Follicular Fluid Estradiol Concentrations Research Articles

Effect of dietary intake on concentrations of insulin-like growth factor-I in plasma and follicular fluid, and ovarian function in heifers

The objective of this study was to determine if alterations in dietary intake of heifers can influence IGF-I concentrations in plasma and(or) follicular fluid (FFL), size of follicles, and steroid concentrations in FFL (as an indicator of steroidogenic capacity). Cyclic heifers [n=23; mean ± SE body weight (BW) = 373 ± 7 kg were individually fed for 10 weeks either: a) 1.8% of BW in dry matter (DM) per d (GAIN; n=7), b) 1.1% of BW in DM per d (MAINT; n=8) or c) 0.7% of BW in DM per d (LOSE; n=8). After 10 wk of treatment, heifers were ovariectomized 36–40 hr after the second injection of prostaglandin F2α analog (2 injections 11 d apart), and plasma and ovaries were collected. Heifers weighed 444 ± 13, 387 ± 8 and 349 ± 9 kg in the GAIN, MAINT and LOSE groups, respectively, at time of ovariectomy. Mean diameter of follicles ≥ 10 mm was greater (P<.05) for GAIN (15.6 mm) than for MAINT (11.0 mm) or LOSE (12.5 mm) heifers. Numbers of follicles and concentrations of IGF-I in plasma and FFL did not differ (P>.20) between LOSE, MAINT and GAIN heifers. Progesterone concentrations were greater (P<.05) in small and medium follicles of GAIN than MAINT or LOSE heifers, but were unaffected by diet in large follicles. Estradiol concentrations in FFL in small, medium and large follicles were unaffected (P>.20) by dietary treatment. However, estradiol concentrations in estrogen-active follicles were two-fold greater (P<.05) in GAIN heifers than either MAINT or LOSE heifers. In conclusion, IGF-I, of ovarian and(or) systemic origin, was unlikely the primary mediator of dietary alterations in ovarian follicular function of heifers in the present study.

Dynamics of ovarian follicular development in cattle following hysterectomy and during early pregnancy

Three experiments were conducted to evaluate ovarian follicular dynamics and functional activity during pregnancy in cattle. In 11 pregnant Charolais cows of Experiment I, size of largest follicle, number of follicles and accumulated follicle size were reduced by day 27 of pregnancy on the ovary bearing the corpus luteum (CL) but not on the non-CL bearing ovary. In experiment II, local attenuation of ovarian follicular development on the CL bearing ovary of seven pregnant heifers was evident compared to the contralateral ovary without the CL. However, in four hysterectomized heifers, follicular development was sustained on both the CL- and non-CL bearing ovaries when CL maintenance was achieved without presence of the uterus or conceptus. In Experiment III, steroidogenic characteristics of the largest and second largest follicles at 17 d postestrus were evaluated for seven pregnant and six cyclic cattle. Follicle by physiological status interactions were detected for both aromatase activity of the follicle and follicular fluid concentrations of estradiol and progesterone. In cyclic cows, the largest follicle had appreciably more aromatase activity than did the second largest follicle; whereas, aromatase activity of the largest follicle from pregnant cows was less than that of cyclic cows. However, in pregnant cows the second largest follicle became the estrogen-active follicle, and this follicle occurred with a higher frequency on the ovary contralateral to the CL-bearing ovary. These changes in aromatase activity were reflected by parallel changes in estrogen concentrations of follicular fluid. The higher progesterone concentration in follicular fluid of the largest follicle in pregnant cows provided further confirmation of their atretic status. In conclusion, during early pregnancy the conceptus and/or uterus ipsilateral to the conceptus appear to secrete compounds which alter local follicular steroidogenic activity and attenuate subsequent follicular growth between 17 to 34 d of pregnancy on the CL-bearing ovary. This local mechanism acting within the ovary may contribute to the antiluteolytic effects of early pregnancy in cattle.

