Acrosome Research Articles

Motile cilia: Key developmental and functional roles in reproductive systems.

Cilia are specialized microtubule-based organelles that extend from the cell surface and are classified into non-motile and motile types. The assembly and function of cilia are regulated by a complex molecular network that enables motile cilia to generate fluid flow across epithelial surfaces through coordinated beating. These motile cilia are found in the respiratory, nervous, and reproductive systems. In males, motile cilia are found in the efferent ducts and facilitate the transport of sperm from the testis to the epididymis. In females, they are mainly found in the oviducts, where they help to transport, nourish and fertilize eggs, and are also present in the endometrial epithelium. This review compares the common factors that affect motile cilia in both male and female reproductive tracts, discusses the origin and development of multiciliated cell and cilia within the efferent ducts and oviducts, and enumerates the infertility or related reproductive diseases that may arise due to motile cilia defects. In males, motile cilia in the efferent ducts create turbulence through their beating, which keeps semen suspended and prevents ductal obstruction. In females, motile cilia are distributed on the epithelia of the oviducts and the endometrium. Specifically, motile cilia in the infundibulum of the oviduct aid in capturing oocytes, while cilia in the isthmus region have been found to bind to sperm heads, facilitating the formation of the sperm reservoir. Several common factors, such as miR-34b/c and miR-449, TAp73, Gemc1, and estrogen, etc., have been shown to play crucial regulatory roles in motile cilia within the efferent ducts and oviducts, thereby further influencing fertility outcomes. Pathogenic mutations that disrupt ciliary function can impair ciliogenesis or alter the structure of sperm flagella, potentially resulting in infertility. Consequently, motile cilia in both the male and female reproductive tracts are crucial for fertility. There are still numerous unresolved mysteries surrounding these cilia that merit further investigation by researchers, as they hold great significance for the clinical diagnosis and treatment of infertility and related reproductive disorders.

Protective effect of trehalose on sperm chromatin condensation failure and semen quality decline in BDE-209-exposed mice.

BDE-209 exposure induced male reproductive toxicity with sperm quality decline. However, the role of autophagy in this was unclear. The purpose was to evaluate the protective effect and its potential mechanism of trehalose (Tre, autophagy inducer) on reproductive damage during spermiogenesis induced by BDE-209. We used 2% w/v Tre and 75mg/kg/d BDE-209 cotreated mice for 42 days. GC-2 spd cells were cotreated with Tre, chloroquine (CQ, inhibition of autophagic flux), compound C (CC, AMPK inhibitor), and BDE-209. Tre intake significantly recovered decrease in sexual organ ratio and poor sperm quality in BDE-209-exposed mice. Supplementation with Tre rescued sperm head malformation by improving aberrant histone-protamine exchange in BDE-209-exposed mice. However, Tre intake couldn't restore the acrosome biogenesis. In addition, Tre supplementation improved testicular damage induced by BDE-209. BDE-209 blocked autophagic flux with increased P62 and LC3BⅡ/Ⅰ levels. Mechanistically, CQ treatment aggravated elevation of P62 and LC3BⅡ/Ⅰ levels induced by BDE-209, otherwise, CC and Tre treatments inhibited the rise in p-AMPK, p-ULK1, P62 and LC3BⅡ/Ⅰ levels induced by BDE-209. Tre supplementation improved reproductive injury in BDE-209-exposed mice by regulating autophagic flow via AMPK-ULK1 signaling pathways, which providing a new theoretical basis and possible therapeutic targets for male reproductive toxicity.

A novel SEPT12 mutation, T96I, is associated with sperm head and annulus defects.

