M.A. Crowe, 2,5 P. Kelly, 3,6 M.A. Driancourt, 4,7 M.P. Boland, 6 and J.F. Roche 5

Transcription

1 BIOLOGY OF REPRODUCTION 64, (21) Effects of Follicle-Stimulating Hormone With and Without Luteinizing Hormone on Serum Hormone Concentrations, Follicle Growth, and Intrafollicular Estradiol and Aromatase Activity in Gonadotropin-Releasing Hormone-Immunized Heifers 1 M.A. Crowe, 2,5 P. Kelly, 3,6 M.A. Driancourt, 4,7 M.P. Boland, 6 and J.F. Roche 5 Faculties of Veterinary Medicine 5 and Agriculture, 6 University College Dublin, Dublin 4, Ireland Institut National de la Recherche Agronomique, 7 Centre de Recherche de Tours, 3738 Nouzilly, France ABSTRACT To evaluate the roles of FSH and LH in follicular growth, GnRH-immunized anestrous heifers (n 17) were randomly assigned (Day ) to one of three groups (n 5 or 6). Group 1 received i.m. injections of 1.5 mg porcine FSH (pfsh) 4 times/ day for 2 days; group 2 received i.v. injections of 15 g plh 6 times/day for 6 days; group 3 received both pfsh and plh as described for groups 1 and 2. After slaughter on Day 6, measurements were made of follicle number and size, and follicular fluid concentrations of progesterone (P 4 ), estradiol (E 2 ), and aromatase activity. Injection of pfsh increased (P.1) the serum concentrations of FSH between 12 and 54 h. Infusion of plh increased (P.5) mean and basal concentrations of LH and LH pulse frequency. Serum E 2 concentrations were higher (P.5) for heifers given pfsh plh than those given either pfsh or plh alone. There was no difference (P.24) between treatments in the number of small follicles ( 5 mm). Heifers given pfsh or pfsh plh had more (P.2) medium follicles ( mm) than those that were given plh alone (none present). Heifers given pfsh plh had more (P.4) large follicles ( 1 mm) than those given either plh or pfsh alone (none present). Overall, only 1 of 35 small follicles and 2 of 96 medium follicles were E 2 -active (i.e., E 2 :P 4 1.), whereas 18 of 21 large follicles (all in the pfsh plh treatment) were E 2 - active; of these, 8 of 18 had aromatase activity. Concentrations of E 2 and E 2 activity in follicular fluid were correlated (r.57; P.1) with aromatase activity in heifers given plh pfsh. In conclusion, plh failed to stimulate follicle growth greater than 5 mm; pfsh stimulated growth of medium follicles that were E 2 -inactive at slaughter and failed to increase serum E 2 concentrations; whereas pfsh plh stimulated growth of medium follicles and E 2 -active large follicles, and a 1- to 14- fold increase in serum E 2 concentrations. follicle, follicular development, FSH, LH, ovary, pituitary hormones 1 This research was supported by a University College Dublin, Presidents Research Award to M.A.C. An abstract containing some of these data was presented at the 88th annual meeting of the American Society of Animal Science, Rapid City, SD, Correspondence: Mark A. Crowe, Faculty of Veterinary Medicine, University College Dublin, Ballsbridge, Dublin 4, Ireland. FAX: ; mcrowe@ucd.ie 3 Current address: Kildalton Agricultural College, Piltown, Co. Kilkenny, Ireland. 4 Current address: Intervet Pharma Research and Development, Rue Olivier de Serres, BP 67131, 4971 Beaucouze, France. Received: 23 June 2. First decision: 25 July 2. Accepted: 31 August by the Society for the Study of Reproduction, Inc. ISSN: Downloaded from on 9 February 218 INTRODUCTION The growth, development, and maturation of ovarian follicles is a fundamental process for effective reproduction in farm animals. In heifers, there are two to three periods of dominant follicle growth during the estrous cycle [1 4], each of which involves emergence, selection, and dominance followed by either atresia or ovulation. The key hormone that regulates follicular growth in cattle is FSH. A transient increase in FSH is associated with emergence of each period of follicle growth [5, 6]. The precise role of LH in controlling follicular dynamics is unclear, although it has been implicated in final maturation and ovulation [7, 8] and is required to stimulate androgen biosynthesis [9]. The aromatase enzyme system (in the granulosa cells of the follicle) converts androgens to estradiol-17 (E 2 ) under the regulation of FSH [1]. It has been demonstrated that dominant follicles have higher E 2 to progesterone (P 4 ) concentrations in the follicular fluid [11, 12] and higher aromatase activity in the follicular walls compared with subordinate follicles [13]. High estrogen activity associated with high aromatase activity is therefore a good indicator of physiological dominance and can be used to distinguish between healthy dominant follicles and atretic subordinate follicles. Immunization against GnRH prevents pulsatile secretion of LH and significantly decreases FSH concentrations, resulting in anestrus with follicular growth arrested at 4mm in diameter [14, 15], thus providing a gonadotropin-deficient model to study folliculogenesis. Furthermore, Crowe et al. [14] demonstrated that 24 mg of recombinant bovine FSH (rbfsh) administered over 4 days to GnRH-immunized heifers induced eight to nine large follicles ( 1 mm) to grow, while 12 mg of rbfsh over 6 days induced two to three large follicles, but a single dominant follicle was not selected in either treatment. This supported the hypothesis that pulsatile LH in addition to FSH may be required for selection of a single dominant follicle. The objective of this study was to use GnRH-immunized anestrous heifers to evaluate the specific roles of FSH and LH in the process of dominant follicle selection. The following hypotheses were tested: 1) high serum FSH concentrations over 2 days followed by decreasing FSH concentrations and 2 days of basal FSH concentrations would stimulate follicular growth; 2) the decreasing serum FSH concentrations alone would not stimulate selection of a dominant follicle; 3) pulsatile administration of LH in association with decreasing FSH concentrations would be required to induce selection of a single dominant follicle similar to that during the normal cycle; and 4) follicles from heifers treated with FSH only would be estrogen-inactive and lack aromatase activity, whereas follicles from heifers treated with FSH and LH would be estrogen-active and

2 GONADOTROPIN CONTROL OF FOLLICULOGENESIS AND STEROIDOGENESIS 369 have aromatase activity that would indicate physiological dominance. MATERIALS AND METHODS GnRH Immunization Seventeen cross-bred beef heifers were immunized against Cys-Gly-GnRH conjugated to human serum albumin (HSA) as described by Prendiville et al. [15]. The conjugate consisted of.1 mg HSA-Cys-Gly-GnRH dissolved in 2.5 ml saline (.9% NaCl). The adjuvant was made up using.25 g diethylaminoethyl-dextran dissolved in 2.5 ml sterile distilled water, with the ph adjusted to using TRIS (5 g/l) as described previously [16]. Equal volumes of conjugate and adjuvant were mixed and the final dosage was 5 ml. All immunizations were given s.c., divided into two injection sites in the lower neck area. Immunization consisted of a primary injection followed by two boosters (28 and 85 days later). Gonadotropin Treatments Ninety days after the primary immunization, heifers were blocked (n 2) by GnRH antibody titers and, within block, were randomly assigned to one of three treatments beginning on Day (92 days after primary immunization): 1) heifers (n 5) received 1.5 mg pfsh i.m. (Folltropin; Vetrepharm Inc., London, ON, Canada) every 6 h for 48 h; 2) heifers (n 6) received 15 g plh i.v. (Lutropin; Vetrepharm) every 4 h for 132 h; 3) heifers (n 6) received a combination of pfsh for 48 h and plh injections for 132 h as in treatments 1 and 2. Blood Sampling and Ultrasound Scanning Blood samples were taken via jugular venipuncture once daily from Day 4 today 1 and via jugular catheter every 6 h beginning at 6 h on Day until Day 6. Intensive blood samples were collected (every 15 min for 8 h) on Day 15 (n 4 at random) and on Days 1, 3, and 5(n 17). Blood samples were stored at room temperature for approximately 1 