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.

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.

Sperm ultrastructure in the ocean quahog Arctica islandica (Arcticidae) and Neotrapezium sublaevigatum (Trapezidae), with a discussion of relationships within the Arcticoidea and with other Euheterodonta (Bivalvia)

ABSTRACT Sperm ultrastructure is described for the ocean quahog Arctica islandica (Linnaeus, 1767) (Arcticidae), a long-lived, and commercially and phylogenetically important marine bivalve from the North Atlantic, and for Neotrapezium sublaevigatum (Lamarck, 1819), an Indo-Pacific member of the only other family of Arcticoidea (Trapezidae). Spermatozoa of A. islandica consist of (in anterior to posterior sequence): an elongate-conical, deeply invaginated, acrosomal vesicle (length 2.0 ± 0.2 μm; invagination occupied by a granular subacrosomal material); a straight, anteriorly-tapered, rod-shaped nucleus (length 6.6 ± 0.4 μm); a short (approximately 0.8 μm) midpiece consisting of two orthogonally arranged centrioles, surrounded by four (approximately 75% of spermatozoa observed) or, less commonly, five (approximately 25% of spermatozoa observed) spherical mitochondria; nine satellite fibres connecting the distal centriole to mitochondria and the plasma membrane; and a flagellum (length 60 ± 5.0 μm, with 9+2 axoneme), originating from the distal centriole. Contents of the acrosomal vesicle of A. islandica are differentiated into a very electron-dense basal ring (with reticulate structure) and two less electron-dense zones. Spermatozoa of N. laevigatum (Lamarck, 1819) differ substantially from those of A. islandica and are characterized by: a rounded-conical, deeply invaginated, acrosomal vesicle (length 0.43 ± 0.2 μm), with a curved basal ring and two less conspicuous components; a barrel-shaped nucleus (length 1.6 ± 0.5 μm) with a broad apical depression accommodating the base of the acrosomal vesicle; a midpiece composed of five (approximately 80% of spermatozoa observed) or four (approximately 20% of spermatozoa observed) mitochondria. Centriolar and flagellar details are essentially as for A. islandica, and putative glycogen deposits are associated with the distal centriole and mitochondria in both species. Sperm data corroborate recent transcriptomic analyses separating Arcticidae and Trapezidae in different imparidentian clades. Based on sperm morphology, A. islandica would appear more closely related to the Glauconomidae of the Cyrenoidea than to the Trapezidae, Veneroidea or any other previously examined group of euheterodonts, suggesting that it could be the only living member of the Arcticoidea. The relationships of the Trapezidae remain uncertain, with apparent sperm similarities to members of several groups of euheterodonts (e.g. Tellinoidea, Pholadoidea, Galeommatoidea), while several potentially closely related key taxa (e.g. Glossidae) remain unstudied for sperm characters.

A 50-bp enhancer of the mouse acrosomal vesicle protein 1 gene activates round spermatid-specific transcription in vivo†

Enhancers are cis-elements that activate transcription and play critical roles in tissue- and cell type-specific gene expression. During spermatogenesis, genes coding for specialized sperm structures are expressed in a developmental stage- and cell type-specific manner, but the enhancers responsible for their expression have not been identified. Using the mouse acrosomal vesicle protein (Acrv1) gene that codes for the acrosomal protein SP-10 as a model, our previous studies have shown that Acrv1 proximal promoter activates transcription in spermatids; and the goal of the present study was to separate the enhancer responsible. Transgenic mice showed that three copies of the −186/−135 fragment (50 bp enhancer) placed upstream of the Acrv1 core promoter (−91/+28) activated reporter expression in testis but not somatic tissues (n = 4). Immunohistochemistry showed that enhancer activity was restricted to the round spermatids. The Acrv1 enhancer failed to activate transcription in the context of a heterologous core promoter (n = 4), indicating a likely requirement for enhancer-core promoter compatibility. Chromatin accessibility assays showed that the Acrv1 enhancer assumes a nucleosome-free state in male germ cells (but not liver), indicating occupancy by transcription factors. Southwestern assays (SWA) identified specific binding of the enhancer to a testis nuclear protein of 47 kDa (TNP47). TNP47 was predominantly nuclear and becomes abundant during the haploid phase of spermatogenesis. Two-dimensional SWA revealed the isoelectric point of TNP47 to be 5.2. Taken together, this study delineated a 50-bp enhancer of the Acrv1 gene for round spermatid-specific transcription and identified a putative cognate factor. The 50-bp enhancer could become useful for delivery of proteins into spermatids.

Ultrastructural Localization of Intracellular Calcium During Spermatogenesis of Sterlet (Acipenser ruthenus)

Calcium regulates many intracellular events such as growth and differentiation during different stages of gamete development. The aim of this study was to localize and quantify the intracellular distribution of calcium during different developmental stages of spermatogenesis in sterlet, Acipenser ruthenus, using a combined oxalate–pyroantimonate technique. The distribution of calcium was described in spermatogonium, spermatocyte, spermatid, and spermatozoon stages. In the spermatogonium and spermatocyte, calcium deposits were mainly localized in the nucleus and cytoplasm. The spermatid had calcium in the nucleus, developing acrosomal vesicle, and cytoplasm. Intracellular calcium transformed from scattered deposits in spermatogonia and spermatocyte stages into an unbound form in spermatid and the spermatozoon. The proportion of area covered by calcium increased significantly (p<0.05) from early to late stages of spermatogenesis. The largest proportion of area covered by calcium was observed in the nucleus of the spermatozoon. In conclusion, although most of the intracellular calcium is deposited in limited areas of the spermatogonium and spermatocyte, it is present an unbound form in the larger area of spermatids and spermatozoa which probably reflects changes in its physiological function and homeostasis during the process of male gamete production in spermatogenesis.