Infertility affects around 8%-12% of reproductive-aged couples and is a major health concern. Both genetic and environmental factors influence male infertility. SEPTIN12 is a crucial testis-specific gene essential for the final differentiation of male germ cells and is strongly linked to male infertility due to numerous detected mutations. The present study identified a novel SEPTIN12 T96I mutation that causes male infertility. Immunofluorescence staining and transmission electron microscopy (TEM) analysis of T96I sperm revealed co-localization of SEPT12 and SEPT7 in the obliquely positioned annulus. In addition, the sperm carrying the T96I mutation demonstrated large nuclear vacuoles, irregular swelling, and decondensation of the acrosomal cap. The overexpression of SEPT12 T96I in NT2/D1 cells impaired the formation of SEPT7 filaments, emphasizing the significance of SEPT12 filaments for sperm morphology and function. Our results demonstrate the importance of SEPTIN12 T96I in male infertility and offer valuable insights for future detection in infertile men.

Cep78 knockout causes sterility and oligoasthenoteratozoospermia in male mice

Oligoasthenoteratozoospermia (OAT) is a common cause of infertility among males, and the majority of cases of idiopathic OAT are thought to be attributed to genetic defects. In this study, the role of the CEP78 protein in spermatogenesis was initially investigated using Cep78 knockout (Cep78−/−) mice. Notably, the male Cep78−/− mice exhibited the OAT phenotype and sterility. To elucidate the mechanisms underlying the functions of the Cep78 gene in spermatogenesis, the histomorphology of germ cells was investigated during different stages of mitosis, meiosis, and spermiogenesis. Apoptotic assays and RNA-sequencing analyses were additionally performed using the testicular tissue samples of control and Cep78−/− mice. The findings strongly suggested that defects in the Cep78 gene can lead to male infertility with OAT and that the CEP78 protein is essential for acrosomal biogenesis, sperm head shaping, and formation of flagella during spermiogenesis. The findings are expected to expand the spectrum of genetic defects in OAT and enhance the accuracy of genetic screening and clinical diagnosis.

Studies on ameliorative potentials of quercetin nanoparticles against imidacloprid induced subacute genotoxicity and histopathological alteration in Swiss albino mice

ABSTRACT Objective Genotoxicity assays include micronucleus test, comet assay, and malformed sperm head used to investigate the protective potential of quercetin and quercetin nanoparticles against imidacloprid-induced genotoxicity in Swiss albino mice. Method The ionic gelation procedure was used to synthesize the quercetin nanoparticles and characterized for their hydrodynamic diameter, zeta potential, SEM, TEM, FT-IR, and encapsulation efficiency. Total 48 mice were taken in eight groups with six animals in each group. Group 1, 2, 3 and 4 received 3% gum acacia, 22 mg/kg imidacloprid (IMI), 25 mg/kg quercetin (Que) and 25 mg/kg quercetin nanoparticles high dose [QNPs (HD)], respectively. Group 5, 6, 7 and 8 received 22 mg/kg imidacloprid + 25 mg/kg quercetin (IMI + Que), 22 mg/kg imidaclopid + 25 mg/kg quercetin nanoparticles [IMI + QNPs (HD)], 22 mg/kg imidacloprid + 12.5 mg/kg quercetin nanoparticle medium dose [IMI + QNPs (MD)] and 22 mg/kg imidacloprid + 6.25 mg/kg quercetin nanoparticles low dose [IMI + QNPs (LD)], respectively. Result The imidacloprid causes genotoxicity in bone marrow cells by increasing the frequency of micronuclei and the comet tail length. Additionally, imidacloprid is mutagenic to germ cells, as determined by a test for aberrant sperm heads. Both quercetin and quercetin nanoparticles lessen the genotoxicity that imidacloprid induces when administered together or separately. Histopathological findings also revealed degenerative changes in bone marrow and testes in imidacloprid administered group as compared to control. Conclusion Quercetin and quercetin nanoparticles showed marked ameliorative effect by restoring the degenerative changes produced by imidacloprid.

A deficiency screen identifies genomic regions critical for sperm head-tail connection.

