Molecular cloning of the cDNA encoding follicle-stimulating hormone β subunit of the Chinese soft-shell turtle Pelodiscus sinensis, and its gene expression

(1)

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Molecular cloning of the cDNA encoding follicle-stimulating

hormone

subunit of the Chinese soft-shell turtle Pelodiscus sinensis,

and its gene expression

Jung-Tsun Chien

a,b

, San-Tai Shen

a

, Yao-Sung Lin

b

, John Yuh-Lin Yu

a,¤

a _{Endocrinology Laboratory, Institute of Zoology, Academia Sinica, Taipei, 115 Taiwan, ROC}

b _{Institute of Ecology and Evolutionary Biology, College of Life Science, National Taiwan University, Taipei, 106 Taiwan, ROC} Received 9 July 2004; revised 22 December 2004; accepted 23 December 2004

Abstract

Follicle-stimulating hormone (FSH) is a member of the pituitary glycoprotein hormone family. These hormones are composed of

two dissimilar subunits,

and . Very little information is available regarding the nucleotide and amino acid sequence of FSH in

reptilian species. For better understanding of the phylogenetic diversity and evolution of FSH molecule, we have isolated and

sequenced the complementary DNA (cDNA) encoding the Chinese soft-shell turtle (Pelodiscus sinensis, Family of Trionychidae)

FSH

precursor molecule by reverse transcription-polymerase chain reaction (RT-PCR) and rapid ampliWcation of cDNA end

(RACE) methods. The cloned Chinese soft-shell turtle FSH cDNA consists of 602-bp nucleotides, including 34-bp nucleotides of

the 5

⬘-untranslated region (UTR), 396-bp of the open reading frame, and 3⬘-UTR of 206-bp nucleotides. It encodes a 131-amino acid

precursor molecule of FSH

subunit with a signal peptide of 20 amino acids followed by a mature protein of 111 amino acids.

Twelve cysteine residues, forming six disul

Wde bonds within -subunit and two putative asparagine-linked glycosylation sites, are

also conserved in the Chinese soft-shell turtle FSH

subunit. The deduced amino acid sequence of the Chinese soft-shell turtle FSH

shares identities of 97% with Reeves’s turtle (Family of Bataguridae), 83–89% with birds, 61–70% with mammals, 63–66% with

amphibians and 40–58% with Wsh. By contrast, when comparing the FSH with the -subunits of the Chinese soft-shell turtle

lutein-izing hormone and thyroid stimulating hormone, the homologies are as low as 38 and 39%, respectively. A phylogenetic tree

includ-ing reptilian species of FSH

subunits, is presented for the Wrst time. Out of various tissues examined, FSH mRNA was only

expressed in the pituitary gland and can be up-regulated by gonadotropin-releasing hormone in pituitary tissue culture as estimated

by

Xuorescence real-time PCR analysis.

 2005 Elsevier Inc. All rights reserved.

Keywords: Follicle-stimulating hormone; cDNA cloning; Amino acid sequence; Chinese soft-shell turtle; Pituitary gland; Gonadotropin-releasing

hormone; mRNA expression

1. Introduction

All vertebrate species are characterized by possession

of a pituitary gland that secretes glycoprotein

hor-mones—gonadotropins and thyrotropin, synthesized,

respectively, by gonadotrops and thyroptrops. There are

two structurally and functionally distinct types of

gonadotropin (GTH): luteinizing hormone (LH) and

follicle-stimulating hormone (FSH). All three pituitary

glycoprotein hormones are composed of two structurally

dissimilar subunits,

and ; subunits are identical for

all three glycoprotein hormones in a species, while

sub-units are speci

Wc and determine the hormonal activity

and species speci

Wcity (

Pierce and Parsons, 1981

). The

¤ _{Corresponding author. Fax: +886 2 2785 8059.}

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subunits are synthesized as separate proteins translated

from di

Verent mRNAs expressed by diVerent genes.

Fol-lowing glycosylation, they are associated by

non-cova-lent bonding to form the biologically active hormone

molecules. The main role of the

subunit is to confer

biological action through the signal transduction

path-way, and that of the

subunit is to convey hormone and

species speci

Wcity. Both subunits are needed for receptor

interaction (

Fox et al., 2001; Lapthorn et al., 1994; Ryan

et al., 1987

).

