This Article Abstract Full Text (PDF) All Versions of this Article: 145/12/5688    most recent Author Manuscript (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, S. P. Articles by Mitchell, G. A. Search for Related Content PubMed PubMed Citation Articles by Wang, S. P. Articles by Mitchell, G. A.

Expression of Human Hormone-Sensitive Lipase (HSL) in Postmeiotic Germ Cells Confers Normal Fertility to HSL-Deficient Mice

Service of Medical Genetics (S.P.W., K.S., H.B., G.A.M.), Hôpital Sainte-Justine, Montréal, Québec H3T 1C5, Canada; Department of Anatomy and Cell Biology (S.C., L.H.), McGill University, Montréal, Québec, Canada, H3A 2B2; and Departments of Pediatrics, Human Genetics, and Pharmacology and Therapeutics (J.T.), McGill University and the Montreal Children’s Hospital, Montréal, Québec, Canada H3H 1P3

Address all correspondence and requests for reprints to: Grant A. Mitchell, M.D., Service of Medical Genetics, Hôpital Sainte-Justine, 3175 Chemin Côte Ste-Catherine, Montréal, Québec, Canada H3T 1C5. E-mail: grant.mitchell{at}recherche-ste-justine.qc.ca.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hormone-sensitive lipase (HSL, Lipe, E.C.3.1.1.3) is a multifunctional fatty acyl esterase that is essential for male fertility and spermatogenesis and that also plays important roles in the function of adipocytes, pancreatic ß-cells, and adrenal cortical cells. Gene-targeted HSL-deficient (HSL–/–) male mice are infertile, have a 2-fold reduction in testicular mass, a 2-fold elevation of the ratio of esterified to free cholesterol in testis, and unique morphological abnormalities in round and elongating spermatids. Postmeiotic germ cells in the testis express a specific HSL isoform. We created transgenic mice expressing a normal human testicular HSL cDNA from the mouse protamine-1 promoter, which mediates expression specifically in postmeiotic germ cells. Testicular cholesteryl esterase activity was undetectable in HSL–/– mice, but in HSL–/– males expressing the testicular transgene, activity was 2-fold greater than normal. HSL transgene mRNA became detectable in testes between 19 and 25 days of age, coinciding with the first wave of postmeiotic transcription in round spermatids. In contrast to nontransgenic HSL–/– mice, HSL–/– males expressing the testicular transgene were normal with respect to fertility, testicular mass, testicular esterified/free cholesterol ratio, and testicular histology. Their cauda epididymides contained abundant, normal-appearing spermatozoa. We conclude that human testicular HSL is functional in mouse testis and that the mechanism of infertility in HSL-deficient males is cell autonomous and resides in postmeiotic germ cells, because HSL expression in these cells is in itself sufficient to restore normal fertility.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
HORMONE-SENSITIVE LIPASE (HSL, gene symbol Lipe, E.C.3.1.1.3) is a multifunctional fatty acyl esterase expressed in postmeiotic male germ cells of the testis (1, 2) as well as several tissues involved in energy homeostasis, including white and brown adipose tissue, adrenal cortex, and pancreatic ß-cells. The testicular isoform of HSL has two domains. The C-terminal region corresponds to the complete, catalytically active sequence identical with that of the major HSL isoforms of nontesticular tissues. The 303-residue N-terminal domain, encoded by a testis-specific exon (see Fig. 1) is of unknown function. HSL accounts for a major fraction of triglyceride hydrolase activity and for all detectable cholesteryl esterase activity in the testis (3, 4).

