Effect of melatonin on daily LH secretion in intact and ovariectomized ewes during the breeding season.

This study was conducted to find out whether daily LH secretion in ewes may be modulated by melatonin during the breeding season, when the secretion of both hormones is raised. Patterns of plasma LH were determined in luteal-phase ewes infused intracerebroventricularly (icv.) with Ringer-Locke solution (control) and with melatonin (100 microg/100 microl/h). Response in LH secretion to melatonin was also defined in ovariectomized (OVX) ewes without and after estradiol treatment (OVX+E2). Basal LH concentrations by themselves did not differ significantly before, during and after both control and melatonin infusions in intact, luteal-phase ewes. However, single significant (P<0.05) increases in LH concentration were noted during the early dark phase in the control and 1h after start of infusion in melatonin treated ewes. In both OVX and OVX+E2 ewes, melatonin decreased significantly (P<0.01, P<0.05, respectively) mean plasma LH concentrations as compared to the levels noted before the infusions. In OVX+E2 ewes, a single significant (P<0.05) increase in LH occurred 1h after start of melatonin treatment, similarly as in luteal-phase ewes. No significant differences in the frequencies of LH pulses before, during and after melatonin infusion were found in all treatments groups. In conclusion, melatonin may exert a modulatory effect on daily LH secretion in ewes during the breeding season, stimulating the release of this gonadotropin in the presence of estradiol feedback and inhibiting it during steroid deprivation. Thus, estradiol seems to be positively linked with the action of melatonin on reproductive activity in ewes.     

Does seasonal reproductive state affect the neuroendocrine response of the ewe to a long-day pattern of melatonin?

This study examined whether or not the reproductive response of female sheep to photoperiod varies with seasonal reproductive state. The specific objective was to test the hypothesis that the reproductive response to a long-day pattern of melatonin varies with the reproductive state of the ewe. The response examined was the synchronization of reproductive neuroendocrine induction (rise in serum luteinizing hormone, or LH) following nocturnal infusion of melatonin into pinealectomized ewes for 35 consecutive nights. This infusion restored a pattern of circulating melatonin similar to that in pineal-intact ewes maintained in a long photoperiod (LD 16:8). The ewes had been pinealectomized and without melatonin replacement for 16-25 months prior to the study. They were in differing reproductive states at the start of the infusion, as their endogenous reproductive rhythm had become desynchronized among individuals and with respect to time of year. Noninfused pinealectomized ewes served as controls. Regardless of the reproductive state at the start of the 35-day infusion of the long-day pattern of melatonin, all treated ewes exhibited the same reproductive neuroendocrine response after the infusion was ended. This consisted of a synchronized rise in LH some 6-8 weeks after the infusion was terminated, the maintenance of a high level of serum LH for some 15 weeks, and a subsequent precipitous fall in LH to a very low level. These results provide evidence that a long-day pattern of melatonin can synchronize reproductive neuroendocrine induction in the ewe, regardless of reproductive condition, and thus do not support the hypothesis that this response differs with seasonal reproductive state.     

Reproductive response and LH secretion in ewes treated with melatonin implants and induced to ovulate with the ram effect

