Parathyroid hormone-related protein (PTHrP) production sites in elasmobranchs

J. Anat. (2002) 201, pp41–52

© Anatomical Society of Great Britain and Ireland 2002

Blackwell Science, Ltd

Parathyroid hormone-related protein (PTHrP) production sites in elasmobranchsM. K. Trivett,1,2 T. I. Walker,3 D. L. Macmillan,2 J. G. Clement,4 T. J. Martin1 and J. A. Danks1

1St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria, Australia, 3065 2Department of Zoology, and 4School of Dental Science, University of Melbourne, Parkville, Victoria, Australia, 3052 3Marine and Freshwater Resources Institute, Queenscliff, Victoria, Australia, 3225

Abstract

This study describes the distribution of parathyroid hormone-related protein (PTHrP) antigen and its mRNA in

seven species of cartilaginous fish from six elasmobranch families. Antigen was detected using antibodies to

synthetic human PTHrP and the mRNA with a riboprobe to human PTHrP gene sequence. The distribution pattern

of PTHrP in the cartilaginous fish studied, reflected that observed in mammals but PTHrP further occurs in some

sites unique to cartilaginous fish. Of particular note was the demonstration of PTHrP in the shark skeleton, which

although considered not to contain bone, may form by a process similar to that forming the early stages of

mammalian endochondral bone. The distribution of PTHrP in the elasmobranch skeleton resembled the distribu-

tion of PTHrP in the developing mammalian skeleton. Differences in the staining pattern between antisera to

N-terminal PTHrP and mid-molecule PTHrP in the brain and pituitary suggested that the PTHrP molecule might

be post-translationally processed in these tissues. The successful use of antibodies and a probe to human PTHrP in

tissues from the early vertebrates examined in this study suggests that the PTHrP molecule is conserved from

elasmobranchs to humans.

Key words calcium-regulating hormone; cartilaginous; fish; immunohistochemistry; in situ hybridization.

Introduction

PTHrP is a mediator of humoral hypercalcaemia of

malignancy (HHM), a condition in which restriction of

calcium excretion by the kidney and release of calcium

from bone results in high plasma calcium levels. Cloning

(Suva et al. 1987) and sequencing (Moseley et al. 1987)

revealed that PTHrP had N-terminal homology with

parathyroid hormone (PTH), the main hypercalcaemic

factor in higher vertebrates, which is produced by the

parathyroid glands. Although little primary sequence

homology exists between the two peptides beyond

residues 1–13, conformational similarities over residues

1–34 allow PTH and PTHrP to activate a common PTH/

PTHrP receptor in mammals (Jüppner et al. 1991). Aspects

of the gene structure of PTH and PTHrP and their chro-

mosomal localization suggest that these two proteins

arose from an ancient gene duplication event (Ingleton

& Danks, 1996). Subsequent studies showed that non-

neoplastic tissues such as skin, kidney, muscle, bone,

mammary tissue and neuroendocrine tissues in

mammals also produce PTHrP (Ingleton & Danks, 1996;

Philbrick et al. 1996). The widespread distribution of

PTHrP in mammalian and avian (Schermer et al. 1991)

tissues suggests multiple physiological roles. These

appear to include the regulation of growth and differ-

entiation of many cell types, relaxation of smooth muscle,

skeletal development and the regulation of calcium

transport across the placenta (Martin et al. 1997).

Fish lack encapsulated parathyroid glands, but

PTH-like substances have been detected in fish plasma

and brain (Harvey et al. 1987; Kaneko & Pang, 1987).

However, fish PTH has not been isolated. More recently,

immunohistochemical and radioimmunoassay data

indicated that bony fish contain PTHrP (Danks et al.

1993). Little is known about the presence of PTH-like

Correspondence Dr Janine Danks, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, 3065, Australia. Tel.: + 61 39288 2480; fax: + 61 39288 2480; e-mail: [email protected]

Accepted for publication 22 April 2002

Tissue distribution of PTHrP in elasmobranchs, M. Trivett et al.


42

peptides in cartilaginous fish (Chondrichthyes), as

bony fish have, until recently, been the main focus of

research in the lower vertebrates. The cartilaginous

fish are a phylogenetically ancient group that includes

the sharks and rays. Two reports indicate that PTHrP

peptides exist in Chondrichthyes. The dogfish, Scylio-

rhinus canicula, contains DNA that hybridizes with an

oligonucleotide probe for chicken PTHrP (Chailleux

et al. 1995) and an independent study demonstrated

the presence of immunoreactive PTHrP in tissues from

the same species (Ingleton et al. 1995).

The present study examined the distribution of

immunoreactive PTHrP and PTHrP mRNA expression in

tissues from seven species of Chondrichthyans from six

different elasmobranch families. The aim of this study

was to gain insight into PTHrP’s tissue distribution in

early vertebrates and determine whether that distribu-

tion was conserved from elasmobranchs to mammals.

