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Brown Science Center, Transylvania University (H.I., S.K.T.), Lexington, Kentucky 40508; Reproductive Sciences Program, Departments of Obstetrics and Gynecology (H.I., D.L.F.) and Biology (D.L.F.), University of Michigan, Ann Arbor, Michigan 48109; and the Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati College of Medicine (M.N.L), Cincinnati, Ohio 45267
Address all correspondence and requests for reprints to: H. I’Anson, Department of Biology, Washington and Lee University, Lexington, Virginia 24450. E-mail: ianson{at}wlu.edu
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Abstract |
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20 kg BW), hypothalamic GnRH is present and
releasable, suggesting that central mechanisms limit the release of
GnRH during chronic growth restriction. Our study compared the number
and distribution of GnRH-containing neurons in growth-restricted
(n = 5) and rapidly growing (n = 5) ovariectomized
prepubertal female lambs at 24 weeks of age (normal age of puberty is
about 30 weeks). Immunoreactive cells were labeled using LR-1 antiserum
(R. Benoit) and an avidin-biotin-immunoperoxidase procedure. GnRH
neurons were localized in 60-µm coronal sections from the level of
the diagonal band of Broca to the mammillary bodies. The estimated
total number of GnRH neurons in the growth-restricted and rapidly
growing lambs was similar (3364.8 ± 513.8 vs.
3151.2 ± 279.8, respectively). In addition, the percent
distributions of GnRH neurons in the diagonal band of Broca, the
anterior hypothalamus, the lateral hypothalamus, and the posterior
hypothalamus were not different. A trend (P = 0.07)
toward a smaller percent distribution in the preoptic area was noted in
growth-restricted lambs (30.6 ± 3.6) compared to rapidly growing
lambs (44.0 ± 5.2). By contrast, the percent distribution of GnRH
neurons in the medial basal hypothalamus was significantly greater in
the growth-restricted lambs compared with the rapidly growing lambs
(17.7 ± 2.2 vs. 6.7 ± 1.4, respectively;
P < 0.005). It is of interest that the percent
distribution of GnRH-containing neurons in the medial basal
hypothalamus of the hypogonadotropic growth-restricted lamb is similar
to that observed in the fetal lamb, whereas the eugonadotropic rapidly
growing lamb is more similar to the adult female. In this context,
decreased GnRH secretion and delayed puberty during diet-induced growth
restriction may arise from alterations in the GnRH neurosecretory
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Introduction |
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To investigate how nutritional and growth-related cues time the increase in GnRH and LH secretion that occurs at puberty (8, 9, 10), we used an experimental model in which ovariectomized lambs were maintained on a restricted diet after weaning to retard growth, and thus to delay puberty (7, 12). Such lambs remain hypogonadotropic even in the absence of inhibitory ovarian steroid feedback (7). They respond normally to physiological doses of GnRH, indicating that the pituitary can function adequately (13). Hypothalamic GnRH content is similar in lambs on a restricted diet with a low LH pulse frequency (less than one pulse per 4 h) and in lambs fed ad libitum with a high LH pulse frequency (four or five pulses per 4 h) (13). Thus, it is unlikely that a decrease in GnRH synthesis underlies hypogonadotropism during chronic low nutrition. Rather, the secretion of GnRH is limited, as evidenced by a low GnRH pulse frequency compared to that observed in rapidly growing lambs (8). In the current study, we asked if variations in the number and distribution of GnRH neurons are responsible for decreased GnRH release during chronic growth restriction. Our approach was to compare these parameters of GnRH-containing neurons in growth-restricted and rapidly growing female lambs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The usual diet (300 g/day, 500 Cal) was
approximately 25% of the nutrient requirement for a rapidly growing
20-kg lamb (National Research Council, 1985). The young females were
weighed weekly, and feed intake was adjusted to maintain the 20-kg
target weight. As evidenced from observations made several times daily,
the lambs remain active and healthy despite the reduced caloric intake.
The rapidly growing group was fed alfalfa hay and the same commercial
pelleted ration, and they exhibited rapid growth, as evidenced from
measurements of body weights at weekly intervals.
