Clinical significance of estrogen receptor phosphorylation.

Multiple sites of phosphorylation on human estrogen receptor α (ERα) have been identified by a variety of methodologies. Now with the emerging availability of phospho-site-specific antibodies to ERα, the relevance of phosphorylation of ERα in human breast cancer in vivo is being explored. Multiple phosphorylated sites in ERα can be detected in multiple breast tumor biopsy samples, providing evidence of their relevance to human breast cancer in vivo. Published data suggest that the detection in primary breast tumors of phosphorylation at some sites in ERα is associated with a better clinical outcome while phosphorylation at other sites is associated with a poorer clinical outcome most often in patients who have been treated with tamoxifen. This suggests the hypothesis that phospho-profiling of ERα in human breast tumors to establish an 'ERα phosphorylation code', may be a more accurate marker of prognosis and/or response to endocrine therapy in human breast cancer.

Markers of ovarian antral follicular development in sheep: comparison of follicles destined to ovulate from the final or penultimate follicular wave of the estrous cycle.

Treatment of non-prolific western white-faced ewes with prostaglandin F(2α) (PGF(2α)) and medroxyprogesterone acetate (MAP) increases the ovulation rate as a result of ovulations from the penultimate wave in addition to the final wave of the cycle. The objective of the current study was to evaluate the expression of markers of vascularization/angiogenesis, a marker of intercellular communication, and cellular proliferation and apoptosis in follicles from the penultimate and final waves. On day 8 of the estrous cycle, 15 ewes were administered a single injection of PGF(2α) and an intravaginal MAP sponge, which remained in place for 6 days. Two days after sponge removal, ovaries which contained follicles from the penultimate and final waves were collected and processed for immunohistochemistry followed by image analysis, and for quantitative real-time RT-PCR. Expression of factor VIII (marker of vascularization), proliferating cell nuclear antigen, and GJA1 (Cx43; marker of gap junctional communication) was greater (P<0.05) in follicles from the final wave compared with follicles from the penultimate wave. For theca cells, mRNA expression for vascular endothelial growth factor (VEGF) was greater (P<0.05) and tended to be greater (P≤0.1 and ≥0.05) for GJA1 and endothelial nitric oxide synthase in follicles from the final wave compared with follicles from the penultimate wave. For granulosa cells, the mRNA expression for GJA1 was greater (P<0.05) and tended to be greater (P≤0.1 and ≥0.05) for VEGF in follicles from the final wave compared with follicles from the penultimate wave. In conclusion, extension of the lifespan of follicles in the penultimate wave reduces follicular viability in the ewe.

Effects of the rate and duration of physiological increases in serum FSH concentrations on emergence of follicular waves in cyclic ewes.

Large antral follicles grow in waves in the ewe, with each wave triggered by a peak in serum FSH concentrations. In this study, our objectives were to determine if the slope of the rise in the FSH peak affects the ability of the peak to trigger wave emergence (experiment 1), and whether increasing serum FSH concentrations and holding them at peak concentrations would provide a stimulus for constant emergence of large antral follicles (experiment 2). In experiment 1, cyclic ewes received ovine FSH (n = 6; 0.1 µg/kg, s.c.) or vehicle (n = 6; control) every 6 h for 42 h. This treatment created a peak in serum FSH concentrations (P < 0.05) during the early growth phase of the first follicular wave of the interovulatory interval and enhanced the growth of follicles in that wave (P < 0.05), but did not trigger emergence of a follicular wave. In experiment 2, cyclic ewes were infused constantly with oFSH (1.98 µg/h; n = 6) or vehicle (control; n = 6) for 60 h starting at the time of the second endogenously driven FSH peak of the interovulatory interval. Infusion of oFSH resulted in a super-stimulatory effect, with a peak in the mean number of large follicles (≥5 mm) on Day 2 after the start of FSH infusion (13 ± 1.2 large follicles per ewe, 1.8 ± 0.2 in control ewes; P < 0.001). In conclusion, exposing early growing antral follicles in a wave to a gradual increase in serum concentrations of FSH enhanced their growth, but did not trigger the expected new follicular wave, and infusion of a dose of oFSH within the physiological range caused a super-ovulatory response in cyclic ewes.

Pulsatile LH secretion and ovarian follicular wave emergence and growth in anestrous ewes.