This information may be used to elucidate possible

physiological roles in this group of vertebrates.

Methods

Tissue collection

All animals were obtained by researchers at the

Marine and Freshwater Resources Institute (MAFRI)

(Queenscliff, Victoria, Australia) from Port Phillip Bay

(Victoria, Australia). The study was carried out within

the guidelines of the St Vincent’s Hospital Animal Ethics

Committee. Animals were anaesthetized in a solution of

50 mg L−1 MS-222 (Sigma Chemical Company, St Louis,

MO, USA) then decapitated. Fresh samples of skin, kidney

and liver were dissected from gummy sharks, Mustelus

antarcticus (n = 10, one male, one female, remainder

undetermined), school sharks, Galeorhinus galeus

(n = 2, one male, one female), banjo sharks or Southern

fiddler rays, Trygonorrhina fasciata (n = 5, three males,

two females) and common spotted stingarees, Urolo-

phus gigas (n = 2, unknown sex). An expanded range

of tissues including gill, rectal gland, vertebrae, jaw,

pancreas, spleen, heart and whole brain (in most cases

including the pituitary) were collected from gummy

sharks (n = 8, five males, three females), Australian

angel sharks, Squatina australis (n = 6, two males, four

females), southern eagle rays, Myliobatis australis

(n = 4, two males, two females) and Port Jackson

sharks, Heterodontus portusjacksoni (n = 3, two males,

one female). Tissues were fixed in either 10% neutral

buffered formalin (Orion Laboratories, Welshpool,

Australia) for 12–24 h, or Bouin Hollande Sublimate

(BHS) (Kracier et al. 1967) for 48–72 h. After dehydration

and clearing, all tissues were embedded in paraffin.

Immunohistochemistry (IHC)

Sections for immunohistochemistry were cut at 5 µm

and mounted on slides coated with 2% triethyoxy-

propyl silane (Sigma) in acetone. Rabbit antisera raised

to synthetic human N-terminal PTHrP(1–14) and (1–16),

and to the mid-molecule region of synthetic human

PTHrP(67–84) were used. PTHrP IHC followed a stand-

ard immunoperoxidase technique (Sternberger et al.

1970; Danks et al. 1989). The antiserum to PTHrP(1–14)

has previously been used on fish tissues (Danks et al.

1993; Ingleton et al. 1995). N-terminal antiserum used

in the current study showed no cross-reactivity with

human PTH either in Western blot or radioimmunoassay

(Danks et al. 1989). All incubations were conducted at

room temperature. Briefly, sections were dewaxed in

two changes of xylene then washed in 100% ethanol.

Immersion of sections in methanol with 1% hydrogen

peroxide for 30 min blocked endogenous peroxidase

activity. Sections were washed in three changes of

phosphate-buffered saline, pH 7.6 (PBS), 60 s each.

Incubation in 10% normal swine serum (Institute of

Medical and Veterinary Sciences, Adelaide, Australia),

diluted in PBS/5% new born calf serum (NCS) (Common-

wealth Serum Laboratories, Melbourne, Australia),

blocked non-specific binding sites. Excess swine serum

was tipped off the slides after 30 min and the primary

PTHrP antiserum applied at dilutions between 1 : 25 and

1 : 200 for 60 min. Human skin was used as a positive

control in each assay and was stained at the same

dilutions as the fish tissues. Unbound antiserum was

washed off the slide in three changes of 5% NCS/PBS,

10 min each. The secondary antibody, swine anti-rabbit

immunoglobulins (Dako, Glostrup, Denmark), was

diluted 1 : 40 in 5% NCS/PBS and incubated with the

sections for 30 min. After two 5-min washes in 5% NCS/

PBS and one 5-min wash in PBS, the peroxidase anti-

peroxidase complex (Dako), diluted to 1 : 80 in PBS, was

applied to the sections for 30 min. Sections were washed

twice in PBS, 5 min each, then equilibrated in 0.05 M

Tris buffer (pH 7.6). The slides were immersed in 200 mL

0.05 M Tris buffer with 100 mg 3′,3′-diaminobenzidine

(Sigma) and 1 mL hydrogen peroxide, for 7 min, to

detect the antibody complex. After three washes in



43

distilled water, each of 5 min, the sections were

counterstained in Harris’s haematoxylin, washed in

distilled water and dehydrated through a graded

ethanol series. Slides were cleared in xylene and

mounted in DePeX®.

Tissues fixed in BHS were washed to remove mercuric

chloride from the sections, and the above protocol was

modified as follows. After dewaxing and washing in

100% ethanol, sections were immersed in 1% iodine,

and then 5% sodium thiosulphate in distilled water,

and finally washed in running tap water. The steps

including and after the endogenous peroxidase block

were not modified.

