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Deficits in Reproduction and Pro-Gonadotropin-Releasing Hormone Processing in Male Cpe^fat Mice

Departments of Psychiatry and Behavioral Sciences, Medicine (Endocrinology), and Cell Biology and Mouse Behavioral and Neuroendocrine Analysis Core Facility (S.S., R.M.R., R.L.R., G.X.L., W.C.W.), Duke University Medical Center, Durham, North Carolina 27710; Department of Molecular Pharmacology, Albert Einstein College of Medicine (Y.F., L.D.F.), Bronx, New York 10461; Gamete Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences (D.O.B., E.M.E.), Research Triangle Park, North Carolina 27709; and U.S.-Japan Biomedical Research Laboratories, Tulane University Herbert Center (M.L., A.A.), Belle Chase, Louisiana 70037

Address all correspondence and requests for reprints to: Dr. William C. Wetsel, Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, Box 3497, 028 CARL Building, Durham, North Carolina 27710. E-mail: wetse001{at}mc.duke.edu.

	Abstract

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Cpe^fat/fat mice are obese, diabetic, and infertile. These animals have a point mutation in carboxypeptidase E (CPE), an exopeptidase that removes C-terminal basic amino acids from peptide intermediates. The mutation renders the enzyme unstable, and it is rapidly degraded. Although the infertility of Cpe^fat/fat mice has not been systematically investigated, it is thought to be due to a deficit in GnRH processing. We have evaluated this hypothesis and found hypothalamic GnRH levels to be reduced by 65–78% and concentrations of pro-GnRH and C-terminal-extended intermediates to be high. Basal serum gonadotropin contents are similar among wild-type, heterozygous, and homozygous mice. Testis morphology and function are abnormal in older obese Cpe^fat/fat mice. Matings between homozygous mutants yield a 5% pregnancy rate. By comparison, when 50-d-old Cpe^fat/fat males are paired with heterozygous females, rates increase to 43%, and they rapidly decrease to negligible levels by 120 d. As fertility declines without accompanying changes in the hypothalamic-pituitary-gonadal axis and before obesity is evident, reproduction is more complex than originally thought. This suspicion is confirmed in 90-d-old Cpe^fat/fat males, who readily interact with females, but rarely mount and fail to show intromission or ejaculation behaviors. Together, these findings show that CPE is a key enzyme for pro-GnRH processing in vivo; however, the reproductive deficits in Cpe^fat/fat males appear to be due primarily to abnormal sexual behavior.

	Introduction

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NEURAL AND HORMONAL peptides first are biosynthesized as protein precursors that must undergo limited proteolysis to produce biologically active peptides (1, 2). Typically, these conversions involve at least two different steps. In the first, excision of the propeptide may occur by a member of the prohormone convertase (PC) family of subtilisin-like enzymes. These enzymes recognize monobasic, dibasic, or tetrabasic amino acids and cleave the precursor at the C-terminus of these residues. The second step is followed by sequential removal of the basic amino acids by an exopeptidase that may be carboxypeptidase E (CPE) (3). In many cases, peptides are subjected to additional processing steps or modifications to achieve full biological activity.

The importance of peptide processing in physiology has been shown through identification of spontaneous mutations in humans (4) and laboratory animals (5), as well as by the generation of mice with deletions of genes for these enzymes (6, 7, 8, 9, 10) and their interacting proteins (11). The first mutation in a processing enzyme arose spontaneously in a mouse at The Jackson Laboratory (Bar Harbor, ME) (5). This animal was obese, diabetic, and infertile, and the mutation was ascribed to the fat locus on chromosome 8. Subsequent genetic studies have revealed that fat/fat mice harbor a point mutation in the Cpe gene (12). In these mice, a single Ser²⁰² to Pro²⁰² substitution renders the enzyme catalytically inactive and subject to rapid degradation in the endoplasmic reticulum (12, 13). Due to this finding, the fat/fat animals have now been renamed Cpe^fat/fat (12).

CPE is an enzyme that is expressed almost exclusively in neural and endocrine tissues and is responsible for removing basic amino acids from the C terminus of peptide intermediates (3). In Cpe^fat/fat mice, processing of proinsulin is aberrant (12). Plasma levels of the prohormone and intermediates are increased, whereas concentrations of fully processed insulin are reduced by at least 70%. This deficiency is postulated to contribute to diabetes in the mutants. Aside from insulin homeostasis, fuel metabolism may be further perturbed because pro-TRH processing is aberrant (14). Functional impairments and processing alterations in other peptides (e.g. proopiomelanocortin and promelanin concentrating hormone) may also contribute to the obesity in Cpe^fat/fat mice (15, 16).

As obesity and diabetes in Cpe^fat/fat animals develop after puberty, several investigators have used these mice as a model for studies of adult-onset obesity and diabetes (5, 17). By contrast, the report of their infertility is anecdotal, and it has not been investigated systematically. Successful reproduction requires coordination and feedback among GnRH, gonadotropins, and sex steroids within the hypothalamic-pituitary-gonadal (HPG) axis. Of these hormones, the only one that is proteolytically processed is GnRH. It has been proposed that processing of pro-GnRH begins with endoproteolysis to yield a GnRH intermediate peptide and GnRH-associated peptide (GAP; see Fig. 1). Next, processing of the intermediate proceeds through sequential excision of the C-terminal basic amino acid residues and amidation of the C-terminus, whereas the N terminus can be converted to pyroglutamate at any step in this process (18). Each of these processing steps is mediated by separate enzymes that may include PC, CPE, peptidylglycine -amidating monooxygenase, and glutaminyl cyclase (19, 20). Transcripts for all four enzymes are found in the preoptic area and anterior hypothalamus, regions containing GnRH cell bodies, as well as in the immortalized hypothalamic GnRH neuronal cell lines (21).

