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Meiotic metaphase II chromosomes with two chromatids and single chromatid sperm chromosomes in an inactivated secondary oocyte after ICSI with an epididymal spermatozoon from a sterile T/T ¢ male. A small chromatin fragment of likely male descent is visible (arrow).
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Male mice, heterozygous for two semi-identical reciprocal translocations T(1;13)70H and T(1;13)1Wa are usually sterile. We have investigated this oligoasthenoteratozoospermic mouse model using ICSI.
B6D2F1 oocytes were injected with epididymal or testicular sperm from fertile or sterile translocation carriers and from chromosomally normal fertile c...
Context in source publication
Context 1
... 18 oocytes, an intact or slightly decondensed sperm head was visible next to 20 metaphase II chromosomes. In a further 18 oocytes, premature chromosome condensation (PCC) of the sperm chromosomes was observed (Figure 1). In these oocytes, the sperm nucleus had transformed into discrete chromosomes. ...
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Citations
... Studies on mouse oocytes inseminated with sperm with induced DNA damage indicated that the resulting early embryos were lacking in traditional checkpoints (Shimura et al., 2002a;Baart et al., 2004;Adiga et al., 2007;Derijck et al., 2008) (Supplementary Fig. S1). Analysis of cell cycle progression in zygotes resulting from insemination with damaged sperm revealed no delay in cell cycling prior to the Sphase, indicating that the traditional G1/S checkpoint is not activated in these zygotes harboring sperm DNA damage (Shimura et al., 2002b;Adiga et al., 2007;Derijck et al., 2008;Xiao et al., 2012;Gawecka et al., 2013;Wang et al., 2013) (Supplementary Fig. S1). ...
Sperm DNA damage is considered a predictive factor for the clinical outcomes of patients undergoing ART. Laboratory evidence suggests that zygotes and developing embryos have adopted specific response and repair mechanisms to repair DNA damage of paternal origin. We have conducted a systematic review in accordance with guidelines from Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) to identify and review the maternal mechanisms used to respond and repair sperm DNA damage during early embryonic development, how these mechanisms operate and their potential clinical implications.
The literature search was conducted in Ovid MEDLINE and Embase databases until May 2021. Out of 6297 articles initially identified, 36 studies were found to be relevant through cross referencing and were fully extracted. The collective evidence in human and animal models indicate that the early embryo has the capacity to repair DNA damage within sperm by activating maternally driven mechanisms throughout embryonic development. However, this capacity is limited and likely declines with age. The link between age and decreased DNA repair capacity could explain decreased oocyte quality in older women, poor reproductive outcomes in idiopathic cases, and patients who present high sperm DNA damage. Ultimately, further understanding mechanisms underlying the maternal repair of sperm DNA damage could lead to the development of targeted therapies to decrease sperm DNA damage, improved oocyte quality to combat incoming DNA insults or lead to development of methodologies to identify individual spermatozoa without DNA damage.
... mutagenesis and the attendant dysregulation of cell differentiation and development in the zygote. Despite this, the zygote has several unique features that influence its capacity for effective DNA repair, including: a complete absence of transcription-coupled translation (Schultz, 2002;Hamatani et al., 2004;Bianchi and Sette, 2011); TOP2 mediated remodelling of sperm derived chromatin resulting in the formation of extensive, albeit transient, DSBs (Adenot et al., 1991;Bizzaro et al., 2000;Derijck et al., 2006;Cortes-Gutierrez et al., 2014); and the absence of a traditional G1-S checkpoint following gamete fusion (Shimura et al., 2002;Baart et al., 2004;Adiga et al., 2007;Carbone and Chavez, 2015). ...