All tissues were assayed in duplicate and the controls

included: (i) human skin as a positive tissue control in

each experiment; (ii) non-immune rabbit serum sub-

stituted for the primary antibody served as the negative

control in each experiment; (iii) the deletion of alternate

layers of the antibody sandwich served as a method

control; (iv) confirmation of the specificity of staining

for the PTHrP antigen, by staining randomly selected

sections with antiserum to human PTH(1–34) (BioGenex,

San Raman, USA) or with an antiserum to chum salmon

growth hormone as described by Danks et al. (1993).

In situ hybridization (ISH)

A 420-base-pair riboprobe to Exon VI of human PTHrP

was labelled with digoxigenin (DIG) (Roche Molecular

Biochemicals, Mannheim, Germany) to examine PTHrP

mRNA expression. The probe spans a region that shows

conservation among known PTHrP sequences and has

been used to examine PTHrP mRNA expression in

mammalian tissues (Kartsogiannis et al. 1997). The

protocol employed an alkaline phosphatase detection

system as described by Zhou et al. (1994) and Kartsogiannis

et al. (1997). Steps before hybridization required sterile

glassware and solutions diluted with water treated

with diethylpyrocarbonate (DEPC) (Calbiochem-

Novabiochem Corporation, La Jolla, CA, USA) to remove

RNase contamination. All steps were conducted at

room temperature unless specified otherwise. Briefly,

sections were dewaxed in three changes of xylene,

hydrated through a graded ethanol series, and then

washed in DEPC-water, 5 min at each step. Treating

sections with 0.2 M HCl for 20 min blocked endogenous

alkaline phosphatase activity. Slides were washed in

DEPC-water, two 15-min washes, and then equilibrated

in 100 mM Tris-HCl pH 8.0/50 mM EDTA for 5 min. The

crosslinks formed during fixation were digested by

incubation with 2–4 µg Proteinase K (Roche), diluted

in 100 mM Tris-HCl pH 8.0/50 mM EDTA, for 30 min at

37 °C. Washing the sections in 2 mg mL−1 glycine in

DEPC-PBS for 5 min stopped the reaction. After wash-

ing in DEPC-PBS, two washes, each of 15 min, the

sections were post-fixed in cold 4% paraformaldehyde

(Sigma)/PBS for 15 min. Sections were washed twice,

15 min each, in DEPC-PBS before prehybridization

for 60 min at 42 °C in a buffer of 50% deionized

formamide/5× SSC/2% blocking reagent (Roche)/0.02%

sodium dodecyl sulphate (SDS) (Bio-Rad Laboratories,

Hercules, USA)/0.1% N-laurosarcosine (Sigma). Pre-

hybridization buffer was tipped off the slide and the

hybridization buffer, containing the same constituents

as the prehybridization buffer but with the addition of

4 ng mL−1 of labelled riboprobe, was added. Slides were

hybridized overnight at 42 °C in a humid chamber.

Steps after hybridization did not require sterile

glassware or Rnase-free solutions. Unbound probe was

removed by a 15-min wash in 2× SSC at 37 °C and by

incubation with 25 µg mL−1 RNase A (Roche)/2× SSC

at 37 °C for 30 min. Stringency washes, 15 min each,

continued at 37 °C in one wash of 2× SSC, two changes

of 1× SSC and two changes of 0.1× SSC. Slides were

equilibrated for 5 min in buffer 1 (0.1 M maleic acid/

0.15 M NaCl) and non-specific binding sites blocked by

a 30-min incubation in a blocking medium of PBS/30%

non-immune rabbit serum/3% bovine serum albumin

(Commonwealth Serum Laboratories)/0.1% Triton X-

100. The primary antibody, sheep alkaline phosphatase-

conjugated anti-DIG immunoglobulins (Roche), was

diluted 1 : 500 in blocking medium and applied for

60 min. Unbound antibody was removed with two 15-min

washes in 0.3% Tween 20/buffer 1, and then a further

15-min wash in buffer 1 alone. Sections were equilibrated

in buffer 3 (100 mM Tris pH 9.5/100 mM NaCl/50 mM MgCl2)

for 3 min. Slides were then placed in a chromogen with

45 µL 4-nitroblue tetrazolium chloride (Roche) and

35 µL 5-bromo-4-chloro-3-indoyl-phosphate (Roche) as

the substrate, diluted in 10 mL buffer 3 that had been

filtered through a 0.2-µm filter (Acrodisc, MI, USA).

Slides were left overnight in the dark for colour to

develop. They were then washed in distilled water and

counterstained in Nuclear Fast Red (Sigma) or Fast

Green (Sigma). Sections were mounted in Clearmount

(Zymed, San Francisco, CA, USA), then DePeX.

