Effect of Leptin on In Vitro Nuclear Maturation and Apoptosis of Buffalo (Bubalus bubalis) Oocyte.
BACKGROUND
Leptin, as a 16 kDa adipokine, is a pleiotropic cytokine-like hormone that primarily secreted from adipose tissue. It also involves in the regulation of energy homeostasis, neuroendocrine function, immunity, lipid and glucose homeostasis, fatty acid oxidation, angiogenesis, puberty and reproduction. The aim of this study was to investigate the effects of in vitro addition of leptin to in vitro maturation (IVM) medium on buffalo oocyte maturation and apoptosis.


METHODS
In this experimental study, Ovaries from apparently normal reproductive organs of slaughtered adult buffaloes (Bubalus bubalis) with unknown breeding history were collected from Urmia Abattoir, Urmia, Iran, and were transported immediately to the laboratory in a thermos flask containing sterile normal saline with added antibiotics. Oocytes were aspirated from 2-8 mm visible follicles of the ovaries using an 18-G needle attached to a 10 ml syringe. IVM medium included tissue culture medium-199 (TCM-199), 10% fetal bovine serum (FBS), 22 µg/ml sodium pyruvate, 0.5 IU/ml ovine follicle-stimulating hormone (oFSH), 0.5 IU/ml ovine luteinizing hormone (oLH), 1 μg/ml oestradiol, 50 μg/ml gentamycin, and leptin [0 (control), 10, 50, and 100 ng/ml]. The good quality buffalo oocytes (batches of 10 oocytes) were placed in a culture plate containing six 50 μl droplets of maturation medium, covered with sterilized mineral oil, and then incubated at 38.5˚C with 5% CO2 in air for 24 hours. The maturation of oocytes was evaluated under a stereomicroscope by detecting the first polar body extrusion of oocytes. FITC-Annexin V propidium iodide (PI) staining method was used to detect oocyte apoptosis.


RESULTS
From a total of 115 collected ovaries, 1100 oocytes were recovered among which 283 oocyte were suitable for IVM. In the groups of leptin treated with 0 (control), 10, 50 and 100 ng/ml, the percentage of oocytes maturation was 74.65, 83.81, 77.85, and 75.40%, while the percentage of oocytes apoptosis was 9.83, 9.54, 9.93, and 10.42%, respectively. Our results showed that addition of 10 ng/ml leptin to buffalo IVM medium increased oocyte maturation, significantly, as compared with that in control group. However, addition of leptin to IVM medium had no significant influence on buffalo oocyte apoptosis.


CONCLUSIONS
Our findings suggested that addition of 10 ng/ml leptin to IVM medium of buffalo oocyte can improve oocyte nuclear maturation. Furthermore, we showed that there is no relation between in vitro addition of leptin to buffalo oocyte IVM medium and oocyte apoptos.


CONCLUSIONS
Our findings suggested that addition of 10 ng/ml leptin to IVM medium of buffalo oocyte can improve oocyte nuclear maturation. Furthermore, we showed that there is no relation between in vitro addition of leptin to buffalo oocyte IVM medium and oocyte apoptosis.
Department of Clinical Sciences, Division of Theriogenology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
Amir
A
Khaki1
Department of Clinical Sciences, Division of Theriogenology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
Rouzali
R
Batavani
Department of Basic Sciences, Division of Embryology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
Gholamreza
G
Najafi
Department of Clinical Sciences, Division of Theriogenology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
Hamid
H
Tahmasbian
Department of Clinical Sciences, Division of Theriogenology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
Abolfazl
A
Belbasi
Department of Immunology, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Irann.
Aram
A
Mokarizadeh
Apoptosis; Buffalo; In Vitro Maturation; Leptin; Oocyte