... As this example illustrates, the zygote is capable of mounting diverse protective strategies to mitigate the risk imposed by an elevated mutational load being carried by the offspring. Nevertheless, early stage mammalian embryogenesis lacks several traditional elements of the canonical somatic cell DNA damage response pathways, including a G1/S and G2/M checkpoint, as well as an ability to undergo programmed cell death (Shimura et al., 2002;Baart et al., 2004;Adiga et al., 2007). Recent evidence now suggests that auxiliary to DNA repair, the preimplantation embryo has adopted a series of non-conventional cell 'removal' processes. ...
Background:
DNA integrity and stability are critical determinants of cell viability. This is especially true in the female germline, wherein DNA integrity underpins successful conception, embryonic development, pregnancy and the production of healthy offspring. However, DNA is not inert; rather, it is subject to assault from various environment factors resulting in chemical modification and/or strand breakage. If structural alterations result and are left unrepaired, they have the potential to cause mutations and propagate disease. In this regard, reduced genetic integrity of the female germline ranks among the leading causes of subfertility in humans. With an estimated 10% of couples in developed countries taking recourse to ART to achieve pregnancy, the need for ongoing research into the capacity of the oocyte to detect DNA damage and thereafter initiate cell cycle arrest, apoptosis or DNA repair is increasingly more pressing.
Objective and rationale:
This review documents our current knowledge of the quality control mechanisms utilised by the female germline to prevent and remediate DNA damage during their development from primordial follicles through to the formation of preimplantation embryos.
Search methods:
The PubMed database was searched using the keywords: primordial follicle, primary follicle, secondary follicle, tertiary follicle, germinal vesical, MI, MII oocyte, zygote, preimplantation embryo, DNA repair, double-strand break and DNA damage. These keywords were combined with other phrases relevant to the topic. Literature was restricted to peer-reviewed original articles in the English language (published 1979-2018) and references within these articles were also searched.
Outcomes:
In this review, we explore the quality control mechanisms utilised by the female germline to prevent, detect and remediate DNA damage. We follow the trajectory of development from the primordial follicle stage through to the preimplantation embryo, highlighting findings likely to have important implications for fertility management, age-related subfertility and premature ovarian failure. In addition, we survey the latest discoveries regarding DNA repair within the metaphase II (MII) oocyte and implicate maternal stores of endogenous DNA repair proteins and mRNA transcripts as a primary means by which they defend their genomic integrity. The collective evidence reviewed herein demonstrates that the MII oocyte can engage in the activation of major DNA damage repair pathway(s), therefore encouraging a reappraisal of the long-held paradigm that oocytes are largely refractory to DNA repair upon reaching this late stage of their development. It is also demonstrated that the zygote can exploit a number of protective strategies to mitigate the risk and/or effect the repair, of DNA damage sustained to either parental germline; affirming that DNA protection is largely a maternally driven trait but that some aspects of repair may rely on a collaborative effort between the male and female germlines.
Wider implications:
The present review highlights the vulnerability of the oocyte to DNA damage and presents a number of opportunities for research to bolster the stringency of the oocyte's endogenous defences, with implications extending to improved diagnostics and novel therapeutic applications to alleviate the burden of infertility.
... All institutional and national guidelines for the care and use of laboratory animals were followed and mouse experiments were approved by the local committee on animal experiments, DEC Consult. B6D2 F1 female mice (Harlan) were used as oocyte donors and superovulation was induced by intraperitoneal injection of 7.5 IU pregnant mare's serum gonadotrophin (Intervet) followed by 7.5 IU human chorionic gonadotropin (Intervet) 48 h later 60,61 . Oocytes were isolated from the oviducts 13 h after human chorionic gonadotropin and cumulus cells were removed by brief incubation in G-MOPS medium containing 80 IU ml À 1 hyaluronidase (Sigma). ...