View larger version (14K): [in this window] [in a new window]   FIG. 1. Proposed processing scheme for mammalian pro-GnRH. Excision of the pro-GnRH by PC yields [Gln1]GnRH-[Gly11Lys12Arg13] and GAP-(1–56). After this step, the intermediate can be processed at the C or N terminal. In the former route, the C-terminal basic amino acids are sequentially removed by carboxypeptidase to give [Gln1]GnRH-[Gly11]. The intermediate is converted by amidating enzyme to [Gln1]GnRH, and this intermediate is processed by glutaminyl cyclase to GnRH. Alternatively, the N-terminal glutamine of [Gln1]GnRH-[Gly11Lys12Arg13], [Gln1]GnRH-[Gly11Lys12], or [Gln1]GnRH-[Gly11] can be converted by glutaminyl cyclase to yield GnRH intermediates with a C-terminal extension. These intermediates are processed as described above to yield GnRH. The amino acids are given as three-letter designations; the pro-GnRH, GAP, and GnRH are shown in bold; the processing enzymes are italicized. See the report by Wetsel and co-workers (18 21 ) for details.

	Materials and Methods

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Animals
The BKS.HRS-Cpe^fat/J mice were obtained as a gift from Dr. Edward Leiter (The Jackson Laboratory). The fat mutation initially arose on the HRS/J murine background at The Jackson Laboratory (5) (www.jax.org). It was crossed onto the C57BLKS/J stain, and at N10, the BKS.HRS-Cpe^fat/J mice showed a more pronounced diabetes and obesity phenotype than the parent strain. These mice were propagated by heterozygous pairings due to their infertility. In our experiments, Cpe^fat littermates were weaned between 21 and 30 d of age and were segregated by sex and genotype at two to four males or four to five females per cage. Animals were genotyped by PCR as previously described (14).

For sexual behavior studies, 60-d-old Swiss-Webster females (Taconic Farms, Germantown, NY) were ovariectomized within 1 wk after arrival. Mice were housed four females per cage. Beginning 2 wk later, animals were administered 10 µg estradiol benzoate (Sigma-Aldrich Corp., St. Louis, MO) in sesame oil (sc) for 3 consecutive d. On the early evening of the fourth day, they were given 500 µg progesterone (Sigma-Aldrich Corp.) in sesame oil (sc) and 5–7 h later were paired with wild-type (WT) or Cpe^fat/fat males for assessment of sexual behavior during the dark phase of the light/dark cycle. The WT and Cpe^fat/fat males were housed individually as described below (Sexual behavior).

Animals were maintained on a 14-h light, 10-h dark cycle in a humidity- and temperature-controlled room with water and standard laboratory chow supplied ad libitum. All experiments were conducted in accordance with NIH guidelines for the care and use of animals and under an approved protocol from the institutional animal care and use committee at Duke University.

Chromatography
Animals were euthanized by decapitation, and peptides were extracted as previously described (21); the pellet was reserved for protein assay (22). Separation of prohormone, GAP-(1–56), and GnRH-like peptides was achieved by size exclusion chromatography (23). Briefly, lyophilized samples were reconstituted in ice-cold buffer containing 10% formic acid-0.1% ß-mercaptoethanol-0.001% BSA, sonicated, boiled in water for 10 min, cooled on ice for 5 min, and loaded onto a column containing Sephadex G-50 resin (Pharmacia Biotech, Piscataway, NJ). Fractions were collected, lyophilized, reconstituted in RIA buffer, and analyzed separately for pro-GnRH- and GAP-like immunoreactivity (IR) with the MC-2 antiserum and for GnRH-like IR with the B9 antiserum. The molecular mass markers included blue dextran (Pharmacia Biotech), cytochrome c, aprotinin, vitamin B-12, and tryptophan (Sigma-Aldrich Corp.), whereas synthetic rat GAP-(1–56) (Peninsula Laboratories, Belmont, CA) and mammalian GnRH (Phoenix Pharmaceuticals, Belmont, CA) served as peptide standards. The chromatograms were corrected for column recoveries that were approximately 83% and 72% for GnRH- and GAP-like IRs, respectively. The concentrations of pro-GnRH-, GAP-, and GnRH-like IRs were calculated by subtracting the baseline values from the area under each peak of IR.

