J. Anat. (1975), 120, 2, pp. 275--288 With 7 figures Printed in Great Britain
275
Localization of luteinizing hormone-releasing hormone in rat hypothalamus using radioimmunoassay J. C. KING, T. H. WILLIAMS * AND A. A. ARIMURAt
*Department of Anatomy, University of Iowa, Iowa City, Iowa 52242, t Department of Medicine, Tulane University and Endocrine and Polypeptide Laboratories, Veterans Administration Hospital, New Orleans, La. 70114 (Accepted 8 February 1975) INTRODUCTION
For several years biochemists and physiologists have made great advances in the study of hypothalamic hormones which regulate pituitary function through the use of purification and synthesizing techniques; but anatomists working on structural correlates - including precise localization of these hormones - have been held back because of the lack of accurate and reliable techniques. We cannot attain the desired understanding of these important brain mechanisms until the morphological aspects have been clarified. However, modern techniques for assaying hypothalamic hormones, such as that for LH releasing hormone (LH-RH) (Arimura et al. 1973), now make morphological advances possible. The production of regulatory substances within the hypophysiotropic area of the hypothalamus (Halasz, Pupp & Uhlarik, 1962) is a part of the master programme that implements orderly manufacture and release of many endocrine secretions. Mechanisms of synthesis, storage, and release of these regulatory hormones need to be investigated. Luteinizing hormone (LH) and follicle stimulating hormone (FSH) releasing capabilities were discovered in hypothalamic extracts by McCann, Taleisnik & Friedman (1960) and Igarashi & McCann (1964). The work of localizing these regulating hormones has been started in other laboratories. Efforts have been made to map the distribution of releasing hormones for LH and FSH using an in vitro bioassay technique (Crighton, Schneider & McCann, 1970; Watanabe & McCann, 1968). The separateness of the releasing hormones LH-RH and FSH-RH has been challenged by Schally et al. (1971) who consider that one releasing hormone possesses the dual capability. Luteinizing hormone-releasing hormone, characterized as a decapeptide (Baba, Matsuo & Schally, 1971; Matsuo et al. 1971 a), and synthesized (Matsuo, Arimura, Nair & Schally, 1971 b), has been shown to bring about release of LH in doses of 0-2 to 0-5 ng/rat (Arimura & Schally, 1972). LH-RH also releases FSH (Kastin et al. 1970). Antisera to synthetic LH-RH have been made, and this has facilitated the development of a radioimmunoassay (RIA) method for presump-
276
J. C. KING, T. H. WILLIAMS AND A. A. ARIMURA
Table 1. Summary of procedures used for radioimmunoassays RIA nos. 1-8 Ind., individual samples; C, coronal; H, hcrizontal; S, sagittal.
Subjects
Extracted with
Samples
Plane of section
-''-o
RIA
no.
Male Female
1 2 3 4 5 6 7 8
33 3 2 - 3 5 6 5
HCI HOAc - x x - x -x x x x x
Pooled Ind. x x x x x
-
-
x x x
C H x x x
S
-
x x
x
- x -
x
tive LH-RH (Arimura et al. 1973; Nett et al. 1973). Radioimmunoassay has been employed by Brownstein et al. (1974) to localize LH-RH within specific hypothalamic nuclei of rats. The individual hypothalamic nuclei were 'punched out' of 300 ,m thick sections with needles, according to the procedure outlined by Palkovits (1973). The present investigation was carried out to define hypothalamic and related telencephalic areas rich in LH-RH, by sectioning a series of brains in each of three planes, carrying out radioimmunoassays for LH-RH on 150 pm sections, and correlating the findings with the morphology (as observed in adjacent sections). In this way the coordinates for high presumed LH-RH activity could be correlated with the topographic anatomy. Other assays mentioned previously involved male rats. In the present investigation we have studied LH-RH localization and levels in both male and female rats, and integrated information across the three planes of section. Comparisons between animals sacrificed at different times on day 2 of dioestrus clarified the dynamic picture of the redistributions of LH-RH. MATERIALS AND METHODS
Eight sets of radioimmunoassays (RIA) were performed on brain sections from adult male and 4 day cycling female Sprague-Dawley strain rats. Male rats weighed between 200 and 250 g. Female rats for the assays of pooled sections ranged in age from 78 to 85 days (RIA nos. 3-5). Slightly older female rats (95-115 days) were used in individual assays (RIA nos. 6-8). A summary of the procedures used in each experiment is given in Table 1. Animals were anaesthetized with ether, their brains removed, placed on a planchet, and frozen in an international cryostat at quick-freeze temperatures. 'Cryokwik' was sprayed over the surface of the brain to assist freezing. The brain was sectioned in one of three planes: coronal, horizontal or sagittal. Sections 150 ,um thick were homogenized with 1 ml of 2 N acetic acid or 0 5 ml of 0-1 N-HCI. Sections at corresponding levels from different animals were either pooled or analysed individually (see Table 1). In addition, a 10 pm section was taken rostral and caudal to each thick section and
Localization of LH-RH in rat hypothalamus
277
Fig. 1. Levels of the arcuate-median eminence (ME) complex are indicated in coronal sections with reference to infundibular recess (IR), tuberoinfundibular sulcus (TI) and pituitary stalk (s). In (a), the most rostral section, large neurons of the anterior arcuate nucleus (stippled area) are clustered medially at the base of the IlIrd ventricle. Slightly more caudally (b), the infundibular recess has begun to form and the rostral tip of the preinfundibular median eminence is apparent. The arcuate nuclei (stippled) surround the IR at this level. In (c) a slight invagination appears lateral to the median eminence in the region of the tuberoinfundibular sulcus.The arcuate nuclei (stippled) are more extensive. In (d) the prominence of the tuberoinfundibular sulcus heralds the beginning of pituitary stalk formation (separated off (s) in (e)). By (f) the ventricle (v) has become small and triangular. Arcuate neurons, indicated by the stippled area, are assembled beside lateral borders of the ventricle.
stained with toluidine blue for morphological correlations. Following centrifugation of acid extracts, aliquots of the supernatant were lyophilized and dissolved in 1 ml of 1 % egg white-phosphate buffered saline (PBS), at pH 7*5. Duplicate samples were then analysed for LH-RH by RIA (as described previously by Arimura et al. 1973). RESULTS
Radioimmunoassays for the LH-RH content of brain tissue were performed on male and female rats (for details see Table 1). For each plane of section, results will be given separately: coronal, parasagittal, and, finally, horizontal. After the general descriptions of LH-RH distribution, some exceptions - possibly attributable to individual variation or sex difference - will be noted. Finally, putting together the
278
J. c. KING, T. H. WILLIAMS AND A. A. ARIMURA 7
6
E
-r
3
~
l. 2
~ ~
....
dm
0 3
1
2
9 11 13 15 17 8 10 12 14 16 18 Sample number 7
5 4
6
Fig. 2. Results of assays of pooled coronal sections from three dioestrous females. Values of LH-RH are given in nanograms (ng) per sample. Values for each sample are superimposed over a sagittal oLtline of hypothalamic nuclei. Sample 1 was taken just rostral to the anterior commissure. ar, arcuate nucleus; dm, dorsomedial nucleus; ha, anterior hypothalamic area; m, mammillary nuclei; pa, preoptic area; ph, posterior hypothalamic area, sc, suprachiasmatic nucleus; vm, ventromedial nucleus.
information gained from each plane separately, regions of high LH-RH content will be summarized. The analyses are displayed in tables and figures. The assay of pooled samples of coronal sections from dioestrus females are displayed in Fig. 2. Fig. 3 shows the results of individually analysed coronal sections from dioestrus females. Figs. 4 and 5 illustrate data from pooled and individually analysed parasagittal sections, respectively. Finally, Fig. 6, in conjunction with Table 2, illustrates LH-RH content of pooled horizontal sections, while Fig. 7 shows the values obtained from individually analysed horizontal sections from dioestrous females. Coronal sections When the values obtained from coronal sections are studied, the LH-RH levels are conspicuously high across the rostrocaudal extent of the arcuate nucleus. The plotted LH-RH values obtained for this zone rise to form a bell-shaped curve (Fig. 2) which begins just caudal to the supraoptic decussations and just where the arcuate nucleus appears (see Fig. 1 a). As the values (represented in Fig. 2) are followed in a caudal direction, the LH-RH concentration is seen to increase sharply as the infundibular recess (IR) begins to appear (Fig. 1 b). After a slight recession close to the summit of the curve (see Fig. 2), the highest values are reached. The summit of the curve coincides consistently with the median eminence (ME) and the middle third of the arcuate nucleus. This region (corresponding to Figs. 1 c, d) includes the pre-infundibular ME as well as the most voluminous portion of the arcuate nucleus. LH-RH values fall off dramatically as the stalk separates (Fig. 1 e) and approximate to base levels as the third ventricle becomes smaller and triangular (Fig. If).
