Effects of post‐ovulatory aging on centromeric cohesin protection in murine MII oocytes

Abstract Purpose Post‐ovulatory aging causes a high frequency of aneuploidy during meiosis II in mouse oocytes, irrespective of maternal age. In this study, we evaluated the effects of post‐ovulatory oocyte aging on the protection of chromosomal cohesion involved in aneuploidy and verified the relationship between PP2A or SGO2 expression and the phosphorylation level of REC8 in oocytes. Methods Murine ovulated oocytes were incubated for 6 or 12 h in vitro after collection and denoted as the aged group. The oocytes examined immediately after collection were used as the control group. Immunofluorescent staining was used to detect the localization of PP2A, SGO2, BUB1, AURORA B, and MAD2 in the chromosomal centromere. Immunoblotting was used to quantify the expression of proteins describe above and REC8 in the oocytes. Results PP2A expression involved in the de‐phosphorylation of REC8 decreased over time in oocytes, suggesting a deficiency in PP2A in centromeres. This indicated an increase in the level of phosphorylated REC8, which destabilizes centromeric cohesion in oocytes. In contrast, SGO2 expression was significantly high in aged oocytes. Conclusions The findings show that post‐ovulatory aging destabilizes the centromeric cohesin protection in oocytes and can cause aneuploidy, which is often observed in aged oocytes during meiosis II.


| INTRODUC TI ON
It is well known that mammalian oocyte quality degrades over time after ovulation in vitro. 1 To gain insight into oocyte aging, the aging process needs to be considered on two different axes of time. One is maternal aging, which involves an inter-annual change in oocytes that advances with age. This is also described as pre-ovulatory aging, which progresses in the ovary. The other is post-ovulatory aging, the temporal alterations in oocytes that progress in the oviduct or can be induced in vitro. Most studies that investigated oocyte aging have focused on the effect of maternal age, that is, pre-ovulatory aging. However, as the in vitro manipulation time for ovulated oocytes used in reproductive medicine and ova research lengthens, post-ovulatory oocyte aging would become a problem that must be overcome. For example, rescue ICSI, which is often carried out in reproductive medicine, is a treatment program in which ICSI is performed on oocytes judged to be unfertilized after IVF to obtain fertilized oocytes. We believe that target oocytes used for rescue ICSI are already aged by the time of re-insemination and are therefore essentially different from fresh unfertilized oocytes.
Chromosomal aneuploidy is a well-known problem caused by oocyte aging. [1][2][3] Chromosomal aneuploidy in oocytes, which is a major cause of poor developmental competence and loss of pregnancy, 1,[3][4][5][6][7] results from chromosomal segregation errors during meiosis. 8,9 It is known that chromosomal segregation errors such as early segregation and nondisjunction occur during meiosis I in oocytes aged in ovaries with maternal aging. 10,11 Our previous study showed that post-ovulatory aging also produces a high frequency of aneuploidy during meiosis II, irrespective of maternal age. 12 The spindle assembly checkpoint (SAC), which functions during cell division, is one of the checkpoint mechanisms that function during the cell cycle.
SAC is a monitoring system that equally distributes chromosomes by correctly attaching spindle microtubules to the chromosome kinetochore. We have reported that oocyte aging in vitro leads to the destabilization of SAC signaling and causes segregation errors in sister chromatids following the metaphase II (MII). 12 Mammalian oocytes arrest meiosis in the MII stage before ovulation. In ovulated oocytes, it is necessary to align chromosomes on the equatorial plane and maintain adhesion between sister chromatids until meiosis is resumed by fertilization. The cohesin complex, which has a ring-shaped protein structure, acts as an adhesion factor that maintains sister chromatid connections. [12][13][14] In meiosis I, cohesin between sister chromatids in the arms of the two homologous chromosomes that form the divalent chromosome is resolved by degradative enzymes, whereas cohesin between sister chromatids in the centromere is maintained. 13,14 This mechanism ensures that sister chromatid pairs are correctly recognized and distributed to both poles during meiosis II. In MII oocytes, cohesin complexes that localize to the chromosomal centromere provide adhesion between sister chromatids. REC8, which contains a cleavage recognition site for separase, is one of the protein subunits of the meiotic cohesion complex. Degradation of REC8 by activated separase following the release of the SAC signal results in the dissociation of cohesin from the chromosomal centromere. We have shown that time-dependent deterioration of REC8 occurs in murine oocytes aged in vitro after ovulation, despite before activation. 12 This suggests that in vitro aging after ovulation weakens cohesion between sister kinetochores and causes a high frequency of aneuploidy after MII in oocytes.
Shugoshin protein was identified as a factor that protects cohesin REC8 on the centromere from separase so that sister chromatids do not segregate until the anaphase during meiosis. 15 Two paralogs of Shugoshin, Sgo1 and Sgo2, have been identified in fission yeast. 16 Sgo1, which is specifically expressed during meiosis I, localizes to the centromere and prevents sister chromatids from dissociation until meiosis I. 16 In contrast, Sgo2, which is constantly expressed on the centromere during cell division (M-phase), contributes to the accurate distribution of sister chromatids. 17 It has been reported that Shugoshin regulates the phosphorylation level of the target protein during the M-phase by interacting with protein phosphatase 2A (PP2A), which is a dephosphorylate enzyme. 18 Cohesin protection is accomplished by the de-phosphorylation of REC8, which is enabled by the recruitment of PP2A via Shugoshin to the chromosomal centromere. 15,18,19 Shugoshin has been widely conserved from yeast to humans in eukaryotes, and an SGO-like protein has been found in various eukaryotes 18 ; this has been defined as the Shugoshin family protein. In humans and mice, two SGO-like proteins, SGO1 and SGO2, have been found. Although the role of Shugoshin during meiosis in mammals seems to be similar to that in fission yeast, the behavior of Shugoshin is not fully understood.
Functional analysis of Shugoshin using murine oocytes has indicated that SGO1 and SGO2 are expressed during both meiosis I and II, in contrast to the process in fission yeast. 20 In addition, SGO2 is more highly expressed than SGO1 during the meiosis process, and it has been clarified that SGO2 is essential for the protection of centromeric cohesion. 15,20 Therefore, SGO2 and PP2A may be involved in the reduced expression of cohesin REC8 in MII oocytes, as found in previous studies.
The purpose of this study was to examine the effect of postovulatory aging on the expression and localization of SGO2 and PP2A in murine oocytes, and to verify its relationship with the phosphorylation level of cohesin REC8.