All tissues were assayed in duplicate and a panel of

controls was included in each experiment: (i) human



44

skin served as the positive control tissue in each experi-

ment; (ii) sections treated with 150 µg mL−1 RNase A

(Roche)/2× SSC, 2 h at 37 °C prior to hybridization acted

as a negative control; (iii) the specificity of the anti-DIG

sheep polyclonal antibody (Boehringer Mannheim)

was confirmed using no probe controls with antibody,

and endogenous alkaline phosphatase activity was

assessed using no probe slides with antibody omitted as

a standard.

Von Kossa staining

Von Kossa staining (Bancroft & Stevens, 1990) of

undecalcified sections identified areas of calcified

cartilage in elasmobranch vertebrae. Safranin O (Sigma),

1%, was used as a counterstain.

Analysis of IHC and ISH results

Results from immunohistochemistry and in situ hybrid-

ization were examined by light microscopy. Sections

were considered to stain positive for the PTHrP antigen

if brown staining was observed or, in the case of the

PTHrP mRNA, if purple/blue hybridization signal was

visible. Scores for the intensity of staining for the PTHrP

antigen and hybridization for PTHrP mRNA were given.

Scores were presented as: –, no staining (or hybridiza-

tion) in the tissue; +, weak; ++, moderate; and +++,

strong. In tissues where different cell types showed

different intensities of staining or hybridization, scores

were given for each major cell type. The variation in

staining or hybridization intensity between different

experiments was accounted for by comparing the

human skin positive controls included in every experi-

ment for both the immunohistochemical and the

hybridization studies.

Results

The distribution of immunoreactivity and hybridization

signal was similar in all the elasmobranchs studied.

Unless otherwise stated, PTHrP immunoreactivity and

hybridization signal were confined to the cell cyto-

plasm and the patterns of staining between the N-

terminal and mid-molecule antibodies were the same,

except in the case of the brain and pituitary. Table 1

summarizes the distribution of PTHrP antigen and

mRNA in elasmobranch tissues, and presents the scores

given for the intensity of staining or hybridization in

each tissue. No staining was observed when PTH anti-

serum or an unrelated rabbit antiserum was used.

Skin

Moderate PTHrP immunoreactivity was observed in

the epidermal cells immediately next to the dermal

denticles in elasmobranch skin but was weak through-

out the remainder of the epidermis (Fig. 1a). In animals

Table 1 Summary of PTHrP distribution in elasmobranch tissues by IHC and ISH

Tissue Cell/structure type IHC ISH

Skin epidermal cells + +mature denticles – –developing denticles ++ +++dermis – –

Muscle skeletal ++ ++cardiac + +

Gill lamellar epithelium + +interlamellar epithelium ++ +++

Kidney proximal and distal tubules ++ ++neck segments – +glomeruli – +

Rectal gland tubular epithelium ++ +canal epithelium + ++

Liver hepatocytes – –vessel and duct epithelium + +

Pancreas acinar cells – –vessel and duct epithelium + +

Spleen erythrocyte-rich area + +lymphocyte-rich area – –

Notochord inner sheath cells – –outer sheath cells + +epithelial cells + ++vacuolated notochordal cells ++ ++

Vertebrae cartilage matrix – –perichondrium + +uncalcified chondrocytes ++ ++calcified chondrocytes – –

Jaw and teeth epithelium ++ not doneodontoblasts ++ not donechondrocytes – not done

Brain neurones in tectum + +choroid epithelium + +saccus vasculosus epithelium + +Purkinje cells + +preoptic neurones* ++ –

Spinal cord grey matter + ++white matter – –canal epithelium + +

Pituitary neurointermediate lobe* + +pars distalis + +

Scores: –, no staining (or hybridization) in the tissue; +, weak; ++, moderate; and +++, strong. *Staining pattern varied between N-terminal and mid-molecule antisera.



45

Fig. 1 (a) Gummy shark skin immunostained with PTHrP(1–14) antiserum. PTHrP antigen is seen in the epidermis (E), immediately adjacent to the spine (S) of the mature dermal denticle, with weaker staining throughout the remainder of the epidermis, but not in the dermis (D). Pigment (black) is visible. (×110). (b) In situ hybridization of gummy shark skin showing PTHrP mRNA in the epithelium (E) and pulp cavity (P) of developing denticles. Weak signal for the PTHrP mRNA occurs throughout the remainder of the epidermis but not in the dermis (D) (×110). (c) Section of shark gill immunostained with N-terminal PTHrP antiserum showing PTHrP antigen in the crypt or ‘chloride’ cells (Cr), epithelium of the interlamellar (I) and secondary lamellar (S) epithelium. The PTHrP antigen is not seen in the gill ray (R). (×110). (d) Tubules (T) in angel shark rectal gland are tightly packed and interspersed with connective tissue (CT). PTHrP immunoreactivity is seen in tubular epithelial cells and in scattered blood cells (B) but not in the connective tissue. (×110). (e) PTHrP immunohistochemistry showing PTHrP antigen in proximal (P) and distal (D) tubules of the kidney. Antigen is absent from the neck segments (N) and glomeruli (G). (×55). (f) Non-immune control of 1e, showing absence of PTHrP staining (lettering as in 1e, ×55).



