help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2003-1442
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Srinivasan, S.
Right arrow Articles by Wetsel, W. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Srinivasan, S.
Right arrow Articles by Wetsel, W. C.
Endocrinology Vol. 145, No. 4 2023-2034
Copyright © 2004 by The Endocrine Society

Deficits in Reproduction and Pro-Gonadotropin-Releasing Hormone Processing in Male Cpefat Mice

Sudha Srinivasan, Donna O. Bunch, Yun Feng, Ramona M. Rodriguiz, Min Li, Roneka L. Ravenell, Guo X. Luo, Akira Arimura, Lloyd D. Fricker, Edward M. Eddy and William C. Wetsel

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
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cpefat/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 Cpefat/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 Cpefat/fat mice. Matings between homozygous mutants yield a 5% pregnancy rate. By comparison, when 50-d-old Cpefat/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 Cpefat/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 Cpefat/fat males appear to be due primarily to abnormal sexual behavior.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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 Cpefat/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 Cpefat/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 Cpefat/fat mice (15, 16).

As obesity and diabetes in Cpefat/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. 1Go). 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 {alpha}-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.

 
Some time ago, we proposed that CPE is involved in processing GnRH intermediates to the mature decapeptide (18, 19, 20, 21). As GnRH is a major regulator of reproduction in mammals, it has been presumed that the infertility in Cpefat/fat mice is due to abnormal pro-GnRH processing (17). Alternatively, the infertility in the Cpefat animals could be due to alterations at other sites within the HPG axis or nervous system. Within this context, Cpefat mice represent an excellent animal model in which to evaluate the role of CPE in pro-GnRH processing in vivo as well as in reproduction. In the present study, only fertility in males was examined, because preliminary studies in females indicate that their loss of reproductive function may occur though additional mechanisms.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The BKS.HRS-Cpefat/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-Cpefat/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, Cpefat 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 Cpefat/fat males for assessment of sexual behavior during the dark phase of the light/dark cycle. The WT and Cpefat/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 Cpefat/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 {gamma}-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 Cpefat/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 Cpefat/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 Cpefat/fat males were conducted with heterozygotes as controls. As fertility was very low for homozygous mutants, subsequent studies compared rates between Cpe+/fat and Cpefat/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 Cpefat/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 Cpefat/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). Cpefat 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 Cpefat/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 {chi}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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hypothalamic GnRH levels
One of the major phenotypes initially ascribed to the Cpefat/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 Cpefat/fat mice permit us not only to evaluate the effects of the Cpefat mutation on GnRH processing, but also to examine in detail their reputed infertility. Peptides were extracted from hypothalami of WT, Cpe+/fat, and Cpefat/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; Cpefat/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; Cpefat/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; Cpefat/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 Cpefat/fat males compared with WT or Cpe+/fat animals (Fig. 2Go, 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. 2Go, 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 Cpefat/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 {square}, WT; , Cpe+/fat; {blacksquare}, Cpefat/fat males. *, P < 0.05 vs. WT and Cpe+/fat mice.

 
Pro-GnRH processing
To examine pro-GnRH processing in Cpefat/fat hypothalamus in greater detail, extracts were sequentially fractioned by size exclusion chromatography and HPLC. Because GnRH levels, as measured by the A772 and B9 antisera, were relatively constant across age, hypothalamic materials from only 90-d-old WT, Cpe+/fat, and Cpefat/fat animals were examined. Samples were separated by size exclusion chromatography and screened with MC-2 antiserum for analyses of prohormone and GAP contents and with B9 antiserum for inclusion of GnRH and all its intermediates. Three peaks of GAP-like IR were discerned, and these materials eluted in the same positions for all three genotypes (Fig. 3Go). One peak eluted in the void volume representing pro-GnRH bound to higher molecular weight proteins (19, 20, 21, 23). Another appeared at approximately 8200 kDa, corresponding to the predicted size of pro-GnRH, and a third coeluted with synthetic rat GAP-(1–56) at approximately 6500 kDa. When the same fractions were screened with B9 antiserum, one peak of GnRH-like IR could be clearly visualized at approximately 1000 kDa from WT and Cpe+/fat hypothalami (Fig. 3Go, top and middle). By comparison, materials from the homozygous mutants eluted earlier at approximately 1300 kDa (Fig. 3Go, bottom). Aside from differences in chromatographic profiles, the molar concentrations of the 6500-kDa GAP-like IR and the 1000- to 1300-kDa GnRH-like IRs were reduced in Cpefat/fat mice. By comparison, levels of 8200-kDa pro-GnRH in Cpefat//fat hypothalami were increased approximately 2-fold over those in the other two genotypes (compare Fig. 3Go, top and middle with bottom).



