|Ahead of print publication
Age-cumulative effect of REC8 reduction on meiotic chromosome segregation errors in mice
Li-Yuan Tian1, Ling Zhang1, Cheng-Qiu Tao2, Xiao-Qi Lin2, Feng Zhang3, Bin Zhang1
1 Department of Obstetrics, Obstetrics and Gynecology Hospital of Fudan University, Shanghai 200011, China
2 Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai 200011, China
3 Department of Obstetrics, Obstetrics and Gynecology Hospital of Fudan University; Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai 200011, China
|Date of Submission||06-Apr-2021|
|Date of Decision||27-Jul-2021|
|Date of Acceptance||06-Sep-2021|
|Date of Web Publication||09-Oct-2021|
Department of Obstetrics, Obstetrics and Gynecology Hospital of Fudan University, 419 Fangxie Road, Shanghai 200011
Source of Support: None, Conflict of Interest: None
Objective: This study aimed to explore the relationship between cohesin subunit REC8 reduction and meiosis chromosome segregation errors in the ovary.
Methods: Rec8+/− mice were generated using CRIPSR/Cas9 gene editing. The association between age and REC8 expression levels in the ovary was determined by Western blotting. Chromosome segregation errors were investigated by immunofluorescence imaging of superovulated oocytes. Wild-type and Rec8+/− female mice at 5, 8, 20, 36, and 40 weeks were used to evaluate ovarian reserve by ovarian clearing and immunolabeling.
Results: Ovary REC8 expression levels gradually decreased with age, while chromosome segregation errors increased with age. Segregation errors were more common in Rec8+/− mice, suggesting an association with REC8 expression. The ovarian reserve capacity decreased significantly with age. The ovarian reserve in Rec8+/− mice was inferior to that of age-matched wild-type mice, indicating important roles of age and REC8 levels in the ovarian reserve.
Conclusions: REC8 reduction has an age-cumulative effect on meiotic chromosome segregation errors in mouse ovaries. Rec8 haploinsufficiency poses a major challenge in generating normal and reproductive oocytes in aging mice.
Keywords: Chromosome Segregation Errors; Cohesin; Meiosis; REC8
|How to cite this URL:|
Tian LY, Zhang L, Tao CQ, Lin XQ, Zhang F, Zhang B. Age-cumulative effect of REC8 reduction on meiotic chromosome segregation errors in mice. Reprod Dev Med [Epub ahead of print] [cited 2021 Dec 8]. Available from: https://www.repdevmed.org/preprintarticle.asp?id=327880
| Introduction|| |
The incidence of chromosomal diseases increases significantly with advanced maternal age. Adverse outcomes of pregnancy and delivery attributed to abnormal fetal chromosomes are psychological and economic burdens to mothers, families, and the wider society. However, the mechanisms by which chromosome segregation errors occur during advanced maternal age remain unclear.
Chromosomal abnormalities can occur in embryos during mitosis or meiosis due to chromosome segregation errors originating from the maternal or paternal lineage. Approximately 25% of embryo chromosomal abnormalities are caused by mitotic errors during early embryo development. Meiotic errors account for the majority of events (approximately 75%). Therefore, majority of researches have focused on the biology of female germ cells.
Cohesin is a highly conserved protein located in close proximity to the centromeres and chromosome arms. The principal role of cohesin is to maintain chromosomal stability during mitosis and meiosis. Cohesin plays an important role in correct chromosomal segregation. Ishiguro reported aberrant chromosome axis formation, homolog association, meiotic recombination, and centromeric cohesion for sister kinetochore geometry as mechanisms of chromosomal abnormalities. REC8 is a meiosis-specific subunit of cohesin. Weakened cohesion in the chromosome of Rec8+/− mice leads to errors during meiosis, including premature chromosomal segregation. Therefore, it is important to examine the relationship between REC8 levels in the ovary and chromosome segregation errors. In addition to REC8, cohesin requires further regulatory proteins for proper formation and release from chromosomes. However, the specific functions of molecules, such as WAPL and SAC (cohesin release-related factors), remain unclear. Here, the relationships between age, REC8 expression, and the occurrence of chromosomal abnormalities were examined in aging wild-type and Rec8+/− mice.
| Methods|| |
Rec8+/− mice were generated using CRIPSR/Cas9 gene editing (Biogle Genome Editing Center, Changzhou, China), as previously described. Rec8 (NCBI reference sequence NM_020002.3; Ensembl ENSMUSG00000002324) is located on chromosome 14 in mice and comprises 20 exons. Exons from 7 to 16 were used as targets for gene editing. Cas9 and genomic RNA (gRNA) were injected into fertilized eggs to produce Rec8 knockout mice. Mouse colonies were raised in a specific pathogen-free environment with a room temperature of 21°C and controlled 12 h light/dark cycle. All mice had free access to food and water. The mice were maintained at the Animal Laboratory of the School of Life Sciences, Fudan University, Shanghai, China. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Fudan University.
