Supplementation of culture medium with nutrients, growth factors and hormones has crucial aspect for the culture of ovarian follicle. The medium considerably support both of the cell survival and proliferation. FIVG needs an optimized culture condition which can support the growth of CCs and finally oocyte maturation and follicle growth. In the present study we compared the effect of different basal and conditioned mediums, supplements, FFs and hormones on the in vitro growth of CCs. We tried to examine the majority of required components.
In the present study, different basal mediums with FBS (10% & 20%) and Alb (2% & 4%) on OCs and CCs, were compared. Regarding the OCs, our results showed that FBS is more efficient than Alb for in vitro proliferation of OCs and CCs. Also, FBS 20% was more superior to FBS 10%, while increases in Alb concentration could not support the growth of ovarian and CCs, alone. For OCs, in the presence of 10% or 20% FBS, α-MEM was the best medium. Whereas for CCs, if FBS 10% was used, α-MEM was the best medium, but if FBS 20% was applied, so DMEMF12 was superior. Also, the combination of both FBS and Alb had positive effect on OCs and CCs growth.
The mediums for FIVG are two kinds; basal medium and maturation medium. These mediums should support cell survival, proliferation and function. It should support both oocyte and CCs requirements. The applied media in FIVG are classified to: 1) basal media like MEM, Waymouth’s medium, DMEM and McCoy’s 5a medium 2) balanced salt solutions like Earle’s balanced salt solutions (EBSS) and 3) mixed media like DMEM + F12 and α-MEM + Glutamax (11).
Many studies on FIVG used α-MEM medium for short and long term culture in human and different animals (12) (13). While in our study we concluded that (DMEMF12 + 20%FBS) or (DMEMF12 + 20%FBS + 2%Alb) and (DMEM HG + 20%FBS + 2%Alb) showed higher cell viability and proliferation for CCs than α-MEM. We proposed (α-MEM + 20%FBS), DMEMF12 and DMEM LG for OCs and DMEMF12 and DMEM HG (+ FBS + Alb) for CCs. Regarding the DMEM medium, for OCs low glucose is superior but for CCS high glucose is better.
The culture mediums have some basic component including: amino acids, proteins and peptides, carbohydrates, fatty acids and lipids, vitamins, inorganic salt, serum, buffering systems [N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES), phenol red, co2 bicarbonate], trace elements and antibiotic. The details of five used mediums are presented in Table 3 (Table 3). α-MEM has the minimum essential material for cell culture, while DMEM and DMEMF12 have more components. We concluded that α-MEM + 20% FBS and DMEMF12 + 20%FBS can be used for the culture of ovarian and CCs, respectively. So, it seems that OCs can grow with the minimum essential materials but CCs need more constituents. Our results are parallel with some previous studies, that used DMEMF12 as the best media for OCs and CCs (14) (15). As well, in line with our results, Bukovsky et al. used DMEM HG and DMEMF12 in the presence of 20% FBS for in vitro oogenesis (16). Also, Yang et all used DMEMF12 supplied with 15% FBS for in vitro culture of porcine CCs (17). But unlike our study some other studies used α-MEM for the culture of ovarian follicles (18).
It is proposed that phenol red free DMEMF12 medium is superior, because phenol red is a weak estrogen with obvious biological effects (19). Also, it has shown detrimental effect on cat ovarian cryopreserved tissue (16). So, some studies proposed RPMI media for in vitro culture of CCs, because it is mostly free of phenol red (16). But our results did not show any beneficial effect with RPMI on CCs growth. RPMI1640 is the most enriched medium, followed by DMEMF12. Many studies suggested RPMI for the culture of normal and tumor cells. But in our study, we concluded that this enriched medium is not suitable for ovarian and CCs culture. Compare to DMEM, it has lower concentration of calcium (0.8 mM) and higher concentration of phosphate (5 mM) (20) and mostly it is phenol red free. Altogether, the main difference between RPMI and the other media is the absence of phenol red and calcium chloride (CaCl2). In cat, Brito et all reported that phenol red may led to follicular degeneration, especially in vitrification process (21). The domestic cats and dogs are good models for studying in vitro human ovarian follicular development. From this side, RPMI is a good medium but our results on CCs growth showed no advantages for this medium.
