The reaggregation of normal granulosa-cumulus cells and mouse oocytes with polycystic ovarian syndrome in vitro: An experimental study

Abstract Background The dialogue between oocytes and their surrounding cells plays a major role in the progress of oocyte meiosis and their developmental potential. Objective This study aimed to evaluate the effect of co-culture of normal granulosa-cumulus cells (GCCs) with oocytes from polycystic ovarian syndrome (PCOS) mice. Materials and Methods Normal GCCs were collected from 10 virgin adult Naval Medical Research Institute female mice (30-35 gr, 7-8 wk old), and were cultured in an alpha-minimum essential medium supplemented with 5% fetal calf serum for 24-48 hr (1 × 10 6 cells/well). Then, germinal-vesicle oocytes from PCOS mice were cultured in the presence of cultured normal GCCs (experimental group) and without GCCs (control group). The maturation rate and quality of the PCOS oocytes were examined by evaluating TFAM and Cx43 gene expression (real-time PCR) and the connection among PCOS oocytes and normal GCCs after 24 hr of culture. Results The co-culture of normal GCCs and PCOS oocytes in the experimental group led to the formation of a complex called a PCOS oocyte-normal GCCs complex. The maturation rate of these complexes was significantly increased compared to that of the control group (p ≤ 0.001). A significant difference was also found in the expression of Cx43 (p ≤ 0.001) and TFAM (p < 0.05) genes in the experimental group compared with the control group. The connection between PCOS oocytes and normal GCCs was observed in the scanning electron microscope images. Conclusion Co-culture with normal GCCs improves the capacity of PCOS oocytes to enter meiosis, which may result in the promotion of assisted reproduction techniques.


Introduction
Maturation and developmental competence of oocytes and their quality are important criteria in successful reproductive outcomes of assisted reproduction technologies. Polycystic ovarian syndrome (PCOS) is known as the most common cause of female infertility during reproductive age. It is distinguished by oligomenorrhea, bilateral polycystic ovaries, hyperinsulinemia, biochemical and/or hyperandrogenism, and oligo-or chronic anovulatory (1)(2)(3). One of the most important symptoms in PCOS patients is follicular development arrest and dysregulation of paracrine follicle activity (4). As a result, one of the main important concerns in PCOS patients is the poor competence of oocyte development (3,5) and weak response to ovarian stimulation (6,7).
For this reason, the use of assisted reproductive technologies such as in vitro maturation (IVM) for such patients is suggested.
During the IVM procedure, the maturation medium usually replaces the ovarian follicular environment. Therefore, it is necessary to create an appropriate culture medium for improving the competence of immature oocyte development for fertilization and embryogenesis (7). The development and growth of oocytes are supported by granulosa cells (GCs) from primordial to antral stages (8); therefore, one of the strategies to increase in vitro oocyte maturation is co-culture with GCs (9).
In a study, the developmental rate of porcine oocytes derived from early antral follicles improved using reconstructed oocyte-GC complexes (10). In another study, the maturation and development competence of buffalo oocytes derived from preantral follicles was evaluated in the presence of antral follicles and the survival and growth of the preantral follicles improved in this co-culture system (11).
Many studies have reported oocytes as an avascular environment. Normal folliculogenesis relies on the crosstalk among GCs, cumulus cells, and oocytes via gap junction intercellular communication (12,13). Two matchable hemichannels (connexin, Cx) in the plasma membrane of adjacent cells are involved in the formation of gap junctions. Cx43 is mainly localized to the membranes of the GCs and cumulus cells (12). It has been reported that Cx43 expression decreases in the presence of high androgen levels and hence gap-junctional intercellular communication between human GCs is impaired (14). GCs in PCOS patients were induced to produce more androgens, which resulted in the enhancement of hyperandrogenemia and anovulation (1). High androgen levels may also impair folliculogenesis in PCOS patients (14). Therefore, the co-culture of PCOS oocytes with normal cumulus GCs may improve the maturation rate and competence of oocyte development.
To the best of our knowledge, there have been no published studies on the use of normal cumulus GCs to improve the maturation of PCOS oocytes in co-culture conditions. Therefore, the purpose of this study was to answer the following questions: (i) Will a connection be made between normal cumulus-GCs and PCOS immature oocytes in a co-culture medium? (ii) How will a connection between PCOS immature oocytes and normal granulosa-cumulus cells (GCCs) affect the maturation rate of PCOS oocytes and their quality?

