Effect of LH on progesterone and oestradiol production in vivo and in vitro by preovulatory rat follicles

Temporal changes in follicular oestradiol production induced in vitro and in vivo by LH were studied. In-vitro changes were measured by incubating preovulatory rat follicles for 12 h, changing the medium every 2 h. Follicles isolated at various intervals after an injection of 10 i.u. hCG were incubated for 2 h to measure changes in oestradiol production in vivo. In both studies there was an increase in oestradiol production lasting 4 h followed by a sharp decline. Progesterone production was also increased by LH in vitro or hCG in vivo, but remained high. A second exposure to LH did not raise oestradiol synthesis, but increased progesterone synthesis in vitro only. The decline in oestradiol production is most probably due to a decrease in C17-20 lyase activity, because addition of testosterone, but not of 17 alpha-hydroxyprogesterone, increased oestradiol production. Incubation of preovulatory follicles in the absence of LH or incubation of follicles derived from animals in which the spontaneous LH surge was blocked by an injection of pentobarbitone sodium also resulted in a decrease of oestradiol and an increase in progesterone production. This oestrogen-progesterone shift was also caused by a decrease in C17-20 lyase activity. The results demonstrate that the changes in steroid production in vivo and in vitro are similar and occur in the presence and absence of LH. It is concluded that the decrease in oestradiol production is dependent on the decrease in the activity of enzymes converting progesterone to aromatizable androgens.     

The role of ovarian proteases and their inhibitors in ovulation

To assess the role of inhibitors of proteolytic enzymes, such as plasminogen activator (PA) and collagenase in the ovulatory process, inhibitor activity and mRNA levels were examined in periovulatory rat and human ovaries. In the rat, immature animals received 20 IU of pregnant mare serum gonadotropin (PMSG) followed 52 h later by 10 IU of hCG. Ovaries were removed at intervals from 0 to 20 h after human chorionic gonadotropin (hCG) administration. Inhibitor activity for metalloproteinases, such as collagenase, increased from 60.5 +/- 4.1 inhibitor units/ovary at 0 h (i.e., time of hCG treatment) to a maximum of 218.2 +/- 11.4 units/ovary at 8 h after hCG before decreasing at 12 h (time of ovulation) and 20 h (122.2 +/- 7.9 and 71.6 +/- 8.1 units/ovary, respectively). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in our in vitro fertilization program. Metalloproteinase inhibitor activity was evaluated in follicular fluid as well as the levels of PA and PA inhibitor (PAI) mRNA by Northern analysis. Increasing metalloproteinase inhibitor activity was positively correlated with follicular levels of estradiol (p less than 0.001) and progesterone (p less than 0.02, N = 26). Chromatographic separation of follicular fluid resulted in two peaks of metalloproteinase inhibitor activity. The large molecular weight (MW) inhibitor had an approximate size of 700 kilodaltons (kDa) and may represent alpha 2-macroglobulin, a serum-derived inhibitor. The small MW inhibitor shared many of the characteristics of tissue-derived inhibitors of metalloproteinases. Partial purification of the small MW inhibitor by Concanavalin A-Sepharose and Heparin-Sepharose chromatography demonstrated the inhibitor to be a glycoprotein with an approximate MW = 28-29 K. Northern analysis of human granulosa cell total RNA from preovulatory follicles showed little or no detectable tissue-type PA or urokinase-type PA mRNA. In contrast, two species of PA inhibitor type-1 mRNA were detected in relative abundance. The present findings demonstrate the presence of proteolytic inhibitors in periovulatory ovaries of the rat and human. These ovarian inhibitors may play a role in regulating connective tissue remodeling during follicular rupture.     

Interleukin-1 alpha increases thecal progesterone production of preovulatory follicles in cyclic hamsters

