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Understanding Gene Expression: Case Study

This case study explores how scientists used small animal models to gather evidence about how the food that the animal mothers were given to eat during pregnancy affected the age at which their offspring entered puberty.

Early Puberty: A Scientific Investigation to Explain Why

  • Small animal models are used to enable scientists to study situations in controlled environments. Strict ethical regulations are adhered to in the design of animal models. 
  • In modern western societies, both under-nutrition and high-fat diets are of concern during pregnancy. Both these types of diets were studied by scientists looking into the relationship between early-life environment and the risk of early puberty and adult disease.

Stage 1: Designing a Model to Mimic Trends in Human Population Data



To find out whether maternal diet during pregnancy affects the age at which offspring enter puberty.



  • Pregnant Wistar rats were fed one of three diets (balanced, undernourished or high-fat)
  • At the age of 22 days (weaning), the offspring were separated from their mothers and split into two groups ‐ balanced diet or high-fat diet
  • The phenotype of the offspring was observed and recorded
  • Dexa scans measured body fat; plasma and tissue samples were taken to measure physiological factors



Both maternal high-fat and maternal under-nutrition during pregnancy lead to changes in phenotype.


Key changes in the phenotype were:

  • Pups born to mothers who had a balanced diet, but were fed a high-fat diet from weaning (22 days) became fat and started puberty early (Fig 4 and 5, dark grey bars)
  • Pups born to mothers who were undernourished during pregnancy were born small, grew quickly, started puberty early and became obese (Fig 4, pink and red bars; Fig 6; Fig 7)
  • Pups from mothers who were undernourished during pregnancy were more likely to eat more, exercise less, show signs of type two diabetes and high blood pressure as adults (graphs not shown)
  • Pups born to mothers who ate high-fat diets during pregnancy were born small, grew quickly, started puberty early and became obese (Fig 5; Fig 8)




The results show that the maternal diet does have an impact on the phenotype. The offspring of both undernourished mothers and high-fat diet mothers entered puberty earlier than the control animals. 


Next Steps

  • What is happening at a molecular level? 
  • Are there changes in hormone levels?
  • Are there changes in the ovaries of the female offspring? 
  • Can we confirm which genes are being affected by a change in gene expression?

Stage 2: Molecular and Gene Studies



To find out what changes are happening to the offspring entering puberty early in terms of:

  • Hormone levels
  • Changes in the ovaries
  • Gene expression of key ovarian regulatory genes


Tissue and plasma samples from the animal models were used to observe:

  • Hormone levels
  • Changes in the ovaries were measured via microscope studies
  • Polymerase Chain Reaction (PCR) studies were used to measure mRNA levels in key ovarian regulatory genes ‐ this tells us whether the genes are being expressed or not  


Changes in Progesterone Levels

The puberty phenotype of the pups from undernourished mothers and high-fat-fed mothers was the same ‐ they all entered puberty early.  However, Fig 9 shows that when it comes to levels of progesterone (a key hormone in the female cycle) we have opposite results. The offspring of the undernourished mothers have very low levels of progesterone while the offspring of the high-fat-fed mothers have very high levels of progesterone. Changes in the level of the hormone in the plasma mean that there are likely to be changes in the level of gene expression. The fact that we have two opposite effects producing the same phenotype suggests that two different pathways are involved ‐ but that they both lead to the same phenotype ‐ early puberty. 




Changes in the Ovaries

Ovarian follicles are densely packed clusters of cells that contain the immature oocyte (egg). A female is born with immature, primordial follicles. During the lifetime of the female, hormonal cues determine if and when these follicles will mature and potentially release an oocyte (egg).  


Offspring of undernourished mothers had fewer primordial follicles in the ovaries. Offspring of high-fat mothers had more primordial follicles in their ovaries.



Free Radicals, Anti‐Oxidants and Oxidative Stress

In an attempt to find out why these changes were occurring, the science team measured the levels of a number of different markers that can indicate oxidative stress.


Oxidative stress is the term used to describe damage to cells, tissues and organs that is caused by a group of chemicals known as reactive oxygen species. These are chemicals such as free radicals or peroxidises which are produced as a result of the metabolism of oxygen and therefore exists in all aerobic species. Free radicals are highly reactive because they have at least one unpaired electron. The level of oxidative stress is determined by the balance between the rate at which free radicals cause damage and the rate at which anti‐oxidants repair damage.


The science team found that the offspring of undernourished mothers had high levels of a molecule called Protein Carbonyl, a marker of oxidative stress, in their ovaries (Fig 10). They also found that the offspring of both undernourished and high-fat-fed mothers had altered levels of an enzyme that regulates levels of hydrogen peroxide - a chemical that is known to cause oxidative stress.


Developmental Programming: The Predictive Adaptive Response Theory

The prenatal environment is having an effect on adult phenotype. Professor Sir Peter Gluckman of the Liggins Institute and Professor Mark Hanson of the University of Southampton have proposed that the effects that scientists have observed are due to a mismatch between the environment that the fetus experiences in the womb and the environment that the child experiences once born. In the womb, the fetus has experienced “hard times” (i.e. there is not enough food) and will make a series of metabolic adaptations in order to survive. As a result of this, the fetus is well adapted for a life of under-nutrition. When it is born into a world where there is plenty of food, the child is not well adapted. This can result in a series of potential problems including obesity, diabetes, heart disease and early puberty.  


