Steroidogenesis, Reactive Oxygen Species and Female Reproduction
Date
2023
Authors
Tang, Feng
Editors
Advisors
Rodgers, Raymond
Hummitzsch, Katja
Hummitzsch, Katja
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Thesis
Citation
Statement of Responsibility
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Abstract
Ovarian steroidogenesis is the process of synthesising the steroid hormones oestradiol and progesterone in granulosa cells (GCs) and thecal cells (TCs) of the follicle, and is crucial for female reproduction. Follicle stimulating hormone (FSH) and luteinizing hormone (LH) from the pituitary gland regulate follicle growth and differentiation, follicular steroidogenesis as well as oocyte maturation and release (ovulation) for fertilisation. The synthesis of oestradiol and progesterone involves enzymes, cytochrome P450 enzymes, which require electron transport chains for their reactions and can therefore lead to the production of reactive oxygen species (ROS) and if not scavenged by antioxidants to oxidative stress. Aging has been connected to increased oxidative stress in the cells, and previous studies have shown that the main enzyme for the oestradiol production, cytochrome P450 aromatase, is increased in advanced aged women (> 40 years). Furthermore, oocyte quality and therefore fertility declines with a woman’s age. On these grounds, it was imperative to study the interplay between ovarian steroidogenesis, ROS and antioxidants for my thesis, especially regarding oocyte maturation and ovarian aging. The tumor-like granulosa cell line KGN is widely used as model for granulosa cell steroidogenesis, even so hormone levels are 100 times lower than in the primary cells. KGN show also an extremely high expression of thioredoxin reductase 1 (TXNRD1), an enzyme involved in scavenging reactive oxygen species, compared to granulosa cells. This opened up the question whether KGN cells regulate the mRNA expression of their antioxidant enzymes differently to granulosa cells in response to increased steroidogenesis. We showed that dibutyryl cyclic adenosine monophosphate or forskolin induced steroidogenesis in KGN cells resulted in steroidogenesis related genes and antioxidant enzyme genes being differentially expressed. Knockdown of TXNRD1 led to changes in the expression of other antioxidant enzyme classes. Our analysis indicated that KGN cells have a different regulation of steroidogenic and antioxidant genes during steroidogenesis compared to human GCs. However, on the other hand KGN cells are similar to primary GCs, and show a downregulation of steroidogenic activity when their antioxidant system is impaired. Granulosa cells are the nurturing cells of the oocyte in the follicle, and regulate its development in a paracrine fashion. Oocytes have been shown to be susceptible to damage by ROS, especially during oocyte maturation. Maturation is characterised by the transition of germinal vesicle (GV) oocytes to meiosis I (MI) oocytes and finally MII oocytes. However, the key genes and transcripts required for this process are still not known. Furthermore, the antioxidant enzymes available in the oocyte at the GV and MII stage to protect itself against ROS from surrounding granulosa cells and follicular fluid are not fully identified either. We generated and studied the RNA sequencing profiles of pig oocytes at three major developmental stages, the GV stage and MII stage as well as oocytes, which did not complete the transition to MII (‘damaged’), therefore suggesting a lower oocyte quality or developmental delay. No differences in gene expression were identified between MII and the ‘damaged’ pig oocytes. Additionally, we compared our findings for the pig oocyte with public human oocyte data. This identified conserved regulators that ensure correct chromosome segregation in the first meiosis and therefore provide an overview of molecular regulation of oocyte maturation. Women’s aging is associated with a decline in fertility as result of a decline in oocyte quality and developmental competence. This is problematic as more women decide to become a mother later in life. Using public spatial RNA sequencing data from young and advanced aged mouse ovaries, we tried to decipher how ovarian aging affects the expression of steroidogenic and antioxidant genes in follicular cells, especially immature and mature GCs and the oocytes. Ovarian aging results in an impaired antioxidant system in different cell type in the advanced aged ovary including vascular, lymphatic, thecal, and granulosa cells. Simultaneously, ovarian aging also significantly increased steroidogenic gene Cyp19a1 expression in mature granulosa cells and resulted in increased aneuploidy rates in oocytes from advanced aged ovaries compared to younger counterparts. Our results suggests that ageing impairs the antioxidant response in the follicular environment and leads to increased steroidogenesis in mature granulosa cells. This imbalance of antioxidant and ROS might account for the poor oocyte health observed during ageing. In conclusion, our findings confirm firstly that KGN cells can be a good model for some aspects of primary granulosa cell behaviour. However, scientist have to be aware of the limitations and differences of this cell line. Secondly, oocyte which have not completely transitioned into MII oocytes show no gene expression differences compared with MII oocytes suggesting that these oocyte might be still able to fully mature but in a delayed time frame. And we identified key regulators involved in important oocyte maturation events including chromosome segregation, spindle assembly and mitochondrial oxidative phosphorylation at both gene and isoform transcript level. Lastly, ovarian aging seems connected with changes to main antioxidant enzymes in the cell populations related to follicles, which could result in ROS damage due to increased steroidogenesis.
School/Discipline
School of Biomedicine
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Biomedicine, 2023
Provenance
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