Cloning of FSHb, and glycoprotein subunits from the Russian...

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General and Comparative Endocrinology 140 (2005) 61–73

GENERAL AND COMPARATIVE

ENDOCRINOLOGY

Cloning of FSHb, LHb, and glycoprotein a subunits fromthe Russian Sturgeon (Acipenser gueldenstaedtii), b-subunit mRNAexpression, gonad development, and steroid levels in immature fish

Avshalom Hurvitza,b, Gad Deganib,c, Doron Goldbergb, Svetlana Yom Dina,b,Karen Jacksonb,c, Berta Levavi-Sivana,*

a Faculty of Agricultural, Food and Environmental Quality Sciences, Department of Animal Sciences, The Hebrew University of Jerusalem,

Rehovot 76100, Israelb MIGAL–Galilee Technology Center, P.O. Box 831, Kiryat Shmona 10200, Israel

c School of Science and Technology, Tel-Hai Academic, College, Galilee, Israel

Received 8 April 2004; revised 21 September 2004; accepted 22 September 2004Available online 23 November 2004

Abstract

The Russian sturgeon, Acipenser gueldenstaedtii, is a late-maturing Acipenseriformes. To elucidate the role of FSH and LH in itsreproduction, we cloned its glycoprotein a-subunit (GPa) and gonadotropin b-subunits (FSHb and LHb) using 5 0 and 3 0 RACE-PCR. The nucleotide sequences of the Russian sturgeon (st) GPa, FSHb, and LHb are 345, 384, and 411 bp long, encoding peptidesof 91, 115, and 114 amino acids, respectively. The deduced amino acid sequence of each mature subunit showed high similarity withthose of other teleosts. Sequence analysis showed that stFSHb is more similar to higher vertebrate FSHbs (35–37%) than to highervertebrate LHbs (26–30%). The next objective of this work was to compare the development of sturgeon gonads at the very firststages of their growth with the expression of their gonadotropins. Sturgeons at ages 1, 2, 3 or 4 years were sacrificed. The expressionof their gonadotropin b-subunits was determined using quantitative real-time PCR, and their gonads were examined histologically,followed by a determination of the plasma levels of estradiol in females and 11-ketotestosterone (11-KT) in males. The expressionlevels of stFSHb subunit was found to be higher in fish at 3 and 4 years of age than in 1-year olds. mRNA levels of stLHb werehigher than those of stFSHb in both genders. Moreover mRNA levels of stFSHb detected in females were significantly higher thanthose found in males. Even at age 4 years, all female Russian sturgeons tested contained gonads at the pre-vitellogenic stage, withsmall oocytes and very low levels of estradiol in the plasma. However, among the males, at ages 3 and 4 years, we found testes thatcontained spermatids and spermatozoa. Those males were found to have significantly high GSI (gonadosomatic index; gonadalweight as a percentage of BW) levels, stLHb expression and 11-KT levels.� 2004 Published by Elsevier Inc.

Keywords: Gonadotropin; Estradiol; 11-Ketotestosterone; Real-time PCR; Sturgeon; Gene expression; Puberty

1. Introduction

Sturgeons (st) are one of the most ancient groups ofOsteichthyes. They are found along the coasts of the

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* Corresponding author. Fax: +972 8 9465763.E-mail address: [email protected] (B. Levavi-Sivan).

Atlantic and Pacific oceans, in the Mediterranean andBlack seas, and in many rivers, lakes, and inland seas(Dettlaff et al., 1993). The decline in sturgeon popula-tions in their native habitats, mainly the Caspian Sea,due to over-fishing for meat and production of caviar,destruction of their spawning grounds, and pollutionof the water, has led to their introduction into aquacul-ture. White sturgeon (Acipenser transmontanus) and the

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62 A. Hurvitz et al. / General and Comparative Endocrinology 140 (2005) 61–73

Siberian sturgeon (Acipenser baeri) are the leading spe-cies which have been adapted to aquaculture, and assuch they are the most investigated sturgeons with re-gard to culturing aspects. A severe problem in sturgeonmanagement is slow and asynchronous ovarian matura-tion. While most males mature at the age of 4 years, fe-males reach first sexual maturity between 6 and 12 yearsand have biennial ovarian cycles (Doroshov et al.,1997).

Gonadotropins (GtHs) are glycoproteins (GPs) con-sisting of two noncovalently bound subunits, a and b,which have been intensively studied, especially in fishadapted to aquaculture conditions [reviewed by Yaronet al. (2003)]. As in mammals, both GtHs are heterodi-meric GPs, sharing the same a-subunit and distinct b-subunits, the latter conferring their biological specificity.cDNA sequences encoding GtH subunits have been iso-lated and characterized from more than 19 fish species,representing seven teleostean orders [reviewed by Yaronet al. (2003)]. Recently, Querat et al. (2001) establishedthat the duality of GtHs occurred at the time of thechondrichthyans� emergence, prior to the split betweenactinopterygians and sarcopterygians.

Moberg et al. (1991) showed the existence of twoGtHs in the white sturgeon (A. transmontanus) termedstGtH-I and stGtH-II. Pituitary and plasma concentra-tions of stGtH-I were found to be higher than those ofstGtH-II during vitellogeneis and the early stages ofspermatogenesis. Conversely, pituitary and plasma con-centrations of stGtH-II were greater than those ofstGTtH-I during ovulation and spermiation. A gonado-tropin-releasing hormone analog (GnRHa) was effectivein stimulating the release of both GtHs in mature malesand pre-ovulatory females, with a maximal responseoccurring in the spring. Collectively, the data supportthe view that sturgeons possess a dual GtH system con-trolling reproduction (Moberg et al., 1995). Querat et al.(2000) cloned the b-subunits of stGtH-I and stGtH-II ofA. baeri and, based on their phylogenetic tree, suggestedthat they should be termed FSH and LH, respectively.