The different configurations of maternal and paternal chromatin, acquired during oogenesis and spermatogenesis, have to be rearranged after fertilization to form a functional embryonic genome. In the paternal genome, nucleosomal chromatin domains are re-established after the protamine-to-histone exchange. We investigated the formation of constitutive heterochromatin (cHC) in human preimplantation embryos. Our results show that histones carrying canonical cHC modifications are retained in cHC regions of sperm chromatin. These modified histones are transmitted to the oocyte and contribute to the formation of paternal embryonic cHC. Subsequently, the modifications are recognized by the H3K9/HP1 pathway maternal chromatin modifiers and propagated over the embryonic cleavage divisions. These results are in contrast to what has been described for mouse embryos, in which paternal cHC lacks canonical modifications and is initially established by Polycomb group proteins. Our results show intergenerational epigenetic inheritance of the cHC structure in human embryos.
... When spermatogenesis is compromised, for instance, in the oligo-astheno-teratospermia (OAT) syndrome, variation increases. (de Boer et al., 1976; Baart et al., 2004; Yamauchi et al., 2009). In human beings, OAT samples appear to be less well matured compared to spermatozoa from normospermic control samples. ...
... In sixteen cases, ICSI (and sometimes IVF) were used to test the developmental capacity of mutant sperm. Interpretation of data is hampered by the negative effect of epididymal maturation on sperm genetic integrity in conditions of impaired spermatogenesis (Baart et al., 2004; Suganuma et al., 2005b; Yamauchi et al., 2010). Generally, mutants that affect chromatin remodeling were less successfully propagated than the mutants from the other classes. ...
Mouse mutants that show effects on sperm head shape, the sperm tail (flagellum), and motility were analysed in a systematic way. This was achieved by grouping mutations in the following classes: manchette, acrosome, Sertoli cell contact, chromatin remodelling, and mutations involved in complex regulations such as protein (de)phosphorylation and RNA stability, and flagellum/motility mutations. For all mutant phenotypes, flagellum function (motility) was affected. Head shape, including the nucleus, was also affected in spermatozoa of most mouse models, though with considerable variation. For the mutants that were categorized in the flagellum/motility group, generally normal head shapes were found, even when the flagellum did not develop or only poorly so. Most mutants are sterile, an occasional one semi-sterile. For completeness, the influence of the sex chromosomes on sperm phenotype is included. Functionally, the genes involved can be categorized as regulators of spermiogenesis. When extrapolating these data to human sperm samples, in vivo selection for motility would be the tool for weeding out the products of suboptimal spermiogenesis and epididymal sperm maturation. The striking dependency of motility on proper sperm head development is not easy to understand, but likely is of evolutionary benefit. Also, sperm competition after mating can never act against the long-term multi-generation interest of genetic integrity. Hence, it is plausible to suggest that short-term haplophase fitness i.e., motility, is developmentally integrated with proper nucleus maturation, including genetic integrity to protect multi-generation fitness. We hypothesize that, when the prime defect is in flagellum formation, apparently a feedback loop was not necessary as head morphogenesis in these mutants is mostly normal. Extrapolating to human-assisted reproductive techniques practice, this analysis would supply the arguments for the development of tools to select for motility as a continuous (non-discrete) parameter.
© 2014 American Society of Andrology and European Academy of Andrology.
... It is not yet clear how the mammalian zygote responds to DNA damage. Studies on zygotes with induced DNA damage have demonstrated that they do not have traditional G1/S or G2/M checkpoints [1,2], suggesting that alternative mechanisms are in place to ensure the integrity of the genome in developing embryos. Both non-homologous end joining (NHEJ) and homologous recombination (HR) repair pathways are active in the zygotes and some DNA repair is possible [3][4][5]. ...
