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1.
PLoS Genet ; 19(1): e1010600, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36634107

RESUMO

In lepidopteran insects, dichotomous spermatogenesis produces eupyrene spermatozoa, which are nucleated, and apyrene spermatozoa, which are anucleated. Both sperm morphs are essential for fertilization, as eupyrene sperm fertilize the egg, and apyrene sperm is necessary for the migration of eupyrene sperm. In Drosophila, Prmt5 acts as a type II arginine methyltransferase that catalyzes the symmetrical dimethylation of arginine residues in the RNA helicase Vasa. Prmt5 is critical for the regulation of spermatogenesis, but Vasa is not. To date, functional genetic studies of spermatogenesis in the lepidopteran model Bombyx mori has been limited. In this study, we engineered mutations in BmPrmt5 and BmVasa through CRISPR/Cas9-based gene editing. Both BmPrmt5 and BmVasa loss-of-function mutants had similar male and female sterility phenotypes. Through immunofluorescence staining analysis, we found that the morphs of sperm from both BmPrmt5 and BmVasa mutants have severe defects, indicating essential roles for both BmPrmt5 and BmVasa in the regulation of spermatogenesis. Mass spectrometry results identified that R35, R54, and R56 of BmVasa were dimethylated in WT while unmethylated in BmPrmt5 mutants. RNA-seq analyses indicate that the defects in spermatogenesis in mutants resulted from reduced expression of the spermatogenesis-related genes, including BmSxl, implying that BmSxl acts downstream of BmPrmt5 and BmVasa to regulate apyrene sperm development. These findings indicate that BmPrmt5 and BmVasa constitute an integral regulatory module essential for spermatogenesis in B. mori.


Assuntos
Bombyx , Animais , Masculino , Feminino , Bombyx/genética , Sêmen , Espermatogênese/genética , Espermatozoides/metabolismo , Fertilização , Drosophila
2.
J Cell Biol ; 222(2)2023 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-36656648

RESUMO

The molecular mechanism of sperm-egg fusion is a long-standing mystery in reproduction. Brukman and colleagues (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202207147) now provide evidence that the sperm surface protein IZUMO1, which is essential for mammalian fertilization, can induce membrane fusion in cultured cells.


Assuntos
Fusão de Membrana , Proteínas de Membrana , Interações Espermatozoide-Óvulo , Animais , Masculino , Fertilização/genética , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Mamíferos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Sêmen/metabolismo , Interações Espermatozoide-Óvulo/genética , Espermatozoides/metabolismo , Células Cultivadas
3.
Sci Adv ; 9(4): eade7607, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36696506

RESUMO

Spermatozoa need to undergo an exocytotic event called the acrosome reaction before fusing with eggs. Although calcium ion (Ca2+) is essential for the acrosome reaction, its molecular mechanism remains unknown. Ferlin is a single transmembrane protein with multiple Ca2+-binding C2 domains, and there are six ferlins, dysferlin (DYSF), otoferlin (OTOF), myoferlin (MYOF), fer-1-like 4 (FER1L4), FER1L5, and FER1L6, in mammals. Dysf, Otof, and Myof knockout mice have been generated, and each knockout mouse line exhibited membrane fusion disorders such as muscular dystrophy in Dysf, deafness in Otof, and abnormal myogenesis in Myof. Here, by generating mutant mice of Fer1l4, Fer1l5, and Fer1l6, we found that only Fer1l5 is required for male fertility. Fer1l5 mutant spermatozoa could migrate in the female reproductive tract and reach eggs, but no acrosome reaction took place. Even a Ca2+ ionophore cannot induce the acrosome reaction in Fer1l5 mutant spermatozoa. These results suggest that FER1L5 is the missing link between Ca2+ and the acrosome reaction.


Assuntos
Proteínas Musculares , Testículo , Masculino , Feminino , Animais , Camundongos , Membrana Celular/metabolismo , Proteínas Musculares/metabolismo , Testículo/metabolismo , Fusão de Membrana , Fertilidade , Espermatozoides/metabolismo , Mamíferos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo
4.
Biol Res ; 56(1): 2, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36653814

RESUMO

BACKGROUND: The testes are highly susceptible to the adverse effects of chemotherapy and radiation at all stages of life. Exposure to these threats mainly occurs during cancer treatment and as an occupational hazard in radiation centers. The present study investigated the regenerative ability of adipose-derived mesenchymal stem cells (ADMSCs) against the adverse effects of cisplatin on the structure and function of the testes. METHODS: New Zealand white rabbits (N = 15) were divided into three groups of five: a negative control group (no treatment), a cisplatin group (single dose of cisplatin into each testis followed three days later by a PBS injection), and a cisplatin + ADMSCs group (cisplatin injection followed three days later by an ADMSC injection). On day 45 post-treatment, serum testosterone levels were evaluated, and the testes and epididymis were collected for histology, oxidative stress examination, and epididymal sperm analysis. RESULTS: Cisplatin caused damage to the testicular tissue and decreased serum testosterone levels, epididymal sperm counts, and oxidants. An antioxidant imbalance was detected due to increasing malondialdehyde (MDA) and reduced glutathione (GSH) levels in testicular tissue. The ADMSC-treated group displayed a moderate epididymal sperm count, adequate antioxidant protection, suitable hormone levels, and enhanced testicular tissue morphology. CONCLUSIONS: ADMSCs treatment repaired damaged testicular tissue, enhanced biochemical parameters, and modified pathological changes caused by cisplatin.


