Oligonucleotides

MiR-105–1 mimics (double-stranded RNAs that mimic mature endogenous miR-105–1 and enhance miRNA activity, representing a gain-of-function assay), miR-105–1 inhibitor (single-stranded 2-O-methyl-modified oligonucleotide fragments that reduce endogenous miRNA activity, representing a loss-of-function assay), and their respective negative controls (NC) (Table 1) were purchased from GenePharma Co., Ltd. (Shanghai, China). Synthesized and purified through high-performance liquid chromatography, these oligonucleotides exhibited a purity exceeding 97%, validated by mass spectrometry.

Table 1 The sequences of miRNA mimics and miRNA inhibitorPreparation, Transfection, and Culture of Ovarian Granulosa Cells

Twenty porcine ovaries were collected from Landrace prepubertal gilts (6–8 months of age) at the slaughterhouse of Chovmat F.U. in Rastislavice (Slovakia). The ovaries were individually preserved in a thermos containing a physiological solution and processed within 6 h of slaughter.

Ovarian granulosa cells were isolated from porcine ovarian (4.5–6.5 mm diameter) follicles without visible signs of atresia (including weak vascularization, thin follicular walls, and pale follicular fluid) by using aspiration with a syringe. Following aspiration and cell isolation via centrifugation for 10 min at 1.500 rpm, the granulosa cells were rinsed in sterile DMEM/F12 1:1 medium (cat. no. 31331093; Thermo Fisher Scientific, Waltham, MA, USA) and resuspended in the same medium with 10% fetal calf serum (cat. no. 092910154; MP Biomedicals, Santa Ana, California, USA) and 1% antibiotic–antimycotic solution (cat. no. 091674049; MP Biomedicals). Cells were counted by using a Buerker chamber, and their concentration was adjusted to the required volume (106 cells/ml medium). The cell suspension was dispensed in 24-well culture plates (cat. no. 142475; Thermo Fisher Scientific; 1 ml suspension/well) for ELISA procedures, 96-well culture plates (cat. no. 781962; Brand®, Wertheim, Germany; 200 μl/well) for XTT (sodium 3′-[1- (phenylaminocarbonyl)- 3,4-tetrazolium]-bis (4-methoxy6-nitro) benzene sulfonic acid hydrate), TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling), and BrdU assays, or 16-well chamber slides (cat. no. 16260681; Thermo Fisher Scientific; 200 μl/well) for immunocytochemistry. Preculturing the cells at 37.5 °C in 5% CO2 allowed the formation of a 75% confluent monolayer within 2–3 days.

Experimental Setup

In the first series of experiments, investigations focused on the effects of miR-105–1 mimics and inhibitor on miR-105–1 levels, basic ovarian functions, and kisspeptin occurrence. Transfection of ovarian granulosa cells with miR-105–1 mimics, miR-105–1 inhibitor, and their corresponding negative controls was facilitated using Lipofectamine® RNAiMAX Transfection Reagent (cat. no. 13778150; Thermo Fisher Scientific), according to the manufacturer's protocol. A final oligonucleotide concentration of 25 nM was employed, with nontransfected and negative control-transfected cells serving as control groups.

In the second series of experiments studying the effects of miR-105–1 mimics, kisspeptin, and their combinations on basic ovarian functions, after 24 h of transfection, granulosa cells were cultured with and without kisspeptin-10 (0, 1, and 10 ng/ml; cat. no. AS-64240; Eurogentec, Seraing, Belgium). These doses of the shortest biologically active kisspeptin were comparable with the effective doses used in previous similar in vitro experiments [15,16,17]. Control groups included nontransfected or transfected cells without kisspeptin treatment.

After culture, the culture medium and cells were processed for immunocytochemistry, XTT, TUNEL, and BrdU assays, RT‒qPCR, and enzyme immunoassay (ELISA). Furthermore, cell concentration and viability were determined by using the Trypan blue exclusion test and a hemocytometer.

Cell Viability Test

The Trypan blue exclusion test (0.4%) assessed cell viability as per established protocols [20, 21]. Briefly, the medium was removed from the culture plates after incubation of the granulosa cells. Subsequently, the cell monolayer was subjected to Trypan blue staining (cat. no. 091691049; MP Biomedicals) for 15 min. After Trypan blue treatment, cells were fixed for 30 min in 4% paraformaldehyde. After fixation, plates were washed with a physiological solution and subjected to microscopic inspection (magnification: 400 ×). The ratio of dead (stained) cells to the total cell count was calculated.

XTT Assay

The cell proliferative activity was evaluated by using the XTT Cell Proliferation Assay Kit (cat. no. ab232856; Abcam, Cambridge, UK) according to the manufacturer's instructions. XTT is a yellow tetrazolium salt that is reduced to an orange formazan product by mitochondrial reductases in metabolically active proliferating cells [22]. Briefly, the activated-XTT solution was added to each well and incubated for two hours at 37 °C and 5% CO2. Absorbance (abs) was read at 450 nm by using an ELISA reader (cat. no. BS-050108-A02; Biosan, Riga, Latvia). The percentage of proliferative active cells was calculated by using the following equation:

$$\%\;\mathrm o\mathrm f\;\mathrm X\mathrm T\mathrm T\;\mathrm f\mathrm o\mathrm r\mathrm m\mathrm a\mathrm z\mathrm a\mathrm n-\mathrm\;\mathrm=\frac\times100$$

BrdU Assay

Cell proliferation, based on the measurement of 5-bromo-2′-deoxyuridine (BrdU) incorporation during DNA synthesis, was determined by using colorimetric cell proliferation ELISA (cat. no. 11647229001; Sigma-Aldrich, Saint-Louis, MO, USA) according to the manufacturer's instructions. The reaction products were quantified by measuring the absorbance at 450 nm using an ELISA reader (cat. no. BS-050108-A02; Biosan).

