Biomarker-based human and animal sperm phenotyping: the good, the bad and the ugly.

Conventional, brightfield-microscopic semen analysis provides important baseline information about sperm quality of an individual; however, it falls short of identifying subtle subcellular and molecular defects in cohorts of "bad", defective human and animal spermatozoa with seemingly normal phenotypes. To bridge this gap, it is desirable to increase the precision of andrological evaluation in humans and livestock animals by pursuing advanced biomarker-based imaging methods. This review, spiced up with occasional classic movie references but seriously scholastic at the same time, focuses mainly on the biomarkers of altered male germ cell proteostasis resulting in post-testicular carryovers of proteins associated with ubiquitin-proteasome system. Also addressed are sperm redox homeostasis, epididymal sperm maturation, sperm-seminal plasma interactions and sperm surface glycosylation. Zinc ion homeostasis-associated biomarkers and sperm-borne components, including the elements of neurodegenerative pathways such as Huntington's and Alzheimer's disease, are discussed. Such spectrum of biomarkers, imaged by highly specific vital fluorescent molecular probes, lectins, and antibodies, reveals both obvious and subtle defects of sperm chromatin, DNA and accessory structures of the sperm head and tail. Introduction of next generation image-based flow cytometry into research and clinical andrology will soon enable the incorporation of machine and deep learning algorithms with the end point of developing simple, label-free methods for clinical diagnostics and high throughput phenotyping of spermatozoa in humans and economically important livestock animals.

Regional abundances of binder of sperm (BSP) proteins are negatively associated with the quality of frozen-thawed bovine spermatozoa.

In brief: The localization and abundance of the sperm BSP proteins correlate with in vitro fertility in domestic bulls used in artificial insemination service. Abstract: Binder of sperm (BSP) proteins, secreted mainly by the accessory sex glands, are the major protein family present in bovine seminal plasma and on the sperm surface after ejaculation. In vivo, BSP proteins facilitate sperm capacitation and sperm reservoir formation; however, their impact on sperm function within the in vitro systems is less clear. Therefore, this biomarker-based study aimed to characterize the localization and abundance of BSP proteins from in vitro processed frozen-thawed bovine spermatozoa. Using image-based flow cytometry and Western blotting, BSP protein localization, abundance, membrane and acrosomal integrity were investigated in the supernatant (nonmotile) and pellet (motile) fractions of gradient-separated bull spermatozoa. Spermatozoa from the supernatant fraction had high enrichment of all BSP proteins investigated (BSP1, BSP3, BSP5; P < 0.05) when compared to the pellet fraction. In the pellet fraction, BSP1 and BSP3 bound predominately to the acrosomal region, whereas BSP5 had a high affinity for the midpiece. However, in the supernatant fraction, BSP proteins predominately coated the entire sperm surface resulting in the loss of regional specificity. High BSP protein abundance in the spermatozoa also correlated with acrosome and membrane damage. Whereas a high abundance of BSP5 correlated with low embryo cleavage rates, high abundance of BSP1 on the sperm head coincided with a high blastocyst rate. Therefore, changes in the quantity and localization of specific BSP proteins could act as potential biomarkers of sperm quality and fertility.

Impact of sex and gender on axSpA diagnosis and outcomes.

Axial spondyloarthritis (axSpA) was historically considered a disease of men, largely due to the recognition of a more severe, progressive phenotype, ankylosing spondylitis (AS; or radiographic axSpA, r-axSpA) aiding the clinical diagnosis [1,2]. Data demonstrating the near equal prevalence of axSpA in women only started to emerge in the last decades, highlighting intrinsic differences in disease phenotype, and clinical and imaging characteristics between sexes, which partly explain the issue of underdiagnosis in women. Similar to the evolving understanding of spondyloarthritis and the diseases that term describes, the concepts of gender and sex also warrant further clarification to accurately assess their potential role in disease pathophysiology and phenotypic expression. This narrative review delves into the most recent evidence from the literature on the true prevalence of sex differences in axSpA, and the impact of sex and gender on diagnosis, disease characteristics and treatment response in this, still underserved, chronic disease.

Cell-Responsive Shape Memory Polymers.

