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Endocrinology Vol. 139, No. 2 696-702
Copyright © 1998 by The Endocrine Society

ARTICLES

Absence of Increased Neuropeptide Y Neuronal Activity before and during the Luteinizing Hormone (LH) Surge May Underlie the Attenuated Preovulatory LH Surge in Middle-Aged Rats1

Abhiram Sahu and Satya P. Kalra

Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; and the Department of Neuroscience, University of Florida College of Medicine (S.P.K.), Gainesville, Florida 32610

Address all correspondence and requests for reprints to: Abhiram Sahu, Ph.D., Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, S-329 Biomedical Science Tower, 3500 Terrace Street, Pittsburgh, Pennsylvania 15261.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A large body of evidence suggests that the neuroendocrine axis plays a major role in the reproductive aging of female rats. Since increased hypothalamic neuropeptide Y (NPY) neurosecretion is crucial in the preovulatory LH discharge in young rats, we tested the hypothesis that diminution in the preovulatory LH surge in middle-aged (MA) rats may be due to altered neurosecretory activity in NPYergic neurons. In Exp 1, we examined NPY levels in six microdissected hypothalamic nuclei, including median eminence (ME), arcuate nucleus (ARC), and medial preoptic area (MPOA), at 1000, 1200, 1400, 1600, 1800, 2000, or 2200 h on the day of proestrus in young (2.5- to 3-month old) and MA (7- to 9-month old) regularly cycling rats. At 1000 h, ME NPY levels in young rats were significantly lower than those in MA rats. In young rats, the ME NPY levels were significantly increased at 1400 h before the LH surge in the afternoon and thereafter decreased progressively during the interval of the LH surge. In MA rats, however, ME NPY levels decreased in the afternoon in association with an attenuated LH surge. In addition, in the ARC and MPOA, the other hypothalamic sites associated with induction of LH surge, NPY levels increased before and during the LH surge in young rats, no change in NPY levels in these nuclei was observed in association with the attenuated LH surge in MA rats. Also, NPY levels in the ARC and MPOA during the afternoon were significantly lower in MA compared with those in young animals. These results demonstrated the absence of an antecedent increase in NPY levels, specifically in the ME and ARC, during the afternoon of proestrus in MA animals.

In a second experiment, we evaluated whether the absence of dynamic changes in NPY levels in the ME and ARC in MA rats was due to altered hypothalamic NPY gene expression. Regularly cycling young (2.5- to 3-month-old) and MA (8- to 10-month-old) rats were killed at 1000, 1200, 1400, 1600, 1800, 2000, or 2200 h on the day of proestrus. The medial basal hypothalamus was processed for prepro-NPY messenger RNA (mRNA) measurement by ribonuclease protection assay. In young rats, prepro-NPY mRNA levels were significantly increased at 1200 h and remained elevated throughout the afternoon. In contrast, in MA rats prepro-NPY mRNA levels remained unchanged before and during the attenuated LH surge. These results clearly indicate that the augmentation in NPY neuronal activity before and during the LH surge seen in young rats fails to manifest itself in middle-aged rats. As hypothalamic NPY participates in the induction of LHRH surge, our results suggest that reduced LHRH and LH surges in MA rats may be due to diminution in NPY secretion in these animals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FEMALE reproductive aging in rodents involves a gradual transition from regular reproductive cycles to irregular cycles and ultimately an acyclic condition (1, 2). Although this transition from regular to irregular cyclicity may involve impairments at several levels in the hypothalamo-pituitary-ovarian axis, the defects at the hypothalamic level are thought to be the primary factor in the decline of reproduction in rat (3, 4). In middle-aged (MA), regularly cycling rats, the LH surge on proestrus or that induced by ovarian steroids in ovariectomized (ovx) animals is delayed and attenuated (5, 6). The cause(s) of this delayed and attenuated LH surge is not clearly understood. In this regard several laboratories have examined the status of LHRH neurons during aging (1, 2, 7). At present there is no consensus on changes in LHRH levels in the medial basal hypothalamus (MBH) or median eminence (ME) (7, 8, 9, 10, 11, 12). Interestingly though, before cessation of cyclicity, MA female rats fail to show the characteristic increases in hypothalamic LHRH content observed in young female rats on the afternoon of proestrus (7). In addition, in vivo LHRH release from the MBH as measured with the push-pull perfusion technique, decreased in association with the LH surge in MA vs. young steroid-primed ovx rats (10). Despite this evidence of attenuated LHRH release, no major loss of LHRH neurons was detected in MA cycling rats (13) or acyclic aging rats (8, 11), and the capacity of the hypothalamus to release LHRH in response to secretogogues was normal (9, 14, 15, 16). Thus, it is likely that neurochemical signals that normally provoke LHRH hypersecretion may fail to operate in an appropriate time-dependent fashion.