46

where prominent dermal denticles were absent, such

as the spotted stingaree, PTHrP antigen was observed

in cells in the basal layers of the epidermis (not shown).

Hybridization signal for PTHrP mRNA was evenly dis-

tributed throughout the epidermal cells in sharks and rays

(Table 1). There was no immunoreactivity or hybridiza-

tion signal in mature dermal denticles and the dermis

(Fig. 1a,b and Table 1). The epithelial cells and dermal

cells (odontoblasts) of developing denticles contained

PTHrP immunoreactivity (Table 1) and mRNA (Fig. 1b).

Muscle

PTHrP antigen and mRNA were seen in skeletal and

cardiac muscle but not within connective tissue dis-

persed within the muscle (Table 1).

Gill

PTHrP immunoreactivity (Fig. 1c) and hybridization

signal (Table 1) occurred in the interlamellar epithelium,

and the crypt or ‘chloride’ cells of the gill. Weaker stain-

ing was seen in the epithelial cells of the secondary

lamellae (lamellar epithelium). The gill rays and carti-

lage in the gill bar showed little or no PTHrP immuno-

reactivity, and did not hybridize the PTHrP riboprobe.

Rectal gland and kidney

Epithelial cells of the secretory tubules and the central

canal of the rectal gland reacted with PTHrP antisera

(Fig. 1d) and the PTHrP riboprobe (Table 1). Staining

and hybridization were not seen in the connective

tissue between tubules and the mucous cells in the

central canal epithelium. The proximal and distal

tubules in the kidney displayed PTHrP immunoreactiv-

ity while the glomeruli, collecting tubules and neck

segments reacted weakly, if at all, with the PTHrP

antiserum (Fig. 1e). The pattern of mRNA distribution

was more diffuse with weak to moderate hybridization

signal seen throughout the kidney (Table 1).

Liver, pancreas and spleen

PTHrP immunoreactivity and hybridization signal

were not found in hepatocytes but were observed in

the epithelium of the vessels and ducts within the liver

(Table 1). PTHrP antigen and mRNA were found in the

epithelium of small vessels and ducts throughout

the pancreas but not in acinar cells in the pancreas

(Table 1). Islet tissue was not visible in the samples

studied. PTHrP immunoreactivity and hybridization

signal was observed in the erythrocyte-rich regions and

in the epithelium of small vessels in the spleen, but not

in the lymphocyte-rich regions (Table 1).

Skeletal tissues

PTHrP was observed in elasmobranch skeletal tissues,

such as the notochord, vertebrae and teeth (Table 1).

The elasmobranch vertebral column contains remnants

of the notochord and von Kossa staining identified

sites of calcification in the elasmobranch vertebral

column (Fig. 2a). The elasmobranch vertebral column

is composed of non-articulated biconcave vertebral

bodies (centra) which are linked together by the remains

of the notochord and by intervertebral cartilages (Leake,

1975). Each vertebra has a neural arch to protect the

spinal column and a haemal arch that surrounds the

dorsal aorta (Leake, 1975). PTHrP antigen and mRNA

were present in the vacuolated cells and epithelial cells

of the notochord (Table 1). PTHrP was not seen in the

matrix of the elastic sheath but was observed in the

cells within calcified regions of the outer notochordal

sheath (Table 1). PTHrP immunoreactivity and hybrid-

ization signal were observed in chondrocytes that had

not yet become incorporated into the calcified cartilage

in gummy shark vertebrae and in the perichondrium

(Fig. 2b). These chondrocytes, scattered throughout the

uncalcified matrix close to calcifying front, contained

PTHrP, but most of the chondrocytes deep within this

matrix did not (Fig. 2b). PTHrP immunoreactivity was

absent in the cartilage of the jaw but was observed in

the epithelia and odontoblasts of developing teeth

(Fig. 2c). PTHrP was not seen in the dentine of the teeth

or in the underlying dermis Fig. 2(c).

Nervous system

PTHrP immunoreactivity occurred in a number of

discrete sites in the brain whereas sites of mRNA

expression were more diffuse. Cerebellar Purkinje cells

displayed PTHrP immunoreactivity and hybridization

signal but cells in the granular and molecular layers did

not (Table 1). Large neurones adjacent to the ventricle

in the tectum stained for the PTHrP antigen (Fig. 3a,b)

but the hybridization signal was diffuse in this region.

Epithelial cells of the choroid plexus (Fig. 3c) and saccus



47

vasculosus (an organ unique to some bony and carti-

laginous fish) produced PTHrP protein and mRNA

(Table 1). PTHrP antigen and mRNA were observed

within grey matter of the spinal cord and in ependymal

cells lining the central canal (Fig. 3d) but not in white

matter (Table 1). The pre-optic area in the brain reacted

differently with the N-terminal and mid-molecule PTHrP

antisera. PTHrP-positive neurones were observed when

a mid-molecule antibody was used but when an N-

terminal antibody was substituted, the staining was

diffuse, as was the hybridization signal.

