Control of seasonal reproduction by photoperiod involves a complex interplay between various areas near or in the hypothalamus, such as the PMR, RCH, ARC, and POA, with hormones, such as estradiol, melatonin, and thyroid hormones [19
]. The work presented in the present study supports the hypothesis that an additional area, the lPOA/rAHA, is an important component of the circuitry of seasonal neuroendocrine control. Specifically, we demonstrate that following ablation of cells within this region, female sheep exhibit resistance to the inhibitory effects of long-day photoperiods on ovarian cycles. Furthermore, although our tract-tracing results raise the possibility of cells within the lPOA/rAHA directly influencing GnRH neurons, they also clearly show that this region is anatomically connected with other areas already implicated in mediating the effects of photoperiod on reproduction. Thus, the reproductive outcomes observed in our study likely involve not only neurons of the lPOA/rAHA, but those in other regions as well.
Our previous work showed that radiofrequency lesions placed in the lPOA/rAHA extended the period of estrous cycles following exposure to long-day photoperiods [9
]. Interestingly, these lesions had no influence on the timing of estrous cycle onset following exposure to short-day photoperiods, and effects persisted as similar responses were exhibited through two complete short-day/long-day photoperiod cycles [9
]. The present study builds on that initial study through the use of NMA as a lesioning agent. This has the advantage of selectively destroying cell bodies without damaging neural fibers of passage [10
], a critical distinction that we could not make previously. Similar to our earlier study, a profound resistance to the inhibitory effects of a long-day photoperiod occurred in lesioned ewes. Taken together, these studies clearly indicate that neuronal cell bodies in the lPOA/rAHA play a role in mediating the inhibitory effects of long days.
Although the results from this study indicate that the lPOA/rAHA is involved in photoperiodic regulation of reproduction, the precise mechanism whereby this occurs is not clear. We have now identified GnRH afferents from this region, which raises the possibility of direct control of GnRH release. However, this finding must be interpreted with caution because these associations were identified at only the light microscope level in one animal, and evaluation of the extent to which GnRH neurons may be contacted is limited by the fact that tract-tracing injections were only done on one side of the lPOA/rAHA. Interestingly, cells in the lPOA/rAHA bind melatonin [6
] and express estrogen receptor-alpha (ESR1) [22
], raising the possibility that these hormones act locally on cells within the lPOA/rAHA to influence GnRH release. Although we did not identify the phenotype of such cells within the lPOA/rAHA, neurons expressing tyrosine hydroxylase [24
] and dynorphin [26
] in this area have been reported in the sheep. The tyrosine hydroxylase-containing neurons are likely part of the A14 dopamine cell group, and limited data in the sheep are consistent with a role for these neurons in the photosuppression of LH secretion [25
]. With regard to dynorphin, neurons expressing preprodynorphin mRNA are localized to the AHA, and GnRH neurons receive synaptic input from dynorphin neurons [15
]. Preprodynorphin mRNA expression in the AHA is decreased by ovariectomy and restored by progesterone treatment in ewes [28
], and is increased during a long-day photoperiod in rams by testosterone [29
]. An additional population of dynorphin neurons in the ARC also plays a role in regulating GnRH/LH secretion [15
], but it is not known whether they are involved in responsiveness to long-day photoperiods.
As mentioned above, control of seasonal breeding undoubtedly involves a complex interaction of a number of different areas in or near the hypothalamus with the GnRH system [21
]. Our tract-tracing results now reveal the connectivity of the lPOA/rAHA with several areas of known importance to seasonal breeding. Heavy innervations and high numbers of labeled cells were found in the mPOA. This is not entirely surprising because this region lies very close to the injection site; thus, some caution is required in interpreting this result. In sheep, this area contains the majority of GnRH cell bodies and a high number of ESR1-containing neurons [22
]. Indeed, previous work showed that estradiol implants in the ventral mPOA, an area just below the region of our lesions, suppressed LH in ovariectomized ewes during a long-day photoperiod [13
]. Interestingly, melatonin implants placed in this region, which should provide a short-day signal, did not reverse long-day photosuppression in ewes [32
], suggesting that this is not an area responsible for initiation of GnRH/LH secretion in response to stimulatory photoperiods. However, this does not rule out a role for this area in melatonin-driven changes in GnRH/LH secretion during photosuppression.
