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. 2024 Nov 21;15(11):851.
doi: 10.1038/s41419-024-07242-z.

MLKL deficiency elevates testosterone production in male mice independently of necroptotic functions

Affiliations

MLKL deficiency elevates testosterone production in male mice independently of necroptotic functions

Shene Chiou et al. Cell Death Dis. .

Abstract

Mixed lineage kinase domain-like (MLKL) is a pseudokinase, best known for its role as the terminal effector of the necroptotic cell death pathway. MLKL-mediated necroptosis has long been linked to various age-related pathologies including neurodegeneration, atherosclerosis and male reproductive decline, however many of these attributions remain controversial. Here, we investigated the role of MLKL and necroptosis in the adult mouse testis: an organ divided into sperm-producing seminiferous tubules and the surrounding testosterone-producing interstitium. We find that sperm-producing cells within seminiferous tubules lack expression of key necroptotic mediators and thus are resistant to a pro-necroptotic challenge. By comparison, coordinated expression of the necroptotic pathway occurs in the testicular interstitium, rendering cells within this compartment, especially the lysozyme-positive macrophages, vulnerable to necroptotic cell death. We also uncover a non-necroptotic role for MLKL in regulating testosterone levels. Thus, MLKL serves two roles in the mouse testes - one involving the canonical response of macrophages to necroptotic insult, and the other a non-canonical function in male reproductive hormone control.

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Conflict of interest statement

Competing interests: KMP, JMH, KEL, ALS, and JMM contribute to or have contributed to a project developing necroptosis inhibitors in collaboration with Anaxis Pharma. The other authors declare no competing interests. Ethics declaration: All experiments were approved by the WEHI Animal Ethics Committee following the Prevention of Cruelty to Animals Act (1996) and the Australian National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Scientific Purposes (1997).

Figures

Fig. 1
Fig. 1. Co-expression of Caspase-8, RIPK1, RIPK3 and MLKL occurs in interstitial cells of the mouse testis.
A Immunoblot of Mlkl−/−Ripk3/ and wildtype mouse tissue lysates, n = 4 for wildtype and n = 4 Mlkl/−Ripk3−/. B Schematic diagram of mouse testis, not to scale. C Immunostaining of Caspase-8 (Casp8), RIPK1, RIPK3, and MLKL in knockout mice and wildtype mouse testes. For Caspase-8, RIPK1, and RIPK3 stain: n = 3 for Casp8−/Ripk1−/−Ripk3/ mice and n = 3 for wildtype mice. For MLKL stain: n = 3 for Mlkl−/− mice and n = 4 for wildtype mice. Scale bars in first two columns are 50 µm, scale bars in third column are 20 µm. Representative immunohistochemistry for Caspase-8, RIPK1 and RIPK3 shown in brown with haematoxylin counterstain in blue. Representative immunofluorescence for MLKL shown in magenta with nuclear Hoechst staining in white. Sertoli cells (➤), spermatogonium (#), blood vessel (*) and interstitial or Leydig cells (Red and white arrowheads) shown.
Fig. 2
Fig. 2. Mlkl−/− male mice have higher testosterone levels.
Each dot represents the abundance of testosterone in serum from one mouse analysed via LC-MS/MS. Male Mlkl+/+ wildtype n = 25, male Ripk3−/ n = 20, male Mlkl−/ n = 30, female wildtype n = 16, and female Mlkl−/ n = 19. Mice were between the age of 2–9 months for all groups. Mean ± SEM is presented. * P-value ≤ 0.05 and ** P-value ≤ 0.005. All other comparisons are non-significant. Data were analysed with one-way ANOVA with Tukey’s multiple comparisons test.
Fig. 3
Fig. 3. Spermatogenesis in wildtype mouse testes was only mildly affected by necroptotic challenge.
A Method of blinded categorization of seminiferous tubules. B Prevalence (%) of empty, partially full and full seminiferous tubules in TSZ treated and untreated testes in wildtype and Mlkl−/ mice. Number of wildtype and Mlkl−/ mice are n = 9 and n = 10 respectively. Mean ± SEM graphed. Data were analysed with ratio paired t test. * P-value ≤ 0.05, ** P-value ≤ 0.005. C Representative images of haematoxylin and eosin (H&E) stains of testes from wildtype and Mlkl−/ mice. Number of untreated wildtype, TSZ treated wildtype, untreated Mlkl−/ and TSZ treated Mlkl−/ mice testes were n = 9, n = 9, n = 10, and n = 10 respectively. D Representative micrographs of immunohistochemistry for SOX9 in wildtype and Mlkl−/ TSZ treated testes. Number of untreated wildtype, treated wildtype, untreated Mlkl−/, and treated Mlkl−/ mice testes stained for SOX9 were n = 5, n = 5, n = 4, and n = 4 respectively. E Quantification of SOX9+ Sertoli cells. Number of SOX9-stained untreated wildtype, treated wildtype, untreated Mlkl−/, and treated Mlkl−/ mice testes were n = 5, n = 5, n = 4. and n = 4 respectively. Data were analysed with one-way ANOVA with Tukey’s multiple comparisons test. Mean ± SEM graphed. No other comparisons reached statistical significance. Scale bars in (C, D) are 100 μm.
Fig. 4
Fig. 4. Loss of macrophages post-TSZ treatment in mouse testis.
A Immunohistochemistry for F4/80 and lysozyme in untreated wildtype, TSZ-treated wildtype and TSZ-treated Mlkl−/ mouse testes. Micrographs are representative of n = 3 mice/genotype. Scale bar represents 100 μm. B Immunohistochemistry for RIPK1 in testicular interstitial cells. Arrowheads indicate intracellular clusters of RIPK1 near the TSZ injection site. Scale bars represent 10 μm. C Summary of Figs. 1C, 3, and 4A. Red in diagram represents MLKL expression, purple represents RIPK1 expression, orange represents RIPK3 expression, and blue represents Caspase-8 expression in mouse testis. Serum testosterone comments are relative to wildtype male mice based on Fig. 2. TSZ injection conclusions are based on data presented in Fig. 3.

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