REPRODUCTIVE AGEING: BGP-15 mitigates adverse impacts of aging on sperm quality, fertility, and offspring health in male mice.

In Brief: Aging in men is associated with diminished sperm quality and a higher incidence of altered fetal development and miscarriage in resultant pregnancies. This study in mice identifies a therapeutic compound that, when administered to aged males, improves sperm quality, subsequent embryo development and post-natal offspring health. Abstract: Aging in men is associated with diminished sperm quality and a higher incidence of altered fetal development and miscarriage in resultant pregnancies. We used a mouse model of advanced paternal age to characterize embryonic development in older male mice and tested whether pre-conception treatment with the mitochondrial activator BGP-15 improves reproductive outcomes in old males. Like older men, reproductively old male mice had higher levels of sperm DNA damage and delayed pre-implantation development, associated with a reduced fetal weight and placental weight. Analysis of neonatal outcomes of in vivo-conceived offspring found that pups sired by old males were smaller, had delayed locomotor development, and increased mortality. BGP-15 treatment for 5 days prior to conception reduced sperm DNA oxidation levels and improved on-time embryo development after IVF and pup survival. BGP-15 treatment for 3 weeks prior to conception improved on-time pre-implantation embryo development and fetal viability and increased fetal size in pregnancies sired by old males. These results validate that ageing negatively affects male fertility and offspring physiology and indicates that pre-conception treatment with BGP-15 has the potential to improve sperm quality as well as early embryo development and post-natal health.

Impacts of obesity, maternal obesity and nicotinamide mononucleotide supplementation on sperm quality in mice.

Male fertility and sperm quality are negatively impacted by obesity. Furthermore, recent evidence has shown that male offspring from obese rat mothers also have reduced sperm quality and fertility. Here, we extend work in this area by comparing the effects of both maternal obesity and offspring post-weaning diet-induced obesity, as well as their combination, on sperm quality in mice. We additionally tested whether administration of the NAD+-booster nicotinamide mononucleotide (NMN) can ameliorate the negative effects of obesity and maternal obesity on sperm quality. We previously showed that intraperitoneal (i.p.) injection of NMN can reduce the metabolic deficits induced by maternal obesity or post-weaning dietary obesity in mice. In this study, female mice were fed a high-fat diet (HFD) for 6 weeks until they were 18% heavier than a control diet group. Thereafter, HFD and control female mice were mated with control diet males, and male offspring were weaned into groups receiving control or HFD. At 30 weeks of age, mice received 500 mg/kg body weight NMN or vehicle PBS i.p. for 21 days. As expected, adiposity was increased by both maternal and post-weaning HFD but reduced by NMN supplementation. Post-weaning HFD reduced sperm count and motility, while maternal HFD increased offspring sperm DNA fragmentation and levels of aberrant sperm chromatin. There was no evidence that the combination of post-weaning and maternal HFD exacerbated the impacts in sperm quality suggesting that they impact spermatogenesis through different mechanisms. Surprisingly NMN reduced sperm count, vitality and increased sperm oxidative DNA damage, which was associated with increased NAD+ in testes. A subsequent experiment using oral NMN at 400 mg/kg body weight was not associated with reduced sperm viability, oxidative stress, mitochondrial dysfunction or increased NAD+ in testes, suggesting that the negative impacts on sperm could be dependent on dose or mode of administration.

The presence of a truncated base excision repair pathway in human spermatozoa that is mediated by OGG1.

