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CHAPTER 3
Differences in Seasonal Expression of Neurohypophysial Hormone Genes in Ordinary
and Precocious Male Masu Salmon
ABSTRACT
Our previous study showed the seasonal variations in expression of vasotocin (VT) andisotocin (IT) genes in preoptic magnocellular neurons of female masu salmon (Oncorhynchusmasou). The changes in the level of VT mRNA were coincident with those in the plasmatestosterone and estradiol levels. In the present study, generality of this phenomenon insalmonid was tried to confirm in males. We thus examined changes in expression of VT andIT genes by an in situ hybridization technique and an immunohistochemical avidin-biotin-complex method in the preoptic nuclei of ordinary and precocious male masu salmon. Theplasma levels of testosterone and estradiol were measured by enzyme immunoassay. Fishwere sampled in March, May, August and November 1994 and January 1995. Both theintensity of hybridization signals for VT and IT mRNAs and immunoreactivity of VT and ITshowed seasonal variations, although the patterns were different between the ordinary and theprecocious males. In the ordinary males, the intensities of hybridization signals for VT andIT mRNAs were high in the winter. These strong hybridization signals, representingelevation of VT and IT gene expression, were accompanied by the increase in plasma levelsof testosterone and estradiol. However, in the precocious males, the changes in VT and ITmRNA levels were not coincident with the variation of the plasma levels of sex steroidhormones. The sensitivity to sex steroid hormones of VT and IT gene expression may bedifferent between the ordinary and precocious male masu salmon.
Introduction
In the previous study, we showed seasonal changes in expression of genes encodingvasotocin (VT) and isotocin (IT) precursors in preoptic neurosecretory cells (NSC) in thehypothalamus of immature female masu salmon (Oncorhynchus masou) (Ota et al., 1996b).
Since the high levels of VT mRNA in the autumn was accompanied by the elevation ofplasma testosterone and estradiol levels, our previous study suggested involvement of sexsteroid hormones in seasonal regulation of VT and IT gene expression. In mammalian NSCs,sex steroid hormones have regulatory influences on vasopressin (VP) and oxytocin (OT) geneexpression (see Adan and Burbach, 1992). VT has been considered to be involved inreproductive behavior in teleosts (see Urano et al., 1994) as well as in other vertebrates (seeMoore, 1992). Sexually dimorphic expression of VT and IT genes in the hypothalamus ofmatured chum salmon (O. keta) (Ota et al., 1996a; Hiraoka et al., 1996, 1997) supports arelevance of reproductive function with seasonal variation of VT and IT gene expression, andfurther suggest that seasonal variations in VT and IT gene expression are sexually different.
Many of male masu salmon precociously mature in yearling autumn, in contrast tofemales which usually spawn in the autumn of 3 years of age. In amago salmon (O.
rhodurus), which is claimed to be a variant of masu salmon, immature ordinary malesconsistently showed low plasma androgen levels, whereas precocious males exhibitedelevation and then the peak of plasma androgen levels in late August through October (Uedaet al., 1983). Similar differences in plasma testosterone levels may occur between inimmature ordinary males and precocious males of masu salmon. If so, what expectable isthat the patterns of seasonal changes in VT and IT gene expression differ between ordinaryand precocious male masu salmon.
One of the important physiological function of VT and IT in teleosts is considered to beosmoregulation (see Urano et al., 1994). Changes in the level of VT mRNA in preopticNSCs of rainbow trout (O. mykiss), decrease after transfer of fish from fresh water (FW) to80% seawater (SW) and increase after transfer back to FW, were concordant with diureticfunction of VT in teleosts (Hyodo and Urano, 1991a). Although a seasonal correlationbetween the plasma Na+ level and expression of VT and IT genes were not clear in femalemasu salmon (Ota et al, 1996b), a presence of such correlation cannot be excluded in malemasu salmon.
The primary question in the present study was whether seasonal changes in VT and ITgene expression differed between ordinary and precocious male masu salmon, then, whensuch differences occurred, whether they reflected seasonal changes in plasma steroid hormonelevels. In addition, we compared present result with the previous data in the females toexamine sexual dimorphism of seasonal changes in VT and IT gene expression, and theircorrelation with plasma Na+ levels. To clarify these questions, we analyzed changes inexpression of VT and IT genes in individual NSCs in immature ordinary and precocious malemasu salmon using an in situ hybridization technique and an immunohistochemical avidin-biotin-complex (ABC) method. Plasma concentrations of testosterone and estradiol weremeasured by enzyme immunoassay.
