Mature Sperm Research Articles

Clinical outcomes of round spermatid injection versus intra-cytoplasmic sperm injection: The potential role of hormonal pretreatment in male patients with non-obstructive azoospermia.

Round spermatid injection (ROSI) into oocytes offers men with non-obstructive azoospermia (NOA) the opportunity to have biological offspring in cases where mature spermatozoa are not detected. However, the clinical outcomes of ROSI remains poor. This study compares the outcomes of ROSI with intracytoplasmic sperm injection (ICSI) and investigates the effect of hormonal pretreatment. This retrospective cohort study enrolled infertile couples undergoing either ROSI or ICSI at the reproductive center in Taipei Veterans General Hospital. The administration of selective estrogen receptor modulators, gonadotropins, and aromatase inhibitors in male patients were collected. Relevant hormonal markers and biochemical parameters were determined. The outcomes of ROSI and ICSI were assessed based on fertilization rate, implantation rate, and live birth rate. A total of 36 couples were recruited in the ROSI group, whereas 39 couples were recruited in the ICSI group for the analysis. Patients in each group demonstrated similar characteristics, except for a higher proportion of male patients in the ROSI group who were pretreated with anastrozole. The fertilization rate and implantation rate were similar between ROSI and ICSI groups after adjusting for confounding variable. The live birth rate was significant lower in the ROSI group (8.3%) than in the ICSI group (30.8%) before and after adjusting for confounding variable. ROSI demonstrated fertilization and implantation rates comparable to those of ICSI for male patient with NOA undergoing testicular sperm extraction surgery. Further studies evaluating the effect of anastrozole administration on ROSI outcomes are warrant.

Nereidid polychaetes (Annelida) inhabiting the inside of decaying fronds of the mangrove palm Nypa fruticans in a tropical estuary in Malaysia, with special reference to the life history of the dominant species, Namalycastis sp

ABSTRACT Since relatively dense assemblages of nereidid worms were discovered from a unique habitat within the decaying fronds of the mangrove palm Nypa fruticans, we conducted a one-year survey from September 2015 to September 2016 at six sites in Setiu Wetlands, Malaysia, to clarify the species composition of the nereidid assemblages and the life history of the dominant species. The majority of specimens collected from the plant body (507 out of a total 678 specimens) belonged to Namalycastis sp. with remaining specimens belonging to Namanereis sp. (n=168), Namalycastis rhodochorde (n=1), and Perinereis aibuhitensis (n=2). Specimens of P. aibuhitensis (n=2) and Dendronereis sp. (n=2) were also collected from sediments around the palm trees. The taxonomic accounts of two Namalycastis spp. and Perinereis aibuhitensis are provided here. The life history of the dominant species Namalycastis sp. is evaluated based on the population characteristics. This species is gonochoric, with the sex ratio close to 1:1. The ovigerous females were found throughout the year, with their body width (BW) ranging from 1.3 to 4.8 mm. The ovigerous rate (no. of ovigerous females/no. of worms larger than the minimum ovigerous female) was lowest in February and March 2016 (8–9%) and highest from July to November 2016 (23–29%), when the ovigerous females had the largest coelomic oocytes of 100–120µm in diameter. Probable epitokes with additional neuropodial sesquigomph spinigers occurred most abundantly in November 2015 (21% of adults), which was also the only month when a mature male with mature spermatozoa appeared. Few juveniles with BW of less than 1.3 mm occurred from February to July, whereas large adults with BW of more than 3.5 mm occurred from May to November. These results suggest semelparous reproduction with peak spawning in November, coinciding with low salinity during the north-east monsoon.

Successful formation of sperm cells from transplanted primordial germ cells in sterile interspecific avian recipients.

Primordial germ cells (PGCs) are stem cells, from which only gametes develop. In birds, the female sex is heterogametic, thus female gene conservation necessitates preservation of PGCs. PGC transplantation can generate germline chimeras in a host organism and develop into gametes. However, competition between host and transplanted PGCs hinder efficiency of germline chimera generation. We hypothezised that in sterile hybrid recipients with no germ cells of its own, transplanted donor PGCs may exclusively form gametes. Advantages of sterile hybrids as host for PGCs is compliant with many national regulations on genetically modified organisms and technically simpler procedure than the use of busulphan. Therefore, we investigated whether sterile interspecific hybrids may be suitable as recipients for supporting donor PGCs by injecting green fluorescent protein-labelled chicken PGCs into 3-day-old Guinea fowl and domestic fowl hybrid embryos and monitoring PGC development. The injected PGCs colonized almost 100% of the recipient gonads and produced mature spermatozoa after 44 weeks. However, gamete production in these hybrids was initiated much slower than in domestic fowls. This delay may be caused by suboptimal hormonal regulation of gametogenesis in the hybrids. Our results suggest that sterile interspecific hybrids may be suitable hosts for PGCs for efficient gamete production.

