Study Design and Study Area

Experimental preclinical study of cases and controls in the field of experimental biology.

Population and Sample

The sample consisted of 20 male mice (Mus musculus) of the Swiss Rockefeller albino strain, 6-7 weeks old, obtained from the animal facility of the National Institute of Health in Lima, Peru. The experimental group (cases) consisted of 10 mice, to whom an aqueous extract was administered orally via nasogastric tube No.18 (Fisher Scientific, Pittsburgh, PA, USA) at a concentration of 1000 mg/kg of body weight for 21 days. The specimens were kept in the animal facility under conditions of 22-24 ºC ambient temperature, with 14 hours of light and 10 hours of darkness, with free access to a pellet diet (Purina, Peru) and water ad libitum.

Plants

“Mashua” tubers were acquired from local markets (Huancayo, Peru). In the laboratory, the plants were certified by the Department of Botany of the Universidad Nacional Mayor de San Marcos (UNMSM).

Chemicals

Ethyl alcohol 70º, distilled water, PBS solution (Phosphate buffer saline - Sigma), DNA-freeTM Kit (Ambion), SYBR Green I, Trizol.

Extract Obtaining

The tuber was weighed, liquefied, and immediately macerated with 70º alcohol for 2 days, and then the liquid phase was separated from the solid part. The liquid part was taken to the oven to evaporate the alcohol, obtaining a paste that was considered a standard compound.

Experimental Design

The mice were divided into 4 treatment groups. One group served as the Negative Control (CN) and was treated with distilled water. The treatment groups (T7, T14, and T21) were administered the aqueous extract (20%) of T. tuberosum orally for 7, 14, and 21 days, respectively. The total weight (g) of the mice was recorded at the start of the treatment and on the evaluation day. After each treatment, the mice were euthanized by cervical dislocation, and samples of testicles and epididymis were obtained. One testicle and one epididymis were placed in Trizol inside a cryovial (100mg/ml) and stored at -196º C in liquid nitrogen until RNA extraction. Spermatozoa were obtained from the second epididymis to measure sperm parameters (sperm concentration and motility) according to WHO (2010) guidelines. RNA extraction was performed using Trizol, following the protocol for RNA isolation from Invitrogen Cat. No. 15596-18. Once the RNA was obtained, contaminating DNA was removed using the DNA-freeTM Kit from Ambion, following the protocol described in Cat. No. AM 1906. From the DNA-free RNA, cDNA synthesis was performed using RT-PCR, as described in Cat. No. 18080-051. Quantification was done by real-time RT-PCR using the LightCycler® 2.0 system from Roche Applied Science, where the PCR product can be detected and measured by the fluorescent signal of SYBR Green I. The principle is that SYBR Green I binds to the minor groove of the DNA double helix and intercalates in the DNA helix¹⁰. In solution, the dye exhibits very low fluorescence, but fluorescence (at 530 nm wavelength) is enhanced upon binding to DNA. Thus, during PCR, the increase in SYBR Green I fluorescence is directly proportional to the amount of double-stranded DNA generated, and this emission is detected by the optical filter of the LightCycler® 2.0 system. Quantification was relative to the housekeeping gene, beta-actin, which is theoretically expressed constantly in the testicular tissue of mice and served as a reference point for measuring the expression of the target genes. This relative measurement is given by the ratio of the expression of the target genes to the expression of the housekeeping gene in the treated groups at different time periods with the aqueous extract of “mashua” compared to the control group.

Statistical Analysis

Homoscedasticity (equal variances) of the data was tested using Levene's test. To check if the data obtained for the different variables being evaluated were normally distributed, the Shapiro-Wilk test was performed. When the variable being evaluated showed a normal distribution, the means were compared using t-Student test; otherwise, the Mann-Whitney U test was applied for non-parametric tests. The results were expressed as mean ± SE (standard error). The statistical program SPSS Ver 17.00 was used, considering the significance level of p≤0.05.

Ethical Aspects

The care and handling of the animals were carried out in accordance with the ethical guidelines of our institution and the National Research Council for the care and use of laboratory animals¹¹.

Body Weight and Reproductive Organs

No significant differences were observed in the increase in body weight and the weight of the testicles, epididymis, and prostate (p>0.05) among the analyzed groups. The results are summarized in Table 1.

The results of gene expression quantification of two out of four genes (Cytochrome P450 17α-hydroxylase/17,20-lyase, Cyclin) showed no significant difference compared to the control group in relative quantification to Beta Actin expression (data not shown).