Hypertrophy of Gonadotropin Releasing Hormone- Containing Neurons after Castration in the Teleost, Haplochromis burtoni

Richard C. Francis,',* Ben Jacobson,2 John C. Wingfield,3 and Russell D. Fernald'

'Neuroscience Program and Department of Psychology, Stanford University, Stanford, California 94305; 'Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403; and 3Department of Zoology, University of Washington, Seattle, Washington 981 95

SUMMARY

In the African cichlid fish, Haplochrornis burtoni, males are either territorial or nonterritorial. Territorial males suppress reproductive function in the nonterritorial males, and have larger gonads and larger gonadotropin- releasing hormone- (GnRH ) containing neurons in the preoptic area (POA). We describe an experiment de- signed to establish the causal relationship between large GnRH neurons and large testes in these males by deter- mining the feedback effects of gonadal sex steroids on the GnRH neurons. Territorial males were either cas- trated or sham-operated, 4 weeks after which they were sacrificed. Circulating steroid levels were measured, and the GnRH-containing neurons were visualized by stain- ing sagittal sections of the brains with an antibody to

salmon GnRH. The soma areas of antibody-stained neu- rons were measured with a computer-aided imaging sys- tem. Completely castrated males had markedly reduced levels of circulating sex steroids 11 I-ketotestosterone ( I I K T ) and testosterone (T)] , as well as 17P-estradiol ( E2). POA GnRH neurons in castrates showed a signifi- cant increase in mean soma size relative to the intact territorial males. Hence, in mature animals, gonadal ste- roids act as a brake on the growth of GnRH-containing neurons, and gonadal products are not responsible for the large GnRH neurons characteristic of territorial males. Keywords: GnRH, terminal nerve, preoptic area, gonad- ectomy, testosterone.

(0 1992 John Wiley & Sons, Inr.

INTRODUCTION

In vertebrates, gonadotropin-releasing hormone ( GnRH) regulates the secretion of gonadotropin (GtH) from the anterior pituitary gland. GtH, in turn, controls the secretion of gonadal steroid hor- mones including estradiol and testosterone. In- creased levels of these hormones down-regulate GtH. Whether this negative feedback action occurs in the hypothalamus via the GnRH system, or else- where, is a matter of some dispute (reviewed in Kalra and Kalra, 1989). For example, following

Received June 11 , 1992; accepted July 24, 1992 Journal of Neurobiology. Vol. 23, No. 8. pp. 1084-1093 (1992) 0 I992 John Wiley & Sons, Inc. CCC 0022-3034/92/08 1084- 10

I To whom correspondence should be addressed.

1084

gonadectomy in rats, circulating GnRH levels are reported to increase (Root, Reiter, Duckett, and Sweetland, 1975; Gross, 1980; Kalra, Kalra, and Mitchell, 1977; Roselli, Kelly, and Ronnekleiv, 1990), decrease (Yanaihara et al., 1975), or not change (Chowers and McCann, 1965). Similarly, in gonadectomized rats, immunoreactive-GnRH cell number has been reported to increase (King et al., 1987), decrease (Shivers, Harlan, and Morrell, and Pfaff, 1983), or not change (Witkin, 1989; Ro- selli et al., 1990). Finally, GnRH mRNA levels in gonadectomized rats are reported to increase (Tor- anzo et al., 1989; Zoeller, Seeburg, and Young, 1988), decrease (Pfaff, 1986; Rothfeld, Hejtman- cik, Conn, and Pfaff, 1987; Park, Park, Cho, and Kim, 1988; Roberts, Dutlow, Jakubowski, Blum, and Millar, 1989), or not change (Kelly et al., 1989; Wiemann, Clifton, and Steiner, 1990).