Further analysis of GnRH-like IR was achieved by HPLC as previously described (19, 21). Fractions from the size exclusion column immediately surrounding and including the GnRH peak were pooled, an aliquot was taken for recovery, and samples were filtered through Sep-Pak C₁₈ cartridges (Waters Corp., Milford, MA). Samples were lyophilized, reconstituted in 5% acetonitrile-0.05% trifluoroacetic acid buffer, and separated on a C₁₈ column. The elution positions of the various pro-GnRH intermediates were determined with custom-synthesized [Gln¹]GnRH-[Gly¹¹Lys¹²Arg¹³], [Gln¹]GnRH-[Gly¹¹Lys¹²], [Gln¹]GnRH-[Gly¹¹], [Gln¹]GnRH, GnRH-[Gly¹¹Lys¹²Arg¹³], GnRH-[Gly¹¹Lys¹²], GnRH-[Gly¹¹], and [hydroxy-Pro⁸]GnRH as well as mammalian GnRH (Phoenix Pharmaceuticals). Samples were lyophilized and reconstituted in RIA buffer. Chromatograms were corrected for recoveries (97%) of GnRH-like IR materials.

For analysis of pituitary adenylate cyclase-activating polypeptide (PACAP), WT and Cpe^fat/fat testes were dissected, weighed, frozen in liquid nitrogen, and stored at -80 C. Peptides were extracted as previously described (24). Samples were reconstituted in 10% acetonitrile-1% trifluoroacetic acid and separated on a 0.46 x 250-mm TSK-ODS column (Tosohaas Corp., Montgomeryville, PA) at 1 ml/min. One-milliliter fractions were collected, lyophilized, and reconstituted in RIA buffer. Synthetic PACAP27 and PACAP38 (American Peptide Co., Sunnyvale, CA) served as elution standards. The chromatograms were corrected for recoveries of PACAP27-like (60%) and PACAP38-like (30%) IRs.

RIAs
The procedures for iodination of [Tyr⁰]human GAP-(1–56) (Peninsula Laboratories) and the GAP RIA have been described previously (20). The MC-2 antiserum binds both pro-GnRH and GAP-(1–56) (20, 25). The intraassay variability was approximately 7%; all samples were run in one assay. The protocol for GnRH iodination has also been described previously (19). The A772 antiserum binds [Gln¹]GnRH and GnRH (26); the B9 antiserum binds mammalian GnRH and its intermediates, and has very low recognition for pro-GnRH (21). The intra- and interassay variabilities for the two GnRH RIAs were approximately 6% and 9%, respectively.

In younger mice, sera from individual mice were assayed for LH and testosterone or for FSH and testosterone, whereas sera from older animals were assayed for all three hormones. The reagents for the mouse LH and FSH assays were provided by Dr. A. F. Parlow (Harbor-University of California-Los Angeles Medical Center, Torrance, CA). The [¹²⁵I]rat FSH was purchased from Covance (Vienna, VA); the mouse LH was iodinated as previously described (27). The standards for the LH RIA were from 2.4–625 pg, whereas those for the FSH assay were from 5–640 ng. Standards and duplicate samples were incubated with primary antiserum (LH, 1:18,000 final dilution; FSH, 1:12,000 final dilution) in 300 µl for 4 h at room temperature. At the end of this period, 50 µl 2% normal rabbit serum and 50 µl 20,000 cpm mouse [¹²⁵I]LH or rat [¹²⁵I]FSH were added, and the assays were incubated at 4 C for approximately 24 h. The next day, 100 µl antirabbit serum (1:25 dilution; Antibodies, Inc., Davis, CA) were added, and tubes were incubated at 4 C for 4 h. Ice-cold 15% polyethylene glycol was added, tubes were incubated for 2 h at 4 C and centrifuged at 2,500 x g for 30 min at 4 C, and the supernatants were discarded. Tubes were inverted to dry for 24 h and were counted in a -counter. The minimal detectable doses for the LH and FSH assays were 3.0 pg and 2.3 ng, respectively. All samples were run in one assay; the intraassay variabilities were 6% for LH and 8% for FSH. The testosterone RIA kit was purchased from Pantex (Santa Monica, CA). The minimal detectable dose was 0.2 pg; the intraassay variability was approximately 6%.

The PACAP27 and PACAP38 RIAs have been described previously (24, 28). Antiserum 88111-3 was generated against synthetic PACAP-(24–38), and it bound PACAP38; it did not recognize PACAP27 or its precursor. Antiserum 88123-3 was raised against synthetic PACAP27, and it recognized this peptide, but not PACAP38. The intraassay variabilities for both assays were approximately 4%.

Anterior pituitary cell cultures
The brains were removed from WT, Cpe^+/fat, and Cpe^fat/fat males; the membrane overlying the pituitary was ruptured with forceps; and the posterior pituitary and neurointermediate lobe were removed under stereomicroscope. The anterior pituitary was dissected and sliced in half with a scalpel, and the cells were dispersed as described for rats (19, 29). Cell viability was tested by trypan blue exclusion. The cells were plated at a density of 5 x 10⁵ cells into 24-well plates coated with Matrigel (Collaborative Research, Inc., Bedford, MA). Cells were incubated for 3 d in DMEM containing 5% horse serum, 1.25% heat-inactivated fetal bovine serum, 12.5 ng/ml amphotericin B, 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate (Life Technologies, Inc., Gaithersburg, MD) at 37 C in an atmosphere of 95% O₂ and 5% CO₂. At the end of that period, cells were washed twice in DMEM containing 0.25% BSA and incubated in fresh medium (basal conditions) or in presence of 10 nM synthetic mammalian GnRH (Phoenix Pharmaceuticals) or 1 µM A23187 (Calbiochem, La Jolla, CA) for 2 h. Medium was collected and submitted to the LH and FSH RIAs.