Although the sturgeon is a well-studied fish and thereare many articles describing its steroid levels during ad-vanced stages of oocyte maturation and spawning, verylittle is known about the first stages of puberty in thisspecies.

Due to its high production rates and caviar quality,the Russian sturgeon (A. gueldenstaedtii), originallyfrom the Caspian Sea, was chosen to enrich the varietyof commercial fish species in Israel. As a prerequisitefor a study of its reproduction, we cloned the genesencoding the a- and b-subunits of the GtHs and ana-lyzed the expression of the b-subunits in immature maleand female sturgeons grown under local aquacultureconditions. In addition, we characterized the plasma ste-roid levels of the sturgeons during their first 4 years inculture.

2. Materials and methods

2.1. Fish and sampling procedure

Russian sturgeons (A. gueldenstaedtii) originated inthe Caspian Sea were brought from Russia and rearedat ‘‘Dan Fish Farms’’ (Upper Galilee, Israel; 31�30 0N,34�45 0E), under aquaculture conditions, from eggs. Fishwere maintained in 250- to 500-m3 concrete tanks atambient temperature (22–24 �C) and photoperiod, andwere fed twice a day with 4 mm pelleted feed (trout feed;Zemach Feed Mills, Zemach, Israel; containing 50%protein and 18% fat), at 0.5–1% of their biomass,depending on the season. One-, two-, three-, and four-year-old sturgeons were sampled in August, when watertemperature was 22 ± 0.5 �C. Each fish was anesthetizedin a clove oil bath (0.25 mg/L), weight and length wererecorded, and blood was taken from the caudal vascula-ture into heparinized syringes. After centrifugation, theplasma was stored at �20 �C until processing. The pitu-itary gland was removed and stored in RNA Later buf-fer (Ambion, Austin, TX). The gonads were removed,weighed and a portion taken for histology. Total RNAwas extracted from freshly excised pituitaries of stur-geon females and males from each age group (1, 2, 3,and 4 years) by means of the RNeasy total RNA kit(Qiagen, Alameda, CA), according to manufacturer�srecommendations.

2.2. cDNA cloning of sturgeon GtHs

Total RNA was extracted from four pituitaries ofsturgeon, freshly excised, by means of RNeasy totalRNA kit (Qiagen, Alameda, CA), according to manu-facturer�s recommendations. First strand cDNA wassynthesized from 2 lg of the total RNA by SuperscriptSystem (Gibco). The second-strand cDNA were synthe-sized for 3 0 and 5 0 rapid amplification of cDNA ends(RACE)-PCR using a RACE kit (Roche Applied Sci-ence, Mannheim, Germany). Gene-specific primers forthe 3 0 RACE were designed according to the sequencesof relative genes of A. baeri (Accession numbers inGenBank—GPa: AJ310342; LHb: AJ251656; FSHb:AJ251658). Gene-specific primers: P9 (Table 1) forthe GPa subunit, P1 (Table 1) for the FSHb subunit,and P5 (Table 1) for the LHb subunit, were used for3 0 RACE, along with the primer provided in the kit.PCR was carried out in a volume of 50 ll using2.5 U of Taq polymerase (Promega, Madison, WI),5· buffer (Promega), 1.5 mM MgCl2, dNTPs (0.2 mMfinal concentration of each nucleotide), 100 pmol ofeach primer, and 4 ll of cDNA for the b-subunits or2 ll for the GPa. Cycling parameters were: 3 min dena-turation at 94 �C, followed by 40 cycles of 1 min dena-turation at 94 �C, 2 min annealing at 52 �C and 3 minextension at 72 �C, for FSHb; or followed by 34 cycles

Table 1Primers used in this study

Primer Subunit Position Primer sequence

P1 FSHb 367–387 5 0-ACTGACTGTGGCACCCTAAGC-3 0

P2 FSHb 462–446 5 0-CCAGCAGGGTACTAATT-3 0

P3 FSHb 7–30 5 0-CAGAAGTCAACTACCCTGCAGTAT-3 0

P4 FSHb 1015–998 5 0-CAAATTGTTTGCAATGTGC-30

P5 LHb 410–427 5 0-CCTCGGACTGTACCATTC-30

P6 LHb 489–472 5 0-GGAGTGTCAGTAGTTTTG-30

P7 LHb 18–37 5 0-AGAGAGAGACGCTGCCTGAG-3 0

P8 LHb 551–535 5 0-CATGTAATGTGGGGGAG-3 0

P9 GPa 284–307 5 0-CCCAAGAACATTACTTCAGAGGC-3 0

P10 GPa 333–316 5 0-TAAAATCCTTCGCTACAC-30

P11 GPa 57–76 5 0-ATGGCTTGCTATGGGAAGTG-30

P12 GPa 401–382 5 0-GGTTTTATGGTAGTAGCAGG-3 0

P13 FSHb 139–158 5 0-GGCTGCGGAAACTGTGTATC-3 0

P14 FSHb 328–309 5 0-CCACGGGGTAGGTGTAAAAA-3 0

P15 LHb 207–188 5 0-GATGAGGAGGCACTTTGGAC-30

P16 18S 1408–1427 5 0-CCACACGAGATGGAGCAATA-30

P17 18S 1607–1588 5 0-GCTGATGACCCGCACTTACT-30

A. Hurvitz et al. / General and Comparative Endocrinology 140 (2005) 61–73 63

of 1 min denaturation at 94 �C, 1 min annealing at52 �C and 1 min extension at 72 �C for LHb; or fol-lowed by 10 cycles of 15 s denaturation at 94 �C, 30 sannealing at 52 �C and 40 s extension at 72 �C forthe GPa subunit. All PCRs were terminated with anadditional extension at 72 �C for 7 min. For the 5 0

