Follicle growth and associated changes in circulating hormone concentrations were evaluated after follicle ablation in mares (n = 13) during four 13 day periods beginning at means of -98.5, -61.4, -26.0 and 10 days from the first ovulation and corresponding to mid-anovulatory, early-transitional, late-transitional and ovulatory periods, respectively. During each period, all follicles > 5 mm in diameter were ablated (day 0) followed by no subsequent ablations (all-follicle group) or the ablation of all follicles > 5 mm when a new follicle reached > 10 mm (0-follicle group). A follicular wave emerged at means of days 1.5-2.5 in all mares of the all-follicle group during each period. Follicle activity increased between the mid-anovulatory and the transitional periods, as indicated by increases in the diameter of the largest follicle and the number of follicles in the all-follicle groups; nonetheless, there were no differences in the FSH concentrations of the wave-stimulating surge throughout the study. Circulating total inhibin increased between day 3 and day 6 in the all-follicle groups but not in the 0-follicle groups, and was higher during the late-transitional and ovulatory periods than during earlier periods in the all-follicle groups. In the all-follicle groups, circulating FSH decreased between day 3 and day 6. In the 0-follicle groups, FSH did not decrease and was higher during the late-transitional and ovulatory periods than during the mid-anovulatory period. Circulating LH was higher during the late-transitional and ovulatory periods than during earlier periods and was not different between the all-follicle and 0-follicle groups. On the basis of temporal relationships, it was concluded that the follicles of a wave secreted inhibin during all periods and the follicular inhibin suppressed circulating FSH. An increase in circulating inhibin induced by the growth of follicles > 21 mm in diameter during the late-transitional and ovulatory periods counteracted a stimulatory effect on FSH mediated by season.
FX Donadeu and OJ Ginther
Follicular waves and associated circulating hormone concentrations were studied during the anovulatory season in pony mares (n=8). Follicles were monitored by ultrasonography and a blood sample was taken daily from 29 January until ovulation (mean, 28 April). A mid-anovulatory period (largest follicle, 16.0+/-0.5 mm in diameter) and transitional period (largest follicle, 22.4+/-0.5 mm) were distinctive in each mare. The two periods were delineated by an increase in the diameter of the largest follicle to >/=21.0 mm. Follicular waves, identified by significant increases in the mean diameter of the second to sixth largest follicles, were detected during both the mid-anovulatory and transitional periods. The mean number of follicles >/=15.0 mm in diameter and the diameter of the second to sixth largest follicles increased in association with statistically identified FSH surges. The pattern of the FSH concentration changes during surges did not change during the mid-anovulatory and transitional periods. During the declining portion of the FSH surge, follicle growth continued and circulating total inhibin increased, indicating suppression of FSH by inhibin from the growing follicles. Circulating oestradiol or LH did not change relative to wave emergence. Results indicated that follicular waves occurred during the second-half of the anovulatory season, even during the period of lowest follicular activity. On a temporal basis, follicular wave emergence was stimulated by surges in circulating FSH. However, the increase in follicle growth to >/=21.0 mm in diameter for the wave at the beginning of the transitional period and for the subsequent waves was not attributable to a change in the characteristics of the associated FSH surges.
FX Donadeu and OJ Ginther
The role of the number of follicles and circulating immunoreactive inhibin in the decrease in plasma FSH concentrations that occurs during development of a follicular wave was studied in mares. All follicles > or = 6 mm in diameter were ablated by ultrasound-guided transvaginal aspiration of follicular fluid on day 10 after ovulation. During the subsequent wave, all follicles, the three largest follicles (three follicle group), the largest follicle (single follicle group) or no follicles were retained and the remaining follicles were ablated before they reached > 10 mm in diameter (n = 10-11 mares per group). Ablation of new follicles was continued until the day on which the largest follicle of the new wave reached 25 mm in diameter (day 18 after ovulation in the 'no follicle' group). Diameters of retained follicles were measured once a day by transrectal ultrasonography. Plasma samples were taken once a day and analysed by radioimmunoassay for concentrations of FSH and immunoreactive inhibin (includes dimeric inhibin as well as free alpha-subunit forms). Data were normalized to the day of the expected start of the decrease in plasma FSH concentrations (day 0: largest follicle 13 mm in diameter in the follicle-retained groups). A simultaneous increase in circulating concentrations of FSH (P < 0.05) and immunoreactive inhibin (P < 0.05) occurred before the largest follicle reached 13 mm in diameter, which indicates that immunoreactive inhibin produced by follicles < 13 mm in diameter did not suppress FSH. Plasma concentrations of FSH decreased (P < 0.05) and immunoreactive inhibin concentrations increased (P < 0.05) after day 0 in the follicle-retained groups. A slower decrease in FSH concentrations was associated temporally with a delay in the increase in immunoreactive inhibin concentrations in the 'single follicle' group relative to the 'three follicle' and 'all follicle' groups. All follicle-retained groups had similar plasma concentrations of FSH and immunoreactive inhibin after the expected beginning of deviation in growth rates between the two largest follicles (largest follicle 22-23 mm in diameter). These results indicated that the decrease in plasma FSH concentrations from the start of the decrease until the expected day of deviation was a function of multiple follicles of a wave and was attributable to the secretion of inhibin. Thereafter, the largest follicle alone accounted for the continued FSH suppression.