Testis morphology and function
The seminal vesicles and coagulating glands, right testis, combined epididymis and vas deferens, and prostate were trimmed of adipose tissue, blotted, and weighed. The left testis was frozen in liquid nitrogen for PACAP assays. The right testis was fixed for histology in Bouin’s solution for approximately 16 h, dehydrated with increasing concentrations of alcohol, and embedded in paraffin. Six-micrometer sections were cut and stained with hematoxylin and eosin. For immunocytochemistry, 5-µm sections of paraffin-embedded testis from WT and Cpe^fat/fat mice were deparaffinized with two changes of xylene and rehydrated. The slides were bathed in 3% H₂O₂ for 15 min at 37 C to quench endogenous peroxidase and were heated in a microwave oven in a citrate antigen-retrieval solution [0.1 M sodium citrate (pH 6.0) with 0.01% Triton X-100] to unmask the epitopes. Sections were incubated for 2 h at room temperature with rabbit anti-pro-CPE or anticarboxypeptidase D (CPD) sera [each diluted 1:1000 in blocking buffer (NEN Life Science Products Life-Science, Boston, MA)]. As controls, rabbit preimmune serum was used. Sections were washed in a PBS-0.2% Tween 20 solution (pH 7.4), incubated with biotinylated antirabbit Ig (Dako, Carpinteria, CA) for 20 min, washed, incubated with streptavidin-horseradish peroxidase for 20 min, and washed in the same buffer. Staining was visualized with 3,3'-diaminobenzidine for 2 min and was stopped by washing in water. Slides were counterstained with hematoxylin and viewed with a Axioskop light microscope (Zeiss, New York, NY). Images were captured on Ektachrome 64T film (Eastman Kodak Co., Rochester, NY).

To investigate testis function, sperm counts and motility were determined (30). Epididymal sperm from cauda epididymides were expelled into M16 medium (Specialty Media, Lavalette, NJ) containing 20 mg/ml BSA (Life Technologies, Inc.). After sperm expression, the tissue was minced and incubated for 15 min to allow sperm dispersal. Thereafter, aliquots of sperm (without tissue fragments) were removed and diluted 1:5 and 1:10. Sperm were capacitated by incubation at 37 C for 90 min under 95% O₂ and 5% CO₂ atmosphere. Sperm counts, estimated percentages of motile sperm, and flagellar movements were determined by phase microscopy.

Male fertility studies
In preliminary experiments, fertility rates for WT and Cpe^+/fat males were virtually identical. Hence, fertility studies on Cpe^fat/fat males were conducted with heterozygotes as controls. As fertility was very low for homozygous mutants, subsequent studies compared rates between Cpe^+/fat and Cpe^fat/fat males when they were paired with Cpe^+/fat females. Single naive males (45–300 d of age) were housed with two naive heterozygous females. Body weights were recorded at the beginning of each experiment. Males resided in the same cage for 30 d or until the female showed detectable signs of pregnancy, whichever occurred first. Pregnancy was monitored daily by visual inspection, palpation, and weight gain.

Analysis of sexual behavior
WT and Cpe^fat/fat males were first tested for discrimination of olfactory stimuli. In this test, 80 µl of either saline or urine from ovariectomized estrogen-progesterone-primed females were spotted onto 2 x 2-cm² filter paper and placed into separate white 3 x 4 x 0.4-cm Tissue-Tek cassettes (VWR Scientific, Bridgeport, NJ). Cassettes were fixed to opposite ends of a mouse cage. Males were observed for 8 min using the Observer program (Noldus Information Technology, Leesburg, VA). The behaviors included latency to approach and total time spent sniffing and interacting with each cassette.

WT and Cpe^fat/fat males were tested for sexual behavior using ovariectomized, estrogen-progesterone-primed females as partners. Swiss-Webster females were used because they have been shown to be excellent partners for sexual behavior (31). Cpe^fat mice were housed individually in clear Plexiglas observation cages (20 x 39 x 19 cm) at least 4 d before testing. Males were supplied with soiled bedding from their respective home cages as well as with food and water. On 1 d preceding each testing, males were allowed 5 min of social interaction with nonsteroid-treated, ovariectomized, Swiss-Webster females. Animals were transferred to the testing room at least 12 h before behavioral testing. All observations were made more than 1 h after onset of the dark cycle. A female was introduced into the chamber with the male 5–7 h after steroid priming, and sexual behavior was filmed (32). The Noldus Observer program was used to score behaviors, including the duration and frequency of social contact with the female as well as the latencies and frequencies of mounting, intromission, and ejaculation (31). Mounting behaviors were classified as full mounts, which involved the male approaching and grasping the female from behind while making rapid thrusting movements, and incomplete mounts, which included all instances of mounting from the side or front.

To determine whether the steroid-treated females could sexually arouse the males, a separate experiment was run with another group of WT and Cpe^fat/fat males. Experiments were conducted as described above, except the males were examined for evidence of penile erections. As erections are preceded by a change in posturing, an arching of the spine with an elevation of the pelvic area, and attempts to climb upon or mount the female, males were rapidly removed from the test chamber and examined for evidence of penile erection. If no erection was evident, the male was immediately returned to the test cage. Testing was terminated upon the first incidence of penile erection. Behavior was scored in terms of the latency to climb upon or mount (partially or fully) the female and for erection.

