Ultrastructural observations of spermatogenesis in the fungivorous nematode Paraphelenchus myceliophthorus Goodey, 1958 (Rhabditida: Aphelenchoidea, Aphelenchidae)

Summary Spermatogenesis in Paraphelenchus myceliophthorus is similar to that of the ‘rhabditid’ nematodes. The sperm development includes formation of complexes of fibrous bodies (FB) and membranous organelles (MO), which appear in spermatocytes; the complexes dissociate in the spermatids and the immature sperm contains separate FB and MO. Mature spermatozoa from the uterus are bipolar cells subdivided into anterior pseudopod and posterior main cell body containing a nucleus without a nuclear envelope, numerous mitochondria and peripheral MO opening to the exterior via pores. The mature spermatozoa in the uterus form chains of more than ten flattened cells. Spermatogenesis characters, such as nuclear satellites, presence of FB in immature spermatozoa, small knobbles of MO, specific dense inclusions, absence of filopodia and sperm conjugation, make spermatozoa of P. myceliophthorus morphologically specific for comparative analysis with representatives of Aphelenchoidea and other rhabditids.

Early cleavage of preimplantation embryos is regulated by tRNAGln-TTG–derived small RNAs present in mature spermatozoa

tRNA-derived small RNAs (tsRNAs) from spermatozoa could act as acquired epigenetic factors and contribute to offspring phenotypes. However, the roles of specific tsRNAs in early embryo development remain to be elucidated. Here, using pigs as a research model, we probed the tsRNA dynamics during spermatogenesis and sperm maturation and demonstrated the delivery of tsRNAs from semen-derived exosomes to spermatozoa. By microinjection of antisense sequences into in vitro fertilized oocytes and subsequent single-cell RNA-seq of embryos, we identified a specific functional tsRNA group (termed here Gln-TTGs) that participate in the early cleavage of porcine preimplantation embryos, probably by regulating cell cycle–associated genes and retrotransposons. We conclude that specific tsRNAs present in mature spermatozoa play significant roles in preimplantation embryo development.

RNA-Guided Genomic Localization of H2A.L.2 Histone Variant

The molecular basis of residual histone retention after the nearly genome-wide histone-to-protamine replacement during late spermatogenesis is a critical and open question. Our previous investigations showed that in postmeiotic male germ cells, the genome-scale incorporation of histone variants TH2B-H2A.L.2 allows a controlled replacement of histones by protamines to occur. Here, we highlight the intrinsic ability of H2A.L.2 to specifically target the pericentric regions of the genome and discuss why pericentric heterochromatin is a privileged site of histone retention in mature spermatozoa. We observed that the intranuclear localization of H2A.L.2 is controlled by its ability to bind RNA, as well as by an interplay between its RNA-binding activity and its tropism for pericentric heterochromatin. We identify the H2A.L.2 RNA-binding domain and demonstrate that in somatic cells, the replacement of H2A.L.2 RNA-binding motif enhances and stabilizes its pericentric localization, while the forced expression of RNA increases its homogenous nuclear distribution. Based on these data, we propose that the specific accumulation of RNA on pericentric regions combined with H2A.L.2 tropism for these regions are responsible for stabilizing H2A.L.2 on these regions in mature spermatozoa. This situation would favor histone retention on pericentric heterochromatin.

A comparative study of the ultrastructural characteristics of the mature spermatozoa of two fellodistomids Tergestia clonacantha and T. laticollis and contribution to the phylogenetic knowledge of the Gymnophalloidea

The ultrastructure of the mature spermatozoa of Tergestia clonacantha and T. laticollis collected from the digestive tracts of fishes from New Caledonia is described using transmission electron microscopy and compared to that of related species. The spermatozoa of the two species exhibit the general pattern described in most digeneans, namely two axonemes with the 9 + “1” pattern of the Trepaxonemata, nucleus, mitochondrion, cortical microtubules, an external ornamentation of the plasma membrane, spine-like bodies and granules of glycogen. The spermatozoa of T. clonacantha and T. laticollis show the same ultrastructural model with some specificities in each case, particularly in the disposition of the structures in the posterior extremities of the spermatozoon. This study confirms that ultrastructural characters of the mature spermatozoon are useful tools for the phylogenetic analysis of the Digenea.