Adult cyclic hamsters were used to study the effects of interleukin-1 alpha (IL-1 alpha) on in vitro steroidogenesis in preovulatory follicles. IL-1 alpha increased progesterone secretion by preovulatory follicles during a 24-h incubation in RPMI-1640 medium containing hCG (100 mIU/ml) (progesterone levels: 17.5 +/- 2.2 vs. 10.6 +/- 1.9 ng/follicle/ml, p less than 0.05). IL-1 alpha alone had no effect on follicular steroidogenesis. The source of increased progesterone secretion was the thecae (9.8 +/- 1.0 vs. 5.8 +/- 0.4 ng/2 thecae/ml, p less than 0.01) and not the granulosa cells (6.6 +/- 0.2 vs. 6.8 +/- 0.5 ng/20,000 viable granulosa cells/ml). IL-1 alpha also stimulated production of testosterone in thecae of preovulatory follicles. The follicular progesterone increase was dependent on the time of incubation and dose of IL-1 alpha. IL-1 alpha at 5-50 U/ml maximally stimulated progesterone production in the preovulatory follicles, and no significant effect of IL-1 alpha was observed until the 12th hour of incubation. The effects of IL-1 alpha on in vitro steroidogenesis in preantral follicles, experimentally induced atretic preovulatory follicles, and newly formed corpora lutea were examined. IL-1 alpha in the presence of hCG also significantly increased progesterone secretion by atretic preovulatory follicles. In the incubation of preantral follicles or newly formed corpora lutea, however, IL-1 alpha did not alter steroidogenesis. These results indicate that IL-1 alpha stimulates progesterone secretion by preovulatory follicles and that the target tissue for this effect is the thecal layer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Permeability of rat ovarian follicles to LH during development and luteinization

A 'double isotope' technique has been used to describe the temporal relationship between plasma and follicular concentrations of LH after injection of 51Cr and 125I-rat LH into immature rats. Radiolabelled LH was detectable in all follicles 1 min after injection. Concentrations in small antral and large preovulatory follicles were not significantly different at any time and reached a maximum of 34.2 +/- 3.0% of plasma concentrations at 40 min. Concentrations of LH in preovulatory follicles exposed to an ovulatory dose of hCG 4 h previously were significantly greater (P less than 0.05) than those in small antral and preovulatory follicles at all times, and reached a maximum of 46.2 +/- 1.7% of plasma concentrations after 1 h. Polyacrylamide gel electrophoresis and immunoprecipitation with an antibody specific for rat LH indicated that radioactivity in plasma and follicular fluid represented radio-iodinated LH. Steroidogenic activities, light microscopy and measurements of follicular volume of each class of follicle confirmed that small antral, preovulatory follicles and preovulatory follicles exposed to an ovulatory dose of hCG in vivo could be isolated specifically. Based on these findings it is possible to calculate that, during an endogenous pulse of LH secretion, follicular concentrations of LH never exceed 20% of peak plasma concentrations. Pronounced increases in functional activities during antral growth were not correlated with increased follicular permeability. Only after acute exposure to an ovulatory dose of hCG in vivo was permeability significantly increased. We conclude that entry of LH into antral follicles is restricted and that exposure to an ovulatory dose of hCG results in greater amounts of LH entering preovulatory follicles.     

Follicular and hormonal dynamics during the first follicular wave in heifers

A few days after the first follicular wave emerges as 4-mm follicles, follicular deviation occurs wherein 1 follicle of the wave continues to grow (dominant follicle) while the others regress. The objectives of this study were to characterize follicle growth and associated changes in systemic concentrations of gonadotropins and estradiol at 8-h intervals encompassing the time of follicle deviation. Blood samples from heifers (n = 11) were collected and the ovaries scanned by ultrasound every 8 h from 48 h before to 112 h after the maximal value for the preovulatory LH surge. The follicular wave emerged at 5.8 +/- 5.5 h (mean +/- SEM) after the LH surge, and at this time the future dominant follicle (4.2 +/- 0.8 mm) was larger (P < 0.001) than the future largest subordinate follicle (3.6 +/- 0.1 mm). There was no difference in growth rates between the 2 follicles from emergence to the beginning of the deviation (0.5 mm/8 h for each follicle), indicating that, on average, the future dominant follicle maintained a size advantage over the future subordinate follicle. Deviation occurred when the 2 largest follicles were 8.3 +/- 0.2 and 7.8 +/- 0.2 mm in diameter, at 61.0 +/- 3.7 h after wave emergence. Diameter deviation was manifested between 2 adjacent examinations at 8-h intervals. Mean concentrations of FSH decreased, while mean concentrations of LH increased 24 and 32 h before deviation, respectively, and remained constant (no significant differences) for several 8-h intervals encompassing deviation. In addition to the increase and decrease in circulating estradiol concentrations associated with the preovulatory LH surge, an increase (P < 0.05) occurred between the beginning of deviation and 32 h after deviation. The results supported the hypotheses that deviation occurs rapidly (within 8 h), that elevated systemic LH concentrations are present during deviation, and that deviation is not preceded by an increase in systemic estradiol.     

