The effects of recombinant bovine somatotrophin (BST) on the dynamics of ovarian follicular growth and development and peripheral insulin concentrations were investigated. Initially, studies were carried out in a population of Hereford × Friesian heifers to validate the ultrasound technique. In the first experiment, 12 heifers were injected daily with either 25 mg BST or vehicle for two oestrous cycles, and the effects on follicular dynamics and peripheral insulin were determined. In a second experiment, 12 heifers were given a single injection of 10 ml saline or 320 mg BST in a sustained-release formulation to examine the temporal relationships between growth hormone (GH), insulin-like growth factor-1 (IGF-1), insulin and the number of follicles. The validation studies demonstrated that small follicles (<5 mm in diameter) could be clearly detected by real-time ultrasound, and that 75% (9 of 12) of heifers showed three waves of dominant follicle development during the oestrous cycle, whereas the remainder had only two waves. The changes in the numbers of follicles of the three size categories (<5 mm, small; 5−10 mm, medium-sized and > 10 mm, large) also displayed a wave pattern similar to that of the dominant follicle, with a marked reduction in the number of subordinate follicles as the dominant follicle grew and reached its maximum size. In Expt 1, BST treatment increased the number of small follicles and caused a rise in peripheral insulin concentrations (P < 0.01) throughout the treatment period. However, there was no effect of BST on the timing for the pattern of follicular waves during the oestrous cycle, nor on the number of medium-sized or large follicles at each follicular wave. In the second experiment, the temporal change in the number of small follicles following BST treatment was positively correlated with the changes in the peripheral IGF-1 and insulin concentrations. IGF-1 and insulin concentrations increased 48 h after BST injection, and the number of small follicles had increased 24 h later and remained higher during the period when peripheral IGF-1 and insulin concentrations were high. These results demonstrate first, that the number of small follicles was reduced as the dominant follicle grew and reached its maximum size and second that BST treatment could enhance the recruitment of small follicles in heifers. This increase in the number of small follicles was positively correlated with peripheral IGF-1 and insulin concentrations. Third, BST did not affect the turnover of follicular waves, nor the inhibitory action of the dominant follicle on its subordinate follicles. We conclude that BST affects the recruitment of small follicles by increasing peripheral concentrations of IGF-1, or of insulin or both concentrations. Moreover, the effect of BST on the small follicle population was not mediated through the mechanism(s) by which the dominant follicle inhibits subordinate follicles.
J. G. Gong, T. A. Bramley and R. Webb
J. G. Gong, G. Baxter, T. A. Bramley and R. Webb
Treatment with recombinant bovine somatotrophin (bST) can enhance the development of ovarian antral follicles in cattle. The underlying mechanism was examined further by performing a dose–response study to investigate the effects of bST on peripheral concentrations of somatotrophin, insulin-like growth factor I (IGF-I), insulin, FSH and LH, and ovarian follicle development. Twenty mature heifers were randomly divided into five groups and injected s.c. at 6 h intervals for 7 days with 25% of one of the following daily doses of bST: 0, 3.13, 6.25, 12.5 or 25.0 mg. Ovarian follicular dynamics were monitored by real-time ultrasonography. Blood samples were collected daily during the experiment, and every 15 min for 8 h on days 1 and 5 of bST treatment. Treatment with bST increased (P < 0.01) peripheral concentrations of somatotrophin in a dose-dependent manner. Serum concentrations of both IGF-I and insulin were significantly (P < 0.01) increased in all heifers given 12.5 or 25.0 mg bST per day. Peripheral concentrations of IGF-I and insulin in all animals in the group given 3.13 mg bST and two heifers in the group given 6.25 mg bST were not different from those in the control group, while concentrations in the other two heifers given 6.25 mg bST were significantly (P <0.01) higher. The number of ovarian follicles < 5 mm in diameter was increased (P < 0.05) in response to bST, but only in heifers (n = 10) with significantly increased serum concentrations of IGF-I and insulin. There were no effects of treatment on peripheral concentrations of FSH, LH and progesterone, and on the numbers of follicles > 5 mm in diameter. In conclusion, this study has demonstrated in vivo that the effect of treatment with bST on ovarian follicle development appears to be mediated through an increase in circulating IGF-I or insulin concentrations, rather than via an alteration in the secretion of pituitary gonadotrophins or a direct effect of bST on ovarian follicles.
L. A. Penny, D. Armstrong, T. A. Bramley, R. Webb, R. A. Collins and E. D. Watson
Immune cells and their cytokine products have powerful local effects within body tissues. There has been great interest in the potential role of these cells, not only during destruction of the corpus luteum but also during its functional lifespan. In this study, lymphocytes, macrophages and major histocompatibility complex class II molecules were quantified using immunohistochemistry and the reverse transcription–polymerase chain reaction was used to detect mRNA for tumour necrosis factor α and interferon γ within corpora lutea from three groups of cows: (1) corpora lutea collected at an abattoir and assessed visually into four stages (stage I (days 1–5), stage II (days 6–12), stage III (days 13–18) and stage IV (days 19–21) of the oestrous cycle); (2) corpora lutea collected around natural luteolysis (days 14–20); and (3) corpora lutea collected 6, 12 and 24 h after prostaglandin F2α-induced luteolysis. The numbers of T lymphocytes (CD5+ and CD8+) were significantly higher (P < 0.05) at stage IV and from day 16 onwards, before functional luteolysis. There were significantly higher numbers (P < 0.01) of macrophages at stages I, III and IV compared with stage II in visually staged tissue. Major histocompatibility complex class II molecules were increased (P < 0.05) at stages I and IV compared to stage II and at all times after induced luteolysis. Using reverse transcription–polymerase chain reaction, mRNA encoding tumour necrosis factor α and interferon γ was detected in all luteal tissue collected around natural luteolysis and after induced luteolysis. These findings, particularly the increase in T lymphocytes before functional luteolysis, provide further evidence of a significant role for the immune system in affecting reproductive function in cows.
T A Bramley, D Stirling, G S Menzies and D T Baird
Seasonally anoestrous Welsh Mountain ewes received 250 ng gonadotrophin-releasing hormone (GnRH) every 2 h, with (Group 1; n = 13) or without (Group 2; n = 14) progesterone priming for 48 h. Fourteen control ewes (Group 3) were studied during the luteal phase in the breeding season. Animals in Group 4 (n = 12) received progesterone priming followed by 250 ng GnRH at increasing frequency for 72 h, while ewes in Group 5 (n = 13) were given three bolus injections of 30 μg GnRH at 90-min intervals. All treatment regimens induced ovulation. However, only corpora lutea (CL) from ewes in Group 3 (breeding season) or Group 4 exhibited normal luteal function. Luteal luteinizing hormone (LH) receptor levels were significantly higher on day 12 than day 4, and CL from groups with adequate CL (3 and 4) had significantly higher 125I-human chorionic gonadotrophin (hCG)-binding levels than the three groups with inadequate CL on day 12. LH-binding affinity was unchanged. Exogenous ovine LH (10 μg) in vivo on days 3 or 11 after ovulation induced a pulse of progesterone in ewes with adequate CL: however, ewes in Groups 1, 2 and 5 showed no significant response. Basal progesterone secretion in vitro was significantly greater on day 4 than on day 12. Maximal steroidogenic responses of adequate and inadequate CL to hCG and to dibutyryl cyclic-3′,5′-AMP were similar at both stages of the luteal phase. However, the EC50 for hCG on days 4 and 12 was 10-fold lower for groups with an adequate CL (0.1 IU hCG/ml) than for inadequate-CL groups (1 IU hCG/ml; P <0.05). Thus, in addition to the well-characterized premature sensitivity of GnRH-induced inadequate CL to endometrial luteolysin, we have shown (1) a marked decrease in total number of cells in the CL, a profound reduction in vascular surface area, and a decrease in mean large luteal cell volume (with no change in large luteal cell numbers), (2) decreased luteal LH receptor and progesterone content compared with adequate CL and (3) that CL that were becoming, or were destined to become, inadequate failed to respond to ovine LH in vivo and were 10-fold less sensitive to hCG in terms of luteal progesterone secretion in vitro.