Recent decades have seen substantial interest in the development and application of biocompatible shape memory polymers (SMPs), a class of "smart materials" that can respond to external stimuli. Although many studies have used SMP platforms triggered by thermal or photothermal events to study cell mechanobiology, SMPs triggered by cell activity have not yet been demonstrated. In a previous work, we developed an SMP that can respond directly to enzymatic activity. Here, our goal was to build on that work by demonstrating enzymatic triggering of an SMP in response to the presence of enzyme-secreting human cells. To achieve this phenomenon, poly(ε-caprolactone) (PCL) and Pellethane were dual electrospun to form a fiber mat, where PCL acted as a shape-fixing component that is labile to lipase, an enzyme secreted by multiple cell types including HepG2 (human hepatic cancer) cells, and Pellethane acted as a shape memory component that is enzymatically stable. Cell-responsive shape memory performance and cytocompatibility were quantitatively and qualitatively analyzed by thermal analysis (thermal gravimetric analysis and differential scanning calorimetry), surface morphology analysis (scanning electron microscopy), and by incubation with HepG2 cells in the presence or absence of heparin (an anticoagulant drug present in the human liver that increases the secretion of hepatic lipase). The results characterize the shape-memory functionality of the material and demonstrate successful cell-responsive shape recovery with greater than 90% cell viability. Collectively, the results provide the first demonstration of a cytocompatible SMP responding to a trigger that is cellular in origin.

Sperm Redox System Equilibrium: Implications for Fertilization and Male Fertility.

Structural and regulatory requirements of mammalian spermatozoa in both development and function make them extremely unique cells. Looking at the complexity of spermatozoon structure and its requirements for both motility and quick breakdown within the post-fertilization environment, as well as its functional needs as an extremely streamlined cell with high energy requirements, demonstrate the high importance of oxidative-reductive processes. The oxidative state of the testis and epididymis during sperm development and maturation highly influences sperm structure, with a high dependence on disulfide bond formation, facilitated by thiol mediated processes. However, once functionally active, sperm transition to a new high-risk functional paradigm requiring low levels of reactive oxygen species (ROS) while also being highly susceptible to oxidative damage due to the high proportion of polyunsaturated fatty acids within the lipid bilayer of the plasmalemma and the lack of cytosolic antioxidant defenses. This chapter highlights how glutathione and thioredoxin systems mediate the oxidative environment of the male reproductive tract and facilitate the successful development, maturation and function of mammalian spermatozoa.

A Non-Synonymous Point Mutation in a WD-40 Domain Repeat of EML5 Leads to Decreased Bovine Sperm Quality and Fertility.

This study is part of a concerted effort to identify and phenotype rare, deleterious mutations that adversely affect sperm quality, or convey high developmental and fertility potential to embryos and ensuing progeny. A rare, homozygous mutation in EML5 (EML5 R1654W ), which encodes a microtubule-associated protein with high expression in testis and brain was identified in an Angus bull used extensively in artificial insemination (AI) for its outstanding progeny production traits. The bull's fertility was low in cross-breeding timed AI (TAI) (Pregnancy/TAI = 25.2%; n = 222) and, in general, AI breeding to Nellore cows (41%; n = 822). A search of the 1,000 Bull Genomes Run9 database revealed an additional 74 heterozygous animals and 8 homozygous animals harboring this exact mutation across several different breeds (0.7% frequency within the 6,191 sequenced animals). Phenotypically, spermatozoa from the homozygous Angus bull displayed prominent piriform and tapered heads, and outwardly protruding knobbed acrosomes. Additionally, an increased retention of EML5 was also observed in the sperm head of both homozygous and heterozygous Angus bulls compared to wild-type animals. This non-synonymous point mutation is located within a WD40 signaling domain repeat of EML5 and is predicted to be detrimental to overall protein function by genomic single nucleotide polymorphism (SNP) analysis and protein modeling. Future work will examine how this rare mutation affects field AI fertility and will characterize the role of EML5 in spermatogenesis.

The complete mitochondrial genome of the plumed worm Diopatra cuprea (Annelida: Onuphidae).