Harvested ovaries were lysed in protein loading buffer and heated at 95°C for 5 min. REC8 protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred via an electric current to a polyvinylidene fluoride membrane. The membrane was blocked in 5% nonfat milk for 60 min and incubated with primary antibody (Rabbit Anti-Mouse-REC8, Proteintech, USA, 1:1,000 dilution) at 4°C overnight. The membrane was then washed in Tris-buffered saline containing Tween and incubated with a 1:2,000 dilution of horseradish peroxidase-conjugated secondary antibody (Goat-Anti-Rabbit-IgG; Proteintech) for 1 h and washed three times prior to imaging.
Cumulus–oocyte complexes removed following stimulation were placed into a 1 mg/mL solution of hyaluronidase and incubated at 37°C for 30 min to remove granulosa cells. Oocytes were blocked in 10% phosphate-buffered saline-Tween (PBST) containing 10% (v/v) Triton and 10% (v/v) donkey serum for 60 min. Primary antibodies (mouse anti-α-tubulin, Alexa Fluor 488 conjugate; mouse anti-H3S10P, Alexa Fluor 647 conjugate; anti-4′,6-diamidino-2-phenylindole [DAPI]; all 1:1,000 dilution) were added and the follicles were incubated at 37°C for 5 h and washed three times with PBS prior to confocal microscopy imaging.
Ovarian clearing and immunolabeling
The principle of ovarian clearing is to homogenize the refractive index of the tissue using inorganic phase reagents without changing the internal structure. Estrus mice were selected based on the vaginal cell smears. The chest was opened using ophthalmic scissors, and a needle was inserted along the left ventricle from the apex of the heart. Perfusion was performed using PBS precooled to 4°C, followed by 24-h perfusion with 4% paraformaldehyde at 4°C. The following processes were performed at 37°C: (i) dehydration of the ovaries in 30% sucrose solution for 24 h; (ii) blocking using 10% PBST containing 10% Triton and 10% donkey serum with shaking for 3 d; (iii) incubation with primary antibodies (Rabbit-Anti-Mouse-DDX4, Abcam, UK, 1:1,000 dilution; Rabbit-Anti-Mouse-P63, Abcam, 1:5,000 dilution) for 3 d; (iv) washing with PBS solution for 1 d with replacement of the wash medium at least seven times throughout the washing at >1 h intervals; (v) incubation with secondary antibody (Goat-Anti-Rabbit–cy3, Abcam, 1:1,000 dilution) and DAPI for 3 d); (vi) washing with PBS; (vii) clearing with CUBIC-1 solution (25% urea + 25% NNNN + 35% distilled water +15% triton), with the solution changed daily until the tissue was completely transparent; (viii) washing with PBS followed by addition of half-strength CUBIC-2 solution for 3 h; and (ix) addition of CUBIC-2 solution (50% sucrose + 15% distilled water + 25% urea + 10% triethanolamine) and incubation on a shaker for 1 d. Each tissue was mounted and imaged by confocal microscopy.
Data were presented as the mean ± SEM. All data were the results of three independent experiments. Experimental groups were compared using two-tailed unpaired Student's t-test. Statistical significance was set at P < 0.05.
| Results|| |
REC8 expression in ovary decreases with age
REC8 expression levels within the ovaries were investigated by Western blotting. REC8 expression was significantly lower in 37-week-old mice than in 5-week-old mice [Figure 1]a. REC8 expression within the ovary was extremely low at 44 weeks and was undetectable at 48 weeks. Expression levels were lower in Rec8+/− mice than in age-matched wild-type mice [Figure 1]b. These data suggest that REC8 expression within the ovary declines gradually with age, weakening REC8-mediated cohesion.
