Endocrinology Vol. 139, No. 4 2007-2014
Copyright © 1998 by The Endocrine Society
Neonatal Immunization against Gonadotropin-Releasing Hormone (GnRH) Results in Diminished GnRH Secretion in Adulthood
I. J. Clarke,
B. W. Brown,
V. V. Tran,
C. J. Scott,
R. Fry,
R. P. Millar and
A. Rao
Prince Henry’s Institute of Medical Research (I.J.C., V.V.T.,
A.R.), Clayton, Victoria 3168, Australia; Commonwealth Scientific and
Industrial Research Organisation Division of Animal Production
(B.W.B.), Blacktown, New South Wales 2148, Australia; Department of
Physiology (C.J.S.), Monash University, Clayton, Australia; Victorian
Institute of Animal Science (R.F.), Department of Natural Resources and
Energy, Werribee 3030, Australia; and Medical Research Council Research
Unit for Molecular Reproductive Endocrinology (R.P.M.), Regulatory
Peptides Research Unit, Department of Chemical Pathology, University of
Cape Town, Cape Town, South Africa
Address all correspondence and requests for reprints to: I. J. Clarke, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail:
iain.clarke{at}med.monash.edu.au
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Abstract
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The effects of neonatal immunization against GnRH were studied in sheep
after they had reached adulthood (3–4 yr) and the antibody titers had
fallen to undetectable levels. The immunized animals had small gonads,
and the females did not have large follicles (>3 mm) or corpora lutea
in their ovaries. Compared with controls, the immunized animals had low
or nondetectable levels of LH and FSH in peripheral plasma, and the
immunized animals generally failed to respond to a single iv GnRH
challenge. After ovariectomy, the control ewes, but not the immunized
ewes, showed an elevation in plasma LH and FSH levels. The sampling of
hypophysial portal blood, with a newly described method, showed that
the secretion of GnRH was reduced in the immunized animals, but the
amount of GnRH in the median eminence was similar in the control and
immunized ewes. The pituitary content of LH and FSH was reduced in the
immunized ewes as was messenger RNA for the gonadotropin subunits and
the GnRH receptor. These data indicate that neonatal immunization does
not affect the synthesis of GnRH in adulthood but reduces the secretion
of GnRH, causing long-term sterility in these animals.
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Introduction
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GnRH IS THE primary brain hormone that
drives the reproductive system. Pulsatile release into the hypophysial
portal system promotes synthesis of the gonadotropins (LH and FSH) (1),
and there is a tight relationship between pulsatile GnRH secretion and
pulsatile LH secretion (2). Accordingly, active immunization of adult
animals against GnRH causes the loss of synthesis and secretion of the
gonadotropins and cessation of gonadal function, as long as the
antibody titers remain elevated (3). Reproductive activity recommences
as the antibodies dissipate (3). On the other hand, immunization of
neonatal or prepubertal sheep has a prolonged effect on reproductive
function, despite the progressive reduction in GnRH antibodies in
plasma to nondetectable levels (4, 5). These latter studies suggest
that there may be some lesion at the hypothalamo-pituitary level in
these animals. Indeed, similar studies in pigs (6) show that
immunization against GnRH in early life causes a reduction in the
number of GnRH cells in the hypothalamus and structural changes in the
median eminence, although these animals were studied when antibody
titers were still high. The earlier studies (4, 5) of the neonatally
immunized sheep presented limited information on the secretion of
gonadotropins, and the study in pigs presented no information in this
regard. We hypothesized that neonatal immunization against GnRH would
reduce GnRH secretion and have studied the synthesis and secretion of
GnRH, LH and FSH in such animals during adulthood. We have also studied
the expression of the GnRH receptor in the pituitary gland.
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Materials and Methods
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Baseline secretion of gonadotropins and GnRH in rams
A group of rams from the original flock that was studied by
Brown et al. (4) were used in the present study. These rams
were 4–6 yr of age at the time of study. Four control animals and four
GnRH immunized rams were used. The treated animals were immunized at
3–4 weeks of age and received a booster injection at 13–14 weeks of
age (4). None of the rams in the present experiment had detectable GnRH
antibodies at the start of the study (4). A cannula (Dwellcath, Tuta
Labs, Melbourne, Australia) was inserted into one external jugular vein
and connected to a manometer line, and the cannula was kept patent with
heparinized (75 U/ml) saline. Serial blood samples (4 ml) were drawn at
10-min intervals for 12 h on the following day and the plasma
harvested for LH and FSH assay. The samples were stored at -15 C until
assayed. Four immunized animals and six control animals were then
selected for hypophysial portal blood sampling to measure GnRH
secretion. This procedure has been described previously (2). Following
portal sampling, the animals were killed by overdose of barbiturate
(Lethabarb, Virbac Aust. Pty. Ltd., Peakhurst, New South Wales,
Australia) and the pituitaries and median eminence were collected for
further analysis (see below).
