Transferrin Receptors of Isolated Rat Seminiferous Tubules Bind Both ...

BIOLOGY

OF

35, 1227-12

REPRODUCTION

Transferrin

34 (1986)

Receptors Both

of Isolated Rat Seminiferous Rat and Human Transferrin

J. J.

PAULETTE

and

WAUBEN-PENRIS,’ HANS

Department

DER

of Cell

School

J.

GER

A. VAN

Tubules

Bind

STROUS,

DONK

Biology

of Medicine

University

of

Utrecht,

The

Utrecht

Netherlands

ABSTRACT The

binding

and

During the isolation Iron-saturated but

the

uptake and

and

dissociation

The affinity binding sites

of

rat

incubation iron-free

constant

and

human

transferrin

of the tubules, (apo-) transferrin is about

two

by

the blood-testis use the same

times

higher

for

isolated

rat

barrier binding

apotransferrin

of the receptors is equal for rat and human transferrin, but (2.6 x 1010 per 10 cm tubule length) than rat transferrin

seminiferous

remained sites on than

tubules

intact. the surface

for

was

studied.

the

tubules,

of

iron-saturated

human transferrin (1.1 x 1010 per

transferrin.

binds to more surface 10cm tubule length) at

0#{176}C. At 33#{176}Cequal numbers of human and rat transferrin molecules are taken up (about 8 x 1010) per 10 cm tubule length. The quantitative difference between 0#{176}C and 33#{176}Cis caused by the fact that at 33#{176}Creceptormediated endocytosis and recycling occur. As a consequence, both surface and intracellular transferrin receptors are

detected

at

330 C. The

dissociation

constants

are

not

temperature-dependent.

INTRODUCTION

Sertoli cells control within the seminiferous for spermatogenesis complexes between barrier

and

the (Fritz, Sertoli

exclude

electrolytes

from

tubule,

unless

Sertoli Gladwell,

cells

all the

they

composition tubule, which 1973). Tight cells create the

macromolecules luminal

pass

through

(Setchell and 1982; Russell

compartment the

role of transferrin in iron transport and to cells is well established (Baker and Morgan, Aisen and Listowsky, 1980; Perez-Infante and

several

Mather, 1982; Klausner require iron as a constituent and other heme-containing

of

physiological

junctional blood-testis and

cytoplasm

Wallace, 1972; and Peterson,

The delivery 1969;

of the fluid is important

the

of the

Waites 1985).

and In

conditions

et

al., 1983a). All cells of respiratory enzymes proteins; but, under

free

and Listowsky, function for

1980). Cells iron utilization,

(hemoglobin

synthesis)

iron

can

be toxic

expressing such as

and

syncytial

(Aisen

a specialized reticulocytes trophoblasts

addition to their function as a selective barrier, Sertoli cells contribute actively to the composition of the tubular content (Skinner and Griswold, 1980; Wright et al., 1981; Kissinger et a!., 1982; Waites and Gladwell, 1982). The glycoprotein transferrin is a major Sertoli cell product (Skinner and Griswold,

(iron transport across the placenta to the fetus), are exceptionally rich in transferrin receptors (Enns et al., 1981; Harding et a!., 1983). The transferrin receptor is also expressed in significant amounts in mitotically active cells, both normal and neoplastic

1980),

and

1981;

under 1982)

hormonal control and dependent on

(Perez-Infante

the

rate

et al.,

of

its synthesis

1982;

is reported

(Skinner and the surrounding Ritzen,

to

be

Griswold, germ cells

(Karin high

and

Mintz,

Klausner

1981;

et al.,

concentration

of

Trowbridge

1983a),

and

transferrin

and the

receptor

Omary,

presence

of a

molecules

on the cell surface is believed to be a specific marker for cellular proliferation (Larrick and Cresswell, 1979; Karin and Mintz, 1981; Sutherland et al., 1981; Trowbridge and Omary, 1981). Iron bound to transferrin (FeTf) enters the cell by receptor-mediated endocytosis. The iron then dis-

1983).

