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Wisconsin Regional Primate Research Center (M.M., M.G., E.T.) and Department of Pediatrics (E.T.), University of Wisconsin, Madison, Wisconsin 53715-1299
Address all correspondence and requests for reprints to: Ei Terasawa, Ph.D., Wisconsin Regional Primate Research Center, 1223 Capitol Court, Madison, Wisconsin 53715-1299. E-mail: terasawa{at}primate.wisc.edu
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Abstract |
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Introduction |
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It has been shown in rats and rabbits that neuropeptide Y (NPY) in the hypothalamus plays an important role in the modulation of pulsatile LHRH release and the preovulatory LH surge (10, 11). In primates as well, we have reported that infusion of NPY in the stalk-median eminence (S-ME) stimulates LHRH release (12), whereas infusion of an NPY antibody or antisense oligodeoxynucleotide for NPY messenger RNA (mRNA; AS NPY) into the S-ME suppresses LHRH pulses (13, 14), and NPY pulses precede LHRH pulses in OVX monkeys as well as during the progesterone-induced LH surge (9, 13). Moreover, the pulse frequency of NPY pulses increases along with an increase in the LHRH pulse frequency when progesterone induces a LHRH surge in estrogen-treated OVX monkeys (9), suggesting that NPY neurons may mediate positive feedback effects of progesterone in primates. Nonetheless, it is unclear whether NPY neurons play an obligatory or a permissive role in the control of steroid-induced LHRH release in primates. As little information on the mechanism of the preovulatory LHRH surge in primates is available, and there are distinct species differences in neuroendocrine characteristics between primates and rodents (6, 15), it is important to study the triggering mechanism of the steroid-induced LHRH surge in nonhuman primates. Therefore, the present study investigates the effects of an antisense oligodeoxynucleotide for NPY mRNA (AS NPY) on progesterone-induced LHRH release in estrogen-primed OVX female rhesus monkeys. This study expands upon our previous work demonstrating that AS NPY infusion suppresses both NPY and LHRH pulses in OVX female monkeys (14).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Steroid treatments and blood sampling
The steroid treatment protocol used was similar to that reported
previously (6, 7). A 4-cm SILASTIC brand capsule (Dow Corning Corp., Midland, MI) containing 17ß-estradiol
(E2) was implanted sc 14 days before sc injection
of 30 µg estradiol benzoate (EB). Twenty-four hours after EB
treatment, progesterone (2.5 mg in 0.25 ml corn oil) or oil (0.25 ml
corn oil or sesame oil), as a control for progesterone, was injected
sc. Serum samples (1.0 ml) were collected through indwelling saphenous
catheters at time points relative to progesterone (or oil) injection
(-3, 0, 3, 6, 9, and 12 h) for evaluation of LH levels.
Push-pull perfusion
At least 8 weeks before push-pull perfusion experiments began, a
cranial pedestal was implanted under halothane anesthesia (16, 17). The
method for push-pull perfusion of the S-ME in conscious monkeys was
similar to that previously described (16). Briefly, a concentric
cannula with double lumens was stereotaxically inserted into the S-ME
of anesthetized monkeys 3 days before the start of the perfusion
experiments. The placement of the tip of the cannula was confined to an
area 1.0 mm posterior and ventral to the tip of the infundibular recess
and laterally within 1.0 mm of the midline. On the third day after
cannula insertion, modified Krebs-Ringer phosphate buffer solution
(artificial cerebrospinal fluid) with bacitracin (4 U/ml;
Sigma, St. Louis, MO) was infused into the S-ME through
the push cannula at a rate of 23 µl/min using a peristaltic pump
(Minipulse 3, Gilson, Inc., Middleton, WI). Perfusate was collected (on
ice) from the pull cannula at 10-min intervals using an identically
calibrated pump. Each perfusate sample was aliquoted into two vials
(150 µl for LHRH and 75 µl for other neuromodulators) before
storage at -70 C.
