Janerys Rodríguez Montalvo
INTERNSHIP AT THE UNIVERSITY OF WISCONSIN-MADISON
Genitourinary Development and Hormonal Influences; Urinary Bladder Tissue Engineering
OBJECTIVES
Our main objectives are identifying specific hormonal pathways critical for urogenital development in Sprague Dawley during puberty. In addition, we aim to assess the impact of gonadotropin-releasing hormone (GnRH) blocker treatment, Leuprolide, on the development of specific urogenital structures, such as the testes and penis.
JUSTIFICATION
Understanding the intricate relationship between genitourinary development and hormonal influences is crucial for improving healthcare, particularly for transgender youth. During puberty, a complex interaction of hormones shapes the urogenital tract. This research delves into this process in rats, tracing their urogenital development through puberty and into adulthood. By examining the effects of specific hormonal blockers on urogenital morphology, we aim to gain valuable insights into how these medications impact the developing genitourinary system. A broader understanding of hormonal influences on other organ systems during development will be possible through any breakthroughs or contributions during experimental methodology.
Figure 1: Vector
Within this approach, it holds significant potential for improving transgender healthcare. If hormonal blockers, such as gonadotropin-releasing hormone (GnRH) are found to cause unforeseen morphological changes, the findings can start the development of alternative treatment strategies or more targeted blocker use. Additionally, the knowledge acquired on the impact of hormones on urogenital development could pave the way for bioengineering solutions. These solutions could potentially assist with urogenital function in individuals who have undergone hormonal therapy, offering a novel approach to address potential complications with the use of different biomaterials. Ultimately, this could lead to more informed treatment options and improved quality of life for transgender individuals and patients in general suffering from congenital urological issues.
EQUIPMENT AND PROCEDURES
Understanding the equipment involved in various research techniques is crucial for a successful and reliable data collection. This is particularly important when designing and conducting studies that combine multiple procedures, such as PCR (Polymerase Chain Reaction) and behavioral testing. By outlining the necessary equipment, we can ensure proper execution and concise planning within the lab. This will be a complete guide to understanding the key questions as to what, where, and why to every essential in this research. All these procedures are done with personal protection equipment.
a. MARKING,MORPHOLOGY, AND WEIGHT: (MMW)
Within this protocol, the rats are subjected to a scale every day. This is to track their growth throughout the weeks, as the shots of Leuprolide are administered by weight. After weigh-ins, they are inserted into a towel to mark their tails with Sharpie to identify them (example: two-dot rat from box 3). Finally, in morphology, the rats stay in the towel as we place one hand on the rat's pelvis. With the other hand, two fingers are placed around the penile-scrotum area to expose the penis. This is done to examine their genital development.
Figure 2: Rat in scale
Figure 3: Rat in the towel for marking and morphology
An example video, where the hand placement and exertion of the penis is done. This is the morphology part of the research to assess their development throughout the weeks, which is then written down on an official document:
Video 1 : Morphology
Table 1: Document for MMW assessment
b. BLOOD DRAWS
Blood draws are important to recognize within the study, as they can show us how the rats are complying with the administered hormone Leuprolide. This process involves a specific protocol so the blood collection can be completed. Weighing the rats is required, as it tells us how much blood must be extracted. Then one of the rat's legs is shaved to expose the skin and facilitate the puncture. Vaseline is smeared across the skin that was shaved to help see the vein and so the blood doesnt get caught up in the fur.
Table 2: Weight to blood ratio sheet for the rats
Figure 4: Puncture preparation (shaving)
Once the vein is found, the rat is punctured. Sometimes the rats may move a lot, so extra pressure must be put on their arms for them to stay still. Usually, the needles that are used are in the 20G1-22G1 needle range. Then, the blood that is collected will be found inside a capillary instrument specialized for small amounts of bodily fluid. When the capillary is full, we put a cotton ball on the blood area so it can clot and seal. There won't always be a successful collection of 200μL of blood, so sometimes it is necessary to redo most of the process. Finally, the used needles and capillaries are discarded in the bio-hazard bin.
Figure 5: 21G1 needle and biohazard bin
Figure 6: 200μL capillary blood collector
Finally, the samples are put into a centrifuge for 10 minutes to seperate the particles in the blood(plasma, red blood cells, and white blood cells). We want the serum that is present in the blood sample for further experimentation on its biochemical composition and the influences Leuprolide has taken on the rats. After it is extracted, the sample is put in a freezer to preserve said serum for analysis on its interaction with the specific GnRH drug.
Figure 7: Centrifuge utilized at 25° C
Figure 8: Extraction of serum
c. HORMONAL INJECTION
The hormonal injections of Leuprolide are administered in the early stages of development of the rats. It is important to note that there was no specific dosage, as mentioned earlier it is all dependent on their weight. Every rat will grow at a different rate so the dosage is described as ((n.) mL). The injections were administered the first three weeks after their date of birth. It is not necessary afterwards because they reach a full sexual maturity at the six week mark, so it is assumed that by the fourth week they are in the pubescent stage where urogenital change is expected to occur . ( As seen in Table 1 )
d. POLYMERASE CHAIN REACTION (PCR)
Polymerase Chain Reaction or PCR is utilized in hormonal research to identify possible mutations within the model organism (in this case, the rats) . This helps us distinguish unbalances present to measure hormone gene activity. This provides clues on how much hormone a cell is programmed to make. Techniques like PCR can indirectly tell us if a hormone receptor is upregulated or downregulated. By looking at gene expression, we can see if a cell is making more or less receptors for a specific hormone, influencing how sensitive the cell is to that hormone's signal.