Aglycone Analysis and Quantitative Tracking of Correlated Terminal Glycan Pair on Spermatozoa.

Mammalian sperm glycans directly mediate several key life events. However, previous studies have not focused on two key factors that regulate these processes, the terminal glycan pattern and the anchoring sites. Herein, we group the capping monosaccharide sialic acid (Sia) and its capping substrates galactose/N-acetylgalactosamine (Gal/GalNAc) into a "correlated terminal glycan pair" (glycopair) and, for the first time, reveal the differences in the aglycone pattern of this pair on spermatozoa using glyco-selective in situ covalent labeling techniques. Sia is mainly found in glycoproteins, whereas terminal Gal/GalNAc is mainly found in glycolipids. We quantitatively track the dynamic changes of the glycopair during sperm epididymal migration and find that the Sia capping ratio decreases with the increased expression of the glycopair; caudal upswim spermatozoa also show a lower Sia capping ratio than down spermatozoa. We thus propose two new parameters reflecting the terminal glycoforms of spermatozoa, which can well distinguish the maturity of spermatozoa. By fluorescence imaging of the glycopair in different regions of the sperm, we find that different parts of the sperm contribute differently to the overall glycan changes.

Synchronously Mature Intersex Japanese Flounder (Paralichthys olivaceus): A Rare Case.

Japanese flounder is usually gonochoristic, with gonads that are either testes or ovaries. Here, we report an unusual case of hermaphroditism in Japanese flounder captured from the Bohai Sea. In the intersex flounder, the membrane of the upper ovary was closely connected to the abdominal muscles and internal organs, and the eggs filled the entire abdomen. The lower ovary was small and closely connected to the testes. The testes contained few fully mature sperm. Both eggs and sperm were capable of fertilization. The levels of several reproduction-related hormones (17β-estradiol, 11-ketotestosterone, 17α, 20β-dihydroxyprogesterone, luteinizing hormone, follicle-stimulating hormone, and testosterone) in the intersex flounder were intermediate, between those in females and males. The results showed that the heterozygosity of the intersex flounder was 0.632, and there were 28 single-nucleotide polymorphisms in the cyp21a gene. Compared with that of wild flounder, the activity of 21-hydroxylase was reduced by approximately 20.0%, and expressions of cyp19a, amh, and dmrt1 differed. We present the first report of its kind, detailing the anatomy, hormonal endocrinology, molecular biology, and physiology of the intersex Japanese flounder.

Current summer heat waves impair rainbow trout (Oncorhynchus mykiss) spermatogenesis: Implications for future fish farming management practices in South Europe

Freshwater environments provide different ecosystem services for human well-being, and are one of the most important natural sources of healthy animal protein. Climate change and extreme temperature events, among other factors, represent a growing concern for their sustainable use. In contrast to the research effort to elucidate how global warming affects these aquatic environments, few studies pay attention to the potential effects of heat waves, a current phenomenon with direct effects on contemporaneous organisms. Here, the potential effects of a single heat wave event on rainbow trout (Oncorhynchus mykiss) males, mimicking a recorded environmental event, have been evaluated by means of biometric, redox and stress response, plasma testosterone, histopathology and transcriptomic analyses. Environmental monitoring showed how, during the last 4 years, mean water temperature increased (2–4 °C; from April to October), heat wave duration progressively increased (from 15 to 100 days), and mean intensities ranged 1.7–3.8 °C with a maximum of 8.0 °C. An experimentally recreated heat wave lasting only 16 days, with mean and maximum intensities of 2.7 and 6.7 °C, induced a decrease in body weight and total antioxidant status, but increased blood plasma cortisol levels and redox enzyme activity. The induced heat wave decreased testosterone plasma levels and sperm quality, arrested spermatocyte type I and II differentiation and increased spermatozoa apoptosis. These effects were correlated with a differential expression of 1156 genes (116 up- and 1040 down-regulated genes) in exposed fish when compared to Control fish, reflecting an alteration in cell apoptosis and spermatozoa maturation processes. These results not only increase concern about current heat wave events, recreating a real scenario, but also unveil for the first time the particular molecular mechanisms of subsequent spermatogenesis disruption, opening new avenues for designing urgent and necessary strategies to mitigate the effects of extreme climate change conditions on freshwater aquaculture.

