Phill Rasnick TA: Nathan Garvin Biology 473/Section 008 April 12, 2013 The Effect of Varying Reproductive Hormone Levels on the Brain-Ovarian Axis in Female Rats Introduction
The regulation of reproduction and the hormones involved takes place through hormonal feedback loops between the brain and gonads; in females, this relationship is known as the brain-ovarian axis and is under control by the hormones gonadotropic releasing hormone (GnRH), follicle stimulating hormone (FSH), luteinizing hormone (LH), estrogen, progesterone, and inhibin. The menstrual cycle is initiated by an increase in gonadotropins from the anterior pituitary including FSH and LH. Under the influence of FSH follicles within the ovary begin to grow and develop. FSH and LH also respectively stimulate the granulosa cells and thecal cells of the follicles to secrete steroid hormones which are converted to estrogen by the granulosa cells. Increasing estrogen levels has a negative feedback on the pituitary FSH FSH and LH secretion, secretion, preventing the development development of additional follicles. Despite Despite the the decreasing FSH and LH levels, the increasing estrogen levels stimulate a further increase of estrogen through a positive feedback pathway with the granulosa cells. Uterine endometrium proliferates under the influence of estrogen by increasing in cell number and enhancing blood supply to prepare for the implantation of a fertilized egg. Once follicular development reaches a particular point, there a large surge in estrogen levels with an accompanying secretion of inhibin and progesterone. During this time, estrogen and progesterone enhance the pituitary’s responsiveness to GnRH. While estrogen provides negative feedback on gonadotropin release through inhibition of the hypothalamus and anterior pituitary, persistently high estrogen estrogen levels levels provide positive positive feedback, feedback, leading leading to a surge in GnRH followed followed by largely increasing section of LH and FSH. Inhibin secretion, however, inhibits FSH secretion resulting in the FSH peak to be significantly reduced when compared to the LH peak. The high levels of LH are responsible for ovulation to occur. This is marked by the egg being released from the follicle and entering the fallopian tubes. Without the egg, the follicle develops into a
2
structure known as the corpus luteum, responsible for producing increasing amounts of progesterone and estrogen. These hormones exert negative feedback on the hypothalamus and pituitary suppressing gonadotropin secretion in addition to the suppression by inhibin production. The corpus luteum has a lifespan of only a few weeks and spontaneously undergoes apoptosis if pregnancy does not occur. Estrogen and progesterone levels decline, stopping the negative feedback on the gonadotropins and causes the levels of FSH and LH to rise again as the menstrual cycle restarts. Because the endometrium is maintained by the presence of progesterone, decreasing levels of progesterone cause the endometrium to break down resulting resulting in menstrual discharge. discharge. In this experiment, unilateral and bilateral ovariectomies are carried out in addition to sham operations to observe the roles of these circulating reproductive hormones as well as the effect of varying hormone levels on the female sexual structures in a rat. It is hypothesized that performing a unilateral ovariectomy on the rat will result in an increased size of the second ovary when compared to the control group. This is expected because with removal of an ovary, the other ovary may need to overcompensate to ensure levels of estrogen are sustained. The single ovary in the unilateral rat will be exposed to a higher concentration of gonadotropins because the other ovary is not there to make use of them. This should result in the single ovary producing more estrogen than normal. Additionally, the decreased estrogen levels should also result in less negative feedback on gonadotropin production, further increasing circulating gonadotropin levels and also estrogen production in that single ovary. In terms of the uterine horn, it is hypothesized that there should be no significant change in the size between sham operation and unilateral ovariectomy groups because levels of estrogen in both groups are expected to be elevated enough to sustain the endometrium. However, when comparing the bilateral ovariectomy results to the control and unilateral data, it is expected that there should be a significant decrease in size of the uterine horn of the bilateral experimental group due to estrogen production being diminished. When considering the effect of varying reproductive hormone levels on the pituitary gland, it is expected that there will be an indirect relationship between pituitary size and levels of circulating estrogen due to a decrease in negative feedback on the pituitary resulting from diminished estrogen levels. As a result, it is expected
3
that rats post sham operation will have a slightly smaller pituitary glands when compared to the unilateral or bilateral ovariectomy groups. Also, because of the decreased negative feedback, it is expected that rats from the unilateral test group will have pituitary glands larger than the control group but smaller than the bilateral ovariectom ovariectomy y group. Thus, pituitary size in post post bilateral bilateral ovariectomy ovariectomy rats is is expected expected to be greater in size than the pituitary gland for both the control and unilateral ovariectomy groups. From a clinical standpoint, the data collected during this experiment can be useful in identifying the effect of menopause on females. This is because conducting an ovariectomy is the surgical equivalent to menopause, marked by the time when the ovaries can no longer be receptive of gonadotropin signals thus producing no estrogen. Methods
In this experiment ovariectomies were carried out to provide information on the effect of varying levels of circulating reproductive hormones. Data was collected for both unilateral and bilateral ovariectomies, ovariectomies, with a sham operation providing results for the control. The term ovariectomy refers to the surgical removal of an ovary. Unilateral ovariectomies revolved around the removal of one ovary, while the bilateral procedure removed both ovaries from the rats. Experimental protocol was followed exactly from the biology 473 Rodent 473 Rodent Survival Survival Surgery Surgery Protocol Protocol Handout. Sterility Handout. Sterility was maintained throughout the entire procedure, and local anesthetics were used to ensure proper handling of the rats. Pre surgery, Ketamine was first used to bring the animal to a dream-like state of mind followed by use of xylazine as an analgesic and muscle relaxant. Bupivacaine was additionally added post-surgery to the sutured muscle wall to provide further pain relief. To determine determine the effect of varying levels of reproductive hormones on the brain-ovarian axis, the animals were euthanized 3 weeks post-surgica weeks post-surgicall procedure, procedure, and gonad, gonad, uterine uterine horn, and pituitary weights were collected. Two tailed t-test analysis was used to identify the significance of the results. To identify significance, an experimental alpha value of 0.05 was used. Because 3 different tests are being carried out, the Bonferroni method was used to correct the alpha comparison comparison level to a value of 0.01.