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.

 
To identify materials in the 1000- to 1300-kDa GnRH peaks, fractions from WT, Cpe+/fat, and Cpefat/fat mice were pooled separately and subjected to HPLC. The chromatographic profiles from WT and heterozygous animals were very similar, whereas those from homozygous mutants were quite different (Fig. 4Go). Compared with WT hypothalami, detailed analyses of the chromatograms revealed that the molar percentages of GnRH intermediates with C-terminal basic amino acids were increased in Cpefat/fat mice by approximately 3- to 284-fold, respectively (Table 1Go). By comparison, the molar percentages of GnRH-[Gly11], [hydroxy-Pro8]GnRH, and GnRH were reduced between 2- to 4-fold, respectively. The distortions in processing were also evident when the molar ratios were considered (Table 1Go). These findings demonstrate that multiple steps in the pro-GnRH processing cascade are affected by the Cpefat mutation.



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.

 

View this table:
[in this window]
[in a new window]
  		TABLE 1. Analyses of processing of pro-GnRH intermediates in hypothalami from WT, Cpe+/fat, and Cpefat/fat mice

 
Anterior pituitary status
The dramatic decrease in hypothalamic GnRH levels in Cpefat/fat mice suggested that anterior pituitary function might be altered. Serum gonadotropins were measured from WT, heterozygous, and homozygous mutant males before the onset of obesity (50 d), at the beginning of weight gain (90 d), and when obesity was apparent in Cpefat/fat animals (200 d). Although basal LH and FSH levels were reduced (25% and 15%, respectively) in Cpefat/fat males only at 200 d of age, concentrations were not statistically different among genotypes (data not shown). These findings demonstrate that basal gonadotropin levels are similar among animals regardless of age.

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. 5Go). Synthetic mammalian GnRH (10 nM) significantly stimulated more LH and FSH secretion in male Cpefat/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 Cpefat/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.

 
Gonadal status
As basal serum gonadotropin levels did not differ among the three genotypes across age, one might suspect that sex hormone contents in blood would be comparable. Although serum testosterone levels were similar among genotypes at 50 and 90 d, they were significantly reduced in Cpefat/fat animals by 200 d and did not decrease beyond this point at 300 d of age (Fig. 6Go). Despite its decline in serum, it should be emphasized that the testosterone concentrations in Cpefat/fat animals were still within the range considered normal for mice (33).



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.

 
To further investigate the infertility phenotype of the Cpefat/fat mice, WT and homozygous mutants were examined before (90 d), during (180 d), and after obesity had reached a maximum (300 d of age). Body weights of the genotypes were not different at the earliest age (WT, 21.9 ± 0.54 g; Cpefat/fat, 22.7 ± 0.67 g), whereas the homozygous mutants were significantly heavier at 180 d (35.9 ± 1.89 g) and 300 d (43.7 ± 2.53 g)of age than WT controls (26.4 ± 0.68 and 27.1 ± 0.83 g, respectively). Testis weights as well as weights of combined seminal vesicles and coagulating glands were significantly lower in Cpefat/fat males at 180 d, and this effect became more pronounced at 300 d of age (Table 2Go). By comparison, no group differences were noted in epididymal and vas deferens weights up to 300 d.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Organ weights and sperm function in WT and Cpefat/fat male mice

 
As gross evaluation might not be sensitive to cellular changes that could contribute to the Cpefat phenotype, testis morphology was examined by light microscopy. Because gross changes in testis status were observed only at the oldest age, testis sections were studied at the beginning of weight gain (90 d) and after it had reached a maximum (300 d). At 90 d of age, testis morphology was similar between WT and Cpefat/fat males (Fig. 7Go, A and B). By 300 d of age, however, some of the seminiferous tubules in homozygous mutants were atrophic and had enlarged lumen, a thin seminiferous epithelium, and a reduction in spermatocytes and spermatids (Fig. 7Go, C and D). Sperm counts and motility were also depressed in the older Cpefat/fat males (Table 2Go). Collectively, these data show that testicular function is decreased, but only in older, obese Cpefat/fat males.



