Anatomical features of the canine C2-C3 spinal cord vascular environment

OBJECTIVE Interarcuate branch (IAB) is a vascular structure, particularly developed in C2-3 intervertebral space, forming a dorsal bridge that connects ventral venous plexi in the vertebral canal. While precisely described in the human, the precise anatomical features of IABs have not been reported in the veterinary literature. The purpose of this study is to describe the features and relations of IABs in the C2-3 vertebral canal. ANIMALS 10 dogs were enrolled; 5 dogs for necropsy and 5 dogs for histology. PROCEDURES The ventral venous plexi in the cervical spine of 5 dogs were injected with latex and underwent vertebral canal dissection for visual assessment of the IAB. Two out of 5 dogs were injected with the addition of barium sulfate and underwent a CT scan. The C2-3 regions of 5 small-breed dogs were harvested for histological examinations. RESULTS IABs arose from the ventral venous plexus at the level of the intervertebral vein; they originated from 2 separate branches located caudally and cranially to the intervertebral foramen, forming a ventrodorsal triangle surrounding the spinal nerve root. No dorsal anastomosis was observed on the CT scan nor at dissection but were observed histologically. A cervical fibrous sheath was observed all around the vertebral canal. CLINICAL RELEVANCE IABs are voluminous venous structures at the C2-3 intervertebral space in dogs and found within a split of the cervical fibrous sheath, which is adherent to the interarcuate ligament and the ligamentum flavum. This anatomical description is paramount when planning an approach to the C2-3 intervertebral space.

Novel Loss-of-Function Mutations in DNAH1 Displayed Different Phenotypic Spectrum in Humans and Mice

Male infertility is a prevalent disorder distressing an estimated 70 million people worldwide. Despite continued progress in understanding the causes of male infertility, idiopathic sperm abnormalities such as multiple morphological abnormalities of sperm flagella (MMAF) still account for about 30% of male infertility. Recurrent mutations in DNAH1 have been reported to cause MMAF in various populations, but the underlying mechanism is still poorly explored. This study investigated the MMAF phenotype of two extended consanguineous Pakistani families without manifesting primary ciliary dyskinesia symptoms. The transmission electron microscopy analysis of cross-sections of microtubule doublets revealed a missing central singlet of microtubules and a disorganized fibrous sheath. SPAG6 staining, a marker generally used to check the integration of microtubules of central pair, further confirmed the disruption of central pair in the spermatozoa of patients. Thus, whole-exome sequencing (WES) was performed, and WES analysis identified two novel mutations in the DNAH1 gene that were recessively co-segregating with MMAF phenotype in both families. To mechanistically study the impact of identified mutation, we generated Dnah1 mice models to confirm the in vivo effects of identified mutations. Though Dnah1△iso1/△iso1 mutant mice represented MMAF phenotype, no significant defects were observed in the ultrastructure of mutant mice spermatozoa. Interestingly, we found DNAH1 isoform2 in Dnah1△iso1/△iso1 mutant mice that may be mediating the formation of normal ultrastructure in the absence of full-length protein. Altogether we are first reporting the possible explanation of inconsistency between mouse and human DNAH1 mutant phenotypes, which will pave the way for further understanding of the underlying pathophysiological mechanism of MMAF.

Characterization of different oligomeric forms of CRISP2 in the perinuclear theca versus the fibrous tail structures of boar spermatozoa

Mammalian sperm carry a variety of highly condensed insoluble protein structures such as the perinuclear theca, the fibrous sheath and the outer dense fibers, which are essential to sperm function. We studied the role of cysteine rich secretory protein 2 (CRISP2); a known inducer of non-pathological protein amyloids, in pig sperm with a variety of techniques. CRISP2, which is synthesized during spermatogenesis, was localized by confocal immunofluorescent imaging in the tail and in the post-acrosomal region of the sperm head. High resolution localization by immunogold labeling electron microscopy (EM) of ultrathin cryosections revealed that CRISP2 was present in the perinuclear theca and neck region of the sperm head, as well as in the outer dense fibers and the fibrous sheath of the sperm tail. Interestingly, we found that under native, non-reducing conditions CRISP2 formed oligomers both in the tail and the head but with different molecular weights and different biochemical properties. The tail oligomers were insensitive to reducing conditions but nearly complete dissociated into monomers under 8 M urea treatment, while the head 250 kDa CRISP2 positive oligomer completely dissociated into CRISP2 monomers under reducing conditions. The head specific dissociation of CRISP2 oligomer is likely a result of the reduction of various sulfhydryl groups in the cysteine rich domain of this protein. The sperm head CRISP2 shared typical solubilization characteristics with other perinuclear theca proteins as was shown with sequential detergent and salt treatments. Thus, CRISP2 is likely to participate in the formation of functional protein complexes in both the sperm tail and sperm head, but with differing oligomeric organization and biochemical properties. Future studies will be devoted to the understand the role of CRISP2 in sperm protein complexes formation and how this contributes to the fertilization processes.