J. R. McNeilly, N. P. Evans, T. A. Bramley, P. Brown, A. J. Clark and R. Webb
Selection of the luteinizing hormone (LH) response to exogenous gonadotrophin-releasing hormone (GnRH) in sheep has resulted in the establishment of two lines (High and Low) with a fivefold difference in pituitary sensitivity to GnRH. The effect of selection on gonadotrophin gene expression in the presence or absence of an exogenous gonadotrophin-releasing hormone (GnRH) challenge in twenty-week-old ram lambs from both lines was examined. Before treatment with either GnRH or saline, LH and follicle-stimulating hormone (FSH) concentrations were significantly higher in the High line than in the Low line animals (LH and FSH: P < 0.01). One hour after either GnRH or saline, all animals were slaughtered. In the absence of a GnRH challenge, there were significantly higher concentrations of all three gonadotrophin subunit mRNAs in the High line compared with the Low line, corresponding to the higher basal concentrations of LH and FSH. When comparing treatments between the lines, following a GnRH challenge, LHβ subunit mRNA was significantly (P < 0.001) higher in both lines than before the GnRH, whereas there was no significant change in either α or FSHβ subunit mRNA. These results indicate that the differences in basal gonadotrophin secretion are related to differences in gonadotrophin subunit mRNAs with the High line animals having an inherently greater amount of all three gonadotrophin subunit mRNAs. Selection has not altered the differential amounts of gonadotrophin subunit mRNAs, since there is an overall increase in all three gonadotrophin subunits. GnRH appears to preferentially control LHβ mRNA in both High and Low line animals.
J. G. Gong, T. A. Bramley, C. G. Gutierrez, A. R. Peters and R. Webb
The effect of chronic treatment with a gonadotrophin-releasing hormone agonist (GnRHa) on ovarian function in cattle was investigated by injecting heifers i.m. twice a day with saline, 5 μg GnRHa (Buserelin) or 10 μg GnRHa (n = 7) for 21 days. Blood samples were taken twice a day during the treatment period, and then three times a day for 7 days and once daily for a further 4 days. Frequent samples were also collected on day 1, day 10 and day 21 of treatment. The ovaries of all heifers were examined daily using real-time ultrasonography throughout the experimental period. No significant differences in the response were observed between two doses of GnRHa. The first GnRHa injection produced a large LH and FSH surge and this acute response was still present by day 21 of treatment, but both the magnitude and duration of response were significantly attenuated (P < 0.01). After an initial increase, LH returned to the basal concentration, which was maintained until the termination of treatment, when concentrations increased significantly, with a preovulatory surge occurring approximately 6 days later. Peripheral FSH concentrations during the oestrous cycle in control animals displayed a pattern of three waves, each of which closely preceded a wave of follicular development. Concentrations of FSH in GnRHa-treated heifers showed a normal pattern for the first wave after the start of treatment. During the next wave, concentrations increased and remained at the peak values until about 4 days after the end of treatment. An additional ovulation was induced in 11 of 14 GnRHa-treated heifers within 2–3 days of the start of treatment, and a significant (P < 0.05) increase in serum progesterone concentrations was detected 2 days later. All GnRHa-treated heifers then showed a normal follicular wave, with the development and regression of a dominant follicle. The dominant follicles from the next wave grew to only 7–9 mm in diameter and remained at this size until the end of treatment, when they resumed growth, ovulated approximately 7 days later and formed corpora lutea. We conclude that chronic treatment of heifers with GnRHa for 3 weeks suppresses pulsatile secretion of LH and blocks the development of dominant follicles beyond 9 mm in diameter, preventing the preovulatory LH surge and ovulation. However, GnRHa did not suppress the secretion of FSH within the 3 week treatment period. The maintenance of the dominant follicles for an extended period should provide an ideal model to study the control of follicular atresia in cattle in vivo.
C. Broadley, G. S. Menzies, T. A. Bramley and E. D. Watson
Corpora lutea were obtained from mares at days 3, 10 and 14 after ovulation, and examined histologically. The morphology of isolated luteal cells obtained by either mechanical or collagenase dissociation of the tissue was examined and the cells stained to detect the steroidogenic enzyme Δ5, β-hydroxysteroid dehydrogenase. The ratio of large:small cells was significantly higher for cells obtained from mechanically dissociated luteal tissue than for cells obtained by collagenase dissociation (P < 0.01). Cells obtained by both mechanical and collagenase dissociation secreted progesterone, although neither cell population responded to exogenous gonadotrophin with an increase in progesterone secretion. Homogenates of equine luteal tissue bound 125I-labelled human LH with high affinity and specificity, and the specific activity and binding affinity of luteal LH receptors did not change significantly from day 3, to days 10 and 14 after ovulation. However, mechanically dissociated cells on days 10 and 14 bound significantly more LH than did collagenase-dissociated cells on these days (P < 0.05). These results indicate that (i) collagenase dissociation of mare luteal tissue yields a population of cells that is unrepresentative of the corpus luteum, and (ii) the mare corpus luteum is not responsive to LH in vitro at the stages examined.