4
Results Figure 1: The Effect of Ovariectomy on Ovary Size in Control and Post Unilateral Overiectomy Rats
Effect of Ovariectomy on Ovary Size 0.180 0.160 0.140
) 0.120 g ( t 0.100 h g i e 0.080 W 0.060
Control Unilateral
0.040 0.020 0.000 Experimental Group
Figure 1: Displays the mean ovarian weights for collected from sham operated control groups and unilateral ovariectomy groups. The Ovariectomy appeared to have a positive effect on ovary size in the post unilateral unilateral experimental experimental group. Average Average ovary size in the control was was found to be 0.109 grams grams while the ovary in post ovariectomy rats averaged 0.148. Standard error was additionally calculated to provide the approximate margin of error for each experimental group.
Based on the results displayed in figure 1, performing the ovariectomy resulted in an increase of size for the remaining ovary of the unilateral ovariectomy group when compared to control data. T-test calculations were used to indicate that the ovary size in the unilateral ovariectomy test group was significantly greater than ovary size for the control treatment (t=3.065, df=9, α2=0.01).
5
Figure 2: The Effect of Ovariectomy on Uterine Horn Weight
Effect of Ovariectomy on Uterine Horn Size 0.700 0.600 0.500
) g ( t 0.400 h g i e 0.300 W
Control Unilateral Bilateral
0.200 0.100 0.000 Experimental Group
Figure 2: Presents experimentally collected data for uterine horn weights post unilateral ovariectomy, bilateral ovariectom ovariectomy, y, and sham sham operated operated rats. No significance significance was found found between between weight differences among control and unilateral experimental groups, however, the bilateral ovariectomy resulted in a significant decrease in uterine horn weights when compared to either control or unilateral test groups. Average weight of the uterine horn post sham operation, post unilateral ovariectomy, and post bilateral ovariectomy were 0.602 grams, 0.569 grams, and 0.154 grams respectively. Standard error was additionally calculated to determine the error interval for each experimental group.
Results from figure 2 indicate that unilateral ovariectomies had little to no effect on the size of the uterine horn when compared to control groups. Statistical calculations show no significance difference in uterine horn weights between control and unilateral operated groups (t= 0.503, df=30, α2=0.01). Although no difference was observed between the unilateral unilateral and control groups in terms of uterine horn size, t-test analysis demonstrated that the uterine horn size had been significantly decreased in the bilateral group when compar ed ed to the control (t=7.232, df=23, α2=0.01). Additionally, there was a significant decline in uterine horn size in the bilateral experimental group in comparison to the unilateral test group (t=5.767, df=23, α2=0.01)
6
Figure 3: The Effect of Ovariectomy on Pituitary Gland Weight
Effect of Ovariectomy on Pituitary Size 0.020 0.018 0.016 0.014 ) g 0.012 ( t h 0.010 g i e W0.008 0.006
Control Unilateral Bilateral
0.004 0.002 0.000 Experimental Group
Figure 3: Presents the experimentally collected data for average pituitary size in the experimental rat populations for the unilateral unilateral ovariectomy ovariectomy,, bilateral ovariectomy, ovariectomy, and sham operation test test groups. For the control, unilateral, and bilateral test groups, average pituitary weight collected was 0.016, 0.017, and 0.014 grams respectively. Standard error was calculated to approximate error bars and the interval for error for each experimental experimental group.