O-094 Utilization of ultrastructural analysis and genomics of spermatozoa to better characterize subtle forms of male factor infertility

Study question Can sequencing the sperm genome provide insight into the various forms of male factor infertility caused by sperm organelle ultrastructural defects? Summary answer A comprehensive genomic assessment of spermatozoal DNA is able to identify genetic causes of ultrastructural defects visualized by transmission electron microscopy (TEM). What is known already To evaluate a man’s reproductive potential, a conventional semen analysis through the assessment of concentration, motility, and morphology can indicate the proficiency of male gametes. Among those, conventional morphology assay can only provide indirect information on the different components of the sperm cell. The assessment of nanoscopic details such as chromatin, centriolar, mitochondrial, and axonemal components can only be observed by TEM. Indeed, TEM has been used to identify defects in the acrosome, chromatin compaction, and axonemal/periaxonemal structures. Furthermore, exome sequencing of spermatozoal DNA may identify novel causes and candidate genes for these ultrastructural defects. Study design, size, duration In the past 2 years, 20 men with history of fertilization failure or severe astheno-/terato-zoospermia were selected for TEM analysis of their spermatozoa, while 3 fertile men served as controls. Sperm head characteristics, intactness of fibrous sheath, and axonemal/periaxonemal structure were examined by diagnostic TEM. For consenting patients, NGS assessment was concurrently performed to identify mutations responsible for the structural abnormalities identified by TEM. Participants/materials, setting, methods TEM was performed on the ejaculates of 20 infertile patients and 3 fertile controls. Post-centrifugation cell pellets were resuspended, fixed, and dehydrated to be infiltrated and embedded onto the resin. Fixed specimens were sliced by ultramicrotome to 100-nm sections, then viewed by JEOL-1400 electron microscope at 300,000X magnification. At least 100 spermatozoa were evaluated by TEM. For consenting patients, DNA was extracted and amplified from at least 500 spermatozoa for concurrent NGS analysis. Main results and the role of chance Four types of sperm ultrastructural defects were observed, including globozoospermia, dysplasia of fibrous sheath (DFS), proximal centriole defect, and primary ciliary dyskinesia (PCD). One combined case of globozoospermia and DFS was identified. In globozoospermic patients (n = 13), 97-100% of the spermatozoa displayed characteristic spherical heads with absence of acrosomes, dispersed chromatin, and perinuclear theca deformities. Centrosomal and axonemal structures were conserved. NGS identified gene deletions (DPY19L, PICK1, SPATA16) directly related to the globozoospermic phenotype. In patients with DFS (n = 4), complete absence of flagellum was observed in 90-100% of spermatozoa. These defective gametes also displayed mitochondria disorganization, microtubular deformities, and cytoplasmic residues containing coiled flagellum with deformed capitulum within the plasma membrane. Contrary to the globozoospermia, acrosomes and nuclei appeared normal, indicating incomplete late spermiogenesis. Indeed, NGS confirmed gene deletions involved in flagellar development/function (AKAP4, SPAG16, CATSPER1). For the patient with proximal centriole defect (n = 1), sperm nucleus, fibrous sheath, and flagellar structure were conserved. However, 90% of proximal centrioles assessed exhibited microtubular disorganization, confirmed by ODF2 mutation per NGS. In the PCD patient (n = 1), chaotic flagellar microtubule arrangement and absence of outer dynein arms were prevalent in 90% of axonemal cross-sections examined, which was explained by a DNAH5 gene deletion. Limitations, reasons for caution While TEM can overcome the limitations of conventional semen analysis by providing direct visualization of the inner organelle arrangement of spermatozoa to accurately diagnose rare sperm pathologies, it is not routinely applied in clinics due to its high cost and technical specifications. Therefore, confirmatory NGS can provide additional diagnostic value. Wider implications of the findings Ultrastructural analysis with a concurrent genomic assessment characterized phenotypes and genotypes of rare sperm pathologies in infertile men. The utilization of TEM, corroborated by genomic assay, is therefore crucial for clinical and translational reproductive medicine to better characterize male factor infertility. Trial registration number N/A

