Introduction
Fertility preservation is a major concern in cancer patients subjected to chemotherapy or radiotherapy as these treatments cause severe depletion in the ovarian reserve pool, which culminates for patients in ovarian failure (Molina et al., Reference Molina, Barton and Loprinzi2005; Perez-Andujar et al., Reference Perez-Andujar, Newhauser, Taddei, Mahajan and Howell2013). Oocyte and embryo cryopreservation could be considered as strategies to preserve the fertility of adult patients, however these techniques could not be applied for prepubertal patients (Jeruss and Woodruff, Reference Jeruss and Woodruff2009; Amorim and Shikanov, Reference Amorim and Shikanov2016). In prepubertal cancer patients, ovarian tissue could be cryopreserved and autotransplanted following recovery, but contamination of cryopreserved tissue with malignant cells and cancer reoccurrence are the risks pertaining to this technique (Bockstaele et al., Reference Bockstaele, Tsepelidis, Dechene, Englert and Demeestere2012; Xiao et al., Reference Xiao, Zhang, Romero, Smith, Shea and Woodruff2015; Amorim and Shikanov, Reference Amorim and Shikanov2016). Alternatively, cryopreserved ovarian tissue could be cultured to activate primordial follicles, which could further be used for in vitro production of embryos (Telfer et al. Reference Telfer, McLaughlin, Ding and Thong2008; Amorim and Shikanov, Reference Amorim and Shikanov2016; McLaughlin et al., Reference McLaughlin, Albertini, Wallace, Anderson and Telfer2018; Atrabi et al., Reference Atrabi, Akbarinejad, Khanbabaee, Dalman, Andrade Amorim, Najar-Asl, Valojerdi and Fathi2019). Although in vitro activation of primordial follicles and in vitro production of metaphase II oocyte have been achieved successfully in postpubertal women (Telfer et al., Reference Telfer, McLaughlin, Ding and Thong2008; McLaughlin et al., Reference McLaughlin, Albertini, Wallace, Anderson and Telfer2018), in vitro activation of primordial follicles in prepubertal girls remains to be defined (Telfer et al., Reference Telfer, McLaughlin, Ding and Thong2008; Luyckx et al., Reference Luyckx, Scalercio, Jadoul, Amorim, Soares, Donnez and Dolmans2013; Telfer and Zelinski, Reference Telfer and Zelinski2013). Therefore, studies that identify the factors involved in the process of primordial follicle activation could help not only to improve the current technique for postpubertal women but also suit the technique for prepubertal girls (Adhikari et al., Reference Adhikari, Flohr, Gorre, Shen, Yang, Lundin, Lan, Gambello and Liu2009).
Phosphatase and tensin homologue (PTEN) is the main negative regulator of phosphoinositide 3-kinase (PI3K), thereby contributing to perpetuation of dormancy in primordial follicles (Kim, Reference Kim2012; Zhang and Liu, Reference Zhang and Liu2015). Upon inhibition of PTEN, PI3K/AKT signalling is activated, which would suppress tuberin/tuberous sclerosis complexes 1 and 2 (TSC1 and TSC2) (Kim, Reference Kim2012). TSC1 and TSC2 serve as negative regulators of the mammalian target of rapamycin (mTOR) pathway and, in turn, their inhibition triggers the mTOR pathway (Kim, Reference Kim2012; Zhang and Liu, Reference Zhang and Liu2015), which controls cell metabolism, growth, proliferation and survival (Laplante and Sabatini, Reference Laplante and Sabatini2009), and plays a pivotal role in activation of dormant primordial follicles (Adhikari et al., Reference Adhikari, Risal, Liu and Shen2013; Tong et al., Reference Tong, Li, Lu, Cao, Gao and Liu2013; Zhang et al., Reference Zhang, Risal, Gorre, Busayavalasa, Li, Shen, Bosbach, Brännström and Liu2014; Cheng et al., Reference Cheng, Kim, Li and Hsueh2015; Sun et al., Reference Sun, Su, He, Zhang, Liu, Zhang, Hou, Liu and Li2015). In this context, activation of mTOR has been reported to stimulate primordial follicle activation (Cheng et al., Reference Cheng, Kim, Li and Hsueh2015; Sun et al., Reference Sun, Su, He, Zhang, Liu, Zhang, Hou, Liu and Li2015). Conversely, inhibition of mTOR has been observed to prevent primordial follicle activation (Adhikari et al., Reference Adhikari, Risal, Liu and Shen2013; Tong et al., Reference Tong, Li, Lu, Cao, Gao and Liu2013; Zhang et al., Reference Zhang, Risal, Gorre, Busayavalasa, Li, Shen, Bosbach, Brännström and Liu2014), even in Pten-deficient mice, which are prone to massive and accelerated activation of primordial follicles (Chen et al., Reference Chen, Breen and Pepling2009).