Assuntos
Azoospermia , Células-Tronco Mesenquimais , Masculino , Animais , Coelhos , Humanos , Testículo/metabolismo , Cisplatino/efeitos adversos , Antioxidantes/farmacologia , Azoospermia/induzido quimicamente , Azoospermia/metabolismo , Azoospermia/patologia , Sêmen , Espermatozoides/metabolismo , Espermatozoides/patologia , Estresse Oxidativo , Testosterona/farmacologia
5.
Oxid Med Cell Longev ; 2023: 4923614, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36686378

RESUMO

Background: Infertility is a global medical and social problem that affects human health and social development. At present, about 15% of couples of the right age in the world are infertile. As all we know, genetic defects are the most likely underlying cause of the pathology. ATP5D is also known as the delta subunit of mitochondrial ATP synthase. Mitochondria maintain sperm vitality, capacitation, acrosome reaction, and DNA integrity through ATP. Mitochondrial damage can trigger energy synthesis disorders, resulting in decreased sperm quality and function or even disappearance. The specific role of ATP5D in regulation of the male reproductive system remains elusive. Methods: In this study, semen from normal and infertile males were collected and their indicators were examined by analysis of routine sperm parameters; ATP5D protein content in semen was examined by ELISA. Singer sequencing was used to detect whether there was a mutated of ATP5D in semen. Meanwhile, ATP5D knockout (KO) and knockin (KI) male mice were selected at 8-12 weeks of age and mated with adult wild-type (WT) female mice for more than two months to assess their fertility and reproductive ability. Morphological changes in tissues such as testes and epididymis were observed by HE staining; spermatozoa were taken from the epididymis of the mice; sperm counts were performed and morphological changes were observed by Diff-Quik staining. Results: The results showed that the expression of ATP5D in infertile males was significantly lower than that in normal males (P < 0.001) and the normal morphology rate of spermatozoa was much lower than that of normal males, and the sequencing results showed no mutations. The animal reproductive experiments showed no significant changes in the number of fertility in KO/KI mice compared with WT mice, but the duration of fertility was significantly longer (P = 0.02). The testicular cells in KO mice were loosely arranged and disorganized, the lumen was larger, the interstitial cells were atrophied, and the number of spermatozoa was reduced and the malformation rate was higher in WT males. This suggests that ATP5D is an essential protein for sperm formation and fertility in male mice and may be used as a biomarker of male fertility. Conclusion: This study found ATP5D correlated with male infertility and the expression levels were significantly reduced in the seminal plasma of all male infertile patients without gene mutations. KO male significantly prolonged fertility time and impaired testicular histomorphology. This suggests that ATP5D may be associated with spermatogenic function and fertility in male mice and may be used as a biomarker for male fertility. Future studies are required to elucidate the potential mechanisms. The trial registration number is KLL-2021-266.


Assuntos
Infertilidade Masculina , Sêmen , Adulto , Humanos , Masculino , Feminino , Animais , Camundongos , Sêmen/metabolismo , Espermatozoides/metabolismo , Testículo , Infertilidade Masculina/diagnóstico , Fertilidade , Biomarcadores/metabolismo , Motilidade Espermática
6.
BMC Genomics ; 24(1): 8, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36624393

RESUMO

BACKGROUND: Exosomes are nanosized membranous vesicles secreted by various types of cells, which facilitate intercellular communication by transporting bioactive compounds. Exosomes are abundant in biological fluids including semen, and their protein composition and the potential of seminal plasma exosomes (SPEs) as fertility biomarkers were elucidated in humans, however, little information is available regarding buffalo (Bubalus bubalis). Here, we examined protein correlation between spermatozoa, seminal plasma (SP), and SPEs, and we compared and analyzed protein differences between high-motility (H-motility) and low-motility (L-motility) SPEs in buffalo. RESULTS: SPEs were concentrated and purified by ultracentrifugation combined with sucrose density gradient centrifugation, followed by verification using western blotting, nanoparticle tracking analysis, and transmission electron microscopy. Protein composition in spermatozoa, SP and SPEs, and protein difference in H- and L-motility SPEs were identified by LC-MS/MS proteomic analysis and were functionally analyzed through comprehensive bioinformatics. Many SPEs proteins originated from spermatozoa and SP, and nearly one third were also present in spermatozoa and SP. A series of proteins associated with reproductive processes including sperm capacitation, spermatid differentiation, fertilization, sperm-egg recognition, membrane fusion, and acrosome reaction were integrated in a functional network. Comparative proteomic analyses showed 119 down-regulated and 41 up-regulated proteins in L-motility SPEs, compared with H-motility SPEs. Gene Ontology (GO) enrichment of differentially expressed proteins (DEPs) showed that most differential proteins were located in sperm and vesicles, with activities of hydrolase and metalloproteinase, and were involved in sperm-egg recognition, fertilization, single fertilization, and sperm-zona pellucida binding processes, etc. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that differential proteins were mainly involved in the PPRP signaling pathway, calcium signaling pathway, and cAMP signaling pathway, among others. Furthermore, 6 proteins associated with reproduction were validated by parallel reaction monitoring analysis. CONCLUSION: This study provides a comprehensive description of the seminal plasma exosome proteome and may be of use for further screening of biomarkers associated with male infertility.


