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Endocrinology Vol. 140, No. 2 791-799
Copyright © 1999 by The Endocrine Society

ARTICLES

Luteinizing Hormone-Releasing Hormone Quantified in Tissues and Slice Explant Cultures of Postnatal Rat Hypothalami

Jennifer A. Maurer and Susan Wray

Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Susan Wray, Section Chief, Cellular and Developmental Neurobiology, Laboratory of Neurochemistry, NINDS, NIH, Building 36, Room 4D20, Bethesda, Maryland 20892. E-mail: swray{at}codon.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
LH-releasing hormone (LHRH) peptide from postnatal rat preoptic area (POA)/hypothalamic tissues in vivo and slice explant cultures maintained in vitro was quantitated using an enzyme-linked immunosorbant assay. Moreover, messenger RNA (mRNA) copy number was calculated in LHRH neurons maintained in culture using in situ hybridization histochemistry with autoradiographic film analysis. POA/hypothalami from postnatal day 5–6 pups averaged 1250 pg of LHRH, with approximately 28% of peptide residing within rostral tissues where most LHRH perikarya reside. Explant cultures maintained 18 days in vitro contained 30.4–92.0 pg/slice with a whole animal total of 244.8 pg. Considering cell numbers in vivo and in vitro, LHRH neurons in whole animal produce 1.0 pg of LHRH/cell, whereas those in culture average 2.0 pg/cell. Furthermore, LHRH mRNA copies/cell in organotypic culture was estimated conservatively at 1410 copies/cell, a relatively high number. This work shows that, compared with whole animal, cultures have substantial LHRH stores, indicating maturation of synthetic activity and/or formation of new terminals in vitro. High LHRH mRNA copy number also suggests a high rate of peptide biosynthesis. Our analysis, demonstrating the dynamic potential of LHRH neurons, suggests that subtle changes in LHRH mRNA expression in all cells or a subpopulation can dramatically alter the LHRH system biosynthetic capacity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LH-RELEASING HORMONE (LHRH), a decapeptide secreted into the hypophysial portal circulation from nerve terminals in the median eminence, controls gonadal function by regulating LH and FSH release from the pituitary. Two distinctive features characterize regulated neuroendocrine secretion of LHRH from the median eminence. First, to maintain reproductive function, the release is pulsatile with interpulse intervals of 30–70 min and peak decays of 10–24 min in rats (1, 2, 3). Second, in mammals, ovulation and the preovulatory rise in circulating LH and FSH are preceded by a surge of LHRH (2, 4).

LHRH perikarya in rat [numbering approximately 1200; (5, 6)], although scattered in a continuum from the olfactory bulbs to the median eminence, are most highly localized at the level of the organum vasculosum lamina terminalis (OVLT). These neurons, most of which project to the median eminence (7, 8, 9), must transport their secretory product 4.0–6.4 mm [neonate (see Fig. 3Go); adult, (6)] to the site of exocytosis in an orchestrated manner. If, like other hypothalamic neuroendocrine secretion, LHRH has an axonal transport of approximately 5.8 mm/h (10) and newly synthesized secretory granules release their contents before older stores (reviewed in Refs. 11, 12), LHRH release at the median eminence should be tightly coupled to LHRH biosynthesis and gene expression in the perikarya (13, 14). Indeed, in hypothalamic slices, phorbol ester increased LHRH messenger RNA (mRNA) levels and peptide release (15) and, in vivo, elevated LHRH mRNA in medial preoptic area (mPOA) cell bodies was followed by increased LHRH peptide in the median eminence (16). Therefore, activity of the LHRH system, with a dispersed population remote from the site of neuroendocrine secretion, seemingly converges to produce coordinated release of LHRH.



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  		Figure 3. Distribution of LHRH within the POA/hypothalamus as measured in acute slices. Background panel, LHRH-immunostained parasagittal section of a neonatal rat POA/hypothalamus. Arrows indicate location of three immunopositive neurons. Rostral is to the left. Bar graph, Whole POA/hypothalamic regions were blocked, and 400 µm coronal sections were made using a McIlwain tissue slicer. The anatomical origin of acute slices 2–11 is indicated by the parasagittal tissue section (each bar = 400 µm). The tissue was acid extracted and processed for EIA. Results are expressed as mean ± SE of 22 pups from five litters. Effect of slice on LHRH amount was significant (P < 0.0001) using a one-way ANOVA.

