[CANCER RESEARCH 33, 721-723,
April 1973]
Plasma Gonadotropin Levels during Early Stages of Ovarian
Tumorigenesis in Mice of the WX/WV Genotype1
Edwin D. Murphy and Wesley G. Beamer2
The Jackson Laboratory, * Bar Harbor. Maine 04609
SUMMARY
The ovaries of (C57BL/6J
X C3H/HeJ)
Fi-W/W"
mice
contain few oocytes at birth, rapidly lose the follicular
apparatus, and develop a 95% incidence of bilateral complex
tubular adenomas by 5 months of age. Plasma levels of the
pituitary gonadotropic hormones, luteinizing hormone and
follicle-stimulating hormone, determined by radioimmunoassay, were significantly elevated between 30 and 210 days of
age in W/W" mice in comparison with congenie +/+ controls.
Both hormones were increased at least 4-fold throughout the
period except for luteinizing hormone, which was increased
2-fold at 30 days. Ovariectomy at 60 days of age induced the
same maximum levels of luteinizing and follicle-stimulating
hormones within 2 weeks in females of both genotypes. These
findings provide quantitative confirmation of the hypothesis
that prolonged stimulation by gonadotropic hormones induces
ovarian tumors.
INTRODUCTION
Mutant alÃ-elesat the W locus produce the pleiotropic effects
sterility, macrocytic anemia, and lack of hair pigmentation
(11). The mechanism of gene action on the gonads involves
interference with the migration and proliferation of the
primordial germ cells (6). (C57BL/6J X C3H/HeJ) F1-WX/WV
females (hereafter called B6C3F]) have less than 1% of the
normal complement of oocytes at birth, followed by an
exponential decrease until none are present at 13 weeks (7).
The sequence of morphological changes in the ovaries during
the 1st 6 months of life is loss of follicular apparatus,
interstitial cell hyperplasia and ingrowth of "germinal"
epithelial tubules, and formation of complex tubular ade
nomas composed of invasive "germinal" epithelial tubules and
interstitial cells (7). Bilateral ovarian tubular adenomas
develop in over 95% of E6C3FÕ-WX/WVfemales by 5 months
of age but not in congenie +/+ females by 24 months of age (7,
8). Granulosa cell tumors and luteomas develop in 38% of
mice of the W*/W" genotype surviving 16 months of age (8).
'Supported in part by NIH Research Grant CA-05985 from the
National Cancer Institute, NIH Research Grants HD-05523 and
HD-04083 from the National Institute of Child Health and Human
Development, and a contribution from Dr. and Mrs. Clarence C. Little.
3Postdoctoral Fellow, National Cancer Institute Training Grant
CA-05013.
'Fully accredited by the American Association for Accreditation of
Laboratory Animal Care.
Received December 11, 1972; accepted January 5, 1973.
The major hypothesis for the cause of ovarian tumorigenesis
in rodents postulates prolonged stimulation by gonadotropic
hormones (2—4). This study reports persistent high plasma
levels of LH4 and FSH, measured by radioimmunoassay,
during the age period of development of complex tubular
adenomas in the ovaries of B6C3Fi -Wx/W"mice.
MATERIALS AND METHODS
Mice. Congenie B6C3F{-WX/W» and +/+ mice were
produced by ma tings of C57BL/6J W/+ females with
C3H/HeJ W"/+ males. Female mice were weaned between 3
and 4 weeks of age and placed in groups of 2 to 4 in stainless
steel cages (10 x 14 x 24 cm) with food and water continually
available. A photoperiod of 14 hr with lights on between 5
a.m. and 7 p.m. was maintained. Experiment 1 is a survey of
circulating gonadotropin levels in +/+ and W/W" females at
30, 60, 90, 120, and 210 days of age. The animals were killed
by decapitation for plasma samples, which were then stored at
—¿20°.
