The addition of 5-aminolevulinic acid to HBSS protects testis grafts during hypothermic transportation: a novel preservation strategy

Abstract

The aim of this investigation was to determine the optimal storage medium for testicular hypothermic transportation and identify the ideal concentration for the application of the protective agent 5-aminolevulinic acid (5-ALA). Furthermore, this study aimed to explore the underlying mechanism of the protective effects of 5-ALA. First, we collected and stored mouse testicular fragments in different media, including Hank’s balanced salt solution (HBSS; n = 5), Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; n = 5), and alpha-minimum essential medium (αMEM; n = 5). Storage of testicular tissue in HBSS preserved the integrity of testicular morphology better than that in the DMEM/F12 group (P < 0.05) and the αMEM group (P < 0.01). Testicular fragments were subsequently placed in HBSS with various concentrations of 5-ALA (0 [control], 1 mmol l⁻¹, 2 mmol l⁻¹, and 5 mmol l⁻¹) to determine the most effective concentration of 5-ALA. The 2 mmol l⁻¹ 5-ALA group (n = 3) presented the highest positive rate of spermatogonial stem cells compared with those in the control, 1 mmol l⁻¹, and 5 mmol l⁻¹ 5-ALA groups. Finally, the tissue fragments were preserved in HBSS with control (n = 3) and 2 mmol l⁻¹ 5-ALA (n = 3) under low-temperature conditions. A comparative analysis was performed against fresh testes (n = 3) to elucidate the underlying mechanism of 5-ALA. Gene set enrichment analysis (GSEA) for WikiPathways revealed that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was downregulated in the 2 mmol l⁻¹ 5-ALA group compared with that in the control group (normalized enrichment score [NES] = −1.57, false discovery rate [FDR] = 0.229, and P = 0.019). In conclusion, these data suggest that using 2 mmol l⁻¹ 5-ALA in HBSS effectively protected the viability of spermatogonial stem cells upon hypothermic transportation.

INTRODUCTION

Fertility preservation is highly important for patients receiving antineoplastic treatments, as these therapies frequently result in gonadotoxic adverse effects and subsequent infertility. To address this issue, gamete cryopreservation has become a standard procedure offered to patients.1 Adult males have the option to cryopreserve their sperm at local human sperm banks for future fertility treatment. Prepubescent boys are unable to undergo sperm cryopreservation as adult men.2 Spermatogonial stem cell (SSC) transplantation3 and testicular tissue grafting4 have been identified as potential techniques to assist prepubescent patients in preserving their fertility. Currently, cryopreservation of testicular tissue is used as a temporary technique until more clinically available options emerge.5 Fertility preservation for prepubertal boys, which presents a practical challenge, has been carried out in several countries.6 This challenge arises from the increasing number of patients undergoing testicular surgery outside of reproductive centers, which necessitates the hypothermic transportation of testicular tissue to cryopreservation facilities for subsequent controlled slow freezing and SSC or testicular tissue grafting.

Hypothermic storage at temperatures ranging from 1°C to 35°C is commonly used for temporary preservation and transportation of biological samples.7,8 In the practice of transporting testes of immature animals and humans, a temperature of 4°C is usually used for low-temperature transportation.9,10 This technique decelerates the progression of the cell cycle.11,12 However, irreversible cellular injuries still occur because of cooling (hypothermia-induced injury) and the loss of extracellular matrix (ECM) support (ECM loss injury).11,13 Hypothermia-induced injury is caused by adenosine triphosphate (ATP) depletion, reduced enzyme activities, and imbalances in the concentrations of some divalent ions as major contributors.11 Additionally, the absence of the ECM is proposed as another critical factor contributing to cell death during hypothermic preservation.14,15,16

Five-aminolevulinic acid (5-ALA), a naturally occurring delta amino acid found in the human body, is synthesized from glycine and succinyl coenzyme A (CoA) by mitochondrial ALA synthase in animal cells.17,18 The addition of 5-ALA to a hypothermic preservation solution has been shown to counteract mitochondrial stress in human hepatocyte-like cells after exposure to cold temperatures and subsequent rewarming.19 These findings suggested that 5-ALA could mitigate damage and facilitate metabolic recovery in isolated perfused rat livers that have undergone prolonged cold storage.

Consequently, we developed a short-term hypothermic preservation model for testicles in mice, which closely resembled the methodologies used in previous studies.9 The aim of this model was to investigate whether treatment with 5-ALA in a basic storage medium could protect against testicular tissue damage and elucidate the underlying mechanisms involved.

