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.

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