Naked mole-rat ovaries allow investigation of ovarian reserve, in vitro germ cell expansion, and oocyte IVM within a single sample

Abstract

Recently, we described that in the naked mole-rat ovary it is possible to study the ovarian reserve and the mitotic expansion of the germ cell postnatally. Herein, we show oocyte in vitro maturation and in vitro germ cell expansion using the same ovary.

The female reproductive system is the first system to age in humans, beginning for women in their mid-thirties (Quesada-Candela et al., 2020, Shirasuna and Iwata, 2017). Female reproductive aging is characterized by a loss of follicles, the functional units of the ovary, consisting of oocytes surrounded by companion granulosa cells. The quality of the oocytes remaining in the ovary also deteriorates with age. Reproductive aging is associated with adverse reproductive outcomes, including infertility, miscarriages, and birth defects, with such consequences becoming more frequent as women, globally, are delaying childbearing (Gruhn et al., 2019). The adult human ovary is devoid of definitive germline stem cells. As such, female reproductive senescence largely results from the depletion of a finite ovarian follicle pool that is produced during embryonic development. Because the ovarian reserve, which dictates reproductive lifespan, is established in utero, the reproductive aging timeline, in effect, is slated before birth.

Research groups around the world have explored different models to study mammalian ovarian development and aging. However, these models only allow us to study specific times in development, and, up to now, it has been impossible to evaluate in the same ovary both the early stages of development and aging. Nevertheless, to each rule, there is an exception. The naked mole-rat (Heterocephalus glaber; NMR) is the longest-lived rodent, with a maximum lifespan of >37 years (Ruby et al., 2018, Buffenstein and Jarvis, 2002). Female NMRs show no decline in fertility or fecundity during their entire lifespan, meaning that the dominant breeding females (also known as “Queens”) will breed until they die (Jarvis, 1981). Recently, we reported that in the NMR ovary there is an in vivo and in vitro mitotic expansion of the primordial germ cells in early postnatal life and also in reproductively active 3-year-old females (Brieño-Enriquez et al., 2023). NMR ovaries also show the co-existence of germ cells at every major stage of development in the postnatal NMR ovary, including pre-meiotic cells, cells at meiotic prophase I, and follicles. Although exhaustion of the oocyte pool or ovarian senescence has not yet been proven in the NMR ovary, the fact that they have very asynchronous oogenesis suggests we can find 37-year-old oocytes as well as younger oocytes in the same animal. This unique trait allows us to compare how age and/or environment affect oocytes, as well as to study the protective mechanisms used by NMR. We capitalized on this opportunity and asked if it is possible to induce in vitro maturation of oocytes from the same ovarian samples that were previously used to evaluate ovarian reserve thru histological analysis and in vitro germ cell expansion (Brieño-Enriquez et al., 2023). To answer this question, we analyzed ovaries from 3-year-old subordinate adults (SUB 3yr; n=10) and 3-year-old reproductively activated adults (EX-SUB 3yr; n=10). To induce reproductive activation in non-breeding females, 3-year-old females were removed from their colonies and housed with a male in a new cage for 4 weeks (Brieño-Enriquez et al., 2023). After this period, the animals were euthanized, following federal and institutional guidelines (MWRI, IACUC #20117234; and U. of Toronto IACUC # 20011632). Ovaries were collected in phosphate-buffered saline (PBS) within a laminar flux hood, washed three times with PBS and cleared of oviducts and connective tissue. After dissection, ovaries were placed in collection medium (Waymouths’s medium, 10% Fetal calf serum (FCS), 1% penicillin-streptomycin and 0.1% sodium pyruvate) at 32°C. Oocytes were released from the ovaries using 30-gauge needles. The residual tissue was used for culture as was described previously (Brieño-Enriquez et al., 2023) (Figure 1a). First, we evaluated if reproductive status affects the number of oocytes obtained per ovary. A total of 573 denuded oocytes were obtained, 226 from SUB 3yr and 347 from EX-SUB 3yr, a reduction of 34.84% was observed in SUB 3yr ($\stackrel{-}{x}=22.60\pm 5.98$, per ovary) compared to EX-SUB 3yr ($\stackrel{-}{x}=36.29\pm 9.74$, per ovary) (Figure 1b). During the obtention of the oocytes, we observed that the SUB 3yr oocytes were smaller than the EX-SUB 3yr oocytes, so we decided to evaluate the diameter and the zona pellucida (ZP) thickness in both groups (Figure 1c and 1d). Using 89 oocytes, we confirmed the diameter of the SUB 3yr oocytes ($\stackrel{-}{x}=63.50\pm 8.99$; n=45) was 22.77% smaller that the EX-SUB 3yr oocytes ($\stackrel{-}{x}=82.23\pm 9.95$; n=44) (Figure 1c). Analysis of ZP thickness showed that SUB 3yr oocytes ($\stackrel{-}{x}=6.64\pm 1.34$; n=45) had a 14.55% thinner ZP compared to EX-SUB 3yr oocytes ($\stackrel{-}{x}=7.78\pm 1.28$; n=44) (Figure 1d).