Decreased follicular steroids and insulin-like growth factor-I and increased atresia in diabetic gilts during follicular growth stimulated with PMSG

Four streptozotocin-diabetic gilts (maintained on exogenous insulin for 3 months) and 4 normoglycaemic gilts were treated with 600 i.u. PMSG. Diabetic gilts had insulin therapy removed at the time of PMSG administration. Plasma glucose averaged 463 +/- 5 mg/100 ml for diabetic gilts and 82 +/- 4 mg/100 ml for control gilts over the 72-h sampling period. Serum insulin was lower in diabetic than in normoglycaemic gilts (glycaemic state by time interaction; P less than 0.0001). At ovary removal 75 h after PMSG, numbers and percentages of large (greater than or equal to 7 mm) and medium (3-6 mm) non-atretic follicles were similar for diabetic and control gilts (31 vs 68%; s.e.m. = 7; P less than 0.05). Diabetic gilts had a greater percentage of atretic follicles over all size classes (50 vs 21%; s.e.m. = 7; P less than 0.03). After PMSG, LH was suppressed within 12 h in control gilts and remained similar to values in diabetic gilts until 72 h, when LH was elevated in 2 diabetic gilts (glycaemic state by time interaction; P less than 0.001). Pulsatile LH patterns during 52-55 h after PMSG were not affected by glycaemic state. Serum concentrations of IGF-I tended (P less than 0.1) to be lower in diabetic gilts. Concentrations of oestradiol and FSH in serum were similar in diabetic and control gilts. Follicular fluid concentrations of oestradiol in follicles greater than or equal to 7 mm were lower in diabetic than normoglycaemic gilts (341 vs 873 ng/ml; s.e.m. = 86; P less than 0.05). Testosterone was higher in follicles 3-6 mm in diameter in diabetic than in normoglycaemic gilts (142 vs 80 ng/ml; s.e.m. = 26; P less than 0.05). Progesterone concentrations in follicular fluid were not affected by glycaemic state. Concentrations of IGF-I in follicles greater than or equal to 7 mm were lower in diabetic than control gilts (150 vs 200 ng/ml; s.e.m. = 13; P less than 0.05). We conclude that follicles of diabetic gilts respond to external gonadotrophic stimulation with decreased hormone production and increased ovarian follicular atresia, despite an absence of effects on circulating gonadotrophin and oestradiol concentrations.

Histological evidence for heterogeneity in the development of preovulatory pig follicles.

Ovaries were collected from naturally cycling gilts during the preovulatory period and the stage relative to the LH surge estimated by measurement of oestradiol and progesterone concentrations in follicular fluid. Many of the follicles recovered had become flaccid with an associated increase in follicular fluid viscosity. Marked infolding of both the granulosa and theca tissue in some follicles suggested early luteinization. However, these morphological changes did not necessarily occur simultaneously in the same follicle, or in all follicles within an ovary. Moreover, they were not consistently related to characteristic differences in the concentration of follicular fluid steroids, suggesting either that the morphological and biochemical aspects of the luteinization of follicles may be independently controlled, or may respond at different rates to the same signal.

Prostaglandin F 2α, progesterone and estradiol concentrations in human follicular fluid and their relation to success of in vitro fertilization

Prostaglandin F 2α (PGF 2α), progesterone (P 4) and estradiol-17β (E 2) in follicular fluid were measured by radioimmunoassay in patients undergoing in vitro fertilization and embryo transfer. Follicular growth was induced using clomiphene citrate-hMG-hCG (15 patients) and FSH-hMG-hCG (4 patients). There was no significant difference in follicular fluid PGF 2α and P 4 concentrations relative to oocyte maturity as assessed morphologically. The highest PGF 2α concentration was found in fluid from FSH-hMG-hCG cycles where fertilization occurred. The value is significantly higher (p < 0.002) than in fluid from clomiphene-hMG-hCG cycles whether fertilization took place or not. There was no significant difference in P 4 and E 2 levels in relation to the type of ovarian induction or success in fertilization. Positive correlation between P 4 and E 2 in follicular fluid was found (r = 0.404). The positive correlation between total dose of hMG given to the patients and PGF 2α concentration in their preovulatory follicular fluid (r = 0.434) suggests that PGF 2α is secreted locally as the result of hMG and hCG stimulation. It is proposed that PGF 2α could be a biochemical marker for assessing the success of in vitro fertilization.

