BIOLOGY

OF REPRODUCTION

46, 817-828

Distribution

(1992)

of the

Microtubule-Dependent and

ERIC

S. HALL,

Department

JTJLIA

EVELETH,

of Pathology

Kinesin

CHENGYU

and

in Rat

JIANG,

Laboratory

Motors

Dynein

Testis1

DARLENE

Medicine,

Cytoplasmic

M.

REDENBACH,

University,

Brown

and

KIM

Providence,

BOEKELHEIDE2

Rhode

Island

02912

ABSTRACT To

the possible

examine

to study

the

motor)

presence kinesin

and

in Sertoli

cells

role

of microtubule-based

localization

and

(a fast-growing

during

of the

transport

microtubule

end-directed

all stages

motor)

of spermatogenesis,

in testicular

a peak

was

also localized

within

Sertoli

cializations.

In germ

cells,

cytoplasmic

dynein

immunofluorescence

spermatids,

and

VI.

Kinesin

apical cell

small,

cell

Sertoli

the

dynein that

dynein

seen

toward

in the

cytoplasmic and

dynein

and

(stages

cytoplasm

peak

of step

stages germ

17 and

the

lX-XIV.

cell

previously

current

with

observed dynein

1S-17

X-VI).

reported and

stages

V

of Sertoli

and

transferrin,

secretion.

Further,

concerning

hypotheses

I-IV)

stage-dependent

orientation

protein

transport

during

The

spe-

(stages

18 spermatids

(stages

protein

was

ectoplasmic

of steps

step

end-directed

Cytoplasmic

cell-associated

of androgen-binding

in Sertoli

techniques

slow-growing

immunofluorescence

spermatids

with

is consistent

(a

in manchettes

10-18

secretion

immunofluorescent

during

IX-XIV)

observed

of steps

is involved

to manchettes

kinesin

9-14

in conjunction

lumen)

used

dynein

dynein

cytoplasm

was

in manchettes

the

Cytoplasmic

in apical

to steps

immunofluorescence,

ends

hypothesis

of cytoplasmic

were

was observed

(slow-growing

with

localization

structures

circular

cytoplasmic

microtubules

consistent

hollow

immunofluorescence

cells

we

cytoplasmic

rat testis.

within

with

immunofluorescence

and

function,

mechanoenzymes

manchette

is the func-

tion.

Mammalian monal, and togenesis.

Sertoli structural Specialized

INTRODUCTION

ented

cells provide environment

the nutritional, hornecessary for sperma-

[-1

tight

between

junctions

adjacent

creting number

seminiferous of Sertoli

tubule fluid (STF) cell-secreted proteins

lease

of mature

cell

of

IX-XII

during Protein

volving

rat seminiferous distinct stages

sperm

secretion

stages els

The

at the

[11],

and

of stage

endoplasmic

is secreted

[11]. secretion

reticulum,

vesicles, cell Microtubules

[10].

Golgi

process

apparatus,

are

can

levin-

tively, nesin

and the secre-

gans

in Sertoli

cells

are

December

Received

October

‘Supported

2, 1991.

by PHS

‘Correspondence Laboratory

Medicine,

testes,

19, 1991. ROl ES05033 Kim Box

and

Boekeiheide, G-B518,

K04

E500193.

M.D.,

Ph.D.,

Providence,

Department

RI 02912.

FAX

of Pathology (401)

and

and 817

ends

transthat can

Cytoplasmic

dynein motors

in

the

along

presence the

of AlP,

surface

of the

from squid axoplasm [24, 25] from Drosophila [26], sea urtissue [28], chick fibroblasts

rat

liver

chains

[31].

It is composed

of 124

and

64 kDa,

of respec-

[32].