 
An array of peptides (17, 18), neurotransmitters (19, 20, 21), and hormones (22, 23) appear to regulate LHRH neuroendocrine function transsynaptically. Intracellularly, regulation may be coordinated by transcriptional (24, 25, 26), posttranscriptional (25, 27, 28, 29), and posttranslational (13) mechanisms in vivo. Nevertheless, the manner in which extracellular and intracellular signals produce the unique secretory profile of LHRH neurons remains unclear. To investigate factors that regulate secretory function, neuropeptide processing, and gene expression in primary, differentiated LHRH neurons, yet circumvent difficulties arising from examination of the LHRH system in vivo, our laboratory utilizes organotypic slice explant cultures of postnatal rat POA/hypothalami (30, 31, 32, 33). Using a newly established competitive enzyme-linked immunosorbant assay (EIA), the present study quantitates LHRH peptide within, a) whole postnatal POA/hypothalami, b) rostral/caudal tissue blocks, c) freshly cut slices before culturing, and d) long-term organotypic slice explant cultures. Differences in LHRH peptide levels are discussed within the context of LHRH neuronal subpopulations and maturational effects. In addition, cellular LHRH mRNA copy number was estimated in slice explant cultures to understand more fully the relationship between mRNA levels, peptide content, and system dynamics.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
For tissue preparation and culturing, D-glucose, apotransferrin, putrescine, sodium selenite, bovine insulin, and L-ascorbic acid were purchased from Sigma Chemical Co. (St. Louis, MO). The supplier of Gey’s balanced salt solution, Eagle’s basal medium, Earle’s balanced salt solution, Ham’s F-12 nutrient mixture, L-glutamine, and PSN antibiotic mixture was Gibco BRL (Grand Island, NY). Horse serum, BSA, and chicken plasma were purchased from Biofluids, Inc. (Rockville, MD), Boehringer Mannheim (Indianapolis, IN), and Cocalico Biologicals, Inc. (Reamstown, PA), respectively.

For the EIA, LHRH acetate salt, D-[lys6]-LHRH, and p-nitrophenyl phosphate (PNPP) tablets were purchased from Sigma Chemical Co., Co. (St. Louis, MO). Pierce Chemical Co. (Rockford, IL) and Amersham (Arlington Heights, IL) supplied F(ab')2 fragment goat antirabbit IgG and streptavidin-alkaline phosphatase, respectively. MaxiSorp microwell plates were supplied by Nunc, Inc. (Naperville, IL). Rabbit antisera directed against conjugated rat LHRH, in equal volumes from three separate bleeds (SW1, SW2, and SW3), was affinity purified by Lofstrand Labs Limited (Gaithersburg, MD); this antisera immunocytochemically stains LHRH in perikarya and fibers (30). Biotinylated-LHRH was generated by reacting D-[Lys6]-LHRH in a 1:2 molar ratio with sulfosuccinimidyl-6-(biotinamido) hexanoate sodium salt (Vector Laboratories, Inc., Burlingame, CA) in 0.1 M NaHCO3, pH 8.5, and purified using HPLC. The stock concentration of LHRH peptide standard was quantitatively analyzed by Harvard Microchemistry Facility (Cambridge, MA).

Tissue preparation
Postnatal day 5–6 rat pups were weighed and, in accordance with NIH guidelines, brains were removed and whole POA/hypothalamic regions were blocked. For rostral/caudal analysis of the POA/hypothalamic region, the tissue was divided into rostral and caudal sections by cutting the tissue block at the optic chiasm. For acute slices, 400 µm coronal sections were made using a McIlwain tissue slicer, and separated in Gey’s balanced salt solution enriched with 0.5% glucose. Tissues were placed in 1.7 ml siliconized microtubes on ice containing 100–500 µl 0.1 N HCl, homogenized for 10 sec, frozen on dry ice, and stored at -80 C until assay. LHRH standards were prepared in 0.1 N HCl and kept on ice; striatal tissues, approximating sizes of 400 µm acute slices, rostral/caudal tissue blocks, or whole POA/hypothalamic blocks were added to 100–500 µl aliquots of standards and processed as described for unknowns. Standards and unknowns, before assay, were thawed, and microfuged for 5 min to remove precipitate. Supernatants were aliquoted into fresh, siliconized tubes and frozen at -20 C for assay at a later date. On the day of assay, standards and unknowns were neutralized and diluted (1.5–5x) in 1 M phosphate buffer to a final pH of 6.2–6.4 before use in the EIA.