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To determine pulsatile patterns of LH secretion, concentrations of the hormone were measured in blood samples collected by jugular venipuncture. Samples were allowed to clot overnight at 4 C; serum was decanted after centrifugation and stored at -20 C until assay.
Experimental design
The study was performed at approximately 24 weeks of age
(September). On the day before the perfusions, the pattern of LH pulses
between growth-restricted and rapidly growing lambs was compared by
measuring LH in blood samples collected every 12 min for 4 h.
On the following day, before the animals were killed, the lambs were injected with heparin (3 ml, 10,000 U/ml) to minimize blood clotting in subsequent procedures. The lambs were then killed with 5 ml Beuthanasia (Schering-Plough Animal Health Corp., Kenilworth, NJ) (containing 390 mg sodium pentibarbital and 50 mg sodium phenytoin/ml), and the heads were perfused bilaterally via the carotid arteries with 0.5 liters 0.9% saline with 0.1% sodium nitrite, followed by 3 liters 2% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.3) containing 0.1% sodium nitrite (for vasodilation) and heparin (100 U/ml). After perfusion, the brain was carefully removed from the cranium, and a block of tissue containing the preoptic area and hypothalamus was microdissected out.
Tissue processing
Brains were processed according to the methods of Lehman
et al. (14). Briefly, the tissue block was postfixed in 2%
paraformaldehyde at 4 C for the next 24 h, and subsequently
incubated in 30% sucrose in 0.1 M phosphate buffer (PB)
with 0.1% sodium azide at 4 C for 5 days (the solution was changed
daily). Thereafter, the brains were frozen at -90 C until sectioning.
All subsequent procedures were performed simultaneously on paired
brains, one from a growth-restricted lamb and one from a rapidly
growing lamb.
Sixty-micron coronal sections were cut on a freezing microtome and washed overnight in PB, pH 7.3, with 0.1% Triton X-100 at 4 C. Every fourth section was processed for GnRH immunocytochemistry and counterstained with cresyl violet to identify nuclear boundaries.
For visualization of GnRH-containing neurons, the free floating sections were submerged for 30 min in 0.1 M glycine to remove excess aldehydes, washed three times for 10 min each time in PB with 0.1% Triton X-100, and then submerged for 10 min in 0.5% hydrogen peroxide with 1% Triton X-100 to eliminate endogenous peroxidase activity. Sections were washed four times for 10 min each time in PB with Triton X-100 and incubated in 1% normal donkey serum for 1 h. After these procedures, the sections were exposed to primary antiserum (rabbit LR-1, generously provided by Dr. Robert Benoit) at a dilution of 1:20,000 in PB with 1% normal donkey serum, 0.1% Triton X-100, and 0.1% sodium azide for 72 h at 4 C. This antiserum has been validated previously for staining GnRH perikarya and fibers in sheep (14), and it recognizes amino acids 3, 4, 7, 8, 9, and 10 of the decapeptide but no other identified neuropeptide. The sections were then treated at room temperature for 1 h with biotinylated donkey antirabbit IgG, followed by a 1-h incubation at room temperature with avidin-biotin-horseradish peroxidase complex (Vectastain universal kit, Vector Laboratories). Horseradish peroxidase was visualized using 3,3'-diaminobenzidine as the chromogen, with glucose oxidase to generate the hydrogen peroxide substrate (15). This reaction was allowed to proceed for approximately 30 min. Sections were mounted on gelatin-coated slides, dehydrated, counterstained with cresyl violet, dehydrated, cleared, and coverslipped with Permount (Fisher Scientific).
LH assay
LH was measured in duplicate 50- to 200-µl aliquots of serum
using a modified RIA (13, 16) developed by Niswender et al.
(17). Assay sensitivity (n = 2 assays), defined as 2
SD from maximum binding, averaged 0.6 ng/ml for 200 µl
serum, expressed relative to NIH LH-S12. Intra- and interassay
coefficients of variation for 200-µl samples of a serum pool
containing 2.8 ng/ml LH, which inhibited binding of labeled ligand to
55%, averaged 11.3% and 12.1%, respectively.