Assuntos
Exossomos , Sêmen , Animais , Masculino , Humanos , Sêmen/metabolismo , Búfalos , Motilidade Espermática , Cromatografia Líquida , Exossomos/metabolismo , Proteômica , Espectrometria de Massas em Tandem , Espermatozoides/metabolismo , Proteoma/metabolismo
7.
Int J Mol Sci ; 24(2)2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36674454

RESUMO

Despite its importance in somatic cells and during spermatogenesis, little is known about the role that autophagy may play in ejaculated spermatozoa. Our aim was to investigate whether the molecular components of autophagy, such as microtubule-associated protein 1 light chain 3 (LC3), are activated in stallion spermatozoa during the capacitation and acrosome reaction and if this activation could modulate these biological processes. To analyze the autophagy turnover, LC3I and LC3II proteins were assessed by western blotting, and the ratio between both proteins (LC3II/LC3I) was calculated. In somatic cells, this ratio indicates that autophagy has been activated and similar LC3 processing has been described in mammalian spermatozoa. The subcellular localization of autophagy-related proteins was assessed by immunofluorescence with specific antibodies that recognized Atg16, Beclin-1, and LC3. The colocalization of acrosomal membranes (PNA) and LC3 was studied by confocal microcopy, and the acrosome reacted cells were quantified by flow cytometry. The incubation of stallion sperm in capacitating conditions (BWW; 3 h) significantly increased LC3 processing. This increment was three to four times higher after the induction of the acrosome reaction in these cells. LC3 was mainly expressed in the head in mature ejaculated sperm showing a clear redistribution from the post-acrosomal region to the acrosome upon the incubation of sperm in capacitating conditions (BWW, 3 h). After the induction of the acrosome reaction, LC3 colocalized with the acrosome or the apical plasmalemma membranes in the head of the stallion spermatozoa. The inhibition or activation of autophagy-related pathways in the presence of autophagy activators (STF-62247) or inhibitors (E-64d, chloroquine) significantly increased LC3 processing and increased the percent of acrosome reacted cells, whereas 3-methyladenine almost completely inhibited LC3 processing and the acrosome reaction. In conclusion, we found that sperm capacitation and acrosome reaction could be regulated by autophagy components in sperm cells ex vivo by processes that might be independent of the intraluminal pH of the acrosome and dependent of LC3 lipidation. It can be speculated that, in stallion sperm, a form of noncanonical autophagy utilizes some components of autophagy machinery to facilitate the acrosome reaction.


Assuntos
Reação Acrossômica , Acrossomo , Masculino , Cavalos , Animais , Acrossomo/fisiologia , Reação Acrossômica/fisiologia , Capacitação Espermática/fisiologia , Sêmen , Espermatozoides/metabolismo , Autofagia , Mamíferos
8.
Int J Mol Sci ; 24(2)2023 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-36675176

RESUMO

Heparin, a class of glycosaminoglycans (GAGs), is widely used to induce sperm capacitation and fertilization. How heparin induces sperm capacitation remains unclear. Olfactory receptors (ORs) which are G protein-coupled receptors, have been proposed to be involved in sperm capacitation. However, the interaction between ORs and odor molecules and the molecular mechanism of ORs mediating sperm capacitation are still unclear. The present study aimed to explore the underlying interaction and mechanism between heparin and ORs in carrying out the boar sperm capacitation. The results showed that olfactory receptor 2C1 (OR2C1) is a compulsory unit which regulates the sperm capacitation by recognizing and binding with heparin, as determined by Dual-Glo Luciferase Assay and molecular docking. In addition, molecular dynamics (MD) simulation indicated that OR2C1 binds with heparin via a hydrophobic cavity comprises of Arg3, Ala6, Thr7, Asn171, Arg172, Arg173, and Pro287. Furthermore, we demonstrated that knocking down OR2C1 significantly inhibits sperm capacitation. In conclusion, we highlighted a novel olfactory receptor, OR2C1, in boar sperm and disclosed the potential binding of heparin to Pro287, a conserved residue in the transmembrane helices region 7 (TMH7). Our findings will benefit the further understanding of ORs involved in sperm capacitation and fertilization.