Evidence that hypothalamic neuropeptide Y (NPY), a 36-amino acid peptide of the pancreatic polypeptide family (17), is a crucial signal for the generation of the LHRH/LH surge (18) and that hypothalamic NPY levels and release decrease in association with decreases in serum LH and testosterone levels in aging male rats (19, 20) led us to hypothesize that alterations in hypothalamic NPY neuronal activity in MA rats may be responsible for the attenuated and delayed LH surge in MA rats. To test this hypothesis, we examined NPY levels in six microdissected hypothalamic nuclei and NPY gene expression in the hypothalamus at different times on the day of proestrus in young and MA regularly cycling rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Sprague-Dawley CD female rats of two different age groups (young, 2–3 months old; MA, 7–10 months old) were obtained from Charles River Laboratories (Wilmington, MA). Rats were maintained on a daily 14-h light, 10-h dark cycle (lights on, 0500–1900 h) at 23 C, with food (rat chow) and water available ad libitum.

Exp 1: NPY levels in microdissected hypothalamic nuclei in young and MA rats on proestrus
Young and MA rats showing regular 4-day estrous cycles were killed at 1000, 1200, 1400, 1600, 1800, 2000, or 2200 h on the day of proestrus. Brains were quickly dissected out, frozen on dry ice, and stored at -80 C until processing. Trunk blood was collected, and plasma was stored at -20 C until LH determination by RIA.

Three hundred-micron thick brain coronal sections were made in cryocut at -10 C. Six hypothalamic nuclei, viz. medial preoptic area (MPOA), suprachiasmatic nuclei, dorsomedial nucleus (DMN), ventromedial nucleus (VMN), arcuate nucleus (ARC), and ME, were microdissected as described previously (19, 20, 21). NPY levels in the microdissected sites were measured by RIA (21). There were seven to nine rats per group at each time point.

Exp 2: prepro-NPY messenger RNA (mRNA) levels in the MBH in young and MA rats on proestrus
This experiment is similar to that described in Exp 1, except that in this study the MBH were dissected out from the brain, frozen on dry ice, and stored at -80 C until processed for RNA extraction as described below. Trunk blood was also collected, and plasma was stored at -20 C for LH determination by RIA. There were six to eight rats per group at each time point.

NPY and LH RIAs
NPY levels in the microdissected tissue were determined by RIA as described previously (21). Porcine NPY (Peninsula Laboratories, Belmont, CA) was used as the reference standard. Iodinated NPY was purchased from Amersham Corp. (Arlington Heights, IL). NPY antibody R-31, raised against porcine NPY, was provided by Dr. Harold Spies (Oregon Regional Primate Research Center, Beaverton, OR). All samples were analyzed in the same assay, with an assay sensitivity of 1.95–3.90 pg/tube and an intraassay coefficient of variation of 8.4%. NPY levels were expressed as picograms per µg protein. Serum LH levels were determined at the RIA Core Facility in the Center for Research in Reproductive Physiology, Department of Cell Biology and Physiology, University of Pittsburgh, using reagents provided by Dr. G. W. Niswender and the National Hormone and Pituitary Program, NIDDK. The levels were expressed in terms of LH RP-3.