C

B

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Figure 1 Parasagittal section through brain of Huplochrumis bzirtoni. AC = anterior commis- sure: CV = cerebral valvula; H = habenula: MB = mammillary body: OLT = olfactory tract; ON = optic nerve; OT = optic tectum: PC = posterior commissure; POA = preoptic area: PT = pallium of telencephalon: VS = vascular sac. For more complete description of E-I. biirfoni brain anatomy, see Fernald and Shelton, 1985.

Interpreting these disparate results is difficult for two reasons. First, neurons in the hypothala- mus expressing GnRH probably constitute a het-

erogeneous population of which many do not proj- ect to the pituitary. Thus changes observed in those GnRH-containing neurons regulating leutenizing

1086 Francis et ul

Table 1 Levels of Steroids'

Mean (k S.E.) Postoperative Circulating

Group T KT E, Sham

Partial

Complete

( n = 4) 36.69 (k2.63) 3.56 (k0.60) 14.85 (k4.34)

( n = 3 ) 18.82 (23.59) 2.93 (k1.01) 5.49 (t2.84)

( n = 3) 3.62 (k2.02) 0.34 (k0.05) 2.58 (k0.67)

' Nanograms per milliliter.

hormone (LH) secretion may be obscured by non- parallel responses in GnRH-containing neurons which do not affect LH levels. Because GnRH sub- serves a variety of functions other than gonadotro- pin regulation, its level may not accurately reflect its activity with respect to the brain-pituitary- gonadal axis. Second, the conflicting results re- ported may have arisen from comparing animals which were not in comparable physiological states or had genetic or developmental differences. To understand how androgen feedback affects the hy- pothalamus, analysis of GnRH neurons directly re- sponsible for LH regulation is necessary.

Teleost fish offer several advantages for under- standing the relationship between androgens and GnRH. GnRH neurons in teleosts are restricted in number and confined to two or three distinct loci, in contrast to a somewhat diffuse distribution within the mammalian hypothalamus. Moreover, a large proportion of those neurons in the preoptic area (POA) project directly to pituitary gonado- tropes (Schreibman, Halpern, Goos, and Margolis- Kazan, 1979; Munz, Stumpf, and Jennes, 1981), there being no hypophyseal portal system in te- leosts.

To date, however, there have been relatively few studies on the feedback interactions of gonadal ste- roids and GnRH in teleosts. In a study on catfish, Habibi, De Leeuw, Nahorniak, Goos, and Peter ( 1989) demonstrated that GnRH receptor levels in the pituitary are regulated by gonadal steroids. Kobayashi and Stacey ( 1990) reported that ovari- ectomized goldfish had significantly higher levels of GtH than their intact counterparts and that these levels were reduced following testosterone and estradiol replacement. Neither study investi- gated the role of hypothalamic GnRH neurons in this regulation. Schreibman, Margolis-Nunno, Halpern-Sebold, Goos, and Perlman (1986) did find an effect of androgen levels on GnRH biosyn- thesis in Xiphophorus maculatus. In this species,

the timing of the appearance of GnRH-containing neurons in three distinct brain nuclei is closely re- lated to the onset of maturation ( Halpern-Sebold and'schreibman, 1983). Intact juveniles experi- enced accelerated maturation after receiving an- drogen treatments, presumably as a result of effects on GnRH levels (Schreibman et al. 1986).

In the African cichlid, Haplochromis burtoni, GnRH neurons in the POA also play a central role in the timing of maturation (Davis and Fernald, 1990; Francis et al., submitted). These neurons are magnocellular and hence easily located, counted, and measured. In addition, they exhibit pro- nounced plasticity in soma size (Davis and Fer- nald, 1990; Francis et al., submitted), providing a useful assay for the effects of gonadal steroids.