Experimental design
Exp 1. To determine the effects of progesterone on LHRH
release and LH release, push-pull perfusion at 10-min intervals and
blood samplings at 3-h intervals were conducted simultaneously. Twelve
female OVX monkeys that had been implanted with
E2 capsules 2 weeks before the experiment and
injected with 30 µg EB 24 h before the experiment were used.
After 4 h of control perfusion, 2.5 mg progesterone in corn oil or
corn oil alone were injected sc, and perfusate collection was continued
until 12 h after progesterone or oil injection. The time of
progesterone or oil injection was designated time zero. All 12 monkeys
received progesterone injection (n = 12), whereas 11 of the 12
monkeys received oil injection (n = 11) in random order in
successive experiments. LHRH in perfusates, but not NPY, was measured
in this experiment.
Exp 2. To determine whether suppression of NPY release alters the progesterone-induced LHRH surge in conscious OVX rhesus monkeys, we infused an antisense oligodeoxynucleotide for NPY mRNA (AS NPY) into the S-ME using a push cannula, whereas perfusate samples were collected through a pull cannula, as described previously (14). The AS NPY (5'-CCA GTC GCT TGT TAC CTA GC-3') is a 20-base oligonucleotide corresponding to the N-terminus of human NPY immediately downstream from the initiation codon (GenBank accession no. M.15789), as described previously (14). An oligonucleotide (5'-CTC CGC TTC AGT ACG CTA GT-3') containing the same bases in scrambled sequence (SC NPY) was used as a control. Both AS NPY and SC NPY were synthesized at the Biotechnology Center, University of Wisconsin (Madison, WI). We examined the specificity of the AS NPY and SC NPY using the GenBank Primate Database (FASTA Program, Genetics Computer Group, Madison, WI) as described previously (14). The AS NPY and SC NPY were then desalted and resuspended in artificial cerebrospinal fluid under sterile conditions. LHRH levels in perfusates were measured by RIA. NPY levels were not measured in this experiment.
Exp 3. To determine whether the effects of AS NPY on LHRH release in the progesterone-treated group differ from those in the oil-treated group, AS NPY was infused into the S-ME of animals that received oil injection. We did not examine the effects of SC NPY on LHRH release in animals treated with oil, because SC NPY did not cause any significant effect on LHRH release in a previous study (14). LHRH and NPY levels in perfusates were measured by RIAs.
Exp 2 and 3 were conducted concurrently on 17 OVX monkeys, in which E2 capsules were implanted 2 weeks before and EB was injected 24 h before the initiation of the experiment. After 2 h of control perfusion, AS NPY or SC NPY at 10 µM was continuously infused for 10 h, followed by an additional 4 h of control perfusion. Progesterone in corn oil or corn oil alone was injected sc 2 h after the initiation of infusion of AS NPY or SC NPY, and the time point at which progesterone or oil injection was designated time zero. Eleven of the 17 monkeys were used for progesterone with AS NPY (n = 11), 12 of the 17 monkeys were used for progesterone with SC NPY (n = 12), and 5 of the 17 monkeys were used for oil with AS NPY (n = 5). Eleven of the 17 monkeys were assigned to 2 different treatments, and none of animals was tested twice with the same treatment.
Hormone assays
LHRH concentrations in the perfusate fractions were measured by
RIA, using anti-LHRH serum (R1245), as described previously (17). A
150-µl aliquot of perfusate was used for the LHRH assay. The
sensitivity of the LHRH assay was 0.05 pg/tube at 95% binding. The
intra- and interassay coefficients of variation were 7.9% and 10.1%,
respectively.
NPY concentrations in the perfusate fractions were measured by RIA, using anti-NPY serum, provided by Dr. K. Chihara, Kobe University (Kobe, Japan) (18). This assay was modified from a previously reported assay (19). A 75-µl aliquot of perfusate was used for NPY assay. The sensitivity of the NPY assay was 0.7 pg/tube at 95% binding, and the intra- and interassay coefficients of variation were 10.8% and 13.3%, respectively.
LH concentrations in serum samples were measured in duplicate (100 µl) by RIA as described previously (7), using anti-LH serum (R13, pool D), provided by Dr. G. Niswender. The reference standard used was WDP-x81–1720, provided by the NICHHD. The sensitivity of the assay was 2.0 ng/tube at 90% binding. The intra- and interassay coefficients of variation were 4.2% and 5.9%, respectively.