I
Localization of LH-RH in rat hypothalamus 175
279
-
100 _ 2
._o0 v
125
3
I
1254 -J
100
5
R
C
Fig. 3. LH-RH values for coronal sections individually analysed are shown (animals 1-5). Animal 1 was sacrificed early in the afternoon of dioestrus day 2. Animal 5 was sacrificed late the same afternoon. Each profile shows a rostral component (R) and a caudal component (C). The smaller rostral hump becomes more prominent as dioestrus progresses.
In addition to this high curve of LH-RH levels there is a much smaller anterior (rostral) hump which varies in precise location from assay to assay. This secondary hump occurred between the anterior commissure (in the septal region) and the supraoptic decussations. Group and individual variations in coronal section assays. The prominent high curve in the arcuate-ME region was a consistent finding in all pooled and almost all individual assays. The profiles of LH-RH values for five individually assayed dioestrous females are shown in Fig. 3 (1-5). In these animals, the afternoon of day 2 of dioestrus is progressing. The prominent curve of LH-RH values seen in 1 seems to become narrower and smaller in animals 2-5.
280
J. C. KING, T. H. WILLIAMS AND A. A. ARIMURA
d
7 6 E
-3 1
0 3
1 2
5 4
12 14 16 18 10 13 15 17 Sample number 7
6
9
8
Fig. 4. The results of assays of pooled parasagittal sections from two dioestrous females are presented. The values of LH-RH are given in nanograms (ng) per sample. Values for each sample are superimposed over a coronal outline at the level of the arcuate-median eminence region. ar, arcuate nucleus; dm, dorsomedial nucleus; vm, ventromedial nucleus.
The rostral smaller hump was more variable in size and location. When present in the male brain it appeared in the region of the suprachiasmatic area. In dioestrous females (pooled) this hump appeared in the medial preoptic area (Fig. 2, sample 2). In individually analysed dioestrous females the rostral hump increased (Fig. 3) until it became quite high (100 pg in animal 5) in the animal sacrificed latest in the day. This hump was sometimes broad as in animals 2 and 4 and at other times narrow as in 3 and 5. In females, peak values for the rostral hump were in the septal (2 and 5) or medial preoptic (3 and 4) area. In pooled males the rostral hump was in the suprachiasmatic region. Parasagittal sections The LH-RH values for parasagittal sections are shown in Fig. 4, in which a coronal section is given as a frame of reference. High concentrations of LH-RH are restricted to a small area on either side of the midline. LH-RH values on either side of the midline appear to be symmetrical, if some allowance is made for experimental error (e.g. variation in plane of section). Group and individual variations in parasagittal section assays. No parasagittal analysis was carried out on males. Pooled and individual parasagittal sections from dioestrous females were assayed. A region of high concentration occurred between 300 and 450 ,um from the midline. Within this region are the periventricular and arcuate nuclei, and the most medial portions of the medial preoptic and anterior hypothalamic areas, and of the suprachiasmatic, paraventricular, ventromedial and dorsomedial nuclei. In four of the five animals assayed (Fig. 5), LH-RH values showed an M-shaped profile, with a dip in the curve that corresponds to the sagittal plane. In one animal this dip in the curve was not represented, but in this case the
Localization of LH-RH in rat hypothalamus
281
400
500
0 Li
ba
400I
-
IC
-i 300
300
Fig. 5. The results of assays of parasagittal sections individually analysed are shown (animals 1-5). Profiles show an M-shaped curve for 4 of the 5 animals. The exception (animal 2), which gave a higher LH-RH peak value and no dip, is attributed to oblique sectioning. Toluidine blue stained frozen sections from the same animals confirm this explanation. The dip is close to the sagittal plane indicated by the triangle. Values fall off rapidly on both sides of the midline.
peak value was very high, suggesting a summation effect due to sections being cut obliquely.
Horizontal sections Sections yielding high LH-RH content again appeared close together, but in horizontal sections the LH-RH rich zones were very close to the ventral surface of the brain. All the LH-RH assayed was restricted to a ventral slab of tissue 450 um thick above the tip of the broken stalk of the pituitary. This zone includes the ME and the arcuate nucleus. Group and individual variations in horizontal section assays. No male horizontal
282
^=g1 ~ ~ ~ ~ ~ 4
J. C. KING, T. H. WILLIAMS AND A. A. ARIMURA
_
_
_
_
__
~~~~~~~~~~~~p
(Sample number 10 9
7
_
_
_
__ha
_
6
Fig. 6. Levels used for obtaining sections from three dioestrous females are shown, using a parasagittal outline as a frame of reference. The results of the pooled assays appear in Table 3. ar, arcuate nucleus; din, dorsomedial nucleus; ha, anterior hypothalamic area; m, mammillary nuclei; pa, preoptic area; ph, posterior hypothalamic area ; sc, suprachiasmatic nucleus; yin, ventromedial nucleus.
sections were assayed. Both pooled and individually analysed sections from dioestrous females showed LH-RH concentrated close to the ventral surface of the brain. However, an interesting discrepancy was found between pooled and individual analyses. In the case of pooled samples, a small satellite zone of LH-RH activity was found lying dorsal to the main area of concentration. This satellite zone encompasses the most dorsal portion of the supraoptic nucleus and of the arcuate nucleus. It also includes ventral and medial portions of the medial preoptic and anterior hypothalamic areas and parts of the suprachiasmatic and ventromedial nuclei. Because of the small amount of tissue utilized for individual assays, the amount of LH-RH in the satellite zone may have been insufficient for its detection in these samples.
Composite analysis across planes By correlating results from all three planes of section it can be inferred that regions most rich in LH-RH are confined to a ventral and medial core extending from the rostral to the caudal tip of the arcuate-ME complex. This zone contains the entire arcuate nucleus and the median eminence. The median eminence contains the largest amount of LH-RH, but the arcuate nucleus also contains a significant amount. The arcuate nucleus can be subdivided according to LH-RH content. The region of highest concentration is the middle third (see Figs. 1 c, d). A second region of high concentration is the anterior pole of the nucleus, beginning just caudal to the supraoptic decussations (Fig. 1 a). The third zone of high concentration is in the region of the post-infundibular ME where the stalk is now separate (Fig. 1 e). The rostral hump of LH-RH activity is much less significant than the caudal curve. There was an indication of a difference in the location of this zone in male and female rats. In the male this subsidiary rostral zone of activity was small to marginal as compared with females, and was located in the suprachiasmatic region, In the pooled
Localization of LH-RH in rat hypothalamus
283
900 1
2
3
4
5
6
0
Li 0
0.
o
C
1-J
Sample number
1900
c
0
LI
/
I -J
1~I 0, Sample number
Fig. 7. LH-RH values for horizontal sections individually analysed are indicated for each female studied in this plane (animals 1-6). Animals 1-3 were sacrificed in the early afternoon of dioestrus day 2; animals 4 and 5 were sacrificed late in the same afternoon. Animal 6 was sacrificed in the early evening of dioestrus day 2.