Two experiments were conducted to examine the effects of treating seasonally anoestrous ewes with melatonin before ram introduction on reproductive response, and on LH secretion in anoestrous ewes induced to ovulate by rams.In Experiment 1, a total of 667 ewes from three flocks involving Merino (Flock 1, N = 149), Merino entrefino (Flock 2, N = 325) and Rasa Aragonesa (Flock 3, N = 203) breeds were used. Within each flock, ewes isolated from rams since the previous lambing were assigned at random to receive melatonin implants of Regulin (75, 175 and 105 in Merino, Merino entrefino and Rasa Aragonesa flocks, respectively) or to serve as untreated controls (74 in Merino, 150 in Merino entrefino and 98 in Rasa Aragonesa flocks). Fertile rams were introduced into all flocks 5 weeks after implantation in March (Flocks 1 and 2) or April (Flock 3), and remained with the ewes for a 50 day mating period. Percentage of ewes with luteal activity at ram introduction did not differ between melatonin treated and control ewes in any flock. There were no significant differences in either the mean interval from ram introduction to lambing or the distribution of lambing. Implantation with melatonin resulted in an improvement of prolificacy in all three flocks, although this only reached statistical significance in the Merino flock (1.15 vs. 1.03 in treated and control ewes, respectively, P < 0.05). Fertility was increased significantly (P < 0.05) in the Merino entrefino flock (64.5% in treated vs. 51.3% in control ewes).In Experiment 2, two trials were undertaken utilizing a total of 63 ewes. Trial 1 involved 24 mature Manchega ewes and Trial 2 involved 39 Merino ewe lambs. Half of the animals in each trial received a Regulin implant on 28 February (Trial 1) or 12 March (Trial 2) and the remaining half acted as controls. Rams were introduced 5 weeks after implantation and remained with the ewes for a 25 day period. In both trials, anoestrous ewes at ram introduction were bled at 20 min intervals for 3 h before and 5 h after ram introduction and then at 3 h intervals over the next 24 h for assessment of plasma concentrations of LH. Secretion of LH before or following introduction of rams was not affected by melatonin. Both treated and control anoestrous ewes in each trial responded to introduction of rams with an increase in the frequency of the LH pulses (P < 0.05), but no significant changes were detected in pulse amplitude or mean levels of LH. A preovulatory surge of LH was detected between 8 and 26 h after ram introduction, but neither mean interval from ram introduction to the peak of LH surge, nor the magnitude of the LH peak, was influenced by melatonin treatment.Results from this study show that: (1) melatonin implants administered during early seasonal anoestrus have the potential to improve reproductive performance in Spanish breeds of sheep, but the response is conditioned by breed, management system and environmental factors; (2) melatonin did not modify the secretion of LH in anoestrous ewes induced to ovulate by the ram effect under our experimental conditions.     

Nightly duration of pineal melatonin secretion determines the reproductive response to inhibitory day length in the ewe

The pineal controls the reproductive response of ewes to both stimulatory (short) and inhibitory (long) day lengths. Melatonin, a pineal hormone whose nocturnal secretion is entrained by photoperiod, mediates the effect of stimulatory photoperiod. We now report that melatonin also mediates the effect of inhibitory day length, monitored as response to estradiol negative feedback on luteinizing hormone (LH) secretion. Ovariectomized, estradiol-implanted ewes were pinealectomized and intravenously infused with melatonin to restore the nightly melatonin rise. Following transfer from short to long days, and a concurrent switch from short- to long-day melatonin patterns, LH dropped precipitously in pinealectomized ewes, matching the photoinhibitory response of pineal intact controls. LH dropped similarly in pinealectomized ewes when long-day melatonin was infused under short days. Pinealectomized ewes transferred from long to short days displayed a marked LH rise, provided melatonin was also switched to the short-day pattern. LH remained suppressed if long-day melatonin was infused following transfer to short days. These data indicate the nighttime melatonin rise mediates reproductive responses to inhibitory, as well as stimulatory photoperiods; they further suggest the duration of this rise controls suppression of LH under long days. Rather than being strictly pro- or antigonadal, the pineal participates in measuring day length.     

Effect of frontal hypothalamic deafferentation on duration of breeding season and melatonin secretion in the ewe