PI3K/AKT signalling is not the only mechanism that activates the mTOR pathway as it also could be regulated by PI3K/AKT-independent mechanisms (Memmott and Dennis, Reference Memmott and Dennis2009; Li et al., Reference Li, Lee, Liu, Banasr, Dwyer, Iwata, Li, Aghajanian and Duman2010). In fact, the mTOR pathway integrates signals from nutrients, including amino acids, and growth factors to coordinately regulate cell growth and the cell cycle (Shamji et al., Reference Shamji, Nghiem and Schreiber2003; Fingar and Blenis, Reference Fingar and Blenis2004). In this regard, there is in vitro and in vivo evidence that some amino acids including leucine, arginine and glutamine can influence the mTOR pathway (Nakajo et al., Reference Nakajo, Yamatsuji, Ban, Shigemitsu, Haisa, Motoki, Noma, Nobuhisa, Matsuoka and Gunduz2004; Yao et al., Reference Yao, Yin, Chu, Liu, Deng, Li, Huang, Zhang, Tan and Wang2008; Durán et al., Reference Durán, Oppliger, Robitaille, Heiserich, Skendaj, Gottlieb and Hall2012; Jewell et al., Reference Jewell, Kim, Russell, Yu, Park, Plouffe, Tagliabracci and Guan2015; Chantranupong et al., Reference Chantranupong, Scaria, Saxton, Gygi, Shen, Wyant, Wang, Harper, Gygi and Sabatini2016; Ma et al., Reference Ma, Han, Li, Hu, Gilbreath, Bazer and Wu2017). Nevertheless, the effects of these amino acids on primordial follicles development are poorly understood. Therefore, the present study was conducted to evaluate the effects of leucine, arginine, glutamine or their combination on activation of primordial follicles and gene expression of corresponding factors in in vitro cultured ovaries of 1-day-old mice.
Materials and methods
Experimental groups and study design
Ovaries were harvested from 1-day-old mice following sacrifice by decapitation and were allocated randomly to different experimental groups. The ovaries were cultured in vitro for 11 days using different media including base medium (BM), base medium containing arginine (ARG), base medium containing glutamine (GLU), base medium containing leucine (LEU) or base medium containing a combination of arginine, glutamine and leucine (AGL). Histological and molecular assessments were implemented on days 5 and 11 of culture.
Ovarian tissue culture
The BM consisted of α-MEM supplemented with 10% fetal bovine serum (FBS) (Gibco), 50 μg/ml penicillin (Sigma, USA) and 50 μg/ml streptomycin (Sigma, USA). Amino acids including arginine (Sigma, USA), glutamine (Sigma, USA) and leucine (Sigma, USA) were added to the BM at a concentration of 200 µmol/l. Following preparation of the medium, ovaries were cultured in 96-well plates at 37°C under 5% CO2 and 95% air. Every other day, 75% of the medium of each well was changed.
Histological evaluation of the mouse ovaries
To calculate the rate of formation and activation of primordial follicles, cultured ovaries (n = 7) were fixed in Bouin’s solution and then in formalin. Subsequently, the fixed ovaries were dehydrated, embedded in paraffin, serially sectioned (6-μm thick sections) and stained with haematoxylin and eosin. For each ovary, the sections were serially assessed using a light microscope (Nikon Eclipse E200, Tokyo, Japan). Follicles were classified as primordial (oocytes with one layer of flattened granulosa cells), transitional (oocytes with one layer of flattened and cuboidal granulosa cells), primary (oocytes with a single layer of cuboidal granulosa cells) or preantral (oocytes with two or more layers of cuboidal granulosa cells). Additionally, the numbers of multi-oocyte follicles and follicles containing disproportionately large oocytes considering follicular stage were recorded.
