Production of calves with high genetic paternity
is an increasingly important area for
Leptin is defined as a 16 kDa adipokine, primarily secreted by adipose tissue, and a multifunctional
Leptin is expressed in murine (
There are evidences that addition of leptin into
IVM medium can stimulate oocyte maturation in
Researches in and around apoptosis, the programmed cell death, have increased substantially since the early 1990s. It has been proved
that treatment with exogenous leptin in ob/ob
mice and human with congenital defect in leptin
producing can reintegrate the immune response
With our knowledge, there is no report about the
All chemicals and reagents were purchased from Sigma Chemical Co., St. Louis, Mo, USA, unless otherwise stated. Plastic dishes and six-well plates were obtained from Petes Co., USA.
In this experimental study, ovaries from apparently normal reproductive organs of adult
buffaloes (Bubalus bubalis) of unknown breeding history slaughtered in Urmia Abattoir, Urmia, Iran (37˚ 33΄ N, 45˚ 4΄ E) were collected
within 10 minutes after slaughter and transported to the laboratory in a thermos flask (32-
33˚C) containing sterile normal saline supplemented with antibiotics (1000 IU/ml penicillin
G and 1 mg/ml streptomycin) within 1 hour of
Due to remaining of some follicles embedded
in the ovary, aspiration of oocytes from the buffalo ovaries is considered as a big challenge,
so, in the first step, oocytes were aspirated from
2-8 mm visible follicles of the ovaries using
an 18-G hypodermic needle attached to a 10
ml disposable plastic syringe containing aspiration medium [TCM-199 fortified with 10%
fetal bovine serum (FBS; Invitrogen, USA)].
In the second step, the ovaries were dissected
and washed with aspiration medium to recover
the remaining oocytes. The aspirated fluid was
transferred to the 37˚C pre-warmed petridish.
Cumulus oocyte complexes were isolated under
a low-power magnification zoom stereo microscope (Nikon, Japan). For assessment of oocytes
quality, the classification of Yadav et al. (
Recovered buffalo oocytes; good quality oocyte for IVM (magnification ×125) (A); poor quality oocyte for IVM (B).
The collected oocytes were washed two time in fresh pre-warmed 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered Tyrode’s medium (TL-HEPES) followed by two washings in culture medium containing TCM-199 supplemented with 10% FBS, and were then subjected to a final wash with IVM medium before transferring to the drops (45).
This study was performed between April and June (2012), two times in a week.
In vitro maturation medium included TCM-199, 10% 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 (mouse recombinant leptin) [0 (control), 10, 50, and 100 ng/ml] (45, 46). Good quality buffalo oocytes (batches of 10 oocytes) were placed in a culture plate containing six droplets of 50 μl of maturation medium, covered with sterilized mineral oil, and then incubated at 38.5˚C with 5% CO2 in air for 24 hours. Oocytes maturation was evaluated under a stereomicroscope by detecting the first polar body extrusion which is the indicator of oocyte attaining the metaphase II stage (44) (Fig 2).
Mature buffalo oocytes; arrows show polar body indi- cating buffalo oocyte maturation (magnification All-10×200, 10 ×100).
Fluorescein isothiocyanate-Annexin V/propidium iodide (FITC-Annexin V/PI) double staining method was used to detect apoptosis (
In Annexin-V staining, the membranes containing phosphatidylinositol binded to fluorescent dye
due to inversion of oocytes membrane and apoptosis, so under the fluorescence microscope is detectable as a green staining. Non apoptotic oocytes are
not stained (
Annexin–V staining for detecting oocyte apoptosis (magnification ×250); apoptotic oocyte (A), non-apoptotic oocyte (B).
Data on maturation and apoptosis were analyzed using software package used for statistical analysis (SPSS) (Version 19; SPSS Inc., Chicago, IL, USA). Statistical mean and standard error of mean (SEM) were calculated for each group and were compared by one-way analysis of variance (ANOVA). Duncan’s test was used for the multiple comparison and least significant difference (LSD) values were calculated for significant difference between control group and treatment groups. Differences were considered significant when p≤0.05.
Out of the 1100 oocytes recovered from a total of 115 collected ovaries, 238 were suitable for IVM (Table 1).
The number of used ovaries, recovered oocytes, and good quality oocytes for IVM, while showing apoptosis in different leptin treated groups
|Leptin concentration ng/ml||Number of ovaries||Recovered oocytes||Good quality oocyte|
The percentage of oocyte maturation in control group and leptin treated groups is mentioned in figure 4. Addition of 10 ng/ml leptin to buffalo IVM medium increased oocyte maturation, significantly (p<0.05).
Effect of different leptin concentrations on oocyte maturation. There is a significant difference (p<0.05)
The percentage of oocyte apoptosis in control group and leptin treated groups is mentioned in fig 5. There was no significant difference in buffalo oocytes apoptosis between control group and the other leptin treated groups (p>0.05).
Effect of different leptin concentrations on oocyte apoptosis.
The present study was carried out to investigate
the effects of different concentrations of leptin
added during the
Apoptosis has an important role in mammalian
development as a quality control mechanism for
eradicating damaged, non-functional, and abnormal cells, as well as those cells that are in incorrect place (
The reason for these opposing responses in different cell types is unknown, but differences in the expression patterns of leptin receptors and as-
sociated signaling molecules may play an important role (
Our finding showed that leptin had no significant
effect on oocyte apoptosis after IVM in comparison with that in control group. But, there is an in
vivo study which reported that leptin administration in rats can rescue oocytes and follicles from
atresia by attenuation of apoptosis (
Ikeda and co-workers reported that an increase
in the extent of apoptosis may alter connectivity
between the cells of the cumulus-oocyte, and
subsequently reduces the quality of oocytes,
while the degree of apoptosis has also negative
correlation with the developmental competence
of bovine cumulus-oocyte complexes (
Our findings showed that addition of 10 ng/ml leptin to IVM medium of buffalo oocytes can increase oocyte nuclear maturation, and we recommend adding this hormone to IVM medium for improving oocyte maturation of this merit mammal. Also, our study showed that leptin has no effect on buffalo oocyte apoptosis after IVM.