There is no neo-oogenesis in the adult mammalian ovary.

In 2004 and 2005, Johnson et al. published two very provocative studies (1, 2), in which they claimed that in the adult mouse ovary, neo-oogenesis takes place and originates either from the ovarian surface epithelium (OSE) (1) or from the bone marrow (BM) via circulating blood cells (2). These studies were provocative since they challenged the long-held view that mammals are born with a finite number of eggs that declines with ageing. Consequently, an intensive discussion has developed among experts in the field, some of whom are proponents of neo-oogenesis, while others are opponents (Table 1). Table 1 Some articles written by proponents and opponents of neo-oogenesis in the adult ovary Neo-oogenesis is a very complex issue that has led to many questions. The aim of our lecture is to explain why we believe that spontaneous neo-oogenesis does not take place in the adult ovary by addressing three of these questions. Is there any evidence for spontaneous neo-oogenesis in adult rodent and human ovaries? Johnson et al. (1) claimed that, in mice, atresia in the immature follicle pool (i.e. including follicles from the primordial to the preantral stage) is so high that complete exhaustion of the pool would be predicted for young adults. Consequently, according to Johnson et al., only the renewal of oocytes as generated by neo-oogenesis can explain the fact that mice are still fertile after the advanced age of 1 year. To estimate the rate of this atresia, Johnson et al. (1) scored as atretic those immature follicles exhibiting a “condensed, involuted or fragmented oocyte”, and consequently counted around 2700 healthy follicles and 200 to 400 atretic immature follicles at postnatal day 30 PN in C57/Bl6 mice. Meanwhile, Bykov et al. (3) counted between 1810 and 3280 healthy and 235–480 atretic follicles, i.e. similar numbers to the data of Johnson et al. (1). However, except for 3 atretic primordial follicles, all immature (primordial, primary and preantral) follicles were healthy, which is in agreement with previous published data showing that atresia of immature follicles is very low in rodents and human ovaries. Among the atretic follicles, antral atretic follicles exhibited a healthy looking oocyte, whereas degenerated and fragmented oocytes were only observed in atretic follicles at a late stage of atresia and which were previously antral follicles. Thus, it emerges that Johnson et al. (1), misattributed as atretic immature follicles those 200 to 400 atretic follicles that were already present at least 8 days earlier, as shown by their BrdU labeling. How can we explain such a misinterpretation? Returning to the criteria used to categorize atresia above, whereas condensation of oocytes (Figure 1) constitutes the normal fate of atretic resting follicles, and oocyte degeneration the normal fate of early growing follicles, fragmented oocytes are only seen in antral follicles at a late stage of atresia (Figure 2). When antral follicles undergo atresia, they progressively lose their antrum and shrink to the size of preantral follicles. This is the likely reason why Johnson et al. (1) mistook these follicles as being “immature”. Figure 1 Some examples of atretic resting follicles exhibiting a condensed oocyte in the mouse ovary (bar=20 μm) Figure 2 Some examples of involuted and fragmented oocytes from atretic antral follicles at a late stage of atresia in the mouse ovary (bar=80 μm) Consequently, it cannot be deduced (1) that between postnatal days 30 and 42, 10% to 33% of the immature pool is atretic. These percentages apply to antral follicles that degenerate instead of becoming preovulatory, with the oocyte being one of the last of the constituent cells to disappear. This issue is crucial since the rate of follicle depletion in the postnatal mouse ovary provided by Johnson et al. (1), which was deduced from the percentages of immature atretic follicles at different ages, was calculated based on a follicular clearance rate of between 3 and 18h rather than the more appropriate estimate for antral follicles of more than 8 days. Consequently, the ovarian reserve would not be completely exhausted by young adulthood and adult female mice would not need neo-oogenesis for maintaining a normal ovarian function. In the rat, meanwhile, Meredith et al. (4) have shown, using BrdU incorporation, that approximately 60% of resting follicles present at a given time are still present 5 months later. Also, in this species, Zhang et al. (5), failed to detect early meiosis-specific proteins at the transcriptional (SCP1, SCP3, SPO11) or translational (SCP1, STRA8) levels in the post natal rat ovary. Together, the long half-life of the resting follicles and the absence of cells in early meiosis argue against the existence of spontaneous neo-oogenesis in the adult rat ovary. If, as required by the neo-oogenesis concept, oogonia exists in the adult ovary as intermediates between stem cells and oocytes, they will enter meiosis at various times and progress through leptotene, pachytene and zygotene stages to reach diplotene (dictyate oocyte) stage, at which meiosis is blocked. If Johnson’s calculation for follicle renewal were correct (1), around 60 oocytes would be in transit through meiosis every day. Figure 3 shows that it is very easy to discriminate between oocytes in the intermediate (pre-dictyate) stages of meiosis and those that are arrested (dictyate). Despite the hundreds of thousands of primordial follicles that have been analysed for quantification and quality assessment purposes, pre-dictyate-stage meiotic oocytes have never been observed in either primates (at least 250 human and adult macaca ovaries examined by A. Gougeon) or rodent ovaries. Also, Liu et al. (6), failed to observe early meiotic oocytes and proliferating germ cells, or to detect mRNA for early meiosis-specific or oogenesis-associated genes (SPO11, PRDM9, SCP1, TERT and NOBOX), in adult human ovaries. In addition, Byskov et al. (7) observed that, in 82 human ovaries (from the embryonic stage to the age of 32 years), almost all oogonia stained exclusively for SSEA4, NANOG, OCT4 and c-kit, whereas only a small fraction stained for oogonia-specific MAGE-A4. These few oogonia disappeared from the ovary before 2 years of age, leaving only dictyate oocytes that stained for c-kit. Figure 3 Leptotene, pachytene, zygotene (black arrows) and diplotene (white arrow) stages of meiosis in the foetal monkey (Macaca fascicularis). Notice the difference between the primordial follicle at the diplotene (dictyate) stage and the intermediate stages ... Consequently, it can be concluded that oogonia, the bona fide female germline stem cells, do not persist to support spontaneous neo-oogenesis in the adult human ovary.