In this study, we describe the complete mitochondrial genome of Diopatra cuprea (Bosc, 1802). The mitogenome was found to contain 14,990 base pairs (67.53% A + T content), with a total of 37 genes (13 protein coding, 22 transfer RNAs, and 2 ribosomal RNAs). This study also examined mitogenome phylogenetics relationships of closely related species and recovered that D. cuprea is closely related to eunicids. This work has added to the genetic resources for furthering evolutionary studies of Annelida.

Core Histones Are Constituents of the Perinuclear Theca of Murid Spermatozoa: An Assessment of Their Synthesis and Assembly during Spermiogenesis and Function after Gametic Fusion.

The perinuclear theca (PT) of the eutherian sperm head is a cytoskeletal-like structure that houses proteins involved in important cellular processes during spermiogenesis and fertilization. Building upon our novel discovery of non-nuclear histones in the bovine PT, we sought to investigate whether this PT localization was a conserved feature of eutherian sperm. Employing cell fractionation, immunodetection, mass spectrometry, qPCR, and intracytoplasmic sperm injections (ICSI), we examined the localization, developmental origin, and functional potential of histones from the murid PT. Immunodetection localized histones to the post-acrosomal sheath (PAS) and the perforatorium (PERF) of the PT but showed an absence in the sperm nucleus. MS/MS analysis of selectively extracted PT histones indicated that predominately core histones (i.e., H3, H3.3, H2B, H2A, H2AX, and H4) populate the murid PT. These core histones appear to be de novo-synthesized in round spermatids and assembled via the manchette during spermatid elongation. Mouse ICSI results suggest that early embryonic development is delayed in the absence of PT-derived core histones. Here, we provide evidence that core histones are de novo-synthesized prior to PT assembly and deposited in PT sub-compartments for subsequent involvement in chromatin remodeling of the male pronucleus post-fertilization.

Sperm Cohort-Specific Zinc Signature Acquisition and Capacitation-Induced Zinc Flux Regulate Sperm-Oviduct and Sperm-Zona Pellucida Interactions.

Building on our recent discovery of the zinc signature phenomenon present in boar, bull, and human spermatozoa, we have further characterized the role of zinc ions in the spermatozoa's pathway to fertilization. In boar, the zinc signature differed between the three major boar ejaculate fractions, the initial pre-rich, the sperm-rich, and the post-sperm-rich fraction. These differences set in the sperm ejaculatory sequence establish two major sperm cohorts with marked differences in their sperm capacitation progress. On the subcellular level, we show that the capacitation-induced Zn-ion efflux allows for sperm release from oviductal glycans as analyzed with the oviductal epithelium mimicking glycan binding assay. Sperm zinc efflux also activates zinc-containing enzymes and proteases involved in sperm penetration of the zona pellucida, such as the inner acrosomal membrane matrix metalloproteinase 2 (MMP2). Both MMP2 and the 26S proteasome showed severely reduced activity in the presence of zinc ions, through studies using by gel zymography and the fluorogenic substrates, respectively. In the context of the fertilization-induced oocyte zinc spark and the ensuing oocyte-issued polyspermy-blocking zinc shield, the inhibitory effect of zinc on sperm-borne enzymes may contribute to the fast block of polyspermy. Altogether, our findings establish a new paradigm on the role of zinc ions in sperm function and pave the way for the optimization of animal semen analysis, artificial insemination (AI), and human male-factor infertility diagnostics.

GSTO2 Isoforms Participate in the Oxidative Regulation of the Plasmalemma in Eutherian Spermatozoa during Capacitation.

In addition to perinuclear theca anchored glutathione-s-transferase omega 2 (GSTO2), whose function is to participate in sperm nuclear decondensation during fertilization (Biol Reprod. 2019, 101:368-376), we herein provide evidence that GSTO2 is acquired on the sperm plasmalemma during epididymal maturation. This novel membrane localization was reinforced by the isolation and identification of biotin-conjugated surface proteins from ejaculated and capacitated boar and mouse spermatozoa, prompting us to hypothesize that GSTO2 has an oxidative/reductive role in regulating sperm function during capacitation. Utilizing an inhibitor specific to the active site of GSTO2 in spermatozoa, inhibition of this enzyme led to a decrease in tyrosine phosphorylation late in the capacitation process, followed by an expected decrease in acrosome exocytosis and motility. These changes were accompanied by an increase in reactive oxygen species (ROS) levels and membrane lipid peroxidation and culminated in a significant decrease in the percentage of oocytes successfully penetrated by sperm during in vitro fertilization. We conclude that GSTO2 participates in the regulation of sperm function during capacitation, most likely through protection against oxidative stress on the sperm surface.

