scholarly journals Protamine-2 Deficiency Initiates a Reactive Oxygen Species (ROS)-Mediated Destruction Cascade during Epididymal Sperm Maturation in Mice

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1789
Author(s):  
Simon Schneider ◽  
Farhad Shakeri ◽  
Christian Trötschel ◽  
Lena Arévalo ◽  
Alexander Kruse ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Blastocyst Stage ◽  
Sperm Maturation ◽  
Preimplantation Embryos ◽  
Dna Integrity ◽  
Label Free ◽  
Epididymal Sperm ◽  
Treatment Regimens ◽  
Protamine 2

Protamines are the safeguards of the paternal sperm genome. They replace most of the histones during spermiogenesis, resulting in DNA hypercondensation, thereby protecting its genome from environmental noxa. Impaired protamination has been linked to male infertility in mice and humans in many studies. Apart from impaired DNA integrity, protamine-deficient human and murine sperm show multiple secondary effects, including decreased motility and aberrant head morphology. In this study, we use a Protamine-2 (Prm2)-deficient mouse model in combination with label-free quantitative proteomics to decipher the underlying molecular processes of these effects. We show that loss of the sperm’s antioxidant capacity, indicated by downregulation of key proteins like Superoxide dismutase type 1 (SOD1) and Peroxiredoxin 5 (PRDX5), ultimately initiates an oxidative stress-mediated destruction cascade during epididymal sperm maturation. This is confirmed by an increased level of 8-OHdG in epididymal sperm, a biomarker for oxidative stress-mediated DNA damage. Prm2-deficient testicular sperm are not affected and initiate the proper development of blastocyst stage preimplantation embryos in vitro upon intracytoplasmic sperm injection (ICSI) into oocytes. Our results provide new insight into the role of Prm2 and its downstream molecular effects on sperm function and present an important contribution to the investigation of new treatment regimens for infertile men with impaired protamination.

scholarly journals Prm2 deficiency triggers a Reactive Oxygen Species (ROS)-mediated destruction cascade during epididymal sperm maturation in mice

2020 ◽  
Author(s):  
Simon Schneider ◽  
Farhad Shakeri ◽  
Christian Trötschel ◽  
Lena Arévalo ◽  
Alexander Kruse ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Male Infertility ◽  
Molecular Mechanisms ◽  
Sperm Maturation ◽  
Preimplantation Embryos ◽  
Sperm Function ◽  
Epididymal Sperm ◽  
Small Proteins ◽  
Infertile Men ◽  
New Treatment

AbstractProtamines are the safeguards of the paternal sperm genome. They replace most of the histones during spermiogenesis, resulting in DNA hypercondensation, thereby protecting its genome from environmental noxa. Impaired protamination has been linked to male infertility in mice and humans in many studies. Apart from impaired DNA integrity, protamine-deficient human and murine sperm show multiple secondary effects, including decreased motility and aberrant head morphology. In this study, we use a Prm2-deficient mouse model in combination with label-free quantitative proteomics to decipher the underlying molecular processes of these effects. We show that loss of the sperm’s antioxidant capacity, indicated by downregulation of key proteins like SOD1 and PRDX5, ultimately initiates an oxidative stress-mediated destruction cascade during epididymal sperm maturation. This is confirmed by an increased level of 8-OHdG in epididymal sperm, a biomarker for oxidative stress-mediated DNA damage. Prm2-deficient testicular sperm are not affected and initiate the proper development of blastocyst stage preimplantation embryos in vitro upon intracytoplasmic sperm injection (ICSI) into oocytes. Our results provide new insight into the role of Prm2 and its downstream molecular effects on sperm function and present an important contribution to the investigation of new treatment regimens for infertile men with impaired protamination.Significance statementSexual reproduction requires the successful fertilization of female eggs by male sperm. The generation of functional sperm is a complex, multi-step differentiation process known as spermatogenesis that takes places in the male testis. One important step for physiological sperm function is the incorporation of small proteins, known as protamines into the DNA. Defects within this process are common causes of male infertility. However, the underlying molecular mechanisms still remain largely unknown, thus preventing targeted therapies. Here, we identify the molecular cascade being initiated in protamine-deficient murine sperm that ultimately impedes fertilization. Our findings have broad implications for the development of new treatment options for infertile men with faulty protamination that seek medical advice.

