NORMAL RAT PITUITARY CELLS:
3mm or PREMARY CULTURE.
3:50me or moms m AND

moms 0F PROLACTIN- 2530mm;

Thesis for the Degree of M. S.
MECHIGAN STATE UNWERSBTY
PATRlClA ANNE PAYNE
1975

 

IHESLS,

 

 

 

 

 

 

 

ABSTRACT

NORMAL RAT PITUITARY CELLS: STUDY OF PRIMARY CULTURE,
SECRETION OF PROLACTIN, AND BINDING OF
pRDLACTIN-lZSIoDINE
BY

Patricia Anne Payne

Dispersed cells of normal pituitaries from three ages
of female rats: immature or 14 days old, mature or 45
days old, and retired breeders or greater than six months
of age were cultured for periods extending to 14 days.
These cultures were characterized for their ability to
release prolactin in XlEEE relative to their protein and
deoxyribonucleic acid (DNA) concentrations. A significant
difference in quantities of prolactin released into the
medium by the pituitary cell cultures was demonstrated by
radioimmunoassay. Immature pituitary cells released the
lowest levels of prolactin, mature pituicytes secreted
midrange values, and retired breeder pituicytes released
the highest levels of prolactin. These normal rat pituitary
cells were demonstrated to specifically bind prolactin-1251,
thus illustrating the presence of receptors for prolactin on
pituicytes. A high positive correlation was demonstrated
between per cent specific binding of prolactin and prolactin
released in 113:9 among cultures of pituicytes. Secretion

rates of prolactin and the number of binding sites may be

Patricia Anne Payne

related. As binding material for prolactin receptor assays,

this study has demonstrated the utility of cell cultures.

NORMAL RAT PITUITARY CELLS: STUDY OF PRIMARY
CULTURE, SECRETION OF PROLACTIN, AND BINDING

OF PR0LACTIN-125IODINE

BY

Patricia Anne Payne

A THESIS
Submitted to
Michigan State University

in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Physiology

ACKNOWLEDGMENTS

I extend thanks to the many people who have supported
me throughout my research. My thanks for their advice,
help, laughter, time and caring. These people were
critical for the completion of this study and thesis.

I would especially like to thank Dr. William L. Frantz
for his guidance and encouragement during my research. Ap-
preciation is also extended to Drs. C. W. Welsch, E. M.
Convey and J. B. Scott, members of my advisory committee.
Thanks is expressed for the use of the facilities of Dr.
Clifford Welsch. Without the use of his equipment, this
research would have been impossible.

To Olan Dombroske goes special thanks for his in-
valuable help and understanding. Gratitude is expressed
to Charles Brooks for the instruction and utilization of
his radioimmunoassay procedure and to Karen Kowalski for
the hours spent in preparation of this manuscript. To
my parents, family and R.C.B., my thanks are dimensionless
and inadequate.

I am grateful for the financial support from Dr.
William Frantz through the National Science Foundation,

Grant No. GB-30686 and the Department of Physiology.

ii

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

INTRODUCTION

LITERATURE REVIEW

1.

II.

III.

IV.

V.
VI.

The Development of In Vitro Systems
for Pituitary Study— .

A. Organ Cultures of the Rat Pituitary
B. Cell Cultures of the Rat Pituitary

 

Effects of Age and Sex on Prolactin Secretion

Control of Prolactin Synthesis and Release

A. Hypothalamic Regulation: Inhibiting and
Releasing Factors and the Biogenic Amines

B. Steroids

C. Stimulating and Inhibiting Compounds,
Thyroid Hormones and Gonadotropins

D. Prolactin and Growth Hormone

Correlation of Cell Number, Protein, and
Deoxyribonucleic Acid

Radioimmunoassay

Radioreceptor Assay

MATERIALS AND METHODS

I.
II.

Animals
Method of Pituitary Gland Cell Culture

A. Growth Medium Preparation
B. Method of Cell Dispersion and Culture

iii

Page
vi

vii

10
11
12
14
16
19
19
19

19
20

TABLE

III.

OF CONTENTS cont'd

Methods of Cell Number, Protein and

Deoxyribonucleic Acid: Assay and Correlation

A. Measurement of Cell Number and Viability

B. Measurement of Protein and Deoxyribonucleic
Acid (ENA)

IV. Method of Radioimmunoassay
A. Sample Collection
B. Prolactin Radioimmunoassay
V. Prolactin Radioreceptor Assay
A. Ovine Prolactin Iodination
B. Radioreceptor Cell Preparation
C. Method of Prolactin Receptor Assay
VI. Prolactin Binding Analysis . . . . . . . . .
A. Calculation of Per Cent Total, Non-Specific
and Specific Binding . . . . . . . .
B. Scatchard Analysis
EXPERIMENTAL
1. Correlation of Deoxyribonucleic Acid and
Protein Content to Cell Number
A. Objective
B. Procedure
C. Results
D. Discussion
II. Concentrations of Prolactin of the Culture
Medium as a Function of Age of Animal and Days
in Culture
A. Objectives
B. Procedures
1. Culture .
2. Assay Procedure
C. Results
D. Discussion
III. The Binding of Prolactin-lzslodine to Normal

Rat Pituicytes and Its Correlation to Secretion
of Prolactin In Vitro
A. Objectives
B. Procedures
1. Culture
2. Binding Assay
C. Results
D. Discussion

iv

Page

22
22

23

40
40

41
41

43

TABLE OF CONTENTS cont'd Page

GENERAL DISCUSSION . . . . . . . . . . . . . . . . . . 54
APPENDICES . . . . . . . . . . . . . . . . . . . . . . 59

I. Composition of Dulbecco' 5 Modified Eagle (DME)
Medium Powder . . . . . . . . . 59

II. Concentrations of Prolactin: Three Way Nested
Analysis of Variance of the Means for Unequal
Sample Sizes . . . . . . . . . . . . . . . . . . 61

III. Analysis of Variance for Prolactin Release and
Age of Animal . . . . . . . . . . . . . . . . . 62

IV. Tukey's T-Test for Analysis of Differences in
Medium Prolactin Concentrations for Immature,
Mature and Retired Breeder Female Rats . . . . . 63

V. Scheffe's Paired Contrasts for Analysis of
Effect of Day in Culture on Concentrations of
Prolactin Released into the Medium . . . . . . . 64

VI. Scheffe's Analysis of Prolactin Release vs

Day in Culture . . . . . . . . . . . . . . . . . 65
VII. Product-Moment Correlation Coefficient . . . . . 66
VIII. Linear Regression Analysis . . . . . . . . . . . 67
REFERENCES . . . . . . . . . . . . . . . . . . . . . . 68

LIST OF TABLES

Table Page

1. DNA Content of Tumor Rat Pituitary Cells
(C llRAP) and Normal Rat Retired Breeder
Piguitary Cells . . . . . . . . . . . . . . . . . 35

II. Protein Content of Tumor Rat Pituitary Cells
(CgllRAP) and Normal Rat Pituitary Retired
Breeder Cells . . . . . . . . . . . . 36

III. Medium Prolactin Concentrations of Immature,
Mature, and Retired Breeder Female Rat
Pituitary Gland Cell Cultures . . . . . . . . . . 44

IV. Analysis of Factor Significance: Age, Day in
Culture, and the Interactions of Age and Day
in Culture . . . . . . . . . . . . . . . . . . . 45

V. The Binding of Prolactin-1251 to Cultured
Normal Pituitary Cells of Immature, Mature,
and Retired Breeder Female Rats . . . . . . . . . 49

VI. The Correlation of Per Cent Specific Binding
and Prolactin Secreted by Three Ages of Rat
Pituitcytes . . . . . . . . . . . . . . . . 51

VII. Analysis of Variance for Prolactin Release
and Age of Animal . . . . . . . . . . . . . . . . 62

VIII. Scheffe's Analysis of Prolactin Release vs
Day in Culture . . . . . . . . . . . . . . . . . 65

vi

Figure

LIST OF FIGURES

Page

Mean concentrations + standard error of the

mean for DNA (ug) and log of prolactin mean
concentrations (mg) . . . . . . . . . . . . . . 37

Correlation of per cent specific binding of
prolactin-1251 and prolactin secreted (ng

per ml) for immature, mature and retired

breeder pituitary cells . . . . . . . . . . . . 52

vii

Introduction

 

Synthesis and release of prolactin are regulated by
the interplay of genetic and environmental factors. The
former are slow in develOpment and more profound in ex-
pression, as illustrated by the fluctuations in prolactin
serum concentrations in sexual development. The latter
may be characterized as "fine tuning" and produce transient
and regulatory effects as exhibited by prolactin in the
development of mammary glands prior to lactation. Releas-
ing and inhibiting factors (hormones) are elicited by the
neural or physical stimulation of other hormones. The
possibility of direct "feedback control" or autoregulation
of a hormone and possibly prolactin has a good theoretical
basis in kinetic analysis. This system entails the specific
recognition of prolactin by receptors on or in the pituitary
producing and releasing the hormone.

Such a mechanism of autoregulation has been demonstrated
with the secretion of thyroid hormones in rats and mice, and
the regulatory factors for thyroid hormones: thyrotropin
releasing hormone (TRH) and thyroid stimulating hormone (TSH)
(Bowers et_al., 1967; Vale gt 31., 1967; Averill, 1969). To
date, no definitive studies have shown specific binding of
prolactin to normal mammotrophic cells of the anterior
pituitary gland as a mechanism for prolactin regulation
upon its own synthesis and secretion. This thesis describes
the development and initial findings of an in vitrg con-

trolled experimental system for prolactin regulation.

