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S. J. Dieleman and D. M. Blankenstein

Summary. Preovulatory bovine follicles (n = 28) were collected at different times after the onset of standing oestrus until shortly before ovulation. In-vitro conversion of tritiated androstenedione in the presence of NADPH by homogenates of the follicular wall was compared in phases relative to the LH peak. During phase 0 (before the LH surge) conversion into oestradiol-17β was high and production of oestrone was about 8-fold lower. During phases 1 (0–6 h after the LH peak) and 2A (6–14 h after the LH peak) the production of oestradiol and oestrone remained constant; the percentage of remaining androstenedione increased. In phase 2B (14–20 h after the LH peak) conversion into oestradiol and oestrone had decreased to about one third correlating with a higher percentage of remaining androstenedione. In phase 3(20 h after the LH peak until ovulation) conversion into oestradiol and oestrone remained constant. The ratio between the production of oestrone and oestradiol remained constant throughout the phases of preovulatory development (0·13), indicating a concurrent inhibition of aromatase and 17β-hydroxysteroid dehydrogenase activities. Conversion into 19-hydroxyandrostenedione showed a pattern similar to that of oestradiol, and testosterone was produced in minute quantities. The results indicate that in preovulatory bovine follicles eventual inhibition of aromatization takes place at about 14 h after the preovulatory LH peak.

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S. J. Dieleman and D. M. Blankenstein

Summary. Preovulatory cow follicles (n = 34) were collected at different times after the onset of oestrus until shortly before ovulation. In-vitro conversion of tritiated pregnenolone in the presence of NAD+ by homogenates of the follicular wall was compared in phases relative to the LH peak. During phase 0 (before the LH surge) a moderate conversion into progesterone occurred, but it was subsidiary to that into 17α-hydroxypregnenolone and other unidentified steroids. During phases 1 (0–6 h after the LH peak), 2A (6–14 h) and 2B (14–20 h) the production of progesterone and 17α-hydroxypregnenolone remained constant; at phase 2B the percentage of remaining pregnenolone was higher than in the preceding phases. In phase 3 (20 h after the LH peak until ovulation) conversion into progesterone had increased about 4-fold to the highest levels observed (97% after 2 h incubation), and production of 17α-hydroxypregnenolone and unidentified steroids was low. In an additional experiment, homogenates of the wall of 3 follicles at phase 3 were also incubated with tritated progesterone in the presence of NADPH. The percentage of remaining progesterone was high, and a moderate conversion into 17α-hydroxyprogesterone occurred. In the main experiments, however, production of this steroid was not observed.

The results indicate that steroid synthesis in the preovulatory follicle of the cow changes to the production of progesterone shortly before ovulation.

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S. J. Dieleman and M. M. Bevers

Summary. Normally cyclic heifers received 2500 i.u. PMSG i.m. at Day 10 of the oestrous cycle and 15 mg prostaglandin (PG) i.m. 48 h later. From 30 h after PG the LH concentration in the peripheral blood was estimated every hour using a rapid RIA method which allowed the LH concentration to be known within 4 h. Monoclonal antibody against PMSG was injected in the jugular vein of 29 heifers at 4·8 h after the maximum of the preovulatory LH peak; 28 heifers were not treated with anti-PMSG (controls). Peripheral blood concentrations of PMSG, LH, progesterone and oestradiol were compared. Ovaries were collected by ovariectomy at fixed times, 22–30 h after the LH peak, and numbers were counted of small (2–10 mm), large (> 10 mm) and ovulated follicles, and of follicles with a stigma.

In anti-PMSG-treated cows, the PMSG concentration fell sharply to non-detectable levels within 2 h of the treatment, indicating that PMSG was neutralized in these cows at the onset of final follicular maturation. In all cows, the concentration of oestradiol showed a significant decrease at about 8 h after the LH peak. After anti-PMSG treatment ovulations took place from 24 until 30 h after the LH peak, whereas in control cows follicles had already ovulated at or before 22 h and ovulations continued until 30 h. At 30 h 90% of the follicles had ovulated in anti-PMSG-treated cows vs 72% in the controls, resulting in 15 and 8 ovulations per cow respectively (P < 0·05). Also, administration of monoclonal antibody against PMSG synchronized final follicular maturation and shortened the period of multiple ovulations.

In conclusion, neutralization of PMSG shortly after the preovulatory LH peak suppresses adverse effects of PMSG on final follicular maturation, leading to an almost 2-fold increase of the ovulation rate.

