EFFECTS OF ACTIVE I M M U N I Z A T I O N AGAINST GONADOTROPIN
RELEASING HORMONE ON GONADOTROPIN SECRETION
AFTER O V A R I E C T O M Y AND TESTOSTERONE
PROPIONATE A D M I N I S T R A T I O N TO MARES 1
F. Garza, Jr., D. L. T h o m p s o n , Jr. 2 ,
P. S. M i t c h e l l a n d J. J. Wiest
Louisiana State University Agricultural Center 3
Baton Rouge 70803
ABSTRACT
Five lighthorse mares were actively immunized against gonadotropin releasing hormone (CnRH)
conjugated to bovine serum albumin (BSA) to study the involvement of GnRH in luteinizing
hormone (LH) and follicle stimulating hormone (FSH) secretion following ovariectomy (OVX) and
after administration of testosterone propionate (TP). Five mares immunized against BSA served as
controls. Immunizations were started on November 1, and OVX was performed in June (d 1). All
mares were treated with TP from d 50 to 59 after OVX. On the day of OVX, concentrations of LH
were lower (P<.05) in GnRH-immunized mares than in BSA-immunized mares and were generally
nondetectable; FSH concentrations were reduced (P<.05) by 50% in GnRH-immunized mares relative to BSA-immunized mares. In contrast to BSA-immunized mares, plasma concentrations of LH
or FSH did not increase after OVX in GnRH-immunized mares. The LH response to GnRH analog
(<.1% cross-reactive with GnRH antibodies) on d 50 was reduced (P<.05) by 97% in GnRH-immunized mares relative to BSA-immunized mares, whereas the FSH response was similar for both
groups. Treatment with TP for 10 d reduced (P<.01) the LH response and increased (P<.01) the
FSH response to GnRH analog in BSA-immunized mares, but it had no effect (P>.l) on the response of either gonadotropin in GnRH-immunized mares. We conclude that normal GnRH input
to the pituitary gland is necessary in the mare for the post-OVX increase in LH and FSH secretion
and for the TP-induced increase in FSH secretion after GnRH analog administration.
(Key Words: Mares, Ovariectomy, Testosterone, LH, FSH, Gonadotropin Releasing Hormone.)
Introduction
In t h e horse, as in o t h e r species, g o n a d o t r o p i n releasing h o r m o n e ( G n R H ) a p p e a r s t o b e
the hypothalamic peptide responsible for maint e n a n c e o f n o r m a l luteinizing h o r m o n e (LH)
a n d follicle s t i m u l a t i n g h o r m o n e ( F S H ) secret i o n b y t h e p i t u i t a r y . E v i d e n c e f o r this i n c l u d e s
1 Approved for publication by the Director of the
Louisiana Agric. Exp. Sta. as manuscript no. 8 6 - 1 1 - 0272. We thank L. E. Reichert, Jr., Albany Medical
College, for purified oLH and H. Papkoff, Univ. of
California, San Francisco, for purified equine gonadotropins.
2Send reprint requests to D. L. Thompson, Jr.,
Dept. of Anita. Sci., Louisiana State Univ., Baton
Rouge, 70803.
3 Dept. of Anim. Sci., Louisiana Agric. Exp. Sta.
Received June 24, 1987.
Accepted September 16, 1987.
479
t h e o b s e r v a t i o n s t h a t 1) a d m i n i s t r a t i o n o f G n R H
t o m a r e s a n d stallions causes a rapid increase in
LH a n d F S H c o n c e n t r a t i o n s in jugular b l o o d
( G i n t h e r a n d W e n t w o r t h , 1 9 7 4 ; Evans a n d Irvine, 1 9 7 6 ; T h o m p s o n et al., 1 9 8 3 c ; A s h l e y et
al., 1 9 8 6 ) , 2) seasonal f l u c t u a t i o n s in p i t u i t a r y
LH c o n t e n t in t h e m a r e are generally c o r r e l a t e d
w i t h c o n c e n t r a t i o n s of G n R H in t h e h y p o t h a l a m u s ( H a r t et al., 1 9 8 4 ) , 3) e p i s o d e s o f G n R H
s e c r e t i o n d e t e c t e d in m e d i a l basal h y p o t h a l a m u s
via p u s h - p u l l c a t h e t e r i z a t i o n are generally associa t e d w i t h s u b s e q u e n t pulses in LH c o n c e n t r a t i o n s in j u g u l a r b l o o d ( S h a r p a n d G r u b a u g h ,
1 9 8 7 ) a n d 4) active i m m u n i z a t i o n o f m a r e s a n d
stallions against G n R H r e d u c e s s e c r e t i o n o f LH
and, t o a lesser degree, F S H ( S c h a n b a c h e r a n d
P r a t t , 1 9 8 5 ; G a r z a et al., 1986a).