Pituitary

The elasmobranch pituitary demonstrated differential

staining with the N-terminal and mid-molecule PTHrP

antibodies. These differences appeared to be independ-

ent of the sex and species of the animal. Mid-molecule

PTHrP immunoreactivity was detected in the nervous

tissue between endocrine cells of neurointermediate

lobe (pars intermedia and pars nervosa) and in discrete

sites within the infundibulum (Fig. 3e). N-terminal

PTHrP immunoreactivity occurred in the endocrine

cells defined as such by their histology and anatomical

relationship, of the neurointermediate lobe and pars

distalis (Fig. 3f). Hybridization signal was observed in

the pars distalis and the pars intermedia (Table 1).

Discussion

Data from this study demonstrate that PTHrP is widely

distributed among elasmobranchs. It extends the

Fig. 2 (a) Von Kossa staining shows the sites of calcification (brown) in the gummy shark vertebrae. The outer notochordal sheath (O) is calcified but the inner sheath (I) is not. Vaculoated notochordal cells are not visible. Calcified cartilage (CC) occurs at the periphery of the vertebra and in association with the outer notochordal sheath. Uncalcified cartilage appears orange to red. Associated muscle (M) is visible. (×14). (b) PTHrP immunoreactivity is seen in the cytoplasm of chondrocytes (arrow) at the edge of the calcification front that lies between the calcified cartilage (CC) and perichondrium (P). Chondrocytes within the uncalcified cartilage (C) stain for PTHrP antigen, as does skeletal muscle (M). (×137.5). (c) Staining for the PTHrP antigen in the developing tooth is seen in the odontoblasts (O), and the epithelial cells (E) overlying and adjacent to the developing tooth. Some scattered cells in the pulp cavity (P) stain, but the dentine (D) does not. (×110). (d) An adjacent section stained with antiserum to PTH (1–34) shows no immunoreactivity (lettering as in 2c, ×110).



48

evolutionary history of PTHrP past teleosts where there

has been the identification of a receptor that recogn-

izes human PTHrP (Rubin & Jüppner, 1999) and the

isolation and analysis of the fish (Fugu rubripes) PTHrP

gene (Power et al. 2001). The presence of PTHrP in

bony fish may not be relevant to its localization in

elasmobranchs, as there is some controversy about

the evolution of these two groups. Some researchers

Fig. 3 (a) Immunohistochemistry shows staining in neurones (N) in the tectum that border the ventricle (V). The ventricle is lined by a layer of ependymal cells (Ep). The surrounding tissue is negative. (×110). (b) No staining of the neurones, ependymal cells lining the ventricle is seen with the PTH antiserum (lettering as in 3a, ×110). (c) Immunohistochemistry shows PTHrP antigen in epithelial cells (E) of the choroid plexus and in ependymal cells (Ep). Only scattered cells in the capillaries (C) stain for the PTHrP antigen. (×55). (d) In the spinal cord, hybridization signal for the PTHrP mRNA is seen in the grey matter (G) and, to a lesser extent, in ependymal cells lining the central canal (C). No hybridization is seen in white matter (W). (×55). (e) Angel shark pituitary incubated with mid-molecule PTHrP antiserum shows staining at the edge of the neurointermediate lobe (NL) and the stalk (S) connecting the pituitary to the diencephalon (D). Part of the saccus vasculosus (SV) is visible. (×55). (f) An adjacent section incubated with N-terminal PTHrP antiserum shows staining in the endocrine cells of the neurointermediate lobe but not in the stalk or diencephalon. Part of the saccus vasculosus is visible (lettering as in 3e, ×55).



49

suggest that elasmobranchs arose from a common

stock that gave rise to the Chondrichthyes and the

bony fish (Compagno, 1977; Schaeffer & Williams,

1977) but others suggest that they arose from a differ-

ent stock to the bony fishes (Gilbert, 1993).

The presence of PTHrP antigen and mRNA in sites

such as the gill, kidney and rectal gland suggests

involvement in osmoregulation. PTHrP antigen and

mRNA was located in the intraepithelial lamellae as

well as in the crypt or ‘chloride’ cells. The gill inter-

lamellar epithelium in teleosts has roles in osmoregula-

tion and chloride cells have been identified in the

interlamellar epithelium in elasmobranchs (for review

see Evans, 1993) but their numbers are lower than

those seen in teleosts (Bone et al. 1995). However,

there is no direct evidence that these cells function in

salt secretion in elasmobranchs (Evans, 1979) and the

branchial Na+/K+-ATPase activity is low. If new data do

provide evidence that the gill epithelium participates

in iono- or osmo-regulation in elasmobranchs, it is

possible that PTHrP could be involved in this process.