View larger version (24K): [in this window] [in a new window]   FIG. 1. The Lipe gene, HSL mRNAs, and the testicular HSL transgene. a, The numerous alternative first exons of HSL are designated as described (29 ) plus the main testicular exon (T1) and the orthologous sequence to the testicular exon reported in humans, designated T2. b and c, The testicular exon and all nontesticular exons splice to a common site 20 nucleotides upstream of the ATG start codon in exon 1. Exons T1 and C have in-frame upstream start codons and encode unique N-terminal extensions to the coding region shared by all known HSL isoforms. d, The human testicular HSL cDNA transgene. The protamine 1 gene promoter (hatched), 5' untranslated region (UTR, white), testicular exon (dark gray), and the rest of the HSL cDNA coding sequence (light gray), 3' UTR and polyadenylation signal (PA, white) are indicated, as are important restriction sites (see Materials and Methods). The 1-kb size marker refers to d.

To better understand the mechanisms underlying the infertility seen in HSL deficiency, we created transgenic mice expressing a normal human testicular HSL cDNA from the protamine-1 promoter, which has been shown to be specific for postmeiotic male germ cells (12). We then bred this transgene onto a HSL-deficient background.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals
Mice were maintained on a 12-h light, 12-h dark photoperiod and were provided with food and water ad libitum. The gene-targeted HSL-deficient mouse strain has been reported (4). The gene-targeting event causes the removal of the 498 amino-terminal codons and results in undetectable HSL activity and protein levels in tissues, including testis. F₁ heterozygotes were bred into a C57BL/6J background for at least eight generations. Genotyping was by PCR analysis (13) of tail DNA as described (4). Three-month-old mice were used for all studies reported here (n = 4/genotype). Organs were weighed in 3-month-old HSL–/– mice and controls.

Vector construction
A human HSL cDNA was cloned downstream of the mouse protamine-1 promoter in the pPrCExV-1 vector (14), a gift from Robert E. Braun (Fig. 1). A full-length testicular HSL cDNA was assembled in three cloning steps. In HSL cDNAs, the adenine of the initiation methionine codon in exon 1 is designated as position +1. Using the clone hHSL(1.3)/blue (15), a gift from C. Holm, which contains 1.3 kb from the 3' region of the human HSL cDNA, we introduced two point mutations, A1250C and C1253A. These mutations do not change the predicted amino acid sequence but create an NdeI site at nucleotide 1250. Second, to obtain a full-length adipose tissue HSL cDNA, we amplified a fragment corresponding to residues –18 to 1260 of the human HSL cDNA using RT-PCR, using a sense primer 5'-TCAAGGCTCATCCACAACAT-3' (residues –18 to 2) and an antisense primer 5'-CGCAAGGCAtATgCGTTCCCCTGTTTGA-3' (1236–1262) and containing the C1250/A1253 mutations, shown in lowercase. This amplicon was cloned into the NdeI site of the cDNA and the SalI site of the pBluescript (SK) polylinker (Stratagene, La Jolla, CA), creating a full-length human HSL cDNA corresponding to the major adipose HSL isoform. Finally, a cDNA containing the coding sequence from the testicular HSL exon, plus 88 bp of exon 1, was amplified from human testis total RNA (Clontech, Pal Alto, CA) and inserted into the naturally occurring XhoI site at position 63 of the human adipose HSL cDNA clone and a cloning XhoI site in pBluescript, creating a full-length human testicular HSL cDNA. This was cloned 3' to the protamine-1 promoter, in the BamH1 site of pPrCExV-1. In the resulting vector, designated pPrHSLt, the sequence and orientation of inserts were verified by sequencing on both strands.

Genotyping of transgenic mice
Isolation of mouse tail genomic DNA was as described (16). We used 200 ng of genomic DNA for amplification. The sense primer, Prt-1 (5'-ACCCCTGCTCACAGGTTGGC-3') corresponds to residues –57 to –77 upstream of the adenine of the initiation methionine codon of mouse protamine-1 cDNA. The antisense primer, LipH68 (5'-CCTGGCTCGAGAAGAAGGCT-3'), is complementary to nucleotides 956–976 of the human testicular HSL cDNA. PCR conditions were as described (16) except that Prt-1 and LipH68 were used as primers. Amplification of a 1.1-kb fragment was diagnostic for the presence of the transgene.

