Release the sperm!

While preparing a class about synthetic biology, I came across this older paper that actually shows a practical application for synthetic biology.  Kemmer et al. describe a new technique for artificial insemination of cows in the Journal of Controlled Release (in 2011).  I’m not condoning these practices in cows; that is a debate for another day.  I am much more interested in the biology behind this ingenious way of improving the timing of artificial insemination.  Let’s get into it.

Luteinizing hormone

Before I describe the synthetic circuits, we have to go over what the luteinizing hormone (LH) does.  LH is released from the pituitary gland in the brain and travels through the blood to the gonads (in males and females).  In females, there is a huge surge of LH release once a month, which triggers the release of an oocyte (an egg) from the mature follicle in the ovaries.  In other words, increased LH causes ovulation.  The LH hormone binds to LH receptors (LHR) which are expressed on the surface of the target cells in the ovary.  When LH binds its receptors, it triggers a molecular cascade inside the target cell, which leads to the production of another molecule called cyclic AMP (cAMP).  cAMP is a versatile molecule that can initiate lots of cellular responses, like changes in gene expression or activation of enzymes.

The current practice in cow farming is to keep an eye on the female cows and when they appear to be in estrus, then the farmers inject sperm into the cow and hope for the best.  Different cows, though, will have different durations of estrus, so it is sort of a guessing game to time the insemination perfectly.  The LH surge regulates release of the oocytes, so what if you could design a synthetic system that also releases sperm in response to LH?  The sperm will be encapsulated and inert until the LH surge initiates the release of the sperm from their holding cell.  The farmer could inseminate the female cow when estrus appears to be close at hand and the female’s own LH will release the sperm at just the right time when the oocyte is naturally released. 

How can the researchers design a holding cell for sperm that is responsive to LH?

The synthetic circuit

The holding cell is going to be a little hollow bead of cellulose (diameter = 350-400 um).  Cellulose is a naturally occurring molecule made up of lots of glucose sugars hooked together.  The cellulose beads will stay intact unless there is an enzyme called cellulase to break all those bonds between the sugars.  The researchers envelop living sperm and modified mammalian cells inside the microbeads and these get injected into the uterus of the female cow.  The sperm seem to be happy inside the cellulose and are still functional when they are later released.

 

The modified cells have two engineered transgenes:

1) We want these cells to be responsive to LH, so the cells must express the LH receptor.  The researchers find that the rat LHR actually works best, so these cells will have the gene for making the rat LHR.

2) Remember that when LH binds to LHR, there will be a rise of cAMP inside the cell.  cAMP will activate a protein called CREB that binds to DNA and activates expression of genes (I’m skipping a few steps here).  Okay, so LH will bind LHR, cAMP levels will increase, CREB will be activated and will bind to specific DNA sequences in front of genes.  The researchers put the cellulase gene right after a CREB binding sequence in the second transgene.  CREB should bind to the DNA and activate expression of the cellulase gene.

Hopefully you can see where this going now.  When LH is released during ovulation, it will also bind to these modified cells and cause expression of cellulase (the enzyme that breaks down cellulose).  The cellulose surrounding the sperm will be destroyed and the sperm will be released at the same time as the egg.  Bam!

The two pathways initiated by the LH surge.  On the left is one of the modified cells inside the cellulose capsule.

Does it work?  The researchers inserted the cellulose implants into the uterus of Swiss dairy cows.  Next they injected the cows with a hormone that triggers release of LH.  The capsules were degraded and sperm released at the same time as the cow naturally released an oocyte.  Fertilization occurred and embryos developed via this well-timed artificial insemination.  The sperm capsules significantly increase the time window for artificial insemination, which takes the guess work out of insemination. 

Look, synthetic biology working in a useful setting, rather than in bacteria or mice.

Probiotics for autism

The human microbiome is a hot topic in biology these days.  It is becoming clear that the microbes living in and on our body can have major consequences for our health and happiness.  In fact, abnormalities in the gut microbiome may underlie one of the great medical mysteries of our time: autism.   That some bacteria in our intestines could affect our behaviors and brain development is mind blowing.

Hsiao et al. recently published a study in the journal Cell that investigated the connection between the gut microbiome and autism using a mouse model of autism.  They were drawn to this subject based on the fact that individuals with autism spectrum disorder (ASD) often have gastrointestinal abnormalities, like irritable bowel syndrome and increased intestine permeability.

Autistic mice?

Apparently you can produce mice that exhibit the “core communicative, social and stereotyped impairments” associated with ASD, by injecting their pregnant mothers with a molecule that stimulates an immune response.  In humans, maternal infection is linked to increased risk of autism in their children.  The production of these mice was the most questionable part of the paper in my opinion.  They never call these mice autistic, and the mice do show impairments associated with neurological diseases.  So perhaps we should think of it as a model of a generic neurological disorder.  For the sake of simplicity, though, I will refer to them as “autistic mice”, but remember that it is not a perfect model system.

They find that the autistic mice have various defects in their gastrointestinal (GI) tract.  For instance, their intestinal walls are leaky, so molecules that are not supposed to be absorbed can cross from the gut into the blood stream.  This problem seems to be caused by the fact that these mice express less of the proteins that make the tight junctions between cells.  Think of these as fences between cells, so molecules can’t sneak through there into the body.  In an ideal situation, all molecules that are absorbed from the gut must go through the cells, a process which is highly regulated. 

Tight junctions prevent molecules from passing from the gut into the blood.  Image adapted from dbriers.com

They find a number of metabolites that are produced in the intestine from bacteria, which end up in the blood of autistic mice, but not in the normal mice.  In other words, these are potentially toxic molecules that they need to get rid of, but the toxins are leaking into the blood of the autistic mice.  That’s not good.  In fact, if you inject one of these molecules into a normal mouse, it will become more anxious, similar to the autistic mice.  They couldn’t reproduce all of the behaviors of the autistic mice just with this one molecule, but it’s a good proof of principle.  Presumably it’s the build up of all of these metabolites in the blood that cause impairments of the nervous system.

Dysbiosis of the intestinal flora

I love that word “dysbiosis”.  It means that the intestinal microbiome is out of whack.  The wrong types of bacteria are in there messing stuff up.  Hsiao et al. found a number of species present in the autistic mice that were not in normal mice and vice versa.  Presumably this imbalance in the microbiome is what is making the gut leaky. 

To prove this, the authors fed the autistic mice a probiotic (a “good” type of bacteria) called Bacteroides fragilis (B. frag).  Interestingly, B. frag never actually colonized the guts of the mice, but just having it pass through helped to restore the normal microbiome.  Some of the species that were only present in autistic mice disappeared after they consumed B. frag.  The leakiness of the gut was almost completely reversed, including expression of tight junction proteins.  It wasn’t a perfect reversal, but a number of those metabolites in the blood decreased back to normal.

Behavior affected by microbiome

To review: when a pregnant mouse has an infection, her offspring show signs of autism (a mouse-version).  Somehow this infection causes the wrong bacteria to colonize the guts of the offspring.  The dysbiosis leads to changes in gene expression and a leaky gut that allows toxic molecules into the blood stream, thus affecting the development of the nervous system.  Consumption of a probiotic at weaning age fixes a lot of the gut issues.  Does it also reverse some of the behavior impairments associated with autism?

The short answer is yes!  Autistic mice fed B. frag were less anxious, less obsessive, more communicative and interacted more with other mice.  The test for obsessive behavior was kind of cute.  The mice were put in a cage filled with sand with marbles sitting on top.  The autistic-like mice bury a greater percentage of the marbles, demonstrating a stereotyped behavior.

Yogurt from everyone!

If I had an autistic child and read this paper, I would start them on probiotics right away.  I mean probiotics are good for everyone, right, so it definitely seems worth trying.  In fact, the authors say that B. fragilis is depleted in human ASD children compared to matched controls.  Furthermore, probiotics have already been shown to be beneficial in treating chronic fatigue syndrome.   

The authors end their paper with this bold statement: “We propose the transformative concept that autism, and likely other behavioral conditions, are potentially diseases involving the gut that ultimately impact the immune, metabolic, and nervous systems, and that microbiome-mediated therapies may be a safe and effective treatment for these neurodevelopmental disorders.”

Throw another adipocyte on the fire

Humans are able to live in so many different climates, in a wide range of temperatures and yet our inner core body temperature remains nearly constant.  This ability to thermoregulate has something to do, of course, with clothing and the ability to cool and heat our living spaces, but our bodies also offer many adaptations to regulate body temperature.  If it’s too hot, we sweat, releasing excess heat through evaporative cooling.  If it’s too cold, we shiver, producing heat in our working muscles.  The production of heat through physiological mechanisms is called thermogenesis and also includes a non-shivering version.  Today’s paper is about non-shivering thermogenesis, which is when our fat cells produce heat.