Data analyses
LH pulses were identified using a pulse detection algorithm,
Pulsefit (13, 18). Undetectable hormone concentrations were assigned a
value equivalent to the limit of detection of the assay. Hypothalamic
nuclei were identified using the atlas of the sheep brain developed by
Welento et al. (19). Using outlines of the cresyl
violet-stained hypothalamic nuclei, each section was drawn, and the
location of each GnRH-containing neuron was plotted. The number of GnRH
neurons in each section was counted. The sections from each brain were
subdivided according to the position of nuclei and major nerve tracts
into the region of the diagonal band of Broca (DBB), the preoptic area
(POA), the anterior hypothalamus (AH), the lateral hypothalamus, the
mediobasal hypothalamus (MBH), and the posterior hypothalamus (PH).
Morphometric analysis was performed on the MBH because this was the
only region that showed a difference in the distribution of
GnRH-containing neurons between groups. The neurons were classified as
unipolar, bipolar, or multipolar. Neurons in which the processes
emanating from the cell soma were difficult to identify due to damage
during sectioning were not classified morphologically. The total number
of neurons in each brain, the distribution of neurons within the
aforementioned brain regions, and the percentages of different
morphological subtypes within the MBH from growth-restricted and
rapidly growing lambs were compared using ANOVA (Statview
512+, Brainpower, Las Calabas, CA). Percentages were
arcsine transformed before ANOVA, and the Scheffe test was used in any
post-hoc comparisons made among means. In all analyses,
P < 0.05 was considered significant. Data are
presented as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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depicts serum LH concentrations from the
growth-restricted lambs (left panel) and rapidly growing
lambs (right panel). As previously reported in the
growth-restricted lambs, no LH pulses were observed during the 4-h
frequent sampling period, whereas in the rapidly growing lambs, a LH
pulse frequency of two to four pulses per 4 h was detected.
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)3300, estimated from counting every fourth section) was slightly
higher than that reported for the adult female sheep [666–2418
neurons (14) and 2500 neurons (20)] or the fetal lamb (1800
neurons) (21).
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-6) depicts drawings of schematic sections in the POA and
hypothalamus from representative growth-restricted and rapidly growing
lambs upon which the location of GnRH-containing neurons at similar
levels was plotted (closed circles). GnRH-containing neurons
were found scattered throughout the POA and hypothalamus from the
region of the DBB to the mammillary bodies (Fig. 4). The majority of
GnRH cell bodies were found in the POA, at the level of the organum
vasculosum of the lamina terminalis (Fig. 4B). Rostrally, GnRH cell
bodies were observed medially in the region of the DBB/medial septum
(Fig. 4A) and caudally in the AH and MBH, with the cells spread
medially and laterally along the ventral surface of the brain (Fig. 4, C and D). Few GnRH cell bodies were observed in the PH at the level of
the mammillary bodies. The distribution and appearance of GnRH fiber
tracts and terminal fields were similar to those previously described
for the adult female sheep (14, 20, 22).
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. Despite the range of the
total number of GnRH-containing neurons observed among lambs, the
relative percent distribution of GnRH-containing neurons in each brain
region within an individual lamb remained similar. There was no
difference between the growth-restricted and rapidly growing lambs in
the percent distribution of GnRH-containing neurons in the region of
the DBB, AH, lateral hypothalamus, or PH. There appeared to be a trend
toward a smaller percent distribution of GnRH-containing neurons in the
POA of the growth-restricted lambs compared with the rapidly growing
lambs (30.6 ± 3.6 vs. 44.0 ± 5.2, respectively;
P = 0.07). In contrast, the percent distribution of
GnRH-containing neurons in the MBH was nearly 3 times greater in the
growth-restricted lambs compared with that in the rapidly growing lambs
(17.7 ± 2.2 vs. 6.7 ± 1.4, respectively;
P < 0.005). Photomicrographs showing GnRH-containing
neurons in the MBH from a representative growth-restricted and rapidly
growing lamb are presented in Fig. 6.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There are several considerations that must be taken into account to explain these differences in population distribution of GnRH-containing neurons. It is of interest to note that a related study from our laboratory reported no differences in total GnRH content in the POA/AH and MBH/arcuate nucleus of growth-restricted and rapidly growing lambs (13). However, this study may not be comparable with the present data, because total content also measures GnRH contained in fibers as well as cell bodies. In addition, the data were not expressed per mg tissue, but rather per region. Thus, any differences in content may have been obscured because the sizes of these regions are very different in such lambs. In the protein-deficient growing male lamb, GnRH content within the median eminence is increased (23), whereas in the rat, hypothalamic GnRH content is not altered in the prepubertal growth-restricted female (24), but is increased during acute undernutrition in the adult male (25). GnRH messenger RNA analysis in lambs during development or when maintained in different metabolic states has yet to be reported.