Quantitative real-time PCR
The expression levels of genes associated with Pten, Pi3k, Tsc1, Tsc2, mTor, Gdf9, Bmp15, Zp3 and Cx37 were evaluated on days 5 and 11 of culture using quantitative real-time PCR (Table 1). Ovaries (n = 4) were collected and stored in RNAlater® at −80°C. Total RNA was extracted using the RNeasy plus Micro Kit – QIAGEN (Qiagen, Hilden, Germany), and was reverse-transcribed to cDNA using a standard oligo-dT reverse-transcription protocol in a reaction volume of 20 µl. From each cDNA sample, a 2 µl aliquot was used as a template for real-time PCR analysis. Each sample was run in duplicate. The Power SYBR Green PCR master mix (ABI) was used according to the manufacturer’s instructions. Real-time PCR was performed on an ABI Step One Plus real-time PCR machine (Applied Biosystems, Warrington, UK). The protocol was 40 cycles at 94°C for 10 min, 94°C for 30 s, and 60°C for 1 min. Fluorescence detection data were analyzed and normalized for mRNA levels to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels.
Mouse primer sequences for real-time PCR
Statistical analysis
Initially, binary variables including data associated with the proportion of different categories of follicles were arcsine-transformed prior to analysis. Data were analyzed using the GLM procedure. LSMEANS statement was used for multiple comparisons. All analyses were conducted using SAS version 9.4 software (SAS Institute Inc., Cary, NC, USA). Differences were considered statistically significant at P < 0.05.
Results
Development of ovarian follicles
Follicular formation was higher on day 5 than day 11 in BM, ARG and GLU groups (P < 0.05). On day 5, the number of follicles in the GLU group was greater than that in the AGL group (P = 0.043; Fig. 1). The proportion of primordial follicles was not influenced by the interaction of group and time (P > 0.05). The proportion of primordial follicles decreased from day 5 to day 11 (P < 0.0001). The proportion of primordial follicles in the LEU group was lower than in the the BM group regardless of day of culture (P = 0.017; Fig. 1). The proportion of transitional follicles was influenced by the main effect of time and increased from day 5 to day 11 (P = 0.001). On days 5 and 11, the proportion of transitional follicles was higher in the BM group compared with the ARG, GLU, LEU and AGL groups (P < 0.05; Fig. 1). The proportion of primary follicles was affected by the main effect of time and increased from day 5 to day 11 (P < 0.0001). On days 5 and 11, the proportion of transitional follicles was lower in the BM group compared with all other experimental groups (P < 0.01; Fig. 1). The proportion of preantral follicles in the ARG group increased from day 5 to day 11 (P = 0.007). Activation rate was not affected by the interaction of groups and time (P > 0.05). The proportion of activated follicles was higher on day 11 than day 5 (P < 0.0001). Regardless of day of culture, activation rate in the GLU and LEU groups was higher than that in the BM group (P < 0.05; Fig. 1).
(A) Ovarian structure of mice ovaries in culture media as well as their histological sections in base medium (BM), arginine (ARG), glutamine (GLU), leucine (LEU) and the combination of arginine, glutamine and leucine (AGL) groups on days 5 and 11 of culture. (B) Proportion of primordial, transitional, primary and preantral follicles in addition to follicular formation and activation in the BM, ARG, GLU, LEU and AGL groups on days 5 and 11 of culture.
The percentages of multi-oocyte follicles and follicles containing disproportionately large oocytes were not affected by treatment, time and interaction of treatment and time (P > 0.05; Fig. 2).
