Search Results (14)

The Mediterranean soft-shell turtle (Trionyx triunguis) is classified as critically endangered by the IUCN. Effective conservation requires a clear understanding of its reproductive strategies and population structure. By combining mitochondrial DNA tandem repeat-region profiling with genome-wide SNP data obtained through 3RADseq, we gained high-resolution insights into the genetic composition and breeding behavior of Mediterranean populations. Our results revealed complex reproductive dynamics, including multiple paternity, sperm storage, and repeated nesting within a single season—strategies that enhance genetic diversity in small, fragmented populations. Using SNP-based kinship inference, we estimated the number of breeding females and identified full and half-sibling groups, offering a robust genomic framework for assessing population size and structure. Genetic similarity patterns highlighted moderate differentiation among Israeli river populations, suggesting some connectivity, while samples from Türkiye were clearly distinct, reflecting long-term geographic and genetic separation. This integrative approach provides a scalable, repeatable tool for long-term monitoring. The combined use of maternal and biparental markers enables detailed tracking of genetic diversity, breeding contributions, and demographic trends—key elements for designing informed, adaptive conservation strategies. Full article

Effective species identification is crucial for the conservation and management of marine mammals, particularly in regions such as the Mediterranean Sea, where several cetacean populations are endangered or vulnerable. In this study, we developed and validated a High-Resolution Melting (HRM) analysis protocol for the rapid, cost-effective, and reliable identification of the four representative marine cetacean species that occur in the Mediterranean Sea: the bottlenose dolphin (Tursiops truncatus), the striped dolphin (Stenella coeruleoalba), the sperm whale (Physeter macrocephalus), and the fin whale (Balaenoptera physalus). Species-specific primers targeting mitochondrial DNA regions (cytochrome b and D-loop) were designed to generate distinct melting profiles. The protocol was tested on both tissue and fecal samples, demonstrating high sensitivity, reproducibility, and discrimination power. The results confirmed the robustness of the method, with melting curve profiles clearly distinguishing the target species and achieving a success rate > 95% in identifying unknown samples. The use of HRM offers several advantages over traditional sequencing methods, including reduced cost, speed, portability, and suitability for degraded samples, such as those from the stranded individuals. This approach provides a valuable tool for non-invasive genetic surveys and real-time species monitoring, contributing to more effective conservation strategies for cetaceans and enforcement of regulations against illegal trade. Full article

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with a highly variable expression of phenotypes (restricted interest or activity and repetitive behavior in communication and social interactions), genes (mutation), markers (alteration of transcription) and pathways. Loss of function of the CC2D1A gene appears to primarily affect the brain, leading to a range of behavioral problems in humans. In our study published in 2020, we found that the expressions of miR-19a-3p, miR-361-5p, miR-150-5p, miR-3613-3p, miR-126-3p and miR-499a-5p were downregulated in the serum samples of autistic patients, their families and mouse models (Cc2d1a +/− and valproic acid treated males). Here, acquired non-Mendelian hereditary character in a genetically defined mouse model of autism (Cc2d1a +/−) correlates with the transcriptional alteration of five miRNAs. We seek to test the hypothesis that miRNA levels vary by changes in RNA/DNA structure during development, thereby creating transcription alteration and cell memory. Behavioral tests were conducted on the offspring of Cc2d1a (+/−) mutant and control mice, such as novel object, social interaction, marble burying and tail suspension behavior. Two RNA fractions were isolated from mouse hippocampal tissues and sperm cells via standard TRIzol extraction: free RNA and the fraction of RNA bound to DNA in the form of a DNA/RNA hybrid (R-loop). The expression levels of miR-19a-3p, miR-361-5p, miR-150-5p, miR-126-3p and miR-499a-5p were investigated by quantitative real-time RT-PCR. We report differences in the distribution of five miRNAs in the hippocampus between male and female mice, particularly in colonies of Cc2d1a (+/−) mice. Furthermore, the number of miRNAs engaged in the DNA/RNA hybrid fraction is generally higher in the mutant pedigree than in the control group. On the other hand, in sperm, both fractions are at lower levels than in controls. R-loops contribute to the physiology and pathology of organisms including human disease. Here, we report a variation in five miRNA levels between gender and tissue. Our results suggest that the transcription levels of these five miRNAs are directly regulated by their RNA. Full article