POA/hypothalamic tissues were cultured as slice explants by the roller-tube method as previously described (30, 31, 32, 33). Briefly, cut slices were refrigerated for at least 1 h in Gey’s balanced salt solution enriched with glucose, adhered onto coverslips by a plasma/thrombin clot, placed in test tubes, and rotated in a roller drum. Explant cultures were also generated from striatal tissues and grown in parallel with the POA/hypothalamic slice cultures for EIA standards. For optimal thinning, cultures were initially grown in serum-containing media (32). Seven days before experimentation, cultures were transferred to defined media and fed every 2 days with defined media (31). On culture day 18, POA/hypothalamus explant cultures were extracted into 1.7 ml siliconized microtubes on ice containing 85–100 µl 0.1 N HCl, homogenized for 10 sec, frozen on dry ice, and stored at -80 C until assay. Striatal tissue explant cultures were extracted into 85–100 µl LHRH standards prepared in 0.1 N HCl and processed as described for unknowns. Standards and unknowns, before assay, were thawed, and microfuged for 5 min to remove precipitate. These standards and unknowns were processed as described for the fresh tissues before assay.

Enzyme-linked immunosorbant assay protocol and validation
Microwell plates were coated with 100 µl goat antirabbit IgG (1:500, overnight at 4 C) in 563 mM NaCO3 and 215 mM Na2CO3, pH 9.6. The following day, all procedures were performed at room temperature. After washing with wash buffer (0.2% Tween 20 in PBS, pH 7.4), 50 µl affinity purified anti-LHRH (1:600) in standard diluent (0.1% BSA in PBS, pH 7.4) were incubated in the wells for 2 h. After washing, 50 µl of neutralized and diluted standards and unknowns were incubated in wells for 2 h. Then, 50 µl of biotinylated-LHRH (1:1,000,000) was added to all wells and allowed to compete for anti-LHRH binding sites for 30 min. Wells were washed and 100 µl streptavidin-alkaline phosphatase (1:1000) was incubated for 30 min. After washing, 100 µl p-nitrophenyl phosphate (1 mg/ml) in 1 mM MgCl2, 16.2 mM NaHCO3, and 17 mM Na2CO3, pH 10, was aliquoted into each well, allowed to react for 50–90 min in the dark, and read at 405 nm in a MRX Microplate Reader (Dynatech Corp. Laboratories, Chantilly, VA). All samples were measured in duplicate or triplicate. Unknowns were calculated from standards plotted as a sigmoidal concentration-response curve by a nonlinear least squares fit (Revelation Software version 2, Dynatech Corp. Laboratories). Measurements from repeated assays of the same original tissue sample were meaned.

Preliminary experiments revealed shifts in the standard curve with freeze/thawing or the inclusion of protein or tissue, especially at low amounts of LHRH (data not shown). Therefore, standards and unknowns were treated identically with regard to freeze/thawing, tissue content, extraction volumes, and sample dilution factors. Furthermore, parallel preparation of the standards and unknowns controlled for extraction yield. The assay was then validated for each sample type (i.e. whole POA/hypothalami blocks, half rostral/caudal blocks, acute slices, and explant cultures). Mean responses (OD units) of duplicate or triplicate samples from the standards of 4–6 experiments were combined and plotted against LHRH concentration (pg/well); sigmoidal dose-response curves were best-fitted through each set of points to create calibration curves (Fig. 1Go). Response error relationships for each sample type were generated by plotting the SD in response of duplicate or triplicate samples against the response and then fitting the points with a straight line using linear regression analysis (not shown). Points within the concentration range of the assay were selected, and calibration curves were used to calculate the response at each selected point. Response error relationships were used to determine SD associated with each selected point. Concentration limits at each point (i.e. that which corresponds to response ± 1 SD) were determined and the concentration gradient [(c.g.) = (response + 1 SD) - (response - 1 SD)] was calculated. The error (SD) at each point was divided by its concentration gradient to calculate concentration error; each concentration error was divided by its corresponding concentration and multiplied by 100 to determine the percent coefficient of variation and plotted against concentration to generate a precision profile. In Table 1Go, for each data type, minimal detectable and maximal secure concentrations were calculated as 2.5x SD below the upper mean OD response plateau, and above the lower mean OD response plateau, respectively, on each calibration curve. Intraassay coefficients of variation were estimated as the average of mean coefficients of variation from each standard curve (n = 4–6). Coefficients of variation at 10 and 1000 pg/well (working limits of the assay) reflect interassay variation. EC50s are reported as geometric means with 95% confidence intervals (n = 4–6).



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  		Figure 1. Calibration curves of each sample type. Standards were prepared identically to unknowns with regard to freeze/thawing, tissue content, extraction volumes, and sample dilution factors. Control tissues, approximating acute slices, rostral/caudal tissue blocks, whole POA/hypothalamic blocks or explant cultures, were added to 100–500 µl aliquots of standards and processed for EIA. Mean responses (OD units) of duplicate or triplicate samples from the standards (7–10/curve) of n experiments were combined, plotted against LHRH concentration (pg/well), and best-fitted with sigmoidal dose-response curves. For whole POA/hypothalami blocks (n = 5), half rostral/caudal blocks (n = 6), acute slices (n = 6), and explant cultures (n = 4), R2 values of the fitted curves were 0.85, 0.96, 0.91, and 0.95, whereas Hill slopes were -1.22, -1.07, -1.02, and -1.11, respectively.