(A) Histological sections indicating the presence of multi-oocyte follicles (black arrows) in base medium (BM), arginine (ARG), glutamine (GLU), leucine (LEU) and the combination of arginine, glutamine and leucine (AGL) groups on days 5 and 11 of culture. (B) Histological sections indicate the presence of follicles that contained disproportionately large oocyte (white arrowheads) in base medium (BM), arginine (ARG), glutamine (GLU), leucine (LEU) and the combination of arginine, glutamine and leucine (AGL) groups on days 5 and 11 of culture. (C) Proportion of multi-oocyte follicles and follicles containing disproportionately large oocytes in BM, ARG, GLU, LEU and AGL groups on days 5 and 11 of culture.
Viability of different stages of follicles
The viability of primordial follicles on day 5 and day 11 was lower in the BM group compared with all other experimental groups (P < 0.0001; Fig. 3). The viability of transitional follicles in the ARG group was higher on day 11 than day 5 (P = 0.015). On day 11, the viability of transitional follicles in the BM group was greater than that in the GLU, LEU and AGL groups (P < 0.05; Fig. 3). The viability of primary follicles in all experimental groups was higher on day 11 than on day 5 (P < 0.001). On day 11, the viability of primary follicles in the BM group was greater than that in the ARG, GLU, LEU and AGL groups (P < 0.0001; Fig. 3). On day 5, no preantral follicle was observed in the BM and ARG groups and the viability of preantral follicles did not differ among the GLU, LEU and AGL groups (P > 0.05). On day 11, the viability of preantral follicles was lower in the BM group than in the ARG, GLU, LEU and AGL groups (P < 0.0001; Fig. 3).
Viability of primordial, transitional, primary and preantral follicles based on histological examination in base medium (BM), arginine (ARG), glutamine (GLU), leucine (LEU) and the combination of arginine, glutamine and leucine (AGL) groups on days 5 and 11 of culture.
Gene expression
The expression of Pi3k increased over the course of the culture in the GLU group (P = 0.002). In addition, Pi3k gene expression was greater in the GLU and AGL groups compared with the BM and ARG groups (P < 0.01; Fig. 4). On day 11, Gdf9 expression was greater in the LEU group compared with the BM, ARG, GLU and AGL groups (P < 0.05; Fig. 4). On day 11, the expression of Bmp15 in the LEU group was higher compared with that in the BM group (P = 0.0009; Fig. 4). On day 11, Zp3 expression was higher in the LEU group than in the BM and AGL groups (P < 0.05; Fig. 4). Nonetheless, treatment, time and interaction of treatment and time did not affect the expression levels of Pten, Tsc1, Tsc2, mTor and Cx37 (P > 0.05; Fig. 4).
Gene expression of Pten, Pi3k, Tsc1, Tsc2, mTor, Gdf9, Bmp15, Zp3 and Cx37 in base medium (BM), arginine (ARG), glutamine (GLU), leucine (LEU) and the combination of arginine, glutamine and leucine (AGL) groups on days 5 and 11 of culture.
Discussion
The present study revealed that incorporation of leucine, glutamine, arginine and their combination in culture medium promoted in vitro activation of primordial follicles in 1-day-old murine ovaries and this acceleration of primordial follicle activation did not culminate in any significant elevation in the proportion of multi-oocyte follicles or follicles containing disproportionately large oocytes. This finding is of significance, as in vitro activation of primordial follicles is still a serious issue in prepubertal girls that are subjected to oophorectomy and ovarian tissue cryopreservation prior to cancer therapy (Telfer et al., Reference Telfer, McLaughlin, Ding and Thong2008; Luyckx et al., Reference Luyckx, Scalercio, Jadoul, Amorim, Soares, Donnez and Dolmans2013; Telfer and Zelinski, Reference Telfer and Zelinski2013).