Founders testing positive by PCR were also genotyped by Southern blotting. Mouse genomic DNA (5 µg) was digested with HindIII and probed with a 1.3-kb fragment spanning residue 1263 to the 3' extremity of the human HSL cDNA. In normal C57BL/6 mice (not shown), this probe hybridizes neither to genomic DNA nor to adipose tissue mRNA. Using this probe, an 8-kb HindIII fragment was detectable in the transgenic mice, corresponding to an internal fragment of the transgenic insert.

Production of transgenic mice expressing human testicular HSL in male germ cells
The vector was digested with HindIII to remove plasmid sequences, then purified with a kit (no. 20021, QIAGEN, Valencia, CA). Microinjections were performed on 450 C57BL/6 mouse embryos, which were transferred to pseudopregnant females. Founders were bred with HSL-deficient or heterozygous mice. The testicular HSL transgene (ttg) was bred onto a HSL-deficient background to obtain males with no endogenous HSL expression (HSL–/– ttg).

Assessment of fertility
Three-month-old males of three different genotypes (HSL+/+ ttg, HSL–/– ttg, and HSL–/–; n = 6/genotype) were each mated with one to two 2-month-old CD-1 females (Charles River Laboratories, Inc., St. Constant, Canada). Males and virgin females were housed together for 14 d. Females were evaluated for the presence of copulation plugs, pregnancy, and litter size. Females failing to become pregnant after mating with HSL–/– males were later demonstrated to be fertile by mating with HSL+/+ males.

Northern blot and enzymatic studies
For Northern analysis, RNA was extracted from testes of HSL–/–ttg mice aged 10, 19, and 25 d and 3 months, as well as the testes of 3-month-old controls. To test for testis-specific transgene expression, we also extracted RNA from other organs, including white and brown fat, spleen, heart, and skeletal muscle. Northern blotting was as described (17) using the above-mentioned 3' 1.3-kb HSL cDNA probe and a control oligonucleotide (5'-CGGAACTACGACGGTATCTG-3') that hybridizes to 18S rRNA (18).

Cholesteryl esterase was measured as described (19). The contents of cholesteryl ester and of free cholesterol in testes were determined as described (20).

Antibody production and Western blots
An N-terminal human HSL cDNA fragment containing codons 1–323 of adipose HSL was cloned into pEt-30a(+) (Novagen, Madison, WI). After addition of isopropylthio-ß-galactoside, cultures were incubated to an OD of 0.6, and then the expressed HSL peptide was isolated from the inclusion body. The expressed peptide contained an N-terminal His tag and was purified as recommended by the company. The His tag was removed by enterokinase digestion. The HSL peptide was solubilized in 0.1 M Tris-Cl (pH 8.0) containing 0.1% L-sarcosine, 20% glycerol, and 10 mM 1,4-dithioerythritol. Polyclonal rabbit anti-HSL antibodies were produced (Invitrogen, Carlsbad, CA).

For Western blots, 20 µg of fat-free infranatant proteins, resolved using SDS-PAGE, were electroblotted to a polyvinylidene difluoride membrane in ice-cold 25 mM Tris, 192 mM glycine (pH 8.3) at 100 V constant voltage for 1 h. After transfer, the membrane was blocked overnight at 4 C with a solution of Tris-buffered saline (TBS) containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5% (wt/vol) skim milk, and 0.1% Tween 20 and then incubated at room temperature with a 1:8000 dilution of HSL antiserum in TBS containing 1% skim milk and 0.1% Tween 20 for 1 h. The membrane was washed three times for 10 min each with TBS containing 0.1% Tween 20 and then incubated for 1 h in a 1:10,000 dilution of peroxidase-conjugated goat antirabbit IgG in the above buffer. The membrane was washed three times for 10 min each with TBS containing 0.1% Tween 20. The bound antibody was detected by the BM chemiluminescence blotting substrate peroxidase (POD) detection system according to instructions from the manufacturer (Roche Diagnostics, Laval, Canada).