Males of this species fall into two easily distin- guishable categories: territorial and nonterritorial. Territorial males exhibit significantly larger soma sizes in their preoptic-immunoreactive GnRH (irGnRH) neurons and larger testes than their nonterritorial counterparts, which they aggres- sively dominate (Davis and Fernald, 1990; Francis et al., submitted). Upon losing their territories, these males exhibit a reduction in both mean soma size of POA irGnRH neurons and size of testes (Francis et al., submitted). Conversely, when non- territorial males acquire a territory, they experi- ence growth in both the testes and in the size of their POA-ir GnRH neurons. This plasticity of neu- ronal soma size is not observed in the terminal nerve (TN) population of irGnRH neurons of the ventral telencephalon. Hence, it is not a general property of GnRH neurons in this species, but rather confined to the POA population, which is the major source of fibers to the pituitary.

In this study we sought to determine whether the POA-irGnRH neurons also exhibit plastic changes in soma size in response to alterations in the endocrine environment, and if so, could be then used to assay the effects of androgen feedback at the level of the hypothalamus. Ultimately, we seek to understand cause and effect with respect to large gonads and large POA-irGnRH neurons in the territorial males; whether, that is, testicular growth or enlargement of POA-irGnRH neurons is causally primary.

MATERIALS AND METHODS

Subjects

All fish in these experiments were derived from wild- caught stock from Lake Tanganyika (Fernald and Hir-

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Figure 3 Mean cross-sectional area of i rGnRH neurons in POA o f intact (shams SYMBOL) and completely castrated (SYMBOL) territorial males plotted as a function ofstandard length. Each data point represents between SO and 100 measurements on an animal.

ata, 1977a) and reared in the laboratory under con- trolled conditions. Fish wcre maintained in seminatural environments that emulate essential features of the natu- ral Lake Tanganyika habitat (Fernald and Hirata. 1977b): temperatureof29OC, pH 8.0and 12: 12 h light/ dark cycle with full-spectrum lights. Fish were fed once daily on cichlid chow (Wardley).

Each community tank (249 I: 9 I X 6 I X 45 cm) held three to five territorial males, five 01- more nonterritorial males. and 10-15 mature females at various reproduc- tive stages. Gravel covered the bottom to allow digging. an important component of reproductive behavior (Fer- nald, 1977). Terracotta pot shards served as spawning sites and territorial landmarks.

General Protocol

Only territorial males were used in this study. All sub- jects were removed from their community tanks and placed in 41 1 ( 5 1 X 26 X 30 cm) aquaria in one of two treatment groups: sham-operated or gonadectomized. Though physically isolated, subjects could and did inter- act visually. They remained in the 41-1 tanks for 1 week for behavioral observations (see below), after which the fish were either sham-operated or gonadectomized.

Prior to the operation the fish were anesthetized with trieaine methanosulfate ( I : 10,000; MS-222). Anesthesia was maintained by occasional administration of MS-222 to the gills throughout the operation. An incision was made beginning anterior to the vent and extending to the clavicle. In half the fish the gonads were removed (cas-

trates). whereas in the others the incision was sewn shut immediately after it was made (shams). Following the operation, fish were revived and returned to their individ- ual aquaria and treated with antibiotics (Kanamyacin sulphate and nitrofurazone, Spectrogram).

One month after the operation. fish were sacrificed. weighed, standard length measured and the body cavity checked for residual gonads which were weighed ifpres- ent. The brains were removed and processed for anti- body staining (see below).

For data analysis. gonadectomized males were sorted into two categories based on residual testes found at sacri- fice: those for which the gonadectomy was complete and those for which the gonadectomy was incomplete.

Histology

Brains were immersion-fixed 4-6 h in 4% paraformalde- hyde buffered with sodium phosphate and then cryopro- tected by immersion in 30% sucrose overnight. Tissue was sectioned in the sagittal plane on a cryostat at a thickness of 40 pm. Immunoreactive-GnRH cells were labeled with a polydonal antibody (GF-5, kindly pro- vided by Dr. N. Shenvood), which has a high affinity for salmon GnRH, the dominant form in teleosts, and a much lower affinity for chicken GnRH-I1 (Shenvood, Doroshov, and Lance, 199 1 ). Antibody-labeled neurons were visualized through a diaminobenzadine (DAB) re- action (see Davis and Fernald 1990, for details).