h and at 4 C for 18 to 24 h. Serum was decanted after centrifugation at 16 g for 15 min and stored at 2 C for subsequent hormonal analysis. Ovaries of heifers were examined daily by ultrasound from Day 4 to Day 6 with a transrectal 7.5 MHz linear transducer (Dynamic Imaging Ltd., Livingston, Scotland) as described previously [2, 17] to monitor the number and size of follicles. Follicles were categorized as medium (5- to 9-mm diameter) or large ( 1-mm diameter) based on ultrasound analysis. Animal experimentation was performed in compliance with protocols approved by the ethics committee of University College Dublin, the Cruelty to Animals Act (Ireland, 1876), and European Union Directive 86/69/EC. Follicle Recovery and Processing Heifers were slaughtered on Day 6 ( h after initiation of gonadotropin treatments). Ovaries were recovered within 2 min of slaughter and placed in M199 (Gibco BRL, Life Technologies Ltd., Paisley, Scotland) and stored in thermos flasks until they were processed in the laboratory. Individual follicles were dissected from each ovary and number and size were recorded. They were categorized into small ( 4-mm diameter), medium (5- to 9-mm diameter), and large ( 1-mm diameter) size classes. Follicular fluid was aspirated from all individual follicles and stored at 2 C until assayed for E 2 and P 4. Aromatase activity in follicular walls was determined by conversion of tritiated testosterone to tritiated water and estradiol as described by Badinga et al. [13]. Briefly, hemispheres of follicle walls were incubated in minimal essential medium (Sigma Chemical, Poole, Dorset, UK) supplemented with.5 Ci [1, 2-3 H(n)] testosterone (New England Nuclear, Boston, MA) for 3 h at 38.5 C in an atmosphere of 95% air and 5% CO 2. Aliquots of culture medium (.5 ml) were percolated through PrepSep C18 columns (Waters, Milford, CT) that had been previously washed with 1 ml methanol and 1 ml deionized water. Following sample percolation, the columns were eluted sequentially with 3.5 ml deionized water and 4 ml methanol to separate 3 H 2 O from steroids. Aliquots of water eluate (.5 ml) were added to liquid scintillation fluid and counted. Aromatase activity was calculated from the fractional conversion of 3 H from [1, 2-3 H(n)] testosterone into 3 H 2 O. Hormonal Analysis Serum GnRH antibody titers were measured by radioimmunoasssay [18] in samples collected 4 days after the primary injection followed by samples at 1- to 14-day intervals after primary immunization until commencement of FSH injections. Results are presented as the percentage total 125 I-GnRH bound at a serum dilution of 1:256. The intraassay coefficient of variation (CV; n 3 4) for a serum pool with 45.6% binding at a dilution of 1:256 was 2.8% and the interassay CV (n 5) for the same sample was 3.1%. Concentrations of P 4 were measured in oncedaily serum samples from Day 4 to slaughter and in follicular fluid using a previously validated radioimmunoassay [19]. Mean interassay CV (n 5) for samples containing.2,.82, and 2.44 ng P 4 /ml were 14.%, 15.9%, and 11.2%, respectively. Intraassay CV (n 4 6) for the same samples were 14.2%, 7.4%, and 7.%, respectively. The sensitivity of the P 4 assay was.5 ng/ml. Concentrations of E 2 were measured in serum samples at 12-h intervals from Day to Day 6 and in follicular fluid by a modification for bovine sera [15] of the method described by Evans et al. [2] using the Serono estradiol MAIA assay kit (Biodata S.p.A., Montecelio, Italy). Mean interassay CV (n 5 7) for samples containing.78, 2.38, and 5.89 pg/ ml were 22.5%, 15.6%, and 19.1%, respectively. Intraassay CV (n 4 6) for the same samples were 11.8%, 14.1%, and 11.5%, respectively. The sensitivity of the E 2 assay was.2 pg/ml. Concentrations of FSH were measured in serum samples collected every 6 h from Day until slaughter using a heterologous assay as described by Crowe et al. [21] using an NIDDK-anti-oFSH antibody (AFP- C ; National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD) and a bovine FSH standard preparation (bfsh B1; U.S. Department of Agriculture, Beltsville, MD). Mean interassay CV (n 9) for samples containing 16.6, 23.3, and 51.4 ng/ml were 7.2%, 5.7%, and 7.9%, respectively. Intraassay CV (n 4 6) for the same samples were 4.6%, 5.3%, and 9.3%, respectively. The sensitivity of the FSH assay was 1.6 ng/ml. Serum LH concentrations were determined using the method described by Cooke et al. [22]. Mean interassay CV (n 6 13) for samples containing 2.2, 6.5, and 1.2 ng LH/ml were 11.6%, 12.1%, and 11.