| Oocyte collection and in vitro aging
To obtain MII oocytes, female mice were induced to super-ovulate by consecutive injection of 5 IU pregnant mare serum gonadotropin (ASKA Animal Health Co., Ltd.) and 5 IU human chorionic gonadotropin (hCG; ASKA Animal Health) at 48-h intervals. The superovulated mice were euthanized 15 h after the hCG injection, and their oviductal ampullae were broken to release the cumulus-oocyte complexes (COCs) into TYH medium under paraffin liquid (Nacalai Tesque, Inc.). The collected COCs were incubated for a certain time at 37.5 °C under a humidified atmosphere with 5% CO 2 and used as in vitro aged oocytes. Two experimental groups were designated in this study. One was an aged group, comprising oocytes incubated for 6 or 12 h in vitro after oocyte collection. In our previous study, it has already reported that aging time over 6 h in vitro increases numerical chromosome aberrations in oocytes during meiosis II.
Especially, 40% of aged oocyte for 12 h in vitro has caused chromosome aneuploidy. 12 The other was a fresh group, which functioned as the control, comprising oocytes that were immediately examined after collection.

| Chromosome analysis
To eliminate the male genome factor, chromosome analysis of oocytes was performed after activating treatment by SrCl2 without fertilization. After the activation treatment, oocytes were cultured in mKSOM for 20 h. Six hours before termination of the culture, the activated oocytes were treated with mKSOM supplemented with 0.1 µg/ml demecolcine (Wako Pure Chemical Industries, Ltd.).
Chromosome spreads were prepared according to the method re-   ImageJ was used to quantify the signal intensity of the detected target protein. The captured RGB images containing the signal of target protein were converted to grayscale, and the sum of the gray values of all the pixels in the region indicated by the signal was taken as the brightness value.