OJ Ginther, BG Woods, C Meira, MA Beg and DR Bergfelt
Follicle growth and circulating hormone concentrations were compared between an interovulatory interval and the first 60 days of the anovulatory season in pony mares. Daily observations were made from November of three groups: (i) ablation of follicles of >/=6 mm in diameter at day 10 after an ovulation that initiated an interovulatory interval, as determined retrospectively (ovulatory group, n=8), (ii) ablation at day 10 after the last ovulation of the year (anovulatory-10 group, n=6); and (iii) ablation at day 60 after the last ovulation of the year (anovulatory-60 group, n=6). Follicular waves were defined as major (dominant follicle) and minor (no dominant follicle). The percentage of mares with major waves after ablation for the ovulatory, anovulatory-10 and anovulatory-60 groups was 100, 33 and 0%, respectively, and the percentage with minor waves was 0, 67 and 100%, respectively. Minor waves were also detected in 83% of anovulatory mares between day 20 and day 60. Growth of the largest follicle was similar for major waves and minor waves but only until the beginning of deviation in the major waves. FSH surges after ablation were similar for all groups and for surges detected during days 20-60. Concentrations of LH were greater in association with major waves than with minor waves. Both diameter of the largest follicle and LH concentrations for minor waves were greater after ablation at day 10 after the last ovulation of the year than after ablation at day 60. The results of this study indicate that major follicular waves developed in some mares early in the anovulatory season and that minor waves developed throughout the first 2 months. Despite similarities in the wave-stimulating FSH surge, differences in follicle growth occurred and were attributable, on a temporal basis, to differences in LH concentrations. A minor wave developed into a major wave when the largest follicle reached a diameter characteristic of the beginning of deviation in the presence of an adequate LH stimulus for continued growth of a dominant follicle.
OJ Ginther, DR Bergfelt, MA Beg and K Kot
The intervals between emergence of follicular waves 1 (first wave of an oestrous cycle) and 2, and between the associated FSH surges (surges 1 and 2), were studied in control (n = 7) and recombinant bovine (rb)FSH-treated (n = 7) heifers. The expected start of the deviation in follicle diameter between the two largest follicles of wave 1 was defined as the day on which the largest follicle reached 8.5 mm (day 0). In the control heifers, circulating concentrations of FSH decreased and oestradiol increased between day 0 and day 1.5 or day 2.0 in a reciprocal relationship. The opposite reciprocal relationship between an FSH increase and an oestradiol decrease occurred during the next 3 days. This temporal result is consistent with a negative systemic effect of oestradiol on FSH at this time. rbFSH was administered in a dosage regimen that was expected to result in a similarity between FSH surge 2 in the rbFSH-treated group and surge 2 in the control group. On average, surge 2 and wave 2 occurred approximately 2 days earlier in the rbFSH-treated group than in the control group, and characteristics of the FSH surge and follicular wave were similar (no significant differences) between groups. These results support the hypothesis that low circulating FSH concentrations after the deviation in follicle diameter control the interval to emergence of the subsequent follicular wave. However, in one of seven rbFSH-treated heifers, the largest follicle from the apparent stimulation of rbFSH reached only 5.7 mm; therefore, the possibility of involvement of additional mechanisms cannot be dismissed.
SJ Tsai, K Kot, OJ Ginther and MC Wiltbank
There is growing evidence to indicate that PGF(2alpha)-induced luteolysis involves altered gene expression in the corpus luteum. Concentrations of mRNA encoding nine different gene products were quantified at three time points from corpora lutea in situ. Serial luteal biopsies (2.1-5.5 mg per biopsy) were collected using an ultrasound-guided transvaginal method and mRNA concentrations were quantified with standard curve quantitative competitive RT-PCR. In the first experiment, three luteal biopsies were collected from three heifers and analysed in multiple assays to evaluate the repeatability of the methods. Concentrations of mRNA for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), PGF(2alpha) receptor (FP receptor) and LH receptor were found to be highly repeatable between assays, between multiple biopsies and between animals (coefficients of variation 1.3-17.3%). In the second experiment, heifers on days 9-11 after ovulation were assigned randomly to receive saline only (n = 6), saline with biopsies taken at t = 0, 0.5 and 4.0 h after injection (n = 6), PGF(2alpha) only (n = 6) or PGF(2alpha) with biopsies taken at t = 0, 0.5 and 4.0 h after treatment (n = 7). Biopsy alone did not change corpus luteum diameter, serum progesterone concentrations or days to next ovulation within the saline- or PGF(2alpha)-treated groups. Concentrations of mRNA for steroidogenic acute regulatory protein, FP receptor, 3beta-hydroxysteroid dehydrogenase, cytosolic phospholipase A(2) and LH receptor were decreased at 4.0 h after PGF(2alpha) injection. In contrast, PGF(2alpha) increased mRNA concentrations for prostaglandin G/H synthase-2, monocyte chemoattractant protein-1 and c-fos but the time course differed for induction of these mRNAs. Concentrations of mRNA for GAPDH did not change after PGF(2alpha) treatment. In conclusion, the techniques allowed analysis of multiple, specific mRNAs in an individual corpus luteum at multiple time points without altering subsequent luteal function. Use of these techniques confirmed that luteolysis involves both up- and downregulation of specific mRNA by PGF(2alpha).
