Distal Centriole Research Articles

A disease-associated PPP2R3C-MAP3K1 phospho-regulatory module controls centrosome function

Centrosomes have critical roles in microtubule organization, ciliogenesis, and cell signaling.1,2,3,4,5,6,7,8 Centrosomal alterations also contribute to diseases, including microcephaly, cancer, and ciliopathies.9,10,11,12,13 To date, over 150 centrosomal proteins have been identified, including several kinases and phosphatases that control centrosome biogenesis, function, and maintenance.2,3,4,5,14,15,16,17,18,19,20,21 However, the regulatory mechanisms that govern centrosome function are not fully defined, and thus how defects in centrosomal regulation contribute to disease is incompletely understood. Using a systems genetics approach, we find here that PPP2R3C, a poorly characterized PP2A phosphatase subunit, is a distal centriole protein and functional partner of centriolar proteins CEP350 and FOP. We further show that a key function of PPP2R3C is to counteract the kinase activity of MAP3K1. In support of this model, MAP3K1 knockout suppresses growth defects caused by PPP2R3C inactivation, and MAP3K1 and PPP2R3C have opposing effects on basal and microtubule stress-induced JNK signaling. Illustrating the importance of balanced MAP3K1 and PPP2R3C activities, acute overexpression of MAP3K1 severely inhibits centrosome function and triggers rapid centriole disintegration. Additionally, inactivating PPP2R3C mutations and activating MAP3K1 mutations both cause congenital syndromes characterized by gonadal dysgenesis.22,23,24,25,26,27,28 As a syndromic PPP2R3C variant is defective in centriolar localization and binding to centriolar protein FOP, we propose that imbalanced activity of this centrosomal kinase-phosphatase pair is the shared cause of these disorders. Thus, our findings reveal a new centrosomal phospho-regulatory module, shed light on disorders of gonadal development, and illustrate the power of systems genetics to identify previously unrecognized gene functions.

P-095 Towards deciphering the molecular architecture of the human sperm connecting piece

Abstract Study question How can we make the human sperm connecting piece accessible for structural analysis by cryo-electron tomography (cryo-ET)? Summary answer Preparation of sperm by chemical thinning or production of lamellae via cryo-focused ion beam (cryo-FIB) milling allows direct imaging of the human sperm connecting piece. What is known already Previous studies from our group and others have demonstrated the versatility of cryo-ET to advance our understanding of sperm biology. Structures of macromolecular complexes have been resolved in vitro and in situ, enabling a functional assessment at molecular or even atomic level. Resolving structures at high resolution further allowed identification of proteins previously not known to have a role in mediating male fertility. Such insights are instrumental to further improve diagnosis and treatment of male infertility. Study design, size, duration In our research study we use sperm samples from patients undergoing IVF treatment or donor material, which we receive through an ongoing collaboration with the Division of Woman and Baby, University Medical Centre Utrecht (UMCU). These samples are processed in our own laboratory and subsequently imaged by cryo-ET at the Electron Microscopy Centre of Utrecht University. Participants/materials, setting, methods Due to its thickness, the connecting piece cannot be directly analyzed by cryo-ET. Thus, we designed two strategies to achieve thinning of this area and allow collection of tomograms for structural analysis. While the first approach involves incubation of sperm with a detergent, a reducing agent and heparin, the second approach utilizes cryo-FIB milling to produce thin lamellae of the connecting piece. Main results and the role of chance Both strategies developed to access the connecting piece with cryo-ET are effective and allowed us a glimpse of the molecular landscape of the link between sperm head and tail. Chemical thinning of the connecting piece enabled imaging of the proximal and distal centriole while still embedded in the segmented columns or outer dense fibers, respectively. Furthermore, electron density within the microtubules of both centrioles indicates that they are decorated by microtubule inner proteins. Data obtained from lamellae produced by cryo-FIB milling complements these findings, displaying regular densities along the microtubules extending from the proximal centriole. Analyzing tomograms collected from cryo-FIB lamellae we further found a regular protein density that appears to be connecting the nuclear envelope to the base plate. Moreover, cryo-ET of lamellae allowed a detailed view of nuclear pore complexes and the postacrosomal sheath in their native environment. Limitations, reasons for caution Altering the native state of the sperm cell by chemical preparation is a limitation of this approach. Cryo-FIB milling retains the native state of the cell but is limited in throughput. To improve our cryo-FIB milling approach, we will implement automated milling procedures. Wider implications of the findings Chemical preparation and cryo-FIB milling of sperm enable the in-depth analysis of the connecting piece by cryo-ET. These strategies can now be applied to provide structural details at the molecular level and thus advance our understanding of male infertility related to defects of the head-tail junction. Trial registration number not applicable

CP110 and CEP135 Localize Near the Proximal Centriolar Remnants of Mice Spermatozoa.