Light microscopy
Three 3-month-old mice of each genotype (HSL+/+ ttg, HSL–/– ttg, and HSL–/–) were anesthetized with sodium pentobarbital (Somnitol, MTC Pharmaceuticals, Hamilton, Canada) and perfusion fixed through the heart with 5.0% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). After perfusion, tissues were collected, cut into 1-mm³ pieces, processed, embedded in Epon, and prepared for light microscopy as previously described (21).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Results of microinjections and characterization of the human testicular HSL transgene
Oocyte microinjection of the vector containing the protamine-1 promoter and the entire coding region of the human testicular HSL cDNA (Fig. 1) yielded 32 pups. Three (9%) were transgenic, two males and one female. One male transmitted the transgene. His progeny was used for further studies. Genomic Southern blotting with a probe specific to the human HSL cDNA revealed the predicted 8-kb HindIII fragment in testicular HSL transgenic mice (Fig. 2A), which segregated as an autosomal trait (not shown). Transgenic HSL mRNA was easily detectable in testes of HSL ttg mice (Fig. 2B). In contrast, transgene expression was undetectable in white adipose tissue (Fig. 2B) and also in brown fat, spleen, heart, and skeletal muscle (not shown). Similarly, by Western blotting, transgenic human testicular HSL was detectable in the testis but not in white adipose tissue (Fig. 3).

View larger version (58K): [in this window] [in a new window]   FIG. 2. Genomic integration and expression of the human testicular HSL transgene. A, Southern blot of genomic DNA digested with HindIII and revealed by the human 3' HSL cDNA probe. An approximately 8-kb fragment identifies the transgene. B, Northern blot of white adipose tissue and testicular homogenates using the 1.3-kb HSL cDNA probe specific to human HSL, showing testis-specific expression of the human HSL transgene. Tissues and genotypes are indicated.

View larger version (36K): [in this window] [in a new window]   FIG. 3. Western analysis of transgenic HSL expression in tissues of 3 month-old mice. The antibody detects a region common to testicular and nontesticular HSL isoforms. Tissues and genotypes are indicated.

View larger version (69K): [in this window] [in a new window]   FIG. 4. Effect of age on expression of HSL ttg mRNA in testes. The probe is a 1.3-kb HSL cDNA fragment that detects only human HSL. HSL genotypes and ages are indicated. 18S, 18S rRNA subunit.

The gross examination of the male genital tract in 3-month-old HSL–/– ttg mice was normal, including the weights of testes (Table 1) and seminal vesicles (data not shown). In contrast, the masses of brown fat depots were abnormally enlarged in transgenic HSL–/– mice, as in nontransgenic HSL-deficient mice (Table 1).

Seminiferous tubules at stage VI of the seminiferous epithelial cycle are shown in Fig. 5. Histologically, compared with HSL +/+ ttg mice (Fig. 5A) and HSL +/+ mice (not shown), the testes of HSL–/– ttg males were also histologically normal (Fig. 5B). In contrast, HSL–/– mice showed few if any elongating spermatids in the seminiferous epithelium (Fig. 5C). All stages of the seminiferous tubule cycle (I–XII) were studied and were similar in HSL–/– ttg, HSL+/+ ttg and HSL+/+ mice (not shown). Abundant mature spermatozoa were present in the epididymides of both HSL–/– ttg (Fig. 5E) and HSL+/+ ttg mice (Fig. 5D), whereas in HSL–/– mice, spermatozoa were absent, and only round, degenerating cells were noted (Fig. 5F).