8030 Sexually Dimorphic Metabolic Consequences Of CCN5/WISP2 Gene Deletion Revealed A Male-Specific Pattern Of Gene Expression

Abstract Disclosure: M. Yang: None. V. Xega: None. M. Zakikhani: None. J. Liu: None. Matricellular protein CCN5/WISP2 is a novel target of IGF-1 and WNT signaling within the pancreatic islets. Our previous research suggests CCN5 promotes β-cell proliferation and survival through Akt/PKB and focal adhesion kinase signaling pathways. More recently, we have reported sexual dimorphic features of CCN5 gene deletion and overexpression. Specifically, male knockout mice demonstrated improved insulin sensitivity and glucose tolerance upon high-fat diet (HFD)-induced obesity, while females were unaffected (ENDO 2022). On the other hand, male aP2-CCN5 transgenic mice that overexpress CCN5 in adipose tissues were partially protected from HFD-streptozotocin induced diabetes, with improved glucose tolerance; females exhibited impaired glucose tolerance and exacerbated diabetes (ENDO 2023). Our findings indicate that an increased level of CCN5 protects β-cells, but only in male mice. This protection cannot be solely attributed to the pro-islet effect and suggests the involvement of extra-pancreatic and sex-specific roles. This study indirectly screened normal CCN5 gene expression by detecting β-galactosidase activity, measured in X-gal-stained tissues before fixation and sectioning. In order to create the gene deletion through homologous recombination, LacZ was inserted between exons 2 and 5 of the CCN5 gene. As a result, male knockout mice exhibited a distinct blue staining in the pseudostratified columnar epithelium with stereocilia of the epididymis and vas deferens. This type of epithelium comprises a single layer of cells but appears to have multiple layers due to the nuclei being at different levels. The epithelium possesses long projections made of actin filaments and plays a crucial role in absorbing, transporting, and maturing sperm cells. The staining was also clear in Leydig cells of the testis, which produce testosterone, which was confirmed using immunohistochemistry and authentic CCN5 antibody. In female mice, however, only a few cells in the ovary and scattered staining was observed in the endometrial glands of the uterus. Since androgens promote energy expenditure, lipolysis, and insulin sensitivity, knockout or overexpression of CCN5 may potentially affect androgen and/or sperm production, leading to metabolic consequences in males. Our results from in vitro, knockout, and overexpression models support the notion that CCN5 promotes β-cell growth and survival against diabetes and further suggest that it possesses sex-specific, metabolic effects. Presentation: 6/2/2024

Macroanatomy, Histomorphometry, and Androgen Receptor Expression in the Epididymis of Kacang Goats Aged 4, 8, and 12 Months

The epididymis is a crucial component of the goat's reproductive structure. The epididymis is responsible for the transportation, concentration, and maturation of sperm. This study aimed to examine the differences in the structure of macro and microanatomy, tissue composition, and the existence of androgen receptors in the epididymis of Kacang goats aged 4, 8, and 12 months. The assessment of macroscopic organ growth was done immediately after sampling, whereas microscopic measurements were carried out following histological preparations using hematoxylin-eosin (HE) and immunohistochemical (IHC) procedures. The results on the macroscopic anatomy of the epididymis indicated a significant association between age with the width of the caput dexter and sinister and the circumference of the right cauda. However, no significant relationship was found between age with the corpus length and the left cauda's circumference. Significant variations were observed in the diameter and concentration of the agglutinated spermatozoa in the lumen during histomorphometry of the epididymis in three age groups of Kacang goats. There were no statistically significant variations in the expression of androgen receptors among the three age groups. This study showed that the correlation coefficient test reveals a positive relationship between age and the caput width and corpus length dimensions, indicating that these measurements tend to grow as age increases. On the other hand, the diameter of the agglutinated spermatozoa in the epididymal lumen exhibits significant variations between the ages of 4 months, with the ages of 8 months and 12 months, suggesting that the sperm becomes fully matured by the age of 8 months.