Baseline gonadotropin levels in control and hypogonadal ewes before
and after ovariectomy and the response to GnRH challenge
A group of Corriedale ewes were immunized at 4 weeks of age with
4 ml (im) of Vaxstrate (Arthur Webster Pty. Ltd., Queensland,
Australia) and were given the same treatment as a booster at 8 weeks of
age. These animals were studied at 3–4 yr of age. Jugular venous blood
samples were collected from 5 immunized animals and five controls at 10
min intervals for 6 h. Immediately following this, the animals
were injected (iv) with 10 µg GnRH, and blood samples were taken 5,
10, 20, 35, and 50 min after injection. The animals were then
ovariectomized (OVX) and a further series of blood samples (as above)
were collected 7 days later.
GnRH secretion in control and hypogonadal ewes following
ovariectomy
Forty days after ovariectomy, the ewes were prepared for portal
blood sampling using an modified version of the original method as
follows. The animals were prepared for surgery, and the pituitary gland
approached by the transnasal, transsphenoidal route described
previously (2). The nose was opened on the left side of the animal, and
the face of the pituitary gland was exposed by drilling through the
sphenoid bone, along the midline. A small horizontal incision was made
in the dura mater across the midline at the level where the portal
blood vessels are prominently displayed in this species on the surface
of the pituitary gland. A single 12-cm long 12G needle was prepared to
access to the portal vessels as follows. The end of the needle was
filed so that it would approximate the angle of the pituitary face when
the needle was inserted. A length of polyethylene tubing (PE205, Clay
Adams, Parsippany, NJ) was inserted into the 12G needle and melted onto
a luer fitting at the top of the needle. A length of polyethylene
tubing that was protruding from the other end of the needle was melted
by flame and, while liquid, was forced back into the needle by tamping
on a cold surface. This provided an obturator that fitted into the
needle. The needle was introduced through the nonoperative side of the
nose, along a track made by stainless steel wire. The needle was guided
through the dura mater so that it was almost touching the pituitary
gland. The dura mater was then drawn close around the needle and a
small piece of sc fat was used to cover the dura mater at the end of
the tunnel. The tunnel in the sphenoid bone was then filled with dental
acrylic (RR self cure repair material, Dentsply Ltd., Weybridge,
Surrey, UK). The operative side of the nose was closed. A piece of skin
was removed around the hub of the needle, and two screws were inserted
in to the nasal bone. The hub of the needle and the screws were then
covered in dental acrylic, and the animal allowed to recover. This
surgical procedure took between 50–60 min. The animals were allowed
one week for recovery and then sampled. For portal sampling, the
animals were heparinized as previously described (7) through a jugular
venous cannula. After 2 h of heparinization, the obturator was
slowly removed from the 12G needle; no bleeding was observed during
this procedure and it was assumed that the portal vessels were not
damaged. A lesion maker was made from a 16G needle such that it
protruded 2–3 mm beyond the end of the implanted needle, and this was
inserted and repeated stabs were made through 360°. A length of
polyethylene tubing was inserted into the 12G needle after it had been
heated and shaped to fit the animal’s head. This tubing was
connected to a variable speed peristaltic pump (Pharmacia, Uppsala,
Sweden), and samples were collected onto ice, into tubes that
contained 0.5 ml 5 mM Bacitracin. Portal samples were
generally 2–2.5 ml. Plasma was harvested from paired jugular and
portal samples collected at 5-min intervals; the samples were frozen at
-15 C until assayed. At the end of portal sampling, the animals were
killed, and the pituitaries and median eminence were collected for
further analysis (see below).
GnRH antibody detection
Anti-GnRH antibody titer was determined on jugular blood samples
of both control and immunized sheep. The samples were diluted to 1/10
and 1/100 of serum concentrations and GnRH antibody binding assay was
performed. The diluted samples were incubated with GnRH tracer
(specific activity: 1600 µCi/µg) in buffer at 4 C for 2 days. Then
normal sheep serum, donkey antisheep antibody, and polyethylene glycol
were added, mixed, and incubated at 4 C for 3 h. After that, the
tubes were centrifuged for 30 min at 3000 rpm. Then 5% potato starch
was added, and the tubes were centrifuged for a further 5 min. The
supernatants were aspirated, and the tubes counted to measure the
amount of tracer bound. A positive control in which plasma sample was
replaced with rabbit anti-GnRH antibody (EL 14A, courtesy of Dr. J.