Statistics
The data are presented as the mean and SE. Pregnancy rates were analyzed with ² tests. Data from other experiments were evaluated by t tests, or one- or two-way ANOVAs. A posteriori comparisons were performed by Bonferroni or Newman-Keuls tests. In cases where homogeneity of variance was violated for the t test (e.g. via Levine’s test), the data were analyzed by the Mann-Whitney U test. P < 0.05 was considered significant.

	Results

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Hypothalamic GnRH levels
One of the major phenotypes initially ascribed to the Cpe^fat/fat mice was infertility (5). It has been suggested that this feature may be due to a deficiency in pro-GnRH processing (17). Experiments in Cpe^fat/fat mice permit us not only to evaluate the effects of the Cpe^fat mutation on GnRH processing, but also to examine in detail their reputed infertility. Peptides were extracted from hypothalami of WT, Cpe^+/fat, and Cpe^fat/fat males at different ages and screened with specific antisera that recognized the fully processed peptide (A772 antiserum) or that bound GnRH and its intermediates (B9 antiserum). Hypothalamic GnRH levels were measured at approximately 50 d of age before the onset of obesity (WT, 19.1 ± 0.27 g; Cpe^+/fat, 19.4 ± 0.41; Cpe^fat/fat, 18.6 ± 0.38 g), at 90 d when the homozygous mutants were just beginning to gain weight (WT, 22.5 ± 0.39 g; Cpe^+/fat, 21.9 ± 0.53 g; Cpe^fat/fat, 23.2 ± 0.98 g), and at 180 d after obesity was evident (WT, 25.9 ± 0.62 g; Cpe^+/fat, 26.7 ± 0.51 g; Cpe^fat/fat, 37.1 ± 2.36 g). When samples were assayed with A772 antiserum, GnRH-like IR in the hypothalamus was reduced by 65–78% in Cpe^fat/fat males compared with WT or Cpe^+/fat animals (Fig. 2, top). There was a tendency for the GnRH-like IR to be more depressed in homozygous mutants at 180 d than at 50 or 90 d of age. In contrast, the B9 antiserum revealed that GnRH-like IR was not distinguished by genotype across these same ages (Fig. 2, bottom). As the A772 and B9 antisera bind to different regions of GnRH-containing peptides, these findings demonstrate that the composition of hypothalamic GnRH-like IR materials from WT and Cpe^+/fat mice is different from that in Cpe^fat/fat males regardless of age, and that CPE activity in heterozygotes is sufficient for proper GnRH processing.

View larger version (20K): [in this window] [in a new window]   FIG. 2. GnRH-like IR from hypothalami of WT, Cpe+/fat, and Cpefat/fat males at approximately 50, 90, and 180 d of age. Top, GnRH-like IR was quantitated by the A772 antiserum that recognized the decapeptide and [Glu1]GnRH. Bottom, GnRH-like IR was measured by the B9 antiserum that binds to mammalian GnRH and its intermediates, but has very low recognition for pro-GnRH. Concentrations in hypothalami from , WT; , Cpe+/fat; , Cpefat/fat males. *, P < 0.05 vs. WT and Cpe+/fat mice.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Size exclusion chromatography of materials in hypothalamic extracts from WT, Cpe+/fat, and Cpefat/fat males at approximately 90 d of age. GAP-like IR materials that include the pro-GnRH and GAP-(1–56) are displayed as a thin line for WT (top), Cpe+/fat (middle), and Cpefat/fat (bottom) animals, whereas GnRH-like IR materials are presented as a thick line and measured from the same samples. The molecular weight markers are located at the top of each chromatogram. The MC-2 antiserum was used to measure pro-GnRH- and GAP-like IR, whereas GnRH-like IR was quantitated with the B9 antiserum. Note, the B9 antiserum shows slight cross-reactivity to pro-GnRH (see bottom panel). The chromatograms are corrected for column recoveries.

View larger version (20K): [in this window] [in a new window]   FIG. 4. HPLC separation of GnRH-like IR materials from the prior fractionation by size exclusion chromatography. GnRH intermediates and decapeptide from hypothalami of WT (top), Cpe+/fat (middle), and Cpefat/fat mice (bottom). The elution positions of the synthetic peptide standards are shown at the top of each chromatogram. The chromatograms are corrected for column recoveries. Q, Glutamine; G, glycine; K, lysine; R, arginine; Pro8, hydroxyproline.

To evaluate responses under stimulated conditions, primary anterior pituitary cell cultures from mice at 90 d of age were used. Basal LH secretion was not distinguished by genotype, whereas FSH release was reduced in homozygous mutants (Fig. 5). Synthetic mammalian GnRH (10 nM) significantly stimulated more LH and FSH secretion in male Cpe^fat/fat gonadotropes than in WT or heterozygous cultures. When cells were activated with 1 µM Ca²⁺ ionophore (A23187), secretion was similar across genotypes. These findings show that pituitaries of Cpe^fat/fat males are especially responsive to synthetic GnRH.