The objective of this study was to determine if pulsatile LH secretion was needed for ovarian follicular wave emergence and growth in the anestrous ewe. In Experiment 1, ewes were either large or small (10 x 0.47 or 5 x 0.47 cm, respectively; n = 5/group) sc implants releasing estradiol-17 beta for 10 d (Day 0 = day of implant insertion), to suppress pulsed LH secretion, but not FSH secretion. Five sham-operated control ewes received no implants. In Experiment 2, 12 ewes received large estradiol-releasing implants for 12 d (Day 0 = day of implant insertion); six were given GnRH (200 ng IV) every 4 h for the last 6 d that the implants were in place (to reinitiate pulsed LH secretion) whereas six Control ewes were given saline. Ovarian ultrasonography and blood sampling were done daily; blood samples were also taken every 12 min for 6 h on Days 5 and 9, and on Days 6 and 12 of the treatment period in Experiments 1 and 2, respectively. Treatment with estradiol blocked pulsatile LH secretion (P < 0.001). In Experiment 1, implant treatment halted follicular wave emergence between Days 2 and 10. In Experiment 2, follicular waves were suppressed during treatment with estradiol, but resumed following GnRH treatment. In both experiments, the range of peaks in serum FSH concentrations that preceded and triggered follicular wave emergence was almost the same as control ewes and those given estradiol implants alone or with GnRH; mean concentrations did not differ (P < 0.05). We concluded that some level of pulsatile LH secretion was required for the emergence of follicular waves that were triggered by peaks in serum FSH concentrations in the anestrous ewe.

Pulsed GnRH secretion and the FSH secretory peaks that initiate ovarian antral follicular wave emergence in anestrous ewes.

In ewes, immunization against GnRH blocks LH pulses but mean serum FSH concentrations are only partly reduced; the fate of the FSH peaks that precede ovarian follicular waves has not been studied. In this study, we used immunization against GnRH to examine the need for pulsed GnRH secretion in the genesis of FSH peaks in the anestrous ewe. Six anestrous ewes were given a GnRH immunogen on Day 0 and a booster injection on Day 28. Control ewes (n=6) received adjuvant only. Transrectal ultrasonography was performed daily for 2 days prior to and 10 days following both the primary (Days -2 to 10) and booster (Days 26-38) injections and for a 13-day period beginning 26 days after booster injection (Days 54-66). Blood samples were collected daily. Intensive bleeding (every 12min for 7h) was performed on Days 9, 37, and 65 of the experimental period to characterize the pulsatile pattern of LH secretion. GnRH antibody titers were increased and LH pulses were abolished immediately after booster immunization (P<0.05). The number of FSH peaks, FSH peak concentration and amplitude and basal FSH concentrations were only decreased in immunized ewes in the period of observations starting 26 days after booster immunization (P<0.05); however, some peaks were still seen. The number of follicular waves was decreased in the period around booster immunization and no follicular waves were seen during the period starting 26 days after booster immunization in immunized ewes (P<0.05). In summary, in anestrous ewes, when pulsed LH secretion was abolished by immunization against GnRH, the peaks in serum concentrations of FSH that trigger ovarian follicular waves continued for a period of time. We concluded that although blocking the effects of GnRH gradually causes a diminution of FSH secretion, there is no acute requirement for GnRH in the regulation of FSH peaks. The existence of FSH peaks in the absence of follicular waves, and pulsed LH secretion, suggests that some endogenous rhythm may drive the occurrence of FSH peaks, independent of both changes in negative feedback by secretory products from ovarian antral follicles and GnRH.

Ovarian follicular dominance and the induction of daily follicular waves in the ewe.

Large antral follicles grow in waves in the ewe, and each wave is triggered by a peak in serum concentrations of FSH. The existence of follicular dominance in the ewe is unclear. The objective of experiment 1 was to determine if an endogenously driven follicular wave could emerge during the growth phase of a wave induced by injection of ovine FSH (oFSH). Cyclic ewes (n = 7) were given oFSH (two injections of 0.5 microg/kg; s.c.; 8 h apart) on 2 separate days equally spaced in the interval between the first two endogenously driven follicular waves of an estrous cycle. Injection of oFSH induced two follicular waves in the interval between the first two endogenously driven waves of the cycle. The second endogenously driven wave of the estrous cycle emerged in the midgrowth phase of a follicular wave induced by injection of oFSH and its day of emergence, and growth pattern did not differ from that of the equivalent wave in control ewes (emerging 5.4 +/- 0.2 and 4.8 +/- 0.5 days after ovulation, respectively; P > 0.05). Experiment 2 was designed to determine if emergence of follicular waves could be induced on a daily basis. Six anestrus ewes were given oFSH (two injections of 0.35 microg/kg; s.c.; 8 h apart) on each of 4 days, starting 24 h after the expected time of an endogenously driven FSH peak. Each injection of oFSH resulted in a discrete peak in serum FSH concentrations and the emergence of a new follicular wave. The present findings provide evidence for the lack of follicular dominance in the ewe.