Centrioles form centrosomes that organize microtubules, assist in cell structure, and nucleate cilia that provide motility and sensation. Within the sperm, the centrosome consists of two centrioles (proximal and distal centriole) and a pericentriolar material known as the striated column and capitulum. The distal centriole nucleates the flagellum. Mice spermatozoa, unlike other mammal spermatozoa (e.g., human and bovine), have no ultra-structurally recognizable centrioles, but their neck has the centriolar proteins POC1B and FAM161A, suggesting mice spermatozoa have remnant centrioles. Here, we examine whether other centriolar proteins, CP110 and CEP135, found in the human and bovine spermatozoa centrioles are also found in the mouse spermatozoa neck. CP110 is a tip protein controlling ciliogenesis, and CEP135 is a centriole-specific structural protein in the centriole base of canonical centrioles found in most cell types. Here, we report that CP110 and CEP135 were both located in the mice spermatozoa neck around the proximal centriolar remnants labeled by POC1B, increasing the number of centriolar proteins found in the mice spermatozoa neck, further supporting the hypothesis that a remnant proximal centriole is present in mice.

Abnormal centriolar biomarker ratios correlate with unexplained bull artificial insemination subfertility: a pilot study

The mechanisms underlying male infertility are poorly understood. Most mammalian spermatozoa have two centrioles: the typical barrel-shaped proximal centriole (PC) and the atypical fan-like distal centriole (DC) connected to the axoneme (Ax). These structures are essential for fertility. However, the relationship between centriole quality and subfertility (reduced fertility) is not well established. Here, we tested the hypothesis that assessing sperm centriole quality can identify cattle subfertility. By comparing sperm from 25 fertile and 6 subfertile bulls, all with normal semen analyses, we found that unexplained subfertility and lower sire conception rates (pregnancy rate from artificial insemination in cattle) correlate with abnormal centriolar biomarker distribution. Fluorescence-based Ratiometric Analysis of Sperm Centrioles (FRAC) found only four fertile bulls (4/25, 16%) had positive FRAC tests (having one or more mean FRAC ratios outside of the distribution range in a group’s high-quality sperm population), whereas all of the subfertile bulls (6/6, 100%) had positive FRAC tests (P = 0.00008). The most sensitive biomarker was acetylated tubulin, which had a novel labeling pattern between the DC and Ax. These data suggest that FRAC and acetylated tubulin labeling can identify bull subfertility that remains undetected by current methods and may provide insight into a novel mechanism of subfertility.

Ultrastructure of Javaen barb fish Systomus orphoides Valenciennes, 1842 spermatozoa (Cypriniformes: Cyprinidae) by electron microscopes.

Javaen barb fish Systomus orphoides Valenciennes, 1842 (Cypriniformes: Cyprinidae) is a freshwater fish whose population is declining and threatened with extinction. In this study, the ultrastructure of spermatozoa of Javaen barb fish (S. orphoides) was studied using transmission and scanning electron microscopy. The spermatozoa of S. orphoides are relatively simple cells composed of a spherical head, a short midpiece, and a flagellum, as in most Cyprinidae. The ultrastructure is characterized by the absence of acrosome, the total length of spermatozoa is 27.16 ± 4.5 μm, and the head has spherical with a length of 1.84 ± 0.10 μm and width of 1.55 ± 0.15 μm containing a nucleus, midpiece region containing the proximal and distal centrioles and mitochondria. Two or three mitochondria surrounding the axoneme (with a 9 + 2 microtubular pattern). Ultrastructural analyses by SEM and TEM of Javaen barb fish spermatozoa cells are very consistent with those of Cyprinidae. This study provides the ultrastructure information of S. orphoides spermatozoa in the Cyprinidae family this research could be useful in increasing reproductive efficiency and further prevent the extinction of this species.