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.

 
The alterations in gonadal status in older homozygous mutants could be attributed to direct effects of the Cpefat mutation on testis or indirectly through the obesity. CPE activity has been measured in testis (34); however, it is not known which cells possess the activity. Our immunocytochemistry studies revealed that pro-CPE-like IR was localized to the acrosomes of spermatids and spermatozoa from both WT (Fig. 7EGo) and Cpefat/fat animals (Fig. 7FGo). No immunostaining was visualized with preimmune serum (Fig. 7Go, I and J). Hence, CPE is located in sperm.

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 Cpefat/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. 8AGo) and PACAP38 (Fig. 8BGo) were decreased in Cpefat/fat testis, whereas they were increased in the 300-d-old mice (Fig. 8Go, C and D). These data indicate that peptide processing of PACAP in sperm is intact at the time when testicular changes are evident in Cpefat/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. 7GGo) and Cpefat/fat (Fig. 7HGo) testis. The staining of tail of sperm was nonspecific because the same staining could be visualized with preimmune serum (Fig. 7Go, 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 Cpefat/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.

 
Fertility studies
The fat/fat mice were reported to be infertile (5). However, there has been no detailed systematic investigation of their reproductive status. We found no differences between fertilities of male and female WT and heterozygous mice (~90%) and, thus, can consider WT and Cpe+/fat mice to be interchangeable as controls. When matings of male and female heterozygotes (65 breeding pairs) were compared with those of homozygous mutants (42 breeding pairs), overall fertility rates were 88% and 5%, respectively. To specifically assess the contribution of the male, Cpefat/fat males were mated with Cpe+/fat females (78 breeding pairs). Only 25.6% of the females became pregnant, suggesting that although Cpefat/fat males displayed reproductive insufficiency, they were not infertile.

A characteristic feature of Cpefat/fat males is that obesity develops after puberty, with diabetes appearing later in life (17). As obesity affects fertility (38), the reproductive competence of Cpefat/fat males was assessed before, at the beginning, and after the development of obesity. To ensure that high rates of fertility would be achieved, Cpefat/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 Cpefat/fat (19.7 ± 0.73 g) males were comparable. Even at this early age, fertility rates (e.g. ~43%) were significantly lower for Cpefat/fat males (Table 3Go). These rates dramatically declined to 9% when Cpefat/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 Cpefat/fat males; the Cpe+/fat males impregnated approximately 85% of the females (Table 3Go). At this older age, Cpefat/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 Cpefat/fat males are subfertile; however, their reproductive performance rapidly declines before obesity develops.


View this table:
[in this window]
[in a new window]
  		TABLE 3. Age-dependent fertility status of male Cpefat mice

 
Sexual behavior
Although the initial decline in fertility of Cpefat/fat males less than 51 d of age is associated with a deficiency in pro-GnRH processing in the hypothalamus, this deficit cannot explain the rapid decline in fertility after this time. As pituitary and gonadal function appear intact at this age, we decided to examine reproductive performance more closely. In our mating studies, evidence of sexual activity was evaluated by inspecting females for vaginal plugs. These data complimented the pregnancy studies by showing that vaginal plugs were readily detected when 50-d-old Cpefat/fat males were bred with Cpe+/fat females; however, they were rarely observed at older ages. These observations suggest that sexual behavior may be dysfunctional in homozygous mutant males.

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 Cpefat/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 Cpefat/fat males to approach the cassette with female urine [WT, 9.8 ± 1.5 sec (n = 10); Cpefat/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; Cpefat/fat, 41.4 ± 19.8 sec). These results demonstrate that WT and Cpefat/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 Cpefat/fat male, both animals engaged in social interaction with the female. Compared with WT males, homozygous mutants spent more time with the female (Table 4Go). Despite this fact, the latencies for Cpefat/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 Cpefat/fat male. These data suggest that although Cpefat/fat males exhibit a high degree of sociability with female mice, they have a profound deficit in functional reproductive behavior.