The Sperm Neck provides a stable connection between the sperm head and tail, which is critical for fertility in species with flagellated sperm. Within the Sperm Neck, the Head-Tail Coupling Apparatus serves as the critical link between the nucleus (head) and the axoneme (tail) via the centriole. To identify regions of the Drosophila melanogaster genome that contain genetic elements that influence Head-Tail Coupling Apparatus formation, we undertook a 2 part screen using the Drosophila Deficiency kit. For this screen, we utilized a sensitized genetic background that overexpresses the pericentriolar material regulatory protein Pericentrin-Like Protein. We had previously shown that Pericentrin-Like Protein overexpression disrupts the head-tail connection in some spermatids, but not to a degree sufficient to reduce fertility. In the first step of the screen, we tested for deficiencies that in combination with Pericentrin-Like Protein overexpression causes a reduction in fertility. We ultimately identified 11 regions of the genome that resulted in an enhanced fertility defect when combined with Pericentrin-Like Protein overexpression. In the second step of the screen, we tested these deficiencies for their ability to enhance the head-tail connection defect caused by Pericentrin-Like Protein overexpression, finding 6 genomic regions. We then tested smaller deficiencies to narrow the region of the genome that contained these enhancers and examined the expression patterns of the genes within these deficiencies using publicly available datasets of Drosophila tissue RNAseq and Drosophila testes snRNAseq. In total, our analysis suggests that some deficiencies may contain single genes that influence Head-Tail Coupling Apparatus formation or fertility, while other deficiencies appear to be genomic regions rich in testis-expressed genes that might affect the Head-Tail Coupling Apparatus through complex, multigene interactions.

Swimming by Spinning: Spinning-Top Type Rotations Regularize Sperm Swimming Into Persistently Progressive Paths in 3D.

Sperm swimming is essential for reproduction, with movement strategies adapted to specific environments. Sperm navigate by modulating the symmetry of their flagellar beating, but how they swim forward with asymmetrical beats remains unclear. Current methods lack the ability to robustly detect the flagellar symmetry state in free-swimming spermatozoa, despite its importance in understanding sperm motility. This study uses numerical simulations to investigate the fluid mechanics of sperm swimming with asymmetrical flagellar beats. Results show that sperm rotation regularizes the swimming motion, allowing persistently progressive swimming even with asymmetrical flagellar beats. Crucially, 3D sperm head orientation, rather than the swimming path, provides critical insight into the flagellar symmetry state. Sperm rotations during swimming closely resemble spinning-top dynamics, with sperm head precession driven by the helical beating of the flagellum. These results may prove essential in future studies on the role of symmetry in microorganisms and artificial swimmers, as body orientation detection has been largely overlooked in favor of swimming path analysis. Altogether, this rotational mechanism provides a reliable solution for forward propulsion and navigation in nature, which would otherwise be challenging for flagella with brokensymmetry.

Homozygous deleterious variants in MYCBPAP induce asthenoteratozoospermia involving abnormal acrosome biogenesis, manchette structure and sperm tail assembly in humans and mice.

