Analysis of human serum fatty acids and sperm quality

During the period from September 2017 to January 2020, serum specimens were collected from thirty subjects who experienced spermatogenesis abnormalities. This group consisted of 27 patients diagnosed with nonobstructive azoospermia (NOA) and 3 with extreme oligospermia (EO). Additionally, 30 individuals without any known fertility issues who were in the process of receiving standard semen assessments at the medical laboratory served as the control group. The participants in this study were between the ages of 20 and 43 (Supplementary Material 1, Table S1-S2), and blood samples were collected between 8:00 and 11:00 am. Subsequently, the samples were centrifuged at 1800×g for 10 min at room temperature to separate the serum. In previous research [22], the serum levels of free fatty acids were evaluated. Shanghai Applied Protein Technology Co. Ltd. utilized GC/MS to measure 39 medium- and long-chain free fatty acids (C6–C24). The sperm quality was assessed with a WLJY-9000 computer-assisted system (WLJY-9000, WeiLi, Beijing, China). The collection of clinical samples was conducted with informed consent and was authorized by the Research Ethics Committee of Jinling Hospital.

Animals and experimental design

This study utilized 60 4-week-old male C57BL/6 mice obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) for the purpose of conducting in vivo experiments. Sigma-Aldrich (Missouri, USA) provided the OCA (C2875) and busulfan (55-98-1). Figure 1A depicts the experimental layout. Following a seven-day acclimatization period, the mice were distributed randomly into six categories: control, busulfan alone, and four OCA therapy sets at doses of 32, 64, 128, and 256 mg/kg BW, with each group containing 10 mice. The control group received Coil oil orally for 3 weeks and then received a single dimethylsulfoxide (DMSO) injection intraperitoneally. The busulfan alone group was given Coil oil orally for 3 weeks and a busulfan injection of 30 mg/kg BW intraperitoneally. The OCA + busulfan group received OCA in Coil oil orally, followed by a busulfan injection of 30 mg/kg BW intraperitoneally. OCA treatment continued for 5 weeks, after which the mice were euthanized for testis sample collection and subsequent analysis.

Fig. 1

Experimental design and the effects of OCA and busulfan on spermatogenesis. (A) Experimental design. All animals were randomly divided into a control group, a busulfan group and four treatment groups with OCA (32, 64, 128, and 256 mg/kg BW every two days). (B-E) Sperm concentration (B), sperm PR (C), organ coefficient of the testis (D) and epididymis (E) of the mice at 9 weeks after OCA treatment (n = 7 ∼ 10). (F) Representative photographs of testicular morphology with HE staining. Scale bars: 100 μm and 20 μm. The black asterisk (*) indicates damaged spermatogenic tubules. SPG, spermatogonia; SPM, spermatocyte; SP, spermatozoa; SC, Sertoli cell. (G-H) The width of the seminiferous epithelium (G) and diameter of the seminiferous tubules (H) were calculated randomly from 50 cross-sections of round or nearly round seminiferous tubules (long axis: short axis < 1.2:1). (I) Proportion of different seminiferous tubules calculated from 10 random fields for each group. The data are presented as the mean ± SD. Statistical analyses were carried out using one-way ANOVA followed by Tukey’s post hoc test. ****P < 0.0001 vs. the control group, ####P < 0.0001 vs. the busulfan group

The initial busulfan concentration was 30 mg/mL in DMSO, which was thoroughly mixed with an equivalent volume of PBS in an ultrasonic device at 37 °C to reduce its toxicity. The mice were first euthanized using ether and then sacrificed through cervical dislocation to collect testis and epididymis samples. Postanesthesia cervical dislocation was considered a humane euthanasia method, as outlined in the Guide for the Care and Use of Laboratory Animals (Eighth Edition) by the Institutional Animal Care & Use Committee. The experimental protocols were conducted in adherence to the standards established by the National Laboratory Animal Care and Use Research Committee.

TM4 cell treatment

To determine the best treatment concentrations of OCA and busulfan, busulfan was dissolved in DMSO, while OCA was dissolved in alcohol, and both were diluted in culture medium. Equal amounts of DMSO and alcohol were added to the control, busulfan, and OCA groups at a final concentration of less than 0.1% based on a previous study [23]. TM4 cells were either pretreated with OCA (100 µM) for 2 h or exposed to busulfan (800 µM) for 24 h. The optimal OCA concentration was established through the CCK-8 assay. Furthermore, in the rapamycin intervention experiments, TM4 cells were preexposed to OCA for 2 h before being subjected to rapamycin (5 µM) (S1842, Beyotime Biotechnology, Shanghai, China), as confirmed by the CCK-8 assay.

Sperm quality analysis

The method for evaluating sperm quality was previously described [22]. The epididymides were recently transferred into human tubal fluid (HTF) medium and sliced into small fragments with ophthalmic scissors. Following incubation at 37 °C for 5 min, a hemocytometer (Qiujing, Shanghai, China) was used to observe 10 µL of the sperm suspension with the assistance of a light microscope (Olympus, Tokyo, Japan). The numbers of forward-moving sperm, nonforward-moving sperm, and immobile sperm were counted separately.

Histology analysis of the testis and epididymis

The testis and epididymis samples were fixed by immersion in Bouin’s solution for 24 h, followed by dehydration using ethanol of varying concentrations. Subsequently, they were embedded in paraffin for sectioning. Five-micron-thick tissue sections were then stained with hematoxylin and eosin (HE) before being imaged and digitized under an optical microscope.