Centriolar satellites expedite mother centriole remodeling to promote ciliogenesis.

Centrosomes are orbited by centriolar satellites, dynamic multiprotein assemblies nucleated by Pericentriolar material 1 (PCM1). To study the requirement for centriolar satellites, we generated mice lacking PCM1, a crucial component of satellites. Pcm1-/- mice display partially penetrant perinatal lethality with survivors exhibiting hydrocephalus, oligospermia, and cerebellar hypoplasia, and variably expressive phenotypes such as hydronephrosis. As many of these phenotypes have been observed in human ciliopathies and satellites are implicated in cilia biology, we investigated whether cilia were affected. PCM1 was dispensable for ciliogenesis in many cell types, whereas Pcm1-/- multiciliated ependymal cells and human PCM1-/- retinal pigmented epithelial 1 (RPE1) cells showed reduced ciliogenesis. PCM1-/- RPE1 cells displayed reduced docking of the mother centriole to the ciliary vesicle and removal of CP110 and CEP97 from the distal mother centriole, indicating compromised early ciliogenesis. Similarly, Pcm1-/- ependymal cells exhibited reduced removal of CP110 from basal bodies in vivo. We propose that PCM1 and centriolar satellites facilitate efficient trafficking of proteins to and from centrioles, including the departure of CP110 and CEP97 to initiate ciliogenesis, and that the threshold to trigger ciliogenesis differs between cell types.

CP110 and CEP135 localize near the proximal and distal centrioles of cattle and human spermatozoa.

Centrosomes play an important role in the microtubule organization of a cell. The sperm's specialized centrosome consists of the canonical barrel-shaped proximal centriole, the funnel-shaped distal centriole, and the pericentriolar material known as striated columns (or segmented columns). Here, we examined the localization of the centriole proteins CEP135 and CP110 in cattle and human spermatozoa. In canonical centrioles, CP110 is a centriole tip protein that controls cilia formation, while CEP135 is a structural protein essential for constructing the centriole. In contrast, we found antibodies recognizing CEP135 and CP110 label near the proximal and distal centrioles at the expected location of the striated columns and capitulum in cattle and humans in an antibody and species-specific way. These findings provide a pathway to understanding the unique functions of spermatozoan centrosome.

O-039 Novel sperm centrosome function, structure, and composition during fertilization

Abstract Sperm centrioles play a critical role in sperm differentiation and embryo development, and they are implicated in infertility and miscarriages. However, the precise way centrioles function and are inherited during fertilization in mammals remains unresolved. A key reason for this lack of understanding is that many proteins are only transiently present in the sperm centriole because of a process known as Centrosome Reduction and that sperm centrioles exhibit dramatic species-specific adaptions. It was long widely accepted that structural degeneration and functional inactivation occur at one or two of the centrioles in mammals, resulting in a vestigial remnant. This led to the belief that human spermatozoa have only one functional centriole (the Proximal Centriole, PC). While Centrosome Reduction happens in mice, our studies suggest that two centrioles are present, and both are essential in non-murine mammals. However, the Distal Centriole (DC) has a highly atypical structure and composition, while the PC is only modestly modified. These findings led us to a new model: Centriole Remodeling, which states that preexisting spermatid centrioles, along with their pericentriolar material (PCM), go through a structural and compositional transformation during differentiation resulting in remodeled centrioles and PCM that functions as a dynamic basal complex (DBC) in the spermatozoon and as a precursor for the embryonic centrioles. A crucial mystery remaining is: what are the centrioles' essential functions? To answer this question, we study the sperm centrioles' function and remodeling process in infertile men and rabbit mutants with partially disabled sperm centrioles. Trial registration number -

Ultrastructural research of spermiogenesis in two sponges, Crellomima imparidens and Hymedesmia irregularis (Demospongiae): New evidence of sperms with acrosome in sponges.