It is unlikely that any differences in immunostaining occurred between the two groups of lambs, as immunocytochemistry was always performed simultaneously on sections from paired brains, one from each group. In addition, there was little possibility of bias in counting the GnRH-containing neurons, as this was performed such that the group identity of each lamb was hidden during this phase of the experiment. It is also unlikely that GnRH-containing neurons migrated from one brain region to another in the growth-restricted lambs. Migration of the GnRH-containing neurons from the nasal placode is well documented during embryonic development in a variety of species (chick, 26–28; mouse, 29–31; rat, 32; primate, 33), but does not appear to continue postnatally.
Several studies have examined the distribution of GnRH-containing neurons in the sheep brain. In the female fetal lamb, 19% of such neurons are located in the MBH and 17% in the POA (21), whereas in the adult female, only 1–5% are found in the MBH (14, 34) and 50–60% in the POA (14). The aforementioned studies were conducted in the animals of the Suffolk breed, as is used in the current study, and all used the same LR-1 antiserum and very similar immunocytochemical procedures. In studies using other sheep breeds and antiserum, the results concerning the distribution of GnRH-containing neurons in the MBH are equivocal. Caldani and co-workers (20) observed 15% GnRH-containing neurons in the MBH of adult females, a percentage similar to that reported in the fetal lamb, whereas Glass and co-workers (22) found no such neurons in the MBH. However, if one compares the results from the studies mentioned above using Suffolk sheep and the same antiserum as the experiment described herein (14, 21, 34), it is interesting to note that the growth-restricted and rapidly growing lambs have a distribution of GnRH-containing neurons intermediate between the fetal and the adult female. Further, in terms of MBH neuron distribution, the growth-restricted lamb is more similar to the fetal lamb (fetus, 19%; growth-restricted lamb, 18%), whereas the rapidly growing lamb appears to be more similar to the adult female (adult, 1–5%; rapidly growing lamb, 7%). Finally, there appears to be a trend toward an increase in GnRH-containing neurons within the POA with age; the neurons are most numerous in the adult ewe (50–60%), fewer neurons were measured in rapidly growing and growth-restricted lambs (44% and 31%, respectively), and even fewer were found in fetal lambs (17%).
Even though the preceding results were not obtained in the same experiment, the differences are striking, and one can suggest several alternative hypotheses to try to explain the picture of GnRH neuron development that appears to be emerging.
One such hypothesis would suggest that the growth-restricted lamb is developmentally delayed in terms of the GnRH neurosecretory system compared to the rapidly growing lamb. This would imply that the GnRH neurosecretory system of the growth-restricted lamb is more similar developmentally to that of the fetal lamb. The ovine fetus appears to possess a functional GnRH neurosecretory system, such that LH pulse frequency is approximately one pulse per 3 h (35), a slow pulse rate similar to that measured in the growth-restricted lamb (normally zero or one pulse per 4 h). These observations would appear to confirm the hypothesis that the growth-restricted lamb is developmentally delayed in terms of the GnRH neurosecretory system. Interestingly, however, hourly LH pulses occur as early as 6 weeks of age in the ovariectomized female lamb (36) after removal of estradiol negative feedback. Thus, even in the young lamb, the GnRH neurosecretory system is functionally competent to produce a LH pulse frequency similar to that observed during the first follicular phase at puberty (10). In the context of our study, the growth-restricted lamb would have already achieved this level of maturity before growth restriction, which was between 10 and 16 weeks of age. Thus, it is unlikely that the GnRH neurosecretory system is developmentally delayed in such lambs, even though GnRH release becomes reduced during growth restriction (8) due to central nervous system inhibition (37) by as yet unknown neurotransmitter pathways.