This would be consistent with its localization to sites

that are known to participate in osmoregulation, such

as the kidney and rectal gland.

PTHrP increases phosphate excretion and restricts

calcium excretion in the mammalian kidney (Ebeling

et al. 1989; Rizzoli et al. 1989; Zhou et al. 1989). The

sites of PTHrP localization in the elasmobranch kidney

suggest that PTHrP may be a regulator of sodium

and phosphate transport, and possibly of other ions

and urea. The kidney in elasmobranchs regulates salt,

urea and water balance (Henderson et al. 1986), and

maintains the plasma of the shark hyperosmotic to

seawater. Marine elasmobranchs maintain high con-

centrations of urea and trimethylamine oxide (TMAO)

in the plasma so that blood osmolarity is close to that

of seawater (review: Pang et al. 1977), and as a result,

suffer little osmotic loss of water. The kidney tubules in

elasmobranchs regulate the excretion of sodium and

chloride and divalent ions and reabsorption of urea

and TMAO (Hickman & Trump, 1969; Pang et al. 1977).

PTHrP increases phosphate excretion in perfused rat

kidneys (Ebeling et al. 1989) and may affect sodium-

dependent phosphate transport (Pizurki et al. 1988)

and regulate sodium transport (Caverzasio et al. 1988)

in opossum kidney cells. Studies of the effects of PTHrP

on the elasmobranch kidney would be necessary to

determine whether it has similar effects in the kidney

of lower vertebrates.

The rectal gland is unique to elasmobranchs. (Evans,

1993). The rectal gland acts as a site of extrarenal secre-

tion of excess NaCl in marine elasmobranchs and

involves an Na/Cl co-transport system (for a review, see

Evans, 1993). This transport system is in turn dependent

upon the Na/K exchange driven by the Na+/K+-ATPase

which is found in this gland (Evans, 1993). Similarities

exist in the NaCl transport system in the rectal gland

with that found in the loop of Henle (thick ascending

limb) in the mammalian kidney. As the rectal gland

represents a novel site of PTHrP distribution within ver-

tebrates, it may be that it has some as yet undefined

roles in this organ.

Results from the current study indicate that the

pattern of distribution of PTHrP in the elasmobranch

rectal gland and gill is conserved between different

families of elasmobranchs. Physiological investigation

is required to determine whether PTHrP has novel roles

in the gill epithelium. Fugu rubripes N-terminus PTHrP

was found to have a stimulatory action on calcium

uptake in sea bream larvae implying that PTHrP might

be a hypercalcaemic factor in teleosts (Guerreiro et al.

2001). They hypothesized that PTHrP might act on the

gill chloride cells and the intestinal epithelium prob-

ably through specific receptors. Whether PTHrP acts in

this manner in elasmobranchs cannot be investigated

until elasmobranch PTHrP has been isolated and its

homology with teleost PTHrP determined.

The results of the current study, together with previ-

ous work on dogfish (Ingleton et al. 1995), suggest that

PTHrP has a generalized distribution in the epithelium

forming the choroid plexus and saccus vasculosus

of elasmobranchs. The demonstration of PTH/PTHrP

receptors in these epithelia in the red stingray (Akino

et al. 1998) suggests that PTHrP may be physiologically

active in these tissues. It has been proposed that the

epithelium of the choroid plexus is secretory and

involved in the regulation of ion gradients between

the cerebrospinal fluid and the blood (Cserr, 1971; Cserr

& Bundgaard, 1984). The saccus vasculosus, a tissue

unique to some bony and cartilaginous fish, may also

represent a transporting epithelium involved in

osmoregulation (Jansen et al. 1981, 1982). PTHrP is

secreted by the saccus vasculosus in bony fish (Devlin

et al. 1995). Ingleton et al. (1995) proposed that

epithelial cells in the saccus vasculosus and the choroid

plexus may be a source of factors, such as PTHrP,

secreted into the cerebrospinal fluid, or provide a

system for transporting neuronal products from the



50

cerebrospinal fluid into the circulation. Distribution

data for PTHrP mRNA from the current study suggest

that the immunoreactive PTHrP in these sites is produced

in situ.

The Chondrichthyes are characterized by an internal

skeleton composed of cartilage (Compagno, 1977;

Schaeffer & Williams, 1977), parts of which may calcify.