View larger version (23K): [in this window] [in a new window]   FIG. 5. In vitro release of gonadotropins from WT, Cpe+/fat, and Cpefat/fat dispersed anterior pituitary cell cultures. LH (top) and FSH (bottom) release into medium under basal conditions or during stimulation with 10 nM synthetic GnRH or 1 µM A23187. *, P < 0.05 vs. WT and Cpe+/fat cultures.

View larger version (18K): [in this window] [in a new window]   FIG. 6. Basal serum testosterone levels in male WT, Cpe+/fat, and Cpefat/fat mice at approximately 50, 90, 200, and 300 d of age. *, P < 0.05 vs. the WT and Cpe+/fat animals.

View larger version (89K): [in this window] [in a new window]   FIG. 7. Morphology and localization of pro-CPE- and CPD-like IR in testis of WT and Cpefat/fat mice. Hematoxylin and eosin staining of testis from approximately 90-d-old WT (A) and Cpefat/fat (B) males or from approximately 300-d-old WT (C) and homozygous (D) mutants. The arrows depict sperm in A–C. D, The arrow to the left shows some shedding of the seminiferous epithelium into the lumen in older Cpefat/fat testis. The arrow to the right depicts a thinning of the seminiferous epithelium with a reduction in spermatocytes and spermatids, such that the tubules appear atrophic in these older mutants. Magnification, x20. Pro-CPE-like IR in testis from WT (E) and Cpefat/fat animals (F), approximately 90 d of age, is shown. CPD-like IR from the same WT (G) and Cpefat/fat testis (H) are shown. The arrows in E and F show pro-CPE-like immunostaining on the acrosomes of sperm; those in G and H reveal CPD-like IR in Sertoli cells. Control sections run for testis from the same WT (I and K) and Cpefat/fat (J and L) animals using preimmune serum for pro-CPE (I and J) and CPD (K and L). Magnification, x40.

To determine whether peptide processing was perturbed in sperm, we examined processing of PACAP (a peptide in sperm) at times that coincided with the onset (90 d) and full appearance of obesity (300 d) in Cpe^fat/fat mice. PACAP-like peptides were separated by HPLC and quantitated by antisera that specifically recognized either PACAP27 or PACAP38 (35). At 90 d of age, levels of PACAP27 (Fig. 8A) and PACAP38 (Fig. 8B) were decreased in Cpe^fat/fat testis, whereas they were increased in the 300-d-old mice (Fig. 8, C and D). These data indicate that peptide processing of PACAP in sperm is intact at the time when testicular changes are evident in Cpe^fat/fat mice. Hence, some other carboxypeptidase-like enzyme must compensate for the loss of CPE. One such candidate is CPD (36). To examine this possibility, testis were evaluated for CDP immunoreactivity. This antiserum immunostained Sertoli cell processes that interdigitated between germ cells in both WT (Fig. 7G) and Cpe^fat/fat (Fig. 7H) testis. The staining of tail of sperm was nonspecific because the same staining could be visualized with preimmune serum (Fig. 7, K and L). Together, the immunocytochemical data show that both pro-CPE- and CPD-like IRs are present in testis, but the enzymes reside in different cells. More recently, an additional member of the metallocarboxypeptidase family of enzymes has been identified in testis (37). As carboxypeptidase A5 is localized on sperm, it might serve as a compensating enzyme in Cpe^fat/fat testis, although the substrate specificity of this enzyme is not predicted to efficiently cleave basic residues.

View larger version (28K): [in this window] [in a new window]   FIG. 8. HPLC separation of PACAP-like peptides in testis. A, HPLC separation of materials in testis from approximately 90-d-old WT and Cpefat/fat males screened with PACAP27 antiserum. B, Fractionation of materials from approximately 300-d-old mice screened with the same antiserum. C and D, The same fractions in A and B were screened with the PACAP38 antiserum. Dark lines, WT; lighter lines, Cpefat/fat.

A characteristic feature of Cpe^fat/fat males is that obesity develops after puberty, with diabetes appearing later in life (17). As obesity affects fertility (38), the reproductive competence of Cpe^fat/fat males was assessed before, at the beginning, and after the development of obesity. To ensure that high rates of fertility would be achieved, Cpe^fat/fat males were mated with Cpe^+/fat females for 30 d. Their fertility rates were compared with those of heterozygous male and female pairings. At 45–50 d of age, body weights of Cpe^+/fat (20.0 ± 0.38 g) and Cpe^fat/fat (19.7 ± 0.73 g) males were comparable. Even at this early age, fertility rates (e.g. 43%) were significantly lower for Cpe^fat/fat males (Table 3). These rates dramatically declined to 9% when Cpe^fat/fat males were bred between 51–120 d of age, even though the body weights of the heterozygous (23.9 ± 1.01 g) and homozygous (25.0 ± 1.27 g) mutants were similar. By comparison, between 121–180 d of age, none of the female heterozygotes became pregnant when mated with Cpe^fat/fat males; the Cpe^+/fat males impregnated approximately 85% of the females (Table 3). At this older age, Cpe^fat/fat males were significantly heavier (34.2 ± 2.55 g) than Cpe^+/fat controls (26.1 ± 0.68 g). Collectively, these data demonstrate that young Cpe^fat/fat males are subfertile; however, their reproductive performance rapidly declines before obesity develops.

