Kisspeptin, gonadotrophin-releasing hormone and oestrogen receptor α colocalise with neuronal nitric oxide synthase neurones in prepubertal female sheep.

Puberty is a process that integrates multiple inputs ultimately resulting in an increase in gonadotrophin-releasing hormone (GnRH) secretion. Although kisspeptin neurones play an integral role in GnRH secretion and puberty onset, other systems are also likely important. One potential component is nitric oxide (NO), a gaseous neurotransmitter synthesised by nitric oxide synthase (NOS). The present study aimed to neuroanatomically characterise neuronal NOS (nNOS) in prepubertal female sheep and determine whether oestradiol exerts effects on this system. Luteinising hormone secretion was reduced by oestradiol treatment in prepubertal ovariectomised ewes. Neurones immunoreactive for nNOS were identified in several areas, with the greatest number present in the ventrolateral portion of the ventromedial hypothalamus, followed by the ventromedial hypothalamus, preoptic area (POA) and arcuate nucleus (ARC). Next, we determined whether nNOS neurones contained oestrogen receptor (ER)α and could potentially communicate oestradiol (E2 ) feedback to GnRH neurones. Neuronal NOS neurones contained ERα with the percentage of coexpression (12%-40%) depending upon the area analysed. We next investigated whether a neuroanatomical relationship existed between nNOS and kisspeptin or nNOS and GnRH neurones. A high percentage of kisspeptin neurones in the POA (79%) and ARC (98%) colocalised with nNOS. Kisspeptin close contacts were also associated with nNOS neurones. A greater number of close contacts were observed in the ARC than the POA. A high percentage of POA GnRH neurones (79%) also expressed nNOS, although no GnRH close contacts were observed onto nNOS neurones. Neither the numbers of nNOS neurones in the POA or hypothalamus, nor the percentage of nNOS coexpression with GnRH, kisspeptin or ERα were influenced by oestradiol. These experiments reveal that a neuroanatomical relationship exists between both nNOS and kisspeptin and nNOS and GnRH in prepubertal ewes. Therefore, nNOS may act both directly and indirectly to influence GnRH secretion in prepubertal sheep.

Neurokinin-3 receptor activation in the retrochiasmatic area is essential for the full pre-ovulatory luteinising hormone surge in ewes.

Neurokinin B (NKB) is essential for human reproduction and has been shown to stimulate luteinising hormone (LH) secretion in several species, including sheep. Ewes express the neurokinin-3 receptor (NK3R) in the retrochiasmatic area (RCh) and there is one report that placement of senktide, an NK3R agonist, therein stimulates LH secretion that resembles an LH surge in ewes. In the present study, we first confirmed that local administration of senktide to the RCh produced a surge-like increase in LH secretion, and then tested the effects of this agonist in two other areas implicated in the control of LH secretion and where NK3R is found in high abundance: the preoptic area (POA) and arcuate nucleus (ARC). Bilateral microimplants containing senktide induced a dramatic surge-like increase in LH when given in the POA similar to that seen with RCh treatment. By contrast, senktide treatment in the ARC resulted in a much smaller but significant increase in LH concentrations suggestive of an effect on tonic secretion. The possible role of POA and RCh NK3R activation in the LH surge was next tested by treating ewes with SB222200, an NK3R antagonist, in each area during an oestradiol-induced LH surge. SB222200 in the RCh, but not in the POA, reduced the LH surge amplitude by approximately 40% compared to controls, indicating that NK3R activation in the former region is essential for full expression of the pre-ovulatory LH surge. Based on these data, we propose that the actions of NKB in the RCh are an important component of the pre-ovulatory LH surge in ewes.

Neurokinin 3 receptor immunoreactivity in the septal region, preoptic area and hypothalamus of the female sheep: colocalisation in neurokinin B cells of the arcuate nucleus but not in gonadotrophin-releasing hormone neurones.