MATERIALS AND METHODS

Animals and testis collection

Three- to five-week-old C57BL/6 male mice were purchased from the Animal Center of Sun Yat-sen University (Guangzhou, China), and the animal experimental protocol was approved by the Committee for Animal Care and Use of Sun Yat-sen University (Approval No. SYSU-IACUC-2023-B1127). All the mice were killed by cervical dislocation, after which both left and right testis samples were collected immediately. The testis was flushed with storage medium and then harvested (Figure 1).

Figure 1.

Schematic illustration of the experimental design. 5-ALA: 5-aminolevulinic acid; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; HBSS: Hanks’ balanced salt solution; DMEM/F12: Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12; αMEM: alpha minimum essential medium; PI: propidium iodide; PLZF: promyelocytic leukemia zinc finger; UCHL1: ubiquitin C-terminal hydrolase L1; ROS: reactive oxygen species.

Short-term storage

In the initial phase of the study, tissue was used to determine the most suitable storage medium. Following the acquisition of testicular biopsies (both left and right testis), each testis was sectioned into fragments measuring 6–20 mm³, which is the appropriate size for cryopreservation.20 The testicular biopsies of fifteen male mice were divided into three different media: Hank’s balanced salt solution (HBSS; catalog 24020-117; Gibco, Grand Island, NY, USA), Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; catalog 11330500BT; Gibco), and alpha minimum essential medium (αMEM; catalog 12571-063; Gibco). The fragments were stored at 4°C for 6 h–8 h.

The subsequent phase of the study aimed to determine the ideal functional concentration of 5-ALA (catalog HY-N0305; Med Chem Express, Monmouth Junction, NJ, USA) for C57BL/6 male mouse tissue samples. For this purpose, fragments were stored in a 4°C refrigerator for 6‒8 h at 5-ALA concentrations of 0 (control), 1 mmol l⁻¹, 2 mmol l⁻¹, and 5 mmol l⁻¹ (n = 5 each) in the identified optimal medium from the initial phase of the study. The pH of each solution was measured. As the concentration of 5-ALA increased, the pH of the solution decreased in a concentration-dependent manner. Specifically, the pH decreased from 8.14 at without 5-ALA to 7.16 at 1 mmol l⁻¹ 5-ALA, 6.83 at 2 mmol l⁻¹ 5-ALA, and 5.46 at the highest concentration of 5-ALA (5 mmol l⁻¹).

In the third part of the study, samples from nine male mice were randomly divided into three groups (n=3 of each group) as follows: a fresh group, a control group (without 5-ALA), and an optimal functional concentration group. Samples from the fresh group were not placed in media and were directly included in the subsequent experiments, whereas those from both the control group and the optimal functional concentration group were stored at 4°C for 6–8 h in the refrigerator prior to further testing. The fresh group represented normal physiologically functioning testicles. The control group represented low-temperature-transported testes. The optimal concentration group represented testicles with the addition of the protective agent 5-ALA during hypothermic transportation.

Histological analysis

After the completion of short-term storage, testicular tissue (n = 5) was collected and washed with cold phosphate buffered saline (PBS; pH = 7.4). The tissue was then fixed with formalin for 48 h and subsequently embedded in paraffin following standard treatment protocols. Thin slices measuring 4 μm were obtained from the resulting wax block using a microtome. These slices were subjected to staining with hematoxylin and eosin and were examined via light microscopy. The morphological appearance of each fragment was evaluated via hematoxylin and eosin staining for histological analysis. Within each section, the seminiferous tubules were assessed on the basis of the following four parameters:9 the tubule structure, basement membrane rupture, tubular cell swelling, and tubular cell loss. Each parameter was assigned a score based on the criteria outlined in Supplementary Table 1, with scores ranging from 0 (minimal) to 3 (maximal). In addition, we performed a quantitative analysis of the seminiferous tubules to determine their diameter and the height of the seminiferous epithelium.21,22,23 The diameters of the seminiferous tubules per testis were measured. A perpendicular line passing through the center of each tubule was used, excluding tubules that appeared less circular in shape. The height of the seminiferous epithelium was measured by constructing three equidistant lines from each transverse section of the seminiferous tubule.

Supplementary Table 1.

