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Department of Pharmaco-Biology (S.Aq., E.M., S.C., C.M., V.P., S.An.), Faculty of Pharmacy; Department of Cell Biology (M.G.); and Centro Sanitario (S.Aq., M.G., E.M., S.C., C.M., V.P., S.An.), University of Calabria, 87030 Arcavacata di Rende (Cosenza), Italy
Address all correspondence and requests for reprints to: Dr. Sebastiano Andò, Faculty of Pharmacy, University of Calabria, Arcavacata-Rende (Cosenza) 87036, Italy. E-mail: aquisav{at}libero.it or sebastiano.ando{at}unical.it.
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Abstract |
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Methods: By RT-PCR, Western blot, and immunofluorescence techniques, we have demonstrated that human sperm express leptin. RIA method evidenced leptin secretion. Phosphatidyl-inositol-kinase-3 (PI3K)/Akt pathway was examined by PI3K activity assay and Western blot. Leptin and insulin regulation of glycogen synthesis was evaluated by glycogen synthase activity (GSA).
Results: The large differences of leptin secretion between uncapacitated and capacitated sperm suggest a functional role for leptin in capacitation. Indeed, in uncapacitated sperm, leptin enhances both cholesterol efflux and protein tyrosine phosphorylation. In uncapacitated sperm, both insulin and leptin increased PI3K activity, Akt S473, and glycogen synthase kinase-3 S9 phosphorylation. Interestingly, during capacitation, concomitantly to the massive release of both hormones, we observed a strong reduction in the phosphorylation of glycogen synthase kinase-3 S9, kinase downstream of Akt that regulates the glycogen synthase. Our results from GSA showed that the enzymatic activity was significantly higher in uncapacitated than in capacitated sperm. Particularly, in uncapacitated sperm, GSA appeared to depend on the hormones concentration, because the enzymatic activity was stimulated at low doses, whereas it was inhibited at high doses. Moreover, both leptin and insulin regulate in autocrine fashion sperm glycogen synthesis.
Conclusion: Leptin secretion by sperm suggests that the male gamete may be able to modulate its metabolism independently by systemic leptin. These data open new considerations about leptin significance in male fertility.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The ob/ob mice (lacking of functional leptin) or OB-R/OB-R mice (lacking of functional leptin receptor) are infertile and fail to undergo normal sexual maturation. Importantly, fertility of ob/ob mice is restored by leptin and not by simply reducing body weight, indicating an effect of the hormone per se on reproductive function (3, 4). Particularly, male mice (ob/ob) had small testes, azoospermia, and multinucleated spermatids. As in the female, hypogonadotropic hypogonadism and infertility are common features in male ob/ob mice (5). In line with results from experimental studies, in humans the absence of endogenous leptin is associated with hypogonadism and absence of pubertal development (6, 7, 8).
Leptin is expressed in the seminiferous tubules and in seminal plasma (9), but its cellular origin in these contexts is not exactly defined. Several studies support the role of serum leptin in the regulation of gonadal functions in men (10) indirectly via the central neuroendocrine system and directly via peripheral tissue membrane receptors (11, 12). Besides, compelling evidence indicates that leptin functional regulation of the male gonadal axis appears to be a tightly regulated action, carried out at different levels of the hypothalamic-pituitary-testicular system, involving not only stimulatory, but also inhibitory, effects. Recently (13), it was hypothesized that the net effect of leptin upon male reproductive function may depend on the circulating level of the molecule. Thus, predominant stimulatory effects, primarily at the hypothalamus, are observed at physiological leptin levels above a minimal threshold. In contrast, direct inhibitory actions at the testicular level may take place in the presence of a significantly elevated leptin concentration, as detected in obesity (2).
Leptin in various cell types has a range of roles, but the principal role is as a lipostat, signaling to other systems the energy reserves available to the body, mediating fuel use, and consequently energy expenditure. Recently, a new target for leptin in the male genital tract was evidenced because leptin receptor was found to be present in human spermatozoa (9). It has long been recognized that capacitated sperm display an increased metabolic rate and overall energy expenditure, presumably to affect the changes in sperm signaling and function during capacitation (14). However, the relationship between the signaling events associated with capacitation and the changes in sperm energy metabolism is poorly understood. Overall, there is a lack of information regarding how mammalian spermatozoa manage their energy status. In somatic cells, both leptin and insulin play a central role in regulation of energy homeostasis (15). Particularly, in vitro and in vivo evidence supports the hypothesis that leptin may mimic insulin action on glycogen synthesis (16). Sperm glycogen metabolism seems to be regulated by modulation of glycogen synthase in a manner similar to that observed in other tissues (17).