View this table:
[in this window]
[in a new window]
  		TABLE 4. Analyses of sexual behavior in WT and Cpefat/fat mice

 
The loss of appropriate sexual responses could be attributed to physiological and/or behavioral defects. To address this issue, another group of 90-d-old naive WT and Cpefat/fat males was paired with steroid-primed, ovariectomized, Swiss-Webster females and examined for evidence of penile erections. Although the latencies for the homozygous mutants to climb upon or display partial mounts of the female were delayed compared with those of WT controls (WT, 281.0 ± 42.17 sec; Cpefat/fat, 522.3 ± 195.8 sec), all mice, regardless of genotype, showed penile erections. Hence, the lack of sexual behavior in Cpefat/fat males cannot be attributed to physiological deficiencies.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major objective of our studies was to use the Cpefat 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 Cpefat/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 Cpefat/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 Cpefat/fat males. Mating studies confirm that fertility is very low with homozygous mutant pairings, but this is significantly improved when young Cpefat/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 Cpefat/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 Cpefat/fat mice.

We were interested in studying Cpefat/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 Cpefat 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 Cpefat 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 Cpefat/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 Cpefat mutation, processing of GnRH intermediates would still be incomplete, and the concentrations of fully processed decapeptide would be reduced, as is observed in Cpefat/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 Cpefat/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 Cpefat/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 Cpefat/fat males was also evaluated. It should be noted that many of the morphological and functional deficits in testis from obese Cpefat/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 Cpefat/fat animals. Our analyses reveal, however, that PACAP processing in sperm is intact in obese Cpefat/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 Cpefat/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 Cpefat/fat males are not completely infertile, rather they are subfertile. Young homozygous mutant males can impregnate WT or heterozygous females, but matings with Cpefat/fat females are rarely successful. Despite this fact, fertility rates of young Cpefat/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 Cpefat/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 Cpefat/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 Cpefat/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 Cpefat/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 Cpefat/fat males. Instead, the deficit appears to be due primarily to a loss in sexual behavior. Inasmuch as the Cpefat 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 Cpefat/fat males.