Recent evidence has implicated neurokinin B (NKB) in the complex neuronal network mediating the effects of gonadal steroids on the regulation of gonadotrophin-releasing hormone (GnRH) secretion. Because the neurokinin 3 receptor (NK3R) is considered to mediate the effects of NKB at the cellular level, we determined the distribution of immunoreactive NK3R in the septal region, preoptic area (POA) and hypothalamus of the ewe. NK3R cells and/or fibres were found in areas including the bed nucleus of the stria terminalis, POA, anterior hypothalamic and perifornical areas, dopaminergic A15 region, dorsomedial and lateral hypothalamus, arcuate nucleus (ARC) and the ventral premammillary nucleus. We also used dual-label immunocytochemistry to determine whether a neuroanatomical basis for direct modulation of GnRH neurones by NKB was evident. No GnRH neurones at any rostral-caudal level were observed to contain NK3R immunoreactivity, although GnRH neurones and fibres were in proximity to NK3R-containing fibres. Because NKB fibres formed close contacts with NKB neurones in the ARC, we determined whether these NKB neurones also contained immunoreactive NK3R. In luteal-phase ewes, 64% +/- 11 of NKB neurones colocalised NK3R. In summary, NK3R is distributed in areas of the sheep POA and hypothalamus known to be involved in the control of reproductive neuroendocrine function. Colocalisation of NK3R in NKB neurones of the ARC suggests a potential mechanism for the autoregulation of this subpopulation; however, the lack of NK3R in GnRH neurones suggests that the actions of NKB on GnRH neurosecretory activity in the ewe are mediated indirectly via other neurones and/or neuropeptides.

Colocalisation of dynorphin a and neurokinin B immunoreactivity in the arcuate nucleus and median eminence of the sheep.

Dynorphin A (DYN)-containing cells play a key role in conveying the negative feedback influence of progesterone upon pulsatile gonadotrophin-releasing hormone (GnRH) secretion in the ewe. A very high percentage of DYN cells in the arcuate nucleus express the progesterone receptor; another population of arcuate nucleus cells that also express steroid receptors in the sheep are those that express the tachykinin peptide, neurokinin B (NKB). Both DYN and NKB fibres have been shown to form close contacts with ovine GnRH cells. Therefore, the present study tested the hypothesis that neurones expressing NKB and DYN represent the same neuronal population in the arcuate nucleus. Confocal microscopic analysis of brain sections processed for dual immunofluorescence revealed that a large majority of DYN neurones in the arcuate nucleus were also immunoreactive for NKB. Likewise, a similar majority of NKB neurones in the arcuate nucleus were immunoreactive for DYN. By contrast, DYN cells in the preoptic area and anterior hypothalamus did not colocalise with NKB, nor did DYN cells in the paraventricular or supraoptic nuclei. Fibres that stained positively for both DYN and NKB were seen in the arcuate nucleus, where they formed close appositions with DYN/NKB-positive neurones, and in the external zone of the median eminence. Taken together with previous findings, these data suggest that a subpopulation of arcuate nucleus neurones coexpressing DYN and NKB mediate the negative feedback influence of progesterone on pulsatile GnRH secretion in the ewe and may also be involved in other feedback actions of gonadal steroids.

Immunocytochemical colocalization of GABA-B receptor subunits in gonadotropin-releasing hormone neurons of the sheep.

GABA has been shown to play an important role in the control of gonadotropin-releasing hormone (GnRH) and luteinizing hormone secretion in many mammals. In sheep, seasonal differences in the ability of GABA-B receptor antagonists to alter pulsatile luteinizing hormone secretion have led to the hypothesis that this receptor subtype mediates the increased inhibitory effects of estradiol on GnRH and luteinizing hormone pulse frequency seen during the non-breeding season (anestrus). The aim of the present study was to use multiple-label immunocytochemistry to determine if ovine GnRH neurons contain the GABA-B receptor subunits R1 and/or R2, and to determine whether there are seasonal differences in the colocalization of these subunits in GnRH neurons. A majority of GnRH cells in the preoptic area, anterior hypothalamic area, and medial basal hypothalamus of both breeding season and anestrous ewes contained either GABA-B R1 or R2 subunits; a subset of GnRH neurons in breeding season (42%) and anestrous ewes (60%) contained both subunits. In contrast to colocalization within cell bodies, GnRH fibers in the median eminence did not colocalize GABA-B receptor subunits. Although the percentage of GnRH neurons expressing GABA-B receptor subunits tended to be higher in anestrus than in the breeding season, there were no significant seasonal differences in R1 and R2 subunit colocalization in GnRH cell bodies. Thus, while GABA may act directly on GnRH cell bodies via GABA-B receptors in the sheep, any role that GABA-B receptors may play in seasonal reproductive changes is likely mediated by other neurons afferent to GnRH cells.