View larger version (154K): [in this window] [in a new window]   FIG. 5. Effect of human testicular HSL transgene expression on testicular histology. A–C, Stage VI tubules from HSL+/+ ttg (A), HSL–/– ttg (B), HSL–/– (C) mice. Seminiferous tubule histology is normal in transgenic mice on both HSL normal (A) and HSL-deficient (B) backgrounds but markedly abnormal in nontransgenic HSL–/– mice (C). Whereas abundant normal-appearing elongating spermatids (arrows) are present in HSL+/+ ttg and HSL–/– ttg mice, few if any are seen in the seminiferous tubules of HSL–/– mice. D–F, Cauda epididymidis from HSL+/+ ttg (D), HSL–/– ttg (E), and HSL–/– (F) mice. Similar concentrations of spermatozoa (S) are present in the lumen (Lu) of epididymides of HSL+/+ ttg and HSL–/– ttg mice, but spermatozoa are absent, and only round cells (curved arrows) are seen in HSL–/– mice. G–I, Brown adipose tissue from HSL+/+ (G), HSL–/– ttg (H), and HSL–/– (I) mice. The brown adipose tissue histology is abnormal and similar in HSL–/– mice and in HSL–/– ttg mice (4 ).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We created transgenic mice that overexpress human testicular HSL in a testis-specific fashion. Our evidence suggests that expression of the testicular transgene, as for the normal endogenous testicular HSL isoform, is confined to postmeiotic germ cells. First, the transgenic protamine promoter used in these mice has previously been shown to mediate postmeiotic germ-cell-specific expression (12). Second, the onset of transgenic HSL expression corresponds to the initial wave of meiotic division and development of round spermatids in mouse testis.

The human transgene is functional in mouse testis, as shown by the normal testicular histology of HSL–/– ttg mice. Although previous immunocytochemical studies have described low-level HSL expression in type B spermatogonia and primary spermatocytes (8), the significance of this is unknown. The demonstration in this article of correction of the infertility and testicular histological abnormalities of the HSL–/– mice by postmeiotically expressed HSL suggests that HSL expression elsewhere than in postmeiotic germ cells is not essential for spermatogenesis.

Before this work, hypotheses for the infertility of HSL–/– mice included 1) a direct effect of HSL deficiency, 2) possible humeral influences resulting from systemic HSL deficiency, and 3) inadvertent interference with other genes near the targeted Lipe locus. Of note, the infertility of HSL–/– mice is not because of androgen deficiency. Seminal vesicle mass and sexual activity, both of which reflect testosterone action, are normal in transgenic HSL–/– males, as in nontransgenic HSL–/– males (7). Furthermore, testosterone levels are normal in HSL–/– mice (3). The observations in testicular transgenic mice clearly demonstrate that male sterility in HSL deficiency arises directly from HSL deficiency in round spermatids and/or later postmeiotic male germ cells.

Although normal spermatogenesis is abolished by complete HSL deficiency, the minimum level of HSL activity necessary for normal spermatogenesis is unknown. A more than 6-fold range of HSL activity from 50% of normal in heterozygous HSL+/–mice (7) to the increased activity of HSL+/+ ttg mice is compatible with normal fertility and testicular morphology.

The striking increases of HSL mRNA and protein in transgenic mice (Figs. 2 and 3) were associated with only a 2-fold increase of cholesteryl esterase activity (Table 1). This may reflect intrinsic differences in specific activity between human and mouse testicular HSL, or perhaps may be related to modification of the human HSL within the mouse germ cells. For instance, cAMP- and MAPK-related phosphorylation of HSL is well documented in adipocytes (22, 23). It is plausible that in the testis of the transgenic mice, HSL phosphorylation may be altered in response to the nature or amounts of the transgenic HSL.

This study also demonstrates that human testicular HSL is functional in mouse cells. This was not previously apparent, because human and mouse HSL differ by numerous amino acid deletions and substitutions throughout their sequences (24) and in the testis-specific N terminal. In adipose tissue, HSL is reported to interact with at least two proteins, adipocyte fatty acid binding protein (25) and lipotransin (26). If testicular HSL also engages in protein-protein interactions, our observations imply that human HSL can interact with the murine counterparts of its normal partners.