180 IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY REPRESENTED TRANSCRIPTS IN PREIMPLANTATION BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 169 ◽  
Author(s):  
C. E. McHughes ◽  
G. K. Springer ◽  
L. D. Spate ◽  
R. Li ◽  
R. J. Woods ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Nuclear Transfer ◽  
Developmental Stages ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Production Techniques

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental stages

The protective effect of melatonin on the in vitro development of yak embryos against hydrogen peroxide-induced oxidative injury

Zygote ◽  
2019 ◽  
Vol 27 (3) ◽  
pp. 118-125 ◽  
Author(s):  
Wei Peng ◽  
Mengtong Lei ◽  
Jun Zhang ◽  
Yong Zhang
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Critical Role ◽  
Underlying Mechanism ◽  
Preimplantation Embryos ◽  
Effective Antioxidant ◽  
Vitro Development ◽  
Oxidation Effect

SummaryMelatonin plays a critical role in several types of cells as an antioxidant to protect intracellular molecules from oxidative stress. The anti-oxidation effect of melatonin in yak embryos is largely unknown. We report that melatonin can protect the development of yak preimplantation embryos against oxidative stress induced by hydrogen peroxide (H2O2). Therefore, the quality of blastocysts developed from zygotes exposed to H2O2 was promoted. In addition, we observed that melatonin reduced H2O2-induced intracellular reactive oxygen species (ROS) levels and prevented mitochondrial dysfunction in zygotes. These phenomena revealed the effective antioxidant activity of melatonin to prevent oxidative stress in yak embryos. To determine the underlying mechanism, we further demonstrated that melatonin protected preimplantation embryos from oxidative damage by preserving antioxidative enzymes. Collectively, these results confirmed the anti-oxidation effect of melatonin in yak embryos that significantly improved the quantity and quality of blastocysts in the in vitro production of embryos in yaks.

Dephosphorylation of protamine 2 at serine 56 is crucial for murine sperm maturation in vivo

2019 ◽  
Vol 12 (574) ◽  
pp. eaao7232 ◽  
Author(s):  
Katsuhiko Itoh ◽  
Gen Kondoh ◽  
Hitoshi Miyachi ◽  
Manabu Sugai ◽  
Yoshiyuki Kaneko ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
Sperm Maturation ◽  
Sperm Cells ◽  
Chromatin Binding ◽  
Wild Type ◽  
Underlying Mechanisms ◽  
Deficient Mice ◽  
Protamine 2

The posttranslational modification of histones is crucial in spermatogenesis, as in other tissues; however, during spermiogenesis, histones are replaced with protamines, which are critical for the tight packaging of the DNA in sperm cells. Protamines are also posttranslationally modified by phosphorylation and dephosphorylation, which prompted our investigation of the underlying mechanisms and biological consequences of their regulation. On the basis of a screen that implicated the heat shock protein Hspa4l in spermatogenesis, we generated mice deficient in Hspa4l (Hspa4l-null mice), which showed male infertility and the malformation of sperm heads. These phenotypes are similar to those of Ppp1cc-deficient mice, and we found that the amount of a testis- and sperm-specific isoform of the Ppp1cc phosphatase (Ppp1cc2) in the chromatin-binding fraction was substantially less in Hspa4l-null spermatozoa than that in those of wild-type mice. We further showed that Ppp1cc2 was a substrate of the chaperones Hsc70 and Hsp70 and that Hspa4l enhanced the release of Ppp1cc2 from these complexes, enabling the freed Ppp1cc2 to localize to chromatin. Pull-down and in vitro phosphatase assays suggested the dephosphorylation of protamine 2 at serine 56 (Prm2 Ser56) by Ppp1cc2. To confirm the biological importance of Prm2 Ser56 dephosphorylation, we mutated Ser56 to alanine in Prm2 (Prm2 S56A). Introduction of this mutation to Hspa4l-null mice (Hspa4l−/−; Prm2S56A/S56A) restored the malformation of sperm heads and the infertility of Hspa4l−/− mice. The dephosphorylation signal to eliminate phosphate was crucial, and these results unveiled the mechanism and biological relevance of the dephosphorylation of Prm2 for sperm maturation in vivo.

