Cell-signalling in sperm midpiece ensures quiescence and survival in cauda epididymis

Sperm in most mammalian species including rat, mice and human are kept completely quiescent (motionless) and viable for up to a few weeks in the cauda epididymis before ejaculation. Vigorous motility is initiated almost instantly upon sperm release from cauda during ejaculation. The molecular mechanisms that suppress sperm motility but increase cell-survival during storage in cauda epididymis are not known. Intracellular signalling via phosphorylation cascades are quick events that may regulate motility and survival of transcriptionally inactive sperm. Pathscan® intracellular signalling array provided the preliminary picture of cell-signaling in quiescent and motile rat sperm, indicating upregulation of cell-survival pathways in quiescent sperm, which were downregulated during motility activation. Interactome of signalling-proteins involved in motility activation was constructed by STRING-software, which identified MAPK-p38, AKT, mTOR and their downstream target p70S6K as the key kinases regulating sperm function. Further validation was achieved by western-blotting and pathway activators/inhibitors. Immunofluorescence localized the kinase proteins in the sperm mid-piece region (mitochondria), a known extra-nuclear target for these signalling pathways. Activators of these kinases inhibited sperm motility but increased viability, and vice-versa was true for inhibitors, in most of the cases. Activators and inhibitors also affected sperm mitochondrial membrane potential, ATP content and ROS levels. Data suggest that sperm motility and survival are inversely complementary and critically regulated by intracellular cell signalling. Aberrant cell signalling in caudal sperm may affect cell survival (sperm concentration) and motility of ejaculated sperm.

A sex chromosome inversion is associated with copy number variation of mitochondrial DNA in zebra finch sperm

The propulsion of sperm cells via movement of the flagellum is of vital importance for successful fertilization. While the exact mechanism of energy production for this movement varies between species, in avian species energy is thought to come predominantly from the mitochondria located in the sperm midpiece. Larger midpieces may contain more mitochondria, which should enhance the energetic capacity and possibly promote mobility. Due to an inversion polymorphism on their sex chromosome TguZ , zebra finches ( Taeniopygia guttata castanotis ) exhibit large within-species variation in sperm midpiece length, and those sperm with the longest midpieces swim the fastest. Here, we test through quantitative real-time PCR in zebra finch ejaculates whether the inversion genotype has an effect on the copy number of mitochondrial DNA (mtDNA). We find that zebra finches carrying the derived allele (correlated with longer sperm midpieces) have more copies of the mtDNA in their ejaculates than those homozygous for the ancestral allele (shorter midpieces). We suggest downstream effects of mtDNA copy number variation on the rate of adenosine triphosphate production, which in turn may influence sperm swimming speed and fertilization success. Central components of gamete energy metabolism may thus be the proximate cause for a fitness-relevant genetic polymorphism, stabilizing a megabase-scale inversion at an intermediate allele frequency in the wild.

Fis1 ablation in the male germline disrupts mitochondrial morphology and mitophagy, and arrests spermatid maturation

Male germline development involves choreographed changes to mitochondrial number, morphology, and organization. Mitochondrial reorganization during spermatogenesis was recently shown to require mitochondrial fusion and fission. Mitophagy, the autophagic degradation of mitochondria, is another mechanism for controlling mitochondrial number and physiology, but its role during spermatogenesis is largely unknown. During post-meiotic spermatid development, restructuring of the mitochondrial network results in packing of mitochondria into a tight array in the sperm midpiece to fuel motility. Here, we show that disruption of mouse Fis1 in the male germline results in early spermatid arrest that is associated with increased mitochondrial content. Mutant spermatids coalesce into multinucleated giant cells (GCs) that accumulate mitochondria of aberrant ultrastructure and numerous mitophagic and autophagic intermediates, suggesting a defect in mitophagy. We conclude that Fis1 regulates mitochondrial morphology and turnover to promote spermatid maturation.