Mammalian egg activation at fertilization is triggered by a long-lasting series of increases in cytosolic Ca²⁺ concentration. These Ca²⁺ oscillations are due to the production of InsP₃ within the egg and the subsequent release of Ca²⁺ from the endoplasmic reticulum into the cytosol. The generation of InsP₃ is initiated by the diffusion of sperm-specific phospholipase Czeta1 (PLCζ) into the egg after gamete fusion. PLCζ enables a positive feedback loop of InsP₃ production and Ca²⁺ release which then stimulates further InsP₃ production. Most cytosolic Ca²⁺ increases in eggs at fertilization involve a fast Ca²⁺ wave; however, due to the limited diffusion of InsP₃, this means that InsP₃ must be generated from an intracellular source rather than at the plasma membrane. All mammalian eggs studied generated Ca²⁺ oscillations in response to PLCζ, but the sensitivity of eggs to PLCζ and to some other stimuli varies between species. This is illustrated by the finding that incubation in Sr²⁺ medium stimulates Ca²⁺ oscillations in mouse and rat eggs but not eggs from other mammalian species. This difference appears to be due to the sensitivity of the type 1 InsP₃ receptor (IP3R1). I suggest that ATP production from mitochondria modulates the sensitivity of the IP3R1 in a manner that could account for the differential sensitivity of eggs to stimuli that generate Ca²⁺ oscillations. Full article

The group X secreted phospholipase A2 (PLA2G10) is present at high levels in mouse sperm acrosome. The enzyme is secreted during capacitation and amplifies the acrosome reaction and its own secretion via an autocrine loop. PLA2G10 also improves the rate of fertilization. In in vitro fertilization (IVF) experiments, sperm from Pla2g10-deficient mice produces fewer two-cell embryos, and the absence of PLA2G10 is rescued by adding recombinant enzymes. Moreover, wild-type (WT) sperm treated with recombinant PLA2G10 produces more two-cell embryos. The effects of PLA2G10 on mouse fertility are inhibited by sPLA₂ inhibitors and rescued by products of the enzymatic reaction such as free fatty acids, suggesting a role of catalytic activity. However, PLA2G10 also binds to mouse PLA2R1, which may play a role in fertility. To determine the relative contribution of enzymatic activity and PLA2R1 binding in the profertility effect of PLA2G10, we tested H48Q-PLA2G10, a catalytically-inactive mutant of PLA2G10 with low enzymatic activity but high binding properties to PLA2R1. Its effect was tested in various mouse strains, including Pla2r1-deficient mice. H48Q-PLA2G10 did not trigger the acrosome reaction but was as potent as WT-PLA2G10 to improve IVF in inbred C57Bl/6 mice; however, this was not the case in OF1 outbred mice. Using gametes from these mouse strains, the effect of H48Q-PLA2G10 appeared dependent on both spermatozoa and oocytes. Moreover, sperm from C57Bl/6 Pla2r1-deficient mice were less fertile and lowered the profertility effects of H48Q-PLA2G10, which were completely suppressed when sperm and oocytes were collected from Pla2r1-deficient mice. Conversely, the effect of WT-PLA2G10 was not or less sensitive to the absence of PLA2R1, suggesting that the effect of PLA2G10 is polymodal and complex, acting both as an enzyme and a ligand of PLA2R1. This study shows that the action of PLA2G10 on gametes is complex and can simultaneously activate the catalytic pathway and the PLA2R1-dependent receptor pathway. This work also shows for the first time that PLA2G10 binding to gametes’ PLA2R1 participates in fertilization optimization. Full article