FSH functions together with LH to promote the

growth and development of gonads, to control

gameto-genesis and to regulate gonadal endocrine functions

(

Moyle et al., 1994

). The structure, function, and

regula-tion of FSH molecules have been investigated most

extensively in mammalian species (

Moyle et al., 1994;

Pierce and Parsons, 1981

). The nucleotide sequences of

cDNA and amino acids of FSH have been studied most

in mammals and Wsh, relatively less in birds and

amphib-ians, and least in reptiles. The cDNAs and deduced

peptide sequence of the FSH

molecule are available for

at least 16 species of mammal (

Belov et al., 1998; Fujiki

et al., 1978; Jameson et al., 1988; Kato, 1988; Koura

et al., 2004; Kumar et al., 1995; Lawrence et al., 1997;

Liao et al., 2003; Maurer, 1987; Sairam et al., 1981;

Schmidt et al., 1999

), 28 species of

Wsh (

Degani et al.,

2003; Han et al., 2004; Hassin et al., 1995; Itoh et al.,

1988; Jackson et al., 1999; Kajimura et al., 2001; Kato

et al., 1993; Lin et al., 1992; Liu et al., 1997; Quérat

et al., 2000, 2001; Suzuki et al., 1988; Weil et al., 1995;

Yoshiura et al., 1997

), 4 species of bird (

Kikuchi et al.,

1998; Koide et al., 1996; Shen and Yu, 2002

; GenBank

Accession No. BAC07314), 4 species of amphibian

(

Hayashi et al., 1992; Komoike and Ishii, 2003; Saito

et al., 2002

; GenBank Accession No. CAC39253), and

one species of reptile (

Aizawa and Ishii, 2003

). These

studies have revealed species diVerences in both

nucleo-tide sequence of FSH cDNAs and the translated

pro-tein sequence of FSH molecule. These studies have also

demonstrated that the intra-animal class homology of

the FSH protein sequence varies greatly between the

classes of vertebrate; for example, the homology among

16 species representative of 8 orders of mammals is 84 %,

while the homology among 24 species representative of 9

orders of

Wsh is only 54%. The Wndings may imply that

the speed of evolution of the FSH

molecule diVers

among di

Verent vertebrate classes. Furthermore, the

inter-class homology of the FSH

protein sequence

var-ies from 40 to 70%. Such high degree of diversity may

imply that the FSH

molecule has undergone

substan-tial alteration of protein sequence during the course of

evolution of vertebrates.

In reptiles, the cDNA encoding FSH

has been

reported, recently, for Reeves’s turtle (

Aizawa and Ishii,

2003

). To our knowledge, this is the only species of

rep-tile whose FSH cDNA has been cloned so far. Reprep-tiles

occupy a key position in evolutionary history of the

ver-tebrates between the amphibians and the birds and

mammals. For better understanding of the phylogenetic

diversity and evolution of the pituitary FSH molecule in

vertebrates, we have cloned FSH

from the Chinese

soft-shell turtle, Pelodiscus sinensis (Family of

Triony-chidae), a reptile that is commercially cultured and easily

accessible in Taiwan. The obtained FSH

cDNA and the

deduced amino acid sequence of the FSH

subunit were

compared to those of Reeves’s turtle (Family of

Batag-uridae) and those of other vertebrates classes. The in

vitro gene expression of FSH mRNA of the Chinese

soft-shell turtle pituitary, as challenged by

gonadotro-pin-releasing hormone was also investigated.

2. Materials and methods

2.1. Animal

Adult male Chinese soft-shell turtles, P. sinensis

(body weight of 700–1000 g), purchased from a local

commercial breeder, were used for this study. All

experi-mental procedures in handing of animals were reviewed

and approved by the Laboratory Animal Ethics

Com-mittee, Academia Sinica.

2.2. Oligonucleotide design

Oligonucleotide primers for the ampli

Wcation of the

Chinese soft-shell turtle FSH

cDNA were designed

based on the conserved region of chicken (

Shen and Yu,

2002

) and quail (

Kikuchi et al., 1998

) FSH

subunits.

The sense primer and antisense primer used for cloning

the Chinese soft-shell turtle FSH

are given in

Table 1

.

