Biology subjects

O'Bryan, M. K.

Publications and source records attributed to O'Bryan, M. K..

9 recordsLinked to original sources

bioRxiv2024

Mammalian heat shock protein A4 family ortholog Hsc70Cb is required for two phases of spermatogenesis in D. melanogaster

Heat shock proteins play essential roles as molecular chaperones, enacting protein folding and preventing of protein aggregation. In a previous study, a predicted damaging homozygous non-synonymous genetic variant was detected in the heat shock protein gene HSPA4L. Here, we used RNA interference in Drosophila melanogaster to explore the role of the heat shock protein A member 4 family (HSPA4) family in male fertility. Expression of the fly orthologue of the mammalian HSPA4 and HSPA4L genes, Hsc70Cb, was ablated in the male germline using two RNAi lines and the Nanos-Gal4 driver. Strong knockdown of Hsc70Cb in male germ cells resulted in male sterility, characterised by the absence of germ cells in testes and the over-proliferation of the testis soma. A less robust knockdown of Hsc70Cb in the male germline resulted in a sperm individualisation defect and a failure of sperm release into the seminal vesicle (analogous to the epididymis). When human HSPA4 or HSPA4L cDNA was introduced into infertile Hsc70Cb mutant flies, a partial rescue was observed, whereby in the robust Hsc70Cb knockdown setting germ cells progressed to the spermatocyte stage before undergoing cell death. Collectively, the absence of sperm in the Hsc70Cb (line 1) is consistent with the infertile man harbouring a homozygous HSPA4L genetic variant, supporting the hypothesis that HSPA4L is required for male fertility in humans and flies and highlighting the utility of the fly as a model of human spermatogenesis. In briefHsc70Cb is essential for spermatogonia survival and sperm individualisation in Drosophila. This study highlights the conserved roles of the HSPA4 family across animals and the utility of flies as a model organism for male fertility research.

genetics↗

bioRxiv2024

A novel CCDC112-dependent process of sperm midpiece formation and epididymal maturation

The sperm mitochondrial sheath has proposed functions in structural support and energy production for motility. Here we define coiled coil domain containing protein 112, CCDC112, as crucial for male fertility, specifically in the assembly and function of the mitochondrial sheath. We unveiled a previously unrecognised process of epididymal mitochondrial sheath maturation. Sperm mitochondrial sheaths are structurally immature upon exiting the testis and continue to mature as they transit from the caput to the cauda epididymis. Data reveal the critical role of CCDC112 in mitochondrial morphogenesis during sheath formation. A lack of CCDC112 lead to significantly reduced mitochondrial respiration capacity, irregular flagellar waveforms, diminished progressive motility and a failure of sperm to traverse the female reproductive tract to the site of fertilisation and penetrate the zonae pellucidae of oocytes. Finally, we identify CCDC112 as a component of the distal appendages of the mother centriole and identify a facilitative role in core tail assembly. Collectively, CCDC112 is a key regulator of sperm mitochondrial sheath assembly while further expanding the understanding of spermatogenesis, energy generation and flagella kinematics.

developmental biology↗

bioRxiv2023

AXDND1 is required to balance spermatogonial commitment and for sperm tail formation in mice and humans

Dynein complexes are large, multi-unit assemblies involved in many biological processes including male fertility via their critical roles in protein transport and axoneme motility. Previously we identified a pathogenic variant in the dynein gene AXDND1 in an infertile man. Subsequently we identified an additional four potentially compound heterozygous variants of unknown significance in AXDND1 in two additional infertile men. We thus tested the role of AXDND1 in mammalian male fertility by generating a knockout mouse model. Axdnd1-/- males were sterile at all ages but could undergo one round of histologically complete spermatogenesis. Subsequently, a progressive imbalance of spermatogonial commitment to spermatogenesis over self-renewal occurred, ultimately leading to catastrophic germ cell loss, loss of blood-testis barrier patency and immune cell infiltration. Sperm produced during the first wave of spermatogenesis were immotile due to abnormal axoneme structure, including the presence of ectopic vesicles and abnormalities in outer dense fibres and microtubule doublet structures. Sperm output was additionally compromised by a severe spermiation defect and abnormal sperm individualisation. Collectively, our data highlight the essential roles of AXDND1 as a regulator of spermatogonial commitment to spermatogenesis and during the processes of spermiogenesis where it is essential for sperm tail development, release and motility.