%, respectively. Intraassay CV (n 4 6) for the same samples were 9.5%, 9.5%, and 14.2%, Downloaded from on 9 February 218

3 37 CROWE ET AL. FIG. 1. Mean SEM serum GnRH antibody titers for GnRH-immunized heifers during the 9 days prior to gonadotropin injections (primary [P] and booster [B1] immunizations were given 28 days apart, with a second booster [B2] given 57 days later). respectively. The sensitivity of the LH assay was.1 ng/ ml. Statistical Analyses Percentage FSH concentrations above baseline were analyzed using multivariate analysis, thus no assumption about the correlation between repeated measures on the same animal was required. When treatment effects were constant over time, differences between treatments were analyzed by a one-way ANOVA [23] applied to the mean levels (over time) of each animal. In the case of a treatment by time interaction, differences at each time were analyzed using one-way ANOVA [23], with significant differences determined after a Bonferroni adjustment for multiple comparisons, to obtain a 5% experiment-wise error. LH pulse frequencies and amplitudes were determined using the pulsar algorithm [24]. Values for peak determination were as follows: G(1) 3.8, G(2) 2.6, G(3) 1.9, G(4) 1.5, and G(5) 1.2. The depth criterion for splitting peaks was 2.7. Data for LH pulse amplitude, ratio of E 2 :P 4 concentrations in follicular fluid, and total amounts of E 2 and P 4 per follicle (transformed to log e ) were analyzed by a weighted ANOVA [23] using the Minitab Statistical Software general linear models procedure (Minitab Inc., State College, PA), with the number of pulses used as the weight factor for LH pulse amplitude and the number of follicles used as the weight factor for the intrafollicular data. Concentrations of LH in serum, the number of LH pulses, and the number of estrogen-active follicles were analyzed by ANOVA [23] using the Minitab general linear models procedure. When differences occurred, specific differences between treatments were determined using Fisher s one-sided least significant difference t-test at the 5% probability level. Correlation coefficients within treatments were determined for aromatase activity with P 4,E 2, and E 2 :P 4 concentrations in follicular fluid. RESULTS Immunization and Reproductive Activity Following primary immunization with the HSA-GnRH conjugate, antibody titers remained low but then rose rapidly after the first booster immunization and reached a peak (32.7% 3.3% at a serum dilution of 1:256) on Day 43 after primary immunization; thereafter, titers declined until 9 days after primary immunization, when they increased after the second booster immunization (Fig. 1). All heifers became anestrus before 9 days postprimary immunization, based on follicles 4 mm in diameter. This was confirmed at the beginning of the injection regime by progesterone concentrations.5 ng/ml. Progesterone concentrations remained.5 ng/ml throughout the experimental period. Hormonal Concentrations Concentrations of FSH were not different (P.5) between heifers that were treated with pfsh alone or in combination with plh throughout the experimental period (Fig. 2). Mean serum FSH concentrations in heifers that were treated with plh only did not increase and were lower FIG. 2. Changes in mean SEM serum FSH concentrations (ng/ml) in GnRH-immunized heifers treated with pfsh alone, pfsh and plh, or plh alone. a, b Means, within time points, with different superscripts are different (P.5). Downloaded from on 9 February 218