 

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  		Table 1. Characterization of enzyme-linked immunosorbant assay

 
Immunocytochemistry
Slices were fixed in 4% formaldehyde in PBS for 2 h, embedded in 10% gelatin, and cut (100 µm) on a vibratome (Lancer, St. Louis, MO). Cultures on coverslips were fixed with 4% formaldehyde in PBS for 1 h. After fixation, samples were washed with PBS, incubated for 1 h in 10% NGS/3% Triton X-100, washed several times in PBS, and incubated in LHRH antibody [1:2500, SW1; (30)] in PBS overnight at 4 C. The next day, vibratome sections or cultures were washed several times in PBS and incubated in biotinylated secondary antibody (1:500) in PBS with 0.3% Triton X-100 for 8 or 1 h, respectively. Vibratome sections or cultures were washed with PBS several times and processed for avidin-biotin-horseradish peroxidase/3'3-diaminobenzidine histochemistry as described previously (33, 34).

In situ hybridization histochemistry and film analysis
Three days before experimentation, slice explant culture medium was supplemented with 1 µM tetrodotoxin, a Na+ conductance inhibitor, to suppress transsynaptic interactions and equilibrate LHRH mRNA baseline levels (32). ISHH was performed as previously described (33, 34). Briefly, slice explants were fixed in 4% formaldehyde, rinsed in PBS, permeabilized in 0.3% Triton-X 100/0.05 M EDTA/0.1 M Tris buffer, rinsed in Tris buffer, washed in 0.25% acetic anhydride/0.1 M triethanolamine hydrochloride-0.9% NaCl, rinsed in 2x SSC, dehydrated through ethanol, delipidated in chloroform, rinsed in ethanol, and air dried. A 48 oligonucleotide probe (5 pmol), complementary to the coding region of rat LHRH precursor within exon 2 (bases 102–149; (35) was 3'end-labeled with [S35]dATP (33). Frozen rat brain sections were used as positive controls. Labeled probe (500,000 cpm) was applied to each culture in 25 µl of hybridization buffer (33) and cultures were hybridized overnight in humid chambers at 37 C. The following day, cultures were rinsed in 1x SSC/65 mM dithiothreitol (DTT), washed at high stringency (33), rinsed in 1x SSC at room temperature, rinsed briefly in water, dehydrated in ethanol, dried, and placed against Hyperfilm-ßmax (Amersham, Arlington Heights, IL) with carbon-14 standards (American Radiolabeled Chemicals, Inc., St. Louis, MO) for 68–71 h. Optical density measurements were not saturated using these exposure conditions. Autoradiographs were digitized, and optical density readings were calibrated using carbon-14 standards. Total area (mm2) of optical density measurements 15% above culture background was highlighted. For explant cultures 2–5, areas equaled 0.044, 0.094, 0.072, and 0.031 mm2, respectively. Mean optical density measurements within the highlighted area, calibrated to carbon-14 standards (µCi/g), were recorded. Measurements were converted to dpm/mm2 using the linear relationship y = 24x + 1.02, where y is [14C] in brain paste standards (dpm/mm2) and x is [14C] in carbon-14 calibration standards (36). Because a linear relationship (1:1) exists between 35S concentration and 14C-labeled brain paste standards under the autoradiographic conditions employed (36), the amount of 35S per culture (dpm/mm2) was estimated. For explant cultures 2–5, the converted, mean optical density measurements equaled 21.20, 21.70, 21.02, and 18.02 dpm/mm2, respectively. Mean radiolabeling (dpm/mm2) multiplied by total radiolabeled area (mm2) produced radiolabeling per culture (dpm). Using 14000 Ci/mmol as the probe specific activity (33) and assuming a hybridization efficiency of 56% (37), LHRH mRNA copy number per culture was estimated.