Resko) was also included.
Pituitary hormone content and messenger RNA (mRNA) levels for
gonadotropin subunits
The pituitary glands were halved; one half was used for hormone
analysis, and the other was extracted for RNA. The content of LH and
FSH was determined following extraction as previously described (8).
The extracts were serially diluted to ensure that the samples ran
parallel to the standard curve of the assay and values were read at the
midpoint of the standard curve. RNA extractions and Northern blotting
procedures were carried out as previously described (9).
Northern blots for the pituitary GnRH receptor were performed on
polyA+ mRNA prepared with the polyAtract Isolation System
IV (Promega, Madison, WI). Five micrograms of polyA+ mRNA
was run on a 1.2% agarose gel, and the Northern blot was probed with a
1.8-kb complementary DNA (cDNA) probe for the ovine GnRH receptor
previously described by Illing et al. (10).
Median eminence GnRH content
The median eminences were homogenized in 2 ml of 0.2
M acetic acid and incubated at 100 C for 5 min. After
cooling on ice, the homogenate was centrifuged at 12,000 x
g (10,000 rpm) for 30 min at 4 C, and the supernatant was
taken for RIA.
Hormone assays
Plasma LH, FSH, and GnRH assays were conducted as previously
described (11, 12, 13). The assays were interpolated by the competitive
binding program of Burger et al. (14). The standards for the
LH assays were prepared from NIH-oLH-S18, and those for the FSH assays
were prepared from NIH-oFSH-S13. The sensitivities of the LH and FSH
assays were 0.17 and 0.20 ng/ml, respectively. The coefficients of
variation within assays were 7.2 ± 0.4% at 6.3 ± 0.3 ng/ml
for LH and 6.0 ± 0.0% at 5.2 ± 0.0ng/ml for FSH. The
coefficients of variation between assays were 20% at 9.7 ng/ml for LH,
and 13% at 6.9 ng/ml for FSH. The GnRH assay had a sensitivity of
0.5–1.4 pg/ml. The coefficient of variation within GnRH assays was
7.4 ± 0.4% at 15.2 ± 0.7 pg/tube and between assays was
18%.
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Results
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Histology of the ovaries
The ovaries of two of the control sheep had corpus lutea present
at the time of removal and showed follicle development at various
stages. In the ovaries of the immunized ewes, there were no follicles
greater than 3 mm. Examples of cross-sections of ovaries from control
and immunized animals are shown in Fig. 1
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Antibodies to GnRH
The antibody levels in the rams that were used in this study were
previously reported by Brown et al. (4). None of the treated
rams had detectable antibody. Likewise, none of the ewes had detectable
GnRH antibodies at the beginning of the study.
Plasma gonadotropin and levels and GnRH secretion in control and
hypogonadal rams
The six control rams had between 1 and 3 pulses of LH secretion
each over the 12 h sampling. One of the four hypogonadal rams had
one LH pulse in the 12-h period. The mean (SEM) amplitude
of LH pulses in control animals was 4.1 ± 0.4 ng/ml, and the
amplitude of the single pulse in one of the hypogonadal rams was 0.6
ng/ml.
During portal sampling (5–7 h), three of the six control rams showed a
single large amplitude GnRH pulse (range 10.7–31.9 ng/ml). No GnRH
pulses were detected in any of the four hypogonadal rams.
Plasma gonadotropin levels in control and hypogonadal ewes before
and after ovariectomy and response to GnRH
Plasma LH and FSH levels before and after ovariectomy are shown in
Table 1. In the control ewes, pulsatile
LH secretion was apparent before ovariectomy, and a clear response to
GnRH was seen in each animal (Fig. 2). In
the hypogonadal ewes, no LH pulses were detected in four out of five
animals. One hypogonadal ewe had small LH pulses (Table 1) occurring
every 68 min. Mean plasma LH levels were lower in four out of five
hypogonadal ewes than in controls before ovariectomy (Table 1).
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Table 1. Plasma LH levels, pulse amplitude and frequency, and
plasma FSH levels in control and neonatally immunized ewes before and
after ovariectomy
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Figure 2. Plasma LH levels in control and immunized ewes and
the LH response to 10 µg GnRH (iv). Arrows show time
of GnRH injection.
