The conserved genetic program of male germ cells uncovers ancient regulators of human spermatogenesis

Reviewer #1 (Public Review):

Summary:

By combining an analysis of the evolutionary age of the genes expressed in male germ cells, a study of genes associated with spermatocyte protein-protein interaction networks and functional experiments in Drosophila, Brattig-Correia and colleagues provide evidence for an ancient origin of the genetic program underlying metazoan spermatogenesis. This leads to identifying a relatively small core set of functional interactions between deeply conserved gene expression regulators, whose impairment is then shown to be associated with cases of human male infertility.

Strengths:

In my opinion, the work is important for three different reasons. First, it shows that, even though reproductive genes can evolve rapidly and male germ cells display a significant level of transcriptional noise, it is still possible to obtain convincing evidence that a conserved core of functionally interacting genes lies at the basis of the male germ transcriptome. Second, it reports an experimental strategy that could also be applied to gene networks involved in different biological problems. Third, the authors make a compelling case that, due to its effects on human spermatogenesis, disruption of the male germ cell orthoBackbone can be exploited to identify new genetic causes of infertility.

Weaknesses:

The main strength of the general approach followed by the authors is, inevitably, also a weakness. This is because a study rooted in comparative biology is unlikely to identify newly emerged genes that may adopt key roles in processes such as species-specific gamete recognition. Additionally, using a TPM >1 threshold for protein-coding transcripts may exclude genes, such as those encoding proteins required for gamete fusion, which are thought to be expressed at a very low level. Although these considerations raise the possibility that the chosen approach may miss information that, depending on the species, could be potentially highly functionally important, this by no means reduces its value in identifying genes belonging to the conserved genetic program of spermatogenesis.

Reviewer #2 (Public Review):

Summary:

This is a tour de force study that aims to understand the genetic basis of male germ cell development across three animal species (human, mouse, and flies) by performing a genetic program conservation analysis (using phylostratigraphy and network science) with a special emphasis on genes that peak or decline during mitosis-to-meiosis. This analysis, in agreement with previous findings, reveals that several genes active during and before meiosis are deeply conserved across species, suggesting ancient regulatory mechanisms. To identify critical genes in germ cell development, the investigators integrated clinical genetics data, performing gene knockdown and knockout experiments in both mice and flies. Specifically, over 900 conserved genes were investigated in flies, with three of these genes further studied in mice. Of the 900 genes in flies, ~250 RNAi knockdowns had fertility phenotypes. The fertility phenotypes for the fly data can be viewed using the following browser link: https://pages.igc.pt/meionav. The scope of target gene validation is impressive. Below are a few minor comments.

(1) In Supplemental Figure 2, it is notable that enterocyte transcriptomes are predominantly composed of younger genes, contrasting with the genetic age profile observed in brain and muscle cells. This difference is an intriguing observation and it would be curious to hear the author's comments.

(2) Regarding the document, the figures provided only include supplemental data; none of the main text figures are in the full PDF.

(3) Lastly, it would be great to section and stain mouse testis to classify the different stages of arrest during meiosis for each of the mouse mutants in order to compare more precisely to flies.

This paper serves as a vital resource, emphasizing that only through the analysis of hundreds of genes can we prioritize essential genes for germ cell development. its remarkable that about 60% of conserved genes have no apparent phenotype during germ cell development.

Strengths:

The high-throughput screening was conducted on a conserved network of 920 genes expressed during the mitosis-to-meiosis transition. Approximately 250 of these genes were associated with fertility phenotypes. Notably, mutations in 5 of the 250 genes have been identified in human male infertility patients. Furthermore, 3 of these genes were modeled in mice, where they were also linked to infertility. This study establishes a crucial groundwork for future investigations into germ cell development genes, aiming to delineate their essential roles and functions.

Weaknesses:

The fertility phenotyping in this study is limited, yet dissecting the mechanistic roles of these proteins falls beyond its scope. Nevertheless, this work serves as an invaluable resource for further exploration of specific genes of interest.