The sperm ultrastructure of a few representative species of Tenebrionoidea was studied. Two species belong to the Mordellidae (Mordellistena brevicauda and Hoshihananomia sp.), one species to Oedemeridae (Oedemera nobilis), and one species to Tenebrionidae (Accanthopus velikensis). It is confirmed that Mordellidae are characterized by the lowest number of spermatozoa per cyst (up to 64), a number shared with Ripiphoridae. In contrast, in the two other families, up to 512 spermatozoa per cyst are observed, the same number present, for example, in Tenebrionidae. Also, as in the other more derived families of tenebrionoids studied so far, during spermatogenesis in O. nobilis and A. velikensis, sperm nuclei are regularly distributed in two sets at opposite poles of the cysts. On the contrary, the Mordellidae species do not exhibit this peculiar process. However, during spermiogenesis, the bundles of sperm bend to form a loop in their median region, quite evident in the Hoshihananomia sp., characterized by long sperm. This process, which also occurs in Ripiphoridae, probably enables individuals to produce long sperm without an increase in testicular volume. The sperm looping could be a consequence of the asynchronous growth between cyst size and sperm length. The sperm ultrastructure of the Mordellidae species reveals that they can be differentiated from other Tenebrionoidea based on the shape and size of some sperm components, such as the accessory bodies and the mitochondrial derivatives. They also show an uncommon stiff and immotile posterior flagellar region provided with only accessory tubules. These results contribute to a better knowledge of the phylogenetic relationship of the basal families of the large group of Tenebrionoidea. Full article

In maturing sperm cells, a major genome re-organization takes place, which includes a global increase in the acetylation of histones prior to their replacement by protamines, the latter being responsible for the tight packaging of the male genome. Understanding the function of the oncogenic BRD4-NUT fusion protein in NUT carcinoma (NC) cells has proven to be essential in uncovering the mechanisms underlying histone hyperacetylation in spermatogenic cells. Indeed, these studies have revealed the mechanism by which a cooperation between BRD4, a bromodomain factor of the BET family, NUT, a normally testis-specific factor, and the histone acetyltransferase p300, induces the generation of hyperacetylated chromatin domains which are present in NC cells. The generation of Nut ko mice enabled us to demonstrate a genetic interaction between Nut and Brdt, encoding BRDT, a testis-specific BRD4-like factor. Indeed, in spermatogenic cells, NUT and p300 interact, which results in an increased acetylation of histone H4 at both positions K5 and K8. These two positions, when both acetylated, are specifically recognized by the first bromodomain of BRDT, which then mediates the removal of histone and their replacement by protamines. Taken together, these investigations show that the fusion of NUT to BRD4 in NUT Carcinoma cells reconstitutes, in somatic cells, a functional loop, which normally drives histone hyperacetylation and chromatin binding by a BET factor in spermatogenic cells. Full article

Postcopulatory sexual selection is credited as a principal force behind the rapid evolution of reproductive characters, often generating a pattern of correlated evolution between interacting, sex-specific traits. Because the female reproductive tract is the selective environment for sperm, one taxonomically widespread example of this pattern is the co-diversification of sperm length and female sperm-storage organ dimension. In Drosophila, having testes that are longer than the sperm they manufacture was believed to be a universal physiological constraint. Further, the energetic and time costs of developing long testes have been credited with underlying the steep evolutionary allometry of sperm length and constraining sperm length evolution in Drosophila. Here, we report on the discovery of a novel spermatogenic mechanism—sperm cyst looping—that enables males to produce relatively long sperm in short testis. This phenomenon (restricted to members of the saltans and willistoni species groups) begins early during spermatogenesis and is potentially attributable to heterochronic evolution, resulting in growth asynchrony between spermatid tails and the surrounding spermatid and somatic cyst cell membranes. By removing the allometric constraint on sperm length, this evolutionary innovation appears to have enabled males to evolve extremely long sperm for their body mass while evading delays in reproductive maturation time. On the other hand, sperm cyst looping was found to exact a cost by requiring greater total energetic investment in testes and a pronounced reduction in male lifespan. We speculate on the ecological selection pressures underlying the evolutionary origin and maintenance of this unique adaptation. Full article