|Figure 1: REC8 expression levels within the ovary of wild-type and Rec8+/− mice. (a) The expression levels of REC8 and its internal control (β-actin) were compared between younger (5 weeks) and older (37 weeks) mice. (b) The expression levels of REC8 and its internal control (β-actin) in the wild-type and Rec8+/− mice at 40, 44, and 48 weeks.|
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REC8 has a maternal age effect on chromosome segregation errors in meiosis
Oocytes in the meiosis II (MII) stage were collected 12 h after the injection of human chorionic gonadotropin (hCG), and imaging was performed to identify the separation of chromosomes by positioning the spindle, DNA, and histones. Normal and abnormal chromosomal separation were clearly visible [Figure 2]a. Chromosome segregation errors were more frequent with increased age. These errors include a variety of polar and multipolar spindles with one or more scattered chromosomes. Increasing forms of chromosome segregation errors were observed in advanced wild-type and Rec8+/− mice [Figure 2]b. Clear differences in the rate of chromosome segregation error were observed between wild-type and Rec8+/− [Figure 3]. The decay of REC8 had a maternal aging effect on chromosome segregation error.
|Figure 2: Chromosome segregation errors in MII oocytes. (a) Fluorescent labeled DNA (blue), spindles (green), histones (red), and chromosome segregation images after merging in MII. The upper image shows a normal chromosome separation. The lower image shows an abnormal chromosome separation image. (b) Observed chromosome segregation errors in advanced Rec8+/− and advanced wild-type mice. Scale bar = 25 μm.|
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|Figure 3: Rate of chromosome segregation errors. *P < 0.05; ns: No significant difference.|
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Rec8 haploinsufficiency is important in ovarian reserve
Estrus wild-type and Rec8+/− mice were used to investigate the impact of Rec8 haploinsufficiency on ovarian reserves. Primary (diameter 0–17 μm and developing follicles (diameter 17–40 μm) were counted after immunolabeling of cleared ovaries [Figure 4]. Primary, developing, and total follicle counts decreased gradually at 5, 8, 20, 36, and 40 weeks of age in both the wild-type and Rec8+/− mice [Figure 4]. The age-related decrease in follicle counts in Rec8+/− mice was significantly greater than that in wild-type mice [Figure 5]. The difference in total follicle count between wild-type and Rec8+/− mice was apparent from the earliest time point (5 weeks). Moreover, developing follicles represented a greater proportion of total follicles as the age of the mice increased, indicating depletion of the ovarian reserve over time [Figure 6]. The proportion of developing follicles was highest in Rec8+/− mice, suggesting that both age and Rec8 haploinsufficiency play an important role in ovarian reserve.
|Figure 4: Three-dimensional imaging of follicles. Imaging was performed using Imaris. Red dots (DDX4 positive cells) indicate follicles (left), developing follicles (middle) and total follicles (right). The upper and lower images represent wild-type mice at 5 weeks and Rec8+/− mice at 40 weeks, respectively. Scale bar = 300 μm.|
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|Figure 5: The number of follicles in wild-type and Rec8+/− mice. Labeled comparisons are between wild-type and Rec8+/− mice of the same age (n = 6). *P < 0.05; †P < 0.001; ns: No significant difference.|
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| Discussion|| |
Pre- and postnatal care of geriatric mothers has been the recent focus of increasing attention. The risk of chromosomal diseases in children increases significantly with maternal age. The mechanisms of these chromosomal abnormalities remain unclear. Only approximately 5% of aneuploidy events in children are attributed to errors in paternal germ cells. Moreover, cell cycle checkpoint mechanisms prevent the majority of male germ cells with chromosomal errors from being passed on, regardless of age. Thus, studies have focused on female germ cells. In the present study, we explored changes within the ovaries of wild-type and Rec8+/− mice of different ages.
The occurrence of oocyte meiosis errors and chromosomal abnormalities in offspring is closely related to the age of mothers and increases exponentially in the ten years prior to menopause., Cohesin is a multisubunit protein complex originally discovered in yeasts. The complex comprises four subunits: two structural maintenance of chromosome complex subunits (SMC1 and SMC3), stromal antigen STAG3, and recombination gene 8 (REC8) kleisin protein. Cohesin is localized near the centromere and chromosome arms. Its multiple roles in cells include chromosome separation, DNA repair, and gene expression. The mechanism of transcriptional regulation has not been fully elucidated. Cohesion during mitosis and meiosis is a crucial role of cohesin. Cohesin is synthesized at the embryonic stage of oocytes. Cohesion occurs in S phase and is coincident with DNA replication and remains unaltered after loading onto the chromosomes., Loss of REC8 can cause meiotic disorders, and REC8 deficiency can lead to reproductive cell failure and infertility.,, In the present study, the ovaries of Rec8−/− mice were abnormal and were unable to ovulate.