Assuntos
Heparina , Receptores Odorantes , Suínos , Masculino , Animais , Heparina/farmacologia , Heparina/metabolismo , Capacitação Espermática , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Sêmen/metabolismo , Simulação de Acoplamento Molecular , Espermatozoides/metabolismo
9.
Int J Mol Sci ; 24(2)2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36675287

RESUMO

In dairy goat farming, increasing the female kid rate is beneficial to milk production and is, therefore, economically beneficial to farms. Our previous study demonstrated that alkaline incubation enriched the concentration of X-chromosome-bearing sperm; however, the mechanism by which pH affects the motility of X-chromosome-bearing sperm remains unclear. In this study, we explored this mechanism by incubating dairy goat sperm in alkaline dilutions, examining the pattern of changes in sperm internal pH and Ca2+ concentrations and investigating the role of the sAC/cAMP/PKA pathway in influencing sperm motility. The results showed that adding a calcium channel inhibitor during incubation resulted in a concentration-dependent decrease in the proportion of spermatozoa with forward motility, and the sperm sAC protein activity was positively correlated with the calcium ion concentration (r = 0.9972). The total motility activity, proportion of forward motility, and proportion of X-chromosome-bearing sperm decreased (p < 0.05) when cAMP/PKA protease activity was inhibited. Meanwhile, the enrichment of X-chromosome-bearing sperm by pH did not affect the sperm capacitation state. These results indicate that alkaline dilution incubation reduces Ca2+ entry into X-sperm and the motility was slowed down through the sAC/cAMP/PKA signaling pathway, providing a theoretical foundation for further optimization of the sex control method.


Assuntos
Sêmen , Motilidade Espermática , Masculino , Feminino , Animais , Espermatozoides/metabolismo , Transdução de Sinais , Cabras
10.
PLoS One ; 18(1): e0273888, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36598915

RESUMO

Methamphetamine (METH) can induce spermatogenesis impairment, testicular apoptosis, and abnormal sperm quality. It also promotes changes in the expression of receptors for sex hormones and neurotransmitters, including GABA receptors in the testis. Proteomic assessment focusing on proteins involved in the calcium signalling pathway in the testis can facilitate diagnostic factors contributing to testicular and sperm functions, especially those related to spermatogenesis and fertilisation. In this study, we proposed to determine the localisation and differential expression of GABA A receptor alpha 1 subunit (GABA A-α1) in the spermatozoa of METH-administered rats. The differential proteomic profile of the testis was also observed by focusing on proteins in the KEGG pathways belonging to the calcium signalling pathway. There were 212 differentially expressed proteins in the rat testis, based on the cut-off value of 1.2-fold change. Most of those proteins, 13 proteins, were classified in the calcium signalling pathway, including 4 down-regulated and 9 up-regulated proteins. An immunolocalisation study of the GABA A-α1 receptor and calbindin revealed their localisation in the equatorial segment of the head in the rat spermatozoa. The expression of calbindin is also found in the middle piece of sperm. An increase in GABA A-α1 receptor in rat spermatozoa was correlated with an increase in abnormal sperm motility and morphology after methamphetamine exposure. Moreover, calbindin expression in sperm decreased in METH-administered rats. All our findings demonstrate that METH influences intracellular calcium homeostasis by acting through the calcium signalling pathway-associated proteins. Moreover, it might disrupt ion homeostasis in sperm through the GABA A-α1 receptor and calbindin, triggering a change in intracellular calcium and chloride ions. These changes may cause abnormalities in spermatogenesis, testicular apoptosis, and sperm quality impairment.


Assuntos
Metanfetamina , Testículo , Masculino , Ratos , Animais , Testículo/metabolismo , Receptores de GABA-A/metabolismo , Metanfetamina/farmacologia , Proteômica , Cálcio/metabolismo , Motilidade Espermática , Sêmen/metabolismo , Espermatozoides/metabolismo , Espermatogênese/fisiologia , Ácido gama-Aminobutírico/metabolismo
11.
Development ; 150(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36633190

RESUMO

Many animals achieve sperm chromatin compaction and stabilisation by replacing canonical histones with sperm nuclear basic proteins (SNBPs) such as protamines during spermatogenesis. Hydrozoan cnidarians and echinoid sea urchins lack protamines and have evolved a distinctive family of sperm-specific histone H2Bs (spH2Bs) with extended N termini rich in SPK(K/R) motifs. Echinoid sperm packaging is regulated by spH2Bs. Their sperm is negatively buoyant and fertilises on the sea floor. Hydroid cnidarians undertake broadcast spawning but their sperm properties are poorly characterised. We show that Hydractinia echinata and H. symbiolongicarpus sperm chromatin possesses higher stability than somatic chromatin, with reduced accessibility to transposase Tn5 integration and to endonucleases in vitro. In contrast, nuclear dimensions are only moderately reduced in mature Hydractinia sperm. Ectopic expression of spH2B in the background of H2B.1 knockdown results in downregulation of global transcription and cell cycle arrest in embryos, without altering their nuclear density. Taken together, SPKK-containing spH2B variants act to stabilise chromatin and silence transcription in Hydractinia sperm with only limited chromatin compaction. We suggest that spH2Bs could contribute to sperm buoyancy as a reproductive adaptation.


