Deletion of ACTRT1 is associated with male infertility as sperm acrosomal ultrastructural defects and fertilization failure in human.

STUDY QUESTION: Could actin-related protein T1 (ACTRT1) deficiency be a potential pathogenic factor of human male infertility? SUMMARY ANSWER: A 110-kb microdeletion of the X chromosome, only including the ACTRT1 gene, was identified as responsible for infertility in two Chinese males with sperm showing acrosomal ultrastructural defects and fertilization failure. WHAT IS KNOWN ALREADY: The actin-related proteins (e.g. ACTRT1, ACTRT2, ACTL7A, and ACTL9) interact with each other to form a multimeric complex in the subacrosomal region of spermatids, which is crucial for the acrosome-nucleus junction. Actrt1-knockout (KO) mice are severely subfertile owing to malformed sperm heads with detached acrosomes and partial fertilization failure. There are currently no reports on the association between ACTRT1 deletion and male infertility in humans. STUDY DESIGN, SIZE, DURATION: We recruited a cohort of 120 infertile males with sperm head deformations at a large tertiary hospital from August 2019 to August 2023. Genomic DNA extracted from the affected individuals underwent whole exome sequencing (WES), and in silico analyses were performed to identify genetic variants. Morphological analysis, functional assays, and ART were performed in 2022 and 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS: The ACTRT1 deficiency was identified by WES and confirmed by whole genome sequencing, PCR, and quantitative PCR. Genomic DNA of all family members was collected to define the hereditary mode. Papanicolaou staining and electronic microscopy were performed to reveal sperm morphological changes. Western blotting and immunostaining were performed to explore the pathological mechanism of ACTRT1 deficiency. ICSI combined with artificial oocyte activation (AOA) was applied for one proband. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a whole-gene deletion variant of ACTRT1 in two infertile males, which was inherited from their mothers, respectively. The probands exhibited sperm head deformations owing to acrosomal detachment, which is consistent with our previous observations on Actrt1-KO mice. Decreased expression and ectopic distribution of ACTL7A and phospholipase C zeta were observed in sperm samples from the probands. ICSI combined with AOA effectively solved the fertilization problem in Actrt1-KO mice and in one of the two probands. LIMITATIONS, REASONS FOR CAUTION: Additional cases are needed to further confirm the genetic contribution of ACTRT1 variants to male infertility. WIDER IMPLICATIONS OF THE FINDINGS: Our results reveal a gene-disease relation between the ACTRT1 deletion described here and human male infertility owing to acrosomal detachment and fertilization failure. This report also describes a good reproductive outcome of ART with ICSI-AOA for a proband. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Chongqing medical scientific research project (Joint project of Chongqing Health Commission and Science and Technology Bureau, 2023MSXM008 and 2023MSXM054). There are no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

The sperm structure of Clinidium canaliculatum (Costa): A contribution to the systematic position of Rhysodidae (Coleoptera: Carabidae).

The systematic position and the phylogenetic relationship of Rhysodidae members is still debated, with some authors considering the group as a separate family of Adephaga, while for others they could be a subfamily of Carabidae. The group have morphological traits quite different from Carabidae and an aberrant behaviour compared to ground beetles being not predaceous. The sperm ultrastructure of C. canaliculatum was studied comparatively with other species of beetles, Carabidae in particular. The results indicate that the sperm structure of this species is similar to that of the Carabinae species. As in these species, C. canaliculatum has sperm conjugates with an apical conical cap protecting the heads and the initial region of flagella. This sperm appearance is also shared by another species of Rhysodidae, Omoglymmius hamatus. The material of the apical cap consists of an electron-dense material with a peculiar outer net configuration. Many species of Carabidae, however, can present a different type of sperm conjugation, the spermatostyle: a long rod-like structure where the individual sperms have only the most apical part inserted in the cortical area and the flagella are completely free. C. canaliculatum sperm are endowed with a mono-layered acrosome, a nucleus of variable shape along its length, a flagellum consisting of a typical axoneme 9 + 9+2, provided with 16 protofilaments in the tubular wall of accessory tubules, two asymmetric mitochondrial derivatives with the left one larger than the opposite one, and the right accessory body elongated and larger than the opposite one. These sperm characteristics, which are shared also by another member of the group, suggest the demotion of the family Rhysodidae to the subfamily Rhysodinae within Carabidae, a result also supported by recent molecular data.