   Acknowledgments
 
We thank Dr. Edward H. Leiter (The Jackson Laboratory, Bar Harbor, ME) for sending us a colony of BKS.HRS-Cpefat/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.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Steiner DF 1998 The proprotein convertases. Curr Opin Chem Biol 2:31–39[CrossRef][Medline]
  2. Seidah NG, Chrétien M 1999 Proprotein and prohormone convertases: a family of subtilases generating diverse bioactive polypeptides. Brain Res 848:45–62[CrossRef][Medline]
  3. Fricker LD 1991 Peptide processing exopeptidases: amino and carboxypeptidases involved in peptide biosynthesis. In: Fricker LD, ed. Peptide biosynthesis and processing. Boca Raton: CRC Press; 199–229
  4. Jackson RS, Creemers JWM, Ohagi S, Raffin-Sanson M-L, Sanders L, Montague CT, Hutton, JC, O’Rahilly S 1997 Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene. Nat Genet 16:303–306[CrossRef][Medline]
  5. Coleman DL, Eichler EM 1990 Fat (fat) and tubby (tub), two autosomal recessive mutations causing obesity syndromes in the mouse. J Hered 81: 424–427
  6. Furuta M, Yano H, Zhou A, Rouille Y, Holst JJ, Carroll R, Ravazzola M, Orci L, Furuta H, Steiner DF 1997 Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2. Proc Natl Acad Sci USA 94:646–6651[Abstract/Free Full Text]
  7. Mbikay M, Tadros H, Ishida N, Lerner CP, De Lamirande E, Chen A, El-Alfy M, Clermont Y, Seidah NG, Chretien M, Gagnon C, Simpson EM 1997 Impaired fertility in mice deficient for the testicular germ-cell protease PC4. Proc Natl Acad Sci USA 94:6842–6846[Abstract/Free Full Text]
  8. Roebroek AJ, Umans L, Pauli IG, Robertson EJ, van Leuven F, Van de Ven WJ, Constam DB 1998 Failure of ventral closure and axial rotation in embryos lacking proprotein convertase furin. Development 125:4863–4876[Abstract]
  9. Constam DB, Robertson EJ 2000 SPC4/PACE4 regulates a TGFß signaling network during axis formation. Genes Dev 14:1146–1155[Abstract/Free Full Text]
  10. Zhu X, Zhou A, Dey A, Norrbom C, Carroll R, Zhang C, Laurent V, Lindberg I, Ugleholdt R, Holst JJ, Steiner DF 2002 Disruption of PC1/3 expression in mice causes dwarfism and multiple peptide processing defects. Proc Natl Acad Sci USA 99:10293–10298[Abstract/Free Full Text]
  11. Westphal CH, Muller L, Zhou A, Zhu X, Bonner-Weir S, Schambelan M, Steiner DF, Lindberg I, Leder P 1999 The neuroendocrine protein 7B2 is required for peptide hormone processing in vivo and provides a novel mechanism for pituitary Cushing’s disease. Cell 96:689–700[CrossRef][Medline]
  12. Naggert JK, Fricker LD, Varlamov O, Nishina PM, Rouille Y, Steiner DF, Carroll RJ, Paigen BJ, Leiter EH 1995 Hyperproinsulinaemia in obese fat/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity. Nat Genet 10:135–142[Medline]
  13. Varlamov O, Leiter EH, Fricker L 1996 Induced and spontaneous mutations at Ser202 of carboxypeptidase E. Effect on enzyme expression, activity, and intracellular routing. J Biol Chem 271:13981–13986[Abstract/Free Full Text]
  14. Nillni EA, Xie W, Mulcahy L, Sanchez VC, Wetsel WC 2002 Deficiencies in pro-thyrotropin-releasing hormone processing and abnormalities in thermoregulation in Cpefat/fat mice. J Biol Chem 277:48587–48595[Abstract/Free Full Text]
  15. Shen F-S, Loh YP 1997 Intracellular misrouting and abnormal secretion of adrenocorticotropin and growth hormone in Cpefat mice associated with a carboxypeptidase E mutation. Proc Natl Acad Sci USA 94:5314–5319[Abstract/Free Full Text]
  16. Rovere C, Viale A, Nahon J, Kitabgi P 1996 Impaired processing of brain proneurotensin and promelanin-concentrating hormone in obese fat/fat mice. Endocrinology 137:2954–2958[Abstract]
  17. Leiter EH, Kintner J, Flurkey K, Beamer WG, Naggert JK 1999 Physiologic and endocrinologic characterization of male sex-biased diabetes in C57BLKS/J mice congenic for the fat mutation at the carboxypeptidase E locus. Endocrine 10:57–66[CrossRef][Medline]
  18. Wetsel WC, Srinivasan S 2002 Pro-GnRH Processing. Prog Brain Res 141:221–241[Medline]
