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BOR - Papers in Press, published online ahead of print March 3, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.025858
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BIOLOGY OF REPRODUCTION 71, 266–272 (2004)
DOI: 10.1095/biolreprod.103.025858
© 2004 by the Society for the Study of Reproduction, Inc.

Neuroendocrinology

Leptin-Receptor Gene Transfer into the Arcuate Nucleus of Female Fatty Zucker Rats Using Recombinant Adeno-Associated Viral Vectors Stimulates the Hypothalamo-Pituitary-Gonadal Axis1

Erin Keen-Rhinehart3,4, Satya P. Kalra4, and Pushpa S. Kalra2,3

Departments of Physiology and Functional Genomics3 Neuroscience,4 McKnight Brain Institute, University of Florida, Gainesville, Florida 32601


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fatty fa/fa Zucker rats with a missense mutation in the leptin receptor (OB-R) are obese and infertile with prolonged estrous cycles. To determine whether their reproductive deficits could be corrected by OB-R installation, we employed viral vectors to introduce the OB-R gene into either the arcuate nucleus (ARC) or the paraventricular nucleus (PVN) of the hypothalamus, sites of OB-R expression in wild-type rats. Recombinant adeno-associated viral (rAAV) vectors encoding the human leptin-receptor gene (rAAV-OB-Rb) were microinjected intraparenchymally to produce doxycycline-regulatable OB-R gene expression. Expression of the OB-R gene in the ARC and PVN was verified using reverse transcription-polymerase chain reaction. Expression of OB-R in the ARC, but not in the PVN, resulted in normalization of estrous cycle length, increased ovarian follicular development, and decreased serum progesterone levels. Compared to saline-injected rats, hypothalamic expression of neuropeptide Y (NPY) and pro-opiomelanocortin were decreased in ARC rAAV-OB-Rb-injected rats. Parallel decreases were noted in NPY and ß-endorphin (ß-END) concentrations in the hypothalamus, whereas luteinizing hormone-releasing hormone (LHRH) levels increased. These studies showed that rAAV vectors can be successfully used to install functional OB-R in the hypothalamus for extended periods. The resultant stimulation of the hypothalamo-pituitary-gonadal (HPG) axis in ARC-injected rats was probably brought about by the observed decreases in NPY and ß-END, which inhibit hypothalamic LHRH. Because these changes were seen in ARC-injected, but not in PVN-injected, rats, the results suggest that the ARC may be the primary site where leptin acts to regulate the HPG axis.

gonadotropin-releasing hormone, hypothalamic hormones, leptin, leptin receptor, neuropeptide Y


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Secreted by white adipocytes, leptin is best known for its role in maintaining energy balance by decreasing food intake and increasing energy expenditure [1–4]. Several reports suggest that leptin also has a role in normal reproductive function. Leptin acts as a permissive signal for the onset of puberty, reverses fasting-induced anestrus and decreases LH in rodents, and counteracts the negative effects of insulin-like growth factor-I on follicular development [5– 10]. Leptin also has been reported to stimulate the release of LH-releasing hormone (LHRH) from the hypothalamus as well as FSH and LH from the pituitary [7, 8, 11–13]. Conversely, animals with mutations in either the leptin or the leptin-receptor (OB-R) gene have nonfunctional reproductive axes [14, 15]. The homozygous (fa/fa) fatty Zucker rat has a mutated OB-R gene that causes several perturbations in the hypothalamo-pituitary-gonadal (HPG) axis [16]. These rats are infertile, with abnormal estrous cycles, decreased plasma sex steroids, hyporesponsiveness to sex-steroid hormones, and attenuated sexual behavior and LH surge [14, 17–20].

The OB-R has been localized to several hypothalamic nuclei, including the ventromedial hypothalamus, the medial preoptic area (MPOA), the paraventricular nucleus (PVN), and the arcuate nucleus (ARC) [21–26]. The OB-R mRNA colocalizes with neuropeptide Y (NPY) as well as pro-opiomelanocortin (POMC) mRNA-expressing neurons in the ARC, but not with LHRH in the MPOA [10, 27–30]. Leptin action in the central nervous system inhibits the activity of both NPY and POMC neurons [31–33].

In a normal gonadal steroid environment, NPY stimulates the release of LHRH [34–37]. Production of NPY in the ARC regulates LHRH secretion by action on LHRH perikarya and dendrites in the MPOA and at the level of LHRH nerve terminals in the median eminence [38]. However, a continuous excess of NPY inhibits LHRH secretion, especially under low levels of sex steroids, as present in the fatty Zucker rat [15, 35, 37–39]. In wild-type rats, leptin inhibits NPY gene expression and stimulates LHRH and LH release [7, 8, 11–13]; however, in fatty Zucker rats, NPY expression in the ARC is upregulated as a consequence of defective leptin signaling [40–44]. Additionally, NPY stimulates the endogenous opioid peptide ß-endorphin (ß-END), which has a tonic inhibitory influence on both LHRH and LH release [39, 45, 46]. Thus, by restoring normal leptin signaling in fa/fa rats, it might be possible to correct the alterations in neuropeptides contributing to the reproductive dysfunction in the female fatty Zucker rat.

