Localization of Luteinizing Hormone-Releasing Hormone in the Preoptic Area and Hypothalamus of the Rat Using Radioimmunoassay J. E. WHEATON, 1 L. KRULICH, AND S. M. M C C A N N 2 Department of Physiology, The University of Texas Health Science Center at Dallas, Medical School, Dallas, Texas 75235

Southwestern

ABSTRACT. To determine the localization of luteinizing hormone-releasing hormone (LHRH), five brains from adult male rats were serially sectioned in a cryostat at - 1 0 C in either the frontal, horizontal or sagittal planes. Acetic acid-ethanol extracts of each section were assayed for LHRH using radioimmunoassay (RIA) and in some cases using bioassay as well. Approximately 0.2 ng LHRH

eminence (ARC-ME) region. In the ARC-ME region, 2.7 ng of LHRH were concentrated primarily in the median eminence. The lateral distribution of LHRH in the ARC-ME region extended beyond the median eminence into tissue corresponding to the lateral aspect of the ventromedial nucleus. Concomitant bioassay and RIA determinations of LHRH were highly correlated. Of the sections bioassayed,

was concentrated in medial basal preoptic (MB-PO)

only those sections containing LHRH

tissue overlying the rostral portion of the optic chiasm. This LHRH appears to be associated with the organum vasculosum of the lamina terminalis and/or adjacent neural tissue. Uniform, low levels of LHRH were detected in hypothalamic tissue between the preoptic area (POA) and arcuate-median

FSH. These results confirm the presence of LHRH in the POA and in the rostral hypothalamus of the rat brain. The possible significance of LHRH in the POA for the regulation of LH release is discussed. (Endocrinology 97: 30, 1975)

D

EFINING the neural localization of LHRH has been the purpose of considerable research since the demonstration of LH-releasing activity in extracts of rat brain over a decade ago (1). Until recently, the usual method to determine the localization of LHRH was based on the release of LH from anterior pituitary glands incubated in vitro with extracts of brain sections. The results indicated that LHreleasing activity of the rat brain is for the most part concentrated in the medial basal hypothalamus (MBH) but also is present to a lesser extent in more rostral preopticsuprachiasmatic tissue (2-4). More recently, the isolation, characterization and subsequent synthesis of sufficient quantities of LHRH to permit its use as an antigen (or hapten) has enabled LHRH localization studies to use sensitive radioimmunoassay and immunohistochemReceived December 26, 1974. 1 Postdoctoral Research Fellow of the Ford Foundation. 2 Supported by grants from NIH (AM 10073 and HD 05151), the Ford Foundation and the Texas Population Research Institute.

released

ical techniques. The present study was conducted to examine the distribution of immunoassayable LHRH in rat brain. Particular attention was paid to its possible presence in the preoptic region. In addition, bioassay of some of the regions for LH and FSH-releasing activity was also performed to determine if the biological activity could be accounted for by the quantity of immunoassayable LHRH which was present. Materials and Methods Tissue sections. Five brains from male rats (Sprague-Dawley strain, 250-300 g) were serially sectioned through the preoptic-hypothalamic region in the frontal horizontal or sagittal planes by a modification of the method previously reported (3). Frozen brains from rats which were decapitated in the morning were initially sliced in the frontal plane approximately 1 mm in front of the optic chiasm and just behind the mammillary bodies in preparation for sectioning at - 1 0 C at 400 ixm using a cryostat. Immediately preceding each section, a 20 fim section was cut for histological placement. Serial frontal sections were cut in a rostrocaudal direction and a 30

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 03:43 For personal use only. No other uses without permission. . All rights reserved.

