Testicular macrophages (tMΦ) are the most abundant immune cells in the testicular interstitium, comprising approximately 20% of interstitial cells. They reside in close proximity to Leydig cells, forming intricate cytoplasmic interdigitations, and are regarded as “guardians of fertility.” Recent single-cell lineage studies have identified two distinct tMΦ subsets:
- Interstitial tMΦ: derived from yolk sac progenitors and seeded during embryogenesis;
- Peritubular tMΦ: bone marrow–derived, infiltrating the testis around two weeks after birth.
The interstitial subset highly expresses IL-10, maintaining local immune suppression and promoting testosterone synthesis. In contrast, peritubular tMΦ are MHCII⁺, capable of antigen presentation and interaction with regulatory T cells, thereby facilitating clearance of autoantigens leaked during spermatogenesis and reinforcing testicular immune privilege.
Moreover, tMΦ secrete bioactive factors—including 25-hydroxycholesterol, M-CSF, and retinoic acid—that directly stimulate steroidogenesis in Leydig cells and support the proliferation and differentiation of spermatogonial stem cells. Depletion models demonstrate that loss of tMΦ leads to a ~50% reduction in testosterone levels and early arrest of spermatogenesis, underscoring their dual pivotal role in both endocrine and reproductive functions.
Previously, we have covered the use of clodronate liposomes for depleting macrophages in the liver, lung, intestine, brain, peripheral blood, and spleen. Today, we present relevant studies and protocols on testicular macrophage depletion using clodronate liposomes.
Representative Literature and Protocols
Literature 1
Source: Functional and phenotypic characteristics of testicular macrophages in experimental autoimmune orchitis
Impact Factor: 5.2 (Q1)
Macrophage Depletion Protocol
Male Sprague–Dawley rats (50–60 days old) were used. Pilot experiments demonstrated that direct intratesticular injection via the tunica albuginea caused testicular parenchymal damage. Consequently, macrophage depletion was performed via intraperitoneal injection. Immunized rats received 2 mL clodronate liposomes every 2–3 days from day 0 post-immunization until sacrifice at days 50–60.
Results
Immunoperoxidase staining of testicular sections from rats immunized with testis homogenate plus adjuvant and treated with either PBS-liposomes (A, C) or clodronate liposomes (B, D) revealed localization of ED1⁺ (A, B) and ED2⁺ (C, D) testicular macrophages. Both ED1⁺ and ED2⁺ macrophage populations were significantly reduced in the clodronate-treated group.
Literature 2
Source: Pituitary–testicular axis in rats lacking testicular macrophages
Impact Factor: 5.2 (Q1)
Macrophage Depletion Protocol
Adult male Wistar rats received bilateral intratesticular injections of 150 μL clodronate liposomes, PBS-liposomes, or saline using a 30 G needle. Blood samples were collected on days 0, 5, 10, and 15 for hormone analysis. Animals were euthanized on day 15 for histological assessment.
Results
Representative interstitial regions of testes on day 15 are shown: In the NaCl control group (A), macrophages contained abundant trypan blue–positive vacuoles and were easily identifiable; Leydig cells and seminiferous tubules appeared normal.
In the clodronate-treated group (B), macrophages were nearly absent (<2% of normal), with counts of 7 ± 5 vs. 430 ± 16 (PBS-lp) and 418 ± 32 (NaCl) macrophages/mm² interstitium (mean ± SEM, N = 5).
Notably, Leydig cell morphology and spermatogenic epithelium remained intact despite macrophage depletion.
Literature 3
Source: The human chorionic gonadotrophin-induced inflammation-like response is enhanced in macrophage-depleted rat testes
Impact Factor: 3.9 (Q2)
Macrophage Depletion Protocol
To deplete testicular macrophages, adult male Wistar rats received an intratesticular injection of 150 μL clodronate liposomes (Cl₂MDP-containing) into the right testis using a 30 G needle, while the left testis received 150 μL of 0.9% NaCl as an internal control. On day 10 post-injection, rats were given an i.p. injection of EDS (75 mg/kg) to induce Leydig cell apoptosis, allowing assessment of the tissue response in the absence of macrophages.
Results
- Figure 5A: Lymphocyte infiltration increased 13-fold (peak at 12 h) in the macrophage-depleted (Cl₂MDP) testis, versus only 2-fold in the saline-injected control side.
- Figure 5B: Macrophage numbers remained near zero on the right side, while the left side showed a 2-fold increase between 24–72 h.
- Figure 5C: Leydig cell clearance efficiency was identical on both sides (complete loss within 24 h), indicating that phagocytic function was compensated by infiltrating monocytes.
Literature 4
Source: In vivo manipulation (depletion versus activation) of testicular macrophages: central and local effects
Impact Factor: 3.9 (Q2)
Macrophage Depletion Protocol
Male Wistar rats were used. Adult rats (70 days old) and juvenile rats (born in-house, litters standardized to 8 pups) were treated at 22 days of age. Under light ether anesthesia, a 30 G needle was used for intratesticular injection of:
(i) 100 μL clodronate liposomes bilaterally (bilateral depletion);
(ii) 100 μL clodronate liposomes in the right testis, 100 μL 0.9% NaCl in the left (unilateral depletion);
(iii) 100 μL MTP-PE liposomes bilaterally (macrophage activation);
(iv) 100 μL 0.9% NaCl bilaterally (control).
Rats were euthanized by decapitation 10 days post-injection. Trunk blood was collected, and serum stored at –20°C. Interstitial fluid was harvested from testes. In each bilateral treatment group (n = 5), 2 mL of 3% trypan blue in saline was administered i.p. 24 h before sacrifice to label macrophages. The right testis was fixed in Bouin-Hollande solution, processed for paraffin embedding, sectioned at 5 μm, and stained with neutral red or H&E.
Results
- Figures 2a–b: In juvenile rats, clodronate treatment led to near-complete macrophage ablation, accompanied by a ~50% reduction in Leydig cell number, though individual Leydig cell size remained unchanged.
- Figures 3A–C: Macrophage-depleted juvenile rats showed significantly reduced numbers of both macrophages and Leydig cells compared to controls.
Complete Solutions for Macrophage Research
Yeasen Biotechnology offers a comprehensive portfolio of reagents for macrophage research—covering every stage from macrophage isolation and polarization, to in vivo depletion model establishment, mechanistic functional studies, and immunological validation assays. Our integrated solutions help researchers accelerate discovery, improve reproducibility, and support publication in high-impact journals.
Related Product
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Name |
Cat. No. |
Size |
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40337ES08/10 |
5 mL/10 mL |
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40338ES08/10 |
5 mL/10 mL |