Cystathionine β‐synthase is required for oocyte quality by ensuring proper meiotic spindle assembly

Abstract Objectives Poor oocyte quality is detrimental to fertilization and embryo development, which causes infertility. Cystathionine β‐synthase (CBS) is one of the key enzymes modulating the metabolism of homocysteine (Hcy). Studies have shown that CBS plays an important role in female reproduction. However, the role of CBS in regulating oocyte quality during meiotic maturation still needs further investigation. Materials and Methods Immunohistochemistry, immunofluorescence, drug treatment, western blot, cRNA construct and in vitro transcription, microinjection of morpholino oligo and cRNA were performed for this study. Results We found that CBS was expressed both in human and mouse oocytes of follicles. In mouse oocytes, CBS was distributed in the nucleus at germinal vesicle (GV) stage and localized to spindle from germinal vesicle breakdown (GVBD) to metaphase II (MII). The expression of CBS was reduced in ovaries and oocytes of aged mice. CBS depletion resulted in meiotic arrest, spindle abnormality and chromosome misalignment, disrupted kinetochore‐microtubule attachments and provoked spindle assembly checkpoint (SAC). CBS was disassembled when microtubules were disrupted with nocodazole, and co‐localized with the stabilized microtubules after taxol treatment. Furthermore, CBS depletion decreased the acetylation of α‐tubulin. Conclusions These results reveal that CBS is required for the acetylation of α‐tubulin to ensure proper spindle assembly in regulating oocyte quality during meiotic maturation.


| INTRODUCTION
Poor oocyte quality means the incapability of oocyte to go through successful maturation, fertilization and embryo development, causing infertility. 1 During meiotic maturation, proper spindle assembly is essential to the correct segregation of chromosomes. 2 Poor oocyte quality with increasing age remains an important issue for female fertility. 3 The level of serum homocysteine (Hcy) is increased in some infertile patients with poor quality oocytes. 4 It is well known that cystathionine β-synthase (CBS) is the key enzyme modulating the metabolism of Hcy, which catalyzes Hcy with serine or cysteine into cystathionine and water or hydrogen sulfide (H 2 S). [5][6][7][8] It has been also reported that the expression of CBS in the liver of infertile rats is reduced, which partly explains the increased serum Hcy level. 4 Until now it is not clear whether the expression of CBS is reduced in oocytes with poor quality. In recent years, increasing evidence has indicated that CBS is important for female reproduction. 9,10 It has been shown that CBS is expressed in mouse follicular cells. 11 However, whether it is expressed in human or mouse oocytes has not been reported yet. The inhibition of CBS in follicular cells hinders oocyte meiotic maturation, as shown by the increased rate of GV oocytes. 12 The role of CBS in regulating oocyte quality during meiotic maturation still needs further exploration.
In this study, we aim to explore the role of CBS in regulating oocyte quality during meiotic maturation and focus on the underlying mechanism in meiotic spindle assembly by participating in acetylation of α-tubulin.

| Oocyte collection and culture
All the procedures of animal care and use were in accordance with Animal Care Commission policies of Capital Medical University.

| Antibodies
Rabbit polyclonal anti-CBS antibody was purchased from Proteintech

| Immunohistochemistry
Human ovarian sections containing normal follicles were collected from patients with ovarian mature teratoma, ovarian seromucinous cystadenoma, luteal cyst, yolk sac tumor and immature teratoma in the Department of Pathology, Luhe Hospital, Capital Medical University. We obtained informed consent for experiments with human subjects and complied with the privacy rights of human subjects. Mouse ovary sections were available from 7-week-old female BALB/C mice. Sections were dewaxed in xylene and hydrated in graded alcohol (100%-70%).
Endogenous peroxidase was blocked in 3% H 2 O 2 for 30 min. Antigen retrieval was performed in citrate buffer (pH 6) at 100 C for 2 min. The sections were washed in phosphate buffer saline (PBS, Sigma) and nonspecific binding was reduced in 5% BSA for 1 h. The sections were incubated with primary antibody (anti-CBS antibody 1: 500) at 4 C overnight.
Ovary sections were washed and incubated with secondary antibody for 1 h. Diaminobenzidine (DAB) and haematoxylin staining were performed.
The sections were dehydrated and mounted in neutral resin.

| Cold treatment
Oocytes were cultured for 8 h in M16 medium and then treated in MEM (1Â) + GlutaMAX-I on ice for 5 min for immunofluorescence.

| Statistical analysis
The data repeated at least three independent experiments were presented as the mean ± standard error of mean (SEM). Statistical differences were evaluated by t test or one-way analysis of variance (ANOVA) using GraphPad Prism 5 software (GraphPad Software) and p < 0.05 was considered significant.