cell biology↗

bioRxiv2023

Epsilon tubulin is an essential determinant of microtubule-based structures in male germ cells

Alpha, beta and gamma tubulins are essential building blocks for all eukaryotic cells. The functions of the non-canonical tubulins, delta, epsilon and zeta, however, remain poorly understood and their requirement in mammalian development untested. Herein we have used a spermatogenesis model to define epsilon tubulin (TUBE1) function in mice. We show that TUBE1 is essential for the function of multiple complex microtubule arrays, including the meiotic spindle, axoneme and manchette and in its absence, there is a dramatic loss of germ cells and male sterility. Through examining axoneme structure, we identify differences in TUBE1 function between somatic and germ cells and potentially between species. Moreover, we provide evidence for the interplay between TUBE1 and katanin-mediated microtubule severing, and for the sub-specialization of individual katanin paralogs in the regulation of specific microtubule arrays.

developmental biology↗

bioRxiv2023

CEP76 is a critical regulator of male germ cell transition zone function and tail composition

The transition zone is a specialised gate at the base of cilia/flagella, which segregates the ciliary compartment from the cytoplasm and strictly regulates protein entry. In this study, we have identified CEP76 as an essential germ cell transition zone protein, involved in the selective entry and incorporation of key proteins required for sperm function and fertility into the ciliary compartment and ultimately the sperm tail. In its absence sperm tails are shorter and immotile as a consequence of deficits in essential sperm motility proteins including DNAH2 and AKAP4, which accumulate at the sperm neck in the mutant. We demonstrate CEP76 is required for sperm tail fibrous sheath formation, outer dense fibre loading and axoneme stability in the principal piece and ultimately sperm motility. Finally, we identify that CEP76 dictates annulus positioning and composition, adding further evidence that the spermatid transition zone and annulus are part of the same functional structure.

cell biology↗

bioRxiv2022

Spastin is an essential regulator of male meiosis, acrosome formation, manchette structure and nuclear integrity

The development and function of male gametes is critically dependent on a dynamic microtubule network, yet how this is regulated remains poorly understood. We have recently shown that microtubule severing, via the action of the meiotic AAA ATPase protein clade, plays a critical role in this process. Here, we sought to elucidate the roles of spastin, an as yet unexplored member of this clade in spermatogenesis. Using a SpastKO/KO mouse model, we reveal that spastin loss resulted in a complete loss of functional germ cells. Spastin plays a critical role in the assembly and function of the male meiotic spindle, and in its absence, apoptosis is significantly increased. Consistent with meiotic failure, round spermatid nuclei were enlarged, indicating aneuploidy, but were still able to enter spermiogenesis. During spermiogenesis, we observed extreme abnormalities in manchette structure, supernumerary acrosome formation, and commonly, a loss of nuclear integrity. This work defines a novel and essential role for spastin in regulating microtubule dynamics during spermatogenesis and is of potential relevance to patients carrying Spastin variants and to the medically assisted reproductive technology industry. Summary statementWe identify an essential role for the microtubule severing enzyme spastin in the regulation of microtubule dynamics during spermatogenesis.

