Dynamic Expression of the SLIT-ROBO Pathway in the Periovulatory Follicle and Corpus Luteum in Primates.

Abstract

The Slit-Robo pathway was initially identified in Drosophila and plays a critical role in axon guidance during development of the nervous system. Recently, three Slit (1-3) ligands and four Robo (1-4) receptor isoforms were identified in mammals, in both non-neuronal and neuronal systems where they may function in tissue growth, remodeling, and development. Members of the SLIT-ROBO system were detected in human and nonprimate ovary, but their regulation and functions are unclear. As an initial step in considering its importance in the primate ovary, experiments were designed to investigate the expression of SLIT-ROBO family members in the pre- and peri-ovulatory follicle and corpus luteum (CL) of rhesus monkeys during the menstrual cycle. Using our previously reported Controlled Ovulation (COv) model, the dominant follicle (n=4/interval) was collected at 0, 12, 24, and 36 (ruptured, or unruptured) hours after injection of an ovulatory dose (1000 IU) of hCG. CL (n>4/stage) were collected at early, mid, mid-late, late, or very late (days 3-5, 6-8, 10-12, 14-16 post-LH surge, and menses respectively) luteal phase. These stages encompass the entire lifespan of the CL, from development (early luteal phase) through functional (late) and structural (very late) regression. Total RNAs were extracted from individual tissues, real-time PCR performed, and mRNA levels analyzed by Sigma Stat software using 18S rRNA as an internal control. The mRNAs for all three SLIT ligands and four ROBO receptors were detected in each tissue sample. Appreciable mRNA levels for SLIT1 were present in the preovulatory follicle at 0 hour before hCG injection, followed by a transient decline (p<0.05) at 12 hours post-hCG. Elevated levels returned at 24-36 hours post-hCG prior to follicle rupture, but then declined (p<0.05) to low-levels in the ruptured follicle, which remained invariant in the CL from early to very late luteal phase. The mRNA levels for SLIT2 were appreciable, but unchanging within the periovulatory follicle or CL, until increasing (p<0.05) in the very late luteal phase. Levels of SLIT3 mRNA were low and not significantly different during the periovulatory interval or luteal phase. In contrast, ROBO1 mRNA levels increased (p<0.05) in the preovulatory follicle within 12 hours post-hCG, and remained elevated through follicle rupture, as well as in CL throughout the luteal phase. The levels of ROBO2 or ROBO4 mRNA did not change in the periovulatory follicle or CL during the menstrual cycle, except for a modest, transient increase (p<0.05) in ROBO4 transcript at mid luteal phase. Remarkably, ROBO3 mRNA levels increased from 12 to 36 hours post-hCG in the periovulatory follicle, and again throughout the CL lifespan to peak at late to very late luteal phase (p<0.05). Thus, the presence and dynamic expression of several members of the SLIT-ROBO family in the preovulatory follicle and corpus luteum suggests that this ligand-receptor system plays a role in ovulatory processes and/or luteal development and regression during the ovarian cycle in primates. Supported by NICHD HD18185 (Proj 3), HD053648, RR00163 (poster)