Recombinant adeno-associated viral (rAAV) vectors are derived from a single-stranded DNA parvovirus that integrates into the host's genome with little, if any, immunogenicity. It has good transduction efficiency in nondividing cells and selectively infects neurons in the brain for long-term, stable transgene expression [47–51]. To our knowledge, the only drawback in using rAAV is its relatively small insert size (4.7 kilobases). We have used rAAV successfully to transfer the leptin gene into the rodent brain [52, 53]. In the present study, we used rAAV to transfer the OB-R gene into the hypothalamus of female fatty Zucker rats, and we determined its effects on the HPG axis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Viral Vectors

The two vectors used in the present study are shown in Figure 1. One rAAV transfer vector carried the transgene under the control of a tetracycline-responsive promoter (tetO). The second vector is a combination of a transcriptional activator and repressor driven by the strong housekeeping promoter, chicken ß-actin promoter (CBA), with a cytomegalovirus enhancer to provide control over expression of the transgene using a tetracycline-like compound, doxycycline (dox). To construct the receptor vector, designated rAAV-OB-Rb, the human long-form OB-R cDNA, cloned from pCDNA3.1-OB-Rb (a gift from Dr. H. Hsiung, Lilly Research Laboratories, Indianapolis, IN) was subcloned into pTR-UF13 plasmid vector downstream of tetO. The promoter vector, designated rAAV-rtTA/ rTS, was constructed to encode two chimeric genes, a reverse tet-regulatable trans-activator (rtTA), which activates gene transcription in the presence of dox, and a tet-controlled trans-repressor (tTS), which silences gene expression in the absence of dox. Expression of both genes is linked by an internal ribosomal entry site into one di-cistronic cassette with a CBA, which is active in all cell types, including neurons. In the absence of dox, the tTS protein product forms a homodimer, binds to tetO, and prevents unregulated gene transcription. However, in the presence of dox, the rtTA protein product undergoes a conformational change, forms a homodimer, and binds to tetO to initiate gene transcription of OB-R. Both vectors, rAAV-OB-Rb and rAAV-rtTA/tTS, were packaged and titered at the University of Florida Powell Gene Therapy Center as previously described [52, 53].



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  		FIG. 1. Diagrammatic representation of rAAV vectors. A) The rAAV-OB-Rb vector encoding the human leptin-receptor gene with a polyadenylated tail (Poly-A) sequence driven by a tetracycline-responsive promoter and flanked by inverted terminal repeats (TR). B) The rAAV-rtTA/rTS vector encoding the regulatory sequences, rtTA and rTS, separated by an internal ribosomal entry site (IRES) sequence, with a Poly-A sequence flanked by terminal repeats and driven by the chicken ß-actin promoter with a cytomegalovirus (CMV) enhancer

Animals

All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Florida. Female fatty (fa/fa) Zucker rats (age, 3–4 wk) were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and housed in individual cages in air-conditioned rooms (22°C) with lights-on from 0500 to 1900 h. Rat chow (Ralston Purina Co., St. Louis, MO) and tap water were available ad libitum. Under ketamine/ xylazine anesthesia (ketamine, 100 mg/kg body weight [BW]; xylazine, 15 mg/kg BW), rats were fitted into a stereotaxic apparatus. With the aid of a Kopf microinjector, four rats were microinjected with 1 µl of saline, and 12 rats were microinjected with 0.5 µl of rAAV-OB-Rb (2.4 x 1010 infectious particles/ml) plus 0.5 µl of rAAV-rTS/rtTA (4.5 x 109 particles/ ml) bilaterally into the ARC (2.7 mm posterior to bregma, 0.2 mm lateral of the sagittal sinus, and 9.8 mm ventral to the dura mater). Seven rats were microinjected with 0.5 µl of rAAV-OB-Rb (2.4 x 1010 infectious particles/ml) plus 0.5 µl of rAAV-rTS/rtTA (4.5 x 109 particles/ml) bilaterally into the PVN (1.8 mm posterior to bregma, 0.4 mm lateral of the sagittal sinus, and 7.5 mm ventral to the dura mater) [54]. Rats were allowed 7–10 days to recover from surgery. Half the rats microinjected with rAAV-OB-Rb in the ARC and all the rats injected with saline or rAAV-OB-Rb in the PVN were provided dox (Clontech, Palo Alto, CA) at 250 mg/L in 0.1% saccharin drinking water. The other half of the group microinjected with rAAV-OB-Rb in the ARC were given drinking water containing 0.1% saccharin during the 60-day experiment. Estrous cycles were monitored by daily examination of vaginal cytology.

Approximately 60 days after the start of the dox treatment, rats in diestrus were killed in the morning by decapitation. The brains were rapidly removed, and the hypothalami were excised. The hypothalamic fragment extended from the optic chiasm rostrally to the mammillary bodies caudally and to the lateral sulci. The hypothalamic fragment was divided longitudinally into halves. One half was homogenized in 0.1 N hydrochloric acid and stored at –20°C for peptide analyses. The other half was snap-frozen on dry ice for mRNA quantitation. In addition, ovaries were removed at the time of death and then weighed and processed for histological evaluation of the numbers of corpora lutea, small follicles (diameter, <300 µm), and large follicles (diameter, >300 µm). Serum from trunk blood was collected and stored at –20°C until analysis of estradiol and progesterone.

Reverse Transcription-Polymerase Chain Reaction for OB-R mRNA Expression

Total hypothalamic RNA was extracted and DNase-treated using the RNeasy Kit (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. Primers for the human OB-R were designed using the sequence of the plasmid pCDNA3.1+hOBR (Lilly Research Laboratories) to generate a 580-base pair product. The OB-R primers were as follows: sense, 5' ATG AGG ACG AAA GCC AGA G; antisense, 5' TGT GAG CAA CTG TCC TGG. The gene was amplified using reagents from Perkin-Elmer Biosystems (Foster City, CA) from a single reverse-transcription (RT) reaction with the following parameters: denaturation, 95°C for 1 min; annealing, 65°C for 1 min; extension, 72°C for 1 min; 38 cycles. All samples also underwent polymerase chain reaction (PCR) for an internal cyclophillin control and were quantified as previously described [50, 51].

