Gonadotropic regulation of follicular maturation in cyclic hamsters using antisera to FSH and. LH. Ph.D. Thesis. Indian. Institute of Science,. Ban- galore,. India.
BIOLOGY
OF
REPRODUCTION
Effect
21,
of Neutralization
117-123
(1979)
of Endogenous
or Luteinizing
Hormone
(LH)
A Histochemical C. S. SHEELA
Follicle
Stimulating
on Ovarian
and
Lipids
Biochemical
Laboratory
of Endocrine
Department
of
Indian
(FSH)
Evaluation
A. K. SUSHEELA2
RANI,’
Hormone
in the Hamster:
and
N. R. MOUDGAL’
Biochemistry, Biochemistry,
Institute
of
Bangalore-560
Science,
India
012, and
Histo
and
Cytochemistry
Department All
India
Institute New
Laboratory,2 of of
Anatomy,
Sciences,
Medical
Delhi-hO
016,
India
ABSTRACT The
effect
of neutralizing the normal cycling hamster ence of sudanophilic lipids reduction
in
the
intensity
FSH or LH on ovarian lipids in the on the day of proestrus, histochemical in the granulosa cells of the follicles of
lipid
staining
was
observed
on
cycling hamster was studied. In examination revealed the presand in the interstitium. A clear
proestrus
in
the
ovary
of
hamsters
treated with FSH antiserum on the previous proestrus. Similar treatment with antiserum to LH, on the other hand, caused an accumulation of lipids in these structures. Estimation of the free and esterified fractions of cholesterol and triglycerides in the nonluteal tissue of the ovary of hamsters on proestrus following treatment with FSH antiserum on the previous proestrus revealed a significant reduction in all 3 lipid components. Even a short term deprivation of FSH caused a similar reduction in these lipids in the ovary. In contrast, treatment with LH antiserum either on the previous proestrus or on the previous day (diestrus-2) resulted in an enhancement in esterifled cholesterol and triglycerides, while it caused a reduction in the free cholesterol fraction of the ovary on proestrus. It is suggested
follicular in
that
maturation,
maintaining
hamster
though
their
normal
treatment
with
antisera
effect
on lipid metabolism and triglyceride levels
sterol
studies
shown
that
luteinizing
from
not
(LH)
only
steroid
sterol metabolism tissue of pregnant hamsters (Moudgal 1972;
Mukku
However,
ovarian
able
interest
the
effect
laboratory of
with
endogenous LH
biosynthesis,
antiserum
but
and
Moudgal, nothing
1975,
is known
Received
or
LH
causes
endogenous
a disruption
in
long
periods
on
examine
if
sterol tissue study
sudanophilic
of
the
and of the
ovary
was
lipids
are
in
influ-
enced by the lack of endogenous FSH or LH. This was later followed by biochemical estimations of ovarian cholesterol (free and esteri-
1976).
fled)
regarding
LH
or
compartments
and
triglycerides.
of LH and follicle in sterol metabolism
of neutralizing
August
short
to
specific
stimulating in nontissue. Hence, it was of consider-’ to examine in the present study
February
for
undertaken
also
MATERIALS Histo
chemical
AND
METHODS
Methods
Regularly cycling female hamsters (Mesocricetus auratus), maintained under a lighting schedule of 14L:1OD (lights on at 0600 h) in a well ventilated room, were either untreated or were given an intracardiac injection of 0.1 ml FSH antiserum or 0.2
or
ml
Accepted
FSH
triglyceride levels in the nonlutea! ovary of the cycling hamster. A preliminary histochemical
have
and biosynthesis in the luteal and pseudopregnant rats and et a!., 1972; Behrman et al.,
virtually
the involvement hormone (FSH) luteal
FSH our
neutralization hormone
affects
either
is indicated. INTRODUCTION
Earlier
to
is different. A positive role for FSH and LH in the nonluteal ovarian tissue of cycling
8, 1979. 23, 1978.
LH
at 1300 trus
117
antiserum
(Sheela
h on the day
(4 days
later),
they
Rani
and
of proestrus. were
killed
Moudgal,
1977a)
On the next proesby decapitation at
118
RANI
h and I ovary from immediately frozen
1000
was Frozen
sections
were
cut
each animal (n in isopentane in a cryostat
(Ames
ET
AL.
3/group)
=
at
RESULTS
-70C. Histochemical
Labtech)
at 14 m and mounted on clean glass slides. After slight air drying, they were fixed for 24 h in Baker’s calcium formal (40% formalin:10% CaCI2 :water [1:1:81 and a piece of chalk for neutralization). The
The higher
cells
sections
siderably
Biochemical
under
with
a microscope.
cells
than
content 1). In
of
in
the
antiserum,
the
as
as
the
antiserum
at
1300
h of proestrus
The
animals treated killed 4 days later
with
or 0900 antisera
on
1
proestrus were on the next proestrus and those given antisera on diestrus-2 were killed on the next day (proestrus) at 1700 h. A control group of hamsters was untreated and killed at 1700 h of proestrus. The ovaries were removed and carefully cleaned of the surrounding fat and other extraneous tissue and weighed to the nearest 0.1 mg. It should be mentioned here that in the cycling hamster by the evening of proestrus the luteal tissue is almost completely regressed and is not discernible even structurally (Lukaszewska and Greenwald, 1970). The method used for the extraction of lipids with
chloroform:methanol silica gel plates
(1:2) with the
and separation by TLC on use of 2 solvent systems
granulosa
cells
was
Effect on
of Antisera
Free
and of
and
in
Administration
Both free and esterjfied cholesterol by the method of Glick et al. (1964).
