Follicular Deviation and Acquisition of Ovulatory Capacity in Bovine Follicles 1

BIOLOGY OF REPRODUCTION 65, 143 149 (21) Follicular Deviation and Acquisition of Ovulatory Capacity in Bovine Follicles 1 Roberto Sartori, 3,4 Paul M. Fricke, 2,3,4 João C.P. Ferreira, 6 O.J. Ginther, 5 and Milo C. Wiltbank 4 Department of Dairy Science 4 and Department of Animal Health and Biomedical Sciences, 5 University of Wisconsin, Madison, Wisconsin 5376 Departamento de Reprodução Animal e Radiologia Veterinária, 6 UNESP, Botucatu, SP, Brazil ABSTRACT Selection of dominant follicles in cattle is associated with a deviation in growth rate between the dominant and largest subordinate follicle of a wave (diameter deviation). To determine whether acquisition of ovulatory capacity is temporally associated with diameter deviation, cows were challenged with purified LH at known times after a GnRH-induced LH surge (experiment 1) or at known follicular diameters (experiments 2 and 3). A 4-mg dose of LH induced ovulation in all cows when the largest follicle was 12 mm (16 of 16), in 17% (1 of 6) when it was 11 mm, and no ovulation when it was 1 mm ( of 19). To determine the effect of LH dose on ovulatory capacity, follicular dynamics were monitored every 12 h, and cows received either 4 or 24 mg of LH when the largest follicle first achieved 1 mm in diameter (experiment 2). The proportion of cows ovulating was greater (P.5) for the 24-mg (9 of 13; 69.2%) compared with the 4-mg (1 of 13; 7.7%) LH dose. To determine the effect of a higher LH dose on follicles near diameter deviation, follicular dynamics were monitored every 8 h, and cows received 4 mg of LH when the largest follicle first achieved 7., 8.5, or 1. mm (experiment 3). No cows with a follicle of 7 mm ( of 9) or 8.5 mm ( of 9) ovulated, compared with 8% (8 of 1) of cows with 1-mm follicles. Thus, follicles acquired ovulatory capacity at about 1 mm, corresponding to about 1 day after the start of follicular deviation, but they required a greater LH dose to induce ovulation compared with larger follicles. We speculate that acquisition of ovulatory capacity may involve an increased expression of LH receptors on granulosa cells of the dominant follicle and that this change may also be important for further growth of the dominant follicle. follicular development, granulosa cells, luteinizing hormone, ovary, ovulation INTRODUCTION The final stages of follicular development in cattle occur in a wave-like pattern [1 3]. Selection of the dominant follicle is associated with a deviation in growth rate between the dominant and largest subordinate follicle, an event termed diameter deviation [4, 5]. The average diameter of the largest follicle of the wave at the onset of diameter deviation is 8.5 mm [4]. The mechanisms that lead to diameter deviation are not yet defined but appear to be closely associated with induction of granulosa cell LH re- 1 Supported in part by the University of Wisconsin-Madison College of Agricultural and Life Sciences, grant 9418 from the U.S. Department of Agriculture, and a scholarship from CAPES of Brazil to R.S. 2 Correspondence: Paul M. Fricke, Department of Dairy Science, University of Wisconsin-Madison, 1675 Observatory Drive, Madison, WI 5376. FAX: 68 263 9412; e-mail: fricke@calshp.cals.wisc.edu 3 Co-first authors. Received: 23 March 21. First decision: 17 April 21. Accepted: 25 June 21. 21 by the Society for the Study of Reproduction, Inc. ISSN: 6-3363. http://www.biolreprod.org 143 Downloaded from https://academic.oup.com/biolreprod/article-abstract/65/5/143/2723423 on 24 November 217 ceptors, an increase in circulating estradiol-17, and a decrease in plasma FSH [4 6]. One recent study tested the relationship between diameter deviation and a number of granulosa cell and follicular fluid characteristics [6]. There was an increase in LH receptor mrna at a diameter.5 mm sooner ( 8 h earlier) than the beginning of deviation in growth rate or increased follicular fluid estradiol concentrations. Other studies did not assess diameter deviation but found that granulosa cells from follicles near 1 mm in diameter had an increase in [ 125 I]-hCG binding [7], increased LH receptor mrna [8, 9], and increased responsiveness to LH based on camp generation [1]. In addition, follicles near 9 1 mm in diameter appear to