4 GONADOTROPIN CONTROL OF FOLLICULOGENESIS AND STEROIDOGENESIS 371 FIG. 3. Changes in mean SEM serum concentrations of estradiol (E 2 ; pg/ml) in GnRH-immunized heifers treated with pfsh and plh, pfsh alone, or plh alone (beginning at h). a, b, c Means, within time points, with different superscripts are different (P.5). (P.5) than the concentrations in heifers treated with pfsh alone or in combination with plh from 12 to 36 h and 48 h after the first pfsh injection. Concentrations of FSH were lower (P.5) in heifers treated with pfsh alone ( and 132 h after the first FSH injection) or in combination with plh ( h after the first FSH injection) compared with heifers that were treated with plh alone. Concentrations of E 2 in serum of heifers treated with pfsh alone did not differ (P.5) from those that were treated with plh alone throughout the experimental period, except at 48 h after the first pfsh injection, when heifers that had been treated with pfsh alone had higher (P.5) E 2 concentrations (Fig. 3). Heifers treated with both pfsh and plh had higher (P.5) mean serum E 2 concentrations than did heifers that were treated with plh alone between 24 and 12 h after the first pfsh injection, and had higher (P.5) mean serum E 2 concentrations than heifers that were treated with pfsh alone at 24 h and from 48 to 12 h after the first pfsh injection. There were no LH pulses observed in any of the four heifers examined on Day 15. There was no interaction between treatment and day in the mean or baseline serum LH concentrations and there was no significant effect of day on mean or basal serum LH concentrations. Both mean and basal serum LH concentrations during the experimental period were not different (P.5) between heifers that were treated with plh alone or in combination with pfsh (Table 1). Heifers treated with pfsh alone had lower (P TABLE 1. Mean ( SEM) number of LH pulses (per 8-h period), serum LH (ng/ml), and baseline LH (ng/ml) concentrations; and LH pulse amplitude (ng/ml) on Days 1, 3, and 5 of the experimental period, in GnRHimmunized heifers treated with plh alone, pfsh alone, or both pfsh and plh. LH Pulses (n) Mean LH concentrations (nm/ml) Baseline LH concentrations (ng/ml) LH pulse amplitude (ng/ml) plh pfsh pfsh plh a a a b b b a a a 5..5 a,b Means within rows with different superscripts are different (P.5)..1) mean and basal serum LH concentrations during the experimental period than did heifers that were treated with plh. There was no interaction between treatment and day in the number of LH pulses (per 8 h) or in the amplitude of LH pulses. Data for LH pulse amplitude from heifers treated with pfsh alone were excluded from the statistical analysis because an insufficient number of heifers in this treatment had LH pulses. Heifers treated with pfsh alone had fewer (P.1) LH pulses per 8 h during the experimental period than did heifers that were treated with plh alone or in combination with pfsh (Table 1). There was no difference (P.5) in the number of LH pulses or LH pulse amplitude during the experimental period in heifers that were treated with both pfsh and plh and in heifers treated with plh alone. Mean SEM LH profiles for the 8-h pulse bleeds on Days 1, 3, and 5 of the experimental period are presented for two representative animals in each treatment in Figure 4. Follicle Numbers Ultrasound scanning data. GnRH-immunized heifers treated with plh only had no follicles 5 mm in diameter. These heifers were not included in the statistical analysis of follicular data. The mean number of medium-sized follicles in heifers treated with pfsh and plh was higher ( vs ; P.5) than for those treated with pfsh only on Day 2 after gonadotropin treatments began (Table 2). The mean number of medium-sized follicles was not different (P.5) between the two treatments on any other day. The mean number of large follicles in heifers treated with pfsh and plh was greater (P.5) than in heifers treated with pfsh only on Days 3, 4, 5, and 6 after the gonadotropin treatments began (Table 2). Follicle numbers dissected at slaughter. Following slaughter, the number of small follicles was not different between treatments (1.8.6, and 2..8 for heifers treated with either pfsh, plh, or pfsh plh). There were greater (P.35) numbers of medium follicles in heifers treated with either pfsh alone (6. 1.) or pfsh plh ( ) than in heifers that received plh alone (none present). Only heifers that received pfsh plh had large follicles ( ) present on the ovaries at slaughter. Downloaded from on 9 February 218

