Mammalian spermatozoa are highly polarized cells characterized by compartmentalized cellular structures and energy metabolism. Adenylate kinase (AK), which interconverts two ADP molecules into stoichiometric amounts of ATP and AMP, plays a critical role in buffering adenine nucleotides throughout the tail to support flagellar motility. Yet the role of the major AK isoform, AK1, is still not well characterized. Here, by using a proteomic analysis of testis biopsy samples, we found that AK1 levels were significantly decreased in non-obstructive azoospermia (NOA) patients. This result was further verified by immunohistochemical staining of AK1 on a tissue microarray. AK1 was found to be expressed in post-meiotic round and elongated spermatids in mouse testis and subsequent mature sperm in the epididymis. We then generated Ak1 knockout mice, which showed that AK1 deficiency did not induce any defects in testis development, spermatogenesis or sperm morphology and motility under physiological conditions. We further investigated detergent-modeled epididymal sperm and included individual or mixed adenine nucleotides to mimic energy stress. When only ADP was available, Ak1 disruption largely compromised sperm motility, manifested as a smaller beating amplitude and higher beating frequency, which resulted in less effective forward swimming. The energy restriction/recover experiments with intact sperm further addressed this finding. Besides, decreased AK activity was observed in sperm of a male fertility disorder mouse model induced by cadmium chloride. These results cumulatively demonstrate that AK1 was dispensable for testis development, spermatogenesis or sperm motility under physiological conditions, but was required for sperm to maintain a constant adenylate energy charge to support sperm motility under conditions of energy stress.

In mammals, testis development is triggered by expression of the sex-determining Y-chromosome gene SRY to commit Sertoli cell (SC) fate at gonadal sex determination in the fetus. Several genes have been identified to be required to promote the testis pathway following SRY activation (i.e. SOX9) in embryo; however, largely remains unknown about the genes and mechanisms involved in stabilizing the testis pathway after birth and throughout adulthood. Herein, we report that postnatal males with SC-specific deletion of Raptor demonstrated absence of SC unique identity and adversely acquired granulosa cell-like characteristics, along with loss of tubular architecture and scattered distribution of SCs and germ cells. Subsequent genome-wide analysis by RNA sequencing revealed a profound decrease in the transcripts of testis genes (i.e. Sox9, Sox8, and Amh) and conversely an increase in ovary genes (i.e. Lhx9, Foxl2, and Fst); these changes were further confirmed by immunofluorescence and quantitative reverse-transcription PCR. Importantly, co-immunofluorescence demonstrated that Raptor deficiency induced SCs dedifferentiation into a progenitor state; the Raptor-mutant gonads showed some ovarian somatic cell features, accompanied by enhanced female steroidogenesis and elevated estrogen levels, yet the ZP3-positive terminally feminized oocytes were not observed. In vitro experiments with primary SCs suggested that Raptor is likely involved in FGF9-induced formation of cell junctions among SCs. Taken together, our results established that Raptor is required to maintain SC identity, stabilize the male pathway, and promote testis development.

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Oligoasthenoteratozoospermia is a human infertility syndrome caused by defects in spermatogenesis, spermiogenesis, and sperm maturation, and its etiology remains unclear.Kelch-like 10 (KLHL10) is a component of ubiquitin ligase E3 10 (KLHL10) and plays an important role in male fertility. Deletion or mutation of the Klhl10 gene in Drosophila or mice results in defects in spermatogenesis or sperm maturation. However, the molecular mechanisms by which KLHL10 functions remain elusive. In this study, we identified a missense mutation (c.1528A→G, p.I510V) in exon 5 of KLHL10, which is associated with oligoasthenoteratozoospermia in humans. To investigate the effects of this mutation on KLHL10 function and spermatogenesis and/or spermiogenesis, we generated mutant mice duplicating the amino acid conversion using the clustered regularly interspaced palindromic repeat/caspase 9 (CRISPR/Cas9) system and designated them Klhl10 I510V mice. However, the Klhl10 I510V mice did not exhibit any defects in testis development, spermatogenesis, or sperm motility at ten-weeks-of-age, suggesting that this mutation does not disrupt the KLHL10 function, and may not be the cause of male infertility in the affected individual with oligoasthenoteratozoospermia.