have the capacity to ovulate [11 13], although no specific evaluation of ovulation response and diameter deviation has been reported. In this study we hypothesized that follicles acquire ovulatory capacity in response to an LH surge (ovulatory capacity) in association with follicular diameter deviation. Expression of LH receptors on granulosa cells may be the mechanism mediating the acquisition of ovulatory capacity; however, this was not directly tested in these experiments. Thus, we postulated that the dominant follicle would acquire ovulatory capacity just after the beginning of diameter deviation, whereas nondominant follicles or follicles before diameter deviation would lack ovulatory capacity. We tested these hypotheses by challenging cows with purified LH at predetermined times after a GnRH-induced LH surge (experiment 1) or at known follicular diameters (experiments 2 and 3). In addition, varying doses of LH were used in the latter experiments on the basis of results from experiment 1. MATERIALS AND METHODS Animal Procedures Primiparous and multiparous Holstein dairy cows ranging from 35 to 75 days of lactation were used for all experiments. Cows were housed in a stanchion barn at the University of Wisconsin-Madison Dairy Cattle Research Center with free access to water, and were managed using standard dairy husbandry practices. The University of Wisconsin Animal Care and Use Committee approved all animal handling and sample collection procedures. Cows were milked twice daily and were fed a total mixed ration balanced to meet or exceed minimum nutritional requirements for dairy cattle. The Ovsynch protocol using GnRH (Cystorelin; Merial Limited, Iselin, NJ) and prostaglandin (PG) F 2 (Lutalyse; Pharmacia Corporation, Peapack, NJ) as described previously [14] was used to synchronize ovulation and the onset of a new follicular wave in lactating dairy cows. Briefly, cows received an i.m. injection of GnRH (1 g; Day 9). Seven days later, cows received an i.m. injection of PGF 2 (25 mg; Day 2) to induce regression of the spontaneous and/or GnRH-induced corpus luteum (CL). Ovulation was synchronized by administration of a second i.m. injection of GnRH (1 g; Day ) 48 h after PGF 2 treatment. Ovulation of a dominant follicle in response to the second GnRH injection occurs in about 85% of lactating cows receiving this protocol [15]. Ovulation occurs within 24 32 h after the second GnRH injection in cows with a synchronized ovulation, and this ovulation is followed by growth of a new follicular wave [14]. Ovarian follicular dynamics were monitored by using an ultrasound

144 SARTORI ET AL. TABLE 1. Ovulatory capacity of bovine follicles in response to 4 mg of LH and diameter (mean SEM) of follicles on Day 3, 4, 5, or 6 of a follicular wave synchronized with GnRH (Day ) in lactating dairy cows. Days after GnRH 3 4 5 6 Ovulatory cows No. (%) /9 () a 3/12 (25) a,b 6/12 (5) b,c 8/8 (1) c Diameter (mm) of the largest follicle of the wave for ovulatory cows 11.7.3 A 12.7.5 A 13.6.4 Diameter (mm) of the largest follicle of the wave for nonovulatory cows 8..3 1.1.3 B 1.3.2 B Replicates 1 and 2 of experiment 1 combined. a,b,c Values with different superscripts within a column differ (P.5). A,B Values with different superscripts within a row differ (P.5). instrument equipped with a transrectal 7.5 MHz linear-array transducer (Aloka 5V; Corometrics Medical Systems, Inc., Wallingford, CT) as described previously [2, 16]. To assure measurements were at fixed intervals of 24 h (experiment 1), 12 h (experiment 2), or 8 h (experiment 3), each cow underwent an ultrasound examination at predetermined times each day, and in a specific sequence. Ovulatory capacity was assessed by i.m. administration of LH (Lutogen; Ausa International Inc., Tyler, TX). The bioactivity of this LH preparation was equivalent to NIH-LH-S19. A preliminary experiment was performed to evaluate the dose of LH that would produce ovulation. A total of 24 cows on Day 7 after the second GnRH injection of the Ovsynch protocol were given either 2 or 4 mg of LH (based on information from the company). Day 7 was chosen because all cows have a functional dominant follicle ( 13 mm) at this time. All cows (8 of 8) ovulated to a 4-mg dose of LH, whereas only 75% (12 of 16) ovulated to a 2-mg dose (unpublished