In addition to stimulation of morphological activation of primordial follicles towards subsequent stages, leucine augmented Zp3 expression, which contributes to interaction of the oocyte with sperm at fertilization and its expression commences concomitantly with activation of primordial follicles (El-Mestrah et al., Reference El-Mestrah, Castle, Borossa and Kan2002; Wassarman et al., Reference Wassarman, Jovine and Litscher2004; Litscher et al., Reference Litscher, Williams and Wassarman2009; Zhou et al., Reference Zhou, Yan, Shen, Cheng, Xi, Yuan, Fu, Ding, Luo and Wang2018). Furthermore, leucine elevated gene expression of Gdf9 and Bmp15, which belong to the transforming growth factor β superfamily and are involved in the development of primordial follicles (Gilchrist et al., Reference Gilchrist, Lane and Thompson2008; Otsuka et al., Reference Otsuka, McTavish and Shimasaki2011). It has been observed that Gdf9-null mice have small ovaries that are devoid of normal follicles beyond the primary stage (Dong et al., Reference Dong, Albertini, Nishimori, Kumar, Lu and Matzuk1996). There is also in vitro evidence indicating that GDF9 promotes the growth and differentiation of primary follicles (Hayashi et al., Reference Hayashi, McGee, Min, Klein, Rose, Duin and Hsueh1999; Nilsson and Skinner, Reference Nilsson and Skinner2002). Although Bmp15-knocked-out ovaries contained all follicle stages, these mice are subfertile due to defective ovulation processes and experience higher rates of follicular atresia (Yan et al., Reference Yan, Wang, DeMayo, DeMayo, Elvin, Carino, Prasad, Skinner, Dunbar and Dube2001). Moreover, it has been reported that GDF9 and BMP15 enhanced the proliferation of granulosa cells (Vitt et al., Reference Vitt, Hayashi, Klein and Hsueh2000; Otsuka et al., Reference Otsuka, Yao, Lee, Yamamoto, Erickson and Shimasaki2000; Huang et al., Reference Huang, Cheung, Zhang, Huang, Auersperg and Leung2009). It is believed that these oocyte-derived factors can act either individually or cooperatively (McNatty et al., Reference McNatty, Juengel, Reader, Lun, Myllymaa, Lawrence, Western, Meerasahib, Mottershead and Groome2005; Mottershead et al., Reference Mottershead, Ritter and Gilchrist2011; Otsuka et al., Reference Otsuka, McTavish and Shimasaki2011) and their signals are mediated via SMAD-dependent and SMAD-independent pathways (Gilchrist et al., Reference Gilchrist, Lane and Thompson2008; Huang et al., Reference Huang, Cheung, Zhang, Huang, Auersperg and Leung2009; Mottershead et al., Reference Mottershead, Ritter and Gilchrist2011; Otsuka et al., Reference Otsuka, McTavish and Shimasaki2011; Herbst et al., Reference Herbst, Baas, Kim, Felip, Pérez-Gracia, Han, Molina, Kim, Arvis and Ahn2016). Hence, it could be surmised that upregulation of Gdf9 and Bmp15 have contributed to acceleration of primordial follicle activation by leucine.
In ovaries treated with glutamine, increased activation of primordial follicles was accompanied with elevated gene expression of Pi3k. At somatic cell level surrounding the oocyte, activation of PI3K in squamous pre-granulosa cells inhibits TSC1 and TSC2, which leads to phosphorylation of mitogen-activated protein kinase (MAPK) culminating in stimulation of the mTOR pathway and upregulation of KIT ligand (Kim, Reference Kim2012; Zhao et al., Reference Zhao, Zhang, Li, Zheng, Xu, Yang, Xia and Zhang2018). Upon binding KIT ligand to its receptor on the oocyte, PI3K would be activated in the dormant oocyte, which triggers phosphorylation of FOXO3a and its subsequent exportation from the oocyte nucleus, which leads to activation of the primordial follicle (John et al., Reference John, Gallardo, Shirley and Castrillon2008; Zhao et al., Reference Zhao, Zhang, Li, Zheng, Xu, Yang, Xia and Zhang2018). Therefore, the positive effect of glutamine on activation of primordial follicles could be partly attributed to upregulation of Pi3k.
Arginine enhanced the activation of primordial follicles as well as leucine and glutamine, however it did not influence the expression of factors assessed in the present study. Accordingly, further research is required to decipher downstream mechanisms whereby arginine could expedite the development of primordial follicles.
Activation of primordial follicles in the combination group was comparable with that in other amino acid-treated groups, in which a single amino acid was added to the medium. Furthermore, the positive effect of glutamine on Pi3k expression was observed in the combination group. Nevertheless, the upregulatory influence of leucine on gene expression of oocyte-derived factors, including Gdf9, Bmp15 and Zp3, was not observed in the combination group. This phenomenon implicates that presence of arginine and/or glutamine might have disrupted the mechanisms through which leucine affected molecular behaviour of follicles.