Materials and methods
Fish Immature (1+) ordinary male masu salmon (fork length 7.7-16.7 cm, body weight 3.9-49.2 g) and precocious male masu salmon (fork length 11.1-15.6 cm, body weight 11.1-39.8g) were obtained from the Toya Lake Station for Environmental Biology. The fish werereared in spring water of constant temperature (9-10oC) in circular 1400-liter outdoor tanksunder natural photoperiod, and were sampled in March, May, August and November 1994 andJanuary 1995 (n=5, each). They were anesthetized with 0.02% tricaine methane sulfonate(MS-222, Sigma), and were weighed and measured of body length. Then blood was collected from the caudal vasculature. Blood samples kept in ice were later centrifuged at3000 rpm for 15 min to obtain plasma. Plasma Na+ concentrations were measured by anAtomic Absorption and Flame Emission Spectrometer (Shimadzu, AA-640-13).
Tissue preparation Immediately after the collection of blood samples, animals were decapitated and tissueblocks from the forebrains that mainly included the hypothalamus were taken out. Theywere then immersed in 4% paraformaldehyde in 0.05 M phosphate buffer (pH 7.3) at 4oC for2 days. After fixation, the tissues were washed in cold 70 % ethanol overnight, dehydratedthrough graded ethanols, and were embedded in paraplast. Serial transverse sections werecut at 8µm, separated into several groups, and were mounted on gelatinized slides.
In situ hybridization (ISH) The procedure for in situ hybridization followed the method previously described byHyodo et al. (1988) and Hyodo and Urano (1991a). For hybridization probes, 46mersynthetic oligonucleotides were also used as was described in the previous paper (Hyodo andUrano 1991a). The nucleotide sequences of probes correspond to the regions in the chumsalmon mRNAs which encode proVT (-5 to 11) and proIT (-5 to 11). Since salmonids so farexamined have at least two genes for each of VT and IT (Heierhorst et al., 1990; Hyodo et al.,1991; Suzuki et al., 1992a; Hiraoka et al., 1993), the VT probe was designed to hybridizewith salmon VT-I and -II mRNAs, whereas the IT probe recognizes salmon IT-I and -IImRNAs.
The probes were labeled at the 3' ends with [α-35S] dATP (DuPont/NEN Products) by a3'-end labeling system (Amersham), and purified with a NENSORB purification cartridge(DuPont/NEN Products). Final specific activities of the probes were 0.9-1.2 x 108 cpm/µg.
The radiolabeled probes were diluted in a hybridization buffer (0.9 M NaCl, 0.6 mM EDTA,0.02% bovine serum albumin (BSA), 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 10 mMdithiothreitol, 100 µg/ml denatured calf thymus DNA, and 10% dextran sulfate in 20 mM Trisbuffer, pH 7.5) so as to apply 7.5 ng/90 µl of the probe to each slide glass. Hybridizationwith the labeled probe was performed at 46 oC for 16 hr. The sections were then washed in1 x standard saline citrate (SSC) at room temperature for 10 min, twice in 1x SSC at 45oC for30 min, and finally in 1 x SSC at room temperature for 10 min. Specificity of hybridizationsignals was previously confirmed by several tests (Hyodo and Urano, 1991a).
Immunohistochemistry Tissue sections adjacent to those used for the ISH were immunohistochemically stainedby the avidin-biotin-complex method as was previously described (Ota et al., 1996). Theprimary antisera (gifts from Dr. S. Kawashima) were used as follows: rabbit anti-vasopressinpreabsorbed with IT was diluted 1:10000 with phosphate buffered saline containing 0.5%BSA (PBS-BSA, pH7.6), and rabbit anti-oxytocin preabsorbed with VT was diluted 1:20000also with PBS-BSA. These values for dilution of the antisera which were determined byserial dilution experiments stained magnocellular NSCs sub-maximally, so that changesupward or downward could be detected. Specificity of immunohistochemical staining wasconfirmed by pre-absorption tests in which the primary antisera were pretreated with antigen-conjugated CNBr-activated Sepharose 4B (Pharmacia).