5 372 CROWE ET AL. FIG. 4. Representative profiles of LH pulses in GnRH-immunized heifers treated with pfsh alone, pfsh and plh, or plh alone. Data are means SEM of LH concentrations in serial blood samples collected every 15 min over an 8-h window on Days 1, 3, and 5 after initiation of gonadotropin treatments. Follicular Fluid TABLE 2. Mean ( SEM) number of medium (5 9 mm) and large ( 1 mm) sized follicles in GnRH-immunized heifers treated with pfsh alone or both pfsh and plh (Day day of initiation of gonadotropin treatments). Day pfsh. 1.4 a Medium follicles pfsh plh b pfsh.6.4 a a a a Large follicles pfsh plh b b b b a,b Means, within follicle class and rows, with different superscripts are different (P.5 for medium follicles; P.1 for large follicles). Only heifers that received a combination of pfsh and plh had medium or large follicles that were estrogen-active (E 2 :P 4 ratio 1.; Table 3). Among the 18 of 21 large follicles in the FSH LH group that were estrogen-active, 8 had positive aromatase activity. For medium follicles in heifers treated with pfsh plh, the two estrogen-active follicles were also aromatase-active, and an additional follicle in this group was aromatase-active but not estrogenactive. Two medium follicles from heifers in the pfsh-only group had aromatase activity in the absence of estrogen activity. Concentrations of E 2 and the ratio of E 2 :P 4 concentrations in follicular fluid were highly correlated (r.57; P.1) with aromatase activity in heifers treated with a combination of pfsh and plh but not in heifers treated with pfsh alone (Table 4). There was no correlation (P.5) between P 4 concentrations in follicular fluid and aromatase activity. Heifers treated with plh alone produced no follicles with aromatase activity and, hence, were excluded from this analysis. DISCUSSION The main findings of this experiment are as follows: 1) GnRH-immunized heifers treated with pfsh alone grew large numbers of medium-sized follicles but few large follicles, all of which were estrogen-inactive; 2) GnRH-immunized heifers treated with pfsh and plh grew substantial numbers of medium and estrogen-active large follicles (some of which had associated aromatase activity), however, selection of a single dominant follicle per se did not occur; and 3) treatment of GnRH-immunized heifers with plh alone did not induce growth of follicles 5 mmin diameter. Serum E 2 concentrations were 1- to 14-fold higher in heifers treated with both pfsh and plh than in heifers treated with either pfsh alone or plh alone. Follicular fluid from heifers treated with a combination of pfsh and plh had E 2 concentrations and E 2 :P 4 ratios that were highly correlated with aromatase activity. This was not the case with follicular fluid from heifers treated with TABLE 3. Proportion of estrogen-active and aromatase-active follicles in GnRH-immunized heifers treated with pfsh alone, plh alone, or both pfsh and plh. Small follicles ( 5 mm) Medium follicles (5 9 mm) Large follicles ( 1 mm) Total Proportion of estrogen-active (and aromatase-active) follicles pfsh plh pfsh plh 1/9 /3 (2/3) / a 1/39 a /14 / / a /14 ab /12 2/66 (3/66) 18/21 (8/21) b 2/99 b a,b Means, within rows, with different superscripts are different (P.2). Downloaded from on 9 February 218