Kinesin

may

mitotic formation

be involved

in fast axonal

spindle movement of tubulovesicular

in sea net-

[29,36]. and

has

dynemn since

and

mitotic

brain

first

isolated

isolated

from

rearrangements

[40]; [43]; and

Cytoplasmic

the [-1 end in organelle

sea

Caenorbabditis

from

[39]; Dictyostelium [42]; HeLa cells

[31, 44, 45].

towards implicated spindle

was

been

[38]; Reticulomyxa [41]; squid axons

unidirectionally and has been

863-1971.

and light

in axons [33,34], [27,35], and the

Cytoplasmic

on-

[-]

with a total length of around 80 nm [24, 25, 27]. Kiis a unidirectional motor moving towards the +] end

transport urchins

cium Accepted

coverslips

[30], two

of microtubules

[37]

be toto Ser-

of microtubule-based

microtubules

cells and

with

their [20].

to microtubules in the absence of AlP and activity. In addition, when these proteins to glass

heavy

(greater

of microtubules

microtubules.

families

transport

HeLa

two

works

93%)

two

oriented

Microtubule-dependent involves molecules

coverslip [23-25]. Kinesin was first isolated and has since been isolated chin eggs [27], bovine adrenal

tion by a number of cell types [12-141, and may act as tracks for vesicle and organelle transport [15-18]. Microtubules are composed of tubulin subunits, which preferentially polymerize to one end of microtubules, giving them fastgrowing [+] and slow-growing [-I ends [19]. The majority than

microtubules [21, 22]. probably

along

are

adsorbed

they

transport

membrane components, are involved in protein

move

kinesin

[29], is a complex

have

that bind tightly possess ATPase

[7], of of

during

at highest

axons

to and

and

Sertoli

peaks

bulk transport of STF constituents must [-] ends of the microtubules. In contrast

cells,

bind

has fourin the re-

VIII

protein

transferrin

stages VIII-XII transport and

and secretory cytoskeleton.

end

the the

[+] ends towards the cell nucleus and the lumen of the seminiferous tubule

towards the nucleus port in Sertoli cells

[3]. STF contains a including andro-

epithelium culminating

androgen-binding

toli

germ fluid by se-

gen-binding protein [4, 5], transferrin [6], ceruloplasmin acidic glycoprotein [7], and inhibin [8,91. The secretion many of these proteins varies during different stages spermatogenesis. teen morphologically

Thus, wards

Ser-

toli cells form the blood-testis barrier, which isolates cells in the adluminal compartment from the interstitial [1, 2]. Sertoli cells provide nutrients and hormones

with their ends towards

dynein

urchins

eleParamerat

liver, moves

of microtubules [23] transport [39,46-48] [49, 50].

Additionally,

TESTOSTERONE

25. Wise

I,

Maurer

of thymosin

RE. Characterization

estrual period: Effects of elevated

esirdiol

134 during

ci and

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AND

hCG

DEPRESSION

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hormones

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HS, McShan

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RM. Gonadotrophin-induced

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UT. Thvmosin 134 and F,, an actin sequestering Chem 1991; 266:4029-4032. 48. Watts JD, Cary PD, Sautiere P. Crane-Robinson C. Thymosins: both nuclear and cvtoplasmic proteins. EurJ Biochem 1991; 192:643-651. 49. Hall NR, Spangelo BL, Farah JM, O’Donohue IL, Goldstein AL. Regulation of neuroendocrine pathways by thymosins. In: Moody 1W (ed), Neural and Endocrine Peptides and Receptors. New York: Raven Press; 1986: 683-694. 50. Rebar RW, Morandini IC, Petze JE, Erickson GF. Hormonal basis of reproductive defects in athymic mice: reduced gonadotrophins and testosterone in males. Biol

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897

THYMOSINS

OF

and

R, Morley

oxvtocin

gene

5, Schmale

and oxvtocin.

Biol

expression

H, Richter Chem

D. The

Hoppe-Seyler

367:695-704. R, Walther hormone

terior Pituitary

N, Morlev genes. Hormones.

5, Brackman In:

Yoshida

Am,sterdam:

B. Gonadal S, Share

L (ed),

expression Recent

Etsevier Pub; 1988:

of hypophyseal Progress

219-225.

in

Pos-

BIOLOGY

OF

46,

REPRODUCTION

Regulation

898-904

of Pulsatile

(1992)

Gonadotropin

Secretion

(Activin-Binding LOUIS

Department

Protein)

V. DEPAOLO,2

of Molecular

by Estrogen,

Inhibin,

in Ovariectomized

MOTOYUKI

SHIMONAKA,

Endocrinology The Whittier La Jolla, Ca4fornia

Follistatin

Rats1

and

Institute

and

NICHOLAS

for

LING

Diabetes

Endocrinology

and

92037

ABSTRACT The

following

on pulsatile pg)

study

of estradiol

and

the

trough

at the pulse

effects only studies

are

EB dose.