Hormonal parameters in follicular fluid and the fertilization rate of in vitro cultured oocytes.

Oocytes and matched samples of follicular fluid were obtained from 52 preovulatory follicles aspirated laparoscopically for in vitro fertilization (IVF). Follicular fluid concentrations of estradiol (E2), progesterone (P), testosterone (T), prolactin (HPRL), prostaglandin F2 alpha (PGF2 alpha), prostaglandin E (PGE), protein content, and collagenolytic activity were measured and related to the fertilization rate of oocytes cultured in vitro. High concentrations of P and low levels of T and HPRL were associated with mature, fertilizable oocytes. Levels of PGF2 alpha, PGE, and follicular fluid protein concentrations were similar in both groups. Mean collagenolytic activity was increased in the fertilized oocytes, although no significant difference could be observed. Our data demonstrate a close association between follicular fluid steroid and HPRL concentrations and successful fertilization of oocytes.

Alteration of human follicular fluid plasminogen activator activity by ovarian hyperstimulation.

Recently, it has been shown that granulosa-cell secretion of plasminogen activator (PA) is responsive to luteinizing hormone (LH)/human chorionic gonadotropin (hCG) as well as follicle stimulating hormone (FSH) in the rat and the pig. Accordingly, we asked whether PA activity in follicular fluid from exogenously stimulated human follicles was different from that of normal cycles and whether or not these activities correlated with follicular maturation as determined by follicular fluid steroid concentration. Follicular aspirates were obtained from women who were participating in an in vitro fertilization protocol. Follicular fluid concentrations of estradiol and progesterone were determined by established radioimmunoassay. PA activity, determined using a modified indirect solid-phase radioassay, was significantly less in follicles from patients treated with human menopausal gonadotropin (hMG) plus clomiphene (P less than 0.05) compared to untreated patients or those receiving hMG or clomiphene alone. Correlations of PA activity and follicular fluid steroid concentrations demonstrated no significant correlation in samples from treated patients. In contrast, untreated spontaneously cycling patients had a significant (r = 0.89, P less than 0.05), positive correlation between follicular fluid estradiol levels. There was no correlation between PA activity and follicular fluid progesterone levels in any of the groups. These results suggest that a subtle balance in granulosa-cell secretion of PA and steroid exists, which appears to be disrupted by follicular hyperstimulation during treatment of patients participating in in vitro fertilization protocols.

Correlation of inhibin and follicle regulatory protein activities with follicular fluid steroid levels in anovulatory patients

Follicular fluid was obtained from anovulatory patients (n = 12), stimulated with human menopausal gonadotropin, clomiphene, and human chorionic gonadotropin to evaluate the relative responses of inhibin, follicle regulatory protein, and steroid levels in follicles from ovaries requiring exogenous stimulation for follicular development. Follicular fluid concentrations of estradiol, progesterone, androstenedione, testosterone, dihydrotestosterone, and 3 alpha-androstenediol were determined by radioimmunoassay. Follicular fluid inhibin activity was determined by suppression of rat pituicyte follicle-stimulating hormone, and follicle regulatory protein activity was determined by suppression of porcine granulosa cell aromatase. The mean level of steroids were progesterone (7529 +/- 1601 ng/ml), estradiol (1082 +/- 158 ng/ml), androstenedione (15.2 +/- 3.17 ng/ml), 3 alpha-androstenediol (0.90 +/- 0.13 ng/ml), testosterone (2.23 +/- 33 ng/ml), and dihydrotestosterone (0.77 +/- 0.11 ng/ml). Follicle regulatory protein activity was 16.6% +/- 4.3% and mean inhibin level was 62.9 +/- 7.52 U. These results are in contrast to reports of follicular fluid steroid levels from normal ovulatory patients treated with exogenous gonadotropin. Although altered levels of hormones were present within these follicles, they clearly were not atretic, as evidenced by elevated estradiol levels and estradiol/androstenedione ratios. Alterations in the normal follicular response to pharmacologic gonadotropin stimulation in the follicles of anovulatory women suggest the presence of granulosa cell dysynchrony .