  19. Wetsel WC, Mellon PL, Weiner RI, Negro-Vilar A 1991 Metabolism of pro-luteinizing hormone-releasing hormone in immortalized hypothalamic neurons. Endocrinology 129:1584–1595[Abstract/Free Full Text]
  20. Wetsel WC, Culler MD, Johnston CA, Negro-Vilar A 1988 Processing of the luteinizing hormone-releasing hormone precursor in the preoptic area and hypothalamus of the rat. Mol Endocrinol 2:22–31[Abstract/Free Full Text]
  21. Wetsel WC, Liposits Z, Seidah NG, Collins S 1995 Expression of candidate pro-GnRH processing enzymes in rat hypothalamus and an immortalized hypothalamic neuronal cell line. Neuroendocrinology 62:166–177[Medline]
  22. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275[Free Full Text]
  23. Wetsel WC, Hill DF, Ojeda SR 1996 Basic fibroblast growth factor regulates the conversion of pro-luteinizing hormone-releasing hormone (pro-LHRH) to LHRH in immortalized hypothalamic neurons. Endocrinology 137:2606–2616[Abstract]
  24. Li M, Mbikey M, Arimura A 2000 Pituitary adenylate cyclase-activating polypeptide precursor is processed solely by prohormone convertase 4 in the gonads. Endocrinology 141:3723–3730[Abstract/Free Full Text]
  25. Culler MD, Negro-Vilar A 1986 Development of specific antisera and a radioimmunoassay procedure for the gonadotropin-releasing hormone associated peptide (GAP) of the LHRH prohormone. Brain Res Bull 17:219–223[CrossRef][Medline]
  26. Arimura A, Nishi N, Schally AV 1976 Delayed implantation caused by administration of sheep immunogamma globulin against LHRH in the rat. Proc Soc Exp Biol Med 152:71–75[CrossRef][Medline]
  27. Wetsel WC, Negro-Vilar A 1989 Testosterone selectively influences protein kinase-C-coupled secretion of proluteinizing hormone-releasing hormone-derived peptides. Endocrinology 125:538–547[Abstract/Free Full Text]
  28. Arimura A, Somogyvari-Vigh A, Miyata A, Mizuno K, Coy DH, Kitada C 1991 Tissue distribution of PACAP as determined by RIA: highly abundant in the rat brain and testis. Endocrinology 129:2787–2789[Abstract/Free Full Text]
  29. Culler MD 1993 In vitro techniques for assessing pituitary secretory function. In: Chapin RE, Heindel JJ, eds. Male reproductive toxicology. New York: Academic Press; 26–51
  30. Eddy EM, Washburn TF, Bunch DO, Goulding EH, Gladen BC, Luban DB, Korach KS 1996 Targeted disruption of the estrogen receptor gene in male mice causes alteration of spermatogenesis and infertility. Endocrinology 137:4796–4805[Abstract]
  31. Ogawa S, Lubahn DB, Korach KS, Pfaff DW 2000 Behavioral effects of estrogen receptor gene disruption in male mice. Proc Natl Acad Sci USA 94:1476–1481
  32. Rissman EF, Wersinger SR, Fugger HN, Foster TC 1999 Sex with knockout models: behavioral studies of estrogen receptor {alpha}. Brain Res 835:80–90[CrossRef][Medline]
  33. Beamer WG, Wilson MC, Leiter EH 1983 Endocrinology. In: Foster HL, Small JD, Fox JG, eds. The mouse in biomedical research. New York: Academic Press; vol 3:166–245
  34. Fricker LD, Berman YL, Leiter EH, Devi LA 1996 Carboxypeptidase E activity is deficient in mice with the fat mutation. Effect on peptide synthesis. J Biol Chem 271:30619–30624[Abstract/Free Full Text]
  35. Arimura A, Somogyvari-Vigh A, Miyata A, Mizuno K, Coy DH, Kitada C 1991 Tissue distribution of PACAP as determined by RIA: highly abundant in the rat brain and testis. Endocrinology 129:2787–2789
  36. Song L, Fricker LD 1996 Tissue distribution and characterization of soluble and membrane-bound forms of metallocarboxypeptidase D. J Biol Chem 271:28884–2888[Abstract/Free Full Text]
  37. Wei S, Segura S, Vendrell J, Aviles FX, Lanoue E, Day R, Feng Y, Fricker LD 2002 Identification and characterization of three members of the human metallocarboxypeptidase gene family. J Biol Chem 277:14954–14964[Abstract/Free Full Text]
  38. Huss-Ashmore R 1980 Fat and fertility: demographic implications of differential fat storage. Yerb Phys Anthropol 23:65–92
  39. Johnston RE, Bronson F 1982 Endocrine control of female mouse odors that elicit luteinizing hormone surges and attraction in males. Biol Reprod 27:1174–1180[Abstract]
