Testis

Microsurgical testicular sperm extraction (microTESE) with intracytoplasmic sperm injection (ICSI) represents the current standard treatment for nonobstructive azoospermia (NOA). However, cures remain unavailable for NOA patients lacking retrievable haploid cells. mRNA supplementation could be a potential treatment for genetic defects leading to impaired spermatogenesis. Lipid nanoparticles (LNPs) have emerged as mRNA delivery vehicles with minimal risk of genome integration; however, their ability to selectively deliver mRNA to specific cell types remains limited. To overcome this, microRNA (miRNA) target sequences were incorporated into mRNA constructs to restrict expression specifically to germ cells. Using pyruvate dehydrogenase E1 subunit alpha 2 (PDHA2) knockout mice as an NOA model with meiotic arrest, we demonstrate that LNP-mediated delivery of Pdha2 mRNA enables the resumption and completion of meiosis, restores sperm production, and facilitates the generation of healthy fertile offspring via ICSI. Whole-genome sequencing of the offspring confirmed the absence of large-scale genomic abnormalities. Our results provide proof of concept for a safe and effective chemically synthesized LNP-based mRNA therapy with miRNA-regulated germ cell specificity, offering a promising therapeutic approach to treating male infertility caused by spermatogenesis arrest.

BackgroundTesticular macrophages (TMs) are key regulators of testicular immune privilege and endocrine function in the testis. However, their age-related heterogeneity and role in testicular degeneration remain poorly characterized.MethodsWe performed spatial transcriptomics and FACS-enriched single-cell RNA sequencing (scRNA-seq) to characterize testicular macrophage heterogeneity across ageing. Key findings were validated through intratesticular injection of recombinant CCL8 protein, organotypic culture of seminiferous tubules, and immunofluorescence analysis.ResultsUsing spatial transcriptomics, we identified pronounced Leydig niche senescence in aged testes, mechanistically linked to TM-mediated inflammatory remodelling. Coupling FACS-enriched TM isolation with scRNA-seq resolved seven transcriptionally distinct subpopulations, including ageing-associated subsets (Ccl8hi and Cxcl13hi). These subsets exhibited inflammatory signalling rewiring (e.g., CCL8-CCR2 axis) and activation of senescence transcriptional regulators (ASCL2, SPI1, CEBPB, JUNB), with conserved mediators (CCL8, TREM2, IL1β, and CXCL2) across murine and human testes. Functional validation showed that intratesticular injection of recombinant CCL8 protein in 3-month-old mice recapitulated ageing phenotypes, such as germ cell apoptosis and steroidogenic decline.ConclusionsOur multi-omics atlas highlights TM heterogeneity as a driver of testicular inflammaging and identifies CCL8 as a conserved target for therapeutic interventions aimed at mitigating age-associated male reproductive decline.Key pointsSpatiotemporal multi‑omics establish inflammaging as a defining feature of testicular ageing. A refined CD74‑based sorting strategy improves enrichment of testicular macrophages. scRNA-seq resolves seven TM subsets with age‑dependent shifts. TM‑secreted inflammatory mediators-especially CCL8-drive testicular inflammaging.

Global climate change exacerbates heat stress, a major threat to male fertility in livestock. As Sertoli cells (SCs) are essential for spermatogenesis but are highly vulnerable to heat, understanding the underlying metabolic dysfunction is critical. This study investigated heat-induced metabolic dysregulation in goat SCs and evaluated the protective effects of melatonin. Through integrated transcriptomics and stable isotope-assisted metabolic flux analysis (MFA), we mapped alterations in gene expression and central carbon metabolism. Heat stress provoked mitochondrial dysfunction and oxidative damage. Metabolic flux analysis revealed suppressed mitochondrial oxidative metabolism, evidenced by reduced glucose-derived carbon incorporation into TCA cycle intermediates. Transcriptomics identified key perturbations in energy metabolism and sphingolipid signaling. Melatonin pretreatment restored mitochondrial function and redox homeostasis, and triggered a protective metabolic reprogramming that enhanced cell viability. Our findings demonstrate that melatonin mitigates heat stress by preserving mitochondrial integrity and redox homeostasis, providing mechanistic insights into male infertility and suggesting melatonin as a promising therapeutic for enhancing livestock reproductive resilience under climate stress.

Testicular organoids (TOs) offer an opportunity to preserve fertility, but current TO protocols are limited by suboptimal maintenance of the organoid structure, meiotic defects, and incomplete in vitro spermatogenesis (IVS). Here, a strategy is developed for self-reconstitution of single-cell suspensions of neonatal mouse testes into TOs containing the major testicular cell types and generation of tubular-like structures and haploid spermatids. Morphological and lineage-specific marker analyses revealed that these TOs met the criteria for organoids, including germ cell differentiation and recapitulation of key events in meiosis (e.g., chromosome recombination and synapsis). Notably, the spatiotemporal characteristics of spermatogenesis in the TOs are comparable to those in testes, and their derived haploids resembled step-4/5-like round spermatids in vivo. Further scRNA-seq analysis confirmed that spermatid-like cells accounted for ≈2.43% of the total germ cells. Next, undifferentiated germ cells are able to develop into haploid spermatids even when chimeric TOs are reconstructed via testicular somatic cells (Sertoli cells or Leydig cells) from mice with different genetic backgrounds. Collectively, the TO-based findings provide a promising platform for studies on testicular microenvironment construction, IVS, and fertility preservation.