An ovulatory gonadotropin stimulus increases cytosolic phospholipase A2 expression and activity in granulosa cells of primate periovulatory follicles.

CONTEXT: Prostaglandins (PGs) produced within ovarian follicles in response to the ovulatory gonadotropin surge are essential for follicle rupture and oocyte release. Arachidonic acid, the common precursor for PG synthesis, is cleaved from membrane phospholipids via the activity of phospholipase A2 (PLA2). OBJECTIVE: The purpose of this study was to determine which PLA2 form(s) is involved in PG production by primate periovulatory follicles. DESIGN AND INTERVENTIONS: Gonadotropins were administered to cynomolgus monkeys to stimulate multiple follicular development; human chorionic gonadotropin (hCG) initiated periovulatory events. Granulosa cells and whole ovaries were obtained before (0 h), and 12, 24, and 36 h after hCG administration. PATIENTS: Granulosa-lutein cells were also obtained from women undergoing infertility treatment. OUTCOME MEASURES AND RESULTS: mRNA for cytosolic (c)PLA2 and secretory (s)PLA2V, but not sPLA2IIA, was expressed by granulosa cells. cPLA2 mRNA levels were low at 0 h, elevated by 12 h, and remained high 24-36 h after hCG administration. sPLA2V mRNA levels were low at 0 h and did not change in response to hCG. cPLA2 and sPLA2V were detected by immunocytochemistry in granulosa cells of periovulatory follicles before and at all times after hCG administration. PLA2 activity was low in lysates of granulosa cells obtained 0-24 h after hCG and was elevated in granulosa cells obtained 36 h after hCG administration. A cPLA2-selective inhibitor decreased both PLA2 activity in monkey granulosa cell lysates and PGE2 accumulation in cultures of human granulosa-lutein cells. CONCLUSIONS: cPLA2 is primarily or exclusively responsible for the gonadotropin-stimulated mobilization of arachidonic acid necessary for PG production by primate periovulatory follicles.

Identification of chemokine receptor CCR4 antagonist.

The present study reports the identification and hits to leads optimization of chemokine receptor CCR4 antagonists. Compound 12 is a high affinity, non-cytotoxic antagonist of CCR4 that blocks the functional activity mediated by the receptor.

Microsomal prostaglandin E synthase-1 (mPGES-1) is the primary form of PGES expressed by the primate periovulatory follicle.

BACKGROUND: Prostaglandin E2 (PGE2) has been identified as the key ovulatory PG in the primate follicle. Follicular PGE2 levels increase just before the expected time of ovulation, suggesting that the midcycle LH surge induces the expression of enzymes involved in PGE2 synthesis. METHODS: To identify the specific form(s) of prostaglandin E synthase (PGES) expressed by the primate periovulatory follicle, we examined granulosa and theca cell expression of the three microsomal (m) and cytosolic (c) forms of PGES (mPGES-1, mPGES-2 and cPGES) identified to date. Monkey granulosa cells and whole monkey ovaries were obtained from animals receiving exogenous gonadotropins to stimulate multiple follicular development; monkeys then received an ovulatory dose of HCG to initiate periovulatory events. RESULTS: Expression of mPGES-1 mRNA and protein by granulosa cells of periovulatory follicles increased in response to HCG administration, peaking just before the expected time of ovulation. Immunocytochemistry showed that mPGES-1 protein was present in both granulosa and theca cells of monkey periovulatory follicles. Monkey granulosa cells also expressed mPGES-2 and cPGES mRNA, but mRNA levels did not change in response to HCG administration. Isolated monkey theca cells expressed both mPGES-1 and cyclooxygenase-2 mRNA, and produced PGE2 in vitro. Human granulosa-lutein cells obtained from women undergoing treatment for infertility expressed mRNAs for mPGES-1, mPGES-2 and cPGES. CONCLUSIONS: These data indicate that mPGES-1 is a gonadotropin-regulated PG synthesis enzyme expressed by granulosa cells of primate periovulatory follicles and suggest that mPGES-1 may be the primary PGES responsible for the increased follicular PGE2 levels necessary for primate ovulation.

Adipose differentiation-related protein: a gonadotropin- and prostaglandin-regulated protein in primate periovulatory follicles.

The midcycle LH surge stimulates a rise in follicular fluid prostaglandin E2 (PGE2), which is necessary for normal ovulation. To examine PGE2-regulated processes in primate follicles, monkey granulosa cells were cultured with hCG alone or with hCG and PGE2, and the resulting total RNA was subjected to microarray analysis. Twenty PGE2-regulated mRNAs were identified, and we selected a lipid droplet protein, adipose differentiation-related protein (ADRP), for further study. To determine whether hCG and PGE2 regulate ADRP expression in vivo, monkeys received gonadotropins to stimulate multiple follicular development. Human chorionic gonadotropin was then administered alone or with the PG synthesis inhibitor celecoxib, and follicular aspirates or whole ovaries were obtained at times that span the 40-h periovulatory interval. Administration of hCG increased granulosa cell ADRP mRNA and protein, with peak levels measured just before the expected time of ovulation. Treatment with hCG and celecoxib decreased granulosa cell ADRP mRNA levels compared with those of animals treated with hCG only. ADRP was detected by immunocytochemistry in many monkey tissues that synthesize prostaglandins but was not consistently expressed by steroidogenic tissues. Granulosa cells of periovulatory follicles immunostained for ADRP after, but not before, hCG administration; ADRP colocalized with large lipid droplets within the granulosa cell cytoplasm. These studies identify ADRP as a novel gonadotropin- and PGE2-regulated protein in the granulosa cells of primate periovulatory follicles. Because ADRP facilitates arachidonic acid uptake in non-ovarian cells, ADRP-associated lipid droplets may enhance arachidonic acid uptake by granulosa cells to provide a precursor for periovulatory prostaglandin production.

Prostaglandin dehydrogenase and prostaglandin levels in periovulatory follicles: implications for control of primate ovulation by prostaglandin E2.

Prostaglandin (PG) E2 produced by the periovulatory follicle in response to the midcycle LH surge is essential for successful ovulation in primates. Granulosa cells express the PG synthesis enzyme cyclooxygenase-2 in response to the LH surge, but elevated cyclooxygenase-2 mRNA levels precede rising follicular fluid PGE2 levels by 24 h. Therefore, PG metabolism may play a significant role in regulating follicular concentrations of PGE2 during the periovulatory interval. To test this hypothesis, granulosa cells, follicular fluid, and whole ovaries were obtained from adult monkeys receiving exogenous gonadotropins to stimulate development of multiple, large follicles at times spanning the 40-h periovulatory interval. Ovarian expression of the NAD+-dependent 15-hydroxy PG dehydrogenase (PGDH) was assessed by RT-PCR, Western blotting, and immunohistochemistry. PGDH mRNA levels were low in granulosa cells obtained 0 h after hCG, rose 10-fold 12 h after hCG, and were not different from 0 h by 24-36 h after hCG administration. Granulosa cell PGDH protein was present 0-12 h after hCG but was low/nondetectable 36 h after hCG administration. Follicular fluid PGE2 levels were low at 0-12 h, slightly higher at 24 h, and then rose 10-fold to peak at 36 h hCG. Levels of biologically inactive PGE2 metabolites in follicular fluid were also low at 0 h but elevated at 12-24 h after hCG, times at which PGE2 levels remain low. Therefore, PGDH is present in the primate periovulatory follicle in a pattern consistent with modulation of follicular PGE2 levels during the periovulatory interval, supporting the hypothesis that gonadotropin-regulated PGDH plays a role in the control and timing of ovulation in primates.