TUNEL Assay

DNA fragmentation induced in the cell culture was measured by using the TUNEL assay (HT TiterTACS™ Apoptosis Detection Kit; cat. no. 4822–96-K; R&D Systems, Minneapolis, MN, USA) following the manufacturer's instructions. The absorbance was measured at 450 nm by using an ELISA reader (cat. no. BS-050108-A02; Biosan) after adding 0.2 N HCl. Negative controls involved cells labeled without transferase terminal deoxynucleotidyl transferase (TdT), while positive controls were generated by using TACS-Nuclease for one hour at 37 °C before hydrogen peroxide treatment.

Immunocytochemical Analysis of the Presence of Kisspeptin, Proliferation, and Apoptosis Markers

The presence of the kisspeptin protein, markers of proliferation (PCNA and cyclin B1), and apoptosis (bcl-2, bax, caspase 3, and p53) were detected via immunocytochemistry [23]. After washing and fixation, the cells were incubated in a blocking solution with 1% bovine serum albumin (cat. no. 9048–46-8; Sigma-Aldrich) at room temperature for 1 h, to prevent non-specific binding of the antiserum. Next, the cells were incubated with primary antibodies listed in Table 2 for 1 h at room temperature. To detect the binding sites of the primary antibody, the cells were incubated with secondary antibody for 1 h at room temperature listed in Table 2. To visualize positive signals, the cells labelled with horseradish peroxidase were stained with 3,3′-diaminobenzidine (DAB) substrate (cat. no. 11718096001; Sigma-Aldrich) for 1 h. Following DAB-staining, the cells on chamber slides were washed with PBS and covered with a drop of Glycergel mounting medium (cat. no. C056330-2; Agilent Technologies, Santa Clara, CA, USA). A coverslip was then attached to the microslide, and the cells were visualized using a light microscope. Cells labelled with CFL 594 were mounted in VECTASHIELD Antifade Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI), a selective stain for cell nuclear DNA (cat. no. H-1200–10; Vector Laboratories, Inc., Burlingame, CA, USA). DAPI and CFL 594-labeled secondary antibodies were detected using fluorescence microscopy. Cells treated without the primary antibody were used as negative controls. The number of stained cells and the location of intracellular molecules were determined based on the brown coloration of DAB peroxidase or the red fluorescence emitted by the CFL 594 label using a light or fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and the IM500 Leica software. The ratio of stained cells to the total number of cells was determined.

Table 2 Antibodies, source, and commercially purchased reagents usedRT‒qPCR for miR-105–1

After treatment with miR-105–1 mimics and miR-105–1 inhibitor for 48 h, total RNA from transfected granulosa cells was extracted by using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. The concentration and quality of total RNA were measured by a Quantus Fluorometer (cat. no. E6150; Promega, Madison, WI, USA). Mature miR-105–1 expression levels were quantified utilizing the Hairpin-it miRNAs qPCR kit (cat. no. QPM-010; GenePharma Co., Ltd.) on an ABI 7500 Fast Instrument (cat. no. 4351104; Thermo Fisher Scientific). The amplification thermocycling conditions were as follows: an initial denaturation at 95 °C for 3 min, followed by 40 cycles at 95 °C for 10 s, annealing and elongation at 60 °C for 10 s, and 60 °C for 60 s. U6 small nuclear RNA (snRNA) was used as an internal control, and relative gene expression was calculated by using the 2–ΔΔCt method [24]. The sequences of the utilized primers (Table 3) were designed and synthesized by GenePharma Co., Ltd. All of the samples were analyzed in triplicate from the same RNA preparation, and the mean values were calculated.

Table 3 The sequences of gene primers for RT-qPCREnzyme-Linked Immunoassay (ELISA)

The concentrations of progesterone (cat. no. FR E-2500), 17β-estradiol (cat. no. FR E-2000), and IGF-1 (cat. no. ME E-0500) were determined in 25 µl aliquots of the incubation medium by using an ELISA according to the manufacturer's instructions (LDN Immunoassays and Services, Nodhorn, Germany). The characteristics of these assays are presented in Table 4. This ELISA was validated for the culture medium samples by using dilution tests.

Table 4 Characteristics of the immunoassays used in experimentsStatistical Analysis

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, each obtained from at least six ovaries. Each experimental group was represented by four culture wells containing ovarian granulosa cells. For the Trypan blue exclusion test, the rates of viability were calculated from at least 100 cells per well. For the immunocytochemical analysis, the proportion of cells containing antigens was calculated from at least 1,000 cells per well. For the ELISA, blank control values were subtracted from corresponding values that were determined for media-containing cells to exclude any nonspecific background (less than 10% of the total values). The rates of substance secretion were calculated per 106 viable cells/day. Significant differences between the groups were determined by using the Shapiro‒Wilk normality and Student's t tests, as well as one-way ANOVA followed by Tukey's tests, with SigmaPlot 11.0 (Systat Software, GmbH, Erkrath, Germany). Differences were compared for statistical significance at P levels less than 0.05 (P < 0.05).