Hypothalamic Peptide Estimation

The acidified hypothalamic samples were neutralized with 0.1 N NaOH, and protein concentration was determined using a Bradford protein assay (Bio-Rad, Richmond, CA). Hypothalamic neuropeptide levels were expressed as pg/µg protein.

Levels of LHRH in the hypothalamus were measured by RIA according to the method previously described by Bonavera et al. [55] using synthetic LHRH (Peninsula Laboratories, Belmont, CA) as both reference standard and iodinated hormone. The LHRH antiserum (EL-14) was obtained from Drs. W.E. Ellinwood and M. Kelly (Oregon Regional Primate Center, Portland, OR). All 50-µl samples were processed in the same assay, and the sensitivity of the assay was 0.2 pg/tube.

The concentration of NPY in the hypothalamus was determined by RIA as described previously [56] using porcine NPY as the reference standard (Peninsula Laboratories). Antiserum to NPY (RA31) was originally supplied by Dr. William Crowley (University of Tennessee, Memphis, TN). Iodinated NPY was purchased from Amersham Corporation (Arlington Heights, IL). All 50-µl samples were processed in the same assay, and the sensitivity of the assay was 1.95 pg/tube.

Levels of ß-END were measured by the RIA procedure described by Koenig et al. [57]. The antiserum (#BN-3) was obtained from Dr. L. Krulich (University of Texas, Dallas, TX). The ß-END (Peninsula Laboratories) was used as both standard and iodinated hormone. All 50-µl samples were processed in the same assay, and the sensitivity of the assay was 2.5 pg/tube.

Serum Steroid Measurements

Serum estradiol was measured in samples extracted with diethyl ether using the third-generation estradiol RIA kit from Diagnostic Systems Laboratories (Webster, TX) according to the manufacturer's instructions. The sensitivity of the assay was 0.6 pg/ml, and all samples were processed in the same assay.

Serum progesterone was measured in nonextracted samples using a kit from Diagnostic Systems Laboratories according to the manufacturer's instructions. The sensitivity of the assay was 0.10 ng/ml, and all samples were processed in the same assay.

Quantitative PCR

Total hypothalamic RNA was extracted and DNase-treated using RNeasy Kit (Qiagen). One microgram of RNA was reverse-transcribed using random hexamer primer Taqman reagents (PE Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Synthesized cDNA corresponding to 50 ng of total RNA was used for real-time quantitative PCR.

Gene-specific primers and probes were designed for POMC and NPY using Primer Express Software (PE Applied Biosystems) according to the manufacturer's guidelines. All primers and Taqman probes were purchased from PE Applied Biosystems. Taqman PCR assays for both genes were performed on cDNA samples in 96-well plates on an ABI Prism 7000 Sequence Detection System (PE Applied Biosystems). The 18S assays, designed and purchased through PE Applied Biosystems, were run in parallel to each different sample. Each 25-µl reaction involved 12.5 µl of Taqman 2x Master Mix (PE Applied Biosystems), 1.0 µl of cDNA, 2.5 µl of sense primer (8 µM), 2.5 µl of antisense primer (8 µM), 0.3 µl of probe (100 nM), and 6.2 µl of PCR-grade water. The PCR parameters were 95°C for 10 min followed by 40 cycles of 60°C for 1 min and 95° C for 15 sec. The gene-specific primers were as follows:

POMC: sense, 5' GGC CTT TCC CCT AGA GTT CAA; antisense, 5' ACG TGC TCC AAG CCA TCA G
POMC probe: 6FAMAGC TGG AAG GCG AGCMGBNFQ
NPY: sense, 5' AAC CAG TCT GCC TGT CCC AC; antisense, 5' CAA GGG AAA TGG GTC GGA
NPY probe: 6FAMATG CAT GCC ACC AGG CTG GTAMRA

Statistical Analysis

All values are expressed as the mean ± SEM. Statistical analyses were performed by one-way ANOVA followed by the Tukey post-hoc test using GraphPad Prism software (GraphPad Software, San Diego, CA). The level of significance was set at P < 0.05 for all analyses. For the real-time quantitative PCR data, statistics were done on values normalized to 18S before the data were transformed into percentage differences.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of OB-R mRNA in hypothalami was assessed by RT-PCR as shown in Figure 2. As expected, no OB-R mRNA was detectable in control rats injected with saline. However, significant expression of human OB-R mRNA was detected in rats microinjected with rAAV-OB-Rb in the ARC or PVN and provided dox in the drinking water. Surprisingly, we also detected some expression of OB-R mRNA in rAAV-OB-Rb-injected rats that were not given dox. This expression, however, was only a fraction of that seen in rats given both vectors along with dox (Fig. 2A).



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  		FIG. 2. Human leptin-receptor mRNA expression in hypothalami of rats microinjected in the ARC or PVN. A) Quantitation of human OB-R mRNA relative to cyclophilin. B) Representative mRNA bands from RT-PCR products run on a 1% agarose gel showing both cyclophilin and leptin-receptor expression. In this and subsequent figures, groups that share the same superscript are not significantly different. CYC, Cyclophillin; hOB-R, human leptin-receptor gene; SAL, saline

The effects of rAAV-OB-Rb on reproductive function are shown in Table 1. In control, saline-injected rats, estrous cycle length averaged 7.8 ± 0.8 days, with several prolonged periods of diestrus (predominantly leukocytic vaginal cytology) ranging from 8 to 20 days. This was essentially unchanged in rats given either of the vectors in the ARC without dox (7.4 ± 0.4 days) or when the vectors were microinjected in the PVN (6.6 ± 0.4 days). However, rats injected with rAAV-OB-Rb in the ARC and given dox showed significantly shorter estrous cycle lengths (4.7 ± 0.4 days, P < 0.05) as well as increased incidence of vaginal estrus (cornified cells) exhibited over the 60-day period of the experiment. Furthermore, ovarian weights were significantly higher in ARC-injected rats. However, uterine weights were significantly reduced in these rats as well as in those not given dox. The BW was measured at the beginning and end of the study, and only rats given rAAV-OB-Rb with dox in the ARC had significantly reduced BW compared to the saline group (388 ± 12 vs. 461 ± 28 g, P < 0.05).