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Gonadosomatic Index Figure 4 [ = {gonad wt/body wt ) * 1001 for all subjects. The best-fit regression line is ( J *

Mean soma area of i rCnRH neurons plotted as a function of gonadosomatic index 409.23

~ 76.814 [log (x ) ] , Y’ = 0.529).

Cell Measurements

Figure I depicts the POA in a parasagittal section of a male If. hzivtoni brain. We measured the soma area of i rCnRH neurons in the POA with the aid of computer- captured video images (Image Measure) from a micro- scope (Zeiss, 600X). A thresholding function was used to delineate the cell body. Soma sizes were measured only for neurons in which the nucleus was visible in the plane of section and for which the entire perimeter could be discerned under the microscope. The treatment group of the subjects was unknown to the person doing the measurements.

Radioimmunoassay

To measure the effect of gonadectomy on circulating an- drogen levels, blood samples were obtained after castra- tion or sham operations from some of the subjects. Blood ( 50-200 P I ) was collected in heparinized capillary tubes from the first branchial artery. The blood was cen- trifuged for 5 min at 14,000 rpm to isolate the plasma, which was then stored at -80°C until assayed. Only sub- jects from which we obtained at least 50 1 of plasma were assayed.

The sex steroid hormones, testosterone and 1 l-oxo- testosterone ( = 1 1 ketotestosterone), as well as 170- estradiol were measured by radioimmunoassay after pu- rification on diatomaceous earth-glycol-water columns.

The procedures generally follow Wingfield and Farner ( 1975) and Ball and Wingfield ( 1987) with the following modifications. Approximately 2000 cpm of respective tritiated steroids were incubated with plasma samples for at least 1 h at 4°C and then extracted with 5 ml of redis- tilled dichlormethane. The organic phase was aspirated off and dried under a stream of nitrogen at 40°C. Dried extracts were dissolved in ( 2 X ) 0.5-ml aliquots of redis- tilled ethyl acetate in iso-octane and transferred to the top of diatomaceous earth: ethylene glycol: propylene glycol: water microcolumns (6: I : l : I , w / v / v / v ) . Steroid fractions were then eluted from the columns using in- creasing concentrations of ethyl acetatc in iso-octane. Testosterone was eluted in 4 ml of 10% estradiol in 4 ml of 20%, and 1 1-KT in 4 ml of 30% ethyl acetate in iso-oc- tane. Partially purified fractions were dried under a stream of nitrogen and reconstituted in phosphate-buf- fered saline prior to radioimmunoassay. Assay, separa- tion of bound and free hormones, and assay perfor- mance criteria were as described previously ( Wingfield and Farner, 1975; Ball and Wingfield, 1987).

The assay reliability after chromatography were within the limits given by Wingfield and Farner ( 1975) for testosterone. The 1 1 -ketotestosterone assay reliabil- ity criteria were as follows: recoveries after extraction and chromatography averaged 86.3% (range: 76%- loo%), and assay blanks for this steroid were below the lowest dose on the standard curve. Intraassay variation determined on standards taken through the entire ex-

traction and chromatography procedure was 8.7% for a low dose (500 pg) and 3.8% for a high dose ( 1000 pg). Interassay variation was 5.4% and accuracy of measure- ment based on assays of 500 pg standards after adjust- ment for recovery was 929%.