Distribution of preprodynorphin mRNA and dynorphin-a immunoreactivity in the sheep preoptic area and hypothalamus.

Endogenous opioid peptides (EOP) are important modulators in a variety of neuroendocrine systems, including those mediating reproduction, energy balance, lactation, and stress. Recent work in the ewe has implicated the EOP, dynorphin (DYN), in the inhibitory effects of progesterone on pulsatile gonadotropin releasing hormone secretion. Although DYN is involved in a number of hypothalamic functions in the sheep, little is known regarding the localization of preprodynorphin (PPD) expression and its major product DYN A (1-17). In this study, we determined the distribution of PPD mRNA and DYN A-containing cell bodies in the brains of ovary-intact, luteal ewes. To detect PPD mRNA, an ovine PPD mRNA was subcloned by reverse transcription-polymerase chain reaction from sheep hypothalamus and used to create a (35)S-labeled riboprobe for in situ hybridization. Neurons that expressed PPD mRNA and DYN A immunoreactivity were widely distributed in the ovine preoptic area and hypothalamus. PPD mRNA-expressing cells were seen in the supraoptic nucleus, paraventricular nucleus, preoptic area, anterior hypothalamus area, bed nucleus of the stria terminalis, ventromedial nucleus (VMN), dorsomedial nucleus of the hypothalamus, and the arcuate nucleus. All of these regions also contained DYN A-positive cell bodies except for the VMN, raising the possibility that PPD is preferentially processed into other peptide products in the VMN. In summary, based on the expression of both mRNA and peptide, DYN cells are located in a number of key hypothalamic regions involved in the neuroendocrine control of homeostasis in sheep.

Seasonal plasticity in the brain: the use of large animal models for neuroanatomical research.

Seasonally breeding mammals display an annual cycle of fertility that is associated with both structural neuroplasticity and functional changes in the activity of the GnRH neurones in the brain. Sheep are valuable models for understanding the hormonal and environmental cues that regulate seasonal reproduction, as well as the brain circuitry that underlies this response. As a result of the large size of sheep, we can tightly correlate the anatomy of GnRH cells and their patterns of gene expression with direct measurements of their neurosecretory output. Tract tracing studies have begun to reveal the pathways by which seasonal changes in response to oestradiol negative feedback affect the function of the reproductive system. Electron microscopic studies have shown that synaptic inputs on to ovine GnRH cells undergo marked seasonal rearrangements that are independent of hormonal changes and may reflect the intrinsic seasonality of the brain. Recent work indicates that the polysialylated form of neural cell adhesion molecule (PSA-NCAM), a marker of neuroplasticity, is well positioned anatomically to contribute to seasonal structural and functional alterations. Applying state-of-the-art neuroanatomical techniques to this model has allowed us to delineate the neural pathways responsible for the seasonal shut down of reproduction in sheep, as well as to begin to uncover the cellular mechanisms underlying seasonal neuroplasticity in the adult mammalian brain.

Estradiol increases multiunit electrical activity in the A15 area of ewes exposed to inhibitory photoperiods.

Seasonal anestrus in ewes results from an increase in response to the negative feedback action of estradiol (E(2)). This increase in the inhibitory effects of E(2) is controlled by photoperiod and appears to be mediated, in part, by dopaminergic neurons in the retrochiasmatic area of the hypothalamus (A15 group). This study was designed to test the hypothesis that E(2) increases multiunit electrical activity (MUA) in the A15 during inhibitory long days. MUA was monitored in the retrochiasmatic area of 14 ovariectomized ewes from 4 h before to 24 h after insertion of an E(2)-containing implant subcutaneously. In six of these ewes, MUA activity was also monitored before and after insertion of blank implants. Three of the 14 ewes were excluded from analysis because E(2) failed to inhibit LH. When MUA was recorded within the A15, E(2) produced a gradual increase in MUA that was sustained for 24 h. Blank implants failed to increase MUA in the A15 area, and E(2) did not alter MUA if recording electrodes were outside the A15. These data demonstrate that E(2) increases MUA in the A15 region of ewes and are consistent with the hypothesis that these neurons mediate E(2) negative feedback during long photoperiods.

