PCOS is a multifactorial disease characterized by endocrine and metabolic anomalies in women. The prevalence of PCOS is seen 6.1% according to diagnostic criteria of NIH, 19.9% according to Rotterdam criteria, 15.3% according to AE PCOS Society criteria in Turkish women (Michelmore et al. 1999; Yildiz et al. 2012). Therefore, there is more than one approach in the treatment of PCOS. It is important to try different treatment options with the PCOS model created experimentally due to the ovulation disorders and oocyte quality problems that occur in PCOS and to understand its effect on the maturation of oocytes. In this study, factors affecting oocyte maturation after administration of clomiphene citrate alone and combined application of metformin and pioglitazone in an experimental PCOS mouse model were investigated in ovarian tissues and oocytes using immunohistochemical and immunofluorescence methods. The data of this study show morphologically and histologically the disruption of the estrus cycle, irregular and different sized follicles, cystic follicles, and estrus cycle induced by PCOS. It also statistically shows the deterioration of glucose tolerance and the increasing body weight of the mice. DHEA-induced PCOS model studies have demonstrated that similar biochemical, statistical and histopathological findings in ovarian tissue (Yaba and Demir 2012; Kaufmann et al. 2015; Jafarzadeh et al. 2018; Bertoldo et al. 2019).
The results of our study show that clomiphene citrate treatment and combined therapy reduce cystic follicles in ovarian tissue and increase the expression levels of all molecules examined in ovarian tissues, especially secondary follicles. It was determined that clomiphene citrate treatment significantly increased Nobox, Foxl2, Cx43 and Cep55 in oocytes, but it was observed that there was no significant difference in the increases of other molecules examined in oocytes. Although combined therapy is therapeutic for both ovarian tissues and oocytes, statistically significant results have not been obtained. Ovulation induction is a treatment method used in PCOS patients to stimulate ovulation. In studies, clomiphene citrate is used alone or in combination with drugs such as metformin for ovulation induction treatment, but data on single or combined use are still controversial (Costello et al. 2019).
Newborn ovary homeobox (Nobox) is an oocyte-specific gene that is expressed in germ cells, follicle development, human and mouse primordial and growing oocytes (Suzumori et al. 2002; Rajkovic et al. 2004). It also plays an important role in determining oocyte quality (Belli et al. 2013). For Nobox, our findings show that the PCOS group decreased in ovaries and oocytes, and its immunoreactivity increased in treatment groups. In addition, a statistically significant increase was found in the oocytes of the clomiphene citrate group. In a study in which Nobox was ablated transcriptional regulatory genes that play a role in oocyte growth and differentiation, it was shown that both gene and protein expression of Nobox were disrupted by impairment of the expression of regulatory genes (Wang et al. 2020). In our study, the increase of Nobox in oocytes and ovarian tissue in the group treated with clomiphene citrate suggested that there was no disruption at the gene level with PCOS and that there was a protein-level repair with the treatment applied. Another study showed that Nobox was not found in developmentally deficient oocytes but found in developmentally competent oocytes, and as a result, it was revealed that Nobox could be a possible marker for oocyte quality in mice (Belli et al. 2013). In our study, we thought that the oocytes obtained from patients with PCOS were developmentally sufficient, the deterioration in oocyte quality after the treatment was correctable and oocyte maturation could be restored. In another study, it was shown that Nobox expression increased by applying a different treatment protocol for follicle development (Monti et al. 2009). According to the results of this study and the literature, Nobox may be one of the underlying indicators of oocyte maturation problems occurring in PCOS, and it should be examined in more detail in PCOS and ovulation induction treatment.
Forkhead box L2 (Foxl2) is necessary for the differentiation of granulosa cells and protection of the ovary (Schmidt et al. 2004). Also, it plays a role in the development of ovaries and sex determination in the early period, as well as in cell differentiation and follicle depletion in the late period (Pisarska et al. 2011, Uhlenhaut and Treier 2011). In our data, it was observed that Foxl2 decreased in ovaries and oocytes in the PCOS group and increased in the treated groups. It was found to be statistically significant in oocytes in the clomiphene citrate group. Interestingly, a study showed that oocytes suppress Foxl2 secreted from cumulus cells (Emori et al. 2020). However, this study is not sufficient to understand the role of Foxl2 in PCOS. To better understand the effect of Foxl2 on oocyte maturation both in PCOS and ovulation induction, Foxl2 secreted from cumulus cells, granulosa cells and oocytes should be examined spatiotemporally. In our study, the decrease of Foxl2 may have caused oocyte maturation to stop in PCOS, but the increase in Foxl2 in oocyte cells, granulosa cells and oocytes in treatment groups suggested that oocyte maturation started again. In two different studies, it has been shown that Foxl2 is increased in granulosa cells (Liu et al. 2020 and Whinship et. al 2020). Kim et al. reported that in fused toes locus mutant mice, Foxl2 is expressed low levels in granulosa cells, and it formed very few numbers primordial or primary follicles containing non-growing oocytes surrounded by granulosa cells (Kim et al. 2011). According to all these results, Foxl2 may be one of the main molecules for the continuation of oocyte maturation after ovulation induction treatment, but more research needs to be done on this subject.