RACE-PCR, the gene-specific reverse primers were de-signed according to the sequences of the cDNA clonedin the 3 0 RACE (P10, P2, and P6 for GPa, FSHb, andLHb, respectively; Table 1) and were used with the pri-mer provided in the kit. PCRs were as alreadydescribed. The cycling parameters were 3 min denatur-ation at 94 �C, followed by 35 cycles of 1 min denatur-ation at 94 �C, 1 min annealing at 54 �C and 1 minextension at 72 �C for FSHb; or followed by 35 cyclesof 1 min denaturation at 94 �C, 1 min annealing at55 �C and 1 min extension at 72 �C for LHb; or fol-lowed by 10 cycles of 30 s denaturation at 94 �C, 40 sannealing at 30 �C, and 50 s extension at 72 �C, forthe GPa subunit. All PCRs were terminated with anadditional extension at 72 �C for 7 min. The resultingamplified DNA was ligated into a pGEM-T VectorSystem (Promega), following the manufacturer�s proto-col. PCR amplification was performed with P3 and P4(Table 1) for stFSHb, P7 and P8 for stLHb, and P11and P12 for GPa as primers to obtain the full-lengthcDNA. Ligations, transformations, and plasmid prepa-rations were according to Sambrook et al. (1989). Thesequencing was carried out at the Center for GenomicTechnologies of the Hebrew University. At least threeindependent clones were sequenced in each case.

2.3. Sequence assembly and analysis

The sequences of each cDNA were assembled usingthe GAP4 software package (Bonfield et al., 1995). Se-quence analysis, molecular weight, and isoelectric point

calculations were carried out by Wisconsin Package10.0, Genetics Computer Group (GCG). The positionof the signal-peptide cleavage site was determined usingthe SignalP V1.1 program (Nielsen et al., 1997). Multi-ple sequence alignments and cluster analysis by theneighbor-joining method were performed using the Clu-stalX program (Higgins and Sharp, 1989).

2.4. Histological analysis

The gonad samples were fixed in Bouin and subse-quently processed for light microscopy. Paraffin sectionsof 6 lm were stained with hematoxylin and eosin.The terminology used for the sturgeon hybrid, thebester (Amiri et al., 1996a,b), was adopted for ourdescriptions.

Mean oocyte diameter was calculated for each fishafter measuring five of the largest oocytes present in ahistological section. Only oocytes sectioned throughthe nucleus were measured. Since ovarian developmentin the sturgeon is very uniform, only several sectionsper fish (n = 6) needed to be examined.

The presence and amounts of spermatogonia, sperma-tocytes, spermatids, and spermatozoa in the testes werestudied semi-quantitatively and randomly over the entiresurface of a mid-longitudinal section of the testes. Testeswere classified into the following categories: immature(or young) testes, containing no germ cells (stage A); tes-tes containing a small number of spermatogonia (stageB); and mature testes, showing active spermatogenesiswith spermatocytes and spermatids (stage C).

2.5. Real-time PCR

To compare the mRNA levels of the b-subunits ofsturgeon FSH and LH, the relative abundance oftheir mRNA was normalized to the amount of an


endogenous reference, the 18S subunit of rRNA, by thecomparative threshold cycle (CT) method, according toLevavi-Sivan et al. (2004b). The relative amount of eachb-subunit�s mRNA was calculated by the formula2�DCT , where DCT corresponds to the difference betweenthe CT measured for stFSHb or stLHb, and that mea-sured for 18S rRNA. To validate this method, serialdilutions were prepared from a pituitary cDNA sample(0.5, 0.1, 0.02, 0.01, and 0.005), and the efficiencies ofeach b-subunit and 18S ribosomal RNA amplificationswere compared by plotting DCT versus log (template),according to the method of Muller et al. (2002). Linearregressions of the plots showed the following R2 valuesand slopes, respectively: 0.976 and �3.00 for 18SrRNA; 0.999 and �3.096 for stFSHb; and 0.992 and�2.80 for stLHb.

Total RNA was prepared from individual pituitariesusing Trizol (Invitrogen, Carlsbad, CA) and each sam-ple was reverse-transcribed at 57 �C using Reverse-iT1st Strand Synthesis Kit (ABgene, Surrey, UK) and ran-dom hexamers, according to the manufacturer�sprotocols.

Gene-specific primers used for the real-time PCRwere designed using Primer3 Software. The primers usedfor stFSHb amplified a 170-bp product and corre-sponded to nt 139–158 and 328–309 (P13 and P14,respectively, Table 1, Accession No. AY519657). Prim-ers for stLHb (P7 and P15, Table 1, Accession No.AY333426) amplified a 190-bp product. Primers for18S rRNA (P16 and P17, Table 1, Accession No.AY188400) amplified a 180-bp product. The PCR mix-ture consisted of 5 ll of dilute cDNA sample, 0.75 pmolof each primer, and 7.5 ll of Mastermix for Syber GreenI (ABgene) in a final volume of 15 ll. Amplification wascarried out in a RotorGene 3000 Sequence DetectionSystem (Corbett Research, Sydney, Australia) underthe following conditions; for 18S rRNA: initial denatur-ation at 95 �C for 10 min, followed by 40 cycles of dena-turation at 95 �C for 15 s and annealing-extension at60 �C for 20 s, and then a final extension at 72 �C for20 s; for stFSHb: initial denaturation at 95 �C for15 min, followed by 40 cycles of denaturation at 95 �Cfor 15 s and annealing at 62 �C for 20 s, and extensionat 72 �C for 15 s; for stLHb: initial denaturation at95 �C for 10 min, followed by 40 cycles of denaturationat 95 �C for 10 s and annealing at 64 �C for 20 s, andextension at 72 �C for 15 s. Amplification of stFSHb,stLHb and 18S rRNA cDNAs was performed simulta-neously in separate tubes and in duplicate, and the re-sults were analyzed with the Q-Gene software(BioTechniques Software Library at: www.BioTechni-ques.com). Dissociation-curve analysis was run aftereach real-time experiment to ensure that there was onlyone product. To control for false positives, a reverse-transcriptase negative control was run for each templateand primer pair.