RESULTS

Individuals with complete gonadectomies had markedly lower circulating levels of all steroids as- sayed than did sham-operated fish (Table 1 ; Mann-Whitney U = 12, n, = 4, 12, = 3, p = 0.05). Although animals with incomplete gonadectomies exhibited generally lower levels of both androgens and estradiol than their sham-operated counter- parts, these differences were not significant. In indi- viduals for which no trace of gonad could be found 4 weeks after the operation, the two androgens and estradiol were not completely eliminated. The source of the residual circulating steroids was pre- sumably the interrenal (adrenal) tissue (Idler and Truscott, 1972: Fontaine, 1975).

irGnRH Neuron Soma Sizes

Fish that received complete gonadectomies had sig- nificantly larger preoptic irCnRH neuron soma sizes than did intact (sham) fish [Fig. 2,3 ( Mann- Whitney U = 45, n1 = 9, n2 = 5 , p < 0.001 ) ] . Mean soma sizes of complete castrates were also larger than for incomplete castrates (63 1.8 m2 and 49 I .2 m2, respectively), though these differences did not reach the critical value for statistical significance ( U = 72, n , = 10. n, = 9, p = 0.10). Partial cas- trates exhibited larger mean soma sizes than did sham-operated fish (491.2 m2 versus 439.2 m2), but again the differences were not significant ( U = 37, /zl = 10, n, = 5 , p = 0.10).

There was a significant negative correlation be- tween the mean soma size of irGnRH neurons and gonadosomatic index (GSI) (Fig. 4). The loga- rithm of the GSI accounts for over 50% of the total variance in mean soma size.

Previous work showed minimal correlation be- tween body size and mean soma size of irGnRH neurons (Davis and Fernald, 1990). We nonethe- less selected individuals of approximately the same-size ranges to serve as shams or as operated fish to minimize body-size variation in these exper- iments. Mean standard length of the sham-oper- ated fish was 83.5 mm, for the partial castrates, 85.5 mm, and for the complete castrates, 85.8 mm.

For all subjects combined there was a positive

though nonsignificant correlation between stan- dard length and mean soma size ( r = 0.210). For sham-operated fish, the Spearman rank correlation coefficient ( r ) was 0.250, for partial castrates, r = 0.062, and for complete castrates, Y = -0.062. Therefore, in the operated fish, standard length ac- counted for virtually none of the variance in mean soma size. In all fish combined, standard length variation accounted for 4% of the variation in mean soma size.

DISCUSSION

Clearly, successful gonadectomy results in a marked hypertrophy of the irGnRH neurons in the preoptic area. Such hypertrophy might result either from passive expansion as the peptide accu- mulates in these neurons, due to suppressed secre- tion of GnRH, or from the up-regulation of GnRH production. We consider the latter more likely. First, we find no evidence in vertebrates that an- drogens stimulate GnRH secretion, so their ab- sence should not suppress it. Moreover, the larger neurons found in the territorial males versus the nonterritorial males (Francis and Fernald, submit- ted) and also in mature versus immature males (Davis and Fernald, 1990) are obviously related to reproductive activity. The large GnRH neurons in the reproductively active territorial males indicate elevated GnRH biosynthesis and not a suppressed GnRH secretion. We expect that the further hyper- trophy of these neurons in castrated territorial males occurs for the same reason.

The plasticity of these is itself noteworthy. The results reported here, together with previous work on the social modulation of the size of these neu- rons (Davis and Fernald, 1990: Francis and Fer- nald, submitted), suggest that these neurons inte- grate sensory and endocrine information. That both sorts of signals induce size changes is some- what novel. There are scattered reports of similar soma-size plasticity in other vertebrate neural sys- tems (Konishi and Akutagawa, 1985; Bottjer, Meisner, and Arnold, 1986; Fishman and Breed- love, 1984). Interestingly, in these cases, too, the neurons are affected either directly or indirectly by endocrine signals. Bottjer et a]. ( 1986) report that neurons in one nucleus involved in the sound pro- duction of a passerine bird, exhibit growth during the song learning process, whereas another nucleus involved in song production exhibits cell addition without neuronal growth. Why these two popula- tions respond so differently under conditions re-

quiring increased activity is an interesting ques- tion. Are there particular types of neurons more prone to changes in soma size? Are neurons sub- serving particular neural functions more likely to experience size alterations? Is size plasticity a means of circumventing constraints on neuro- genesis?