Scoring for histological evaluation

Score	3	2	1	0
Structure	Intact structure	All cell types present, although the structure is slightly disordered	Random distribution of remaining cells	No cells present
Rupture of the basal membrane	No rupture	Partly ruptured	Mostly ruptured	Fully ruptured
Swelling	No swelling	Most cells normal	Some cells normal	No normal cells
Tubular cell loss	No cell loss	Some cell types lost	Most cell types lost	All cell types lost

Flow cytometric analysis

Testicular tissues (n = 3) were dissected into small pieces with microscissors. Subsequently, collagenase type IV (1 mg ml⁻¹; catalog 17104-019; Gibco) was added, and the tissue was digested in water at 37°C for 15–30 min. To terminate digestion, PBS containing 10% bovine serum albumin (BSA) was added. The resulting homogenate was then centrifuged at 200g (Microfuge 20R Centrifuge; Beckman, Brea, CA, USA) for 3 min at 4°C for washing and filtered through a 50-mm filter. The cell precipitate was resuspended in PBS to obtain a suspension of mouse testicular cells. For the detection of cell apoptosis, an Annexin V/PI staining mixture (catalog 70-AP101-100; Multisciences, Hangzhou, China) was added following the manufacturer’s instructions. Finally, the cells were analyzed via flow cytometry (CytoFLEX S; Beckman).

TUNEL staining

Apoptosis rates in testis tissue (n = 3) were tested using a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining kit (catalog C1086; Beyotime, Shanghai, China) according to the manufacturer’s instructions. After staining, the images were analyzed via confocal microscopy (TCS SP8; Leica, Wetzlar, Germany).

Western blot analysis

Testicular tissue (n = 3) weighing 10–20 mg was incubated with ice-cold radio immunoprecipitation assay (RIPA) lysis buffer (catalog 01408/15322; CWBIO, Beijing, China) containing a phosphatase inhibitor cocktail (catalog ab201111; Abcam, Cambridge, UK) for 30 min. The lysate was centrifuged at 12 000g (Microfuge 20R Centrifuge; Beckman) for 15 min, and the resulting supernatant was collected to determine the protein concentration. The concentration of total protein was measured using a BCA protein assay kit (catalog 23228; Thermo Fisher Scientific, Waltham, MA, USA). The protein samples were prepared in sodium dodecyl sulfate (SDS) sample buffer, separated via SDS-polyacrylamide gel electrophoresis (PAGE), and subsequently transferred onto a nitrocellulose membrane. The membranes were blocked with a 5% nonfat powdered milk mixture and then incubated with primary antibodies diluted 1:1000 in the blocking buffer. The bands were visualized via the enhanced chemiluminescence (ECL) method (catalog 4AW011-200B; 4A BIOTECH, Beijing, China).

Immunofluorescence analysis

The paraffin sections were dewaxed and dehydrated before being incubated overnight at 4°C with primary antibodies for immunofluorescence analysis. The tissues were subsequently incubated in the dark with secondary antibodies for 1 h at 37°C. Finally, the nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) for 10 min. The primary and secondary antibodies used in this study are listed in Supplementary Table 2 and 3.

Supplementary Table 2.

The primary antibodies used in this study

Primary antibody	Dilution	Catalog number	Manufacturer	Experiment
Anti-PLZF	1:100	AF2944	R&D Systems	Immunofluorescence
Anti-UCHL1	1:100	13179S	Cell Signaling Technology
Anti-cleaved caspase-3	1:100	9664T	Cell Signaling Technology
Anti-UCHL1	1:1000	13179S	Cell Signaling Technology	Western blot
Anti-PLZF	1:1000	sc-28319	Santa Cruz Biotechnology
Anti-ACTB	1:10 000	66009-1-Ig	Proteintech
Anti-p38	1:1000	8690S	Cell Signaling Technology
Anti-p-p38	1:1000	4511S	Cell Signaling Technology

PLZF: promyelocytic leukemia zinc finger; UCHL1: ubiquitin C-terminal hydrolase L1; ACTB: actin beta; p-p38: phosphorylated p38

Supplementary Table 3.

The secondary antibodies used in this study

Secondary antibody	Dilution	Catalog number	Manufacturer	Experiment
Donkey anti-rabbit IgG H&L (Alexa Fluor® 647)	1:200	ab150075	Abcam	Immunofluorescence
Donkey anti-goat IgG H&L (Alexa Fluor® 488)	1:200	ab150129	Abcam
Goat anti-rabbit IgG H&L (CY3)	1:200	BA1032	BOSTER
Anti-mouse IgG, HRP-linked	1:3000	7076P2	Cell Signaling Technology	Western blot
Anti-rabbit IgG, HRP-linked	1:3000	7074P2	Cell Signaling Technology

Measurement of glutathione (GSH), catalase (CAT), and malondialdehyde (MDA) levels in testicular tissues

After the testes (n = 3) were harvested, the tissues were homogenized and then placed in cold PBS. The homogenate was centrifuged at 600g (Microfuge 20R Centrifuge; Beckman) at 4°C for 10 min, and the supernatant was collected for further analysis of the oxidative stress level. GSH (catalog G4305; Servicebio Company, Wuhan, China), CAT (catalog A700-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and MDA (catalog G4300; Servicebio Company) levels in testicular tissues were assayed using commercial kits. We further analyzed the test data according to the instructions.