developmental biology↗

bioRxiv2022

Fancm regulates meiotic double-strand break repair pathway choice in mammals

Meiotic crossovers are required for accurate chromosome segregation and to produce new allelic combinations. Meiotic crossover numbers are tightly regulated within a narrow range, despite an excess of initiating DNA double-strand breaks. Here, we describe the tumour suppressor FANCM as a meiotic anti-crossover factor in mammals. Crossover analyses with single-gamete and pedigree datasets both reveal a genome-wide increase in crossover frequencies in Fancm-deficient mice. Gametogenesis is heavily perturbed in Fancm loss of function mice, which is consistent with the reproductive defects reported in humans with biallelic FANCM mutations. A portion of the gametogenesis defects can be attributed to the cGAS-STING pathway. Despite the gametogenesis phenotypes in Fancm mutants both sexes were capable of producing offspring. We propose that the anti-crossover function and role in gametogenesis of Fancm are separable and will inform diagnostic pathways for human genomic instability disorders.

genetics↗

bioRxiv2020

Flagellar energetics from high-resolution imaging of beating patterns in tethered mouse sperm

While much is known about the microstructure of sperm flagella, the mechanisms behind the generation of flagellar beating patterns by the axoneme are still not fully understood. We demonstrate a technique for investigating the energetics of flagella or cilia. We record the planar beating of tethered wildtype and Crisp2-knockout mouse sperm at high-speed and high-resolution and extract centerlines using digital image processing techniques. We accurately reconstruct beating waveforms using a Chebyshev-polynomial based Proper Orthogonal Decomposition of the centerline tangent-angle profiles. External hydrodynamic forces and the internal resistance from the passive flagellar material are calculated from the observed kinematics of the beating patterns using a Soft, Internally-Driven Kirchhoff-Rod (SIDKR) model. Energy conservation is employed to further compute the flagellar energetics. We thus obtain the distribution of mechanical power exerted by the dynein motors without any further assumptions about mechanisms regulating axonemal function. We find that, in both the mouse genotypes studied, a large proportion of the mechanical power exerted by the dynein motors is dissipated internally, within the passive structures of the flagellum and by the motors themselves. This internal dissipation is considerably greater than the hydrodynamic dissipation in the aqueous medium outside. The net power input from the dynein motors in sperm from Crisp2-knockout mice is significantly smaller than in corresponding wildtype samples. The reduced power is correlated with slower beating and smaller amplitudes. These measurements of flagellar energetics indicate that the ion-channel regulating cysteine-rich secretory proteins (CRISPs) may also be involved in regulating mammalian sperm motility.

cell biology↗

bioRxiv2020

Bacteriophages targeting Acinetobacter baumannii capsule induce antimicrobial resensitization

Carbapenem-resistant Acinetobacter baumannii is responsible for frequent, hard-to-treat and often fatal healthcare-associated infections. Phage therapy, the use of viruses that infect and kill bacteria, is an approach gaining significant clinical interest to combat antibiotic-resistant infections. However, a major limitation is that bacteria can develop resistance against phages. Here, we isolated phages with activity against a panel of A. baumannii strains and focused on clinical isolates AB900 and A9844 and their phages for detailed characterization. As expected, coincubation of the phages with their hosts in vitro resulted in the emergence of phage-resistant bacterial mutants. Genome sequence analysis revealed that phage-resistant mutants harbored loss-of-function mutations in genes from the K locus, responsible for the biosynthesis of the bacterial capsule. Using molecular biology techniques, phage adsorption assays, and quantitative evaluation of capsule production, we established that the bacterial capsule serves as the primary receptor for these phages. As a collateral phenotype of impaired capsule production, the phage-resistant strains could not form biofilms, became fully sensitized to the human complement system, showed increased susceptibility to beta-lactam antibiotics, and became vulnerable to additional phages. Finally, in a murine model of bacteremia, the phage-resistant A. baumannii demonstrated a diminished capacity to colonize blood and solid tissues. This study demonstrates that phages can be used not only for their lytic activity but, if combined with a posteriori knowledge of their receptors and the mechanism of bacterial resistance, for their potential synergy with other antimicrobial agents, thus providing even broader clinical options for phage therapy.

microbiology↗