All samples were collected between 10 a.m. and 2 p.m.
to avoid proestrous gonadotropins. In Experiment 2, the
functional state of the pituitary-gonadal axis in +/+ and
Wx/W females is evaluated by ovariectomy at 60 days of age
and terminal decapitation for plasma samples 1, 2, and 4
weeks later.
Hormone Assays. All accumulated samples within each
experiment were assayed at the same time for either LH or
FSH. Radioimmunoassays for LH and FSH were carried out
by double-antibody
precipitation
methods previously de
scribed (1). Materials for radioimmunoassay of rat LH and
FSH were supplied by the rat pituitary hormone program of
the National Institute of Arthritis, Metabolism and Digestive
Diseases, Bethesda, Md. '3 ' I was obtained from New England
Nuclear, Boston, Mass., as Nal at greater than 500 mCi/ml for
protein iodination. Mouse LH and FSH used as reference
standard hormones were prepared by Dr. W. G. Beamer. All
values for plasma hormones are expressed as ng equivalents
N1H-LH (or FSH) SI per ml plasma. Plasma samples were
assayed at 1:1 dilution in duplicate; duplicate tubes yielding
values differing by 10% or more were discarded. Insufficient
plasma for 2 assays was frequently encountered in 30-day-old
animals. The data of Experiment 2 were subjected to analysis
of variance, and individual treatments were analyzed by the
Student-Newman-Keuls multiple range test.
*The abbreviations used are: LH, luteinizing
follicle-stimulating hormone.
hormone;
APRIL 1973
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1973 American Association for Cancer Research.
FSH,
721
Edwin D. Murphy and Wesley G. Beamer
RESULTS
Experiment 1. The plasma LH and FSH levels found in
intact B6C3P! -WX¡W"and congenie +/+ female mice are
presented in Chart 1. Throughout the ages examined, both
gonadotropins
were significantly elevated in WX¡WVas
compared with +/+ females. Both hormones were elevated at
least 4-fold, except for LH levels in the 30-day samples, which
showed about a 2-fold increase.
Experiment 2. The effects of ovariectomy at 60 days of age
on plasma gonadotropins
in W*/W and +/+ mice are
summarized in Chart 2. A significant rise (p < 0.001) in
plasma LH of +/+ and WX¡W"
mice occurred by 2 weeks after
ovariectomy, when mean levels had increased 5- and 2-fold,
respectively. On the other hand, mean plasma FSH was
significantly higher (p < 0.001) than intact levels 1 week
postovariectomy for both +/+ and WX/W" mice. Two weeks
after ovariectomy, mean FSH levels were approximately 7-fold
greater for +/+ mice but were only 50% greater for WX/W
mice. Thus mice of both genotypes responded to ovariectomy
with increased levels of circulating gonadotropins and achieved
the same absolute levels of LH and FSH within 2 weeks.
30.024.0OI
40OOOEI/>
21 0
O°-
0o.
Chart 1. Mean plasma levels of LH 18.0i_iCT
and FSH in congenie +/+ (o), and W*/W
(•),female mice between 30 and 210
days of age. Numbers of mice and S.E.
(vertical bars) are indicated.
57
300OEX\1Oi2OOOC10005OO6}8
—¿
II
*IO
ir
'i6T
12.0C9.
06.
'*
g4
03.0lii6{
-
o
ì 5 '
$5,1w*/wv;o
60
30
90
71
v+IO
9o 6
C
\1
30
9O
120
ö
120
ZIO
60
I
210
Age ( days)
n -I-/-I-IO
IO
Li
I
6. 9
99L*;%^Ã-¿#Ã-¿m%a-<
o
i
Chart 2. Mean plasma levels of LH
and FSH in congenie +/+ and WX/WV
female mice before and 1, 2, and 4
weeks after ovariectomy at 60 days of
age. Numbers of mice and S.E. (vertical
bars) are indicated.
^
o.
16.0
|
I4.O
i12-0
-r
E 3OOO
I
co
10.0
0« 8.0
4000
in
O
a.