Figure 1.

Naked mole-rat (NMR) as a model for reproductive aging. a) Experimental design for in vitro maturation, germ cell expansion and analysis of ovarian reserve in NMR. b) Quantification of denuded oocytes obtained per ovary in 3yr subordinate females (SUB 3yr) and 3yr reproductively activated females (EX-SUB 3yr) (n = 10 per group). In all box plots, edges represent 25th and 75th percentiles, the horizontal line inside the box represents the median, and the white ‘+’ represents the mean. Using Mann Whitney test, comparisons were statistically significant (p=0.0014). c) Measurement of oocyte diameter in SUB 3yr (n=44) and EX-SUB 3yr (n=45) at germinal vesicle (GV) stage. Using Mann Whitney test, comparisons were statistically significant (p=0.0001). d) Measurement of zona pellucida thickness in SUB 3yr and EX-SUB 3yr at GV. Using Mann Whitney test, comparisons were statistically significant (p=0.0001). e) Analysis of oocyte in vitro maturation in both SUB 3yr and EX-SUB 3yr. The graph represents the percentage of oocytes at GV, germinal vesicle breakdown (GVBD), metaphase I (MI) and metaphase 2 (MII) at the different times of culture 0, 3, 6, 12, 24 and 30h. f) Representative images of fixed oocytes at different times of culture (0, 2, 4, 6, 8, 10, 12, 18, 20 and 24h) stained with b tubulin (magenta) and DAPI (grey). g) In vitro fertilization from oocytes maturated in vitro.

After analyzing the morphology, we asked whether these changes in diameter and ZP thickness affected in vitro maturation. However, we did not know the timeline for in vitro maturation of NMR oocytes. To establish the timeline, oocytes were cultured in MEMα+ GlutaMAX (Thermo Fisher) supplemented with 15% FCS and covered with light mineral oil (EmbryoMax, Sigma-Aldrich) at 32°C in a MIRI time lapse incubator (Esco). Our cultures showed that, independent of reproductive status, the timeline of in vitro maturation of NMR oocytes was longer than in mouse and closer to the in vitro maturation of human oocytes (Figure 1e) (Holubcova et al., 2015). To confirm these observations, we quantified the percentage of oocytes of each stage at different times of culture. No significant differences were observed during the 3 first hours of culture (Figure 1e). However, after 6h of culture, the percentage of oocytes at MI in SUB 3yr (38%) was significantly lower than in EX-SUB 3y (55%); this delay in SUB 3yr samples was observed in all the later culture times (12, 24, and 30h). In fact, at 12h of culture, 24% of the EX-SUB 3yr oocytes were already in MII whereas none of the SUB 3yr oocytes were. We followed the cultures, and, by 30h, 55% of the SUB 3yr and 75% of the EX-SUB 3yr oocytes were at MII. Interestingly, 20% of the SUB 3yr stayed at germinal vesicle (GV) after 30h of in vitro maturation while only 10% of the EX-SUB 3yr persisted at this stage.