2.6. ELISA for steroids

Estradiol and 11-ketotestosterone (11-KT) weredetermined by enzyme-linked immunosorbent assay(ELISA), according to Cuisset et al. (1994); Nash et al.(2000) and Levavi-Sivan et al. (2004a), using acetylcho-linesterase as a label. The anti-11-KT was donated byDr. D.E. Kime (Sheffield, UK) and was previously de-scribed in Cuisset et al. (1994). The anti-estradiol wasdescribed in Levavi-Zermonsky and Yaron (1986). Allsamples were analyzed in duplicate, and for each ELISAplate, a separate standard curve was run. The lower lim-its of detection were 0.93 and 0.50 pg/ml for 11-KT andestradiol, respectively. The intra- and inter-assay coeffi-cients of variance were less than 7 and 11%, respectively.Steroid levels in the sturgeon plasma, determined byELISA, were validated by verifying that serial dilutionswere parallel to the standard curve.

2.7. Statistical analysis

Data are presented as means ± SEM. The signifi-cance of the differences between group means of hor-mone or mRNA levels was determined by one-wayanalysis of variance (ANOVA) followed by Student–Newman–Keuls (SNK) test using the Graph-Pad Prismsoftware (GraphPad, San Diego, CA) with the level ofsignificance in different groups set at p < 0.05.

3. Results

3.1. cDNA cloning and sequence analysis of the stFSHb,

stLHb, and GPa subunits

The full-length cDNA of stFSHb was compiled fromthe data obtained by the 5 0 and 3 0 RACE, and the nucle-otide and deduced amino acid sequences are shown inFig. 1 (EMBL Accession No. AY519657). The cDNAwas 1080-bp long and had an open reading frame of384 bp, beginning with the first ATG codon at position37 and ending with the stop codon at position 421. Aputative polyadenylation signal, ATTAAA, was recog-nized 25 bp upstream of the poly(A) tail. The positionof the signal-peptide cleavage site was predicted at posi-tion 15, yielding a signal peptide of 14 amino acids and amature peptide of 114 amino acids. The calculatedMr ofsFSHb polypeptide chain is 12173.61 and the polypep-tide has an isoelectric point of 4.25. Two putative N-linked glycosylation sites were found at positions 12and 29 from the N-terminus of the putative maturepeptide.

The full-length cDNA of stLHb was compiled fromthe data obtained by the 5 0 and 3 0 RACE, and the nucle-otide and deduced amino acid sequences are shown inFig. 2 (EMBL Accession No. AY333426). The cDNA

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Fig. 1. The nucleotide and deduced amino acid sequences of cDNA encoding the sturgeon FSHb subunit. The nucleotide numbers are shown onboth sides of the sequence. The first amino acid of the putative mature subunit is numbered +1. Amino acids that comprise the signal sequence areindicated with negative numbers. Putative N-glycosylation site(s) are marked by a triangle. The start and stop codons are indicated in grey, and theconsensus sequence for the polyadenylation signal is boxed.

Fig. 2. The nucleotide and deduced amino acid sequences of cDNA encoding the sturgeon LHb subunit. The nucleotide numbers are shown on bothsides of the sequence. The first amino acid of the putative mature subunit is numbered +1. Amino acids that comprise the signal sequence areindicated with negative numbers. Putative N-glycosylation site(s) are marked by a triangle. The start and stop codons are indicated in grey, and theconsensus sequence for the polyadenylation signal is boxed.


was 599-bp long and had an open reading frame of411 bp, beginning with the first ATG codon at position70 and ending with the stop codon at position 481. Aputative polyadenylation signal, ATAAA, was recog-nized 16 bp upstream of the poly(A) tail. The positionof the signal-peptide cleavage site was predicted at posi-tion 23, yielding a signal peptide of 22 amino acids and amature peptide of 115 amino acids. The calculatedMr ofstLHb polypeptide chain is 14812.18 and the polypep-tide has an isoelectric point of 4.85. One putative

N-linked glycosylation site was found at position 8 fromthe N-terminus of the putative mature peptide.

The full-length cDNA of the stGP a-subunit wascompiled from the data obtained by the 5 0 and 3 0

RACE, and the nucleotide and deduced amino acid se-quences are shown in Fig. 3 (EMBL Accession No.AY519658). The cDNA was 655-bp long and had anopen reading frame of 345 bp, beginning with the firstATG codon at position 57 bp and ending with the stopcodon at position 402 bp. A putative polyadenylation

Fig. 3. The nucleotide and deduced amino acid sequences of cDNA encoding the sturgeon glycoprotein a subunit. The nucleotide numbers areshown on both sides of the sequence. The first amino acid of the putative mature subunit is numbered +1. Amino acids that comprise the signalsequence are indicated with negative numbers. Putative N-glycosylation site(s) are marked by a triangle. The start and stop codons are indicated ingrey, and the consensus sequence for the polyadenylation signal is boxed.


signal, ATAAA, was recognized 21 bp upstream of thepoly(A) tail. The position of the signal-peptide cleavagesite was predicted at aa 25, yielding a signal peptide of24 amino acids and a mature peptide of 91 amino acids.The calculated Mr of the stGPa polypeptide chain is10502.1 and the polypeptide has an isoelectric point of8.84. Two putative N-linked glycosylation sites werefound at positions 55 and 77 from the N-terminus ofthe putative mature peptide.