Asthenoteratozoospermia is a common cause of male infertility. To further define the genetic causes underlying asthenoteratozoospermia, we performed whole-exome sequencing in a cohort of Han Chinese men with asthenoteratozoospermia. Homozygous deleterious variants of MYCBPAP were first identified in two unrelated Chinese cases. Replication analyses in a French cohort revealed an additional asthenoter-atozoospermia-affected case harboring a homozygous nonsense variant in MYCBPAP. All of the identified MYCBPAP variants were absent or extremely rare in the public human genome databases. Further functional assays indicated remarkably reduced abundance of MYCBPAP in the spermatozoa from MYCBPAP-associated cases. Subsequently, we generated a Mycbpap knockout (Mycbpap-/-) mouse model, which also exhibited male infertility with reduced sperm motility and abnormal morphologies in sperm heads and flagella. Further investigations demonstrated that Mycbpap-/- male mice presented disrupted acrosome biogenesis and abnormally elongated manchette during spermiogenesis. Intriguingly, proteomic analyses indicated that the proteins related to spermatogenesis, acrosomal and flagellar functions were significantly down-regulated in the testes from Mycbpap-/- male mice. Endogenous immunoprecipitation combined with mass spectrometry revealed interactions of MYCBPAP with a ribosome elimination related protein ARMC3 and central apparatus proteins including CFAP65 and CFAP70. Furthermore, MYCBPAP-associated male infertility in humans and mice could be partially overcome by using intracytoplasmic sperm injections. Collectively, these findings illustrate the essential role of MYCBPAP in normal spermatogenesis and homozygous deleterious variants in MYCBPAP can be considered as a genetic diagnostic indicator for infertile men with asthenoteratozoospermia. Our study will provide effective guidance for genetic counseling, clinical diagnosis and assisted reproduction treatments of MYCBPAP-associated male infertility.

CCDC113 stabilizes sperm axoneme and head-tail coupling apparatus to ensure male fertility.

The structural integrity of the sperm is crucial for male fertility, defects in sperm head-tail linkage and flagellar axoneme are associated with acephalic spermatozoa syndrome (ASS) and the multiple morphological abnormalities of the sperm flagella (MMAF). Notably, impaired head-tail coupling apparatus (HTCA) often accompanies defects in the flagellum structure, however, the molecular mechanisms underlying this phenomenon remain elusive. Here, we identified an evolutionarily conserved coiled-coil domain-containing (CCDC) protein, CCDC113, and found the disruption of CCDC113 produced spermatozoa with disorganized sperm flagella and HTCA, which caused male infertility. Further analysis revealed that CCDC113 could bind to CFAP57 and CFAP91, and function as an adaptor protein for the connection of radial spokes, nexin-dynein regulatory complex (N-DRC), and doublet microtubules (DMTs) in the sperm axoneme. Moreover, CCDC113 was identified as a structural component of HTCA, collaborating with SUN5 and CENTLEIN to connect sperm head to tail during spermiogenesis. Together, our studies reveal that CCDC113 serve as a critical hub for sperm axoneme and HTCA stabilization in mice, providing insights into the potential pathogenesis of infertility associated with human CCDC113 mutations.

Coevolution and Adaptation of Transition Nuclear Proteins and Protamines in Naturally Ascrotal Mammals Support the Black Queen Hypothesis.

Protamines (PRMs) and transition nuclear proteins (TNPs) are two key classes of sperm nuclear basic proteins that regulate chromatin reorganization and condensation in the spermatozoon head, playing crucial roles in mammalian spermatogenesis. In scrotal mammals, such as humans, cryptorchidism, the failure of the testes to descend into the scrotal sac is generally associated with higher rates of defective spermatozoon quality and function. However, ascrotal mammals, such as cetaceans, with naturally undescended testes, produce normal spermatozoa similar to their scrotal counterparts. This study investigates the evolutionary pattern and functional changes in PRMs and TNPs to explore the potential molecular mechanisms underlying spermatogenesis in naturally ascrotal mammals. Although we found a conserved genomic arrangement for PRM and TNP genes across mammals, the coevolutionary loss of intact PRM2 and TNP2 was observed in several species, correlating significantly with diverse testicular positions. Notably, in cetaceans, which lack intact PRM2 and TNP2, we detected enhanced thermostability and DNA binding in PRM1, along with superior DNA repair capability in TNP1. These findings suggest that gene loss of PRM2 and TNP2, combined with functional enhancements in PRM1 and TNP1 proteins, evolved in response to physiological challenges posed by natural cryptorchidism in most ascrotal lineages. This evolutionary strategy enhances chromatin condensation efficiency and promotes DNA repair during spermatogenesis in natural cryptorchid mammals, supporting the Black Queen Hypothesis.

