The chemicals and accessories were of analytical grade and were collected as per the requirements. Copper (II) acetate monohydrate [Cu(CH3COO)2.H2O], glacial acetic acid (CH₃COOH), polyvinylpyrrolidone (PVP; MW 40,000), and culture media (DMEM) used were from HIMEDIA while sodium hydroxide pellets (NaOH) used were from Rankem. Other chemicals were: Phosphate Buffer Saline (PBS), Deionized water (DI).
Apparatus & Instruments
Conical flasks, magnetic stirrer, funnel, the crucible, spatula, oven, furnace, burette and burette stand, CO2 incubator, culture plates, fine forceps, needle, micropipettes, stereo-microscope, and fluorescent microscope.
Synthesis of Cupric Oxide NPs
Cupric Oxide Nanoparticles (CuONPs) were synthesized using the aqueous precipitation method (Ahamed et al. 2014), a type of chemical method which involves the reduction of a metal salt by reducing agent on the magnetic stirrer. In this method, 0.1 M of Copper (II) acetate monohydrate [Cu(CH3COO)2.H2O)] was dissolved in deionized water to form an aqueous solution. This solution was reduced by another aqueous solution of NaOH drop by drop on a magnetic stirrer in a conical flask at the desired molar ratio of 1:10 under the glacial acetic acidic environment. PVP (Polyvinylpyrrolidone) was used as a stabilizer to control the growth of CuONPs’ size and morphology. During the addition of the reducing agent, the color of the solution changed gradually from blue to sea green then from sea green to blackish-brown after which the reaction was stopped (Fig. 1). The blackish-brown solution indicates the synthesis of Cupric Oxide precipitates in solution. Obtained precipitates were washed with water and alcohol many times to remove the impurities present and dried at 70oC for 5 hrs. (T Group). Particles were divided into 3 groups (E1, E2, and E3) and incubated at different conditions to know the effect of temperature at a constant time on the size and structure of nanoparticles (Table 1).
Collection of ovaries and culture of ovarian follicles in vitro
Ovaries of sexually mature, normal cycling Jamnapari breed of goat were brought to the lab in ice-cold 0.9% normal saline at 4oC from Municipal Slaughter House, Chandigarh (30o70′N, 76o80′S,). Follicles (3-8 mm diameter) from ovaries were manually separated using fine forceps and were classified as healthy, pre-atretic, and atretic follicles on the morphometric basis including vascularity, color, and turbidity of follicular fluid (Sharma and Bhardwaj 2009).
Healthy follicles (pinkish, highly vascularized having amber color follicular fluid) were cultured for 24 hrs. in 5 groups i.e. one control and 4 treatment groups (A, B, C, and D). Each treatment group was subdivided into 2 subgroups (1 and 2), where subgroup 1 had 10 µg ml-1 and 2 had 20 µg ml-1 concentration of respective CuONPs in culture media (Dulbecco’s Modified Eagle Medium) supplemented with 200-units of antibiotics (100 IU/ml penicillin and 100 IU/ml streptomycin). These culture media were incubated in a CO2 incubator (5% CO2, 95% humidity, 38oC temperature). The experimental layout of a follicular culture of goat ovaries is presented in Fig. 2.
Preparation of Granulosa Cell Suspension
The aspiration method was used to make cell suspension. Treated healthy follicles (3-8 mm) were aspirated with the help of a 20-gauge needle in a 2 ml syringe containing Phosphate Buffer Saline (PBS) at pH 7.4. Cumulus–Oocyte Complexes (COCs) were removed with the help of micropipettes under stereo-microscope. The remaining cell suspension was washed 3 times by centrifugation method with the help of (PBS) at 2000 rpm for 5 minutes each to remove any kind of debris.
The morphometric analysis of the apoptotic granulosa cells was done by Broaddus et al. (1996) method. In this method granulosa cells’ apoptosis was evaluated by Acridine Orange (AO) staining. Each cell suspension prepared from treated follicles was mixed with an equal quantity of AO solution. AO solution was made by dissolving 1 µL of AO in 1 ml PBS. Cells were then observed under fluoroscent microscope. Cells appearing green were normal, cells appearing yellow/orange were pre-apoptotic and cells which appeared red were apoptotic. Quantification of healthy, pre-apoptotic and apoptotic granulosa cells were done to measure Apoptotic Percentage Index (API).
Cytotoxicity data are expressed as mean±standard error. The cytotoxicity assay was analyzed with the help of One Way ANOVA with Tukey post-hoc test (all treatments were compared to control as well as to one other). For the statistical analysis, SPSS 16.0 was used. The p-values of less than 0.05 were considered significant.