Nineteen ovaries were collected from noncycling New Zealand White rabbits (3 months of age, live weight 2,2 ± 0,1 kg) at the local rabbit farm of the Research Institute of Animal Production, Nitra, Slovakia. The ovaries were individually stored in a thermos with a physiological solution at room temperature and processed within 6 h of slaughter.

In the first series of our experiments, to analyze ovarian cell viability, proliferation, apoptosis, and secretory activity, we gently scraped the ovarian granulosa cells from the inner surface of the washed ovarian follicles (800–1200 μm) via a lancet and isolated them by centrifugation (10 min at 1500 rpm). Follicles with signs of atresia (opaque, hemorrhagic follicles or follicular cysts) have not been used as a source of granulosa cells. Afterward, the cells were resuspended and cultured in sterile Dulbecco’s modified Eagle’s medium (DMEM/F12) and 1:1 medium (BioWhittaker; Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (Bio-West, Inc., Logan, UT, USA) and 1% antibiotic-antimycotic solution (Sigma‒Aldrich, St. Louis, MO, USA). The cells were counted using an automated cell counter (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the concentration was adjusted to the required volume (106 cells/ml medium). The cell suspension was dispensed in 24-well culture plates (NuncTM, Roskilde, Denmark; 1 ml suspension/well) for enzyme-linked immunosorbent assay (ELISA) and in 96-well culture plates for Cell Counting Kit-8 (CCK-8), BrdU (bromodeoxyuridine), and Cell Death Detection Kit (Brand®, Wertheim, Germany; 100 µl/well) assays. The cells were precultured in medium at 37.5 °C in 5% CO2 until an 80% confluent monolayer was formed (2 days).

In the second series of experiments, we selected follicular fragments as the experimental model because ovarian steroidogenesis under physiological conditions requires the cooperation of both the granulosa and theca layers of the ovarian follicle (see Introduction) [3, 5]. The tissue near the ovary was removed, and the ovaries were opened with scissors at the site of entry of blood vessels. The connective tissue inside the ovary was gently punctured with a lancet to access the follicles. The follicles were pressed down and separated from the surrounding connective tissues. Afterward, the follicular wall was cut into thin lengthwise fragments (2–4 mm in diameter, weight 15 ± 8 mg). Then, these fragments were washed three times in sterile DMEM/F12 and 1:1 medium (BioWhittaker) and cultured in the same medium supplemented with 10% fetal bovine serum (Bio-West, Inc.) and 1% antibiotic-antimycotic solution (Sigma‒Aldrich) in 24-well culture plates (NuncTM; 1 ml/fragment/well) at 37.5 °C in 5% CO2 for 24 h.

After preculture, in both series of experiments, the medium was replaced with fresh medium of the same composition, and granulosa cells and fragments were cultured with and without progranulin (0, 0.1, 1, 10; Active Bioscience GmbH, Hamburg, Germany) or ometin 1 (0, 0.1, 1, 10; Active Bioscience GmbH). The chosen doses of progranulin corresponded the doses of this adipokine, which were efficient in regulation of ovarian cancer cell functions [16, 19,20,21]. The chosen doses of omentin corresponded the doses of omentin used in the previous in vitro experiments on non-ovarian cells [13, 22] and the level of omentin detected in human ovarian follicular fluid [25]. Immediately before administration to cells, adipokines were dissolved in phosphate-buffered saline (PBS). The control groups were composed of cells not treated with progranulin or omentin.

Immediately after culture, the granulosa cells were processed for the CCK-8 viability test and BrdU and cell death detection assays. The cell concentration was determined by counting on an automated cell counter (Thermo Fisher Scientific, Inc.). The medium conditioned by either granulosa cells or ovarian fragments was stored at -14 °C until ELISA was performed (see below).

Cell viability was measured by using a Cell Counting Kit-8 (CCK-8; Abcam, Cambridge, UK) as recommended by the manufacturer. In brief, 10 µL of CCK-8 solution was added to each well, and the plates were incubated at 37 °C for 24 h. The absorbance (abs) was read at 450 nm by using an ELISA reader (Thermo Fisher Scientific, Inc.). The percentage of proliferative active cells was calculated.

Cell proliferation, based on the measurement of 5-bromo-2′-deoxyuridine (BrdU) incorporation during DNA synthesis, was determined by using a colorimetric cell proliferation ELISA (Roche Diagnostics GmbH, Roche Applied Science, Germany) according to the manufacturer’s instructions. The reaction products were quantified by measuring the absorbance at 450 nm using an ELISA reader (Thermo Fisher Scientific, Inc.).

Cell apoptosis was evaluated using a Cell Death Detection Kit (Roche Diagnostics GmbH) according to the manufacturer’s instructions. This assay is based on the measurement of the level of cytoplasmic histone-associated DNA fragments as an index of induced apoptotic cell death. The absorbance was read at 405 nm using an ELISA reader (Thermo Fisher Scientific, Inc.).

The concentrations of progesterone and 17β-estradiol were analyzed in 25 µl aliquots of the incubation medium using an ELISA according to the manufacturer’s instructions (LDN Immunoassays and Services, Nodhorn, Germany). The features of these tests are outlined in Table 1. The accuracy of these ELISAs was tested for culture medium samples through dilution experiments.

The data from this study are reported as the means of values that were obtained in three separate experiments performed on separate days with different groups of granulosa cells and fragments, each obtained from at least six ovaries. Each experimental group included four culture wells containing ovarian granulosa cells and fragments. Significant differences between the groups were determined by using ANOVA followed by Tukey’s test, with SigmaPlot 11.0 (Systat Software, GmbH, Erkrath, Germany). Differences were considered to be significant at P values less than 0.05 (P < 0.05).