The impact of sperm DNA damage in assisted conception and beyond: recent advances in diagnosis and treatment

Abstract Sperm DNA damage is a useful biomarker for male infertility diagnosis and prediction of assisted reproduction outcomes.
It is associated with reduced fertilization rates, embryo quality and pregnancy rates, and higher rates of spontaneous miscarriage
and childhood diseases. This review provides a synopsis of the most recent studies from each of the authors, all of whom have major
track records in the field of sperm DNA damage in the clinical setting. It explores current laboratory tests and the accumulating body
of knowledge concerning the relationship between sperm DNA damage and clinical outcomes. The paper proceeds to discuss the
strengths, weaknesses and clinical applicability of current sperm DNA tests. Next, the biological significance of DNA damage in
the male germ line is considered. Finally, as sperm DNA damage is often the result of oxidative stress in the male reproductive tract,
the potential contribution of antioxidant therapy in the clinical management of this condition is discussed. DNA damage in human spermatozoa is an important attribute of semen quality. It should be part of the clinical work up and properly controlled trials
addressing the effectiveness of antioxidant therapy should be undertaken as a matter of urgency.

Two potential clinical applications of the alkaline single-cell gel electrophoresis assay .1. Human bladder washings and transitional cell carcinoma of the bladder .2. Human sperm and male infertility

Part 1: The alkaline single-cell gel electrophoresis (comet) assay was used to analyse the integrity and DNA content of exfoliated cells extracted from bladder washing specimens from 9 transitional cell carcinoma patients and 15 control patients. DNA damage, as expressed by % tail DNA and tail moment values, was observed to occur in cells from both control and bladder cancer samples. The extent of the damage was, however, found to be significantly greater in the cancer group than in the control group. Comet optical density values were also recorded for each cell analysed in the comet assay and although differences observed between tumour grades were not found to be statistically significant, the mean comet optical density value was observed to be greater in the cancer group than in the control population studied, These preliminary results suggest that the comet assay may have potential as a diagnostic tool and as a prognostic indicator in transitional cell carcinoma, Part 2: Baseline DNA damage in sperm cells from 13 normozoospermic fertile males, 17 normozoospermic infertile males and 11 asthenozoospermic infertile males were compared using a modified alkaline comet assay technique. No significant difference in the level of baseline DNA damage was observed between the 3 categories of sperm studied; however the untreated sperm cells were observed to display approximately 20% tail DNA. This is notably higher than the background DNA damage observed in somatic cells where the % tail DNA is normally less than 5%. Sperm from the 3 groups of men studied were also compared for sensitivity to DNA breakage, using the modified alkaline comet assay, following X-ray irradiations (5, 10 and 30 Gy) and hydrogen peroxide treatments (40, 100 and 200 mu M). Significant levels of X-ray-induced damage were found relative to untreated control sperm in the two infertile groups following 30 Gy irradiation. Significant damage in hydrogen peroxide-treated sperm was observed in sperm from fertile samples, at 200 mu M and in infertile samples at 100- and 200-mu M doses relative to controls. These results therefore indicate that fertile sperm samples are more resistant to X-ray- and hydrogen peroxide-induced DNA breakage than infertile samples. Further studies involving greater numbers of individuals are currently in progress to confirm these findings.

Glycine betaine-mediated protection of peas (Pisum sativum L.) during blanching and frozen storage

We used glycine betaine (5–20% w/v) for blanching green peas (100°C, 60 s), and their subsequent freezing and storage (–20°C, 90 days). Blanching after the addition of glycine betaine at ≥10% (w/v) followed by a 90 day storage period which resulted in the most desirable outcome: higher vitamin C levels, a superior green color, enhanced organoleptic quality and texture, and improved retention of peroxidase and lipoxygenase activity relative to control peas (no glycine betaine added). Microscopic characterizations of control and treated peas revealed that glycine betaine acts as a cryoprotectant which maintains cellular integrity. Glycine betaine (10% w/v) could be used commercially for production of frozen peas with better quality attributes.

Sperm motility in Solea senegalensis: effect of temperature, salinity, pH and ovarian fluid