PP1γ1 is unable to substitute for the mammal-specific PP1γ2 isoform to support male fertility and sperm function.

The serine-threonine phosphatase has four paralogs - PP1α, PP1β, PP1γ1 and PP1γ2 - encoded by three genes, Ppp1ca, Ppp1cb, and Ppp1cc. Protein phosphatase PP1γ2, one of two isoforms of the gene Ppp1cc, is expressed in spermatogenic cells in testis and sperm, while PP1γ1 is found in somatic cells. The two PP1γ isoforms, formed by alternate splicing which occurs only in mammals, are identical except at their C-termini. Global or testis knockout of Ppp1cc in mice results in male infertility due to disrupted spermiation and mid-to-late-spermiogenesis. Transgenic expression of PP1γ2, driven by a testis-specific promoter in differentiating spermatogenic cells, rescues spermatogenesis and fertility in the Ppp1cc-null mice. Why PP1γ2 is essential and present only in mammalian sperm is a mystery. We have generated a knock-in mouse where the Ppp1cc gene is edited to express only PP1γ1. Spermatogenesis was normal in knock-in mice. Testis-expressed PP1γ1 in the knock-in mice and PP1γ2 in the wild-type mice were incorporated in equal amounts in sperm. Sperm bearing PP1γ1 have reduced flagellar beat amplitude and motility, and male mice were severely sub-fertile. Although in wild-type mice PP1γ2 is in both the head and tail, in knock-in mice PP1γ1 is absent from sperm heads, leading to an altered intra-sperm protein phosphatase landscape. Phospho-proteomic analysis of sperm proteins suggested a plausible molecular basis for compromised PP1γ1 functions: it identified GSK3α, a known substrate of PP1, to be dysregulated in knock-in sperm. This study provides a preliminary explanation for the isoform-specific requirement of PP1γ2 for male fertility.

Spatial Organization of the Sperm Cell Glycoproteome.

Sperm cells are terminally differentiated cells that are essential for reproduction in sexually reproducing species. Consistent with their highly specialized function, sperm cells harbor a unique proteome containing many proteins not expressed in somatic cells. In contrast, the post-translational landscape of the sperm proteome remains largely unexplored, limiting our understanding of how modifications such as glycosylation impact sperm function and sperm-egg interactions. Here, we used glycopeptide-centric glycoproteomics to comprehensively characterize protein N-glycosylation in sperm from three mammalian species, revealing clear conservation of glycosylation profiles. We find that glycosylation patterns in sperm proteins are distinct from those in plasma, with as clear distinctive features less sialyation and more paucimannosylation in sperm. Moreover, based on their subcellular location, sperm protein glycosylation varies, with paucimannose species enriched in the acrosomal vesicle, oligomannose species in the sperm head membrane, and complex glycan species in the acrosomal membrane.

Reproductive parameters and sex cell ultrastructure in cinnabar goatfish <i>Parupeneus heptacanthus</i> (Mullidae) from the coastal zone of Nha Trang (Vietnam)

The size composition of the fish, gonadal condition, and oocyte and spermatozoon ultrastructure are studied in Parupeneus heptacanthus from the coastal zone of Nha Trang (southern Central Vietnam). The males are larger than the females, and the body length—body weight relationship is significantly different in the representatives of both sexes. Based on ovarian condition, oogenesis is continuous. Average batch fecundity is 11 056 oocytes. The egg envelope in the end of vitellogenesis (~300 µm in diameter) includes zona radiata 5.0 µm in width and chorion (0.5 µm). Based on spermatozoon head ultrastructure, the species is similar to other species of the genus Parupeneus, but the flagellum is substantially longer reaching on average 63 µm.