samples from dioestrous females the rostral hump was located in the medial preoptic area, rostral to that observed in the males. In the individually analysed females this hump was sometimes observed even more rostrally in the region of the diagonal band of Broca ventral to the septal nuclei. This location variability in females will be discussed. I9
A
ANA 120
284
J. C. KING, T. H. WILLIAMS AND A. A. ARIMURA
Table 2. Values of LH-RH obtained by RIA 5 from horizontal sections with sample no. 1 taken from the ventral surface oJ the brain Sample no. 1 2 3 4 5 6 7 8-16
LH-RH (pglsample) 1800 100
30 425 35
DISCUSSION
LH-RH rich zones were defined by analysing LH-RH content across three planes of sections. Two distinct profiles of LH-RH values emerged: a prominent high caudal curve and a smaller rostral hump. The more conspicuous elevation spans the entire arcuate-median eminence complex. This major complex has come to be considered the 'final common pathway' (Scharrer, 1965) through which hypothalamic control of ovulation is channelled. After cutting all known inputs to this region by microsurgery (Halasz et al. 1962), tonic (but not phasic) release of pituitary gonadotropins is maintained. Ovulation is evoked by stimulation of the arcuate-median eminence region (Critchlow, 1958). The same stimulation causes elevation of plasma LH, and it seems reasonable to suppose that this LH elevation is a consequence of LH-RH release from the arcuate ME region. Some axons of arcuate neurons, named the tuberoinfundibular system by Rethelyi & Halasz (1970), terminate in the palisade zone of the median eminence near the portal capillaries of the hypothalamo-hypophyseal system (Szentagothai, 1964). Recently we described an arrangement of LH-RH immunoreactive processes corresponding to the tuberoinfundibular system (King et al. 1974), but neuron cell bodies specifically immunoreactive with LH-RH were not found. A question remaining is whether arcuate neurons actually synthesize gonadotropin releasing hormones. Some investigators have suggested that tanycytes, specialized ependymal cells, are the actual hormone secreting cells (Vigh et al. 1963). Within the caudal curve representing high LH-RH values, three subdivisions were defined. One area of high LH-RH content corresponds with the anterior extent of the arcuate nucleus. In this portion of the nucleus the neurons are larger than in other regions. The second region, which contains the largest amount of the releasing hormone, is coextensive with the middle third of the arcuate nucleus. Here the arcuate nucleus achieves it greatest dimensions. The rise in LH-RH levels here probably reflects a greater number of neurons containing LH-RH. Between the first and second subdivisions there is a sharp transient dip in the level of activity. Although it is reasonable to try to relate the changing LH-RH values, throughout the subdivisions, to the changing size of the arcuate nucleus at different coronal levels, this reasoning
285
Localization of LH-RH in rat hypothalamus
provides no explanation for the sharp dip. The dip may correspond to the presence of an arcuate cell population with a different constitution. In fact, a rather distinct transition zone exists between the larger arcuate neurons anteriorly and the smaller neurons found in the middle third of the arcuate nucleus. The third subdivision is the region of the postinfundibular ME. This subdivision contains less LH-RH than the middle third of the nucleus. This decrease may be explained in part by the smaller number of arcuate neurons seen in cross section. Caudal to the postinfundibular-ME, LH-RH values rapidly diminish, reaching base levels immediately posterior to the arcuate nucleus. The high curve corresponding to the arcuate-ME region was present in both males and females. One specific feature, however, did appear to be related to the female sex cycle. In dioestrous females, individually analysed, the broad curve corresponding with the arcuate region shrank and almost disappeared as the afternoon of dioestrus progressed (see bottom two profiles in Fig. 3). It can be hypothesized that this drop in LH-RH represents the discharge of LH-RH into the portal capillaries of the median eminence. Chowers & McCann (1965) and Ramirez & Sawyer (1965), using bioassays, have reported changes in titres of LH-RH in the median eminence region during the oestrous cycle. Chowers & McCann (1965) also describe a drop in LH-RH on the evening of dioestrus. On the other hand, Ramirez & Sawyer (1965) specify that the decrease occurs late in the day of proestrus. Our observations are more in accord with those of Chowers & McCann. The arcuate-ME region has been reported as rich in LH-RH by Crighton et al. (1970) and rich in FSH-RH by Watanabe & McCann (1968). These studies utilized the in vitro bioassay procedure. More recently, Brownstein (1974) reported similar results using the radioimmunoassay procedure of Arimura et al. (1973) to assay punched out hypothalamic nuclei, and portions of nuclei. The proposed role of rostral areas - particularly the preoptic - in the control of ovulation has been considered repeatedly. Clemens, Shaar, Kleber & Taudy (1971) demonstrated many sites at which electrical stimulation causes an increase in plasma LH level. Stimulation of the medial preoptic area causes ovulation in proestrous females in which spontaneous ovulation has been blocked by the administration of pentobarbitol (Everett, 1964). There is a lower threshold for producing ovulation by electrical or electrochemical stimulation in the medial preoptic area than in the arcuate nucleus (Terasawa & Sawyer, 1969). Stimulation of the medial preoptic area brings about the release of LH within an hour in proestrous Nembutal-blocked females (Kalra et al. 1971). Furthermore, there seems to be a kind of dose-response relationship between the electrical or chemical stimulation of the medial preoptic area and the incidence of rats ovulating as well as the mean number of ovulations per rat (Velasco & Rothchild, 1973). Only a limited number of experiments substantiate a relationship between rostral brain areas and anterior pituitary activation. The existence of some pathway from rostral areas to drive the arcuate-ME eminence region has been demonstrated by lesion and deafferentation experiments. However, the exact role of these rostral areas in the control of ovulation has not been elucidated. 19-2
286
J. C. KING, T. H. WILLIAMS AND A. A. ARIMURA
We have described the more anterior LH-RH rich zone as 'the rostral hump'. This well-specified hump appeared between the rostral telencephalon (ventral to the septal region) and the suprachiasmatic region. The location of this hump seemed to be related to two factors: the sex of the animal and the phase of the cycle. In male rats the location of the hump was in the suprachiasmatic region, as indicated by two assays. The rostral hump seen in the female exhibited interesting variations. In pooled samples, it appeared in the medial preoptic area rostral to that observed in the male. In individually analysed dioestrous females it was located in even more rostral locations. Sometimes high LH-RH values were noted in the most rostral coronal section taken. This region corresponds to the rostral telencephalon ventral to the septal nuclei. This rostral component gains prominence as the day of dioestrus progresses, broadening and increasing in magnitude. At the same time, the arcuate-ME levels are shrinking. Diminishing levels of LH-RH values in the arcuate-ME complex associated with increasing levels in the rostral hump could reflect an uptake of LH-RH by these more rostral areas. This may be part of a mechanism for initiating female sexual behaviour (Moss & McCann, 1973). We can postulate rostral ward transfer of LH-RH via neurosecretory elements or non-nervous elements, glial or vascular. Alternatively, LH-RH may not be transferred from the caudal to the rostral region, but, rather, manufactured in the latter. Crighton et al. (1970), using bioassays to study male animals, found evidence that LH-RH is present as far rostral as the medial preoptic-suprachiasmatic area. Also, utilizing the immunoperoxidase technique, Zimmerman et al. (1974) found gonadotropin-releasing hormone in the organum vasculosum of the lamina terminalis in one of the two male mice studied. On the other hand, Brownstein (1974), using radioimmunoassay for studying LH-RH localization in male rats, did not report LH-RH rostral to the anterior arcuate. The present investigation is a first step in establishing the dynamics of LH-RH production and discharge in the female, and localizing this regulatory hormone in both sexes. SUMMARY
Radioimmunoassays for LH-RH were performed on frozen rat brain sections cut serially in coronal, parasagittal and horizontal planes. In some of the assays, samples were pooled from corresponding areas in different animals. A clear pattern of distribution of LH-RH rich regions emerged. Two prominent components - a caudal high curve and a rostral smaller hump - were observed, and their variable characteristics discussed. The high curve represents the arcuate-median eminence (ME) region. Our data suggest that this region is not homogeneous, and three different subdivisions of this arcuate-ME region can be distinguished on the basis of LH-RH content. High values were obtained consistently in the arcuate-ME region, except for females in the late afternoon of dioestrus day 2, at which stage the levels in this region dropped until they were little more than base line. The rostral hump of high LH-RH activity varies both in position and amplitude.