None of the amino acids altered mTor expression or its negative regulators including Tsc1 and Tsc2 in the present study. Nevertheless, it could not be concluded that modification of mTOR complex activity in response to treatment with amino acids did not contribute to the promotion of primordial follicle activation. In this regard, stimulation of the mTOR pathway has been observed to enhance primordial follicle activation (Sun et al., Reference Sun, Su, He, Zhang, Liu, Zhang, Hou, Liu and Li2015) and inhibition of this pathway has been reported to attenuate the depletion of primordial follicles (Goldman et al., Reference Goldman, Chenette, Arju, Duncan, Keefe, Grifo and Schneider2017) even in the case of promoted PI3K signalling (Adhikari et al., Reference Adhikari, Risal, Liu and Shen2013). Furthermore, it has been indicated that arginine and leucine have intracellular sensors, namely CASTOR1 (Saxton et al., Reference Saxton, Chantranupong, Knockenhauer, Schwartz and Sabatini2016a; Wolfson et al., Reference Wolfson, Chantranupong, Saxton, Shen, Scaria, Cantor and Sabatini2016) and Sestrin2 (Saxton et al., Reference Saxton, Knockenhauer, Wolfson, Chantranupong, Pacold, Wang, Schwartz and Sabatini2016b; Wolfson et al., Reference Wolfson, Chantranupong, Saxton, Shen, Scaria, Cantor and Sabatini2016), respectively, which activate the mTOR pathway via inhibiting GATOR2 as a negative regulator of the mTOR complex 1 (Saxton et al., Reference Saxton, Chantranupong, Knockenhauer, Schwartz and Sabatini2016a,b; Wolfson et al., Reference Wolfson, Chantranupong, Saxton, Shen, Scaria, Cantor and Sabatini2016; Saxton and Sabatini, Reference Saxton and Sabatini2017). Yet, the influence of treatment with amino acids on the mTOR pathway was not evaluated in the present study, as this study was designed primarily to investigate the effect of these amino acids on primordial follicle activation and gene expression of the main corresponding factors. Indeed, further research is required to assess whether treatment with AGL can influence the mTOR pathway and its upstream regulators in ovarian follicles.
Regardless of the results associated with primordial follicles activation, a lower rate of viability was observed in transitional and primary follicles in the ALG, GLU, LEU and AGL groups compared with the BM group. Although apoptosis is an inextricable property of ovarian function and development (Hussein, Reference Hussein2005), it is believed that the rate of apoptosis in small activated follicles is generally negligible under normal conditions (Tingen et al., Reference Tingen, Bristol-Gould, Kiesewetter, Wellington, Shea and Woodruff2009). However, hyperactivation of primordial follicles through the PI3K/AKT/mTOR signalling pathway has been indicated to dramatically increase apoptosis in growing follicles (Chen et al., Reference Chen, Xia, Guan, Li and Zhang2016). Therefore, escalation of apoptosis in transitional and primary follicles of ovaries that have been treated with amino acids could have simply resulted from the accelerated rate of primordial follicle activation in the respective groups.
In conclusion, the present study revealed that addition of arginine, glutamine, leucine and their combination enhanced the activation of primordial follicles in the ovaries of 1-day-old mice under in vitro condition. Additionally, results associated with quantitative RT-PCR showed that leucine upregulated Gdf9 and Bmp15, while glutamine and the combination of amino acids elevated Pi3k expression. These findings could help to modify the culture medium for ovarian tissue to enhance the activation and development of primordial follicles towards production of metaphase II (MII) oocytes under in vitro condition.
Acknowledgments
The authors would like to express their gratitude to the staff at the Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran for their kind assistance.
Financial Support
Royan Institute supported this project scientifically and financially.
Conflicts of Interest
None
Ethical Standards
One-day-old NMRI mice were kept under controlled conditions (temperature: 20–25ºC; humidity: 40–60%) with a 12-h light cycle. All procedures described in the present study were performed under the approval of the Royan Ethics Committee (IR.ACECR.ROYAN.REC.1396.223).