Solution hybridization/ribonuclease (RNase) protection assay for prepro-NPY mRNA determination
RNA preparation. Total RNA was isolated from the MBH of rats, using RNAzol (RNA STAT-60) followed by precipitation with isopropanol and ethanol washes according to the manufacturer’s instructions (Tel-Test, Friendswood, TX). The integrity of the RNA was checked by visualization of the ethidium bromide-stained 28S and 18S ribosomal RNA bands, and quantitation was performed by measuring absorbance at 260 nm.

Riboprobe synthesis. The antisense NPY complementary RNA was obtained by in vitro transcription of a 511-bp fragment of a rat NPY complementary DNA (cDNA) cloned in pBluscript plasmid (22) (provided by Dr. S. Sabol, NIMH, Bethesda, MD). This plasmid was linearized with PvuII and transcribed with T3 RNA polymerase in the presence of [{alpha}-32P]UTP using a transcription kit (Ambion, Austin, TX). An 117-bp fragment of a rat cyclophilin cDNA (23) (provided by Dr. J. L. Roberts, Mount Sinai School of Medicine, New York, NY) was linearized with EcoRI and transcribed with T7 RNA polymerase in the presence of [{alpha}-32P]UTP to make an antisense probe. As cyclophilin is constitutively expressed in various tissues, including brain, quantitation of cyclophilin mRNA levels was used to normalize the values obtained for prepro-NPY mRNA.

RNase protection assay
RNase protection assays were performed as described previously (24, 25) with some modifications. Briefly, 3 µg rat MBH RNA, 32P-labeled complementary RNA probes (NPY and cyclophilin), and 12 µg yeast transfer RNA (Boehringer Mannheim, Indianapolis, IN) were allowed to hybridize in solution at 45 C overnight, followed by combined RNase A and RNase T1 digestion of nonhybridized probe at 32 C for 1 h. Stable hybrids were phenol/chloroform extracted, ethanol precipitated, and then denatured and separated on 6% polyacrylamide-8 M urea gels. The dried gel was exposed in a Bio-Rad CS Molecular Imaging Screen for 2–3 h, and the image of each gel was acquired using a GS-525 Molecular Imager (Bio-Rad Laboratories, Hercules, CA). The volume analysis of each band obtained from RNase protection assay was performed by Molecular Analyst Software (Bio-Rad).

The values for prepro-NPY mRNA levels were first normalized with cyclophilin mRNA and then presented in relation to 1000 h values.

Statistical analysis
Statistical significance of the data was first determined by two-way ANOVA to evaluate the significant effects of groups (young vs. MA), time, and group vs. time interactions, followed by Student-Newman-Keuls multiple range test post-hoc analysis to compare significant differences between the groups at matched time points. The data were also analyzed by one-way ANOVA with post-hoc testing using the Student-Newman-Keuls multiple range test to compare the levels within a treatment group across time. These statistical tests were conducted using the GB-STAT statistical program for Macintosh (Dynamic Microsystems, Silver Spring, MD). Comparisons with P < 0.05 were considered significantly different.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exp 1
The profile of serum LH concentrations and ME NPY levels in young and MA rats on proestrus are presented in Fig. 1Go. In young rats, LH levels were elevated at 1600 h, remained elevated at 1800 h, and then decreased at 2000 h to the range seen at 1000 h. On the other hand, in MA rats, LH rise was seen only at 1800 h. LH levels in these rats were significantly lower at 1600 and 1800 h compared with those in young rats (P < 0.05), indicating an attenuated LH surge.



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  		Figure 1. Serum LH concentrations and ME NPY levels in young and MA rats on proestrus. Values are the mean ± SEM (n = 7–9 animals/time point·age group). *, P < 0.05 vs. 1000 h values; a, P < 0.05 vs. MA rats at this time point.

