Knockout of cyclin dependent kinases 8 and 19 leads to depletion of cyclin C and suppresses spermatogenesis and male fertility in mice

Reviewer #1 (Public review):

Summary:

In this paper, Bruter and colleagues report effects of inducible deletion of the genes encoding the two paralogous kinases of the Mediator complex in adult mice. The physiological roles of these two kinases, CDK8 and CDK19, are currently rather poorly understood; although conserved in all eukaryotes, and among the most highly conserved kinases in vertebrates, individual knockouts of genes encoding CDK8 homologues in different species have revealed generally rather mild and specific effects, in contrast to Mediator itself. Here, the authors provide evidence that neither CDK8 nor CDK19 are required for adult homeostasis but they are functionally redundant for maintenance of reproductive tissue morphology and fertility in males.

Strengths:

The morphological data on the atrophy of the male reproductive system and the arrest of spermatocyte meiosis are solid and are reinforced by single cell transcriptomics data, which is a challenging technique to implement in vivo. The main findings are important and will be of interest to scientists in the fields of transcription and developmental biology.

Weaknesses:

There are several major weaknesses.

The first is that data on general health of mice with single and double knockouts is not shown, nor are there any data on effects in any other tissues. This gives the impression that the only phenotype is in the male reproductive system, which would be misleading if there were phenotypes in other tissues that are not reported. Furthermore, given that the new data show differing expression of CDK8 and CDK19 between cell types in the testis, data for the genitourinary system in single knockouts are very sparse; data are described for fertility in figure 1E, ploidy and cell number in figure 3B and C, plasma testosterone and luteinizing hormone levels in figure 6C and 6D and morphology of testis and prostate tissue for single Cdk8 knockout in supplementary figure 1C (although in this case the images do not appear very comparable between control and CDK8 KO, thus perhaps wider fields should be shown), but, for example, there is no analysis of different meiotic stages or of gene expression in single knockouts. This might have provided insight into the sterility of induced CDK8 knockout.

The second major weakness is that the correlation between double knockout and reduced expression of genes involved in steroid hormone biosynthesis is portrayed as a likely causal mechanism for the phenotypes observed. While this is a possibility, there are no experiments performed to provide evidence that this is the case. Furthermore, there is no evidence shown that CDK8 and/or CDK19 are directly responsible for transcription of the genes concerned.

Finally, the authors propose that the phenotypes are independent of the kinase activity of CDK8 or CDK19 because treatment of mice for a month with an inhibitor does not recapitulate the effects of the knockout, and nor does expression of two steroidogenic genes change in cultured Leydig cells upon treatment with an inhibitor. However, there are no controls for effective target inhibition shown.

Comments on revisions:

This manuscript is in some ways improved - mainly by toning down the conclusions - but a few major weaknesses have not been addressed. I do not agree that it is not justified to perform experiments to investigate the sterility of single CDK8 knockout mice since this could be important and given that the new data show that while there is some overlap in expression of the two prologues, there are also significant differences in the testis. At the least, it would have been interesting and easy to do to show the expression of CDK8 and CDK19 in the single cell transcriptomics, since this might help to identify the different populations.

The only definitive way of concluding a kinase-independent phenotype is to rescue with a kinase dead mutant. While I agree that the inhibitors have been well validated, since they did not have any effects, it is hard to be sure that they actually reached their targets in the tissue concerned. This could have been done by cell thermal shift assay. In the absence of any data on this, the conclusion of a kinase-independent effect is weak.

Figure 2 legend includes (G) between (B) and (C), and appears to, in fact, refer to Fig 1E, for which the legend is missing the description.