results). Therefore, the 4-mg dose of LH was used in experiment 1. Response to the synchronization of ovulation protocol was assessed for all cows by confirming the presence of a large antral follicle on the day of the second GnRH injection and absence (i.e., ovulation) of that follicle 48 h thereafter. Cows that did not ovulate to the second GnRH injection were excluded from the experiment (n 5 of 46 in experiment 1, 3 of 29 in experiment 2, and 5 of 33 in experiment 3). The day of the second GnRH injection was defined as Day of the synchronized follicular wave. Follicular Deviation For cows exhibiting a single dominant follicle within a wave, the beginning of diameter deviation was defined as the beginning of the greatest difference in growth rates (diameter changes between successive ultrasound examinations) between the largest follicle (i.e., dominant follicle) and the second largest follicle (i.e., largest subordinate follicle) at or before the examination at which the second largest follicle reached its maximum diameter [4]. Two dominant follicles was defined as two follicles of 1 mm that had not yet undergone diameter deviation. For cows exhibiting two dominant follicles within the same follicular wave, time of deviation was defined as the beginning of the greatest difference in growth rates (diameter changes between successive ultrasound examinations) between the two largest follicles (i.e., codominant follicles) and the third largest follicle (i.e., largest subordinate follicle) of the wave. The diameter of the largest of the codominant follicles was used for analysis of size of follicle at treatment. These definitions were applied retrospectively to allow assignment of the time of deviation in a follicular wave for individual cows [4]. Experiment 1 In replicate 1, cows received 4 mg of LH on Day 3 (n 3), Day 4 (n 5), Day 5 (n 5), or Day 6 (n 3) after GnRH treatment (equivalent to 2, 3, 4, or 5 days after ovulation). The size and location of all antral follicles 4 mm in diameter were recorded on the day of LH administration and at 24 and 48 h thereafter to detect ovulation of any follicles that were present at the time of LH administration. In replicate 1 of experiment 1, follicular growth profiles prior to LH administration were not evaluated, but they were in all subsequent experiments. Therefore, the data from these cows could provide information on the size of follicles with ovulatory capacity (similar to previous studies such as 11) but they could not directly test the hypothesis on follicular deviation and ovulatory capacity. In replicate 2, cows were randomly assigned to receive 4 mg of LH on Day 3 (n 6), Day 4 (n 7), Day 5 (n 7), or Day 6 (n 5) after GnRH treatment. Size and location of all antral follicles 4 mmwas monitored every 24 h by using transrectal ultrasonography beginning on Day 1 after the second GnRH treatment and continuing until Day 7. Experiment 2 Based on the failure of 1-mm follicles to ovulate in experiment 1, we attempted to determine the effect of LH dose on ovulation of 1-mm follicles. Cows were randomly assigned to receive an i.m. injection of either 4 (n 13) or 24 (n 13) mg of LH at the time of the ultrasound examination when the largest follicle of the wave first achieved a diameter 1 mm. Size and location of all follicles 4 mm in diameter were monitored every 12 h by ultrasound beginning on the day of the second GnRH injection of the Ovsynch protocol until 48 h after LH administration. Follicular diameter at LH administration was chosen for this study based on results from experiment 1 in which 84% (21 of 25) of 1-mm follicles had undergone diameter deviation. Experiment 3 To determine the relationship between follicular diameter and ovulatory response using a higher dose of LH, cows were randomly assigned to receive 4 mg of LH when the largest follicle of the wave first achieved a diameter of 7. (n 9), 8.5 (n 9), or 1. mm (n 1). The 4-mg dose was chosen as a dose that was 1-fold greater than the dose that ovulated all follicles 12 mm in experiment 1 and the preliminary experiment in order to allow for a definitive ovulatory stimulus. Some follicles failed to ovulate to the 24-mg dose in experiment 2; therefore, a higher dose was used in experiment 3. Diameter and location of all follicles 4 mm in diameter were monitored every 8 h