A análise da mobilidade seminal é uma ferramenta importante para reprodução em aquacultura. Esta é uma técnica in vitro que auxilia a estabulação, manutenção e selecção de lotes de reprodutores. A análise de mobilidade seminal pode tornar-se potencialmente uma ferramenta para o melhoramento das condições do ambiente de fertilização. A utilização do software CASA (Computer Assisted Sperm Analysis) revolucionou a descrição e quantificação específica da mobilidade seminal. A maioria da informação recolhida sobre mobilidade de sémen de peixes baseia-se em espécies de água doce, pelo que é crucial conhecer as condições óptimas de activação da mobilidade de espermatozóides para novas espécies de de água salgada de interesse em aquacultura tal como Solea senegalensis. A optimização das condições de fertilização desta espécie é particularmente importante já que os lotes de reprodutores em cativeiro podem desenvolver disfunções reprodutoras. Este trabalho teve como objectivo realizar a avaliação das condições óptimas de activação da mobilidade do sémen em S. senegalensis em termos de temperatura, salinidade e pH. O segundo objectivo foi realizar a avaliação da influência de fluido ovárico homólogo (S. senegalensis) e heterólogo (Epinephelus marginatus) na mobilidade seminal de S. senegalensis. Deste modo foram realizados dois conjuntos de experiências: 1) mobilidade de sémen de 7 machos analisado através do CASA em diferentes temperaturas, salinidades e pH, 2) mobilidade de sémen de 8 machos activados na presença de diferentes concentrações de fluido ovárico. Os parâmetros do CASA foram registados e posteriormente analisados através de médias e cluster analysis. Concluiu-se que temperaturas mais elevadas (20 ºC) e baixas salinidades (25 ‰ e 30 ‰) da solução de activação ocorre um melhoramento das características de mobilidade seminal, tal como a velocidade. A presença de fluido ovárico em baixas concentrações melhora as características da mobilidade seminal assim como a longevidade dos espermatozóides. O fluido ovárico é consequentemente um factor que estimula a mobilidade seminal que tem sido negligenciado em estudos anteriores. Este estudo demonstrou que durante a época de reprodução a temperatura da água (20 ºC) e a salinidade (25 ‰ e 30 ‰) no tanque são os principais factores que melhoram a activação da mobilidade do sémen, sendo consequentemente uma contribuição importante para compreender a dinâmica do processo de fertilização em S. senegalensis.

Potential biomarkers on gametogenesis process and in sperm quality of european EEL (Anguilla anguilla)

Dissertação de mestrado, Aquacultura, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Development and standardization of a protocol for sperm cryopreservation of two important commercial oyster species

Dissertação de mestrado, Aquacultura e Pescas, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Why do females mate with multiple males ? The sexually selected sperm hypothesis

One debated issues in evolutionary biology is, why in many species females mate with multiple males. Several hypotheses have been put forward, yet the benefits of multiple mating (here defined as mating with several males) remain unclear in many cases. The sperm sexual selection (SSS) hypothesis has been developed to account for the widespread occurrence of multiple mating in females. It argues that multiple mating by females may rapidly spread, when initially a small fraction of the females mate multiply, and if there is a heritable difference among males in one or several of the four characteristics: (1) the quantity of sperm they produce; (2) the success of their sperm in reaching and fertilizing an egg; (3) their ability to displace the sperm that females stored during previous mating; and (4) their ability to prevent any other male from subsequently introducing sperm (e.g., differential efficiency of mating plugs).

Reproduction in the temperate crayfish, Orconectes rusticus: intermale aggression, copulatary behaviour, and sperm competition

The chelipeds of Orconectes rusticus are sexually dimorphic; males possessing the larger. Males use their chelae in intermale aggressive interactions, both to threaten, and assault opponents. In dyadic interactions males with larger chelae were dominant over otherwise physically similar opponents. A high frequency of attack behaviour, coupled with a low frequency of threats during these interactions indicates that actual physical contact is required for opponent assessment. Large clawed males oriented females into the copulatory position faster than small clawed males. Females more frequently escaped the precopulatory-grasp attempts of small clawed males. Additionally, male-female pairs that included a large clawed male remained in copula longer than pairs that included a small clawed male. Sperm of the second male to mate took precedence over the sperm of the primary male. Sperm precedence was incomplete; about 900/0 paternity accrued to the second male.

Sperm utilization patterns in Gryllus integer (orthoptera: gryllidae

Sperm competition is the competition for fertilizations between ejaculates, within a female, following multiple mating. There are four sperm utilization or precedence patterns: first male precedence, where the first male to mate fertilizes most of the eggs laid by a female; last male precedence, where the last male to mate fertilizes most of the eggs laid by a female; "all-or-none" pattern, where sperm from either male fertilizes all the eggs laid by a female but which male's sperm that is used is random; or sperm mixing, where sperm from each male is used equally in fertilizing eggs laid by a female. Intermediate utilization patterns are also possible. Sperm competition occurs in a wide variety of insect species as well as other animals. This study was undertaken to study sperm competition in the field cricket, Gryllus integer. Four experiments were conducted: a radiation and sterilization experiment, a diapause experiment, and 2 competition experiments. It was found that 7,000 rad of gamma radiation sterilized adult ~ integer males. There was no diapause in the laboratory in ~ integer eggs. In the first competition experiment, three groups of females were used: females mated with a normal male, then with a second normal male (NN group); females mated with a normal male, and then with a sterile male (NR group); and females mated with a sterile male, and then with a normal male (RN group). The results obtained from this experiment showed that the mean proportion of eggs hatched was significantly different between 3 groups of females, with the proportion hatched much greater in the NN group than in either the NR or RN groups. The pattern for the proportion of eggs hatched following a double mating most closely resembled a pattern expected if sperm mixing is occurring. Results obtained in the replicate competition experiment showed that the mean proportion of eggs hatched for the females in the NR group was significantly lower than the proportion hatched in the other two groups. This also supports a model of sperm mixing as a precedence pattern. Values calculated following Boorman and Parker (1976), for the proportion of eggs fertilized by the second male to mate following a double mating, were 0.57 in competition experiment 1 and 0.62 in the replicate. These values indicate that sperm mixing occurs in~ integer.

