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Endocrinology Vol. 141, No. 7 2624-2630
Copyright © 2000 by The Endocrine Society

ARTICLES

Expression of the Leptin Receptor during Germ Cell Development in the Mouse Testis1

Talal El-Hefnawy2, Sergey Ioffe and Martin Dym

Department of Cell Biology, Georgetown University Medical Center, Washington, D.C. 20007

Address all correspondence and requests for reprints to: Dr. Martin Dym, Department of Cell Biology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington, D.C. 20007. E-mail: dymm{at}gunet.georgetown.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leptin, a recently identified hormonal product of the ob gene, is known to regulate appetite, body metabolism, and reproductive functions. We investigated the expression of the leptin receptor (Ob-R) in testes from different age groups. The messenger RNA for Ob-R was found in testes from all age groups using RT-PCR. Using immunohistochemistry, we observed age- and stage-dependent distribution of the Ob-R in mouse testis. In testis of 5-day-old mice, its expression was mainly in type A spermatogonia. In the 20- and 30-day-old testis, Ob-R expression was in the spermatocytes; in the adult testis, it was specific to spermatocytes in stages IX and X of the cycle of the seminiferous epithelium. Five main immunoreactive proteins were detected using Western blot (220, 120, 90, 66, and 46 kDa). The 120-kDa protein was evident only in 20-day-old and older testes, whereas the 90-kDa band was present only in the 5- and 10-day-old testis. Leptin treatment induced phosphorylation of signal transducer and activator of transcription-3 in cultured seminiferous tubules from adult and 5-day-old testes. Our results show for the first time age- and stage-specific localization of a functional Ob-R in testicular germ cells. We hypothesize a direct role for leptin, through phosphorylation of signal transducer and activator of transcription-3, in proliferation and differentiation of germ cells, which may partially explain the infertility observed in leptin-deficient mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SPERMATOGENESIS is the process by which type A spermatogonial stem cells divide and differentiate to give rise to mature spermatids (1). Hormones and growth factors are involved in this unique and complex process. Many of these products have been studied extensively, but their exact role in the spermatogenic process remains to be elucidated. Leptin is a newly identified 167-amino acid product of white adipose tissue that was discovered to reduce weight, body fat, and food intake of the genetically obese Ob-/Ob- (ob/ob) mouse (2, 3, 4, 5). These male mice were infertile and had small testes, azoospermia, and multinucleated spermatids. Leptin treatment was able to reverse the reproductive lesions in the Ob mutant mice (6). Female Ob-/Ob- mice were also infertile, and leptin treatment restored ovulation and gave rise to normal pregnancy and parturition in the treated animals (7). The Ob-/Ob- animals had reduced levels of GnRH (8), which was assumed to be the cause of the infertility due to subsequent reduction of LH and FSH levels (9). In these studies, however, it was clear that the LH levels were not dramatically reduced, whereas the FSH levels were 40% less than normal. Thus, the reproductive lesion in the male Ob-/Ob- animals may be due to factors other than reduced gonadotropin levels; in addition, previous studies rebutted the importance of FSH in maintaining spermatogenesis (10, 11).

Leptin acts through binding to a specific obese receptor (Ob-R), of which several isoforms exist that differ mainly in the length of the cytoplasmic domain (12, 13, 14, 15, 16, 17, 18). In common with several other cytokines, leptin was found to stimulate proliferation and differentiation in the hemopoiesis process (19, 20). Upon binding to its receptor, leptin acts through phosphorylation of signal transducers and activators of transcription (STAT3) and mitogen-activated protein kinase (ERK), followed by induction of transcription of several genes, including protooncogenes such as jun-B (15, 21, 22, 23). Activation of the STAT3 signaling pathway is known to be an important modulator of stem cell renewal and differentiation (24, 25, 26).