Based on the results in figure 3, no significant change was found in pituitary size between any test groups. T-test identified no significant difference between pituitary size for control and unilateral test groups (t=0.343, df=27, α2=0.01). Statistical analysis also i dentified no significant difference in pituitary size between control and bilateral ovariectomy ovari ectomy groups (t=0.646, df=20, α2=0.01). There was additionally no significant difference determined for weight size between unilateral and bilateral test groups (t=0.962, df=19, α2=0.01).
Discussion
In support of the hypothesis, the t-test showed that there was a significant difference in ovarian size between the sham operation control group and the unilateral ovariectomy experimental group. Figure 1 indicates that the unilateral ovariectomy had a significant effect on ovary size, resulting in an increase in ovary ova ry size for the group in which unilateral ovariectomy had been performed. This could be due to an increase in estrogen production for that single ovary, resulting from excess stimulation from the gonadotropins. The cause of this could be a
7
decrease of negative feedback on the pituitary resulting from lower levels of estrogen. These results are in strong support of the hypothesis because of the indicated significance between the two groups. The t-test additionally showed strong support for the hypothesis involving the effect of ovariectomies on the size of the uterine u terine horn. A significant difference was observed between the control and bilateral groups as well as between betw een the unilateral and bilateral groups. According to figure 2, the size of the uterine u terine horn was significantly decreased in the bilateral experimental group when compared to both control and unilateral groups. Because estrogen is responsible for maintaining the endothelial lining of the uterus, it was expected that without estrogen, as occurs oc curs in the bilaterally treated group, the endothelium will deg enerate. No difference was observed between unilateral and control groups in this test because the ovary in rats that had undergone the unilateral procedure is still capable of producing e nough estrogen to support the uterine endothelium Opposing the hypothesis, no difference was observed in pituitary weight between any of the tested groups as shown in figure 3. Also known as the “master gland,” the pituitary gland is responsible in controlling numerous processes in the body, secreting more hormones than just the gonadotropins discussed. Some of these hormones include growth hormone for the regulation of growth, thyroid stimulating hormone to regulate the thyroid and metabolism, as well as prolactin which is involved in the production p roduction of breast milk. As a result, the size of the pituitary gland may not be significantly affected by levels of estrogen due to the numerous other othe r processes affected by the gland. Due to the relatively small size of the pituitary gland, and the fact that the feedback loop between the gonadotropins and sex hormones occur on the anterior pituitary, even if a difference did occur it may not have been identifiable. Further tests using a bigger sample size
8
and additional means of measuring size of the anterior pituitary could be better used to in dicate any differences in pituitary size due to chang es in estrogen levels. The main sources of error in this experiment are a limited sample size and issues associated with collecting the organs post procedure for me asurement. For the rats that had undergone ovariectomies, it was identified that many of the uterine horns were inflamed and swollen with excess scar tissue as a result of the procedure. P rior to collecting uterine horn weights, much of the swollen tissue was removed. This could have resulted in a decrease d ecrease in uterine horn weight for the ovariectomy test grou ps, however, based on the results obtained it is unlikely that this slight change accounted for the significant reduction in uterine horn size identified in the bilateral experimental group. Additional error could ha ve occurred with collecting data on the pituitary gland. As indicated by data, several groups were incapable of isolating the pituitary gland. Because of the small size of the pituitary gland, if the organ was not n ot collected intact, there would be a negative effect on the observed weight. As discussed, further experiment should be used to help h elp identify the effect of estrogen on pituitary size. Overall, the results of this data can be used to identify issues that occur in postmenopausal women because an ovariectomy is the surgical equivalent to menopause. After menopause, ovaries lose the ability to be regulated by circulating gonadotropins resulting in an absence of estrogen production. This is carried out in experiment through ovariectomy by removing one or two ovaries causing a respective decrease or termination of estrogen production. Due to the lack of estrogen, menopausal women are susceptible to varying va rying symptoms including hot flashes, genitalia and breast atrophy, and as well as osteoporosis. Because of the issues associated with depletion of estrogen, menopausal women are often subject to hormone replacement therapy which typically consists of both e strogen and progesterone. In more recent
9
years, selective estrogen receptor modulators (SERMs) have been us ed to treat issues associated with menopause by selectively binding to estrogen receptors at different affinities.
10
References
Overview of Reproductive Hormones. PSU, Biol 473 Laboratory Sp 2013 Biol. 473 Rodent Survival Surgery Protocol Handout. PSU, Biol 473 Laboratory Sp 2013 Guidelines for Analyzing Your Reproductive Endocrinology Data. PSU, Biol 473 Laboratory Sp 2013 Silverthorn, Dee. Human Dee. Human Physiology Physiology an Integrated Approach. 6 ed. Glenview, IL: Pearson, 2013. Print.