Chemotactic detection of incipient ovarian follicular atresia.

Chemotactic activity was measured in the follicular fluid collected from normal and atretic Graafian follicles isolated from the rat ovaries. The atresia of Graafian follicles was induced by pentobarbitone injections for 3 days beginning the day of proestrous. The chemotactic activity, as measured by direct morphological evaluation of cellular locomotion of individual cells and Boyden leading front assay, was significantly higher in follicular fluid from atretic follicles and it showed a progressive increase from day 1 to day 3 of blockade of ovulation. In vitro exposure of blocked follicles to PMSG and hCG on day 1, 2 and 3 failed to alter the chemotactic response of leukocytes towards follicular fluid of atretic follicles. Increased chemotaxis in the follicular fluid after 24 hr of blockade of ovulation appears to form an important criterion to identify atretic follicles well in advance, before the morphological symptoms of degeneration become apparent and the incipient change once induced in follicles is not reversed by gonadotropins.     

Comparison of glucose-6-phosphate dehydrogenase activity in healthy and atretic follicles in rat ovary

Changes in the glucose-6-phosphate dehydrogenase activity have been determined in relation to atresia of Graafian follicles in the rat ovary. Induction of atresia in follicles either due to absence of hCG in the hormonally stimulated immature ovaries or by repeated injections of pentobarbitone sodium to proestrous rats caused significant rise in the enzyme activity. Measurement of enzyme activity in isolated follicular compartments of healthy and atretic follicles revealed that it is significantly higher in the thecal tissue than the granulosa. Increase in enzyme activity in the atretic follicles than the healthy ones occurs due to its rise both in theca and granulosa cells. The significance of these changes in the enzyme activity in healthy and atretic follicles are discussed in relation to the precocious luteinization of cells in the follicular envelope with the onset of atresia.     

Progesterone promotes oocyte maturation, but not ovulation, in nonhuman primate follicles without a gonadotropin surge

During the periovulatory interval, intrafollicular progesterone (P) prevents follicular atresia and promotes ovulation. Whether P influences oocyte quality or maturation and follicle rupture independent of the midcycle gonadotropin surge was examined. Rhesus monkeys underwent controlled ovarian stimulation with recombinant human gonadotropins followed by a) experiment 1: an ovulatory bolus of hCG alone or with a steroid synthesis inhibitor (trilostane, TRL), or TRL + the progestin R5020; or b) no hCG, but rather sesame oil (vehicle), R5020, or dihydrotestosterone (DHT). In experiment 1, the majority of oocytes remained immature (65% +/- 20%) by 12 h post-hCG. However, the percentage of degenerating oocytes increased (P < 0.05) with TRL (42% +/- 22% vs. 0% controls), but was reduced (P < 0.05) by progestin replacement (15% +/- 7%). By 36 h post-hCG, the majority of oocytes in all three groups reached metaphase II (MI). In experiment 2, no evidence of follicle rupture was observed in the vehicle, R5020, or DHT groups. Despite the absence of hCG, a significant (P < 0.05) percentage of oocytes resumed meiosis to metaphase I in R5020- (41 +/- 9) and DHT- (36 +/- 15) but not vehicle- (4 +/- 4) treated animals. Only oocytes from R5020-treated animals continued meiosis in vivo to MII. More (P < 0.05) oocytes fertilized in vitro with R5020 (40%) than with vehicle (20%) or DHT (22%). Thus, P is unable to elicit ovulation in the absence of an ovulatory gonadotropin surge; however, P and/or androgens may prevent oocyte atresia and promote oocyte nuclear maturation in primate follicles.     

Concentrations of steroids and gonadotropins in follicular fluid from normal heifers and heifers primed for superovulation