Study animals
This experimental study was conducted over a

Experimental design
To produce a conditioned medium, normal

RNA isolation and real-time polymerase chain reaction (real-time PCR)
At the end of the culture period, the PONC complexes and PCOS oocytes (experimental and control groups, respectively) were collected.

Statistical analysis
All experiments were repeated five times and data were expressed as mean ± S.D. 2 , oneway ANOVA, and Tukey's post-hoc tests were used to analyze differences among the groups and gene expression. The statistical analysis was

PCOS ovaries evaluation
In the PCOS mice, the irregular estrous cycles were confirmed along with the restriction to estrous stages upon estradiol valerate treatment.
The histological examinations showed that the

Discussion
The maturation medium and IVM procedure strongly affected the oocyte quality and maturation. The present study showed that normal GCCs could interact with PCOS oocytes, with a gap junction created among them, and that as a result, PCOS oocyte maturation rates could improve. It seems that co-culturing with normal GCCs provides a suitable 'niche' for PCOS oocyte maturation like the ovary. Therefore, after co-culture, the maturation rate of PCOS oocytes and the profile of gene expression improved.
These results are reasonable because the dialogue between oocytes and the surrounding cells plays a major role in progressing oocyte meiosis and their developmental potential (14).
It is possible that some molecules from normal It has been reported that the cleavage rate of embryos resulting from matured oocytes in a co-cultured condition with cumulus cells can be higher in comparison to matured denuded oocytes (21). In one study, a novel co-culture system was described regarding preantral follicles along with antral follicles. This efficacious co-culture system promoted the development of small preantral follicles (11). This research was in line with the present study's results that the PCOS oocyte quality and meiotic progress was improved in the co-culture with normal GCCs. It has also been reported that the rate of mouse blastocyst formation can be improved in co-culture with cumulus cells (20). However, in a study by Lin and colleagues co-culture of oocytes with GCs did not have a positive effect on mice oocyte maturation (22).
It has been found that some substances are produced by GCs cultured in vitro. These substances could inhibit or delay the meiotic maturation of oocytes (23). The inhibitory effects of GCs on oocyte maturation were observed in a study on cows (24). Another study found that gap junctions among oocytes and cumulus cells could transfer cAMP among cells and the accumulation of cAMP in the oocytes resulted in meiosis inhibition (23). Therefore, there are still contradictory results in relation to the co-culture effects of GCs on oocyte maturation.
It seems plausible that soluble factors, such as the extra-cytoplasmic matrix or extracellular medium, can affect the formation of an oocytecumulus cell complex (25). The co-culture system, and consequently the connection between oocytes and GCs, may allow better coordination between nuclear and cytoplasmic maturation, which promotes maturation potential.
Therefore, the proportion of matured oocytes is higher compared with ones in maturation media without GCs (26). It has also been demonstrated that an extensive production and reorganization of organelles and the replication of the mitochondrial genome occur during oocyte maturation. These alterations are vital for oocyte cytoplasmic maturation (25).
Mitochondrial function is correlated with the mitochondrial DNA. During oocyte maturation, the amount of mitochondrial DNA becomes significantly larger. TFAM is known as an important factor in regulating mitochondrial DNA transcription and replication. Therefore, the relative expression of the TFAM gene and the developmental competence of oocytes are associated (25). In the present study, TFAM gene expression was significantly higher in PCOS oocytes co-cultured with normal GCCs.
Therefore, it could be concluded that co-culture with normal GCCs may promote cytoplasmic maturation and developmental competence of PCOS oocytes.

Conclusion
The co-culture of PCOS oocytes with normal GCCs appears to improve PCOS-related abnormal follicular development. In addition, the connection among PCOS oocytes and GCCs, the higher levels of Cx43 and TFAM gene expression, and the improved maturation of PCOS oocytes after co-culture suggest that the co-culture system using normal GCCs might be a better method for IVM protocols than adding different external factors. This may result in the promotion of assisted reproduction techniques.