-^ u>6iS^v//////////////////////////////.W//////////AVs«NS
i1—¿
e 2000
NX
6,0
«X^Ax^x^s
1000
4.0
2.0
500
\^ÕÕ1Ssw////////////,^1
Intact
I
4
After
722
intact
ovariectomy
(weeks)
CANCER RESEARCH VOL. 33
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1973 American Association for Cancer Research.
LH, FSH, and Ovarian Tumors in W/W Mice
confirmation of the hypothesis that prolonged stimulation by
gonadotropic hormones produces ovarian tumors. Repeated
s.c. implantation of normal ovaries at 6-month intervals in the
W*/W genotype inhibits ovarian tumorigenesis for at least 2
DISCUSSION
years (E. D. Murphy, unpublished data).
The known models for ovarian tumorigenesis in the mouse
The elevation of LH and FSH plasma levels by 30 days of
age in female mice of the WX/WVgenotype and the persistence involve drastic depletion of oocytes and the follicular
of high levels during the 1st 7 months of life correlate well apparatus: genie deletion of oocytes, X-irradiation, transplan
with the morphological changes reported in E6C3F¡-WX/WVtation to the spleen and other sites, treatment with chemical
ovaries during this period (7). By 30 days of age, all of 6 carcinogens, spontaneous tumors of old age (7), and neonatal
control ovaries have produced Graafian follicles and some have thymectomy (9). The depletion of oocytes occurs earlier in
formed the 1st corpora lutea. Approximately one-half of 12 the HMocusmutants than in most other models.
WX/WV ovaries produced only a few Graafian follicles and
The individual roles of LH and FSH in hyperplasia and
none produced corpora lutea. By 60 days of age, one-half of neoplasia of the multiple available target cells are not yet clear.
the WX/WVovaries contain only a few corpora lutea, and The highly predictable model of spontaneous ovarian tumori
hemorrhagic cystic follicles, occurring in about one-sixth of genesis provided by C3B6P! -W*/W mice offers the possibility
the ovaries, are the last large follicles observed. Hyperplasia of of correlation of LH and FSH levels with hyperplasia and
interstitial cells and "germinal" epithelium has begun, fol
neoplasia of interstitial cells, "germinal" epithelium, and
lowed by exponential growth of these tissues, with invasion of stromal cells and with the evolution of granulosa cell tumors
ovarian and periovarian tissues and of blood vessels, progress and luteomas. The sensitivity of radioimmunoassay of gonado
ing to formation of complex tubular adenomas in 95% of the tropins permits direct correlation with the development of a
ovaries by 5 months of age (7).
particular tumor type in individual mice.
The 4- to 5-fold increase in plasma LH and FSH levels in
B6C3F, +/+ mice after ovariectomy is in agreement with the REFERENCES
serum levels reported for the mouse by Kovacic and Parlow (5)
50 days after ovariectomy. FSH was 5 times and LH was 2
1. Beamer, W. G., Murr, S. M., and Geschwind, I. I. Radioimmuno
times the diestrous levels. In previous studies by Parlow (10),
assay of Mouse Luteinizing and Follicle Stimulating Hormones.
Endocrinology, 90: 823-826, 1972.
with bioassay methods, FSH but not LH was detected in the
2. Biskind, M. S., and Biskind, G. R. Development of Tumors in the
serum of ovariectomized mice, and neither hormone was
Rat Ovary after Transplantation into the Spleen. Proc. Soc. Exptl.
found in intact female mice.
Biol. Med., 55: 176-179, 1944.
The mean levels for plasma LH in intact W/W" mice were
3.