Maternal overweight increased sensitivity of mouse preimplantation embryos to oxidative stress in vitro

2021 ◽  
Vol 105 ◽  
pp. 62-71
Author(s):  
Zuzana Šefčíková ◽  
Janka Babeľová ◽  
Veronika Kovaříková ◽  
Juraj Koppel ◽  
Dušan Fabian
Keyword(s):  
Oxidative Stress ◽  
Preimplantation Embryos ◽  
Increased Sensitivity ◽  
Maternal Overweight

The effects of endometritis on the establishment of pregnancy in cattle

2012 ◽  
Vol 24 (1) ◽  
pp. 252 ◽  
Author(s):  
Robert O. Gilbert
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Inflammatory Mediators ◽  
Post Partum ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Breeding Period ◽  
Increased Risk ◽  
Factor Α ◽  
And Function

Endometritis is common in post partum dairy cows and is associated with impaired reproductive performance reflected in reduced first service conception, reduced hazard of pregnancy over the breeding period and increased risk of reproductive culling. The observed effects may be mediated directly by bacterial products, such as lipopolysaccharide (LPS, endotoxin), or indirectly by inflammatory mediators, such as cytokines, eicosanoids, nitric oxide and oxidative stress affecting sperm, ovarian, uterine and embryonic function. An inflammatory milieu in the uterus has been associated with changes in sperm motility and function as well as increased sperm phagocytosis. Zygotes resulting from fertilisation of oocytes with sperm subjected to oxidative stress are less likely to develop to the blastocyst stage. In addition, LPS and tumour necrosis factor-α (TNFα) impair follicular steroidogenesis, growth and ovulation. Oocytes exposed to LPS or prostaglandin (PG) F2α during maturation are less likely to develop to blastocyst stage after fertilisation. Embryos exposed to inflammatory mediators during development have fewer trophoectoderm cells. Nitric oxide impairs development of preimplantation embryos and TNFα increases blastomere apoptosis. Endometritis in women has been associated with higher rates of implantation failure. Extragenital inflammation (e.g. mastitis) is also associated with an increased rate of embryonic loss in cattle. These observations make it clear that direct and indirect effects of endometritis, and inflammation in general, can interrupt successful reproduction at several crucial stages.

scholarly journals Transcription of paternal Y-linked genes in the human zygote as early as the pronucleate stage

Zygote ◽  
1994 ◽  
Vol 2 (4) ◽  
pp. 281-287 ◽  
Author(s):  
Asangla Ao ◽  
Robert P. Erickson ◽  
Robert M.L. Winston ◽  
Alan H Handysude
Keyword(s):  
Mammalian Species ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Human Embryos ◽  
Cell Stage ◽  
Gonad Differentiation ◽  
Embryonic Genome ◽  
Human Preimplantation Embryos

SummaryGlobal activation of the embryonic genome occurs at the 4– to 8–cell stage in human embryos and is marked by continuation of early cleavage divisions in the presence of transcriptional inhibitors. Here we demonstrate, using recerse transcripase–polymerase chin reaction (Rt–PCR), the presence of transcripts for wo paternal Y chromosomal genes, ZFY and SRY in human preimplantation embryos. ZFY transcripts were detected as early as the pronucleate stage, 20–24 h post-insemination In vitro and at intermediate stages up to the blastocyst stage. SRY Transcripts were also detected at 2–cell to blastocyos observed in many mammalian species focuses attention on the role of events in six determination prior to gonad differentiation.

scholarly journals Developmental and molecular correlates of bovine preimplantation embryos

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 895-904 ◽  
Author(s):  
Hakan Sagirkaya ◽  
Muge Misirlioglu ◽  
Abdullah Kaya ◽  
Neal L First ◽  
John J Parrish ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methyltransferase ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Preimplantation Embryos ◽  
Developmental Potential

