day twelve. Re-aggregated samples did not form ovarian cords or follicles during the time course, and TRA98+ oocytes were not detected after day seven in re-aggregated transplants. Active Caspase3 was not detected in oocytes. (C' and C'') Clusters containing FOXL2+ cells were observed following re-aggregation, however, FOXL2+ cells were only detected at the perimeter of these clusters, and neither FOXL2+ nor TRA98+ cells were ever observed at the interior in contrast to intact ovarian follicles. C' and C'' panels: DAPI - blue, FOXL2 - red, and TRA98 - green. 100× magnification.

Figure 5

Re-aggregation impaired intact ovarian cord formation. Low magnification (40×) analysis of ovary structure by TRA98 germ cell and FOXL2 granulosa somatic cell immunofluorescence after five days of e12.5 female genital ridge transplantation revealed the striking difference in ovary organization between intact and re-aggregated samples. Intact sample oocytes and granulosa cells clustered together in defined ovarian cord-like structures (dashed lines), in contrast to re-aggregated samples which did not form ovarian cords containing oocyte clusters. Active Caspase3 was not detected in oocytes.

Figure 6

Oocyte loss was associated with ovary disorganization following re-aggregation. (A, B) Immunofluorescence revealed TRA98+ oocytes and FOXL2+ granulosa cells in ovarian cord-like structures (dashed lines) primarily sequestered from PECAM+ endothelial cells (A) and Laminin+ basment membrane (B) following five days of intact e12.5 female genital ridge transplantation. Along with disrupted ovarian cord formation, re-aggregation also resulted in a uniform distribution of PECAM+ and Laminin+ cells amongst dispersed oocytes and granulosa cells that correlated with subsequent oocyte loss. 100× magnification.

Figure 7

Disruption of intact ovarian structure by re-aggregation resulted in oocyte loss due to an oocyte-specific deficiency. To distinguish between oocyte-specific or somatic cell-specific mechanisms of re-aggregation-induced oocyte loss, germ cells from e12.5 female genital ridges of Oct4-GFP transgenic mice were purified from somatic cells by FACS, and GFP+ and alkaline phosphatase (AP+) germ cells were cultured in vitro for three days to induce meiosis (SYCP3+) and to synchronize germ cells with later ovarian stages. Subsequently, cultured GFP+ germ cells were co-aggregated with permissive e16.5 or P2 stage ovarian tissue and transplanted for three weeks. In contrast to Oct4-GFP transgenic ovary controls with robust oocyte-specific GFP expression, e12.5-derived GFP+ oocytes were not detected after transplantation and indicated a re-aggregation induced oocyte-specific impairment following pre-ovarian genital ridge disruption.

Figure 8

Model of intact ovarian cord formation and maturation promoting oocyte survival and development in follicles. Prior to ovary development on e13.5, germ cells located in cysts in the e12.5 female genital ridge are not yet competent to survive and form follicles when re-aggregated (re-agg). However, ovary maturation on e13.5 and development of intact ovarian cord structures containing oocyte and somatic granulosa cell clusters are sufficient to permit some oocyte survival and follicle formation upon re-aggregation. By e16.5, intact ovarian cord-enclosed meiotic oocyte clusters and granulosa somatic cells are now primed to undergo robust follicle formation and oocyte development in follicles when re-aggregated. The percentage of surviving re-aggregated oocytes was calculated in comparison to intact transplant controls. Oocyte (green) and granulosa cell (blue) contacts and paracrine signaling factors may promote oocyte survival (+) and possibly facilitate programmed ovarian germ cell cyst break down into follicles. The cord and/or oocyte-mediated recruitment of granulosa cell clusters during ovary differentiation may also provide somatic cell signaling factors such as WNT4, R-spondin1, and Follistatin that promote oocyte survival and maturation. In addition, intact ovarian cords may facilitate the protection of oocytes from re-aggregation induced inhibitory signaling from somatic cells in the genital ridge. Thus, we define a critical window from e13.5 to e16.5 of oocyte enclosure in intact fetal ovarian cord structures for the intrinsic programming of oocytes with competence to survive and undergo further development.