beginning at the time of the second GnRH injection of the Ovsynch protocol and continuing until 48 h after LH administration. Statistical Analysis Categorical data (ovulation rate) were analyzed by the Mantel-Haenszel chi-square test or the Fisher exact test [17]. The Fisher exact test was performed in experiments 1 and 3 to compare the percentage of cows ovulating among groups. Differences between continuous variables such as follicular growth rate, and follicle diameter at the time of LH treatment were analyzed on each specific day by a Student t-test. Differences among days of the cycle for mean diameter of the largest follicle of the wave were analyzed using a general linear models procedure [17]. RESULTS Experiment 1 Table 1 summarizes the ovulation results from experiment 1. A total of 17 of the 41 cows ovulated in response to 4 mg of LH. The percentage of cows that ovulated increased with increasing days from second GnRH treatment (P.5). On either Day 4 or 5, cows that ovulated (Table 1) had larger follicles than cows that did not ovulate (P.5). Across all days, mean diameter of responsive (n 17) follicles was greater (P.1) than that of unresponsive (n 24) follicles (12.9.3 vs. 9.3.3 mm, respectively). Overall, no follicle ( of 19) 1 mm (range 7 1 mm), 17% (1 of 6) of 11-mm follicles, and 1% (16 of 16) of follicles 12 mm (range 12 15 mm) ovulated. Three of the cows had double ovulations (7.3% of all cows, or 13.6% of ovulatory cows). Follicular Growth Profiles for Replicate 2 of Experiment 1 No cow on Day 3 ( of 6) had undergone deviation prior to LH and none ovulated after LH. Six of the seven cows for each of Days 4 and 5 exhibited diameter deviation prior to LH; however, only one cow on Day 4 ovulated, whereas five cows on Day 5 ovulated in response to 4 mg of LH. On Day 6, all cows had undergone diameter deviation prior Downloaded from https://academic.oup.com/biolreprod/article-abstract/65/5/143/2723423 on 24 November 217

ACQUISITION OF OVULATORY CAPACITY IN BOVINE FOLLICLES 145 to LH and all cows ovulated in response to LH. Thus, 11 of 17 of the cows that had undergone follicular deviation ovulated in response to 4 mg of LH. No cows ovulated follicles that had not yet undergone follicular deviation after the 4-mg LH treatment. Follicular codominance at the time of LH was exhibited by some cows on Day 4 (n 2), Day 5 (n 3), and Day 6 (n 1). Mean diameter of the largest follicle of the wave increased (P.1) from Day 3 to Day 6 (8..3, 1.9.3, 11.5.4, and 13.6.4 mm for Days 3, 4, 5, and 6, respectively). Using data from cows (n 19) that did not express codominance in replicate 2, the diameter of the two largest follicles was normalized either to the day of second GnRH administration or to the day of diameter deviation (on average, Day 3.5.2 after GnRH). When data were normalized to the day of GnRH administration (Day in Fig. 1A), there was a gradual divergence in mean growth rates of the two largest follicles. When data were normalized to the day of deviation (Fig. 1B), there was no difference (P.1) in growth rate between the two largest follicles until the day of deviation. Diameters of the dominant and largest subordinate follicles at deviation were 9.1.4 versus 7.9.3 mm, respectively, compared with diameters of 11.1.4 versus 7.5.3 mm, respectively, 24 h postdeviation. Several follicle growth profiles for individual cows are shown in Figure 2. Eight of the 25 cows in experiment 2 (32%) ovulated a single follicle after administration of 4 mg of LH. All of these cows had undergone diameter deviation prior to LH administration (Fig. 2A, for example). The largest subordinate follicle in one of the single ovulating cows underwent recovery after LH administration (Fig. 2B). Three cows (12%) ovulated two follicles after administration of 4 mg of LH (Fig. 2C, for example). All of these double-ovulating cows had codominant follicles at the time of LH and all three displayed diameter deviation from the third largest follicle prior to LH administration. Experiments 2 and 3 FIG. 1. Growth profiles (mean SEM) of the dominant and largest subordinate follicles of cows (n 19) in replicate 2 of experiment 1. A) Mean profiles for the follicles normalized to the day of GnRH injection (Day ). B) Mean profiles for the follicles normalized to the day of deviation (Day ) of