Table 1

Primers used for cloning cDNA of the Chinese soft-shell turtle FSH subunit in this study

Primers Nucleotide sequence

Sense primers

P5⬘-1 5⬘-TAC AGG ATG AAG ACA ATT AAC-3⬘ P5⬘-2 5⬘- ATC GCT GTG GAG AAA GAG GAG TGC-3⬘ P5⬘-3 5⬘-GAT ACT GAC AAC ACC GAC TGC ACT-3⬘ 5⬘-AAP 5⬘-GGC CAC GCG TCG ACT AGT ACG GGI

IGG GII GGG IIG-3⬘

Antisense primers

P3⬘-1 5⬘-AAG GAG CAG TAG CTG GGT-3⬘

P3⬘-2 5⬘-AAG GAG CAG TAG CTG GGT-3⬘

P3⬘-3 5⬘-CTC ACA GTG GCA CTC GGT AGC-3⬘ 3⬘-AP 5⬘-GGC CAC GCG TCG ACT AGT AC(T)17-3⬘

AUAP 5⬘-GGC CAC GCG TCG ACT AGT AC-3⬘

-Actin

Sense primer 5⬘-GGT ATT GTG CTG GAC TCT GGT-3⬘ Antisense

primer

(3)

The primers used for ampli

Wcation of the 5⬘ ends of the

Chinese soft-shell turtle FSH

cDNA were designed

from the Chinese soft-shell turtle FSH

subclone

sequence (

Table 1

). The

-actin primers, which served as

a reference for the loading amount of total RNA of the

tissues, were designed based on the Chinese soft-shell

turtle

-actin subcloned sequence (Chien, J.-T. and Yu,

J.Y.-L, unpublished data, Endocrinology Laboratory,

Institute of Zoology, Academia Sinica, Taipei).

2.3. RNA isolation and reverse transcription-polymerase

chain reaction

A total of

Wve experiments were performed for RNA

isolation and reverse transcription-polymerase chain

reaction (RT-PCR) analysis. Three turtles were used in

each experiment. The turtles were sacriWced, pituitary

glands were removed and placed into liquid nitrogen.

Total pituitary cellular RNA was extracted with total

RNA miniprep system kit (Viogene, Sunnyvale, CA).

The quality of RNA was measured at A

_{260 nm}

/A

_{280 nm}

(Pharmacia Biotech UV/visible spectrophotometer,

Ultrospec 3000). Only RNAs with A

_{260 nm}

/A

_{280 nm}

ratios

of 1.6 to 2.0 were used for RT-PCR. Reverse

transcrip-tion was performed using Moloney-murine leukemia

virus (MMLV) reverse transcriptase (MMLV-RT)

(Stratagene, La Jolla, CA), according to the procedure

supplied by the manufacturer. Oligo(dT) primer (100 ng)

and total cellular RNA (500 ng) from pituitary glands

were heated to 65 °C for 5 min. Then, 10 U MMLV-RT

was added to each reaction and the reactions were

incu-bated for 60 min at 37 °C. The PCRs were performed

under hot-start condition (94 °C, 2 min) with pfu Turbo

DNA polymerase (Stratagene, La Jolla, CA) for 35

cycles of 94 °C (0.5 min), 50 and 72 °C (1 min each), and

then 7 min at 72 °C before holding at 4 °C.

2.4. 3

⬘ and 5⬘-rapid ampliWcation of cDNA end

(3

⬘-, 5⬘-RACE)

RACE technique was used to extend the cDNA end

of FSH

sequence including the 3⬘- and 5⬘-UTR in

accordance to the procedures provided by the

manufac-turer (Life Technologies, Gaithersburg, MD). For

3⬘-RACE, 1

g of pituitary RNA was primed with 3⬘

adapter primer (3⬘-AP) and reverse transcribed using

MMLV-RT (Super Script II reverse transcriptase, Life

Technologies, Gaithersburg, MD) as described above.