Sperm models in European Plecoptera.

Systematic issues regarding Plecoptera are still debated, and the molecular data seem to be unable to definitively clarify the relationships within the order. Spermatozoa are under constant evolutionary pressure, and comparative spermatology can be useful in carrying systematic and phylogenetic information. In the present paper we describe the sperm structure, using light, scanning and transmission electron and immunofluorescence microscopy, of six Euholognatha species belonging to genera not analyzed in our previous studies, i.e. Capnopsis, Amphinemura, Rhabdiopteryx, Tyrrhenoleuctra, Zwicknia and Protonemura. The spermatozoa of all the species examined are fîliform and have a flagellum characterized by an axoneme with 9 + 9+2 pattern and two mitochondrial derivatives. Their ultrastructure shows a degree of heterogeneity within the order. On the contrary, morphological features of sperm are well conserved inside a single Euholognathan family, and the species share a general family sperm model, even if different interspecific or intergeneric characters can be identified and used for systematic inferences. Among Nemouroidea, Taeniopterygidae, showing a peculiar sperm model, seems to have an isolated phylogenetic position. Nemouridae, with a mono-layered acrosome, are isolated among the remaining families, while we can hypothesize a sister taxa relationship between Leuctridae and Capniidae. As regards Perloidea, the sperm characters suggest a closer relationship between Chloroperlidae and Perlodidae, rather than between Perlidae and Perlodidae, as commonly hypothesized.

Morphology of male and female reproductive systems in the ground beetle Apotomus and the peculiar sperm ultrastructure of A. rufus (P. Rossi, 1790) (Coleoptera, Carabidae).

Relatively few studies have focused on evolutionary losses of sexually selected male traits. We use light and electron microscopy to study the male and female reproductive anatomy of Apotomus ground beetles (Coleoptera, Carabidae), a lineage that we reconstruct as likely having lost sperm conjugation, a putative sexually selected trait. We pay particular attention to the structure of the testes and spermatheca. Both of these organs share a strikingly similar shape-consisting of long blind canals arranged into several concentric overlapping rings measuring approximately 18 mm and 19.5 mm in total length, respectively. The similarity of these structures suggests a positive evolutionary correlation between female and male genital organs. Males are characterized by unifollicular testes with numerous germ cysts, which contain 64 sperm cells each, and we record a novel occurrence of sperm cyst "looping", a spermatogenic innovation previously only known from some fruit fly and Tenebrionid beetle sperm. The sperm are very long (about 2.7 mm) and include an extraordinarily long helicoidal acrosome, a short nucleus, and a long flagellum. These findings confirm the structural peculiarity of sperm, testis, and female reproductive tract (FRT) of Apotomus species relative to other ground beetles, which could possibly be the result of shifts in sexual selection.

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.

The sperm ultrastructure of members of basal Tenebrionoidea (Coleoptera).