Dopaminergic structures in the ovine hypothalamus mediating estradiol negative feedback in anestrous ewes.

This study examined the role of two dopaminergic (DA) cell groups, the A-14 and A-15 DA groups, in the seasonal shift in the response of LH to estradiol negative feedback in ewes. Radiofrequency lesions were placed bilaterally, in the area of the A-15 or the ventromedial A-14 cell groups of ovariectomized ewes, while control animals underwent sham neurosurgery. The effect of estrogen was tested in anestrus by analyzing LH pulse patterns before and 3 and 10 days after the insertion of estradiol implants. To evaluate the effects of these lesions on DA inhibition of LH secretion, LH pulse patterns were compared before and after an iv injection of the DA antagonist pimozide on day 3 of estradiol treatment. LH pulses were also examined in these ewes during the breeding season before and 3 days after the insertion of estradiol implants. Also, the effect of the DA receptor agonist apomorphine was tested to determine any effect of lesions on DA receptors inhibitory to LH. Lesions in either the A-14 or A-15 area decreased, but did not completely abolish, estradiol inhibition of LH pulse frequency in anestrus. Both types of lesions also blocked the stimulatory effects of pimozide on LH pulse frequency in estradiol-treated ovariectomized anestrous ewes. During the breeding season, estrogen decreased LH pulse amplitude, but not frequency, in all groups. The DA receptor agonist apomorphine decreased LH pulse frequency in all groups. Furthermore, immunohistochemistry for tyrosine hydroxylase revealed catecholaminergic fibers apparently connecting the caudal A-14 and the rostral A-15 areas. These results suggest that both the A-14 and A-15 DA cell groups are involved in the inhibition of LH by estradiol in anestrous, but not breeding season, ewes. Seasonal shifts in the activity of these DA neurons may, thus, play a role in the annual reproductive cycle of the ewe.

Immunocytochemical localization of beta endorphin and gonadal steroid regulation of proopiomelanocortin messenger ribonucleic acid in the ewe.

In the ewe, estradiol and progesterone inhibit luteinizing hormone (LH) secretion during the breeding season. Endogenous opioid peptides (EOP) are also inhibitory to LH secretion, and both estrogen and progesterone have been reported to enhance EOP inhibition of LH release. Which EOP are involved in this inhibition is unclear. In this study, we concentrated on beta-endorphin because evidence for its ability to inhibit LH secretion exists in ewes. We first studied the distribution of beta-endorphin-immunoreactive neurons in 4 cycling ewes using immunocytochemistry. Cell bodies were found only within the medial basal hypothalamus (MBH) and were concentrated in arcuate nucleus and mammillary recess of the third ventricle, with a few in the median eminence. Extensive fiber tracts were seen in preoptic area (POA) and median eminence. We next tested the hypothesis that gonadal steroids increase the synthesis of EOP by measuring levels of mRNA for proopiomelanocortin (POMC), the precursor to beta-endorphin. Ovariectomized ewes were treated with no steroids (n = 7) or given subcutaneous Silastic implants containing either estradiol (n = 6) or progesterone (n = 6). After 4 days of treatment, EOP inhibition of LH secretion was measured by determining the LH response to WIN 44,441-3 (WIN), an EOP antagonist. LH pulse frequency and pulse amplitude were determined in blood samples collected at 12-min intervals for 3 h before and after intravenous administration of 12.5 mg WIN. WIN injection increased (p < 0.01) the LH pulse-frequency only in progesterone-treated and pulse amplitude only in estradiol-treated ewes. After blood sampling, the ewes were killed, and POA, MBH, and pituitary gland were removed. Total RNA was extracted from these tissues and dot blotted onto nitrocellulose membranes for hybridization with a DNA probe complementary to the POMC mRNA. The resulting autoradiographs were quantified densitometrically. Levels of POMC mRNA in the MBH were increased (p < 0.01) by both estradiol and progesterone as compared with the no steroid group. There was no detectable POMC mRNA in the POA. These results suggest that estrogen and progesterone enhance EOP inhibition of LH secretion by increasing POMC mRNA levels and thus synthesis of beta-endorphin.