Statistical analyses
For acute slice and explant culture data LHRH measurements, some unknowns fell below the lower working limit of the assay (10 pg/well). Upon data analysis, our results showed that inclusion of zero readings into the data at these points unfairly decreased population means relative to that of samples with measurements that routinely fell within the working limits of the assay; conversely, omitting this data artificially inflated population means. Therefore, readings below the working limit of the assay were assigned a weighted value between the highest population mean possible (assuming 10 pg LHRH/well) and lowest population mean possible (assuming 0.0 pg LHRH/well) dependent on the fraction of readings within that population that fell within working limits of the assay. The fraction of readings observed within the working assay range varied between 0.32 (acute slice, s2) and 0.95 (explant culture, s3). Statistical comparisons of data were calculated with StatView version 4.1 (Abacus Concepts, Inc., Berkeley, CA) and GraphPad Prism version 2.01 (GraphPad Software, Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A sensitive enzyme-linked immunosorbant assay was developed to quantitate LHRH peptide from mammalian POA/hypothalamic tissues and slice explant cultures (Fig. 1Go and Table 1Go). Whole POA/hypothalamic tissue blocks from postnatal day 5 and 6 rat pups were assayed to determine the total amount of LHRH present and ascertain effects of pup weight, sex, and age on LHRH content, if any (38). On average, postnatal day 5 and 6 pups contained 1250 pg of LHRH and weighed 12 g (Table 2Go). No significant differences (P > 0.05, Student’s t test) were observed between male and female pups or between postnatal day 5 and 6 pups with respect to LHRH content, pup weight, or LHRH content/pup weight. Thereafter, data generated from postnatal day 5 and 6 pups were combined irrespective of pup sex or weight.


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  		Table 2. POA/hypothalamic LHRH content and pup weight

 
LHRH cell bodies reside in the POA and anterior hypothalamus and project to the median eminence (39). To determine the distribution of LHRH within POA/hypothalamus, whole tissue blocks were halved into rostral/caudal sections at the optic chiasm and assayed for LHRH content. Amounts of LHRH contained in these areas differed significantly (Fig. 2Go). The rostral portion of the POA/hypothalamus, location of the majority of LHRH cell bodies in postnatal rat (6), contained only 27.5% of the total LHRH measured. Therefore, nearly three-fourths of all LHRH appears to reside within projections and terminals of LHRH neurons. The sum of LHRH in the rostral and caudal sections was less than that measured in whole POA/hypothalamic tissue blocks (Table 2Go). Because this may have resulted from additional loss of LHRH by leakage from the halved tissue and/or reduced recovery from sample tissue processing, we believe that the quantity measured in whole POA/hypothalamus is a better estimate of amount of LHRH present in the whole animal. Extrapolating from the whole POA/hypothalamus tissue block measurement (Table 2Go), the rostral section contains, on average, 343 pg of LHRH while the caudal contains 906 pg.



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  		Figure 2. Caudal POA/hypothalamic tissue blocks contain more LHRH than rostral blocks. Whole POA/hypothalamic regions were blocked, divided into rostral and caudal sections at the optic chiasm, acid extracted, and assayed for LHRH content by EIA. Results are expressed as mean ± SE of 18 pups from four litters. Significance, calculated using the t test for paired data (P < 0.0001), is indicated by an asterisk.

 
To further define the distribution of LHRH, 400 µm coronal sections through the POA/hypothalamus were examined. These acute slices are comparable to those used to create slice explant cultures from this region (30, 31, 33, 34). In Fig. 3Go, distribution of LHRH measured within the POA/hypothalamus is shown. These data confirm that the majority of LHRH resides in the caudal portion of the hypothalamus at the location of LHRH nerve terminals. Cell number counts and LHRH peptide measurements of slices 2–5, extrapolated from the whole POA/hypothalamus tissue block measurement, are listed in Table 3Go.


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  		Table 3. LHRH content of POA/hypothalamic slices

 
LHRH content of long-term slice explant cultures generated from postnatal day 5 and 6 rat POA/hypothalami was assayed. Cultures were maintained for 7 days in defined medium before acid extraction on day 18. Of the four slice explant cultures, s3, on average, contained the highest amount of LHRH peptide (37.6% of total; Table 3Go). Likewise, s3 explant cultures and acute slices contain the greatest number of LHRH neurons (40.4 and 45.6% of totals, respectively; Table 3Go). These data also show that while only approximately 16% of the originally cultured neurons survive, a substantial quantity of LHRH peptide is present in vitro. Direct comparison between acute slices, explant cultures, and whole animal values reveals an appreciable increase in LHRH content per cell after culturing (Fig. 4Go).



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  		Figure 4. LHRH peptide per cell: acute slices vs. slice explant cultures. LHRH measured per acute slice (left panel) or slice explant culture (right panel) as determined by EIA was divided by the number of immunopositive LHRH neurons/acute slice or slice explant culture, respectively (Table 3Go). Calculations are expressed as means. The dotted line (y = 1.04) indicates the whole animal estimate of LHRH peptide/cell, based upon an approximated population of 1200 LHRH neurons (5 6 ) and a measurement of 1249 pg LHRH (Table 2Go) in the postnatal rat POA/hypothalamus.