Assessment of reactive oxygen species (ROS) levels in the testes

ROS are indicators of the level of oxidative stress, and ROS levels were used to assess the oxidative environment in the testes (n = 3). ROS production was assessed through using a dihydroethidium (DHE) fluorescein diacetate (10 mmol l⁻¹; catalog S0063; Beyotime). Nuclei were stained with DAPI, and the images were captured under a microscope (TCS SP8).

Transmission electron microscopy (TEM)

TEM was used to examine the shape and organization of the mitochondria in the SSCs. The testes (n = 3) were treated with a 2.5% glutaraldehyde fixative (catalog G1102; Servicebio Company) for 4–6 h. The samples were subsequently fixed with 1% osmium tetroxide for 2–4 h. Following fixation, the samples underwent a series of dehydration steps using graded alcohol. Finally, the dehydrated samples were embedded in EPON resin. The prepared samples were then thinly sectioned and placed onto a copper grid. TEM (JEM-1400; Japan Electron Optics Laboratory, Tokyo, Japan) was used to visualize changes in the ultrastructure of SSCs.

RNA-Sequencing

The testes (n = 3) were harvested under fresh conditions (group fresh) and subjected to hypothermic transportation with or without 2 mmol l⁻¹ 5-ALA (control group and 2 mmol l^-1 5-ALA group). Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA sequencing was performed at OE Biotech (Shanghai, China); the TruSeq Stranded mRNA LTSample prep kit (Illumina, San Diego, CA, USA) was used for library generation, and 150-bp paired-end sequencing was performed via an Illumina HiSeq X Ten instrument (Illumina). Quality assessment of each sample was performed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) and an Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA).

Raw gene-level counts were normalized to counts per million (CPM). Principal component analysis (PCA) was performed via NetBID (version 2.0.2) to assess the overall similarity between samples. Nonmetric multidimensional scaling (NMDS) analysis, based on the Bray‒Curtis distance metric, was employed to assess the intergroup distances, whereas analysis of similarities (ANOSIM) was used to perform statistical comparisons of the differences. DESeq2 was used to perform differentially expressed gene (DEG) analysis, with the threshold set to a |fold change| >2 and P-adjusted < 0.05 .24 To conduct functional enrichment analysis of the DEGs, the Metascape tool25 was used, utilizing the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome databases. Gene set enrichment analysis (GSEA) was conducted on the DEGs via the WikiPathways database.

Statistical analyses

The data were presented as the mean ± standard deviation (s.d.). Statistical analysis was performed via either the unpaired Student’s t-test or one-way analysis of variance (ANOVA), followed by Tukey’s test, for normally distributed data. For nonnormally distributed data, the Kruskal‒Wallis test was applied. Statistical analysis and graphical presentation were accomplished via the GraphPad Prism 9.0 software (GraphPad, San Diego, CA, USA). Statistically significant differences between the groups were considered when the P < 0.05.

RESULTS

HBSS was determined to be a suitable storage medium

To investigate the impact of different transportation media on the integrity of testicular tissue during low-temperature transportation, three media were chosen, namely, HBSS, DMEM/F12, and αMEM. The testicular tissue samples were stained with hematoxylin and eosin, and the results indicated that the low-temperature transportation led to impaired integrity of the seminiferous tubules. The average scores for tubular integrity were 2.690 for HBSS, 2.415 for DMEM/F12 (P < 0.05, compared with HBSS), and 2.280 for αMEM (P < 0.01, compared with HBSS), as shown in Figure 2a. We measured the diameter of the tubules and the height of the seminiferous epithelium in the different media. There were no significant differences in these parameters among the different media (P > 0.05; Supplementary Figure 1 ^{(61.2KB, tif)} ). Furthermore, an analysis of germ cell apoptosis revealed significant increases in apoptosis in both the DMEM/F12 (P < 0.05) and αMEM (P < 0.0001) groups compared with the HBSS group (Figure 2b). These findings were confirmed by TUNEL staining, which indicated that the HBSS group exhibited a lower level of apoptosis than the DMEM/F12 (P < 0.05) and αMEM (P < 0.05) groups did (Figure 2c). Based on these findings, HBSS was selected as the preferred transportation medium for further investigation. The next step was to evaluate whether the addition of 5-ALA to HBSS could optimize the transportation efficiency of testicular tissue under hypothermic conditions.

Figure 2.