Mouse zygotes do not activate apoptosis in response to DNA damage. We previously reported a unique form of inducible sperm DNA damage termed sperm chromatin fragmentation (SCF). SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B) followed by irreversible DNA degradation by a nuclease(s). Here, we created zygotes using spermatozoa induced to undergo SCF (SCF zygotes) and tested how they responded to moderate and severe paternal DNA damage during the first cell cycle. We found that the TUNEL assay was not sensitive enough to identify the breaks caused by SCF in zygotes in either case. However, paternal pronuclei in both groups stained positively for γH2AX, a marker for DNA damage, at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. We also found that both pronuclei in SCF zygotes with moderate DNA damage replicated normally, but paternal pronuclei in the SCF zygotes with severe DNA damage delayed the initiation of DNA replication by up to 12 hrs even though the maternal pronuclei had no discernable delay. Chromosomal analysis of both groups confirmed that the paternal DNA was degraded after S-phase while the maternal pronuclei formed normal chromosomes. The DNA replication delay caused a marked retardation in progression to the 2-cell stage, and a large portion of the embryos arrested at the G2/M border, suggesting that this is an important checkpoint in zygotic development. Those embryos that progressed through the G2/M border died at later stages and none developed to the blastocyst stage. Our data demonstrate that the zygote responds to sperm DNA damage through a non-apoptotic mechanism that acts by slowing paternal DNA replication and ultimately leads to arrest in embryonic development.
... In the context of ICSI success it seems clear that in situations where there is increasing DNA damage during epididymal transit, as is the case with NPYq deficiency, it is better to use testicular sperm; however, freezing should be avoided. Improved ICSI success with testicular as compared to epididymal sperm has previously been reported for mice heterozygous for two different but semi-identical translocations[43]and in mice deficient for transition proteins[11,44], as well as in infertile men with sperm DNA damage[45]. A caveat is that in the present study sperm freezing was done without cryoprotectant, but with the method developed to keep DNA damage to a level comparable to freezing with cryprotectant[46,47]. Indeed, substantial freezing-induced DNA damage has been reported even with mouse sperm frozen with cryoprotectant[48]and with cryopreserved human sperm[49][50][51]. ...
Mice with severe non-PAR Y chromosome long arm (NPYq) deficiencies are infertile in vivo and in vitro. We have previously shown that sperm from these males, although having grossly malformed heads, were able to fertilize oocytes via intracytoplasmic sperm injection (ICSI) and yield live offspring. However, in continuing ICSI trials we noted a reduced efficiency when cryopreserved sperm were used and with epididymal sperm as compared to testicular sperm. In the present study we tested if NPYq deficiency is associated with sperm DNA damage - a known cause of poor ICSI success.
We observed that epididymal sperm from mice with severe NPYq deficiency (that is, deletion of nine-tenths or the entire NPYq gene complement) are impaired in oocyte activation ability following ICSI and there is an increased incidence of oocyte arrest and paternal chromosome breaks. Comet assays revealed increased DNA damage in both epididymal and testicular sperm from these mice, with epididymal sperm more severely affected. In all mice the level of DNA damage was increased by freezing. Epididymal sperm from mice with severe NPYq deficiencies also suffered from impaired membrane integrity and abnormal chromatin condensation and suboptimal chromatin protamination. It is therefore likely that the increased DNA damage associated with NPYq deficiency is a consequence of disturbed chromatin remodeling.
This study provides the first evidence of DNA damage in sperm from mice with NPYq deficiencies and indicates that NPYq-encoded gene/s may play a role in processes regulating chromatin remodeling and thus in maintaining DNA integrity in sperm.
... In male-sterile mouse models with characteristics of OAT, it was found that the amount of 2 cell embryos was sharply reduced when more mature cauda epididymal sperm were used contrary to testicular sperm or caput epididymal sperm (Baart et al., 2004;Suganuma et al., 2005). Baart and co-workers showed that in case of zygote arrest, both pronuclei had started DNA replication, the male one being arrested in S-and G2-phase, which is suggestive of a nucleus organization problem. ...