DNA repair has long been considered impossible in human spermatozoa due to the high level of DNA compaction observed in these cells. However, detailed examination of the base excision repair pathway in human spermatozoa has revealed the presence of an enzyme critical to this pathway, 8-oxoguanine DNA glycosylase 1 (OGG1). This glycosylase was associated with the sperm nucleus and mitochondria and could actively excise 8-hydroxy-2'-deoxyguanosine (8OHdG), releasing this adduct into the extracellular space. This activity was significantly reduced in the presence of cadmium (II), a recognized inhibitor of OGG1, in a time- and dose-dependent manner (P<0.001). Remarkably, spermatozoa do not possess the downstream components of the base excision repair pathway, apurinic endonuclease 1 (APE1) and X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1). The absence of these proteins was particularly significant, as APE1 is required to create a 3'-hydroxyl (3'-OH) terminus at the apurinic site created by OGG1, which would be recognized by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. As a result, TUNEL was unable to detect oxidatively induced DNA damage in spermatozoa following exposure to hydrogen peroxide. In the same cells, intracellular and extracellular 8OHdG could be clearly detected in a manner that was highly correlated with the outcome of the sperm chromatin structure assay (SCSA). However, incubation of these cells for 48 hours revealed a time-dependent increase in TUNEL positivity, suggesting the perimortem activation of a nuclease. These results emphasize the limited capacity of mature spermatozoa to mount a DNA repair response to oxidative stress, and highlight the importance of such mechanisms in the oocyte in order to protect the embryo from paternally mediated genetic damage.

Sperm motility is lost in vitro as a consequence of mitochondrial free radical production and the generation of electrophilic aldehydes but can be significantly rescued by the presence of nucleophilic thiols.

The prolonged incubation of human spermatozoa in vitro was found to induce a loss of motility associated with the activation of mitochondrial reactive oxygen species generation in the absence of any change in mitochondrial membrane potential. The increase in mitochondrial free radical production was paralleled by a loss of protein thiols and a concomitant rise in the formation of 4-hydroxynonenal, an electrophilic product of lipid peroxidation that was found to directly suppress sperm movement. These results prompted a search for nucleophiles that could counteract the action of such cytotoxic aldehydes, as a means of ensuring the long-term survival of spermatozoa in vitro. Four nucleophilic compounds were consequently assessed (penicillamine, homocysteine, N-acetylcysteine, and mercaptosuccinate) in three species (human, rat, and horse). The results of this analysis revealed drug and species specificity in the manner in which these compounds affected sperm function, with penicillamine conferring the most consistent, effective support. This prosurvival effect was achieved downstream of mitochondrial reactive oxygen species generation and was associated with the stabilization of 4-hydroxynonenal generation, the preservation of sperm thiols, and a reduction in 8-hydroxy-2'-deoxyguanosine formation. Theoretical calculations of Fe-S and Cu-S bond distances and corresponding binding energies suggested that the particular effectiveness of penicillamine may, in part, reflect the ability of this nucleophile to form stable complexes with transition metals that catalyze lipid peroxidation. The practical implications of these findings were indicated by the effective preservation of equine spermatozoa for 8 days at ambient temperature when the culture medium was supplemented with penicillamine.

Boronate probes for the detection of hydrogen peroxide release from human spermatozoa.

Human spermatozoa are compromised by production of reactive oxygen species (ROS), and detection of ROS in spermatozoa is important for the diagnosis of male infertility. The probes 2',7'-dichlorohydrofluorescein diacetate (DCFH), dihydroethidium (DHE), and MitoSOX red (MSR) are commonly used for detecting ROS by flow cytometry; however, these probes lack sensitivity to hydrogen peroxide (H2O2), which is particularly damaging to mammalian sperm cells. This study reports the synthesis and use of three aryl boronate probes, peroxyfluor-1 (PF1), carboxyperoxyfluor-1, and a novel probe, 2-(2-ethoxyethoxy)ethoxyperoxyfluor-1 (EEPF1), in human spermatozoa. PF1 and EEPF1 were effective at detecting H2O2 and peroxynitrite (ONOO(-)) produced by spermatozoa when stimulated with menadione or 4-hydroxynonenal. EEPF1 was more effective at detection of ROS in spermatozoa than DCFH, DHE, or MSR; furthermore it distinguished poorly motile sperm as shown by greater ROS production. EEPF1 should therefore have a significant role in the diagnosis of oxidative stress in male infertility, cryopreservation, age, lifestyle, and exposure to environmental toxicants.