Isolation of CD63-positive epididymosomes from human semen and its application in improving sperm function.

Extracellular vesicles (EVs) are highly heterogeneous, and different EV subpopulations from various origins mediate different biological effects. The separation of different subpopulations of EVs from mixtures is critical but challenging. Epididymosomes are secreted by the epididymal epithelium and play a key role in sperm maturation and function. However, limited access to human epididymal tissue and epididymal fluid has hampered further study of epididymosomes and their potential clinical applications. Here, we established a novel strategy based on flow cytometry sorting to isolate human CD63-positive epididymosomes from ejaculate. We identified CD52, a membrane-located protein expressed exclusively in the epididymis, as the sorting marker for human epididymosomes. Then, CD63-positive epididymosomes were isolated from human semen using a flow cytometry sorting instrument and concentrated. Additionally, we observed that isolated CD63-positive epididymosomes improved sperm function more than other CD63-positive seminal EV subpopulations did, demonstrating the successful isolation of a subpopulation of epididymosomes from human semen and their potential application in the clinic.

Mapping the Landscape of Obesity Effects on Male Reproductive Function: A Bibliometric Study.

Due to changes in lifestyle and dietary habits, the global population with obesity is increasing gradually, resulting in a significant rise in the number of individuals having obesity. Obesity is caused by an imbalance between energy intake and consumption, leading to excessive fat accumulation, which interferes with normal human metabolism. It is also associated with cardiovascular disease, metabolic syndrome, male reproductive endocrine regulation disorders, systemic and local inflammatory reactions, excessive oxidative stress, and apoptosis. All these factors can damage the internal environment for sperm generation and maturation, resulting in male sexual dysfunction, a decline in sperm quality, and lower fertility. This study analyzes the trends and priorities of the effects of obesity on male reproductive disorders from a bibliometric perspective. This study uses the Web of Science as the statistical source, covering all time spans. Tools like Web of Science, VOSviewer, and CiteSpace are used to analyze countries, institutions, authors, journals, and keywords in the field. Total publications, total citations, and average number of citations are selected for statistics. The results show that the research on the impact of obesity on male reproductive function can be roughly divided into three stages: the initial stage, the slow development stage, and the rapid development stage. Our statistical scope includes 463 highly relevant articles that we have screened. We found that the journal with the most publications in this field is Andrologia, and the institution with the highest total citations is the University of Utah. The most influential countries, institutions, and authors in this field are the United States, the University of Utah, and Carrell, Douglas. Currently, research related to the impact of obesity on male reproduction focuses mainly on three aspects: biochemistry, molecular biology, and reproductive biology. The keyword explosion results indicate that sperm, obesity, and male reproduction are at the forefront and trends of future research in this field. There has been a shift from basic biochemical and molecular research to research on molecular mechanisms relying on omics technologies. However, we have observed that the number of papers published in 2022 is lower than in 2021, indicating a growth interruption during this period. Considering that this deviation may be due to the impact of the COVID-19 pandemic, it may hinder the progress of certain experiments in 2022. In recent years, China has rapidly developed research in this field. However, the average citation rate is relatively low, indicating the need for Chinese scholars to improve the quality of their articles further. Based on our research and in the context of global obesity, men are at risk of increased infertility. Addressing this issue relies on our continued research into the mechanisms of obesity-related male reproductive disorders. Over the past forty-three years, with the contributions of scientists worldwide, research in this field has flourished. The impact of obesity on male reproductive disorders has been extensively studied. Currently, research in this field primarily focuses on male sperm function, sperm quality, and the effects or mechanisms of cells on male reproduction. Future trends in this field should concentrate on the relationship between male fertility and energy metabolism, as well as the endocrine function of adipose tissue. This study comprehensively analyzes the current research status and global trends in obesity and male reproductive disorders. We also discuss the future developments in this field, making it easier for researchers to understand its developmental history, current status, and trends, providing valuable reference for effective exploration in this area.