One of the differences between the cell culture mediums is in the pyruvate and glucose concentration. All mediums have pyruvate and were categorized to high and low glucose mediums. The amount of these components is important because the oocyte of growing follicle preferentially metabolises pyruvate over glucose, but the somatic compartments of ovarian follicles are more glycolytic. Glucose metabolism in cumulus/oocyte complex is very complicated during hormone-stimulated stages (22). The cells need glucose or pyruvate for their growth. It has been confirmed that most of the mammalian oocytes prefer pyruvate as energy substrate and have a low glycolytic activity. While, CCs prefer glucose over pyruvate. Primordial follicles consume 2-fold more pyruvate than glucose, probably due to the lowest numbers of surrounding CCs. In growing follicles (from primary to secondary stage), the CCs proliferated and increased in numbers, so glucose consumption and lactate production increase, as well. This pattern change around the time of antrum formation, with antral follicles become predominantly glycolytic (23). This may be the reason that in our study, DMEM HG was superior than LG for CCs culture.
Beside the basal medium component, CCs growth needs some other ingredients. They are categorized to gonadotropins, serums as the protein source, survival factors like ITS and growth/paracrine factors (Table 4) (11). So, in the present study we compared different kinds of serum (FBS, HSA and BSA), ITS, L-Glutamine and hormones. Since, the growth factors are expensive; we tried to compensate the absence of growth factors with FF and serums, which are enriched of various growth factors.
Another part of our study was on FSH like drugs. We concluded that 300mIU/ml Gonal-F, as a FSH like drug and 3.5IU/ml pregnyl (a FSH and LH like drug) have the most increasing effect on CCs growth. The growing follicles are FSH dependant and supplementation of FSH can improve both the follicle growth and oocyte maturation. So, FSH is not only necessary for CCs growth, but also essential for oocyte nucleus maturation. It causes more growth and differentiation in early antral follicles. Also, it is essential for the steroidogenesis and regulates the connection between oocyte and GCs (24). All the pervious researches increased the rate of follicle survival with addition of FSH to culture medium (11). The controversy is on the FSH dosages. A minimal concentration of 10 mUI/ml of FSH is essential for IVG of intact preantral follicles (24). Javed et all added 10–200 mIU/ml FSH to culture medium. They observed by addition of 100 mIU/ml FSH to culture medium, follicles survival, their diameters, germinal vesicles breakdown and oocyte maturation rates were increased (25). Silvia et al, reported that 10-µg/mL insulin and 100-µg/mL FSH can improve the in vitro meiotic resumption rate of caprine preantral follicles. Barros et al. used FSH with fixed or sequential concentrations. They showed that a sequential concentration of 750 ng/mL recombinant human FSH (compared to control 1000 ng/mL) improved oocyte and follicle growth and maturation (26). We compared 100-500mIU/ml Gonal-f and concluded that just 300 and 400mIU/ml concentrations increased CCs growth and viability, significantly.
Also, we compared several dosages of pregnyl. It is composed of alpha and beta subunits. The alpha is identical to LH, FSH and alpha subunit of thyroid stimulating hormone (TSH). So, its function is similar to both LH and FSH. LH plays an important role in the maturation of follicles, supports follicle development and induces ovulation. But it was not consider as necessary component of FIVG culture medium, but recently its positive effect on follicle growth is being actively debated (27). HCG and LH bind to the same receptor as FSH but activate different signalling pathway (28). Mediums supplemented with HCG stimulate in vitro maturation of oocyte. HCG has a more significant luteinizing effect compare to LH (18), while LH shows larger follicular diameters than HCG (29). Most studies used 1.5 IU/ml HCG (27), but our results showed that 3.5 IU/ml of HCG has more growing effect on CCs. It was shown that FSH and LH supplementation in a serum free medium caused the decrease of DNA fragmentation in GCs and increase of DNA fragmentation in theca cells (30). We concluded that HCG can lead to higher CCs proliferation than FSH, alone. The reason may be due to its slight LH like effect.