One microliter (out of 50

l) of RT product was then

ampliWed by PCR with nest forward primer (P5⬘-2) of

the obtained sequence of the Chinese soft-shell turtle

FSH

and abridge universal ampliWcation primer

(AUAP). For 5

⬘-RACE, the Wrst-strand cDNA was

syn-thesized from 1

g of pituitary RNA using the obtained

cDNA sequence (P3

⬘-2) of FSH with MMLV-RT. The

original mRNA template was then removed by

treat-ment with RNase, and the cDNA was puri

Wed by spin

cartridge. A homopolymeric tail of dCTP was added to

the 3

⬘-end of the Wrst-strand DNA by terminal

deoxynu-cleotide transferase. PCR ampli

Wcation was

accom-plished with 5

⬘ ampliWed anchor primer (5⬘-AAP) and a

nest sequence primer (P3

⬘-3). The PCR protocols for

3⬘-end were performed under hot-start condition (94 °C,

2 min) with Taq DNA polymerase (Life Technologies,

Gaithersburg, MD) for 35 cycles of 94 °C (0.5 min), 54

and 72 °C (1 min each), and then 7 min at 72 °C and nest

PCR for 35 cycles of 94 °C (0.5 min), 50 and 72 °C (1 min

each), and then 7 min at 72 °C before holding at 4 °C;

and 5⬘-end were performed under hot-start condition

(94 °C, 2 min) with Taq DNA polymerase (Life

Technol-ogies) for 35 cycles of 94 °C (0.5 min), 50 and 72 °C

(1 min each), and then 7 min at 72 °C and nest PCR for

35 cycles of 94 °C (0.5 min), 60 and 72 °C (1 min each),

and then 7 min at 72 °C before holding at 4 °C.

2.5. Nucleotide and amino acid sequence analysis

Nucleotide sequences of cloned cDNAs or direct

PCR cDNA products were commercially determined by

Xuorescence dye termination reaction (BigDye

Termina-tor Cycle Sequencing Ready Reaction Kit,

Perkin-Elmer, Foster City, CA) and analyzed by automated

DNA sequencer (Perkin-Elmer, Foster City, CA).

Multi-ple protein sequence alignment of FSH

subunits was

performed using ClustalW program (

Aiyar, 2000

).

2.6. Tissue speci

Wcity of FSH gene expression

Two experiments were conducted and two turtles

were used in each experiment. For the tissue speci

Wcity

study of FSH

gene expression, total RNA was isolated,

as described above, from various tissues including

pitui-tary, brain, adipose tissue, thyroid, muscle, liver, heart,

and testis. The same amount of total RNAs (100 ng)

from each tissue was reverse transcribed to

Wrst-strand

DNA, and then subjected to PCR ampli

Wcation (35

cycles) of the entire coding region of the Chinese

soft-shell turtle FSH cDNA by using the primer set P5⬘-1,

P3⬘-1, and the Wrst strand DNA of each tissue was also

subjected to PCR ampliWcation of the Chinese soft-shell

turtle

-actin (35cycles) as a reference for the loading

amount of total RNA. PCR products of cDNA were

revealed by 2.5% agarose gel electrophoresis.

2.7. Regulation of the Chinese soft-shell turtle FSH

mRNA expression

The eVects of gonadotropin releasing hormone

(GnRH) on FSH mRNA levels of the Chinese

soft-shell turtle pituitary were investigated under in vitro

conditions. Mammalian LHRH (acetate salt,

pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH

₂

, Sigma–Aldrich,

(4)

USA) was used in this experiment. Pituitaries were

removed from adult male turtles and washed

immedi-ately in 1£ Hank’s bu

Ver (Sigma–Aldrich, USA) twice,

then placed in sterile serum-free M199 medium plus

antibiotic–antimycotic (Life Technologies, Grand

Island, NY) on ice. Extraneous tissue was removed from

pituitary. Each pituitary was sliced into 4 pieces,

pre-incubated for 1 h at 28 °C, and then pre-incubated with

GnRH at doses of 10

¡8

and 10

¡6

M in 1.0 ml M199

medium plus antibiotics–antimycotic under aeration of

95% O

₂

and 5% CO

₂

for 6 h at 28 °C. The control groups

were without hormonal treatment. At the end of

incuba-tion, pituitary tissues were collected for isolation of

RNA. The RNA was reverse transcribed to the

Wrst-strand DNA as described above. The products were then

subjected to Xuorescence real-time quantitative PCR

analysis to examine the expression level of the Chinese

soft-shell turtle FSH mRNA. The FSH cDNA

con-centration in each sample was Wrst calculated from the

standard curve of FSH cDNA and then divided by the

concentration of -actin to correct the diVerence of

RNA amount in each sample. Fluorescence real-time

PCR was performed to examine the relative mRNA

lev-els using

Xuorescence dye SYBR Green I for continuous

observation of the ampli

Wed cDNA level (

Morrison et

al., 1998

). Such assay allows rapid and accurate quanti

W-cation of initial transcript copy number.