In the present study, we showed that leptin is expressed in, and secreted from, human ejaculated spermatozoa, providing evidence for a role of the hormone in sperm physiology.
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Subjects and Methods |
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PMN cell isolation medium was from BIOSPA (Milan, Italy). Total RNA Isolation System kit, enzymes, buffers, and nucleotides 100-bp ladder used for RT-PCR were purchased from Promega Corp. (Milan, Italy). Moloney murine leukemia virus (M-MLV) was from Life Technologies Italia (Milan, Italy). Oligonucleotide primers were made by Invitrogen (Milan, Italy). DMEM-F12 medium, BSA protein standard, Laemmli sample buffer, prestained molecular weight markers, Percoll (colloidal polyvinylpyrrolidone-coated silica for cell separation), sodium bicarbonate, sodium lactate, sodium pyruvate, dimethylsulfoxide, Earle’s balanced salt solution, and all other chemicals were purchased from Sigma Chemical (Milan, Italy). Acryl amide bisacrylamide was from Labtek Eurobio (Milan, Italy). Triton X-100, Eosin Y was from Farmitalia Carlo Erba (Milan, Italy). ECL Plus Western blotting detection system, Hybond ECL, [
-32P]ATP, and HEPES sodium salt were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Human leptin RIA kit was from Linco Research, Inc. (St. Charles, MO-Biogemini Sas, Catania, Italy). Cholesterol-oxidase (CHOD)-peroxidase (POD) enzymatic colorimetric kit was from Inter-Medical (Biogemini Sas, Catania, Italy). Monoclonal mouse p85-regulatory subunit of phosphatidyl-inositol-kinase-3 (PI3K) antibody, goat polyclonal actin antibody, polyclonal rabbit antileptin (A-20) antibody, rabbit antiinsulin antibody, rabbit antiphosphotyrosine antibody (PY99), rabbit anti-p-Akt1/Akt2/Akt3 S473 antibody, POD-coupled antirabbit and antigoat, antirabbit IgG fluorescein isothiocyanate-conjugated were from Santa Cruz Biotechnology (Heidelberg, Germany). Rabbit anti-p-glycogen synthase kinase 3 (-GSK-3) S9 antibody was from Cell Signaling (Milan, Italy). Nylon membranes were provided by Roche Diagnostics Corp. (Indianapolis, IN). Uridine diphosphate (UDP) [U-14C]glucose (25 µCi/ml) was from Amersham.
Semen samples and spermatozoa preparations
Ejaculates were collected from healthy volunteers undergoing semen analysis, by masturbation (18), after 3 d of sexual abstinence. The study was approved by the local medical-ethical committees, and all participants gave their informed consent. Sperm samples with normal parameters of semen as volume, sperm count, motility, vitality, and morphology, according to the World Health Organization Laboratory Manual (18), were included in this study. In each experiment, three normal samples were pooled. Spermatozoa preparations were performed as previously described (19).
Evaluation of sperm viability
Viability was assessed using Eosin Y to evaluate potential toxic effects of different treatments. A blinded observer scored 100 cells for stain uptake (dead cells) or exclusion (live cells). Viability was evaluated before and after pooling the samples. There were no adverse effects among the different treatments on human sperm viability (data not shown).