Pulse analysis
LHRH pulses in Exp I were analyzed using the Pulsar algorithm
(20). Pulses of LHRH were depicted using parameters identical to those
previously reported, i.e. cut-off criteria for
G1, G2,
G3, G4, and
G5 were 3.8, 2.6, 1.9, 1.6, and 1.2, respectively
(7, 17).
Statistical analysis
Exp 1. To determine changes in mean LHRH release, pulse
amplitude, and interpulse intervals during the progesterone-induced LH
surge, the results from the six 2-h periods after progesterone or oil
injection were compared with those from the two 2-h periods before
injection using two-way ANOVA with repeated measures in which the
variables were treatments and time. Post-hoc analysis of
changes occurring during specific time periods after progesterone
treatment was conducted using the Student-Newman-Keuls multiple range
test. Effects of progesterone on LH release were similarly examined by
two-way ANOVA followed by post-hoc analysis using the
Student-Newman-Keuls multiple range test.
Exp 2 and 3. To determine whether the progesterone-induced LHRH surge was blocked by AS NPY, 2 h means of LHRH release after the AS or SC NPY treatments were compared with 2 h means of LHRH before the treatments using two-way ANOVA, followed by the Student-Newman-Keuls post-hoc analysis. Similarly, to examine whether AS NPY effects with the progesterone treatment differ from those with the oil treatment, the 2 h means of LHRH release after AS NPY in the progesterone and oil treatment groups were compared with the 2 h means before AS NPY using two-way ANOVA followed by the same post-hoc analysis. The effects of AS NPY on NPY release in the oil-treated group were similarly examined using one-way ANOVA.
The statistical analysis was conducted using raw values, and significance was attained at P < 0.05. For graphic presentation, mean LHRH and mean NPY levels were expressed with normalized data, i.e. mean LHRH release and mean NPY release during a 2-h period before the injection of progesterone or oil (Exp 1) or the initiation of AS NPY or SC NPY infusion (Exp 2 and 3) were designated 100% in each animal, and the remaining data were calculated accordingly.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The pulse frequency and/or pulse
amplitude of LHRH pulses started to increase approximately 3 h
after progesterone. A higher frequency of LHRH release continued,
whereas a large pulse amplitude was not sustained in most cases (Fig. 1). In contrast, oil injection did not alter LH release or LHRH release
(Fig. 1).
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). Post-hoc
analysis indicated that mean LH levels after progesterone were
significantly higher than those before progesterone and the
corresponding mean LH levels in the oil control (P <
0.01 to 0.05).
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, A and
B). Post-hoc analysis indicated that mean LHRH levels and
mean pulse amplitude during the 4–12 h after progesterone were
significantly larger than those before progesterone and the
corresponding LHRH means in the control group (P <
0.05). Similarly, the overall mean of the interpulse interval after
progesterone was significantly shorter than that in the oil control
(P < 0.05; Fig. 2C), and mean interpulse intervals
during the 2–10 h after progesterone were significantly shorter than
that before progesterone or the corresponding means in the oil
control.
Effects of AS NPY on progesterone-induced LHRH release
Progesterone induced a robust LHRH increase in animals infused
with SC NPY (Fig. 3, A and B; overall
significance, P < 0.05). Although the first LHRH
increase was observed between 2–4 h in 7 of 12 animals in this group,
post-hoc analysis indicated that the LHRH increase did not
reach statistical significance until 4–6 h after progesterone
injection, and the LHRH peak occurred at 6–8 h (P <
0.01). The progesterone-induced LHRH increase lasted for at least
12 h after progesterone injection (P < 0.05),
when the experiment was terminated.
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, A and B), compared with the results
from the SC NPY group. Overall significance between AS NPY and SC NPY
was P < 0.01. Mean LHRH levels after progesterone
injection in the AS NPY group did not significantly fluctuate over time
(P > 0.1). Post-hoc analysis indicated that
mean LHRH levels at 4–12 h after progesterone injection in the AS NPY
group were significantly lower than corresponding LHRH means in the SC
NPY group (P < 0.05).
