P.F. Daels

Induction of reproductive function in anestrous mares using a dopamine antagonist

We investigated the role of dopamine in the regulation of seasonal reproductive activity in mares. Nine seasonal anestrous mares, maintained under a natural photoperiod, were treated daily with a dopamine D2 antagonist, [-]-sulpiride (200 mg/mare, im), beginning February 5 (day of year = 36) until the first ovulation of the year or for a maximum of 58. Nine untreated anestrous mares were maintained under the same conditions. The ovaries were examined by ultrasonography twice a week, and blood was collected three times a week for progesterone, LH, FSH and prolactin determinations. Mean day of first ovulation was significantly advanced for [-]-sulpiride-treated mares than control mares (mean day of year +/- SEM = 77.3 +/- 7.9 and 110.0 +/- 6.8, respectively; P < 0.01). Eight mares ovulated during [-]-sulpiride treatment while one mare failed to ovulate. Ovulation occurred 91 d after the start of treatment or on Day 127. All mares continued to have normal estrous cycles after the first ovulation. First cycle length and luteal progesterone concentrations did not differ between [-]-sulpiride-treated and control mares. Plasma prolactin concentrations were significantly increased at 2 and 9 h after [-]-sulpiride administration (P < 0.05), and had returned to basal levels by 24 h. At the time of the LH surge associated with the first ovulation, mean LH and FSH secretion was significantly higher in [-]-sulpiride-treated mares than in control mares (P < 0.05). These results suggest that dopamine plays a role in the control of reproductive seasonality in mares and exerts a tonic inhibition on reproductive activity during the anovulatory season.

Pregnancy-specific elevations in fecal concentrations of estradiol, testosterone and progesterone in the domestic dog (Canis familiaris).

Estradiol (E2), testosterone (T) and progesterone (P4) concentrations were determined by enzyme-immunoassay in aqueous extracts of fecal samples obtained during anestrus, proestrus, estrus and metestrus of 11 nonpregnant and 11 pregnant bitches. Fecal hormone concentrations (ng/g) changed in relation to stage of cycle. Mean fecal steroid concentrations in 22 anestrous bitches and 3 ovariectomized bitches were low and similar for E2 (53 +/- 5 and 27 +/- 2), T (60 +/- 7 and 36 +/- 6), and P4 (62 +/- 6 and 86 +/- 15). Within 0 to 3 d of the ovulatory LH surge fecal E2 reached peak concentrations (301 +/- 38). The T peaks (281 +/- 41) were coincident or 1 to 3 d later. Fecal P4 was then elevated for approximately 2 m.o. Between Days 26 and 45 after ovulation, mean fecal P4 concentrations were higher (P < 0.05) in pregnant (401 +/- 60) than in nonpregnant bitches (164 +/- 23) and peak fecal P4 concentrations in individual animals were higher (P < 0.01) in pregnant (812 +/- 121) than in nonpregnant bitches (425 +/- 97). In the same period mean concentrations of E2 (117 +/- 13 vs 61 +/- 5) and T (102 +/- 10 vs 70 +/- 6) were also higher (P < or = 0.05) in pregnant than in nonpregnant bitches. Serum E2, T and P4 concentration were positively correlated (P = 0.1) with concentration in fecal samples obtained one day after serum collection. Although serial fecal ovarian steroid concentrations demonstrate the time course of ovulatory cycles, the diagnostic value of individual fecal samples appears limited. The ratios of peak to basal values were approximately 6, 5 and 7 for E2, T and P4, respectively, and were considerably lower than ratios of 12 to 50 previously reported for serum or plasma concentrations. The results demonstrate that there are pregnancy-specific increases in P4, E2 and T production reflected in fecal concentrations. While such increases are reflected in fecal samples, they are generally not evident in serum or plasma concentrations because of increased hemodilution, metabolism and clearance in pregnant bitches. The physiological stimulus for these increases, presumably ovarian in origin, or the potential role of prolactin is not known.