 
To understand more fully the relationship between peptide levels and gene expression, the amount of LHRH mRNA per slice explant culture was estimated using ISHH, autoradiography, and quantitative film analysis (36). Because tetrodotoxin treatment of organotypic cultures reduces variability of LHRH mRNA levels between single cells and therefore results in a more normal distribution of ISHH measurements (32), explant cultures were treated with 1 µM tetrodotoxin before fixing. Examples of autoradiographic films exposed to slice explants processed for ISHH using a radiolabeled synthetic deoxynucleotide antisense probe for LHRH mRNA are shown in Fig. 5Go. Note the distribution of LHRH neurons in slice explant cultures mirrors that of LHRH-immunostained neurons in vivo. In comparison to the other explants, s3 cultures, in addition to containing more LHRH neurons and peptide (Table 3Go), expressed the highest amount of phenotypic mRNA (Table 4Go).



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  		Figure 5. LHRH immunostained neurons in POA/hypothalamic acute slices and autoradiographic images of slice explant cultures processed for LHRH mRNA ISHH. Left panels, Coronal views of representative vibratome sections (100 µm) from slices 2–5 immunostained for LHRH. These sections show tissue used for explant cultures on the day of culturing. Arrows indicate individual LHRH neurons. Bar, 250 µm. Right panels, Autoradiographic images of slice explant cultures 2–5 maintained for 18 days in culture and processed for LHRH mRNA using a synthetic, radiolabeled deoxynucleotide antisense probe. Arrowheads indicate images of individual LHRH neurons. Bar, 250 µm. Note the distribution of LHRH neurons in slice explant culture mirrors that of LHRH-immunostained neurons in vivo.

 

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  		Table 4. Estimates of LHRH mRNA copy number

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present investigation quantitates LHRH peptide from POA/hypothalamic tissues in vivo and slice explant cultures maintained in vitro using an enzyme-linked immunosorbant assay. We report that postnatal day 5 and 6 rat pups contain (independent of pup weight, sex, and age) approximately 1250 pg of LHRH. Only one-quarter of LHRH peptide was measured within tissue rostral to the optic chiasm, site of the majority of LHRH cell bodies. About three-quarters of peptide was measured caudally in the region of LHRH terminals, indicating that in the neonate in vivo, a large repository of peptide is positioned at or near release sites. Assuming a static, homogenous population of 1200 LHRH neurons (6), each neuron in the neonatal animal is responsible for approximately 1.0 pg of LHRH. In contrast, LHRH neurons in organotypic slice explant cultures derived from neonatal tissues averaged 2 pg of peptide/cell. Using in situ hybridization histochemistry with autoradiographic film analysis, LHRH mRNA copy number in explant cultures was estimated conservatively at approximately 1410 copies/cell, a relatively high copy number for a mRNA (40, 41). Therefore, neonatal LHRH neurons maintained for 18 days in vitro possess abundant template for a high rate of peptide synthesis in the perikarya. The present findings in organotypic slice explants suggest maturation of LHRH peptide synthetic activity and/or formation of new LHRH terminals allowing for accumulation of peptide in culture.

Within acute slices 2–5, the apparently low cellular LHRH content (<=1 pg/cell) reflects the finding that the majority of peptide measured resides in projections and terminals in tissues caudal to these slices. However, between individual acute slices 2–5, a disparity exists in the average peptide/cell. Although acute slice 3 contains the OVLT, greatest number of LHRH cell bodies, and largest amount of peptide, it has the lowest average LHRH peptide/cell (Fig. 4Go). Acute slice 3 does contain fewer tracts from rostral cell bodies than do slices 4 and 5 (42); however, estimations of peptide within those projections cannot account for the 2- to 4-fold increase in LHRH peptide/cell observed in acute slices 4 and 5 over that observed in slice 3. Furthermore, slice 2 neurons, which likely contain fewer tracts than slices 3–5 (42), have over 50% more LHRH on a cellular basis than slice 3 neurons. Ultrastructural analysis of LHRH-immunolabeled tissues from postnatal day 8 rats revealed secretory granules in median eminence and OVLT terminals, as well as in LHRH perikarya (43). Immunohistochemical staining of LHRH nerve endings in the OVLT, but not the median eminence, appeared to plateau 1–2 weeks after birth (44). Therefore, LHRH neurons at the OVLT may supply a greater number of terminals, release a larger proportion, and retain a lesser amount of peptide compared with LHRH neurons in other regions, which could account for the lower LHRH peptide/cell measured in these studies in slice 3. Alternatively, neurons at the level of the OVLT could synthesize LHRH at a relatively lower rate; however, subpopulations restricted to neonatal OVLT have not been reported (5, 45).