BTB integrity analysis

The evaluation of BTB integrity was carried out with a biotin tracer, as detailed in previous studies [24, 25]. After receiving 5 weeks of busulfan treatment, three mice were chosen at random from each experimental group for additional analysis. Then, the mice were given 1% sodium pentobarbital injection at a dosage of 5 mL/kg BW via intraperitoneal administration for anesthetization. Incisions were created in the lower abdomen of the mice to expose their testes. Subsequently, 20 µL of freshly prepared EZ-Link Sulfo-NHS-LC-Biotin (21,335, Thermo Fisher Scientific, Massachusetts, USA) in PBS supplemented with 1 mM CaCl2 was injected into the stroma of the upper, middle, and lower testicular regions. The testes were extracted after 30 min of diffusion and promptly frozen in liquid nitrogen for cryosectioning. The slices, 10 μm in thickness, were immersed in 4% paraformaldehyde for 10 min and then treated with Alexa Fluor 488-labeled streptavidin (S32354, Thermo Fisher Scientific, Massachusetts, USA) for 1 h at room temperature. The specimens were then stained with DAPI and examined using a fluorescence microscope to visualize the seminiferous tubules. BTB damage was assessed using the following equation: A total of 50–60 round or oval-shaped cross-sections of the seminiferous tubules from each group were randomly examined. For oval-shaped tubules, Dbiotin was computed as the average of the long and short axes of the tubule.

$$The\ extent\ of\ BTB\ damage=\frac_}_}\times 100\%$$

(Dbiotin: the diffusion distance of biotin; Dradius: the radius of the tube)

Western blot

Testicular samples and cells were subjected to different treatments and lysed in RIPA buffer. The proteins were extracted by sonication on ice, followed by centrifugation at high speed and low temperature. Following the quantification of protein concentrations, proteins (20 µg) were resolved via SDS‒PAGE and subsequently transferred to PVDF membranes (Millipore, Massachusetts, USA). Following the blocking process with BSA, both primary and secondary antibodies were added to the membranes. The following primary antibodies were used in the present study: ZO-1 rabbit pAb (21773-1-AP, ProteinTech, Wuhan, China), Claudin11 rabbit pAb (AF5364, Affinity, Ohio, USA), Claudin5 rabbit pAb (AF5216, Affinity, Ohio, USA), Occludin rabbit pAb (13409-1-AP, ProteinTech, Wuhan, China), HO1/HMOX1 rabbit pAb (10701-1-AP, ProteinTech, Wuhan, China), NQO1 rabbit pAb (11451-1-AP, ProteinTech, Wuhan, China), SQSTM1/P62 rabbit mAb (ab109012, Abcam, Shanghai, China), and LC3B rabbit mAb (ab192890, Abcam, Shanghai, China). The visualization of the target protein bands was carried out using a chemiluminescent imaging system. The protein bands were subsequently quantified using ImageJ.

Analysis using real-time PCR

Gene expression levels related to oxidative stress were measured through RT‒PCR analysis in this study. After total RNA was isolated from the testes using a Total RNA Purification Kit (082001, BEI-BEI Biotech, Zhengzhou, China), cDNA synthesis was conducted utilizing HiScript III RT SuperMix for qPCR (R323, Vazyme Biotechnology, Nanjing, China) following the evaluation of RNA concentration and integrity. The quantitative analysis was carried out on a Roche LightCycler 96 Real-time PCR machine (Roche Diagnostics, Basel, Switzerland) using qPCR SYBR Green Master Mix (Q121, Vazyme Biotechnology, Nanjing, China). The reference gene β-actin served for uniform labeling, and gene expression values were calculated using the 2-ΔΔCq formula [26]. The sequences of primers used in the present study are listed in Table S3 (Supplementary Material 1).

Measurement of SOD and MDA

In brief, the tissue samples were homogenized and then centrifuged to extract protein. Following the prescribed protocol, the activity of SOD and the concentration of MDA were measured using specific assay kits.

Intracellular ROS quantification

The levels of ROS within the cells were quantified utilizing a DCFH-DA fluorescent probe (S0033 M, Beyotime Biotechnology, Shanghai, China) followed the manufacturer’s guidelines. Following treatment, 10 µM DCFH-DA solution in medium devoid of FBS was added to each corresponding well. Afterward, the cells were incubated in a lightless setting at 37 °C for 20 min prior to being rinsed twice with medium devoid of FBS. The brightness of the fluorescence was captured with a fluorescence microscope.

Transepithelial electrical resistance (TER) evaluation

To evaluate cell barrier performance in a controlled environment, TER was measured daily in three specific regions of the samples using a Milli-cell electrical resistance system (Millipore, Massachusetts, USA). Sertoli cells were initially inoculated in MilliCell Hanging Cell Culture Inserts (PET 0.4 μm, Millipore, Massachusetts, USA) at a concentration of 0.5 × 106 cells/cm2 and allowed to develop for three days to establish cellular barriers. After treatment, a Millicell electrical resistance system (Millipore, Massachusetts, USA) was used to measure the TER. The TER was calculated by the following formula: TER (Ω·cm2) = (resistance from treatment (Ω) - initial resistance (Ω)) × surface area of membrane (cm2).

Statistical analysis

The data illustrated in this investigation were sourced from a minimum of three distinct in vivo specimens and three separate in vitro trials. The outcomes were visualized utilizing GraphPad Prism 7 (GraphPad Software, California, USA) and are reported as the means ± standard deviations (SDs). Statistical significance across various groups was scrutinized utilizing SPSS 19.0 software (SPSS, Illinois, USA) through independent t tests and one-way analyses of variance (ANOVAs) followed by post hoc assessments of least significant divergence (matching variances) or Games-Howell (mismatching variances). A significance level of P < 0.05 was used to determine statistical significance.