We first found that SRSF1 expression showed fluctuations during spermatogenesis (Fig. S1 online). To define the specific involvement of SRSF1 in meiosis, we studied the physiological roles of SRSF1 in vivo using a Stra8-GFPCre Srsf1Fl/Fl mouse model (mouse experiments were approved by the Ethics Committee of China Agricultural University (ethical approval No. AW80401202-3-3)) (Fig. S2a–c online) [7], [8]. These data showed that conditional knockout (cKO) of Srsf1 in mouse germ cells impaired meiotic progression and led to non-obstructive azoospermia (NOA) (Fig. 1a, b and S2d–n online). To further evaluate the detailed phenotype, we observed spermatocytes using mouse germ cell surface spreading by co-immunostaining with antibodies against synaptonemal complex protein 3 (SYCP3) and phosphorylated histone H2AX (γH2AX) (Fig. 1c). The data indicated that loss of SRSF1 led to meiotic arrest at the pachytene stage. Meanwhile, we found all chromosomally abnormal synapsis. To further confirm the phenotype, SIX6OS1/C14ORF39, SYCP3, and CREST co-staining was performed during mouse germ cell surface spreading [9]. The data showed that homologous chromosomes were unable to undergo synapsis in cKO spermatocytes (Fig. 1d). In addition, HORMAD1, SYCP3, and CREST co-staining results revealed that all chromosomes failed to undergo synapsis during the pachytene stage (Fig. 1e). Successful homologous pairing relies on initial homology recognition and chromosome movement, and telomere-nuclear envelope attachment (TNEA) is essential for chromosome movement [10], [11]. Thus, intact synaptonemal complex (SC) and TNEA are the keys to synapsis formation [11], [12]. To explore the underlying molecular mechanisms, the expression of SC-related and TNEA-related genes was measured by RT-qPCR. The results showed that the expression of SC-related genes (e.g., Syce1l, Sycp2, Scre, Stag3, Syce2, Tex12, and Smc1b) and TNEA-related genes (e.g., Spdya, Terb1, Kash5, Cdk2, Majin, Terb2, and Sun1) was significantly reduced in cKO STRA8+ cells (Fig. 1f, g). To further explain the abnormal TNEA, transmission electron microscopy was performed on spermatocytes from 14 days post-partum (dpp) mouse testes. The results showed that telomeres were not anchored to the nuclear envelope due to the abnormalities of TNEA (Fig. 1h). To further evaluate this phenotype, TRF1, SYCP3, and Lamin B1 co-staining was performed in 14 dpp mouse testis sections (Fig. 1i). Quantitative results showed a decrease in telomere distribution on the nuclear periphery and an increase in the intra-nuclear domain (Fig. 1j).