Studying factors influencing meiosis progression in the mouse oocyte

Liu, Ning

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https://hdl.handle.net/2142/120567 Copy

Description

Title

Studying factors influencing meiosis progression in the mouse oocyte

Author(s)

Liu, Ning

Issue Date

2023-04-28

Director of Research (if dissertation) or Advisor (if thesis)

Qiao, Huanyu

Doctoral Committee Chair(s)

Qiao, Huanyu

Committee Member(s)

• Flaws, Jodi
• Spinella, Michael
• Marko, John

Department of Study

Comparative Biosciences

Discipline

VMS - Comparative Biosciences

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• meiosis
• oocyte
• chromosome
• spindle

Abstract

Meiosis is an essential process for eukaryotic organisms to create gametes, including oocytes or spermatocytes. Compared to typical mitosis, it is much more complicated since it involves two types of chromosome separation and two consecutive cell divisions. Meiosis I is very special process which contains a lot of meiosis specific activities such as pairing, synapsis, and recombination. This division is also prone to errors, which means a high rate to cause some problems during this process. Any defects occurred at this stage may cause disease like infertility, miscarriage, or congenital malformation. As a result, it is crucial to study the mechanisms involved and investigate factors that may cause abnormalities in this process. Oocytes are produced in female ovaries and undergo a special process called oogenesis to become matured oocytes. The quantity and quality of oocytes determine the fertility of female. Oogenesis begins during fetal development, and oocytes are arrested at meiosis I prophase stage until puberty. After puberty, some oocytes could periodically resume to activate the further maturation process, which would be arrested at meiosis II metaphase stage waiting for fusing with sperm to form a zygote. Oocyte in vitro culture is a popular method to study its maturation, which allow us to collect ovaries from mouse and isolate individual oocytes. The prepared oocytes could be cultured in petri dishes, and the in vitro timeline has been well-studied. Therefore, it is easy to track the oocyte progression under the microscope and obtain the oocytes at certain stage, providing a platform for investigating meiotic mechanisms and structures involved. Spindles and chromosomes are two important structures that are formed only during mitosis or meiosis. These structures work together to ensure the even distribution of genetic materials, so each daughter cell gets equal amounts of genetic information from their parents after cell division. Although spindle and chromosome structures have been discovered and studied for over a century, many questions remain unanswered. In this study, we applied micromanipulation system to investigate their mechanics and decipher its structure and mechanisms. The micromanipulation system is a powerful tool that have been widely used in different fields, including biology, chemistry, and physics. By combining in vitro oocyte culture with micromanipulation, we were able to isolate small objects like cells or even some components inside the cell and measure them with high precision. In this study, we combined oocyte in vitro culture with micromanipulation to isolate spindles and chromosomes from mouse oocytes. After isolation, the spindle and chromosome structures exhibited high stability in PBS solution, which allowed us to to manipulate them delicately. Our goal was to utilize this method to study isolated spindles and chromosomes. First, we isolated fresh, intact spindles from mouse oocytes. We discovered that this spindle isolation method could be applied to both meiosis I and meiosis II at different substages. However, when we attempted to isolate spindles from somatic cells at metaphase, it was impossible to separate the spindles from the cell. We also attempted to isolate chromosomes at different substages and found that centrosomes could be isolated at early stages, but when cells entered metaphase stage, the centrosome formed a strong connection with the cell membrane preventing the spindle isolation in such somatic cells. Since spindles formed in oocytes do not depend on centrosome, these spindles can be separated from cells. Therefore, spindle isolation is very unique in oocytes and cannot be applied to other somatic cells. Since we are able to isolate spindles from cells, we also used this phenomenon to study spindle physical properties, which is difficult to conduct in vivo. According to results, we found that isolated spindles are stable and not dependent on liquid-liquid phase separation. Then we found that meiotic spindles behave as if the elastic microtubules are embedded in a stiff spindle matrix. Furthermore, we measured the stiffness of the MI spindle and observed a length difference for the same spindle before and after isolation. Under this circumstance, our findings allowed us to answer a long-standing question regarding whether the spindle migration during meiosis is regulated by pulling force or pushing force. We observed that the spindle is stretched inside cells, providing direct evidence that the spindle migration is driven by pulling force. In addition, we estimated the pulling force to be approximately 680 pN. Finally, we go further studying factors that could influence the spindle stiffness. The results indicated that the spindle length and stiffness are regulated by microtubule stability. We also wanted to apply this method to the field of toxicology as there are numerous chemicals and drugs that are claimed to influence spindle length, and it is reasonable to assume that the spindle stiffness is also altered. Previously we found that IAA, a water disinfection byproduct, could increase the spindle length. Therefore, we isolated spindles after IAA treatment and measured the spindle stiffness. As expected, the spindle stiffness is significantly increased in IAA treatment group. Our findings suggest that some environmental toxicants could impact spindle stiffness, which means spindle stiffness could be a characteristic to evaluate the safety of newly invented chemicals or drugs. In addition to spindles, we also isolated chromosomes from oocytes. A previous paper published by our lab showed that chromosome stiffness from meiotic cells is 10 times higher than that from mitotic cells. However, the factors lead to this high chromosome stiffness in meiotic cells have not yet been figured out. Since SYCP1 has been proved not to contribute to the chromosome stiffness, we turned our attention to studying the meiosis-specific cohesins. Surprisingly, we found that the meiosis-specific cohesin REC8, STAG3 and RAD21L do not contribute to the chromosome stiffness. We also found that chromosomes from old MI oocytes have higher stiffness than those from young MI oocytes. This is consistent with a published paper that shows chromosomes from old MII oocytes are much stiffer than those from young MII oocytes. Therefore, age is an important factor influencing chromosome stiffness. We gave hypothesis that DNA damage-repairing proteins cause the increase in chromosome stiffness, given that old oocytes contain more DNA damage. However, our results disapproved this hypothesis, as DNA damage actually reduced the chromosome stiffness in oocytes. Therefore, further investigation is needed to determine the factors contributing to the high chromosome stiffness in meiosis I cells. Environmental toxicants such as PFAS, have been widely used in various fields and are gaining more and more attention due to their potential toxicities. We aimed to study one type of PFAS, PFNA, since their effects on oocytes maturation have not been completely studied. We used oocyte in vivo culture to incubate collected oocytes with different concentrations PFNA. Our results showed that PFNA can inhibit oocyte maturation by decreasing the GVBD rate and PBE rate. Furthermore, PFNA treated oocyte had significantly higher rates of early apoptosis, indicating that oocyte quality would be impaired when exposed to PFNA.

Graduation Semester

2023-05

Type of Resource

Thesis

Copyright and License Information

Copyright 2023 Ning Liu

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