 
In young rats, ME NPY levels showed a significant time-related change in association with the LH surge on proestrus. Compared with those at 1000 h, ME NPY levels were significantly increased at 1400 h when LH levels were still basal. Thereafter, in association with LH hypersecretion, NPY levels progressively decreased to reach a nadir at 1800 h. In MA rats, in contrast, ME NPY levels decreased at 1200 and 1600–2200 h compared with those at 1000 h. ME NPY levels in MA rats were significantly higher at 1000 h and lower at 1400 and 1600 h compared with those in young rats (Fig. 1Go).

NPY levels in the ARC and MPOA are presented in Fig. 2Go. Compared with those at 1000 h, ARC NPY levels in young rats were significantly increased at 1200, 1600, and 1800 h. There was no change in the morning and afternoon levels of ARC NPY in MA rats. NPY levels were also significantly lower at 1200, 1600, and 1800 h in MA rats compared with those in young rats. In the MPOA of young rats, NPY levels were significantly increased at 1600 h compared with those at 1000 h. There was no afternoon increase in MPOA NPY levels in MA rats. In both age groups, MPOA NPY levels were significantly decreased at 2000 and 2200 h compared with those at 1000 h. VMN NPY levels in young rats remained stable during the afternoon, but showed a decrease (P < 0.05) only in MA rats (Fig 3Go). In the DMN, compared with morning levels, a significant decrease in afternoon levels at 2000 and 2200 h was seen in young and MA rats (Fig. 3Go).



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  		Figure 2. NPY levels in the ARC and MPOA in young and MA rats on proestrus. Values are the mean ± SEM (n = 7–9 animals/time point·age group). *, P < 0.05 vs. 1000 h values; a, P < 0.05 vs. MA rats at this time point.

 


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  		Figure 3. NPY levels in the VMN and DMN in young and MA rats on proestrus. Values are the mean ± SEM (n = 7–9 animals/time point·age group). *, P < 0.05 vs. 1000 h values; a, P < 0.05 vs. middle-aged rats at this time point.

 
Exp 2
In this experiment serum LH concentrations in both young and MA rats were significantly increased at 1600 h, with a peak level at 1800 h in young rats and at 2000 h in MA rats (Fig. 4Go). However, the serum LH concentrations in MA rats were significantly lower at 1600 and 1800 h compared with those in young rats (P < 0.05). In Fig. 4Go, hypothalamic prepro-NPY mRNA levels are presented relative to 1000 h values for each age group. Compared with 1000 h, prepro-NPY mRNA levels in young rats were significantly increased at 1200 h; thereafter, prepro-NPY mRNA levels remained elevated throughout the afternoon despite decreases in LH levels between 2000–2200 h. In MA rats, there was no significant change in prepro-NPY mRNA levels between morning and afternoon (Fig. 4Go). Although it appeared from the data presented in Fig. 4Go that relative changes in NPY gene expression in MA rats were lower between 1200–2200 h compared with those in young rats, the mRNA levels in the two groups were not comparable because the levels were presented as the fold change relative to 1000 h values.



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  		Figure 4. Serum LH levels (lower panel) and hypothalamic prepro-NPY mRNA levels in young and MA rats on proestrus. Prepro-NPY mRNA levels are presented relative to the 1000 h values of the individual age group. Values are the mean ± SEM (n = 6–8 animals/time point·age group). *, P < 0.05 vs. 1000 h values.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results of the present study suggest an age-related alteration in hypothalamic NPY neuronal activity in regularly cycling middle-aged rats on the day of proestrus. Specifically, enhanced hypothalamic NPY neuronal activity, as assessed by an increase in NPY levels in the hypothalamic nuclei, particularly in the ME and ARC, and an increase in MBH NPY gene expression before and during the LH surge in young rats were not evident in MA cycling rats.