Details of spermatogenesis and sperm organization are often useful for reconstructing the phylogeny of closely related taxa of invertebrates. Here, the spermiogenesis and the ultrastructure of sperm were studied in two marine demosponges, Crellomima imparidens and Hymedesmia irregularis (order Poecilosclerida). In C. imparidens and H. irregularis, we found bundles of microtubules arranged along the nucleus during spermiogenesis. These bundles derived from the basal body of axoneme, reaching the apical pole of the cell. In C. imparidens, the microtubules surround the nucleus, forming the manchette. In H. irregularis, the microtubules pass along only one side of the cell periphery. During spermiogenesis, the nucleus stretches and elongates. In both species, the nucleus is twisted into a spiral structure. We suppose that the manchette of microtubules could be responsible for controlling the elongation and shaping of the sperm nucleus to a helical form and for the twisting and/or condensation of chromatin in these sponges. The spermatozoon of both species has an elongated shape. Its apical part has an acrosome, which is dome-shaped in C. imparidens and flattened and lenticular in H. irregularis. The cytoplasm of the spermatozoa contains some small mitochondria, and proximal and distal centrioles arranged at an angle to each other. There is a small volume of residual cytoplasm with dark glycogen-like granules. The axoneme of the spermatid and the flagellum of the sperm of both sponges is located in the deep tunnel-like cytoplasmic depression. The comparison of spermatozoa morphology of different species of the order Poecilosclerida demonstrates that the knowledge of variation within genera and families can give valuable insights into the significance of many characters proposed for phylogenetic studies of this order.

Spermatology and sperm ultrastructure in farmed coho salmon (Oncorhynchus kisutch)

Fish sperm quality assessment is an essential process for commercial production. The goal of this study was to evaluate the quality of fresh milt of farmed coho salmon using a functional and structural approach. A total of 37 males were used to determined parameters such as sperm concentration, pH and osmolality of seminal plasma, viability, mitochondrial membrane potential and fertility. In addition, ultrastructure and morphology were assessed through transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The results show that coho salmon release a large volume of milt (55.08 mL ± 20.14 mL) with a mean concentration of 13.45 ± 3.40 × 109 spermatozoa mL−1. A positive correlation was found between sperm concentration and spermatocrit (P < 0.05; r = 0.8447). Milt contained a high percentage of motile sperm (85.00–95.00%) with a mean motility period of 45.25 ± 2.56 s, viability of 95.24 ± 2.60% and mitochondrial membrane potential of 91.33 ± 4.03%. The fertility rate was also high (96.00 ± 4.16%). SEM and TEM images revealed that mature spermatozoa of coho salmon have a total length of 32.85 ± 1.25 μm, are anacrosomal, and consist of an ovoid head, a short cylindrical mid-piece and a long flagellum which can be differentiated into a long main-piece and a short end-piece. The posterior part of the nucleus contains the nuclear fossa in which are located the distal and proximal centrioles, perpendicular to each other. There is only one mitochondrion, positioned as a ring in the mid-piece. The flagellum has two lateral side-fins, apparently of unequal length, and shows the typical “9 + 2” axoneme. These findings indicate that most of milt and sperm traits are within the range reported for other salmonids. Similarly, the results of morphology and ultrastructure are consistent with other salmonids, especially rainbow trout and brown trout.

The Transformation of the Centrosome into the Basal Body: Similarities and Dissimilarities between Somatic and Male Germ Cells and Their Relevance for Male Fertility.