Male transgenerational epigenetic effects have been discovered in the discipline of mouse radiation genetics, using genetic and non-genetic readouts. The mechanism to explain the origin of the transmission of epigenetic and genetic instability is still unknown. In a search for a hypothesis that could satisfy the data, we propose that regulation of chromosome structure in the germline, by the occupancy of matrix/scaffold associated regions, contains molecular memory function. The male germline is strikingly dynamic as to chromatin organization. This could explain why experience of irradiation stress leaves a persistent mark in the male germline only. To be installed, such memory requires both S-phase and chromatin reorganization during spermatogenesis and in the zygote, that likely also involves reorganization of loop domains. By this reorganization, another layer of information is added, needed to accommodate early embryonic development. Observations point at the involvement of DNA repair as inducer of transgenerational epigenetic modulation. Nuclear structure, chromatin composition and loop domain organization are aspects of human sperm variability that in many cases of assisted reproduction is increased due to inclusion of more incompletely differentiated/maturated sperm nuclei. Adjustment of loop domains in early embryo development can be anticipated and zygotic and cleavage stage S-phase repair activity will have to deal with potential paternal DNA lesions. Therefore, by changing male nucleus structure due to reproduction from impaired spermatogenesis, the transgenerational information content could be changed as well. We discuss aspects of male reproductive performance in the context of this hypothesis.
... Total acrosin activity and acrosome reaction play an important part in fertilization (26,27). Reduced oocyte activation and first-cleavage rate after ICSI was reported in cases of chromosomal translocations associated with OAT (28). Abnormal semen analyses often are associated with abnormal chromatin. ...
To evaluate the electrical activation of oocytes in patients with previously failed or limited fertilization after intracytoplasmic sperm injection (ICSI) and in patients with possible failure of fertilization.
Prospective randomized study.
A private IVF center in Egypt.
Two hundred forty-six patients with severe oligoasthenospermia or nonobstructive azoospermia with total teratospermia or totally immotile spermatozoa were selected for the study. Patients who previously had total failure or limited fertilization after ICSI also were included.
Sibling oocytes were randomly divided after ICSI into two groups: the study group (n = 1,640) was subjected to electroactivation, and the control group (n = 1,435), to no electroactivation. Electroactivation was performed by using a double-square direct-current pulse. Embryo transfer was performed with the best available embryos.
Fertilization rate, degeneration rate, and pregnancy outcome.
Two hundred forty-one ICSI cycles were included in the study. The fertilization rate was statistically significantly higher in the electroactivated group as compared with in the control group (68% vs 60%, odds ratio = 1.397, 95% confidence interval = 1.197 to 1.629). The oocyte degeneration rate was not statistically significantly different between the two groups (5.9% vs 4.9%, odds ratio = 0.96, 95% confidence interval = 0.73 to 1.26). In total, 112 clinical pregnancies resulted (pregnancy rate = 46.5%). Total fertilization failure occurred in 5 cycles in the control group, and none failed in the study group.
Oocyte electroactivation after ICSI significantly improved the fertilization rate in severe oligoasthenoteratospermia and nonobstructive azoospermia.
... The zygote has several unique features influencing DNA repair such as: (i) lack of transcription coupled translation, thereby relying on mRNAs and proteins stored in the oocyte (24,25), (ii) Topoisomerase II mediated remodeling of sperm derived chromatin resulting in transient DSBs (26 -28), (iii) start of the cell cycle after gamete fusion, lacking a known G1-S checkpoint (29,30), (iv) male and female chromatin is physically separated and epigenetically dissimilar (31 -33). Finally, in the genesis of chromosome aberrations (CA), the zygote shows a bias for chromosome type over chromatid-type abnormalities (22,23,34). ...
... Zygotes derived from either C.B17 or scid oocytes fused with 3 Gy irradiated sperm show a comparable reduction in mitotic index ( Fig. 5A and Table 4) and an increase in residual gH2AX foci at first mitotic cleavage ( Fig. 5B and Supplementary Material, Table S1). As no G1 S checkpoint is known in the mouse zygote (29,30), the reduction in mitotic index likely occurs at S-phase as does the repair of the late G1 gH2AX foci in especially C.B17 (CA frequency is back to control level, Table 1). Somehow, the balance between NHEJ and HR in C.B17 is such that at S-phase, repair suffices to avoid the extra generation of chromosome type CA, at the expense of an increase in chromatid-type yH2AX foci (Fig. 5B). ...