Expression and Binding Activity of Luteinizing Hormone/Chorionic Gonadotropin Receptors in the Primary Corpus Luteum During Early Pregnancy in the Mare

Abstract Luteal steroids are necessary to maintain the first 70–90 days of pregnancy in the mare. At 35 days postovulation, the resurgence of the primary corpus luteum (CL) coincides with the secretion of the fetal hormone eCG. In order to study the responsiveness of the primary CL to eCG, we have examined levels of luteal equine LH/CG receptors (eLH/CG-R) mRNAs by Northern blot analysis and measured concentrations of eLH/CG binding sites on luteal membranes using 125I-eLH saturation binding assays at three stages of gestation: before the onset of eCG secretion (Days 14–31), from onset to maximum eCG secretion (Days 38–62), and during decline of eCG secretion (Days 83–101). Multiple transcripts of eLH/CG-R (7, 5.7, 4.9, 3.9, 2.8, 1.8, 0.6 kilobase [kb]) were identified in the primary CL at all stages examined. Three of them (5.7, 2.8, 0.6 kb) coded for truncated eLH/CG-R lacking the transmembrane domain. The relative intensities of the four major transcripts tended to decrease (5.7 and 3.9 kb) or were steadily expressed (7 and 1.8 kb) during pregnancy. The affinity of eLH/CG binding sites did not change during pregnancy whereas the number of eLH/CG binding sites decreased significantly after the onset of eCG secretion. Nevertheless, levels of binding sites were still at 44.6% (Days 38–62) to 24.7% (Days 83–101) of those measured before the onset of eCG secretion. Taken together, the presence of eLH/CG-R mRNAs and of a substantial part of eLH/CG binding sites with high affinity suggest that the primary CL still expresses a high number of eLH/CG-R and remains responsive to eCG during early pregnancy.

Testosterone secretion during early pregnancy in mares

We have characterized the testosterone secretion pattern during the first 80 d of pregnancy in mares and determined the sources that contribute to circulating testosterone levels during this period. Ten untreated, pregnant mares (Group 1), 10 altrenogest-treated, pregnant mares (Group 2), and 10 altrenogest-treated, pregnant mares in which the CL was eliminated by administration of PGF-2alpha on Day 16 (Group 3) were used in this study. Complete luteolysis occurred following PGF-2alpha administration in all mares in Group 3. Six of the 10 mares in Group 3 did not have an active CL until after Day 60 of pregnancy (Group 3a) and were included in the analysis. The remaining four mares developed a new CL on Days 32, 40, 43 and 49 of pregnancy and were excluded from analysis. Mares without a functional CL (Group 3a) had significantly lower testosterone concentrations than mares with a functional CL (Groups 1 and 2), during the period before equine chorionic gonadotropin (eCG) secretion. At the onset of eCG secretion, testosterone concentrations increased rapidly but the rate of increase decreased with time in mares with a functional CL (Groups 1 and 2). In mares without a functional CL (Group 3a), testosterone concentrations did not increase at the onset of eCG secretion but increased at a gradually increasing rate after Day 50. The lower testosterone concentration in mares without a functional CL before eCG secretion suggests that the CL contributes significantly to the circulating testosterone concentration during the period before eCG secretion. The close time relationship between the onset of eCG secretion and the increase in testosterone secretion in mares with a functional CL and the lack of a testosterone increase in pregnant mares without a functional CL suggest that the increase in testosterone secretion after Day 35 of pregnancy is the result of eCG-stimulated, luteal testosterone synthesis.

Expression of 3β-hydroxysteroid dehydrogenase, cytochrome P450 17α-hydroxylase/17,20-lyase and cytochrome P450 aromatase enzymes in corpora lutea of diestrous and early pregnant mares☆

In the pregnant mare, luteal estrogen production increases at the onset of equine chorionic gonadotropin (eCG) secretion by endometrial cups. In previous studies, we have demonstrated that eCG stimulates luteal androgen and estrogen production in pregnant mares. To further elucidate the regulation of steroidogenesis within the equine corpus luteum (CL) of pregnancy, we examined the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/17,20 lyase (P450(17alpha)) and cytochrome P450 aromatase (P450(arom)) in luteal tissue samples collected during diestrus (Days 7 to 10) and pregnancy before (Days 29 to 35) and after (Days 42 to 45) the onset of eCG secretion. Immunoblot analyses revealed a single protein per enzyme with molecular weights of 48 kDa (3beta-HSD), 58 kDa (P450(17alpha)) and 56 kDa (P450(arom)). Steady-state levels of 3beta-HSD were lower in luteal tissue of diestrus than pregnancy, but expression did not change during pregnancy. Steady-state expression of P450(17alpha) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, P450(17alpha) expression was significantly higher after the onset of eCG secretion. Steady-state expression of P450(arom) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, luteal expression of P450(arom) was significantly lower after the onset of eCG secretion. These data support the hypotheses that eCG has a differential effect on the expression of luteal steroidogenic enzymes, that the eCG-induced increase in luteal estrogen production is the result of an increase in available aromatizable androgen due to an increase in P450(17alpha) expression and activity, and that increased luteal estrogen production is not due to an increase in aromatase expression.

