Establishing the sperm and seminal plasma mechanisms of paternal programming

Our health as an adult can be influenced by how we develop in the womb. Poor maternal diet during pregnancy can increase the risk her offspring will become overweight, have type 2 diabetes and heart disease as adults. While the importance of a good maternal diet is well understood, the importance of the father's diet has been overlooked. However, studies in humans and animals indicate that if the father eats a poor quality diet (too much or too little) at the time of conception, this not only affects the quality of his sperm, but can also affect the long-term cardiovascular and metabolic health of his offspring.

Animal studies have played a significant role in helping us understand how parental diet may affect offspring health. Here, mice have proved especially informative as their reproduction and development are similar to humans. We have shown that feeding male mice either a low protein diet (LPD, under-nourished) or high fat diet (HFD, over-nourished) affects the expression of hundreds of genes within their testes, accelerates the rate their embryos develop and increases the size of their offspring during pregnancy. Importantly, all of these factors have been associated with increased risk of obesity, type 2 diabetes and heart disease in adult life. Indeed, we have shown that adult offspring from male mice fed LPD become over weight and developed symptoms of heart disease and type-2 diabetes. Interestingly, we observed that both the sperm and the fluid they are carried in (seminal plasma) were able to drive offspring ill-health. These observations suggest that a father can affect the health of his offspring in two separate ways; first through information carried in the sperm and second through the seminal plasma. It is these two pathways that we will explore in detail in this project. We are uniquely positioned to use our established mouse LPD and HFD models to define the mechanisms linking a father's diet with the development of his offspring.

Under this proposal, we will first determine how a father's diet affects the genetic information passed on in his sperm. Specifically, we will define the sperm's RNA content, molecules known to influence gene expression within the embryo shortly after fertilisation. We will also study how these RNA molecules are packaged into the sperm as they develop and mature in the testis. Complementing these studies we will explore the cellular structure of the testes to determine how a poor quality diet affects the way they make sperm. Our second objective will study the composition of the seminal plasma and how it interacts with the sperm and the maternal reproductive tract. These interactions are important as the seminal plasma adds additional RNA molecules to the sperm after they leave the testes, preparing the sperm for fertilisation. Additionally, the seminal plasma initiates a range of immune and inflammatory responses within the uterus that prepares it ahead of embryo implantation. Therefore we will study how the seminal plasma modifies the RNA cargo of the sperm and how it affects the proteins, immune cells and blood vessels within the uterus. Following this, we will characterise the interaction between the uterus and the embryo to see if their normal communication is disrupted by poor paternal diet. Finally, we want to understand how quickly the sperm and seminal plasma can return to normal once the LPD and HFD males have been placed onto a control diet. These studies will be important for informing how permanent the effects of a poor quality diet might be for male reproductive health.

This work is timely in its focus on the role of a father's diet. In addition, our focus on the role of the seminal plasma as well as the sperm means our study is also novel. We believe that understating how a father can affect the health of his offspring is critical for both informing men on how improve their chances of becoming a father and on the benefits for the health of their children.

The link between maternal nutrition, embryo development and adult health is well established. However, the impact of a father's diet for the health of his offspring remains poorly defined. We have shown that feeding male mice either an under-nourished low protein diet (LPD) or over-nourished high fat diet (HFD) affected reproductive parameters such as testicular morphology and gene expression, maternal uterine inflammatory status, embryonic development and fetal growth. Underling these, we observed that adult offspring ill-health is programmed equally by both the seminal plasma and the sperm from dietary manipulated male mice. These data link the quality of paternal diet with semen composition, sperm epigenetic status and offspring ill-health. However, the underlying mechanisms require elucidation.

Under this proposal, we will feed male C57BL6 mice either a control diet, LPD or HFD. First, we will define sperm total RNA content by RNA-seq, analysing testicular RNA (coding and non-coding) processing pathways as well as testicular morphology. We will also determine the role of sperm RNA's in directing embryo development and metabolism. Second, we will characterise the impact of paternal diet on the seminal plasma proteome and epididymal extra-cellular vesicle RNA content. Following this, we will determine how the epididymal extra-cellular vesicles and seminal plasma modulate the RNA content of the sperm as well as the maternal uterine immune and vascular environment and the effects these have on embryo-uterine interaction. Finally, we will establish the speed, and extent, to which male reproductive health is restored after returning to a control diet.

This study represents a significant shift for developmental programming, being the first to detail the separate roles of the sperm and seminal fluid and define how they respond to male diet. Findings from this proposal will have translational relevance for human health, being of significance to intending fathers.