Quantification of hybridization signals and immunoreactivity Both of VT and IT neurons are localized mainly in the magnocellular part of preopticnucleus (PM). The PM can be divided into three loci, pars parvocellularis (PMp), parsmagnocellularis (PMm) and pars gigantocellularis (PMg) (Braford and Northcutt, 1983).
The soundness of such nomenclatorial usage was recently confirmed by their sexuallydimorphic characteristics (Ohya et al., 1998). The intensity of hybridization signals and the magnitudes of immunoreactive stainability in individual NSCs in the PMp, PMm and PMgwere analyzed with a computer program for image analysis (Image Pro Plus, MediaCybernetics). Photographs of NSCs that contained hybridization signals andimmunoreactivity were taken under dark- and light- field microscopy, respectively. Imagesto be analyzed were then captured into a computer with a film scanner (POLASCAN,Polaroid). The area and the mean density per unit area were measured of individual NSCs inthe PMp, PMm and PMg. For statistical analysis, Student's t-test was applied after Scheffe'sF test for variance.
Enzyme immunoassay The plasma estradiol and testosterone levels were measured by enzyme immunoassay.
Plasma samples were extracted with diethyl ether. The extracts were then evaporated todryness with nitrogen gas, and assayed by use of testosterone and estradiol enzymeimmunoassay kits (CAYMAN CHEMICAL). Range of intra-assay variations was 1.8 to7.8%, and that of inter-assay variations was 4.1 to 13.7%.
The localization of neurons that included hybridization signals for VT and IT mRNAsgenerally coincided with the distribution of VT and IT-immunoreactive (-ir) neurons,respectively. The PMp consists of relatively small cells in the rostroventral part of the PM,the PMm consists of large neurons in the middle part of the PM, and the PMg is composed oflarger cells which are localized caudal to the PMm. NSCs in these subnuclei showedseasonal variations in the intensity of VT and IT hybridization signals and immunoreactivityof VT and IT. The patterns of changes were characteristics for each of VT and IThybridization signals, and also for VT and IT immunoreactivity, whereas the changes inparticular parameters were concordant in all of the PMp, PMm and PMg.
The intensity of hybridization signal for VT mRNA and the immunoreactivity of VT The intensity of hybridization signals for VT mRNA in NSCs of the PMm and PMgwas higher than that in the PMp in the ordinary males regardless of seasons. Theimmunoreactivity of VT in the PMm and PMg tended to be higher than that in the PMp Both the intensity of hybridization signals for VT mRNA (Fig.1) and immunoreactivityof VT showed apparent seasonal variations in the ordinary and precocious males (Fig. 2).
The intensity of hybridization signals for VT mRNA was low in the autumn, and thenelevated to the highest level in the winter (Fig. 2A) in the ordinary male. In the precociousmales, the intensity of hybridization signals for VT mRNA was elevated in the late spring,and then reached the highest level in the autumn (Fig. 2B). Almost all of precocious malesmatured and died in the autumn. The immunoreactivity of VT in the autumn was higherthan that in the summer in the ordinary and precocious males (Fig. 2C and D). The changesin the immunoreactivity of VT did not necessarily coincide with those in the intensity ofhybridization signals for VT mRNA in the ordinary males, whereas the profiles were rathersimilar in the precocious males.
The intensity of hybridization signal for IT mRNA and the immunoreactivity of IT The intensity of hybridization signals for IT mRNA and also immunoreactivity of IT inNSCs did not differ so much among the three PM subnuclei in any of particular seasons.
Obvious seasonal changes were observed in the intensity of hybridization signals for ITmRNA and also immunoreactivity of IT, although the patterns were different from thosefound in VT neurons (Fig. 3). The intensity of hybridization signals for IT mRNA was highin the late spring and then gradually decreased to the autumn, and then, in turn, showed sharpincrease from the autumn to the winter in the ordinary males (Fig. 3A). In the precocious males, the intensity of hybridization signals for IT mRNA was decreased in the late springand then gradually increased toward the autmn (Fig. 3B). The immunoreactivity of IT waselevated in the late spring. The levels were left rather unchanged in the ordinary males (Fig.