Assuntos
Histonas , Hidrozoários , Animais , Masculino , Histonas/metabolismo , Cromatina/metabolismo , Hidrozoários/genética , Sêmen/metabolismo , Espermatozoides/metabolismo , Protaminas/metabolismo
12.
Clin Epigenetics ; 15(1): 5, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36611168

RESUMO

BACKGROUND: Combination chemotherapy has contributed to increased survival from Hodgkin disease (HD) and testicular cancer (TC). However, questions concerning the quality of spermatozoa after treatment have arisen. While studies have shown evidence of DNA damage and aneuploidy in spermatozoa years following anticancer treatment, the sperm epigenome has received little attention. Our objectives here were to determine the impact of HD and TC, as well as their treatments, on sperm DNA methylation. Semen samples were collected from community controls (CC) and from men undergoing treatment for HD or TC, both before initiation of chemotherapy and at multiple times post-treatment. Sperm DNA methylation was assessed using genome-wide and locus-specific approaches. RESULTS: Imprinted gene methylation was not affected in the sperm of HD or TC men, before or after treatment. Prior to treatment, using Illumina HumanMethylation450 BeadChip (450 K) arrays, a subset of 500 probes was able to distinguish sperm samples from TC, HD and CC subjects; differences between groups persisted post-treatment. Comparing altered sperm methylation between HD or TC patients versus CC men, twice as many sites were affected in TC versus HD men; for both groups, the most affected CpGs were hypomethylated. For TC patients, the promoter region of GDF2 contained the largest region of differential methylation. To assess alterations in DNA methylation over time/post-chemotherapy, serial samples from individual patients were compared. With restriction landmark genome scanning and 450 K array analyses, some patients who underwent chemotherapy showed increased alterations in DNA methylation, up to 2 to 3 years post-treatment, when compared to the CC cohort. Similarly, a higher-resolution human sperm-specific assay that includes assessment of environmentally sensitive regions, or "dynamic sites," also demonstrated persistently altered sperm DNA methylation in cancer patients post-treatment and suggested preferential susceptibility of "dynamic" CpG sites. CONCLUSIONS: Distinct sperm DNA methylation signatures were present pre-treatment in men with HD and TC and may help explain increases in birth defects reported in recent clinical studies. Epigenetic defects in spermatozoa of some cancer survivors were evident even up to 2 years post-treatment. Abnormalities in the sperm epigenome both pre- and post-chemotherapy may contribute to detrimental effects on future reproductive health.


Assuntos
Doença de Hodgkin , Neoplasias Testiculares , Humanos , Masculino , Epigenoma , Sêmen , Metilação de DNA , Neoplasias Testiculares/tratamento farmacológico , Neoplasias Testiculares/genética , Doença de Hodgkin/tratamento farmacológico , Doença de Hodgkin/genética , Doença de Hodgkin/metabolismo , Espermatozoides/metabolismo
13.
J Proteomics ; 273: 104791, 2023 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-36538967

RESUMO

Cryopreservation may reduce sperm fertility due to cryodamage including physical-chemical and oxidative stress damages. As a powerful antioxidant, melatonin has been reported to improve cryoprotective effect of sperm. However, the molecular mechanism of melatonin on cryopreserved ram sperm hasn't been fully understand. Give this, this study aimed to investigate the postthaw motility parameters, antioxidative enzyme activities and lipid peroxidation, as well as proteomic, metabolomic changes of Huang-huai ram spermatozoa with freezing medium supplemented with melatonin. Melatonin was firstly replenished to the medium to yield five different final concentrations: 0.1, 0.5, 1.0, 1.5, and 2.0 mM. A control (NC) group without melatonin replenishment was included. Protective effects of melatonin as evidenced by postthaw motility, activities of T-AOC, T-SOD, GSH-Px, CAT, contents of MDA, 4-HNE, as well as acrosome integrity, plasma membrane integrity, with 0.5 mM being the most effective concentration (MC group). Furthermore, 29 differentially abundant proteins involving in sperm functions were screened among Fresh, NC and MC groups of samples (n = 5) based on the 4D-LFQ, with 7 of them upregulated in Fresh and MC groups. 26 differentially abundant metabolites were obtained involving in sperm metabolism among the three groups of samples (n = 8) based on the UHPLC-QE-MS, with 18 of them upregulated in Fresh and MC groups. According to the bioinformatic analysis, melatonin may have positive effects on frozen ram spermatozoa by regulating the abundance changes of vital proteins and metabolites related to sperm function. Particularly, several proteins such as PRCP, NDUFB8, NDUFB9, SDHC, DCTN1, TUBB6, TUBA3E, SSNA1, as well as metabolites like L-histidine, L-targinine, ursolic acid, xanthine may be potential novel biomarkers for evaluating the postthaw quality of ram spermatozoa. In conclusion, a dose-dependent replenishment of melatonin to freezing medium protected ram spermatozoa during cryopreservation, which can improve motility, antioxidant enzyme activities, reduce levels of lipid peroxidation products, modify the proteomic and metabolomic profiling of cryopreserved ram spermatozoa through reduction of oxidative stress, maintenance of OXPHOS and microtubule structure. SIGNIFICANCE: Melatonin, a powerful antioxidant protects ram spermatozoa from cryopreservation injuries in a dose-dependent manner, with 0.5 mM being the most effective concentration. Furthermore, sequencing results based on the 4D-LFQ combined with the UHPLC-QE-MS indicated that melatonin modifies proteomic and metabolomic profiling of ram sperm during cryopreservation. According to the bioinformatic analysis, melatonin may have positive effects on frozen ram spermatozoa by regulating the expression changes of vital proteins and metabolites related to sperm metabolism and function. Particularly, several potential novel biomarkers for evaluating the postthaw quality of ram spermatozoa were acquired, proteins such as PRCP, NDUFB8, NDUFB9, SDHC, DCTN1, TUBB6, TUBA3E, SSNA1, as well as metabolites like L-histidine, L-targinine, ursolic acid, xanthine.