The objectives of this study were to determine if ewes subjected to frontal hypothalamic deafferentation (FHD) during anestrus remained anestrus or began to have estrous cycles, and if melatonin secretion was disrupted by FHD. Ovary-intact ewes in Group 1 were subjected to either FHD (n = 10) or sham FHD (n = 5) in early July 1983. Estrous cycles were monitored by measuring circulating progesterone concentrations from before FHD until September 1985. Group 2 ewes (n = 4) were subjected to FHD in October 1984. In late April 1985, blood samples were taken from all ewes at 1- to 4-h intervals from 1100 h to 0700 h of the following day to monitor diurnal changes of melatonin. Hypothalami were collected for histological evaluation of lesions. All Group 1 ewes (sham FHD and FHD) initiated normal estrous cycles in August and September 1983, and all ceased cycles by mid-February 1984. All sham FHD and 4 FHD ewes remained anestrus until August or September of 1984 and then resumed normal cycles. In contrast, 5 FHD ewes resumed cycles as early as April 1984 and then cycled intermittently or almost continuously. Two Group 2 ewes cycled continuously after FHD and 2 cycled infrequently. FHD ewes that showed prolonged breeding seasons had cuts that damaged the suprachiasmatic nucleus (SCN) and adjacent structures. Mean nocturnal (2000 h-0500 h) melatonin concentrations did not differ (p greater than 0.05) between sham FHD, FHD "normal season," and FHD "continuous cycle" ewes. In summary, damage to the SCN region by FHD during anestrus had no detectable effect on either onset or cessation of the next breeding season but greatly prolonged subsequent breeding seasons. Thus, the environmental signals that both initiated and terminated the 1983 breeding season apparently had been given before FHD was performed in midsummer. Damage to the SCN region during the breeding season caused some ewes to cycle continuously. The effects of FHD apparently were not due to disruption of melatonin secretion. FHD ewes that showed prolonged breeding seasons had normal seasonal changes of plasma prolactin concentrations. This suggests that different neural structures control seasonal patterns of gonadotropin and prolactin secretion.     

Effects of Superior Cervical Ganglionectomy and Melatonin Replacement on Intra-hypothalamic LHRH Content and Pulsatile Luteinizing Hormone Release in the Mink

Long-term effects of subcutaneous melatonin implants on intrahypothalamic LHRH content and on pulsatile luteinizing hormone release have been investigated in ganglionectomized male mink. Animals were submitted to bilateral removal of the superior cervical ganglion in mid-April. A preliminary study revealed that plasma LH concentrations remain at a basal level throughout the year following ganglionectomy. In a second experiment, one month after ganglionectomy and transfer from the natural photoperiod environment to short daylengths (LD 4:20), melatonin pellets were subcutaneously implanted to overcome deafferentation of the pineal. Progressive effects of treatment were studied 7 days, 15 days, and one, two and three months after insertion of the melatonin implants. The intra-hypothalamic LHRH content in ganglionectomized mink was at a basal level similar to that observed during seasonally sexual quiescence, or after exposure to inhibitory long days (LD 20:4). A significant and transient elevation in LHRH content was observed already after fifteen days, and also one month after insertion of melatonin implants. This resulted in mean values similar to those observed during the breeding season, or after exposure to stimulatory short days (LD 4:20). A decrease in hypothalamic LHRH content started after two months. No pattern of pulsatile LH secretion was recorded in ganglionectomized untreated mink. A significant increase in all parameters of pulsatile LH secretion was observed fifteen days after the elevation of LHRH content induced by melatonin treatment, and maximum values were reached after two months. Pituitary activity tended to decrease after three months, characterized in particular by a significant decrease in the mean frequency of LH pulses. In addition, the increase in pulsatile characteristics of LH release occurred two months before the peripheral renewal of testicular activity. Apparently, the reproductive endocrine function in ganglionectomized mink treated with melatonin implants is restored more rapidly at the hypothalamic level than at the pituitary or testicular levels.     

Effects of Superior Cervical Ganglionectomy and Melatonin Replacement on Intra-hypothalamic LHRH Content and Pulsatile Luteinizing Hormone Release in the Mink