... When too heavily damaged, the sperm nucleus either disintegrates at chromatin remodeling (63) after sperm entry or chromatin collapses at the onset of replication (64), which could be the consequence of the absence of a DNA damage checkpoint at this stage (29,30). ...
In the human, the contribution of the sexes to the genetic load is dissimilar. Especially for point mutations, expanded simple tandem repeats and structural chromosome mutations, the contribution of the male germline is dominant. Far less is known about the male germ cell stage(s) that are most vulnerable to mutation contraction. For the understanding of de novo mutation induction in the germline, mechanistic insight of DNA repair in the zygote is mandatory. At the onset of embryonic development, the parental chromatin sets occupy one pronucleus (PN) each and DNA repair can be regarded as a maternal trait, depending on proteins and mRNAs provided by the oocyte. Repair of DNA double-strand breaks (DSBs) is executed by non-homologous end joining (NHEJ) and homologous recombination (HR). Differentiated somatic cells often resolve DSBs by NHEJ, whereas embryonic stem cells preferably use HR. We show NHEJ and HR to be both functional during the zygotic cell cycle. NHEJ is already active during replacement of sperm protamines by nucleosomes. The kinetics of G1 repair is influenced by DNA-PK(cs) hypomorphic activity. Both HR and NHEJ are operative in S-phase, HR being more active in the male PN. DNA-PK(cs) deficiency upregulates the HR activity. Both after sperm remodeling and at first mitosis, spontaneous levels of gammaH2AX foci (marker for DSBs) are high. All immunoflurescent indices of DNA damage and DNA repair point at greater spontaneous damage and induced repair activity in paternal chromatin in the zygote.
... Superovulation was induced by i.p. injection of 7.5 IU pregnant mare's serum gonadotrophin (PMSG, Intervet, Boxmeer, The Netherlands ) around 9 pm, followed by 7.5 IU hCG (Intervet) after 48 h. Oocytes were harvested from the oviducts 13 h after administration of hCG and stored without cumulus cells at 37°C for up to 5 h in complemented Mem-α [20]. ...
... Microinjection was performed as described in [21] and [20] with some adaptations. The temperature in the injection droplet was kept at 24°C. ...
... Sperm were selected for normal morphology and motility. After injection, oocytes were kept on the injection platform for 5 minutes, then gradually warmed to 37°C and placed in culture medium at 37°C, 5% CO2 in air [20]. ...
about 15% to 30% of the DNA in human sperm is packed in nucleosomes and transmission of this fraction to the embryo potentially serves as a mechanism to facilitate paternal epigenetic programs during embryonic development. However, hitherto it has not been established whether these nucleosomes are removed like the protamines or indeed contribute to paternal zygotic chromatin, thereby potentially contributing to the epigenome of the embryo.
to clarify the fate of sperm-derived nucleosomes we have used the deposition characteristics of histone H3 variants from which follows that H3 replication variants present in zygotic paternal chromatin prior to S-phase originate from sperm. We have performed heterologous ICSI by injecting human sperm into mouse oocytes. Probing these zygotes with an antibody highly specific for the H3.1/H3.2 replication variants showed a clear signal in the decondensed human sperm chromatin prior to S-phase. In addition, staining of human multipronuclear zygotes also showed the H3.1/H3.2 replication variants in paternal chromatin prior to DNA replication.
these findings reveal that sperm-derived nucleosomal chromatin contributes to paternal zygotic chromatin, potentially serving as a template for replication, when epigenetic information can be copied. Hence, the execution of epigenetic programs originating from transmitted paternal chromatin during subsequent embryonic development is a logical consequence of this observation.