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The mean (± SEM) area under the curve of the LH
vs. time profile following injection of GnRH was 685 ±
100 ng/ml·min, whereas four out of five of the hypogonadal ewes
showed no response to GnRH. In the one hypogonadal ewe that had small
LH pulses, an LH response was seen to GnRH (Fig. 2
), and the area under
the response curve was 71 ng/ml·min.
Plasma FSH levels were significantly (P < 0.03) lower
in the four hypogonadal ewes that did not respond to GnRH (Table 1).
The animal that did respond to GnRH had a plasma FSH level of 1 ng/ml
compared with 0.86 ng/ml in the control animals. Ovariectomy caused a
significant increase in the plasma LH (P < 0.02) and
FSH (P < 0.03) levels in the control animals (Table 1), but there was no significant increase in plasma gonadotropin levels
in four out of five of the hypogonadal animals (Fig. 3). In the hypogonadal ewe that had shown
a response to GnRH, plasma LH levels were doubled after ovariectomy,
but the plasma FSH levels were only slightly increased (Table 1). This
animal was not included in further studies.
GnRH secretion in the OVX control and hypogonadal ewes
The modified method of portal sampling was successful in all of
the experiments carried out. All five of the control ewes showed
regular pulsatile GnRH secretion and concordance of GnRH and LH pulses;
two examples from each group of animals are shown in Fig. 4. In the OVX hypogonadal ewes, the
pulses of GnRH that were detected in the hypophysial portal plasma were
significantly (P < 0.05) smaller than those in the OVX
controls with no significant difference between the groups in the
interpulse interval for GnRH pulses (Fig. 5).
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Figure 4. Examples of GnRH and LH secretion in control and
immunized ewes. The arrowheads denote pulses of
secretion as defined by Clarke and Cummins (1982).
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Figure 5. Mean (± SEM) amplitude and frequency
of GnRH pulses in OVX control and hypogonadal ewes.*,
P < 0.05.
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Median eminence content of GnRH
The mean (± SEM) content of GnRH in the median
eminence of control and hypogonadal rams was 62.8 ± 18.5 ng and
43.8 ± 16.5 ng respectively (not significantly different).
Similarly, the values for the ewes were 77.0 ± 27.5 ng in the
controls and 64.7 ± 19.4 ng in the immunized animals (not
significantly different).
Pituitary gonadotropin content and mRNA content
Pituitary gonadotropin content of LH and FSH in control and
hypogonadal rams and ewes is given in Table 2. There was significantly less
gonadotropin in the pituitaries of the hypogonadal animals. Figure 6 shows Northern blots for the
gonadotropin subunits and the GnRH receptor in the hypogonadal and
control OVX ewes. The amount of mRNA for the gonadotropin subunits was
generally so low that densitometry could not provide meaningful
results. The GnRH receptor blot shows multiple transcripts of the
expected size (10); mRNA levels were lower in the hypogonadal animals
(Fig. 6b).
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Figure 6. Northern blots for the gonadotropin subunits in
the pituitary glands of control and hypogonadal OVX ewes (A) and a
Northern blot for the GnRH receptor in the same animals using
polyA+ mRNA (B). Lanes 1–5 are control animals and 6–10
are neonatally immunized ewes. The transcript sizes for the GnRH
receptor are shown on the right.
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Discussion
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The present studies show that sheep that are immunized against
GnRH shortly after birth have markedly lowered secretion of GnRH in
adulthood, although there is a normal amount of GnRH detectable in the
median eminence. There is a marked reduction in the synthesis of
gonadotropins in the pituitary gland with reduced secretion of
gonadotropins, and the outcome is permanent hypogonadotropic
hypogonadism. It seems most likely that the reduced secretion of GnRH
is the result of some permanent alterations within the hypothalamus or
the median eminence.
The hypogonadotropic condition was only seen in 4/5 ewes studies,
showing that there is a variation in response to the immunization.
