Leptin Supplementation Stimulates Synergism with Growth Factors and Hormones to Express Its Receptor in Cultured Preantral Follicles of Sheep

Leptin, a metabolic hormone plays an important role in reproductive functions like ovarian folliculogenesis, oocyte maturation (Kamalamma et al., 2016) and modulation of steroidogenesis (Anil Kumar et al., 2019). Recently its effect on development of preantral follicles was studied and found that optimum dose of Leptin that supported the growth of sheep PFs’ in vitro was 10ng/mL (Kamalamma et al., 2016).  It favored the expression of antiapoptotic genes and suppressed apoptotic gene during different stages of follicle development and also improved aromatase gene expression, an enzyme essential for conversion of Androgens to Estrogens (Lakshminarayana et al., 2014).
       
Leptin receptor (LepR) is a transmembrane receptor with six isoforms (Ob-Ra, Ob-Rb, Ob-Rc, Ob-Rd, Ob-Re, Ob-Rf), of which Ob-Rb is longest form and its mRNA expression was studied in preovulatory follicles and corpus luteum (Smolinska et al., 2013) indicating its ability to regulate reproduction at molecular level. Since for action of any hormone on target cell and to have local action on any tissue, expression of its own receptor is necessary and also it is not known whether such improvement in ovarian follicular development by Leptin is mediated through presence of its homologous receptors in the sheep ovaries as a local factor. Therefore present study is undertaken with an idea of understanding the evolution of Leptin receptors as the follicular development progresses from preantral follicles to ovulation stage. Moreover this will enable designing of appropriate media to culture preantral follicles by inclusion of Leptin along with different growth factors and hormones.

All the methods employed in this study were described in detail in several earlier publications from Embryo Biotechnology Laboratory, College of Veterinary Science, Sri Venkateswara Veterinary University, Tirupati. However, a brief description with respect to isolation and culture of PFs’ (Arunakumari et al., 2010), RNA isolation (Lakshminarayana et al., 2014), reverse transcription and RT-PCR (Kona et al., 2016; Srividya et al., 2017; Anil kumar et al., 2019) is given hereunder. 
 
Collection of ovaries and isolation of different stages of ovarian follicles
 
A total of 480 ovaries collected from sheep slaughter house located in Gandhi road, Tirupati on different days during 2018 were used in this study. Ovaries recovered were transported to the laboratory within 1h after slaughter in sterile, warm (37oC) phosphate buffered saline.
       
In vivo grown intact preantral (PFs’), early antral, antral, large antral follicles and cumulus oocyte complexes (COCs) were mechanically isolated (Fig 1 A, C, E, G) by micro dissection method from ovarian cortex (Group I; 25 replicates) under a stereo-zoom microscope (SMZ 2T, Nikon corporation, Japan) according to the methods developed in our laboratory (Arunakumari et al., 2010) and different development stages of follicles were shown in Fig 1.
 

 
Culture media
 
The in vitro groups were divided based on the type of medium used to culture the preantral follicles (PFs’). PFs’ cultured in TCM 199B without any supplementation (Group II; 30 replicates), TCM 199B supplemented with 10ng/ml of ovine leptin (My Biosource Cat No. MBS142116, USA) (Group III; 30 replicates) and TCM199B supplemented with  gentamycin sulphate (50µg/ml), Thyroxine (1µg/ml), Follicle stimulating hormone (2.5µg/ml), Insulin like Growth factor-1 (10ng/ml), of growth hormone (1MIU/ml) and ovine Leptin (10ng/ml) (Group IV; 30 replicates).
 
Selection and culture of the preantral follicles and in vitro maturation (IVM) of the oocytes obtained from the in vivo grown and cultured large antral follicles
 
Preantral follicles in the size range of 250-400µm with intact basement membrane were selected for the culture (Fig  1A). All the subsequent methods of culture and medium preparation were followed as in previous studies in the laboratory (Arunakumari et al., 2010; Kona et al., 2016).
       
The cumulus cells and oocytes were collected from different stages of in vivo grown follicles (Group I; Fig 1 A, E, I, M, Q, I) and their corresponding in vitro stages i.e., PFs’ cultured in Group II (Fig 1 B, F, J, N, R), Group III (Fig 1 C, G, K, O, S), Group IV (Fig 1 D, H, L, P, T) for 3min, 2, 4 or 6 day cultured follicles and COCs from 6 day cultured follicles (Fig 1 J).
 
Isolation and quantification of total RNA
 
Isolation of RNA was carried out by pooling cumulus cells and oocytes from 30-50 follicles at each development stages of follicles using patented Medox-Easy spin column Total RNA Mini prep Kits (13, 15, 16) according to the manufacturer’s instructions (Medox Biotech India Pvt. Ltd., Chennai, India). Concentration and purity of RNA was determined using Nanodrop lite (Thermo scientific S.No.1354). RNA samples having purity (Absorbance at 260/280) in the range of 1.8-2.1 only were used in the expression studies. The RNA sample was stored at -70oC till analyzed.
 
Reverse transcription (RT) and real-time PCR
 
High capacity reverse transcription kit (Applied Biosystems part no: 4368814) was used for the reverse transcription. RT reaction was carried out for 10 minutes at 25oC, for 120 minutes at 37oC and for 5 minutes at 85oC in a thermocycler (Eppendorf Mastercycler Gradient) according to the manufacturer’s instructions.
       