T h e effects o f o v a r i e c t o m y ( O V X ) a n d
s u b s e q u e n t a d m i n i s t r a t i o n of t e s t o s t e r o n e prop i o n a t e (TP) o n LH a n d FSH s e c r e t i o n in t h e
m a r e have b e e n well d o c u m e n t e d ( G i n t h e r ,
1 9 7 9 ; Wallace, 1 9 8 1 ; Reville-Moroz et al.,
J. Anim. Sci. 1988. 66:479--~86
480
GARZA AND THOMPSON
1984; Thompson et al., 1984). Moreover,
Reville-Moroz et al. (1984) showed that the
TP-induced increase in FSH secretion after
exogenous GnRH in mares after OVX was due
in part to increased de novo production of
FSH. Whether endogenous GnRH mediates
these effects of OVX or TP treatment in the
mare is unknown. Thus, the purpose of the
present experiment was to determine the
involvement of GnRH in post-OVX increases in
LH and FSH secretion and in TP-induced
increase in FSH secretion after exogenous
GnRH in the mare. To do this, we have used
the technique of active immunization against
GnRH, an experimental approach that has been
described previously for several species (Fraser
et al., 1975; Clarke et al., 1978; Fraser and
McNeilly, 1982; Esbenshade and Britt, 1985;
Adams and Adams, 1986; Garza et al., 1986a).
Materials and Methods
Ten lighthorse mares (>2,5 yr old and
weighing between 364 and 510 kg) that had
displayed normal estrous cycles during the
previous breeding season were used. All mares
were kept on pasture and were fed grass hay as
needed to maintain good b o d y condition
throughout the experiment. Five mares were
immunized with 4.0 mg of a conjugate prepared from GnRH 4 and bovine serum albumin
(BSA) as described by Fraser et al. (1974). The
remaining five mares were immunized with 4.0
mg of BSA (controls). The immunization
regimen from the start (November 1) until
OVX (June) has been described (Garza et al.,
1986a). Additional secondary immunizations
(2.0 mg antigen) were given on d 36 and 48
following OVX. Effects of immunization on
estrous, ovarian and endocrine characteristics
up to OVX have been reported (Garza et al.,
1986a).
GnRH-immunized mares did not exhibit
estrous cycles (Garza et al., 1986a). Thus, when
a BSA-immunized mare reached d 7 or 8 of
diestrus in June, she was randomly paired with
one GnRH-immunized mare, and both were bilaterally ovariectomized (d 1) via flank incisions
under local anesthesia. Samples of jugular blood
were drawn from each mare daily thereafter
through d 60.
On d 50, an indwelling catheter was placed
in one jugular vein of each mare for frequent
blood sampling. Three samples of blood (12 ml)
were drawn from each mare at 15-min intervals.
Gonadotropin releasing hormone analog (des ~
Glyl~
4) was then administered through the catheter at a dosage of
40 ng/kg of b o d y weight (which is approximately equivalent to 1.0 pg/kg of GnRH; Garza
et al., 1986a). Blood samples were drawn at 15,
30, 45, 60, 75, 90, 120, 150, 180, 210, 240,
270 and 300 min after GnRH analog injection.
Relative to GnRH, the GnRH analog was <.1%
cross-reactive with antibodies present in the
GnRH-immunized mares (Garza et al., 1986a).
After the 300-rain blood sample had been
collected on d 50, each mare was administered
TP (175 /lg/kg of b o d y weight) via sc injection
in corn oil. Treatment with TP was repeated
daily through d 59. On d 60, GnRH analog was
again administered and blood drawn as described
for d 50.
Heparinized plasma was harvested via
centrifugation and stored at --15 C. Concentrations of LH and FSH in plasma were measured
by radioimmunoassay as described (Thompson
et al., 1983a,b). Sensitivities of the LH and
FSH assays were .5 and 1.8 ng/ml, respectively;
intra-assay and inter-assay coefficients of
variation were <10% and <12%, respectively,
for both gonadotropins. Tritiated GnRH
binding was determined as described by Garza
et al. (1986a). Dilution rate was calculated b y
dividing the total volume per tube (.4 ml) by
the volume of serum required to bind 20% of
48,000 dpm of tritiated GnRH (453 pg).