Since testosterone, 1 I -ketotestosterone, and es- tradiol all were markedly reduced following gonad- ectomy, it remains to be established which steroid is responsible for the change in GnRH cell size. Selective androgen replacement in completely gonadectomized males should provide the answer.

Whichever gonadal steroid is the main actor, the hypertrophy of POA-ir GnRH neurons after gonad- ectomy indicates that androgens limit the growth of these neurons, even in the territorial males for which the social environment typically promotes such neuron growth. Hence, the positive correla- tion between irGnRH neuron size and gonadal de- velopment observed in intact fish must result from the stimulation of gonadal development by the irGnRH neurons and not the reverse.

In contrast to these results, Schreibman et al. ( 1986) reported that externally administered an- drogens caused an increase in GnRH biosynthesis and /or secretion, resulting in precocious matura- tion of intact juvenile Xiphophorzis maczdutzrs. It is possible that these seemingly contrary results re- flect differences in androgen feedback effects re- lated to age. During early development this nega- tive feedback loop might not yet be established. Indeed, androgens may exert a positive-feedback effect during much of prematurational develop- ment.

The castration-induced hypertrophy of GnRH neurons accords with the results of Toranzo et al. (1989) and Zoeller et al. (1988) on gonadecto- mired rats, in which increased GnRH mRNA lev- els were identified through in silzi hybridization. They concluded that gonadal steroids exert a nega- tive feedback effect on GnRH production. As noted above, others (Pfaff, 1986; Rothfeld et al., 1987; Park et al., 1988; Roberts et at., 1989) have found lower GnRH levels subsequent to gonadec- tomy, whereas Kelly et al. ( 1989) and Wiemann et al. ( 1990) reported no change in mRNA levels.

It is difficult to reconcile these disparate results. Technical and methodological differences aside, they suggest a possible heterogeneity of GnRH neu- ron populations. We have identified at least one discrete population of these neurons implicated in testicular development that responds dramatically to testis removal. This is consistent with a negative

feedback effect of gonadal steroids on GnRH lev- els. But which steroid is responsible remains to be determined, as well as the aspect of GnRH biosyn- thesis that it affects: transcription, mRNA process- ing, or posttranslational modification.

Interpreting this negative feedback effect is complicated by the fact that steroid hormones prob- ably do not act directly on the GnRH neurons (Morrell, McGinty, and Pfaff, 1985), but rather through the mediation of one or more neurotrans- mitter types (Cicero, Schainker, and Meyer, 1979; Bhanot and Wilkinson, 1983). One such class of neurotransmitters is the endogenous opioid ( EOP) system. @-Endorphin, which is derived from the larger precursor molecule, proopiomelanocortin ( POMC), has been implicated in the steroid regula- tion of GnRH in rats (Eipper and Mains, 1980; Chowen-Breed et al., 1989a,b) and other mam- mals (Gilbeau, Hosobuchi, and Lee, 1987; Brooks, Lamming, Lees, and Haynes, 1986), as well as te- leost fish (Rosenblum and Peter, 1989).

We have demonstrated that castration results in a significant hypertrophy of GnRH neurons in the POA. These findings suggest that in male Hap- lochrornis burtoni, gonadal steroids affect the nega- tive feedback control of gonadotropins at least in part through the inhibition of GnRH production and secretion in these neurons.

Our results also indicate that the association be- tween large GnRH neurons and large gonads in the territorial males results from stimulation of go- nadal development by the preoptic neurons, and not from the stimulation of neuron growth by the gonads. In adult males, gonadal steroids probably set an upper limit on the size of the POA-GnRH neurons, which are otherwise socially modulated. We predict that the nonterritorial males will experi- ence this postcastrations hypertrophy only if the inhibitory social stimuli provided by territorial males is removed.

The authors thank Dr. T. Takahashi for use of his image analysis system, S. Kroger for expert technical help. and Drs. B. Evans and C. Heller for comments on the manuscript. This study was supported by NIH H D 23799 to R.D.F. and M H 09485 to R.C.F.

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