could

form

by iv.

after

alone levels

influenced

FSI

additive with

combined

with

basis

for

exploring

novel

contraceptive

INTRODUCTiON

from the brain Without exception

important steroids

feedback [11. On the

steroids

and

pears given that

the

in

of LH secretion are the the relative importance

regulating

are

negative the

feedback

nonsteroidal

signals

LH and

rat,

there

of nonsteroids is ample

of steroids

in regulating

evidence

clearly

FSH

shown

that

restoration

to ovariectomy

of

requires

demonstrating

nonsteroidal

materials

cine

fluid)

follicular

Accepted

December

Received

October

‘This work 2902 (to N.L). Whittier

(provided

to levels with by

[4, 5]. Likewise,

may

(rh)

a role

18, 1991.

Jolla, CA 92037.

FAX:

Louis

for

Diabetes

(619)

These is more

the

mornings

castrated

at the the

time

of diestrus-

to inject

steroid-free

diestrous-2

rats

of ovariectomy

pre-

in order

LH hypersecretion

that

follows

LH release [7). above, studies nonsteroids using isolation

actions in vivo data comparing and

purified

in ovariectomized here was performed treatment pulsatile

to examine the effects of animal models were con-

and

characterization

inhibin

and

of the

follistatin

these proteins have to permit evaluation

FSH-

(activinbecome of their

[9-14]. Based on our initial dosethe effects of recombinant human porcine

follistatin on

(OVX) rats [12], to investigate

with estrogen and LH and FSH release

AND

the the

FSH study effects

sereof

rh-inhibin-A or folin OVX rats.

METHODS

Animalc

by PHS Grant

Dr.

acutely

MATERIALS

P0l-HD-09690

and Contract

NO1HD-0-

age Institute

inhibin

is necessary

protein) [8]. Recently, in sufficient quantities

combined listatin on

and por-

exert

into estrogen

to the

inhibin-A

cretion ported

prior

estrogen

steroid-stripped

inhibin

both

in the regit has been

measured

both

additive

levels.

that both steroids and noneffects of these compounds

polypeptides

biological response

female

that

LH

and

between it

prevent

prior

binding available

8, 1991.

was supported

2Correspondence: The

levels

FSH

replacement

ducted

in regulat-

steroids and nonsteroids are probably involved ulation of both gonadotropins. For example,

fluid given

suppressing

in-

in the

since

in controlling As indicated steroids and

FSH

hormones,

FSH

The

castration [6]. More recently, it was shown that circulating LH levels increase after passive immunization of diestrous1 rats with an antiserum directed against a fragment of the inhibin-a chain, suggesting a role for endogenous inhibin

ap-

regulating

secretion

to completely

sex of

secretion

polypeptide

hibin in particular [2, 3]. Despite the apparent importance ing

FSH

indicating additive

inhibin

LH release. EB were

and

mean

follistatin

in rats. The

W

follicular

directly on of signal

of inhibin

nonsteroids

thereby

and

suppressed

of pulsatile

suppressed

proestrus

viously

among species and between sexes within a With regard to the female rat, it would seem

principal

secretion

modulators other hand,

the

follistatin

inhibin,

2

mean

interventions.

2 and

to regulate the output of gonadotroacross mammalian species, the most

nonsteroids

to vary species.

that act either via modulation

not

for

intervals

suppressed

Both

but

the effects

significantly

and

in abating

One component of a humoral communication link between the gonad and anterior pituitary gland is the secre-

input pins.

whereas

EB dose

estrogen

at 10-miss

LH release.

5, 20

(0.5,

10 jag recombinant

dose-dependently

or any parameter

release than treatment with either agent alone, in the physiological regulation of FSH secretion the

tions emanating from the gonads the pituitary gland or indirectly

lowest

doses

follistatin,

Follistatin,

frequency

or in combination,

alone

or various

collected

was

of pulsatile (40%).

at all EB doses, the

treatment

blood

of EB alone

extent pulse

of oil

porcine

purified

nonsteroids,

either

given

injections

s.c.

