In vitro progesterone production by luteinized human mural granulosa cells is modulated by activation of AMPK and cause of infertility.

BACKGROUND: Mural granulosa cells from IVF patients were provided by the West Virginia University Center for Reproductive Medicine in Morgantown, WV. The effect of adenosine monophosphate activated protein kinase (AMPK) activation, primary cause of infertility, age, BMI, and pregnancy outcome on production of progesterone were examined separately. METHODS: Isolated mural sheets from IVF patients (n = 26) were centrifuged, supernatant discarded, and the pellet re-suspended in 500 µl of DMEM/F12. Mural granulosa cells were plated at 10,000 cells/well in triplicate per treatment group with 300 µl DMEM/F12 media at 37 °C and 5% CO2 in a humidified incubator to permit luteinization. Four days after initial plating, cells were treated with either an AMPK inhibitor, DM; an AMPK activator, AICAR; or hCG. Cells were cultured for 24 h after treatment when medium was collected and frozen at -20 °C until assayed for P4 by radioimmunoassay. RESULTS: The AMPK activator, AICAR, inhibited P4 production (P < 0.001), whereas the AMPK inhibitor, DM, did not affect basal P4 (P < 0.05). Progesterone production increased when cells from patients whose primary cause of infertility was a partner having male infertility were treated with hCG compared to control (P = 0.0045), but not in patients with other primary infertility factors (P > 0.05). Additionally, hCG increased P4 production in patients between the ages 30-35 (P = 0.008) and 36-39 (P = 0.04), but not in patients ages 25-29 (P = 0.73). Patients with normal BMI had increased P4 production when treated with hCG (P < 0.0001), however there was no change in P4 production from cells of patients who were overweight or obese (P > 0.05). Cells from patients who became pregnant to IVF had greater P4 production when stimulated with hCG than those who did not become pregnant when compared to controls (P > 0.05). CONCLUSIONS: Understanding how AMPK activation is regulated in ovarian cells could lead to alternative or novel infertility treatments. Human mural granulosa cells can serve as a valuable model for understanding how AMPK affects P4 production in steroidogenic cells. Additionally, when stimulated with hCG, P4 production by mural granulosa cells differed among infertility type, age, BMI, and pregnancy outcome.

Low peripheral progesterone and late embryonic/early fetal loss in suckled beef and lactating dairy cows.

Pregnancy failure during placentation in lactating dairy cows was associated with low concentrations of serum progesterone. Beef cows have greater serum progesterone and less pregnancy failure. Experiment 1 determined that reduction of serum progesterone affected late embryonic/early fetal loss in suckled beef cows. Cows (n=40) received progesterone from two new or used controlled internal drug releasing devices, replaced every 5d, beginning on Day 28 of gestation (mating=Day 0); CL were enucleated on Day 29. Retention of pregnancy was 77% in treated cows and 97% in 78 control cows (P<0.05). Experiment 2 determined how pregnant, lactating dairy cows with high or low progesterone concentrations during Days 28-34 differed in luteal function or in serum progesterone during replacement therapy. Luteal tissue from such cows was assayed for progesterone and expression of mRNA for genes of endothelin and prostaglandin (PG) systems. Secretion of progesterone and prostaglandins by dispersed luteal cells was determined during incubation with LH, endothelin-1, or arachidonic acid. Neither luteal progesterone nor mRNAs for endothelin or prostaglandin systems differed. Endothelin-1 inhibited secretion of progesterone more (P<0.05) in luteal cells from cows with low versus high serum progesterone, when incubated with arachidonic acid. Secretion of prostaglandin F(2)alpha was increased and that of 6-keto-PGF(1)alpha decreased by endothelin-1 in vitro. Serum progesterone during replacement was lower (P<0.05) for cows with low than high serum progesterone at lutectomy. Thus, clearance, more than luteal production, determined peripheral progesterone in pregnant, lactating dairy cows.

Association of fertility with numbers of antral follicles within a follicular wave during the oestrous cycle in beef cattle.

The association between conception rate at first service and numbers of follicles developed during a follicular wave was examined in 102 suckled beef cows and 14 heifers. Follicular development was monitored using ultrasonography for either two (trial 1) or three (trial 2) consecutive oestrous cycles (pre-breeding, breeding and post-breeding equivalent). Animals were examined on alternate days from day 6 after first oestrus (day 0) until ovulation and from day 6 after insemination until next ovulation or day 24 of pregnancy and were observed for oestrus twice daily and inseminated artificially at either the second (trial 1) or third oestrus (trial 2). Cows were classified as having two or three waves of follicular development for each oestrous cycle. Numbers of follicles >or=4 mm per wave were determined, and based on the maximum diameter they attained, were classified as small (4-6 mm), medium (7-10 mm) or large (>or=11 mm) follicles. Total numbers of follicles, and primarily numbers of small and medium follicles, were affected by trial and within trial by cow, oestrous cycle and follicular wave. Heifers had more small and total numbers of follicles, but fewer large follicles than cows in trial 1 (p < 0.05). The average number of antral follicles per wave in the breeding cycle or post-breeding period did not affect conception rates, which averaged 84%. Repeatability of the total numbers of antral follicles between and among oestrous cycles and follicular waves ranged from 0.01 to 0.97. In conclusion, fertility was not affected by the numbers of antral follicles >or=4 mm in diameter in a single follicular wave.