Abnormal leptin levels are associated in many cases with reproductive defects (27, 28) and polycystic ovaries (29, 30). In the ovary, where Ob-R is expressed (31, 32), leptin was shown to regulate granulosa cell steroidogenesis (33, 34, 35) and oocyte maturation through the STAT3 signaling pathway. Ob-R messenger RNA (mRNA) was shown to be present in the adult rat testis (36) and in mouse Leydig tumor cells (mLTC-1) using RT-PCR (37). Leptin down-regulates LH/hCG-induced steroidogenesis, however, it stimulates hCG-induced cAMP production in cultured rat Leydig cells and cultured rat testis (37, 38). In addition, another recent study demonstrated the ability of leptin to cross the blood-testis barrier (39), allowing it to access possible target cells in the seminiferous epithelium. In the present study we investigated the age- and stage-specific distribution of the Ob-R as well as the potential direct actions of leptin on the signaling transduction pathways in mouse seminiferous epithelium.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissue preparations
C57/BL6 mice of different ages (see Results) were obtained from Charles River Laboratories, Inc. (Wilmington, MA). The animals were provided with a standard laboratory diet and normal light hours (14 h of light, 10 h of darkness) and temperature (21–22 C). Mice were killed in the morning by decapitation 2 days after their arrival from the supplier, and tissues were frozen directly on dry ice for total protein/RNA isolation or were fixed in 4% paraformaldehyde (pH 7.2) at 4 C for immunostaining. On the following day, the fixed tissues were placed in 10% sucrose in PBS and a day later in 20% sucrose at 4 C. The tissues were then embedded in AOC freezing medium and stored at -60 C before sectioning. Mice treated with busulfan received 40 mg/kg BW, ip, at 6 weeks of age, and a month later the testes were isolated and snap-frozen for protein isolation.

RNA extraction, RT-PCR reactions, and Southern hybridization
Total RNA was obtained from different mouse tissues (two independent isolations from two mice of each age group) using the single step method of Chomczynski and Sacchi (40). Total RNA was treated for 20 min with ribonuclease-free deoxyribonuclease (RQ-DNase, Promega Corp., Madison, WI) to eliminate contaminating genomic DNA. One microgram of the total RNA was reverse transcribed using reverse transcriptase (Superscript, Life Technologies, Inc., Paisley, Scotland) and hexamer primers mix (Life Technologies, Inc.) for 1 h at 42 C. Two microliters of the first strand reaction were then included as template for the PCR reactions using Taq polymerase (Life Technologies, Inc.). The oligonucleotide primers (forward primer, 5'-CCAAAATTCTGACTAGTGTTGGATCGA-3'; reverse primer, 5'-AGCCGTCTCTCTGTAAGACGCAGT-3') for Ob-R were designed to flank two introns and to amplify a 470-bp fragment from nucleotides 1067–1537 of the published sequence for the mouse leptin receptor. Full-length Ob-R was used as a positive control for the PCR reaction. The PCR reaction started at 94 C for 3 min, followed by 39 cycles (94 C for 40 sec, 56 C for 50 sec, and 72 C for 50 sec), and ended with a 10-min incubation at 72 C. The complementary DNA (cDNA) amplicons were resolved on a 2% agarose gel, denatured, and transferred to nylon membrane (Amersham Pharmacia Biotech, Aylesbury, UK).

Southern hybridization was performed using a 32P end-labeled antisense nested oligonucleotide probe (5'-TGATTGGATTGTGCTGGGTGACCATCTGCAAGTCAT TTTAGTTAA-3', which corresponds to nucleotides 1422–1378 of the complete cDNA sequence for mouse Ob-R) (14). Hybridization was performed at 44 C for 18 h, then the membranes were washed at room temperature with decreasing salt concentrations containing 0.1% SDS. The membranes were exposed to autoradiography, and the sizes of the bands were determined by comparison with molecular size markers on the ethidium bromide-stained agarose gel pictures.

Immunolocalization of Ob-R in mouse testis
The frozen fixed tissues (from two different animals) were cut at 8 µm and processed immediately for immunostaining using an AEC kit (Histostain-Plus, Zymed Laboratories, Inc., San Francisco, CA) according to the manufacturer’s protocol. Ob-R polyclonal antibody [Ob-R (H300): sc-8325, Santa Cruz Biotechnology, Inc., Santa Cruz, CA] was used at a dilution of 1:50 for incubation with testis sections for 1 h at 37 C. The H300 antibody was raised against a recombinant protein corresponding to amino acids 541–840 mapping within an internal domain of human Ob-R. Validation of the H300 specificity was performed on sections from adult mouse ovary and brain. After the reactions, tissue sections were counterstained with hematoxylin and examined with a Carl Zeiss light microscope (New York, NY).