Hypothalamic NPY plays an important role in generation of the LH surge in rat (18, 26). As seen in the current study, previous investigations demonstrated that NPY levels in the ME increase before the LH surge on proestrous (27) and that induced by ovarian steroids in ovx rats (28). NPY release in the ME-ARC region also increases before the LH surge (29). Similarly, the NPY concentration in hypophyseal portal plasma is significantly higher on the afternoon of proestrus (30). Furthermore, in association with increases in NPY levels and release, hypothalamic NPY gene expression increases before the LH surge at proestrus (25) and that induced by ovarian steroids in ovx rats (24). These findings, showing an overall increase in hypothalamic NPY neuronal activity before and during the LH surge together with evidence that central NPY antibody infusion (31) or NPY antisense oligodeoxynucleotide administration (32) abolished the LH surge, clearly suggest that enhanced NPY synthesis and release, and thereby NPY neuronal activation, are obligatory for activation of the LHRH-LH axis on the afternoon of proestrus. Thus, it is possible that the alteration in NPY neurosecretion observed in the present study in MA rats may be responsible for the attenuated LH surge and may lead to abolition of LH surges in old rats.

Furthermore, several lines of evidence showing that 1) NPY stimulates LHRH release in vivo (33, 34) and in vitro (35); 2) NPY neurons are synaptically linked with LHRH neurons (36); 3) NPY injection into the preoptic area induced LHRH gene expression (37); and 4) NPY potentiated the action of LHRH at the level of the pituitary (38, 39, 40, 41, 42) by increasing the number of LHRH-binding sites (43) strongly suggest that disruption in any of these sequelae in the NPYergic systems would adversely impact the generation of LHRH and LH surges. Thus, it is likely that the lack of NPY stimulation during the afternoon may be responsible for the reduced number of activated LHRH neurons (13, 44) and the reduced LHRH release (10) reported in MA regularly cycling rats.

Although we did not evaluate hypothalamic NPY release, based on previous studies demonstrating a good correlation among hypothalamic NPY gene expression, ME NPY levels, and NPY release (24, 25, 27, 29, 45, 46), we are tempted to speculate that there was a decreased NPY release in MA rats. In agreement with this inference are the reports showing decreased NPY release in the MBH during aging in male rats (19). There is a remote possibility that NPY release in MA rats is, indeed, increased on proestrus, as seen in young rats (29), and one can suggest that an altered postsynaptic action of NPY at the hypothalamic and/or pituitary levels may underlie the diminution in the LH surge.

Besides the ME and ARC, NPY levels were also examined in the MPOA, VMN, and DMN in young and MA rats. An increase in NPY levels in the MPOA in association with the LH surge was evident only in young rats. As a synaptic link between NPY neurons and LHRH cell bodies exists in this region (36), and NPY injection into this area increases LHRH gene expression (37), the lack of an increase in NPY levels in the MPOA may also contribute to an alteration in LHRH neuronal activity in MA rats. As expected, NPY levels in the VMN and DMN, the sites that play little role in the preovulatory LH discharge (47, 48), remained unchanged before and during the LH surge in young and MA rats. Overall, there was a decrease in NPY levels in most of the nuclei at 2000–2200 h in both young and MA rats. The significance of this decrease is unclear at present and is most likely not related to LH secretion, but could be related to other functions of NPY, including its role in feeding behavior (49).

The factors responsible for the age-related alteration in NPY neurosecretion are unknown. However, NPY neuronal activity is known to be regulated by various central and peripheral signals, including metabolic factors (49, 50) and steroids of adrenal (51, 52, 53) and gonadal origin (18, 26). Among these, the role of gonadal steroids is particularly important in the context of reproductive senescence. NPY neurons contain steroid receptors (54), and gonadal steroids modulate hypothalamic NPY gene expression (24, 25, 46, 55); NPY levels in those hypothalamic nuclei, including ME and ARC (28, 56, 57), that are known to be involved in the LH surge and reproduction; and NPY release in vivo (46) and in vitro (26, 56, 57, 58). Stimulation of LHRH and LH release by NPY itself is dependent on gonadal steroids (26). Furthermore, in male rats, aging alters the response of NPY to gonadal steroids in the hypothalamic nuclei (20). Changes in steroidal concentration, particularly of progesterone has been demonstrated on the afternoon of proestrus in MA rats (59, 60). In young rats, increased progesterone secretion (61) has been shown to advance and amplify LH surges (62), possibly by recruiting activated LHRH neurons (63, 64) and enhancing pituitary sensitivity to LHRH (65). Progesterone has been shown to increase both LHRH and NPY levels in the ME in estrogen-primed ovx rats (28). Furthermore, progesterone enhanced NPY gene expression in the afternoon in estrogen-primed ovx rats (24). Progesterone was also shown to be necessary for potentiating with LHRH-induced LH release at the level of the pituitary by NPY (42). Thus, altered steroidal milieu and/or a differential response of NPY neuronal system to gonadal steroids may result in altered NPY neurosecretion in MA rats.