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N J Beijerink, H S Kooistra, S J Dieleman, and A C Okkens

Dopamine agonists decrease plasma prolactin concentration and shorten the duration of anoestrus in the bitch. In order to determine whether this shortening results from decreased prolactin release or is due to another dopamine agonistic effect on the pulsatile release of FSH and LH, eight anoestrous beagle bitches were treated with a low dose of the serotonin antagonist metergoline (0.1 mg per kg body weight, twice daily) starting 100 days after ovulation. Six-hour plasma profiles of LH and FSH were obtained 7 days before, immediately before, 1 week after, and then at 2-week intervals after the start of the treatment with the serotonin antagonist until signs of pro-oestrus appeared. Plasma prolactin concentration was measured three times weekly from 75 to 142 days after ovulation and thereafter once weekly until the next ovulation, and was observed to decrease significantly after the start of treatment. The length of the interoestrous interval in the treated dogs was, however, not different from that in the preceding pretreatment cycle or from that in a group of untreated bitches. During the first weeks of treatment no changes were observed in the pulsatile plasma profiles of FSH and LH. Four weeks after the start of the treatment with the serotonin antagonist there was an increase in the mean basal plasma FSH concentration and the mean area under the curve for FSH, without a concurrent increase in LH secretion. The increase in FSH secretion continued until late anoestrus. In conclusion, the serotonin antagonist-induced lowering of plasma prolactin concentration was not associated with shortening of the interoestrous interval. The plasma profiles of LH and FSH were similar to those observed during physiological anoestrus, but different from those observed during anoestrus shortened by treatment with a dopamine agonist. Hence the prematurely induced oestrus observed during administration of dopamine agonists cannot be explained by a decreased plasma prolactin concentration but must be due to some other dopamine agonistic effect, probably increased FSH secretion. The observations in this study further strengthen the hypothesis that an increase in circulating FSH is essential for ovarian folliculogenesis and consequently the termination of anoestrus in the bitch.

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S. J. Dieleman, M. M. Bevers, J. Poortman, and H. T. M. van Tol

Summary. Preovulatory bovine follices (n = 73) were collected at different times after the onset of oestrus until shortly before ovulation, which occurred at 24 ± 1 ·4 h after the peak concentration of LH in the peripheral blood. Non-atretic antral follicles (n = 9) of 15–19 mm were also collected from cows during the luteal phase of the oestrous cycle. Follicular fluid concentrations of dehydroepiandrosterone, androstenedione and oestrone, and of LH, FSH and prolactin were compared in 2-h periods relative to the LH plasma peak. Before the LH surge the concentrations of the steroids were much higher than in non-atretic luteal-phase follicles of similar size. From 0 to 6 h after the LH peak the steroid concentrations decreased sharply to remain low until ovulation; only that of androstenedione increased again after 14 h to remain constant. The ratio between the concentrations of androstenedione and dehydroepiandrosterone remained constant until 14 h after the LH peak; at 14 h it increased about 4-fold and remained high until ovulation. The ratio between the oestrone and androstenedione concentration increased gradually to a 10-fold higher value until at 14 h an abrupt decrease was observed. These changes indicate that after the LH peak androgen production is directly inhibited and, at a slower rate, the aromatizing activity. Androstenedione appeared to be the major aromatase substrate.

Before the plasma LH peak the follicular fluid concentration of FSH was higher than in luteal-phase follicles; the concentrations of LH and prolactin were not different from those in luteal-phase follicles. About 4 h after the LH plasma peak the LH concentration in follicular fluid reached a maximum, which was one seventh of that in peripheral blood; it remained elevated until 20 h. The FSH concentration was higher after the LH plasma peak than before; just before ovulation it was still 2-fold higher. The concentration of prolactin fluctuated throughout the preovulatory development from the onset of oestrus until ovulation. It is suggested that oestradiol biosynthesis in bovine preovulatory follicles is terminated by an inhibitory action of the preovulatory LH peak on androgen production.