In the course of this work, a second testicular HSL isoform was described in humans (27). In this isoform, use of an alternate nontranslated first exon produces a distinct HSL mRNA, but the predicted translation product is identical to the main adipose HSL isoform, lacking the 303-residue N-terminal sequence of the major testicular isoform and of the testicular transgene described here. Based on published reports, the mRNA of this isoform appears to be absent or expressed at a low level in rodent testis (21, 28, 29). By homology searching (not shown), we identified the murine ortholog of the other human HSL testicular exon, between residues –6659 and –6502 of the mouse Lipe gene (AF179427), designated T2 in Fig. 1. Our study does not define the role, if any, of the other testicular HSL isoform. However, we demonstrate that normal fertility is possible in animals expressing only the major testicular HSL isoform exclusively in postmeitotic germ cells. Therefore, expression from the recently described exon and/or expression of HSL isoforms elsewhere than in postmeiotic germ cells is not essential for fertility.

These observations illustrate the usefulness of transgenic manipulation for studies of testicular metabolism. The study of testicular metabolism is technically challenging because of the complex cytoarchitecture of the testis and the small amounts of tissue available. Traditional cell separation techniques (30) can be used for biochemical studies of isolated germ cells in normal mice but may be difficult to interpret in mice with abnormalities in the seminiferous epithelium such as those seen in HSL–/– mice (7).

There is considerable interest worldwide in the development of efficient male contraception (31). Although most current strategies are centered on suppression of male hormone action (32), postmeiotic germ-cell-specific peptides represent potential targets for contraceptive development (21, 33, 34). The postmeiotic germ-cell-specific testicular HSL isoform may prove to be an attractive target for male contraception, given that its absence causes absolute male infertility in a cell autonomous fashion.

	Acknowledgments

 
We thank Linge Pan and Robert Sztrolovics for technical assistance.

	Footnotes

 
This work was funded by Canadian Institutes for Health Research Grant MOP38045 to G.M. and J.T. and by the Canadian Genetic Diseases Network. J.T. is a Scholar of the Fonds de recherche en santé du Québec and a William Dawson Scholar of McGill University.

Abbreviations: HSL, Hormone-sensitive lipase; HSL ttg, human testicular HSL transgene; TBS, Tris-buffered saline.

Received July 15, 2004.