2.8. Construction of a phylogenetic tree of vertebrate

FSH

subunits

A phylogenetic tree of selected vertebrate FSH

s was

constructed based on the aligned amino acid sequences,

and analysed by the neighbor-joining method

(Molecu-lar Evolutionary Genetic Analysis, MEGA, Ver 2.1). For

deriving the con

Wdence value for this analysis, bootstrap

trials were replicated 1000 times. GenBank accession

numbers and references of FSH sequences analyzed in

this study are shown in

Table 2

.

2.9. Statistical analysis

The data obtained from Xuorescence real-time PCR

analysis for the regulation of FSH mRNA expression

Table 2

Species and references of FSHs used for sequence comparison in this study

“—” The sequences were submitted to GenBank only.

Animal class/species ScientiWc name Order Family GenBank Accession No. Reference

Reptiles

Chinese soft-shell turtle P. sinensis Testudinoidea Trionychidae This study

Reeves’s turtle Chinemys reevesii Testudinoidea Bataguridae BAB92948 Aizawa and Ishii (2003) Birds

Chicken Gallus domesticus Galliformes Phasianidae NP989588 Shen and Yu (2002)

Quail Coturnix japonica Galliformes Phasianidae BAC01164 Kikuchi et al. (1998)

Crested ibis Nipponia nippon Ciconiiformes Threskiornithidae BAC07314 —

Ostrich Struthio camelus Struthioniformes Struthionidae P80663 Koide et al. (1996)

Mammals

Ovine Ovis aries Cetartiodactyla Bovidae P01227 Sairam et al. (1981)

Porcine Sus scrofa Cetartiodactyla Suidae AAA31039 Kato (1988)

Equine Equus caballus Perissodactyla Equidae Fujiki et al. (1978)

Human Homo sapiens Primates Hominidae NP000501 Jameson et al. (1988)

Rat Rattus norvegicus Rodentia Muridae BAA00455 Maurer (1987)

Opossum Trichosurus vulpecula Didelphimorphia Phalangeridae AAC71065 Lawrence et al. (1997) Amphibians

Newt Cynops pyrrhogaster Caudata Salamandridae BAB92958 Saito et al. (2002)

Bullfrog Rana catesbeiana Anura Ranidae Q9PS36 Hayashi et al. (1992)

Marsh frog Rana ridibunda Anura Ranidae CAC39253 —

Japanese toad Bufo japonicus Anura Bufonidae BAB93559 Komoike and Ishii (2003)

Chondrichthyan

DogWsh Squalus acanthias Carcharhiniformes Squalidae AJ310344 Quérat et al. (2001)

Chondrostean

Sturgeon Acipenser baerii Acipenseriformes Acipenseridae CAB93504 Quérat et al. (2000) Teleosts

LungWsh Neoceratodus forsteri Ceratodontiformes Ceratodontidae CAE17337 Quérat et al. (2004) European eel Anguilla anguilla Anguilliformes Anguillidae AAN64352 Degani et al. (2003)

GoldWsh Carassius auratus Cypriniformes Cyprinidae BAA13530 Yoshiura et al. (1997)

Rainbow trout Oncorhynchus mykiss Salmoniformes Salmonidae BAB17686 —

Chum salmon Oncorhynchus keta Salmoniformes Salmonidae AAA49408 Itoh et al. (1988)

Striped bass Morone saxatilis Perciformes Moronidae Hassin et al. (1995)

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by GnRH were subjected to one way analysis of

vari-ance (one-way ANOVA). Di

Verences between doses of

the GnRH treated group and the controls (without

hor-monal treatment) were tested by Newman–Keuls’ test

and considered signi

Wcant at P 6 0.05.

3. Results

3.1. Cloning and sequence analysis of FSH

cDNA for the

Chinese soft-shell turtle pituitary

A PCR product containing 396 bp was obtained and

appeared as a single band on a 2.5% agarose gel; it was

identi

Wed to encode the Chinese soft-shell turtle FSH

subunit precursor molecule from its nucleotide sequence.