Data collected over time were analyzed b y
analysis of variance that accounted for the
repetitive sampling (split-plot design; Gill and
Hafs, 1971). Differences between groups for
each period were assessed for significance by
the Least Significant Differences test (Steel and
Torrie, 1980). Net areas under the LH and FSH
response curves after GnRH analog injection
were calculated as described by Thompson and
Nett (1984). Because variances associated with
means for LH and FSH areas and for GnRH
titers were found heterogeneous via Bartlett's
test (Steel and Torrie, 1980), these data were
transformed to logx0 (x + 1). The transformed
data were analyzed by one-way analysis of
variance (Steel and Torrie, 1980); actual means
and SE are presented.
Results
4 Sigma Chemical Co., St. Louis, MO.
The dilution rate (D) of plasma in vitro
averaged 896 -+ 352 on day of OVX and 1,001
TESTOSTERONE
TO GnRH-IMMUNIZED
+- 448 on d 50 for GnRH-immunized mares. In
contrast, there was no significant GnRH binding
by up to 200 /al of undiluted plasma from
BSA-immunized mares (D<2; P<.001 between
groups based on analysis of log-transformed
data).
Prior to OVX on d 1, plasma concentrations
of LH and FSH were lower (P<.05) by approximately 95% and 50%, respectively, in GnRHimmunized mares relative to BSA-immunized
mares (figure 1). During the 50 d following
OVX, plasma concentrations of LH and FSH
increased (P<.01) in BSA-immunized mares,
but not in GnRH-immunized mares (figure 1).
Plasma LH concentrations in GnRH-immunized
mares remained lower (P<.05) relative to
BSA-immunized mares and were generally
nondetectable. Concentrations of FSH in
plasma, although easily detectable, also remained
constant in GnRH-immunized mares and were
approximately 65% lower (P<.05) than in
BSA-immunized mares by d 50.
When mares were administered GnRH analog
on d 50 following OVX, the FSH response, assessed by the net area under the curve, was similar ( P > . l ) for GnRH-immunized and BSA-immunized mares (figure 2). In contrast, the LH
response to GnRH analog was essentially non-
481
MARES
existent in GnRH-immunized mares and was
lower (P<.05) relative to BSA-immunized
mares (figure 3). Following TP treatment, the
FSH response to GnRH analog was increased
(P<.01) by approximately threefold in BSAimmunized mares compared with the pretreatment response on d 50 (figure 2). There was no
change in the FSH response to GnRH analog in
GnRH-immunized mares due to TP treatment.
The LH response to GnRH analog was decreased
(P<.05) in BSA-immunized mares by TP treatment (figure 3); the LH response to GnRH
analog in GnRH-immunized mares remained
low and did not differ (P>.I) from the response
on d 50.
Concentrations of FSH in daily blood
samples were not affected ( P > A ) by administration of TP in either group of mares (figure
4); FSH concentrations remained lower (P<.05)
in GnRH-immunized mares relative to BSAimmunized mares throughout the TP-treatment
period (d 50 to 59). Concentrations of LH in
daily blood samples during this same time decreased (P<.01) in BSA-immunized mares such
that they were similar to those in GnRH-immunized mares by d 57 (figure 4). Concentrations
of LH in GnRH-immunized mares remained
nearly undetectable throughout this period.
O GNRH-IMMUNIZED
9 CONTROL
~
80 ,
~
60,
"r
40
2O
2
|
|
|
|
10
20
30
40
50
DAYS
Figure 1. Mean concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in plasma of mares immunized with bovine serum albumin (BSA) (control) and mares immunized with gonadotropin
releasing hormone (GnRH) immediately before (d 1) and through d 50 after ovariectomy. Pooled SE from the
analyses of variance were 3.4 and .76 ng/ml for FSH and LH concentrations, respectively. The vertical line witbin
each panel indicates the least-significant difference value (P<.05) for comparison between groups within each
day.
482
G A R Z A A N D THOMPSON
140
100
i
100
/
GNRH-IMMUNIZED
100
I
1
b
60 ~
1
0
X
0
~
20
0
1
2
3
4
5
HOURS
Figure 2. Mean concentrations o f follicle stimulating h o r m o n e (FSH) in plasma of mares i m m u n i z e d with
bovine serum albumin (BSA) (control) and mares i m m u n i z e d with gonadotropin releasing h o r m o n e (GnRH)
immediately before and for 5 b after injection of GnRH analog (time 0). The pretreatment injection was given
on d 50, and the p o s t t r e a t m e n t injection was given after 10 d of t r e a t m e n t of all mares with testosterone propionate. Mean (+- SE) n e t areas under the GnRH response curves are indicated by t h e bar graphs. Means with different superscripts differ (P<.05).