OJ Ginther, DR Bergfelt, MA Beg and K Kot
Progesterone was used to reduce LH concentrations starting at the time when the largest follicle was > or = 5.7 mm in diameter or well before the expected start of follicle deviation (largest follicle > or = 8.5 mm in diameter). Plasma concentrations of LH, FSH and oestradiol were determined at 4 h intervals in control and progesterone-treated heifers (n = 8 per group). Concentrations of LH were lower (P < 0.05) in the progesterone-treated group, reflecting an absence of the transient increase in LH concentrations that encompasses follicle deviation. An increase in oestradiol and a continued decrease in FSH occurred at the start of follicle deviation in the control cows but not in the treated heifers. In a second experiment, follicular fluid of the two largest follicles of control and progesterone-treated heifers was sampled at the expected start of deviation (n = 8--10 per group). The concentrations of oestradiol, but not androstenedione and free insulin-like growth factor I (IGF-I), in follicular fluid were higher (P < 0.001) in the largest follicle than in the second largest follicle. Progesterone treatment reduced (P < or = 0.02) the concentrations of all three factors in follicular fluid and increased (P < 0.05) the concentrations of insulin-like growth factor binding protein 2 (IGFBP-2). These results confirm that oestradiol contributes to the continued decrease in FSH concentrations after the start of follicle deviation. Prevention of the transient LH increase, the oestradiol increase and the continued FSH decrease did not significantly alter the mean time or follicle diameters characteristic of expected follicle deviation. However, in some treated individuals (three of eight), the observed follicle deviation was delayed. In addition, these results indicate that the secretion of oestradiol into the circulation and the increase in oestradiol and IGF-I and decrease in IGFBP-2 concentrations in the follicular fluid at the start of deviation are functions of the transient increase in LH concentrations that encompasses follicle deviation.
MA Beg, C Meira, DR Bergfelt and OJ Ginther
Follicle deviation is characterized by continued growth of the largest (developing dominant) follicle and reduced growth of the smaller (subordinate) follicles. The aim of the present study was to test the following hypotheses: (1). oestradiol contributes to the depression of circulating FSH encompassing follicle deviation and (2). oestradiol plays a role in the initiation of deviation. Heifers were treated with progesterone (n = 5) or antiserum against oestradiol (n = 7) or given no treatment (control; n = 6). On the basis of previous studies, progesterone treatment would decrease LH and thereby the circulatory and intrafollicular concentrations of oestradiol and the antiserum would reduce the availability of oestradiol. Progesterone was given in six 75 mg injections at 12 h intervals beginning when the largest follicle of wave 1 first reached >or=5.7 mm (t = 0 h). Oestradiol antiserum (100 ml) was given in a single injection at t = 12 h. Follicles of the wave were defined as F1 (largest) and F2, according to the diameter at each examination. Blood samples were collected at 12 h intervals during t = 0-72 h. Treatment with progesterone lowered the circulatory concentrations of LH by 12 h after the start of treatment (P < 0.05), and concentrations remained low compared with those of controls during the treatment period. Treatment with oestradiol antiserum had no effect on LH. Both progesterone and the antiserum treatments increased the FSH concentrations compared with controls (P < 0.05), which supports the first hypothesis. The interval from t = 0 h to the beginning of deviation was longer in the progesterone- (51.0 +/- 7.6 h; P < 0.06) and antiserum (51.4 +/- 6.3 h; P < 0.05)-treated groups than in the controls (38.0 +/- 3.7 h), which supports the second hypothesis. There was no difference among groups in the diameters of F1 and F2 at deviation. Reduced diameter (P < 0.05 or P < 0.06) of both F1 and F2 occurred in both the progesterone- and antiserum-treated groups at t = 36 h and 48 h, compared with controls. Follicle retardation occurred in both the progesterone- and antiserum-treated groups despite the high FSH concentrations, whereas LH was altered only in the progesterone-treated group. Therefore, the follicle effect can be attributed to inadequate intrafollicular oestradiol. This interpretation implies a functional local role for oestradiol in the deviation process, independent of the systemic negative effect on FSH.