Our data demonstrate that REC8 expression levels decrease with age in mice, falling below detectable levels at 48 weeks. Chiang et al. proposed a functional cohesion threshold for REC8 levels. The authors argued that cohesion between homologous chromosomes and sister chromatids cannot be effectively maintained at REC8 levels below this threshold, leading to significant increases in the error rate during meiosis. Aneuploidy mostly originates from oocytes. However, it is not clear whether reduction in REC8 is a major risk factor for chromosome segregation errors.
The main causes of chromosome segregation errors are crossover defects, loss of cohesin, spindle assembly defects, spindle assembly checkpoint failure, insufficient energy, overacetylation of protein, and DNA damage, among others., Cohesin has attracted significant attention owing to its structure, attachment site, and metabolism. Cohesin is essential for early embryonic development. Homozygous deletion of mitotic cohesin can be lethal to embryos, while heterozygous knockout mice develop a distinct phenotype., Fu et al. collected MII stage follicles 12 h after hCG injection and reported that the number of follicles in the young age group was significantly higher than that in the aged group. These results correspond with our data regarding depletion of the ovarian reserve. By observing the morphology of the spindle and DNA, we demonstrated that chromosome segregation errors increased with age, and that the types of chromosome segregation errors observed also increased.
Cohesin is required to maintain the stability of chromosome crossover and regulate chromosome structure and shape. During meiosis, chromosome cohesion gradually weakens, and is lost via two distinct pathways. The characteristic pathway is the destruction and cleavage of REC8 by protease and separase. The other pathway involves cleavage-independent removal., Regulation of cohesion metabolism is modulated by complex mechanisms, including the hyperactivity of separase, abnormal Shugoshin protein degradation, oxidative damage, increased intracellular oocyte pH, abnormal metabolism of spindle assembly checkpoint, anaphase-promoting complex, and age-related reproductive hormones.,,,,
During MI transitions from metaphase to anaphase, REC8 dissociates from the arms of homologous chromosomes, resulting in the separation of homologous chromosomes. However, centromeric REC8 persists until anaphase in MII.,, Segregation errors of chromosomes include nonsegregation, presegregation, and reverse segregation. Within the normal cell cycle, the loading and dissolution of cohesin are strictly regulated. Errors caused by premature segregation of sister chromatids are more common than those caused by nonseparation during meiosis.,
Currently, it is not possible to accurately measure REC8 expression in the human ovary. Studies have indicated that REC8 attenuation is an important risk factor for chromosomal abnormalities in the elderly. The distance between the centromeres increases with age, with values of 0.25, 0.38, and 0.82 μm in mice 3, 12, and 15 months of age, respectively. These findings also indicate that cohesion weakens with age.,, Our data demonstrate that REC8 decreased in the ovary overall. However, we did not determine the levels of REC8 within single oocytes. Subsequently, experiments were conducted to determine the number of oocytes in the ovaries of mice in different genotype and age. However, further investigations are required to determine the changes in REC8 levels in individual oocytes.
There has been no reported difference in three-dimensional image quantification between automated Imaris software and manual counting from hematoxylin and eosin-stained slides. We applied automated quantification to immunolabeled ovaries after ovarian clearing using the Imaris software to automatically count DDX4-positive cells. We observed a decrease in primordial follicles and developing follicles in wild-type mice with increased age, with fewer total follicles in age-matched Rec8+/− mice. The proportion of developing follicles in the total follicles gradually increased, indicating a decline in the ovarian reserve. Quantification of REC8 levels within single oocytes represents a future avenue of research, which warrants further investigation.
The loss of cohesion may act as a “molecular clock” limiting the reproductive capacity of females via the introduction of specific chromosomal errors. Improving the maintenance of oocyte quantity and quality remains a significant challenge in reproductive medicine. It remains unclear how loss of cohesion may be alleviated to reduce chromosome segregation errors that correlate with maternal aging.
Financial support and sponsorship
This study was supported by the Shanghai Municipal Science and Technology Major Project (2017 SHZDZX01).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]