Accepted for publication August 24, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Holm C, Osterlund T, Laurell H, Contreras JA 2000 Molecular mechanisms regulating hormone-sensitive lipase and lipolysis. Ann Rev Nutr 20:365–393[CrossRef][Medline]
2. Holst LS, Langin D, Mulder H, Laurell H, Grober J, Bergh A, Mohrenweiser HW, Edgren G, Holm C 1996 Molecular cloning, genomic organization, and expression of a testicular isoform of hormone-sensitive lipase. Genomics 35:441–447[CrossRef][Medline]
3. Osuga JI, Ishibashi S, Oka T, Yagyu H, Tozawa R, Fujimoto A, Shionoiri F, Yahagi N, Kraemer FB, Tsutsumi O 2000 Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity. Proc Natl Acad Sci USA 97:787–792[Abstract/Free Full Text]
4. Wang SP, Laurin N, Himms-Hagen J, Rudnicki MA, Levy E, Robert MF, Pan L, Oligny L, Mitchell GA 2001 The adipose tissue phenotype of hormone-sensitive lipase deficiency in mice. Obes Res 9:119–128[Medline]
5. Roduit R, Masiello P, Wang SP, Li H, Mitchell GA, Prentki M 2001 A role for hormone-sensitive lipase in glucose-stimulated insulin secretion: a study in hormone-sensitive lipase-deficient mice. Diabetes 50:1970–1975[Abstract/Free Full Text]
6. Li H, Brochu M, Wang SP, Rochdi L, Côté M, Mitchell G, Gallo-Payet N 2002 Hormone-sensitive lipase deficiency in mice causes lipid storage in the adrenal cortex and impaired corticosterone response to corticotropin stimulation. Endocrinology 143:3333–3340[Abstract/Free Full Text]
7. Chung S, Wang SP, Pan L, Mitchell GA, Trasler J, Hermo L 2001 Infertility and testicular defects in hormone-sensitive lipase-deficient mice. Endocrinology 142:4272–4281[Abstract/Free Full Text]
8. Blaise R, Guillaudeux T, Tavernier G, Daegelen D, Evrard B, Mairal A, Holm C, Jegou B, Langin D 2001 Testis hormone-sensitive lipase expression in spermatids is governed by a short promoter in transgenic mice. J Biol Chem 276:5109–5115[Abstract/Free Full Text]
9. Blaise R, Grober J, Rouet P, Tavernier G, Daegelen D, Langin D 1999 Testis expression of hormone-sensitive lipase is conferred by a specific promoter that contains four regions binding testicular nuclear proteins. J Biol Chem 274:9327–9334[Abstract/Free Full Text]
10. Arenas MI, Lobo MV, Caso E, Huerta L, Paniagua R, Martin-Hidalgo MA 2004 Normal and pathological human testes express hormone-sensitive lipase and the lipid receptors CLA-1/SR-BI and CD36. Hum Pathol 35:34–42[CrossRef][Medline]
11. Kabbaj O, Holm C, Vitale ML, Pelletier RM 2001 Expression, activity, and subcellular localization of testicular hormone-sensitive lipase during postnatal development in the guinea pig. Biol Reprod 65:601–612[Abstract/Free Full Text]
12. Zambrowicz BP, Harendza CJ, Zimmermann JW, Brinster RL, Palmiter RD 1993 Analysis of the mouse protamine 1 promoter in transgenic mice. Proc Natl Acad Sci USA 90:5071–5075[Abstract/Free Full Text]
13. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H 1986 Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp Quant Biol 51 Pt1:263–273
14. Braun RE, Behringer RR, Peschon JJ, Brinster RL, Palmiter RD 1989 Genetically haploid spermatids are phenotypically diploid. Nature 337:373–376[CrossRef][Medline]
15. Holm C, Kirchgessner TG, Svenson KL, Fredrikson G, Nilsson S, Miller CG, Shively JE, Heinzmann C, Sparkes RS, Mohandas T, Lusis AJ, Belfrage P, Schotz M 1988 Hormone-sensitive lipase: sequence, expression, and chromosomal localization to 19 cent-q13.3. Science 241:1503–1506[Abstract/Free Full Text]
16. Wang SP, Marth JD, Oligny L, Vachon M, Robert M-F, Ashmarina L, Mitchell GA 1998 3-Hydroxy-3-methylglutaryl-CoA lyase (HL): gene targeting causes prenatal lethality in HL-deficient mice. Hum Mol Genet 7:2057–2062[Abstract/Free Full Text]
17. Wang S, Nadeau JH, Duncan A, Robert M-F, Fontaine G, Schappert K, Johnson KR, Zietkiewicz E, Hruz P, Miziorko H, Mitchell GA 1993 3-Hydroxy-3-methylglutaryl coenzyme A lyase (HL): cloning and characterization of a mouse liver HL cDNA and subchromosomal mapping of the human and mouse HL genes. Mamm Genome 4:382–387[CrossRef][Medline]