Alpha-1-B glycoprotein (A1BG) inhibits sterol-binding and export by CRISP2

Proteins belonging to the CAP superfamily are present in all kingdoms of life and have been implicated in various processes, including sperm maturation and cancer progression. They are mostly secreted glycoproteins and share a unique conserved CAP domain. The precise mode of action of these proteins, however, has remained elusive. Saccharomyces cerevisiae expresses three members of this protein family, which bind sterols in vitro and promote sterol secretion from cells. This sterol-binding and export function of yeast Pry proteins is conserved in the mammalian CRISP proteins and other CAP superfamily members. CRISP3 is an abundant protein of the human seminal plasma and interacts with alpha-1-B glycoprotein (A1BG), a human plasma glycoprotein that is upregulated in different types of cancers. Here we examined whether the interaction between CRISP proteins and A1BG affects the sterol-binding function of CAP family members. Co-expression of A1BG with CAP proteins abolished their sterol export function in yeast and their interaction inhibits sterol-binding in vitro. We map the interaction between A1BG and CRISP2 to the third of five repeated immunoglobulin-like (Ig) domains within A1BG. Interestingly, the interaction between A1BG and CRISP2 requires magnesium, suggesting that coordination of Mg2+ by the highly conserved tetrad residues within the CAP domain is essential for a stable interaction between the two proteins. The observation that A1BG modulates the sterol-binding function of CRISP2, has potential implications for the role of A1BG and related Ig domain containing proteins in cancer progression and the toxicity of reptile venoms containing CRISP proteins.

CEP112 coordinates translational regulation of essential fertility genes during spermiogenesis through phase separation in humans and mice

Spermiogenesis, the complex transformation of haploid spermatids into mature spermatozoa, relies on precise spatiotemporal regulation of gene expression at the post-transcriptional level. The mechanisms underlying this critical process remain incompletely understood. Here, we identify centrosomal protein 112 (CEP112) as an essential regulator of mRNA translation during this critical developmental process. Mutations in CEP112 are discovered in oligoasthenoteratospermic patients, and Cep112-deficient male mice recapitulate key phenotypes of human asthenoteratozoospermia. CEP112 localizes to the neck and atypical centrioles of mature sperm and forms RNA granules during spermiogenesis, enriching target mRNAs such as Fsip2, Cfap61, and Cfap74. Through multi-omics analyses and the TRICK reporter assay, we demonstrate that CEP112 orchestrates the translation of target mRNAs. Co-immunoprecipitation and mass spectrometry identify CEP112’s interactions with translation-related proteins, including hnRNPA2B1, EEF1A1, and EIF4A1. In vitro, CEP112 undergoes liquid-liquid phase separation, forming condensates that recruit essential proteins and mRNAs. Moreover, variants in patient-derived CEP112 disrupt phase separation and impair translation efficiency. Our results suggest that CEP112 mediates the assembly of RNA granules through liquid-liquid phase separation to control the post-transcriptional expression of fertility-related genes. This study not only clarifies CEP112’s role in spermatogenesis but also highlights the role of phase separation in translational regulation, providing insights into male infertility and suggesting potential therapeutic targets.

Ultrastructural Characteristics of the Mature Spermatozoon of Artyfechinostomum malayanum (Digenea: Echinostomatidae), an Intestinal Parasite of Rattus norvegicus (Rodentia: Muridae) in Vietnam.

The study of sperm characteristics has proven useful for elucidating interrelationships in several groups of Platyhelminthes, such as digeneans. Thus, in the present work, the ultrastructural organization of the mature spermatozoon of the digenean Artyfechinostomum malayanum (Echinostomatidae), a parasite of Rattus norvegicus (Rodentia: Muridae) from Dong Thap Province, Vietnam, was investigated for the first time using transmission electron microscopy. The male gamete of A. malayanum exhibits two axonemes of different lengths, showing the 9 + '1' pattern of the Trepaxonemata, a nucleus, two mitochondria, two lateral expansions, two bundles of parallel cortical microtubules, external ornamentation, spine-like bodies, and granules of glycogen. Thus, the mature spermatozoon follows a Type V sperm model proposed for digeneans. We also highlight some noteworthy characteristics in Echinostomatidae with possible phylogenetic implications, such as two lateral expansions in the anterior region of the spermatozoon and two mitochondria.

Perspective in the Mechanisms for Repairing Sperm DNA Damage.

