Role of Melatonin as a Survival Factor for In vitro Development of Sheep Preantral Follicles

It was well established that mammalian preantral follicles do not grow in vitro as well as they do in in vivo (Arunakumari et al., 2010; Anil kumar et al., 2019). Two major reasons for such compromised development of preantral follicles are:1) Oxidative stress encountered during in vitro culture due to free radicals generated by endogenous process such as normal cellular metabolism and exogenous factors such as chemicals added to culture media, hyperoxia, exposure to light etc. (Natarajan et al., 2010) and 2) Frequent apoptosis of granulosa cells in culture. This indicates the need for presence of antioxidants in culture medium like Melatonin, a powerful free radical scavenger. Melatonin, a tryptophan containing hormone is synthesized predominantly in pineal gland, retina, gut and its production is also observed in the ovary in both cumulus cells and oocytes (Cassone et al., 1997; Soares et al., 2003). Earlier reports evidenced the presence of melatonin receptors in the ovarian follicular cells and follicular fluid (Soares et al., 2003) indicating the role of Melatonin in folliculogenesis (Taketani et al., 2011), oocyte quality and maturation capacity (Adriaens et al., 2006; Rodriguez et al., 2007) and maintenance of follicular integrity (Barros et al., 2013). Previous studies also indicated that melatonin was potentially useful in culture condition as it was a powerful free radical scavenger and a broad-spectrum antioxidant (Reiter et al., 2001; Nakamura et al., 2003; Tan et al., 2007) and it may directly affect ovarian function thereby serving as a local regulator in rats (Sakaguchi et al., 2013). Although existing information suggests that melatonin might control ovarian folliculogenesis, only meager reports are available indicating its direct role, especially on preantral follicles development to ovulatory stage in sheep. Accordingly the objective of the present study includes a) Standardization of optimum concentration of Melatonin that supports better development of sheep PFs’ in vitro and b) Interaction of Melatonin with other hormones and growth factors and their influence on in vitro growth of sheep PFs’.

Unless otherwise stated, culture media, hormones, growth factors, FCS and all the other chemicals used in this study were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and plastics from Nunclon (Roskilde, Denmark). All the methods described briefly hereunder are routinely employed in the culture of PFs’ in the laboratory and described in detail in several earlier publication from the laboratory (Arunakumari et al., 2010; Kamalamma et al., 2016; Kona et al., 2016; Srividya et al., 2017; Anil kumar et al., 2019; Sravani pragna et al., 2020).
 
Isolation of preantral follicles (PFs’)
 
A total of 175 ovaries and 790 preantral follicles collected on different days were used in this study. Ovaries recovered after sheep slaughter were transported to the laboratory within 1h after slaughter in sterile, warm (37^oC) phosphate buffered saline.

Intact preantral (PFs’) in the size range of 250-400 µm with intact basement membrane were mechanically isolated (Fig 1A) by micro dissection method from ovarian cortex under a stereo-zoom microscope (SMZ 2T, Nikon corporation, Japan) and cultured them for six days (Fig 1B, 1C, 1D) according to the methods developed in our laboratory.


 
Determination of optimum concentration of Melatonin for in vitro culture of Sheep PFs’ (Experiments 1 and 2)
 
Preantral follicles were cultured in bicarbonate buffered tissue culture medium 199 supplemented with 50 mg/mL Gentamycin sulfate (TCM 199B) and different concentrations of Melatonin (M 5250) chosen on the basis of the results obtained in earlier studies on IVM of mouse oocytes (Nabiuni et al., 2013).  Two separate experiments with different ranges of concentrations of Melatonin (Experiment: 1- 0, 5, 10, 25, 50, 100, 500 pM and Experiment: 2- 25, 50, 100, 250, 500 pM) were conducted.
 
Evaluating the effect of Melatonin in combination with other growth factors and hormones (Experiment 3)
 
To ascertain whether Melatonin supplementation to the culture medium encourage reasonable development and further improvement of cultured PFs’ in sheep three differently supplemented media were used to culture the PFs’: (1) TCM 199 B medium containing 50 mg/mL gentamycin sulphate (control), (2) control medium supplemented with 1 mg/mL T4, 2.5 mg/mL FSH, 10 ng/mL IGF-1, 1 mIU/mL of GH (Arunakumari et al., 2010) (referred to as the standard medium hereafter) and (3) standard medium supplemented with 100 pM of Melatonin (M 5250).
 
Evaluation of growth and In vitro maturation of oocytes obtained from cultured PFs’
 
The cultured PFs’ were morphologically evaluated every 24 h for growth, increase in the diameter and antrum formation using an inverted microscope (Leica, DMIRB, Germany).  The cumulus oocyte complexes from PFs’ cultured for six days (Fig 1E) were further subjected to In vitro maturation for another 24 h.  Subsequently the oocytes were washed in Hoechst 33342 fluorescent stain solution (B 2261) and incubated in a 50 µl droplet of the same solution for 15 minutes at 39^oC (Rao et al., 2002) which were examined under fluorescent light on an inverted microscope (Leica, Germany; excitation: 352-455 nm and emission 460-490 nm) for M II stage (Fig 1F).
 
