Relative Ovarian Resistin Expression in Normal Cycling Rats and Rats with Cystic Ovaries. (2009)

Resistin (RETN) is a cytokine that has been identified in adipose tissue and macrophages and implicated in insulin resistance. Recently, RETN mRNA was identified in Leydig cells of male rats. Some studies have shown elevated concentrations of serum RETN in women with polycystic ovaries. These findings led us to hypothesize that RETN may be expressed in the rat ovary. The objectives were to determine if: 1) RETN mRNA is expressed in the rat ovary, 2) RETN mRNA fluctuates throughout the estrous cycle, and 3) RETN mRNA expression changes in rats with induced ovarian cysts. Experiment 1 - RETN Expression During the Estrous Cycle Long-Evans rats were monitored by daily vaginal cytology to determine cycle stage. After five weeks, rats were assigned to one of three groups: diestrus I & II (D, n=5), proestrus (P, n=5), and estrus (E, n=6). Animals were anesthetized, decapitated, and ovaries dissected and frozen at -70°C for later analyses. RNA was extracted from ovaries and reverse transcribed using RNeasy Midi and QuantiTect Reverse Transcription kits (Qiagen). Ovarian RETN mRNA was quantified by real time PCR using Taqman probes for rat RETN and rat B-actin mRNA (internal control). The threshold cycle number (Ct, when sample signal exceeds background) was used to quantify mRNA. Data were normalized to B-actin by calculating the delta Ct (RETN Ct - actin Ct). Lower values indicated higher RETN mRNA expression. The average delta Cts were: D=10.7, P=11.8, and E=11.1. We determined fold changes between groups using an algorithm that accounts for the doubling of cDNA with each PCR cycle (P vs E=1.6, P vs D=2.1, E vs D=1.4). While RETN mRNA was detected in all rat ovaries throughout the cycle, we were unable to detect significant differences between stages. Experiment 2 - RETN Expression in Cystic Ovaries: To induce ovarian cyst formation, the experimental group (n=4) received 0.04% propylthiouracil in drinking water for 30 days to cause hypothyroidism. On days 11-30, they also received daily s.c. injections of 10 IU hCG in 0.1 mL of saline. Control animals (n=4) received normal drinking water for 30 days and daily injections of 0.1 mL of saline on days 11-30. All ovaries from rats in the hypothyroid/hCG group had large cystic follicles; whereas none of the euthyroid ovaries exhibited evidence of cysts. This was supported by higher ovarian weight in hypothyroid/hCG-treated rats (mean=505 ± 52mg) compared to controls (mean=105 ± 17mg). Results of RT-PCR showed an average delta Ct of 7.2 in rats with cystic ovaries compared with 9.1 in control animals, indicating that the hypothyroid/hCG-treated group had 3.8-fold higher RETN mRNA expression. Although we were able to detect RETN mRNA in rat ovaries, we could find no significant differences in RETN expression throughout the estrous cycle. RETN expression, as quantified by RT-PCR, was significantly higher in the induced-cyst rats than in the normally cycling controls. Our results indicate that RETN is normally expressed at a basal level throughout the estrous cycle and is elevated in animals with induced ovarian cysts. The potential involvement of RETN in normal ovarian physiology, as well as the process of cyst induction, clearly merit further investigation. Future studies should include localization of RETN in the ovary, quantification of RETN protein and studies of its regulation by the endocrine syste. (poster)