Once we established the NMR in vitro maturation timeline, we endeavored to describe spindle formation in NMR oocytes. To do so, we cultured oocytes in 25 μl-drops of culture media (described above) on the bottom of a 60-mm sterile plastic Petri dish and then covered the dish with light mineral oil. The petri dishes were incubated at 5% CO₂ and 32°C. Oocytes were collected at 2, 4, 6, 8, 10, 12,18, 20, 24 and 30h, and fixed in 4% paraformaldehyde pH=9.2 for 10 min. To evaluate spindle formation, we performed immunofluorescence against β-tubulin. After fixation, oocytes were washed 3 times with PBS containing 0.4% Kodak photoflo for 10 min, followed by 0.1% PBS-Triton X and blocked in 1X PBS-antibody dilution buffer (ADB) and incubated over night at 4°C. The next day, oocytes were washed with 0.4% Kodak photoflo and incubated for 2 hours at room temperature with a secondary antibody (Alexafluor 488, Jackson Immunoresearch). Oocytes were washed, mounted, and imaged using a confocal microscope (Leica Stellaris). We were able to identify from GV to polar body extrusion and bipolar MII spindle (Figure 1f). Like in humans or mice before the germinal vesicle break down (GVBD), NMR chromosomes were highly condensed and clustered around the nucleolus. Surprisingly like human oocytes (Holubcova et al., 2015), NMR chromosome aggregate had a very limited number of microtubules, and they were clearly observed as an aster at ~4–5 hours. Later, around 6 hours after GVBD, the chromosomes appear individualized and start to be oriented. Around 8 to 10h, an early bipolar spindle can be observed and chromosome congression occurred at ~12h. After 18 to 20h, the oocytes showed features of polar body abscission, and finally, 24hr later, bipolar MII spindle/polar body extrusion was observed. Taken altogether, our results confirm that it is possible to induce oocyte in vitro maturation in the naked mole-rat, and that spindle morphology and the timeline required for maturation is closer to humans than mice.

Finally, we asked if oocytes maturated in vitro were able to be fertilized. To answer this question, we decided to perform classic in vitro fertilization (IVF). We obtained sperm from 3 male breeders, and the sperm was collected and capacitated in HTF for 1 hour at 32°C. In agreement with previous publications(van der Horst et al., 2011), we observed that NMR sperm had a very low number of motile spermatozoa (only ~3.75%) and a high number of morphologically abnormal spermatozoa. We used a total of 100 oocytes, 41 oocytes from SUB 3yr and 59 from EX-SUB 3yr. Oocytes were placed in the fertilization dishes with the sperm, kept at 32°C for 12h, washed in HTF, and moved to KSOM for culture. Two cell stage was reached 24h post in vitro fertilization; however, only 11 oocytes from SUB 3yr and 24 from EX-SUB 3yr reached this stage. In later stages, we observed 4 cells (36h) and 16 cells (72h); however, the embryos were highly fragmented. These results show that it is possible to fertilize in vitro matured NMR oocytes. Nevertheless, the conditions of the culture and the fertilization method need to be improved to obtain healthy embryos.

Defects in human gametogenesis are associated with age and a high risk of defects in chromosome segregation, leading to miscarriage. For years, we sought to study in the same model early stages of oogenesis such as meiotic prophase I as well as oocyte maturation. Herein, we present the naked mole-rat as a model that allows mitotic expansion of germ cells in vitro as well as oocyte in vitro maturation from the same ovary. This model will allow us to theoretically study a 37-year-old oocyte and a brand-new primordial germ cell from the same ovary, letting us test how age, environmental factors, or drugs can disrupt the genome, cohesion, segregation, etc. Although many details do need to be worked out, we are confident of the incredible value of such a model system.