Statistical analysis
 
The dependent variables are the development parameters of the follicles, and independent variables are the different treatments in the experiments. Comparison of the proportion of PFs’ exhibiting growth, average increase in diameter, antrum formation and maturation to MII stage of the oocytes from the in vitro cultured PFs’ among different treatment groups were undertaken separately by one way ANOVA followed by Duncans multiple range test using SPSS 20 software.

To our knowledge, this is the first study to address the influence of melatonin on in vitro development of preantral follicles. A total of seven hundred and ninety (790) preantral follicles were cultured in vitro in control, standard medium and melatonin treated groups. In experiment I, six different concentrations of melatonin (5-500pM) on in vitro development of PFs’ were investigated individually and it appeared that melatonin at a concentration of 100 pM favorably influenced the growth of PFs’, average increase in diameter, exhibiting antrum formation and meiotic maturation percentage of oocytes to metaphase II from cultured PFs’.  (Table 1). Previous studies by Shi et al., (2009) and El-Raey et al., (2011) support this observation wherein they indicated that higher levels of melatonin in the fluid of preovulatory follicles in porcine and bovine ovaries respectively might play an important role in process of ovulation. In contrast Ganji et al., (2015) reported that addition of 100 pM melatonin in the culture medium had negative effect on their survival, growth and diameter in mouse which might be due to species variation. The proportion of PFs’ exhibiting growth and average increase in diameter was similar in the PFs’ cultured in TCM199B supplemented with 50pM and 100pM but the proportion of PFs’ exhibiting antrum formation was significantly greater at 100 pM of Melatonin (Table 1). Although 25, 50 and 250 pM of melatonin supplementation appeared to induce greater increase in growth, diameter and higher proportion of the follicles exhibiting antrum formation, it was not statistically significant. Therefore another experiment involving the concentrations of 25, 50, 100, 250 and 500pM was undertaken. However, even in experiment II, 100pM of melatonin supported the best development thereby confirming that this might be the optimum concentration for in vitro development of preantral follicles (Table 2).





In our study, among the melatonin treatments only the oocytes collected from preantral follicles cultured in 100 pM of melatonin reached to MII stage and interestingly 5, 10, 25, 50, 250 and 500 pM concentrations of melatonin failed to support oocytes from cultured follicles to reach MII stage (Table 2). It may be speculated from previous studies that melatonin at 100 pM could promote better expression of MT1 and MT2 receptors via c-AMP mediated signaling in the cumulus oocyte complexes or stimulation of maturation promoting factor of oocytes and in cumulus cells that positively affected the cytoplasmic and nuclear maturation (Soares et al., 2003; Chattoraj et al., 2005; El-Raey et al., 2011).

Further in Experiment III, 100 pM concentration of melatonin was used in combination with growth factors and hormones to evaluate its action in inducing preantral follicular development with respect to parameters studied. While the average increase in diameter was similar in the PFs’ cultured in standard medium and standard medium supplemented with 100 pM of Melatonin, the proportion of PFs’ exhibiting growth, antrum formation and meiotic maturation of oocytes to M-II were found to be significantly higher in standard medium supplemented with 100pM of Melatonin than in standard medium alone or in control medium (Table 3).  It is remarkable to note that inclusion of melatonin (100 pM) in the standard medium (i.e., TCM 199B supplemented with FSH, T4, IGF-I and GH) supported better development and also resulted in highest proportion of oocytes reaching M-II from cultured follicles than in all other treatments. This could be due to the protective ability of melatonin on the cultured PFs’ by modulating the growth factor expression (Schaeffer et al., 1997), up-regulating the anti-apoptotic genes (Fu et al., 2014) or by defending the oxidative damage to M-II oocyte spindle (Banerjee et al., 2012), therefore, indicating the positive influence of melatonin when added in combination with growth factors on the follicular development as was reported earlier (Wang et al., 2013; He et al., 2016).



From this study we observed that melatonin supplementation at an optimum dose of 100 pM significantly improved the in vitro development of sheep PFs’ and the yield of fertilizable oocytes from cultured PFs’. Concomitant supplementation of melatonin in the standard medium supported better growth of cultured preantral follicles. Therefore, it is concluded that melatonin (100 pM) acts as survival factor for in vitro development of preantral follicles in sheep.

From this study we observed that melatonin supplementation at an optimum dose of 100 pM significantly improved the in vitro development of sheep PFs’ and the yield of fertilizable oocytes from cultured PFs’. Concomitant supplementation of melatonin in the standard medium supported better growth of cultured preantral follicles. Therefore, it is concluded that melatonin (100 pM) acts as survival factor for in vitro development of preantral follicles in sheep.

The authors thank Sri Venkateswara Veterinary University (S.V.V.U), Tirupati for providing the chemicals and laboratory facilities to conduct this work.

All authors declare that they have no conflict of interest.