The Role of LHX9 in the Formation of the Ovarian Reserve and its Implication in Female Reproductive Health

Premature ovarian insufficiency (POI), defined as the loss of ovarian function before the age of 40, affects approximately 1 in 100 women and represents a significant cause of ovulation-related infertility. Despite this, the underlying genetic causes of POI remain unknown in 70% of affected individuals. The ovarian reserve dictates the reproductive lifespan of a female mammal, and its maintenance is crucial in retaining fertility. During its formation, newly determined oocytes will cluster into ‘germ cell nests’ within the developing ovaries. Later in embryonic development, these nests will undergo scheduled breakdown through the ingression of somatic granulosa cells derived from the ovarian surface epithelium, resulting in a pool of discrete oocytes contained within primordial follicles. This intricate process requires the precise coordination of several signalling pathways. Recent findings implicated the role of the transcription factor LIM-homeobox gene 9 (LHX9) as a key regulator of germ cell nest breakdown. Previous research revealed LHX9’s essential role in promoting somatic cell proliferation in the early developing gonads. A recent study in the Wilson lab demonstrated how a female heterozygous (Lhx9^+/-) mouse model exhibited fertility decline, producing fewer pups per litter with age, as well as a significant reduction in the proportion of oocytes within nests compared to primordial follicles. Additionally, published patient variants within LHX9 have been linked to gonadal agenesis/dysgenesis phenotypes; one study reported a de novo missense variant (c.947A>G, p.Q316R) in LHX9 from a 46,XY child with finger and toe abnormalities, female external genitalia and absent uterus and ovaries. Another study described a nonsense mutation in LHX9 (c.35C>A, p.S12*) in a patient who presented with micropenis and hypospadias.

This project first investigated nest formation in wild-type and heterozygous (Lhx9^+/-) E12.5 mouse ovaries in culture. MitoTracker dye was utilized to fluorescently stain the ovarian surface epithelium cells and track their ingression into the ovary over 48 hours. This method was initially optimized before the amount of fluorescence within the ovary, corresponding to ingressed granulosa cells, could be quantified and compared. This revealed that an increased amount of fluorescence was present in the heterozygous ovary compared to the wildtype, corresponding to an increase in granulosa cell ingression into the ovary. The proportion of cells undergoing apoptosis in post-natal day 1, 5-month and 9-month-old wildtype and Lhx9^+/- ovaries were also quantified and compared using a TUNEL assay, providing evidence to suggest that heterozygous ovaries contain an increased proportion of apoptotic cells when compared to the wildtype ovary at the same stage. Furthermore, the implications of the c.947A>G and c.35C>A LHX9 patient variants were explored to examine how these alterations could impact its function and interactions with associated proteins, such as LDB1. The co-expression of LHX9 with LDB1 in the ovary was revealed through single-cell RNA sequencing analysis, and the predicted effects of the patient variants within the alternative LHX9 isoform structures were explored using various databases. While the cloning of LHX9 isoform 1 and LDB1 into their respective expression vectors was unsuccessful, the expression of LHX9 isoform 3 was able to be observed in an ovarian cell line. These attempts provided a foundation for future work to build upon to examine the co-expression of LHX9 and LDB1 in a cell line. Site-directed mutagenesis was successfully used to induce the c.35C>A variant in the isoform 3 of LHX9.

This research aimed to elucidate the role of LHX9 in ovarian reserve formation and its contribution to conditions such as POI through an integrative approach combining mouse models, cellular assays, and genetic analysis. Ultimately, the findings could enhance genetic screenings for infertility and gonadal dysgenesis, improving our understanding and management of these conditions while working to reduce the incidence of premature ovarian insufficiency among women.