Susanne Linderman and Dr. Eric Wilson, Microbiology and Molecular Biology
The purpose of my project was to determine whether or not the hormone prolactin had an effect on CCL28 gene expression. CCL28 is a chemotactic protein (chemokine) that attracts IgA antibody secreting cells (ASC) to the lactating mammary gland. Receptor proteins on the surface of IgA antibody secreting cells recognize the surrounding CCL28 concentrations, and cause these cells to migrate towards the source of CCL28. Since the passive immunity that is passed from mother to infant depends on the IgA that is secreted by these cells, CCL28 plays an important role in infant health. In 2004, Wilson and Butcher established that CCL28 levels increase dramatically in pregnant and lactating mice . However, the question of how this increase is mediated remains unaddressed. In the past, hormones have not been shown to play a role in chemokine gene expression. However, the expression of CCL28 in the mammary gland mirrors that of the hormone prolactin. I hypothesized that prolactin plays an important role in the expression of CCL28. Prolactin is a hormone that is up-regulated during pregnancy and lactation. It plays an important role in milk production, restructuring the mammary tissues, and increasing expression of pIgR in the mammary gland. pIgR is a polymeric Ig receptor that allows IgA to cross over from the mother to the infant. If prolactin is involved in the expression of CCL28, a decrease in prolactin should also cause a decrease in CCL28.
In order to test my hypothesis, I began treating lactating mice with bromocryptine. Bromocryptine is a drug that reduces the level of prolactin. In order to see whether the drug was working or not, beta casein gene expression was monitored as a positive control. Beta casein levels have been shown to be dependent on prolactin, and therefore mirror the concentration of prolactin.
My first experiment showed promising results. I used RT Q-PCR on RNA that I extracted from mouse mammary glands to determine the concentration of CCL28 and beta casein (and therefore prolactin). CCL28 seemed to be dramatically reduced after treatment with bromocryptine, but there was variability between individual mice, and I did not have enough replicates to draw any conclusions. In order to limit variability, I set about optimizing my experimental approach. First I tried injecting the mice for various periods of time. Then I used various dosages of bromocryptine, and prepared the bromocryptine in different manners. It was difficult to find the correct dosage, because most of the work done previously with bromocryptine was done on rats, and I was using mice. In addition, bromocryptine is a powder that must first be dissolved in a solution before it can be injected, and each paper prepared a different solution for injection. I also started injecting twice daily, instead of once daily, and started collecting the mammary gland tissue sooner after the last injection in order to try to achieve the greatest effect. However, after all of these attempts, I was still plagued with variability.
Prolactin did not seem to be consistently reduced after bromocryptine treatment. I hypothesized that this might be due to the effect of suckling on prolactin levels. Studies show that prolactin levels increase dramatically shortly after suckling . I tried separating the mothers from pups for a short period of time before collecting the mammary glands in order to equalize the time after suckling. The time of separation could not be too long, or the prolactin levels of all mice would sink too dramatically, but it could not be too short, or I would have the variability I thought was due to suckling. I even tried separating them for a while, and then reuniting them so that they would all be the same, but that was not effective.
Since reducing prolactin levels with bromocryptine was giving me so much trouble, I decided to try to raise prolactin levels instead. 1-methyl-3,4-dihydroquinoline hydrochloride hydrate is a structural analogue of salsolinol and stimulates prolactin release. My hypothesis was that an increase in prolactin levels would cause an increase in CCL28 levels in the mammary gland.
In the end, I was unable to get consistently reproducible results. Although I tried changing many parameters of my experiment, there are still many reasons why I still had variability. One reason is that prolactin is produced at several locations in the body. Although I was blocking the prolactin production at the anterior pituitary gland with bromocryptine injections, the prolactin made at the mammary gland, may have made up for that decrease in many of the mice. Another reason is that prolactin levels have also been shown to be related to stress. If some mice are more stressed than others, this might be reflected in prolactin levels. Although I always tried to make the entire process as comfortable as possible for the mice, it was impossible to eliminate stress entirely.
Because I was unable to draw concrete conclusions from my data due to variability, I was unable to reject my hypothesis. This means that prolactin might have an effect on CCL28 levels in the mammary gland after all. However, as I have learned, hormones are tricky to work with. They are very dynamic and levels can change very quickly. I have also come to realize that nothing is as simple as it seems. The complexity of regulation in the mammary gland made it much more difficult to control than anticipated. Even with all of the adjustments to treatment times, dosages, drug preparation, and pup separation I was unable to eliminate the variability inherent in my experiments. In order to be able to draw conclusions, further research on this topic will have to be done.
References
- Eric Wilson, Eugene Butcher. 2004. CCL28 controls immunoglobulin (ig)A plasma cell accumulation in the lactating mammary gland and IgA antibody transfer to the neonate. Journal of Experimental Medicine 200(6):805.
- Bodnár I, Mravec B, Kubovcakova L, Tóth EB, Fülöp F, Fekete MIK, Kvetnansky R, Nagy GM. 2004. Stress- as well as suckling-induced prolactin release is blocked by a structural analogue of the putative hypophysiotrophic prolactin-releasing factor, salsolinol. J Neuroendocrinol 16(3):208-13.