The concentrations of six steroids and of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in follicular fluid from preovulatory and large atretic follicles of normal Holstein heifers and from preovulatory follicles of heifers treated with a hormonal regimen that induces superovulation. Follicular fluid from preovulatory follicles of normal animals obtained prior to the LH surge contained extremely high concentrations of estradiol (1.1 +/- 0.06 micrograms/ml), with estrone concentrations about 20-fold less. Androstenedione was the predominant aromatizable androgen (278 +/- 44 ng/ml; testosterone = 150 +/- 39 ng/ml). Pregnenolone (40 +/- 3 ng/ml) was consistently higher than progesterone (25 +/- 3 ng/ml). In fluid obtained at 15 and 24 h after the onset of estrus, estradiol concentrations had declined 6- and 12-fold, respectively; androgen concentrations had decreased 10- to 20-fold; and progesterone concentrations were increased, whereas pregnenolone concentrations had declined. Concentrations of LH and FSH in these follicles were similar to plasma levels of these hormones before and after the gonadotropin surges. The most striking difference between mean steroid levels in large atretic follicles (greater than 1 cm in diameter) and preovulatory follicles obtained before the LH surge was that estradiol concentrations were about 150 times lower in atretic follicles. Atretic follicles also had much lower concentrations of LH and slightly lower concentrations of FSH than preovulatory follicles. Hormone concentrations in follicles obtained at 12 h after the onset of estrus from heifers primed for superovulation were similar to those observed in normal preovulatory follicles at estrus + 15 h, except that estrogen concentrations were about 6-40 times lower and there was more variability among animals for both steroid and gonadotropin concentrations. Variability in the concentrations of reproductive hormones in fluid from heifers primed for superovulation suggests that the variations in numbers of normal embryos obtained with this treatment may be due, at least in part, to abnormal follicular steroidogenesis.     

Androgen synthesis during follicular development: evidence that rat granulosa cell 17-ketosteroid reductase is independent of hormonal regulation

Although androgens have been implicated in follicular atresia, ovarian follicular androgen synthesis is required for preovulatory follicular growth. To localize the site(s) of androgen biosynthesis and to obtain a better understanding of the regulation of the androgenic pathway(s) in rat ovarian follicles we examined the relative abilities of developing follicles to accumulate specific androgens [testosterone (T) and dihydrotestosterone (DHT)] using both radioimmunoassay (RIA) and 3H-substrate metabolism techniques. Small antral and preovulatory follicles were obtained from control or human chorionic gonadotropin (hCG)-primed immature rats, respectively (Richards and Bogovich, 1982). Small antral follicles, theca and granulosa cells produced little immunoassayable androgen (T + DHT) when incubated with or without 8-bromo-cAMP. In contrast, preovulatory follicles and theca produced more androgen than small antral tissues and in a manner acutely stimulable by cAMP. Granulosa cells produced little androgen under these conditions. Inclusion of [3H] androstenedione in the incubates yielded increased accumulation of [3H] T and [3H] DHT for all small antral and preovulatory tissues. Indeed, granulosa cells from both small antral and preovulatory follicles possessed a remarkable ability to accumulate [3H] T. This ability was not altered by hypophysectomy or subsequent treatment with estradiol and/or follicle-stimulating hormone (FSH). These results suggest that 17-ketosteroid reductase may be a constitutive enzyme in granulosa cells.     

Effect of GnRH antagonist-induced prolonged follicular phase on follicular atresia and oocyte developmental competence in vitro in superovulated heifers

A GnRH antagonist (Antarelix) was used to suppress endogenous pulsatile secretion of LH and delay the preovulatory LH surge in superovulated heifers to study the effect of a prolonged follicular phase on both follicle and oocyte quality. Oestrous cycles were synchronized in 12 heifers with progestagen (norgestomet) implants for 10 days. On day 4 (day 0 = day of oestrus), heifers were stimulated with 24 mg pFSH for 4 days and luteolysis was induced at day 6 with PGF2 alpha (2 ml Estrumate). Animals in the control group (n = 4) were killed 24 h after the last FSH injection. At this time, heifers in group A36h (n = 4) and group A60h (n = 4) were treated with 1.6 mg of Antarelix every 12 h for 36 and 60 h, respectively, and then killed. After dissection of ovarian follicles, oocytes were collected for individual in vitro maturation, fertilization and culture; follicular fluid was collected for determination of steroid concentrations, and granulosa cells were smeared, fixed and stained for evaluation of pycnosis rates. Granulosa cell smears showed that 90% of follicles were healthy in the control group. In contrast, 36 and 58% of the follicles in group A36h showed signs of early or advanced atresia, respectively, while 90% of the follicles in group A60h showed signs of late atresia. Intrafollicular concentrations of oestradiol decreased (P < 0.0001) from healthy follicles (799.14 +/- 40.65 ng ml-1) to late atretic follicles (3.96 +/- 0.59 ng ml-1). Progesterone concentrations were higher (P < 0.0001) in healthy follicles compared with atretic follicles, irrespective of degree of atresia. Oestradiol:progesterone ratios decreased (P < 0.0001) from healthy (4.58 +/- 0.25) to late atretic follicles (0.07 +/- 0.009). The intrafollicular concentrations of oestradiol and progesterone were significantly higher (P < 0.0001) in the control than in the treated groups. The oestradiol:progesterone ratio was higher (P < 0.0001) in the control (4.55 +/- 0.25) than in the A36h (0.40 +/- 0.05) and A60h (0.07 +/- 0.009) groups. Unexpectedly, the cleavage rate of fertilized oocytes, blastocyst rate and number of cells per blastocyst were not significantly different among control (85%, 41% and 95 +/- 8), A36h (86%, 56% and 93 +/- 5) and A60h (88%, 58% and 79 +/- 4) groups. In addition, there were no significant differences in the blastocyst rates from oocytes derived from healthy (45%), early atretic (54%), advanced atretic (57%) and late atretic follicles (53%). In conclusion, the maintenance of the preovulatory follicles in superovulated heifers with a GnRH antagonist induced more atresia and a decrease in oestradiol and progesterone concentrations. However, the developmental potential in vitro to day 8 of the oocytes recovered from these atretic follicles was not affected.     