Accepted May 12, 1986. Received February 20, 1986. ‘Reprint requests: Paulette J. J. Wauben-Penris, Department of Cell Biology, School of Medicine, University of Utrecht, Nic. Beetsstraat 22, 3511 HG Utrecht, The Netherlands.

sociates from prelysosomal CURL 1227

transferrin, compartment

(Compartment

probably previously of

Uncoupling

in a low designated Receptor

pH as and

1228

WAUBEN-PENRIS

Ligand) Yamashiro unknown

(Ciechanover et al., way to

Listowski,

1980;

ferrin recycles

(apoTf), back

et al., 1983; Geuze et al., 1983; 1984), and is delivered in a yet iron storage proteins (Aisen and Klausner

et

the

medium

to

and

Mintz,

1981;

a!.,

1983;Klausneretal.,

be reutilized

1983b).

iron

et al.,

carrier

Bucks,

(Karin

Harding

et

1983a,b). from be iron

cross

the

the general a continuous is lost from

pool.

To

taken ported

up by the Sertoli cells through the cytoplasm

blood-testis

germ

cells.

which

the

There

are

several

seminiferous It could

barrier,

possible

tubules be

that

iron

must

iron,

be

transto the

mechanisms

could

accomplish

once

delivered

stages

review, possibility

see

Sertoli transferrin niferous

cells,

of

spermatogenic

by this

(for

are

be internalized round spermatids

further secreted Evidence

transported by Sertoli into the lumen of the exists that transferrin

by pachytene (Holmes et al.,

by using

and

lodo-beads

IL).

rat

transferrin

(Pierce

Unbound

fungizone, amino Scotland)

Darmstadt,

Chemical

was

125

mM

L-glutamin

acids, and

West

15

laboratory a

cell semican

spermatocytes 1983; Steinberger

MO),

and et

a!.,

(2

vitamins NaHCO3

Germany)

HEPES

removed

mM),

non-

(all from (0.85 g/l,

and

buffered

(Merck).

were of the highest available Male Wistar rats were

Russell and Peterson, 1985). Another is that iron ions, after being released in the

and tubule.

Louis,

Rockford,

streptomycin,

with

to the

cells

England)

Company,

essential Paisley,

Sertoli cells via serum diferric-transferrin and transferrin receptors, proceeds to the germ cells by gapjunctions that exist between Sertoli cells and some maturation

(St.

by gel filtration through a Sephadex G-25 column (Pharmacia, Uppsala, Sweden). Incubation medium was Eagles Minimum Essential Medium (MEM), supplemented with penicillin,

circulation to the process because the testicular iron

at the basal side, and forwarded

Co.

from Jackson Laboratories (Avondale, PA). These were made iron-free according to Aisen (1966) or were saturated with iron according to Klausner et al. (1983b). Proteins were iodiated with 125 I-Na (Amersham,

Apotrans-

1983;

AL.

Chemical

transferrin receptor, and is released into

as an

Ciechanover

Iron transfer germ cells must sperm-associated

transport.

al.,

bound to the to the cell surface

ET

All

Gibco, Merck,

to

other

quality. kept under

pH

7.4

chemicals standardized

conditions.

Isolation and Incubation Testes incubated

from with

of Seminiferous

mature 10 mg

rats were collagenase

decapsulated and (CLS II; Worthing-

ton, Malvern, PA) in 20 ml Hanks’ Solution (HBSS) for 5 mm in a shaking 33#{176}C(150

cycles

three

times

with

and

incubated

per

mm).

HBSS, for

The

teased

mm

30

Balanced waterbath

tubules

apart at

Tubules

were

with

fine

33#{176}Cto

Salt at

washed tweezers,

release

any

1984; Sylvester and Griswold, 1984). We have developed a system to study the binding and uptake of transferrin by intact, isolated rat seminiferous tubules wherein cell associations within

transferrin bound to the receptors 1983). After cooling on ice the ferred to the incubation medium. about 10-5 0 cm in length.