  40. Vandenbergh JG 1994 Pheromones and mammalian reproduction. In: Knobil E, Neill JD, eds. The physiology of reproduction. New York: Raven Press; 343–359
  41. Fischer WH, Spiess J 1987 Identification of a mammalian glutaminyl cyclase converting glutaminyl into pyroglutaminyl peptides. Proc Natl Acad Sci USA 84:3628–3632[Abstract/Free Full Text]
  42. Busby Jr WH, Quackenbush GE, Humm J, Youngblood WW, Kizer JS 1987 An enzyme(s) that converts glutaminyl-peptides into pyroglutaminyl-peptides. J Biol Chem 262:8532–8536[Abstract/Free Full Text]
  43. Berman Y, Mzhavia N, Polonskaia, Devi LA 2001 Impaired prohormone convertases in Cpefat/Cpefat mice. J Biol Chem 276:1466–1473[Abstract/Free Full Text]
  44. Dong W, Fricker LD, Day R 1999 Carboxypeptidase D is a potential candidate to carry out redundant processing functions of carboxypeptidase E based on comparative distribution studies in the rat central nervous system. Neuroscience 89:1301–1317[CrossRef][Medline]
  45. Varlamov O, Eng FJ, Novikova EG, Fricker LD 1999 Localization of metallocarboxypeptidase D in AtT-20 cells: potential role in prohormone processing. J Biol Chem 274:14759–14767[Abstract/Free Full Text]
  46. Fricker LD, Snyder SH 1982 Enkephalin convertase: purification and characterization of a specific enkephalin synthesizing carboxypeptidase localized to adrenal chromaffin granules. Proc Natl Acad Sci USA 79:3886–3890[Abstract/Free Full Text]
  47. Okada T, Kitamura K, Baba Y, Arimura A, Schally AV 1973 Luteinizing hormone-releasing hormone analogs lacking N-terminal pGLU ring structure. Biochem Biophys Res Commun 53:1180–1186[CrossRef][Medline]
  48. Fujino M, Kobayashi S, Obayashi M, Shinagawa S, Fukuda T, Kitada C, Nakayama R, Yamazaki I 1972 Structure-activity relationships in the C-terminal part of luteinizing hormone releasing hormone (LH-RH). Biochem Biophys Res Commun 49:863–869[CrossRef][Medline]
  49. Asnicar MA, Köster A, Heiman ML, Tinsley F, Smith DP, Galbreath E, Fox N, Ma YL, Blum WF, Hsiung HM 2002 Vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating peptide receptor 2 deficiency in mice results in growth retardation and increased basal metabolic rate. Endocrinology 143:3994–4006[Abstract/Free Full Text]
  50. Jones N, Ainsworth-Harrisson G 1957 Genetically determined obesity and sterility in the mouse. Stud Fertil 9:51–64
  51. Hellman B 1966 Some metabolic aspects of the obese-hyperglycemic syndrome in mice. Diabetologia 3:222–229[CrossRef]
  52. Mounzih K, Lu R, Chehab FF 1997 Leptin treatment rescues the sterility of genetically obese ob/ob males. Endocrinology 138:1190–1193[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Cell Sci.Home page
M. Hosaka, T. Watanabe, Y. Sakai, T. Kato, and T. Takeuchi
Interaction between secretogranin III and carboxypeptidase E facilitates prohormone sorting within secretory granules
J. Cell Sci., October 15, 2005; 118(20): 4785 - 4795.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
L. Marzban, G. Soukhatcheva, and C. B. Verchere
Role of Carboxypeptidase E in Processing of Pro-Islet Amyloid Polypeptide in {beta}-Cells
Endocrinology, April 1, 2005; 146(4): 1808 - 1817.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
P.-S. Tsai, S. M. Moenter, H. R. Postigo, M. El Majdoubi, T. R. Pak, J. C. Gill, S. Paruthiyil, S. Werner, and R. I. Weiner
Targeted Expression of a Dominant-Negative Fibroblast Growth Factor (FGF) Receptor in Gonadotropin-Releasing Hormone (GnRH) Neurons Reduces FGF Responsiveness and the Size of GnRH Neuronal Population
Mol. Endocrinol., January 1, 2005; 19(1): 225 - 236.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
N. X. Cawley, J. Zhou, J. M. Hill, D. Abebe, S. Romboz, T. Yanik, R. M. Rodriguiz, W. C. Wetsel, and Y. P. Loh
The Carboxypeptidase E Knockout Mouse Exhibits Endocrinological and Behavioral Deficits
Endocrinology, December 1, 2004; 145(12): 5807 - 5819.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Srinivasan, S.
Right arrow Articles by Wetsel, W. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Srinivasan, S.
Right arrow Articles by Wetsel, W. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals