Analysis of the Effect of Leukemia Inhibitory Factor on Follicular Growth in Cultured Murine Ovarian Tissue

Abstract

Leukemia inhibitory factor (LIF) is expressed in the ovary and controls follicular growth. LIF has been reported to accelerate the primordial to primary follicle transition, the growth of cultured preantral follicles, and the maturation of oocytes. Previous reports on factors that regulate follicular growth have largely employed cultured follicles. However, there are several types of follicles and somatic cells in the ovary that are likely to interact with one another to regulate follicular growth. Therefore, a novel approach is essential for understanding the function of factors that regulate follicular growth in the ovary. In this study, we evaluated the function of LIF using cultured ovarian tissue. Ovarian tissue slices were cultured in the presence or absence of recombinant LIF and neutralizing anti-LIF antibody to enable continuous monitoring of follicular growth within the context of the ovary as well as analysis of the process of follicular growth. The results revealed that LIF inhibited the growth of primary, secondary, and antral follicles. Furthermore, we verified the inhibitory function of LIF using the neutralizing antibody, which accelerated follicular growth. These results suggest that LIF is likely to coordinate follicular growth in the ovary. The culture and analysis methods employed in this study are thus effective for clarifying the tissue-level functions of factors that regulate follicular growth within the ovary.

The concentration-dependent effect of progesterone on follicle growth in the mouse ovary

“…The cultured ovarian tissue slices were treated with 100 mIU/ml of LH for 12 h every 4 days to reproduce the physiological LH surge. Concentrations of FSH and LH used were based on previous experiments [10, 11]. The effect of P 4 on follicle growth was evaluated after adding P 4 (10 ng/ml, 100 ng/ml, and 1 μg/ml) (Sigma-Aldrich) to the culture medium, followed by culture for 18 days.…”

“…Based on these results, we established the criteria for the classification of follicles based on the area. The detailed information is presented in our previous report [11]. …”

“…While follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are known to regulate the growth of antral follicles, the regulation of early stages of follicle growth is complex and unclear [1,2,3]. The early stage in follicle growth is independent of FSH and LH and controlled by many factors such as the kit ligand, growth differentiation factor 9 (GDF-9), leukemia inhibitor factor (LIF), insulin-like growth factor 1 (IGF-1), and bone morphogenetic protein-4 (BMP-4) [4, 5, 6, 7, 8, 9, 10, 11]. The development of follicles in the ovary is regulated by several mechanisms; understanding of these mechanisms necessitates simultaneous observation of the growth dynamics of various follicles.…”

“…The development of follicles in the ovary is regulated by several mechanisms; understanding of these mechanisms necessitates simultaneous observation of the growth dynamics of various follicles. Our previous study reported a new method for culturing mouse ovarian tissue to observe the dynamics of follicle growth [11]. This method allows simultaneous observation of dynamics of many follicles to evaluate the effect of a given factor at the tissue level.…”

“…The concentration of P 4 in blood transiently increases during phase 2 and is the highest during phase 3 [29]. We tested our hypothesis by treating cultured mouse ovarian tissues with different concentrations of P 4 (10 ng/ml, 100 ng/ml, and 1 μg/ml) and analyzed the effect of P 4 on follicle growth [11]. Our study revealed new effects of P 4 on the follicle growth and ovulation at tissue level.…”