Figure 10: PCR setup
To start the procedure, an additional initial setup is required. First, a bucket of ice is needed to put the solutions inside, including 300μL of Sybr dye, 179.1 μL of water, and 0.45 μL of primers for genes involved in GnRH signaling. A tube rack is also found within the ice bucket to maintain a cool temperature since most solutions could melt at room temperature but freeze under contact with ice. Once the microtubes are inserted with the samples of interest they are inserted into a thermal cycler and revised the next work day.
Figure 11: Bucket of ice with the solutions
Figure 12: Thermal cycler utilized
Subsequently, the thermal cycler will present the data that was retrieved onto the CFW Maestro Software. This instrument facilitates data import, visualization of amplification curves, quantification of target DNA, and basic statistical analysis. This helps assess reaction efficiency and identify potential issues within the samples retrieved from the rat's hormone levels. It is also used to compare gene expression levels and ensure the statistical significance of the results between the rats. The analysis involves two main pages of recovered data, which will be further explained in the 'Conclusions & Results' section.
Figure 13: CFW Maestro Software
e. BEHAVIORAL TESTING
This research leverages Leuprolide's ability to disrupt the natural hormonal homeostasis. By employing established paradigms like the Elevated Plus Maze (EPM), which exploits a rodent's inherent aversion to open spaces. For instance, Leuprolide-induced suppression of testosterone in males is expected to translate to decreased anxiety-like behaviors within the EPM. Furthermore, observing social interactions between rats within treatment and control groups can reveal how Leuprolide affects their behaviors, providing valuable insights into the social ramifications of hormonal disruption.
Figure 14: Elevated Plus Maze
This process needs to be done under particular circumstances, the room must be lit with a red light to facilitate lab staff visibility but to detour the rats to focus on the maze. The rats are put under a bucket for 10 seconds, then the bucket is lifted with a polley system and they have 4 minutes to find a box under the table by getting into one of the holes. As the maze is cleaned with EtOH, no trace of smells are left behind, so the rats are on their own to find the box. If after 4 minutes the rats cannot find the box, they are pushed with a black board to the box. Then, we measure one minute of them inside of the box. After the procedure is completed, we immediately take the rat back to the cage and clean the maze area once again to proceed with the next rat.
Video 2: Example of Elevated Plus Maze
Conclusions and relationship with previous investigations
When we put into consideration the relationship of this project with other research, we see that there are still things to understand, such as the different aspects of hormonal influences in the urogenital developement. Advancements in this field hold immense promise for regenerating damaged or diseased urogenital tissues, potentially offering new avenues for treatment. Wihth that said, further investigation is needed to fully understand the relationship between hormones and genitourinary structures. This project has explored the connection between genitourinary development, hormonal influences, and the potential applications of tissue engineering.
Through thorough analysis, some key aspects will be discussed by itself and in comparison with other projects. This comparative analysis will not only highlight the strengths of our current project but also identify areas where future research efforts can be most impactful. Ultimately, such insights can pave the way for the development of more targeted and effective tissue engineering strategies for urogenital tissue regeneration and hormonal development of urogenital organs.
This project focused on the processes of physiological and urogenital changes during puberty. A major determination was in how hormonal influences act strongly for physical maturation. LH (Luteinizing) and FSH(Follicle-stimulating) hormones stimulate the gonads to produce sex hormones, with rising testosterone levels acting as a brake on GnRH production in the brain, maintaining hormonal balance. GnRH agonists disrupt this rhythm by constantly stimulating GnRH receptors, leading to high and constant testosterone levels. This effectively pauses puberty by suppressing the natural pulsing of GnRH and reducing LH stimulation of the gonads . While initial testosterone exposure is crucial for testicular development, ongoing functionality doesn't require sustained levels. Long-term use of GnRH agonists like Leuprolide, which rely on androgens, can even lead to tissue regression.
Figure 16: GnRH process, physiological and gonad effects (made by me)
GnRH agonists affect GnRH signaling in the brain and how this translates to gene expression changes in the urogenital organs, providing a comprehensive picture of the drug's impact (A) . Here, molecular and histological markers would be essential to definitively determine if this process is altered (B) . Additionally, analyzing gene expression in urogenital organs and keratin holds promise for developing predictive models for UTI risks, potentially leading to more personalized medicine (C). It's important to note that for some aspects, tissue analysis might provide more informative data compared to blood draws. Now, regarding the PCR analysis of Anti-Mullerian Hormone AMH protein levels in the testes after Leuprolide administration, it wouldn't directly tell you about successful puberty development (D), where different levels or hormones may be present.