Assuntos
Melatonina , Preservação do Sêmen , Animais , Masculino , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Criopreservação/métodos , Histidina/metabolismo , Histidina/farmacologia , Melatonina/farmacologia , Melatonina/metabolismo , Proteômica , Sêmen , Preservação do Sêmen/métodos , Ovinos , Motilidade Espermática , Espermatozoides/metabolismo , Xantinas/metabolismo , Xantinas/farmacologia , Metabolômica
14.
Gen Comp Endocrinol ; 330: 114148, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36272447

RESUMO

BACKGROUND: While many testis-enriched genes have been identified as important regulators of the spermatogenic process, the specific roles played by several of these genes and their functional importance has yet to be fully clarified. METHODS: We employed a CRISPR/Cas9 approach to introduce a 5 bp in-frame deletion within the Spdye4a gene (Exon 2) of C57BL/6 mice (Spdye4a-/-). Fertility and sperm counts were evaluated. Testes tissues and cell suspensions were analyzed via histological and immunofluorescence staining. mRNA and protein levels of candidate genes were assessed through qPCR and Western blotting. In vitro fertilization was used to assess the ability of sperm cells to bind to egg cells. RESULTS: Spdye4a-/- mice did not exhibit any reduction in fertility, and exhibited comparable sperm counts, morphology and motility to those of wildtype littermates. Functionally, Spdye4a-/- sperm exhibited normal sperm-egg binding activity in vitro. Furthermore, the testes of Spdye4a-/- mice exhibited a full range of germ cells from spermatogonia to mature spermatozoa. No differences in the progression of meiotic prophase I were observed when comparing Spdye4a-/- and wildtype mice, indicating that the loss of Spdye4a had no adverse effect on spermatogenesis. DISCUSSION: Spdye4a is dispensable in the context of mice fertility and spermatogenesis. This study will prevent other laboratories from expending repeated efforts to generate similar knockout mice.


Assuntos
Meiose , Testículo , Masculino , Camundongos , Animais , Testículo/metabolismo , Camundongos Endogâmicos C57BL , Sêmen , Espermatogênese/genética , Espermatozoides/metabolismo , Fertilidade/genética , Espermatogônias , Camundongos Knockout
15.
Biomater Adv ; 145: 213242, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36549152

RESUMO

The swimming forces exerted by mammalian spermatozoa during the pathway to the ovary and during the interaction with the oocyte are thought to play a fundamental role in the fertilization of the egg. In particular, a process named capacitation is of key relevance for its success. Capacitation enables spermatozoa to undergo the acrosome reaction and to exhibit different motility called hyperactivation with a change in the sperm cell tail motion from symmetric to a more asymmetric beating, characterized by wider flagellar bending at lower frequencies. Despite several studies about the mechanism that underlies capacitation, no quantitative information is available about the forces associated with sperm motility. Sperm cell motility has been widely studied with digital imaging tools and video microscopy, but these methodologies cannot provide information about the forces exerted by spermatozoa during the motion and the contribution of every single frequency of flagellar beating to the sperm cell movement. For this purpose, fluidic force microscopy was used to trap single swimming spermatozoa allowing to evaluate these parameters. We observe significant differences between capacitated and non-capacitated spermatozoa in terms of force exerted and beating frequencies. The description of the dynamics of this process is of great interest in the field of reproductive medicine. Such information could be useful to clarify unknown causes of male infertility or for the development of novel methods to assess the quality of semen samples.