Furth,
J. Hormones and Neoplasia. In: A. Engel and T. Larsson
lower at 60 days of age in Experiment 2 than they were in
(eds.), Cancer and Aging, pp. 131-151. Stockholm: Nordiska
Experiment 1. At 60 days of age, there is large variance in LH
Bokhandelns Forlag, 1968.
levels in W^/W mice (Chart 1) and in the degree of depletion
4. Gardner, W. U. Some Studies on Ovarian Tumorigenesis. In: G. E.
W. Wolstenholme and M. O'Connor (eds.), Hormone Production in
of the follicular apparatus and of interstitial cell hyperplasia in
the ovaries (7). The small samples may not have been fully
Endocrine Tumours, pp. 153-169. Boston: Little, Brown and Co.,
representative of the population range.
1958.
5. Kovacic, N., and Parlow, A. F. Alterations in Serum FSH/LH
After ovariectomy, there was a significant increase of both
Ratios in Relation to the Estrous Cycle, Pseudopregnancy, and
LH and FSH levels in mice of the W/W genotype, indicating
Gonadectomy in the Mouse. Endocrinology, 91: 910-915, 1972.
that the ovaries were producing sufficient quantities of sex
6. Mintz, B., and Russell, E. S. Gene-induced Embryological
hormones to exercise some feedback control of the pituitary
Modifications of Primordial Germ Cells in the Mouse. J. Exptl.
gonadotropins. The presence of a small number of corpora
Zoo!., 134: 207-237, 1957.
lutea and occasional growing follicles in some of the ovaries at
7. Murphy, E. D. Hyperplasia and Early Neoplastic Changes in the
60 days of age can account for limited production of
Ovaries of Mice after Genie Deletion of Germ Cells. J. Nati. Cancer
progestins and estrogens. The continuation of LH and FSH
lnst.,48: 1283-1295, 1972.
levels in the WX/WUfemales at approximately the 60-day level
8. Murphy, E. D., and Russell, E. S. Ovarian Tumorigenesis following
suggests similar continuing feedback control. In spite of the
Genie Deletion of Germ Cells in Hybrid Mice. Acta. Univ. Intern.
Contra Cancrum, 19: 779-782, 1963.
loss of the follicular apparatus and the subsequent disappear
9. Nishizuka, Y., Tanaka, Y., Sakakura, T., and Kojima, A. Frequent
ance of corpora lutea, the vaginal epithelium and uterine horns
of WX/WVdo not approach the castrate state (7). The
Development of Ovarian Tumors from Dysgenetic Ovaries of
Neonatally Thymectomized Mice. Gann, 63: 139-140, 1972.
hyperplasia of interstitial cells, well advanced by 60 days of
age, provides a logical source of sex hormones. Shin et al. (12) 10. Parlow, A. F. Effect of Ovariectomy on Pituitary and Serum
Gonadotropins in the Mouse. Endocrinology, 74: 102-107, 1964.
have demonstrated histochemically the presence of 3 ß11. Russell, E. S. Review of the Pleiotropic Effects of W-series Genes
hydroxy steroid dehydrogenase in the hyperplastic interstitial
on Growth and Differentiation. In: D. Rudnick (ed.), Aspects of
cells of the ovaries of W/W" mice, using pregnanolone as a
Synthesis and Order in Growth, pp. 113-126. Princeton, N. J.:
substrate.
Princeton University Press, 1954.
The association of high plasma levels of LH and FSH with 12. Shin, M. L., Hendrickson, G., and Murphy, E. D. Ultrastructural
and Histochemical Studies of Ovarian Interstitial Cells in W-Series
the development of complex tubular adenomas in the ovaries
of mice of the WX/W" genotype provides quantitative
SterUe Mice. Am. J. Pathol., 66: 45a, 1972.
Analysis of variance indicated no difference between the 2
populations in response to ovariectomy.
APRIL 1973
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1973 American Association for Cancer Research.
723
Plasma Gonadotropin Levels during Early Stages of Ovarian
Tumorigenesis in Mice of the Wx/Wv Genotype
Edwin D. Murphy and Wesley G. Beamer
Cancer Res 1973;33:721-723.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/33/4/721
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1973 American Association for Cancer Research.