Long-term effects of subcutaneous melatonin implants on intrahypothalamic LHRH content and on pulsatile luteinizing hormone release have been investigated in ganglionectomized male mink. Animals were submitted to bilateral removal of the superior cervical ganglion in mid-April. A preliminary study revealed that plasma LH concentrations remain at a basal level throughout the year following ganglionectomy. In a second experiment, one month after ganglionectomy and transfer from the natural photoperiod environment to short daylengths (LD 4:20), melatonin pellets were subcutaneously implanted to overcome deafferentation of the pineal. Progressive effects of treatment were studied 7 days, 15 days, and one, two and three months after insertion of the melatonin implants. The intra-hypothalamic LHRH content in ganglionectomized mink was at a basal level similar to that observed during seasonally sexual quiescence, or after exposure to inhibitory long days (LD 20:4). A significant and transient elevation in LHRH content was observed already after fifteen days, and also one month after insertion of melatonin implants. This resulted in mean values similar to those observed during the breeding season, or after exposure to stimulatory short days (LD 4:20). A decrease in hypothalamic LHRH content started after two months. No pattern of pulsatile LH secretion was recorded in ganglionectomized untreated mink. A significant increase in all parameters of pulsatile LH secretion was observed fifteen days after the elevation of LHRH content induced by melatonin treatment, and maximum values were reached after two months. Pituitary activity tended to decrease after three months, characterized in particular by a significant decrease in the mean frequency of LH pulses. In addition, the increase in pulsatile characteristics of LH release occurred two months before the peripheral renewal of testicular activity. Apparently, the reproductive endocrine function in ganglionectomized mink treated with melatonin implants is restored more rapidly at the hypothalamic level than at the pituitary or testicular levels.     

Importance of duration of nocturnal melatonin secretion in determining the reproductive response to inductive photoperiod in the ewe

The pineal gland, through its nocturnal melatonin secretion, mediates the effects of inhibitory (long) and stimulatory (short) photoperiod on reproduction in female sheep. Earlier studies revealed that duration of the nighttime melatonin rise is important in determining the inhibitory effect of day length on reproduction in the ewe. The present study tested whether the duration is also important in mediating the inductive response to short days. Pinealectomized ewes, housed under long days, received a short-day melatonin infusion (16-h duration) for 90 days. Reproductive status was monitored from the response to estradiol negative feedback of luteinizing hormone (LH) secretion. This short-day melatonin pattern led to unambiguous reproductive induction, despite the exposure to inhibitory long days. The increase in serum LH was comparable, in terms of latency and magnitude, to that in pinealectomized controls receiving the same short-day melatonin pattern under short days, and in pineal-intact controls transferred from long to short days. Since the reproductive status conformed to the length of time that melatonin was elevated each day rather than to photoperiod, these results support the conclusion that duration of the nighttime melatonin rise mediates the reproductive response of the ewe to an inductive photoperiod. In all, the melatonin rhythm is considered an integral component of the physiologic mechanism measuring day length; through duration of its nocturnal secretion, melatonin encodes both inhibitory and stimulatory photoperiods.     

Influence of breed and season on ovarian and pituitary response in progestogen-PMSG-treated ewes

Breed and seasonal effects on LH release, ovarian steroid secretion, and ovulation were evaluated in mature Finnish Landrace (Finn) and Hampshire ewes that received either a progestogen-PMSG treatment in May, July and November (experiment 1) or estradiol-17beta (50 mug) in May and July (experiment 2). The progestogen-PMSG treatment increased plasma estradiol within 12 hr after the PMSG injection at all three treatment periods and resulted in plasma LH and estradiol profiles similar to those during proestrus in cyclic ewes. Season, but not breed, affected the time from PMSG injection to preovulatory LH surge (56.5+/-1.4 hr in November vs 77.1+/-3.4 hr in July). Ovulation rate was higher in Finn than Hampshire ewes except in July when it decreased in Finn ewes. Magnitude of the estradiol-mediated LH release was decreased in July in Finn but not Hampshire ewes. Seasonal effects on reproduction in progestogen-PMSG treated ewes appear to be mediated through pituitary gonadotropin secretion with breed differences as to time and/or intensity of the seasonal effect(s).     