Pituitary cell cultures are easily manipulated and
conceivable for assaying radioreceptor activity and hor-
monal concentrations of the incubation medium. Utiliza-
tion of cultures of normal anterior pituitary cells from
female rats at various stages of sexual development pro-
vides a means to compare the prolactin binding activity
with synthesis and release of prolactin in_vi£rg. These
findings compared to the in 1112 serum and pituitary con-
centrations of prolactin of female rats of the same ages
would aid in supporting or negating the in_vi££g results.
This research was designed to elucidate this relationship
of ontogeny, prolactin secretion and specific prolactin
binding to cells of the female rat anterior pituitary
after cultivation. Pituitary cells from three ages of
female rats: 14 days of age, 45 days of age, and greater
than six months of age were cultured and assayed for their

ability to secrete prolactin and to bind prolactin-1251.

Literature Review

 

I. The Development of In Vitro Systems for Pituitary Study
A. Organ Cultures of the Rat Pituitary
Organ culture of the bisected pituitaries has been a
widely used method to study the regulatory mechanisms for
prolactin (PRL) synthesis, storage, and release. When
isolated from the hypothalamus, the pituitary in 11319
will continue to secrete PRL (Meites £3 31., 1959; 1961).

Addition of crude hypothalamic extracts to the culture
medium decreased PRL release (Talwalker, 1963). Kragt and
Meites (1967) demonstrated a negative dose response effect
of hypothalamic extract on the release of PRL into the medium
in vitrg. These findings support previous studies in 1113,
which suggested the existence of a hypothalamic prolactin
inhibiting factor (PIF) (Grosvenor _t _l., 1964; Amenomori
and Meites, 1970).

Lesions in the median eminence, the pathway for nerve
tracts and blood vessels from the hypothalamus to the
pituitary, increased rat serum PRL levels (Chen et 31.,
1970; Welsch EE.il-: 1971). Anterior pituitary glands,
transplanted under the renal capsule, also produced higher

concentrations of serum PRL (Everett, 1954; 1956).

B. Cell Cultures of the Rat Pituitary
Use of anterior pituitary cells in long term cul-
tures has been limited to tumor cell cultures. Tashjian

(1968) cloned or isolated several pituitary cell lines from

X-ray irradiated Wistar-Furth rats. Each cell line was
further characterized as to growth hormone (GH) and PRL
secretion (Tashjian gt gt., 1970), karyotype (Sonnenschein
gt gt., 1970) and hormone release in response to hydro~
cortisone (Tashjian gt gt., 1970). In 1973, studies by
Gautvik and Tashjian demonstrated a dose dependence be-

2 ions in the extracellular medium and increases

tween Ca+
in medium hormone concentration. Magnesium ions had the
reverse effect. A similar increase in hormone release was
noted with high levels of K+ in the presence of calcium
(Gautvik and Tashjian, 1973b).
A similar cell line was derived from Wistar-Furth

rats with anterior pituitary tumors induced with estradiol
benzoate by Sonnenschein (1973). Anterior pituitary tumor

cells were dispersed with viokaseR

, a pancreatic enzyme
mixture and with a microliter dilution technique cloned
for a specific cell type. The morphology of the individual
lines of cloned cells varied in terms of the percentages of
dendrite-fibroblast type cells and clusters of epitheliod
cells in suspension. Morphology was not, however, indica-
tive of function as cells of different morphology were able
to secrete GH and PRL (Sonnenschein gt gt., 1974).

Hopkins and Farquhar (1973) utilized a variety of
dissociating agents and inhibitors i.e. trypsin, col-

lagenase, neuraminidase, and soybean trypsin inhibitor.

With isolated anterior pituitary cells from Sprague-Dawley

ViokaseR, Viobin Corporation

male rats that were cultured for short periods, they were

able to quantitate 3

H-leucine incorporation into prolactin
as seen by gel electrophoresis and autoradiographic elec-
tron microscopy. Other methods of trypsin dispersions of
anterior pituitary cells have been develOped by Ishikawa
(1969) for cell morphology and classification studies and
for the effects on release of adrenocorticotropin (ACTH)
by hypothalamic extracts. Portanova gt 21- (1970) and
Sayers and Portanova (1974) isolated anterior pituitary
cell types from a trypsin dispersed preparation by velocity
sedimentation at unit gravity. This technique also pro-
vided a useful way to study the pituitary mammotrophs or
acidophils (Hymer gt gt., 1973).

Recently, the collagenase—viokase dispersion method
of Vale gt gt. (1972) has been widely used. This is an
adaptation of a system for studying thyrotropin releasing
factor (TRF) or thyrotrOpin releasing hormone (TRH) and
luteinizing releasing factor (LRF) as a challenge for
thyroid stimulating hormone (TSH) and luteinizing hormone
(LH), respectively. Baker gt gt. (1974) extended the use
of viokase to disperse pituitary cells for immunochemical
study of monolayer cultures. Over a 32 day culture period,
the cells persisted and PRL and ACTH regularly were present
in the medium.

With these methods of anterior pituitary gland dis-
persion, it is possible to maintain viable cells which

retain many 13 vivo characteristics. Application of these

procedures to long term culture of normal rat pituitary
cells is feasible if structure and function are maintained
and replication provides a sufficient number of cells for

study.

II. Effects of Age and Sex on Prolactin Secretion

Serum concentrations and pituitary contents of pro-
lactin vary significantly with age and sex of the animal.
Yamamoto gt_gt. (1970) utilized t3 tttg incorporation of
14C-leucine into PRL for the study of female rats of 12
to 140 days of age. Prolactin synthesis increased at days
37 to 45 relative to 13 to 21 day old animals continued to
increase with age. In males, a similar increase at day 45
was noted but the rate of increase with age was not as
great as noted for female rats. Prolactin concentrations
of serum and per 100g body weight for all age groups of
rats were higher in females than in males.

Voogt gt gt. (1970) found similar age-related con-
centrations of PRL in pituitaries and serum of female rats.
At ages prior to vaginal opening, serum concentrations
were 13-21 ug per m1, and a three-fold increase was noted
on the day of vaginal opening. From 41-45 days of age,
prolactin levels were similar to those found in 3-month-
old estrous rats (70-80 ug/ml). Pituitary prolactin con-
centration trends paralleled serum amounts except on the

day of vaginal opening. Voogt also produced higher levels

of prolactin in pre-pubertal females with daily injections
of 0.50 ug estradiol benzoate.

In support of a stimulatory effect of estrogen on
prolactin release, Ieiri E£.il° (1971) demonstrated that
pituitary prolactin content and release is increased at
proestrus and estrus in rats. Using male rats, Negro-
Vilar gt gt. (1973) found fluctuations in secretion rates
but reported lower values for prolactin concentrations
similar to those reported by Ieiri gt gt. (1971). They
found significant elevations of serum PRL during periods
that coincided with rapid growth of the testes and acces-
sory sex organs.

In comparing 21-month-old rats in constant estrus with
3-month-old cycling rats, the older females had approximately
a two-fold greater anterior pituitary prolactin content by
the modified pigeon crop assay of Reece and Turner (1937)
by Clemens and Meites (1971). Simultaneously, high con-
centrations of follicle stimulating hormone (FSH) and low
pituitary luteinizing hormone (LH) were also noted. As
the rat matured, the synergistic and antagonistic effects
of the gonadotropins and steroids in the maturation process
may stimulate prolactin synthesis and release as the re-
quirements for prolactin in sexual development increase.
Whether this age related prolactin secretion was maintained
tg ttttg was of interest in the study undertaken. The
regulation of prolactin by gonadotropins and steroids was

however not investigated.

III. Control of Prolactin Synthesis and Release

A. Hypothalamic Regulation: Inhibiting and Releasing
Factors and the Biogenic Amines

The presence of a hypothalamic prolactin inhibiting
factor (PIF) was reported by Meites E£.§l- (1959), Pasteels
gt gt. (1961), Talwalker gt gt. (1963). Others have sug-
gested the existence of an avian hypothalamic prolactin
releasing factor (PRF) (Kragt and Meites, 1965). In mam-
malian species, a PRF has been termed to explain initiation
of lactation following injection of crude hypothalamic
extracts in estrogen primed rats (Meites gt a1., 1960).

In vivo, catecholamines and biogenic amine precursors

 

have been shown to inhibit rat prolactin release by in-
creasing release of PIF or possibly by inhibiting TRH at
the pituitary, thus acting as an antagonist to the TRH
effect on the release of PRL (Chen and Meites, 1975).
Kamberi gt _t. (1971) observed no direct inhibition by the
catecholamines on release of prolactin with perfused hemi-
pituitary glands. Biogenic amines placed in the ventricular
nucleus were inhibitory and were dose and class dependent.
Dopamine inhibited prolactin at 2.5 to 5 ug levels but
epinephrine and norepinephrine at the same dose had no
effect (Shaar and Clemens, 1974). Ojeda and McCann (1974)
suggested a control mechanism that depends upon a balance
in development of stimulating (estrogen sensitive) and
inhibiting (dopaminergic) receptors for prolactin during

sexual development.

Organ culture studies of anterior pituitary glands
indicate that biogenic amine inhibition of prolactin re-
lease is dose dependent. With 80 to 640 ng per ml doses
of dopamine, prolactin inhibition is significant, 200 to
1000 ng per m1 doses of norepinephrine or epinephrine are
required for similar inhibition (Koch gt gt,, 1970).

These findings report the need for pharmacological doses

of catecholamines for inhibition of the release of pro-
lactin from pituitary glands ifl.!i££2- Implants of acetyl-
choline into the third ventricle have also been shown to

inhibit PRL release as has pilocarpine, a stimulant of the

cholinergic receptor (Grandison gt gt., 1974).

B. Steroids

Increase in serum prolactin concentrations at puberty
has been attributed to antecedent increases in estrogen at
puberty (Minaguchi gt gt., 1968; Voogt, 1971). Prolactin
secretion ifl.Xi££2 was increased by estrogen and progesterone
but to a greater degree with estrogen. Estrogen and testos-
terone, but not 17 a-hydroxyprogesterone injections stimu-
lated 3H-leucine incorporation into prolactin in castrated
and intact female rats, MacLeod gt gt. (1969).