Western hybridization for Ob-R
Testes from different age groups (5, 10, 20, and 30 days old and adult) were lysed in ice-cold Tris-buffered saline (TBS) with 1% Nonidet P-40 and 10% glycerol. Two independent lysates were obtained from two animals from each age group. Phenylmethylsulfonylfluoride, aprotinin, leupeptin, and sodium vanadate (all from Sigma, St. Louis, MO) were added to inhibit proteinase activity. One hundred micrograms of protein from each lysate were used for SDS-PAGE. The proteins were first reduced with sample buffer [4% SDS, 20% glycerol, 0.5% mercaptoethanol (Sigma), and 125 mmol/liter Tris, pH 6.8], then boiled for 5 min before loading on 12% polyacrylamide gels. Rainbow markers (Bio-Rad Laboratories, Inc., Richmond, CA) were used to assess the sizes of proteins of interest. Samples were resolved for 1 h in a minigel apparatus (Bio-Rad Laboratories, Inc.), then transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc.). Ponceau stain (Sigma) was used to confirm equal loading and transfer of the proteins. The membranes were washed with TBS containing 0.1% Tween (TBS-T), then incubated for 1 h with the blocking solution (Blockin, Bio-Rad Laboratories, Inc.). Membranes were washed twice with TBS-T, then incubated overnight at 4 C with the primary antibody [Ob-R (H300)/sc-8325, 1:2,000 dilution] in TBS-T containing 1% BSA. The membranes were washed and incubated for 1 h with peroxidase-conjugated antirabbit IgG (Amersham Pharmacia Biotech; 1:10,000 dilution). Immunoreactive bands were detected using the ECL system (Amersham Pharmacia Biotech) and exposed to autoradiography films (Amersham Pharmacia Biotech) for 1–5 min. Membranes were stripped and reused for detection of {alpha}-actin using a specific monoclonal antibody (AC-40, Sigma).

Phosphorylation of STAT3 and ERK1 and -2
Seminiferous tubules (ST) were isolated and pooled from testes of 5-day-old (20 animals), 30-day-old (4 animals), or adult (4 animals) mice. Experiments with adult and 5-day-old mice were independently repeated 3 times. Testes were decapsulated, and the tubules were separated using collagenase digestion (1 µg/µl) for 20 min in a shaking water bath at 34 C. The tubules were then washed with DMEM-Ham’s F-12 medium to remove the interstitial cells. The isolated tubules were treated for 0, 5, 10, 15, or 20 min with 8, 80, and 800 µg/liter recombinant leptin (PeproTech, Inc., Rocky Hill, NJ) in a shaking water bath at 34 C. The interstitial cells from 5-day-old testis were also investigated for the effect of leptin treatment on ERK1, ERK2, and STAT3 phosphorylation. For all time courses, the total culture period for all samples was equal (for example, 5-min stimulation was 15-min culture without leptin and 5-min culture with leptin) to avoid time-related artifacts. After incubations, the tubules were chilled on ice and centrifuged immediately at 1000 x g for 5 min at 4 C, then lysed on ice (see above for the lysis buffer). STAT3, ERK1, and ERK2 were precipitated overnight at 4 C using corresponding antibodies [STAT3 (C-20) rabbit polyclonal IgG and ERK2 (D-2) mouse monoclonal IgG2b] and IgG/A Plus (all from Santa Cruz Biotechnology, Inc.). After the incubations, the gel beads were recovered by centrifugation, washed twice with the lysis buffer and once with sterile water, then resuspended in 20 µl sample buffer (see above). Proteins were denatured at 100 C for 5 min and chilled on ice, and after a brief centrifugation the supernatants were loaded onto 8% polyacrylamide gels. Transfer and immunodetection were performed as described above using a monoclonal tyrosine phosphate antibody (1:1000 dilution; Upstate Biotechnology, Inc., Lake Placid, NY); for controls, we used C-20 (STAT) or D-2 (ERK) as the primary antibody. Peroxidase-conjugated antimouse or antirabbit antibody (Amersham Pharmacia Biotech) was used as the secondary antibody. The bands were visualized using enhanced chemiluminescence (ECL) or a colorimetric detection kit (Bio-Rad Laboratories, Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Detection of Ob-R mRNA in mouse testis
To investigate the presence of mRNA for the leptin receptor (Ob-R), we used RT-PCR, followed by Southern hybridization to an antisense oligonucleotide probe for the mouse Ob-R mRNA. Ob-R (470-bp amplicon) was detected in 5- to 30-day-old testis, adult testis, and adult brain (Fig. 1Go) as well as in adult ovary (data not shown). No amplicons were detected in RT-free samples or in the buffer-only negative control tubes (data not shown). Southern hybridization to the specific antisense oligonucleotide probe was performed to confirm the specificity of the PCR amplicons. The nested end-labeled oligoprobe recognized its complementary sequence corresponding to the major part of exon 8 of the Ob-R cDNA. The sizes of the amplicons from the different tissues were related to mol wt markers (100-bp ladder; Life Technologies, Inc.) and also to the positive control, where the full-length Ob-R cDNA plasmid was used.



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  		Figure 1. Southern hybridization of the Ob-R for the 470-bp RT-PCR amplicons from 5-, 10-, 20-, and 30-day-old and adult (A) testis and 7-day-old brain RNA. Full-length cDNA for Ob-R was used as positive control (+ve). An antisense nested 32P end-labeled oligonucleotide probe was used to detect the amplicons transferred on the nylon membranes. After hybridization, the membranes were exposed to autoradiography overnight at 4 C on x-ray films obtained from Amersham Pharmacia Biotech.

 
Localization of Ob-R in mouse testis during development
Using immunohistochemistry, we examined Ob-R expression in mouse testis at different developmental ages. The receptor was expressed in germ cells at all ages and in some interstitial cells, whereas Sertoli cells did not show Ob-R staining in any age group (Fig. 2Go). The type of germ cells expressing Ob-R was both age and stage specific. In the 5-day-old testis, Ob-R was mainly expressed on type A spermatogonia (SpgA; Fig. 2Go, a and b). The Ob-R was also detected in the 10-day-old testis in SpgA, and some expression was also detected in type B spermatogonia (data not shown). In 20- and 30-day-old testes, Ob-R expression in germ cells was confined mainly to the spermatocytes and appeared to be related to the presence of a lumen in the seminiferous tubules (Fig. 2Go, c and d). Also, some SpgA stained positively for Ob-R in both 20-day-old (Fig. 2dGo) and 30-day-old testis. As expected, positive staining was observed in adult ovarian oocytes, some granulosa cells, and adult brain cells (data not shown).



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  		Figure 2. Immunohistochemistry for Ob-R in 5-day-old (a and b) and 20-day-old (c and d) testis. Frozen sections (8 µm) were treated first with peroxidase blocker, and then incubated for 1 h in the absence or presence of a polyclonal antibody against Ob-R. Immune reaction was detected using an AEC kit, Zymed Laboratories Inc. (South San Francisco, CA). The results shown are representative of three independent experiments, using different tissues in each. In a and b, note the staining for leptin in SpgA. In c and d, the staining is in the pachytene spermatocytes. In c, there appears to be more leptin staining in the tubules that have a lumen. The arrow in d points to a stained SpgA. Magnification: a and c, x150; b and d, x400.

 
Stage-specific expression of Ob-R in adult mouse testis
In the adult testes, the leptin receptor was mainly located on the spermatocytes at stages IX and X of the cycle of the seminiferous epithelium, the stages following sperm release (Fig. 3Go). Sporadic expression of Ob-R was also found in SpgA in some tubules (data not shown). Weak or no expression was found in adult interstitial cells (Fig. 3Go).



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  		Figure 3. Stage-specific expression of Ob-R in adult mouse testis. Tissues were fixed in 4% formaldehyde, then embedded in O.C.T. compound (Tissue-Tek, Saukurus-Finetek, Inc., Torrance, CA) AOC freezing medium and cut at 8 µm. For detection, the tissues were treated first with peroxidase blocker, then incubated for 1 h in the absence or presence of a polyclonal antibody against Ob-R. The immune reaction was detected using an AEC kit. Pictures were taken using a Carl Zeiss light microscope with magnifications of x10 (upper panel) and x20 (lower panel). The results shown here are representative of three independent experiments using different tissues in each. Note the leptin staining in certain tubules only. These tubules correspond to stages IX and X of the cycle of the seminiferous epithelium. Magnification: a, x150; b, x300.

 
Identification of five immunoreactive forms of Ob-R in the immature and adult mouse testis
The presence of Ob-R in the different age groups was further confirmed by Western hybridization using the same antibody as that used for immunohistochemistry (Fig. 4Go, upper panel). Five different immunoreactive isoforms (240, 120, 90, 60, and 40 kDa) appeared to be present in all age groups. Whereas the 240-, 60-, and 40-kDa bands were constitutively expressed throughout the different ages of development, two bands showed significant age-dependent variation. The 90-kDa band was strong in 5- and 10-day-old mice, but almost disappeared in 20-day-old or older mice. On the other hand, the 120-kDa band was strong only in 20-day-old and older mice and it disappeared in the testis from busulfan-treated animals. As expected, {alpha}-actin used to assess equal loading and transfer was equal in all age groups (Fig. 4Go, lower panel).