The role of nutrition in the regulation of LH secretion during anestrus by the serotoninergic and dopaminergic systems in Mediterranean ewes treated with melatonin

The steroid-dependent inhibition of LH secretion involves dopaminergic and serotoninergic systems but it is unclear how the plane of nutrition affects this inhibition during anestrus in melatonin treated ewes. Melatonin implants (18 mg) were inserted (Day 0) into ovariectomized, estradiol treated adult Rasa Aragonesa ewes on a high (H; n = 8) or low energy diet (L; n = 6) which were applied in early anestrus (Day 29-57) and late anestrus (Day 90-104). Cyproheptadine (0.1 mg/ kg), a serotoninergic (SHT2) receptor antagonist, was administered in early and late anestrus (Day 50 and 107) followed by pimozide (0.08 mg/kg), a dopaminergic2 receptor antagonist (Day 57 and 114). The H ewes had significantly higher LH concentrations (P < 0.05) before cyproheptadine treatment in early anestrus. The H and L ewes responded in a similar way to the antagonists in both early and late anestrus, except for L ewes who had a higher LH pulse amplitude after pimozide treatment in both periods (P < 0.05). During early anestrus, cyproheptadine tended to increase (P = 0.06) LH pulse frequency in L ewes and LH concentrations in H ewes. The LH secretion also increased in L ewes after pimozide administration during early anestrus (P < 0.05 for mean LH concentrations and LH pulse frequency and amplitude). However, pimozide dramatically increased LH secretion during late anestrus (Day 114) irrespective of the plane of nutrition (P = 0.06-0.08 for LH pulse frequency and P < 0.05 for LH concentrations and pulse amplitude). In melatonin treated Mediterranean ewes, the plane of nutrition appeared to modify the effect of dopaminergic and serotoninergic systems on the steroid-dependent inhibition of LH secretion throughout anestrus.     

Kisspeptin mediates the photoperiodic control of reproduction in hamsters

The KiSS-1 gene encodes kisspeptin, the endogenous ligand of the G-protein-coupled receptor GPR54. Recent data indicate that the KiSS-1/GPR54 system is critical for the regulation of reproduction and is required for puberty onset. In seasonal breeders, reproduction is tightly controlled by photoperiod (i.e., day length). The Syrian hamster is a seasonal model in which reproductive activity is promoted by long summer days (LD) and inhibited by short winter days (SD). Using in situ hybridization and immunohistochemistry, we show that KiSS-1 is expressed in the arcuate nucleus of LD hamsters. Importantly, the KiSS-1 mRNA level was lower in SD animals but not in SD-refractory animals, which spontaneously reactivated their sexual activity after several months in SD. These changes of expression are not secondary to the photoperiodic variations of gonadal steroids. In contrast, melatonin appears to be necessary for these seasonal changes because pineal-gland ablation prevented the SD-induced downregulation of KiSS-1 expression. Remarkably, a chronic administration of kisspeptin-10 restored the testicular activity of SD hamsters despite persisting photoinhibitory conditions. Overall, these findings are consistent with a role of KiSS-1/GPR54 in the seasonal control of reproduction. We propose that photoperiod, via melatonin, modulates KiSS-1 signaling to drive the reproductive axis.     

Overexpression of suppressor of cytokine signaling 3 in the arcuate nucleus of juvenile Phodopus sungorus alters seasonal body weight changes

The profound seasonal cycle in body weight exhibited by the Djungarian hamster (Phodopus sungorus) is associated with the development of hypothalamic leptin resistance during long day photoperiod (LD, 16:8 h light dark cycle), when body weight is elevated relative to short day photoperiod (SD, 8:16 h light dark cycle). We previously have shown that this seasonal change in physiology is associated with higher levels of mRNA for the potent inhibitor of leptin signaling, suppressor of cytokine signaling-3 (SOCS3), in the arcuate nucleus (ARC) of LD hamsters relative to hamsters in SD. The alteration in SOCS3 gene expression preceded the body weight change suggesting that SOCS3 might be the molecular switch of seasonal body weight changes. To functionally characterize the role of SOCS3 in seasonal body weight regulation, we injected SOCS3 expressing recombinant adeno-associated virus type-2 (rAAV2-SOCS3) constructs into the ARC of leptin sensitive SD hamsters immediately after weaning. Hamsters that received rAAV2 expressing enhanced green fluorescent protein (rAAV2-EGFP) served as controls. ARC-directed SOCS3 overexpression led to a significant increase in body weight over a period of 12 weeks without fully restoring the LD phenotype. This increase was partially due to elevated brown and white adipose tissue mass. Gene expression of pro-opiomelanocortin was increased while thyroid hormone converting enzyme DIO3 mRNA levels were reduced in SD hamsters with SOCS3 overexpression. In conclusion, our data suggest that ARC-directed SOCS3 overexpression partially overcomes the profound seasonal body weight cycle exhibited by the hamster which is associated with altered pro-opiomelanocortin and DIO3 gene expression.     

