Study subjects and grouping

Ten SPF grade SD rats, 6 ~ 8 weeks old, weighing 200 ~ 220 g, were purchased and housed in the experimental animal center of Beijing Jinglai Huake Biotechnology Co., Ltd. The center was set at a temperature of 24 ± 2 °C and humidity of 50% ~ 70%. Free drinking and eating, as well as regular cleaning and disinfection, were provided. Male Sprague-Dawley rats (6–8 weeks old) were purchased from Jinglai Huake Biotechnology Co., Ltd, Beijing, China and housed in our institution’s specific pathogen-free (SPF) facility. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Jinglai Huake Biotechnology Co., Ltd (Protocol No. SYXK(Jing)-MZQ-2023001).

The ten rats were divided into two groups: the VC rat model (5 rats) and the sham control (5 rats) [1,2]. After the rats were anesthetized, a median incision was made in the abdomen to fully expose and separate the left renal vein segment between the adrenal vein and the inferior vena cava on the left retroperitoneal. A metal rod with a diameter of about 0.6 mm was placed parallel to the left renal vein segment and ligated with 4−0 silk thread. After apparent left renal congestion was observed, the metal rod was then pulled out. When the left renal vein dilation and congestion disappeared, the diameter of the left renal vein ligation was halved. At about four weeks, there was no significant difference in the size and weight between the left and right kidneys. An obvious left VC indicates successful VC modeling. VC modeling success was defined as >50% increase in spermatic vein diameter versus sham controls (measured by ocular micrometer).The sham group received the same treatment as the VC group except for no ligation.Sample size (n = 5 per group) was determined based on prior rodent studies of VC models [2,10] and pilot data showing significant effect sizes (Cohen’s d > 1.5) in key endpoints (e.g., TFRC expression).

Rats were anesthetized via intraperitoneal injection of sodium pentobarbital (40 mg/kg body weight). Depth of anesthesia was confirmed by absence of pedal reflex.

Postoperative analgesia (buprenorphine, 0.05 mg/kg) was administered every 12 hours for 48 hours.After experimental endpoints, rats were euthanized under deep anesthesia (sodium pentobarbital, 80 mg/kg) by cervical dislocation. This method was selected to minimize suffering and comply with AVMA guidelines for humane euthanasia.

Detection indicators and methods

First, the pathological indicators related to VC were detected as follows:

1. (1) The anesthetized rats underwent laparotomy, and their left and right spermatic vein diameters were measured using an operating microscope with an eyepiece micrometer.
2. (2) After the left testis was isolated, the morphological changes were observed by H&E staining, and spermatogenic function was evaluated according to Johnsen’s score.

Then, the left cremaster muscle was isolated and tested as follows:

1. (3) H&E staining was used to observe the damage.
2. (4) ROS content was detected by the kit.
3. (5) The number of SCs in different states was detected by immunofluorescence.

Finally, SCs were isolated from the left cremaster muscle and detected as follows:

1. (6) The expression level of TFRC was detected by qPCR and Western blot.
2. (7) The m6A modification level of TFRC mRNA was detected by MERIP-qPCR.
3. (8) The expression levels of m6A modification key enzymes were detected by qPCR and Western blot.
4. (9) The morphology of iron death ferroptosis was observed by transmission electron microscopy, including cell membrane, mitochondrial outer membrane rupture, mitochondrial size, mitochondrial membrane density, and other changes.
5. (10) The expression levels of GPX4 and FTH1 and indicators of ferroptosis were detected by qPCR and Western Blot.
6. (11) The content of MDA, Fe²⁺, GSH, and GSSG were detected by corresponding kits.

Statistical analysis

SPSS v.22.0 was used for data analysis. Categorical variables were expressed by numbers and rates, and continuous variables were described by means ± standard deviations. The differences in continuous variables between the two groups were analyzed using a t-test for normally distributed variables and a non-parametric test for non-normally distributed variables. Data normality was assessed by Shapiro-Wilk test before selecting parametric or non-parametric tests.The differences in categorical variables between the two groups were analyzed using Fisher’s exact test or chi-square test.

