Effects of Prolactin on the Hypothalamic Pituitary Adrenal Axis in Postpartum Female Rats

A thesis submitted to the Miami University Honors Program in partial fulfillment of the requirements for University Honors with Distinction

By : Meredith Beck Faculty Advisors: Dr. Phyllis Callahan and Dr. James Janik

May 2011 Oxford, OH 2

Abstract: Little is known about how the hormone prolactin (PRL) interacts with the Hypothalamic Pituitary Adrenal (HPA) axis during the stress response. In this study, postpartum female rats, which have elevated PRL levels due to lactation, were used to determine how the physiological state of lactation affects the resting sensitivity of the HPA axis. This will help us to further understand how PRL affects HPA axis activity and its potential role in the stress response. I used postpartum females housed with their pups, females separated from their pups for 24 hours or for 8 days prior to experimentation. I quantified levels of Corticotrophic Releasing Hormone (CRH) in the hypothalamus, Adrenocorticotrophic Hormone (ACTH) in the anterior pituitary gland, and circulating Corticosterone (CORT) in all three groups of postpartum females as well as in virgin female rats, which served as my comparison, control group. Compared to virgin females, all postpartum females had elevated, circulating CORT levels whether they remained housed with their pups or were separated from them. Hypothalamic CRH levels among the different treatment groups were not different, but, after 8 days of separation from pups, CRH levels did decrease to approximately half the levels detected in virgin females. Virgin females and postpartum females after 8 days of pup separation had very similar ACTH content in the anterior pituitary while animals housed with pups and those separated from them for 24 hours, had lower ACTH content. We concluded that the postpartum state has the effect of decreasing HPA axis sensitivity by increasing the level of circulating stress hormones. After 8 days of pup separation, CORT levels, a biomarker of HPA axis activity, returned to the pre-pregnancy state, i.e. the levels detected in virgin females.

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Effects of Prolactin on the Hypothalamic Pituitary Adrenal Axis in Postpartum Female Rats

By Meredith Beck

Approved by:

______, Advisor Dr. Phyllis Callahan

______, Reader Shweta Nayar

______, Reader Manu Kalyani

Accepted by:

______, Director, University Honors Program

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Acknowledgements:

Undergraduate Research Award Department of Zoology Honors Programs Dr. Callahan & Dr. Janik Manu Kalyani and Shweta Nayar

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Introduction: Stress is a real or perceived threat to an organism’s steady, resting state (homeostasis), and it stimulates a neural and endocrine response. In order to understand the mammalian response to stress, we must understand the complexities and interactions of both the neurological and endocrine components involved in that response. Activation of the Hypothalamic-Pituitary-Adrenal (HPA) axis is one of the important neuroendocrine responses that occur following stress and it helps an organism reestablish homeostasis (Herman et al, 2003). When an individual is exposed to stress, neural signals are sent to the hypothalamus in the brain and it secretes Corticotrophin Releasing Hormone (CRH) (for review Engelmann et al, 2004). This hormone activates the anterior pituitary gland to produce AdrenoCorticoTrophic Hormone (ACTH) (reviewed in Engelmann et al, 2004). ACTH stimulates the adrenal cortex to secrete glucocorticoid hormones (GCC), of which the most prominent form in rats is corticosterone (CORT). CORT enters the blood and targets the liver, muscle and fat cells to provide the metabolic substrates, primarily glucose, required to generate the metabolic energy necessary for the individual to launch a response to the stress (Herman et al., 2003). Prolactin (PRL) is an anterior pituitary hormone that is best characterized as the hormone of lactation. It promotes milk synthesis and secretion in female mammals (Freeman, et al., 2000). However, circulating PRL levels are also increased by stress (Herman et al., 2003), and this causes increased uptake of PRL into the brain (Mangurian, et al., 1992) through specific PRL receptors (PRL-R) that are expressed in a particular part of the brain, the choroid plexus (Fujikawa, et al., 1995). The physiological role of PRL in the stress response and its possible effects on the HPA axis are not well understood. While the stress-induced activation of the HPA axis is a well-characterized and vitally important process, the gender and physiological state of the individual responding to the stress affects this activation. For example, during pregnancy and lactation, the stress response is reportedly attenuated (Torner et al, 2002), likely due to the profound neuroendocrine changes that take place in females. One major change that occurs in postpartum, lactating females is that they have sustained, elevated PRL levels and increased PRL-R expression in the choroid plexus (Grattan et al, 2001) due to suckling- induced hyperprolactinemia (Pi and Voogt, 2000). This postpartum hyperprolactinemia seems to cause attenuation of the HPA axis to a stressor (Torner et al, 2002). Interestingly, research in our laboratory has shown that resting levels of CRH, ACTH and CORT are elevated in the postpartum condition. It is not known how elevated basal levels of these important stress hormones affect the HPA axis response to stress, nor how long it takes for resting levels to return to the pre-pregnancy state. A better understanding of how lactation affects HPA axis sensitivity will help us to understand how much PRL affects an individual’s ability to respond to stress. It has been proposed that the HPA axis attenuation in postpartum females is essential in order to reduce anxiety so that maternal care is optimized (Bosch et al, 2007). The purpose of this study was to determine how the physiological state of lactation in postpartum females affects the resting state sensitivity of the HPA axis. To accomplish this goal, I quantified CRH levels in the hypothalamus, ACTH levels in the anterior pituitary gland, and CORT levels in the blood of four groups of animals: postpartum female rats that remain housed with their pups, postpartum female rats 24 6 hours after pup separation, postpartum female rats eight days after pup separation, and virgin female rats (controls). The hypothesis tested is that compared to virgin female rats, lactating females have higher basal CORT and lower basal CRH and ACTH, indicating that the hormones of the HPA axis are already elevated l, and therefore, the ability to respond to stress is attenuated. By removing pups from two groups of postpartum females, the circulating PRL levels were decreased, and as the prolactinemic state decreased, the prediction was that its attenuating effects on the female would diminish as well. The results of this study help advance our understanding of neuroendocrine sensitivity in lactating females and has potential implications for helping identify the source of postpartum mood disorders which may result, in part, from reduced HPA axis sensitivity during the postpartum period (Bosch et al, 2007).

