fertilization

today i had an almost perfect fertilization with my super fertile fresh testes! in a petri dish of hundreds of xenopus eggs, only 2 did not fertilize. i'm so good at fertilizing eggs...muahahahah!

here's part of my job (i do this 3 times a week):

• ~one week before fertilization, i inject a low dose of human chorionic gonadotropin into a female xenopus. this is to prime her, or get her eggs ready and at the same stage. (at first, i was really nervous about giving frogs injections, because you can't be like, "okay don't move, or it'll hurt more." but it's okay, seems like they usually can't feel the injection at all!)
  
• the night before fertilization, i inject a high dose of human chorionic gonadotropin into the primed female.
• the morning of fertilzation, i squeeze and massage the female, while aiming her butt at a petri dish. eggs come squirting out into the dish! (easier said than done. you have to grab both legs of the frog, split them apart and stretch them out so she can't kick herself out of your hands. and it's best if you cover her eyes so she feels safe. and you have to massage and squeeze her while you are at it. and you only have 2 hands. i am now an EXPERT at this!)
  
• meanwhile, i drugged a male frog, cut it open, dissected out its testes, and stored the testes in the cold room. they can be used for up to 1 month!
• i cut a small piece of a testis, mash it up with teethed forceps, and touch all the eggs with my testis.
• i wait 3 minutes, allowing all the sperm to attach to the eggs.
• then, i flood the dish with water. the osmotic shock makes the eggs take in the sperm!
• ~1.5 hours later, the single cell eggs should have begun to divide into two cells!
• and my embryos are on their way to growing up!

these xenopus embryos are in two cell stage. i stole this picture, but this is pretty much what my embryos look liked. unfortunately, many times, instead of looking like this, the the embryos are all rotten and smelly.

two jewels of wisdom

i've realized that shauna's blog is (at times) more informative than my blog. so, i've decided to contribute two jewels of wisdom that i have gathered during my time in biomedical research purgatory.

1. specialize in anything chronic

lauren, a dentist turned phd student in a lab that shares space with my lab, studies chronic pain. as a dentist, she specialized in chronic jaw pain. her patients had been in pain for years by the time they saw her. generally, they had no problem waiting another two weeks to see lauren. in her line of work, there are few emergent cases, which means much less oncall type situations and less "sure i'll squeeze you in" type appointments. as opposed to dentists who specialize in acute pain...

2. pick animal models that grow slowly

in my research i work with xenopus laevis (african clawed frog) embryonic spinal neurons. meaning these xenopous neurons are a model for the human neurons we want to know more about. xenopus embryos develop fast and their neurons grow fast, which makes them a convenient model. the neural tube forms in xenopus embryos less than 24 hours post fertilization. and...to culture (cut off a piece of tissue from the back including spinal cord, dissociate the cells, and plate out single cells/neurons on glass) neurons at the stage i want, i have to catch the xenopus embryos in a 1-2 hour window. which means my schedule in the lab revolves around the development of my xenopus babies. AND...because the neurons grow so fast, i have to do my experiments 14-20 hours after plating out my cells. i have to get back to lab and i can't be late! trying to do experiments on neurons much after 20 hours means dying neurons or neurons that are no longer growing (using growing neurons is important for us!!). and thus, my days revolve around xenopus embryo development and neuron growth. as opposed to a rat or mice model. rat and mice development is more or less measured in days. while their neural tube forms closer to ten days post fertilization (more waiting), i can culture embryonic rat hippocampus neurons any time on day 18. and to catch them while they are still growing fast (but not as fast as xenopus neurons unfortunately), i can do experiments on them anytime within the first 3 days after plating!

better yet, skip animal models altogether and join the growing field of computational neurobiology and make the computer work around your schedule.

hmm...seems my two points are actually pretty similar...