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The Myc network’s multiple functions in spermatogenesis

From the Eisenman Lab, Basic Sciences Division

Sperm – in theory, you only need one good one to get the job done. Nevertheless, the average pair of human testicles is estimated to crank out about a trillion of these little cells over a lifetime, leading them to be considered perhaps the most expendable cells in our bodies. But sperm don’t come cheap, and their plenitude is not without its limits – sperm production is a metabolically intensive process, and defects in the development or maintenance of the germline progenitor cells responsible for their production can have serious consequences for fertility. In a new paper in PLOS Biology, Dr. Bob Eisenman, a professor in Fred Hutch’s Basic Sciences division and member of the Fred Hutch/UW Cancer Consortium, and colleagues identify a role for the MYC protein network in cellular metabolism, differentiation, and survival during sperm production.

Dr. Eisenman’s longstanding interest in the MYC network of proteins is largely focused on their role in cancers. His group’s work took an unexpected turn, however, when they sought to understand the role of the Myc network member MLX in normal mouse development. Surprisingly, explained lead author Dr. Patrick Carroll, they found that “loss of MLX in mice has no apparent effect on embryonic development yet leads to complete male infertility”. While females lacking MLX (MLXKO) bred normally, the sperm of MLXKO males were markedly deficient in number and quality. Male mice lacking the MLX binding partner and fellow MYC network member MondoA were also infertile, though in this case sperm were generated at appropriate numbers but were nonmotile, suggesting at least partially distinct functions for these partners. Eisenman noted that "before this study, we really didn't know anything about sperm development. However, with considerable help from Chip Muller in the UW Male Fertility Lab we were able to jump-start this project". Pei-Feng Cheng, a staff associate in the Eisenman Lab, also generated the mice and made major contributions to the analysis.

Because the MYC network proteins are transcriptional regulators, the authors next performed gene expression profiling of the testes to better understand the root causes of the defective sperm production. While markers for mature sperm were decreased, markers for immature germ cells were increased, indicating that germ cell differentiation is blocked at an early stage of sperm development in MLXKO mice. These data also revealed signatures of altered metabolism, and metabolic testing confirmed disruptions in both glucose and lipid metabolism in the seminiferous tubules, suggesting that MLX controls the energy production process involved in making sperm. Finally, the authors observed signatures of increased cell stress and apoptosis, including upregulation of the Fas cell death receptor, consistent with their observation that many immature germ cells appeared to undergo apoptosis in MLXKO testes. These characteristics of MLXKO testes could be compounding, the authors note, as altered glucose levels might promote Fas-dependent cell death.

Finally, the authors sought to understand how MLX works with other MYC network proteins to regulate germ cell gene expression. Chromatin immunoprecipitation showed that MLX and MAX, MYC’s obligate binding partner, bind many shared genes in the testes, including those known to regulate sperm differentiation, and that MAX binding is shifted in MLXKO testes, particularly to genes involved in stress response. They further found that loss of the MLX-binding transcriptional repressor MNT also promotes apoptosis, suggesting that these two proteins may normally work together to suppress cell death. In conclusion, the authors propose that MLX plays a complex and multifaceted role in sperm cell development, working with several partner proteins to coordinately regulate differentiation, metabolism, and cell survival (Figure 1).

While this foray into reproductive biology may seem quite the deviation from Dr. Eisenman’s general focus on cancer mechanisms, it is not so great a shift as it appears. His group has previously found, for instance, that MLX facilitates tumor growth by promoting metabolic reprogramming and suppressing apoptosis. And in this study, they found that MLX’s roles in  male germ cell tumors (MGCTs) are similar to those it plays in normal sperm development – namely metabolic support and suppression of cell death, two properties that, while we may value them in our sperm, we certainly do not in our tumors. Thus, this work provides a clear example of the close relationship between normal development and cancer, and of the ways in which research in these two areas can synergize.

Following up on this work, Dr. Carroll is keen to probe deeper into MLX’s transcriptional regulation of the various cellular activities revealed here. “While we have demonstrated a role for MLX in suppression of stress and apoptosis in cells within the testes and MGCTs as well as primary immune cells, understanding the detailed genomic basis of this activity, and particularly its involvement with MYC's roles in immunoregulation, metabolism and oncogenesis will be broadly relevant. I anticipate that a deeper understanding of the MLX arm of the MYC network will provide new insights into therapeutic modulation of MYC activity in a wide range of cancers. My immediate plans involve in depth mapping of the MYC network genomic occupancy and determination of its role in higher order chromatin structure and epigenetics in both normal and transformed immune cells, as well as other cancer types.” Much like development itself, while this work began with sperm, it appears primed to expand into a wealth of diverse cell types.

MLX funtions in spermatogenesis
MLX functions with several MYC network proteins to regulate a variety of cellular functions during sperm development. Image provided by Patrick Carroll.

This work was supported by the National Institutes of Health and the Howard Hughes Medical Institute.

Fred Hutch/UW Cancer Consortium members Bob Eisenman, Dan Raftery and Jay Shendure contributed to this work.

Carroll PA, Freie BW, Cheng PF, Kasinathan S, Gu H, Hedrich T, Dowdle JA, Venkataramani V, Ramani V, Wu X, Raftery D, Shendure J, Ayer DE, Muller CH, Eisenman RN. The glucose-sensing transcription factor MLX balances metabolism and stress to suppress apoptosis and maintain spermatogenesis. PLoS Biol. 2021 Oct 20;19(10):e3001085. doi: 10.1371/journal.pbio.3001085. PMID: 34669700; PMCID: PMC8528285.