Hypomorphic and hypermorphic mouse models of Fsip2 indicate its dosage-dependent roles in sperm tail and acrosome formation

ABSTRACT Loss-of-function mutations in multiple morphological abnormalities of the sperm flagella (MMAF)-associated genes lead to decreased sperm motility and impaired male fertility. As an MMAF gene, the function of fibrous sheath-interacting protein 2 (FSIP2) remains largely unknown. In this work, we identified a homozygous truncating mutation of FSIP2 in an infertile patient. Accordingly, we constructed a knock-in (KI) mouse model with this mutation. In parallel, we established an Fsip2 overexpression (OE) mouse model. Remarkably, KI mice presented with the typical MMAF phenotype, whereas OE mice showed no gross anomaly except for sperm tails with increased length. Single-cell RNA sequencing of the testes uncovered altered expression of genes related to sperm flagellum, acrosomal vesicle and spermatid development. We confirmed the expression of Fsip2 at the acrosome and the physical interaction of this gene with Acrv1, an acrosomal marker. Proteomic analysis of the testes revealed changes in proteins sited at the fibrous sheath, mitochondrial sheath and acrosomal vesicle. We also pinpointed the crucial motifs of Fsip2 that are evolutionarily conserved in species with internal fertilization. Thus, this work reveals the dosage-dependent roles of Fsip2 in sperm tail and acrosome formation.

Variation in Tensile Properties of Single Vascular Bundles in Moso Bamboo

Moso bamboo (Phyllostachys edulis), an apt example of an anisotropic, functionally graded composite material, is the most important commercial bamboo species of China. This species has excellent mechanical properties due to its unique vascular bundle structure. This article examines the variation in mechanical properties of single vascular bundles with respect to their location within a bamboo culm. The mechanical exfoliation method was used to prepare the single vascular bundle. This study found that moso bamboo has superior stiffness and strength. Additionally, the variation in properties was large in the radial direction but minimal in longitudinal direction. The large variation in mechanical properties of vascular bundles can be ascribed to the synergistic effect of the fibrous sheath and parenchyma rather than to changes in fibrous sheath properties. This study provides a basis for the structure application for moso bamboo.

New Insights Into Sperm Ultrastructure Through Enhanced Scanning Electron Microscopy

The analysis of spermatozoa morphology is fundamental to understand male fertility and the etiology of infertility. Traditionally scanning electron microscopy (SEM) has been used to define surface topology. Recently, however, it has become a critical tool for three-dimensional analysis of internal cellular ultrastructure. Modern SEM provides nanometer-scale resolution, but the meaningfulness of such information is proportional to the quality of the sample preservation. In this study, we demonstrate that sperm quickly and robustly adhere to gold-coated surfaces. Leveraging this property, we developed three step-by-step protocols fulfilling different needs for sperm imaging: chemically fixed monolayers for SEM examination of the external morphology, and two high-pressure freezing-based protocols for fast SEM examination of full cell internal morphology and focused ion-beam SEM tomography. These analyses allow previously unappreciated insights into mouse sperm ultrastructure, including the identification of novel structures within the fibrous sheath and domain-specific interactions between the plasma membrane and exosome-like structures.

Parsonage turner syndrome: a common syndrome usually missed

Background: Parsonage-turner syndrome (PTS) is a rare syndrome that can occur in normal healthy individuals with sudden, rather abrupt, unilateral shoulder pain that may begin rather insidiously but quickly amplifies in severity and intensity. The acute period of pain is subsequently replaced over a course of a few days to weeks with progressive weakness, reflex changes, and sensory abnormalities in varying presentations that typically involve the shoulder girdle musculature and proximal upper limb muscles. There are no definite etiological factors present in causation of this syndrome.Methods: Prospective multicentric cohort study covering period from 2010 to 2020. 10 young male patients were included in the study group, all of them presented with winging of scapula and pain. Initial evaluation was done at neurology department and after failure of conservative treatment of average period of six month they were referred to plastic surgery department. All patients had ENMG findings preoperatively.Results: 10 male patients were studied with average age of 24.8 years. Partial paralysis present for an average of 1 year. Repeated micro trauma was the most common aetiology. Intraoperatively 7 patients had anomalous vascular compression of long thoracic nerve (LTN) and three had compression due to fibrous sheath. All patients had complete recovery at end of one year.Conclusion: Isolated LTN paralysis is a rare condition that is not well known. If conservative management fails, then neurolysis of the distal segment of LTN gives good result if performed within 6-12 months of paralysis.