PRETREATMENT
Of.~.lPOST-TR EATM ENT
1~68coNT oj.__
,18
.15
,,12
,,9
f-"r
.6
--~
E
2
3
m
X
GNRH-IMMUNIZED
3
,.i,. 1 2
I
2~
e
-
I
o
1
:~
:~
I
I
4
s
"r"
i--
i
HOURS
Figure 3. Mean concentrations of luteinizing h o r m o n e (LH) in plasma of mares i m m u n i z e d with bovine serum
a l b u m i n (BSA) (control) and mares i m m u n i z e d with gonadotropin releasing h o r m o n e (GnRH) immediately before and for 5 h after injection of GnRH analog (time 0). The p r e t r e a t m e n t injection was given on d 50, and the
p o s t t r e a t m e n t injection was given after 10 d of t r e a t m e n t of all mares with testosterone propionate. Mean (+ SE)
net areas u n d e r the G n R H response curves are indiqated by the bar graphs. Means with different superscripts
differ (P<.05).
TESTOSTERONE TO GnRH-IMMUNIZEDMARES
O GNRH-IMMUNIZED
9 CONTROL
~" 60
I
$
4
DAYS
Figure 4. Mean concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in
plasma of mares immunized with bovine serum albumin (BSA) (control) and mares immunized with gonadotropin releasing hormone (GnRH) during 10 d of
TP treatment. First injection of TP is indicated by the
arrow. Pooled SE from the analyses of variance were
5.1 and .38 ng/ml for FSH and LH concentrations,
respectively. The vertical line within each panel indicates the least-significant difference value (P<.05) for
comparison between groups within each day.
Discussion
The effects of active immunization against
GnRH on LH secretion in the horse are similar
to those reported for the rat, ewe and gilt
(Fraser et al., 1975; Clarke et al., 1978; Fraser
and McNeilly, 1982; Esbenshade and Britt,
1985; Adams and Adams, 1986; Garza et al.,
1986a). In all these species, immunization
against GnRH reduced daily LH secretion by
90% to 100% in intact and OVX females. In
contrast, the response of FSH secretion varies
among species. In rats and gilts, FSH secretion
in intact and OVX females was reduced by 93%
to 98% (or to undetectable concentrations),
whereas in ewes the reduction was between 0%
and 50% in intact animals and 90% in OVX
animals. Thus, the FSH response in the horse is
more similar to that of the ewe than of the rat
or gilt. However, in contrast to horses, in
which the average response was not altered, the
FSH response to a GnRH analog injection in
ewes was reduced by about 75% by active
immunization against GnRH (Adams and
Adams, 1986).
Given the relative effects of active immunization against GnRH on LH and FSH concentrations in these mares before OVX (Garza et
483
al., 1986a) and subsequently in the present
experiment, we conclude that LH secretion,
and probably production, is highly dependent
on the normal complement of GnRH reaching
the pituitary gland. However, there appear to
be at least two components of FSH secretion in
mares, one being highly dependent on normal
GnRH input to the pituitary gland and the
other being relatively independent of normal
input. This relative independence might be a
complete independence from GnRH input, or
it might be a dependence on much lower concentrations than normal reaching the pituitary.
An independent component of FSH production
and secretion is well documented for gonadotropes maintained up to 21 d in cell culture
(Miller et al., 1977; Miller and Wu, 1981). Although we do not know how much endogenous
GnRH actually reached the gonadotropes in
GnRH-immunized mares, we do know that the
FSH response to authentic GnRH (not analog)
administered via the jugular vein in these same
mares prior to OVX (Garza et al., 1986a) at a
dose of 1.0/ag/kg of body weight was reduced
by 85% in GnRH-imrnunized mares relative to
BSA-immunized mares. Because 1.0 /.tg/kg of
GnRH is approximately 100-fold greater than
the a m o u n t required to produce an LH pulse
similar in size to endogenous pulses (Alexander
and Irvine, 1986), the anti-GnRH antibodies
in GnRH-immunized mares likely had an even
greater neutralizing effect on endogenously
secreted GnRH than was indicated from GnRH
administration at this large dose.