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  		TABLE 1. Effects of rAAV-OB-Rb treatment on reproductive function.a

Representative hematoxylin and eosin-stained ovarian sections of rats given saline or the vectors with dox are shown in Figure 3, A and B. The quantified effect of rAAV-OB-Rb on follicular development and on the number of corpora lutea is listed in Table 1. The ovaries of saline-injected rats and of vector-treated rats not given dox contained mostly corpora lutea and a few small follicles. Large antral follicles were rarely seen. In contrast, the ovaries of rats treated with rAAV-OB-Rb plus dox contained both normal corpora lutea and follicles at various stages of development, including large antral follicles. Expression of rAAV-OB-Rb in the ARC significantly decreased the number of corpora lutea (P < 0.05) and increased the number of both large and small follicles per ovary. The number of small follicles (diameter, <300 µm) was doubled, and several large antral follicles (diameters, >300 µm) were observed. Some evidence of ovarian follicular development was also seen in ARC-injected rats not provided dox.



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  		FIG. 3. Effects of rAAV-OB-Rb treatment on ovarian histology. A and B) Representative hematoxylin and eosin-stained ovarian sections (thickness, 40 µm; magnification 20x) from (A) saline treated and (B) rAAV-OB-Rb microinjected in rats treated with ARC plus dox. C–E) Quantitation of (C) corpora lutea, (D) small follicles (diameter, <300 µm), and (E) large follicles (diameter, >300 µm). CL, Corpora lutea

Serum estradiol and progesterone levels are also shown in Table 1. Estradiol levels were relatively low in fatty Zucker rats, and no effect of viral vector treatment on serum estradiol levels measured at diestrus was found. The control groups of rats had elevated serum progesterone levels characteristic of diestrus; however, rats microinjected in the ARC had significantly lower progesterone levels, regardless of dox treatment. No effect of rAAV-OB-Rb microinjection into the PVN was observed on any parameter of reproductive function (Table 1).

The effect of rAAV-OB-Rb on LHRH concentration in the hypothalamus is shown in Figure 4. Expression of rAAV-OB-Rb in the ARC significantly elevated hypothalamic LHRH peptide levels in the female fatty Zucker rats. In control rats injected with saline, the LHRH concentration was 1.27 ± 0.23 pg/µg protein. No effect of rAAV-OB-Rb injection without dox (1.40 ± 0.62 pg/µg protein) or of rAAV-OB-Rb injection in the PVN with dox (1.57 ± 0.28 pg/µg protein) was found. However, in rats treated with rAAV-OB-Rb plus dox, LHRH concentrations were twofold higher (2.48 ± 0.48 pg/µg protein, P < 0.05).



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  		FIG. 4. Effects of rAAV-OB-Rb treatment on hypothalamic LHRH levels

The effects of rAAV-OB-Rb on NPY are shown in Figure 5. Expression of OB-R in the ARC caused significant decreases in hypothalamic NPY peptide and mRNA levels. Expression of NPY mRNA, as assessed by quantitative real-time PCR, is represented as the percentage change from the saline control group. Injection of rAAV-OB-Rb plus dox decreased NPY mRNA (Fig. 5A) by 77%. The decrease in NPY mRNA was accompanied by a corresponding decrease in hypothalamic NPY peptide levels (10.2 ± 1.7 vs. 29.9 ± 4.7 pg/µg protein in saline-injected rats, P < 0.05) (Fig. 5B). No change was observed in NPY mRNA expression in rats given the vectors without dox or given the vectors with dox in the PVN; however, a small decrease in NPY concentration in ARC-injected rats without dox (19.3 ± 2.4 pg/µg protein) was noted.



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  		FIG. 5. Effect of rAAV-OB-Rb treatment on hypothalamic (A) NPY mRNA expression and (B) peptide concentration

Expression of POMC mRNA was markedly reduced in rats injected with rAAV-OB-Rb (Fig. 6). However, only the rats given rAAV-OB-Rb with dox in the ARC had significantly decreased hypothalamic ß-END levels compared to all other treatment groups, regardless of dox treatment. As shown in Figure 6B, ß-END levels were significantly reduced in ARC-injected rats given dox compared to saline-injected rats. However, no difference was observed in ß-END levels of ARC-injected rats not given dox or PVN-injected rats compared to the saline-treated rats.



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  		FIG. 6. Effect of rAAV-OB-Rb treatment on hypothalamic (A) POMC mRNA and (B) ß-END peptide levels


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates, to our knowledge for the first time, the feasibility of using rAAV vectors to site-specifically express functional OB-R in the hypothalami of female fatty Zucker rats for extended periods. The vector system used to install OB-R in the hypothalamus was designed to be regulatable using a tetracycline-like antibiotic, dox. However, RT-PCR analysis of hypothalamic tissue showed that some gene transcription occurred with this system even in the absence of dox. Several explanations are possible for this unexpected outcome, which has been observed in other studies as well [58, 59]. Residual affinity of the rtTA for tetO is possible even in the absence of dox [58]. It has also been shown that the terminal repeats necessary for AAV vector packaging can mediate gene expression [58]. Alternatively, some cells in the ARC could have been transduced only by the rAAV-OB-Rb vector and not by the promoter vector, causing gene expression to occur without regulation. Although we discovered a partial inability to regulate gene expression during the present study in rats given the vector without dox, rats that were given both the vector and dox showed significantly higher levels of OB-R expression.

