Androglobin, a chimeric mammalian globin, is required for male fertility
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Evaluation Summary
This manuscript demonstrates that male mice lacking androglobin, a poorly understood heme-containing protein, are infertile and have defects in late stage spermatogenesis. A variety of molecular techniques were used to delineate the mechanism of spermatogenesis defect. A strength of the data is the identification of the androglobin interacting partner septin 10. With some clearer data on the mechanism underlying the connection between androglobin and septin, the paper will be of interest to researchers studying spermatogenesis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Spermatogenesis is a highly specialised process, involving multiple dedicated pathways and regulatory check-points. Defects ultimately lead to male sub-fertility or sterility, and numerous aspects of mammalian sperm formation remain unknown. The predominant expression of the latest globin family member, androglobin (Adgb) in mammalian testis tissue prompted us to assess its physiological function in spermatogenesis. Adgb knockout mice display male infertility, reduced testis weight, impaired maturation of elongating spermatids, abnormal sperm shape and ultrastructural defects in microtubule and mitochondrial organisation. Epididymal sperm from Adgb knockout animals display multiple flagellar malformations including coiled, bifide or shortened flagella, and erratic acrosomal development. Following immunoprecipitation and mass spectrometry, we could identify septin 10 (Sept10) as interactor of Adgb. The Sept10-Adgb interaction was confirmed both in vivo using testis lysates, and in vitro by reciprocal co-immunoprecipitation experiments. Furthermore, absence of Adgb leads to mislocalisation of Sept10 in sperm, indicating defective manchette and sperm annulus formation. Finally, in vitro data suggest that Adgb contributes to Sept10 proteolysis in a calmodulin (CaM)-dependent manner. Collectively, our results provide evidence that Adgb is essential for murine spermatogenesis and further suggest that interdependence between Adgb and Sept10 is required for sperm head shaping via the manchette and proper flagellum formation.
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Evaluation Summary
This manuscript demonstrates that male mice lacking androglobin, a poorly understood heme-containing protein, are infertile and have defects in late stage spermatogenesis. A variety of molecular techniques were used to delineate the mechanism of spermatogenesis defect. A strength of the data is the identification of the androglobin interacting partner septin 10. With some clearer data on the mechanism underlying the connection between androglobin and septin, the paper will be of interest to researchers studying spermatogenesis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This study convincingly shows that Androglobin (Adgb) merits its name. The authors show that the absence of Adgb from mice is associated with infertility of the males and gross defects in late-stage spermatogenesis. By various rigorous analyses, including mRNA sequencing, immunoblotting, immunoprecipitation, and microscopy, they further show that Adgb influences the levels of several septins, most notably Sept 10, interacts with the protein, and contributes to its cleavage. This is a well-written and thorough paper that significantly advances our understanding of mammalian spermatogenesis.
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Reviewer #2 (Public Review):
Keppner and colleagues report the physiological role of androglobin in spermatogenesis. The group has been working on androglobin for years. By generating KO mice, the authors identified an essential role of this chimeric globin in spermatogenesis and male fertility. Abundant data were presented, but it remains unclear to this reviewer how ablation of Adgb leads to the structural defects observed and which defects are primary and which ones are secondary. Although attempts were made to reveal the underlying mechanism, the authors failed to explain how the absence of ADGB causes disruptions and how ADGB-interacting septins are involved.
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Reviewer #3 (Public Review):
The manuscript of Keppner et al is a novel preliminary characterization of a testes specific noncanonical globin protein called Androglobin. This work builds off of the authors prior findings over the last decade on the androglobin gene, generating the first Adgb knockout mouse model to explore its physiological functions. Herein they convincingly demonstrate that the loss of Adgb results a striking reproductive phenotype, where knockout male mice are infertile and azoospermic due to complete disruption of spermatid elongation and maturation. They attempt to explain how this phenotype may manifest in these mice and show that Adgb binds with a number of critical proteins for spermatogenesis including septins. However, the only mechanism they are able to implicate (primarily via in vitro over expression …
Reviewer #3 (Public Review):
The manuscript of Keppner et al is a novel preliminary characterization of a testes specific noncanonical globin protein called Androglobin. This work builds off of the authors prior findings over the last decade on the androglobin gene, generating the first Adgb knockout mouse model to explore its physiological functions. Herein they convincingly demonstrate that the loss of Adgb results a striking reproductive phenotype, where knockout male mice are infertile and azoospermic due to complete disruption of spermatid elongation and maturation. They attempt to explain how this phenotype may manifest in these mice and show that Adgb binds with a number of critical proteins for spermatogenesis including septins. However, the only mechanism they are able to implicate (primarily via in vitro over expression studies) is that Adgb may be capable of proteolytic cleavage of septins via a calmodulin dependent interaction. Though certainly an exciting theory, the reasoning and mechanisms behind these phenomena still are unexplored and warrant further investigation.
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