To determine the remaining 3

⬘ and 5⬘ portions of the

Chinese soft-shell turtle FSH

cDNA sequence, 5⬘ and

3⬘ rapid ampliWcation of cDNA end (RACE) were

per-formed. The cloned Chinese soft-shell turtle FSH

cDNA contained 602-bp nucleotides, including 34-bp

nucleotides of 5

⬘ untranslated region (UTR), 396-bp of

the open reading frame, and 206-bp of 3

⬘ UTR (

Fig. 1

).

No polyadenylation site was identi

Wed in the FSH

cDNA. The precursor protein of the Chinese soft-shell

turtle FSH

subunit contained a putative signal peptide

of 20 amino acids and a mature protein of 111 amino

acids (

Fig. 1

). The mature protein of the Chinese

soft-shell turtle FSH

is compared to FSHs of Reeves’s

tur-tle, avian, mammalian, amphibian, and

Wsh species (

Fig.

2 ). As indicated, 12 cysteine residues of the Chinese

soft-shell turtle FSH are conserved as for other tetrapod

vertebrates at positions 1, 15, 18, 26, 30, 49, 64, 80, 82, 85,

92, and 102. One asparagine-linked glycosylation site,

located at position 5 between the 1st cysteine and the

2nd cysteine, is conserved in the Chinese soft-shell turtle

as in other vertebrates from Wsh through mammals,

while the other asparagine-linked glycosylation site,

located at position 22 between the 3rd cysteine and the

4th cysteine is also conserved in the Chinese soft-shell

Fig. 1. The nucleotide sequence of the Chinese soft-shell turtle FSH cDNA includes 34 bp of 5⬘-untranslated region, 396 bp of coding region, and 206 bp of nucleotide sequence of 3⬘-untranslated region. The predicted open reading frame encodes a precursor protein of 131 amino acid with a sig-nal peptide (SP) of 20 amino acids and a mature protein of 111 amino acids as shown under the nucleotide sequence. The start codon (ATG) and stop codon (TAA) are shown as boxed and shaded. The signal peptide (residues 1–20) is shown by underline.

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turtle as in other tetrapod vertebrates and certain more

primitive Wsh.

3.2. Tissue speciWcity of FSH gene expression

To examine the tissue speciWcity of FSH gene

expression, the entire open reading frame of the Chinese

soft-shell turtle FSH cDNA was ampliWed by RT-PCR

of total RNA from various tissues. As shown in

Fig. 3

,

FSH

mRNA was only expressed in the pituitary, but

not in brain, adipose tissue, thyroid, muscle, liver, heart,

and testis. The nucleotide sequence of FSH cDNA

cloned from pituitary is identical to the FSH nucleotide

sequence described above.

3.3. Regulation of the Chinese soft-shell turtle FSH

mRNA expression by GnRH

To study the regulation of FSH mRNA expression

by GnRH in the Chinese soft-shell turtle, pituitary

Fig. 2. Multiple sequence alignments of FSH subunits. The deduced amino acid sequence of the Chinese soft-shell turtle FSH cDNA are aligned with FSH subunit protein sequences from selected species of diVerent vertebrate groups (see Table 2 for references). For convenience, all FSHs are numbered in accordance with the Chinese soft-shell turtle FSH from the putative N-terminus. Residues identical to the Chinese soft-shell turtle FSH are presented as dots (·). Hyphens (-) have been inserted to show deletion of amino acids in order to obtain maximum homology. Twelve cys-teine residues, forming six disulWde linkages, are shaded. Two putative N-linked glycosylation sites of tetrapods are denoted by 䉲 and lightly shad-owed in gray. The numericals at the right column are the total numbers of amino acids of FSH precursor proteins of the selected vertebrate species. The signal peptides are underlined. * For alignment of maximal homology, the extra Gly residue originally appeared immediately after the 7th Cys was deleted (Saito et al., 2002).

(7)

fragments were cultured and treated for 6h with di

Verent

doses of GnRH. As shown in

Fig. 4

, the FSH mRNA

levels of the Chinese soft-shell turtle pituitaries treated

with GnRH at doses of 10

¡8

and 10

¡6

M as analyzed by

Xuorescence real-time PCR were 151 and 265%,

respec-tively, in comparison to the controls (100%).