ARMC12 regulates spatiotemporal mitochondrial dynamics during spermiogenesis and is required for male fertility

The mammalian sperm midpiece has a unique double-helical structure called the mitochondrial sheath that wraps tightly around the axoneme. Despite the remarkable organization of the mitochondrial sheath, the molecular mechanisms involved in mitochondrial sheath formation are unclear. In the process of screening testis-enriched genes for functions in mice, we identified armadillo repeat-containing 12 (ARMC12) as an essential protein for mitochondrial sheath formation. Here, we engineered Armc12-null mice, FLAG-tagged Armc12 knock-in mice, and TBC1 domain family member 21 (Tbc1d21)-null mice to define the functions of ARMC12 in mitochondrial sheath formation in vivo. We discovered that absence of ARMC12 causes abnormal mitochondrial coiling along the flagellum, resulting in reduced sperm motility and male sterility. During spermiogenesis, sperm mitochondria in Armc12-null mice cannot elongate properly at the mitochondrial interlocking step which disrupts abnormal mitochondrial coiling. ARMC12 is a mitochondrial peripheral membrane protein and functions as an adherence factor between mitochondria in cultured cells. ARMC12 in testicular germ cells interacts with mitochondrial proteins MIC60, VDAC2, and VDAC3 as well as TBC1D21 and GK2, which are required for mitochondrial sheath formation. We also observed that TBC1D21 is essential for the interaction between ARMC12 and VDAC proteins in vivo. These results indicate that ARMC12 uses integral mitochondrial membrane proteins VDAC2 and VDAC3 as scaffolds to link mitochondria and works cooperatively with TBC1D21. Thus, our studies have revealed that ARMC12 regulates spatiotemporal mitochondrial dynamics to form the mitochondrial sheath through cooperative interactions with several proteins on the sperm mitochondrial surface.

Pre- and post-copulatory traits working in concert: sexual dichromatism in passerines is associated with sperm morphology

Passerine birds produce costly traits under intense sexual selection, including elaborate sexually dichromatic plumage and sperm morphologies, to compete for fertilizations. Plumage and sperm traits vary markedly among species, but it is unknown if this reflects a trade-off between pre- and post-copulatory investment under strong sexual selection producing negative trait covariance, or variation in the strength of sexual selection among species producing positive covariance. Using phylogenetic regression, we analysed datasets describing plumage and sperm morphological traits for 278 passerine species. We found a significant positive relationship between sperm midpiece length and male plumage elaboration and sexual dichromatism. We did not find a relationship between plumage elaboration and testes mass. Our results do not support a trade-off between plumage and sperm traits, but may be indicative of variance among species in the strength of sexual selection to produce both brightly coloured plumage and costly sperm traits.

The novel testicular enrichment protein Cfap58 is required for Notch-associated ciliogenesis

Cilia and flagella are critical organelles with conserved internal structures and diverse developmental and physiological processes according to cell type. Although the core components of structures are shared with thousands of associated proteins involved in cilia or flagella formation, we hypothesized that some unknown proteins, such as outer dense fiber 2 (Odf2/Cenexin) perform distinct functions in these organelles. In the present study, we identified several uncharacterized proteins through mass spectrometry interactome analysis of Odf2/Cenexin proteins. We further examined the expression patterns and functions of a protein named cilia and flagella associated protein 58 (Cfap58) in cultured astrocytes and sperm flagella. The results of a combination of biochemical analyses and drug administration studies reveal that Cfap58 is a testis-enrichment protein that exhibits similar localization to Odf2/Cenexin proteins and is required for the elongation of the primary cilium and sperm midpiece via modulation of the Notch signaling pathway. However, the cell cycle-related functions and localization of Odf2/Cenexin in the mother centriole were not altered in Cfap58 knockdown cells. These findings indicate that Cfap58 may be partially recruited by Odf2/Cenexin proteins and is indispensable for the cilia and flagellar assembly. These data provide us with a better understanding of ciliogenesis and flagellar elongation and may aid in identifying new targets for diseases caused by Notch-mediated ciliopathies and flagellar abnormalities.

