Tissue culture supports physiological development of fetal mouse ovaries
A culture technique to support development of fetal mouse ovaries from E13.5 was developed, to allow effects of etoposide to be examined both before and after follicle formation in a consistent manner, in vitro, with neonatal mouse ovary culture already an established method. Over the 12 days of culture, germ cells progressed through prophase I of meiosis, followed by formation of PFs, with some follicles subsequently initiating follicle growth to the transitional and primary follicle stage (Fig. 2Ai,ii). Cultured follicles were morphologically healthy, similar to that observed in uncultured postnatal day 4 (P4) in vivo ovaries (Fig. 2Aiii). Although there was a significant reduction in follicle numbers within cultured ovaries compared to in vivo P4 ovaries (Fig. 2Bi; p < 0.01, n = 5, from 3 independent cultures), health and follicle developmental stage were both comparable to that found in vivo (Fig. 2Bii,iii). Progression of cultured oocytes through prophase I of meiosis to diplotene was analysed through visualisation of Sycp3. Sycp3 is a component of the axial/lateral element of the synaptonemal complex (SC) that assembles during meiotic prophase I [35, 36]. In the female mouse embryo, germ cells enter meiosis at E13.5 and progress through prophase I to diplotene over the next 6–7 days [37]. Images here show that meiosis progressed in vitro as in vivo: oocytes progressed through leptotene/zygotene, pachytene, and diplotene, as assessed by SC assembly and disassembly (Fig. 3Ai-iii). In vitro oocytes were observed in all stages of prophase I, with 57.1 % still leptotene/zygotene and 42.3 % having reached pachytene by Day 2 of culture. By Day 4 of culture, the vast majority (99.2 %) had reached pachytene, and by Day 6 of culture, the majority (82.5 %) of oocytes had progressed through to the diplotene stage of prophase I (Fig. 3B, upper panel): by Day 6 of culture, no leptene/zygotene oocytes remained, as would be expected at the equivalent E19.5 point in vivo [37]. Together, these results validate the use of this fetal ovary culture system for investigations into meiotic progression and follicle formation.
Germ cells are more vulnerable to etoposide exposure prior to follicle formation
We first tested whether etoposide might be impairing the ability of fetal oocytes to progress through meiotic prophase. Fetal mouse ovaries were immunostained for Sycp3 after culture in control medium, or in the presence of etoposide (150 ng ml−1) for 2, 4 or 6 days to assess progression through early prophase I. There was a difference between etoposide-treated and control oocytes at Day 2 of culture (p < 0.01), but the effect was no longer observed by Days 4 or 6 of culture (Fig. 3B; p = 0.4 at Days 4 and 6). The effect at Day 2 could be due to an initial ‘acceleration’ in meiosis in germ cells exposed to etoposide, or because the germ cells are more sensitive to etoposide at mitotic and/or pre-leptotene stages: increased sensitivity is perhaps more likely given the reduction in follicle number after etoposide exposure (see results below). Overall, results show that female germ cells are able to progress through meiosis to the diplotene stage of prophase I in the presence of etoposide.
E13.5 fetal mouse ovaries were then cultured for twelve days either in control medium throughout (Days 0–12), or exposed to a range of etoposide doses (50, 100 or 150 ng ml−1) for the first six days of culture (Days 0–6), followed by a further six days in control medium (Days 6–12): Day 7 of culture, when follicles have begun to form in the cultured fetal mouse ovaries, was considered as equivalent to the day of birth in vivo. Etoposide exposure occurred, therefore, prior to follicle formation, spanning entry into meiotic prophase and progression through to diplotene stage.
Over the twelve days of culture, oocytes entered meiotic prophase and formed follicles, with some follicles then initiating growth to the primary stage. At the end of culture, histological sections of cultured ovaries were examined (Fig 4Ai-iii), follicles counted and assessed for health. Etoposide had a markedly detrimental effect on total follicle numbers, with a dose-dependent loss of follicles of 72.5 % and 89.7 % at the medium and high doses respectively (Fig. 4Bi, p < 0.01 at 100 ng ml −1, p < 0.001 at 150 ng ml−1; n = 6, 2 independent cultures). The observed follicle reduction was due to a loss in PF and transitional follicles within these ovaries (Fig. 5Ai, p < 0.01; n = 6). The loss was particularly marked for PFs, which constitute over 75 % of the follicles in control ovaries, with numbers reduced to 3.7 % of controls after exposure to 150 ng ml−1, compared to 25 % of transitional follicles remaining. The percentage of follicles assessed as unhealthy also increased in a dose dependent manner with increasing etoposide dose, reaching significance at the highest dose of etoposide (Fig. 4Ci, p < 0.05; n = 6). Again, when each follicle stage was examined, this was seen to be due to a significant increase in the percentage of PF and transitional follicles assessed as unhealthy (Fig. 5Bi, p < 0.05; n = 6). Ovaries were then examined histologically at day 6 of culture, considered as equivalent to the last day of gestation, a time point at which germ cells are beginning to form follicles (Fig. 6Ai,ii). Ovaries cultured in the presence of the highest dose of etoposide (150 ng ml−1) had markedly and significantly fewer germ cells than controls, indicating that the reduced number of follicles observed at the end of culture (Figs. 4Bi and 5Ai) was due to germ cell loss prior to follicle formation (Fig. 6B, p < 0.0001, n = 5 for controls, n = 6 for treatment group).