Serum is another component of the culture media. It causes the decrease of estradiol secretion with an unknown reason. It has been shown that during FIVG of in vitro preantral follicle, FSH steroidogenic and mitogenic effects changed (31). In the present study various kinds of serums were evaluated. They containe various growth factors and hormones. They help cell attachement to other cells and surfaces, so they act as spreading factor. They could be as a buffering agent and/or a binding protein. Finally they decrease the mechanical damages to the cells. Also, they have some disadvatages including different compositions in various samples. It may induces some inhibitory factors and the risk of contamination (31).
We evaluated three kinds of serums for CCs growth (FBS, HSA and BSA). FBS has some advantages in cell culture including stimulatory factors and low concentration of immunoglobulin (32). BSA is shown to improve in vitro development of follicles (33). In simplified medium, replacement of FBS with BSA creates a defined medium that provides better conditions for oocyte in vitro maturation (24). Regarding HSA, it was shown that HSA + ITS cause the decrease of atretic follicles and increase of healthy follicles rate and follicle size. Also, HSA promotes cell proliferation and act as free-radical and reactive oxygen species (ROS) scavengers (24). We added some concentrations of HSA and BSA from 3–25 mg/ml. Compared to control, all the concentrations increase CCs proliferation and viability. Unlike HSA, 3 and 6 mg/ml of BSA increased the CCs growth, significantly. Also, we concluded that the HSA alone had no beneficial effect on the growth of ovarian and CCs, whereas in combination with FBS it caused considerable growth.
Also, the CCs need amino acids. L-Glutamine is an essential amino acid for both ovarian and CCs. Generally, its amount is adjusted to be around 2 mM, but it can vary from 0.5 to 10mM, depending on the cell and media types (vary from 0.68 mM in medium 199 to 4mM in DMEM). It was shown that arginine, glutamine and leucine, used in the ovarian tissue culture, can accelerates in vitro activation of primordial follicles in 1-day-old mouse ovary (34). Some combinations of glutamine are available including: L-glutamine, GlutaMax or GlutaGro. L-glutamine is nearly unstable and degrades over time in refrigerator and more rapidly in incubator, but GlutaMax is more stable. So, addition of an extra amount of L-glutamine to culture medium wouldn't harm the cells, even it may be necessary component especially for media close to its expire date (35). Glutamine supports the growth of cells which have high energy requirements. Also, it synthesizes large amounts of nucleic acids and proteins. It acts as an alternative energy source for the cells that use glucose inefficiently or for rapidly dividing cells. So, when glucose level is low but energy demand is high, cells can metabolize amino acids like glutamine, as the most readily available amino acids for use as energy (https://www.sigmaaldrich.com/life-science/cell-culture/learning-center/media-expert/glutamine.html). Most of the mediums are supplemented with 2mM/ml L-Glutamine (36) (37). In the current study we used 4-37mM/ml L-Glutamine. The results showed that 12 and 17 mM/ml L-Glutamine caused higher CCs growth compare to 2mM/ml. But more than 22mM/ml was detrimental for CCs growth. Altogether, L-Glutamine accelerate in vitro growth of CCs and in vitro activation of primordial follicles (37).
ITS or insulin, transferrin and selenium are other components that used in FIVG. Glucose is an essential factor for CCs growth. Its metabolism is influenced by ovarian growth factors and insulin. Insulin also increases the uptake of metabolic precursors like amino acids. So, for FIVG insulin is added to culture medium at the dosage of 5 mg/ml (supra physiological concentration). Insulin together with selenium and transferrin act as survival factor. But, high amount of insulin, mimics insulin resistance model, encouraged apoptosis in GCs (38).
Selenium is another essential trace mineral which is relevant to various patho-physiological processes (38). It is added to IVG medium, as well. But high doses of selenium reduce the rate of proliferating primordial follicles. It regulate the 17β-estradiol bio-synthesis and the growth of the GCs in adult ovaries (39). Heat stress induces apoptosis in various cells. Selenium has effects on maintaining the cellular physiologic functions and protects the cells against chronic heat stress induced apoptosis in GCs (40).