Autoradiographic analysis of proestrous changes in the binding of 125I-labeled prolactin to the hamster ovary

Autoradiographic histochemistry was employed to examine changes in the binding of 125I-labeled prolactin (Prl) to ovaries from proestrous hamsters before (at 1200 h), during (at 1600 h), and after (at 2000 h) the preovulatory gonadotropin surge. In untreated control hamsters, there was a marked and progressive loss of Prl binding, first in the interstitial cells and follicular thecae by 1600 h, and then in the granulosa cells of the preovulatory follicles by 2000 h. When proestrous hamsters were treated with ergocryptine to significantly lower serum Prl, or injected with exogenous Prl, Prl binding to their ovaries did not differ from controls, suggesting that decreased Prl binding was due to neither increased occupancy of binding sites by endogenous Prl nor down regulation of Prl receptors by Prl itself. Conversely, when proestrous hamsters were treated with phenobarbital to block the luteinizing hormone (LH)/follicle-stimulating hormone (FSH) surge, the loss of Prl binding sites in the ovaries was prevented, suggesting that the LH/FSH surge might initiate a down regulation of Prl receptors in the ovary. Such a down regulation of Prl receptors may serve as a mechanism by which the ability of Prl to affect periovulatory events in the ovary might be regulated.     

Effects of the antiandrogen hydroxyflutamide on progesterone secretion by preovulatory rat follicles in vivo and in vitro.

Serum and ovarian progesterone levels and in vitro production of progesterone by preovulatory follicles were measured on proestrus in pregnant mare's serum gonadotropin (PMSG) primed immature rats in which the luteinizing hormone (LH) surge and ovulation were blocked by administration of the antiandrogen hydroxyflutamide. Serum progesterone levels observed at 12:00 on proestrus were significantly elevated, twofold above those observed in vehicle-treated controls, by in vivo administration of 5 mg hydroxyflutamide 4 h earlier. In control rats, proestrous progesterone did not increase until 16:00, in parallel with rising LH levels of the LH surge. No LH surge occurred in the hydroxyflutamide-treated rats, ovulation was blocked, and serum progesterone declined throughout the afternoon of proestrus, from the elevated levels present at 12:00. Administration of human chorionic gonadotropin (hCG) at 11:00 advanced the elevation of serum progesterone by 2 h in vehicle-treated controls and prevented the decline in progesterone levels in hydroxyflutamide-treated rats. The patterns of change in ovarian tissue concentrations with time and treatment were essentially similar to those observed for serum progesterone. In in vitro experiments, progesterone secretion during 24 h culture of preovulatory follicles obtained on PMSG-induced proestrus was significantly increased, sixfold, by addition to the culture media of 370 microM but not of 37 microM hydroxyflutamide. Testosterone (50 nM) and hCG (20 mIU/mL) caused 26- and 14-fold increases, respectively, in progesterone secretion by cultured follicles. Hydroxyflutamide significantly reduced the stimulatory effect of testosterone but not of hCG on progesterone secretion in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the secretion of inhibin and steroid hormones during induced follicular atresia after hypophysectomy in the rat