6 GONADOTROPIN CONTROL OF FOLLICULOGENESIS AND STEROIDOGENESIS 373 TABLE 4. Correlation coefficients (r) and associated significance levels (P) of aromatase activity with progesterone (P 4, estradiol-17 (E 2 ), and E 2 : P 4 concentrations in follicular fluid of GnRH-immunized heifers treated with pfsh alone or both pfsh and plh. P 4 E 2 E 2 :P 4 pfsh.21 (P.221).3 (P.886).4 (P.825) Aromatase activity pfsh plh.1 (P.332).6 (P.1).57 (P.1) pfsh alone. Follicles taken from heifers treated with plh alone had lower follicular fluid concentrations of P 4 than follicles from heifers treated with pfsh alone or both pfsh and plh. In a previous study using GnRH-immunized heifers as a model, Crowe et al. [14] demonstrated that low doses of rbfsh induced a small cohort of follicles to grow and develop, whereas high doses of rbfsh resulted in large numbers of both medium and large follicles. However, in the present study, pulsatile plh together with pfsh induced a much higher number of large follicles to grow than pfsh on its own, despite the fact that heifers in both treatment groups received the same dosage of pfsh and that heifers treated with plh alone grew no follicles greater than 5 mm in diameter. Hence, it appears that pulsatile LH acts synergistically with FSH to stimulate the growth of large follicles and is required to induce secretion of estradiol from large ovarian follicles. This is consistent with data from GnRH antagonist hypogonadal ewes in which FSH alone or in combination with LH pulses stimulated follicle growth, but estradiol secretion occurred only when FSH was supplemented with additional LH pulses [25]. Luteinizing hormone stimulates androgen biosynthesis [9], which is required as a precursor for follicular aromatase enzyme activity. Therefore, a reduction in androgen availability for aromatization may occur when LH pulses are absent, leading to morphological growth of large follicles that lack estrogen synthesis. Several investigators have suggested that the dominant follicle is less dependent on FSH than others in a cohort, or is more sensitive to available concentrations of FSH through increased induction of FSH receptors [1, 5, 26]. The substantial numbers of large follicles that grew in heifers treated with both pfsh and plh in comparison with pfsh alone may be due to their being more physiologically active and more sensitive, or availing more readily of the reduced serum concentrations of FSH observed from 48 h onward. Recent data suggest that follicles up to 7 mm in diameter are likely to be FSH-dependent because they contain FSH receptor mrna and not LH receptor mrna in granulosa cells with LH receptor mrna present in the theca. It is only when healthy follicles reach a size of 8 mm that LH receptor mrna is expressed in the granulosa cells [7]. This is consistent with an earlier literature report that LH binding to granulosa cells was greater in large follicles collected on Day 7 of the estrous cycle compared with follicles collected on Days 3 and 5 of the cycle [12]. Heifers treated with an agonist of GnRH by injection or infusion lacked LH pulses and follicles were arrested at 7 9 mm in diameter [27, 28]. If treatment with GnRH agonist was discontinued after 28 days, LH pulse frequency recovered, and one of the 7- to 9-mm follicles was selected to become dominant and ovulated [28]. These studies, in association with the fact that large follicles in the current study only secreted appreciable amounts of E 2 when stimulated to grow with FSH and LH rather than FSH alone, supports the hypothesis that in cattle, healthy large follicles switch their dependency from predominantly FSH to predominantly LH at 8 9 mm in size and require pulsatile LH to develop their normal steroidogenic capacity. Selection of a dominant follicle has been defined in cattle as the time when an estrogen-active follicle promotes its own growth and inhibits the growth of other follicles [11, 12]. Concentrations of E 2 in follicular fluid and aromatase activity of follicular walls are higher in dominant follicles than in subordinate follicles [13]. The present study demonstrated a high correlation between E 2 and E 2 : P 4 concentrations with aromatase