3.2. Comparison of stFSHb, stLHb, and stGPa

The deduced amino acid sequences of the Russiansturgeon�s FSHb, LHb, and GPa subunits were com-pared with homologous subunits from a number ofother fish species (Fig. 4, Table 2). Gaps marked bydashes are introduced to maximize the alignments ofthe cysteine residues between the subunits (Fig. 4). Thehighest degree of identity was found with A. baeri (91,98, and 100% identity for stGPa, stLHb, and stFSHb,respectively), followed by the subunits of Anguilliformes(48–72%), and cyprinids (45–72%). The percent identi-ties of the sturgeon b-subunits were 40–59% with thoseof the Perciformes and 42–59% with those of the salmo-nid species. The lowest level of identity was observedwith Fundulus heteroclitus (53, 46, and 39% for stGPa,stLHb, and stFSHb, respectively). The identity withother percomorph fishes was in the range of 40–47%for stFSHb, 46–59% for stLHb, and 54–62% for thestGPa subunits. In both stFSHb and stLHb subunits,the positions of all 12 cysteines are conserved, and soare the N-glycosylation sites. The identity betweenstFSHb and stLHb is very low (43%), and can be attrib-uted to the 12 conserved cysteines and other conservedregions.

3.3. Expression of stFSHb and stLHb in males and

females

Real-time quantitative PCR, which enables the spe-cific and sensitive detection of transcripts, was used tostudy the expression of the GtH b-subunits at differentages and in different genders. mRNA levels of stFSHbwere significantly higher in females than in males (Fig.5A), while those of stLHb were similar in both genders(Fig. 5B). mRNA levels of stFSHb in females were sig-nificantly lower in their first year of life, increased dra-matically during the second year, and then did notchange significantly until the fourth year. The mRNAlevels of stLHb did not increase significantly until theage of 4 (Fig. 5). In both genders, the levels of stLHbwere significantly higher than those of stFSHb.

3.4. Gonad development and steroid levels

No significant difference was found during the firstthree years between males and females in respect to theirweight. However, the female�s weight was significantlyhigher than that of the males after 4 years of growth(6.8 kg ± 1.53 and 4.23 kg ± 1.0 for females and males,respectively).

In this study, we used four fish groups at the ages of 1year (n = 19), 2 years (n = 14), 3 years (n = 19), and 4years (n = 18). Histological analysis and steroid-leveldeterminations were performed on all fish. Fish fromthe age of 2 years on had differentiated gonads, enablingestradiol determination in females and 11-KT levels inmales. Levels of both steroids were determined in allyounger (1-year-old) fish.

Histological analysis showed that most females, irre-spective of their age, were at the pre-vitellogenic stage,

Fig. 4. Alignment of the amino acid sequences of the FSHb, LHb, and glycoprotein a (GPa) subunits of sturgeon and other actinopterygian species. The putative N-linked glycosylation sites areboxed. The sequences were extracted from the GenEMBL and Swiss-Prot databases, or taken from published articles.

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Table 2Amino acid identities between sturgeon GtH subunits and other vertebrate GtH subunits

Class/order Species stFSHb (%) stLHb (%) stGPa (%) Accession numbers in GenBank

Chondrichthyes/Carcharhiniformes

Scyliorhinus canicula 51 53 64 FSH: AJ310344, LH: AJ310345, GPa: AJ310343

Chondrostei/Acipenseriformes

Acipenser baeri 100 98.5 91 FSH: AJ251658, LH: AJ251656, GPa: AJ310342

Teleostei/Anguilliformes Anguilla anguilla 49 63 72 FSH: AY169722,LH: X61039, GPa: X61038Anguilla japonica 48 63 FSH: AB016169, LH: AY082379

Cypriniformes Cyprinus carpio 47 68 70 FSH: AB003583, LH: X59888, GPa: X56497Carassius auratus 45 65 69 FSH: AB015483, LH: AB015596,GPa : D86551Danio rerio 45 58 72 FSH: AY424303, LH: AY424304,GPa : AY424306

Siluriformes Clarias gariepinus 50 59 67 FSH: AF324541, LH: X97761, GPa: X97760Ictalurus punctatus 53 58 69 FSH: AF112191, LH: AF112192, GPa: AF112190

Osmeriformes Plecoglossus altivelis 42 56 FSH: AY124336, LH: AY124337Salmoniformes Coregonus autumnalis 42 58 FSH: L23432, LH: L23431

Oncorhynchus masou 44 59 62 FSH: S69275, LH: S69276, GPa: S69274Oncorhynchus keta 44 59 62 FSH: M27153, LH: M27154, GPa: M27152

Cyprinodontiformes Fundulus heteroclitus 39 46 53 FSH: M87014, LH: M87015, GPa: U12923Perciformes Oreochromis niloticus 40 54 58 FSH: AF289174, LH: AY294016, GPa: AF303087

Pagrus major 40 59 56 FSH: AB028212,LH: AB028213, GPa: AB028211Morone saxatilis 40 58 58 FSH: L35070, LH: L35096, GPa: L35071

Pleuronectiformes Paralichthys olivaceus 41 49 57 FSH: AB042422, LH: AB042423, GPa: AF268692Hippoglossus hippoglossus 42 47 54 FSH: AJ417768, LH: AJ417769, GPa: AJ417770


according to the classification of Van Eenennaam andDoroshov (1998) for the Atlantic sturgeon and that ofAmiri et al. (1996a,b) for hybrid sturgeon. Gonadal his-