A plethora of studies have shown that leptin signaling in the hypothalamus is crucial for normal function of the HPG axis [5–8, 10–13, 60–62]. The OB-R has been found in several nuclei in the hypothalamus [21–24, 26]; however, to our knowledge, the target site specific for leptin's effects on the HPG axis has yet to be conclusively identified. In the present study, many significant improvements were observed in the HPG axis of rats expressing OB-R in the ARC, whereas OB-R expression in the PVN was virtually without effect on function of the HPG axis.

In the female fatty Zucker rats lacking a functional OB-R, estrous cycles are irregular, with long periods of diestrus [19, 20]. The presence of several corpora lutea in the ovaries verified this observation. In rats with OB-R expression in the ARC, but not in the PVN, estrous cycle length was normalized, and a decrease in the number of corpora lutea was observed. These rats also showed evidence of follicular activity, indicated by a large number of ovarian follicles at various stages of development. Additionally, the ovaries contained corpora lutea, suggesting that the rAAV-OB-Rb-treated rats were, in fact, ovulating. Based on the observed changes in ovarian function, it is reasonable to assume that serum LH and FSH levels were increased. This was supported by the observed increase in hypothalamic LHRH concentration. Our results indicate that functional OB-R in the ARC is both necessary and sufficient to produce normal estrous cycles in the female fatty Zucker rat, especially considering that OB-R installation in the PVN had no such effect. Leptin receptors have also been localized in both the pituitary and the ovary, and leptin can have direct effects on these tissues [63]. Therefore, to restore fertility, we may need to install OB-R at other sites in the HPG axis.

The literature contains conflicting reports about the status of the uterus in the fatty Zucker rat. The initial description of the Zucker rat [14] reported underdeveloped uteri, whereas in 1986, Chelich and Edmonds [64] reported that the uteri appear to be more normal than once thought. In the present study, we observed a decrease in uterine weight in response to OB-R installation in the ARC. Prolonged periods of diestrus suggest that the fatty Zucker rats undergo periods of pseudopregnancy characterized by high progesterone levels and enlarged uteri. Installation of the OB-R in the ARC, but not in the PVN, normalized the estrous cycle length and reduced the number of days in diestrus as well as the number of corpora lutea in the ovary. The resultant reduction in serum progesterone levels and, possibly, endometrial proliferation would explain the observed reduction in uterine weight. Similarly, reductions in progesterone levels and uterine weight were also seen, although to a smaller extent, in the ARC-injected rats without dox that showed partial expression of OB-R.

Leptin receptors have been localized to many nuclei in the brain, but to our knowledge, they have not been colocalized with LHRH neurons in the rat [24, 26, 29, 32, 65, 66]. Therefore, other neuropeptides in the hypothalamus may function as interneurons to signal leptin's effects to LHRH neurons in the MPOA. One candidate, NPY, has bimodal effects on the HPG axis depending on the steroidal state of the animal [38]. In a normal steroid environment, NPY is stimulatory to the HPG axis, causing increases in LHRH and LH; however, in the fatty Zucker rat, in which high NPY levels continuously stimulate NPY receptors, it is inhibitory to LHRH release [35]. Another neuropeptide that inhibits LHRH secretion is ß-END, a product of the POMC gene [46]. It would follow that decreasing these two neuropeptides in the hypothalamus might stimulate the reproductive axis in these rats. Indeed, we found that functional leptin signaling in the ARC, but not in the PVN, reduced these inhibitory neuropeptides and that removal of this inhibition resulted in upregulation of LHRH. These results support the recent report that functional leptin signaling in the ARC of OB-R-deficient Koletsky rats reduced NPY mRNA expression [67]. Clearly, the ARC NPY perikaryon is the site where leptin acts to decrease NPY gene expression and, thereby, to decrease the supply of NPY at terminals in the MPOA where they are linked to LHRH neurons and also to LHRH terminals in the median eminence, where LHRH is stored before release into the hypophysial portal veins.

Although Morton et al. [67] reported enhanced expression of POMC and, presumably, its protein products following transfer of the OB-R gene into the ARC of Koletsky rats, we observed a decrease in POMC mRNA as well as ß-END concentration in fatty Zucker rats. This discrepancy in the effects on POMC mRNA may have resulted from the shorter time course of effectiveness when adenovirus was used for gene transfer [67], in contrast to the longer-lasting effectiveness of AAVs (present study). The decrease in hypothalamic ß-END levels likely reduces the tonic inhibition of LHRH by endogenous opioid peptides and contributes to the observed increase in LHRH levels.

In summary, using viral vector gene therapy, we have successfully expressed the OB-R in the hypothalami of fatty Zucker rats that lack functional OB-R. That this receptor was functional is evidenced by normalization of estrous cycles and ovarian activity. We also observed decreases in hypothalamic NPY and ß-END peptide levels as well as in their gene expression. These neuropeptidergic changes may be responsible for the observed increase in LHRH levels resulting in activation of the HPG axis. While it remains necessary to examine other nuclei shown to express OB-R in the hypothalamus to identify other target sites involved in leptin's effects on the HPG axis, it would appear that the ARC is the major site of leptin's actions on the HPG axis.


   ACKNOWLEDGMENTS
 
The help of Dr. Sergei Zolotukhin for viral vector design and Dr. Michael Dube for technical instruction is gratefully acknowledged.