Correlation of human follicular fluid inhibin activity with spontaneous and induced follicle maturation.

We sought to correlate the inhibin activity of individual ovarian follicles (greater than 16 mm in diameter) from untreated (7 patients; 7 follicles), clomiphene-stimulated (150 mg/day; menstrual cycle days 5-9; 9 patients, 14 follicles), and human menopausal gonadotropin (hMG)-stimulated (150 IU/day; menstrual cycle days 3-11; 8 patients; 23 follicles) ovarian cycles and to correlate these results with the follicular fluid (FF) steroid concentration. Follicular aspirates were obtained via laparoscopy from 24 regularly menstruating patients when the diameter of the largest follicle reached 20 mm, as determined by serial ultrasonography. FF concentrations of estradiol, progesterone, testosterone, 17-hydroxyprogesterone, and androstenedione were determined by RIA. Inhibin activity was determined using the inhibition of basal 24-h FSH secretion by dispersed rat anterior pituitary cells. Inhibin values were highest among the follicles aspirated from those patients who received hMG [277 +/- 31 (+/- SE) U/ml] compared to untreated subjects (51 +/- 13 U/ml) or those who received clomiphene (96 +/- 14 U/ml). Estradiol was highest in FF from untreated patients (2295 +/- 1155 ng/ml) compared to levels in patients who received hMG (368 +/- 1.76 micrograms/ml) or clomiphene (1049 +/- 174 ng/ml). FF progesterone values were highest in untreated patients (9.4 +/- 2.59 micrograms/ml) compared to those in hMG-treated (5.04 +/- 1.76 micrograms/ml) and clomiphene-treated patients (7.82 +/- 1.24 ng/ml). FF 17-hydroxyprogesterone values (7.82 +/- 1.24 ng/ml). FF 17-hydroxyprogesterone values were similarly higher in the untreated (1.55 +/- 0.21 micrograms/ml) and clomiphene-treated (2.54 +/- 0.27 micrograms/ml) patients than in the hMG-treated group (0.73 +/- 0.09 micrograms/ml). FF androstenedione (untreated, 50.7 +/- 30 ng/ml; clomiphene-treated, 73.4 +/- 23.4 ng/ml; hMG-treated, 60.2 +/- 19.8 ng/ml) and testosterone (6.66 +/- 2.45, 5.98 +/- 1.46, and 6.39 +/- 2.16 ng/ml, respectively) concentrations in all three patient groups were similar. In untreated patients, there was a highly significant positive correlation between intrafollicular inhibin activity and FF estradiol, testosterone, and androstenedione concentrations and a statistically significant negative correlation between intrafollicular inhibin activity and FF progesterone concentrations. Patients receiving clomiphene therapy demonstrated at least two different response patterns, one with a positive and one a negative correlation between intrafollicular inhibin activity and FF steroid concentrations. The patients receiving hMG therapy had no statistically significant correlation between intrafollicular inhibin

Growth and differentiation of large antral follicles after spontaneous luteolysis in heifers: changes in concentration of hormones in follicular fluid and specific binding of gonadotropins to follicles.

