Sperm motility in mice with Oligo-astheno-teratozoospermia restored by in vivo injection and electroporation of naked mRNA

Reviewer #1 (Public Review):

The authors assess the effectiveness of electroporating mRNA into male germ cells to rescue the expression of proteins required for spermatogenesis progression in individuals where these proteins are mutated or depleted. To set up the methodology, they first evaluated the expression of reporter proteins in wild-type mice, which showed expression in germ cells for over two weeks. Then, they attempted to recover fertility in a model of late spermatogenesis arrest that produces immotile sperm. By electroporating the mutated protein, the authors recovered the motility of ~5% of the sperm, although the sperm regenerated was not able to produce offspring using IVF.

This is a comprehensive evaluation of the mRNA methodology with multiple strengths. First, the authors show that naked synthetic RNA, purchased from a commercial source or generated in the laboratory with simple methods, is enough to express exogenous proteins in testicular germ cells. The authors compared RNA to DNA electroporation and found that germ cells are efficiently electroporated with RNA, but not DNA. The differences between these constructs were evaluated using in vivo imaging to track the reporter signal in individual animals through time. To understand how the reporter proteins affect the results of the experiments, the authors used different reporters: two fluorescent (eGFP and mCherry) and one bioluminescent (Luciferase). Although they observed differences among reporters, in every case expression lasted for at least two weeks.

The authors used a relevant system to study the therapeutic potential of RNA electroporation. The ARMC2-deficient animals have impaired sperm motility phenotype that affects only the later stages of spermatogenesis. The authors showed that sperm motility was recovered to ~5%, which is remarkable due to the small fraction of germ cells electroporated with RNA with the current protocol. The 3D reconstruction of an electroporated testis using state-of-the-art methods to show the electroporated regions is compelling.

The main weakness of the manuscript is that although the authors manage to recover motility in a small fraction of the sperm population, it is unclear whether the increased sperm quality is substantial to improve assisted reproduction outcomes. The quality of the sperm was not systematically evaluated in the manuscript, with the endpoints being sperm morphology and sperm mobility.

Some key results, such as the 3D reconstruction of the testis and the recovery of sperm motility, are qualitative given the low replicate numbers or the small magnitude of the effects. The presentation of the sperm motility data could have been clearer as well. For example, on day 21 after Armc2-mRNA electroporation, only one animal out of the three tested showed increased sperm motility. However, it is unclear from Figure 11A what the percentage of sperm motility for this animal is since the graph shows a value of >5% and the reported aggregate motility is 4.5%. It would have been helpful to show all individual data points in Figure 11A.

The expression of the reporter genes is unambiguous; however, better figures could have been presented to show cell type specificity. The DAPI staining is diffused, and it is challenging to understand where the basement membranes of the tubules are. For example, in Figures 7B3 and 7E3, the spermatogonia seems to be in the middle of the seminiferous tubule. The imaging was better for Figure 8. Suboptimal staining appears to lead to mislabeling of some germ cell populations. For example, in Supplementary Figure 4A3, the round spermatid label appears to be labeling spermatocytes. Also, in some instances, the authors seem to be confusing, elongating spermatids with spermatozoa, such as in the case of Supplementary Figures 4D3 and D4.

The characterization of Armc2 expression could have been improved as well. The authors show a convincing expression of ARMC2 in a few spermatids/sperm using a combination of an anti-ARMC2 antibody and tubules derived from ARMC2 KO animals. At the minimum, one would have liked to see at least one whole tubule of a relevant stage.

Overall, the authors show that electroporating mRNA can improve spermatogenesis as demonstrated by the generation of motile sperm in the ARMC2 KO mouse model.

Reviewer #2 (Public Review):

Summary:

Here, the authors inject naked mRNAs and plasmids into the rete testes of mice to express exogenous proteins - GFP and later ARMC2. This approach has been taken before, as noted in the Discussion to rescue Dmc1 KO infertility. While the concept is exciting, multiple concerns reduce reviewer enthusiasm.

Strengths:

The approach, while not necessarily novel, is timely and interesting.

Weaknesses:

Overall, the writing and text can be improved and standardized - as an example, in some places in vivo is italicized, in others it's not; gene names are italicized in some places, others not; some places have spaces between a number and the units, others not. This lack of attention to detail in the preparation of the manuscript is a significant concern to this reviewer - the presentation of the experimental details does cast some reasonable concern with how the experiments might have been done. While this may be unfair, it is all the reviewers have to judge. Multiple typographical and grammatical errors are present, and vague or misleading statements.

Reviewer #3 (Public Review):

Summary:

The authors used a novel technique to treat male infertility. In a proof-of-concept study, the authors were able to rescue the phenotype of a knockout mouse model with immotile sperm using this technique. This could also be a promising treatment option for infertile men.

Strengths:

In their proof-of-concept study, the authors were able to show that the novel technique rescues the infertility phenotype in vivo.

Weaknesses:

Some minor weaknesses, especially in the discussion section, could be addressed to further improve the quality of the manuscript.

It is very convincing that the phenotype of Armc2 KO mice could (at least in part) be rescued by injection of Armc2 RNA. However, a central question remains about which testicular cell types have been targeted by the constructs. From the pictures presented in Figures 7 and 8, this issue is hard to assess. Given the more punctate staining of the DNA construct a targeting of Sertoli cells is more likely, whereas the more broader staining of seminiferous tubules using RNA constructs is talking toward germ cells.

Further, the staining for up to 119 days (Figure 5) would point toward an integration of the DNA construct into the genome of early germ cells such as spermatogonia and/or possibly to Sertoli cells. Given the expression after RNA transfection for up to 21 days (Figure 4) and the detection of motile sperm after 21 days (Figure 11), this would point to either round spermatids or spermatocytes.

These aspects need to be discussed more carefully (discussion section: lines 549-574).

It would also be very interesting to know in which testicular cell type Armc2 is endogenously expressed (lines 575-591).