Changes of male urogenital system in Cdk8/19 knockout mice. (A) Crossing of Cdk8fl/fl, Cdk19−/− and Cre/ERT2 mice and formation of experimental (Cdk8fl/fl/Cdk19−/−/Rosa-Cre/ERT2 + tamoxifen, Cdk8fl/fl/Rosa-Cre/ERT2 + tamoxifen and Cdk19−/−) and control (Cdk8fl/fl/Cdk19−/−/Rosa-Cre/ERT2 without tamoxifen and wild-type + tamoxifen) groups. (B) Confirmation of tamoxifen-induced CDK8 iKO in testes by Western blot. (C) After 2 months of KO induction iDKO mice had significantly lower body weight. (D) Male urogenital system atrophy in iDKO mice.

Cdk8/19 knockout blocks spermatogenesis in mice. (A) H&E staining of prostate, epididymis and testes of iDKO mice and tamoxifen-treated control. 100X magnification (B) H&E staining of WT and iDKO seminiferous tubules. 400X magnification. (G) Sexual behavior and fertility of tamoxifen treated control, single KOs and iDKO male mice. (C) Time course of experiments. CDK8 iKO 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. (D) Western blot analysis of Cre/ERT2 (Cre+Tam), single (CDK8 iKO and CDK19 KO) and double (iDKO) knockout testes 2 months after tamoxifen injections. CcnC protein is absent in iDKO, but not single KO in the testes. pSTAT727 is independent of CDK8/19 KO. Stars mark nonspecific staining by CDK19 antibodies. (E) CDK8 and CDK19 IHC staining of testes sections, 200x (upper row) and 630x magnification (bottom row) showing staining in various types of testicular cells. (F-K). Flow cytometry analysis of CDK8/CcnC expression in different testicular cell types. Figures F and I show major CDK8 (50.68%) and CcnC populations (44.52%), figures G and J show that 1n (round, but not elongated spermatids), 2n and 4n cells can be CDK8 and CcnC positive, figures H and K indicate, not only cKit+ cells among 2n-4n can be CDK8 and CcnC positive.

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. iDKO 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. M - meiotic entry spermatocytes; L - leptotene; Ph - pachytene; PM - post-meiotic stages.

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 iDKO samples. Number of secondary spermatocytes is significantly decreased and spermatids are almost absent in iDKO samples. (B) UMAP projection and relative cell numbers for spermatogonia and primary spermatocytes. Post-pachytene spermatocytes are severely depleted in iDKO 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 iDKO lose terminal differentiation and re-enter cell cycle. Percentage of cells in G1-S and G2-M transitions are increased in iDKOs. (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 iDKOs.

Confirmation of scRNA sequencing data by 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 iDKOs. (B) Western blot for CYP17A1 confirms disappearance of the protein in iDKOs, but not in other genotypes. (C) Testosterone blood level is decreased only in iDKO mice. (D) Luteinizing hormone production is not impaired by CDK8 iKO and CDK19 KO or iDKO.

Limited recovery of spermatogenesis 5 months after iDKO induction. (A) Round and elongated spermatids become detectable by flow cytometry 5 months after iDKO. (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 CDK8/19 inhibitor Senexin B (1 μM) or hydroxytamoxifen-induced CDK8/19 iDKO.