Localization of LH-RH in rat hypothalamus
287
These variations are associated with (1) the sex of the animal and (2) the stage of the female cycle. In males this hump appeared in the region of the suprachiasmatic nucleus, while in dioestrous females it appeared in the medial preoptic area, rostral to the male location. Some changes in LH-RH levels are thought to be related to the stage in the female sex cycle. During the afternoon of dioestrus, the caudal high curve representing the arcuate-ME region shrank, whereas the rostral smaller hump (preoptic region) showed much higher levels. Some feed-back take-off may occur from the LH-RH released by the arcuate-ME region. Instead of synthesizing its own LH-RH, the preoptic area may concentrate some of the LH-RH released from the arcuate-ME region, thereafter initiating sexual behaviour as suggested by Moss & McCann (1973). REFERENCES ARIMURA, A., SATO, H., KUMASKA, T., WOROBEC, R. B., DEBELIUK, L., DUNN, J. & SCHALLY, A. U. (1973). Production of antiserum to LH-releasing hormone (LH-RH) associated with gonadal atrophy in rabbits: Development of radioimmunoassays for LH-RH. Endocrinology 93, 1092-1103. ARIMURA, A. & SCHALLY, A. V. (1972). Physiological and clinical studies with natural and synthetic luteinizing hormone-releasing hormone (LH-RH). Medical Journal of Osalea University 23, 77-100. BABA, Y., Matsuo, H. & SCHALLY, A. V. (1971). Structure of porcine LH- and FSH-releasing hormone. IX. Confirmation of proposed structure by conventional sequential analyses. Biochemical and Biophysical Research Communications 44, 459-463. BROWNSTEIN, M. (1974). Distribution of neurotransmitters in discrete hypothalamic nuclei. Program: Neurobiology of CNS-hormone interaction. Anatomical Neuroendocrinology. Sponsored by the University of North Carolina Neurobiology Program. Chapel Hill, North Carolina, 14-16 May. CHOWERS, I. & MCCANN, S. M. (1965). Content of luteinizing hormone-releasing factor and luteinizing hormone during the estrous cycle and after changes in gonadal steroid titers. Endocrinology 76, 700-708. CLEMENS, J. A., SHAAR, C. J., KLEBER, J. W. & TAUDY, W. A. (1971). Areas of the brain stimulatory to LH and FSH secretions. Endocrinology 88, 180-184. CRIGHTON, D. B., SCHNEIDER, H. P. G. & MCCANN, S. M. (1970). Localization of LH-releasing factor in the hypothalamus and neurohypophysis as determined by an in vitro method. Endocrinology87, 323-329. CRITCHLOW, B. V. (1958). Ovulation induced by hypothalamic stimulation in the anesthetized rat. American Journal of Physiology 195, 171-174. EVERETT, J. W. (1964). Preoptic stimulative lesions and ovulation in the rat: 'threshold' and LH-release time in late diestrus and pro-estrus. In Major Problems in Neuroendocrinology (Ed. E. Bajusz and G. Jasmin). Basel: Karger. HALASZ, B., Pupp, L. & UHLARIK, S. (1962). 1Hypophysiotrophic area in the hypothalamus. Journal of Endocrinology 25, 147-154. HARRIS, G. W. & JACOBSEN, D. (1952). Functional grafts of the anterior pituitary gland. Proceedings ofthe Royal Society B 139, 263-276. IGARASHI, M. & MCCANN, S. M. (1964). A hypothalamic follicle stimulating hormone-releasing factor. Endocrinology 74, 446-452. KALRA, S. P., AJIKA, K., KRULICH, L., FAUCETT, C. P., QUIJADA, M. & MCCANN, S. M. (1971). Effects of hypothalamic and preoptic electro-chemical stimulation on gonadotropin and prolactin release in proestrus rats. Endocrinology 88, 1150-1158. KASTIN, A. J., SCHALLY, A. V., GUAL, C., MIDGLEY, A. R., BOWERS, C. Y. & GOMEZ-PEREZ, F. (1970). Administration of LH-releasing hormone to selected subjects. American Journal of Obstetrics and Gynaecology 108, 177-182. KING, J. C., PARSONS, J. A., ECLAUDSEN, S. L. & WILLIAMS, T. H. (1974). Luteinizing hormone releasing hormone (LIH-RH) pathway of the rat hypothalamus revealed by the unlabelled antibody peroxidase - antiperoxidase method. Cell and Tissue Research 153, 211-217. MATSUO, H., BABA, Y., NAIR, R. M. G., ARIMURA, A. & SCHALLY, A. V. (1971 a). Structure of the porcine LH and FSH-releasing hormone. 1. The proposed aminoacid sequence. Biochemical and Biophysical Research Communications 43, 1334-1339. MATSUO, H., ARIMURA, A., NAIR, R. M. G. & SCHALLY, A. V. (1971 b). Synthesis of the porcine LHand FSH-releasing hormone by the solid-phase method. Biochemical and BiophysicalResearch Communications 45, 822-827.
288 J. C. KING, T. H. WILLIAMS AND A. A. ARIMURA MCCANN, S. M., TALEISNIK, S. & FRIEDMAN, H. M. (1960). LH-releasing activity in hypothalamic extracts. Proceedings of the Society for Experimental Biology and Medicine 104, 432-434. Moss, R. L. & MCCANN, S. M. (1973). Induction of mating behaviour in rats by luteinizing hormonereleasing factor. Science 181, 177-179. MOTTA, M., PERIA, F., TIMA L., ZANISI, M. & MARTINI, L. (1971). Intrahypothalamic localization of the nuclei synthesizing the gonadotropin releasing factors. Journal of Neurovisceral Relations Supp. X, pp. 32-40. NETT, T. M., AKBAR, A. M., NISWENDER, G. D., HEDLUND, M. T. & WHITE, W. F. (1973). A radioimmunoassay for gonadotropin-releasing hormone (Gn-RH) in serum. Journal of Clinical Endocrinology and Metabolism 36, 880-885. PALKOvITs, M. (1973). Isolated removal of hypothalamic or other brain nuclei of therat. Brain Research 59, 449-450. RAMIREZ, V. D. & SAWYER, C. H. (1965). Fluctuations in hypothalamic LH-RF (luteinizing hormone, releasing factor) during the rat estrous cycle. Endocrinology 76, 282-289. RETHELYI, M. & HALASZ, B. (1970). Origin of the nerve endings in the surface zone of the median eminence of the rat hypothalamus. Experimental Brain Research 11, 145-158. SCHALLY, A. V., ARIMURA, A., BABA, Y., NAIR, R. M. G., MATSUO, H., REDDING, T. W., DEBELIUK, L. & WHITE, W. F. (1971). Isolation and properties of the FSH and LH-releasing hormone. Biochemical and Biophysical Research Communications 43, 393-399. SCHARRER, E. (1965). The final common path in neuroendocrine integration. Archives d' anatomie microscopique et de morphologie experimentale 54, 359-370. SZENTAGOTHAI, J. (1964). The parvicellular neurosecretory system. In Lectures on the Diencephalon (Ed. W. Bargmann and J. P. Schade), Amsterdam: Elsevier. TERASAWA, E. I. & SAWYER, C. H. (1969). Changes in electrical activity in the rat hypothalamus related to electrochemical stimulation of adenohypophysial function. Endocrinology 85, 143-149. VELASCO, M. E. & ROTHCHILD, I. (1973). Factors influencing the secretion of luteinizing hormone and ovulation in response to electrochemical stimulation of the preoptic area in rats. Journal of Endocrinology 58, 163-176. VIGH, B., AROS, B., WENGER, T., KORITSANZKY, S. & CEGLEDI, G. (1963). Ependymosecretion (ependymal neurosecretion). IV. The Gomori-positive secretion of the hypothalamic ependyma of various vertebrates and its relation to the anterior lobe of the pituitary. Acta biologica Academiae scientiarum hungaricae 13, 407-419. WATANABE, S. & MCCANN, S. M. (1968). Localization of FSH-releasing factor in the hypothalamus and neurohypophysis as determined by in vitro assay. Endocrinology 82, 664-673. ZIMMERMAN, E. A., Hsu, K. C., FERIN, M. & KOZLOWSKI, G. P. (1974). Localization of gonadotropinreleasing hormone (Gn-RH) in the hypothalamus of the mouse by immunoperoxidase technique. Endocrinology 95, 1-8.