Sar and Meites (1968) reported that progesterone in-
creased pituitary prolactin content tg tttg. In contrast
Jones gt gt. (1965) and MacLeod gt 1. (1969), found no

corresponding PRL increases with progesterone treatment.

10

These discrepancies however appear to be dose and system

dependent (Blake gt gt., 1972; Kalra gt gt., 1973).

C. Stimulating and Inhibiting Compounds, Thyroid
Hormones and Gonadotropins

Much of the work on stimulation and inhibition of
prolactin secretion by other hormones has concerned the
effects of thyroid hormones and TRF. Evidence for a two-
to-five-fold increase in PRL secretion by pituitary cells
treated with TRH in culture was noted by Tashjian gt gt.
(1971). Triiodothyronine (T3) inhibited the stimulatory
effects of TRH on PRL secretion but did not increase the
cell replication rate (Tsai and Samuels, 1974). Nicoll
and Meites (1963) found that thyroid hormones directly
increased prolactin release. tg tttg, Rivier and Vale
(1974) have reported increased PRL secretion with TRF and
its analogs. Using a bovine pituitary cell culture,

Machlin gt gt. (1974) noted a release of PRL, GH and TSH
by TRH in a bovine pituitary cell incubation.

The precursor of serotonin, S-hydroxytryptophan (S-HTP),
stimulated release of PRL and TSH. Alone and in combination
with the biogenic amine inhibitors, reserpine and a-methyl-
metatyrosine (<1MMT), serotonin had similar PRL and TSH
effects. Simultaneous administration of dopamine and TRH
to male rats elicited only TSH release without a PRL

elevation (Chen and Meites, 1975).

11

Ergot drug derivatives have also been reported to
suppress serum prolactin and block the stimulatory ef-
fects of estradiol benzoate (Lu gt _t., 1972; Brooks and
Welsch, 1974). Welsch gt gt, (1971) linked the increased
incidence of rat mammary tumors to elevated serum prolactin
concentrations and increased estrogen serum concentrations.
Reduction of mammary tumor incidence was possible with the
inhibition of PRL release by 2-bromo-a-ergocryptine (Welsch
and Gribler, 1973).

Prolactin implanted in the median eminence terminated
rat pregnancies in early gestation (Clemens and Meites,
1968) and caused resumption of cycling in postpartum dies-
trous rats. Voogt gt gt. (1969) showed prolactin implants
to stimulate FSH release. Increased FSH and LH concen-
trations were also noted when pseudopregnancy was terminated
with prolactin implants (Voogt and Meites, 1971). In ovari-
ectomized virgin rats, LH and FSH concentrations are in-
creased while prolactin quantities were depressed. Per-
phenazine, a PRL releaser, with methallibure, a gonadotropin
suppressor, also served to decrease PRL secretion (Ben-David
gt _t., 1971). From this data, one might propose the exis-
tence of an antagonism between PRL and gonadotropin secre-

tions that is related to the stage of sexual development

of the rat.

D. Prolactin and Growth Hormone
The role of prolactin on its own regulation is un-

clear. In female rats, implants of 250 ug of PRL into

12

the median eminence significantly increased LH and FSH but
failed to elevate serum PRL. No change in levels of PIP
or PRF was noted (Voogt and Meites, 1971). MacLeod and
Abad (1968) showed a direct inhibition of prolactin syn-
thesis, as measured in the pituitaries of females with
multiple pituitary tumor explants, by an tg ttttg_assay of
3H-leucine incorporation.

There appears to be no effect of GH on PRL secretion.
Both hormones are produced by the acidophilic cells and
are similar in chemical structure (1i gt gt., 1969).

These common factors may explain the similarities in their
functional responses. McGarry gt gt. (1968) have noted
increased glucose utilization, greater nitrogen retention,
increase in the calcium levels of the urine and stimulation
of body growth by both GH and PRL.

It is evident with the effects of hormones and re-
lated compounds upon prolactin synthesis and release that
a more defined and controllable system for the study of
PRL would provide greater insight into PRL regulation.
Pituitary cells ifl.!i££2.WOUId provide such a system for
study of this mechanism on a cellular level, especially,
in the areas of dose-dependent and direct effects of hor-

mones on the pituitary.

IV. Correlation of Cell Number, Protein, and Deoxyribo-
nucleic Acid
A standardized method of cell counting using a

Neubauer hemocytometer and the vital stain crystal violet

13

has proven to be one of the most useful procedures. Absher
(1974) has reported a 10% counting error with multiple
samplings for counting and a standardized counting method.
In evaluation of the dye exclusion technique for viability
with trypan blue, Tennant (1964) found 85% of the counted
cells were capable of replication under optimal conditions.

Lowry 23.31- (1951) determinations for protein have
been used in conjunction with cell number to study cellular
growth phases and hormone regulation by a specific pituitary
cell population (Tashjian gt gt., 1968, 1970, 1971). A
more sensitive and accurate assay for determining cell
numbers with respect to hormone synthesis and release in-
volves DNA analysis. If a random method of cell sampling
is maintained, all phases of mitosis will be represented
with equal probability.

The diphenylamine DNA assay of Burton (1959) has been
further modified by Giles and Myers (1965) to increase
sensitivity to a DNA concentration of 5 ug and convenience
of assay. The methods for extractions and determinations
of Schmidt and Thannhause (1945) and Schneiter (1945) are
more time consuming.

In 1955, Leslie found the DNA concentrations in male
rat liver to increase with age (0.59 pg per cell at 10
days to 1.14 pg per cell at day 182). Values were ap-
proximately 25% higher in females of corresponding ages.
Hepatectomy initially decreased DNA content per cell but

average values increased during regeneration supporting

14

hyperplasia. Hepatomas exhibited a DNA content per cell
greater than control (1.0 pg per cell versus 0.913 pg per
cell). Cunningham gt gt. (1950) reported DNA content for
normal liver cells to be 6.1 pg per nucleus and 8 pg per
nucleus in hepatomas. The studies of Thomson gt gt. (1953)
indicated the DNA content of kidney, heart, bone, and spleen
consistently within a range of 6.46 pg per nucleus to 6.90
pg per nucleus. Studies of Leavitt S£.§l' (1973) with
isolated pituitary cells from ovariectomized rats reported

a higher DNA content per cell (10.82 pg) than the 6.6 pg

per normal pituitary cell reported by Vendrely (1955).

V. Radioimmunoassay
An excellent review of the aspects of protein hormone
receptor bindings for the radioimmunoassay (RIA) may be

found in Principles of Competitive Binding Assays (1971)

 

edited by Odell and Daughaday. The initial binding studies
of labeled protein hormone were developed for insulin by
Berson (1953). In 1959, the first studies of competitive
binding inhibition of antibody and labeled insulin (antigen)
competing with human plasma insulin were performed by Yalow
and Berson. The binding or complexing of antigen to anti-
body assumes that labeled and unlabeled hormones are
identical in their binding capabilities, and that antigen-
antibody recognition is specific. The specific antigen-
antibody recognition model of immunology has been applied
to other protein including prolactin (antigen) binding to

specific gamma globulins (antibodies).

15

Concentrations of serum rat prolactin have been assayed
by the biological pigeon crop proliferation method (Lyons,
1937; Reece and Turner, 1937), the densitometric analysis
(Nicoll gt gt., 1969) and the double antibody radioimmuno-
assay of Niswender gt gt. (1969). The latter method utilizes
rabbit antisera for rat prolactin and a second ovine gamma
globulin for precipitation of the antigen-antibody complex.
Neill and Reichert (1971) have compared the values of the
rat prolactin influence of the NIAMD Rat Prolactin Radio-
immunoassay kit of Parlow with values obtained using
pituitary extracted and purified rat prolactin. All mea-
sured levels of PRL were virtually identical except in
serum from hypophysectomized rats. In 1972, Gala and Kuo
compared serum prolactin levels measured by pigeon crop
assay, electrophoretic microdensitometry (ED) and RIA in
rat anterior pituitary organ culture medium. Measurement
by bioassay yielded greater PRL values than those demon-
strated by ED or RIA. Densitometer and RIA measurements
corresponded closely in value.

The conversion of per cent labeled hormone binding to
concentrations of PRL for unknowns is calculated by com-
parison to a standard curve of per cent specific PRL bind-
ing plotted against known concentrations of a reference
prolactin. In application of the basic assumption,
caution must be exercised with interpretation of the
standard curve reliability, the sensitivity or range of

hormone detection, the specificity of the antigen-antibody

16

reaction, the precision of standard curve values and the
accuracy of the calculated mean values (Midgley gt gt.,
1969).

Feldman and Rodbard (1971) have developed a mathematical
model on the basic RIA assumptions: 1) antigen and antibody
are separate homogeneous chemical forms; 2) a one to one
complex of antigen and antibody without cooperativity or
allosteric factors exists; 3) labeled and unlabeled hormone
react identically; 4) antigen-antibody reaction will con-
tinue until a steady state or equilibrium is attained; 5)
bound (B) and free (F) antigen may be separated without
equilibrium disruption and the B/F ratio may be measured.
With the curves generated by the ratio of B/F plotted
against B, Feldman and Rodbard have been able to detect
nonequilibrium and cross reactivity in RIA systems. With
analysis, radioimmunoassay provides a specific rapid means

for determination of PRL levels in serum and culture medium.

VI. Radioreceptor Assay

The basic assumption which apply to the radioimmunoassay
also apply to the radioreceptor assay (RRA) for membrane
binding sites. The major consideration in the binding of
a hormone to its binding site on a membrane is the biological
activity or binding ability of the hormone. Immunoreactivity
cannot always be equated with biological activity. The
reverse is also true. To be considered a true "receptor"
the binding site-hormone complex must initiate or elicit

a specific function or reaction (Cuatrecasas, 1974).