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P. L. A. M. Vos, M. M. Bevers, A. H. Willemse, and S. J. Dieleman

After synchronization of oestrus, normally cyclic heifers (n = 31) received 2500 iu pregnant mares' serum gonadotrophin (PMSG) i.m. and had a progesterone-releasing intravaginal device (PRID) without the oestradiol capsule inserted on day 10 of the oestrous cycle and received 15 mg prostaglandin (PG) i.m. 48 h later. PRIDs were removed 96 h after insertion and 16 heifers received 1.0 mg GnRH i.m. while the controls (n = 15) received 10 ml saline i.m. All heifers were injected with anti-PMSG i.v. 10 h later. Peripheral blood concentrations of PMSG, progesterone, oestradiol and LH were compared. Ovaries were collected on death 7 days after the GnRH or saline injection and the number of corpora lutea counted. Heifers were considered to have responded well (> 60 pmol l−1) or poorly (< 60 pmol l−1) to superovulation on the basis of the oestradiol concentration 24 h after PG administration. During PRID treatment, LH concentrations remained at basal values. In the heifers treated with GnRH, a single LH surge occurred 2.3 ± 0.1 h (sd) after the GnRH injection with a maximum concentration of 14.6 ± 2.3 (sem) μg l−1 and a duration of 6–8 h. In 12 of the 15 control heifers, LH concentrations remained low (range 0.10–1.94 μg l−1) during the 72 h following the saline injection; three controls showed a spontaneous LH surge at 18, 23 and 23 h after the saline injection, respectively, with a maximum concentration of 6.0–12.5 μg l−1 and a duration of 10–12 h. The oestradiol concentration increased continuously during PRID treatment until the injection of GnRH or saline, when it was four times higher in the heifers that responded well than in the heifers that responded poorly. It decreased sharply 6 h after GnRH indicating that the follicles still responded normally to a preovulatory LH signal, whereas in control heifers a similar decrease took place 4 h later following anti-PMSG treatment. In the GnRH-treated heifers, the heifers responding well showed a significantly higher number of corpora lutea than did the animals showing a poor response, 16.4 ± 2.2 (n = 9) and 5.4 ± 1.4 (n = 7), respectively. Five of the 12 control heifers without an immediate LH surge showed a single corpus luteum, and seven heifers did not have a corpus luteum. For the three controls with an LH surge, 33.0 ± 8.5 corpora lutea were observed. In conclusion, the preovulatory LH signal can be effectively postponed in PMSG/PG-superovulated heifers using a PRID. However, the PRID treatment has to be followed by GnRH to obtain the LH surge at a defined time of preovulatory follicular development. Follicular function with regard to oestradiol secretion and the potential to ovulate remains unchanged.

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B. W. Knol, S. J. Dieleman, M. M. Bevers, and W. E. van den Brom

Dose–response relationships between GnRH and LH, and between GnRH and testosterone, were investigated in six male dogs by intravenous administration of a GnRH analogue at different doses. Each dose of GnRH analogue induced an immediate rise in the plasma concentration of LH and then a rise in plasma testosterone concentration. Irrespective of the dose used, the rise in testosterone began 10 min after the GnRH injection. Administration of GnRH at doses of 0.01, 0.1, 1, 10 and 100 μg kg−1 resulted in maximum LH concentrations in plasma (mean ± sem; n = 6) of 22 ± 7, 27 ± 6, 40 ± 7, 57 ± 13 and 56 ± 10 μg l−1, respectively. These doses induced maximum concentrations of testosterone in plasma (mean ± sem; n = 6) of 16 ± 4, 20 ± 4, 22 ± 3, 22 ± 4 and 24 ± 7 nmol l−1, respectively. The lag time between peak concentrations of LH and testosterone varied from 35 to 55 min. The calculated maximum response of testosterone to LH, secreted by the anterior pituitary after GnRH injection, was 1.8 times higher than to GnRH. It was concluded that intravenous administration of GnRH induced marked and dose-dependent increases in plasma concentrations of LH and testosterone, and that there does not appear to be a direct effect of GnRH on Leydig cells in male dogs.

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R. van den Hurk, E. R. Spek, G. Dijkstra, C. J. A. H. V. van Vorstenbosch, S. C. J. Hulshof, and S. J. Dieleman

Summary. Five Dutch–Friesian heifers were injected i.m. with 3000 iu pregnant mares' serum gonadotrophin (PMSG) on day 10 of the oestrous cycle, to study the effects on the number and micromorphological quality of antral follicles (⩾0·3 mm in diameter). The ovaries were collected 48 h after PMSG injection.