Sequential changes in the function of antral follicles during the period of follicular atresia were investigated after hypophysectomy (Hypox) at 1100 hr on proestrus. Within 6 hours after Hypox, concentrations of progesterone (P), testosterone (T) and estradiol-17 beta (E) decreased abruptly in ovarian venous plasma (OVP) and follicles showed a reduced ability to ovulate. Six hours after Hypox, ovulation was still induced by human chorionic gonadotropin (hCG) in all animals but with significantly fewer number of oocytes compared to the group given hCG at 1100 hr on the day of proestrus. Nine hours after Hypox, several granulosa cells of all large follicles (greater than 400 microns in diameter) exhibited morphological signs of atresia. Twelve hours after Hypox, all large and medium sized (200-400 microns in diameter) follicles showed advanced stages of atresia and almost all follicles failed to ovulate in response to hCG. Inhibin activity in OVP declined more slowly compared to the profiles of steroid hormones and 53% of the initial inhibin activity was still maintained at 18 hours after the operation. Inhibin activity further decreased to 7% of the initial level at 24 hours and was undetectable by 48 hours after Hypox. These results suggest that fully developed Graafian follicles gradually lose their ability to secrete inhibin in contrast to the rapid decrease in secretion of steroid hormone during the process of atresia.     

Gonadotropin regulation of glutamate cysteine ligase catalytic and modifier subunit expression in rat ovary is subunit and follicle stage specific

We have observed that levels of the antioxidant glutathione (GSH) and protein levels of the catalytic and modifier subunits of the rate-limiting enzyme in GSH synthesis, GCLc and GCLm, increase in immature rat ovaries after treatment with gonadotropin. The goals of the present studies were to delineate the time course and intraovarian localization of changes in GSH and GCL after pregnant mare's serum gonadotropin (PMSG) and after an ovulatory gonadotropin stimulus. Twenty-four hours after PMSG, there was a shift from predominantly granulosa cell expression of gclm mRNA, and to a lesser extent gclc, to predominantly theca cell expression. GCLc immunostaining increased in granulosa and theca cells and in interstitial cells. Next, prepubertal female rats were primed with PMSG, followed 48 h later by 10 IU of hCG. GCLm protein and mRNA levels increased dramatically from 0 to 4 h after hCG and then declined rapidly. There was minimal change in GCLc. The increase in gclm mRNA expression was localized mainly to granulosa and theca cells of preovulatory follicles. To verify that GCL responds similarly to an endogenous preovulatory gonadotropin surge, we quantified ovarian GCL mRNA levels during the periovulatory period in adult rats. gclm mRNA levels increased after the gonadotropin surge on proestrus and then declined rapidly. Finally, we assessed the effects of gonadotropin on ovarian GCL enzymatic activity. GCL enzymatic activity increased significantly at 48 h after PMSG injection and did not increase further after hCG. These results demonstrate that gonadotropins regulate follicular GCL expression in a follicle stage-dependent manner and in a GCL subunit-dependent manner.     

Follicular dynamics around the recruitment of the first follicular wave in the cow

The present study aimed to test the generally accepted view that a follicular wave starts with follicles newly recruited from the population smaller than 3 mm, which later compete for dominance. According to this view, subordinate follicles are expected to be too atretic to join the next follicular wave. Ten cows were ovariectomized shortly prior to the LH surge, thus around the start of the first follicular wave of the cycle. Per cow, on average, 14.4 follicles of >/=3 mm were dissected. Follicular health was determined on the basis of four parameters: 1) judgment of the degree of atresia by stereomicroscope, 2) incidence of apoptotic nuclei among the granulosa cells, 3) estradiol and progesterone concentrations, and 4) insulin-like growth factor-I (IGF-I) binding proteins (IGFBPs)-2, -4, and -5 concentrations in the follicular fluid. In addition to the preovulatory follicle, 3.1 other follicles, mainly sized 3-4.5 mm, were found to be healthy based on the proportion of apoptotic nuclei, and concentrations of estradiol/progesterone, and IGFBPs. The ability of these follicles to respond with growth on the preovulatory and periovulatory FSH surges was supported by a comparison to the follicular population of four cows 31-68 h after the LH surge. The present results point to an alteration of the view on the follicular wave. The larger follicles during the first days of the follicular wave are, in general, derived from follicles that also joined the previous wave. A portion of these growing follicles are estradiol active and compete for dominance. Other growing follicles lack estradiol production and are probably derived from rather atretic follicles. The first newly recruited follicles do not reach the size of 3 mm before 31 h after the preovulatory FSH surge. At that time, the larger follicles are already competing for dominance.