17

In the development of the binding assay of ovine pro-
lactin, a lactoperoxidase iodination produced a biologically
active hormone (Frantz and Turkington, 1972; Posner, 1974).
Ovine prolactin was found to bind specifically to mouse
mammary where its function in lobular-alveolar development
and lactation were documented (Frantz and Turkington, 1972).
Specific binding has also been demonstrated in mouse liver,
kidney, midbrain, and primary sex organs and characterized
for time and temperature dependent equilibrium conditions
and competitive hormone binding (Frantz gt gt., 1974).

From data obtained in prolactin binding to mammary carcinoma
cells, Turkington (1974) has proposed that the number of
receptors is directly related to the prolactin dependency

of the carcinoma. Shiu and Friesen (1974a) have reported
specific prolactin binding in rabbit mammary gland, liver,
ovary, and kidney.

Kelly gt gt. (1974) have demonstrated an ontogenic,
sex and physiological state interaction of prolactin bind-
ing in the livers of rats, rabbits, and guinea pigs.

Binding increased as the age of the animal and its re-
quirement and levels of PRL increased. This correlation
may suggest that prolactin induced its own receptors.

Recently Frantz gt gt, (1975) have shown a high de-
gree of specific binding of prolactin to the cells of cul-
tured tumor and normal rat pituitary glands. Shiu and
Friesen (1974b) have also been able to solubilize and

purify prolactin receptors from rabbit mammary epithelial

18

cells. Development of this method of solubilization of
receptors will enable further study on the mechanisms of
the receptor for prolactin. Study of the specific bind-
ing values of prolactin to levels of prolactin secretion
may also aid in knowledge of the prolactin receptor and a

possible autoregulation for prolactin.

Materials and Methods

 

I. Animals

Female Sprague-Dawley rats of the desired age groups:
13 day old or immature, 45 day old or mature, and 180 day
old or retired breeders were obtained from Spartan Research
Animals, Inc. (Haslett, MI). To decrease effects of trans-
portation stress, all rats were caged and maintained for
at least 24 hr after arrival under 250:1o and 15 hr
artificial illumination on a standard Purina Rat Chow diet

(Ralston Purina Co., St. Louis, MO) and tap water gg libitum.

II. Method of Pituitary Gland Cell Culture
A. Growth Medium Preparation
Pituicyte Dulbecco's Modified Eagle Medium (PDME) for
cell culture maintenance was prepared using a modification
of the medium of Sonnenschein (1974). The constituents of
PDME are: 13.47 g per L Dulbecco's Modified Eagle Medium
Powder (Appendix I) (GIBOC, Grand Island, NY), 15% Horse
Serum (Difco, Detroit, MI), 2.5% Fetal Calf Serum (Difco),
812 ml triple distilled water, 5 mM N'-2-Hydroxyethyl-
piperazine-N'-Ethanesulfonic Acid (HEPES) buffer (GIBCO).
Antibiotic-Antimycotic mixture (GIBCO) is equivalent to
100 units per ml pencillin-G, 100 ug per ml streptomycin
and 0.25 ug per ml fungizoneR, a mycostatin, and 72 ug
Anti-PPLO-agent (GIBCO).
R
Fungizone, E. R. Squibb 8 Sons
19

20

The medium is adjusted to pH 7.2 with 7.5% sodium
bicarbonate (GIBCO). Vacuum filtration through a 47mm,
0.45 N pore filter (Gelman Instrument Co., Ann Arbor, MI)
and a sterile Pyrex Millipore-filtering apparatus
(Millipore Corp., Bedford, MA) produced a medium suitable

for cell culture. The medium is stored at 40 until used.

B. Method of Cell Dispersion and Culture

Groups of 10-12 rats of a specified age class were
guillotined within 20 sec after removal from their cages.
Heads were washed with 95% ethanol under a laminar flow
hood (Type WS Series 300, Westinghouse, Grand Rapids, MI)
before the anterior pituitary glands were removed asep-
tically.

A medial cephalic incision was made through the skin
from the region of the foramen magnum ventrally to the
orbitals with small scissors and the skin retracted. With
the aid of bone cutters or scissors any remaining spinal
cord was removed. Lateral incisions through the skull
along parietal temporal sutures were made and the bone flap
was reflected anteriorly. The cerebral hemispheres were
reflected dorsally and the optic chiasma severed. In
45 day old or mature females and retired breeder females,
anterior pituitaries were teased from the neurohypophysis
and the surrounding connective tissue tg_gttg with small
surgical forceps. In the case of immature females, the

entire pituitary gland was removed.

21

In all cases, pituitary fragments were placed in a
sterile 100 X 15 mm Falcon petri dish (Bioquest, Cockeys-
ville, MD) containing 10 m1 sterile Puck's Saline A solution
and isolation of the adenohypophysis completed. The pitui-

tary tissue was minced (1 mm3

blocks), and washed twice
with 10 ml sterile Puck's Saline A, then a final saline
solution containing 0.25% trypsin (Difco Laboratories,
Detroit, MI) was added. This tissue suspension was trans-
ferred via a 10 ml sterile siliconized pipette to a 125 ml
screw capped Pyrex Erlenmeyer flask containing a 5 cm
Teflon stirring bar. Ten ml 1:5000 Ethylenediaminetetra-
acetic acid or versene (GIBCO, Grand Island, NY) were added
to the flask.

Two 20 min incubations were performed at 370 in a
Model 805 incubator (Precision Scientific Co., Chicago, IL).
The tissue fragments were dispersed during incubation by
the agitation provided by a magnetic stirrer (Fischer
Scientific, USA) set below the foaming point of the dis-
persing mixture. After 20 min of incubation, 10.0 ml of
the trypsinized cell suspension were removed and 5 ml
placed in each of two 13 X 100 mm screw capped Pyrex test
tubes. The suspensions were diluted 1:1 (v/v) with
Pituicyte Dulbecco's Modified Eagle Medium (PDME) and
centrifuged at 500 X g for 10 min at room temperature
(25:10C) in a clinical chemical centrifuge (International
Equipment Co., Needham Heights, MA). The enzyme-medium

supernatant was decanted, and the cell pellet was

22

resuspended in PDME. Viable cell number was determined

by staining with 0.1% trypan blue or 0.02% crystal violet
and counting by the method of Absher (1974) (see Materials
and Methods, Section III below). The final suspension of
cells was diluted to a concentration of 2.0 X 105 cells
per m1. One ml fractions were transferred to 30 m1 Falcon
tissue culture flasks (Bioquest) and an additional 2 ml

of PDME was added. Eight to ten flasks were prepared from
a single dispersion of pituitary glands.

Flasks were incubated at 370 in sealed plexiglass
chambers under constant aeration in a 95% 02:5% C02
humidified atmosphere for maintenance of a pH of 7.4.
Cells were observed on day of culture for morphology and
density and each subsequent day. Medium was replaced

every 48 hr with fresh PDME medium at 370.

III. Methods of Cell Number, Protein and Deoxyribonucleic
Acid: Assay and Correlation

A. Measurement of Cell Number and Viability

A fraction of the cell suspension obtained from cells
of a specific culture and flask was assayed for cellular
population. Cells contained in the medium and adhering
to the flask were counted. A 0.01% trypan blue solution
dissolved in 0.02 M citric acid is excluded by viable
cells. This technique of dye-exclusion required incubation
for 20-30 min at 37°. A 0.02% crystal violet or vital

dye solution dissolved in 0.02 M citric acid stains only

23

the viable cells. The latter stain permitted rapid count-
ing without incubation. Cell number was determined by
the method of Absher (1974) with a Neubauer hemocytometer.
The number of observed viable cells was corrected for
factors of dilution and volume to obtain the total number

of cells in suspension.

B. Measurement of Protein and Deoxyribonucleic Acid
(DNA)

Protein concentrations were measured by light ab-
sorption by the standard method of Lowry gt gt. (1951)
and quantitated using a standard bovine serum albumin
curve with a concentration range of 5-500 ug. These assays
were performed in duplicate with samples of each cell cul-
ture (varying cell numbers).

The concentrations of DNA were measured by the di-
phenylamine modification of Giles and Myers (1965). Cells
of different populations and cultures were dissolved in
hot 10% perchloric acid prior to assay. Absorbance read-
ings at 595 mu were compared with a calf thymus DNA
standard curve of 1-50 ug. Values for DNA, protein, and
cell number were also applied to prolactin 1251 binding
values, expressing final counts in dpm per ug protein or

DNA and dpm per cell number.

24

IV. Method of Radioimmunoassay

A. Sample Collection

At each 48 hour medium change, two medium samples,
each from the decanted pool of medium of three randomly
chosen 30 ml flasks of a specified culture, were collected.
The pooled medium samples were centrifuged at 500 X g for
20 min at 40 in a Sorvall RCZ-B (Sorvall, Newtown, CT)
to remove cells and debris. The medium was decanted and
stored at -200 until prolactin concentration was determined

by the double antibody radioimmunoassay (Niswender gt gt.,

1969).

B. Prolactin Radioimmunoassay

Prolactin concentrations in the culture medium were
determined by a modification of the technique of Niswender
gt gt. (1969). This double antibody radioimmunoassay was
established by Charles L. Brooks (Department of Anatomy,
Michigan State University, East Lansing, MI 48824) from a
standard Radioimmunoassay Rat Prolactin Kit distributed by
the National Institute for Arthritis and Metabolic Diseases
(NIAMD).

All samples were assayed at three different concen-
trations in a final volume of 800 ul. For each sample
480, 460, and 420 ul of phosphate buffer saline-1% bovine
serum albumen (PBS-BSA) were pipetted into three 12 X 75 mm
glass tubes followed by the addition of 20, 40, and 80 ul

of the sample to each tube respectively. Two hundred ul

25

of rabbit anti-rat prolactin (AB) diluted l:25,000 were
added per tube and the mixture incubated at 40 for 24 hr.
One hundred ul of the I125 chloramine-T labeled NIH R-P-l
rat prolactin I125 (*H), approximating 30,000 cpm, were
added to each tube and the samples were agitated and
stored at 4° for 24 hr. Thereafter, 200 ul of a second
antibody ovine anti-rabbit gamma globulin (1:100), were
added and incubation at 40 continued for 72 hr. The
double antibody prolactin complex was precipitated on

day five by combining 3.0 m1 of phosphate buffered saline
(PBS) per culture tube and was centrifuged at 1000 X g for
30 min at room temperature. The tubes were decanted of
supernatant, inverted for 30 min, and permitted to air
dry prior to gamma counting on a Nuclear Chicago Autogamma
Counter (Model No. 1185, Nuclear Chicago/Searle, Chicago,
IL) with a 50% counting efficiency.