Non-shivering thermogenesis

To understand how non-shivering thermogenesis works, we need to take a step back and discuss cellular respiration.  The cells of our body store energy from food in the chemical bonds of a molecule called ATP.  During cellular respiration, a cell will convert glucose or fat into carbon dioxide, while slowly tapping into the energy in those food molecules in order to make ATP.  The final step of cellular respiration is that the energy from the electrons in glucose are passed from protein to protein, releasing energy that is used to pump protons into a membrane-bound cellular space.  You can think of these protons as a form of potential energy, like stuffing a closet full of balls.  When you open up the closet door, all the balls come tumbling out, releasing their potential energy in the process.  During cellular respiration, this potential energy is used by an enzyme to make ATP.  During non-shivering thermogenesis, though, the potential energy stored in all those protons stuffed into a small space is released by the cell as heat.  Thus, the energy from food is used to heat the body rather than being stored in ATP.

The main type of cell that does non-shivering thermogenesis is brown adipocytes, or fat cells.  Brown fat is very common in infants, but is also found in adult humans in the upper chest and neck.  The purpose of brown fat is to provide heat for the body.  Thus, non-shivering thermogenesis is activated by a drop in body temperature.  The cold temperature is sensed by the brain, which activates the sympathetic nervous system (the “fight or flight” response), which signals to the brown fat cells to express the genes necessary to bypass ATP production and release heat instead.  In a recent paper published in PNAS, Ye et al. describe how a different type of fat cell is able to skip all the nervous system steps and sense the cold directly (red arrow in diagram).  It is pretty cool that the fat cells are able to sense temperature, as if they were neurons, and can act autonomously to remedy the situation.  No need for a brain here!

Independent thermogenesis

Through a series of experiments, the authors demonstrate that a particular type of fat cell will express genes necessary for non-shivering thermogenesis when exposed to cold, independent of sympathetic nervous system activation.

In one experiment, they grew fat cells at different temperatures and measured gene expression using a technique called quantitative PCR (qPCR).  The idea behind this technique is that if a gene is highly expressed, there will be a lot of mRNA in the cell (remember the “central dogma” of molecular biology) and qPCR is a method for measuring the concentration of mRNA for a particular gene.  They focused their measurements on thermogenic genes that are known to be part of the non-shivering thermogenesis mechanism, such as Ucp1, which is the enzyme that actually allows the protons to fall back across the membrane, thereby releasing their energy as heat.  They found that these fat cells that were exposed to the cold expressed more Ucp1 mRNA, even in the absence of any nervous system.  These are just cells in a dish, so this must be an intrinsic property of fat cells.

It wasn’t just any fat cell that had this response.  In fact, brown adipocytes did not express more Ucp1 in the cold.  It was a different type of fat cell called a white adipocyte.  What is white fat?  The majority of fat in our body is white fat and its purpose is to store fat for energy (for cellular respiration) and to act as a thermal insulator, so we don’t lose as much heat through our skin.  There is one subtype of white fat that has been shown to do non-shivering thermogenesis and it was this type that could express thermogenic genes, like Ucp1, in the cold, independent of the nervous system.

Okay, so these white fat cells don’t need input from the nervous system, but do they still use the same intracellular pathway to turn on expression of these genes?  Normally, when a fat cell is activated by the sympathetic nervous system, it sets off a molecular cascade of events inside the cell, which involves activation of molecules in a pathway called the cAMP pathway (as shown in the diagram).  The authors inhibited this pathway in various ways and found that the cells could still respond to the cold as before, so this effect must use a different pathway.

There are still a number of open questions, such as: how do fat cells sense temperature?  Do they use the same types of receptors as temperature-sensitive neurons?  Why are some white fat cells independent, but brown fat cells need the nervous system to activate thermogenesis?  One thing that is clear, however, is that white fat cells are clearly important for temperature regulation as well as fat storage.  The authors suggest that tapping into thermogenesis might be a good way to help obese patients get rid of excess energy storage by releasing it as heat.  This pathway that is independent of the sympathetic nervous system could allow medications to target only the fat cells without involving the sympathetic nervous system which controls so many other functions in the body.

Something to think about as the cold Bay Area summer sets in.