the dominant and subordinate follicles in individual waves. In experiment 2, mean follicle diameter (1.2.8 mm) on the day of LH treatment did not differ (P.1) between cows receiving 4 mg versus 24 mg of LH. The percentage of cows ovulating was greater (P.5) for cows receiving 24 mg of LH (9 of 13 69.2%) than for cows receiving 4 mg of LH (1 of 13 7.7%). Twenty percent (2 of 1) of the cows that ovulated had a double ovulation. In experiment 3, actual mean follicle diameter was similar to the expected diameter at the time of LH treatment. No cows in which the largest growing follicle was 7. mm ( of 9) or 8.5 mm ( of 9) ovulated in response to 4 mg of LH. However, 8% (8 of 1) of cows with a 1.-mm follicle ovulated in response to 4 mg of LH. Of the cows that ovulated, 12.5% (1 of 8) had a double ovulation. Follicle growth patterns for cows in experiments 2 and 3 are shown in Figures 3 and 4. Mean diameter of the dominant and second largest follicle for all the cows (n 14) having a single ovulation after the 24- or 4-mg LH dose is shown in Figure 3A. Ten single-ovulating cows (71.4%) had detectable diameter deviation before LH administration (Fig. 3B), whereas four single-ovulating cows (28.6%) had no detectable diameter deviation prior to LH (Fig. 3C). Three of 18 (17%) ovulating cows had double ovulation in experiments 2 and 3. All of these cows had codominant follicles prior to LH and had exhibited diameter deviation from the third largest follicle prior to LH. For all single-ovulating cows (Fig. 3A) or cows that had diameter deviation prior to LH treatment (Fig. 3B), the growth rate of the dominant follicle was greater (P.5) compared with the largest subordinate follicle between Day.5 and Day. (i.e., the day the follicle attained 1 mm). This difference in growth rate was not observed for cows that had not undergone diameter deviation prior to LH treatment (Fig. 3C). Growth patterns for the two largest follicles in cows that did not ovulate after 24 or 4 mg of LH are shown in Figure 4A. Although diameter deviation was not detected by the time of LH injection in four of six cows that did not ovulate, the average follicle growth rate for all six cows tended to differ (P.64) between the dominant versus the largest subordinate follicle between Day.5 and Day. after LH (Fig. 4A). This appeared to be primarily due to differences in growth for the dominant and largest subordinate follicle for the two cows that underwent diameter deviation prior to LH. Of the cows that did not ovulate after 24 or 4 mg of LH, only 33.3% (2 of 6) underwent deviation Downloaded from https://academic.oup.com/biolreprod/article-abstract/65/5/143/2723423 on 24 November 217

146 SARTORI ET AL. FIG. 2. Growth profiles of the dominant and largest subordinate follicles and response to administration of 4 mg exogenous LH for individual cows in experiment 1. Arrows denote the day of LH administration; Ov denotes ovulation of a follicle within 24 h of the last ultrasound examination. A) Profile from an individual cow that had a single ovulation in response to LH. B) Profile from an individual cow that had recovery of the largest subordinate follicle following ovulation of the largest follicle. C) Profile of an individual cow that exhibited codominant follicles and in which two follicles ovulated after LH. FIG. 3. Growth patterns (mean SEM) of the ovulatory and largest subordinate follicles of cows treated with 24 or 4 mg of LH when the largest follicle first reached 1 mm in diameter (experiments 2 and 3). Data were normalized to the day of LH injection (i.e., Day ). A) Average size of follicles from all cows (n 14) that had a single ovulation after LH treatment. B) Average size of follicles from cows (n 1) that had a single ovulation after LH treatment and had detectable diameter deviation prior to the LH treatment. C) Average size of the follicles from cows (n 4) that had a single ovulation after LH treatment and no detectable diameter deviation prior to the LH treatment. Downloaded from https://academic.oup.com/biolreprod/article-abstract/65/5/143/2723423 on 24 November 217

