Dataset Information

Cryopreservation Induces Alterations of miRNA and mRNA Fragment Profiles of Bull Sperm.

ABSTRACT: Although cryopreservation of bull semen is widely used commercially in the livestock breeding industry, cryopreservation results in low fertility of bull sperm. As an important regulatory factor, the alteration of small non-coding RNA (sncRNA) profile during cryopreservation of bull sperm is not yet completely known. In the present study, we sequenced sncRNAs of frozen and fresh sperm to study the link of alteration of the sncRNA profiles (particularly in miRNAs and mRNA fragments) with low sperm fertility caused by cryopreservation. We identified 55 miRNAs and 526 mRNA fragments differentially expressed (DE) between frozen and fresh sperm. Subsequently, the functional analysis revealed that targeted genes of DE miRNAs in sperm had roles in the fertilization, ATP, and apoptosis. Instead, targeted genes of DE miRNAs in cow metaphase II oocyte were significantly enriched in the MAPK signaling pathway, autophagy-animal pathway, and mitophagy-animal pathway. Additionally, biological processes of DNA repair, spermatid development, response to temperature stimulus, and cellular response to DNA damage stimulus were enriched by mRNA fragments. In conclusion, we found that DE miRNAs or DE mRNA fragments in cryopreservation may influence the fertility of sperm, these findings will provide the reference to improve the cryopreservation technology of bull semen.

SUBMITTER: Shangguan A

PROVIDER: S-EPMC7214931 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Cryopreservation Induces Alterations of miRNA and mRNA Fragment Profiles of Bull Sperm.

Shangguan Aishao A Zhou Hao H Sun Wei W Ding Rui R Li Xihe X Liu Jiajia J Zhou Yang Y Chen Xing X Ding Fengling F Yang Liguo L Zhang Shujun S

Frontiers in genetics 20200505

Although cryopreservation of bull semen is widely used commercially in the livestock breeding industry, cryopreservation results in low fertility of bull sperm. As an important regulatory factor, the alteration of small non-coding RNA (sncRNA) profile during cryopreservation of bull sperm is not yet completely known. In the present study, we sequenced sncRNAs of frozen and fresh sperm to study the link of alteration of the sncRNA profiles (particularly in miRNAs and mRNA fragments) with low sper ...[more]

PMID: 32431726

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Project description:The present study quantitatively characterized the proteomic changes in bull spermatozoa induced by the cryopreservation process. We performed high-throughput comparative global proteomic profiling of freshly ejaculated (before cryopreservation), equilibrated (refrigerated storage; during cryopreservation), and frozen (ultralow temperature; after cryopreservation) bull spermatozoa. Using the liquid chromatography–mass spectrometry (LC-MS/MS) technique, a total of 1,692, 1,415, and 1,286 proteins were identified in fresh, equilibrated, and cryopreserved spermatozoa, respectively. When the proteome of fresh spermatozoa was compared with equilibrated spermatozoa, we found that 166 proteins were differentially expressed. When equilibrated spermatozoa were compared with cryopreserved spermatozoa, we found that 147 proteins were differentially expressed between them. Similarly, we found that 156 proteins were differentially expressed between fresh and cryopreserved spermatozoa. Among these proteins, the abundance of 105 proteins was lowered during the equilibration process itself, while the abundance of 43 proteins was lowered during ultralow temperature preservation. Remarkably, the equilibration process lowered the abundance of sperm proteins involved in energy metabolism, structural integrity, and DNA repair and increased the abundance of proteins associated with proteolysis and protein degradation. The abundance of sperm proteins associated with metabolism, cGMP-PKG (cyclic guanosine 3′,5′-monophosphate-dependent protein kinase G) signaling, and regulation of the actin cytoskeleton was also altered during the equilibration process. Collectively, the present study showed that the equilibration step in the bull sperm cryopreservation process was the critical point for sperm proteome, during which a majority of proteomic alterations in sperm occurred. These findings are valuable for developing efficient protocols to minimize protein damage and to improve the quality and fertility of cryopreserved bull spermatozoa.

Project description:Although sperm preservation is a common means of personal fertility preservation, its effects on embryonic development potential need further investigation. The purpose of this study was to identify key microRNA (miRNA) in cryopreserved sperm and determine the changes of these miRNAs and their target genes during embryonic development using cryopreserved sperm. Moreover, the embryonic development potential of cryopreserved sperm was estimated in assisted reproductive technology (ART), where key miRNAs and target genes were validated in sperm and subsequent embryos. Clinical data of embryonic development from cryopreserved sperm indicated a significant decrease in fertilization rate in both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cases, as well as a reduction in blastocyst formation rate in ICSI cases. Meanwhile there was a significant increase in blocked embryo ratio of Day1, Day2, and Day3.5 embryos when frozen-thawed mouse sperm was used, compared with fresh mouse sperm, suggesting a potential negative effect of sperm cryopreservation on embryonic development. From frozen-thawed and fresh sperm in humans and mice, respectively, 21 and 95 differentially expressed miRNAs (DEmiRs) were detected. miR-148b-3p were downregulated in both human and mouse frozen-thawed sperm and were also decreased in embryos after fertilization using cryopreserved sperm. Target genes of miR-148b-3p, Pten, was identified in mouse embryos using quantitative real-time PCR (qRT-PCR) and Western blot (WB). In addition, common characters of cryopreservation of mouse oocytes compared with sperm were also detected; downregulation of miR-148b-3p was also confirmed in cryopreserved oocytes. In summary, our study suggested that cryopreservation of sperm could change the expression of miRNAs, especially the miR-148b-3p across humans and mice, and may further affect fertilization and embryo development by increasing the expression of Pten. Moreover, downregulation of miR-148b-3p induced by cryopreservation was conserved in mouse gametes.

