A great deal of evidence has accumulated in recent years to suggest that there has been a gradual increase in male reproductive pathology over the past 30–40 years, as evidenced by increased rates of testicular cancer and declining semen quality. The hypothesis is advanced that this phenomenon is causally related to the ability of male germ cells to generate reactive oxygen metabolites. When produced in low levels, such metabolites are thought to enhance sperm function by stimulating DNA compaction and promoting a redox-regulated cAMP-mediated pathway that is central to the induction of sperm capacitation. When produced in excessive amounts, the same metabolites stimulate DNA fragmentation and a loss of sperm function associated with peroxidative damage to the sperm plasma membrane. Free radical-induced mutations in the male germ line may also be involved in the aetiology of childhood cancer and recent increases in the incidence of seminoma. In light of these considerations, establishing the mechanisms for free radical generation by the male germ line and determining the factors that influence this activity are important objectives for future research in this area.
R John Aitken
There has never been a greater need for scientists trained in reproductive science. Most developed countries are witnessing unprecedented rates of recourse to assisted conception sitting cheek-by-jowl with high rates of induced abortion. This article addresses these two incongruous faces of reproductive healthcare. Every year at least 44 million abortions are performed worldwide, many under unsafe and insanitary conditions that carry a significant risk to the lives of women deprived of safe, effective methods for controlling their fertility. Although birth control is a complex issue involving myriad social and political factors, the technical vacuum in this area is significant. Through no fault of the family planning authorities, there have been no radically new methods of fertility control since the oral contraceptive pill was introduced in 1960 and even this contribution to planned parenthood has its roots in the biochemistry of the 1920s and 1930s. Moreover, the pharmaceutical industry has, by and large, turned its back on fundamental research activities in this area. At present, our major investment in reproductive healthcare involves treating ever-increasing numbers of couples with assisted reproductive technologies (ART). However, these treatments are often delivered without critically considering the underlying causes of this condition or seriously contemplating the long-term consequences of the current enthusiasm for such therapy. Significantly, the clinical factors underpinning the commitment of couples to ART include advanced maternal age and a variety of lifestyle factors, such as smoking and obesity, which are known to compromise the developmental potential of the oocyte and DNA integrity in spermatozoa.
Tessa Lord and R John Aitken
With extended periods of time following ovulation, the metaphase II stage oocyte experiences deterioration in quality referred to as post-ovulatory oocyte ageing. Post-ovulatory ageing occurs both in vivo and in vitro and has been associated with reduced fertilization rates, poor embryo quality, post-implantation errors and abnormalities in the offspring. Although the physiological consequences of post-ovulatory oocyte ageing have largely been established, the molecular mechanisms controlling this process are not well defined. This review analyses the relationships between biochemical changes exhibited by the ageing oocyte and the symptoms associated with the ageing phenotype. We also discuss molecular events that are potentially involved in orchestrating post-ovulatory ageing with a particular focus on the role of oxidative stress. We propose that oxidative stress may act as the initiator for a cascade of events that create the aged oocyte phenotype. Specifically, oxidative stress has the capacity to cause a decline in levels of critical cell cycle factors such as maturation-promoting factor, impair calcium homoeostasis, induce mitochondrial dysfunction and directly damage multiple intracellular components of the oocyte such as lipids, proteins and DNA. Finally, this review addresses current strategies for delaying post-ovulatory oocyte ageing with a particular focus on the potential use of compounds such as caffeine or selected antioxidants in the development of more refined media for the preservation of oocyte integrity during IVF procedures.
R John Aitken
Male and female germ lines are vulnerable to oxidative stress. In spermatozoa, such stress triggers a lipid peroxidation cascade that culminates in the generation of electrophilic lipid aldehydes that bind to DNA and a raft of proteins involved in the delivery of functionally competent cells. One set of targets for these aldehydes are the proteins of the mitochondrial electron transport chain. When this interaction occurs, mitochondrial ROS generation is enhanced leading to the sustained generation of oxidative damage in a self-perpetuating cycle. Such damage affects all aspects of sperm function including motility, sperm-egg recognition, acrosomal exocytosis and sperm-oocyte fusion. Oxidative stress in the male germ line also attacks the integrity of sperm DNA with potential impacts on the developmental capacity of embryos and the health and wellbeing of the offspring. Potential pathways of reactive oxygen species (ROS) generation in male germ cells could involve enhanced lipoxygenase activity, activation of NADPH oxidase and/or electron leakage from mitochondria. Similarly, in the female germ line, both the induction of oocyte senescence following ovulation and the deterioration of oocyte quality with maternal age appear to involve the generation of oxidative damage. In this case, the mitochondria appear to be a particularly important source of ROS compromising the viability and fertilizability of the oocyte and interfering with the normal segregation of chromosomes during meiosis. In light of these considerations, antioxidants should have some role to play in the preservation of reproductive function in both men and women; however, we still await appropriate trials to test this hypothesis.