Attenuation of the estradiol-induced surge release of luteinizing hormone by antihistamine in ovariectomized ewes

Ovariectomized ewes received intramuscular (i.m.) injections of an H₁-histamine receptor antagonist, diphenhydramine, or saline during the anestrous and breeding seasons to determine if histamine may regulate the estradiol-induced surge release of LH in ewes. In addition, concentrations of histamine and GnRH in hypothalamic regions and histamine and LH in the pituitary gland were determined during the estradiol-induced surge of LH. Pretreatment mean, basal, and estradiol-induced secretion of LH did not differ (P > 0.05) among seasons. However, the quantity of LH (ng) measured during the estradiol-induced surge of LH was less (P < 0.05) in ewes treated with diphenhydramine (411 ± 104) than saline (747 ± 133). Treatment with diphenhydramine did not (P > 0.05) influence steady-state concentrations of histamine in hypothalamic or pituitary gland tissues, hypothalamic concentrations of GnRH, or anterior pituitary concentrations of LH during the estradiol-induced surge of LH. It is concluded that histamine may modulate the estradiol-induced surge release of LH in ewes by affecting the secretion of GnRH.     

Ovulatory activity and plasma prolactin concentrations in wild and domestic ewes exposed to artificial photoperiods between the winter and summer solstices

Mouflon and domestic Manchega sheep differ in the timing of their reproductive season under natural photoperiod (NP) conditions. This study examines whether they also differ in their reproductive responses to artificial photoperiod cues. For this, mouflons (n=24) and ewes (n=24) were exposed between the winter and summer solstices to artificial long days (LD: 16 h light/day), to short days (SD) simulated via the use of melatonin implants, or to NP conditions (controls), and their ovulatory activity monitored. The effects of these treatments on annual changes in prolactin concentration were also recorded. In the LD mouflon ewes, the offset and onset (7 March ± 5 and 2 October ± 4, respectively) of cyclic ovulatory activity occurred earlier (P<0.001) than in the NP animals (26 April ± 6 and 20 October ± 2, respectively), but no differences were seen (P>0.05) between the SD and NP mouflon ewes in either the onset of anoestrus (12 May ± 6 and 26 April ± 6, respectively) or the onset of subsequent ovulatory activity (13 October ± 8 and 20 October ± 2, respectively); however the duration of the anoestrus period was significantly reduced in the SD. In LD Manchega ewes, the onset of anoestrus was advanced (2 February ± 5 vs 15 March ± 11), but ovulatory activity started at the same time as in NP Manchega ewes (16 July ± 4 vs 5 July ± 8). In the SD Manchega ewes, two animals showed continuous cyclic ovulatory activity over the course of the experiment while the remainder entered anoestrus two months later (16 May ± 6, P<0.001) than their NP counterparts. In these SD ewes, the onset of cyclic ovarian activity was very variable. An annual rhythm of plasma prolactin concentration was seen in both the mouflon and Manchega ewes under all three photoperiod conditions. However, the amplitudes of the changes seen in prolactin concentration were smaller in both the LD and SD animals than in the corresponding NP animals (P<0.001). In conclusion, the results show that these two types of Mediterranean sheep differ in their ovulatory response when subjected to artificial photoperiods. The results also suggest that refractoriness to SDs may be the most important physiological mechanism regulating the onset of anoestrus in highly seasonal breeds, but not in less seasonal breeds.     