In addition to triplicate samples of medium containing
unknown amounts of prolactin, five tubes containing only
100 ul of labeled hormone were prepared. Five samples
with normal rabbit serum (NRS) were made with 500 ul
PBS-BSA and 200 ul 0.3% normal rabbit serum (NIAMD Anti-
Rat Prolactin Serum—2) to account for the antiserum bind-
ing. The determinations of unknown prolactin concentra-
tions were calculated by comparing triplicate samples of
medium to a standard curve of per cent specific binding
of prolactin plotted as a function of rat prolactin

reference concentrations (NIAMD-RP-l) ranging from 0.2-20

26

ng which were assayed for *H binding. The degree of total
antibody binding is also calculated by excluding prolactin
or unknowns from a series of nine assay tubes.

The sigmoid curve generated from the plot of per cent
prolactin binding against prolactin concentration (ng) was
used for determining assay sensitivity and the prolactin
content of the unknowns. All sample PRL concentrations

were expressed as ng PRL per ml medium.

V. Prolactin Radioreceptor Assay

A. Ovine Prolactin Iodination

In a 2.0 ml polyethylene serum vial, 5 ug of ovine
prolactin (NIH-S-ll; 25.6 IU/mg) were iodinated using the
modified lactoperoxidase method of Frantz gt gt, (1974).
To 100 ul of 40 mM diethylbarbituric acid buffer (Mallin-
ckrodt Chemical Works, St. Louis, MO) pH 7.0, was added
0.5 mCi of carrier free Na 1251 (New England Nuclear,
Boston, MA or Amersham/Searle, Chicago, IL). In immediate
succession were added the PRL (5 ug in 25 ul of HOH) 10 ug
lactoperoxidase (Calbiochem, La Jolla, CA) in 25 ul of
40 mM diethylbarbituric acid and 250 ug of hydrogen
peroxide (Mallinckrodt Chemical Works) diluted 1:30,000
with distilled water. Reaction time was 5 min at 25°
followed by the addition of 0.5 ml of chilled 16% sucrose
solution. The mixture was immediately transferred to a
0.9 X 14 cm Sephadex G-75 column (Pharmacia Fine Chemicals,

Piscataway, NJ) in a 40 cold room. The column had been

27

previously washed with 1% bovine serum albumin (Sigma
Chemical Co., St. Louis, MO) and equilibrated with 20 ml
40 mM diethylbarbituric acid solution (barbital buffer).
Elution of the prolactin-125 iodine was performed at 4°
with mM barbital buffer and one m1 fractions were col-
lected. Radioactivity of the fraction was determined by
counting 1 ul volumes spotted on filter paper. The first
peak of radioactivity was transferred to a 1.4 X 15 cm
DEAR-cellulose (Diethylaminoethyl cellulose, Sigma Chemical
Co.) column equilibrated with 1% bovine serum albumin and
5 mM potassium phosphate buffer (Mallinckrodt Chemical
Works), 5-500 mM gradient of potassium phosphate buffer
pH 7.0. Fractions of one ml were collected and the first
major peak of radioactivity was tested for biological
activity with mouse mammary homogenates (Frantz gt al.,
1974) or cultured tumor pituitary cells (Frantz gt al.,
1975).

In later prolactin iodinations used for prolactin
binding studies, the following procedural changes were
made. Prolactin was dissolved in 0.1M ammonium bicar-
bonate, pH 8.1, and diluted with 4 ml glass distilled
water to give a final 0.01M ammonium bicarbonate concen-
tration (Vanderlaan, personal communication). Lacto-
peroxidase was dissolved with 0.4M sodium acetate (J.
Baker Chemical Co., Phillipsburg, NJ) at a pH of 5.6.

The Sephadex G-75 column was washed and the iodinated

28

preparation was eluted with 0.1M sodium acetate buffer.
All other buffers, concentrations, volumes and procedures

were the same.

B. Radioreceptor Cell Preparation

When a specific cell culture had reached approximately
2.0 X 106 cells per 30 m1 flask, cultures were terminated
and the cells collected for prolactin radioreceptor assay
(PRRA). The medium was stored for RIA as previously de-
scribed (see Materials and Methods; Section IV, Method of
Radioimmunoassay). The cell monolayer was washed three
times with Puck's Saline A solution, pH 7.4 and then scraped
into 3.0 ml of Puck's Saline A solution with the aid of a
rubber policeman. A 0.1% trypan blue vital dye exclusion
cell count was performed on the dispersed cells. The dis-
persed cells and medium were then centrifuged separately
at 480 X g for 20 min at 40 in a Sorvall "Superspeed"
(Sorvall Co., Newtown, CT). The supernatant was decanted
and the cells resuspended and pooled in Puck's Saline A
solution to the desired cell concentration for individual
binding experiments. If not used immediately, cells were

stored at -20° (until used for binding).

C. Method of Prolactin Receptor Assay
In the radioreceptor assay of Frantz _t _t. (1974)

total binding (TB) and non-specific binding (NSB) tubes were

determined by the direct competition of 1251 PRL with

29

o-LH and o-PRL respectively. Each sample to test for non-
specific binding sample [in a 500 ul polyethylene micro-
tube (Beckman, USA)] consisted of: (1) 200 ul of incuba-
tion medium (1% bovine serum albumin, 0.4 mM NaCl and 2

mM tris (hydroxymethyl) aminoethane pH 7.2); (2) 2 ug
cold prolactin (PRL); (3) radiolabeled prolactin-1251 in
the range of 10,000 1000,000 cpm; and (4) 100 ul of cell
suspension (approximately 1 X 106 cells).

The total binding samples contained the above quanti-
ties of incubation medium and prolactin-1251, but had 2 ug
of ovine luteinizing hormone (o-LH) instead of o-PRL.

Cell samples were counted for two min prior to binding in
a gamma counter to determine total available, iodinated
prolactin as disintegrations per min (dpm). After this
precount, the desired number of cells (1.0 X 104 - 1.0 X
106 cells) was added in 100 ul volumes to each tube.

After agitating samples by vortex and incubation at 40 for
30 min, the incubation was terminated by centrifugation at
10,000 X g in a Beckman Model 152 Microfuge (Beckman Corp.)
for 5 min also at 4°. The supernatant was aspirated and
the cell pellet was washed with 100 ul of cold glass dis-
tilled water which was quickly aspirated. The incubation
tubes were cut at the level of the cell pellet and re-
suspended in Lowry C. After the final gamma count for 20
min the protein content of the reacted cells was assayed in

a standard protein determination (Lowry gt gt., 1951).

30

VI. Prolactin Binding Analysis

A. Calculation of Per Cent Total, Non-Specific and
Specific Binding

From precount, final count and ug protein per sample,
it is possible to calculate per cent total, non-specific,
and specific binding of 125I PRL for anterior pituitary
cells. Per cent total binding (TB) per sample is defined
as the final count of the total binding LH tubes in dpm
per 100 ug protein divided by the mean of the TB precount
of the sample series X 100. Non-specific percentages
are calculated as the quotient of final dpm of PRL bind-
ing (NSB) tubes per 100 ug protein divided by the average
NSB precount sample series X 100. The per cent of specific
binding (SB) is calculated as the difference between the
averages of TB final counts (dpm per 100 ug protein) and
NSB final counts (dpm per 100 ug protein) divided by the
average TB final count (dpm per 100 ug protein) multiplied
by 100.

B. Scatchard Analysis

The binding of a protein hormone to a binding site
assumes many features of the antigen-antibody binding
complex theory: (1) homogeneous form of the hormone;
(2) achievement of chemical equilibrium without disruption
after reaction; (3) labeled and unlabeled hormone react
with the same affinity; (4) little or no binding site

cooperativity; and (5) a one to one correlation of hormone

31

molecule and binding site (Kahn gt gt,, 1974).
The latter three assumptions apply to the first law
of mass action. In the steady state of the hormone (H)-

receptor (R) binding complex (HR) the equation:

V

K2

describes the equilibrium and the K values are the reaction
rate constants. The affinity or equilibrium constant for
i

dissociation (Kd) may be defined in terms of the second

order chemical kinetic reaction:

K H (R-HR) K
K1 HR
Expanding and isolating the hormone-receptor term the con-

centration of HR equals:

HR - H (HR) or H-R 1 (R-HR)
Kd H Kd

 

Kd may also be defined as the inverse the association con-

stant, K
a

32

from which the Scatchard (1949) expression of bound hormone

(B) to free hormone (F) is derived:

Ka (R-HR)

HR

R
assuming one free binding site per one free binding site
molecule. According to the terminology of Scatchard,this

expression extends to number of binding sites (q) and bound

hormone (B) and

- = Ka (CI-B)

By plotting the concentration of bound, labeled hormone
against free labeled hormone a steady state reaction curve
is generated. By standard Scatchard notation, the plot of
the concentration ratio of bound labeled hormone to the free
labeled hormone (B/F) in cpm against bound hormone (B) in
cpm produces a straight line for the idealized system with
an abscissa intercept equivalent to the number of binding
sites (q) and the slope interpreted as the negative Ka

(-Ka), equivalent to the Kd.

33

Experimental

 

1. Correlation of Deoxyribonucleic Acid and Protein Content
to Cell Number

A. Objective

In order to better quantify per cent specific binding
and cell number, an assay more sensitive to cell number and
indicative of a random cell population was chosen. A study
of the correlation of cell number with the concentration
of deoxyribonucleic acid (DNA) and protein was performed

on two rat pituitary cell cultures.