Endogenous opioid suppression of luteinizing hormone pulse frequency and amplitude in the ewe: hypothalamic sites of action.

Evidence suggests that endogenous opioid peptides (EOP) inhibit pulsatile luteinizing hormone (LH) secretion during both the luteal and follicular phases of the ovine estrous cycle. Further data from sheep and other species indicate that the hypothalamus is the primary site of action for this EOP inhibition. The purpose of the following experiments was to determine which areas of the hypothalamus are involved in the EOP inhibition of pulsatile LH secretion. Regularly cycling ewes (n = 10) were stereotaxically implanted with guide tubes into the preoptic area (POA) and medial basal hypothalamus (MBH). Implants containing the EOP antagonist WIN 44,441-3 (WIN) were placed into each of these areas. Blood samples were collected at 12-min intervals for 3 h before and during WIN administration in the luteal phase and for 4 h before and during WIN administration in the follicular phase of the estrous cycle. During the luteal phase, WIN implants in either area increased (p less than 0.01) LH pulse frequency (POA 1.4 +/- 0.3/3 h before vs. 3.1 +/- 0.4/3 h during; MBH 1.1 +/- 0.2/3 h before vs. 2.8 +/- 0.5/3 h during). There was no effect on LH pulse amplitude. In contrast, during the follicular phase, WIN implants selectively increased (p less than 0.01) LH pulse frequency when implanted in the POA (3.2 +/- 0.4/4 h before vs. 5.2 +/- 0.6/4 h during) while increasing (p less than 0.05) only LH pulse amplitude when placed in the MBH (0.7 +/- 0.2 ng/ml before vs. 1.4 +/- 0.3 ng/ml during).(ABSTRACT TRUNCATED AT 250 WORDS)

Hypothalamic sites of catecholamine inhibition of luteinizing hormone in the anestrous ewe.

Norepinephrine (NE) and dopamine (DA) actively inhibit the release of LH in anestrous ewes. This can be detected as an increase in LH pulse frequency following i.v. injection of NE and DA antagonists. The objective of this study was to determine the sites of these inhibitory actions in the ovine hypothalamus by using local administrations of the NE antagonist, phenoxybenzamine (PBZ), or the DA antagonist, pimozide (PIM), into specific hypothalamic areas. Each neurotransmitter antagonist was administered via a chronically implanted steel guide tube into either the preoptic area (POA), retrochiasmatic area (RCh), or the median eminence region (ME). Blood samples were taken every 15 min for 2 h before and 4 h during implantation of these drugs and were analyzed for LH and prolactin by RIA. Control (no treatment) samples were obtained similarly from the same animals on another day. Placement of PBZ into the POA significantly increased LH pulse frequency and mean LH concentrations over control values whereas PIM did not. In contrast, PIM significantly increased LH pulse frequency and mean LH concentrations when placed in the ME or in the RCh, but PBZ did not. No effects of PIM on prolactin concentrations were detected. These results suggest that an NE neural system operates in the POA and that a DA system acts in the medial basal hypothalamus (RCh or ME) to suppress GnRH pulse frequency in the ovary-intact anestrous ewe.

Integration of full-dose adjuvant chemotherapy with definitive radiotherapy for primary breast cancer: four-year update.

