https://www.clevelandclinic.org/ReproductiveResearchCenter/docs/agradoc114.pdf

Role of reactive oxygen species in the pathophysiology of human reproduction

Aphids are important agricultural pests and also biological models for studies of insect-plant interactions, symbiosis, virus vectoring, and the developmental causes of extreme phenotypic plasticity. Here we present the 464 Mb draft genome assembly of the pea aphid Acyrthosiphon pisum. This first published whole genome sequence of a basal hemimetabolous insect provides an outgroup to the multiple published genomes of holometabolous insects. Pea aphids are host-plant specialists, they can reproduce both sexually and asexually, and they have coevolved with an obligate bacterial symbiont. Here we highlight findings from whole genome analysis that may be related to these unusual biological features. These findings include discovery of extensive gene duplication in more than 2000 gene families as well as loss of evolutionarily conserved genes. Gene family expansions relative to other published genomes include genes involved in chromatin modification, miRNA synthesis, and sugar transport. Gene losses include genes central to the IMD immune pathway, selenoprotein utilization, purine salvage, and the entire urea cycle. The pea aphid genome reveals that only a limited number of genes have been acquired from bacteria; thus the reduced gene count of Buchnera does not reflect gene transfer to the host genome. The inventory of metabolic genes in the pea aphid genome suggests that there is extensive metabolite exchange between the aphid and Buchnera, including sharing of amino acid biosynthesis between the aphid and Buchnera. The pea aphid genome provides a foundation for post-genomic studies of fundamental biological questions and applied agricultural problems.

/pdf/genome-sequence-of-the-pea-aphid-acyrthosiphon-pisum-2opiyalluh.pdf

Genome Sequence of the Pea Aphid Acyrthosiphon pisum

https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.2002.tb02599.x

Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques.

Sperm DNA integrity is essential for the accurate transmission of genetic information. It has a highly compact and complex structure and is capable of decondensation-features that must be present in order for a spermatozoon to be considered fertile. Any form of sperm chromatin abnormalities or DNA damage may result in male infertility. In support of this conclusion, it was reported that in-vivo fecundity decreases progressively when > 30% of the spermatozoa are identified as having DNA damage. Several methods are used to assess sperm chromatin/DNA, which is considered an independent measure of sperm quality that may yield better diagnostic and prognostic approaches than standard sperm parameters (concentration, motility and morphology). The clinical significance of this assessment lies in its association not only with natural conception rates, but also with assisted reproduction success rates. Also, it has a serious impact on the offspring and is highly prognostic in the assessment of fertility in cancer patients. Therefore, screening for sperm DNA damage may provide useful information in cases of male idiopathic infertility and in those men pursuing assisted reproduction. Treatment should include methods for prevention of sperm DNA damage.

/pdf/role-of-sperm-chromatin-abnormalities-and-dna-damage-in-male-5a2riixlg7.pdf

Role of sperm chromatin abnormalities and DNA damage in male infertility

Reactive oxygen metabolites are known to disrupt sperm-oocyte fusion, sperm movement, and DNA integrity; however, the relative sensitivities of these elements to oxidative stress are unknown. In this study these factors were assessed in human spermatozoa exposed to increasing levels of oxidative stress achieved through the stimulation of endogenous oxidant generation with NADPH or direct exposure to hydrogen peroxide. At low levels of oxidative stress, DNA fragmentation was significantly reduced while the rates of sperm-oocyte fusion were significantly enhanced. As the level of oxidative stress increased, the spermatozoa exhibited significantly elevated levels of DNA damage (p < 0.001) and yet continued to express an enhanced capacity for sperm-oocyte fusion. At the highest levels of oxidative stress, extremely high rates of DNA fragmentation were observed but the spermatozoa exhibited a parallel loss in their capacities for movement and oocyte fusion. These studies emphasize how redox mechanisms can either enhance or disrupt the functional and genomic integrity of human spermatozoa depending on the intensity of the oxidative stimulus. Because these qualities are affected at different rates, spermatozoa exhibiting significant DNA damage are still capable of fertilizing the oocyte. These results may have long-term implications for the safety of assisted conception procedures in cases associated with oxidative stress.

/pdf/relative-impact-of-oxidative-stress-on-the-functional-57r02fvx60.pdf

Relative Impact of Oxidative Stress on the Functional Competence and Genomic Integrity of Human Spermatozoa

The molecular basis of many forms of male infertility is poorly defined. One area of research that has been studied intensely is the integrity of the DNA in the nucleus of mature ejaculated spermatozoa. It has been shown that, in men with abnormal sperm parameters, the DNA is more likely to possess strand breaks. However, how and why this DNA damage originates in certain males and how it may influence the genetic project of a mature spermatozoon is unknown. Two theories have been proposed to describe the origin of this DNA damage in mature spermatozoa. The first arises from studies performed in animal models and is linked to the unique manner in which mammalian sperm chromatin is packaged, while the second attributes the nuclear DNA damage in mature spermatozoa to apoptosis. One of the factors implicated in sperm apoptosis is the cell surface protein, Fas. In this review, we discuss the possible origins of DNA damage in ejaculated human spermatozoa, how these spermatozoa arrive in the ejaculate of some men, and what consequences they may have if they succeed in their genetic project.

Origin of dna damage in ejaculated human spermatozoa

During spermiogenesis, mammalian chromatin undergoes replacement of nuclear histones by protamines, resulting in a DNA that is highly condensed in the mature sperm. We have previously demonstrated that a percentage of human spermatozoa exhibit 1) positivity to the guanine-cylosine-specific chromomycin A3 (CMA3) fluorochrome and 2) the presence of endogenous nicks in their DNA. In situ protamination of mature human sperm limits the percentage of sperm positive to CMA3 and exhibiting endogenous nicks. In this study, we report further investigations that aim to clarify the relationship existing between levels of CMA3 stainability and the presence of endogenous nicks in the DNA of mature human spermatozoa. Human spermatozoa from 25 different samples showed values of sensitivity to the CMA3 fluorochrome ranging from 13% to 75%. The same samples showed a percentage of sensitivity to endogenous nick translation ranging from 1% to 38%. A strong correlation (r = 0.86) was evident between these two parameters. Prior staining of sperm with the CMA3 fluorochrome drastically reduced sensitivity to nick translation. In contrast, previously nick-translated sperm stained with CMA3 showed very little difference from samples that had not been pretreated. The presence of nicked sperm in the ejaculate may indicate anomalies during spermiogenesis and be an indicator of male infertility.

Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessibility.

A major event in enhancing sperm chromatin stability is the replacement of the histones by protamines during spermiogenesis. In this study, we present results indicating that chromomycin A3 (CMA3 ) can be used to show protamine deficiency in sperm chromatin. Fixed chromatin of mature mouse spermatozoa showed high fluorescence after treatment with ethidium bromide (EB), but was completely unstained after treatment with CMA3. The same chromatin was found to be highly resistant to in situ nick-translation. In contrast, a substantial fraction of human spermatozoa were positive for CMA3. The accessibility of CMA to the DNA of human sperm was eliminated if the slides were previously treated with protamine in situ. This treatment did not affect the accessibility of EB to the chromatin. Individual human sperm samples revealed a substantial frequency of spermatozoa with endogenous nicks, which was found to be the same as the frequency of spermatozoa responding positively to CMA3 staining. Treatment of preparations with protamines prevented the identification of the endogenous nicks. These data as a whole suggest that CMA3 could represent a useful tool for the detection of protamine deficiency in sperm chromatin. Furthermore, confirmation of experiments relating sensitivity to nick translation and positivity to CMA3 may allow an indirect in situ visualization of nicked and partially denatured DNA, which could correlate with certain forms of male factor infertility.

/pdf/effect-of-deoxyribonucleic-acid-protamination-on-432sepjsoq.pdf

Effect of deoxyribonucleic acid protamination on fluorochrome staining and in situ nick-translation of murine and human mature spermatozoa.

Human semen is heterogeneous in quality, not only between males but also within a single ejaculate. Differences in quality are evident, both when examining the classical parameters of sperm number, motility and morphology and in the integrity of the sperm nucleus. The aim of this study was to determine the efficiency of the PureSperm((R)), Percoll((R)) and swim-up preparation techniques to eliminate spermatozoa with nuclear anomalies. Semen samples were collected, washed and one part of the semen spread on a slide, the remainder was prepared using the swim-up, PureSperm((R)) or Percoll((R)) techniques. Spermatozoa from different fractions were fixed on slides and assessed. Sperm samples (n) from different men were stained using the chromomycin A(3) (CMA(3)) fluorochrome, which indirectly demonstrates a decreased presence of protamine (n = 31 for swim-up; n = 45 for PureSperm((R)); n = 39 for Percoll((R))). Spermatozoa prepared using PureSperm((R)) (n = 35) and Percoll((R)) (n = 37) were also examined for the presence of endogenous DNA nicks. Good quality spermatozoa should not possess DNA nicks and not stain (i.e. fluoresce) with CMA(3). When prepared using the swim-up technique the spermatozoa recovered showed no significant improvement with the CMA(3) staining. When spermatozoa were prepared using the PureSperm((R)) and Percoll((R)) techniques, a significant (P < 0.001) decrease in both CMA(3) positivity and DNA strand breakage was observed. These results indicate that both the PureSperm((R)) and Percoll((R)) techniques can enrich the sperm population by separating out those with nicked DNA and with poorly condensed chromatin.

The use of two density gradient centrifugation techniques and the swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies

Mammalian spermiogenesis involves the replacement of histones by protamines, resulting in a highly compacted chromatin. Upon fertilization, the reverse process occurs. We have previously shown that the chromomycin A 3 (CMA3) fluorochrome represents a useful tool for detecting protamine deficiency in spermatozoa. In this study we investigated CMA3 fluorochrome accessibility and the presence of endogenous nicks in maturing and fertilizing mouse sperm. Testicular sperm of stages 1-7 and 8-14 showed high positivity (> 96% ) to CMA3, decreasing to 63% in stage 15-16 spermatids. In situ protamination of stage 15-16 spermatids saw an inhibition of CMA3 accessibility. Only 8% of the mature spermatozoa in the efferent ducts were CMA3positive; this value decreased to 0% in the caput epididymidis. At fertilization, CMA3 fluorescence reappears in decondensing sperm. Fluorescein isothiocyanate (FITC) fluorescence, identifying endogenous nicks, was evident in 6% of stage 1-7 spermatids, increased to 22% in stage 8-14 spermatids, and disappeared in stage 15-16 spermatids. During fertilization, endogenous nicks were not observed in decondensing sperm. We propose that 1) the presence of nicks in mouse testicular spermatids suggests that DNA cutting and ligating occurs prior to completion of protamination and 2) the absence of nicks during fertilization indicates that decondensation is not simply the reversal of the initial chromatin packaging process.

U. Bianchi

Papers

Origin of dna damage in ejaculated human spermatozoa

Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessibility.

Effect of deoxyribonucleic acid protamination on fluorochrome staining and in situ nick-translation of murine and human mature spermatozoa.

The use of two density gradient centrifugation techniques and the swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies

Relationship between the presence of endogenous nicks and sperm chromatin packaging in maturing and fertilizing mouse spermatozoa.