B. Procedure

Triplicate samples of the different cell concentrations
utilized for prolactin binding studies were performed on
two readily available rat pituitary cell cultures: C811RAP
(Sonnenschein, 1974) and a normal retired breeder rat
pituitary cell culture. Both cell types were collected
several weeks prior to use and were stored at -200 in 10%
glycerol-PDME, a cryoprotective agent. As previously
described (see Materials and Methods, Section III), cell
number, protein and DNA were determined.

DNA concentrations of unknowns were compared with a
standard curve of triplicate samples of calf thymus DNA
(Sigma, St. Louis, M0) at the concentrations: 1.0, 2.5,
5.0, 10.0, 25.0, 50.0, 100.0 ug per sample. Protein con-
centrations were determined by the method of Lowry gt al.

(1951) on aliquots of carefully counted prolactin binding

34

cells from C811RAP and retired breeder cultures. A
standard protein curve was constructed for absorbance
plotted against 10, 40, 50, 80, 100, 120, 150, 180, 200

ug per sample of bovine serum albumin (Sigma Chemical Co.).

C. Results

The relationships of DNA and protein concentrations
to cell number for C811RAP and retired breeder cells are
illustrated in Figure l with the ratios of DNA (pg) per
cell: protein (ng) per cell. Protein and DNA content
increased with increasing cell number as expected. The

mean values and the standard error of the mean (SEM) as

illustrated in Figure 1 are given in Tables I and II.

D. Discussion

With both cell cultures, protein and DNA concentra-
tions increased with increasing cell number. The C811RAP
tumor cells had significantly greater DNA concentrations
as indicated by the Student's t test at the 0.95 con-
fidence interval in comparison to the values of DNA for
the cells of immature and retired breeder rats (Table 1).
Protein contents, however, were not significant by the
Student's t test at the 0.95 confidence interval greater
for the normal retired breeder cultures vs C811RAP cells
(Table II). These relationships were also illustrated in
the ratios of DNA: protein for both cell types (Figure

l). The tumor derived cell had a ratio of 0.2643 compared

35

Table I.

 

DNA Content of Tumor Rat Pituitary Cells (C811RAP) and

Normal Rat Retired Breeder Pituitary Cells

 

Type Cell Number Average DNA :SEM Sample
(per ml) ug Size (n)
C811RAP 1.0 x 105 1.33 0.17 6
2.0 x 105 6.13 0.07 6
5.0 x 105 10.23 0.63 6
1.0 x 106 13.0 1.34 6

** Average DNA content per cell

I +

SEM = 19.27 pg 1 4.1674

4

Retired 3.4 x 10 1.17 0.19 6
Breeder 5
1.75 x 10 4.87 0.13 6
S
3.4 x 10 5.16 0.12 5
5.0 x 105 8.17 1.74 6

** Average DNA content per cell

|+

SEM = 9.79 pg 1 1.54

 

* Immature

Average DNA content per cell 1 SEM = 14.97 pg 1 4.418

 

* Insufficient number of cells to assay a range of cell
concentrations. Abgve value obtained for a concen-
tration of 1.0 x 10 cells.

** Significant difference in DNA per cell between C811RAP
and Retired Breeder cells at 0.05 level.

36

Table II.

 

Protein Content of Tumor Rat Pituitary Cells (C811RAP)

and Normal Rat Pituitary Retired Breeder Cells

 

Cell Type Cell Number Mean 1 SEM Sample
(per m1) (mg) Size (n)
C811RAP 2.2 x 105 29.50 5.06 6
3.6 x 105 35.60 3.08 11
1.0 x 106 49.0 2.91 10
1.5 x 106 141.0 4.86 7

** Average protein per cell = 72.90 ng : 18.79

Retired

Breeder 1.0 x 105 24.67 1.52 6
1.6 x 105 28.71 2.23 5
2.2 x 105 36.38 3.02 8
1.6 x 106 58.60 7.58 5

** Average protein per cell = 122.83 ng 1 35.77

 

** Non-significant difference in protein per cell be-
tween Retired Breeder and C llRAP cells at 0.05
level. 8

37

Figure 1.

Mean concentrations + standard errors of
the mean for DNA (ugT and log protein
mean concentrations (mg) of C811RAP and
Retired Breeder cells.

Ratio DNA per cell: Protein per cell:
C811RAP = 0.2643

Retired Breeder = 0.0797

38

32.00 a
«noun..-

 

 

 

 

ouc‘uc O .8 (:0
M B m u D 9 o 7 6 5
P _ _ P _ k P P p P
1 _ _ . . d _ . a _
11w
1. 11m
4'
II"
It!
l1m
I I'm
Ifi‘e
J'
116
'1"
t
. Ill-2
+ m .1 w m w m _1 m
u o 5 o 5 n m 5
a 2. H w. w. m o. 0 u
Senna-.3 - 2.. 2.32... 3..

35.5.. 0

lol5

NUMBER OF C ELLS

FIGURE I

39

with a ratio of 0.0797 for the normal cell. These values
were indicative of the activity of the individual cell
types.

The neoplastic C llRAP cell replicated at a faster

8
rate than the normal pituitary culture. The higher DNA
values for C811RAP were indicative of this rate. A
heteroploid or aneuploid state existing in the tumor cell
was possible. In 1970, Sonnenschein gt gt. demonstrated
karotype differences in a rat pituitary tumor derived cell
culture. The average protein concentration of normal
pituitary cell was greater than that of tumor cells.
These results may be correlated to the high prolactin
levels released into the medium by normal cells. With
a greater prolactin synthesis and release than C811RAP
(Payne, unpublished data), retired breeder cells might be
expected to have a higher protein content (see Experimental,
Section II). The high standard errors of the mean for
protein may partially be attributed to the different days
of cell harvest or collection and the stage of prolactin
synthesis and release. As previously shown, prolactin
concentrations of the medium varied during the period of
collection.

The DNA concentrations per cell (Table I) do not
correspond to the normal diploid chromosome content of
6.6 pg per cell as determined by Vendrely gt gt. (1955).

These values more closely correspond to the 10.82 pg per

cell levels obtained by Leavitt gt gt. (1973) for isolated

40

rat anterior pituitary cells. This higher value was at-
tributed to cellular aneuploidy or polyploidy. The
greater DNA content of the immature rat (14.97 pg 1 4.418)
in comparison with the retired breeder cell (9.79 pg 1 1.54)
followed the trend observed in other ontogenic studies
(Leslie, 1955) and is indicative of the faster replication
and deve10pmenta1 rates of the younger animal. The average
DNA contents per cell for normal and neoplastic pituitary
cells were also greater than the levels obtained for other
tissues of the normal rat. Thomson gt gt. (1953) indicated
a DNA range of 6.46 pg per nucleus to 6.90 pg per nucleus.

The greater DNA content for normal rat pituitary cells
may be the result of a greater susceptibility of chromo-
somes to damage during trypsinization or culture i.e.
translocation of chromatin during mitosis producing aneu-
ploidy and/or polyploidy. It would be of interest to
extend this study to all age groups at various days of
culture and to compare protein, DNA, and cell number to

prolactin secretion tg vitro.

II. Concentrations of Prolactin of the Culture Medium as
a Function of Age of Animal and Days in Culture

A. Objectives

In female rat serum and pituitary glands, concen-
trations of prolactin remain low until day 37, when a
sharp increase in pituitary content is noted with a
doubling in rate on approximately day 45 of vaginal open-

ing and again between 46-78 days with a sustained rate

41

until day 400 (Yamamoto gt gt., 1970; Voogt gt gt., 1970).
Since concentrations of prolactin in both the serum and
the anterior pituitaries differ according to the stage of
sexual development, the study to measure the release of
prolactin by cells cultured from anterior pituitary glands
of sexually immature, mature and old (retired breeder)
female rats was undertaken. Whether the tg ttttg situation
increased, maintained or decreased the amount of prolactin
release without chronic hypothalamic inhibition or stimu-
lation was also studied. The concentrations of prolactin
of specific cultures were then correlated with the degree

of specific binding of these cells.

B. Procedures
1. Culture

Cell cultures of pituitary glands for each age were
maintained in a humidified 95% 02:5% C02 atmosphere at 37°.
The incubation was changed at 48 hr intervals (see Materials

and Methods, Section II).

2. Assay Procedure

For each culture derived from a specific age group,
samples of medium from two pools of medium of four flasks
per pool for each 48 hr period were collected and frozen
at -200 until radioimmunoassays were performed within one
month of collection. Culture medium (PDME) samples not

used in culture were also assayed. The period of cultivation

42

for cells for a specific age of animal was dependent upon
the individual culture dispersion or pituitary gland cell
separation. Cultivation ended with cell death or termina-
tion of the culture for prolactin binding studies. The

average length of the incubation period was ten days with

two day intervals of medium collection.

C. Results

The concentrations of prolactin found in the culture
medium of each age for 10-14 day periods of culture are
shown in Table 111. Mean concentrations of prolactin :
standard error of the mean (SEM) for individual cultures
are also listed.

A three-factor nested analysis of variance for un-
equal sample sizes for the factors contributing to pro-
lactin secretion concentrations. Each of the following
factors were accounted for in the analysis: (1) the
fixed effect of age for individual cultures; (2) the
random effect of culture units or flasks as samples with-
in a culture; (3) the fixed effect of day in culture; and
(4) the interaction of culture units and day in culture
which contribute to the differences in levels of prolactin
and inseparable from experimental error (Appendix II).
The significance of the contribution of each factor was
tested by the F-distribution. If calculated F values
proved significant (Table II) and the tested hypothesis

of "no factor effect" (H:E=O) was rejected, further

43

analysis was performed on the interaction of age and day
in culture. Individual levels of prolactin for specific
days in culture were compared for single cultures by

Scheffé's analysis of contrasts (Appendix V).