Controversy exists over the effect of definitive radiotherapy on the ability to administer full doses of adjuvant chemotherapy in primary breast cancer. Ninety-six consecutive women with clinical stage I and II breast cancer were treated with radiotherapy plus chemotherapy. Three combinations of drugs were used: cyclophosphamide and 5-fluorouracil (CF); cyclophosphamide, methotrexate, and 5-fluorouracil (CMF); or cyclophosphamide, methotrexate, 5-fluorouracil, and prednisone (CMFP). Chemotherapy consisted of two cycles of CF (cyclophosphamide at a dosage of 100 mg/m2 orally on days 1-14+5-fluorouracil at 600 mg/m2 iv on days 1 and 8) during concurrent radiotherapy, followed by six cycles of CMFP (same CF dosages+methotrexate at 40 mg/m2 iv on days 1 and 8+prednisone at 40 mg/m2 orally on days 1-14). The study included 63 premenopausal and 33 postmenopausal patients; 72 had 1-3 positive nodes, had greater than or equal to 4 positive nodes, and 9 had negative nodes and negative estrogen receptors. The mean CF doses delivered during concurrent radiotherapy were 95% of the optimal doses, and the mean CMF doses administered during the six cycles after radiotherapy were 89%. The CMF was delivered at level I (greater than or equal to 85% of optimal doses) to 73% of the patients. With a median follow-up of 36 months, 16 relapses have been observed. Two of these patients had treatment failure only in the breast or axilla and are disease free after mastectomy. Of the 72 patients with 1-3 positive nodes, 10 relapsed in distant sites, while 4 of 15 patients with greater than or equal to 4 positive nodes have had distant failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Current status of high linear energy transfer irradiation.

Based on laboratory investigations, high linear energy transfer (LET) particle irradiation is capable of more efficient cell kill than that associated with conventional or low LET irradiation. The advantages of high LET irradiation include: (1) a greater ability to damage hypoxic cells; (2) a lesser ability for repair of sublethal and potentially lethal radiation-induced damage; (3) less variation in radiation sensitivity relative to the cell cycle; and (4) a greater ability to deposit the radiation dose in the region of the tumor as opposed to the normal surrounding tissue (neutrons do not have this advantage compared to other particle therapy). Despite these laboratory advantages, it has been difficult to demonstrate any advantage of high LET irradiation in the clinic. A number of new developments have occurred to test the role of high LET: (1) sophisticated technology to enable treatment delivery with higher dose rate and improved depth dose; (2) the construction of hospital-based facilities; and (3) the development of randomized studies involving diseases in which the risk of early metastasis is minimized. It is hoped that careful study in the clinic over the next decade will elucidate the role of high LET particle therapy.

The effect of adjuvant chemotherapy on cosmesis and complications in patients with breast cancer treated by definitive irradiation.

From 1978 to 1981, 46 patients received primary radiotherapy following excisional biopsy and axillary staging procedure for Stages I and II carcinoma of the breast. The patients were divided into 2 groups: 27 patients who received radiation and completed 12 cycles of adjuvant chemotherapy (CMF or CMFP) and 19 patients who received radiation alone. All patients received radiation to the breast and regional nodes (4600-5000 rad) and a boost to the site of the primary tumor (1500-2000 rad). Median follow-up from completion of radiation was 26 months in the non-adjuvant and 24 months in the adjuvant group with a range of 12 to 49 months. Cosmesis was judged to be good to excellent in 89% (17/19) of the patients receiving radiation alone and 81% (22/27) of the patients receiving adjuvant chemotherapy. Fair to poor cosmesis in the adjuvant group was attributed primarily to increased fibrosis and reduction of breast size. The single complication for which there was an increased incidence in the adjuvant group was arm edema (22 vs. 0%). The incidence of arm edema was unrelated to T stage, type of axillary surgical procedure, number of positive nodes, addition of prednisone or sequencing of chemotherapy. Further efforts should be directed towards minimizing complications and maximizing cosmesis without sacrificing relapse-free survival in patients receiving primary radiotherapy and adjuvant chemotherapy for early breast cancer.

The sites of action of ovarian steroids in the regulation of LH secretion.

In the monkey, estradiol appears to exert both its negative and positive feedback actions on gonadotropin secretion primarily at the hypophysial level. In the rat, inhibitory and stimulatory effects of estradiol on the pituitary are also evident, but additional actions on the medial basal hypothalamus (MBH) and preoptic area must be postulated to fully account for the negative and positive feedback effects of estradiol in this species. The available evidence points to the MBH as a site for the negative feedback action of progesterone in the rat. In both species, progesterone probably acts in the central nervous system to block the positive feedback action of estradiol while the facilitation of luteinizing hormone release by this steroid appears to be at a hypophysial level.