Because a component of FSH secretion
persisted for several months before OVX (Garza
et al., 1986a), and for at least 50 d after OVX
(figure 1), in GnRH-immunized mares, we
conclude that there is also a component of FSH
production that is relatively independent of
normal GnRH input to the pituitary gland.
That is, assuming a half-time of 170 min for
FSH concentrations (Thompson et al., 1986a)
and a constant plasma concentration of 25
ng/ml (figure 1), the average a m o u n t of FSH in
the pituitary glands of nonpregnant mares
(about 3 mg; Thompson et al., 1986b) would
be sufficient for no more than 2 to 3 d of FSH
secretion. Even if these calculations were in
error by a factor of 10, it is apparent that FSH
would still have to be produced to maintain the
observed plasma concentrations for more than
140 d.
In conjunction with the constant, albeit reduced, daily FSH secretion in GnRH-immunized
484
GARZA AND THOMPSON
mares, the FSH response to GnRH analog 50
d after OVX did not differ from that of BSAimmunized mares. Thus, in addition to the
aforementioned secretion and production, there
appears to be a component of FSH storage in
the pituitary gland that is relatively independent of normal GnRH input. This FSH was
released in response to a GnRH analog. Thus we
conclude that the cells that stored it maintained
normal GnRH responsiveness, even though they
received a reduced GnRH input for several
months.
The post-OVX increases in LH and FSH
secretion observed in BSA-immunized mares
were not present in mares actively immunized
against GnRH. In the case of LH, there appeared to be little or no LH available for
secretion in response to OVX; if some factor(s)
other than an increase in endogenous GnRH
secretion happened to be responsible for
the normal post-OVX rise, then we would not
have been able to detect it. In the male rat
(Berardo and DePaolo, 1986), administration of
anti-GnRH serum at the time of castration
prevents the normal LH rise, and we suspect
that a similar result would be found in the mare.
In contrast to LH, FSH in the pituitary was
available for release (as evidenced by the
response to GnRH analog). It is generally
assumed that the increases in LH and FSH
secretion after OVX are due to removal of
negative feedback from ovarian factors (mainly
progesterone for LH and estrogen and/or
inhibin for FSH; Garcia and Ginther, 1978;
Ginther, 1979; Thompson et al., 1983c; Garza
et al., 1986b; Wiest et al., 1987). In other
species, this is mediated by increased pulsatile
GnRH secretion after OVX as evidenced by
increased LH pulse frequency (Gallo, 1980;
Goodman and Karsch, 1980; Anderson et al.,
1985). Similar high-frequency pulses in LH concentrations have been observed in OVX mares
but not in intact mares (Thompson et al.,
1987). From all the above information, we
conclude that the normal post-OVX increases in
LH and FSH secretion in mares are likely due
to increased GnRH secretion by the hypothalamus.
The TP-induced increase in FSH response to
GnRH analog was also prevented by active
immunization against GnRH, indicating that
normal GnRH input to the pituitary gland is
required for this response. Because FSH concentrations in daily blood samples were not affected by testosterone treatment, we suspect
that the greater than threefold increase in FSH
response to GnRH analog in BSA-immunized
mares was at least partially due to increased
FSH production, as we have shown directly in a
previous experiment (Reville-Moroz et al.,
1984). Also as we described previously (RevilleMoroz et al., 1984), daily LH secretion and the
LH response to GnRH analog were suppressed
by TP treatment in BSA-immunized mares. If
the reduction in LH secretion reflects a reduction in short-term GnRH secretion as suggested
for other species (Clarke and Cummins, 1982;
Levine et al., 1982; Goodman and Meyer,
1984), then the GnRH-dependency of the FSH
response to TP ( a stimulatory effect) would be
a long-term dependency (for example, involving cell number or cell type) rather than an immediate requirement for GnRH input (as has
been described for LH secretion). This may indicate that the FSH-producing cells normally involved with the TP-induced stimulation of FSH
response were absent (or alternatively, unresponsive) in GnRH-immunized mares.
In conclusion, the normally expected
increases in LH and FSH secretion after OVX
and the TP-induced increase in FSH secretion
after GnRH analog injection are absent after
long-term immunization against GnRH in the
mare. It appears that LH secretion and likely
production are highly dependent on endogenous
GnRH in the mare. In contrast, there appear to
be two components of FSH production, storage
and secretion; one is relatively independent of
normal GnRH input and the other is relatively
dependent. Whether these two components of
FSH secretion are a result of two separate
FSH-producing cell types within the pituitary
or a result of differential response of a single
cell type to two different levels of GnRH input
(normal vs reduced) needs to be determined.
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