Several other hypothalamic neurotransmitter systems have been shown to exhibit age-related changes in the rat (2, 7). Among these, catecholaminergic neurons have been studied extensively (7). It is interesting to note that changes in ME NPY levels in MA rats observed in this study are very similar to those seen in norepinephrine (NE) turnover in the ME (66). NPY and NE are colocalized in a number of neuronal subpopulations in the brain stem (67), and these cells project into the areas containing the LHRH network (67, 68). Direct contact between NPY and NE in the ARC has been reported (69, 70). NPY and NE have similar effects on the LHRH-LH axis (26, 71), and the two neurotransmitters act synergistically to release LH in vivo (72). Thus, altered neurosecretion of both NPY and NE in MA rats could potentially affect synergistic as well as individual effects of each on the LHRH-LH axis, resulting in reduced LHRH and LH surges.

It is well established that normal reproductive cycles in females are dependent on an intricate balance in pulsatile, diurnal, and cyclic release of LHRH, gonadotropins, and ovarian steroids (73). Various hypothalamic neurotransmitters have been advocated to play a critical role in the maintenance of these rhythms (18). Several lines of evidence suggest that the diurnal rhythm in various members of the neural circuitry that control the LHRH/LH surges is abolished in MA and old female rats (7). For example, the diurnal rhythm in NE turnover, {alpha}1-adrenergic receptor densities, and serotonin turnover in discrete hypothalamic nuclei seen in young rats is abolished in MA rats (7). In our study, a loss of diurnal rhythm in NPY levels in discrete hypothalamic nuclei and in NPY gene expression was also observed in MA rats. Previous studies have shown that NPY secretion in vivo is pulsatile (46, 74), and endogenous NPY is involved in the pulsatile release of LHRH and LH (74, 75, 76). The loss of diurnal rhythm in NPY neuronal activity in MA rats may impair the normal pulse-generating circuitry responsible for the the initiation and maintenance of LHRH and LH surges on the afternoon of proestrus, thus resulting in an attenuated LH surge in these rats. In view of the recent demonstration that continuous infusion of NPY may impair the reproductive axis (77), it will be important to demonstrate whether NPY neuronal activity remains relatively constant in MA rats. It is, however, also possible that NPY neuronal activity in MA rats may be enhanced earlier than 1000 h. A new study will address these questions.

In summary, these results show that the normally occurring pattern of increased NPY neurosecretion is absent on the afternoon of proestrus in regularly cycling MA rats. As NPY is involved in the initiation and sustenance of the LH surge in young rats, we suggest that altered NPY neurosecretion may underlie the delayed and attenuated LH surge in MA rats.


   Acknowledgments
 
Thanks are due to Drs. S. Sabol and J. L. Roberts for supplying cDNA for NPY and cyclophilin mRNA, respectively, and to Dr. H. G. Spies for supplying NPY antibody.


   Footnotes
 
1 Presented at the 77th Annual Meeting of The Endocrine Society, June 14–17, 1995, Washington DC (Abstract P3–680), and at the 79th Annual Meeting of The Endocrine Society, June 11–14, 1997, Minneapolis, MN (Abstract P2–381). This work was supported by NIH Grant AG-10868 (to A.S.). Back

Received August 12, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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