| CBS was expressed both in human and mouse oocytes
In order to determine whether CBS was expressed in human oocytes, immunohistochemistry and immunofluorescence were performed with human ovarian sections. Our results showed that CBS was expressed in oocytes of primordial follicles, primary follicles, secondary follicles, and mature follicles ( Figure 1A,B). In addition, we also found that CBS was expressed in mouse oocytes of primordial follicles, primary follicles and secondary follicles by immunohistochemistry ( Figure 1C), which was consistent with the distribution pattern of CBS in human oocytes of follicles. Then, we used mice as a model for further study. Western blot showed a high level of CBS protein from GV to MII stage ( Figure 1D,E).
Immunofluorescence revealed that CBS was punctately localized in the nucleus at GV stage. Strikingly, at MI stage CBS was distributed in the cytoplasm, similar to the spindle shape ( Figure S1). GV oocytes developed to GVBD, Pre-MI, MI, AI/TI, MII stage, and CBS was colocalized with the spindle microtubules ( Figure 1F), suggesting that CBS may be involved in meiotic spindle formation.
HL-7702 was selected for immunofluorescence to explore the distribution of CBS during mitosis. In HL-7702, CBS was uniformly distributed in the cytoplasm during interphase, prophase, metaphase, anaphase and telophase ( Figure S2), which was different from the distribution in oocytes and suggested CBS may play a different role in meiosis and mitosis.

| Expression of CBS was reduced in ovaries and oocytes of aged mice
To investigate the relationship between the CBS and oocytes with poor quality, the ovaries of 2-and 10-month-old female mice were selected for western blot and immunohistochemistry. We observed that the expression of CBS was reduced in ovaries (Figure 2A

| Depletion of CBS impaired meiotic progression
In order to further evaluate the role of CBS in oocyte quality regulation during meiotic maturation, specific Cbs morpholino was uti-

| CBS was required for acetylation of α-tubulin in oocytes
Abnormal spindle assembly urged us to assess the role of CBS in the microtubule stability. MI oocytes were treated with a microtubuledepolymerizing drug nocodazole and we found as microtubules were depolymerized, the CBS signal localized to the spindle disappeared completely. After treatment with a microtubule-stabilizing reagent taxol, broad spindles were observed in MI oocytes, CBS was colocalized with spindle microtubules (Figure 5A), which suggested that the subcellular localization of CBS was related with microtubule dynamics. The acetylation level of a-tubulin on lysine 40 (K40) has been reported to be a marker of stabilized microtubules. 20 CBSdepleted oocytes showed a decreased fluorescence intensity of acetylated α-tubulin ( Figure 5B,C), implying CBS may ensure spindle assembly by participating in the acetylation of α-tubulin to stabilize microtubules. In order to further verify this hypothesis, oocytes were lysed for western blot with acetylated α-tubulin, the results revealed that the expression of acetylated α-tubulin was remarkably decreased in CBS-KD oocytes ( Figure 5D,E). In addition, the fluorescence intensity of α-tubulin was not significantly different in CBS-depleted oocytes ( Figure S6). Next, to confirm whether the abnormal spindle was due to the defective acetylation of α-tubulin, TubK40Q-Myc or TubK40R-Myc cRNA was injected into CBS-depleted oocytes. As displayed in Figure 5F,G, it was TubK40Q acetylmimic mutant not TubK40R nonacetylatable mutant that rescued the abnormal spindle, indicating that CBS was required for spindle assembly by participating in the acetylation of α-tubulin to stabilize microtubules.