Frozen Niagara Falls

The image is described in German. It appears to be Niagara Falls frozen in winter with many tourists and onlookers at the base.

Étude du mécanisme de protection des spermatozoïdes de mammifères par le lait

Le lait écrémé est utilisé depuis plus d’un demi-siècle comme diluant protecteur des spermatozoïdes de mammifères. Depuis quelques années, il existe une demande grandissante pour des diluants exempts de produits d’origine animale. Toutefois, le mécanisme par lequel le lait protège les spermatozoïdes n’est pas connu, ce qui rend difficile de lui trouver un substitut. Les protéines majeures du plasma séminal de taureau, les protéines « Binder of SPerm » (BSP), sont néfastes lors de la conservation de la semence. Les spermatozoïdes sont en contact avec une grande concentration de protéines BSP qui stimulent une extraction continuelle de cholestérol/phospholipides de leur membrane plasmique. Les lipoprotéines de faible densité (LDL) du jaune d’oeuf, un autre composé utilisé dans les diluants, empêcheraient les protéines BSP de se lier à la membrane des spermatozoïdes de taureaux et de stimuler un efflux des lipides membranaires, ce qui les protégerait durant la conservation. Notre hypothèse était que les protéines du lait protègent les spermatozoïdes durant la conservation en séquestrant les protéines BSP. Premièrement, nous avons démontré par filtration sur gel qu’il y a une interaction entre les protéines BSP bovines et les protéines du lait. Le lait écrémé a été fractionné en trois fractions : F1 (alpha-lactalbumine, bêta-lactoglobuline et caséine kappa), F2 (toutes les protéines du lait) et F3 (sels, sucres et petits peptides). Les protéines BSP1 et BSP5 ont une affinité plus grande pour F1 que BSP3, tandis que toutes les protéines BSP ont une affinité pour F2. Le titrage calorimétrique isotherme a permis de confirmer l’interaction entre les protéines BSP et les protéines du lait. L’association entre la protéine BSP1 bovine et les micelles de caséines est caractérisée par une constante d’affinité (Ka) de 3.5 × 10^5 M-1 et un paramètre stoichiométrique (n) de 4,5 BSP1 pour une caséine. L’association entre la protéine BSP1 bovine et l’alpha-lactalbumine (une protéine du sérum principale), est caractérisée par un Ka de 2.4 × 10^5 M-1 et une valeur “n” de 0,8. Ces résultats indiquent que le lait protège les spermatozoïdes bovins en séquestrant les protéines BSP grâce à une interaction protéine : protéine, tandis que le jaune d’oeuf les protège grâce à une interaction protéine : lipoprotéine. Deuxièmement, nous avons démontré par filtration sur gel que les protéines homologues aux BSP bovines retrouvées dans le plasma séminal de porc, d’étalon et de bélier ont une affinité avec les protéines du lait, ce qui suggère que le mécanisme de protection des spermatozoïdes par le lait pourrait être le même chez ces espèces. Troisièmement, nous avons caractérisé l’interaction entre BSP1 bovine et les LDL du jaune d’oeuf qui a un Ka de 3.4 ± 0.4 × 10^6 M-1 et une valeur de « n » de 104 BSP1 pour une particule de LDL, indiquant qu’il existe des différences entre le mécanisme de protection des spermatozoïdes par le lait et le jaune d’oeuf. Nous croyons que les résultats présentés dans cette thèse aideront à créer de nouveaux diluants ne contenant pas de produits d’origine animale afin de cryoconserver les spermatozoïdes des mammifères.

Influence of Processing Variables on Protein Quality and Frozen Storage Stability of Two Commercially Important Species of Squid (Loligo duvaucelii and Doryteuthis sibogae )

Cephalopods are utilized as an important food item in various countries because of its delicacy as raw consumed food. Mainly sepia and loligo are consumed raw by Japanese and Russians. The freshness of the products is very important when the product is consumed raw. The major species that dominate our squid catch are Loligo duvaucelii and Doryteuthis sibogae. There is a noticeable difference in the quality of both the species. The needle squid (Doryteuthis sibogae ) contributes about 35% of the total squid landing. Due to the fast deterioration , a major portion of the needle squid, which is caught during the first few hauls, is thrown back to sea. The catch in the last hauls only are taken to the landing centers. At present the needle squid is processed as blanched rings and the desired quality is not obtained if it is processed as whole, whole cleaned or as tubes. In this study an attempt is made to investigate the biochemical characteristics in both the species of squid in relation to their quality and, the process control measures to be adopted. The effect of various treatments on their quality and the changes in proteolytic and lysosomal enzymes under various processing conditions are also studied in detail.Thus this study can provide the seafood industry with relevant suggestions and solutions for effective utilization of both the species of squid with emphasis on needle squid.