Optimization of different storage media. (a) Hematoxylin and eosin staining of the testes treated with different media. Morphological scores were evaluated according to the following four parameters: the structure of tubules, ruptures of the basement membrane, swelling of tubular cells, and tubular cell loss (n = 5). (b) Apoptosis rates in testicular tissue after using different media were determined by flow cytometry with Annexin V/PI staining and quantitative analysis of the frequency of early apoptotic cells (n = 3). (c) TUNEL staining after incubation in different media (n = 3). The data are presented as the mean ± standard deviation. ^*P < 0.05, ^**P < 0.01, and ^****P < 0.0001. 5-ALA: 5-aminolevulinic acid; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; HBSS: Hanks’ balanced salt solution; DMEM/F12: Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12; αMEM: alpha-minimum essential medium; PI: propidium iodide.

Effects of different 5-ALA concentrations on SSC viability during short-term hypothermic preservation

At each concentration of 5-ALA, the early apoptosis rate of testicular tissue decreased during low-temperature transportation, as determined via flow cytometry (Figure 3a), which indicated that the testicular tissue was rescued through the addition of 2 mmol l⁻¹ (P < 0.05) or 5 mmol l⁻¹ 5-ALA (P < 0.001). However, whether 2 mmol l⁻¹ or 5 mmol l⁻¹ was the optimal concentration of 5-ALA remained uncertain. Considering that the preservation of testicular fertility primarily depends on maintaining the viability of SSCs, our study determined the most suitable concentration of 5-ALA from this perspective. In subsequent experiments, we assessed the effects of different concentrations of 5-ALA. Compared with the other groups, the group supplemented with 2 mmol l⁻¹ 5-ALA presented significantly greater tubular integrity scores (P < 0.01; Figure 3b). We measured the diameter of the tubules and the height of the seminiferous epithelium at different 5-ALA concentrations. There were no significant differences in these parameters (Supplementary Figure 2 ^{(54.8KB, tif)} ). To further investigate the impact of 5-ALA on SSCs, we examined the expression of the promyelocytic leukemia zinc finger (PLZF) and ubiquitin carboxyl terminal hydrolase L1 (UCHL1), which are markers of SSCs. Western blot analysis revealed significant increases in the expression of both PLZF and UCHL1 in the group treated with 2 mmol l⁻¹ 5-ALA (Figure 3c). Additionally, immunofluorescence staining revealed greater proportions of PLZF⁺ (P <0.001) and UCHL1⁺ (P <0.001) cells in the 2 mmol l⁻¹ 5-ALA group than those in the other groups, indicating that the loss of SSCs could be effectively reversed through the treatment of testicular tissue with 5-ALA (Figure 3d and 3e). Based on these findings and with the aim of maximizing the retention of SSCs, we selected 2 mmol l⁻¹ as the optimal concentration of 5-ALA for use in the low-temperature transportation of testicular tissue.

Figure 3.

Identification of the optimal concentration of 5-ALA. (a) Apoptosis rates in testicular tissue after treatment with different 5-ALA concentrations were determined via flow cytometry and quantitative analysis of the frequency of early apoptotic cells (n = 3). (b) Hematoxylin and eosin staining of the testes treated with different 5-ALA concentrations. Morphological scores were evaluated according to the following four parameters: the structure of tubules, ruptures of the basement membrane, swelling of tubular cells, and tubular cell loss (n = 5). (c) Western blot was used to analyze proteins extracted from the testes of the mice. PLZF and UCHL1 are markers of SSCs. Immunostaining of testicular tissues for SSC markers (d) PLZF and (e) UCHL1. The data are presented as the mean ± standard deviation. ^*P < 0.05, ^**P < 0.01, and ^***P <0.001. NS: not significant; 5-ALA: 5-aminolevulinic acid; HBSS: Hanks’ balanced salt solution; DMEM/F12: Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12; αMEM: alpha-minimum essential medium; PI: propidium iodide; PLZF: promyelocytic leukemia zinc finger; UCHL1: ubiquitin C-terminal hydrolase L1; ACTB: actin beta.

