Adiponectin and its receptors modulate granulosa cell and cumulus cell functions, fertility, and early embryo development in the mouse and human

Abstract

Objective: To study the expression and function of adiponectin and its receptors in mouse and human follicle cells and in early embryo development.

Design: Whole ovaries, granulosa cells, and cumulus-oocyte complexes isolated from immature mice before and during hormone-induced ovulation were used to analyze the expression of adiponectin, its receptors, and ovulation-related genes; human cumulus cells and granulosa cells were isolated from patients undergoing in vitro fertilization (IVF) procedures.

Setting: Multicenter.

Patient(s): Women in IVF programs in Japan and the United States.

Intervention(s): None.

Main outcome measure(s): Expression of adiponectin receptors and fertility.

Result(s): Adiponectin expression is absent/low in mouse and human granulosa cells and cumulus cells. Adiponectin receptors are hormonally regulated in mouse granulosa and cumulus cells in vivo and in culture. Adiponectin differentially alters the expression of Adipor1/Adipor2 as well as genes related to steroidogenesis, ovulation, and apoptosis in cumulus cells versus granulosa cells. Adiponectin enhances oocyte maturation and early embryo development in mouse and human IVF procedures.

Conclusion(s): Adiponectin can modulate not only follicle growth but also embryo development in mice and humans.

Figure 1. Adiponectin receptors are differentially regulated by eCG and hCG in ovarian compartments

Whole ovaries (WO). granulosa cells (GC) and cumulus cell-oocyte complexes (COC) were isolated from immature (im) mice prior to and after treatment with eCG for 48h (0h) and hCG for 16 and/or 48h. A) Total RNA was prepared and used for real-time RT-PCR analyses presented as fold-induction relative to that observed in immature mice for WO. B) In situ hybridization localized adiponectin mRNA to adipose tissue adherent to the ovary but not to any ovarian cells. Different letters denote P<0.05 between samples. Tissues from at least 3 mice were used.

Figure 2. Regulated expression of selected genes by adiponectin, forskolin/FSH or the combination in granulosa cells and COCs

Granulosa cells and COCs were cultured in the presence of adiponectin (20µg/ml), forskolin (10µM) or FSH (100ng/ml)(recombinant FSH from Organon) or the combination for 24h. A) Total RNA was prepared for real-time RT-PCR analyses. B) Media samples were collected for measuring progesterone. C) Analyses of IVF of oocytes and their development. All treatments and assays were run in triplicate. Different letters denote P<0.05 between samples. A= adiponectin, F=forskolin for granulosa cells and FSH for COCs. A/F means the combined treatment of adiponect/forskolin for granulosa cells and adiponectin/FSH for COCs.

Figure 3. Regulation of FOXO transcription factors by adiponectin

Granulosa cells were cultured in the presence of adiponectin (ADIPOQ), forskolin or the combination for 24h. A) Total RNA (as in Figure 2) was prepared for real-time RT-PCR analyses and B) cell lysates were prepared for Western blots. Each assay was run at least three times. C) Granulosa cells were also transfected with adenoviral vectors for 4h, washed and cultured overnight as in Materials and Methods (21). Total RNA was prepared for analyzing the expression of Adipor1 and Adipor2 by quantitative RT-PCR analyses.

Figure 4. Expression of ADIPOR1, ADIPOR2, FOXO1 and FOXO3 in cumulus cells samples obtained from human IVF patients

A) Cumulus cell samples were collected from patients in Japan undergoing IVF procedures and total RNA was prepared. FOXO1, ADIPOR1 and ADIPOR2 were amplified by RT-PCR in selected samples (patient#7,8,15,14,16) using 50ng RNA, 35 cycles and Comparative Quantitation. These may not be in the linear range. Expression of ADIPOQ was low or undetectable. B) The expression levels of FOXO1 and FOXO3 mRNA in each sample were compared to expression of ADIPOR1, ADIPOR2 and PTEN using quantitative RT-PCR conditions in the linear range as described in methods. C) Correlation of gene expression levels of ADIPOR1 and ADIPOR2 in cumulus cells with oocyte maturation, fertilization and embryo development in the same patients. These data were collected in conjunction with the IVF samples in Japan. * denotes P<0.05.