| Immunoblotting
The expression of REC8, PP2A, SGO2, BUB1, AURORA B, and MAD2 proteins was detected in MII oocytes using immunoblotting. In this study, parthenogenetic oocytes were excluded from the analyzed samples of oocytes. 120 oocytes/tube were sampled for each experimental group, and immunoblotting was repeated 5 times Santa Cruz) secondary antibody for 1 h at room temperature. The membranes were washed thrice with PBS-T, and proteins were detected using an ECL Prime western blotting detection kit (GE Healthcare). After the detection of target proteins, the membranes were washed twice and re-blocked. As an internal control, the expression of α-tubulin or β-actin was detected, as described above, using mouse antiα-tubulin antibody (1:1,000 dilution, 017-25031; Wako) or mouse antiβ-actin antibody (1:1,000 dilution, 013-24553; Wako) as the primary antibody and HRP-conjugated anti-mouse IgG as the secondary antibody (1:2,000 dilution; GE Healthcare). ImageJ was used to quantify the intensity of the protein bands of interest, and band intensities were normalized to an internal control.

| Statistical analysis
The Chi-square test was used to determine the statistical signifi-

| Expression of p-REC8 and t-REC8
The expression levels of phosphorylated REC8 (p-REC8) and total REC8 (t-REC8) in MII oocytes are shown as ratios, relative to β-actin expression, in Figure 2A,B. The expression levels of p-REC8 were 0.64 ± 0.01 and 1.03 ± 0.08 in the 6-and 12-h aged groups, respectively. The p-REC8 level of MII oocytes increased over time in vitro, with a significant difference between the 12-h aged group and the fresh group (0.48 ± 0.10, p < 0.05). The expression levels of t-REC8, including un-phosphorylated REC8, were 1.29 ± 0.06 and 1.33 ± 0.04 in the 6-and 12-h aged groups, respectively. There were significant differences between the aged groups and the fresh group (1.82 ± 0.18, p < 0.05).

| Expression of PP2A
The immunofluorescent staining results of PP2A-A in MII oocytes are shown in Figure 3A. PP2A localization was clearly detected in chromosome kinetochores in the fresh group. Although PP2A signals were also detected in chromosome kinetochores in the aged group, the brightness values of PP2A signal were significantly lower than that in the fresh group (p < 0.05, Figure 3B). The expression levels of PP2A in MII oocytes are shown in Figure 3C,D. In this study, we assessed the expression of PP2A-A, which is a PP2A structural A subunit containing two isoforms (Aα and Aβ), as an indicator of PP2A complex expression. The expression levels of PP2A-A are shown as ratios, relative to β-actin expression, and were 0.62 ± 0.20 and 0.66 ± 0.28 in the 6-and 12-h aged groups, respectively. There were significant differences between the aged groups and the fresh group (1.29 ± 0.22, p < 0.05, Figure 3C).

| Expression of SGO2
Immunofluorescent staining results for SGO2 in MII oocytes are shown in Figure 4A. SGO2 localization was clearly detected in chromosome kinetochores, and there was no difference of brightness values of SGO2 signal between the aged group and the fresh group ( Figure 4B). The expression levels of SGO2 in MII oocytes are shown in Figure 4C,D. In post-ovulatory aged oocytes, the expression levels of SGO2 are shown as ratios, relative to α-tubulin expression, and were 1.14 ± 0.22 and 2.75 ± 0.11 in the 6-and 12-h aged groups, respectively. There was a significant difference between the 12-h aged group and the fresh group (1.02 ± 0.22, p < 0.05, Figure 4C).

| Expression of BUB1
Immunofluorescent staining results for BUB1 in MII oocytes are shown in Figure 5A. The brightness values of BUB1 signal in the aged group tend to be lower than that in the fresh group, but there was no difference of brightness values of BUB1 signal between the aged group and the fresh group ( Figure 5B). The expression levels of BUB1 in MII oocytes are shown in Figure 5C,D. In post-ovulatory aged oocytes, the expression levels of BUB1 are shown as ratios, relative to α-tubulin expression, and were 0.27 ± 0.09 and 0.30 ± 0.10 in the 6-and 12-h aged groups, respectively. There was a significant difference between the aged group and the fresh group (0.68 ± 0.07, p < 0.05, Figure 5C).