Transcriptomic assessment indicated that 5-ALA alleviated testicular injury

The transcriptome sequencing was used to explain the protective effect of the 5-ALA addition on testicular tissue (Figure 4a). The fresh group represented normal physiologically functioning testicles without hypothermic injury. The control group (without 5-ALA) represented low-temperature-transported testes. The 2 mmol l^-1 5-ALA group represented testicles with the addition of 5-ALA during hypothermic transportation. NMDS analysis revealed the spatial distributions of samples in the fresh, control, and 2 mmol l^-1 5-ALA groups on the basis of Bray‒Curtis dissimilarity (Figure 4b). The obvious separation between the fresh and control groups indicated that the testicular tissue was severely damaged by cryogenics. ANOSIM revealed no significant within-group differences (P = 0.909; Figure 4c). Furthermore, the 2 mmol l^-1 5-ALA group presented a similar degree of dissimilarity to that of the fresh group but was significantly different from the control group. These findings further support that 5-ALA administration can alleviate cryogenic damage to testicular tissue. The PCA results revealed that the distribution dispersions of the fresh and 2 mmol l^-1 5-ALA groups on the first principal component (PC1) axis were close to but smaller than that of the control group (Figure 4d). Differential gene expression analysis revealed gene expression signatures among the three groups. The number of DEGs between the control group and the fresh group was greater than that between the 2 mmol l^-1 5-ALA group and the fresh group (Figure 4e). We presented the results of functional enrichment analysis of the DEGs as three color patches. The unique DEGs (n = 54) between the control and 2 mmol l^-1 5-ALA groups were significantly enriched in the GO term of regulation of the mitochondrial membrane potential. The unique DEGs between the control and fresh groups (n = 114, representing genes that can be changed by 5-ALA addition) were enriched in the terms of Ca²⁺-activated K⁺ channels and the Rap1 signaling pathway. The 17 DEGs in the overlapping area presented opposite trends between the two comparison groups and were mainly involved in functions such as translation and negative regulation of the neuroinflammatory response (Figure 4e). GSEA showed that oxidative phosphorylation was upregulated in the control group, whereas the p38 mitogen-activated protein kinase (MAPK) signaling pathway was downregulated in the 2 mmol l^-1 5-ALA group (Figure 4f). In summary, damage to testicular tissue during transportation is closely related to impaired mitochondrial function, and the addition of 5-ALA can play a protective role by inhibiting the p38 MAPK pathway.

Figure 4.

Transcriptomic assessment of the effects of 5-ALA treatment on mouse testes. (a) Flowchart of the bulk mRNA transcriptome sequencing (n = 3). (b) NMDS of plots in the fresh, control and 2 mmol l^-1 5-ALA groups. The plot is based on Bray‒Curtis dissimilarity and illustrates the spatial distribution of the samples. (c) ANOSIM was used to assess the similarities between the fresh, control and 2 mmol l^-1 5-ALA groups. “Overall” represents the difference among the groups, and the other labels represent differences within the groups. (d) PCA of the variability of the data among the three groups. (e) Venn diagram showing the results of the GO enrichment analysis. (f) The GSEA for WikiPathways plots show the enrichment of the oxidative phosphorylation signaling pathway in the control group versus the fresh group and that of the p38 MAPK signaling pathway in the 5-ALA-treated group versus the control group. 5-ALA: 5-aminolevulinic acid; ANOSIM: analysis of similarities; NMDS: nonmetric multidimensional scaling; PCA: principal component analysis; MAPK: mitogen-activated protein kinase; GO: Gene Ontology; mRNA: messenger RNA; PC1: first principal component; NES: normalized enrichment score; FDR: false discovery rate; ES: enrichment score.

5-ALA reduces testicular apoptosis by dampening the oxidative stress-activated p38 MAPK pathway

To assess the antioxidant status of the testicles, classical redox markers were measured (Figure 5a). The level of MDA was significantly greater in the control group than those in the fresh group (P < 0.01). However, the levels of GSH and the CAT activity were not significantly different between the control and fresh groups. The administration of 5-ALA significantly mitigated these effects. The level of MDA was significantly lower in the 2 mmol l^-1 5-ALA group than that in the control group (P < 0.05). The levels of GSH (P < 0.01) and CAT (P < 0.05) were greater in the 2 mmol l^-1 5-ALA group than those in the control group. The MAPK pathway is closely involved in oxidative stress-related apoptosis. Compared with that in the fresh group, the phosphorylation of p38 in the MAPK pathway was markedly greater in the control group. However, in the 5-ALA treatment group, the level of p38 phosphorylation in the MAPK pathway was lower than that in the untreated group (Figure 5b). The levels of ROS in the testes were measured via flow cytometry with DHE, a commonly used fluorescent probe for detecting intracellular superoxide anion levels. Excessive production of ROS was observed during short-term hypothermic transportation in the control group compared with that in the fresh group (P < 0.0001). In contrast, the level of ROS in the 5-ALA-treated group was significantly lower than that in the control group (P < 0.001; Figure 5c). Immunofluorescence staining of DHE and PLZF revealed that 5-ALA treatment reduced ROS production (P < 0.01), especially in the SSCs (Figure 5d). ROS trigger the intrinsic apoptosis pathway. As shown in Figure 5e, the number of TUNEL⁺ cells in testicular tissue was significantly lower in the 2 mmol l^-1 5-ALA group than that in the control group (P < 0.01). Furthermore, the percentage of apoptotic SSCs was significantly lower in the 2 mmol l^-1 5-ALA group than that in the control group, as shown by immunofluorescence staining of cleaved caspase-3 and PLZF in Figure 5f (P < 0.001). Mitochondria are the primary source of ROS in mammalian cells. TEM revealed that the mitochondria of the SSCs swelled and vacuolated during low-temperature transportation without 5-ALA. However, the addition of 5-ALA significantly alleviated these effects (Figure 5g). Taken together, these results indicate that 5-ALA protects the testes and their SSCs during hypothermic transportation by reducing the production of mitochondrial ROS and inhibiting the p38 MAPK pathway.