Materials and Methods: All animals were housed in the Miami University animal facility and all procedures were approved by the Miami University Institutional Animal Care and Use Committee (IACUC). Experiments were performed using female Sprague Dawley rats during mid-lactation (day10-14), and virgin females were used as controls. For mating, one female was housed with one male, and daily vaginal smears were taken between 0800-0900h; the appearance of sperm in the smear was recorded as the first day of pregnancy. Pregnant females were removed from the male’s cage and housed individually. Two days after females delivered their pups, the litter was culled to 8 pups (to ensure a relatively equal suckling stimulus) and the dams were separated into three groups. In one group, dams remained housed with their pups until the morning of the experiment. In a second group, dams were separated from their pups for 24 hours before the experiment. In a third group, dams were separated from their pups for 8 days before the experiment. A separate, fourth group of virgin females were included as controls. On the day of the experiment, animals were maintained in a stress-free environment until they were sacrificed. Immediately after sacrifice, trunk blood was collected into heparanized (1000 U/ml) tubes and the brain was removed from the cranium and placed in ice-cold saline. The anterior pituitary gland, which is located within the sella turcica of the cranial vault, was separately collected. The brain was transferred to a cold plate and the hypothalamus was microdissected. Tissue was transferred to1.5 ml eppendorf tubes and frozen on dry ice. The tissues were stored in a -80oC freezer until assayed for CRH or prepared for western blot. The blood samples were centrifuged (3000Xg, 5 minutes) and the plasma was collected and kept frozen at -20ºC until subjected to radioimmunoassay.

Radioimmunoassay (RIA): Circulating levels of CORT in the blood plasma and CRH levels in the hypothalamus were quantified by Radioammunoassay (RIA) . The CORT RIA (MP Biomedicals, Santa Ana, CA) was performed on duplicate samples following manufacturer’s instructions. The CRH RIA (Phoenix Pharmaceuticals, Inc., Burlingame, CA) was performed on duplicate samples following manufacturer’s instructions. 7

Western Blot: The anterior pituitary gland was homogenized by sonication in homogenizing buffer (100µl of 1M Tris, 1% SDS and protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO)). A small aliquot (10µl) was collected to determine the protein content (BCA protein assay kit, Pierce, Rockford, IL). The proteins in the sample were separated based on molecular weight by polyacrylamide gel electrophoresis (PAGE, 12 % SDS- polyacrylamide, 1 hr, 200V). After separation, the samples were transferred (80A, overnight) to Immobilon- P transfer membranes (Millipore Corp, Chelmsford, MA). Membranes were treated with blocking solution (8% Non-fat dry milk (NFDM); 5% Bovine Serum Albumin (BSA), 3 hrs). Primary antibodies were then applied to the membranes (rabbit anti-ACTH 1:25,000 and rabbit anti-actin 1:5,000 in 8% NFDM and 5% BSA, overnight). A second, conjugated antibody (goat anti rabbit (GAR) 1:5,000 in 8% NFDM, 2hrs) was added. Then, the ACTH and actin bands were visualized using SuperSignal® West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and developed on Kodak biomax film (Fisher, Pittsburgh, PA). ACTH was specifically identified on the membrane and Actin, another protein unaffected by lactation, was identified and used as an internal control. Optical density of ACTH was quantified using ImageQuant 5.2 analysis software.