275
Localization of luteinizing hormone-releasing hormone in rat hypothalamus using radioimmunoassay J. C. KING, T. H. WILLIAMS * AND A. A. ARIMURAt
*Department of Anatomy, University of Iowa, Iowa City, Iowa 52242, t Department of Medicine, Tulane University and Endocrine and Polypeptide Laboratories, Veterans Administration Hospital, New Orleans, La. 70114 (Accepted 8 February 1975) INTRODUCTION
For several years biochemists and physiologists have made great advances in the study of hypothalamic hormones which regulate pituitary function through the use of purification and synthesizing techniques; but anatomists working on structural correlates - including precise localization of these hormones - have been held back because of the lack of accurate and reliable techniques. We cannot attain the desired understanding of these important brain mechanisms until the morphological aspects have been clarified. However, modern techniques for assaying hypothalamic hormones, such as that for LH releasing hormone (LH-RH) (Arimura et al. 1973), now make morphological advances possible. The production of regulatory substances within the hypophysiotropic area of the hypothalamus (Halasz, Pupp & Uhlarik, 1962) is a part of the master programme that implements orderly manufacture and release of many endocrine secretions. Mechanisms of synthesis, storage, and release of these regulatory hormones need to be investigated. Luteinizing hormone (LH) and follicle stimulating hormone (FSH) releasing capabilities were discovered in hypothalamic extracts by McCann, Taleisnik & Friedman (1960) and Igarashi & McCann (1964). The work of localizing these regulating hormones has been started in other laboratories. Efforts have been made to map the distribution of releasing hormones for LH and FSH using an in vitro bioassay technique (Crighton, Schneider & McCann, 1970; Watanabe & McCann, 1968). The separateness of the releasing hormones LH-RH and FSH-RH has been challenged by Schally et al. (1971) who consider that one releasing hormone possesses the dual capability. Luteinizing hormone-releasing hormone, characterized as a decapeptide (Baba, Matsuo & Schally, 1971; Matsuo et al. 1971 a), and synthesized (Matsuo, Arimura, Nair & Schally, 1971 b), has been shown to bring about release of LH in doses of 0-2 to 0-5 ng/rat (Arimura & Schally, 1972). LH-RH also releases FSH (Kastin et al. 1970). Antisera to synthetic LH-RH have been made, and this has facilitated the development of a radioimmunoassay (RIA) method for presump-
276
J. C. KING, T. H. WILLIAMS AND A. A. ARIMURA
Table 1. Summary of procedures used for radioimmunoassays RIA nos. 1-8 Ind., individual samples; C, coronal; H, hcrizontal; S, sagittal.
Subjects
Extracted with
Samples
Plane of section
-''-o
RIA
no.
Male Female
1 2 3 4 5 6 7 8
33 3 2 - 3 5 6 5
HCI HOAc - x x - x -x x x x x
Pooled Ind. x x x x x
-
-
x x x
C H x x x
S
-
x x
x
- x -
x
tive LH-RH (Arimura et al. 1973; Nett et al. 1973). Radioimmunoassay has been employed by Brownstein et al. (1974) to localize LH-RH within specific hypothalamic nuclei of rats. The individual hypothalamic nuclei were 'punched out' of 300 ,m thick sections with needles, according to the procedure outlined by Palkovits (1973). The present investigation was carried out to define hypothalamic and related telencephalic areas rich in LH-RH, by sectioning a series of brains in each of three planes, carrying out radioimmunoassays for LH-RH on 150 pm sections, and correlating the findings with the morphology (as observed in adjacent sections). In this way the coordinates for high presumed LH-RH activity could be correlated with the topographic anatomy. Other assays mentioned previously involved male rats. In the present investigation we have studied LH-RH localization and levels in both male and female rats, and integrated information across the three planes of section. Comparisons between animals sacrificed at different times on day 2 of dioestrus clarified the dynamic picture of the redistributions of LH-RH. MATERIALS AND METHODS
Eight sets of radioimmunoassays (RIA) were performed on brain sections from adult male and 4 day cycling female Sprague-Dawley strain rats. Male rats weighed between 200 and 250 g. Female rats for the assays of pooled sections ranged in age from 78 to 85 days (RIA nos. 3-5). Slightly older female rats (95-115 days) were used in individual assays (RIA nos. 6-8). A summary of the procedures used in each experiment is given in Table 1. Animals were anaesthetized with ether, their brains removed, placed on a planchet, and frozen in an international cryostat at quick-freeze temperatures. 'Cryokwik' was sprayed over the surface of the brain to assist freezing. The brain was sectioned in one of three planes: coronal, horizontal or sagittal. Sections 150 ,um thick were homogenized with 1 ml of 2 N acetic acid or 0 5 ml of 0-1 N-HCI. Sections at corresponding levels from different animals were either pooled or analysed individually (see Table 1). In addition, a 10 pm section was taken rostral and caudal to each thick section and
Localization of LH-RH in rat hypothalamus
277
Fig. 1. Levels of the arcuate-median eminence (ME) complex are indicated in coronal sections with reference to infundibular recess (IR), tuberoinfundibular sulcus (TI) and pituitary stalk (s). In (a), the most rostral section, large neurons of the anterior arcuate nucleus (stippled area) are clustered medially at the base of the IlIrd ventricle. Slightly more caudally (b), the infundibular recess has begun to form and the rostral tip of the preinfundibular median eminence is apparent. The arcuate nuclei (stippled) surround the IR at this level. In (c) a slight invagination appears lateral to the median eminence in the region of the tuberoinfundibular sulcus.The arcuate nuclei (stippled) are more extensive. In (d) the prominence of the tuberoinfundibular sulcus heralds the beginning of pituitary stalk formation (separated off (s) in (e)). By (f) the ventricle (v) has become small and triangular. Arcuate neurons, indicated by the stippled area, are assembled beside lateral borders of the ventricle.
stained with toluidine blue for morphological correlations. Following centrifugation of acid extracts, aliquots of the supernatant were lyophilized and dissolved in 1 ml of 1 % egg white-phosphate buffered saline (PBS), at pH 7*5. Duplicate samples were then analysed for LH-RH by RIA (as described previously by Arimura et al. 1973). RESULTS
Radioimmunoassays for the LH-RH content of brain tissue were performed on male and female rats (for details see Table 1). For each plane of section, results will be given separately: coronal, parasagittal, and, finally, horizontal. After the general descriptions of LH-RH distribution, some exceptions - possibly attributable to individual variation or sex difference - will be noted. Finally, putting together the
278
J. c. KING, T. H. WILLIAMS AND A. A. ARIMURA 7
6
E
-r
3
~
l. 2
~ ~
....
dm
0 3
1
2
9 11 13 15 17 8 10 12 14 16 18 Sample number 7
5 4
6
Fig. 2. Results of assays of pooled coronal sections from three dioestrous females. Values of LH-RH are given in nanograms (ng) per sample. Values for each sample are superimposed over a sagittal oLtline of hypothalamic nuclei. Sample 1 was taken just rostral to the anterior commissure. ar, arcuate nucleus; dm, dorsomedial nucleus; ha, anterior hypothalamic area; m, mammillary nuclei; pa, preoptic area; ph, posterior hypothalamic area, sc, suprachiasmatic nucleus; vm, ventromedial nucleus.
information gained from each plane separately, regions of high LH-RH content will be summarized. The analyses are displayed in tables and figures. The assay of pooled samples of coronal sections from dioestrus females are displayed in Fig. 2. Fig. 3 shows the results of individually analysed coronal sections from dioestrus females. Figs. 4 and 5 illustrate data from pooled and individually analysed parasagittal sections, respectively. Finally, Fig. 6, in conjunction with Table 2, illustrates LH-RH content of pooled horizontal sections, while Fig. 7 shows the values obtained from individually analysed horizontal sections from dioestrous females. Coronal sections When the values obtained from coronal sections are studied, the LH-RH levels are conspicuously high across the rostrocaudal extent of the arcuate nucleus. The plotted LH-RH values obtained for this zone rise to form a bell-shaped curve (Fig. 2) which begins just caudal to the supraoptic decussations and just where the arcuate nucleus appears (see Fig. 1 a). As the values (represented in Fig. 2) are followed in a caudal direction, the LH-RH concentration is seen to increase sharply as the infundibular recess (IR) begins to appear (Fig. 1 b). After a slight recession close to the summit of the curve (see Fig. 2), the highest values are reached. The summit of the curve coincides consistently with the median eminence (ME) and the middle third of the arcuate nucleus. This region (corresponding to Figs. 1 c, d) includes the pre-infundibular ME as well as the most voluminous portion of the arcuate nucleus. LH-RH values fall off dramatically as the stalk separates (Fig. 1 e) and approximate to base levels as the third ventricle becomes smaller and triangular (Fig. If).