4. Discussion

The cloned Chinese soft-shell turtle FSH

cDNA

contains 396-bp nucleotide of the open-reading frame

(

Fig. 1

). It encodes a putative precursor protein molecule

of 131 amino acids with a signal peptide of 20 amino

acids and a mature protein of 111 amino acids. The

deduced amino acid sequence of the Chinese soft-shell

turtle FSH

mature protein shares identities of 94% with

Reeve turtle, 83–89% with birds, 61–70% with mammals,

63–66% with amphibians, and 40–58% with

Wsh (

Table

3 ). The present study demonstrated that the number and

position of 12 cysteine residues and two asparagine

linked glycosylation sites have been conserved in the

Chinese soft-shell turtle FSH and all other tetrapod

vertebrates so far studied (

Fig. 2

).

As indicated in

Fig 2

, the positions of the 12 cysteine

residues in FSH are all conserved in amphibians,

rep-tiles, birds, and mammals so far studied. In certain Wsh

(Elasmobranch: dogWsh; Chondrostean: sturgeon; and

less derived teleosts: goldWsh, salmon, and rainbow

trout) the same 12 cysteine residues are also conserved.

However, in more highly derived teleosts such as

Perci-formes: stripe bass and snakehead

Wsh, the position of

the 3rd cysteine residue is shifted to the N-terminus. By

contrast, the positions of the 12 cysteine residues are all

Fig. 3. The tissue speciWcity of FSH mRNA expression analyzed by

RT-PCR 100 ng of total RNA each from pituitary (lane 1), brain (lane 2), adipose tissue (lane 3), testis (lane 4), heart (lane 5), thyroid (lane 6), liver (lane 7), and muscle (lane 8) was subjected to RT-PCR for FSH cDNA ampliWcation and also -actin ampliWcation (35 cycles) which served as a reference of the loading amount of total RNA for each tis-sue. PCR products of cDNAs were revealed by 2.5% agarose gel elec-trophoresis.

Fig. 4. The eVects of GnRH treatment on the gene expression of the Chinese soft-shell turtle FSH mRNA. (A) A representative Xuores-cence real-time PCR of FSH cDNA for cultured Chinese soft-shell turtle pituitary tissues treated with medium, 10¡8_{M GnRH, and} 10¡6M GnRH, respectively. (B) The increased percentage of FSH mRNA level of the Chinese soft-shell turtle pituitary cultured with two doses of GnRH. Values represent means § SD of three separate experiments (*P < 0.05, **P < 0.01), see Section 2 for further details.

Table 3

Comparison of the percentage identity of mature protein of pituitary FSH subunit nucleotide and amino acid sequences between the Chi-nese soft-shell turtle and other selected vertebrates

The references for FSHs of vertebrates used for comparisons are indicated in Table 2. (a) cDNAs were not cloned; their amino acid res-idues were obtained from the chemical analysis of the isolated FSH.

Species Nucleotide

(Identity, %)

Amino acid (Identity, %)

Chinese soft-shell turtle 100 100

Reeves’s turtle 93 97 Chicken 82 86 Quail 78 85 Crested ibis 79 86 Ostrich (a) 84 Ovine 69 68 Porcine 73 69 Equine (a) 68 Human 73 68 Rat 70 68 Opossum 71 67 Newt 68 63 Bullfrog 61 60 Marsh frog 61 61 Japanese toad 61 61 LungWsh 66 62 DogWsh 60 52 Sturgeon 55 50 European eel 50 44 GoldWsh 47 40 Rainbow trout 48 37 Chum salmon 47 38 Striped bass 40 32 Snakehead Wsh 42 29

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conserved in LH

and TSH in both Wsh and tetrapods

so far studied (

Hsieh et al., 2000; Han et al., 2004;

Komoike and Ishii, 2003; Pierce and Parsons, 1981;

Qué-rat et al., 2001

). Two conserved asparagine (Asn)

N-linked glycosylation sites (Asn-X-Ser/Thr) of FSH

s

observed in mammals, birds, amphibians and certain Wsh

are also found in the Chinese soft-shell turtle FSH

sub-unit (Asn

₅

-Ile-Thr and Asn

₂₂

-Ala-Thr). However, there

is only one N-linked glycosylation site present in certain

other

Wsh. A phylogenetic tree of vertebrate FSHs,

including a reptile for the Wrst time, is presented based

on the homology of their amino acid sequences (

Fig. 5

).

