PARENT SESSION
GRANULOSA CELL FUNCTION AND REGULATION
Laurel
7:30 AM-10:00 AM

(281) RELATIONSHIPS AMONG LEVELS OF GRANULOSA CELL INSULIN-LIKE GROWTH FACTOR BINDING-PROTEIN (IGFBP) mRNAS AND FOLLICULAR FLUID (FFL) IGFBPs AND STERIODS IN DOMINANT AND SUBORDINATE FOLLICLES OF PREOVULATORY CATTLE.

Voge, Justin¹, Allen, Dustin¹, Malayer, Jerry², Spicer, Leon¹, ¹ Dept. of Animal Science, Stillwater, OK² Physiological Sciences, Stillwater, OK

ABSTRACT- The objectives of this study were to determine if 1) IGFBP mRNA levels in granulosa cells of dominant preovulatory and subordinate follicles change during the follicular phase of an estrous cycle and 2) if these changes are associated with changes in FFL concentrations of IGFBPs and steroids. Estrous cycles of non-lactating dairy cows (n = 10) were synchronized with two injections of prostaglandin (PGF₂) 11 days apart. Follicular growth was monitored daily via transrectal ultrasonagraphy starting 4 days after the first PGF₂ injection. FFL and granulosa cells were collected either 24 or 48 h after the second injection of PGF₂ via transvaginal aspiration from dominant [largest follicle at time of aspiration and greatest estradiol (E₂) level in FFL] and the largest subordinate follicles. Granulosa cells were obtained after centrifugation of FFL and total RNA extracted. FFL E₂ and progesterone (P₄) levels were determined by RIA and IGFBP levels by ligand blotting. Levels of IGFBP-2, -3, and -4 mRNAs were quantitated using real-time polymerase chain reaction performed in the ABI Prism® 7700 sequence detection system. 18S rRNA controls were used to normalize samples for RNA loading, and relative values were calculated using the comparative threshold cycle (Ct) method. All values were expressed as the Ct relative to the sample within each IGFBP exhibiting the highest Ct. FFL from dominant follicles had severalfold greater (P < 0.001) levels of E₂ than subordinate follicles regardless of time collected. FFL P₄ levels in subordinate but not dominant follicles increased (P < 0.005) between 24 and 48 h. At both 24 and 48 h, FFL levels of IGFBP-2, -4, and -5 were severalfold greater (P < 0.05) in subordinate than dominant follicles. FFL levels of IGFBP-3 did not differ (P > 0.10) among follicle types. IGFBP-2, -3, and -4 mRNA levels did not differ (P > 0.10) among follicle classes regardless of time of collection. However, IGFBP-2 mRNA (but not IGFBP-3 or -4 mRNA) levels were negatively correlated (r = -0.52, P < 0.05) with FFL E₂ levels. We conclude that the decreased levels of IGFBP-2 found in FFL of dominant follicles may be regulated in part by increased levels of E₂, whereas changes in IGFBP-4 levels in FFL cannot be attributed to changes in IGFBP-4 mRNA.

KEY WORDS: insulin-like growth factor binding-proteins, folliculogenesis, granulosa cells