NAADP and the two-pore channel protein 1 participate in the acrosome reaction in mammalian spermatozoa

The functional relationship between the formation of hundreds of fusion pores during the acrosome reaction in spermatozoa and the mobilization of calcium from the acrosome has been determined only partially. Hence, the second messenger NAADP, promoting efflux of calcium from lysosome-like compartments and one of its potential molecular targets, the two-pore channel 1 (TPC1), were analyzed for its involvement in triggering the acrosome reaction using a TPCN1 gene–deficient mouse strain. The present study documents that TPC1 and NAADP-binding sites showed a colocalization at the acrosomal region and that treatment of spermatozoa with NAADP resulted in a loss of the acrosomal vesicle that showed typical properties described for TPCs: Registered responses were not detectable for its chemical analogue NADP and were blocked by the NAADP antagonist trans-Ned-19. In addition, two narrow bell-shaped dose-response curves were identified with maxima in either the nanomolar or low micromolar NAADP concentration range, where TPC1 was found to be responsible for activating the low affinity pathway. Our finding that two convergent NAADP-dependent pathways are operative in driving acrosomal exocytosis supports the concept that both NAADP-gated cascades match local NAADP concentrations with the efflux of acrosomal calcium, thereby ensuring complete fusion of the large acrosomal vesicle.

Spermatogenesis and sperm morphology in Trophon geversianus (Gastropoda: Muricidae)

The ultrastructure of spermatogenesis, the euspermatozoa and paraspermatozoa, is investigated in Trophon geversianus. Spermatogenesis follows the general developmental pattern of caenogastropods. Paraspermatid development is characterized by elongation of the cell, concurrent with the appearance of a cytoplasmic elongation at the apex of the cell and the breakdown of the nucleus into small round fragments (caryomerites). Euspermatozoa consist of: a tall, conical acrosomal vesicle (with a invagination); a rod-shaped, highly electron-dense nucleus with an internal axoneme; an elongate midpiece consisting of the axoneme sheathed by helical mitochondrial elements; an elongate glycogen piece; and a short free-tail region. Paraspermatozoa of T. geversianus are vermiform. They contain approximately 12–16 axonemes arranged peripherally, numerous oblong dense vesicles, numerous less dense (round) vesicles, and scattered mitochondria. Most of the euspermatozoal features of T. geversianus are also observed in many neogastropods. However, the presence of the axoneme continuously located inside of the nucleus has not been reported before, and may prove to be a diagnostic feature of the Muricidae.

Spermatophore and spermatozoal ultrastructure of the Mediterranean hermit crab Pagurus excavatus (Paguridae: Anomura: Decapoda)

The ultrastructure of the spermatophores and spermatozoa of the Mediterranean hermit crab Pagurus excavatus are described, using transmission electron microscopy. The size of the different parts of the spermatophore and spermatozoa are given and their ultrastructure described and compared to similar data already present in the literature for other hermit crabs. The morphology and ultrastructure of the spermatophore and spermatozoa of P. excavatus are species-specific, clearly distinguishing the species from the others already described. The spermatophore and spermatozoa show some similarities with those produced by other representatives of the genus. In particular, the tripartite spermatophore is divided into two halves by the lateral ridge and, as with the spermatophores produced by other species belonging to the genus Pagurus, it is morphologically very different from any other Paguroidea. The spermatozoa are composed of an ovoidal acrosomal vesicle capped by the operculum; the acrosome has a length:width ratio of approximately 1.75, therefore larger than 1 as reported for all anomurans studied to date. At the base of the acrosomal vesicle, there is the thin cytoplasm, the large nucleus and three arms positioned to form a 120° angle between each other. The present description is an important additional step allowing for better understanding of the relationships among the different hermit crab taxa.

Sperm morphology of two species of Olivancillaria (Gastropoda: Olividae) from the south-western Atlantic

Sperm ultrastructure in two species of the marine snail family Olividae is examined. Euspermatozoa of both species are composed of a conical, membrane-bound acrosomal vesicle; an axial rod and a basal plate similar in both species; a solid and highly electron-dense nucleus; an elongate midpiece consisting of the axoneme sheathed by helical mitochondrial elements; an elongate glycogen piece; a double electron-dense ring at the junction of the midpiece and glycogen piece; and a free tail region. The slight narrowing in the acrosomal vesicle invagination is situated in different levels between Olivancillaria deshayesiana and Olivancillaria carcellesi. This morphology could be considered as a specific character. The length of the nucleus in O. carcellesi and in O. deshayesiana is shorter than that of other neogastropods, and could be diagnostic at family level.