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; P < 0.01). Further,
the AS NPY-induced LHRH suppression tended to be more prominent in the
oil-treated group than that in the progesterone-treated group, although
statistical significance between the two treatment groups was not
attained (P = 0.08). Post-hoc analysis
indicated that mean LHRH levels after AS NPY in the oil-treated group
were significantly lower than those before AS NPY, except for the first
2 h (P < 0.05 to 0.001), but mean LHRH levels at
2–12 h after oil injection in the AS NPY group were not significantly
different from the corresponding LHRH means after progesterone
injection in the AS NPY group.
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). Post-hoc analysis
indicated that mean NPY levels at all time points after the initiation
of AS NPY infusion were lower than those before infusion
(P < 0.05–0.001).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results of this and previous (14) studies suggest that infusion of AS NPY into the S-ME consistently suppresses both NPY and LHRH in rhesus monkeys. The infused AS NPY was presumably taken into NPY neurons in the S-ME and retrogradely transported from neuroterminals to the cell body, where AS NPY interferes with NPY synthesis, resulting in a reduction of NPY release and, subsequently, LHRH release. A study from this laboratory (17) further indicates that the diameter of diffusion in the S-ME with push-pull perfusion is approximately 700 µm. Thus, the NPY neuroterminals affected by AS NPY infusion are in a relatively small area within the S-ME. Alterations of LH pulses (21), steroid-induced LH surges (22), and food intake behavior (23) after treatment with AS NPY in the hypothalamus have been reported in rats.
It has been extensively shown that NPY is a major modulator for pulsatile LHRH release. NPY infusion directly into the S-ME in vivo stimulates LHRH pulses (12, 13, 24, 25), whereas infusion of an antibody to NPY or an AS NPY into the ME suppresses LHRH pulses in rats, rabbits, and monkeys (13, 14, 21, 22, 26). Further, NPY stimulates LHRH release in ME fragments of rats (27, 28, 29). As NPY was directly infused into the S-ME in our studies (12, 24), where abundant LHRH neuroterminals, but only a few LHRH cell bodies, are present in primates (30, 31), and as the rat ME does not contain LHRH cell bodies (32), the stimulatory action of NPY appears to occur at the level of LHRH neuroterminals. In fact, a recent study using confocal microscopy with double labeled immunofluorescence has demonstrated colocalization of NPY Y1 receptors on LHRH neuroterminals (33). However, LHRH cell bodies may also be involved in the stimulatory action of NPY, as NPY results in LHRH release in GT-1 cells in a dose-responsive manner (34), which is strikingly similar to that observed in the monkey S-ME in vivo (12, 24). Cell bodies of LHRH neurons are innervated by NPY neurons in rats and sheep (35, 36), but not in monkeys (37). Although there are several reports indicating that ventricular infusion of NPY suppresses LHRH/LH release (26, 38, 39), NPY infused into the ventricular system may diffuse elsewhere into the brain, resulting in inhibitory effects on LHRH release through interneurons. Nonetheless, a direct inhibitory role of NPY in LHRH release cannot be excluded, because NPY mRNA, measured by ribonuclease protection assay, decreased at the onset of puberty in castrated male monkeys concomitant with an increase in LHRH mRNA (40, 41). NPY is inhibitory to hippocampal and suprachiasmatic neurons via presynaptic or postsynaptic receptors (42, 43, 44, 45), and studies in NPY knockout mice indicate that NPY is an endogenous antiepileptic agent through presynaptic inhibition of glutamate neurons (46).