Sperm cryopreservation by vitrification is a promising approach for small-bodied animals such as zebrafish (Danio rerio). However, most vitrification tools adopted in aquatic research were initially designed for applications other than sperm (such as human embryo freezing) and, thus, pose challenges for adoption to sperm vitrification. Three-dimensional (3D) printing combined with open hardware sharing is an emerging strategy to address challenges in the development of cryopreservation tools. The goal of this study was to develop a 3D printed Vitrification Device for Cryo-Vials (VDCV) that can be integrated with the existing vial storage systems. The VDCV combined the vitrification and handling components to achieve functions of sample handling, vitrification, storage, and identification. The vitrification component featured a base, a stem, and a loop. A total of 36 configurations with various loop lengths (8, 10, and 12 mm); loop widths (2.0, 2.5, 3.0, and 3.5 mm); and support structures (open, transverse, and axial) of the VDCD prototypes were designed, fabricated, and tested. Device handling orientations (horizontal and vertical holding angles prior to and during freezing) were also investigated. Computer simulations estimated that the cooling rate of the samples ranged from 0.6–1.5 × 10⁵ °C/min in all the configurations. Prior to freezing, loops with axial supports produced a minimum of 92% film retention. The overall trends of full vitrification occurrence were observed: horizontal plunging > vertical plunging, and axial support > transverse support and open loop. A loop length of 8 mm had the highest overall vitrification occurrence (86–100%). No significant differences (p = 0.6584) were shown in a volume capacity (5.7–6.0 µL) among the three supporting configurations. A single unit of VDCV can provide loading efficiencies of about 6 × 10⁷ sperm/vial, pooling of samples from 3–6 males/vial, and fertilization for 1800 eggs/vial. The VDCV are low-cost (<$0.5 material cost per unit) and can be customized, standardized, securely labeled, and efficiently stored. The prototypes can be accessed by user communities through open-fabrication file sharing and fabricated with consumer-level 3D printers, thus facilitating community-level standardization. Full article

Local three-stranded DNA/RNA hybrid regions of genomes (R-loops) have been detected either by binding of a monoclonal antibody (DRIP assay) or by enzymatic recognition by RNaseH. Such a structure has been postulated for mouse and human telomeres, clearly suggested by the identification of the complementary RNA Telomeric repeat-containing RNA “TERRA”. However, the tremendous disparity in the information obtained with antibody-based technology drove us to investigate a new strategy. Based on the observation that DNA/RNA hybrids in a triplex complex genome co-purify with the double-stranded chromosomal DNA fraction, we developed a direct preparative approach from total protein-free cellular extract without antibody that allows their physical isolation and determination of their RNA nucleotide sequence. We then define in the normal mouse and human sperm genomes the notion of stable DNA associated RNA terminal R-loop complexes, including TERRA molecules synthesized from local promoters of every chromosome. Furthermore, the first strong evidence of all telomeric structures, applied additionally to the whole murine sperm genome compared to the testes, showed reproducible R-loop complexes of the whole genome and suggesting a defined profile in the sperm genome for the next generation. Full article

The defective human survival motor neuron 1 (SMN1) gene leads to spinal muscular atrophy (SMA), the most common genetic cause of infant mortality. We previously reported that loss of SMN results in rapid differentiation of Drosophila germline stem cells and mouse embryonic stem cells (ESCs), indicating that SMN also plays important roles in germ cell development and stem cell biology. Here, we show that in healthy mice, SMN is highly expressed in the gonadal tissues, prepubertal spermatogonia, and adult spermatocytes, whereas low SMN expression is found in differentiated spermatid and sperm. In SMA-like mice, the growth of testis tissues is retarded, accompanied with gamete development abnormalities and loss of the spermatogonia-specific marker. Consistently, knockdown of Smn1 in spermatogonial stem cells (SSCs) leads to a compromised regeneration capacity in vitro and in vivo in transplantation experiments. In SMA-like mice, apoptosis and accumulation of the R-loop structure were significantly elevated, indicating that SMN plays a critical role in the survival of male germ cells. The present work demonstrates that SMN, in addition to its critical roles in neuronal development, participates in mouse germ cell and spermatogonium maintenance. Full article