Transferrin is the carrier of iron, a mandatory requirement of the cells. These cells should have transferrin receptor for intracellular transport of iron. It was expressed in a subpopulation of human granulosa lutein cells which are isolated from follicular puncture. Normally, transferrin and iron concentration increase in FF with advance in follicular maturation. Transferrin insufficiency causes iron overload and leads to oocyte dysmaturity. Small growing follicles with 1–2 layers of GCs have cytoplasmic transferrin in their cuboidal GCs (41).
Previous studies used various dosages of ITS from 10 ng/ml to 10µg/ml. We used four concentrations of 5 & 10 ng/ml to 5 & 10µg/ml. Our results showed that 10 ng/ml followed by 5µg/ml caused to higher GCs viability and growth.
Also, we compared the effect of FF on CCs growth. FFs were isolated from 6 groups of patients. The results showed that 50% and 75% FFs, isolated from male factor infertility patients (healthy women) increased CCs growth significantly, followed by 50% FF of unknown cause infertility patients. Also, 50% FF was superior to 75%. FF has estradiol, progesterone and testosterone. The level of steroids is correlated with follicular diameter. According to Wed et al. study, FF progesterone level is 6100 times more than estradiol and 16900 times higher than testosterone. FF has two kinds of vesicles, [exosomes (50–150 nm) and microvesicles (100–1000 nm)]. The content of these vesicles is bioactive components such as proteins, mRNAs, lipids and miRNAs (42). FF is a plasma filtrate with a large dynamic range of proteins. These proteins are involved in metabolic processes, cellular processes, cellular communication and immune responses (43). Also, FF has some kinds of GCs, called immortalized GCs (44). Although the FFs from PCO patients, endometriosis, advanced aged patients and poor responders did not have positive effect of CCs growth and viability, they did not cause negative effect, compared to control group, as well.
In the present study, the CMs, collected from ovarian cortical cells significantly improve the CCs growth. It is not far from mind, because cortex is the natural place of ovarian follicles which makes its micro environment. But it is interesting that the mixture of medullar and hilus CMs also caused the CCs growth as much as the cortical CMs. This data proposes this idea that probably ovarian medulla and hilum are as important as ovarian cortex for follicular growth.
Also, our results demonstrated that the isolated cells of SB, cortex, medulla and hilum expressed GDF9. It is essential factor for follicular symphony and acts as a regulators of ovulation, folliculogenesis and oocyte quality (45). Interestingly, its expression was higher in medulla and hilum. Regarding the poor responder patients, it was shown that the expression of GDF9 is decreased by age in FF and GCs which results to decrease in assisted reproductive outcomes (46). Also, GDF9 enhances the proliferation and metabolism of CCs and GCs and acts as a CCs expansion factor (47). In mouse, GDF9 promotes the development of CCs by help of oestrogen (48). In our study the addition of ovarian cells CMs could enhance the in vitro proliferation of CCs. One of the suggested reasons may be related to GDF9. In our studies the cells of cortex, medulla and even hilum expressed GDF9 and the results of MTT of CMs on CCs demonstrated that both cortex and a combination of (hilum and medulla) CMs gave the same results.
Also, CMs, collected from ADMSCs + AFDCs has positive effect on CCs growth, but this effect was not as much as that of the OCs. In parallel to our study, the previous studies used CM, collected from mesenchymal stem cells, for in vitro maturation and subsequent development of oocyte (49). It was shown that ATDCs can promote the early stages of follicles survival, growth and maturation. They secret factors which promote the early stages of follicle growth (50). Also, AFDCs have shown to preserve ovarian follicle after chemotherapy (51) and they have potential to differentiate into primordial follicle (52). In a study on the expression of ovarian cells mesenchymal markers (still not published data), we concluded that the cells of all part of ovary expressed mesenchymal markers. So, probably another reason that CM of OCs could support CCs growth is the mesenchymal nature of these cells.
They are many unknown factors which are involved in ovarian follicular development. By applying FFs and CMs, which are enriched of growth factors, hormones and the other efficient elements, we can better support the follicular growth, with the lower cost.