The total amount of LHRH measured within the four slice explant cultures is approximately 59% of that found in the original tissues plated (see Table 4Go). However, after considering percent LHRH cell survival (~16%), a gain in total LHRH content occurs during the 18 day culture period. The culture process itself may precipitate such changes, perhaps as the result of severing efferents which normally decrease synthesis and/or increase secretion of LHRH. Alternatively, the apparent increase subsequent to culturing could result from normal maturation of postnatal tissues in vitro. Hypothalamic LHRH peptide content increases steadily between birth [50–100 pg; (46, 47)] and adulthood [6000–8000 pg; (47, 48)] in rat. Extrapolation of total LHRH measured in neonatal tissues maintained for 18 days in vitro (244.8 pg), accounting for the fraction of total cells cultured and cell survival, equals 2350 pg. This value approaches LHRH measured in postnatal day 23 hypothalami [approximately 2500–3000 pg; (47)], suggesting maturation of the biosynthetic capacity of LHRH neurons in culture and that this maturational process is an intrinsic property of these neurons.

On culture day 18, slice explant cultures have a mean LHRH content of >=1 pg/cell. However, as seen in acute slices, disparity exists in the average peptide/cell between explant cultures. Slice explant 3 contains 1.8-fold as much LHRH/cell, whereas slices 4 and 5 have nearly 3-fold the peptide. Because these differences are observed under baseline, serum-free conditions, intrinsic differences may exist in the biosynthetic or secretory activity of these LHRH neurons. Indeed, LHRH terminals in acute tissues at the level of the OVLT are likely maintained throughout the culturing process and may facilitate additional secretion from these explants. Certainly the chemo- and cytoarchitecture of the slices differ, and transsynaptic influences, such as those evoked by norepinephrine (21), opiates (49), or estrogen (50) may underlie the variations in LHRH/cell between slice explant cultures. In support of this, only neurons in slice culture 3 decreased LHRH mRNA levels in response to estrogen, indicating the presence of estrogen-sensitive, inhibitory, presynaptic efferents (31). Evidence from adult rat in vivo also indicates the presence of LHRH subpopulations distributed within regions of the POA/hypothalamus. The neuropil surrounding POA LHRH neurons appears to have fewer synaptic inputs than that in the anterior hypothalamus (13) and differential immunocytochemical labeling of the POA/hypothalamus detected the highest degree of heterogenous biosynthetic activity between LHRH neurons residing within the OVLT (51). In the proestrous rat, increased numbers of neurons expressing detectable levels of LHRH mRNA (52) and protein (51) further indicate heterogenous biosynthetic activity in this region. Likewise, subpopulations of LHRH neurons within the region of the OVLT coexpress the immediate early gene c-fos (53) and galanin (54, 55, 56) during proestrous. Our results using organotypic slice explant cultures of neonatal hypothalami may reflect the presence of these LHRH subpopulations observed in vivo, especially if the tissues experience a degree of maturation in vitro.

Gene expression and protein levels are thought to be coupled (57, 58, 59, 60), a relationship that has been observed in the LHRH system (15, 16, 61). In vivo, LHRH secretion occurs at sites spatially discrete from peptide synthesis in perikarya (7, 8, 9) and consists only of a small fraction of total peptide stores (48, 62, 63). With axonal transport able to replenish readily releasable peptide stores after one hourly pulse [5.8 mm/h; (10)], LHRH gene expression may be integral to ongoing neurosecretory activity, if like other neuroendocrine secretion, newly synthesized secretory granules release their contents before older stores (reviewed in Refs. 11, 12). Recently, we reported that primary LHRH neurons maintained in organotypic culture, in addition to exhibiting a LHRH mRNA stabilization mechanism, have a rapid LHRH mRNA turnover component [<= 15 min; (33)]. As indicated by mathematical modeling of neuropeptide homeostasis (64), changes in LHRH transcription can therefore be quickly reflected in gene expression levels. To elucidate further the relationship between LHRH gene expression and protein levels, we have included an examination of baseline, unstimulated LHRH mRNA in addition to quantification of LHRH peptide content in slice explant cultures. An average mRNA is present in <= 50 copies within a cell, assuming an average cell expresses approximately 10,000 different mRNAs and contains a total of 250,000–500,000 mRNA molecules (40, 41). A high copy number mRNA, such as that for the housekeeping protein actin, may be present at >= 1000 copies (37, 65). Using ISHH, autoradiography, and quantitative film analysis in explant cultures 2–5, estimates of LHRH mRNA copies/cell ranged from 1040–1860, indicating that LHRH is a high copy mRNA. These values, however, likely underestimate actual copy number because hybridization efficiency used in our calculations was based upon that of dissociated cells in culture (37); slice explant cultures containing LHRH neurons are typically several cells thick (30), thus reducing access and penetration of probe relative to that of monolayer cell culture ISHH. Using a quantitative ribonuclease protection assay (24), previous estimates of LHRH mRNA copy number per adult rat POA/hypothalamus were approximately 8,000,000. Assuming 800-1200 LHRH neurons in this brain region, the average neuron in the female adult also expresses a high LHRH mRNA copy number: 6700–10000 copies. Furthermore, examining single cell complementary DNA libraries derived from primary LHRH neurons of cultured embryonic mouse tissues, our laboratory observed 4,000–30,000 copies/cell (66). Thus, data generated by three dissimilar methods (ISHH with autoradiography, ribonuclease protection assay analysis, and complementary DNA library screening) show that LHRH is a high copy number message in primary neurons. In addition, this characteristic was observed in three disparate tissue types (postnatal rat POA/hypothalamic slice explant cultures, in vivo adult rat tissues, and embryonic mouse nasal explant cultures).