As sexual behavior in male rodents is highly dependent upon olfaction (39, 40), this sense was tested at 90 d of age when fertility rates were rapidly declining in the Cpe^fat/fat mice. At this age, body weights of the homozygous mutants (23.9 ± 1.2 g) were similar to those of WT controls (23.1 ± 0.9 g). In the olfactory discrimination test, mice were given a choice of interacting with a small plastic cassette that contained saline or urine from ovariectomized, estrogen-progesterone-primed females. The latencies of both WT and Cpe^fat/fat males to approach the cassette with female urine [WT, 9.8 ± 1.5 sec (n = 10); Cpe^fat/fat, 10.8 ± 2.3 sec (n = 10)] were not different between genotypes. By comparison, the latencies to engage the saline cassette were protracted, and this was especially evident for the homozygous mutants (WT, 18.8 ± 8.7 sec; Cpe^fat/fat, 41.4 ± 19.8 sec). These results demonstrate that WT and Cpe^fat/fat mice are capable of discriminating between saline and estrous urine, and that both groups are equally attracted to urine from females.

After the olfactory discrimination test, these same mice were evaluated for sexual behavior. When an ovariectomized, estrogen-progesterone-primed female was placed into the cage with either a WT or Cpe^fat/fat male, both animals engaged in social interaction with the female. Compared with WT males, homozygous mutants spent more time with the female (Table 4). Despite this fact, the latencies for Cpe^fat/fat males to demonstrate full mounting behavior were very protracted, and these behaviors were either incomplete (e.g. thrusting movements were abruptly terminated, with the male dismounting the female) or inappropriate (e.g. mounting the female from the side or front). No instances of intromission or ejaculation were observed with any Cpe^fat/fat male. These data suggest that although Cpe^fat/fat males exhibit a high degree of sociability with female mice, they have a profound deficit in functional reproductive behavior.

	Discussion

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The major objective of our studies was to use the Cpe^fat mouse as a model for evaluating two aspects in the potential neuroendocrine control of reproduction. First, we wanted to test whether CPE is a critical enzyme for GnRH processing in vivo. Second, we wished to examine whether failure to appropriately process GnRH intermediates to mature GnRH inhibits fertility in the Cpe^fat/fat mouse. Consistent with the known enzymatic activity of CPE, we show that there is a large increase in the abundance of hypothalamic GnRH intermediates containing C-terminal basic amino acids. Despite this fact, a small concentration of fully processed GnRH is still detected in Cpe^fat/fat hypothalamus. Nevertheless, this small amount of decapeptide appears sufficient to maintain gonadotrope function under basal conditions. Analyses of gonadal status show that testis morphology and sperm counts are abnormal in obese Cpe^fat/fat males. Mating studies confirm that fertility is very low with homozygous mutant pairings, but this is significantly improved when young Cpe^fat/fat males are paired with WT or heterozygous females. Fertility rates, however, decline rapidly for the homozygous mutants before their obesity is apparent. As accompanying changes in the HPG axis are not evident at the time of this decline, our results indicate that the infertility of Cpe^fat/fat males is more complex than originally envisioned. Additional studies suggest that a loss of sexual behavior is the major contributor to reproductive dysfunction in male Cpe^fat/fat mice.

We were interested in studying Cpe^fat/fat animals because we had previously postulated that CPE is involved in processing GnRH intermediates in vivo (18, 19, 20, 21). The present studies provide the first confirmation of this hypothesis by showing that the Cpe^fat mutation leads to an accumulation of Arg- and/or Lys-extended C-terminal GnRH intermediates in the hypothalamus. Unexpectedly, abnormalities are also found in additional processing steps that do not rely directly upon CPE. For instance, levels of [Gln¹]GnRH intermediates are increased. As this intermediate is processed to GnRH by glutaminyl cyclase in vitro (41, 42), and because the enzyme is expressed in brain regions containing GnRH neurons and in the immortalized GnRH cells (21), the enhancement in these intermediates shows that the Cpe^fat mutation interferes with this reaction in vivo. Besides this conversion, the dramatic increase in pro-GnRH levels indicates that endoproteolysis is also disturbed in homozygous mutants. Both PC1/3 and PC2 have been implicated in endoproteolysis of the GnRH prohormone (4, 21). Interestingly, the activities of both enzymes are altered in Cpe^fat/fat mice (43). In whole brain, PC1/3 activity is reduced due to an overall decrease in biosynthesis, and most of the enzyme is in a precursor form. Changes in PC2 activity have been attributed to prolonged association with the 7B2 chaperone protein and with intermediates from processed peptide precursors.

Although we show that CPE is a key enzyme involved in processing GnRH intermediates in vivo, the presence of a small amount of the fully processed decapeptide indicates that some additional enzyme can partially compensate for the loss of CPE. One such candidate is CPD, an enzyme that is expressed in brain regions that contain GnRH neurons (36, 44). In AtT-20 pituitary cells, the enzyme is localized to the trans-Golgi network and immature secretory granules (45). By comparison, CPE resides in the same organelles, but it is also present in mature secretory granules (46). Hence, if CPD could compensate for the Cpe^fat mutation, processing of GnRH intermediates would still be incomplete, and the concentrations of fully processed decapeptide would be reduced, as is observed in Cpe^fat/fat mice.