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  		Figure 4. Western hybridization for Ob-R (upper panel) and {alpha}-actin (lower panel) in 5-, 10-, 20-, and 30-day-old and adult (A) testis. Nitrocellulose membranes were incubated overnight at 4 C with a polyclonal Ob-R antibody or mouse monoclonal antibody against {alpha}-actin. Immunoreactive proteins were visualized using the ECL kit from Amersham Pharmacia Biotech, and the membranes were exposed for 1 min to autoradiography. The sizes of the bands (shown on the right with arrowheads) were correlated to the molecular weight marker (M). The Western blot shown here is one of three independent experiments using different tissue lysates in each blot. Note that the 90-kDa band is present in 5- and 10-day-old testes but absent in 20-day-old and older testis. The 120-kDa band was strong only in 20-day-old and older mice. {alpha}-Actin was equal in all age groups.

 
Induction of STAT3 phosphorylation in mouse seminiferous tubules by leptin
In some systems, leptin is known to act through induction of phosphorylation of STAT and mitogen-activating protein (MAP/ERK). To investigate the hypothesis that leptin acts in a similar way in the testis, we treated isolated seminiferous tubules from adult testis with leptin, then looked at the phosphorylation of STAT3, ERK1, and ERK2. Whereas clear induction (2- to 3-fold stimulation) of STAT3 phosphorylation was evident after a 5- to 10-min stimulation with leptin, no changes were observed in the phosphorylated ERK1 and ERK2 levels in seminiferous tubules from adult testes (Fig. 5Go). Similar results were obtained for the phosphorylation of STAT3, ERK1, and ERK2 in tubules from 5- and 30-day-old testis (data not shown). Relatively similar results were obtained when using 80 or 800 ng/ml leptin in the incubation medium. However, the 800 ng/ml concentration was used in our experiments because it gave us stronger results than the lower concentrations.



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  		Figure 5. Induction of STAT3 phosphorylation in adult mouse seminiferous tubules after 0, 5, 10, 15, and 20 min of incubation with recombinant leptin (800 ng/ml). Immunoprecipitation for STAT3, ERK1, and ERK2 was carried out using the corresponding antibodies and protein A/G agarose. The precipitated proteins were denatured and resolved on 8% PAGE. Proteins were transferred, and a mouse monoclonal antibody against tyrosine phosphate was used to detect the phosphorylated proteins. The immunoreactive bands were visualized using peroxidase-labeled antimouse antibody and the ECL detection system from Amersham Pharmacia Biotech. The lower panel shows the densitometric quantification of three independent experiments; each bar represents the mean ± SEM.

 
Leptin induces ERK1, ERK2, and STAT3 phosphorylation in isolated immature mouse interstitial cells
As Ob-R expression in testicular interstitial cells was most obvious in immature testis, we investigated the possibility of a leptin signaling pathway in these cells through activation of the ERK or STAT system. We studied the levels of phosphorylated ERK1, ERK2, and STAT3 in 5-day-old interstitial cells after 0- to 20-min treatment with leptin (800 ng/ml). Clear induction of both ERK1 and ERK2 as well as in STAT3 phosphorylation was observed within 5–10 min of culture in the presence of leptin (Fig. 6Go). No change was observed on the total STAT3, ERK1, or ERK2 levels (data not shown).



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  		Figure 6. Effect of leptin treatment for 0, 5, 10, and 20 min on STAT3, ERK1, and ERK2 phosphorylation in isolated 5-day-old interstitial cells. After treatment, the cells were collected and homogenized in lysis buffer. Immunoprecipitation for STAT3, ERK1, and ERK2 was carried out using the corresponding antibodies and protein A/G agarose. The lower panel shows the densitometric quantification of two independent experiments; each bar represents the mean ± SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Factors governing the process of proliferation and differentiation of male germ cells are not fully understood. Leptin is a newly identified hormone from white adipose tissue that has been shown to have direct implications on fertility in both male and females. In a number of clinical cases where Ob-R is not functioning properly and in cases with hypo/hyperleptinemia, patients present with infertility (27, 28, 41, 42), decreased LH and FSH secretion (43), or polycystic ovaries (29, 30). Because of its relation to body weight and the ability to reduce appetite (3) as well as its actions in reproductive functions, the importance of leptin and its receptor has grown rapidly in the past few years.