A second possibility is that the differences in distribution of GnRH-containing neurons in growth-restricted vs. rapidly growing lambs may reflect a difference in the functionality of these neurons. The larger population of GnRH-containing neurons in the MBH of growth-restricted lambs compared to rapidly growing lambs could be responsible at least in part for the reduced release of GnRH in such lambs. GnRH is known to exert ultrashort loop feedback on its own release (39, 40, 41, 42, 43). Thus, activation of such an inhibitory GnRH neuronal population could be induced during growth restriction, thereby decreasing GnRH release. A similar hypothesis has been suggested for the decrease in arcuate nucleus GnRH-containing neurons observed after puberty in the male ferret (44).
Finally, the LR-1 antiserum recognizes GnRH within its precursor as well as the decapeptide itself (Benoit, R., personal communication) (45). Thus, a third possibility for the increase in GnRH-containing neurons within the MBH of growth-restricted lambs is that this population of neurons produces GnRH, but that it is not released due to increased activity of inhibitory neurotransmitter pathways or to decreased activity of stimulatory pathways. Previous studies have revealed that although GnRH secretion is decreased in the growth-restricted lamb, it can be induced repeatedly by hourly administration of the glutamate/aspartate neurotransmitter agonist, N-methyl-D,L-aspartate (13). Thus, GnRH is synthesized, but not normally secreted, in the growth-restricted lamb. A similar inverse relationship between distribution of GnRH-containing neurons and GnRH/LH release has been observed in a study that compared pre- and postpubertal male ferrets (44). There were 50% fewer GnRH-containing neurons in the arcuate nucleus of postpubertal ferrets, which is correlated with an increase in LH release compared with that in the prepubertal ferret. However, in this study the smaller number of GnRH-containing neurons did not reflect a higher secretory activity of these neurons, as colchicine treatment of postpubertal (25-week-old) ferrets did not increase the number of GnRH-containing neurons to levels observed in prepubertal ferrets. Such colchicine treatment has not been attempted in lambs to address the issue of changes in secretory activity as they relate to our study.
The percent distribution of GnRH-containing neurons within the POA of growth-restricted and rapidly growing lambs (31% and 44%, respectively) is intriguing in light of the differences measured in this region in the adult ewe compared to the fetal lamb (50–60% and 17%, respectively) (14, 21, 34). As previously mentioned, both the fetal lamb and the growth-restricted lamb secrete GnRH and LH at a low pulse frequency [one pulse per 3 h (35) vs. zero to one pulse per 4 h (8)]. Thus, it is conceivable that the decreased number of GnRH-containing neurons within the POA of such lambs may reflect a decrease in the synthesis of GnRH and its precursor in this region. GnRH messenger RNA analysis in lambs during development and when maintained in different metabolic states would provide some insight into this possibility. Another possibility is that the differences in GnRH-containing neurons in this region reflect the ability of such lambs to secrete surge levels of GnRH at ovulation. The POA has been implicated in induction of the GnRH, and hence LH, surge that induces ovulation (46, 47, 48, 49, 50). The potential to respond to the stimulatory feedback action of estradiol and produce a LH surge is attained a few weeks after birth (51), and the capacity to produce a LH surge is still present in the growth-restricted lamb (7). Because a GnRH surge has been observed to induce a LH surge in the prepubertal female (52) and adult sheep (53, 54, 55), one assumes that a GnRH surge is required to induce a LH surge in the sheep. If this is the case, then it is tempting to suggest that the relatively low percentage of GnRH-containing neurons in the fetal lamb POA (17%) reflects an immature surge-inducing GnRH neurosecretory system. Perhaps the increased GnRH immunostaining in the POA of the other three groups of sheep reflects maturation of the surge-inducing GnRH neurosecretory system to a level where a GnRH surge can now be induced.
In conclusion, the distribution of GnRH-containing neurons in the growth-restricted lamb and that in the rapidly growing lamb were similar, except in the regions of the MBH and POA. The differences in GnRH immunostaining in these regions suggest that growth retardation induced by dietary restriction produces changes in the GnRH neurosecretory system that may be responsible for the decreased release of GnRH, and thus delayed puberty, in such lambs.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Biology, Washington and Lee
University, Lexington, Virginia 24450.
3 Current address: University of Louisville School of Medicine,
Louisville, Kentucky 40292.
Received July 9, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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