ACQUISITION OF OVULATORY CAPACITY IN BOVINE FOLLICLES 147 TABLE 2. Number (n) and percentage (of N) of cows in which the largest follicle of the synchronized wave first achieved a diameter of 7., 8.5, and 1. mm relative to the time of GnRH injection.* Days after GnRH 1. 2. 2.1 3. 3.1 4. 4.1 5. 5.1 6. 7. mm (N 54 cows) n 2 29 4 1 % of N 37. 53.7 7.4 1.9 * Experiments 2 and 3 combined. Follicle diameter 8.5 mm (N 45 cows) n 2 23 14 6 % of N 4.4 51.1 31.1 13.3 1. mm (N 36 cows) n 22 1 4 % of N 61.1 27.8 11.1 Eight of the 12 cows (67%) that did not ovulate after the 4-mg dose of LH in experiment 2 had detectable deviation prior to the LH treatment (Fig. 4B). The growth rate of the dominant follicle was greater than the largest subordinate follicle (P.5) from Hour.5 to Hour when analyzing either all cows (data not shown) or only the cows that underwent deviation prior to LH treatment (Fig. 4B). Table 2 shows the variability among cows in the time from the second GnRH treatment to the time the follicle grew to a size of 7., 8.5, or 1. mm in diameter, combined for experiments 2 and 3. Most cows (9.7%) attained a follicle diameter of 7. mm between 1 and 3 days after GnRH; however, four cows required between 3.1 and 4 days, and one cow required more than 5 days. Most cows (82.2% 88.9%) attained a follicle diameter of 8.5 mm between 2.1 and 4 days after GnRH, and a 1-mm follicle diameter about 1 day later (3.1 5 days). FIG. 4. Growth patterns (mean SEM) for the largest and second largest follicles of the wave for cows that did not ovulate after treatment with LH when the largest follicle first reached 1 mm in diameter (experiments 2 and 3). Data were normalized to the day of LH injection (i.e., Day ). A) Average size of follicles from cows (n 4) that did not ovulate after 24 or 4 mg of LH. B) Average size of follicles from cows (n 8) that had no ovulation after the 4-mg dose of LH and detectable diameter deviation prior to the LH treatment. prior to LH treatment, as compared to 76.5% (13 of 17) of cows that had single or double ovulation after LH. The effect of treatment with LH on growth of the largest subordinate follicles was analyzed for experiments 2 and 3. In a fashion similar to the cow shown in Figure 1B, 6 of 17 cows (35%) that ovulated with 24 or 4 mg of LH had recovery of a subordinate follicle. All of the cows with a 1-mm dominant follicle that did not ovulate to the 24- or 4-mg dose of LH (n 6) exhibited either two (n 5) or more than two (n 1) dominant follicles during this follicular wave. In experiment 2, 12 of 13 cows did not ovulate to the 4-mg dose of LH, and six of them developed two or more dominant follicles during this follicular wave. In experiment 3, none of the nine cows treated with 4 mg of LH at a follicle size of 8.5 mm had ovulation; however, seven of the nine cows (77.8%) exhibited two or more dominant follicles during this follicular wave (two with two dominant follicles and five with more than two dominant follicles). DISCUSSION Selection of a dominant follicle in cattle involves endocrine and cellular events that are still being defined. Several physiological changes have been associated with follicular selection, including deviation in follicular growth rates, decreased circulating FSH, increased circulating estradiol-17, increased expression of LH receptors, and increased free follicular fluid insulin-like growth factor (IGF)-1 [4 6, 8, 9]. In this study we evaluated the temporal relationship between follicular diameter deviation, as assessed by transrectal ultrasound, with LH responsiveness of follicles in vivo, as assessed by the ability of the follicle to ovulate in response to an exogenous LH surge (ovulatory capacity). We found that ovulatory capacity generally followed diameter deviation with a shorter delay between these two events when a higher dose of LH was utilized. A comparison of studies from the scientific literature that used other measures of LH responsiveness to our measurements of in vivo ovulatory capacity provides novel insight into the mechanisms involved in follicle growth near the critical time of diameter deviation. In experiment 1, a dose of LH (4 mg) was chosen that had been found in a preliminary study to ovulate all large ( 13-mm diameter) follicles on Day 7 after GnRH treatment. The 4-mg dose of LH was insufficient to ovulate 1- mm follicles even though many of these follicles had undergone diameter deviation, whereas all growing follicles greater than 12 mm and 17% of 11-mm follicles responded with ovulation. In contrast, a greater dose of LH (24 or 4 mg) ovulated 7% 8% of 1-mm follicles, and those 1- mm follicles that failed to ovulate had generally not yet Downloaded from https://academic.oup.com/biolreprod/article-abstract/65/5/143/2723423 on 24 November 217