As a member of the centrosomal protein family, Cep55 is expressed in mouse oocytes from germinal vesicle to metaphase II stages and plays an important role in the meiotic maturation of mouse oocytes, spindle organization and cell cycle progression (Xu et al. 2015). In our study, it was shown that Cep55 decreased in the PCOS group and increased in the treated groups. A statistically significant difference was found between PCOS group and the clomiphene citrate group in oocytes. It is known that oocytes exposed to high androgen during follicle development stages in PCOS and obesity resulting from PCOS disrupts meiotic maturation in oocytes (Reis and Honorato-Sampio 2018; Snider and Wood 2019). The re-increase of Cep55 in the treatment groups suggested that there was no abnormal situation in the division of spindle strands, which is important in oocyte maturation and later division stages. Xu et al. showed that Cep55 is important in the integrity of the spindle in oocytes, chromosome pairing and, accordingly, in metaphase anaphase transition. This important role of Cep55 suggests that chromosomes do not cause damage during crossing ovary, so that the meiotic maturation of the oocyte occurs smoothly. In the light of our findings and literature, it will be an important protein in the maturation and chromosomal integrity of oocytes after detailed molecular studies are carried out to elucidate the molecular mechanism underlying the decrease of Cep55 in PCOS and its increase after ovulation induction therapy.
Connexin 43 (Cx43) is expressed in granulosa cells in the ovary and responsible for the communication between granulosa cells (Teilmann 2005). Cx43 protein expression level increases during follicle development and decreases with follicular atresia (Ackert et al. 2001). The roles of connexins in oocyte maturation have mostly been investigated in knockout mouse models (Carabatsos et al. 2000; Li et al. 2007). In mouse ovaries, Cx43 binds granulosa cells (Simon et al. 1997; Gittens and Kidder 2005). Oocytes collected from Cx43-null mice fail to reach meiotic maturation, resulting in infertility (Ackert et al. 2001). In mouse oocytes, Cx43 protein is expressed in cumulus cells, transzonal extensions and oocytes (Kidder and Mhawi 2002; Simon et al. 2006). It has been shown that excessive Dihydrotestosterone (DHT) reduces Cx43 expression and disrupts the communication between granulosa cells (Wu et al. 2010). In our study, it was observed that the Cx43 ovarian tissue increased in the PCOS group, especially in the groups treated with cystic follicles. In oocytes, statistically significant differences were found in the control group, PCOS group and treatment groups. This situation suggested that Cx43 is an effective molecule in ovulation induction treatment in polycystic ovary syndrome. In other studies, it was reported that the expression level of Cx43 decreased significantly in women with PCOS, Cx43 could play an important role in the development of oogenesis arrest during oogenesis and oocyte maturation, and electroacupuncture upregulated the disrupted androgen receptor and Cx43 expression levels in PCOS created with letrozole in rats (Liu et al 2020; Xu et al.2020). In addition, some studies have shown that Cx43 and Gap junction-linked intercellular communication may contribute to the pathogenesis of polycystic ovary syndrome, and that Cx43 expression from granulosa cells of patients triggered by the GnRH agonist and human chorionic gonadotropin (hCG) for oocyte maturation is reduced, which may be associated with oocyte and embryo quality (Talhouk et al. 2012; Haas et al. 2016). In another study, when Lycopene was given to diabetic rats, it was shown that Cx43 increased primordial, secondary, and large antral follicles in rats (Yildiz et al. 2016). Yang et al. Administered testosterone to mice and reported that the androgen receptor (AR) increased significantly and Cx43 decreased significantly (Yang et al. 2015). As a result, studies have shown that Cx43 is a very important protein in oocyte maturation after ovulation induction therapy.