The homologies between amniote vertebrate (reptiles,

birds, and mammals) FSHs are much greater than

between LHs. This fact indicates that FSH subunit is

more conserved than LH subunit during evolution of

amniote vertebrates. These observations agree well with

the LH and FSH bioassay results from our laboratory:

LHs from various tetrapod species show remarkable

variation of potency (>10,000-fold) in the LH bioassays

employing the stimulation of testicular androgen in vitro

(

Yu et al., 1995, 1996; Yu and Wang, 1987

); by contrast,

FSHs from various tetrapod species show less variation

of potencies (»1000-fold) in a FSH bioassay stimulating

17 -estradiol formation in immature rat Sertoli cells in

vitro (

Yu et al., 1996

).

In studies of the GTH-receptor interaction,

Moyle

et al. (1994)

illustrated that hCG/ hFSH chimeras

con-taining human FSH

subunit residues between cysteines

11 and 12 were able to bind FSH receptors with high

a

Ynity and elicited signal transduction. They also found

that the chimeras containing human FSH subunit

resi-dues between cysteines 10 and 11 had low LH activity. It

has been shown that mammalian FSHs, but not LHs,

bind to the FSH receptor on Sertoli cells isolated from

immature male rats, activating aromatase, which is

responsible for estrogen formation from exogenous

androgen (

Dorrington and Armstrong, 1975

). Such a

system has been employed to assay the bioactivity of

mammalian FSHs in vitro (

Padmanabhan et al., 1987;

Shen and Yu, 1991; Van Damme et al., 1979

). We further

demonstrated that both FSHs and LHs of reptiles

(snap-ping turtle) and birds (chicken, ostrich, and turkey) can

stimulate estradiol formation in such an in vitro FSH

bioassay (

Yu et al., 1996

). As indicated in

Fig. 6

, the

amino acids between cysteines 11 and 12 of FSH

sub-units in reptiles, birds, and mammals are identical. It is

interesting to note that the corresponding region of LH

subunits in reptiles and birds is highly similar to that of

FSH

subunits; however, in mammals, such a region of

LH

subunits diVers considerably from that of FSH

subunits. Presumably, the reptilian and avian LHs are

Fig. 5. A phylogenetic tree of vertebrate FSH subunit protein. Data were calculated with Blosum-62-amino-acid substitution matrix and con-structed by neighbor-joining method from the mature protein. The duck pituitary glycoprotein hormone subunit (Hsieh et al., 2001) was used as an outgroup to root the tree. Bootstrap values (100 of 1000 replicant) are indicated. The source and references of the selected FSH data are indicated in Table 2.

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recognized as FSHs by the FSH receptor of Sertoli cells

isolated from immature rat testis. These facts may

explain why both FSH and LH of reptiles and birds are

active in FSH bioassay employing immature rat Sertoli

cells (

Yu et al., 1996

).

The formation and secretion of FSH are mainly

regu-lated by hypothalamic and gonadal factors, such as

GnRH, activin, inhibin, and gonadal steroid hormone.

Hypothalamic GnRH acts directly on synthesis and

release of pituitary LH and FSH in mammals (

Schally

et al., 1971, 1972; Yu et al., 1979

). In reptiles, it has been

demonstrated that chicken-I, chicken II, and

mamma-lian GnRH stimulated in vitro the release of LH in three

species of turtle, and the potencies of these GnRHs in

the stimulation of LH release were similar (

Licht and

Porter, 1985a,b; Licht et al., 1987; Tsai and Licht, 1993

).

The stimulatory action of GnRH on pituitary FSH

release has not been reported previously in reptiles.

GnRH enhances the mRNA levels of FSH

subunits in

mammals (

Attardi and Winters, 1993; Dalkin et al.,

1999

), bird (

Shen and Yu, 2002

), and

Wsh (

Dickey and

Swanson, 2000; Gur et al., 2002; Kandel-K

Wr et al.,

2002

). The present study, has demonstrated that

hypo-physial FSH

mRNA levels of the Chinese soft-shell

turtle pituitary are also promoted by GnRH under static

culture conditions (

Fig. 4

). This is the

Wrst

demonstra-tion in reptiles that GnRH upregulates FSH

mRNA

expression. Our

Wndings together with the observations

reported by others on mammals, birds, and

Wsh support

the proposal that hypothalamic GnRH up-regulation of

FSH

mRNA gene is common to all vertebrates.

Acknowledgments

This study was supported by grants from National

Science Council and Academia Sinica, Taipei, Taiwan,

Republic of China.

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