A sex chromosome inversion is associated with copy number variation of mitochondrial DNA in zebra finch sperm

Propulsion of sperm cells via movement of the flagellum is of vital importance for successful fertilization. Presumably, the energy for this movement comes from the mitochondria in the sperm midpiece. Larger midpieces may contain more mitochondria, which should enhance the energetic capacity and hence promote mobility. Due to an inversion polymorphism on their sex chromosome TguZ, zebra finches (Taeniopygia guttata castanotis) exhibit large within-species variation in sperm midpiece length, and those sperm with the longest midpieces swim the fastest. Here, we test through quantitative real-time PCR in zebra finch ejaculates whether the inversion genotype has an effect on the copy number of mitochondrial DNA. Taking the inversion genotype as a proxy for midpiece length, we find that zebra finches with longer midpieces indeed have more copies of the mitochondrial DNA in their ejaculates than those with shorter midpieces, with potential downstream effects on the rate of ATP production and sperm swimming speed. This study sheds light on the proximate cause of a fitness-relevant genetic polymorphism, suggesting the involvement of central components of gamete energy metabolism.Data availabilitySupplementary data file

High-energy diet enhances spermatogenic function and increases sperm midpiece length in fallow deer ( Dama dama ) yearlings

Nutrition is a major factor involved in the sexual development of livestock ruminants. In the male, a high-energy diet enhances the reproductive function, but its effects on the underlying processes such as spermatogenic efficiency are not yet defined. Moreover, the possible changes in sperm size due to a supplemented diet remain poorly investigated. The main goal of this study was to evaluate whether a high-energy diet affects the spermatogenic activity, epididymal sperm parameters (concentration, morphology, morphometry and acrosome integrity) and blood testosterone levels in fallow deer yearlings. For this purpose, 32 fallow deer were allocated into two groups according to their diet: control (pasture) and experimental (pasture and barley grain) groups. Fallow deer from the experimental group showed a significant increase in the Sertoli cell function and sperm midpiece length, together with a higher testicular mass, sperm concentration and percentage of normal spermatozoa than the control group ( p < 0.05). We also found a tendency for higher blood testosterone levels in the animals fed with barley grain ( p = 0.116). The better sperm quality found in the experimental group may be related to their higher efficiency of Sertoli cells and to an earlier onset of puberty. The results of the present work elucidate the mechanisms by which dietary supplementation enhances the male sexual development and might be useful for better practices of livestock management in seasonal breeders.

Increased F2-Isoprostane Levels in Semen and Immunolocalization of the 8-Iso Prostaglandin F2α in Spermatozoa from Infertile Patients with Varicocele

Polyunsaturated fatty acid damages lead to alterations in sperm function. This study aimed to investigate the involvement of F2-isoprostanes (F2-IsoPs), oxidized lipid products from arachidonic acid, in sperm quality impairment. For this purpose, F2-IsoP levels in semen and F2-IsoP localization in spermatozoa were explored in infertile subjects affected by idiopathic infertility or varicocele, as well as in fertile men. As compared to fertile men, in the idiopathic infertility and varicocele groups, sperm concentration, motility, morphology, viability, and fertility index were significantly lower and the mean scores concerning sperm apoptosis, necrosis, and immaturity were significantly higher. The idiopathic infertile group showed a reduction in sperm motility and fertility index, as well as an increase of apoptosis and necrosis percentages, in comparison to the varicocele group. The varicocele group showed the highest levels of F2-IsoPs, a significant increase of sperm immaturity, and a significant correlation between F2-IsoP levels and sperm immaturity. 8-Iso Prostaglandin F2α, biomarker of in vivo F2-IsoP, was clearly localized in sperm midpiece and cytoplasmic residues. Data show that F2-IsoP formation is relevant in semen and sperm from infertile patients with varicocele and high percentage of immaturity, suggesting that a correct fatty acid integrity is needed for sperm maturation.