The sperm flagellum is essential for the transport of the genetic material toward the oocyte and thus the transmission of the genetic information to the next generation. During the haploid phase of spermatogenesis, i.e., spermiogenesis, a morphological and molecular restructuring of the male germ cell, the round spermatid, takes place that includes the silencing and compaction of the nucleus, the formation of the acrosomal vesicle from the Golgi apparatus, the formation of the sperm tail, and, finally, the shedding of excessive cytoplasm. Sperm tail formation starts in the round spermatid stage when the pair of centrioles moves toward the posterior pole of the nucleus. The sperm tail, eventually, becomes located opposed to the acrosomal vesicle, which develops at the anterior pole of the nucleus. The centriole pair tightly attaches to the nucleus, forming a nuclear membrane indentation. An articular structure is formed around the centriole pair known as the connecting piece, situated in the neck region and linking the sperm head to the tail, also named the head-to-tail coupling apparatus or, in short, HTCA. Finally, the sperm tail grows out from the distal centriole that is now transformed into the basal body of the flagellum. However, a centriole pair is found in nearly all cells of the body. In somatic cells, it accumulates a large mass of proteins, the pericentriolar material (PCM), that together constitute the centrosome, which is the main microtubule-organizing center of the cell, essential not only for the structuring of the cytoskeleton and the overall cellular organization but also for mitotic spindle formation and chromosome segregation. However, in post-mitotic (G1 or G0) cells, the centrosome is transformed into the basal body. In this case, one of the centrioles, which is always the oldest or mother centriole, grows the axoneme of a cilium. Most cells of the body carry a single cilium known as the primary cilium that serves as an antenna sensing the cell’s environment. Besides, specialized cells develop multiple motile cilia differing in substructure from the immotile primary cilia that are essential in moving fluids or cargos over the cellular surface. Impairment of cilia formation causes numerous severe syndromes that are collectively subsumed as ciliopathies. This comparative overview serves to illustrate the molecular mechanisms of basal body formation, their similarities, and dissimilarities, in somatic versus male germ cells, by discussing the involved proteins/genes and their expression, localization, and function. The review, thus, aimed to provide a deeper knowledge of the molecular players that is essential for the expansion of clinical diagnostics and treatment of male fertility disorders.

DISCO is key to successful centriole maturation.

Centriole maturation is essential for ciliogenesis, but which proteins and how they regulate ciliary assembly is unclear. In this issue, Kumar et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202011133) shed light on this process by identifying a ciliopathy complex at the distal mother centriole that restrains centriole length and supports the formation of distal appendages.

A ciliopathy complex builds distal appendages to initiate ciliogenesis.

Cells inherit two centrioles, the older of which is uniquely capable of generating a cilium. Using proteomics and superresolved imaging, we identify a module that we term DISCO (distal centriole complex). The DISCO components CEP90, MNR, and OFD1 underlie human ciliopathies. This complex localizes to both distal centrioles and centriolar satellites, proteinaceous granules surrounding centrioles. Cells and mice lacking CEP90 or MNR do not generate cilia, fail to assemble distal appendages, and do not transduce Hedgehog signals. Disrupting the satellite pools does not affect distal appendage assembly, indicating that it is the centriolar populations of MNR and CEP90 that are critical for ciliogenesis. CEP90 recruits the most proximal known distal appendage component, CEP83, to root distal appendage formation, an early step in ciliogenesis. In addition, MNR, but not CEP90, restricts centriolar length by recruiting OFD1. We conclude that DISCO acts at the distal centriole to support ciliogenesis by restraining centriole length and assembling distal appendages, defects in which cause human ciliopathies.

A dynamic basal complex modulates mammalian sperm movement

Reproductive success depends on efficient sperm movement driven by axonemal dynein-mediated microtubule sliding. Models predict sliding at the base of the tail – the centriole – but such sliding has never been observed. Centrioles are ancient organelles with a conserved architecture; their rigidity is thought to restrict microtubule sliding. Here, we show that, in mammalian sperm, the atypical distal centriole (DC) and its surrounding atypical pericentriolar matrix form a dynamic basal complex (DBC) that facilitates a cascade of internal sliding deformations, coupling tail beating with asymmetric head kinking. During asymmetric tail beating, the DC’s right side and its surroundings slide ~300 nm rostrally relative to the left side. The deformation throughout the DBC is transmitted to the head-tail junction; thus, the head tilts to the left, generating a kinking motion. These findings suggest that the DBC evolved as a dynamic linker coupling sperm head and tail into a single self-coordinated system.

Sperm ultrastructure of Ruditapes variegata and Tapes literatus (Mollusca, Bivalvia, Veneridae, Tapetinae) from Pescadores, Taiwan