   FOOTNOTES
 
1 Supported by NIH (NS32727) and AHA (0110128B). Presented in part at the 30th Annual Meeting of the Society for Neuroscience, New Orleans, LA, November 4–9, 2000, and the 31st Annual Meeting of the Society for Neuroscience, San Diego, CA, November 10–15, 2001. Back

2 Correspondence: Pushpa S. Kalra, Department of Physiology and Functional Genomics, University of Florida, Box 100274, Gainesville, FL 32601. FAX: 352 294 0191; pkalra{at}phys.med.ufl.edu Back

Received: 11 December 2003.

First decision: 26 December 2003.

Accepted: 24 February 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Caro JF, Sinha MK, Kolaczynski JW, Zhang PL, Considine RV. Leptin: the tale of an obesity gene. Diabetes 1996 45:1455-1462[Medline]
  2. Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P. Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks [see comments]. Science 1995 269:546-549[Abstract/Free Full Text]
  3. Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Lallone RL, Burley SK, Friedman JM. Weight-reducing effects of the plasma protein encoded by the obese gene. Science 1995 269:543-546[Abstract/Free Full Text]
  4. Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Collins F. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 1995 269:540-543[Abstract/Free Full Text]
  5. Schneider JE, Goldman MD, Tang S, Bean B, Ji H, Friedman MI. Leptin indirectly affects estrous cycles by increasing metabolic fuel oxidation. Horm Behav 1998 33:217-228[CrossRef][Medline]
  6. Schneider JE, Zhou D, Blum RM. Leptin and metabolic control of reproduction. Horm Behav 2000 37:306-326[CrossRef][Medline]
  7. Nagatani S, Guthikonda P, Thompson RC, Tsukamura H, Maeda KI, Foster DL. Evidence for GnRH regulation by leptin: leptin administration prevents reduced pulsatile LH secretion during fasting. Neuroendocrinology 1998 67:370-376[CrossRef][Medline]
  8. Nagatani S, Zeng Y, Keisler DH, Foster DL, Jaffe CA. Leptin regulates pulsatile luteinizing hormone and growth hormone secretion in the sheep. Endocrinology 2000 141:3695-3975
  9. Kalra SP, Xu B, Dube MG, Moldawer LL, Martin D, Kalra PS. Leptin and ciliary neurotropic factor (CNTF) inhibit fasting-induced suppression of luteinizing hormone release in rats: role of neuropeptide Y. Neurosci Lett 1998 240:45-49[CrossRef][Medline]
  10. Finn PD, Cunningham MJ, Pau KY, Spies HG, Clifton DK, Steiner RA. The stimulatory effect of leptin on the neuroendocrine reproductive axis of the monkey. Endocrinology 1998 139:4652-4662[Abstract/Free Full Text]
  11. Carro E, Pinilla L, Seoane LM, Considine RV, Aguilar E, Casanueva FF, Dieguez C. Influence of endogenous leptin tone on the estrous cycle and luteinizing hormone pulsatility in female rats. Neuroendocrinology 1997 66:375-377[Medline]
  12. Kalra SP, Pu S, Horvath TL, Kalra PS. Leptin and NPY regulation of GnRH secretion and energy homeostasis. In: Bourguignon JP, Plant TM (eds), The Onset of Puberty in Perspective. Pittsburgh: Elsevier Science; 2000:317–327
  13. Lebrethon MC, Vandersmissen E, Gerard A, Parent AS, Junien JL, Bourguignon JP. In vitro stimulation of the prepubertal rat gonadotropin-releasing hormone pulse generator by leptin and neuropeptide Y through distinct mechanisms. Endocrinology 2000 141:1464-1469[Abstract/Free Full Text]
  14. Zucker LM, Zucker TF. Fatty, a new mutation in the rat. J Hered 1961 52:275-278[Free Full Text]
  15. Bivens CL, Olster DH. Abnormal estrous cyclicity and behavioral hyporesponsiveness to ovarian hormones in genetically obese Zucker female rats. Endocrinology 1997 138:143-148[Abstract/Free Full Text]
  16. Phillips MS, Liu Q, Hammond HA, Dugan V, Hey PJ, Caskey CJ, Hess JF. Leptin receptor missense mutation in the fatty Zucker rat. Nat Genet 1996 13:18-19[CrossRef][Medline]
  17. Marin-Bivens CL, Kalra SP, Olster DH. Intraventricular injection of neuropeptide Y antisera curbs weight gain and feeding, and increases the display of sexual behaviors in obese Zucker female rats. Regul Pept 1998 75: –76 327-334
  18. Marin-Bivens CL, Olster DH. Food restriction neither improves nor exacerbates reproduction in obese female Zucker rats. Physiol Behav 1999 66:893-897[CrossRef][Medline]
  19. Saiduddin S, Bray GA, York DA, Swerdloff RS. Reproductive function in the genetically obese "fatty" rat. Endocrinology 1973 93:1251-1256[Abstract/Free Full Text]
  20. Whitaker EM, Robinson AC. Circulating reproductive hormones and hypothalamic estradiol and progestin receptors in infertile Zucker rats. J Endocrinol 1989 120:331-336[Abstract/Free Full Text]
  21. Yarnell DO, Knight DS, Hamilton K, Tulp O, Tso P. Localization of leptin receptor immunoreactivity in the lean and obese Zucker rat brain. Brain Res 1998 785:80-90[CrossRef][Medline]