(Insert mildly provocative title here)

Ever seen a pair of pigeons going at it?  And did you notice a penis on the male pigeon?  The answer is no, because most birds do not have external genitalia large enough for penetration.  And yet birds reproduce via internal fertilization.  Why would evolution favor male genitalia too small to actually enter into the female?  This just seems so inefficient. 

There are a few birds that do have well developed phalluses, such as the duck and goose.  What happened during evolution that caused some birds to retain a phallus, whereas most other birds lost it?  A paper appeared this week in Current Biology by Herrera et al., which addresses these questions from a developmental point of view.

Developmental arrest

The authors started this study by comparing the development of the phallus in embryos of two different birds.  They chose to look at (1) chick embryos, which are part of the galliformes group of birds and have reduced phalluses and (2) duck embryos, which are part of the anseriforms group, which have well developed, penetrating penises.  They followed the growth of the genital tubercle, the tissue that will form the penis.  As the duck and chick embryos grow, so do their genital tubercles, with no noticeable difference between the two species during the early stages of development.  At a later time period, though, the tubercle stops growing and regresses in the chicks, while the duck keeps on growing.  This shows that the tissue that makes the two different types of phalluses has the same developmental origin.

Why does the genital tubercle stop growing in the chick?

From a molecular stand point, the chick embryos could either lose the “growth” signal or they could gain expression of a “stop” signal not present in ducks.  From work in other animals, the authors knew that there are two major growth signals responsible for guiding the development of the external genitalia – Sonic Hedgehog (Shh) [see my other post about this protein] and Hox13.  These two genes are strongly expressed in the duck genital tubercle throughout embryonic development, as expected.  Surprisingly, though, they are also strongly expressed in the chick embryos.  This means that the chickens haven’t lost the growth signal.

The authors then investigated if there is some sort of a “stop” signal in the chicks.  They found that in chicks and quails, with reduced phalluses, there is a lot of cell death in the genital tubercle in the later stages of development.  This could account for the regression of the genital tubercle.  They then found that the chicks highly express a protein called BMP4 at the tip of the tubercle, which induces cell death, whereas ducks do not. 

In fact, by overexpressing BMPs in the duck, they induced cell death in the genital tubercle.  In the opposite experiment, they inhibited BMPs in the chick and their genital tubercles increased growth, as if they were ducks.

In summary:

            Chicken: + BMP --> increased cell death --> reduced phallus

            Duck:       - BMP --> no cell death, so continued tissue development --> large phallus

Evolution of reduced phallus

So what does this mean?  Chicks and quails have reduced phalluses, because during development, they express BMP4, which tells the developing cells of the penis to die off.  One really cool thing that the authors did next was to look at cell death in the closest relative to birds-- the alligator.  Yah, they got alligator embryos for this research!  Alligators have developed phalluses and they show hardly any cell death in the genital tubercle.  From this work, they could create an evolutionary tree, which shows that chicks and quails most likely evolved the BMP4 signal after their group separated from ducks.  Although the authors didn’t test any members (ha ha) from the neoaves group, which includes most other birds, we can presume that they also have a similar cell death mechanism to reduce the development of their phalluses.

Phylogenetic tree of birds, showing when the BMP signal evolved. (Adapted from Herrera et al., 2013)

This still begs the question of why would natural selection favor a reduced phallus so much so that it evolved independently in different lineages?  The authors propose two different theories, both of which may have occurred:

1) Sexual selection – sure, it may not be favorable for the males to have reduced phalluses, but it might be advantageous for the females.  In order for insemination to occur in these species, the female has to be a willing participant to allow the male to shimmy up next to her and release the sperm in very close proximity.  This gives the females the power to select their mates.  As opposed to species with large penises, where the male could basically rape the female and still successfully pass on his genes to the next generation.

2) Pleiotropy – this term refers to when a single gene mutation can lead to multiple noticeable changes in the body.  BMPs are a major signal during development of animals.  BMPs are involved in a number of bird-only innovations such as feathers and beaks.  Maybe increased BMP expression gave an advantage to these birds, but also lead to reduced phalluses, as a secondary effect.  This may have occurred first in evolution, but sexual selection may have stabilized this characteristic in the population.

This article was so clear and interesting.  I’m sure it will catch people’s attention because of the subject matter, but it’s a great example of using development to solve an evolutionary question.  Plus it gives reviewers and bloggers a great opportunity to think up clever titles and puns for their articles.  The review that was published alongside this article was titled “Cock-a-doodle-don’t”. How can I compete with that?