Expression and localization of adiponectin and its receptors in ovarian follicles during different stages of development and the modulatory effect of adiponectin on steroid production in water buffalo

Abstract

Adiponectin is an adipocyte-derived hormone regulating energy metabolism, insulin sensitivity and recently found to regulate reproduction. The current study was carried out to investigate gene and protein expression, immunolocalization of adiponectin and its receptors AdipoR1 and AdipoR2 in ovarian follicles of different developmental stages in water buffalo (Bubalus bubalis) and to investigate the effect of adiponectin on steroid production in cultured bubaline granulosa cells. qPCR, western blotting and immunohistochemistry were applied to demonstrate mRNA expression, protein expression and immunolocalization, respectively. The results indicate that adiponectin, AdipoR1 and AdipoR2 were present in granulosa cells (GC) and theca interna (TI) of ovarian follicles and the expression of adiponectin, AdipoR1, AdipoR2 in GC and AdipoR1 and AdipoR2 in TI increased with increase in follicle size (p < .05). Expression of adiponectin was high in small and medium size follicles in TI. The adiponectin and its receptors were immunolocalized in the cytoplasm of GC and TI cells. Further, in the in-vitro study, GCs were cultured and treated with recombinant adiponectin each at 0, 1 and 10 µg/ml alone or with follicle stimulating hormone (FSH) at 30 ng/ml) or Insulin-like growth factor I (IGF-I) at 10 ng/ml for 48 hr after obtaining 75%-80%s confluency. Adiponectin at 10 µg/ml increased IGF-I-induced estradiol (E 2 ) and progesterone (P 4 ) secretion and FSH-induced E 2 secretion from GC and also increased the abundance of factors involved in E 2 and P 4 production (cytochrome P45019A1 [CYP19A1] and 3-beta-hydroxysteroid dehydrogenase [3β-HSD]). In conclusion, this study provides novel evidence for the presence of adiponectin and its receptors in ovarian follicles and modulatory role of adiponectin on steroid production in buffalo.

​Localization of Orexin and its Receptor in Corpus Luteum and in vitro Effect of Orexin on Progesterone Production from Luteal Cells in Buffalo

“…For separation of CL, ovaries (n=40), each with the CL, were used (10 CL per group) for studies. The CLs from each ovary were classified in the following stages of functionality: CL1, early luteal phase (days 1-4); CL2, mid-luteal phase (days 5-10); CL3, late luteal phase (days 11-16) and CL4, regressing CL (days >17) of the estrous cycle (Gupta et al, 2019).…”

“…The intact CL of each stage was carefully dissected from the ovaries. Fixation and processing of the slide were performed as per the method described by Gupta et al (2019). Five representative CLs of each group were used for immunohistochemistry.…”

“…Luteal cells were cultured for determining the effect of orexin on progesterone by the method described by Gupta et al (2019). The minced luteal tissue was washed three times for 5 min at 350g with a dispersing medium.…”

“…The RT-qPCR procedure was used to detect CYP11A1, StAR and HSD3B1 in luteal cells of buffalo. Published primers were used for StAR, HSD3B1 (Kumar et al 2012), β-actin (ACTB), ribosomal protein 15 (RPL15) and cytochrome P450 (CYP11A1) (Gupta et al 2019). Details of the primers used, primer efficiencies and annealing temperature are provided in Table 1.…”

“…Pituitary gonadotropins well regulate the functions of CL. However, a number of local factors have also been found to regulate luteal dynamics (Gupta et al, 2014;Gupta et al, 2019;Thakre et al, 2021).…”