In the ovary, leptin acts directly on granulosa cell steroidogenesis and oocyte maturation (10, 23, 34). Our immunohistochemistry results showed specific staining in oocytes and some granulosa cells (data not shown). Similarly, our results on the induction of ERK and STAT3 in Leydig cells are in accord with recent results that demonstrated the up-regulatory effects of leptin on hCG-induced cAMP production in cultured rat Leydig cells (37). The effective concentration of leptin in our in vitro experiments (80 µg/liter = 5 nmol/liter) was 20 times higher than the normal blood level. However, in vivo conditions are different, because the stimulation is continuous compared with our in vitro 5- to 20-min incubation. The doses used in our study, however, are in accord with other leptin in vitro studies, where typically 10–120 nmol/liter leptin were used (17, 37).

In the adult testis, the stage-specific expression of Ob-R was very evident in spermatocytes at stages IX and X of the cycle of the seminiferous epithelium. Other genes, including FSH receptor, stem cell factor, and several others, also show seminiferous tubule expression in adult testis in a stage-specific manner (44, 45, 46, 47). This stage specificity may be the result of cross-talk between germ cells and the neighboring Sertoli cells (46, 48). These paracrine factors probably regulate signals that allow for the orderly process of spermatogenesis and the extremely precise organization of the seminiferous epithelium into 12 cellular associations (49).

We observed a shift in the main type of germ cells expressing Ob-R with age from SpgA in the 5-day-old mouse to mainly spermatocytes in 20-day-old, 30-day-old, and adult mice. Our Western hybridization results demonstrated that the 90-kDa band was present during early testis development (5 and 10 days), whereas the 120-kDa band was present in later development (20 days to adult). We hypothesize that the 120-kDa Ob-R is a spermatocyte-specific isoform, whereas the 90-kDa band may arise mainly from SpgA as well as from fetal Leydig cells in 5- and 10-day-old testis. In accord, the 120-kDa band was absent in testicular lysates from busulfan-treated animals, where germ cells are almost absent (data not shown). Whether all receptor isoforms identified act as functional receptors or as negative/positive regulators of Ob-R needs to be explored.

The STAT3 signaling pathway is associated with differentiation pathways in several cell types (24, 50). Studies have shown that activation (phosphorylation) of STAT3 in stem cells is able to prevent differentiation of these cells, thus allowing them to replicate and stay in a nondifferentiating phase as stem cells (24, 26). The activation of STAT3 by leptin is possibly the mechanism through which leptin may regulate the proliferation and differentiation of testicular germ cells. Leptin may act on the nondifferentiated cells (SpgA) to allow for their renewal, whereas in spermatocytes, leptin may assist the cells through full differentiation and maturation to spermatids (Fig. 7Go).



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  		Figure 7. The hypothesized leptin actions on male germ cell renewal and differentiation are depicted in this schematic drawing. For spermatogonial stem cells, leptin may act through STAT3 to prevent differentiation, thus allowing the cells to undergo stem cell renewal. On the other hand, in spermatocytes, leptin directs the cells to full maturation into spermatids. Negative feedback regulators from spermatids or other germ cells may contribute to the absence of Ob-R from germ cells in stages other than IX and X of the spermatogenic cycle.

 
The different downstream results of leptin signaling in SpgA and spermatocytes may also be due to the different Ob-R isoforms expressed in these two different cell types. In addition, the absence of ERK1 and ERK2 phosphorylation in response to leptin stimulation of the seminiferous tubules (compared with that in 5-day-old interstitial cells) might be due to the fact that we used whole tubules in our experiments instead of isolated germ cells; thus, the paracrine ERK inducers from Sertoli and myoid cells may mask the leptin effect.

In conclusion, we present here for the first time the distribution of Ob-R in mouse testis and its correlation with germ cells at specific steps of development and differentiation. We observed a variation in the type of Ob-R isoform present in different age groups, and this may contribute to the outcome of leptin stimulation in different cell types. The results also demonstrate induction of the STAT3 signaling pathway in cultured tubules by leptin, through which this hormone may act on renewal and differentiation of spermatogenic cells, similar to its role in hemopoiesis (19, 20). The absence of Ob-R from stages of the cycle other than IX and X suggests a regulatory role for the spermatids in controlling the expression of Ob-R in spermatocytes. Interestingly, the Ob-/Ob- mouse is infertile, and the spermatogenetic arrest observed in testis is at the spermatocyte stage (6) where the highest expression of Ob-R in adult testis was observed. Hence, our results may offer an additional explanation of reduced GnRH levels for the observed infertility in Ob mutant mice.


   Footnotes
 
1 This work was supported in part by NIH Grant RO1-HD-33728. Back

2 Present address: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261. Back

Received December 2, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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