LHRH IN PREOPTIC AREA

31

central, basal rectangular piece of tissue extend- natant were dispensed into assay tubes. These ing 3 mm bilaterally from the midline and about aliquots and the remainder of the supernatant 3 mm dorsally from the base of the brain was were placed in a vacuum desiccator where they excised from each section for extraction. Konig were dried and stored under reduced pressure and Klippel's rat brain atlas (5) was used as for subsequent LHRH assay. reference. Prior to sectioning in the horizontal plane, the LHRH radioimmunoassay (RIA). Assay protocol brain was trimmed as described above and was conducted according to the procedure dedivided into rostral and caudal blocks by remov- scribed by Nett et al. (6) with slight modificaing a frontal section of tissue approximately 1 tion. Sample residues were dissolved in 200 ix\ mm in width just behind the optic chiasm to of 1% egg albumin-PBS pH 7.0, which was separate any LHRH in the preoptic region from substituted for the gelatin-PBS diluent used by that in the MBH which had the same dorsoven- Nett et al. (6). Approximately 30 pg of tral distribution. [125I]LHRH (approx. SA 300 iiCi/fig) were Beginning at the ventral limit of the optic added to each sample followed by 100 fx\ of chiasm, serial horizontal sections were cut antiserum to BSA-LHRH (#42). The antiserum through the rostral block, but 20 /xm sections was a gift from Dr. G. D. Niswender (Colorado were only cut for histological study between State University) and was diluted to 1:32,000, 125 every other thick section in the case of all which generally bound 40% of the [ I]LHRH added to control tubes. Synthetic LHRH horizontal sections. The procedure was similar for the caudal block except that sectioning (Beckman, lot. no. 04702) served as material for began at the ventral limit of the ME and reference and iodination. Dose-response curves for increasing levels of synthetic LHRH and sections were 200 /urn in thickness. Rostral and caudal tissue blocks were acetic acid/ethanol extracts of tissue from either likewise sectioned serially at 200 ^m in the the preoptic-suprachiasmatic region (PO-SC) or sagittal plane starting at the lateral edge and MBH were tested for parallelism (7). continuing to the midline which had been Three LHRH RIA's were conducted during previously etched with a clean razor blade. the course of the experiment, each one containHistological sections were air dired for 2 days ing all the samples from a single plane. Intraand then stained for 20 sec in a 0.5% (wt/vol) assay and interassay coefficients of variation for 30 pg of LHRH were 7 and 11%, respectively. solution of toluidine blue. To further define the dorsoventral distribution of LHRH in the MBH, 8 male rats were LHRH bioassay. The procedure was similar to decapitated and the ME from each brain was that previously reported (8) with a notable removed using fine scissors and a dissecting exception being the use of immature, 30-day-old microscope. The ME and the rest of the brain female rats of the Holtzman strain (Madison, were then frozen on dry ice. A small block of Wisconsin) as donors of anterior pituitary tissue corresponding to the arcuate region of the glands. Pituitary halves were randomized MBH was removed from the brain by making among incubation flasks so that each flask cuts at the anterior and posterior border of the contained 5 hemipituitaries. One of the disthird ventricle, 1 mm on either side of the third solved residues to be bioassayed was added to the ventricle and dorsally 1 mm from the base of the incubation medium. At the conclusion of the brain. This block of tissue and the ME were incubation period, the medium was assayed for subjected to the tissue extraction and assay LH and FSH. LH was measured according to procedures described for the serial tissue sec- the RIA protocol of Niswender et al. (9).3 Rat tions. LH (NIAMD-RP-1) was used as reference mateTissue extraction. The frozen sections were freeze-dried and stored under vacuum until each of the 5 brains for a particular plane had been sectioned (about 5 days). At this time, each section was homogenized in 1 ml of a 0.2N acetic acid/absolute ethanol (1:1) solution. Following centrifugation (8,000 x g for 20 min), duplicate 50 and 100 fi\ aliquots of the super-

3

The authors express gratitude to Dr. G. D. Niswender (Colorado State University) for the supply of ovine-LH (#15) antiserum and LHRH antiserum (#42), and to Dr. Leo Reichert (Emory University) for purified ovine LH used for iodination. Rat LH reference material and kits for measurement of FSH were provided through the NIAMDD Pituitary Hormone Program.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 03:43 For personal use only. No other uses without permission. . All rights reserved.