The sperm ultrastructure of some beetles of Tenebrionoidea was studied with particular attention to those of the Ripiphoridae, Mordellidae, and Meloidae. These three groups are often thought to form a clade, which is the sister group of the remaining Tenebrionoidea. The testes of the two former families have thinner but longer spermatic cysts containing fewer and longer sperm. Within each cyst all sperm cells have the same orientation, but cross sections showed that the orientation of the axonemes alternate between adjacent cysts, possibly due to the cysts bending on themselves. In both families the sperm has a bilayered acrosome and the flagellum, which shows mitochondrial derivatives starting laterally to the nuclear base, has a typical 9 + 9+2 axoneme with accessory tubules provided with 16 protofilaments in their wall, and well-structured triangular shaped accessory bodies. In Mordellistena sp (Mordellidae) sperm, both mitochondrial derivatives and accessory bodies are somewhat asymmetrical. Moreover, the flagellum shows a very thin and long tail end provided with only accessory tubules. Meloidae species have testes with thicker sperm cysts containing numerous shorter sperm. Within the individual cysts the sperm flagella exhibit an alternating orientation of their axonemes as consequence of a peculiar spermatogenetic process. The flagellar structure is similar to that of the above-mentioned species, but the accessory bodies are not well defined and constituted by fuzzy material. In Mylabris hieracii (Meloidae) sperm, the acrosome is flat with a conspicuous perforatorium and its nucleus has a peculiar quadrangular section. Berberomeloe majalis sperm has a large acrosome with an unusual pentagonal perforatorium. The centriolar structure of Mylabris variabilis shows a complex of dense radial links connecting the microtubular structures to the plasma membrane. These results suggest that Ripiphoridae have a closer relationship with Mordellidae than with Meloidae. These findings are in agreement with results obtained with molecular data.

Membrane lipid replacement with nano-micelles in human sperm cryopreservation improves post-thaw function and acrosome protein integrity.

RESEARCH QUESTION: Membrane lipid replacement (MLR) of oxidized membrane lipids can restore sperm cellular membrane functionality and help improve surface protein stability during cryopreservation. What are the effects of MLR with nano-micelles made from a glycerophospholipid (GPL) mixture and cholesterol-loaded cyclodextrin (CLC), on the cryosurvival and expression of acrosome-related proteins in thawed human spermatozoa? DESIGN: Twenty samples were used to determine the optimum level of nano-micelles by incubation of semen with different concentrations of GPL (0.1 and 1%) and CLC (1 and 2 mg/ml) (including GPL-0.1, GPL-1, CLC-1, CLC-2, CLC-1/GPL-0.1, CLC-2/GPL-0.1, CLC-1/GPL-1 and CLC-2/GPL-1) before cryopreservation. Then, 30 semen samples were collected, and each sample was divided into the following three aliquots: fresh, frozen control and frozen incubated with optimum level of nano-micelles (0.1% GPL and 1 mg/ml CLC). RESULTS: CLC-1/GPL-0.1 and GPL-0.1 significantly increased motility parameters. CLC-1, GPL-0.1 and CLC-1/GPL-0.1 significantly improved viability rate compared with frozen control group. Significantly higher mitochondrial activity and acrosome integrity, and a lower rate of apoptosis, were observed in the CLC-1/GPL-0.1 compared with the frozen control group. The expression ratios of arylsulfatase A (ARSA), serine protease 37 (PRSS37), serine protease inhibitor Kazal-type 2 (SPINK2) and equatorin (EQTN) significantly increased compared with the frozen control group. CONCLUSIONS: Modification of membrane cholesterol and GPL mixtures in spermatozoa enhances their acrosome protein integrity by inhibiting early apoptotic changes and spontaneous acrosome reactions.

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.

Deletion of the Spata3 Gene Induces Sperm Alterations and In Vitro Hypofertility in Mice.

Thanks to the analysis of an Interspecific Recombinant Congenic Strain (IRCS), we previously defined the Mafq1 quantitative trait locus as an interval on mouse Chromosome 1 associated with male hypofertility and ultrastructural abnormalities. We identified the Spermatogenesis associated protein 3 gene (Spata3 or Tsarg1) as a pertinent candidate within the Mafq1 locus and performed the CRISPR-Cas9 mediated complete deletion of the gene to investigate its function. Male mice deleted for Spata3 were normally fertile in vivo but exhibited a drastic reduction of efficiency in in vitro fertilization assays. Mobility parameters were normal but ultrastructural analyses revealed acrosome defects and an overabundance of lipids droplets in cytoplasmic remnants. The deletion of the Spata3 gene reproduces therefore partially the phenotype of the hypofertile IRCS strain.

Ultrastructure of sperm and complete mitochondrial genome in Meretrix sp. (Bivalvia: Veneridae) from Taiwan.