Predictive Adaptive Responses

We know from examples in nature that some animals are capable of predicting their adult environment and adjusting their phenotype during development to ensure they are well adapted for the predicted adult environment, improving survival. These animals demonstrate a PREDICTIVE ADAPTIVE RESPONSE. An example of this is the meadow vole, a small animal (similar to a mouse) found in Alaska, Canada and the northern United States. With a life span of around six months, the meadow vole will experience either summer or winter conditions in its life - not both. Coat thickness is determined before birth by maternal signals (the hormone melatonin), related to whether the day length is shortening or lengthening. Meadow voles born at a time when they will live through winter (short days) develop a thick coat, whereas those born to live during summer (long days) will develop a thin coat. This is an example of DEVELOPMENTAL PLASTICITY.  


The phenotype is being determined by gene‐environment interactions, determining which genes are turned on to produce the phenotype most suitable for the predicted environment.  


Developmental plasticity offers a survival, and therefore an evolutionary advantage if the predicted environment matches the actual adult environment as in the case of the meadow vole. However, what we have seen in the evidence relating to fetal origins of adult disease is a mismatch between predicted environment and actual adult environment. When there is this mismatch, problems occur and the results of the plasticity are animals that are maladapted to their environment and therefore have reduced chances of survival for the individual. The animals that experienced poor fetal nutrition are programmed to have an increased chance of early puberty and adult disease. 


Developmental Plasticity

These changes in phenotype are permanent, yet they do not change the genotype. It is likely that these changes in the phenotype have been made in order to maximise the potential of the individual to survive. The fetus develops in a way which will best fit the environment, maximising its potential to survive and reproduce. Scientists believe that there are probably only a few stages during development where the environment is capable of influencing the phenotype in this way. During these stages, we say the development is very “plastic”, it is mouldable. In early life, when the differentiation of cells into specialised tissues is not complete, there tends to be a high potential for plasticity. 


Gene Environment Interactions and Epigenetics

The genotype of an organism, found in the DNA, is expressed when genes are turned on and proteins are synthesised. In order to be expressed, a gene must be unpacked from the condensed state in which it sits wrapped around histone proteins within the chromosome (the DNA—Protein complex is chromatin). This allows the transcription factors access to the gene so that the process of transcription can occur.   


In some gene‐environment interactions, a process called EPIGENESIS is occurring. This process involves non‐genetic factors that do not change the genes themselves but can change the behaviour of the genes. These factors can change the ability of a gene to be accessed within the chromatin and expressed. Epigenetic factors can include changes to the chromatin structure such as the addition of methyl (CH3) groups to the DNA (methylation). Scientists know that environmental influences early in life in a number of organisms cause epigenetic changes to the DNA and therefore change the phenotype, without changing the sequence of bases in the DNA. There is evidence that the packaging of the DNA in the chromosome is changed. This alters the ability of the gene to be unpackaged and transcribed, therefore changing the phenotype.


It is possible that the responses seen in the animal models of poor fetal nutrition and increased likelihood of early puberty and adult disease are caused by epigenetic changes to the DNA. If this is found to be correct, the poor fetal environment is causing an EPIGENETIC change in the DNA packaging which is altering the ability of specific genes to be expressed and therefore altering the phenotype.  



Extension Reading

A number of human and animal studies have suggested that epigenetic effects may be passed on to the next generation, despite the fact that there is no change in the sequence of the DNA. In 2006, scientists showed that epigenetic changes could be passed on to the next generation using a mouse model and investigating the agouti coat colour gene (Cooney, 2006). 


The inheritance of coat colour in the agouti mouse is well known as an epistatic inheritance pattern resulting from interaction of genes on three different loci. The agouti allele (A) is dominant over the non‐agouti (a). The full-colour allele (C) is dominant over the albino allele (c) and the black allele (B) is dominant to the brown allele (b). The agouti gene also controls appetite. A number of variations in the agouti gene exist including Ay=lethal dominant yellow and Avy=viable yellow. Genes A, B  and C show epistatic interaction. No colour is developed unless the dominant C allele is present in the genotype.


Scientists crossed mice carrying the avya alleles (striped‐obese) with mice that were aa (black lean). They found that the offspring of these mice had a range of phenotypes from agouti‐lean brown, to striped obese, and yellow‐obese. This range of phenotype is a result of epigenetic interactions. The more DNA methylation that occurs in the avy allele, the more lean‐brown and the less‐obese yellow offspring there will be in the litter. Supplementing the mother’s diet with vitamin B12, folic acid, choline and betaine will increase the level of methylation on the gene. These supplements are known as methyl donors. Offspring of both mothers and grandmothers who had been fed supplemented diets were more likely to be lean‐agouti brown mice and less likely to be yellow or striped‐obese mice. The fact that the supplemented diet could be that of the mother or the grandmother shows that the methylation effect is being passed onto the next generation - the diet is affecting the germ line developing in the F1 generation and these effects are being carried through to the F2 offspring.


Most epigenetic changes are not passed on. However, evidence that some epigenetic modifications caused by environment that change phenotype are passed on via the gametes now exists.