The extracellular vesicle tetraspanin CD63 journey from the testis through the epididymis to mature bull sperm

The important role of extracellular vesicles, which are considered key mediators of intercellular communication under physiological and pathological conditions, in various cellular processes, including those crucial for mammalian reproduction, has been increasingly studied. Tetraspanins, including CD63, are widely used as markers of extracellular vesicles, but they may also play a role in their biogenesis, cargo selection, cell targeting, and uptake. This study aimed to map the journey of the extracellular vesicle protein tetraspanin CD63 from the testis through the epididymis into mature bull sperm via an approach that included immunohistochemistry (immunofluorescence and immunoperoxidase staining), Western blot analysis, and immunoprecipitation analysis. We described the presence of CD63 in bull testicular and epididymal tissues, extracellular vesicles produced in these organs and spermatozoa during epididymal transit and after ejaculation. In addition, we revealed the nonuniform distribution of potential CD63 partners, such as CD9, integrin αV and syntenin-1, in the sperm head and tail and in extracellular vesicles. These findings contribute to understanding the complex mechanisms underlying sperm maturation and point to the possible involvement of tetraspanins and their associated partners, either as part of extracellular vesicles or sperm membranes, in these processes.

EXPERIMENTAL EVALUATION OF THE COMPARATIVE EFFECTS OF PANASA (ARTOCARPUS HETEROPHYLLUS LAM.) PAKWA PHALA AND BEEJA MAJJA ON SPERMATOGENESIS IN WISTAR ALBINO RATS.

Ayurveda is a holistic medicinal system from India that emphasises the balance of mind, body, and spirit for health maintenance. It heavily relies on local flora, herbs, and natural remedies, focusing on personalised treat-ments. One significant plant in Ayurvedic medicine is Artocarpus heterophyllus Lam, commonly known as jackfruit or ‘Panasa’ in Sanskrit. This plant is widely utilised for its culinary and medicinal properties, including applications such as Brmhana, Mamsavardhaka, Balya, Sukrala, and Vrana Ropana. Jackfruit is rich in pro-teins, fats, iron, carbohydrates, minerals, calcium, thiamine, niacin, riboflavin, vitamin C, and vitamin A. Moreover, many classical texts have documented the Vrshya action of Panasa Pakwa Phala and Beeja Majja. This study aimed to conduct a comparative experimental evaluation of the Vrshya effects of Panasa Pakwa Phala and Beeja Majja on spermatogenesis. Objective: Comparative Experimental study to evaluate Spermato-genic activity of Pakwa Phala (Fruit) and Beeja Majja (Seed) of Panasa (Artocarpus heterophyllus Lam.).Materials and method: The collection of Artocarpus heterophyllus fruits and seeds was done from the natural habitat of the Udupi district and authenticated. The experimental study assessed the comparative sper-matogenic activity of Panasa Pakwa Phala and Beeja Majja upon internal administration. Result and Discus-sion: In the experimental study, rats given Panasa Pakwa Phala exhibited a significant increase in body weight, a non-significant increase in sperm count, sluggish motility, and a decrease in non-motile sperm. Notably, there was a substantial reduction in amorphous head sperm and a non-significant rise in hookless sperm. Conversely, rats treated with Panasa Beeja also showed increased body weight but had different results in sperm motility and morphology, with a significant decrease in normal sperm morphology. This activity profile indicates the presence of a spermatogenesis effect more in the test drug Panasa Pakwa Phala than in Panasa Beeja. Con-clusion: The experimental study demonstrated statistically significant results regarding the internal use of Panasa Pakwa Phala and Beeja Majja, showing positive effects on various parameters. The spermatogenesis effect is more important with the Panasa Pakwa Phala test drug than Panasa Beeja.