Effects of breed, ovarian steroids and season on the pulsatile secretion of LH in ovariectomized ewes

The effects of season and of oestradiol and progesterone on the tonic secretion of LH were studied in ovariectomized Merino and Suffolk ewes, two breeds which differ markedly in the seasonal pattern of their reproductive activity. In the absence of exogenous steroids, the frequency of LH pulses was lower and the amplitude of the pulses was higher in anoestrus than in the breeding season for Merino and Suffolk ewes 30 days after ovariectomy. In long-term (190 days) ovariectomized ewes, this seasonal change in LH secretion was observed in Suffolk ewes only. During seasonal anoestrus, treatment of ewes with subcutaneous oestradiol-17 beta implants (3, 6 or 12 mm in length) decreased the frequency of LH pulses in a dose-dependent manner, with Suffolk ewes being far more sensitive to the inhibitory effects of oestradiol than Merino ewes. The lowest dose of oestradiol (3 mm) had no effect on the secretion of LH in Merino ewes, but reduced secretion in Suffolk ewes. Treatment of ewes with the highest dose of oestradiol (12 mm) completely abolished LH pulses in Suffolk ewes, whereas infrequent pulses remained evident in Merino ewes. During the breeding season, oestradiol alone had no effect on the pulsatile release of LH in either breed, but in combination with progesterone there was a significant reduction in LH pulse frequency. Progesterone effectively decreased LH secretion in both breeds in both seasons. It was concluded that differences between breeds in the 'depth' of anoestrus could be related to differences in the sensitivity of the hypothalamus to both negative feedback by oestradiol and the direct effects of photoperiod.     

Oestradiol and the preovulatory surges of luteinising hormone and follicle stimulating hormone in ewes during the breeding season and transition into anoestrus

This study was designed to see if giving exogenous oestradiol, during the follicular phase of the oestrous cycle of intact ewes, during the breeding season or transition into anoestrus, would alter the occurrence, timing or magnitude of the preovulatory surge of secretion of luteinising hormone (LH) or follicle stimulating hormone (FSH). During the breeding season and the time of transition, separate groups of ewes were infused (intravenously) with either saline (30 ml h⁻¹; n = 6) or oestradiol in saline (n = 6) for 30 h. Infusion started 12 h after removal of progestin-containing intravaginal sponges that had been in place for 12 days. The initial dose of oestradiol was 0.02 μg h⁻¹; this was doubled every 4 h for 20 h, followed by every 5 h up to 30 h, to reach a maximum of 1.5 μg h⁻¹. Following progestin removal during the breeding season, peak serum concentrations of oestradiol in control ewes were 10.31 ± 1.04 pg ml⁻¹, at 49.60 ± 3.40 h after progestin removal. There was no obvious peak during transition, but at a time after progestin removal equivalent to the time of the oestradiol peak in ewes at mid breeding season, oestradiol concentrations were 6.70 ± 1.14 pg ml⁻¹ in ewes in transition (P < 0.05). In oestradiol treated ewes, peak serum oestradiol concentrations (24.8 ± 2.1 pg ml⁻¹) and time to peak (41.00 ± 0.05 h) did not differ between seasons (P > 0.05). During the breeding season, all six control ewes and four of six ewes given oestradiol showed oestrus with LH and FSH surges. The two ewes not showing oestrus did not respond to oestrus synchronisation and had persistently high serum concentrations of progesterone. During transition, three of six control ewes showed oestrus but only two had LH and FSH surges; all oestradiol treated ewes showed oestrus and gonadotrophin surges (P < 0.05). The timing and magnitude of LH and FSH surges did not vary with treatment or season. In blood samples collected every 12 min for 6 h, from 12 h after the start of oestradiol infusion, mean serum concentrations of LH and LH pulse frequency were lower in control ewes during transition than during mid breeding season (P < 0.05). Oestradiol treatment resulted in lower mean serum concentrations of LH in season and lower LH pulse frequency in transition (P < 0.05). We concluded that enhancing the height of the preovulatory peak in serum concentrations of oestradiol during the breeding season did not alter the timing or the magnitude of the preovulatory surge of LH and FSH secretion and that at transition into anoestrus, oestradiol can induce oestrus and the surge release of LH and FSH as effectively as during the breeding season.     