| DISCUSSION
In this study, we demonstrated CBS was expressed both in human and mouse oocytes. In mouse oocytes, CBS was co-localized with spindle microtubules after meiosis resumption. The expression of CBS was decreased in ovaries and oocytes of aged mice. Depletion of CBS led to meiotic arrest, disrupted the spindle assembly by the defective acetylation of α-tubulin, destroyed the kinetochore-microtubule attachments and provoked SAC.
Infertility has become a public health problem worldwide. 21 With the change of people's lifestyle, the incidence of infertility is gradually increasing. 22 It has been reported that the rate of fertility all over the world will decline year by year before 2050. 23 Oocyte quality is a key factor to pregnancy success. 24 However, the molecular mechanism involved in regulating oocyte quality during meiotic maturation still needs further studies. CBS is a key enzyme in metabolizing Hcy, which catalyzes the formation of cystathionine and water or H 2 S from Hcy with serine or cysteine. 8 In recent years, it has been found that CBS has an important role in female reproduction. [9][10][11][12] The role of CBS in oocyte quality regulation during meiotic maturation still needs to be further explored.
We found that CBS was expressed in human oocytes of primordial follicles, primary follicles, secondary follicles, and mature follicles.
As 99% of human genes have mice homologues, mice were generally used in reproductive research. 25,26 Immunohistochemistry showed CBS was also expressed in mouse oocytes of primordial follicles, primary follicles and secondary follicles, which is similar to the distribution of CBS in human oocytes of follicles. Therefore, we used mice as a research model for the next discussion. We found stable expression of CBS during meiotic maturation. The localization of CBS to spindle from GVBD to MII stage implied that CBS may be involved in meiotic spindle formation. Meanwhile in HL-7702, different from that in oocytes, CBS was uniformly distributed in the cytoplasm, which implied that CBS may play a different role in meiosis and mitosis. For women, after 30 years old, oocyte quality is gradually decreased along with age. 27,28 Compared with 2-month-old female mice, the oocyte quality of 10-month-old female mice is significantly decreased. 29 Our data showed the CBS expression in ovaries and oocytes was decreased in aged mice. Specificity protein 1 (Sp1) is a transcription factor binding to CBS promoter. 30  tional activity during ischemia/reperfusion, which leads to a reduction of CBS protein. 30 In aged ovaries, the increase of ROS may increase phosphorylation of Sp1 leading to decreased transcriptional activity, which partly explains the decreased expression of CBS in our study.
Therefore, CBS was reduced in ovaries and oocytes of aged mice, which may be due to the decreased expression and increased phosphorylation of Sp1. The decreased expression of CBS in aged oocytes indicated CBS may be related with oocyte quality regulation. The perivitelline space is higher in oocytes of Cbs knockout mice, 34 suggesting that the quality of these oocytes is low 35  MI oocytes developing to AI is inseparable from the correct segregation of chromosomes. When correct attachments are established between the spindle microtubule and the kinetochore, mitotic arrest deficient protein 2 (Mad2) is removed from the kinetochore and cell division cycle protein 20 (Cdc20) is released from mitotic checkpoint complex (MCC). Anaphase promoting complex/cyclosome (APC/C) is activated and then securin and Cyclin B are degraded. After that, separase cleaves the cohesion between chromosome arms. Then homologous chromosomes are separated. [36][37][38] Therefore, any factor that impacts spindle formation may hinder the correct segregation of chromosomes, leading to meiotic arrest. A great quantity of disorganized spindles and misaligned chromosomes in CBS-depleted oocytes demonstrated that CBS was involved in meiotic spindle assembly.
Moreover, depletion of CBS destroyed the K-MT attachments and provoked SAC. Oocytes treated with nocodazole and taxol showed CBS was related with microtubule dynamics. In our study, IF and western blot showed the fluorescence intensity and expression of acetylated α-tubulin were decreased in CBS-depleted oocytes.
TubK40Q is a mutant of α-tubulin, in which lysine 40 is substituted with glutamine to mimic acetylated tubulin. TubK40R is also a mutant of α-tubulin, in which lysine 40 is substituted with arginine to mimic nonacetylated tubulin. 39,40 In our study, it was TubK40Q acetylmimic mutant not TubK40R non-acetylatable mutant that rescued the abnormal spindle, demonstrating that CBS was essential for spindle assembly by participating in the acetylation of α-tubulin. It has been reported that histone deacetylase 6 (HDAC6) and sirtuin 2 (SIRT2) are the deacetylases of α-tubulin K40. α-tubulin acetyltransferase 1 (α-TAT1) is the acetylase of α-tubulin K40. 41 Li et al. have reported histone deacetylase 3 (HDAC3) can modulate the acetylation of ɑtubulin. 42 Maybe the depletion of CBS can impair the acetylation modification of α-tubulin by affecting some of the above-mentioned deacetylases and acetylase, which needs further effort to be examined in the future. In summary, CBS is required for the acetylation of α-tubulin to ensure spindle assembly in oocyte quality regulation during meiotic maturation. In aged oocytes, the acetylation levels of tubulin are elevated. 43 It has been reported the expression of both HDAC3 and SIRT2 is reduced in aged oocytes, which results in hyperacetylation of α-tubulin in these aged oocytes. 43,44 The elevated acetylation levels of tubulin in aged oocytes may be due to hyperacetylation of α-tubulin by the reduced expression of HDAC3 and SIRT2 being stronger than hypoacetylation by the reduced CBS expression.
Altogether, CBS is involved in oocyte quality regulation during meiotic maturation. CBS can participate in the acetylation of α-tubulin to ensure proper spindle assembly, kinetochore-microtubule attachments and the removal of SAC from the kinetochores. Eventually, chromosomes are correctly segregated. Consequently, our findings will provide a theoretical reference for improving oocyte quality.