To investigate the effects of etoposide on folliculogenesis after germ cells are already enclosed in follicles, neonatal mouse ovaries were cultured for six days in control medium or exposed to etoposide (50, 100, 150 or 200 ng ml−1) for the duration of culture. Over the six days of culture, some PFs initiated growth to the transitional or primary stage, with a few reaching the secondary stage. In contrast to the fetal ovary culture experiments, when oocytes were exposed to etoposide after follicle formation, no effect was seen on total follicle number (Fig. 4Bii, p = 0.092; n = 6, 4 independent cultures), or the percentage of unhealthy follicles (Fig. 4Cii, p = 0.082), despite exposing neonatal ovaries to a higher concentration of etoposide (200 ng ml−1, compared with the highest dose of 150 ng ml−1 for fetal ovary cultures). When each follicle stage was examined individually, the only effect seen was at the transitional stage, which accounts for 15 % of follicles in control ovaries. Here, etoposide significantly decreased transitional follicle numbers at 100, 150 and 200 ng ml−1 doses (Fig. 5Aii, p < 0.05, p < 0.01 and p < 0.05 respectively; n = 6) with a corresponding increase in the percentage of transitional follicles assessed as unhealthy only at the highest dose (Fig. 5Bii, p < 0.01; n = 6). There was no significant effect on the number or health of PFs (p = 0.101, p = 0.173 respectively) or primary follicles (p = 0.604, p = 0.129 respectively).
Overall, results show a marked effect of etoposide on follicle number only when ovaries are exposed prior to follicle formation (fetal ovary culture), with significantly fewer follicles remaining even when exposed to doses as low as 100 ng ml−1. In contrast, exposure of ovaries to etoposide only after follicle formation (neonatal ovary culture), has no effect on overall follicle number or health even up to the highest dose of 200 ng ml−1, with the only significant effect found specifically on transitional stage follicles.
Etoposide affects different follicular cell types depending on the time of exposure
Figure 4 showed the appearance of unhealthy follicles with unhealthy oocytes in cultured fetal ovaries, exposed to etoposide only prior to follicle formation (Fig. 4Aiii: red arrows), in contrast to the unhealthy follicles with unhealthy granulosa cells in cultured neonatal ovaries, exposed to high concentrations of etoposide only after follicle formation (Fig 4Avi: yellow arrows). In order to examine this in more detail, all histological sections were assessed further, with each unhealthy follicle classified as having: (i) an unhealthy oocyte only; (ii) unhealthy granulosa cells only; or (iii) unhealthy oocyte and granulosa cells. Exposure of fetal ovaries to etoposide resulted in a significant increase in the proportion of follicles with morphologically unhealthy oocytes (Fig. 7Ai, p < 0.05), with no effect observed either on the proportion of follicles with unhealthy granulosa cells only or unhealthy oocyte and granulosa cells (Fig. 7Aii,iii, p = 0.429 and 0.470 respectively). In contrast, neonatal cultured ovaries exposed to etoposide only after follicle formation, exhibited a significant increase in the proportion of follicles assessed as unhealthy due to unhealthy granulosa cells, or where both the oocyte and granulosa cells were unhealthy (Fig. 7Bii, Biii, p < 0.05; n = 6), with no significant effect on the proportion of follicles assessed as unhealthy due to an unhealthy oocyte (Fig. 7Bi, p = 0.069; n = 6).
Topo IIα is expressed in the female germ cell only prior to follicle formation
Lastly, we sought to investigate why etoposide was having more pronounced effects on oocyte rather than granulosa health in fetal ovaries, but less predominantly affecting the somatic granulosa cells rather than the oocyte in neonatal ovaries. Etoposide functions by inhibiting Topo II, preventing it from re-ligating the double-stranded DNA breaks that Topo II introduces [22, 24, 25]. TopoIIβ has been previously reported to be expressed in oocytes at all developmental stages, and is also expressed in granulosa cells of PF and growing follicles [29]. TopoIIα plays a more widespread role in resolving replication-induced topological structures than TopoIIβ [revieved in Refs 24 and 28] and could also be contributing to etoposide sensitivity in the developing ovary. Immunohistochemistry was used to assess Topo IIα expression within the developing mouse ovary, both in vivo from E13.5 through to P6 and in vitro throughout the course of the fetal ovary culture. It was also investigated in a second trimester fetal human ovary, a developmental stage at which the ovary contains female germ cells both prior to and subsequent to follicle formation. In the mouse, both in vivo and in vitro, Topo IIα expression changed from a germ cell-only location prior to follicle formation, to a somatic cell-only location after follicle formation, primarily in granulosa but also expressed in some stromal cells (Fig. 8a, b). Similarly, in the developing human ovary, Topo IIα was expressed in germ cells only prior to follicle formation, although here Topo IIα was no longer expressed after follicle formation (Fig. 8c). The damaging effect of etoposide on ovarian germ cells (Figs. 4, 5 and 6) is, therefore, pronounced only when etoposide exposure is coincident with Topo IIα germ cell-specific expression. Thus the developmental regulation of Topo IIα expression could be one of the factors contributing to the more pronounced effect of etoposide on fetal ovaries, when germ cells express Topo IIα.