Figure 5.

5-ALA reduces testicular apoptosis by dampening the oxidative stress-activated p38 MAPK pathway. (a) GSH and MDA levels and CAT activity (n = 3). (b) The relative levels of p-p38 and p38 were assayed via western blot. (c) Representative flow cytometry profiles showing the levels of ROS in the testes and the quantification of the relative mean fluorescence intensity (n = 3). (d) ROS probe DHE staining and immunostaining for PLZF in testicular tissues (n = 3). (e) Representative TUNEL staining profiles showing the impact of treatment with 2 mmol l^-1 5-ALA (n = 3). (f) Immunostaining for cleaved caspase-3 and PLZF in testicular tissues (n = 3). The white arrows indicate the degree of SSC apoptosis. (g) Mitochondrial swelling and vacuolation in SSCs were characterized via TEM. Red arrows indicate mitochondrial swelling and vacuolation; black arrows indicate normal mitochondria. The bars represent the mean ± standard deviation. ^*P < 0.05, ^**P < 0.01, ^***P <0.001, and ^****P <0.0001. 5-ALA: 5-aminolevulinic acid; GSH: glutathione; MDA: malondialdehyde; p-p38: phosphorylated p38; ACTB: actin beta; CAT: catalase; ROS: reactive oxygen species; DHE: dihydroethidium; PLZF: promyelocytic leukemia zinc finger; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; SSC: spermatogonial stem cell; TEM: transmission electron microscopy.

DISCUSSION

In this study, we used a mouse model to investigate the effects of two testis hypothermic transportation parameters, namely, the storage medium and the storage protective agent 5-ALA, in tissue donation sites and end users. Our findings demonstrated that the supplementation of HBSS with 2 mmol l⁻¹ 5-ALA positively influenced the preservation of testes during low-temperature transportation.

Transporting testicular tissue samples is a crucial step in the process of testis cryopreservation. Proper transportation is necessary to ensure the quality of the biopsied tissue. In certain cases, such as surgical emergencies, including testicular torsion26 and fertility preservation for adolescent transgender male patients,27 it may be necessary to transport the removed testicular tissue to a nearby reproductive center for cryopreservation when locally available medical resources may be limited. Optimal transportation conditions provide more people with the chance for fertility preservation and the potential for future fertility restoration.

Hypothermia-induced injuries primarily result from ATP depletion and reduced enzyme activities, leading to metabolic and redox imbalances within cells.11,28,29 These imbalances contribute to cellular damage as a consequence of the cooling process. Under normal physiological conditions, the redox balance is maintained by the elimination of harmful ROS through the endogenous antioxidant defense systems of cells.30,31 During hypothermic preservation, the cellular antioxidant capacity decreases, whereas the production of ROS may increase owing to mitochondrial damage, thereby inducing oxidative stress. Importantly, these injuries caused by hypothermic conditions are interconnected and mutually exacerbating. In line with these findings, our study revealed that hypothermic testicular tissue exhibited morphological impairments, increased production of ROS, leading to oxidative stress, and compromised mitochondrial function, ultimately resulting in the apoptosis of germ cells (Figure 6).

Figure 6.

The mechanism of action of 5-ALA in HBSS in alleviating spermatogonial stem cell injuries during short-term hypothermic preservation. 5-ALA: 5-aminolevulinic acid; HBSS: Hanks’ balanced salt solution; ROS: reactive oxygen species; MAPK: mitogen-activated protein kinase; ATP: adenosine triphosphate.

In the initial phase of the study, we assessed three commonly used media in fertility laboratories, HBSS, DMEM/F12, and αMEM, as potential storage media. Fayomi et al.20 employed HBSS as the transportation solution for cryopreserved prepubertal testicular tissues. These tissues were subsequently autologously grafted onto the back skin or scrotal skin of castrated pubertal rhesus macaques, resulting in their maturation and the production of functional sperm. DMEM/F12 and αMEM are frequently utilized as cryoprotectants for testes.32,33,34,35 Our findings demonstrated that HBSS had a more favorable effect on preserving tubular morphology and reducing testicular cell apoptosis (Figure 2). As a result, we proceeded with employing HBSS as the foundation storage medium in subsequent experiments.