RNA isolation, RT-PCR, and Southern blotting
Total RNA was isolated from human ejaculated spermatozoa purified as previously described (19). PCR amplification of cDNA was performed with 2 U TaqDNA polymerase, 50 pmol primer pair (forward, 5'-CAT TGG GGA ACC CTG TGC GGA TTC-3'; reverse, 5'-TGG CAG CTC TTA GAG AAG GCC AGC-3') in 10 mM Tris-HCl (pH 9.0) containing 0.1% Triton X-100, 50 mM KCl, 1.5 mM MgCl2, and 0.25 mM of each deoxynucleotide triphosphate. The conditions for PCR were: denaturation at 95 C for 1 min, annealing at 55 C for 1 min, and extension at 72 C for 2 min (40 cycles). A DNA marker (100-bp DNA ladder) was used to determine the size of amplified product, that is 348 bp. To check out the presence of DNA contamination, a RT-PCR was performed without M-MLV reverse transcriptase (negative control). The identity of the PCR-amplified cDNA fragment of leptin transcript from human spermatozoa was verified using Southern hybridization. A total of 1–2 ng cDNA probe (5'-CACGCAGTCAGTGTCCTCCA-3', which corresponds to nucleotide cDNA sequence for human leptin) was labeled with [-32P]ATP using polynucleotide kinase. Hybridization was performed overnight at room temperature. Then, membranes were washed with decreasing salt concentrations containing 0.1% sodium dodecyl sulfate, and then exposed to autoradiography with intensifying screens.
Western blot analysis of sperm proteins
Sperm samples, washed twice with Earle’s balanced salt solution (uncapacitating medium), were incubated without or with the indicated treatments and then centrifuged for 5 min at 5000 x g. The pellet was resuspended in lysis buffer as previously described (19). Equal amounts of proteins (60 µg) were boiled for 5 min, separated by 10% PAGE, transferred to nitrocellulose sheets, and probed with an appropriate dilution of the indicated antibody. The bound of the secondary antibody was revealed with the ECL Plus Western blotting detection system according to the manufacturer’s instructions. The negative control was performed using a sperm lysate that was immunodepleted of leptin (i.e. preincubate lysate with antileptin antibody for 1 h at room temperature and immunoprecipitate with protein A/G-agarose).
As internal control, all membranes were subsequently stripped (glycine, 0.2 M, pH 2.6, for 30 min at room temperature) of the first antibody and reprobed with antiactin antibody.
Immunofluorescence assay
Sperm cells, recovered from Percoll gradient, were rinsed three times with 0.5 mM Tris-HCl buffer (pH 7.5) and fixed with absolute methanol for 7 min at –20 C. Leptin staining was carried out, after blocking with normal horse serum (10%), using a rabbit polyclonal antihuman leptin as primary antibody and an antirabbit IgG fluorescein isothiocyanate conjugated (1:100) as secondary antibody. Sperm cells incubated without the primary antibodies were used as the negative controls. The slides were examined under a fluorescence microscope (Olympus BX41; Olympus Corp., Milan Italy), and a minimum of 200 spermatozoa per slide were scored.
Measurement of leptin secreted by human ejaculated spermatozoa
A competitive RIA was applied to measure leptin in sperm culture media. Increasing numbers of spermatozoa were washed twice with unsupplemented Earle’s medium (uncapacitating medium) and were incubated in the same medium for 1 h at 37 C and 5% CO2. Besides, some samples were incubated in Earle’s balanced salt solution medium supplemented with 600 mg BSA/100 ml and 200 mg sodium bicarbonate/100 ml (capacitating medium). At the end of the sperm incubation, the culture media were recovered by centrifugation, lyophilized, and subsequently dissolved in 120 µl kit buffer. Human leptin concentrations were determined in duplicate by a human leptin RIA Kit according to manufacturer’s instructions. Leptin standards ranged between 0.2–100 ng/ml. The limit of sensitivity for the assay was 0.05 ng/ml. Inter- and intraassay variations were 5.4% and 5.1%, respectively. Leptin results are presented as the original concentrations of the supernatants and are given as nanograms per milliliter.
Measurement of cholesterol in the sperm culture media
Cholesterol was measured in duplicate by a CHOD-POD enzymatic colorimetric method according to manufacturer’s instructions in the incubation medium of human spermatozoa. Sperm samples, washed twice with uncapacitating medium, were incubated in the same medium or in capacitating medium for 1 h at 37 C and 5% CO2. Other samples were incubated without (control) or in the presence of increasing leptin concentrations (10, 50, and 100 ng/ml). At the end of the sperm incubation, the culture media were recovered by centrifugation, lyophilized, and subsequently dissolved in 1 ml buffer reaction. The samples were incubated for 10 min at room temperature; then the cholesterol content was measured with the spectrophotometer at 505 nm. The cholesterol standard used was 200 mg/dl. The limit of sensitivity for the assay was 0.05 mg/dl. Inter- and intraassay variations were 0.71% and 0.57%, respectively. Cholesterol results are presented as the original concentrations and are given per 10 x 106 number of spermatozoa.