148 SARTORI ET AL. undergone diameter deviation at the time LH was administered. Thus, follicles that had undergone deviation and had reached a diameter of 1 mm had acquired ovulatory capacity and ovulated in response to a high dose of LH, but not a low dose. However, reservation is indicated on the conclusion that ovulatory capacity occurs after deviation until extensive studies are performed on follicles with diameters between 8.5 and 1. mm. Most data are consistent with an increase in LH receptors near the expected time of diameter deviation. One study [6] compared diameter deviation with granulosa cell LH receptor mrna changes as measured by quantitative reverse transcriptase-polymerase chain reaction. The increased difference in LH receptor mrna expression between the two largest follicles occurred, on average, an equivalent of 8 h before any increased difference in either follicle diameter or follicular-fluid estradiol concentration [6]. Other studies have also shown corresponding differences in LH receptor mrna and binding, although diameter deviation was not assessed. Using in situ hybridization, Xu et al. [8] showed an increase in LH receptor mrna on granulosa cells from nondetectable levels on Day 2 (average of 6.7 mm) to highly expressed levels on Day 4 (average of 1.8 mm; Day day of wave emergence). During this same time, LH receptor mrna in thecal cells increased more than fourfold [8]. Another study [9] also found an increase in detectable (by in situ hybridization) LH receptor mrna in granulosa cells at 1.8 mm, and LH receptor mrna increased nearly twofold as the follicle grew to 13.2 mm, and another twofold as the follicle grew to 15. mm. These increases in LH receptor correspond with acquisition of ovulatory capacity in our study and particularly with the decrease in dose of LH that was needed for ovulation of follicles as they grew from 1 to 12 mm. Although not statistically significant, Bodensteiner et al. [7] reported approximately threefold greater granulosa cell LH receptor numbers (measured by [ 125 I]-hCG binding) from the largest follicle on Day 2 after ovulation (average of 8.5 mm) compared with the largest follicle on Day 4 after ovulation (average of 13. mm). In contrast, Evans and Fortune [18] reported no increase in detectable LH receptor mrna (by in situ hybridization) in granulosa cells from follicles on Day 2 ( 9 mm) compared with Day 3 ( 11 mm; Day day of wave emergence), whereas differences in estradiol concentrations between the dominant and largest subordinate follicle were already detectable on both Day 2 and Day 3. However, Jolly et al. [1] measured the in vitro camp response to LH in granulosa cells from follicles of different sizes and found a clear increase in LH responsiveness as follicles grew larger than 9 1 mm. Thus, the time of diameter deviation ( 8.5 mm) generally relates to a variety of measures of LH responsiveness, including granulosa cell camp response to LH [1], increase in LH binding [7], increase in LH receptor mrna [6, 8, 9], and ovulation in response to LH in vivo (this study). Other in vivo studies, although not specifically designed to determine the relationship between diameter deviation and ovulatory capacity, are also consistent with acquisition of ovulatory capacity in a 9- to 1-mm follicle. Silcox et al. [19] reported that treatment with 1 g of GnRH stimulated ovulation of all large-growing dominant follicles (6 of 6, on average 3.8 days after estrus) and some static phase follicles (2 of 6, on average 7.4 days), but did not affect regressing follicles ( of 5 regressing follicles). The authors, however, did not report follicular diameters. Martinez et al. [13] treated beef heifers with either 1 g of GnRH or 25 mg of LH on Day 3 after ovulation and found that ovulation occurred in neither of two 7-mm follicles, in two of four 8-mm follicles, in three of three 9-mm follicles, and in six of six 1-mm follicles. Follicular growth patterns and diameter deviation were not determined in that study and it was not possible to determine whether LH or GnRH was the treatment stimulus