In a previous study, 5-ALA, which is a precursor in heme biosynthesis, increased the activity of mitochondrial complex IV (cytochrome C oxidase) in mouse livers.19 Indeed, 5-ALA is a photosensitizing precursor that has gained approval from the USA Food and Drug Administration for multiple applications.36 5-ALA is employed as an intraoperative imaging agent in fluorescence-guided visualization of malignant tissue during glioma surgery, assisting surgeons in identifying and removing tumor cells more accurately.37 Our observations suggest that the administration of 5-ALA can reverse testicular injuries induced by low temperature. Specifically, we found that the viability of SSCs significantly increased upon the addition of 2 mmol l⁻¹ 5-ALA (Figure 3c–3e). Additional transcriptome analysis revealed that the protective effect of 5-ALA against testicular tissue damage was associated primarily with the preservation of mitochondrial function. Moreover, when 5-ALA was applied, the p38 MAPK pathway was inhibited, as shown by GSEA for WikiPathways (Figure 4f). In addition, we performed western blot to detect p38 MAPK pathway markers (phosphorylation-p38 and p38), which revealed that low temperature induced the activation of the p38 MAPK pathway in testicular tissue and that 2 mmol l⁻¹ 5-ALA reversed this process (Figure 5b). Therefore, supplementation with 5-ALA was suggested to play a protective role by inhibiting the p38 MAPK pathway.

In the subsequent experiments, the administration of 5-ALA resulted in a reduction in oxidative damage to the cells (Figure 5a). This reduction was achieved by increasing the activity of the CAT enzyme, which is involved in antioxidant defense and utilizes GSH as a cofactor. Furthermore, 5-ALA demonstrated a capacity to alleviate apoptosis triggered by the intrinsic apoptotic pathway, thereby safeguarding the mouse testes during low-temperature storage. These effects contribute to the preservation of testicular tissue during freezing and subsequent fertility restoration. Notably, the mechanism underlying the apoptotic pathway may be linked to the activation of the p38 MAPK signaling pathway.38,39 Accumulating evidence consistently suggests that MAPK pathways can be activated in response to oxidative stress in various cells and tissues.40,41 Importantly, MAPK signaling pathways are known to play crucial roles in regulating reproductive functions in the testis.42,43 In mouse testicular tissue, 5-ALA combined with ferrous iron (5-ALA/Fe²⁺) had a positive effect on mitigating the harm caused by scrotal heat stress on spermatogenesis.44 5-ALA/Fe²⁺ was found to upregulate the expression of heme oxygenase-1 (HO-1) and inhibit MAPK-mediated oxidative stress and apoptosis. In the present study, 5-ALA reduced oxidative stress and apoptosis by restraining the activation of the p38 MAPK signaling pathway (Figure 4f and 5b–5f).

It is important to note that our study has certain limitations. First, the clinical safety of the optimal concentration of 5-ALA was not thoroughly assessed in our study. Future studies should focus on evaluating the clinical safety of the selected concentration of 5-ALA. This will provide a better understanding of the potential side effects or risks associated with the use of 5-ALA in this context. Additionally, the storage period in our experiment was approximately 6–8 h. It would be beneficial to extend this time frame to assess the effectiveness of 5-ALA accurately over a longer transportation period. By prolonging the storage period, we can better evaluate the long-term effects and stability of 5-ALA during transportation or preservation processes.

In conclusion, the 5-ALA presence in HBSS effectively protected the viability of SSCs from hypothermia-induced oxidative stress by reducing the production of ROS through the p38 MAPK pathway.

AUTHOR CONTRIBUTIONS

GHL conceived and designed the study. MMH performed the experiments, statistical analysis, and wrote the manuscript. JXH, ZHX, LYL, and YQL assisted in the experiments. HCC, PGC, XYL, and GHL helped with data interpretation and manuscript review. GHL managed and supervised the project. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.

Supplemental Figure 1

(a) Diameters of the tubules and (b) heights of the seminiferous epithelium in different media (n = 5). HBSS: Hank’s balanced salt solution; NS: not significant; DMEM/F12: Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12; αMEM: alpha-minimum essential medium.

Supplemental Figure 2

(a) Diameters of the tubules and (b) heights of the seminiferous epithelium at different 5-ALA concentrations (n = 5). 5-ALA: 5-aminolevulinic acid; NS: not significant.