Spermatozoan ultrastructure and complete mitochondrial genome in the marine bivalve mollusk Meretrix sp. (Taiwan) from Taiwan are described and contrasted with other bivalves, especially within Meretrix. We have examined the features of the mature gonadal spermatozoa of Meretrix sp. (Taiwan) and provided comparisons with the other four Meretrix species (M. petechialis, M. meretrix, M. lyrata, and M. lamarckii). The morphological characteristics of these spermatozoa are diagnostic for each of the species studied here. The most marked interspecific difference was found in the acrosome. Meretrix sp. (Taiwan) is genetically distinct and is a different species from M. petechialis and M. lusoria (Japan) based on complete mitochondrial genome data. Sperm data for Meretrix are limited but show remarkable congruence with the molecular results. We suggest use Meretrix formosa Gwo and Hsu as the scientific name for Taiwanese hard clams, Meretrix sp. (Taiwan). Additional species, particularly the Japanese hard clam (M. lusoria) require examination before this tentative conclusion can be verified.

Sperm structure of the cattle egret (Bubulcus ibis).

It remains a major concern that sperm structure has continued to be poorly investigated and reported in avian species. To our knowledge, sperm structure in the order Pelecaniforme has not been reported. Although McFarlane (1963; Proceedings of the XIII International Ornithological Congress; Ithaca, NY; American Ornithologists' Union) reported the study of spermatozoa in two genera and two species of the family Ardeidae, he did not provide an account, or the names of the species examined. The present report on the sperm structure of the cattle egret, Bubulcus ibis, is, thus, the first in the order Pelecaniformes (this bird has been placed variably under the order Ciconiiformes, or the order Pelecaniformes). Five sexually mature and reproductively active male cattle egrets were obtained from the wild, humanely euthanized, the reproductive organs dissected out, and tissues from the ducti deferentia were prepared for transmission electron microscopy. The sperm structure of this bird is generally similar to that described for most non-passerine birds. However, the acrosome is a short, conical or bullet-shaped, blunt-ending organelle that lacks a perforatorium. The base of the acrosome is flat and makes contact with the nucleus along, a correspondingly flat plane. The nucleus, thus, ends anteriorly in a flat plane devoid of a concavity or a rostrum, and an endonuclear canal. The acrosomal and nuclear features of this bird are, therefore, main deviations from the situation in the non-passerine clade of birds.

Loss of myosin VI expression affects acrosome/acroplaxome complex morphology during mouse spermiogenesis†.

During spermiogenesis in mammals, actin filaments and a variety of actin-binding proteins are involved in the formation and function of highly specialized testis-specific structures. Actin-based motor proteins, such as myosin Va and VIIa, play a key role in this complex process of spermatid transformation into mature sperm. We have previously demonstrated that myosin VI (MYO6) is also expressed in mouse testes. It is present in actin-rich structures important for spermatid development, including one of the earliest events in spermiogenesis-acrosome formation. Here, we demonstrate using immunofluorescence, cytochemical, and ultrastructural approaches that MYO6 is involved in maintaining the structural integrity of these specialized actin-rich structures during acrosome biogenesis in mouse. We show that MYO6 together with its binding partner TOM1/L2 is present at/around the spermatid Golgi complex and the nascent acrosome. Depletion of MYO6 in Snell's waltzer mice causes structural disruptions of the Golgi complex and affects the acrosomal granule positioning within the developing acrosome. In summary, our results suggest that MYO6 plays an anchoring role during the acrosome biogenesis mainly by tethering of different cargo/membranes to highly specialized actin-related structures.

Sperm ultrastructure and spermatogenesis in the Japanese beetle Popillia japonica (Coleoptera, Scarabaeidae).