Evidence for opioidergic inhibition of oxytocin release in periparturient mares

In the horse mare, the onset of parturition is associated with an increase in oxytocin secretion, and it has been suggested that the onset of parturition may be triggered by endogenous oxytocin release. To test the hypothesis that oxytocin secretion is regulated by endogenous opioids in the periparturient period, we have 1) characterized oxytocin secretion in response to vaginocervical stimulation and 2) determined the effect of naloxone, an opioid antagonist, on oxytocin secretion induced by vaginocervical stimulation in prepartum mares and in postpartum mares at estrus and diestrus. During the last 2 months of pregnancy, the first diestrus and subsequent estrus post partum, a total of 66 vaginocervical stimulations were performed. Mares were pretreated with naloxone (0.5 mg/kg i.v.) or saline, administered 20 min before vaginocervical stimulation on subsequent days, using a randomized switchback design in which mares served as their own controls. Plasma was collected from 30 min before until 30 min after stimulation and was analyzed for oxytocin concentrations. Vaginocervical stimulation resulted in a significant increase in oxytocin secretion in all mares. Between Days 30 and 20 prepartum, the total amount of oxytocin secreted (calculated as area under the curve for 0 to 10 min after vaginocervical stimulation) was significantly greater in naloxone-treated than in saline-treated mares. From Day 20 prepartum until parturition, the differences between naloxone and saline-treated mares tended to decrease with approaching parturition, and were no longer statistically different. Peak plasma oxytocin concentrations were greater in naloxone-treated mares than in saline-treated mares during the entire prepartum period. During the postpartum period, total amount of oxytocin secreted following vaginocervical stimulation tended to be greater than during the prepartum period, and stimulated oxytocin secretion was significantly greater in naloxone-treated mares than in saline-treated mares. In conclusion, these data suggest that endogenous opioids suppress oxytocin secretion pre and post partum. It appears that opioid inhibition is not limited to the prepartum period, tends to decrease gradually towards parturition and is reinstated after foaling.

Endogenous prostaglandin secretion during cloprostenol-induced abortion in mares

Abstract Repeated administration of prostaglandin is the treatment of choice for the termination of pregnancy in mares more than 40 days pregnant. Even though it is well documented that PGF-2α or analogue needs to be administered every 12–24 h for successful induction of abortion, little is known about the underlying endocrine changes and the mechanism by which abortion occurs. The aim of this study was to characterize the changes in PGF-2α, progesterone and estrogen secretion during prostaglandin-induced abortion. Six mares, 82–102 days pregnant, were treated daily with 250 μg cloprostenol, blood was collected at 1-h intervals until fetal expulsion and pregnancy examination was performed daily. Four mares, 92–97 days pregnant, received no treatment but were subjected to the same hourly blood collections and daily genital examinations described for cloprostenol-treated mares for 3 days. Mean time from first cloprostenol administration until fetal expulsion was 48.6 ± 5.6 h and required 2.8 ± 0.2 cloprostenol administrations. In all mares, progesterone concentrations decreased in a near linear manner after the first cloprostenol administration and were invariably low (1.3 ± 0.2 ng ml −1 , mean ± SEM) at the time of fetal expulsion. Mean estrogen secretion remained unchanged until 5 h before fetal expulsion and then decreased rapidly to non-pregnant levels. Endogenous PGF-2α secretion rate increased with each cloprostenol administration and culminated in sustained PGF-2α secretion which persisted until fetal expulsion was completed. From these results we conclude that cloprostenol-induced abortion is associated with endogenous PGF-2α secretion, fetal expulsion coincides with sustained PGF-2α secretion and low progesterone concentrations and plasma estrogen concentrations remain unchanged until hours before fetal expulsion.