Sperm-borne glutathione-S-transferase omega 2 accelerates the nuclear decondensation of spermatozoa during fertilization in mice†.

The postacrosomal sheath (PAS) of the perinuclear theca (PT) is the first compartment of the sperm head to solubilize into the ooplasm upon sperm-oocyte fusion, implicating its constituents in zygotic development. This study investigates the role of one such constituent, glutathione-S-transferase omega 2 (GSTO2), an oxidative-reductive enzyme found in the PAS and perforatorial regions of the PT. GSTO2 uses the conjugation of reduced glutathione, an electron donor shown to be compulsory in sperm disassembly within the ooplasm. The proximity of GSTO2 to the condensed sperm nucleus led us to hypothesize that this enzyme may facilitate nuclear decondensation by reducing disulfide bonds before the recruitment of GSTO enzymes from within the ooplasm. To test this hypothesis, we utilized a cell permeable isozyme-specific inhibitor, which fluoresces when bound to the active site of GSTO2, to functionally inhibit spermatozoa before performing intracytoplasmic sperm injections (ICSI) in mice. The technique allowed for targeted inhibition of solely PT-residing GSTO2, as all that is required for complete zygotic development is the injection of the mouse spermatozoon head. ICSI showed that inhibition of PT-anchored GSTO2 caused a delay in sperm nuclear decondensation, and further resulted in untimely embryo cleavage, and an increase in fragmentation beginning at the morula stage. The confounding effects of these developmental delays ultimately resulted in decreased blastocyst formation. This study implicates PT-anchored GSTO2 as an important facilitator of nuclear decondensation and reinforces the notion that the PAS-PT is a critical sperm compartment harboring molecules that facilitate zygotic development.

The perforatorium and postacrosomal sheath of rat spermatozoa share common developmental origins and protein constituents†.

The perinuclear theca (PT) is a cytosolic protein capsule that surrounds the nucleus of eutherian spermatozoa. Compositionally, it is divided into two regions: the subacrosomal layer (SAL) and the postacrosomal sheath (PAS). In falciform spermatozoa, a third region of the PT emerges that extends beyond the nuclear apex called the perforatorium. The formation of the SAL and PAS differs, with the former assembling early in spermiogenesis concomitant with acrosome formation, and the latter dependent on manchette descent during spermatid elongation. The perforatorium also forms during the elongation phase of spermiogenesis, suggesting that like the PAS, its assembly is facilitated by the manchette. The temporal similarity in biogenesis between the PAS and perforatorium led us to compare their molecular composition using cell fractionation and immunodetection techniques. Although the perforatorium is predominantly composed of its endemic protein FABP9/PERF15, immunolocalization indicates that it also shares proteins with the PAS. These include WBP2NL/PAWP, WBP2, GSTO2, and core histones, which have been implicated in early fertilization and zygotic events. The compositional homogeny between the PAS and perforatorium supports our observation that their development is linked. Immunocytochemistry indicates that both PAS and perforatorial biogenesis depend on the transport and deposition of cytosolic proteins by the microtubular manchette. Proteins translocated from the manchette pass ventrally along the spermatid head into the apical perforatorial space prior to PAS deposition in the wake of manchette descent. Our findings demonstrate that the perforatorium and PAS share a mechanism of developmental assembly and thereby contain common proteins that facilitate fertilization.

WBP2 shares a common location in mouse spermatozoa with WBP2NL/PAWP and like its descendent is a candidate mouse oocyte-activating factor.