Thyroid hormones mediate steroid-independent seasonal changes in luteinizing hormone pulsatility in the ewe

Thyroid hormones permit the increase in response to estradiol negative feedback in ewes at the transition to anestrus. In this study, we tested whether the thyroid hormones are also required for steroid-independent seasonal changes in pulsatile LH secretion. In experiment 1, Suffolk ewes were ovariectomized and thyroidectomized (THX) or ovariectomized only (controls) in late November. LH pulse frequency and amplitude were measured for 4 h in December, April, May, June, and August. Pulse frequency was also measured in the presence of estradiol-containing implants during the breeding (December) and early anestrus (March) seasons. As expected, in the presence of estradiol, pulse frequency declined between December and March in control but not THX ewes. In the absence of estradiol, a seasonal decline in frequency and an increase in amplitude occurred in control ewes, concurrent with lengthening photoperiod. A similar trend was seen in THX ewes, but the seasonal changes were lower in magnitude and not significant. In experiment 2, the same protocol was used (pulse measurements in December, May, and June) with a larger THX group size (n = 7). Results were similar to those of experiment 1 for controls. In THX ewes, pulse frequency did not change over time and was significantly elevated relative to that of controls during the summer. Pulse amplitude in THX ewes tended to increase during summer and did not differ from pulse amplitudes in control ewes. These results demonstrate that thyroid hormones are required for steroid-independent cycles in LH pulse frequency; however, some seasonal changes in amplitude still occur in the absence of thyroid hormones. This finding contrasts with the changes in estradiol negative feedback at the transition to anestrus, which are entirely thyroid hormone dependent.     

How does melatonin code for day length in the ewe: duration of nocturnal melatonin release or coincidence of melatonin with a light-entrained sensitive period?

The main objective of the study was to test the hypothesis that the phase of melatonin release with respect to the light-dark cycle mediates the effects of photoperiod on the reproductive response of the ewe. To test the phase hypothesis, we eliminated endogenous melatonin secretion by pinealectomy and then restored physiological levels of serum melatonin with rises of the same duration but at different phases of the light-dark cycle (either at night or in the middle of the day). Serum melatonin patterns were determined by radioimmunoassay in samples taken hourly for 24 h. The reproductive state was monitored by measuring serum luteinizing hormone (LH) in ovariectomized ewes treated with constant-release estradiol implants. Infusion of a long-day pattern of melatonin was equally effective in maintaining reproductive suppression when given during the night or the middle of the day. LH remained low for approximately 150 days and then rose as ewes became refractory to the inhibitory melatonin signal. These results do not support the phase hypothesis. Rather, they are consistent with the hypothesis that the duration of the nocturnal secretion of melatonin codes for day length.     

Effect of melatonin implants on secretion of luteinizing hormone in intact and castrated rams

Rams were treated with melatonin implants in 2 experiments designed to examine the control of reproductive seasonality. In Exp. 1, rams (n = 12) were allocated to 3 treatment groups: 2 groups were treated with 2 melatonin implants per ram for 4 months from 11 November (N) and 9 December (D) and the remaining group was untreated (C). The seasonal increase in luteinizing hormone (LH) pulse frequency and testes size was advanced in Groups N and D. A second seasonal cycle in LH secretion and testes size occurred in Groups N and D after melatonin implants became exhausted. In Exp. 2, rams (n = 20) were allocated to 4 treatment groups: 10 rams were castrated on 6 October and 1 group of entire rams (EM) and one group of castrated rams (CM) were treated with 2 melatonin implants per ram each month from 3 November until 8 January. The other group of entire rams (EC) and castrated rams (CC) was untreated. An increase in LH pulse frequency occurred after castration. Melatonin treatment increased LH pulse frequency in entire rams and reduced LH pulse frequency in castrated rams. The results demonstrated that the advanced reproductive development as a result of treatment with melatonin implants was due to an effect of melatonin on the hypothalamic pulse generator to increase LH pulse frequency. The ability of melatonin to influence LH pulse frequency in entire and castrated rams indicated that an effect of melatonin on the hypothalamic pulse generator is independent of testicular steroids.