Due to the high levels of GnRH intermediates relative to the fully processed decapeptide in the hypothalamus, it may be expected that anterior pituitary gonadotrope function will be affected. With respect to biological activities of the intermediates, it should be emphasized that GnRH analogs that have substitutions at the N-terminal pyroglutamate or carbon extensions at the C terminus fail to release LH or FSH (47, 48). Hence, low levels of fully processed GnRH may be anticipated to lead to reduced contents of gonadotropins in serum. However, we found that baseline levels of gonadotropins in serum are not different among genotypes. The absence of such changes under basal conditions may be a common feature of hypothalamic-pituitary interactions in Cpe^fat/fat mice, because, despite reduced TRH levels in hypothalamus, baseline levels of TSH in serum are not different from those in WT controls (14). By contrast, when primary anterior pituitary cultures are stimulated with synthetic GnRH in vitro, gonadotropes from Cpe^fat/fat males are actually more responsive than those from WT or Cpe^+/fat littermates. These findings suggest that the low levels of fully processed GnRH in the hypothalamus and, presumably, the low concentrations in hypophyseal portal blood lead to an up-regulation of GnRH receptors on the gonadotropes. A similar enhanced responsiveness has been observed for thyrotropes from homozygous mutant mice after exposure to a cold environment (14).

Besides examining pituitary function, the gonadal status of Cpe^fat/fat males was also evaluated. It should be noted that many of the morphological and functional deficits in testis from obese Cpe^fat/fat males are similar to those in null animals lacking either the sperm PC4 endopeptidase (7) or the vasoactive intestinal polypeptide/PACAP (VPAC2) receptor (49). As pro-PACAP in sperm is processed by PC4 and because the only PACAP receptor in testis is the VPAC2, we wanted to determine whether alterations in PACAP processing might be associated with the testis abnormalities in Cpe^fat/fat animals. Our analyses reveal, however, that PACAP processing in sperm is intact in obese Cpe^fat/fat at the time when testis pathologies are present. As obesity in other mouse models can also affect testicular morphology and function (50, 51, 52), the gonadal changes in Cpe^fat/fat males may be secondary to their obesity or due to alterations in the processing of some additional peptide that requires CPE.

In our fertility studies we found that Cpe^fat/fat males are not completely infertile, rather they are subfertile. Young homozygous mutant males can impregnate WT or heterozygous females, but matings with Cpe^fat/fat females are rarely successful. Despite this fact, fertility rates of young Cpe^fat/fat males are significantly below those of WT and Cpe^+/fat males, and by 90 d of age the reproductive success of the homozygous mutants declines rapidly even before their obesity is evident. Although the initial reduction in fertility at an early age may be due to abnormalities in pro-GnRH processing, the absence of additional changes within the HPG axis suggests that other factors play a major role in their reproductive failure. Our analysis of sexual behavior in Cpe^fat/fat males confirms this suspicion. The homozygous mutants readily interact with and are sexually aroused by the females. Some mice even display full mounting behavior. Nevertheless, those few Cpe^fat/fat males that mount do so inappropriately. Additionally, although the homozygous mutants make thrusting movements at the female, this behavioral sequence is always abruptly terminated, and the male remains on the female for a short period before dismounting. The high levels of social interaction, but low incidences of full mounting behavior, suggest that social behaviors are intact, but the threshold for sexual motivation is reduced in Cpe^fat/fat males. Additionally, the abrupt cessation of thrusting movements indicates that sexual behavior, once initiated, cannot be sustained. As a result, over time females cease to interact with the Cpe^fat/fat males, such that the display of sexual behavior becomes even more reduced.

In conclusion, we demonstrate that CPE is a key GnRH-processing enzyme, but neither the deficiency in processing nor their obesity can fully explain the reproductive insufficiency of Cpe^fat/fat males. Instead, the deficit appears to be due primarily to a loss in sexual behavior. Inasmuch as the Cpe^fat mutation disrupts the processing of a number of different peptides, our results suggest that maturation of some peptide(s) in the brain may be an important contributor to the reproductive phenotype in Cpe^fat/fat males.

	Acknowledgments

 
We thank Dr. Edward H. Leiter (The Jackson Laboratory, Bar Harbor, ME) for sending us a colony of BKS.HRS-Cpe^fat/J mice, Dr. A. F. Parlow (Harbor-University of California-Los Angeles Medical Center, Torrance, CA) for the mouse LH and FSH RIA kits, Ms. Kimberly Sue-Ling (Duke University, Durham, NC) for assisting with the size exclusion chromatography, and Dr. Sheila Collins (Duke University) for her comments on the manuscript.

	Footnotes

 
This work was supported in part by NICHHD, NIH Grant HD-36015 (to W.C.W.) and a Summer Fellowship Award from The Endocrine Society (to R.L.R.).

Current address for S.S.: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Box 3175, Durham, North Carolina 27710.

Abbreviations: CPE, Carboxypeptidase E; GAP, GnRH-associated peptide; HPG, hypothalamic-pituitary-gonadal; IR, immunoreactivity; PACAP, pituitary adenylate cyclase-activating polypeptide; PC, prohormone convertase; WT, wild type.

Received October 27, 2003.

Accepted for publication December 29, 2003.

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