Popillia japonica is an invasive scarab beetle native to Japan that in 1916 invaded New Jersey in USA. From that moment onwards, the insect has spread invading several US states, Canada, the Azores, Italy and, recently, Switzerland. It is a severe agricultural pest included in the EU priority pest list being able to feed on more than 300 plant species and having an important biotic potential. The general morphology of the reproductive apparatus shows paired testes, each of them having six testicular lobes grouped in threes. From the ventral part of each testicular lobe, each containing about 20 follicles, an efferent vessel originates that fuses with the other efferent vessels to form the deferent duct. A pair of long tubular accessory glands is present. The deferent ducts and accessory glands fuse together into an ejaculatory duct before entering the aedeagus. The sperm is a typical pterygote sperm, 110 µm long, composed of a head and a tail. In the head a three-layered acrosome of about 6 µm in length and a nucleus of about 18 µm long are present. During sperm maturation two C-shaped structures appear in the cytoplasm from the opposite sides of the nucleus that then disappear in late spermatids. In the tail a typical 9 + 9 + 2 flagellar axoneme and two mitochondrial derivatives are present. Moreover, in the head-tail transition region the centriolar adjunct forms a sheath from which three elongated accessory bodies originate. Two of these accessory bodies are placed alongside the axoneme, whilst the third one is placed beneath the mitochondrial derivatives. Mature sperm are grouped in cysts containing about 256 sperm cells. A morphological comparison with related species is provided.

Relationship of sperm plasma membrane and acrosomal integrities with sperm morphometry in Bos taurus.

To date, sperm morphometric studies have assessed whole sperm populations without considering sperm function. The aim of this study was to evaluate the relationship of sperm membrane and acrosomal integrity with sperm morphometry in liquid semen samples collected from bulls. To this end, sperm morphometry was performed on cryopreserved semen samples from 16 bulls by a combination of fluorescent dyes, including Hoechst 33343, carboxyfluorescein diacetate, and propidium iodide. This allowed discrimination of different subpopulations on the basis of sperm membrane and acrosomal integrity and analysis of the morphometrics of the sperm head, nucleus, and acrosome using a specific plug-in module created on ImageJ. Acrosomal integrity was related to sperm morphometry as the heads of spermatozoa with a damaged acrosome were significantly smaller than those with a normal acrosome (P < 0.001). In the case of spermatozoa with an intact acrosome, those with a damaged plasma membrane had a larger sperm head and acrosome than spermatozoa with an intact plasma membrane (P < 0.001). No significant differences in the sperm head size were observed between sperm subpopulations without an acrosome or in the nuclear sperm morphometry of the different subpopulations. There was a positive correlation between the sperm motility values of the samples and the morphometric parameters for intact spermatozoa. These correlations were particularly strong for the morphometric parameters of the sperm acrosome. We conclude that there are clear differences in the sperm morphometry depending on the status of the sperm membrane and acrosome and this should be considered when performing this kind of analysis.

Sperm ultrastructure of three species of ladybirds (Coleoptera, Coccinellidae).

The sperm of three coccinellid species belonging to the subfamilies Chilocorinae, Coccinellinae and Epilachninae were studied under light and transmission electron microscopy. The basic sperm structure of these ladybirds is common to that of the other previously studied species, especially the acrosome in front of the basal body and not the nucleus, with this latter running parallel with the flagellar components. In the Chilocorinae Platynaspis luteorubra (Platynaspidini) the sperm are of the type 1, as in Scymnini and Coccinellini, since they exhibited a cylindrical basal body with 9 + 0 and then 9 + 2, microtubules continuing further in an initial flagellar portion with the only axoneme devoid of accessory structures. The sperm exhibits a thin nucleus and mitochondrial derivatives. Such uniformity of sperm ultrastructure could be indicative of the occurrence of a close relationship between Platynaspidini and Scymnini as also proposed in the previous studies. Conversely, they differ markedly from the sperm of type 3 observed in the Chilocorini Exochomus quadripustulatus. In the Coccinellini Propylea quatuordecimpunctata the sperm are also of the type 1, but they can be easily differentiated from those of the other Coccinellinae studied so far, because of their very short acrosome without the posterior extension, the relatively thicker mitochondrial derivatives and the cylindrical nucleus. Epilachna clandestina sperm resemble those of E. quadripustulatus but differ from them, because they exhibit an elliptical nucleus which is anteriorly very thin, and the asymmetrical mitochondrial derivatives in the anterior extremity, with the greater one starting at the same level of the basal body, rather than at the nucleus level, as it occurs in E. quadripustulatus. Because of the differences observed in the sperm ultrastructure we propose a new sperm type (Type 4) for E. clandestina. This study on ladybird spermatozoon ultrastructure clearly indicates that the current classifications of coccinellids do not reflect the natural history of this well-known insect family.