  22. Tartaglia LA, Dembski M, Weng X, Deng N, Culpepper J, Devos R, Richards GJ, Campfield LA, Clark FT, Deeds J, Muir C, Sanker S, Moriarty A, Moore KJ, Smutko JS, Mays GG, Woolf EA, Monroe CA, Tepper RI. Identification and expression cloning of a leptin receptor, OB-R. Cell 1995 83:1263-1271[CrossRef][Medline]
  23. Shioda S, Funahashi H, Nakajo S, Yada T, Maruta O, Nakai Y. Immunohistochemical localization of leptin receptor in the rat brain. Neurosci Lett 1998 243:41-44[CrossRef][Medline]
  24. Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, Trayhurn P. Localization of leptin receptor mRNA and the long form splice variant (Ob-Rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization. FEBS Lett 1996 387:113-116[CrossRef][Medline]
  25. Mercer JG, Moar KM, Hoggard N. Localization of leptin receptor (Ob-R) messenger ribonucleic acid in the rodent hindbrain. Endocrinology 1998 139:29-34[Abstract/Free Full Text]
  26. Elmquist JK, Bjorbaek C, Ahima RS, Flier JS, Saper CB. Distributions of leptin receptor mRNA isoforms in the rat brain. J Comp Neurol 1998 395:535-547[CrossRef][Medline]
  27. Baskin DG, Breininger JF, Schwartz MW. Leptin-receptor mRNA identifies a subpopulation of neuropeptide Y neurons activated by fasting in rat hypothalamus. Diabetes 1999 48:828-833[Abstract]
  28. Hakansson ML, Brown H, Ghilardi N, Skoda RC, Meister B. Leptin-receptor immunoreactivity in chemically defined target neurons of the hypothalamus. J Neurosci 1998 18:559-572[Abstract/Free Full Text]
  29. Hakansson ML, Hulting AL, Meister B. Expression of leptin-receptor mRNA in the hypothalamic arcuate nucleus—relationship with NPY neurones. Neuroreport 1996 7:3087-3092[Medline]
  30. Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, Morgan PJ, Trayhurn P. Coexpression of leptin receptor and preproneuropeptide Y mRNA in arcuate nucleus of mouse hypothalamus. J Neuroendocrinol 1996 8:733-735[CrossRef][Medline]
  31. Sahu A. Evidence suggesting that galanin (GAL), melanin-concentrating hormone (MCH), neurotensin (NT), pro-opiomelanocortin (POMC), and neuropeptide Y (NPY) are targets of leptin signaling in the hypothalamus. Endocrinology 1998 139:795-798[Abstract/Free Full Text]
  32. Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG. Identification of targets of leptin action in rat hypothalamus. J Clin Invest 1996 98:1101-1106[Medline]
  33. Stephens TW, Basinski M, Bristow PK, Bue-Valleskey JM, Burgett SG, Craft L, Hale J, Hoffmann J, Hsiung HM, Kriauciunas A, Mackellar W, Rosteck PR Jr, Schnener B, Smith D, Tinsley FC, Zhang X-Y, Heiman M. The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature 1995 377:530-532[CrossRef][Medline]
  34. Allen LG, Kalra PS, Crowley WR, Kalra SP. Comparison of the effects of neuropeptide Y and adrenergic transmitters on LH release and food intake in male rats. Life Sci 1985 37:617-623[CrossRef][Medline]
  35. Kalra PS, Kalra SP. Steroidal modulation of the regulatory neuropeptides: luteinizing hormone releasing hormone, neuropeptide Y, and endogenous opioid peptides. J Steroid Biochem 1986 25:733-740[CrossRef][Medline]
  36. Kalra SP, Karla PS, Sahu A, Allen LG, Crowley WR. The steroid-neuropeptide connection in the control of LHRH secretion. Adv Exp Med Biol 1987 219:65-83[Medline]
  37. Khorram O, Pau KY, Spies HG. Bimodal effects of neuropeptide Y on hypothalamic release of gonadotropin-releasing hormone in conscious rabbits. Neuroendocrinology 1987 45:290-297[Medline]
  38. Kalra SP, Kalra PS. Nutritional infertility: the role of the interconnected hypothalamic neuropeptide Y-galanin-opioid network. Front Neuroendocrinol 1996 17:371-401[CrossRef][Medline]
  39. Horvath TL, Naftolin F, Kalra SP, Leranth C. Neuropeptide-Y innervation of ß-endorphin-containing cells in the rat mediobasal hypothalamus: a light and electron microscopic double immunostaining analysis. Endocrinology 1992 131:2461-2467[Abstract/Free Full Text]
  40. McKibbin PE, Cotton SJ, McMillan S, Holloway B, Mayers R, McCarthy HD, Williams G. Altered neuropeptide Y concentrations in specific hypothalamic regions of obese (fa/fa) Zucker rats. Possible relationship to obesity and neuroendocrine disturbances. Diabetes 1991 40:1423-1429[Abstract]
  41. Kowalski TJ, Ster AM, Smith GP. Ontogeny of hyperphagia in the Zucker (fa/fa) rat. Am J Physiol 1998 275:R1106-R1109
  42. Kowalski TJ, Houpt TA, Jahng J, Okada N, Liu SM, Chua SC Jr, Smith GP. Neuropeptide Y overexpression in the preweanling Zucker (fa/fa) rat. Physiol Behav 1999 67:521-525[CrossRef][Medline]
  43. Fetissov S, Nicolaidis S. Neuropeptide Y in the magnocellular hypothalamic neurons of obese Zucker rats. Neuropeptides 1998 32:63-66[CrossRef][Medline]
  44. Beck B, Burlet A, Nicolas JP, Burlet C. Hypothalamic neuropeptide Y (NPY) in obese Zucker rats: implications in feeding and sexual behaviors. Physiol Behav 1990 47:449-453[CrossRef][Medline]