3C). In the precocious males, the immunoreactivity of IT was gradually increased from thespring toward the autumn (Fig. 3D). The changes in the immunoreactivity of IT did notcoincide with those in the intensity of hybridization signals for IT mRNA.
Changes in plasma concentrations of testosterone, estradiol and Na+ The plasma concentration of testosterone showed gradual increase from the spring tothe summer and decreased in the autumn, and then elevated in the winter in ordinary males.
In the spring, the plasma concentrations of testosterone in the precocious males were about 7-fold higher than that in the ordinary males. Thereafter, the plasma concentration oftestosterone in the precocious males was decreased in the late spring and attained the peak inthe summer (Fig. 4A). The plasma concentrations of estradiol were gradually increased inboth the ordinary and the precocious males (Fig. 4B). The plasma Na+ level tended todecrease in the autumn, however, no significant change was seen (Fig. 4C).
Discussion
In the present study, we found seasonal variations in the intensity of hybridizationsignals for VT and IT mRNAs and immunoreactivity of VT and IT in preoptic NSCs of themasu salmon hypothalamus. In the immature ordinary males, the intensity of hybridizationsignals for VT mRNA was low in the autumn, while immunoreactivity of VT was elevated.
The intensity of hybridization signals for IT mRNA was low in the autumn, however,immunoreactivity of IT was rather unchanged in the ordinary males. In contrast to theseprofiles in the ordinary males, the intensity of hybridization signals for VT mRNA andimmunoreactivity of VT were elevated in the autumn in the precocious males. The intensityof hybridization signals for IT mRNA and immunoreactivity of IT were also elevated in theautumn, whereas the IT hybridization signals were decreased, but the IT immunoreactivitywas elevated in the late spring. These results apparently showed that the seasonal changes inVT and IT gene expression differed between the ordinary and the precocious male masusalmon.
Increases in the hybridization signals for VT and IT generally imply the enhancementof gene expression. Therefore, the increases in the VT and IT hybridization signals from theautumn to the winter observed in the ordinary males probably reflect elevation of VT and ITgene expression in this season. Such increases in gene expression were accompanied by theelevation of plasma levels of testosterone and estradiol levels. Similar results were seen inthe female masu salmon that the high level of VT mRNA in the autumn was accompanied bythe elevation of plasma levels of testosterone and estradiol (Ota et al., 1996b). Since sexsteroid hormones have regulatory influences on VP and OT gene expression in mammalianNSCs (see Adan and Burbach, 1992), sex steroid should regulate VT gene expression in bothimmature male and female masu salmon. Aromatase, which convert testosterone to estradiol,is present in brain tissues of many vertebrates. Aromatase activity is especially high intelencephalon, preoptic and hypothalamic tissues in teleosts (see Callard et al., 1978). It isthus probable that testosterone is converted to estradiol by aromatase in the brain, and exertsactions on NSCs.
Three estrogen responsive elements and four glucocorticoid responsive elements, whichare also recognized by androgen receptors, are present in the 5'-upstream region of the VT-Igene in chum salmon (Satomi et al., 1994), and four estrogen responsive elements and fourglucocorticoid responsive elements are present in the 5'-upstream region of the IT-I gene inchum salmon (Kuno et al., 1995). Further, in the brain of rainbow trout, estrogen receptor-immunoreactive cells and cells which have estrogen receptor mRNA were localized in three regions, the ventral telencephalon, the anterior ventral preoptic region and the mediobasalhypothalamus (Salbert et al., 1991; Anglade et al., 1994). It is thus probable that the sexsteroid hormones modulate VT gene expression.
In the precocious males, the plasma testosterone and estradiol were decreased in the latespring, while the VT hybridization signals were increased and the IT hybridization signalswere decreased. The VT and IT hybridization signals in the precocious males were ratherunchanged in the summer regardless of the high levels of testosterone. In immature ordinarymales, the high levels of VT mRNA were accompanied by the elevation of plasma levels oftestosterone and estradiol. A possible reason of this difference in the relation between theVT mRNA and the plasma levels of sex steroid hormones was that the plasma testosteroneand estradiol levels in the spring were much higher in the precocious males than in theordinary males. In precocious male masu salmon, the pituitary GTH IIβ contents showedseasonal variation, that is, high in autumn and low in winter in underyearing period throughmaturation (Amano et al., 1993). Further, the pituitary GTH IIβ contents in precociousmales were higher than those in immature males in underyearing period. It is thus possiblethat, in the precocious males, the levels of testosterone were elevated before the yearlingspring, and brains were exposed to high concentration of testosterone. NSCs of theprecocious males might be habituated or insensitive to steroid hormones in the yearling spring.