Animals that are actively immunized against GnRH in adulthood show some
recovery of reproductive function with time (3); there is considerable
variation between animals in the period to recovery. The exact reasons
for this are not known, but recovery after active immunization is not
simply related to the antibody titer and other factors appear to be
involved (3). The reproductive status of neonatally immunized animals
can be complete hypogonadotropic hypogonadism but some animals show
complete reproductive competence as adults (4, 5). In the present
series of studies, there was one animal that was clearly less affected
by the neonatal immunization than the other four animals. This may be
related to the titer of antibody that is generated during the neonatal
period but could equally well be due to the requirement for the
treatment to be within a window of development during which the animal
is maximally susceptible to the immunization. In the case of the sheep,
it seems unlikely that this would be the development of the blood-brain
barrier because this matures before birth in this species (15). The
median eminence may be a preferential site at which immune cells might
attack cells of the central nervous system and cause a permanent change
because this tissue is outside the blood-brain barrier (16). It is
possible that the median eminence is relatively more permeable in the
neonatal period and that the variability in pathological response to
immunization may be due to variation in the timing of the development
of the blood vessels; current studies are investigating this
possibility. In support of the concept that immunization could alter
function of the median eminence, Molenaar et al. (6) showed
that there was some anatomical lesions at this level in pigs that were
actively immunized against GnRH at 10 weeks of age with a booster at 18
weeks of age. Their immunized animals had gross alterations in the
anatomy of the median eminence with the structure being swollen and
with thickened blood vessel walls. It was also reported that the tissue
had the appearance of being edematous. To what extent this study is
comparable to the present one is difficult to determine because of the
different age of immunization and the likely differences between the
species in the development of the median eminence and the
reproductive system. Furthermore, the animals in the study of Molenaar
et al. (6) were killed at 26 weeks of age and had detectable
GnRH antibodies at that time. These immunized pigs were reported to
have reduced immunostaining for GnRH in their median eminence, although
no quantification of this effect was presented.
Studies in rats (17, 18) showed that passive GnRH immunization in the
neonatal period caused a reduction in testis size and genitalia that
was evident at 100 days of age, but these animals were able to respond
to GnRH. It was suggested that transitory inhibition of the
reproductive axis during a critical stage of development had an effect
on the development of the testes and genitalia that occurs postnatally
in the rat. Our immunized sheep are fundamentally different to these
passively immunized rats because there is a profound and long-term
impairment of the reproductive axis in the majority of cases.
The present studies show that there is a reduction in, but not
total elimination of, the secretion of GnRH in neonatally immunized
sheep. This was not easy to demonstrate in the rams that were available
for study because we sampled these as intact animals and the GnRH/LH
pulse frequency is low (19). A more exact comparison was able to be
made in the females after ovariectomy, and this clearly showed that
GnRH secretion into hypophysial portal blood was reduced by the
neonatal immunization. In all cases, GnRH secretion in the immunized
animals was apparent, but the level of secretion of GnRH was
significantly lower than in control animals, due to reduced GnRH pulse
amplitude. Given that the amount of GnRH in the median eminence was
normal in the immunized animals, the thickening of blood vessels in the
median eminence, as shown in GnRH-immunized pigs, could be a cause of
reduced pulse amplitude, and this is currently being investigated. The
study of the ewes utilized a newly devised system for the collection of
portal blood. Previous models (2, 20, 21) used an implant that had two
access needles. The system that we have now devised is much simpler,
requires less surgical time, and is less invasive. The new method is
less likely to allow blood clots to form in the vicinity of the lesion
site in the portal capillaries than our earlier model, and the animals
may be left for extended times between the period of surgery and
sampling.
The levels of gonadotropin in the pituitary glands of the control
animals were similar to those previously reported (22). The low levels
of synthesis and secretion of gonadotropins in the immunized animals
and the presence of some GnRH receptor mRNA suggests that the
gonadotropes are present, albiet functioning at a reduced level. Based
on the low level of gonadotropin that was found in the pituitary gland,
it would appear that the gonadotropes in these animals synthesize
hormone, but not at a level required to show a response to GnRH
challenge. This is consistent with previous observations that small
pulses of GnRH can maintain synthesis of gonadotropins without
detectable secretion (23). The level of synthesis must, however, be
very low because mRNA for the subunits of LH and FSH were undetectable
in three quarters of the neonatally immunized animals studied. Attempts
were made to stimulate pituitary function by repeated pulsatile
treatment with GnRH over 2 weeks, but this led to the appearance of
GnRH antibodies in the neonatally immunized animals (data not
shown).
In conclusion, we have shown that neonatal immunization of sheep causes
a long lasting effect on GnRH secretion and impairment of the function
of gonadotropes, beyond the time when GnRH antibodies are detectable.
This occurs in spite of there being a normal amount of GnRH in the
median eminence of immunized animals and evidence of the existence of
gonadotropes in the pituitary gland. The result of the neonatal
immunization of sheep against GnRH is long-term sterility.
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Acknowledgments
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We thank Mr. Bruce Doughton and Mr. Alan Fawcett for the care of
animals and assistance with the experiments. RIA reagents were supplied
by NIH and Dr. J. Resko. Dr. N. Illing and Dr. I. Becker prepared the
GnRH receptor probe.
Received September 2, 1997.
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Abstract
Introduction