| Expression of AURORA B
Immunofluorescent staining results for AURORA B in MII oocytes are shown in Figure 6A. Although AURORA B localization was clearly detected in chromosome kinetochores in both aged and fresh group, the brightness values of AURORA B signal in the 12-h aged group were significantly higher than that in the fresh group (p < 0.05, Figure 6B). The expression levels of AURORA B in MII oocytes are shown in Figure 6C,D. In post-ovulatory aged oocytes, the expression levels of AURORA B are shown as ratios, relative to α-tubulin expression, and were 0.90 ± 0.17 and 1.54 ± 0.08 in the 6-and 12-h aged groups, respectively. There was a significant difference between the 12-h aged group and the fresh group (0.72 ± 0.05, p < 0.05, Figure 6C).

| Expression of MAD2
The immunofluorescent staining results of MAD2 in MII oocytes are shown in Figure 7A. Although MAD2 signals were clearly detected in chromosome kinetochores in both aged and fresh group, the brightness values of MAD2 signal in the aged group were significantly higher than that in the fresh group (p < 0.05, Figure 7B). The expression levels of MAD2 in MII oocytes are shown in Figure 7C Figure 7C).

| DISCUSS ION
In this study, three facts about the effect of post-ovulatory oocyte aging on the protective mechanism of cohesin in MII were molecules that connect sister chromatids during chromosome segregation regulated by the SAC. REC8, which is a meiosis-specific subunit of cohesin, has a separase recognition site that is specifically cleaved by separase. 22 Therefore, the expression of cohesin REC8 is essential to retain proper sister chromatid connections until the terminal phase of chromosome segregation. It is known that Rec8deficient mice show a loss in synapsis in homologous chromosomes and a poor cohesion between sister chromatids. 23 Even in wild-type mice, it has been shown that REC8 levels gradually decrease on the chromosome as mice age. [22][23][24][25] In old mice, low levels of REC8 expression and weakened cohesion of sister chromatids have been recognized in oocytes with poor connections between sister kinetochores. 24 Decreases in the expression of cohesin REC8 in oocytes are not solely due to advances in maternal age. Mammalian oocytes are arrested in MII prior to ovulation and maintain the MII stage until fertilization is complete. In our previous study, we showed that the expression level of REC8 in young murine oocytes decreased over time after ovulation until fertilization was completed. 12 This result indicates that post-ovulatory aging in vitro can reduce REC8 expression in MII oocytes in a time-dependent manner, even at a young age, irrespective of maternal age. The new finding in the present study was that the expression level of p-REC8 increased in a timedependent manner after ovulation, in contrast to that of t-REC8.
To maintain the function of cohesin as an adhesion factor between sister chromatids, REC8 must be de-phosphorylated and protected from degrading enzymes. 15 Therefore, in vitro aging after ovulation promotes the degradation of REC8 by separase following the phosphorylation of REC8 in MII oocytes. Indeed, in this study, we demonstrated that the expression level of t-REC8 in oocytes aged for more than 6 h after collection from the oviduct was significantly lower than that in fresh oocytes immediately after collection, as in a previous study.
Two factors, PP2A and SGO2, are involved in the dephosphorylation of REC8, which is the protective mechanism of cohesin. PP2A is a major intracellular phosphatase of serine/threonine protein, which regulates extensive cell signaling, including cell cycle and apoptosis. 26 PP2A localized in the chromosomal centromere is a factor that performs REC8 de-phosphorylation. In this study, although PP2A was localized on the chromosomal centromere of MII oocytes in both the aged and control groups, the results of brightness analysis indicate that the PP2A signals on the centromere were weaker in aged oocytes than in control oocytes. The accumulation of PP2A on the centromere may be insufficient in in vitro aged oocytes.