Sperm acrosome-associated 1 (SPACA1) mRNA and protein molecules deficiency indicate low fertility and semen quality of Bali bulls (Bos sondaicus)

Molecular-based biomarkers are believed to be more accurate in determining bulls' fertility and sperm's various fertility characteristics. The sperm acrosome-associated 1 (SPACA1) molecule, located in the anterior acrosomal and equatorial segments of the sperm head, is thought to be related to the function of binding and fusion between sperm and oocyte. This study aims to analyze the association of SPACA1 mRNA and protein with the fertility rate and semen quality of Bali bulls (Bos sondaicus) and assess its potential as a potential molecular biomarker determining bull fertility and sperm quality. Frozen semen from 20 Bali bulls was used in the research, which was then divided into two groups: high (HF) and low fertility (LF). SPACA1 mRNA abundance was analyzed using qRT-PCR, and SPACA1 protein abundance was analyzed using EIA. The semen quality parameters analyzed were motility (CASA), plasma membrane integrity (HOS test), sperm head morphology abnormalities (William staining), sperm viability (SYBR 14-PI), sperm capacitation (CTC assay), and sperm acrosome integrity (FITC-PNA). The results showed that there was a significant difference (P < 0.05) between high fertility (HF) and low fertility (LF) bulls, both in the abundance of SPACA1 at the mRNA, protein, and semen quality levels. Overall, the correlation test results showed a close relationship (P < 0.01) between the abundance of SPACA1 mRNA and protein with field fertility and various semen quality parameters tested in the study. A low level of SPACA1 molecules indicates low fertility and semen quality in Bali bulls. The SPACA1 molecule has the potential to be developed through further research to become a biomarker for determining fertility and semen quality in bulls.

Intracellular Mg2+ concentrations are differentially regulated in the sperm head and mid-piece in acrosome reaction inducing conditions.

The sperm ability to fertilize involves the regulation of ATP levels. Because inside cells, ATP is complexed with Mg2+ ions, changes in ATP levels result in changes in intracellular Mg2+ concentration ([Mg2+]i), which can be followed using intracellular Mg2+ sensors such as Mag-520. In this work, we tested conditions known to decrease sperm ATP such as starvation and capacitation. As expected, in these conditions [Mg2+]i increased in all cell compartments. In contrast, when ATP increases, such as adding nutrients to starved sperm, [Mg2+]i significantly decreases in all compartments. On the other hand, when the acrosome reaction was induced, either with progesterone or with ionomycin, [Mg2+]i was differentially regulated in the head and mid-piece. While Mag-520 fluorescence increased in the sperm mid-piece, it decreased in the head. These changes were observed in capacitated as well as in starved sperm but not in sperm incubated in conditions that do not support capacitation. Changes in [Mg2+]i were still observed when the sperm were incubated in high extracellular Mg2+ suggesting that this decrease is not due to Mg2+ efflux. Interestingly, the progesterone and ionomycin effects on [Mg2+]i were abolished on sperm incubated in Ca2+-free media. Altogether, these results indicate that [Mg2+]i is regulated in sperm during capacitation and acrosomal reaction, and suggest that these measurements can serve to evaluate ATP levels in real time.

Detection of Androgen Receptors in Spermatozoa of Small Ruminants: A Putative Modulation Pathway for Cryoresistance Through AQP3.

This work was aimed to identify androgen receptors (AR) in the spermatozoa of wild and domestic ruminants and to assess the effect of testosterone on sperm localization of aquaporin-3 (AQP3) and cryopreservation process. Sperm samples from wild species were incubated with testosterone (T group), 1,3-propanediol (PDO group), phloretin (PHL group), PDO+T group, PHL+T group. Western blot identified the presence of AR as a single band of about 48 KDa. Immunolabelling of AR was located in the equatorial segment of the sperm head. In mouflons, the cryoresistance ratio for acrosome integrity was lower (p < 0.05) in the PHL+T than in Control and T groups. In ibexes, the cryoresistance ratio for acrosome integrity was lower (p < 0.05) in the PHL+T, PHL, and T group than in the Control group; the cryoresistance ratios for sperm kinematic variables were lower (p < 0.05) in PDO+T than in Control. No changes were found among treatments in the proportion of spermatozoa showing AQP3 in the different membrane domains after incubation and thawing in both mouflon and ibex. In conclusion, testosterone negatively affected sperm cryoresistance expressed as acrosome integrity, enhancing the effects of the AQP blocker PHL. Our findings provide a sound knowledge of the molecular mechanisms that explain the seasonal variation in sperm freezability from ruminants.