Decreased number of satellite cells

Clinically, the growth and development of the cremaster muscle may be an important reason for the occurrence of VC. However, the intrinsic mechanism has not been revealed so far. Due to the skeletal muscle nature of cremaster muscle, its regulatory mechanism may be understood with reference to the regulatory mechanism of skeletal muscle growth and development.

Postnatal skeletal muscle growth and development mainly rely on SC-mediated repair of skeletal muscle injury [11]. SCs were first found in frog skeletal muscle by Mauro et al. in 1961. They are undifferentiated muscle precursor cells retained in individual muscle tissue. SCs are located between the basement membrane of muscle fibers and the sarcolemma, similar to the arrangement of satellites of muscle cells, and are thus called satellite cells [12]. SCs play a crucial role in promoting skeletal muscle growth and development, injury repair, and skeletal muscle remodeling after birth. Their quiescent self-renewal and activated proliferation, differentiation, and fusion to form myotubes are key steps in the growth and development of skeletal muscle after birth [13]. Some studies further found that the decline in the number of SCS would inhibit the repair of skeletal muscle injury, thus hindering skeletal muscle growth. For example, lysine deficiency leads to SCS apoptosis by inhibiting the Janus kinase 2-signal transducer and activator of the transcription 3 (jak2-stat3) signaling pathway, resulting in decreased SCs and inhibited skeletal muscle development [14]. Knockdown of the Yin and Yang 1 protein (YY1) gene can prevent the metabolic reprogramming of SCs and inhibit their proliferation, leading to decreased SCs and inhibited muscle repair [15]. Maintaining a certain number of SCs is the premise of skeletal muscle repair after injury, and a decrease in SCs may hinder the growth and development of skeletal muscle.

In previous studies, oxidative damage induced by long-term and high-dose reactive oxygen species (ROS) exposure is an essential cause of skeletal muscle injury [16]. In clinics, the increase of ROS is closely related to the occurrence of VC [10,17]. This suggests that in the process of VC, there may be pathological changes of cremaster muscle injury caused by ROS, which requires SCS to promote injury repair. Therefore, we speculate that when the number of SCS in the cremaster muscle decreases, it may be difficult to repair the damage to the cremaster muscle caused by ROS, ultimately leading to VC.

Downregulated TFRC expression

Our VC rat model showed that there was a correlation between the ferroptosis level in the cremaster muscle and VC. Specifically, the VC model group showed decreased mitochondrial membrane potential of SCs in the cremaster muscle, increased ROS content, increased MDA content, increased Fe²⁺ content, and decreased GSH compared to the control group. These indicators all suggest the occurrence of ferroptosis. Previous studies have shown the existence of ferroptosis in skeletal muscle SCs [7]. Ferroptosis is a form of programmed cell death driven by iron-dependent lipid peroxidation [18]. Phospholipids containing polyunsaturated fatty acids on the cell membrane are prone to peroxidation under conditions rich in Fe²⁺ and ROS. The accumulation of lipid peroxides eventually destroys the integrity of the cell membrane, resulting in ferroptosis [19]. Glutathione peroxidase 4 (GPX4) and cystine glutamate transport receptor (system XC-) are the main signaling pathways associated with ferroptosis [20]. GPX4 inactivation will lead to the accumulation of lipid peroxides and cause a further increase in ROS to promote ferroptosis [21]. Based on the correlation between VC and ROS and previous data, we speculate that there is a correlation between ferroptosis in the cremaster muscle and VC. ferroptosis in SCs of the cremaster muscle may reduce its number, inhibit its growth and development, and may contribute to the occurrence of VC.