(Ciechanover tubules were The tubules

the tubules have not been disturbed testis barrier is intact. If we could use a heterologous

Tubular cubation

after 4 h of inblue exclusion test.

iron

donor,

secretion

we would route

be able

of transferrin

antibodies against (rat) transferrin.

uptake isolated

the

bloodas the

uptake

using

and the transferrin

and

specific

AND

rat

autologous is reported

transferrin

to

transferrin

was

purchased

from

tubules

I-FeTf

binding excess method Mendel albumin up by Wallace, incubation,

METHODS

Materials Human

The 125

integrity was at 33#{176}C with the or

33#{176}C(the ular cells,

donor for nonhuman cells et al., 1979; Perez-Infante compared the binding and

of human transferrin with rat seminiferous tubules. MATERIALS

the

transferrin

to study separately,

the heterologous Since human

to be an efficient iron (Messmer, 1973; Verhoef and Mather, 1982), we

and

Sigma

ice-cold amount counter.

125

checked Trypan

were

incubated

I-apoTf

for

2 h at 0#{176}C, or for

et

for al.,

best temperature Rommerts

was determined of unlabeled of Hamilton

with

rat

or

et al., transwere

human 4 h at

the culture of testic1980). Nonspecific

by incubation with a 20-fold protein, or according to the et al. (1984) and Mendel and

(1985).

Furthermore, the binding of 125 was studied, a molecule which is not taken seminiferous tubules in vivo (Setchell and 1972, the

Christensen tubules were

HBSS and of bound

et al., 1985). washed three times

lysed in 1 N NaOH, I was measured in

125

After with

and the a gamma

TRANSFERRIN

The

amount

of

to Lowry et protein content Values The

were results

(1949)

for

protein

was

al. (1951) of tubules expressed were

the

BINDING

determined

according

and compared of different known

per plotted

10 cm of according

determination

of

BY

with the lengths.

tubule length. to Scatchard

the

only

experiments

characteristic

examples

dissociation

of the

described

described

resulted in associations histologically; Figure the

method

blue) between microscopy

of spermatozoa

which

from the

isolation

and

prevented

entering Sertoli cells (not shown).

formed

at the

substances

the

tubule. were

1229

We studied the binding transferrins to such tubules concentration

in the

excess

of

unlabeled

or, according and Mendel plot above

of

different 125 I-labeled as a function of the

medium.

The nonspecific binding Fig. 2; 2a is the total method of Klausner

by

incubation

intact seminiferous tubules, where cell had not been disturbed (as checked results not shown). As can be seen in

1, a plug tubules,

for

TUBULES

was determined binding plot): et al. (1983b),

transferrin

to Hamilton (1985), the 4 izg/ml was

was

as follows according to a 20-fold

added

et al. (1984) slope of the extrapolated

(Fig.

2b),

and Mendel total binding back through

the origin (Fig. 2c). These methods gave comparable results. In addition, we studied the binding of 125 I.. albumin, which shows no specific binding or uptake

are shown. RESULTS

The

SEMINIFEROUS

(see the

constant (Kd) and the number of binding sites per 10 cm of tubule length (R0). The Student’s t-test was used for statistical analyses of the results. In the figures,

RAT

still

(e.g., Tight

visible

end

seminiferous

Christensen

tubules et al.,

(Setchell

1985;

Fig.

and

Wallace,

1972;

2d).

of

Trypan junctions

by electron 5

0

4

a 3 E U 0

C 2

2

4

6

8 nglml

FIG. 1. After 4 h of incubation at 33#{176}C,seminiferous tubules were transferred to a Trypan blue solution (1% in PBS). Trypan blue did not enter the tubules. At the open end of the tubule, a plug of spermatozoa formed, preventing substances from entering the tubules from this side.

10

12

14

16

F

FIG. 2. Comparison of different methods to determine the nonspecific binding: (a) O-O: total binding of ‘“I-human FeTf for 2 h at 0#{176}C as a function of the concentration in the medium; (b) t--t,: binding of 135 I-human FeTf for 2 h at 0#{176}Cwith a 20-fold excess of unlabeled human FeTf; (c) #{163}-#{149}#{149} -A: slope of the total binding curve extrapolated through the origin; and (d) #{149}-------S: binding of Ialbumin for 2 h at 0#{176}C.

1230

WAUBEN-PENRIS

ET

AL.

determined in Table

a

of

A

according 1. Rat FeTf

binding

length),

sites but

about

to Scatchard (1949), are shown and apoTf used the same number

(about

the

2 times

1010

Kd for

apoTf

higher

than

Mol/l) (p