Protective effects of Sphaeranthus indicus floral extract against BPS-induced testicular damage in rats occurs through downregulation of RIPK1/3-MLK-driven necroptosis and Fas-FasL-mediated apoptosis

AbstractBisphenol S (BPS) is one of the monomers preferred in the manufacturing of polycarbonate plastics. Unfortunately, its estrogenic and genotoxic effects are similar to those of bisphenol A. The protective effects of Sphaeranthus indicus floral extract (SFE) against reprotoxic effects of BPS (50 µg/kg per body weight) in rats exposed to it via drinking water was investigated. Different SFE doses (25, 50, and 100 mg/kg) were administered via oral gavage for 10 weeks. High-performance liquid chromatography (HPLC) results indicated that SFE was rich in polyphenols, with rutin and quercetin being important bioactive molecules modulating BPS-induced necroptosis and apoptosis. Biochemical analyses unveiled that rats administered BPS only exhibited considerable elevation of biomarkers of nitro-oxidative stress, necroptotic (RIPK1/RIPK3 and MLKL), and apoptotic mediators (Fas/FasL and caspase 3/caspase-8). These events caused changes in sperm characteristics (sperm motility, sperm head, and sperm viability), sperm count, and hormonal profile (thyroid stimulating hormone, luteinizing hormone, and follicle-stimulating hormone) of the rats. Histological analysis suggested that there was pronounced sloughing of Sertoli cells, reduced spermatogenic cell density, increased levels of seminiferous tubules, and disorganized morphometric parameters related to seminiferous tubules. The SFE supplementation in rats with BPS-containing water restored nitro-oxidative stress biomarkers, which led to the reduction of necroptosis and apoptosis. Reinstatement of all the biomarkers of oxidative stress, inflammation, necroptosis, and apoptosis after SFE supplementations restored the hormonal profile and normal histoarchitecture of the testes. Virtual screening elucidated that the key regulators of the necroptosis are RIPK3-rutin and RIPK1-quercetin complexes. Further studies are needed to assess its pharmacodynamics, kinetics, and effective concentration for daily use in humans.

Effect of Probiotics on Sperm Quality in the Adult Mouse.

The administration of probiotics for the treatment of different diseases has gained interest in recent years. However, few studies have evaluated their effects on reproductive traits. The objective of this study was to examine the effect of two mixtures of probiotics, a commercial probiotic (Vivomixx®) and a mix of Lacticaseibacillus rhamnosus GG and Faecalibacterium duncaniae A2-165, on sperm quality in a mouse model. Adult male mice (8months old) were used for two experimental and one control groups (n = 5 each). The probiotics or physiological serum (control) was administered orally, twice a week, during 5weeks. Sperm were collected from the cauda epididymis, and their total number, motility, kinematics, morphology, and acrosome integrity were assessed in recently collected samples and after a 60-min in vitro incubation. Results showed a higher percentage of normal sperm in both experimental groups, with fewer head abnormalities than in the control. Differences were found among groups in the morphometry of sperm heads, being more elongated in mice treated with probiotics. Sperm from probiotic-treated mice showed similar total motility when compared to the controls, although the proportion of progressively moving sperm and their vigor of motility were lower. Sperm swimming descriptors were measured with a CASA system. Velocity parameters were similar among groups whereas linearity was higher in mice treated with the commercial probiotic. These results suggest that the administration of probiotics may increase the proportion of sperm with normal morphology and lead to modifications in sperm head shape that may enhance sperm swimming. Studies using a longer administration period would be useful in further characterizing the effect of these probiotic mixtures on sperm quality and fertilization capacity.