Spermatozoa ultrastructure of two basal pilidiophoran nemerteans, Hubrechtella juliae and Sonnenemertes cantelli (Nemertea, Pilidiophora).

Nemertea is a phylum of worms with a simple internal morphology; nemerteans' spermatozoon morphology can be used for their classification and phylogenetic analyses. The aim of the present study was to describe spermatozoa of the nemerteans Hubrechtella juliae and Sonnenemertes cantelli from the basal groups of the class Pilidiophora at the ultrastructure level. Both species have primitive ('compact-head' sensu Stricker and Folsom, 1998) spermatozoa with ovoid head and five mitochondria in the midpiece, but differ in the structure of acrosomal complex: in Hubrechtella juliae, the single lens-shaped acrosomal vesicle contains an area of moderate electron density not enclosed by a separate membrane; in Sonnenemertes cantelli, the acrosome shows a unique morphology and contains a few electron-dense vesicles with irregular shapes and positions and one more electron-lucent elongated vesicle. Such a pattern of the acrosomal complex organization is described for Nemertea for the first time. An assumption is made that the states "two or more mitochondria" and "posterior acrosomal ring component" may be synapomorphies of Hubrechtiiformes+Heteronemertea (class Pilidiophora), whereas "the posterior margin of the acrosomes forms an acrosomal ring component" is presumably an autapomorphy of the family Lineidae s.l. The results suggest that spermatozoa provide a useful source of characters for nemertean systematics.

Ultra-rapid cooling of ibex sperm by spheres method does not induce a vitreous extracellular state and increases the membrane damages.

Sperm cryopreservation by ultra-rapid cooling based on dropping small volumes of sperm suspension directly into liquid nitrogen, has been successful in some wild ruminant species, including the Iberian ibex (Capra pyrenaica). In ultra-rapid cooling, the contents of these droplets are expected to enter a stable, glass-like state, but to the best of our knowledge no information exists regarding the presence or absence of ice formation in the extracellular milieu when using this technique. Different modifications to the extracellular milieu likely inflict different types of damage on the plasmalemma, the acrosome and mitochondrial membranes. The aims of the present work were: 1) to examine the physical state of the extracellular milieu after cryopreservation at slow and ultra-rapid cooling rates-and thus determine whether ultra-rapid cooling vitrifies the extracellular milieu; and 2) to compare, using conventional sperm analysis techniques and scanning and transmission electron microscopy, the damage to sperm caused by these two methods. Sperm samples were obtained by the transrectal ultrasound-guided massage method (TUMASG) from anesthetized Iberian ibexes, and cryopreserved using slow and ultra-rapid cooling techniques. Sperm motility (22.95 ± 3.22% vs 4.42 ± 0.86%), viability (25.64 ± 3.71% vs 12.8 ± 2.50%), acrosome integrity (41.45± 3.73% vs 27.00 ± 1.84%) and mitochondrial membrane integrity (16.52 ± 3.75% vs 4.00 ± 0.65%) were better after slow cooling (P<0.001) than after ultra-rapid technique. Cryo-scanning electron microscopy (Cryo-SEM) suggested that the vitrified state was not achieved by ultra-rapid cooling, and that the ice crystals formed were smaller and had more stretchmarks (P<0.001) than after slow cooling. Scanning electron microscopy revealed no differences in the types of damage caused by the examined techniques, although transmission electron microscopy showed the damage to the plasmalemma and mitochondrial membrane to be worse after ultra-rapid cooling. In conclusion ultra-rapid cooling provoked more membrane damage than slow cooling, perhaps due to the extracellular ice crystals formed.

Micromorphological and ultrastructural description of spermatozoa from squirrel monkeys (Saimiri collinsi Osgood, 1916).