Mu opioid receptor expression and localisation in murine spermatozoa and its role in IVF

The endogenous opioid peptides are reported to be involved in the regulation of reproductive physiology. Many of the studies conclude with statements on the harmful effect of opioids on male fertility but, in fact, there are no studies regarding the real fertilisation potential of spermatozoa that have been exposed to opioids. The aim of the present study was to examine if modulation of mu opioid receptor (OPRM1) in murine spermatozoa during capacitation influenced embryo production after IVF. The presence of OPRM1 in murine mature spermatozoa was analysed by reverse transcription–polymerase chain reaction and immunofluorescence. We analysed the involvement of OPRM1 on IVF and pre-implantational embryo development by incubating the spermatozoa with the opioid agonist morphine and/or antagonist naloxone. We verified the presence of OPRM1 in murine mature spermatozoa, not only at the mRNA level but also the protein level. Moreover, incubation of the spermatozoa with morphine, before IVF, had an effect on the fertilisation rate of the spermatozoa and reduced the numbers of blastocysts, which was reversed by naloxone. Considering that opioids are widely used clinically, it is important to take into account their effect, via OPRM1, on the fertility of patients.

Cleavage of SPACA1 regulates assembly of sperm–egg membrane fusion machinery in mature spermatozoa†

The acrosome reaction is a multi-step event essential for physiological fertilization. During the acrosome reaction, gamete fusion-related factor IZUMO1 translocates from the anterior acrosome to the equatorial segment and assembles the gamete fusion machinery. The morphological changes in the acrosome reaction process have been well studied, but little is known about the molecular mechanisms of acrosome reorganization essential for physiological gamete membrane fusion. To elucidate the molecular mechanisms of IZUMO1 translocation, the steps of the acrosome reaction during that process must be clarified. In this study, we established a method to detect the early steps of the acrosome reaction and subdivided the process into seven populations through the use of two epitope-defined antibodies, anti-IZUMO1 and anti-SPACA1, a fertilization-inhibiting antibody. We found that part of the SPACA1 C-terminus in the periacrosomal space was cleaved and had begun to disappear when the vesiculation of the anterior acrosome occurred. The IZUMO1 epitope externalized from the acrosomal lumen before acrosomal vesiculation and phosphorylation of IZUMO1 occurred during the translocation to the equatorial segment. IZUMO1 circumvented the area of the equatorial segment where the SPACA1C-terminus was still localized. We therefore propose an IZUMO1 translocation model and involvement of SPACA1.

Cyclin–CDK Complexes are Key Controllers of Capacitation-Dependent Actin Dynamics in Mammalian Spermatozoa

Mammalian spermatozoa are infertile immediately after ejaculation and need to undergo a functional maturation process to acquire the competence to fertilize the female egg. During this process, called capacitation, the actin cytoskeleton dramatically changes its organization. First, actin fibers polymerize, forming a network over the anterior part of the sperm cells head, and then it rapidly depolymerizes and disappears during the exocytosis of the acrosome content (the acrosome reaction (AR)). Here, we developed a computational model representing the actin dynamics (AD) process on mature spermatozoa. In particular, we represented all the molecular events known to be involved in AD as a network of nodes linked by edges (the interactions). After the network enrichment, using an online resource (STRING), we carried out the statistical analysis on its topology, identifying the controllers of the system and validating them in an experiment of targeted versus random attack to the network. Interestingly, among them, we found that cyclin-dependent kinase (cyclin–CDK) complexes are acting as stronger controllers. This finding is of great interest since it suggests the key role that cyclin–CDK complexes could play in controlling AD during sperm capacitation, leading us to propose a new and interesting non-genomic role for these molecules.

The contribution of epididymosomes to the sperm small RNA profile

It is now well established that mature spermatozoa harbour a rich and diverse profile of small non-protein-coding regulatory RNAs (sRNAs). There is also growing appreciation that this sRNA profile displays considerable plasticity, being altered in response to paternal exposure to a variety of environmental stressors. Coupled with evidence that upon delivery to the oocyte at the moment of fertilisation, sperm-borne sRNAs are able to influence both early embryonic development and the subsequent health of the offspring, there is now interest in both the timing and degree of change in the composition of the sRNA cargo of sperm. Models in which such epigenetic changes are linked to the spermatogenic cycle are seemingly incompatible with the lack of overt phenotypic changes in the spermatozoa of affected males. Rather, there is mounting consensus that such changes are imposed on sperm during their transit and storage within the epididymis, a protracted developmental window that takes place over several weeks. Notably, since spermatozoa are rendered transcriptionally and translationally silent during their development in the testes, it is most likely that the epididymis-documented alterations to the sperm sRNA profile are driven extrinsically, with a leading candidate being epididymosomes: small membrane enclosed extracellular vesicles that encapsulate a complex macromolecular cargo of proteins and RNAs, including the sRNAs. Here, we review the role of epididymosome–sperm communication in contributing to the establishment of the sperm sRNA profile during their epididymal transit.