I
Localization of LH-RH in rat hypothalamus 175
279
-
100 _ 2
._o0 v
125
3
I
1254 -J
100
5
R
C
Fig. 3. LH-RH values for coronal sections individually analysed are shown (animals 1-5). Animal 1 was sacrificed early in the afternoon of dioestrus day 2. Animal 5 was sacrificed late the same afternoon. Each profile shows a rostral component (R) and a caudal component (C). The smaller rostral hump becomes more prominent as dioestrus progresses.
In addition to this high curve of LH-RH levels there is a much smaller anterior (rostral) hump which varies in precise location from assay to assay. This secondary hump occurred between the anterior commissure (in the septal region) and the supraoptic decussations. Group and individual variations in coronal section assays. The prominent high curve in the arcuate-ME region was a consistent finding in all pooled and almost all individual assays. The profiles of LH-RH values for five individually assayed dioestrous females are shown in Fig. 3 (1-5). In these animals, the afternoon of day 2 of dioestrus is progressing. The prominent curve of LH-RH values seen in 1 seems to become narrower and smaller in animals 2-5.
280
J. C. KING, T. H. WILLIAMS AND A. A. ARIMURA
d
7 6 E
-3 1
0 3
1 2
5 4
12 14 16 18 10 13 15 17 Sample number 7
6
9
8
Fig. 4. The results of assays of pooled parasagittal sections from two dioestrous females are presented. The values of LH-RH are given in nanograms (ng) per sample. Values for each sample are superimposed over a coronal outline at the level of the arcuate-median eminence region. ar, arcuate nucleus; dm, dorsomedial nucleus; vm, ventromedial nucleus.
The rostral smaller hump was more variable in size and location. When present in the male brain it appeared in the region of the suprachiasmatic area. In dioestrous females (pooled) this hump appeared in the medial preoptic area (Fig. 2, sample 2). In individually analysed dioestrous females the rostral hump increased (Fig. 3) until it became quite high (100 pg in animal 5) in the animal sacrificed latest in the day. This hump was sometimes broad as in animals 2 and 4 and at other times narrow as in 3 and 5. In females, peak values for the rostral hump were in the septal (2 and 5) or medial preoptic (3 and 4) area. In pooled males the rostral hump was in the suprachiasmatic region. Parasagittal sections The LH-RH values for parasagittal sections are shown in Fig. 4, in which a coronal section is given as a frame of reference. High concentrations of LH-RH are restricted to a small area on either side of the midline. LH-RH values on either side of the midline appear to be symmetrical, if some allowance is made for experimental error (e.g. variation in plane of section). Group and individual variations in parasagittal section assays. No parasagittal analysis was carried out on males. Pooled and individual parasagittal sections from dioestrous females were assayed. A region of high concentration occurred between 300 and 450 ,um from the midline. Within this region are the periventricular and arcuate nuclei, and the most medial portions of the medial preoptic and anterior hypothalamic areas, and of the suprachiasmatic, paraventricular, ventromedial and dorsomedial nuclei. In four of the five animals assayed (Fig. 5), LH-RH values showed an M-shaped profile, with a dip in the curve that corresponds to the sagittal plane. In one animal this dip in the curve was not represented, but in this case the
Localization of LH-RH in rat hypothalamus
281
400
500
0 Li
ba
400I
-
IC
-i 300
300
Fig. 5. The results of assays of parasagittal sections individually analysed are shown (animals 1-5). Profiles show an M-shaped curve for 4 of the 5 animals. The exception (animal 2), which gave a higher LH-RH peak value and no dip, is attributed to oblique sectioning. Toluidine blue stained frozen sections from the same animals confirm this explanation. The dip is close to the sagittal plane indicated by the triangle. Values fall off rapidly on both sides of the midline.
peak value was very high, suggesting a summation effect due to sections being cut obliquely.
Horizontal sections Sections yielding high LH-RH content again appeared close together, but in horizontal sections the LH-RH rich zones were very close to the ventral surface of the brain. All the LH-RH assayed was restricted to a ventral slab of tissue 450 um thick above the tip of the broken stalk of the pituitary. This zone includes the ME and the arcuate nucleus. Group and individual variations in horizontal section assays. No male horizontal
282
^=g1 ~ ~ ~ ~ ~ 4
J. C. KING, T. H. WILLIAMS AND A. A. ARIMURA
_
_
_
_
__
~~~~~~~~~~~~p
(Sample number 10 9
7
_
_
_
__ha
_
6
Fig. 6. Levels used for obtaining sections from three dioestrous females are shown, using a parasagittal outline as a frame of reference. The results of the pooled assays appear in Table 3. ar, arcuate nucleus; din, dorsomedial nucleus; ha, anterior hypothalamic area; m, mammillary nuclei; pa, preoptic area; ph, posterior hypothalamic area ; sc, suprachiasmatic nucleus; yin, ventromedial nucleus.
sections were assayed. Both pooled and individually analysed sections from dioestrous females showed LH-RH concentrated close to the ventral surface of the brain. However, an interesting discrepancy was found between pooled and individual analyses. In the case of pooled samples, a small satellite zone of LH-RH activity was found lying dorsal to the main area of concentration. This satellite zone encompasses the most dorsal portion of the supraoptic nucleus and of the arcuate nucleus. It also includes ventral and medial portions of the medial preoptic and anterior hypothalamic areas and parts of the suprachiasmatic and ventromedial nuclei. Because of the small amount of tissue utilized for individual assays, the amount of LH-RH in the satellite zone may have been insufficient for its detection in these samples.
Composite analysis across planes By correlating results from all three planes of section it can be inferred that regions most rich in LH-RH are confined to a ventral and medial core extending from the rostral to the caudal tip of the arcuate-ME complex. This zone contains the entire arcuate nucleus and the median eminence. The median eminence contains the largest amount of LH-RH, but the arcuate nucleus also contains a significant amount. The arcuate nucleus can be subdivided according to LH-RH content. The region of highest concentration is the middle third (see Figs. 1 c, d). A second region of high concentration is the anterior pole of the nucleus, beginning just caudal to the supraoptic decussations (Fig. 1 a). The third zone of high concentration is in the region of the post-infundibular ME where the stalk is now separate (Fig. 1 e). The rostral hump of LH-RH activity is much less significant than the caudal curve. There was an indication of a difference in the location of this zone in male and female rats. In the male this subsidiary rostral zone of activity was small to marginal as compared with females, and was located in the suprachiasmatic region, In the pooled
Localization of LH-RH in rat hypothalamus
283
900 1
2
3
4
5
6
0
Li 0
0.
o
C
1-J
Sample number
1900
c
0
LI
/
I -J
1~I 0, Sample number
Fig. 7. LH-RH values for horizontal sections individually analysed are indicated for each female studied in this plane (animals 1-6). Animals 1-3 were sacrificed in the early afternoon of dioestrus day 2; animals 4 and 5 were sacrificed late in the same afternoon. Animal 6 was sacrificed in the early evening of dioestrus day 2.