WHEATON, KRULICH AND McCANN

32

rial and the results were expressed in equivalents of the NIH-LH-S1 standard preparation. FSH was assayed according to the directions in the NIAMD kit and results expressed in terms of the RP-1 FSH preparation.3 Extracts of 4 selected tissue sections from each brain sectioned in the frontal plane were assayed for LHRH using bioassay as well as RIA.

Results Dose-reponse curves for synthetic LHRH and extracts of tissue from either the PO-SC or MBH are shown in Fig. 1. The 3 preparations gave parallel log-dose responses over the levels tested. Thus, it appears that similar, if not identical, immunochemical properties characterize these 3 preparations. Frontal sections. LHRH was present in tissue extending from the rostral edge of the optic chiasm caudally through the infundibulum (Fig. 2). In tissue rostral to the ARC-ME region, LHRH levels were first detectable and invariably highest (X ± SEM:207 ± 28 pg) in the section of

Endo • 1975 Vol 97 , No 1

tissue immediately caudal to the histological section showing the rostral limit of the optic chiasm (Fig. 2, sec. 3; Fig. 5, sec. 3'). This section consists of preoptic tissue that forms the anterior border of the third ventricle. Also included in this section is the circumventricular organ, the organum vasculosum of the lamina terminalis (OVLT) (10). Between this most rostral peak of LHRH and the ARC-ME region, a constant low level of LHRH was detected (Fig. 2, sec. 4-7). These sections contained an average of 50 pg of LHRH and included tissue from the preoptic, suprachiasmatic and anterior hypothalamic areas. As anticipated, the bulk of LHRH in the hypothalamus of the rat was present in the ARC-ME region. Eighty seven percent (2.7 ng) of the total LHRH detected in the frontal plane was concentrated there. A step-wise increase in LHRH was observed through the ARC-ME area with maximum levels (1.10 ± 0.05 ng) being associated with the part of the ME just rostral to the separation of the pituitary stalk (Fig. 2, sec. 11; Fig. 5, sec. 12').

DOSE-RESPONSE CURVES FOR SYNTHETIC AND EXTRACTED LRH

100 90 80 70

FIG. 1. Dose-response curves in the radioimmunoassay for synthetic luteinizing hormone releasing hormone (LRH) and acetic acid-ethanol extract of the medial basal hypothalamus (MBH) and preoptic-suprachiasmatic region (PO-SC).

60 50 40 30 20 10

0.2

0.4

OS

1.6

3.2

6.3

12.5

25

50

100 200

400 500

Pg SYNTHETIC LRH, FRACTION (10~4) OF PO-SC OR MBH

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 03:43 For personal use only. No other uses without permission. . All rights reserved.

LHRH IN PREOPTIC AREA

33

FIG. 2. The bars of the histogram represent the means of the immunoassayable LRH contained in extracts of 400 fim thick frontal sections (1-13). The vertical line above each bar gives the SEM. A thin section (1' —13') was cut before each 400 ft, section and was used for histological study. The millimetric scale of the abscissa corresponds to the stereotaxic coordinates used in Konig and Klippel's Rat Brain Atlas. Frontal sections are diagramatically projected through an outline of a parasagittal section. Abbreviations used for this and subsequent figures: 1.0ar—nucl arcuatus; ha—nucl anterior (hypothal); hdv—nucl 0.8dorsomedialis (hypothal); hpv— c nucl periventricularis (hypothal); hvm—nucl ventromedi- I 0.6alis (hypothal); pom—nucl preopticus medialis; pose—nucl 0.4preopticus, pars suprachiasmatica; mmm—nucl mamillaris medialis, pars medialis; sc— 0.2nucl suprachiasmaticus; CA— commissura anterior; CO—chiasma opticum; F—columna fornicis; I—infundibulum; ME— median eminence; OVLT— Millimeters organum vasculosum lamina terminalis; MI—massa intercalate; TD—tractus diagonalis; VIII—third ventricle.

a:

Horizontal sections. In the rostral block of tissue, horizontal sections cut between 0.8 and 1.2 mm above the ventral edge of the optic chiasm (the zero coordinate) contained the majority of LHRH in this plane (Fig. 3). This distribution of LHRH superimposed with that in the frontal plane demonstrates that LHRH is concentrated in preoptic tissue less than 400 fim above the rostral limit of the optic chiasm. Lesser but detectable quantities of LHRH were found in a band of tissue extending dorsally 1.6 mm from the ventral edge of the chiasm. The localization in the LHRH rich ARC-ME region was primarily confined to the median eminence. Due to the angle of horizontal sectioning, it was not possible to determine precisely the relative amounts of

LHRH in the ARC or ME regions since horizontal sections through the ARC region also included ME tissue in their rostral projections. Therefore, in other rats, the ME was separated from the ARC region to analyze each region separately. Using this procedure, 1.8 ± 0.2 and 0.8 ±0.1 ng of LHRH were detected in the ME and ARC regions, respectively. Sagittal sections. A very limited lateral distribution of LHRH was detected in the rostral block (Fig. 4). Over 80% of the LHRH in this plane was concentrated within 200 jwn of the midline. LHRH levels were undetectable more than 400 /xm from the midline. Consequently, the composite localization of LHRH in the

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 03:43 For personal use only. No other uses without permission. . All rights reserved.

Endo • 1975 Vol 97 • No 1

WHEATON, KRULICH AND McCANN

34

0.1 0.2 LRH ng 9

8

7

1.0

0.8

,

,

6 5 4 3 Millimeters (mm)

0.6

0.4

0.2

, LRH ng 2

FIG. 3. Diagrammatic representation of the immunoassayable LRH present in extracts of horizontal sections, 400 fim in thickness, cut from a rostral block of tissue and 200 firm in thickness cut from a caudal tissue block. Rostral and caudal tissue blocks were separated by removing a frontal section of tissue corresponding to the hatched region on the diagram.

POA points to medial basal tissue associated with OVLT and adjacent regions as a site for the concentration of approximately 0.2 ng of this neurohormone. In the ARC-ME region, substantial

ROSTRAL BLOCK

LHRH was still detectable in tissue extending 800 /um from the midline. This lateral localization of LHRH corresponds with the lateral aspect of the ventromedial nucleus.

CAUDAL BLOCK

OVLT

FIG. 4. Diagramatic representation of the immunoassayable LRH present in extracts of sagittal sections, 200 fim in thickness, cut from rostral and caudal tissue blocks. Rostral and caudal tissue blocks were obtained as described for horizontal sections.

2.0 1.0 0 mm

2.0 1.0 0 mm

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 03:43 For personal use only. No other uses without permission. . All rights reserved.

35

LHRH IN PREOPTIC AREA

FIG. 5. Low power photomicrograph of the histological sections cut immediately preceding the frontal sections numbered 3, 4, 9 and 12 (Fig. 2). Histological sections 3' and 4' bracket the peak of LRH detected in the preoptic area whereas histological sections 9' and 12' bracket that LRH concentrated in the arcuate-median eminence region.

Simultaneous bioassay and RIA measurement of LHRH. Both the LH-releasing activity and immunoassayable LHRH in extracts of frontal sections are shown in Table 1. There was little difference between the amount of LH released by pituitaries incubated with medium 199 alone or with medium containing the dissolved extracts of sections cut from the septal area (corresponding to sec. 1, Fig. 2). Extracts of tissue from the preoptic area (sec. 3) or ARC-ME region (sec. 9), on the other hand, induced a highly significant release of LH. Extracts of anterior hypothalamic tissue (sec. 5) released more LH than controls but less than that released by extracts of preoptic or ARC-ME tissue. The LH-releasing activity as esti-

mated by LH release into the medium and immunoassayable LHRH determined to be present in each section were highly correlated (r = 0.90; P < 0.01). Similarly, FSHreleasing activity was only found in sections which contained LHRH (Table 1) and this was also significantly correlated with immunoassayable LHRH (r = 0.77; P

SEM

U.

X J

RIA

+1 •X

FSH

secti

c o

0)

3

t/5

fro tal

tis

m

c

X

5

*o

X

C/3

CO [fa

rt X 0)

X

c E cc:

5 e

•J

X