Consistent with Ding et al. [17], our data show TFRC downregulation exacerbates iron overload in the VC rat model, which showed that the downregulation of transferrin receptor (TFRC) expression in the cremaster muscle was associated with VC. Specifically, the VC model group had decreased TFRC protein level of cremaster muscle compared to the control group. After entering the blood circulation, Fe²⁺ will bind to TFRC on the cell membrane surface in the form of transferrin (TF) – bound iron and then be endocytosed and absorbed [22]. Therefore, the lack or downregulation of TFRC tends to inhibit ferroptosis [23,24]. While this suggests a protective role, recent paradoxically report that defects in TFRC or TF are associated with increased ferroptosis. For example, the loss of TFRC in skeletal muscle SCs may contribute the absorption of non-transferrin-bound iron and cause Fe²⁺ accumulation, glutathione metabolism disorder, and lipid peroxidation, inducing skeletal muscle ferroptosis [7]. In our VC model, TFRC deficiency likely disrupts iron homeostasis through two mechanisms: (1) Impaired transferrin-bound iron import causes compensatory uptake of non-transferrin-bound iron (NTBI), leading to labile iron pool overload; (2) Dysregulated iron distribution depletes glutathione reserves and inactivates GPX4, as evidenced by decreased GSH/GSSG ratio and GPX4 expression (Fig 4). This dual dysfunction synergistically accelerates lipid peroxidation cascade. Fe²⁺ accumulation and lipid peroxidation exist in patients with liver cirrhosis, but the expression level of TF is low. Studies showed that mice with liver-specific TF deficiency and high iron diets are prone to liver fibrosis caused by ferroptosis [25]. These studies suggest that the downregulation of TFRC expression may contribute ferroptosis and reduce the number of SCs in the cremaster muscle, inhibiting the growth and development of the cremaster muscle and leading to VC.

m6A modification

In this study, the sRAMP online tool predicted that there were potential m6A modification sites on the TFRC mRNA sequence. Further pre-experiments carried out in the VC rat model showed that increased m6A modification in the cremaster muscle was associated with VC. Specifically, the model group had increased m6A modification of the cremaster muscle compared to the control group. N6 methyladenine (m6A) modification is the most common and dynamically reversible RNA modification on eukaryotic cell mRNA. m6A modification is mainly affected by methyltransferases like 3 (METTL3), METTL14, and other methyltransferases (Writers), and fat mass and obesity-associated protein (FTO). It is also affected by the regulation of demethylases, such as alkylation repair homologue 5 (ALKBH5), and recognition proteins, such as insulin-like growth factor 2 mRNA binding protein 1/2/3 (IGF2 BP 1/2/3) and YTH N6 methyladenosine RNA binding protein 1/2/3 (YTHDF 1/2/3) [24]. Under the synergistic action of writer, eraser, and reader, m6A modification can play different regulatory roles.

Studies have shown that m6A modification mainly regulates mRNA splicing, enucleation, degradation, translation, and other processes [26] and is a critical way to regulate gene expression [27]. Recent studies have found that m6A modification can reduce the stability of target mRNA, thereby inhibiting gene expression [28,29]. To explore whether increased m6A modification in the cremaster muscle affected the stability of TFRC mRNA, this study compared the stability of TFRC mRNA in the cremaster muscle of rats in the control group and the model group. We found that the model group had decreased stability of TFRC mRNA in the cremaster muscle compared to the control group. Combined with these studies, we speculate that m6A modification of TFRC mRNA may contribute the degradation and downregulation of TFRC expression. Previous studies have shown that m6A modification is a potential mechanism to regulate SCs during skeletal muscle growth and development [30,31]. Moreover, m6A modification plays a vital role in the process of ferroptosis [8]. Therefore, m6A modification may reduce TFRC mRNA stability and downregulate its expression, thereby promoting ferroptosis in cremaster SCs.

While our data (decreased GPX4, mitochondrial collapse, Fe² ⁺ accumulation) align with ferroptosis [18–20], we acknowledge that apoptosis may co-occur. Future studies should employ ferroptosis inhibitors (e.g., ferrostatin-1) and apoptosis markers (e.g., cleaved caspase-3) to dissect the relative contributions of ferroptosis and apoptosis to satellite cell loss, including:

1. (1) Co-staining for ferroptosis markers (e.g., PTGS2) and apoptosis markers (cleaved caspase-3);
2. (2) Comparative intervention using ferroptosis inhibitors (e.g., ferrostatin-1) versus apoptosis inhibitors (e.g., Z-VAD-FMK).