samples from dioestrous females the rostral hump was located in the medial preoptic area, rostral to that observed in the males. In the individually analysed females this hump was sometimes observed even more rostrally in the region of the diagonal band of Broca ventral to the septal nuclei. This location variability in females will be discussed. I9
A
ANA 120
284
J. C. KING, T. H. WILLIAMS AND A. A. ARIMURA
Table 2. Values of LH-RH obtained by RIA 5 from horizontal sections with sample no. 1 taken from the ventral surface oJ the brain Sample no. 1 2 3 4 5 6 7 8-16
LH-RH (pglsample) 1800 100
30 425 35
DISCUSSION
LH-RH rich zones were defined by analysing LH-RH content across three planes of sections. Two distinct profiles of LH-RH values emerged: a prominent high caudal curve and a smaller rostral hump. The more conspicuous elevation spans the entire arcuate-median eminence complex. This major complex has come to be considered the 'final common pathway' (Scharrer, 1965) through which hypothalamic control of ovulation is channelled. After cutting all known inputs to this region by microsurgery (Halasz et al. 1962), tonic (but not phasic) release of pituitary gonadotropins is maintained. Ovulation is evoked by stimulation of the arcuate-median eminence region (Critchlow, 1958). The same stimulation causes elevation of plasma LH, and it seems reasonable to suppose that this LH elevation is a consequence of LH-RH release from the arcuate ME region. Some axons of arcuate neurons, named the tuberoinfundibular system by Rethelyi & Halasz (1970), terminate in the palisade zone of the median eminence near the portal capillaries of the hypothalamo-hypophyseal system (Szentagothai, 1964). Recently we described an arrangement of LH-RH immunoreactive processes corresponding to the tuberoinfundibular system (King et al. 1974), but neuron cell bodies specifically immunoreactive with LH-RH were not found. A question remaining is whether arcuate neurons actually synthesize gonadotropin releasing hormones. Some investigators have suggested that tanycytes, specialized ependymal cells, are the actual hormone secreting cells (Vigh et al. 1963). Within the caudal curve representing high LH-RH values, three subdivisions were defined. One area of high LH-RH content corresponds with the anterior extent of the arcuate nucleus. In this portion of the nucleus the neurons are larger than in other regions. The second region, which contains the largest amount of the releasing hormone, is coextensive with the middle third of the arcuate nucleus. Here the arcuate nucleus achieves it greatest dimensions. The rise in LH-RH levels here probably reflects a greater number of neurons containing LH-RH. Between the first and second subdivisions there is a sharp transient dip in the level of activity. Although it is reasonable to try to relate the changing LH-RH values, throughout the subdivisions, to the changing size of the arcuate nucleus at different coronal levels, this reasoning
285
Localization of LH-RH in rat hypothalamus
provides no explanation for the sharp dip. The dip may correspond to the presence of an arcuate cell population with a different constitution. In fact, a rather distinct transition zone exists between the larger arcuate neurons anteriorly and the smaller neurons found in the middle third of the arcuate nucleus. The third subdivision is the region of the postinfundibular ME. This subdivision contains less LH-RH than the middle third of the nucleus. This decrease may be explained in part by the smaller number of arcuate neurons seen in cross section. Caudal to the postinfundibular-ME, LH-RH values rapidly diminish, reaching base levels immediately posterior to the arcuate nucleus. The high curve corresponding to the arcuate-ME region was present in both males and females. One specific feature, however, did appear to be related to the female sex cycle. In dioestrous females, individually analysed, the broad curve corresponding with the arcuate region shrank and almost disappeared as the afternoon of dioestrus progressed (see bottom two profiles in Fig. 3). It can be hypothesized that this drop in LH-RH represents the discharge of LH-RH into the portal capillaries of the median eminence. Chowers & McCann (1965) and Ramirez & Sawyer (1965), using bioassays, have reported changes in titres of LH-RH in the median eminence region during the oestrous cycle. Chowers & McCann (1965) also describe a drop in LH-RH on the evening of dioestrus. On the other hand, Ramirez & Sawyer (1965) specify that the decrease occurs late in the day of proestrus. Our observations are more in accord with those of Chowers & McCann. The arcuate-ME region has been reported as rich in LH-RH by Crighton et al. (1970) and rich in FSH-RH by Watanabe & McCann (1968). These studies utilized the in vitro bioassay procedure. More recently, Brownstein (1974) reported similar results using the radioimmunoassay procedure of Arimura et al. (1973) to assay punched out hypothalamic nuclei, and portions of nuclei. The proposed role of rostral areas - particularly the preoptic - in the control of ovulation has been considered repeatedly. Clemens, Shaar, Kleber & Taudy (1971) demonstrated many sites at which electrical stimulation causes an increase in plasma LH level. Stimulation of the medial preoptic area causes ovulation in proestrous females in which spontaneous ovulation has been blocked by the administration of pentobarbitol (Everett, 1964). There is a lower threshold for producing ovulation by electrical or electrochemical stimulation in the medial preoptic area than in the arcuate nucleus (Terasawa & Sawyer, 1969). Stimulation of the medial preoptic area brings about the release of LH within an hour in proestrous Nembutal-blocked females (Kalra et al. 1971). Furthermore, there seems to be a kind of dose-response relationship between the electrical or chemical stimulation of the medial preoptic area and the incidence of rats ovulating as well as the mean number of ovulations per rat (Velasco & Rothchild, 1973). Only a limited number of experiments substantiate a relationship between rostral brain areas and anterior pituitary activation. The existence of some pathway from rostral areas to drive the arcuate-ME eminence region has been demonstrated by lesion and deafferentation experiments. However, the exact role of these rostral areas in the control of ovulation has not been elucidated. 19-2
286
J. C. KING, T. H. WILLIAMS AND A. A. ARIMURA
We have described the more anterior LH-RH rich zone as 'the rostral hump'. This well-specified hump appeared between the rostral telencephalon (ventral to the septal region) and the suprachiasmatic region. The location of this hump seemed to be related to two factors: the sex of the animal and the phase of the cycle. In male rats the location of the hump was in the suprachiasmatic region, as indicated by two assays. The rostral hump seen in the female exhibited interesting variations. In pooled samples, it appeared in the medial preoptic area rostral to that observed in the male. In individually analysed dioestrous females it was located in even more rostral locations. Sometimes high LH-RH values were noted in the most rostral coronal section taken. This region corresponds to the rostral telencephalon ventral to the septal nuclei. This rostral component gains prominence as the day of dioestrus progresses, broadening and increasing in magnitude. At the same time, the arcuate-ME levels are shrinking. Diminishing levels of LH-RH values in the arcuate-ME complex associated with increasing levels in the rostral hump could reflect an uptake of LH-RH by these more rostral areas. This may be part of a mechanism for initiating female sexual behaviour (Moss & McCann, 1973). We can postulate rostral ward transfer of LH-RH via neurosecretory elements or non-nervous elements, glial or vascular. Alternatively, LH-RH may not be transferred from the caudal to the rostral region, but, rather, manufactured in the latter. Crighton et al. (1970), using bioassays to study male animals, found evidence that LH-RH is present as far rostral as the medial preoptic-suprachiasmatic area. Also, utilizing the immunoperoxidase technique, Zimmerman et al. (1974) found gonadotropin-releasing hormone in the organum vasculosum of the lamina terminalis in one of the two male mice studied. On the other hand, Brownstein (1974), using radioimmunoassay for studying LH-RH localization in male rats, did not report LH-RH rostral to the anterior arcuate. The present investigation is a first step in establishing the dynamics of LH-RH production and discharge in the female, and localizing this regulatory hormone in both sexes. SUMMARY
Radioimmunoassays for LH-RH were performed on frozen rat brain sections cut serially in coronal, parasagittal and horizontal planes. In some of the assays, samples were pooled from corresponding areas in different animals. A clear pattern of distribution of LH-RH rich regions emerged. Two prominent components - a caudal high curve and a rostral smaller hump - were observed, and their variable characteristics discussed. The high curve represents the arcuate-median eminence (ME) region. Our data suggest that this region is not homogeneous, and three different subdivisions of this arcuate-ME region can be distinguished on the basis of LH-RH content. High values were obtained consistently in the arcuate-ME region, except for females in the late afternoon of dioestrus day 2, at which stage the levels in this region dropped until they were little more than base line. The rostral hump of high LH-RH activity varies both in position and amplitude.