Fig. 5. GtH b-subunit gene transcription changes at different sturgeonages. Total RNA was reverse-transcribed, and used for quantitativereal-time PCR. The relative abundance of the FSH b-subunit (A) andLH b-subunit (B) mRNA were normalized to the amount of 18SrRNA by the CT cycle method, where 2�CT reflects the relative amountof relative b-subunit transcript. Results are the average ± SEM (n = 4–9). Means marked by different letters differ significantly (p < 0.05).

tology of representative 4-year-old female Russian stur-geons is shown in Fig. 6. The females sampled in thisstudy had oocytes ranging from 50 to 460 m in diameter.Fig. 6B shows an oocyte at the perinucleolar stage-nu-cleoli are distributed as granules surrounding the germi-nal vesicle and only a single cortical vesicle can be seenat the bottom. Vitellogenic granules were not detected.The oocytes have a thin vitelline envelope (Fig. 6). Alow correlation (r2 = 0.06; n = 30) was found betweenestradiol levels and GSI (gonadosomatic index; gonadalweight as a percentage of BW), and between estradiollevels and oocyte diameter (r2 = 0.01; n = 30), indicatingthat the females were at the pre-vitellogenic phase.

Histological analysis of 1-year-old fish showed thatsex in most of them was still undifferentiated. At theage of 2 years, 33% of the females contained oocyteswith a diameter <100 lm, while 67% contained oocyteswith diameters between 100 and 140 lm. At the age of 3years, all females contained oocytes with a diameter of150–180 m. However, at the age of 4 years, only 21%of the females contained oocytes with a diameter<100 lm, whereas 50% contained oocytes of 100–180 lm and 28% already contained oocytes >180 lm.Estradiol levels were very low in all females at all ages(Table 3).

Males differentiated faster than females. As early asthe first year, we found three differentiating males (outof 25 fish). Most of the males (19 out of 25 sampled)were still immature with testicular lobes containing sper-matogonia. However, some of the males, aged 3 and 4years, had already developed gonads containing sperma-tozoa in some of the lobules.

Fig. 6. Stages of gonadal development in Russian sturgeon females and males. (A) Histological section of a juvenile sturgeon ovary typical for a fishat the age of 3 year; scale bar 100 lM; (B) Ovarian section typical of a fish at the age of 4 year; scale bar 100 lM. All stained with hematoxylin andeosin. Labels: GN, oogonia; AC, adipocytes; OC, oocyte; NL, nucleoli; GV, germinal vesicle; and CA, cortical alveoli. (C) Histological section of ajuvenile sturgeon testis typical for a fish at the age of 1 year; Stage A, scale bar 100 lM. (D) Histological section of a juvenile sturgeon testis typicalfor a fish at the age of 3 year; onset of meiosis; Stage B, scale bar 100 lM. (E). Histological section of a juvenile sturgeon testis typical for a fish at theage of 4 year; Stage C, scale bar 100 lM. All stained with hematoxylin and eosin. Labels: AC, adipocytes; GN, spermatogonia; CS, cysts; and SC,spermatocytes.

Table 3Estradiol (E2) levels in females and 11-KT levels in males sturgeon atdifferent ages

Age (year) E2 (ng/ml ± SEM) n 11-KT (ng/ml ± SEM) n

1 0.239 ± 0.034a 19 0.283 ± 0.05a 192 0.41 ± 0.07a 9 5.22 ± 2.14b 53 0.72 ± 0.06a 8 5.92 ± 0.91b 114 0.48 ± 0.11a 13 5.52 ± 1.60b 5

At the first year E2 and 11KT were measured in all the fish. Meansmarked by different letters differ significantly (p < 0.05).


The presence and amounts of spermatogonia, sper-matocytes, spermatids, and spermatozoa in the testeswere studied semi-quantitatively and randomly overthe entire surface of a mid-longitudinal testis section.Testes were classified into the following three catego-ries: quiescent testes containing no germ cells apartfrom primary spermatogonia and the fat pad (stage

A, Fig. 6 C; stage 1 according to the classificationof Van Eenennaam and Doroshov (1998)); testes con-taining small numbers of spermatogonia (stage B, Fig.6 D; stage 3 according to the classification of VanEenennaam and Doroshov (1998)); and mid-spermato-genesis testes where the majority of the cysts containactive spermatogenesis with spermatocytes and sper-matids as well (stage C, Fig. 6E; stage 4 accordingto the classification of Van Eenennaam and Doroshov(1998)). The appearance of meiotic (from stage A tostage B) as well as post-meiotic germ cells (transitionfrom stage B to stage C) was accompanied by a signif-icant increase not only in GSI values, but also in 11-KT levels (Fig. 7). When the males were groupedaccording to their developmental stage of spermato-genesis, a high correlation was found, not only withtheir GSI values, but also with their plasma 11-KTlevels (Fig. 7).

Fig. 7. Relative levels of 11-KT in plasma and GSI values of malesturgeons grouped according to their developmental stage of sper-matogenesis. See Section 2 and Fig. 6 for classification. Results are theaverage ± SEM (n = 3–14). Means marked by different letters differsignificantly (p < 0.05).


4. Discussion

This paper describes the molecular cloning and se-quence analysis of three GtH subunits, stGPa, stFSHb,and stLHb, from the Russian sturgeon (A. gue-

ldenstaedtii). Molecular characterization of the GtHcDNAs allowed the development of a quantitativereal-time PCR and the investigation of changes in pitu-itary GtH mRNA levels during the early stages of pub-erty in male and female sturgeons.