Saimiri collinsi is used as an animal model in biotechnology research for conservation of species from the genus Saimiri. However, the development of biotechnologies depends on a proper knowledge of the sperm morphology to understand the basic aspects of sperm physiology, as potential male fertility depends on different cellular sperm structures. With this purpose, this study characterized the micromorphological and ultrastructural characteristics of squirrel monkeys (Saimiri collinsi) sperm using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM electromyography revealed that a normal Saimiri collinsi sperm measures 71.7 ± 0.7 µm with lateral tail insertion, a paddle-shaped flattened head and an acrosome occupying most of the head. TEM also showed that the middle piece is characterized by a central 9 + 2 microtubule axoneme surrounded by nine dense fibres, and that the mitochondria were juxtaposed, forming the mitochondrial sheath. Here we provide the first micromorphological and ultrastructure description of S. collinsi sperm.

Abnormal fertility, acrosome formation, IFT20 expression and localization in conditional Gmap210 knockout mice.

GMAP210 (TRIP11) is a cis-Golgi network-associated protein and a Golgi membrane receptor for IFT20, an intraflagellar transport component essential for male fertility and spermiogenesis in mice. To investigate the role of GMAP210 in male fertility and spermatogenesis, floxed Gmap210 mice were bred with Stra8-iCre mice so that the Gmap210 gene is disrupted in spermatocytes and spermatids in this study. The Gmap210flox/flox: Stra8-iCre mutant mice showed no gross abnormalities and survived to adulthood. In adult males, testis and body weights showed no difference between controls and mutant mice. Low-magnification histological examination of the testes revealed normal seminiferous tubule structure, but sperm counts and fertility were significantly reduced in mutant mice compared with controls. Higher resolution examination of the mutant seminiferous epithelium showed that nearly all sperm had more oblong, abnormally shaped heads, while the sperm tails appeared to have normal morphology. Electron microscopy also revealed abnormally shaped sperm heads but normal axoneme core structure; some sperm showed membrane defects in the midpiece. In mutant mice, expression levels of IFT20 and other selective acrosomal proteins were significantly reduced, and their localization was also affected. Peanut-lectin, an acrosome maker, was almost absent in the spermatids and epididymal sperm. Mitochondrion staining was highly concentrated in the heads of sperm, suggesting that the midpieces were coiling around or aggregating near the heads. Defects in acrosome biogenesis were further confirmed by electron microscopy. Collectively, our findings suggest that GMAP210 is essential for acrosome biogenesis, normal mitochondrial sheath formation, and male fertility, and it determines expression levels and acrosomal localization of IFT20 and other acrosomal proteins.

Spermatid differentiation, with particular reference to acrosomogenesis, in the passeridan bird, Carib grackle (Quiscalus lugubris).

Only a few studies on the development of the passerine spermatozoon are available, yet species variations in the conformation as well as structure of the generally helical acrosome have been reported. This study of spermiogenesis in the Carib grackle (Quiscalus lugubris) intended to provide a deeper understanding of the development of the sperm, and in particular to investigate the bi-partite nature and development of the acrosome as well as its relationship with the nucleus, in the absence of a perforatorium that is found in most non-passerine birds. The acrosomal vesicle already displays a bi-partite nature in the acrosomal granule within the Golgi complex, and the attachment of the dense granule (future acrosomal core) within the crest part (future acrosomal crest) establishes polarity as it approaches and attaches to the nucleus. Thereafter, they develop variably. The acrosomal crest leads the elongation and spiraling of the acrosome, and the core portion contributes significantly to the formation of the keel of the crest part. The rounded, core-bearing part of the base of the acrosome progressively indents and fits into the concavity, thus formed, at the anterior part of the nucleus. The possible homology of the acrosomal complex (including the perforatorium) and the nucleus between non-passerine and passerine birds was discussed. The centriolar complex comprises both the proximal and distal centrioles in all spermatids and spermatozoa. The mitochondria undergo a number of morphological changes, including size and electron-density, from the round spermatid through to the mature spermatid; changes that are probably influenced by their functional states in the different evolving phases of the spermatids.