and all parameters

were

that

polypeptides,

Administration

to an equivalent

in combination

FSH

and

received of 60 tg

of the

assessed. levels

FSH

rats

administration

injection

were levels

FSH

Follistatin

to demonstrate

first

secretion

pulses)

mean

polypeptide

efficacious in suppressing FSH steroids are probably important on FSH

two

EB on mean

and

middle the

neither

hours release

of estrogen

ovariectomy,

after

I day later

Four

between

the effects

week

hormone

suppressed

while

of follistatin at the

vehicle.

point

employed

amplitude,

One

followed

on pulsatile

(lowest

doses

(EB)

appropriate

effects

to examine

secretion.

benzoate

or the

inhibin,

h, and

conducted

was

gonadotropin

DePaolo, and

Department Endocrinology,

of Molecular 9894

Endocrinology,

Genesee

Avenue,

Female, from

Sprague-Dawley Charles River

rats were purchased at 7 wk of Laboratories (Portage, Ml). Rats were

exposed to 14 h of light (lights-on 0500 h) and temperatures were maintained between 23 and 25#{176}C. Food and tap

La

535-9473.

898

REGULATION

water formed

were on

Handling conducted and

Use

supplied ad ether-anesthetized

libitum. rats

and experimental in strict accord

manipulation with the NIH

of Laboratory

Experimental The

OF

AND

FSH

Ovariectomy 1-2 days after

LI-I BY

was perarrival.

of animals were Guide for the Care

Animals.

AND

Anal

paradigm

used

in this

study

was

based

(CV)

of

scribed reveal

[12]. by Dr.

The Ralph

tech, Inc., South San Francisco, CA) and identical in potency to the preparations study [121. The experimental

paradigm

use

of

groups

The large number of rats was vehicles were used to control (0.9%

saline)

and

inhibin

ether

and

diately jections estradiol At 1000 various tions

0.1% our

necessary for the

outfitted

involved rats per

the group.

because different effects of follistatin

trifluoroacetic

acid

[TFA]

with

TFA

was

as the elution buffer. On the relationships between the of FSH levels reported prethat iv. injection of 10 p.g would suppress serum FSH

extent. ovariectomy,

after this of either

5-10

on secretion of LH and FSH. control since the rh-inhibin

purified by HPLC using TFA basis of in vivo dose-response polypeptides and suppression viously [121, it was determined inhibin and 60 p.g follistatin levels to a similar Six days after

study

with

(0.1%

adjusted to pH 7.2-7.4) was utilized as a vehicle

of Genen-

follistatin used were used in our earlier

of this rats

OVX

preparations Schwall,

rats

were

indwelling

anesthetized

atrial

surgical procedure, 200 p.l sesame oil

with

cannulae.

of the

following:

250

i.l

09%

saline,

250

p.1

TFA,

levels between 4 and 6 h after injection [121, blood samples (0.4 ml) were obtained

intervals of EB lease. mixture

from and Five

1400-1600

h in order

the polypeptides on hundred microliters

consisting

of OVX

in 0.9% saline containing was injected into the rats samples

were

frozen for by RIA.

spun

the

effects

pulsatile LH and FSH of a blood replacement red

10 U/mI after each

at low

subsequent

rat

of polypepat 10-mm

to assess

speed,

determination

blood

cells

the

serum

of LH and

National

LH and FSH concentrations using rat hormones and Hormone

and

Pituitary

was

stored

FSH

levels

were determined in antisera supplied by the Program,

NIDDK

hormone

that

are

algorithm considered

similar

data obtained (lowest point

from each animal, mean hormone between two pulses) levels, pulse

and

of pulses

per

2 h were

group. Statistical comparisons among made by means of a two-factorial analysis

(Student-Newman-Keuls

ison test). The factors (saline or follistatin; interactive

analyzed vehicle

effects

could be uncovered with was considered significant.

this method, if the CV of (LH) basis

or 1.5 of the

and trough amplitude,

calculated

treatment analysis

to

to differences

[161. With pulsatile

pulse was greater than two times the respective assay CV. On the

number

levels

and descending portions of appropriate assay CV as de-

This method has been shown groups in the various parameters

release cluster was

from

concentrations of variation

the suspected times (FSH)

tential

RP-

for

each

groups of variance

were and

multiple-compar-

were EB dose and treatment or inhibin). Accordingly, po-

of estrogen this

and

analysis.

the

A value

nonsteroids of p