were
trus-2
(acute
ovary
on
reduction
erol was estimated after saponifying the samples with 2% alcoholic KOH at 60-70#{176}C for 30 mm. The liberated glycerol was estimated by the method of
previous
of control and using Student’s
FIG.
t
1. Ovary
experimental test.
of untreated hamster cells (arrow).
staining in the interstitial have a fair amount of lipid. FIG. proestrus. intensity FIG. proestrus. granulosa
2.
groups
Sudan
Black
was
determined
was
LH
antiserum,
enhanced
(Fig.
both as in
earlier,
previous other
hand
animals day
prior
The fractions
in
Fig. free
of reduction
less
similar
with on
1; the
esterified in the 4).
the
The
FSH
in
previous
proestrus
triglycerides
with than
proestrus
ovaries
antiserum
of
either 4 or
on
the
on
the
reduction
in
the
FSH antiserum those treated
on on
the the
a greater
treated
showed Inter-
in these lipids
the
ovarian
showed
The
cholesterol
esterified cholesterol significant decrease.
extent
day.
free and triglycerides
(Table
marked
or
antiserum either on of FSH) or on diesresulted in a signif-
to killing. effect of of lipids
antiserum caused free cholesterol dous accumulation
LH antiserum was different.
a significant fraction, there of esterifled
on
these 3 While LH
decrease in the was a tremencholesterol in
on proesetus revealing the pattern of lipid localization. Note the The theca (T) has very little sudanophilic material. The granulosa B staining.
con-
normal in the
Ovaries
in well
treated
days
the
proestrus was
more
By this method, cholesteryl ester was estimated directly without saponification and hence the values are reported as cholesterol equivalents (Mukku and Moudgal, 1975). For the quantitation of triglycerides, glyceride glyc-
Jover (1963). The results are expressed as g glycerol equivalents/mg tissue. The significance of differences between the means
granulosa
or LH
of FSH lack of FSH)
lack
icant reduction cholesterol as
animals
estimated
the
cells
with
term
proestrus (long
the
made.
of
Pro estrus
on
was
were
lipid (Fig. FSH
Cholesterol
Triglycerides Hamster
estingly,
corrections
The
in the interstitial and
to FSH
added
appropriate
a
interstitial
was negligible treated with
markedly
Esterified
whereas though
and
the
3).
fraction, a marginal,
extraction
on
B showed
cells.
treated material
(hexane:ether:acetic acid, 75:25:2 and 90:10:1, respectively, in the same direction) was essentially based on that described by Major et al. (1967) and Pokel et al. (1972). The extent of recovery of sterols was traced using I ‘4C1 -labeled cholesterol oleate
during
hamster
as compared to the (Fig. 2). In contrast,
hamsters
ml
diestrus-2.
in
interstitial
ovaries of
the sudanophilic
LH
lipids
content
the
reduced
ovary
of
Black
granulosa
lipid
well
Groups of regularly cycling female hamsters were given by intracardiac route 50 il FSH antiserum or 0.2 h of
Sudan
in the thecal cells the ovary of hamsters
proestrous
Estimations
ovary
the
concentration
were then washed with several changes of water over a period of 24 h and were then for lipids with Sudan Black B for 15 mm (Baker, 1946). The stained sections were then differentiated in 50% alcohol, washed in water and mounted in glycerine jelly (7.5% gelatin in 50% glycerol). The
examined
of
stained
proestrus
sections distilled stained
were
Observations sections
dense cells
X 1500.
Pattern of lipid localization in the ovary of hamster treated with FSH antiserum on the previous The sudanophilic material is seen in the interstitium as well as in the granulosa cells. However, the of staining of lipids is considerably reduced. Sudan Black B staining. X 925. 3. Pattern of lipid localization in the ovary of hamster treated with LH antiserum on the previous Note the presence of large droplets of sudanophilic material in the interstitial cells (arrow). The cells also show a much higher intensity of staining of the lipid contents. Sudan Black B staining. X 925.
GONADOTROPINS
:
AND
OVARIAN
LIPIDS
#{149}
#{149}(4.I
-
.
j
-
- ‘.. -
-.
-.
7:’
#{149}--: k,....
;_
#{149}-f
.-..--I.
-
S
f..
.
a
‘
I
:
-
-.
‘‘
1:
.
-.
a,
.
IN CYCLING
HAMSTER
119
120
ET AL.
RANI
TABLE triglyceride
1. Effect of treatment with antisera to FSH or LH at proestrus concentration at 1700 h of proestrus. (Mean ± SEM; numbers
or diestrus-2
on
in parentheses
ovarian cholesterol N/group.)a
=
Triglycerides (Mg glycerol/ mg tissue)
.
(Mg! mg tissue)
Cholesterol
and
Treatment
Free
Untreated
8.37
±
1.70(5)
17.34
±
1.04 (11)
1.38
±
0.12(11)
3.85 4.22
±
0.56(8)’
0.32 1.19
0.68 1.16
±
0.02 (7)” 0.01 (7)
4.71 3.83
±
0.56 0.47
1.82 1.45
±
±
control antiserum
FSH
on proestrush on diestruse LH antiserum on proestrusb on diestruse aThe
values
are
significantly
Esterified
± 0.32
different
14.30
±
(7)’
15.68
±
(7)’ (3)’
26.14
±
20.14
± 0.79
from
those
(8)’ (7)”
4.29 (8)’ (3)’
of corresponding
±
controls
at ‘P
