Validations of Estrogen Assays in Baleen of North Atlantic Right Whales (Eubalaena glacialis)

Whale baleen has proven to be an accurate method in the retrospective longitudinal analysis of hormones. Baleen plates, the filter-feeding apparatus attached at the upper jaw in mysticete whales, continuously grow and represent a multi-year endocrine record that remains stable without undergoing post-mortem decomposition. While previous studies have quantified steroid and thyroid hormone concentrations in baleen from multiple species to evaluate different life-history events, the role of estrogens remains relatively understudied. Understanding reproduction in the critically endangered North Atlantic right whale (NARW), for example, is vital for accurate population estimate models.Therefore, archived baleen samples from 2 female NARW baleen plates were drilled every 4 centimeters using a dremel, and pulverized into a fine powder. Hormones were extracted from the baleen powder and Arbor Assays enzyme immunoassays (EIA) were used to quantify hormone concentrations. Three estrogen hormones: estrone (E1), estradiol (E2), and estriol (E3), were all validated for NARW baleen through parallelism tests using a pooled sample from non-pregnant females. This demonstrated a sample curve that was parallel to the standard curve, both of which were serially diluted samples: estrone (F1,8 = 0.09058, P = 0.771, r2 = 0.99), estradiol (F1,8 = 4.482, P = 0.0671, r2 = 0.98), estriol (F1,8 = 0.9084, P = 0.3685, r2 = 0.99). These hormones were quantified and compared to previously collected progesterone, stable isotopes, and confirmed calf-sightings to determine the behavior of these hormones during pregnancy, lactation, and resting periods. The data from these two females showed a spike in E2 at the end of pregnancy (after the progesterone (P4) spike) and was stable before pregnancy, which was the expected result. These estrogens appear to provide valuable insight in the study of reproduction (including gestation length and inter-calving intervals) in baleen whales.

Hi everyone! My name is Sarah, and I will be presenting my project on Validations of Estrogen Assays for Baleen of North Atlantic Right Whales. The samples used for this research came from the two whales pictured here…
Their names are Stumpy and Staccato, and they’re females who both died from vessel strikes in 2004. Ship strikes and entanglement are the two top-killers of NARW, and they are critically endangered with only 370 individuals remaining. Of those, only 70 are reproductively active females, meaning that the rate of population growth is limited to how often these females can have calves. Before these two were killed, they were a part of the breeding population; so they had documented pregnancies from regular calf sightings, and Stumpy also died with a full-term fetus.
Previous research on their baleen also confirmed that certain pregnancy hormones were elevated at the same time as these two were assumed pregnant, and subsequently seen with calves.
So what is baleen?
Baleen is keratin (the same structure as your fingernails and hair), and it is what they use to filter feed. It’s arranged in vertical strips that hang from the upper jaw as shown in these photos.
My mentor, Dr Hunt, was on the team that first determined that these baleen plates contain stable steroid and thyroid hormones, and repeated sampling along the length of a baleen plate can represent an endocrine record that spans multiple years of a whale’s life.
Because there is still debate in the large whale research community regarding length of gestation and exactly what happens during pregnancy, I was interested in re-examining these two females, this time, focusing on three estrogen hormones: estrone, estradiol, and estriol.

One of these hormones has been measured in NARW before (estradiol), but the other two (estrone and estriol) have never been measured in baleen whales before. We assumed that hormone extraction methods previously used would also work with these hormones, so we followed the protocol that Dr. Hunt developed.
Briefly, we measured the length of the baleen plate and used a dremel to generate powder every 4 cm along the length of the plate and then weighed the powder to 20mg. Hormones are then extracted from the powder using a MeOH-based protocol, followed by resuspension in assay buffer. Next, we performed enzyme immunoassays for each target hormone. This test allows us to calculate the target hormone concentration in each sample.

Because only one of these hormones has been previously validated for use in NARW baleen, my first objective was to ensure all three estrogen hormones could be reliably used in these samples. Specifically, I ran a parallelism test in each estrogen, and these are my results for that. On the x-axis of each graph, you see the log of the relative dose, and on the y-axis of each graph, you will see the percent of bound antibody. The goal for parallelism is for the standard curve to match the sample curve- both of which are made with serially diluted samples. I used a pooled dilution of non-pregnant samples from the two females (Stumpy and Staccato), and all three estrogens passed for parallelism. This meant that the sample curve was not significantly different from the standard curve (that means they were parallel to one another). We can see that the sample curve for E3 (estriol) only has 3 points; we did test other samples, but it appears a dilution greater than 1:4 did not have high enough concentration of the hormone to be detectable (but a 1:1 to 1:4 is detectable).

This project will continue into the summer, but I wanted to provide preliminary results of what we have seen so far. Because estradiol is typically a major pregnancy hormone, we wanted to assess it along the length of each baleen plate, providing longitudinal information during pregnancy, lactation, and non-pregnant (or resting) periods. We are working on continuing these assays along the length of the plate, so you will see some missing points, but we do have the results from one full pregnancy (in Staccato). Just to orient you on this graph, the x-axis provides the distance from the base (in cm), which really means time, and time moves forward from left to right (the very right side of the graph represents when the baleen plate was collected, meaning when she died).
On each of these graphs, the left y-axis and in the color blue, we can see the concentration of estradiol, while on the right y-axis and in the color green, is the previously published progesterone longitudinal profiles for each female. Stumpy on the left graph (a), has roughly the second half of a pregnancy shown on the left side of her graph (earlier in time), while Staccato (graph b) has an entire pregnancy and beginning of lactation shown. Though we are still working to fill in gaps, the results so far match what we expected. The hormone estradiol (E2) was relatively stable before pregnancy, but rose and peaked toward the end of pregnancy. Progesterone starts to elevate at the start of pregnancy, and maintains higher levels to the majority of a pregnancy.

So to summarize, assay parallelism validations were successful for E1, E2, and E3, which means that I will be able to analyze all three hormones along the length of both Stumpy and Staccato’s baleen plates. This furthers our understanding of the relationship between progesterone and the estrogens before, during, and after pregnancy. Once this is established, we may find similar patterns in other baleen whales, which will be interesting upon further study. This type of research will contribute to our understanding of large whale reproductive cycles, which is generally unknown, and will hopefully aid in population models and conservation efforts for this endangered species.

This project was funded by the OSCAR Undergraduate Research Student Program at George Mason. I’d also like to give a special thanks to my mentors Dr Hunt and Ms. Jelincic, for providing me with the guidance needed to complete this project. I also would like to acknowledge the Woods Hole Oceanographic Institute, for letting us borrow these archived baleen plates.
Thank you so much for listening and I hope you enjoyed learning about these incredible females, Stumpy and Staccato.