Assuntos
Sêmen , Capacitação Espermática , Animais , Feminino , Masculino , Capacitação Espermática/fisiologia , Motilidade Espermática/fisiologia , Espermatozoides/metabolismo , Cauda do Espermatozoide/fisiologia , Corantes/metabolismo , Mamíferos
16.
Life Sci ; 313: 121306, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36543282

RESUMO

AIMS: Female sub-fertility, a prominent complication due to Type 1 diabetes (T1D), is generally attributed to disturbances in menstrual cycles and/or ovarian defects/disorders. T1D women, however, are high in oxidative stress, although the impact of the same on their reproduction and associated events remains unknown. Therefore, we assessed the repercussions of elevated oxidative stress on the sperm fate (storage/utilization) in the reproductive tract milieu of T1D females and their fertility using the Drosophila T1D model (Df[dilp1-5]), which lacks insulin-like peptides and displays reduced female fertility. MAIN METHODS: We mated Df[dilp1-5] females to normal males and thereafter examined sperm storage and/or utilization in conjunction with oxidative stress parameters in mated Df[dilp1-5] females at different time points. Also, the impact of antioxidant (Amla or Vitamin C) supplementation on the above oxidative stress parameters in Df[dilp1-5] females and the consequences on their sperm and fertility levels were examined. KEY FINDINGS: Df[dilp1-5] females showed elevated oxidative stress parameters and a few of their reproductive tract proteins are oxidatively modified. Also, these females stored significantly fewer sperm and also did not utilize sperm as efficiently as their controls. Surprisingly, amelioration of the oxidative stress in Df[dilp1-5] females' milieu through antioxidant (Amla or vitamin C) supplementation enhanced sperm storage and improved fertility. SIGNIFICANCE: Hyperglycemia coupled with elevated oxidative stress within the female reproductive tract environment affects the sperm fate, thereby reducing female fertility in T1D. In addition, these findings suggest that antioxidant supplementation may substantially aid in the mitigation of sub-fertility in T1D females.


Assuntos
Diabetes Mellitus Tipo 1 , Infertilidade , Animais , Feminino , Masculino , Drosophila/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Sêmen/metabolismo , Drosophila melanogaster/metabolismo , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Espermatozoides/metabolismo , Estresse Oxidativo , Ácido Ascórbico/farmacologia
17.
Reproduction ; 165(3): R91-R102, 2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36538648

RESUMO

In brief: Mouse PIWI-interacting RNAs (piRNAs) are indispensable for spermatogenesis, but whether these small RNAs serve any function beyond gametogenesis is rarely explored. This review summarizes recent findings that demonstrated a requirement for piRNAs in sperm maturation and discusses a potential intergenerational role for paternal piRNAs. Abstract: Unique to animals, PIWI-interacting RNAs (piRNAs) defend organisms against threats to germline integrity evoked by transposons, retroviruses, and inappropriate expression of protein-coding genes. Characterization of mouse piRNAs and studies of more than a dozen piRNA pathway protein mutants detailed in the past 15 years have firmly established an essential role for piRNAs in male fertility. Despite their vital function in spermatogenesis, mammalian piRNAs were thought to be dispensable beyond gamete formation because all piRNA pathway protein mouse mutants are invariably sterile and do not produce sperm. In contrast to the specialized purpose of piRNAs in gamete formation, tRNA-derived fragments and microRNAs have been the focus of research in RNA-mediated paternal contribution, providing additional examples of the versatility of non-coding RNAs. In recent years, the direct elimination of mouse piRNAs using CRISPR/Cas revealed their extended function in post-testicular sperm maturation. An intergenerational contribution from paternal piRNAs has also been proposed. Together with insights into piRNAs in oocytes and early embryos in mice and other mammals, these newly proposed functions of mammalian piRNAs invite further investigations of piRNA dynamics during sperm maturation and fertilization as well as their roles in reproduction beyond gametogenesis.


Assuntos
Sêmen , Masculino , Animais , Camundongos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Sêmen/metabolismo , Espermatozoides/metabolismo , Espermatogênese/genética , Mamíferos/genética
18.
J Assist Reprod Genet ; 40(1): 41-51, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36515799

RESUMO

PURPOSE: The aim of this study is to identify the genetic cause of primary ciliary dyskinesia (PCD) and male infertility in two unrelated Han Chinese families. METHODS: We performed whole-exome sequencing in two unrelated male Han Chinese patients suffering from infertility and PCD to identify the pathogenic variants. Ultrastructural and immunostaining analyses of patient's spermatozoa were performed to characterize the effect of the variants. The pathogenicity of the variants was validated using patient's spermatozoa by western blotting and immunostaining analysis. Intracytoplasmic sperm injection (ICSI) was conducted in the affected families. RESULTS: Three variants in leucine-rich repeat containing 6 (LRRC6) [patient 1(compound heterozygote): NM_012472: c.538C > T, (p.R180*) and c.64dupT, (p.S22Ffs*19); patient 2 (homozygote): c.863C > A, (p.P288H)] were identified in two unrelated patients with PCD and male infertility. These variants were predicated deleterious and were absent or rare in human population genome data. LRRC6-mutant spermatozoa showed a highly aberrant morphology and ultrastructure with lacked inner and outer dynein arms. The LRRC6 protein was present along the normal sperm flagella, and was significantly decreased in the mutated spermatozoa. Interestingly, both patients were able to conceive through ICSI and birthed a healthy baby. CONCLUSION: Our results extend the LRRC6 variant spectrum and provide reproductive guidance to families suffering from PCD-linked infertility caused by LRRC6 variants.