Such priming effects might alter the sensitivity to testosterone of VT and IT gene expression,and induced differences between the ordinary and the precocious male masu salmon.
In the female masu salmon, which were collected concomitantly with the samepopulation used in the present study, the level of VT mRNA was high in the autumn anddecreased toward the winter (Ota et al., 1996b). Contrary, in the immature ordinary males,the level of VT mRNA was low in the autumn and increased toward the winter. The presentresults thus revealed sexually different expression of neurohypophysial hormone genes in thepreoptic nucleus of immature masu salmon.
In the autumn, the level of VT mRNA in the females determined in our previous study(Ota et.al., 1996b) was higher than that in the ordinary males observed in the present study.
In chum salmon, the level of VT mRNA in females was significantly lower than that in malesat the final stage of maturation (Ota et al., 1996a; Hiraoka et al., 1997). A discrepancybetween the result in pre-spawning chum salmon described above and the present results inimmature masu salmon may be caused by the difference in magnitudes of gonadaldevelopment. In the pre-spawning female chum salmon, the decrease in the level of VTmRNA was accompanied by the sharp increase in plasma level of 17α,20β-dihydroxy-4-pregnen-3-one (DHP), suggesting that DHP was involved in regulation of VT gene expression(Ota et al., in preparation).
Changes in IT immunoreactivity was predominant in the PMm and PMg than that in thePMp in the ordinary and the precocious males. In the ordinary males, changes in VThybridization signals also predominant in the PMm and PMg than that in PMp. In pre-spawning chum salmon, changes in the IT mRNA levels were predominant in the PMp andPMm, while changes in the VT mRNA levels were concordant in all the three PM loci. (Otaet al., 1996a). The regionally different changes in VT hybridization signals and ITimmunoreactivity suggest that the regulation of secretory activity may be different in thesethree PM loci. In the mammalian paraventricular nucleus, neurosecretory neurons indiscrete loci have different characteristics among each other, such as functions and afferentinnervations (see Swanson and Sawchenko, 1983).
Several studies indicated involvement of neurohypophysial hormones inosmoregulation in teleosts (see Urano et al., 1994). The levels of VP and OT mRNAs in therat neurosecretory neurons were sensitive to plasma Na+ level and osmolality (Hyodo et al.,1988, 1989). Variations in the extracellular Na+ level were directly detected by supraopticneurons (Brimble and Dyball, 1977; Mason, 1980). Similarly, in the eel brain, magnocellular neurons were responsive to extracellular Na+ in an in vitro electrophysiologicalexperiment (Sugita and Urano, 1986). In immature rainbow trout, the level of VT mRNA inthe magnocellular neurosecretory neurons was decreased by transfer from FW to 80% SW,and was increased by transfer back to FW from 80% SW (Hyodo and Urano, 1991a),suggesting that VT is involved in FW-adaptation in salmonid fish. In the present study, theplasma Na+ levels fluctuated, however, correlation between the intensity of hybridizationsignal for VT mRNA and the plasma Na+ level was not seen. In female masu salmon, aseasonal correlation were also not found between the plasma Na+ level and expression of VTand IT genes (Ota et al, 1996b). Thus, the seasonal changes in the expression of VT gene inmasu salmon could not be explained by the changes in plasma Na+ levels.
In conclusion, seasonal variations were found in the expression of VT and IT genes inimmature ordinary and precocious male masu salmon. The patterns of these changes weredifferent between the ordinary and the precocious males. Further, the patterns of changes ingene expression were different between VT and IT. The expression of VT and IT genesshould be seasonally regulated under different molecular mechanisms due to the difference inthe sensitivities to sex steroid hormones of VT and IT gene expression between the ordinaryand precocious male masu salmon.

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