PP2A holoenzymes are composed of three subunits: a structural scaffold A subunit, a regulatory B subunit, and a catalytic C subunit, forming a heterotrimer complex. 15,26,27 Each subunit of PP2A has several isoforms; therefore, various combinations of subunits play important roles in regulating the localization and specific activity of PP2A. 27 Oocyte-specific deletion of the PP2A-Aα isoform leads to precocious sister chromatid separation. 28 However, unlike the loss of PP2A-Aα, defects caused by double knockout of PP2A-Cα and PP2A-Cβ do not induce premature loss of centromeric cohesion, but cause an MI arrest associated with abnormal spindles and misaligned chromosomes. 27 Thus, depletion of the PP2A-Aα subunit causes the loss of centromere cohesion, 18,29 but the depletion of other subunits has different consequences. Therefore, much consideration should be given to the subunit composition of the relevant PP2A complex.
In this study, the expression levels of PP2A-Aα and PP2A-Aβ in MII oocytes were examined, and it was shown that in vitro aging for 6 h or more significantly reduced the expression levels of PP2A. This supports the consideration that the accumulation of PP2A in the chromosomal centromere of in vitro aged oocytes may be deficient.
It also suggests that a decrease in aging time-related expression of PP2A and the depletion of PP2A in the centromere leads to an increase in the phosphorylation level of REC8, as described above.
SGO2 is a Shugoshin protein required to protect centromere cohesin during MI and MII in oocytes and is responsible for recruiting PP2A to centromeres by binding to it. 18,19,30 In this study, SGO2 localization was observed on the chromosomal centromere in aged oocytes, similar to that in control oocytes. Localization of SGO2 depends on BUB1, a kinetochore kinase, and H2A, which is phosphorylated to BUB1. 31 In fission yeast, it has been shown that inhibition of BUB1 reduces cohesin REC8 levels, and the existence of both BUB1 and SGO2 protects cohesin REC8. 32 These results suggest that BUB1 is important for the activation of the protective mechanism of REC8 by SGO2. It is known that BUB1 expression gradually decreases with advancing maternal age in human oocytes. 32 In this study, it was showed that BUB1 expression in in vitro aged oocytes also significantly decreased with advancing aging time. Despite the decline in PP2A and BUB1 recruitment to chromosome centromeres, the expression level of SGO2 was more than two times higher in the 12 h aged oocytes in vitro than in the control oocytes, contrary to our expectation. Many reports on the association between oocyte aging and SGO2 expression have been discussed from the viewpoint of maternal aging, and it has been suggested that SGO2 expressed in oocytes is gradually depleted in an age-dependent manner, increasing the vulnerability of cohesin protein toward degradation. 22  However, based on the combination of MAD2 localization data and expression levels, it is possible to discern a potential age-related dysfunction in the checkpoint system through which MAD2 monitors the kinetochore-microtubule connection. Comprehensive consideration from these results, it is suggested that unsuitable attachment between kinetochores and microtubules occurs frequently in in vitro aged oocytes. A decrease in PP2A expression in aged oocytes is apparent, an existence of AURORA B kinase, which is highly expressed to correct the misconnections spindlekinetochore, might cause a deficiency in PP2A antagonizing the action of Aurora B. Therefore, it is speculated that the overexpression of SGO2 was caused by active recruitment of CPC to the kinetochores and maintenance of kinetochore proteins at high phosphorylation levels. To assess the overexpression of SGO2 observed in in vitro aged oocytes, it is necessary to verify the interaction between the protective function of cohesin and the attachment mechanism of kinetochores-microtubules.
In conclusion, it was shown that post-ovulatory oocyte aging reduces the expression level of phosphatase PP2A, suggesting a deficiency in PP2A in centromeres. Furthermore, it was revealed that the decrease in PP2A expression over time leads to an increase in the phosphorylation level of cohesin REC8 in MII oocytes. These findings show that post-ovulatory aging destabilizes the protective mechanism of cohesin in oocytes, causing aneuploidy, which is often observed in in vitro aged oocytes during MII.

H U M A N R I G HTS
This article does not contain any studies with human subjects performed by the any of the authors.

A N I M A L S TU D I E S
All institutional and national guidelines for the care and use of laboratory animals were followed.

E TH I C S A PPROVA L
The study design and proposed studies were approved by the Animal