Assuntos
Transtornos da Motilidade Ciliar , Infertilidade Masculina , Humanos , Masculino , Proteínas/genética , Mutação/genética , Sêmen/metabolismo , Infertilidade Masculina/genética , Espermatozoides/metabolismo , Transtornos da Motilidade Ciliar/genética , Proteínas do Citoesqueleto/genética
19.
J Proteomics ; 273: 104793, 2023 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-36535622

RESUMO

The freezability difference between donkey ejaculates is a limiting factor of sperm cryopreservation. Our recent study shows that the freezability of donkey semen is related to the seminal plasma proteome. In this study, we aimed to identify the different abundance sperm proteins in good freezability ejaculates (GFEs) and poor freezability ejaculates (PFEs) using a Tandem Mass Tag (TMT) peptide labeling coupled with LC-MS/MS approach. A total of 2682 proteins were identified, among which 58 were significantly up-regulated in GFEs and 16 were down-regulated compared with PFEs. Bioinformatic analysis results revealed that the majority of different abundance proteins (DAPs) participated in copper and calcium binding, regulation of RNA biosynthetic process, positive regulation of innate immune response, and negative regulation of programmed cell death. KEGG pathway enrichment analysis showed the up-regulated proteins in GF group were mainly involved in N-Glycan biosynthesis and protein processing in endoplasmic reticulum. Our study was the first to analyze the proteome of sperm from donkey ejaculates with different freezabilities. The identified candidate proteins might be used to explore the molecular mechanism related to donkey sperm cryotolerance and might improve the screening of jacks with good sperm freezability. SIGNIFICANCE: Cryopreserved semen has been widely used in assisted reproductive technology. However, semen cryopreservation is a damaging process, which can cause oxidative stress, reduce sperm motility and motility. There are differences in sperm freezability reported to exist between or within breeds, and even between fractions coming from the same ejaculate. The freezability difference between donkey ejaculates is a limiting factor of sperm cryopreservation. The mechanisms that affect the freezing difference in sperm quality remain to be investigated, and freezability differences was found to be related to protein composition of spermatozoa. Some protein markers that can indicate good freezability or poor freezability semen have been identified in mammals. Until now, there is no information about the relationship between donkey spermatozoa proteome and freezability. Additional novel biomarkers of semen freezability in donkey spermatozoa are also needed. The identified candidate proteins might be used to explore the molecular mechanism related to donkey sperm cryotolerance and might improve the screening of jacks with good sperm freezability.


Assuntos
Equidae , Preservação do Sêmen , Animais , Masculino , Proteoma/metabolismo , Proteômica , Cromatografia Líquida , Motilidade Espermática , Espectrometria de Massas em Tandem , Espermatozoides/metabolismo , Criopreservação/métodos , Preservação do Sêmen/métodos
20.
J Proteomics ; 273: 104794, 2023 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-36535621

RESUMO

Cattle breeding approaches are an evolving field of research in veterinary science. Certain factors such as Ejaculate Rejection Rate (ERR) pose a limitation to such approaches. In this regard, we sought to investigate the spermatozoa and seminal plasma proteome of Hallikar bulls with low (n = 3) and high (n = 3) ERR. Through the Tandem mass spectrometry approach, we identified a total of 2409 proteins, in which 828 proteins were common in both the semen components, whereas 375 and 378 proteins were unique to spermatozoa and seminal plasma respectively. Tandem mass tags (TMT) based protein quantification resulted in 75 spermatozoal, and 42 seminal plasma proteins being differentially regulated between high and low ERR bulls. Proteins such as SPADH2, TIMP-2, and PLA2G7 which are negative regulators of motility were upregulated in the seminal plasma of high ERR bulls. Proteins such as OAZ3, GPx4, and GSTM3 whose upregulation leads to reduced motility were upregulated in the spermatozoa of high ERR bulls. Caltrin and ADM proteins that enhance sperm motility were downregulated in the seminal plasma of high ERR bulls. The regulation of ACE, a negative regulator of sperm motility was upregulated in both the spermatozoa and seminal plasma of high ERR bulls. SIGNIFICANCE: The saying "Bull is more than half of the herd" signifies the importance of bull in the genetic improvement of the herd. Traditionally used semen quality tests will provide limited information about the potential fertility of bulls. The proteomics approach is a promising omics technology to understand the factors involved in male fertility. The present study identified the spermatozoal and seminal plasma proteins that are differentially regulated between high and low ERR bulls. Sperm motility-associated proteins are differentially regulated. This study if improved further, can be used to develop markers associated with semen quality which is useful for the selection of bulls.


Assuntos
Análise do Sêmen , Sêmen , Bovinos , Masculino , Animais , Sêmen/química , Análise do Sêmen/métodos , Proteômica , Motilidade Espermática , Espermatozoides/metabolismo , Proteínas de Plasma Seminal/análise
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