In the present study, we have investigated the ultrastructures of the mature gonadal spermatozoa of R. variegata and T. literatus and presented comparisons with the Manila clam, R. philippinarum, sperm ultrastructure examined. Spermatozoa of R. variegata consist of (in anterior to posterior sequence): an elongate conical, deeply invaginated, acrosomal vesicle (length 1.58 ± 0.06 μm; width 0.99 ± 0.07 μm; invagination occupied by a granular subacrosomal material); a barrel-shaped nucleus (length 1.82 ± 0.06 μm; width 1.50 ± 0.03 μm); a midpiece consisting of two orthogonally arranged centrioles, surrounded by four spherical mitochondria; nine satellite fibers connecting the distal centriole to the plasma membrane; and a flagellum originating from the distal centriole. Contents of the acrosomal vesicle of R. variegata are differentiated into a very electron-dense basal ring and a less electron-dense zone (with seven dense transverse layers structure) on the anterior region of the acrosome. Spermatozoa of T. literatus differ from those of R. variegata and are characterized by a rounded-conical invaginated, acrosomal vesicle (length 0.88 ± 0.08 μm; width 0.77 ± 0.06 μm), with a basal ring; and an anteriorly-tapered, barrel-shaped nucleus (length 1.57 ± 0.04 μm; width 1.60 ± 0.09 μm); a midpiece composed of four mitochondria. Centriolar and flagellar details are essential as for R. variegata. Sperm morphology separating R. variegate, R. philippinarum, and T. literatus in different clades. The anterior region of the acrosomal vesicle in R. variegata sperm had the transverse bands structure whereas the apex of the acrosomal vesicle of T. literatus sperm had no such structure. This difference advocated that acrosomal feature could be an important character for taxonomic distinction. Our data supported the previous studies that the ultrastructure of bivalve sperm is species-specific. This advocates that the phyletic relationships of Tapetinae, commonly based on shell morphology, should also add additional and newer approaches.

Fluorescence-Based Ratiometric Analysis of Sperm Centrioles (FRAC) Finds Patient Age and Sperm Morphology Are Associated With Centriole Quality.

A large proportion of infertility and miscarriage causes are unknown. One potential cause is a defective sperm centriole, a subcellular structure essential for sperm motility and embryonic development. Yet, the extent to which centriolar maladies contribute to male infertility is unknown due to the lack of a convenient way to assess centriole quality. We developed a robust, location-based, ratiometric assay to overcome this roadblock, the Fluorescence-based Ratiometric Assessment of Centrioles (FRAC). We performed a case series study with semen samples from 33 patients, separated using differential gradient centrifugation into higher-grade (pellet) and lower-grade (interface) sperm fractions. Using a reference population of higher-grade sperm from infertile men with morphologically standard sperm, we found that 79% of higher-grade sperm of infertile men with substandard sperm morphology have suboptimal centrioles (P = 0.0005). Moreover, tubulin labeling of the sperm distal centriole correlates negatively with age (P = 0.004, R = −0.66). These findings suggest that FRAC is a sensitive method and that patient age and sperm morphology are associated with centriole quality.

Centriolar defects, centrin 1 alterations, and FISH studies in human spermatozoa of a male partner of a couple that produces aneuploid embryos in natural and artificial fertilization

PurposeTo study the potential paternal contribution to aneuploidies in the man of a couple who obtained trisomic embryos with natural and assisted fertilization.MethodsSemen analysis, immunofluorescence for localization of tubulin and centrin 1, transmission electron microscopy (TEM), and fluorescence in situ hybridization (FISH) analysis for chromosomes 18 and 9 were performed. Sperm of fertile men were used as controls.ResultsThe percentages of sperm motility and normal forms were decreased. The percentages of sperm with tail reduced in dimension, headless tails, coiled tails, and altered head-tail junction were significantly higher (P < 0.01) in the patient than in controls, whereas the percentage of sperm with a normal centrin 1 localization (two spots in the centriolar area) was significantly reduced (P < 0.01) in the patient. Immunofluorescence with anti-tubulin antibody showed that in most of the patient’s sperm connecting pieces (83.00 ± 1.78%), two spots were present, indicating prominent proximal centriole/centriolar adjunct and evident distal centriole, whereas controls’ sperm displayed a single spot, indicating the proximal centriole. The percentage of sperm with two spots was significantly higher (P < 0.01) in the patient than in controls. TEM analysis showed that centriolar adjuncts of the patient’s sperm were significantly longer (721.80 ± 122.26 nm) than in controls’ sperm (310.00 ± 64.11 nm; P < 0.001). The aneuploidy frequencies of the patient’s sperm, detected by FISH analysis, were increased with respect to controls.ConclusionA paternal contribution to sperm aneuploidies cannot be excluded since the patient’s sperm showed altered morphology, immature centriolar adjunct, presence of evident distal centriole, scarce presence of centrin 1, and high aneuploidy frequency.