DNA damage in spermatozoa is a major cause of male infertility. It is also associated with adverse reproductive outcomes (including reduced fertilization rates, embryo quality and pregnancy rates, and higher rates of spontaneous miscarriage). The damage to sperm DNA occurs during the production and maturation of spermatozoa, as well as during their transit through the male reproductive tract. DNA damage repair typically occurs during spermatogenesis, oocytes after fertilization, and early embryonic development stages. The known mechanisms of sperm DNA repair mainly include nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), and double-strand break repair (DSBR). The most severe type of sperm DNA damage is double-strand break, and it will be repaired by DSBR, including homologous recombination (HR), classical non-homologous end joining (cNHEJ), alternative end joining (aEJ), and single-strand annealing (SSA). However, the precise mechanisms of DNA repair in spermatozoa remain incompletely understood. DNA repair-associated proteins are of great value in the repair of sperm DNA. Several repair-related proteins have been identified as playing critical roles in condensing chromatin, regulating transcription, repairing DNA damage, and regulating the cell cycle. It is noteworthy that XRCC4-like factor (XLF) and paralog of XRCC4 and XLF (PAXX) -mediated dimerization promote the processing of populated ends for cNHEJ repair, which suggests that XLF and PAXX have potential value in the mechanism of sperm DNA repair. This review summarizes the classic and potential repair mechanisms of sperm DNA damage, aiming to provide a perspective for further research on DNA damage repair mechanisms.

Unraveling the role of sperm retained histones in bull fertility and daughter fertility

During spermatogenesis, a substantial proportion of histones are substituted by protamine to condense the genome within the sperm head. Studies indicate that a minority of histones, typically ranging from 1 to 15 %, persist in mammalian sperm post-substitution. The persistence of histones in the zygote facilitates chromatin accessibility to transcription factors in regions crucial for early embryonic development. Nevertheless, the potential causal relationship between retained histones and fertility phenotypes remains uncertain. This study seeks to investigate this relationship. The results indicate that in mature bovine sperm, regions of DNA associated with fertility that bind to histones are primarily concentrated in promoters and transcription start sites, potentially impacting bull fertility and offspring fertility through the regulation of relevant genes. Furthermore, microRNAs and estradiol/ESR are suggested to be the main regulators of the canonical pathways identified, highlighting the need for additional research to investigate their potential utility as biomarkers.

Mettl1-dependent m7G tRNA modification is essential for maintaining spermatogenesis and fertility in Drosophila melanogaster

Modification of guanosine to N7-methylguanosine (m7G) in the variable loop region of tRNA is catalyzed by the METTL1/WDR4 heterodimer and stabilizes target tRNA. Here, we reveal essential functions of Mettl1 in Drosophila fertility. Knockout of Mettl1 (Mettl1-KO) causes no major effect on the development of non-gonadal tissues, but abolishes the production of elongated spermatids and mature sperm, which is fully rescued by expression of a Mettl1-transgene, but not a catalytic-dead Mettl1 transgene. This demonstrates that Mettl1-dependent m7G is required for spermatogenesis. Mettl1-KO results in a loss of m7G modification on a subset of tRNAs and decreased tRNA abundance. Ribosome profiling shows that Mettl1-KO led to ribosomes stalling at codons decoded by tRNAs that were reduced in abundance. Mettl1-KO also significantly reduces the translation efficiency of genes involved in elongated spermatid formation and sperm stability. Germ cell-specific expression of Mettl1 rescues disrupted m7G tRNA modification and tRNA abundance in Mettl1-KO testes but not in non-gonadal tissues. Ribosome stalling is much less detectable in non-gonadal tissues than in Mettl1-KO testes. These findings reveal a developmental role for m7G tRNA modification and indicate that m7G modification-dependent tRNA abundance differs among tissues.

WDR64, a testis-specific protein, is involved in the manchette and flagellum formation by interacting with ODF1

The WD40 repeat (WDR) domain is present in a wide range of proteins, providing sites for protein‒protein interactions. Recent studies have shown that WDR proteins play indispensable roles in spermatogenesis, such as in spermatocyte division, sperm head formation and flagellar assembly. In this study, we identified a novel testis-specific gene, WDR64, which has the typical characteristics of WD40 proteins with two β-propellers, and is highly conserved in Mammalia. RT-PCR and Western blot results revealed that WDR64 was highly expressed in testis. WDR64 protein was weakly expressed at postnatal Day 7, increased substantially at postnatal Day 28 and maintained at high levels thereafter. Further immunofluorescence demonstrated that WDR64 was localized posterior to the nucleus in steps 8–14 spermatids in line with the dynamic localization of manchette, moved to the flagella in steps 15–16 spermatids, and localized at the midpiece of the flagellum in mature spermatozoa. To explore the function of WDR64, we performed immunoprecipitation‒mass spectrometry (IP‒MS) to screen its interacting proteins and found that WDR64 interacted with ODF1 to form a complex. The WDR64/ODF1 complex is located at the manchette during nucleus shaping and finally at the midpiece of the mature spermatozoa tail, suggesting that it may be involved in the assembly of the manchette and flagella during spermiogenesis. Our findings provide the first understanding of the expression pattern of WDR64 and its potential molecular mechanism in spermiogenesis.

