Spermidine alleviates heat stress-induced testicular dysfunction potentially by modulating oxidative stress, autophagy, and apoptosis in mice
Description
While the detrimental impact of heat stress on male fertility is well-documented, the development of targeted therapeutic interventions remains a critical unmet need. Spermidine, a naturally occurring polyamine found in both plants and animals, possesses diverse biological activities, including potential antioxidant and autophagy-modulating properties. This study aimed to investigate whether spermidine alleviates heat stress-induced impairment of spermatogenesis via its antioxidant effects and/or regulation of autophagy. The accompanying tables present results from semen analyses—including sperm count, motility, and morphology—as well as assessments of testicular spermatogenic function, such as testis-to-body weight ratio and seminiferous tubule integrity.
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Experiment 1: Following a two-week acclimatization period, animals were randomly allocated to either a control (CON, 30°C) or heat stress (HS, 42°C) group. Under anesthesia induced by 1.25% tribromoethanol (200 mL/kg, i.p.), the distal third of each mouse (including the scrotum) was immersed in a temperature-controlled water bath for 20 minutes (Day 0). Subgroups were euthanized at 1, 7, 14, 21, 28, and 35 days post-exposure. Experiment 2: Animals were randomly assigned to one of three groups: CON (30°C + saline), HS (42°C + saline), or HS+SPD (42°C + 5 mg/kg/d spermidine, i.p.). Saline or spermidine treatment began immediately after heat stress and continued for 14 days until sampling. - Following euthanasia via cervical dislocation, body and testes weights were measured using a precision balance (accuracy ±0.01 g). - Testicular and epididymal tissues were collected, immediately fixed in paraformaldehyde, and embedded in paraffin after gradient alcohol dehydration for subsequent histological evaluation. - The cauda epididymis was carefully dissected and incised to release sperm into 0.5 mL of pre-warmed M2 medium, followed by incubation at 37°C for 15 minutes. Sperm parameters were assessed using computer-aided sperm analysis (CASA). - Sperm smears were prepared and stained with 0.5% crystal violet for 2 minutes. - Female mice were superovulated via intraperitoneal injection of 10 IU PMSG (Ningbo Second Hormone Factory), followed by 10 IU hCG 48 hours later. At 12–14 hours post-hCG administration, mice were euthanized, and cumulus–oocyte complexes (COCs) were collected from the ampulla of the oviducts in pre-warmed M2 medium under a stereomicroscope equipped with a 37°C heating stage. Epididymal sperm were collected and capacitated in 100 μL HTF medium under mineral oil at 37°C with 5% CO₂ for 60 minutes. Sperm suspensions were co-incubated with COCs in fertilization medium for 5 hours under the same conditions. After fertilization, presumptive zygotes were washed three times in KSOM medium and cultured under identical conditions. Embryonic development was evaluated at 24 hours post-insemination (hpi). - Total RNA was extracted from testicular tissue using TRIzol reagent (TransGen Biotech) according to the manufacturer’s instructions. - Protein concentrations in testicular tissue were determined using a BCA protein assay kit (Solarbio) as per the manufacturer’s protocol. - Testosterone levels in testicular tissue were quantified using a commercial ELISA kit (Ruixinbio) following the manufacturer’s guidelines.
Institutions
- Sichuan Agricultural University