D. Discussion

Analysis of variance indicated a Significant con-
tribution to prolactin concentrations of the medium by
the different ages of animals used for culturing, the day
in culture, and the interaction of the age of the animal
and the day in culture at the 0.90 confidence interval
(Table IV). With respect to the age-dependence of cultures,
variations in concentrations of prolactin were greatest for
cells derived from female retired breeder rats when com-
pared with immature females, followed by mature vs immature,
and the least age-dependent difference with retired breeder
vs mature females (Appendix IV and Table IV). Significant
differences in the variances from the means for prolactin
concentrations for specific days in culture were found for
Day 4 vs the first day and all subsequent days in culture
and the first two collections (Days 2 and 4) and the latter
periods of Days 8 and 10 (Appendix VI).

Scheffé's analysis of contrasts (Appendix VI) in-
dicated Day 10 as significantly different when compared
with the prolactin values of other days in culture. Day
4, however, exhibited the most significant difference in
prolactin concentration from the mean during the ten day

period of culture.

44

 

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46

The variations in levels of prolactin released into
the medium for the different classes of female rats in
sexual development qualitatively corresponded to the trend
of those for the tg tttg female rat sexual development.
Quantitatively, ifl.li££2 concentrations of PRL exceeded
tg tttg serum quantities of the same age. Concentrations
of prolactin were impossible to compare on a single pitui-
tary gland or weight of pituitary gland basis. The sensi-
tivity of the primary cell culture, its metabolic rate, and
the heterogeneous cell population of epithelial cells and
fibroblasts must be considered in the evaluation of pro-
lactin in the medium. The time required for recovery after
trypsinization and establishment of cells in culture were
additional factors contributing to the variations in pro-

lactin concentrations.

III. The Binding of Prolactin-125 Iodine to Normal Rat
Pituicytes and Its Correlation to Secretion of Prolactin
12112.22

A Objective

The ability of prolactin to inhibit its own synthesis
and release has been suggested by MacLeod gt_gt3 (1970) from
prolactin secretion studies both tg_tttg_and ifl.!i££22 Posner
gt gt. (1974) has suggested prolactin may regulate its own
receptors either by inducing or activating them. The

degree of specific binding of prolactin to pituicytes cor-

related with levels of prolactin in the culture medium may

47

support this hypothesis. This system may provide a model
for the study of the direct action or feedback of prolactin
on the pituitary cells which synthesize and release it and
also for its effect on the regulation of other hormones of

the pituitary.

B. Procedures
1. Culture

Cells of pituitary glands were cultured according to
the ages of female rat: immature, mature, and retired
breeder. At a period of sufficient growth (10 to 14 days
after initial dispersion of the pituitary glands), cells
were collected as previously described. The media of each
group of cultured cells after collection were stored at
-20° until assayed for its concentrations of prolactin

(see Materials and Methods, Section 11, IV, and V).

2. Binding Assay

The methods of preparation and binding of iodinated
prolactin to those cells is cited in the Materials and
Methods (Section V). To all samples the previously de-
scribed binding components were added. Assays varied in
the addition of: (l) the type of cells: immature, mature,
or retired breeder normal rat pituitary cells; (2) the
cell number: 1x104 to 1x106 cells per sample; and (3)

initial counts of prolactin-1251 per sample: 10,000 dpm

48

to 100,000 dpm. In each assay, separate cell cultures of
each age group and different preparations of iodinated
prolactin were used. All binding assays were performed

at 40 for 30 min.

C. Results

The percentages of total, non-specific and specific
binding were calculated for each binding assay of prolactin-
1251 with pituicytes of immature, mature and retired breeder
rats (see Materials and Methods, Section VI). The sig-
nificance of the difference of means of the final counts
for total versus non-specific bindings was analyzed by the
one-tailed Student's t test to ascertain if the mean of
total binding counts was significantly less than the mean
of non-specific binding counts (Table V).

The relationship of the per cent of specific binding
and the concentrations of released prolactin (ng/ml) on
the day of collection of cells (Table VI) was also analyzed
by the method of correlation (Appendix VII). The line
generated by the method of linear regression (Appendix VIII)
of per cent specific binding of prolactin plotted against
prolactin levels of the medium (Figure 2) illustrated the

positive correlation of the two variables.

D. Discussion

The values of per cent specific binding of prolactin

presented in Table V support the theory that receptors

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49

 

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mHHou xpwuwsufim HmEuoz wowsuazo ou HmNH-:fiuowH0Hm mo mcflvcfim one .> magma

50

for prolactin exist on the cells of the pituitary gland.
Calculated t values for the means of total and non-
specifically bound counts (Table V) further illustrated

the significance of these findings. Scatchard plot analysis
of bound and free iodinated preparations, receptor-saturation,
and competitive inhibition analyses Inn; impossible due to

the limited availability of binding material and the
variability in the capabilities of receptor-binding
(biological activity) of the iodinated preparations.

The analysis for correlation between per cent specific
binding and levels of prolactin released indicated a high
positive correlation (r=0.8147:0.2366) for the different
ages (Table VI and Appendix VII) as depicted in Figure 2.
The data obtained from binding assays indicated that pro-
lactin may induce (synthesize or activate) receptors for
prolactin. The cellular mechanism controlled by the
specific binding of prolactin to pituicytes was not

elucidated and requires further study.

51

Table VI. The Correlation of Per Cent Specific Binding
and Prolactin Secreted by Three Ages of

Rat Pituicytes

 

 

Cell % Specific ng PRL
Type Binding (yl) Secreted (yz)
ng/ml
Immature 30 11.0
Mature 48 165.4
Mature 28 127.4
Mature 21 50.5
Mature 29 28.2
Retired Breeder 56 288.0
Retired Breeder 13 63.6

 

0.8147 + 0.2366 = Correlation Coefficient (r) : Standard
Error of the Correlation Coefficient (Sr)

52

Figure 2.

Correlation of er cent specific binding
of prolactin-12 I and prolactin secreted
(ng per ml) for immature, mature and
retired breeder pituitary cells.

Correlation Coefficient = 0.8147:0.2366
significant at 0.01 level.

PERCENT SPECIFIC BINDING

 

53

 

70——
00“”
A
50“ .
I Immune
o NATURE
__ A serum:
‘0 aneeocn
30“ o
r. o.u47¢o.2330
20—-— °
I0---'
1 I l I I I 1 I l I
I I v I I I I l I
50 I00 I50 ZIDO 250 300
PROLACTIN
no per ml

FIGURE 2

General Discussion

 

In the mixed cell cultures of the rat pituitary, the
age of the animal, the number of days in culture and the
interaction of age and day in culture significantly in-
fluenced the levels of prolactin released as determined by
radioimmunoassay. Protein values per cell for retired
breeder cells were found not to be significantly greater
than those for the tumorous pituitary cells of the C811RAP
line. The DNA values were greater in both cell types than
those reported by Vendrely (1955), Leslie (1955) and
Cunningham gt gt. (1950). The concentration of DNA per
cell of the C811RAP culture was, however, significantly
greater than those for the retired breeder and the im-
mature cultures. These levels of DNA suggested chromosomal
abnormalities (polyploidy or aneuploidy) which may have
influenced the levels of prolactin secreted and specific
binding of prolactin. From the study of the release of
prolactin tg_ttttg by normal immature, mature, and retired
breeder female rats and the binding of their respective
cells with iodinated prolactin, cell cultures have demon-
strated their ability.

Cell cultures are advantageous in binding studies of
prolactin because of the ease of collection without
enzymatic or physical damages. Decreased damage to bind-
ing sites and maximized exposure for interaction with
surface area at the membrane were also advantages that

other preparations do not have. Iodinated prolactin bound

54

SS

specifically to normal cultured rat pituitary cells. A
positive correlation (0.8147) of per cent specific binding
and prolactin released into the medium was demonstrated.
Whether this mechanism has an autoregulatory effect in

the pituitary is unclear. The present variability of
individual iodinated preparations in their biological
binding activity and the cultures with varying percentages
of the cell types of the pituitary and different stages

of metabolism make such a conclusion difficult to support
quantitatively. Levels of endogenous prolactin from cul-
tured pituitary cells may bind and saturate binding sites
during assays competing with both added prolactin and

prolactin-125

I for binding sites. However, if the rate
of binding is rapid (Frantz gt_gt,, 1974) and an equil-
ibrium state is attained (Kahn gt gt., 1974), this factor
may be minimized.

The possibility also exists that binding sites located
on pituitary cells may change with aging of the rat. Older
rats may have a greater number of binding sites per cell
or higher binding affinities for prolactin. Baker gt gt.
(1974) have reported the greater persistence of the
acidophil vs the basophil and chromophobe cell population
of the rat pituitary tg_ttttg. Histological studies of
the pituitary have demonstrated a greater number of
acidophils per pituitary in mature female and lactating

rats (Meites gt gt., 1961). If these conditions existed

in cell culture, then the high per cent specific binding

S6

and concentration of prolactin would be expected.

It may also be postulated that prolactin may induce
its own receptors. The function of such a mechanism in
the pituitary is obscure. However, a higher degree of
specific binding of prolactin may be indicative of a con-
trol mechanism for prolactin release by exocytosis (Hopkins
and Farquhar, 1973) rather than a dual synthesis and re-
lease system. If this theory is true, the exhaustion of
prolactin stores by otherwise viable cells would be ex-
pected. Such a condition was not exhibited as decline in
cell number and poor morphology were associated with low
prolactin concentrations.

The presence of a quantal response or attainment of
a threshold level of prolactin binding for release of
prolactin would produce a sigmoid curve for per cent
specific binding plotted against prolactin concentrations.
This quantal response might involve the adjustment of new
threshold points. Therefore, higher levels of specific
binding would release greater amounts of prolactin con-
centration or per cent specific binding plotted against
age of the rat. In respect to normal physiological states,
a positive feedback alone for increase in release of pro-
lactin with binding of prolactin to the pituitary would
be indicative of a pathological rather than a normal
regulatory mechanism.

