Placental Histology

Another experiment that I'm pursuing in collaboration with L~ (an undergraduate here at UM) is to analyze placental histology of hybrid hamsters. She found that there seems to be some striking abnormalities in the layering of the hybrids' placentas. Placentas are very complex and formed from three main embryonic tissue layers (labyrinthine trophoblast, spongiotrophoblast, and trophoblast giant cells) as well as a maternal layer (decidua).

***This figure is taken from Wagschal and Feil (2006) - a great paper on imprinting and the placenta***

So far L~ has found that there seems to be a sex-specific placental abnormalities in the hybrids - only the female hybrids show major disruptions of the boundaries between the layers, especially between the labyrinthine trophoblast and spongiotrophoblast. This is quite exciting as I can find nowhere in the published literature that reports sex-specific hybrid phenotypes. It also complicates the over/under-growth story because overgrowth assorts by cross type, not sex while undergrowth is only found in males, not females. If we assume that abnormal layering of the placenta results in abnormal nutrient transfer, then L~'s defects do not explain the patterns of over/under-growth.

However, the issue we have is that the sample size is quite low - 7 males and 2 females total from both hybrid types. We are now curious if the pattern that both females show abnormal morphology is true or only an artifact of small sample size. To address this, I have just set up 10 more crosses (expectation is ~50 offspring/placentas) that L~ and I will be dissecting and doing histology to next week.

I hope to be able to present the findings at the Evolution Conference in Salt Lake in a couple months.

6-4-13 update: So far I have dissected 5 Sun x Cam (overgrown) crosses and 1 Cam x Sun (undergrown) cross resulting in 10 placentas (7 Sun x Cam and 3 Cam x Sun). I fixed these in 4%PFA overnight and used the histology lab's tissue processer to embed them in parafin. Yesterday I sectioned them and put them on slides. Now I need to stain them and then measure them.  I had forgotten how difficult and touchy sectioning is. Major props to L~ for all the sectioning she did on the earlier samples.

6-18-13 update: I have dissected everything, embedded them, sectioned them, stained them, and imaged them. I'm going to present the results at Evolution this weekend and I'll type up a post afterwards.

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Wagschal, A. and R. Feil. 2006. Genomic imprinting in the placenta. Cytogenetic Genome Research 113:90–98.

PCR amplification and the number of cycles

I had earlier decided that amplifying 8ul for 9 cycles would be good for my libraries. It turned out that 9 cycles was much to few and I increased the cycle number to 12 as per J~'s suggestion. I have now amplified all 40 samples at 12 cycles (still 8ul) and have just finished bioanalyzing the results. It seems that 12 has worked great for many of my samples, but has under-amplified a few (see below).

Of these samples, the first 4 are under-amplified, the middle 4 are fine, and the last 4 are perfect. None are over-amplified which is great. 

Next I need to pool all of these samples into two lanes as per my earlier plans. The pooling will be done so that there are equal molar ratios of each sample in the final mix. The issue I am having is that J~ and I had talked earlier and said that I should amplify each sample the same number of cycles (12) but at this point, 12 cycles has under-amplified some of them and not others. One option is to amplify the low samples a couple times more to bring them up to par with the rest. I am unsure of whether treating some samples differently will cause any catastrophic failures of my experiment. It seems like it will not as the expression analysis will be standardized by the relative expression of a number of housekeeping genes. I would guess that this should remove any effects of cycle number, but I'm not sure. I'll talk to J~ and report back.

Below is a plot of all the samples spread along the X-axis and the final concentration (ng/ul) that the bioanalyzer reported on the Y. The different colors represent different batches of library prep (batch size = 8) and the different shapes represent the different RNA extraction batches (batch size = 4). The horizontal line is at 5ng/ul.

It looks like there are no batch effects for the most part. The one main exception is in the red batch where the squares (males) are lower than the circles (females). Earlier, I had started with unequal amounts of RNA for that batch and I ended up re-doing the males. It seems that something went wrong with the 2nd prep of those, and I need to re-prep them again. As long as I am at it I will also re-prep some of the others that are below 5ng/ul.