for cows that ovulated 8- and 9-mm follicles. Detection of ovulatory capacity in a follicle of 1 mm probably relates to the dominant follicle becoming physiologically regulated by low-amplitude LH pulses that can cause continued growth of the dominant follicle. Gong et al. [2] found that follicles failed to grow beyond 9 mm in diameter in cows in which LH pulses had been suppressed by chronic treatment with a GnRH agonist. Conversely, reduction in circulating progesterone concentrations was found to increase numbers of LH pulses and to prolong growth and increase maximal diameter of the dominant follicle [21 23]. This persistent dominant follicle can also be induced by treatment with small exogenous LH pulses [24]. It is interesting that a transient increase in mean circulating LH concentrations encompasses the expected time of follicular deviation [25], although this transient increase apparently is not required for diameter deviation [26]. Inhibition of LH concentrations by treatment with progesterone did not alter the time or diameter characteristics of deviation. However, the diameter of the developing dominant follicle was reduced a mean of 31 h after the beginning of deviation, or when the follicle was 1 mm. Thus, maximal growth of the dominant follicle required adequate concentrations of LH at about the time that the follicle developed ovulatory capacity in the present study. Here, we chose to measure the readily detectable endpoint of ovulation at known points in follicular growth. These changes in LH responsiveness correspond to a time when most studies have found an increase in LH receptor expression in granulosa cells. This study was not designed to determine whether LH responsiveness is a cause of deviation or an effect of it. If diameter deviation was caused by LH responsiveness, then we would expect some follicles of 8.5 mm to have already acquired LH receptors and ovulatory capacity. However, follicles of 8.5 mm did not ovulate in response to the highest dose of LH (4 mg). This is a 1-fold greater dose than was required to ovulate all larger follicles. If LH responsiveness is an important part of diameter deviation, follicles at the beginning of diameter deviation (8.5 mm) may have an increased LH responsiveness that was adequate for diameter deviation but not for ovulatory capacity. It is interesting that four of the 17 cows that ovulated to the high dose of LH had not yet exhibited follicular diameter deviation. These follicles may have acquired ovulatory capacity but had not yet undergone detectable diameter deviation. Thus, determination of the timing of acquisition of ovulatory capacity can be a useful method for determining at least one aspect of in vivo follicular LH responsiveness. Although not directly related to our hypotheses, we found substantial recovery of subordinate follicles following LH treatment. Ovulation of a 1-mm follicle was associated with recovery of 35% of subordinate follicles. Martinez et al. [13] reported that in three (27.3%) of the 11 heifers that ovulated after treatment with GnRH or LH on Day 3, the second largest follicle continued growing and became the dominant follicle. Recovery of the subordinate follicle after LH is probably similar to the reported Downloaded from https://academic.oup.com/biolreprod/article-abstract/65/5/143/2723423 on 24 November 217

ACQUISITION OF OVULATORY CAPACITY IN BOVINE FOLLICLES 149 recovery of the subordinate follicle after aspiration or cauterization of a dominant follicle on Day 3 after ovulation [27, 28]. In those studies, removal of 1-mm or smaller dominant follicles led to recovery of the largest subordinate follicle in most cows. In our studies, even in the absence of ovulation, treatment of cattle that had 8.5- or 1-mm follicles with high doses of LH was associated with multiple dominant follicles in most of the cows (13 of 15 86.7%). Although not tested in this study, it seems likely that LH treatment, with or without subsequent ovulation, would inhibit aromatase expression [29], reduce circulating estradiol, and thereby allow a rebound in circulating FSH. The increase in circulating FSH, similar to that observed after cauterization of the dominant follicle [3], could stimulate growth of functional subordinate follicles. 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