The sperm-borne oocyte-activating factor (SOAF) resides in the sperm perinuclear theca (PT). A consensus has been reached that SOAF most likely resides in the postacrosomal sheath (PAS), which is the first region of the PT to solubilize upon sperm-oocyte fusion. There are two SOAF candidates under consideration: PLCZ1 and WBP2NL. A mouse gene germline ablation of the latter showed that mice remain fertile with no observable phenotype despite the fact that a competitive inhibitor of WBP2NL, derived from its PPXY motif, blocks oocyte activation when coinjected with WBP2NL or spermatozoa. This suggested that the ortholog of WBP2NL, WBP2, containing the same domain and motifs associated with WBP2NL function, might compensate for its deficiency in oocyte activation. Our objectives were to examine whether WBP2 meets the developmental criteria established for SOAF and whether it has oocyte-activating potential. Immunoblotting detected WBP2 in mice testis and sperm and immunofluorescence localized WBP2 to the PAS and perforatorium of the PT. Immunohistochemistry of the testes revealed that WBP2 reactivity was highest in round spermatids and immunofluorescence detected WBP2 in the cytoplasmic lobe of elongating spermatids and colocalized it with the microtubular manchette during PT assembly. Microinjection of the recombinant forms of WBP2 and WBP2NL into metaphase II mouse oocytes resulted in comparable rates of oocyte activation. This study shows that WBP2 shares a similar testicular developmental pattern and location with WBP2NL and a shared ability to activate the oocyte, supporting its consideration as a mouse SOAF component that can compensate for a WBP2NL.

The developmental origin and compartmentalization of glutathione-s-transferase omega 2 isoforms in the perinuclear theca of eutherian spermatozoa.

The perinuclear theca (PT) is a condensed, nonionic detergent resistant cytosolic protein layer encapsulating the sperm head nucleus. It can be divided into two regions: the subacrosomal layer, whose proteins are involved in acrosomal assembly during spermiogenesis, and the postacrosomal sheath (PAS), whose proteins are implicated in sperm-oocyte interactions during fertilization. In continuation of our proteomic analysis of the PT, we have isolated two prominent PT-derived proteins of 28 and 31 kDa from demembranated bovine sperm head fractions. These proteins were identified by mass spectrometry as isoforms of glutathione-s-transferase omega 2 (GSTO2). Immunoblots probed with anti-GSTO2 antibodies confirmed the presence of the GSTO2 isoforms in these fractions while fluorescent immunocytochemistry localized the isoforms to the PAS region of the bull, boar, and murid PT. In addition to the PAS labeling of GSTO2, the performatorium of murid spermatozoa was also labeled. Immunohistochemistry of rat testes revealed that GSTO2 was expressed in the third phase of spermatogenesis (i.e., spermiogenesis) and assembled in the PAS and perforatorial regions of late elongating spermatids. Fluorescent immunocytochemistry performed on murine testis cells co-localized GSTO2 and tubulin on the transient microtubular-manchette of elongating spermatids. These findings imply that GSTO2 is transported and deposited in the PAS region by the manchette, conforming to the pattern of assembly found with other PAS proteins. The late assembly of GSTO2 and its localization in the PAS suggests a role in regulating the oxidative and reductive state of covalently linked spermatid/sperm proteins, especially during the disassembly of the sperm accessory structures after fertilization.

Age-related changes in the control of perturbation-evoked and voluntary arm movements.

OBJECTIVE: This study examined how handrail location predictability affects perturbation-evoked arm responses in young and older adults and whether age-related changes in perturbation-evoked arm responses are specific to mechanisms associated with reactive postural control. METHODS: Young and older adults reached for a handrail in response to a support surface translation (perturbation-evoked) or to a visual cue (voluntary). For both movement tasks, the handrail location was made predictable or unpredictable to the participant. Electromyographic (EMG) activity and kinematics of the reaching arm were recorded to quantify the arm response. RESULTS: Posterior deltoid EMG activity during perturbation-evoked and voluntary movements were delayed by 15-74 ms (p<0.001) and 16% smaller (p=0.024) when the handrail was in an unpredictable compared to a predictable location. While ageing resulted in a 12-16 ms delayed initiation of EMG activity during perturbation-evoked reaching (p=0.003), the effects of handrail predictability and movement task did not interact with age. CONCLUSIONS: Age-related differences in perturbation-evoked arm responses are independent of both handrail location predictability and movement task. SIGNIFICANCE: Age-related differences in perturbation-evoked arm responses cannot be solely attributed to declines in reactive postural control. Rather, ageing leads to a deterioration of neural mechanisms common to both perturbation-evoked and voluntary arm movements.