Some recent data on sperm morphology and motility kinetics in Atlantic cod (Gadus morhua L.).

Studying biology of sperm provides valuable information to optimize artificial reproduction and is crucial for sustainable aquaculture. Here, we investigated morphology of spermatozoon in Atlantic cod (Gadus morhua) using transmission and scanning electron microscopy. Furthermore, spermatozoa motility kinetics at different osmolalities were studied using computer-assisted sperm analysis software. The spermatozoon lacked an acrosome and consisted of a head, midpiece, and flagellum. The head of spermatozoa was round, oval, and rather elongated in shape, showing high variations in dimensions. There were up to 6 mitochondria that encircled the proximal part of the flagellum. The proximal and distal centrioles were located within the nuclear notch and arranged orthogonal to each other. The axoneme had a typical 9 + 2 microtubule structure. The flagellar length of spermatozoon was 66.94 ± 0.46μm. Spermatozoa were immotile in the seminal plasma. Dilution of sperm with natural seawater (1100mOsmol/kg) resulted in initiation of motility for 91.0 ± 3.4% of spermatozoa with average velocity of 86.2 ±2.3μm/s and beating frequency of 52Hz. The duration of spermatozoa motility was > 6min; however, the percentage of motile spermatozoa decreased at 60s post-activation. When osmolality of natural seawater was modified using distilled water or NaCl, spermatozoa motility was not initiated at ≤ 400 and ≥ 2500mOsmol/kg, and the highest percentage of motility was observed at 730-1580mOsmol/kg. In a sucrose solution, spermatozoa motility was initiated and suppressed at 600 and 1500mOsmol/kg, respectively, and highest percentage of motility was observed at 800-1100mOsmol/kg. Spermatozoon morphology comparisons within Gadiformes showed differences in dimensions of head and mitochondria, flagellar length, and number of mitochondria. The present study provides valuable data that can be used for phylogenetic implications based on spermatozoon morphology and for development of artificial fertilization and sperm cryopreservation protocols based on sperm motility.

Features of Reproductive Biology of Papuan Scorpionfish Scorpaenopsis papuensis (Scorpaenidae)

Gonadal structure, oocyte morphology, sperm motility after the activation, and spermatozoa ultrastructure are studied in Papuan scorpionfish Scorpaenopsis papuensis. The oocytes are attached to peduncles originated from ovigerous stroma. The stroma is located in the center of each of two separated ovaries. Based on oocyte diameter distribution, the type of oogenesis is continuous and oocyte deposition is multiple. Spermatozoa at a concentration of 0.005–0.011 × 109 spz/mL are observed in the hypertrophied urinary bladder. Following the activation, spermatozoa obtained from the testes retain high progressive motility for at least 13 min. Based on spermatozoon shape, Sc. papuensis does not notably different from other representatives of the suborder Scorpaenoidei with external fertilization. The nuclear fossa in the spermatozoon nucleus base is absent, and the proximal and distal centrioles are characterized by orthogonal position.

The WD40-protein CFAP52/WDR16 is a centrosome/basal body protein and localizes to the manchette and the flagellum in male germ cells

Development of spermatozoa requires remodelling and formation of particular structures. In elongating spermatids, the transient microtubular manchette contributes to the formation of the head–tail coupling apparatus (HTCA) and the sperm tail. The HTCA derives from the centrosome in that the proximal centriole inserts into the nuclear indentation and the distal centriole gives rise to the sperm flagellum. Although impairments in the formation of HTCA and sperm tail cause male infertility their molecular constituents are only partially known. The WD40-protein CFAP52 is implicated in motile cilia, but its relevance for male germ cell differentiation is not known. Here we show that CFAP52 is widespread expressed and localizes to a subset of microtubular structures. In male germ cells, CFAP52 is a component of the transient manchette and the sperm tail. However, expression of Cfap52 is not restricted to motile cilia-bearing cells. In NIH3T3 cells, CFAP52 localizes to the centrosome, the basal body, and the mitotic spindle poles, but not to the primary cilium. Our results demonstrate that CFAP52 is not restricted to motile cilia but instead most likely functions in constituting the centrosome/basal body matrix and the sperm tail.