As well as the presence of mitotic figures and the absence of pyknotic nuclei in the granulosa, atypical granulosa cells were found in nonatretic follicles. These cells had an oblong nucleus and stained with toluidine blue. They were characterized by their dark cell matrix, and the presence of numerous free ribosomes and intermediate filaments of varying quantity. Atypical granulosa cells were micromorphologically similar to fibroblast-like cells in the theca. Their presence coincided with the occurrence of degenerative changes in the cytoplasm of nearby granulosa cells and they were more frequent in atretic follicles. The presence of atypical granulosa cells in follicles hitherto called nonatretic is therefore probably associated with the onset of follicular atresia.

In the PMSG-treated heifers, the mean number of large (⩾6·0 mm in diameter) antral follicles was greater than in the control group (18·4 ± 4·0 versus 3·0 ± 1·0), because of an increase in the number of large nonatretic follicles (11·8 ± 4·4 versus 0·4 ± 0·2). After hormone treatment, the mean number of medium-sized (3·0–5·9 mm) nonatretic follicles also increased (6·4 ± 1·3 versus 1·8 ± 1·0). PMSG did not change the mean number of nonatretic follicles < 3·0 mm or that of atretic follicles in the different size categories. However, when follicles hitherto called nonatretic, with atypical granulosa cells, were taken together with the group of atretic follicles, PMSG appeared to increase the mean number of large atretic follicles (13·6 ± 2·4 versus 3·0 ± 1·0). The mean number of medium-sized and large nonatretic follicles without atypical granulosa cells was markedly increased (3·8 ± 1·0 versus 0·2 ± 0·2 and 4·6 ± 1·9 versus 0·0, respectively). The data demonstrate that PMSG stimulates the formation not only of nonatretic follicles ⩾3·0 mm, but also of atretic follicles ⩾6·0 mm.

Keywords: ovary; follicle; micromorphology; pregnant mares' serum gonadotrophin; heifer

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H. S. Kooistra, A. C. Okkens, M. M. Bevers, C. Popp-Snijders, B. van Haaften, S. J. Dieleman, and J. Schoemaker

The secretory profiles of LH and FSH were investigated before and during the administration of bromocriptine in six beagle bitches. Plasma samples were obtained via jugular venepuncture at 10 min intervals for 6 h every 2 weeks until the next ovulation. Bromocriptine treatment was started 100 days after ovulation. Both before and after bromocriptine treatment, LH and FSH pulses occurred together. The mean duration of the FSH pulse (120 min) was significantly longer than that of the LH pulse (80 min). The interoestrous interval in the bitches treated with bromocriptine was significantly shorter than that of the preceding cycle (160 ± 3 versus 206 ± 24 days). The mean basal plasma FSH concentration (7.4 ± 0.6 versus 6.1 ± 0.7 iu l−1) and the mean area under the curve for FSH (46.6 ± 4.7 versus 40.4 ± 4.4 iu l−1 in 6 h) increased significantly after the start of the bromocriptine treatment. In contrast, the differences in mean basal plasma LH concentration (2.1 ± 0.2 versus 2.0 ± 0.2 μg l−1) and the mean area under the curve for LH (19.0 ± 3.1 versus 19.5 ± 2.5 μg l−1 in 6 h) between the day before and 14 days after the start of the bromocriptine treatment were not significant. Bromocriptine administration also lowered the mean amplitude of the FSH pulse and shortened the mean duration of the FSH pulse, without influencing the LH pulse. In addition to demonstrating the concurrent pulsatile secretion of LH and FSH, the results of the present study demonstrate that the bromocriptine-induced shortening of the interoestrous interval in the bitch is associated with an increase in plasma FSH concentration without a concomitant increase in plasma LH concentration. This finding indicates that treatment with the dopamine agonist bromocriptine increases plasma FSH to a concentration that results in the enhancement of follicle development.

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Vera Baumans, S. J. Dieleman, H. S. Wouterse, Leni van Tol, Grietje Dijkstra, and C. J. G. Wensing

Summary. Serum testosterone concentrations ranged from 0·24 to 1·45 nmol/1 between Day 53 post coitum (p.c.) until Day 40 post partum (p.p.) and did not show variations that could be correlated with the process of testicular descent. The intratesticular androgen appeared to be mainly testosterone, its concentration being about 5000-fold higher than that in serum whereas 5α-dihydrotestosterone could not be demonstrated. The intratesticular testosterone concentration at the initiation of gubernacular regression (Day 0) was apparently, but not significantly, higher than at Day 49 p.c. and at Day 40 p.p. The ability of the neonatal canine testis to synthesize testosterone was indicated by increased serum testosterone concentrations after hCG stimulation.