Elizabeth G Bromfield, R John Aitken, Zamira Gibb, Sarah R Lambourne and Brett Nixon
While IVF has been widely successful in many domesticated species, the development of a robust IVF system for the horse remains an elusive and highly valued goal. A major impediment to the development of equine IVF is the fact that optimised conditions for the capacitation of equine spermatozoa are yet to be developed. Conversely, it is known that stallion spermatozoa are particularly susceptible to damage arising as a consequence of capacitation-like changes induced prematurely in response to semen handling and transport conditions. To address these limitations, this study sought to develop an effective system to both suppress and promote the in vitro capacitation of stallion spermatozoa. Our data indicated that the latter could be achieved in a bicarbonate-rich medium supplemented with a phosphodiesterase inhibitor, a cyclic AMP analogue, and methyl-β-cyclodextrin, an efficient cholesterol-withdrawing agent. The populations of spermatozoa generated under these conditions displayed a number of hallmarks of capacitation, including elevated levels of tyrosine phosphorylation, a reorganisation of the plasma membrane leading to lipid raft coalescence in the peri-acrosomal region of the sperm head, and a dramatic increase in their ability to interact with heterologous bovine zona pellucida (ZP) and undergo agonist-induced acrosomal exocytosis. Furthermore, this functional transformation was effectively suppressed in media devoid of bicarbonate. Collectively, these results highlight the importance of efficient cholesterol removal in priming stallion spermatozoa for ZP binding in vitro.
Aleona Swegen, Benjamin J Curry, Zamira Gibb, Sarah R Lambourne, Nathan D Smith and R John Aitken
Stallion spermatozoa continue to present scientific and clinical challenges with regard to the biological mechanisms responsible for their survival and function. In particular, deeper understanding of sperm energy metabolism, defence against oxidative damage and cell–cell interactions should improve fertility assessment and the application of advanced reproductive technologies in the equine species. In this study, we used highly sensitive LC–MS/MS technology and sequence database analysis to identify and characterise the proteome of Percoll-isolated ejaculated equine spermatozoa, with the aim of furthering our understanding of this cell's complex biological machinery. We were able to identify 9883 peptides comprising 1030 proteins, which were subsequently attributed to 975 gene products. Gene ontology analysis for molecular and cellular processes revealed new information about the metabolism, antioxidant defences and receptors of stallion spermatozoa. Mitochondrial proteins and those involved in catabolic processes constituted dominant categories. Several enzymes specific to β-oxidation of fatty acids were identified, and further experiments were carried out to ascertain their functional significance. Inhibition of carnitine palmitoyl transferase 1, a rate-limiting enzyme of β-oxidation, reduced motility parameters, indicating that β-oxidation contributes to maintenance of motility in stallion spermatozoa.
R John Aitken, Jock K Findlay, Karla J Hutt and Jeff B Kerr
Apoptosis is a critical process for regulating both the size and the quality of the male and female germ lines. In this review, we examine the importance of this process during embryonic development in establishing the pool of spermatogonial stem cells and primordial follicles that will ultimately define male and female fertility. We also consider the importance of apoptosis in controlling the number and quality of germ cells that eventually determine reproductive success. The biochemical details of the apoptotic process as it affects germ cells in the mature gonad still await resolution, as do the stimuli that persuade these cells to commit to a pathway that leads to cell death. Our ability to understand and ultimately control the reproductive potential of male and female mammals depends upon a deeper understanding of these fundamental processes.
Miguel J Xavier, Lisa A Mitchell, Kristen E McEwan, Rodney J Scott and R John Aitken
The Big Blue λSelect-cII selection system has been employed along with whole-exome sequencing to examine the susceptibility of the male germ line to mutation in two challenging situations (i) exposure to a chemotherapeutic regime including bleomycin, etoposide and cis-platinum (BEP) and (ii) the ageing process. A 3-week exposure to BEP induced complete azoospermia associated with a loss of developing germ cells and extensive vacuolization of Sertoli cell cytoplasm. Following cessation of treatment, spermatozoa first appeared in the caput epididymis after 6 weeks and by 12 weeks motile spermatozoa could be recovered from the cauda, although the count (P < 0.001) and motility (P < 0.01) of these cells were significantly reduced and superoxide generation was significantly elevated (P < 0.001). Despite this increase in free radical generation, no evidence of chromatin instability was detected in these spermatozoa. Furthermore, embryos obtained from females mated at this 12-week time point showed no evidence of an increased mutational load. Similarly, progressive ageing of Big Blue mice had no impact on the quality of the spermatozoa, fertility or mutation frequency in the offspring despite a significant increase in the mutational load carried by somatic tissues such as the liver (P < 0.05). We conclude that the male germ line is highly resistant to mutation in keeping with the disposable soma hypothesis, which posits that genetic integrity in the germ cells will be maintained at the expense of the soma, in light of the former’s sentinel position in safeguarding the stability of the genome.
Samantha A M Young, Haruhiko Miyata, Yuhkoh Satouh, Masanaga Muto, Martin R Larsen, R John Aitken, Mark A Baker and Masahito Ikawa
IZUMO1 is a protein found in the head of spermatozoa that has been identified as essential for sperm–egg fusion. Its binding partner in the egg has been discovered (JUNO); however, the roles of several domains within IZUMO1 remain unexplored. One such domain is the C-terminus, which undergoes major phosphorylation changes in the cytoplasmic portion of the protein during rat epididymal transit. However, the cytoplasmic tail of IZUMO1 in many species is highly variable, ranging from 55 to one amino acid. Therefore, to understand the role of the cytoplasmic tail of IZUMO1 in mouse, we utilised the gene manipulation system of CRISPR/Cas9 to generate a point mutation resulting in a premature stop codon, producing mice with truncated IZUMO1. Mice without the cytoplasmic tail of IZUMO1 showed normal fertility but decreased the amount of protein, indicating that whilst this region is important for the expression level of IZUMO1, it is dispensable for fertilisation in the mouse.
Neil A Youngson, G Mezbah Uddin, Abhirup Das, Carl Martinez, Haley S Connaughton, Sara Whiting, Josephine Yu, David A Sinclair, R John Aitken and Margaret J Morris
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.