The sturgeon is a bony fish belonging to the ancientsuper order Chondrostei in the class Osteichthyes, andit was somewhat surprising, to find that the sequencesof the GtH b-subunits were grouped together with thoseof the dogfish (Scyliorhinus canicula) belonging to an-other class of fish, the Elasmobranchii, and also withthose of frogs and higher tetrapods rather than to otherosteichthyans such as the more evolved Teleostei. Simi-lar results have been reported in a comparison of thesesubunit sequences in A. baeri (Querat et al., 2000).

The Russian sturgeon, like other Acipenseriformes, isa tetraploid with 250 chromosomes (2n) (Fontana,1994). Despite the tetraploidy of its genome, only onetype of clone was cloned from each of the GtH subunitsin this study. This suggests only one gene copy for eachsubunit, a conclusion which should be verified viaSouthern blot analysis. A comparison of the deducedamino acid sequence of stFSHb with those of otherGP hormone b-subunits revealed two peculiar charac-teristics which differed from the situation in teleostswhere the N-terminus exhibits an unexpected divergenceat sites of N-glycosylation and cysteine (Yaron et al.,2003). The common pattern of 12 cysteines and twoN-linked glycosylation sites characterizing the tetrapodb-subunits of FSH is conserved in the Russian sturgeon.Hormone glycosylation is important for receptor-medi-ated activation of adenylate cyclase upstream ofG-protein activation (Arey et al., 1997; Beitins andPadmanabhan, 1991).

In the a-subunit, the positions of all 10 cysteines andthe two putative N-linked glycosylation sites of the stur-geon are completely conserved relative to other fish andmammalian species. It also appears that the region fromamino acids 33 to 66 is highly conserved, consisting oftwo paired adjacent cysteines and the first putative N-linked glycosylation site. This region is suggested to beinvolved, in both human (Xia et al., 1994) and red seab-ream (Gen et al., 2000), in the processes of subunitassembly and/or receptor binding. stGPa mRNA con-tains a non-consensus polyadenylation signal (AT-TAAA), which has been found to be the most frequentvariant of the AATAAA signal (Sheets et al., 1990).The same signal motif has also been found in the stripedbass a-subunit mRNA (Hassin et al., 1995), in bothcoho salmon a-subunits (Dickey and Swanson, 2000),and in the tilapia GPa (Gur et al., 2001). The dualityof the a-subunit has been suggested to emerge from aduplication of the entire genome in some of the species(Kobayashi et al., 1997). However, although the Rus-sian sturgeon is tetraploid (Fontana, 1994), only a singlea-subunit was cloned in this study. Both sturgeon GtHb-subunits contain the consensus polyadenylation signal(AATAAA), as has been found in the b-subunits ofseabream, tilapia, and catfish (Elizur et al., 1996; Rosen-feld et al., 1997; Vischer et al., 2003, respectively).

Among GtH subunits, the sequences of the GPa sub-unit show the highest degree of conservation across ver-tebrates (53–91%). Typically, sequence alignmentrevealed a lower degree of amino acid identity betweenthe FSHb (39–51%) subunits for every species, as com-pared with the amino acid identity among the fishLHb subunits (49–68%). This may indicate a more rapiddiversification of FSHb than LHb during evolution, ashas been suggested previously (Querat et al., 2000,2001; Yaron et al., 2003).

In the present study, stFSHb mRNA levels were verylow in fish at early stages of testicular development, in-creased at the more advanced stages of spermatogenesis,and appeared to fall in fish with even more advancedstages of spermatogenesis. stLHb mRNA levels didnot change significantly during the first 4 years of life.In contrast, stFSHb mRNA levels were low during thefirst year, but increased thereafter and remained highin 4-year-old females. mRNA levels of stLHb did notchange significantly during the course of the experiment.Nevertheless, the increase in the stFSHb mRNA in fe-males was not accompanied by either an increase inestradiol levels or any sign of vitellogenesis in the ovary.The expression patterns of stFSHb and stLHb havebeen found to differ at different stages of the reproduc-tive cycle in many fish from different groups: salmonids(Swanson et al., 1991), blue gourami (Jackson et al.,1999), tilapia (Yaron et al., 2001), goldfish (Sohnet al., 2001), Europen eel (Degani et al., 2003), andseabream (Elizur et al., 1996). Pituitary and plasma


concentrations of FSH in white sturgeon were found tobe higher than those of LH during vitellogenesis andearly stages of spermatogenesis. Conversely, pituitaryand plasma concentrations of LH were higher thanthose of FSH during ovulation and spermiation (Mo-berg et al., 1995).

The present study shows sexual dimorphism in FSHbgene expression in immature (2- to 4-year-old) Russiansturgeon, whereby the mRNA levels of FSHb were sig-nificantly higher in females than in males. A somewhatsimilar situation has been reported in goldfish, wherethe increase in FSHb transcripts in males during thebreeding season is less pronounced than in females.The non-synchronous pattern of expression of the twob-subunits in male goldfish was explained by the con-stantly high GSI in the mature-stage testis throughoutthe year with only a gradual increase in androgen levelsduring the spawning season (Sohn et al., 1999).

Sexual dimorphism in GtH subunit mRNA levels hasbeen described in seabreams as well. In both giltheadseabream (Sparus aurata; Elizur et al., 1996) and redseabream (Pagrus major; Gen et al., 2000), FSHbmRNA levels were higher in males than in females,whereas those of LHb were similar in both genders.However, a comparison with the present study wouldnot be valid because the investigated seabreams wereadult fish during the spawning season whereas the fishin the present study were still far from maturity.