Project description:In humans and model species, alterations of sperm DNA methylation patterns have been reported in cases of spermatogenesis defects, male infertility and exposure to toxins or nutritional challenges, suggesting that a memory of environmental or physiological changes is recorded in the sperm methylome. The objective of this study was to ascertain if early life plane of nutrition could have a latent effect on DNA methylation patterns in sperm produced post-puberty. Holstein-Friesian calves were assigned to either a high (H) or moderate (M) plane of nutrition for the first 24 weeks of age, then reassigned to the M diet until puberty, resulting in HM and MM groups. Sperm DNA methylation patterns from contrasted subgroups of bulls in the HM (ejaculates recovered at 15 months of age; n = 9) and in the MM (15 and 16 months of age; n = 7 and 9, respectively) were obtained using Reduced Representation Bisulfite Sequencing. Both 15 and 16 months were selected in the MM treatment as these bulls reached puberty approximately 1 month after the HM bulls. Hierarchical clustering demonstrated that inter-individual variability unrelated to diet or age dominated DNA methylation profiles. While the comparison between 15 and 16 months of age revealed almost no change, 580 differentially methylated CpGs (DMCs) were identified between the HM and MM groups. Differentially methylated CpGs were mostly hypermethylated in the HM group, and enriched in endogenous retrotransposons, introns, intergenic regions, and shores and shelves of CpG islands. Furthermore, genes involved in spermatogenesis, Sertoli cell function, and the hypothalamic-pituitary-gonadal axis were targeted by differential methylation when HM and MM groups were compared at 15 months of age, reflecting the earlier timing of puberty onset in the HM bulls. In contrast, the genes still differentially methylated in MM bulls at 16 months of age were enriched for ATP-binding molecular function, suggesting that changes to the sperm methylome could persist even after the HM and MM bulls reached a similar level of sexual maturity. Together, results demonstrate that enhanced plane of nutrition in pre-pubertal calves associated with advanced puberty induced modest but persistent changes in sperm DNA methylation profiles after puberty.

Project description:Semen cryopreservation results in the differential remodeling of the molecules presented in sperm, and these alterations related to reductions in sperm quality and its physiological function have not been fully understood. Given this, this study aimed to investigate the cryoinjury mechanism of goat sperm by analyzing changes of the metabolic characteristics in sperm during the cryopreservation process. The ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) technique was performed to explore metabolite profiles of fresh sperm (C group), equilibrated sperm (E group), and frozen-thawed sperm (F group). In total, 2570 metabolites in positive mode and 2306 metabolites in negative mode were identified, respectively. After comparative analyses among these three groups, 374 differentially abundant metabolites (DAMs) in C vs. E, 291 DAMs in C vs. F, and 189 DAMs in E vs. F were obtained in the positive mode; concurrently, 530 DAMs in C vs. E, 405 DAMs in C vs. F, and 193 DAMs in E vs. F were obtained in the negative mode, respectively. The DAMs were significantly enriched in various metabolic pathways, including 31 pathways in C vs. E, 25 pathways in C vs. F, and 28 pathways in E vs. F, respectively. Among them, 65 DAMs and 25 significantly enriched pathways across the three comparisons were discovered, which may be tightly associated with sperm characteristics and function. Particularly, the functional terms such as TCA cycle, biosynthesis of unsaturated fatty acids, sphingolipid metabolism, glycine, serine and threonine metabolism, alpha-linolenic acid metabolism, and pyruvate metabolism, as well as associated pivotal metabolites like ceramide, betaine, choline, fumaric acid, L-malic acid and L-lactic acid, were focused on. In conclusion, our research characterizes the composition of metabolites in goat sperm and their alterations induced by the cryopreservation process, offering a critical foundation for further exploring the molecular mechanisms of metabolism influencing the quality and freezing tolerance of goat sperm. Additionally, the impacts of equilibration at low temperature on sperm quality may need more attentions as compared to the freezing and thawing process.

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Cryopreservation Induces Alterations of miRNA and mRNA Fragment Profiles of Bull Sperm.

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Cryopreservation Induces Alterations of miRNA and mRNA Fragment Profiles of Bull Sperm.

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