Recent assays in our lab have indicated an increase

in non-specific binding values for iodinated prolactin.

S7

The factor(s) which contributed to increased non-specific
binding and variability of iodinated preparations is (are)
unknown. During the procedure of enzymatic iodination,

the labeling of prolactin with 125

iodine may disrupt the
stearic configuration necessary for its biological ac-
tivity. The o-prolactin provided by NIH may consist of a
mixture of monomers and polymers of prolactin as suggested
by current research in our laboratory. These variable
forms may decrease the number of biologically active
molecules of prolactin.

Study of the biochemistry of the prolactin molecule,
the characterization of the method and degree of the iodina-
tion of prolactin by the lactoperoxidase-hydrogen peroxide
oxidation, and the conditions of ionic concentration and
pH are required for production of a standardized iodinated
prolactin. The kinetics of the binding reaction: its
dependence on time and temperature of incubation must also
be investigated.

From the study of primary cultures of the pituitary
of the female rat, the heterogeneous cell population of
the primary cultures demonstrated the necessity of an
established, cloned or single cell derived acidophilic
culture for more definitive studies of the binding and
secretion of prolactin. This approach appeared most
feasible with dispersion of retired breeder pituitary
glands. These glands had the greatest number of cells

per pituitary and the best growth characteristics in

58

culture. If the addition of a factor for prolonged cell-
ular growth and maintenance would not interfere with
osmotic or ionic regulation or experimental study such a
substance should be investigated.

If a stable and easily standardized radiolabelled
hormone can be developed, a rapid and efficient binding
assay for clinical application may be achieved. Hormonal
dependence of various pathologies and their response to
treatment may be elucidated with binding assays. Cultured
cells would greatly aid in this development because of
the ease of maintenance and use, the availability of
cells, and the relative homogeneity of an established

culture.

 

APPENDICES

APPENDIX 1: COMPOSITION OF DULBECCO'S
MODIFIED EAGLE (DME) MEDIUM

POWDER*
Component mg per L
L-Arginine - HCl 84.00
L-Cystine ' 2HC1 62.57
L-Glutamine 584.00
Glycine 30.00
L-Histidine HCl - H20 42.00
L-Isoleucine 105.00
L-Leucine 105.00
L-Lysine HCl 146.00
L-Methionine 30.00
L-Phenylalanine 66.00
L-Serine 42.00
L-Threonine 95.00
L-Tryptophane 16.00
L-Tyrosine (Disodium salt) 104.20
L-Valine 94.00
CaCl2 (anhydrous) 200.00
Fe (N03)3 - 9H20 0.10
KCl 400.00
Mg 804 (anhydrous) 97.72
NaCl 6400.00
NaH2 PO4 - H20 124.00
Glucose 1000.00

59

60

Component mg per L
Phenol Red (a) 15.00
Sodium Pyruvate 1100.00
D-Ca Pantothenate (b) 4.00
Choline Chloride 4.00
Folic Acid 4.00
i-Inositol 7.20
Nicotinamide 4.00
Pyridoxal HCl 4.00
Riboflavin 0.40
Thiamine HCl 4.00

* GIBCO, Grand Island, NY

61

APPENDIX II:

 

Concentrations of Prolactin: Three Way Nested Analysis
of Variance of the Means for Unequal Sample Sizes1 r.f.

Table I

 

y = u + ai + B(i)j + Yk + (aY)ik + (BY)(i)jk + E(ijk)

Y = prolactin concentration
u = prolactin mean for all samples

a = fixed effect of the age of the 1th group
of rats (culture)

B(i)' = random effect of jth culture flasks or
3 units in ith culture

“k = fixed effect of kth time or day in
culture

(ay)ik = interaction of age and day in culture
(By) . 'k = interaction of culture flasks and day in
(1)3 culture, inseparable from experimental

error

B(ijk) = experimental error

1 Sokal and Rohlf (1969), pp. 274—286.

62

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um

m + um u uoupm

 

 

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OH
Hanan“ u< u eunuflsu CH ken cam
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mNee NN.emm.eN eN.mNN.emm e m u mxmeNm eeseNso

N HUM m

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mopmscm ewe: mopmscm mopmscm m.m.wv Eovompm sewumwum> mo
wouoomxm :mmz mo Esm mo moopmoo N oopsom
.HH> manme

A<2Hz< mo mo< mz< mm<mgmm

ZHHU<Aomm mom muz<Hm<> no mHm>A<z<

"HHH x~mzmmm<

63

APPENDIX IV:

 

Tukey's T—Test for Analysis of Differences in Medium

Prolactin Concentrations for Immature, Mature and Retired

Breeder Female Rats3

 

tT = (5,1. - 92')

 

 

/
1 1
+ -
MSE (; r )
1 2
t = calculated t value
T
91. = mean prolactin level of age group 1

92. = mean prolactin level of age group 2

 

M8 = mean square error of culture flasks = B
E
r1 = number of samples in age group 1
r2 = number of samples in age group 2
Comparison Retired Breeder Mature Retired Breeder
vs vs vs
Neonate Neonate Mature
T Value *22.34 *15.77 *6.57

 

3 Gill, J.L. (1976) Design and Anatysis of Experiments in
the Animal and Medical Sciences, to be published, Iowa
State University Press, Ahes,_Iowa.

 

* Significant at a critical T value (t,v) of 4.325 (3,49)
and 0.01 level of significance from "Upper Percentage
Points Tukey's T (qa,t,v) of Studentized Range", 1969.
Order Statistics and Their Use in Testing and Estimation.
Vol. I. Aerospace Research Lab. US Government Printing
Office, Washington.

64

APPENDIX V:

 

Scheffe's Paired Contrasts for Analysis of Effect of Day
in Culture on Concentrations of Prolactin Released into

the Medium

 

Scheffe's Mean 1 Confidence Interval4

 

v (6],)

51:"-
k (t 1)fa’t-l, n-t

where v (qk) =E. 1(Czik/ri) MSE4
1:

q = mean prolactin concentration of the kth
k contrast

t = number of groups (ages)

n = total number of samples

v(qk) = variance of the mean qk
ri = replication or number of samples per day
Cik = constant (2 Cik = 0)

MSE = mean square error

= F-distribution value for a level of
significance = a and t-l, n-t degrees of
freedom

f
9' t'l, n-t

4 .
G111, (1976), to be published.

 

65

Ne>6N No.0 on“

em eeeeNmNeNNm e

 

NN.NNN H N.NN

mN.me + Nm.H

+l

mN.me No.mov«

+|

mN.mmH No.vmm«

NN.NNNH NN.NNN«

NH Neg

N Neg

NH Nee

N Nee

e Neg

m>

m>

m>

m>

m>

N Neo

N Nem

e Neg

4 Neo

4 Neg

NN.NNN u NN.NNN«

+|
m
l\
03

mN.me
mN.me + m.Nv
o~.mwa + o.ov

NN.NNN H NN.NNN«

NN eem N ANNN

m>

e cam N Nam

0N Nam m> N Nam

w xmn m> N xmm

o Nam m> N zen

v ken m> N Nam

 

Hw>HWucmdmocowwmcoo

ehzuasu :N mxma
mo mpmmhpcou

Hm>HWHcH
oocowfimcou + mxwv cam:
capomHONQ

onsuaso ca mxmn
mo mummuucou

 

+ A V :moz
cfipomaoum
manhqao 2H

.HHH> mqm<9

><Q m> mm<mqmm ZHHU<Aomm mo mHm>A<z< m.mmmmmom NH> XHszmm<

66

APPENDIX VII:

Product-Moment Correlation Coefficients r.f. Figure 2

r12 = Zylyz
V2 22 2
Y1 Y2
r1 = correlation coefficient of per cent specific
2 binding (yl) and levels of prolactin (yz)
Xyly2 = summation of the products of values for each
Y1 and its corresponding y2

£y12 = summation of the squared values of each y1
Zyzz = summation of the squared values of each y2

Standard error of the correlation coefficient

 

3r = /(l-r2)/n-2

S = standard error of correlation coefficient
r = correlation coefficient

n = number of samples considered in the
correlation

5 Sokal and Rohlf (1969), pp. 494-548.

67

APPENDIX VIII:

Linear Regression Analysis6 r.f. Figures 1 and 2

Linear Regression Equation

Y = a + b X
XY

<>
0

estimated value of Y for a given x value
a = Y intercept of the regression line

b = slope of the regression line or regression
coefficient

X = given x value

by-x = Xxy
2x2
x = X - X X = mean of X values
Y = Y 7 X Y = mean of Y values
a = Y - by~xx

. . . . 7
Logar1thm1c Transformat1on of a Dependent Var1able
A

log Y = log a + b (log e) X

Curvilinear Regression by Orthogonal Polynomial8
A
Y = A + Bgl + C52 + D53
A
Y = estimate of Y for a given x
E. = coefficient of orthogonal polynomial

A,B,C,B= regression coefficients corresponding to given
x values

6 Sokal and Rohlf (1969), pp. 404-420.
7 ibid. p. 477.
8 ibid. p. 470.

REFERENCES

 

References

 

Absher, M., 1973. Hemocytometer Counting. In: Tissue

Culture Methods and Applications, 395-397. Academic
Press, New York.

Amenomori, Y. and J. Meites, 1970. Effects of a hypo-
thalamic extract on serum prolactin levels during
the estrous cycle and lactation. P Soc Exp M 134:
492-495.

 

Averill, R.L.W., 1969. Depression of thyrotropin releasing
factor induction of thyrotropin release by thyroxine
in small doses. Endocrinol 85:67-71.

 

Baker, B.L., J.R. Reel, S.D. Van Dewark, Y.Y. Yu, 1974.
Persistence of cell types in monolayer cultures of
dispersed cells from the pituitary pars distalis

revealed by immunohistochemistry. Anat Rec 179:
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