Results and Discussion: Compared to virgin females, all postpartum females had elevated, circulating CORT levels whether they remained housed with their pups or were separated from them (Figure 1) . Postpartum females housed with their pups had the highest CORT levels. After 24 hours of pup separation, dams had less than half the amount of CORT than did the group of females housed with their pups. After 8 days of pup separation, the CORT levels were similar to those in the group separated for 24 hours and were still higher than levels in the virgins (Figure 1). The CRH levels in the hypothalamus were not different among the different treatment groups except for a decrease in females after being separated from their pups for 8 days (Figure 2). I expected CRH levels in virgin females to be relatively greater than in any of the postpartum females, especially those housed with their pups, because of the higher levels of CORT in postpartum animals. The high CORT levels indicate HPA axis activation, which requires CRH stimulation of the anterior pituitary gland, and thus less hypothalamic CRH content. It is likely, however, that as CRH was released, more CRH was synthesized to maintain constant, basal levels, i.e. no net change in CRH content, a common characteristic of neurosecretory cells (Brown, 1994); therefore, levels were similar to virgin females. CRH levels did decrease in postpartum females, but only after 8 days of separation. I only determined CRH levels at one time point, so it is possible that the as the postpartum female resumes normal, pre-pregnancy estrous cyclicity and reproductive function, CRH levels are variable. The effect of this change on the stress-induced activation of the HPA axis is not known, but may be a transient effect. Virgin female rats and postpartum females after 8 days of pup separation had the highest anterior pituitary gland ACTH content. The postpartum females housed with their pups and those separated from them for 24 hours had similar levels and these were decreased compared to the other two groups (Figure 3). These ACTH content values were expressed as a percent of the ACTH content in the anterior pituitary glands of virgin 8

non-stressed female rats, rather than normalizing it to an internal control, as is more typically done when quantify western blots (see Figure 4 for a representative blot). These data were expressed this way because I was not able to reliably and repeatedly visualize the actin band in all of my samples (Figure 5). Although I initially detected actin reliably (Figure 4), I had to purchase new actin antibody during the course of this study which negatively affected my ability to visualize actin. In the future, I would perform some preliminary westerns to validate antibody concentration and to ensure that the protein could be reliably detected and quantified across multiple batches of antibody. Overall, these results indicate that, during the postpartum period, the resting activity of the HPA (non-stressed state) is increased and this may lead to a decreased sensitivity to stress. The HPA axis is most highly activated in the group of postpartum females housed with their pups. Lactating, postpartum animals have sustained hyperprolactinemia because they are suckling (Freeman, 2000). Under resting conditions, they also had the highest circulating CORT levels among all four groups, indicating an increased HPA axis activity even when there was no stress. In these animals, pituitary ACTH levels were decreased, and this is likely due to increased ACTH secretion, resulting in increased circulating levels of ACTH and increased activation of the adrenal glands. Research has shown that in lactating animals the stress response is attenuated (Torner et al, 2002), but this attenuation could be aided by the decreased basal sensitivity of the axis due to high resting levels of stress hormones. After being separated from their pups, HPA axis activity seems to transition back to the pre-pregnancy (virgin) state. After 24 hours of pup separation, the suckling stimulus is removed for a short time, but PRL levels return to circulating levels that are similar to those in virgins (Unpublished results, Callahan/Janik Lab). CORT levels are still elevated compared to virgins, but decreased compared to the group housed with pups. The fact that the group separated from their pups for 24 hours had similar ACTH levels compared to the group with pups indicates that the high levels of CORT in the group with pups is not due to ACTH stimulation, but is likely due to another stimulus for CORT release. With 8 days of pup separation, ACTH levels are similar to the levels in virgins, suggesting over 8 days of pup separation, animals are transitioning back to normal HPA axis sensitivity. Even though the CORT levels are still elevated, they are lower than during suckling and ACTH levels are similar to virgins. The relatively constant levels of CRH in the hypothalamus are likely due to the fact that neurons tend to maintain constant neurotransmitter levels, even when secretory activity is elevated (Brown, 1994). The reason for decreased level of CRH in the dams separated from their pups for 8 days is not known, but it represents only one time point and this may be during a transient phase. One interpretation of low ACTH levels in the anterior pituitary gland is that there is increased release of ACTH from the pituitary resulting in high circulating levels of ACTH. Alternatively, it is possible that less ACTH is being synthesized in the anterior pituitary gland. Future research should focus on determining if more ACTH is actually being released into the circulation in these postpartum females by determining the ACTH concentrations in the blood. If these levels are low as well, examining other factors that regulate CORT secretion in the adrenals would be necessary. Additional times of pup separation should also be examined to determine the time course for postpartum females to return to the prepregnancy level. 9