Expression of embryonic genes is altered in different culture conditions, which influence developmental potential both during preimplantation and fetal development. The objective of this study was to define the effects of culture conditions on: bovine embryonic development to blastocyst stage, blastocyst cell number, apoptosis and expression patterns of a panel of developmentally important genes. Bovine embryos were culturedin vitroin three culture media containing amino acids, namely potassium simplex optimization medium (KSOMaa), Charles Rosenkrans 1 (CR1aa) and synthetic oviductal fluid (SOFaa). Apoptosis in blastocysts was determined by TUNEL assay and expression profiles of developmentally important genes were assayed by real-time PCR.In vivo-produced bovine blastocysts were used as controls for experiments determining gene expression patterns. While the cleavage rates did not differ, embryos cultured in SOFaa had higher rates of development to blastocyst stage (P< 0.05). Mean cell numbers and percentages of apoptotic cells per blastocyst did not differ among the groups. Expression of the heat shock protein 70 (Hsp70) gene was significantly up-regulated in both CR1aa and KSOMaa when compared with SOFaa (P< 0.001). DNA methyltransferase 3a (Dnmt3a) expression was higher in embryos cultured in CR1aa than in those cultured in SOFaa (P< 0.001). Expression of interferon tau (IF-τ) and insulin-like growth factor II receptor (Igf-2r) genes was significantly up-regulated in KSOMaa when compared with CR1aa (P< 0.001). Gene expression did not differ betweenin vivo-derived blastocysts and theirin vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression.

277 EFFECTS OF ERYTHROCYTE AND ERYTHROCYTE HEMOLYSATE ON BOVINE PREIMPLANTATION EMBRYO DEVELOPMENT IN VITRO UNDER REACTIVE OXYGEN SPECIES CONDITION

2010 ◽  
Vol 22 (1) ◽  
pp. 295
Author(s):  
A. Ideta ◽  
K. Tsuchiya ◽  
Y. Nakamura ◽  
M. Urakawa ◽  
M. Murakami ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Embryo Development ◽  
Reactive Oxygen ◽  
Hemoglobin Concentration ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Oxygen Species ◽  
Preimplantation Embryo Development ◽  
Presence And Absence

Reactive oxygen species (ROS) damage preimplantation embryos by increasing DNA fragmentation, leading to early embryonic death. Erythrocytes have been shown to protect other cells and tissues against ROS. In mice, erythrocytes were recently found to improve the early development of embryos by their antioxidant effect. The purpose of the present study was to examine the effect of erythrocytes on the in vitro development of bovine IVF embryos in medium supplemented with ROS. COCs were aspirated from ovaries collected from a local slaughterhouse and were cultured for 22 h in TCM-199 containing 5% fetal bovine serum. IVF was performed using an IVF100 (Research Institute for the Functional Peptides, Yamagata, Japan) according to the manufacturer’s instructions. In experiment 1, IVF embryos were cultured in CR1aa medium supplemented with an oxidizing agent, 0.5 mM hypoxanthine and 0.01 U mL-1 xanthine oxidase (HX/XOD), in the presence and absence of erythrocytes (5 × 104, 5× 105, 5×106, and 5 × 107 erythrocytes mL-1). In experiments 2 and 3, the development of embryos under the condition without ROS was assessed in the presence and absence of erythrocytes (5 × 106 erythrocytes mL-1) or erythrocyte hemolysate (hemoglobin concentration of 1.9 g L-1), respectively. At 7 days after in vitro culture, the development to the blastocyst stage of IVF embryos was examined using a stereomicroscope. Data were analyzed using Fisher’s PLSD test and Student’s t-test In experiment 1, the presence of HX/XOD significantly inhibited embryo development to the blastocyst stage in vitro (P < 0.05). The addition of erythrocytes to medium supplemented with HX/XOD markedly improved preimplantation development (Table 1). In experiments 2 and 3, supplementation of erythrocytes or erythrocyte hemolysate promoted the development of embryos to the blastocyst stage (experiment 2: erythrocyte 42.4 ± 3.1%, control 28.5 ± 5.7%, P < 0.1; experiment 3: erythrocyte hemolysate 39.1 ± 3.3%, control 30.2 ± 1.0%, P < 0.1). In conclusion, we suggest that the addition of erythrocytes to culture medium can counteract the negative effects of ROS on embryo development and blastocyst formation. Table 1.Effect of HX/XOD and erythrocyte supplementation on embryo development to blastocyst stage

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