In contrast to the situation in salmonids and eels,stLHb mRNA is already detectable in juvenile sturgeonof both genders. The presence of LHb transcripts in thepituitary of immature fish has been reported in anotherlate-maturing fish, the black carp (Mylopharyngodon

piceus), more than 4 years before puberty (Gur et al.,2000), and in the common carp as well (Kandel-Kfiret al., 2002). Although this suggests a different gonado-tropic function during early stages of development, fur-ther research is needed to confirm this notion; it ispossible that in addition to its recognized role during fi-nal oocyte maturation, LH has other endocrine func-tions in juveniles. High levels of LHb have also beenfound in juvenile rainbow trout (Oncorhynchus mykiss;Naito et al., 1991), striped bass (Morone saxatilis; Has-sin et al., 1999), seabream (Gen et al., 2001), and re-cently, in the primitive catfish (Vischer et al., 2003).Moreover, in salmon and trout, LH has been found inthe pituitaries of juvenile fish (Ito et al., 1993; Naitoet al., 1991; Suzuki et al., 1988). In this context, it shouldbe noted that previous studies have indicated the pres-ence of two types of receptor for GtH in different teleo-sts (Oba et al., 2001). It has also been shown that FSHreceptor interacts with both of the homologous LH andFSH, whereas LH receptor interacts specifically withLH in catfish (Vischer and Bogerd, 2003) and salmon(Miwa et al., 1994). The mechanisms underlying gen-der-specific patterns of FSHb gene expression in the

pituitary of male and female sturgeon are still unclear.However, the expression of GtH subunit genes is regu-lated by various endocrine factors, including hypotha-lamic releasing hormones, gonadal steroids andpeptides that can contribute to these changes [reviewedby Yaron et al. (2001, 2003)].

Gametogenesis in Russian sturgeon is generally simi-lar to that in other sturgeon species. Sturgeons aregonochoristic, and intersex is rare. Sturgeon females dis-play group-synchronous oocyte development, withdistinct clutches of vitellogenic and pre-vitellogenic folli-cles. In the hybrid sturgeon, bester, seasonal changes inestradiol, testosterone and vitellogenin levels were wellcorrelated with the progress of oogenesis, and oocytesless than 0.6 mm in diameter were at pre-vitellogenicstages (Amiri et al., 1996b). Females white sturgeon, atlate vitelogenesis stage have oocytes with a diameter of3.33 ± 0.05;mm (Linares-Casenave et al., 2003) andestradiol level of 2–4 ng/ml. This is in agreement withthe present study where we found that all of the femaleshad pre-vitellogenic oocytes with a diameter of 0.05–0.45 mm, and low levels of estradiol. Low levels ofestradiol was also found in juvenile fish of anotherlate-maturing fish species, the black carp (Mylopharyng-

odon piceus) (Gur et al., 2000).Male and female Russian sturgeon have distinctly dif-

ferent rates of sexual maturation. Spermatogenesisoccurs rapidly, as suggested by the testis histology, 11-KT levels and GSI values. Some of the four-year-oldmales already have cysts, most of which contain sperma-tocytes and spermatids. Reproductive development infemales is slower: at the age of 4 years, most of the fe-males sampled in this study were still pre-vitellogenic.In other sturgeons as well, males mature faster and ata younger age than females; in the Atlantic sturgeon,all young sturgeon caught in the river that had sexuallydifferentiated gonads were males (Van Eenennaam andDoroshov, 1998). In Acipenser persicus, sampled in theirnatural habitats, mature males were significantly youn-ger than their female counterparts (Safi et al., 1999).

We found a dramatic increase in the level of 11-KT inmales between the first and second year (Table 3). Thiscan be attributed to either sex differentiation or to theonset of spermatogenesis. In male trout, the male-spe-cific expression of P450c11 (11b-hydroxylase; a key en-zyme catalyzing the synthesis of 11-oxygenatedandrogens) is directed to the involvement of 11-oxygen-ated androgens in testicular differentiation (Govorounet al., 2001; Liu et al., 2000). When GtH is secreted fromthe pituitary, spermatogonial mitosis switches fromstem-cell renewal to proliferation toward meiosis. It ap-pears that GtH does not act directly on germ cells, butrather through the gonadal biosynthesis of 11-KT,which is a major androgen in teleosts (Miura et al.,1991a,b). In various teleosts, this steroid has beenshown to be synthesized in the testis following GtH


stimulation, and high levels have been detected in the ser-um during spermatogenesis [reviewed by Miura andMiura (2003)]. 11-KT has been found crucial for sper-matogenesis in the Japanese eel (Miura et al., 1991a,b),goldfish (Kobayashi et al., 1991), and Japanese huchen(Amer et al., 2001).These findings indicate that 11-KTis one of the factors involved in the initiation of sper-matogonial proliferation toward meiosis. In the hybridsturgeon, 11-KT levels have also been found to be higherduring late spermatogenesis and pre-spermiation andlower at the degeneration stage (Amiri et al., 1999).

In conclusion, we show that the expression levels ofboth GtH b-subunits are higher in 3- and 4-year-old fishthan in 1-year-olds. mRNA levels of stLHb are higherthan those of stFSHb in both genders. Moreover,mRNA levels of stFSHb in females are significantlyhigher than those found in males. We also show thatmale and female Russian sturgeon have distinctly differ-ent rates of sexual maturation. While all females exhib-ited gonads at the pre-vitellogenic stage, with smalloocytes and very low levels of estradiol, 3- and 4-year-old males had testes with spermatids and spermatozoa.Those males were found to have significantly high GSIlevels, stLHb expression and 11-KT levels.

Acknowledgments

This study is supported by a research grant from theIsraeli Ministry of Science, Culture and Sport, RegionalR&D, No. 01-18-00372. We thank Dr. David Kime,Sheffield, for providing the detailed ELISA protocol,as well as the 11-KT antiserum.

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Cloning of FSHb, and glycoprotein subunits from the Russian...

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