Antidepressants as new endocrine disruptors? – transcriptomic profiling in gonads of Mytilus trossulus exposed to norfluoxetine

In this study an impact of norfluoxetine (NFLU) on Mytilus trossulus gonads was investigated focusing on sex-related differences in hormonal changes, gene expression, and transcriptomic profiling. Sex-specific differences in gonadal serotonin levels were found. NFLU stimulates serotonin synthesis and/or transport in female gonads, potentially accelerating oocyte maturation and gamete release. In males, NFLU decreases serotonin level what likely leads to impeding sperm maturation and thus spawning delay. Transcriptomic analyses highlighted the presence of NFLU-induced changes in gene expression related to gametogenesis and neurotransmission. In females, NFLU upregulated genes associated with oocyte development and downregulated those involved in sperm maturation. NFLU-treated males exhibited mixed effects in their genes in relation to spermatogenesis. Additionally, sex-related differences in the expression of the CYP450 genes responsible for detoxification were found. Overall, norfluoxetine acts as an endocrine-disrupting chemical and impacts gonadal serotonin levels and gene expression, potentially disrupting reproductive success of M. trossulus.

Sperm Migration and Hyaluronic Acid Binding: Implications for Male Fertility Evaluation.

Mature, vital, and motile spermatozoa are essential for reaching the oocyte and binding to hyaluronic acid (HA) in the cumulus oophorus matrix. This study aims to determine the relationship between sperm-migration ability and HA-binding potential, as well as the relationship between sperm concentration and motility. Semen samples were collected from 702 men aged 20-56 years (median 34.8). We evaluated the sperm concentration and motility from basic semen analysis, the swim-up test (expressed as millions per mL and the migration efficiency percentage), and the hyaluronan-binding assay (HBA). A moderate positive correlation was found between the migration test results and HBA (R = 0.48). The highest correlation was observed between the concentration of motile spermatozoa and the migration test results (R = 0.85) and HBA (R = 0.4). The sperm migration efficiency strongly correlated with progressive motility (R = 0.6). Although significantly higher sperm migration was observed in patients with normal HBA results, the results of the functional tests were found to differ in some cases. For infertility treatment, the current diagnostic algorithm should be enhanced with more comprehensive seminological methods that assess the sperm-migration ability and HA-binding potential. We also recommend incorporating the swim-up method into the diagnostic protocol before planning assisted reproductive technology (ART) treatment.

Expression pattern and functional analysis of kinesin-14 KIFC1 in spermatogenesis of Macaca mulatta

C-terminal kinesin motor KIFC1 is increasingly concerned with an essential role in germ cell development. During the spermatogenesis of mice, rats, and crustaceans, KIFC1 functions in regulating meiotic chromosome separation, acrosome vesicle transportation, and nuclear morphology maintenance. The expression pattern of KIFC1 is conservatively concentrated at the acrosome and nucleus of haploid sperm cells. However, whether KIFC1 has similar functions in non-human primates remains unknown. In this study, we constructed the testis-specific cDNA library and cloned different transcripts of KIFC1 based on the genomic sequence. New variants of KIFC1 were identified, and showed different functional domains from the predicted isoforms. The spatio-temporal expression of KIFC1 proteins in seminiferous tubules of rhesus monkeys showed an obvious nuclear localization, specifically expressed in the spermatocytes and early haploid spermatids. The transcripts of KIFC1 also exhibited considerable expression in the nucleus of rhesus LLC-MK2 cells. Besides, we demonstrated that KIFC1 located at the acrosome and microtubule flagella of the mature sperm, and KIFC1 inhibition resulted in sperm tail deformation as well as increased the instability of head-to-tail connection. In summary, this study filled a gap in the reproductive research of the KIFC1 gene in non-human primates.