Friday, December 27, 2013

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.

Monday, December 23, 2013

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

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.”