Although bone appears to be absent in modern elasmo-

branchs, it was probably present in ancestral carti-

laginous fish (Clement, 1992). Histological data suggest

that the formation of the mineralized cartilaginous

skeleton in elasmobranchs mimics the early stages of

endochondral ossification in mammals (Clement,

1992). The presence of PTHrP in chondrocytes outside

the calcification zone in shark vertebrae resembles its

appearance in chondrocytes during early endochon-

dral bone formation in mammals (Lee et al. 1995). The

absence of PTHrP in chondrocytes embedded in calci-

fied cartilage, and those deep within uncalcified

matrix, is similar to data demonstrating that osteocytes

deep within mammalian bone matrix are not PTHrP

immunoreactive (Kartsogiannis et al. 1997). PTHrP has

a central role in mammalian skeletal development

through modulation of chondrocyte proliferation and

differentiation (Wysolmerski & Stewart, 1998) and

this role may have been conserved during vertebrate

evolution. The similarity between the distribution of

PTHrP in the elasmobranch skeleton and that in the

developing mammalian skeleton is consistent with

the idea that bone existed in ancestral elasmobranchs.

The demonstration of PTHrP within the notochord may

reflect an early evolutionary association of PTHrP with

vertebrate skeletal elements. The functions that PTHrP

has in the growth of the elasmobranch skeleton remain

to be investigated.

Differences in staining patterns between N-terminal

and mid-molecule PTHrP antisera in the elasmobranch

pituitary and pre-optic nucleus suggest that post-

translational processing of the PTHrP molecule may

occur in these sites. The pre-optic nucleus is found in

all fishes and amphibians and is regarded as the

homologue of supra-optic and paraventricular nuclei of

amniotes (Holmes & Ball, 1974). Differences in staining

between antiserum to N-terminal PTHrP(1–16) and C-

terminal PTHrP(107–111) were observed in the flounder

pituitary (Danks et al. 1998) and these data support

the idea of post-translational processing of PTHrP in

fish. Post-translational processing of the PTHrP mole-

cule in mammals yields a family of biologically active

peptides (Philbrick et al. 1996). The pars distalis of the

elasmobranch pituitary displayed N-terminal PTHrP

immunoreactivity, which is consistent with observa-

tions from the dogfish (Ingleton et al. 1995) and the

sea bream (Danks et al. 1993), suggesting that N-

terminal PTHrP released from the fish pituitary may

act as a classical hormone. Another possibility is

PTHrP in the pars distalis could regulate other pituitary

hormones, such as prolactin or growth hormone, via

autocrine or paracrine pathways.

The distribution of PTHrP mRNA shown in this study

indicates that the immunoreactive PTHrP observed in

these tissues is produced in situ. Although immuno-

reactive PTHrP was not found in the dogfish gill (Ingleton

et al. 1995), the demonstration of PTHrP in the gills of

elasmobranchs from six families suggests that it occurs

widely in the elasmobranchs. Additionally, PTHrP has

been demonstrated in the bony fish gill (Danks et al.

1998). It is possible that PTHrP in sites such as the gill,

rectal gland and saccus vasculosus, tissues not found in

higher vertebrates, will perform tasks not yet revealed

by studies in mammals.

The occurrence of PTHrP in elasmobranch tissues

such as skin, muscle, kidney, spleen, teeth and brain

mirrors the production sites in mammals (Ingleton &

Danks, 1996; Philbrick et al. 1996) and amphibians

(Danks et al. 1997), and this suggests that the sites of

PTHrP production have been conserved in the verte-

brates. It is therefore possible that functions such as

the modulation of differentiation and proliferation

that are associated with these mammalian tissues

may also be present in submammalian vertebrates such

as elasmobranchs. In addition, it is possible that the

distribution of PTHrP will vary during development in

elasmobranchs as it does in mammals. The absence of

PTHrP in the adult elasmobranch liver parallels a similar

absence in mammals, so examination of embryonic

elasmobranchs may demonstrate that PTHrP is pro-

duced by fetal hepatocytes, as it is in fetal mammals

(Ingleton & Danks, 1996; Philbrick et al. 1996).

This study demonstrated widespread distribution of

PTHrP antigen and mRNA in cartilaginous fish includ-

ing phylogenetically ancient species such as the Port

Jackson shark. The tissue distribution of PTHrP in this

group of vertebrates showed some similarity to the

tissue distribution in higher vertebrates. The pattern

of PTHrP distribution in the shark vertebra was of

particular interest, as it resembled the pattern of PTHrP

distribution observed during early endochondral bone



51

formation in mammals. The widespread distribution of

PTHrP in lower vertebrates such as elasmobranchs sug-

gests that it may have a similar variety of physiological

roles as it does in mammals. Since PTHrP was also

found in tissues that are not present in higher verte-

brates it may also be found to serve novel functions in

these tissues.

Acknowledgments

National Health and Medical Research Council,

Australia, supported this research. We are grateful to

Dr Patricia Ingleton for her generous assistance with

organ collection and identification. David Paul (Depart-

ment of Zoology) kindly helped with the photomicro-

graphy and production of the figures. Lauren Brown and

Natalie Bridge (Marine and Freshwater Resources

Institute) assisted with the collection of elasmobranch

tissues.

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Parathyroid hormone-related protein (PTHrP) production sites in elasmobranchs

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