LH pulse frequency and the emergence and growth of ovarian antral follicular waves in the ewe during the luteal phase of the estrous cycle.

BACKGROUND: In the ewe, ovarian antral follicles emerge or grow from a pool of 2-3 mm follicles in a wave like pattern, reaching greater than or equal to 5 mm in diameter before regression or ovulation. There are 3 or 4 such follicular waves during each estrous cycle. Each wave is preceded by a peak in serum FSH concentrations. The role of pulsatile LH in ovarian antral follicular emergence and growth is unclear; therefore, the purpose of the present study was to further define this role. METHODS: Ewes (n = 7) were given 200 ng of GnRH (IV) every hour for 96 h from Day 7 of the estrous cycle, to increase LH pulse frequency. Controls (n = 6) received saline. In a second study, ewes (n = 6) received subcutaneous progesterone-releasing implants for 10 days starting on Day 4 of the cycle, to decrease LH pulse frequency. Controls (n = 6) underwent sham surgery. Daily transrectal ovarian ultrasonography and blood sampling was performed on all ewes from the day of estrus to the day of ovulation at the end of the cycle of the study. At appropriate times, additional blood samples were taken every 12 minutes for 6 h and 36 min or 6 h in studies 1 and 2 respectively. RESULTS: The largest follicle of the follicular wave growing when GnRH treatment started, grew to a larger diameter than the equivalent wave in control ewes (P < 0.05). Mean serum estradiol and progesterone concentrations were higher but mean serum FSH concentrations were lower during GnRH treatment compared to control ewes (P < 0.05). The increased serum concentrations of estradiol and progesterone, in GnRH treated ewes, suppressed a peak in serum concentrations of FSH, causing a follicular wave to be missed. Treatment with progesterone decreased the frequency of LH pulses but did not have any influence on serum FSH concentrations or follicular waves. CONCLUSION: We concluded that waves of ovarian follicular growth can occur at LH pulse frequencies lower than those seen in the luteal phase of the estrous cycle but frequencies seen in the follicular phase, when applied during the mid-luteal phase, in the presence of progesterone, do enhance follicular growth to resemble an ovulatory follicle, blocking the emergence of the next wave.

Association of luteinizing hormone receptor gene expression with cell cycle progression in granulosa cells.

During hormonally induced ovarian follicle growth, granulosa cell proliferation increases and returns to baseline prior to the administration of an ovulatory stimulus. Several key genes appear to follow a similar pattern, including the luteinizing hormone receptor (LHCGR), suggesting an association between cell cycle progression and gene expression. The expression of LHCGR mRNA in granulosa cells isolated from immature rats and treated in culture with FSH increased in a time-dependent manner, whereas administration of the cell cycle inhibitor mimosine completely suppressed expression. Although forskolin was able to induce luteinization in cells treated with mimosine, human chorionic gonadotropin had no effect, indicating the functional loss of LHCGR. The effects of mimosine on cell cycle progression and LHCGR mRNA expression were reversible within 24 h of mimosine removal. Cell cycle inhibition did not alter the stability of LHCGR mRNA, indicating that the primary effect was at the transcriptional level. To determine whether the relationship between LHCGR expression and cell cycle were relevant in vivo, immature rats were given a bolus of PMSG, followed by a second injection of either saline or PMSG 24 h later to augment levels of proliferation. The expression of LHCGR mRNA was elevated in the ovaries of animals receiving a supplement of PMSG. Mimosine also blocked cell cycle progression and LHCGR mRNA expression in macaque granulosa cells isolated following controlled ovarian stimulation cycles and in two different mouse Leydig tumor lines. These data collectively indicate that LHCGR mRNA is expressed as a function of the passage of cells across the G1-S phase boundary.