NPY neurons are profoundly involved in the preovulatory LHRH surge or steroid-induced LHRH surge in rats (10, 11, 47). Expression of NPY mRNA, the tissue content of NPY in the ME, and NPY release from the ME increase before or coincident with the preovulatory and steroid-induced LH surges (48, 49, 50), whereas treatment with NPY antiserum or AS NPY suppresses the preovulatory or steroid-induced LH surge (22, 51). Further, the stimulatory effect of NPY on LHRH release is greatly enhanced by estrogen (24, 28, 52), suggesting the role of NPY in the positive feedback action of steroids. The previous studies in rats demonstrating that NPY neurons in the arcuate nucleus contain estrogen receptors (10, 53, 54) and that NPY neurons innervate LHRH cell bodies in the preoptic area (33, 35, 36), further suggest that NPY neurons are important for mediating the steroid action on the LHRH neuronal system. In the present study we found that inhibition of NPY synthesis by its antisense oligodeoxynucleotide blocks the progesterone-induced LHRH surge in estrogen-primed OVX monkeys.
The NPY neuronal system may not be the only contributor to the positive feedback effect of progesterone on LHRH release. The suppression of AS NPY on LHRH release in oil-treated monkeys tended to be more persistent than that in progesterone-treated monkeys, suggesting that progesterone may have stimulated other neuronal systems, such as norepinephrine and glutamate neurons, both of which reportedly contain estrogen receptors (53, 55). Further, preliminary observations in our laboratory suggest that both norepinephrine and glutamate levels increase during the progesterone-induced LHRH surge (Terasawa, E., and L. Luchansky, unpublished observation). The question of whether input from all of these neuronal systems is equally important for the positive feedback effects of ovarian steroids or whether one of them, perhaps NPY, plays a key role in the preovulatory LH surge remains to be answered.
In primates, it has been shown that an increase in serum estradiol precedes the preovulatory LH surge (1). However, a significant increase in progesterone has been observed several hours before the LH peak (2, 3), and administration of the antiprogestin RU486 interferes with the expected preovulatory gonadotropin surge and ovulation in women and monkeys (56, 57). These reports indicate that in primates, estrogen induces the preovulatory LH release, whereas progesterone facilitates or augments it. As the increase in circulating progesterone during the preovulatory phase originates from the primary follicle (58), full maturation of the Graafian follicle appears to give a signal to the hypothalamus to stimulate the release of LHRH, thereby setting the timing of the LH surge to synchronize with ovulation (7).
In primates, the positive feedback effects of progesterone differ from those of estrogen. The progesterone-induced LH surge (3, 5, 6, 7, 59) occurs with constant peak latency (6–9 h) and a relatively short duration (16–18 h), whereas both the estrogen-induced and spontaneous preovulatory LH surges (5, 60, 61) occur with a longer peak latency (36–48 h) and a longer duration (36–48 h). Although the progesterone-induced LH surge can be blocked with pentobarbital anesthesia (5), the estrogen-induced LH surge cannot (15). Although the difference with pentobarbital anesthesia could be explained by the fact that the positive feedback effects of estrogen do not require the presence of the hypothalamus (62), whereas the positive feedback effects of progesterone do (63), an increase in LHRH output during the estrogen-induced LH surge and preovulatory surge has been consistently reported in monkeys (64, 65, 66, 67). Interestingly, similar differential effects of progesterone and estrogen on the LH surge are observed in OVX guinea pigs (68), i.e. the progesterone-induced LH surge was completely blocked by pentobarbital anesthesia, whereas the estrogen-induced LH surge was not blocked by either multiple injections of pentobarbital or a single injection of phenobarbital, a long-acting barbiturate. Because the circadian-dependent mechanism of estrogen action remained in this case, estrogen’s action is not solely at the pituitary level (68); thus, there is a significant difference in the mechanisms of action of progesterone and estrogen (6). It is possible, therefore, that different neuronal substrates may be involved in estrogen and progesterone actions, and that our findings may not represent the entire mechanism of the preovulatory LH surge.
In summary, understanding the positive feedback mechanism of steroid hormones in primates is essential, as the preovulatory gonadotropin surge is the central event in reproduction. In the present study we have examined the role of NPY in the progesterone-induced LHRH surge. Our results demonstrate that suppression of NPY release impairs the progesterone-induced LHRH surge and further suggest that NPY appears to play a pivotal role in the preovulatory gonadotropin surge.
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Footnotes |
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2 Present address: Department of Pathology and Laboratory Medicine
Emory University, Atlanta, Georgia 30322.
Received November 2, 1999.
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