Quantitative expression of Leptin receptor gene was studied at different development stages of the in vivo grown and corresponding stages of the three groups of cultured ovarian follicles (Table 1). The geometric means of RPLPO, HPRT1 and 18SrRNA genes (17) were used as the normalizer in the analysis of Leptin receptor gene expression. The mRNA sequence for reference genes (RPLPO, HPRT1 and 18SrRNA) and target gene (Leptin receptor) were retrieved from Genbank and custom designed 20X gene expression assays mixes for these genes were obtained from Applied Biosystems (Assay on Demand lot nos: 1402507 A2, A3, A8, 1428816). The primers and probe sequence details of reference genes (RPLPO, HPRT1 and 18SrRNA) and target gene (Leptin receptor) are given in the Table 2. Real-time RT-qPCR was performed on Applied Biosystems 7500 machine. Each 25 µl reaction mix contained 12.5 µl of Taq Man Uni-versal PCR Master Mix (2x), 1.25 µl of 20X gene expression assay mixture, 10 ng of cDNA sample in nuclease free water. Thermal cycling conditions were Erase UNG (Uracil N- glycosylase) activation 2 min @ 50oC, Ampli Taq Gold DNA polymerase activation10 min @ 95oC followed by 40 cycles of 15 s @ 95oC and 1 min @ 60oC. For the calculation of the expression levels (RQ values) of different target genes, first the Ct values of target and reference genes were converted to quantity inputs by using the formula 2 minimum Ct-sample Ct.
 

 
Statistical analysis
 
The entire experiment of Leptin receptor gene expression was repeated twice and the data were pooled after Barlett’s test confirmed the homogeneity of variances.  Log RQ values were analysed by two-way ANOVA with unequal number of observations followed by Tukey multiple comparisons test (SPSS version 20). P values ≤ 0.05 were considered significant.

This is the first report on the Leptin receptor gene expression during different development stages of sheep ovarian follicles grown in vivo or preantral follicles (PFs’) cultured in vitro in three different conditions. A total 2460 follicles were isolated to conduct this study. Leptin receptor gene was expressed in both cumulus cells and oocytes collected from all the groups (Table 1).  In group I, cumulus cells from large antral follicles showed highest expression with an increased trend from early antral to large antral follicles.  These results are similar to findings of Gregoraszczuk et al., (2006) in porcine ovary who reported 6 folds increase in the Leptin receptor expression in antral follicles than early antral follicles, indicating a possible involvement of Leptin in follicular development. Batista et al., (2013) also found that Leptin receptor mRNA expression levels were higher in the granulosa cells of large antral follicles than corresponding cells from small antral follicles.  In our laboratory recent study indicated that estradiol concentration followed an amplified fashion from PFs’ to large antral follicles in in vivo grown sheep ovarian follicles (Anil kumar et al., 2019). In this study also the pattern of expression of Leptin receptor gene in the cumulus cells and oocytes from the PFs’ cultured in group II appeared to be similar to gene expression of aromatase, an enzyme essential for synthesis of estrogen hormone (Lakshminarayana et al., 2014). Therefore increase in the expression of Leptin receptor in later stages of follicular development than early stages might be an influence of estrogen hormone on Leptin receptor expression (Meli et al., 2004; Chen et al., 2006; Garofalo et al., 2006).
       
Leptin receptor gene was expressed in all the stages both in the cumulus cells and oocytes when Leptin was added to culture medium (Table 1). In group III Leptin receptor gene expression was down regulated in PFs’ exposed to 3 min and 2 day cultured follicles and expression was similar in four day and six day cultured PFs’ in cumulus cells as compared to group-II. While in oocytes of PFs’ cultured in group III the expression was upregulated as compared to the group II grown follicles except oocytes in PFs’ exposed to medium for 3 min. However, decreased expression in early stages and upregulated expression of Leptin receptor at later stages of both cumulus cells and oocytes in PFs’ grown in group III could be due to differential action of Leptin on its own receptor expression as reported by cordova et al., (2011).
       
The expression levels in PFs’ grown in group III and group IV were almost similar but higher when compared to group I or group II (Table 1). We observed that LepR expression was highest in the cumulus cells from COCs after IVM and oocytes from preantral follicles cultured for four days in group IV as compared to all other groups (Table 1). The expression levels of Leptin receptor of  preantral follicles (PFs’) grown in group I and IV was similar in all stages of development except COCs after in vitro maturation which was higher in group IV (Table 1). Such significantly higher expression of Leptin receptor in antral and large antral follicular stages of PFs’ grown in group IV could be due to synergistic effects among Leptin, IGF, GH, FSH and Thyroxine in the culture medium (Gregoraszczuk et al., 2006). In both cumulus cells and oocytes the Leptin receptor gene was down regulated from preantral stage to early antral stage in all the groups, which might be due to less requirement of Leptin signalling mechanism at this particular stage of transition. Thus supplementation of Leptin along with other hormones or growth factors stimulated its receptor gene expression better than in vivo or other media used to culture the preantral follicles.

It is concluded that Leptin stimulated its own receptor expression in both cumulus cells and oocytes from early antral stage to large antral stage. Further this stimulatory effect of Leptin on its own receptor showed synergism in the preantral follicles cultured in medium containing leptin along with other growth factors and hormones.

This work was supported by a research grant from the council of Scientific and Industrial Research (grant 37 (1612)/13/EMR-II) to A.V.N. Siva Kumar.