18. Deindl E 2001 18S ribosomal RNA detection on Northern blot employing a specific oligonucleotide. Biotechniques 31:1250:1252[Medline]
19. Holm C, Osterlund T 1999 Hormone-sensitive lipase and neutral cholesteryl ester lipase. Methods Mol Biol 109:109–121[Medline]
20. Dutta J, Biswas A, Saha S, Deb C 1976 A method for the estimation of free and esterified cholesterol involving thin-layer chromatography. J Chromatogr 124:29–36[CrossRef][Medline]
21. Trasler J, Saberi F, Somani IH, Adamali HI, Huang JQ, Fortunato SR, Ritter G, Gu M, Aebersold R, Gravel RA, Hermo L 1998 Characterization of the testis and epididymis in mouse models of human Tay Sachs and Sandhoff diseases and partial determination of accumulated gangliosides. Endocrinology 139:3280–3288[Abstract/Free Full Text]
22. Anthonsen MW, Rönnstrand L, Wernstedt C, Degerman E, Holm C 1998 Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro. J Biol Chem 273:215–221[Abstract/Free Full Text]
23. Greenberg AS, Shen WJ, Muliro K, Patel S, Souza SC, Roth RA, Kraemer FB 2001 Stimulation of lipolysis and hormone-sensitive lipase via the extracellular signal-regulated kinase pathway. J Biol Chem 276:45456–45461[Abstract/Free Full Text]
24. Sztrolovics R, Wang SP, Lapierre P, Chen HS, Robert M-F, Mitchell GA 1997 Hormone-sensitive lipase (Lipe): sequence analysis of the 129Sv mouse Lipe gene. Mamm Genome 8:86–89[CrossRef][Medline]
25. Shen WJ, Patel S, Hong R, Kraemer FB 2000 Hormone-sensitive lipase functions as an oligomer. Biochemistry 39:2392–2398[CrossRef][Medline]
26. Syu LJ, Saltiel AR 1999 Lipotransin: a novel docking protein for hormone-sensitive lipase. Mol Cell 4:109–115[CrossRef][Medline]
27. Mairal A, Melaine N, Laurell H, Grober J, Holst LS, Guillaudeux T, Holm C, Jegou B, Langin D 2002 Characterization of a novel testicular form of human hormone-sensitive lipase. Biochem Biophys Res Commun 291:286–290[CrossRef][Medline]
28. Holst LS, Hoffmann AM, Mulder H, Sundler F, Holm C, Bergh A, Fredrikson G 1994 Localization of hormone-sensitive lipase to rat Sertoli cells and its expression in developing and degenerating testes. FEBS Lett 355:125–130[CrossRef][Medline]
29. Laurin N, Wang SP, Mitchell GA 2000 The hormone-sensitive lipase gene is transcribed from at least five alternative first exons in mouse adipose tissue. Mamm Genome 11:972–978[CrossRef][Medline]
30. Bellve AR, Millette CF, Bhatnagar YM, O’Brien DA 1977 Dissociation of the mouse testis and characterization of isolated spermatogenic cells. J Histochem Cytochem 25:480–494[Medline]
31. Weber RF, Dohle GR 2003 Male contraception: mechanical, hormonal and nonhormonal methods. World J Urol 21:338–340[CrossRef][Medline]
32. Wang C, Swerdloff RS 2004 Male hormonal contraception. Am J Obstet Gynecol 190:S60–S68
33. Ivell R, Danner S, Fritsch M 2004 Postmeiotic gene products as targets for male contraception. Mol Cell Endocrinol 216:65–74[CrossRef][Medline]
34. Schultz N, Hamra FK, Garbers DL 2003 A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets. Proc Natl Acad Sci USA 100:12201–12206[Abstract/Free Full Text]

This article has been cited by other articles:

				M. Fex, S. Lucas, M. S. Winzell, B. Ahren, C. Holm, and H. Mulder {beta}-Cell Lipases and Insulin Secretion Diabetes, December 1, 2006; 55(Supplement_2): S24 - S31. [Abstract] [Full Text] [PDF]

				M. Fortier, K. Soni, N. Laurin, S. P. Wang, P. Mauriege, F. R. Jirik, and G. A. Mitchell Human hormone-sensitive lipase (HSL): expression in white fat corrects the white adipose phenotype of HSL-deficient mice J. Lipid Res., September 1, 2005; 46(9): 1860 - 1867. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 145/12/5688    most recent Author Manuscript (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, S. P. Articles by Mitchell, G. A. Search for Related Content PubMed PubMed Citation Articles by Wang, S. P. Articles by Mitchell, G. A.