Finally, Figure S1C appears wrong. Goblet cells are not in the crypt but on the villi (so the graph axis label is wrong), and there are normally between 5 and 15 per villus, so the iDKO figure is normal, but there are a surprisingly high number of goblet cells in the controls. And normally there are 10-15 Paneth cells/crypt, so it looks like these have been underestimated everywhere. I wonder how the counting was done - if it is from images such as those shown here then I am not surprised as the quality is insufficient for quantification. How many crypts and villi were counted? Given the difficulty in counting and the variability per crypt/villus, with quantitative differences like this it is important to do quantifications blind. I personally wouldn't conclude anything from this data and I would recommend to either improve it or not include it. If these data are shown, then data showing efficient double knockout in this tissue should also accompany it, by IF, Western or PCR. Otherwise, given a potentially strong phenotype, repopulation of the intestine by unrecombined crypts might have occurred - this is quite common (see Ganuza et al, EMBO J. 2012).

Reviewer #2 (Public review):

Summary:

The authors tried to test the hypothesis that Cdk8 and Cdk19 stabilize the cytoplasmic CcNC protein, the partner protein of Mediator complex including CDK8/19 and Mediator protein via a kinase-independent function by generating induced double knockout of Cdk8/19. However the evidence presented suffer from a lack of focus and rigor and does not support their claims.

Strengths:

This is the first comprehensive report on the effect of a double knockout of CDK8 and CDK19 in mice on male fertility, hormones and single cell testicular cellular expression. The inducible knockout mice led to male sterility with severe spermatogenic defects, and the authors attempted to use this animal model to test the kinase-independent function of CDK8/19, previously reported for human. Single cell RNA-seq of knockout testis presented a high resolution of molecular defects of all the major cell types in the testes of the inducible double knockout mice. The authors also have several interesting findings such as reentry into cell cycles by Sertoli cells, loss of Testosterone in induced dko that could be investigated further.

Weaknesses:

The claim of reproductive defects in the induced double knockout of CDK8/19 resulted from the loss of CCNC via a kinase-independent mechanism is interesting but was not supported by the data presented. While the construction and analysis of the systemic induced knockout model of Cdk8 in Cdk19KO mice is not trivial, the analysis and data is weakened by systemic effect of Cdk8 loss, making it difficult to separate the systemic effect from the local testis effect.

The analysis of male sterile phenotype is also inadequate with poor image quality, especially testis HE sections. Male reproductive tract picture is also small and difficult to evaluate. The mice crossing scheme is unusual as you have three mice to cross to produce genotypes, while we could understand that it is possible to produce pups of desired genotypes with different mating schemes, such vague crossing scheme is not desirable and of poor genetics practice. Also using TAM treated wild type as control is ok, but a better control will be TAM treated ERT2-cre; CDK8f/f or TAM treated ERT2 Cre CDK19/19 KO, so as to minimize the impact from well-recognized effect of TAM.

While the authors proposed that the inducible loss of CDK8 in the CDK19 knockout background is responsible for spermatogenic defects, it was not clear in which cells CDK8/19 genes are interested and which cell types might have a major role in spermatogenesis. The authors also put forward the evidence that reduction/loss of Testosterone might be the main cause of spermatogenic defects, which is consistent with the expression change in genes involved in steroigenesis pathway in Leydig cells of inducible double knockout. But it is not clear how the loss of Testosterone contributed to the loss of CcnC protein.

The authors should clarify or present the data on where CDK8 and CDK19 as well as CcnC are expressed so as to help the readers to understand which tissues that both CDK might be functioning and cause the loss of CcnC. It should be easier to test the hypothesis of CDK8/19 stabilize CcnC protein using double knock out primary cells, instead of the whole testis.

Since CDK8KO and CDK19KO both have significantly reduced fertility in comparison with wildtype, it might be important to measure the sperm quantity and motility among CDK8 KO, CDK19KO and induced DKO to evaluate spermatogenesis based on their sperm production.

Some data for the inducible knockout efficiency of Cdk8 were presented in Supplemental figure 1, but there is no legend for the supplemental figures, it was not clear which band represented deletion band, which tissues were examined? Tail or testis? It seems that two months after the injection of Tam, all the Cdk8 were completely deleted, indicating extremely efficient deletion of Tam induction by two-month post administration. Were the complete deletion of Cdk8 happening even earlier ? an examination of timepoints of induced loss would be useful and instructional as to when is the best time to examine phenotypes.

The authors found that Sertoli cells re-entered cell cycle in the inducible double knockout but stop short of careful characterization other than increased expression of cell cycle genes.

Overall this work suffered from a lack of focus and rigor in the analysis and lack of sufficient evidence to support their main conclusions.

Comments on revisions:

This reviewer appreciated the authors' effort in improving the quality of this manuscript during their revision. While some concerns remain, the revision is a much improved work and the authors addressed most of my major concerns.
Figure 2E CDK8 and CDK19 immunofluorescent staining images seem to show CDK8 and CDK19 location are completely distinct and in different cells, the authors need to elaborate on this results and discuss what such a distinct location means in line of their double knockout data.

Minor comments:

Supplemental figure 1(C) legend typo : (C) Periodic acid-Schiff stained sections of ilea of tamoxifen treated R26/Cre/ERI2 and DKO mice.

While the effort to identify and generate new antibodies is appreciated, the specificity of the antibodies used should be examined and presented if available.

Author response:

The following is the authors’ response to the original reviews.

The mice crossing scheme is unusual as you have three mice to cross to produce genotypes, while we could understand that it is possible to produce pups of desired genotypes with different mating schemes, such a vague crossing scheme is not desirable and of poor genetics practice.

We thank the reviewer for this suggestion. Indeed, our scheme is not a representation of the actual breeding scheme but just a brief explanation of lineages used for the acquisition of the triple transgenic mice. We will include the full crossing scheme into the revision.

We added to the text the explanation that all used genotypes were maintained as homozygotes and put a full breeding scheme in the supplementary figure S1A

It is worth mentioning that single knockouts seem to show a corresponding upregulation of the level of the paralogue kinase, indicating that any lack of phenotypes might be due to feedback compensation, which would be an interesting finding if confirmed; this has not been mentioned.

We thank the reviewer for raising an important point about the paralog upregulation. Indeed, our data on primary cells (supplementary 1B) suggests the upregulation of CDK19 in CDK8KO and vice versa. We will point this out in discussion. We plan to examine the data for the testis as soon as more tissues are available.

We addressed this question by performing additional western blot (added to the paper fig. 2D) and found no paralogue upregulation in testes. To do that we also manufactured novel rabbit anti-mouse CDK19 antibodies described in Materials and Methods.

The authors should clarify or present the data on where CDK8 and CDK19 as well as CcnC are expressed so as to help the readers understand which tissues both CDK might be functioning in and cause the loss of CcnC.

Due to a limited sensitivity of single cell sequencing (only ~5,000 transcripts are sequenced from total of average 500,000 transcripts per cell, so the low expressed transcripts are not sequenced in all cells) it is challenging to firmly establish CDK8/19 positive and -negative tissues from single cell data because both transcripts are minor. This image will be included in the next version.

In this version we have added staining by CDK8 and CDK19 antibodies on paraffin sections, showing expression in variety of cells. Additionally, we have analyzed Cdk8/CcnC presence in different testicular cell types by flow cytometry. Both methods show that not only spermatogonial stem cells express Cdk8 as was shown in McCleland et al. 2005, but also some 1n cells, 4n cells and a significant part of cKit^- 2n cells. We added a corresponding paragraph and figures (2E-K) to the paper. We consider this a more definitive answer to the question than RNA data.

Furthermore, data for the genitourinary system in single knockouts are very sparse; data are described for fertility in Figure 1H, ploidy, and cell number in Figures 2B and C, plasma testosterone and luteinizing hormone levels in Figures 5C and 5D, and morphology of testis and prostate tissue for single Cdk8 knockout in Supplementary Figure 1C (although in this case the images do not appear very comparable between control and CDK8 KO, thus perhaps wider fields should be shown), but, for example, there is no analysis of different meiotic stages or of gene expression in single knockouts. It is worth mentioning that single knockouts seem to show a corresponding upregulation of the level of the paralogue kinase, indicating that any lack of phenotypes might be due to feedback compensation, which would be an interesting finding if confirmed; this has not been mentioned.

We agree that a description of the single KO could be beneficial, but we expect no big differences with the WT or Cre-Ert. We found neither histological differences nor changes in cell counts or ratios of cell types. Our ethical committee also has concerns about sacrificing mice without major phenotypic changes, without a well formulated hypothesis about the observed effects. We plan to add histological pictures to the next version of the article.

We have updated histological figures with new figures for iDKO and Cre+Tam mice with additional fields of view and better quality staining (2A-B).

The second major weakness is that the correlation between double knockout and reduced expression of genes involved in steroid hormone biosynthesis is portrayed as a causal mechanism for the phenotypes observed. While this is a possibility, there are no experiments performed to provide evidence that this is the case. Furthermore, there is no evidence showing that CDK8 and/or CDK19 are directly responsible for the transcription of the genes concerned.

We agree with the reviewer that the effects on CDK8/CDK19/CCNC could lead to the observed transcriptional changes in multiple indirect steps. There are, however, major technical challenges in examining the binding of transcription factors in the tissue, especially in Leydig cells which are a relatively minor population. We will clarify it in the revision and strengthen this point in the discussion.

We have added corresponding explanation in the Discussion: “We hypothesize that all these changes are caused by disruption of testosterone synthesis in Leydig cells, although, at this point, we cannot definitively prove that the affected genes are regulated by CDK8/19 directly.”

The claim of reproductive defects in the induced double knockout of CDK8/19 resulted from the loss of CCNC via a kinase-independent mechanism is interesting but was not supported by the data presented. While the construction and analysis of the systemic induced knockout model of Cdk8 in Cdk19KO mice is not trivial, the analysis and data are weakened by the systemic effect of Cdk8 loss, making it difficult to separate the systemic effect from the local testis effect.

We agree with the reviewer that the effects on the testes could be due to the systemic loss of CDK8 rather than specifically in the testis, and we will clarify it in the revision. We will also clarify that although our results are suggestive that the effects of CDK8/19 knockout are kinase-independent, and that the loss of Cyclin C is a likely explanation for the kinase independence, but we do not claim that it is *the* mechanism.

In this version we added several caveats indicating that the proposed mechanism is likely, but not the only one possible.

Also using TAM-treated wild type as control is ok, but a better control will be TAM-treated ERT2-cre; CDK8f/f or TAM-treated ERT2 Cre CDK19/19 KO, so as to minimize the impact from the well-recognized effect of TAM.

We used TAM-treated ERT2-cre for most of the experiments, and did not observe any major histological or physiological differences with the WT+TAM. We will make sure to present them in the revision.

The authors found that Sertoli cells re-entered the cell cycle in the inducible double knockout but stopped short of careful characterization other than increased expression of cell cycle genes.

Unfortunately, we were not able to perform satisfactory Ki67 staining to address this point.

Dko should be appropriately named iDKO (induced dKO). We will make the corresponding change.

We performed necropsy ? not the right wording here.

Colchicine-like apoptotic bodies ? what does this mean? Not clear.

We made appropriate changes - all DKO were renamed iDKO, necropsy changed to autopsy and cells designated as “apoptotic”.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Given the proprietary claims of the authors ("We have for the first time generated mice with the systemic inducible Cdk8 knockout on the background of Cdk19 constitutive knockout"), it does not appear acceptable and indeed might be misleading, to not describe the overall phenotypes of the mice. Are mice normal size/weight? Does an autopsy reveal anything other than atrophied genital tissue in males? Do the authors find a phenotype in the intestinal epithelium, as previously reported? (N.B. this could potentially clarify a discrepancy in the literature since the loss of the secretory lineages in double knockouts reported by the Firestein lab was not reproduced by intestinal organoid double knockout in the paper by the Fisher lab).

We have removed the statement “for the first time”, although to the best of our knowledge this is the fact. We did not attempt to describe all the phenotypic effects of the Cdk8/19 knockout in this paper, since some of the phenotypic observations related to mouse weight and behavior varied between different laboratories involved and require additional analysis. The effect on the urogenital system was by far the most striking histological feature observed and it was carefully addressed in this paper. Other findings require additional experiments and are out of the scope of this paper and we plan to focus on them later. As per suggestion of the reviewer we performed histological analysis of DKO intestines and found the same decrease in the Paneth and goblet cells numbers as described by Dannappel et al. We added corresponding figures (Supplemental fig. 1C) to the paper.

If the authors wish to reinforce their claims about causality of steroidogenic gene expression and phenotype, they could try rescuing the phenotype by treating mice with testosterone.

As stated in Discussion, we hypothesized that injection of testosterone would not rescue the phenotype, as the androgen receptor signaling is also affected. However we would like to perform such an experiment, but we were not able to procure testosterone pellets at this time.

If they wish to claim a direct effect of CDK8/19 on the expression of steroidogenic genes, they could also assess CDK8/19 binding to promoters of the genes analysed by ChIP.

There are big technical challenges in examining the binding of transcription factors in the primary tissue, especially in Leydig cells, a minor population, so we cannot perform such an experiment.

In order to conclude that their CDK8/19 inhibitor treatment worked, they could show target engagement by cell thermal shift assay, loss of CDK8/19 kinase-dependent gene expression, or loss of CDK8/19 substrate phosphorylation (eg interferon-induced STAT1 S727 phosphorylation) under the conditions used. Alternatively, they could show rescue with a kinase-dead allele.

As noted in public comments - we thank the reviewer for raising this concern. The target selectivity and target engagement by the inhibitors used in this study (Senexin B and SNX631-6) have been described in other models and published. CDK8/19 engagement and target selectivity of Senexin B, used in our vitro studies, have been extensively characterized in cell-based assays (Chen et al., Cells 2019, 8(11), 1413; Zhang et al., J Med Chem. 2022 Feb 24;65(4):3420-3433.) Similar characterization has been published for SNX631-6 and its equipotent analog SNX631, which showed drastic antitumor activity when used in vivo at the same dosing regimen as in this paper (Li et al., J Clin Invest. 2024;134(10):e176709). The comparison of the pharmacokinetics data obtained in the present study and in vitro activity of SNX631-6 in a cell-based assay suggests that the tissue concentrations of this drug should have provided substantial inhibition of Cdk8/19. Unfortunately, there are no known phosphorylation substrates specific for Cdk8/19 that can be used as pharmacodynamic markers. The widely used STAT1 phosphorylation at S727 is exerted not only by CDK8/19 but also by other kinases and shows variable response to CDK8/19 inhibition (Chen et al., Cells 2019, 8(11), 1413). In the revised MS, we have added a Western blot with pSTAT1 S727 staining of WT, 8KO, 19KO and iDKO testes. Cdk8/19 knockout did not decrease and apparently even increased the level of pSTAT1 S727, which demonstrates that this marker of CDK8/19 activity it is not suitable for our tissue type. While the evidence that Cdk8/19 kinase inhibition in the testes after in vivo drug treatment does not match the phenotype of iDKO is admittedly indirect, the same result has been obtained in the cell culture studies with Sertoli cells, where the inhibitor concentration (1 µM Senexin B) was much higher than needed for the maximal Cdk8/19 inhibition.

Finally, I did not find any legends to supplementary figures anywhere.

We apologize for not including legends for supplementary figures, and will correct that in the next version of the manuscript.

Additionally, we addressed the question about the sufficiency of the lipid supply for steroidogenesis in testes. There was a possibility that steroidogenesis is impossible due to the lack of cholesterol input, but OilRed staining revealed that the situation is the opposite: lipid content in iDKO testes is significantly higher than in WT testes. We added corresponding text to the article and the supplementary Fig. S6.