Our examination in primary LHRH neurons generated molecular estimates of the defining parameters of neuroendocrine cell phenotype, i.e. the neurosecretory peptide product and its mRNA transcript. With this information in hand, we calculated the apparent, yet seldom enumerated, relationship between gene expression and peptide levels; furthermore, we contemplated the importance of this relationship with respect to function of the LHRH system as a whole. In mammalian expression systems, with combinations of vectors, promoters, and cells designed to maximize protein production, amplification can range from 1 x 105–1 x 108 protein molecules/day/plasmid, with copy numbers typically below 200 (for reviews see Refs. 67, 68). We calculated that LHRH neurons average 0.5–1.47 x 109 peptide molecules/cell and 1040–1860 mRNA copies/cell in explant cultures, suggesting an amplification of 4.8 x 105-1.1 x 106. Using LHRH mRNA copy number and peptide levels for the POA/hypothalamus of adult rat (24), we calculate an in vivo peptide/mRNA amplification of 2.89 - 4.3 x 105. The somewhat higher amplification calculated from slice explant culture data may result from underestimation of LHRH mRNA copy number by ISHH/autoradiography, reduction of baseline mRNA levels by tetrodotoxin, or reflect age related biosynthetic differences. Overall, the degree of amplification is staggering, and demonstrates how subtle changes in the expression level of all LHRH neurons, or even a subpopulation of LHRH mRNA expressing cells, can result in dramatic changes in peptide levels. Thus, the situation requiring perhaps the largest increase in LHRH peptide (preparation for a LHRH surge) could easily be accommodated by this degree of amplification, assuming that release consisted of a small percent of total POA/hypothalamic content [rat total, 6–8 ng; surge, 126 pg; (48)]. For example, if 4.5 x 105 LHRH peptides were synthesized from 1 mRNA, and an average LHRH neuron expressed 1400 copies of mRNA, then the total output of 100 LHRH neurons could supply the required peptide for the surge [6.5 x 1010 (65 billion) LHRH peptide molecules]. This analysis demonstrates the dynamic potential of the LHRH system. Thus, by amplification, small changes in numbers of detectable LHRH neurons (54, 69) or subtle changes in levels of gene expression in all LHRH cells (25, 70), could accommodate the peptide requirements of the entire LHRH system.

In summary, slice explants experience a net gain in LHRH content during culture and contain as much as 100 pg of LHRH after 18 days in vitro. Apparent population differences between anatomical regions are maintained in culture, thus enabling examination of these system characteristics in vitro. In addition to quantifying LHRH peptide, the present investigation estimated mRNA copy number in LHRH neurons. Our findings indicate that LHRH neurons express a high copy number and this translates into a high biosynthetic capacity. Additional analyzes are needed to determine if the increase in LHRH peptide levels associated with maturation (46, 47, 71) are paralleled by changes in LHRH mRNA copy number. Overall, LHRH neurons display a unique biosynthetic profile. A high percentage of LHRH RNA exists as nuclear transcripts [30–40%; (27, 72)], LHRH mRNA exhibits a rapid turnover and stabilization component (33), and posttranscriptional mechanisms seem biologically important (25, 28). Continued investigation of transcription, synthesis, transport, and release properties of LHRH neurons at the molecular, cellular, and system levels will be required to elucidate the basis of pulsatility and surges in LHRH neurons.


   Acknowledgments
 
We thank Dr. Howard Jaffe for purifying biotinylated-LHRH by high-performance liquid chromatography and Drs. Harold Gainer and W. Scott Young III for helpful comments on this manuscript.

Received June 3, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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