Localization of LH-RH in rat hypothalamus
287
These variations are associated with (1) the sex of the animal and (2) the stage of the female cycle. In males this hump appeared in the region of the suprachiasmatic nucleus, while in dioestrous females it appeared in the medial preoptic area, rostral to the male location. Some changes in LH-RH levels are thought to be related to the stage in the female sex cycle. During the afternoon of dioestrus, the caudal high curve representing the arcuate-ME region shrank, whereas the rostral smaller hump (preoptic region) showed much higher levels. Some feed-back take-off may occur from the LH-RH released by the arcuate-ME region. Instead of synthesizing its own LH-RH, the preoptic area may concentrate some of the LH-RH released from the arcuate-ME region, thereafter initiating sexual behaviour as suggested by Moss & McCann (1973). REFERENCES ARIMURA, A., SATO, H., KUMASKA, T., WOROBEC, R. B., DEBELIUK, L., DUNN, J. & SCHALLY, A. U. (1973). Production of antiserum to LH-releasing hormone (LH-RH) associated with gonadal atrophy in rabbits: Development of radioimmunoassays for LH-RH. Endocrinology 93, 1092-1103. ARIMURA, A. & SCHALLY, A. V. (1972). Physiological and clinical studies with natural and synthetic luteinizing hormone-releasing hormone (LH-RH). Medical Journal of Osalea University 23, 77-100. BABA, Y., Matsuo, H. & SCHALLY, A. V. (1971). Structure of porcine LH- and FSH-releasing hormone. IX. Confirmation of proposed structure by conventional sequential analyses. Biochemical and Biophysical Research Communications 44, 459-463. BROWNSTEIN, M. (1974). Distribution of neurotransmitters in discrete hypothalamic nuclei. Program: Neurobiology of CNS-hormone interaction. Anatomical Neuroendocrinology. Sponsored by the University of North Carolina Neurobiology Program. Chapel Hill, North Carolina, 14-16 May. CHOWERS, I. & MCCANN, S. M. (1965). Content of luteinizing hormone-releasing factor and luteinizing hormone during the estrous cycle and after changes in gonadal steroid titers. Endocrinology 76, 700-708. CLEMENS, J. A., SHAAR, C. J., KLEBER, J. W. & TAUDY, W. A. (1971). Areas of the brain stimulatory to LH and FSH secretions. Endocrinology 88, 180-184. CRIGHTON, D. B., SCHNEIDER, H. P. G. & MCCANN, S. M. (1970). Localization of LH-releasing factor in the hypothalamus and neurohypophysis as determined by an in vitro method. Endocrinology87, 323-329. CRITCHLOW, B. V. (1958). Ovulation induced by hypothalamic stimulation in the anesthetized rat. American Journal of Physiology 195, 171-174. EVERETT, J. W. (1964). Preoptic stimulative lesions and ovulation in the rat: 'threshold' and LH-release time in late diestrus and pro-estrus. In Major Problems in Neuroendocrinology (Ed. E. Bajusz and G. Jasmin). Basel: Karger. HALASZ, B., Pupp, L. & UHLARIK, S. (1962). 1Hypophysiotrophic area in the hypothalamus. Journal of Endocrinology 25, 147-154. HARRIS, G. W. & JACOBSEN, D. (1952). Functional grafts of the anterior pituitary gland. Proceedings ofthe Royal Society B 139, 263-276. IGARASHI, M. & MCCANN, S. M. (1964). A hypothalamic follicle stimulating hormone-releasing factor. Endocrinology 74, 446-452. KALRA, S. P., AJIKA, K., KRULICH, L., FAUCETT, C. P., QUIJADA, M. & MCCANN, S. M. (1971). Effects of hypothalamic and preoptic electro-chemical stimulation on gonadotropin and prolactin release in proestrus rats. Endocrinology 88, 1150-1158. KASTIN, A. J., SCHALLY, A. V., GUAL, C., MIDGLEY, A. R., BOWERS, C. Y. & GOMEZ-PEREZ, F. (1970). Administration of LH-releasing hormone to selected subjects. American Journal of Obstetrics and Gynaecology 108, 177-182. KING, J. C., PARSONS, J. A., ECLAUDSEN, S. L. & WILLIAMS, T. H. (1974). Luteinizing hormone releasing hormone (LIH-RH) pathway of the rat hypothalamus revealed by the unlabelled antibody peroxidase - antiperoxidase method. Cell and Tissue Research 153, 211-217. MATSUO, H., BABA, Y., NAIR, R. M. G., ARIMURA, A. & SCHALLY, A. V. (1971 a). Structure of the porcine LH and FSH-releasing hormone. 1. The proposed aminoacid sequence. Biochemical and Biophysical Research Communications 43, 1334-1339. MATSUO, H., ARIMURA, A., NAIR, R. M. G. & SCHALLY, A. V. (1971 b). Synthesis of the porcine LHand FSH-releasing hormone by the solid-phase method. Biochemical and BiophysicalResearch Communications 45, 822-827.
288 J. C. KING, T. H. WILLIAMS AND A. A. ARIMURA MCCANN, S. M., TALEISNIK, S. & FRIEDMAN, H. M. (1960). LH-releasing activity in hypothalamic extracts. Proceedings of the Society for Experimental Biology and Medicine 104, 432-434. Moss, R. L. & MCCANN, S. M. (1973). Induction of mating behaviour in rats by luteinizing hormonereleasing factor. Science 181, 177-179. MOTTA, M., PERIA, F., TIMA L., ZANISI, M. & MARTINI, L. (1971). Intrahypothalamic localization of the nuclei synthesizing the gonadotropin releasing factors. Journal of Neurovisceral Relations Supp. X, pp. 32-40. NETT, T. M., AKBAR, A. M., NISWENDER, G. D., HEDLUND, M. T. & WHITE, W. F. (1973). A radioimmunoassay for gonadotropin-releasing hormone (Gn-RH) in serum. Journal of Clinical Endocrinology and Metabolism 36, 880-885. PALKOvITs, M. (1973). Isolated removal of hypothalamic or other brain nuclei of therat. Brain Research 59, 449-450. RAMIREZ, V. D. & SAWYER, C. H. (1965). Fluctuations in hypothalamic LH-RF (luteinizing hormone, releasing factor) during the rat estrous cycle. Endocrinology 76, 282-289. RETHELYI, M. & HALASZ, B. (1970). Origin of the nerve endings in the surface zone of the median eminence of the rat hypothalamus. Experimental Brain Research 11, 145-158. SCHALLY, A. V., ARIMURA, A., BABA, Y., NAIR, R. M. G., MATSUO, H., REDDING, T. W., DEBELIUK, L. & WHITE, W. F. (1971). Isolation and properties of the FSH and LH-releasing hormone. Biochemical and Biophysical Research Communications 43, 393-399. SCHARRER, E. (1965). The final common path in neuroendocrine integration. Archives d' anatomie microscopique et de morphologie experimentale 54, 359-370. SZENTAGOTHAI, J. (1964). The parvicellular neurosecretory system. In Lectures on the Diencephalon (Ed. W. Bargmann and J. P. Schade), Amsterdam: Elsevier. TERASAWA, E. I. & SAWYER, C. H. (1969). Changes in electrical activity in the rat hypothalamus related to electrochemical stimulation of adenohypophysial function. Endocrinology 85, 143-149. VELASCO, M. E. & ROTHCHILD, I. (1973). Factors influencing the secretion of luteinizing hormone and ovulation in response to electrochemical stimulation of the preoptic area in rats. Journal of Endocrinology 58, 163-176. VIGH, B., AROS, B., WENGER, T., KORITSANZKY, S. & CEGLEDI, G. (1963). Ependymosecretion (ependymal neurosecretion). IV. The Gomori-positive secretion of the hypothalamic ependyma of various vertebrates and its relation to the anterior lobe of the pituitary. Acta biologica Academiae scientiarum hungaricae 13, 407-419. WATANABE, S. & MCCANN, S. M. (1968). Localization of FSH-releasing factor in the hypothalamus and neurohypophysis as determined by in vitro assay. Endocrinology 82, 664-673. ZIMMERMAN, E. A., Hsu, K. C., FERIN, M. & KOZLOWSKI, G. P. (1974). Localization of gonadotropinreleasing hormone (Gn-RH) in the hypothalamus of the mouse by immunoperoxidase technique. Endocrinology 95, 1-8.
