Cdk8/19 knockout blocks spermatogenesis in mice. (A) Crossing of Cdk8fl/fl, Cdk19-/- and Cre/ERT2 mice and formation of experimental (Cdk8fl/flCdk19-/-Cre/ERT2 + tamoxifen, Cdk8fl/flCre/ERT2 + tamoxifen and Cdk19-/-) and control (Cdk8fl/flCdk19-/-Cre/ERT2without tamoxifen and wild-type + tamoxifen) groups. (B) Confirmation of tamoxifen-induced CDK8KO in testes by Western blot. (C) Cyclin C protein is absent in DKO, but not single KO in the testes. (D) Time course of experiments. CDK8KO was activated by tamoxifen administration in males of 8-10 weeks old. Urogenital abnormalities became visible in two weeks. Spermatogenesis was analyzed by flow cytometry and immunofluorescence (IF) after 2, 8 and 20 weeks since activation. Single cell RNA sequencing was performed after 7 weeks of KO. (E) Male urogenital system atrophy in DKO mice. (F) H&E staining of prostate, epididymis and testes of DKO mice and tamoxifen-treated control. 100X magnification (G) H&E staining of WT and DKO seminiferous tubules. Yellow arrows indicate colchicine-like figures in the nuclei of dying cells.1000X magnification. (H) Sexual behavior and fertility of tamoxifen treated control, single KOs and DKO male mice.

Absence of postmeiotic 1n cells in DKO 2 months after KO induction. (A) Distinctive histograms of wild-type (left) and DKO (right) mice. Violet - 4n population, red - 2n population, green - round spermatids, blue - elongated spermatids, orange - apoptotic subG1 cells. (B) Quantitative distribution of testes cells between these groups. Wild type with and without tamoxifen as control groups have similar distribution to CDK8 and CDK19 single KO. DKO testes have greatly reduced number of round spermatids and no elongated spermatids. (C) Overall cellularity is also significantly reduced only in DKO testes. (D) IF staining of control and DKO testes frozen sections. Nuclei are stained by DAPI (blue pseudocolor), SYCP3 is depicted as green, γH2A.X - as red. All stages of spermatogenesis are visible in control testes, while pachytene is the last detected stage in DKO. Confocal microscopy, magnification 600X.

Single cell RNA sequencing reveals loss of spermatids due to steroidogenesis failure. (A) UMAP projection and relative cell numbers for all testicular cell types in control and DKO samples. Number of secondary spermatocytes is significantly decreased and spermatids are almost absent in DKO samples. (B) UMAP projection and relative cell numbers for spermatogonia and primary spermatocytes. Post-pachytene spermatocytes are severely depleted in DKO samples. (C) GO Biological Processes pathways enriched among Leydig cells differentially expressed genes (DEGs). Lipid metabolism and steroid biosynthesis is severely perturbed. (D) Violin plots for key Leydig cells’ genes.

DKO Sertoli cells re-enter the cell cycle and lose characteristic cytoskeleton organization. (A) Violin plots for Reactome cell cycle gene sets indicate that Sertoli cells in DKO lose terminal differentiation and re-enter cell cycle. Percentage of cells in G1-S and G2-M transitions are increased in DKOs. (B) Violin plots for key cytoskeleton and intercellular contacts related DEGs. (C) IF staining for vimentin demonstrates blood-testis barrier (BTB) integrity disruption and loss of characteristic striation cytoskeleton patterns in DKOs. Magnification 600X. (D) Enrichment of GO stress pathways in Sertoli cells indicates their dysfunction in DKOs.

Confirmation of single cell RNA sequencing data with other methods. (A) IF staining for CYP17A1 of testes frozen sections, magnification 600X. CYP17A1 is visualized in extratubular space in Leydig cells in control mice and is completely absent in DKOs. (B) Western blot for CYP17A1 confirms disappearance of the protein in DKOs, but not in other genotypes. (C) In agreement with this testosterone blood level is decreased only in DKO mice. (D) Luteinizing hormone production is not impaired by CDK8KO and CDK19KO or DKO.

А minor recovery of spermatogenesis 5 months after DKO induction. (A) Round and elongated spermatids are once again detected by flow cytometry 5 months after DKO, however, (B) overall testes cellularity is only slightly increased. (C) Postmeiotic cells become visible at H&E staining of the tubules, however, epididymal ducts remain empty. (D) Post-pachytene and post-meiotic (PM) cells became visible on the SYCP3 + γH2A.X stained frozen sections, magnification 600X. (E) CYP17A1 level remains at the background level 5 months after KO induction.

Effects of CDK8/19 inhibitor on spermatogenesis in mice (A-C) male C57BL/6 mice were treated with SNX631-6 medicated chow (500 ppm, 40-60 mg/kg/day dosage on average) for three weeks. (A) Representative images of H&E histology analysis of the testes tissues collected from animals in control or treated groups. (B) organ weights of testes (left and right) at endpoint. (C) SNX631-6 concentrations in plasma and testes tissues at endpoint. (D) qPCR analysis of steroidogenic Star and Fads genes in ex vivo cultured Leydig cells in response to CDK8/19i Senexin B (1 μM) or hydroxytamoxifen-induced CDK8/19 DKO.