Mature spermatozoa contain a whole repertoire of the various classes of cellular RNAs, both coding and non-coding. It was hypothesized that after fertilization they might impact development, a claim supported by experimental evidence in various systems. Despite the current increasing interest in the transgenerational maintenance of epigenetic traits and their possible determination by RNAs, little remains known about conservation in sperm and across generations and the specificities and mechanisms involved in transgenerational maintenance. We identified two distinct fractions of RNAs in mature mouse sperm, one readily extracted in the aqueous phase of the classical TRIzol procedure and a distinct fraction hybridized with homologous DNA in DNA-RNA complexes recovered from the interface, purified after DNase hydrolysis and analyzed by RNA-seq methodology. This DNA-associated RNA (D RNA) was found to represent as much as half of the cell contents in differentiated sperm, in which a major part of the cytoplasmic material has been discarded. Stable complexes were purified free of proteins and identified as hybrids (R-loops) on the basis of their sensitivity to RNase H hydrolysis. Further analysis by RNA-seq identified transcripts from all the coding and non-coding regions of the genome, thus revealing an extensive wave of transcription, prior to or concomitant with the terminal compaction of the chromatin. Full article

Background: Genomes are non-randomly organized within the interphase nucleus; and spermatozoa are proposed to have a unique hairpin-loop configuration, which has been hypothesized to be critical for the ordered exodus of the paternal genome following fertilization. Recent studies suggest that the hairpin-loop model of sperm chromatin organization is more segmentally organized. The purpose of this study is to examine the 3D organization and hairpin-loop configurations of chromosomes in human spermatozoa. Methods: Three-color sperm-fluorescence in-situ hybridization was utilized against the centromeres, and chromosome p- and q-arms of eight chromosomes from five normozoospermic donors. Wide-field fluorescence microscopy and 3D modelling established the radial organization and hairpin-loop chromosome configurations in spermatozoa. Results: All chromosomes possessed reproducible non-random radial organization (p < 0.05) and formed discrete hairpin-loop configurations. However, chromosomes preferentially formed narrow or wide hairpin-loops. We did not find evidence to support the existence of a centralized chromocenter(s) with centromeres being more peripherally localized than one or both of their respective chromosome arms. Conclusion: This provides further evidence to support a more segmental organization of chromatin in the human sperm nucleus. This may be of significance for fertilization and early embryogenesis as specific genomic regions are likely to be exposed, remodeled, and activated first, following fertilization. Full article

Stra8 (Stimulated by Retinoic Acid 8) is considered a meiotic gatekeeper gene. Using reverse transcriptase PCR and rapid amplification of cDNA ends (RACE), the complete sequence of the pig Stra8 gene was cloned. Bioinformatics analyses of this sequence were performed. Using semi-quantitative methods, the expression characteristics of Stra8 in Testis, cauda epididymis, body epididymis, caput epididymis, seminal vesicles, prostate gland, Cowper’s gland, heart, liver, spleen, lung, kidney, stomach, hypothalamus, pituitary gland, cerebrum, cerebellum, and hippocampus of adult Meishan boar and sow tissues were examined. The expression pattern in the testis of 2-, 30-, 60-, 90-, and 150-day old Meishan boars were analyzed using real-time PCR. We constructed a eukaryotic expression vector for the Stra8 gene and used it to transfect NIH-3T3 cells and third generation pig spermatogonial stem cells (SSCs) cultured in vitro. Testes weight and sperm count in the cauda epididymis were evaluated at various time points. The results showed that the length of the pig Stra8 gene cDNA was 1444 bp encoding 366 amino acids with one typical helix-loop-helix (HLH) domain. It is testes-specific expression. Expression was first detected in boar testis starting at day 2, and its expression significantly (p < 0.05) increased with age and body weight. When NIH-3T3 cells and pig SSCs were transfected with the eukaryotic expression vector EGFP (enhanced green fluorescent protein)-N1-pStra8, it was expressed in the cytoplasm of NIH-3T3 cells. However, in SSCs, Stra8 was expressed predominantly in cytoplasm and few in nucleus. Our data suggest that perhaps Stra8 acts as a transcription factor to initiate meiosis in young boar. Full article