The objective of the present study was to characterize changes in growth, concentrations of steroids in follicular fluid (FF) and gonadotropin receptors of individual antral follicles after spontaneous luteolysis in heifers and to reexamine our hormonal method of classifying healthy and atretic follicles. Groups of heifers were ovariectomized (OVX) before regression of corpora lutea on d 17, 18 and 19 of an estrous cycle and after regression of corpora lutea at time periods before, during and after preovulatory gonadotropin surges. Each follicle greater than or equal to 6 mm in diameter was examined for concentration of progesterone, androstenedione, testosterone and estradiol-17 beta in FF and capacities of granulosal and thecal cells to specifically bind 125I-bovine follicle stimulating hormone (bFSH) or 125I-human chorionic gonadotropin (hCG). Follicles were separated into two classes, estrogen-active (E-A) or estrogen-inactive (E-I). Concentrations of estradiol in FF were higher than both progesterone and androgens in E-A follicles, whereas either androgens or progesterone was higher than estradiol in E-I follicles. Diameters, volumes of FF and numbers of granulosal cells in E-A follicles increased from d 17 through the end of the preovulatory gonadotropin surges. During this time, concentrations of progesterone, androstenedione and estradiol in FF and specific binding of 125I-hCG to granulosal and thecal cells increased, whereas specific binding of 125I-bFSH to granulosal cells decreased. In E-I follicles, progesterone in FF decreased and androstenedione increased from d 17 through end of gonadotropin surges. The E-I follicles had a higher incidence of granulosal cells with pycnosis than E-A follicles. We concluded that changes in growth, concentration of steroids in FF and gonadotropin receptors in E-A follicles after spontaneous luteolysis were similar to changes we previously reported for E-A follicles after prostaglandin-induced luteolysis, and that ratio of concentration of estradiol in FF to both progesterone and androgens could be used to separate potential ovulatory (E-A) from atretic (E-I) follicles in heifers.

Changes in oestradiol, progesterone and testosterone concentrations in follicular fluid and in the micromorphology of preovulatory bovine follicles relative to the peak of luteinizing hormone.

Preovulatory bovine follicles (n = 58) were collected at different times after the onset of standing oestrus when cows would allow mounting until shortly before ovulation, which occurred 24 +/- 1.4 h after the peak level of LH in the peripheral blood. Non-atretic antral follicles (n = 71) of 3-20 mm were also collected from cows during the luteal phase of the oestrous cycle. The follicular fluid was aspirated for the radioimmunoassay of oestradiol-17 beta, progesterone and testosterone. The follicular wall was examined micromorphologically. Follicular fluid steroid levels were compared in 2-h periods relative to the LH peak. The development of the preovulatory follicles from onset of oestrus to ovulation can be divided into four phases. Phase 0 (after onset of oestrus but before LH surge) was characterized by a high level of oestradiol (6.05 mumol/l); the levels of progesterone and testosterone were lower (0.387 and 0.165 mumol/l respectively) but higher than in non-atretic luteal follicles of similar size. The theca interna (TI) was wider and the membrana granulosa (MG) cells were larger than those of non-atretic antral follicles. During phase 1 (0-6 h after the LH peak) oestradiol remained constant but at a lower level, progesterone increased (0.727 mumol/l) and testosterone was higher from 0 to 2h after the LH peak (0.241 mumol/l). The TI was 40% wider, whereas the size of the MG cells remained the same. In phase 2 (6-20 h after the LH peak) the level of oestradiol dropped rapidly during the period from 6 to 10 h, that of progesterone decreased to the same level as in phase 0 and that of testosterone was low (0.031 mumol/l). The width of the TI decreased to that of preovulatory follicles in phase 0 and the MG cells were slightly larger. In phase 3 (20 h after the LH peak until ovulation) the level of oestradiol decreased further (0.461 mumol/l) and that of testosterone remained low. Progesterone increased to the highest levels observed (1.51 mumol/l), however, and this coincided with a 39% increase in the size of the MG cells, whereas the width of the TI remained the same as in the preceding phases 0 and 2. In phase 3 the basement membrane began to disintegrate and phagocytic cells could be observed. This points to a simultaneous functional and morphological luteinization. It is suggested that these changes in follicular steroid levels and micromorphology are regulated by the preovulatory LH peak.