Understanding the relationship between PRL and the HPA axis under resting conditions is important to understanding the regulation of these hormones during stress activation. 10

References 1. Blume A, Torner L, Liu Y, Subburaju S, Aguilera G, Neumann ID. 2009. Prolactin activates Mitogen-activated protein kinase signaling and Corticotropin Releasing Hormone transcription in rat hypothalamic neurons. Neuroendocrinology 150: 1841-1849. 2. Bosch OJ, Musch W, Bredewold R, Slattery DA, Neumann IG. 2007. Prenatal stress increases HPA axis activity and impairs maternal care in lactating female offspring: Implication for postpartum mood disorder. Psychoneuroendocrinology 32: 267-278. 3. Brown, Richard. An introduction to Neuroendocrinology. New York: Cambridge University Press, 1994. 4. Engelmann M, Landgraf R, Wotjak CT. 2004. The hypothalamic-neurohypophysial system regulates the hypothalamic-pituitary-adrenal axis under stress: An old concept revisited. Frontiers in Neuroendocrinology 25: 132–149. 5. Freeman, M. E., Kanyicska, S., Lerant, A., & Nagy, G. 2000. Prolactin: Structure, function, and regulation of secretion. Physiological Reviews, 80(4), 1523-1631. 6. Fujikawa T, Soya H, Yoshizato H, Sakaguchi K, Doh-Ura K, Tanaka M, Nakashima K. 1995. Restraint stress enhances the gene expression of prolactin receptor long form at the choroid plexus. Endocrinology 136: 5608-5613. 7. Grattan DR, Pi XJ, Andrews ZB, Augustine RA, Kokay IC, Summerfield MR, Todd B, Bunn SJ. 2001. Prolactin receptors in the brain during pregnancy and lactation: implications for behavior. Hormones and Behavior 40:115-124. 8. Herman JP, Figueiredo H, Mueller NK, Ulrigh-Lai Y, Ostrander MM, Choi DC, Cullinan WE. 2003. Central mechanisms of stress integration: hierarchical circuitry controlling hypothalamo-pituitary-adrenocortical responsiveness. Frontiers in Neuroendocrinology 24:151-180. 9. Mangurian LP, Walsh RJ, Posner BI. 1992. Prolactin enhancement of its own uptake at the choroids plexus. Endocrinology 131(2):698-702. 10. Pi XJ, Voogt JL. 2000. Effect of suckling on prolactin receptor immunoreactivity in the hypothalamus of the rat. Neuroendocrinology 71:308-317. 11. Torner L, Toschi N, Nava G, Clapp C, Neumann ID. 2002. Increased hypothalamic expression of prolactin in lactation: involvement in behavioral an neuroendocrine stress responses. European Journal of Neuroscience 15:1381-138. 11

450

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Figure 1: Circulating CORT levels in postpartum females housed with their pups (+ Pups, n=11), postpartum females with pups separated for 24 hours (24 hr -Pups, n=11) or 8 days (8 days –Pups, n=10) before experiment, and virgin females (n=8). Animals were left undisturbed in their home cage for 60 minutes prior to sacrifice. Trunk blood was collected at the time of sacrifice. Data are means ! SEM.

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Figure 2: CRH levels in the hypothalamus of postpartum females housed with their pups (+ Pups), postpartum females with pups separated for 24 hours (24 hr –Pups) or 8 days (8 days –Pups) before experiment, and virgin females (n=6 for all groups). Animals were left undisturbed in their home cage for 60 minutes prior to sacrifice. Hypothalamus was collected at the time of sacrifice. Data are means ! SEM 12

102%

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n + Pups o 94% C 24 hr -Pups 92% % 8 days -Pups 90%

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Figure 3: ACTH levels in the anterior pituitary of postpartum females housed with their pups (+ Pups), postpartum females with pups separated for 24 hours (24 hr –Pups) or 8 days (8 days –Pups) before experiment, and virgin females (n=2 all groups). Animals were left undisturbed in their home cage for 60 minutes prior to sacrifice. Anterior pituitary was collected at the time of sacrifice. ATCH bands from western blot films were quantified using ImageQuant 5.2 analysis software. Samples were run in duplicate. The two bands for each sample were averaged to determine the ACTH content of the anterior pituitary tissue. Each treatment is expressed as a percent of ACTH content in virgin (control) females.

1 2 3 4 5 6 7 8 actin

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Figure 4: This representative western blot film of anterior pituitary tissue in which ACTH bands were quantifiable, but the actin was not uniformly detected. 13 1 2 3 4

actin

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Figure 5: This representative western blot film of the preliminary runs of virgin female rat anterior pituitary tissue with the older batches of actin anti-body. The actin bands were uniform across all samples.