Through experimentation, a possible explanation is to understand that PCR measures gene expression and amplifies DNA sequences, indicating how actively a gene is being transcribed, not the final protein product. That is where the Anti-Mullerian Hormone (AMH) and Puberty come into play: AMH is involved in early testicular development, not directly in testosterone production or puberty progression. Its main role is preventing female reproductive structures from forming in males during embryonic development. However, we can interpret the results in two ways, depending on the treatment goals. With a low AMH, if PCR shows low AMH gene expression after Leuprolide, it might suggest suppressed Sertoli cells in the testes (partially regulated by testosterone). This wouldn't necessarily be a bad outcome if the goal is to suppress testosterone and delay puberty, which is shown in Figure 17, where most of the lines show a tendency that amplifies the AMH marker.
Figure 17: Amplification results
A high AMH expression wouldn't be a typical "good" result. It could indicate incomplete suppression of Sertoli cells, potentially interfering with the intended effect of Leuprolide. With that we have some important considerations: the definition of a "good" result depends on the treatment goals with Leuprolide. Measuring testosterone levels and physical signs of puberty progression would be more informative for assessing puberty development after Leuprolide treatment. As seen in Figure 18, we can see that while GnRHa initially stimulates FSH, continued use leads to a downregulation of GnRH receptors in the pituitary gland.
Figure 18: hormones control vs experimental (made by me)
This decreased sensitivity causes FSH suppression over time, explaining the observed effect in the animals. Testosterone addition may be used to manage side effects but wouldn't directly affect the initial FSH response. So, we can interpret from these results is that GnRHa treatment can lead to infertility by suppressing FSH, a hormone vital for egg development. Additionally, it can cause reduced sex drive and potential changes in body composition in both males and females. Alternatively, the possibility of secondary puberty after GnRH withdrawal by examining epithelial cell changes and gene expression patterns in the phallus could be investigated. From a clinical standpoint, this research has the potential to improve urological care. By investigating the link between GnRH agonists, UTIs, and urination difficulties, the project could develop better treatment protocols and improve patient well-being.
Studies like the one by Motto et al. (2014) [1] investigate how hormonal fluctuations influence postnatal testicular development in different mouse species. Their research demonstrates that hormones play a crucial role in determining testicular weight, seminiferous tubule structure, and ultimately, sperm production. This aligns with the current research exploring how GnRH agonists, which manipulate hormonal balance, might affect testicular development in humans. Understanding the hormonal pathways involved in normal postnatal development serves as a foundation for investigating potential disruptions caused by GnRH agonist therapy.
Another crucial aspect of this project is the influence of hormones on Sertoli cells within the testes. These cells play a vital role in sperm development and maturation. The research by Shinohara et al. (2018) [2] sheds light on how hormones, particularly testosterone, regulate Sertoli cell function in the postnatal mouse testis. Their findings highlight the dynamic nature of the postnatal testis and how hormonal cues impact stem cell number and niche accessibility. This knowledge can be applied to the current project by exploring how GnRH agonists might alter the hormonal environment and consequently affect Sertoli cell function, potentially impacting sperm development during puberty delay.
By seeing the results in Figure 19, we can see that by measuring the relative volume of seminiferous tubules in developing mice, researchers can gain insights into how external factors like GnRH might influence testosterone production and subsequent testicular growth. In Figure 20, Stem cells from healthy mice restored fertility in infertile male mice with a genetic defect. Transplanted into the testes, the donor stem cells matured into sperm, allowing the infertile mice to produce offspring that even inherited some of the donor's genes. With that said, if for any reason or circumstance, this process of external hormone influence must be reversed, a potential human model can restore their biological reproductive functions.
Drawing connections between these studies on hormonal regulation and postnatal testicular development, this research project can better understand how GnRH agonists influence the intricate processes within the testes during puberty delay. This knowledge can contribute to a more comprehensive picture of hormonal regulation in the urogenital system and its impact on urogenital development.
Figure 19: UROGENITAL MASS; by measuring the relative volume of seminiferous tubules in developing mice, researchers can gain insights into how external factors like temperature might influence testosterone production and subsequent testicular growth [1]. (volume realtive to the days, which is not explicitly mentioned)
Figure 20: Transplantation of adult donor cells into testes of both pup and adult recipients revealed greater donor-derived spermatogenesis (blue staining) in pups compared to adults at 5.5 months post-transplantation [2].
References:
Montoto LG; Arregui L; Sánchez NM; Gomendio M; Roldan ER; (n.d.). Postnatal testicular development in mouse species with different levels of sperm competition. Reproduction (Cambridge, England). https://pubmed.ncbi.nlm.nih.gov/22187670/ [1]
Shinohara,T.; Orwig, K.E.; Avarbock, M.R.; and Ralph L. Brinster, R.L. (n.d.). Remodeling of the postnatal mouse testis is accompanied by dramatic changes in stem cell number and niche accessibility. Proceedings of the National Academy of Sciences of the United States of America. https://pubmed.ncbi.nlm.nih.gov/11371640/ [2]
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