Another area of note that received reciprocal input was the RCH. The RCH region contains the A15 group of dopamine neurons, and there is a large amount of evidence that these neurons, located just medial to the optic tract, are involved in mediating steroid-induced suppression of GnRH secretion during inhibitory photoperiods. Lesions of A15 dopamine neurons significantly compromise the ability of estradiol to suppress LH secretion in ewes during the nonbreeding season, but not during the breeding season [27
]. Furthermore, ovariectomized ewes treated with estrogen exhibit an increase in the number of A15 dopamine cells expressing the immediate early gene product FOS, a marker of neuronal stimulation, only during the nonbreeding season [25
]. Finally, estradiol implants placed directly into the RCH of the hypothalamus reduce LH pulse frequency during the nonbreeding season via ESR1, but not ESR2 [14
]. Interestingly, A15 dopamine neurons do not express ESR1 [24
], so estradiol likely influences these neurons indirectly. The identities of these indirect inputs are not completely known but may include ESR1-containing neurons just dorsal to the A15 neurons or the ventral mPOA.
A large number of labeled cells and BDA-labeled fibers were also observed within the mediobasal hypothalamus in the areas of the ARC and dorsomedial nucleus. The role of the ARC in regulating GnRH/LH secretion recently has received attention because of a subset of neurons residing therein that coexpress kisspeptin, neurokinin B, and dynorphin (termed KNDy neurons). KNDy neurons express ESR1 [36
], and kisspeptin is a potent stimulator of GnRH/LH release in several species [37
]. Expression of kisspeptin is inhibited by gonadal steroids [36
] and is suppressed during an inhibitory photoperiod [42
]. These data support an important role for kisspeptin in the seasonal regulation of reproduction. In addition, we have recently shown that NKB stimulates LH release during the follicular phase of the estrous cycle [44
] and in peripubertal female ewe lambs (Hileman and Goodman, unpublished results). With regard to the dorsomedial hypothalamus, cells in this area express the RFamide-related peptide, gonadotropin-inhibiting hormone (GnIH) [42
]. Although the role of GnIH in seasonal reproduction is not entirely clear for sheep [46
], the number of GnIH-containing cells and the percentage of GnRH neurons exhibiting GnIH-containing close contacts are lower during the breeding season than nonbreeding season [42
], consistent with its role as an inhibitor of GnRH/LH secretion [46
]. Thus, reciprocal contacts between these areas and the lPOA/rAHA could potentially be important for regulating the response to long-day photoperiods.
We observed dense reciprocal innervations of the PMR, an area that appears to be very important in the actions of both melatonin and thyroxine (T4
implants in the PMR were effective in inducing anestrus in ewes [47
], and melatonin implants in this site (presumably providing a short-day signal) during long days induced LH secretion [48
]. Further, an area of high melatonin binding in the caudal ARC/PMR [48
] corresponds very well with an area receiving a high density of BDA-labeled fibers in our study. Interestingly, the responses observed in our female sheep are somewhat similar to those observed in the absence of thyroid hormones at the end of the breeding season [20
] (i.e., animals do not enter anestrus). Our previous study [9
] showed that lesions of the lPOA/rAHA do not disrupt circulating T4
levels or melatonin secretory patterns. Thus, input from the lPOA/rAHA may influence the response to T4
and/or melatonin, and therefore affect the timing of anestrus in response to long-day photoperiods.
The data presented in the present study clearly identify the lPOA/rAHA as playing a role in the photoperiodic control of reproduction in the sheep. Neurotoxic lesions in this region clearly interfered with the ability of a long-day photoperiod to induce anestrus in ewes. Furthermore, our tract-tracing results unequivocally show that the lPOA/rAHA is intimately connected with several regions of the hypothalamus that are involved in regulating seasonal reproduction. Additional studies will be required to identify the specific neural substrates involved and the functional relationship of this area with other photoperiod-sensitive regions of the hypothalamus.