activity in follicles taken from heifers treated with both pfsh and plh. There was no significant correlation between aromatase activity and E 2 or E 2 :P 4 concentrations in follicular fluid of heifers treated with pfsh alone. Because FSH appears to be responsible for controlling the aromatase system [29, 3] and LH stimulates the biosynthesis of androgens required for aromatization [9], it is not surprising that follicles induced to grow under the influence of FSH and LH have such correlations, whereas follicles induced to grow under the influence of FSH alone do not. Dominant follicles possess greater inhibin bioactivity than do estrogen-inactive follicles [31]. Both inhibins and E 2 from ovulatory and nonovulatory dominant follicles are believed to act in combination to cause a suppression of FSH secretion from the anterior pituitary gland [32 36]. However, it has been shown that intrafollicular concentrations of inhibins decrease during the growth of dominant ovulatory follicles, but increase during the growth of dominant nonovulatory follicles [37]. In the present experiment, suppression of serum FSH was evident in heifers treated with pfsh alone or in combination with plh, with FSH concentrations decreased below those of heifers treated with plh alone from 96 h after the first pfsh injection. Elevated serum concentrations of E 2 in heifers treated with both pfsh and plh would partly explain such a suppression. However, heifers treated with pfsh alone had no such increases either in serum concentrations or follicular amounts of E 2, over heifers treated with plh alone. Hence, inhibins (if present in high enough concentrations) from the follicles of these heifers may have played a role in the suppression of serum FSH concentrations. This would suggest that some of the large follicles on the ovaries of the heifers treated with pfsh alone may have been actively producing large amounts of inhibins. Whereas the large follicles of heifers treated with both pfsh and plh may have been producing both large amounts of E 2 and possibly inhibins. Indeed, previous work demonstrated increased ovarian secretion of inhibin in hypogonadal ewes stimulated with FSH in the presence or absence of low-amplitude LH pulses [25]. In summary, a greater number of large follicles grew under the influence of pfsh and plh than either gonadotropin on its own. Although a single dominant follicle was not selected in any of the three treatments, follicles from heifers treated with both gonadotropins appeared healthier as indicated by a strong correlation between both E 2 and E 2 :P 4 concentrations with aromatase activity and also higher serum concentrations of E 2 than in heifers from the other two treatments. The suppression of serum FSH observed in heifers treated with both pfsh and plh was probably caused by elevated serum E 2 concentrations and possibly in association with increased concentrations of specific inhibin forms. Downloaded from on 9 February 218

7 374 CROWE ET AL. ACKNOWLEDGMENTS The authors are grateful to T. Harte and the staff at Lyons Research Farm for the maintenance and care of experimental animals; and to N. Hynes for assistance with hormone radioimmunoassays. The authors acknowledge Dr. Philip Joumard of Argene Biosoft, France, for providing progesterone antiserum (Pi 531 B); the NHPP for providing FSH antiserum (AFP-C ) and FSH for iodination (AFP-4177A); D. Bolt (USDA, Beltsville MD) for providing bovine FSH standard; and Dr. J. Roser (Department of Animal Science, University of California, Davis, CA) for providing LH antiserum. REFERENCES 1. Ireland JJ, Roche JF. Hypothesis regarding development of dominant follicles during a bovine estrous cycle. In: Roche JF, O Callaghan D (eds.), Follicular Growth and Ovulation Rate in Farm Animals. The Hague, The Netherlands: Martinus Nijhoff; 1987: Savio JD, Keenan L, Boland MP, Roche JF. Pattern of growth of dominant follicles during the oestrous cycle of heifers. 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