  45. Kalra PS, Norlin M, Kalra SP. Neuropeptide Y stimulates beta-endorphin release in the basal hypothalamus: role of gonadal steroids. Brain Res 1995 705:353-356[CrossRef][Medline]
  46. Sahu A, Crowley WR, Kalra SP. An opioid-neuropeptide-Y transmission line to luteinizing hormone (LH)-releasing hormone neurons: a role in the induction of LH surge. Endocrinology 1990 126:876-883[Abstract/Free Full Text]
  47. Bartlett JS, Samulski RJ, McCown TJ. Selective and rapid uptake of adeno-associated virus type 2 in brain. Hum Gene Ther 1998 9:1181-1186[Medline]
  48. Kaplitt MG, Leone P, Samulski RJ, Xiao X, Pfaff DW, O'Malley KL, During MJ. Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain. Nat Genet 1994 8:148-154[CrossRef][Medline]
  49. Muzyczka N. Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr Top Microbiol Immunol 1992 158:97-129[Medline]
  50. Dhillon H, Kalra SP, Prima V, Zolotukhin S, Scarpace PJ, Moldawer LL, Muzyczka N, Kalra PS. Central leptin gene therapy suppresses body weight gain, adiposity and serum insulin without affecting food consumption in normal rats: a long-term study. Regul Pept 2001 99:69-77[CrossRef][Medline]
  51. Dhillon H, Kalra SP, Kalra PS. Dose-dependent effects of central leptin gene therapy on genes that regulate body weight and appetite in the hypothalamus. Mol Ther 2001 4:139-145[CrossRef][Medline]
  52. Hauswirth WW, Lewin AS, Zolotukhin S, Muzyczka N. Production and purification of recombinant adeno-associated virus. Methods Enzymol 2000 316:743-761[Medline]
  53. Zolotukhin S, Byrne BJ, Mason E, Zolotukhin I, Potter M, Chesnut K, Summerford C, Samulski RJ, Muzyczka N. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther 1999 6:973-985[CrossRef][Medline]
  54. Paxinos G, Watson C. 1986 The rat brain in stereotaxic coordinates. Academic Press: San Diego; 1986
  55. Bonavera JJ, Sahu A, Kalra PS, Kalra SP. Evidence that nitric oxide may mediate the ovarian steroid-induced luteinizing hormone surge: involvement of excitatory amino acids. Endocrinology 1993 133:2481-2487[Abstract/Free Full Text]
  56. Sahu A, Kalra SP, Crowley WR, O'Donohue TL, Kalra PS. Neuropeptide Y levels in microdissected regions of the hypothalamus and in vitro release in response to KCl and prostaglandin E2: effects of castration. Endocrinology 1987 120:1831-1836[Abstract/Free Full Text]
  57. Koenig JI, Meltzer HY, Gudelsky GA. Morphine or capsaicin administration alters the secretion of ß-endorphin into the hypophysial portal vasculature of the rat. Neuroendocrinology 1986 43:611-617[CrossRef][Medline]
  58. Forster K, Helbl V, Lederer R, Urlinger S, Wittenburg N, Hillen W. Tetracycline-inducible expression systems with reduced basal activity in mammalian cells. Nucleic Acids Res 1999 27:708-710[Abstract/Free Full Text]
  59. Olsson M, Nikkhah G, Bentlage C, Bjorklund A. 1995 Forelimb akinesia in the rat Parkinson model: differential effects of dopamine agonists and nigral transplants as assessed by a new stepping test. J Neurosci 1995 15:3863-3872[Abstract]
  60. Diano S, Kalra SP, Horvath TL. Leptin-receptor immunoreactivity is associated with the Golgi apparatus of hypothalamic neurons and glial cells. J Neuroendocrinol 1998 10:647-650[CrossRef][Medline]
  61. Fox AS, Olster DH. Effects of intracerebroventricular leptin administration on feeding and sexual behaviors in lean and obese female Zucker rats. Horm Behav 200; 37:377–387
  62. Yu WH, Walczewska A, Karanth S, McCann SM. Nitric oxide mediates leptin-induced luteinizing hormone-releasing hormone (LHRH) and LHRH and leptin-induced LH release from the pituitary gland. Endocrinology 1997 138:5055-5058[Abstract/Free Full Text]
  63. Zamorano PL, Mahesh VB, De Sevilla LM, Chorich LP, Bhat GK, Brann DW. Expression and localization of the leptin receptor in endocrine and neuroendocrine tissues of the rat. Neuroendocrinology 1997 65:223-228[CrossRef][Medline]
  64. Chelich AM, Edmonds ES. 1986 Pseudopregnancy and decidual response in the obese Zucker rat: a re-examination. Biol Reprod 1986 34:805-808[Abstract]
  65. Grill HJ, Schwartz MW, Kaplan JM, Foxhall JS, Breininger J, Baskin DG. Evidence that the caudal brainstem is a target for the inhibitory effect of leptin on food intake. Endocrinology 2002 143:239-246[Abstract/Free Full Text]
  66. Hoggard N, Hunter L, Duncan JS, Williams LM, Trayhurn P, Mercer JG. Leptin and leptin-receptor mRNA and protein expression in the murine fetus and placenta. Proc Natl Acad Sci U S A 1997 94:11073-11078[Abstract/Free Full Text]
  67. Morton GJ, Niswender KD, Rhodes CJ, Myers MG, Blevins JE, Baskin DG, Schwartz MW. Arcuate nucleus-specific leptin-receptor gene therapy attenuates the obesity phenotype of Koletsky (fak/fak) rats. Endocrinology 2003 144:2016-2024[Abstract/Free Full Text]



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