Caffeine attenuates spermatogenic disorders in mice with induced chronic scrotal hyperthermia

Abstract

Objective: Chronic scrotal hyperthermia (SHT) can lead to serious disorders of the male reproductive system, with oxidative stress playing a key role in the onset of these dysfunctions. Thus, we evaluated the impact of caffeine, a potent antioxidant, on cellular and tissue disorders in mice with chronic SHT.

Methods: In this experimental study, 56 adult male NMRI mice were allocated into seven equal groups. Apart from the non-treated control group, all were exposed to heat stress. Two groups, termed "preventive" and "curative," were orally administered caffeine. The preventive mice began receiving caffeine immediately prior to heat exposure, while for the curative group, a caffeine regimen was initiated 15 consecutive days following cessation of heat exposure. Each treated group was subdivided based on pairing with a positive control (Pre/curative [Cur]+PC) or a vehicle (Pre/Cur+vehicle). Upon conclusion of the study, we assessed sperm characteristics, testosterone levels, stereological parameters, apoptosis, antioxidant and oxidant levels, and molecular markers.

Results: Sperm parameters, testosterone levels, stereological parameters, biochemical factors (excluding malondialdehyde [MDA]), and c-kit gene expression were significantly elevated in the preventive and curative groups, especially the former, relative to the other groups. Conversely, expression levels of the heat shock protein 72 (HSP72) and nuclear factor kappa beta (NF-κβ) genes, MDA levels, and apoptotic cell density were markedly lower in both caffeine-treated groups relative to the other groups, with more pronounced differences observed in the preventive group.

Conclusion: Overall, caffeine attenuated cellular and molecular abnormalities induced by heat stress in the testis, particularly in the mice treated under the preventive condition.

Keywords: Apoptosis; Caffeine; Chronic scrotal hyperthermia; Inflammation; Oxidative stress.

Figure 1.

Overall study timeline for the experimental groups. This schematic shows the time points of scrotal hyperthermia induction, caffeine administration, and sampling. Boxes of the same color indicate similarities in the type of intervention between subgroups of the preventive and curative treatment groups. Cur, curative; PC, positive control.

Figure 2.

Effects of caffeine on sperm characteristics in mice with induced chronic scrotal hyperthermia. (A) Sperm count, (B) viability, (C) motility, and (D) morphology were compared across the study groups. Data are presented as mean±standard deviation. Pre, preventive; PC, positive control; Cur, curative. ^a)p<0.05; ^b)p<0.001; ^c)p<0.0001.

Figure 3.

Effects of caffeine on testosterone levels in mice with induced chronic scrotal hyperthermia. Data are presented as mean±standard deviation. Pre, preventive; PC, positive control; Cur, curative. ^a)p<0.01; ^b)p<0.0001.

Figure 4.

Effects of caffeine on histological structure in mice with induced chronic scrotal hyperthermia. (A) Histological photomicrographs under hematoxylin and eosin staining. (B) Comparison of histological structure across groups based on Johnsen score. Data are presented as mean±standard deviation. Pre, preventive; PC, positive control; Cur, curative. ^a)p<0.01; ^b)p<0.0001.

Figure 5.

Effects of caffeine on stereological parameters in mice with induced chronic scrotal hyperthermia. (A) Testicular volumes calculated using the Cavalieri method. (B) Length density of the seminiferous tubules. (C) Numerical density of spermatogonia, primary spermatocytes, spermatids, Sertoli cells, and Leydig cells. Data are presented as mean±standard deviation. Pre, preventive; PC, positive control; Cur, curative. ^a)p<0.05; ^b)p<0.01; ^c)p<0.001; ^d)p<0.0001.

Figure 6.

Effects of caffeine on cell apoptosis in mice with induced chronic scrotal hyperthermia. (A) Representative micrographs from immunostaining against caspase 3 protein in the testicular tissue. Caspase 3-positive cells are represented by dark brown cytoplasm. (B) Quantitative analysis of caspase 3-positive cells. Data are presented as mean±standard deviation. Pre, preventive; PC, positive control; Cur, curative. ^a)p<0.05; ^b)p<0.01; ^c)p<0.0001.

Figure 7.

Effects of caffeine on testicular oxidative biomarkers and gene expression in mice with induced chronic scrotal hyperthermia. (A) Concentrations of oxidant (malondialdehyde [MDA]) and antioxidant (glutathione [GSH], superoxide dismutase [SOD], and catalase [CAT]) factors in the testicular tissues were evaluated using the biochemical method. (B) The quantities of transcripts for nuclear factor kappa beta (NF-κβ) (a gene involved in inflammation), as well as c-kit and heat shock protein 72 (HSP72) (involved in spermatogonial differentiation, proliferation, and resistance to oxidative stress), were analyzed using quantitative real-time polymerase chain reaction. Data are presented as mean±standard deviation. Pre, preventive; PC, positive control; Cur, curative. ^a)p<0.05; ^b)p<0.01; ^c)p<0.001; ^d)p<0.0001.