PI3K assay
Spermatozoa were washed twice in uncapacitating medium and centrifuged at 800 x g for 20 min. Sperm samples were then incubated for 1 h at 37 C and 5% CO2 without or with the indicated treatments, and the PI3K assay was performed as previously described (20). A p85 regulatory subunit of PI3K was precipitated from 500 µg sperm lysates. The negative control was performed using a sperm lysate, where p110 catalyzing subunit of PI3K was previously removed by preincubation with the respective antibody (1 h at room temperature) and subsequently immunoprecipitated with protein A/G-agarose.
Glycogen synthase activity (GSA)
The GSA was determined by the principle: glycogen + UDP[U-14C]glucose
glycogen(14C) + UDP.
Washed spermatozoa were incubated for 1 h at 37 C under uncapacitating or capacitating conditions as described above. Both uncapacitated and capacitating sperm were treated with leptin (10 and 50 ng/ml), insulin (3.3 or 10 nM), antileptin (or normal) rabbit serum (1:100) alone or combined with 10 ng/ml leptin, antiinsulin (or normal) rabbit serum (1:100) alone or combined with 3.3 nM insulin, wortmannin (10 µM) alone or combined with 10 ng/ml leptin or 3.3 nM insulin, monensin (25 µM), antileptin (1:100) plus antiinsulin (1:100) antibodies, LiCl (2 mM). After treatment, sperm extracts were performed in KCl (100 mM); EDTA (2 mM); HEPES (20 mM), pH 7.1; phenylmethylsulfonyl fluoride (1 mM). Aliquots of 100 µl were then assayed immediately in a reaction mixture containing glycogen (20 mg/ml), EDTA (5 mM), NaF (50 mM), UDP glucose (2.5 mM), Tris/HCl (25 mM), UDP [U-14C]glucose (0.0005 mCi/ml), and glucose 6-phosphate (8 mM). The assay was buffered at pH 8.0. The reaction was performed at 37 C; after 15 min, 50 µl of the homogenate/assay buffer was spotted on a Whatman filter paper (1 cm x 2.5 cm). The filters were dropped into cold (4 C) 66% ethanol for 30 min to precipitate glycogen; in addition, they were washed twice at room temperature for 20 min each in 66% ethanol to remove [14C]glucose 1-phosphate. After being dried, the filters were transferred to scintillation vials; 3 ml scintillation solution was added to each, and mixtures were counted for radioactivity. Data are expressed as milliunits UDP-glucose incorporated per milligram protein.
Statistical analysis
The experiments for RT-PCR were repeated on at least three independent occasions, whereas Western blot analysis was performed in at least six independent experiments. The data obtained from RIA (six replicate experiments using duplicate determinations), CHOD-POD enzymatic colorimetric method (six replicate experiments using duplicate determinations), GSA (eight replicate experiments using duplicate determinations) were presented as the mean ± SEM. The differences in mean values were calculated using a paired t test, with a significance level of P < 0.05. Regression analysis was performed using the SPSS program (SPSS, Inc., Richmond, CA).
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Results |
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To determine whether mRNA for leptin is present in human ejaculated spermatozoa, RNA, isolated from Percoll-separated samples of normal men, was subjected to reverse PCR and then to Southern blot analysis. The RT-PCR products for leptin in sperm were not a result of any DNA contamination, because the RNA samples were subjected to deoxyribonuclease treatment before RT-PCR. The primer sequences were based on the human leptin gene sequence, and the RT-PCR amplification revealed the expected PCR product size of 348 bp (Fig. 1A
). To verify the identity of the amplified products, we performed Southern blot analysis (Fig. 1B). The presence of leptin protein in human ejaculated spermatozoa was investigated by Western blot using an antibody raised against the carboxyl terminus of the mature human leptin protein (Fig. 1C). One immunoreactive band was observed at the same mobility of the adipocytes extract (lane A), used as positive control.
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Using immunofluorescence technique, we identified a positive signal for leptin in human spermatozoa (Fig. 2). Leptin immunoreactivity is specifically compartmentalized in uncapacitated sample at the equatorial segment and at the midpiece (NC), whereas capacitated sperm showed an overall decrease and a more uniform distribution in the signal intensity (CAP).
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The RIA method was used to evaluate whether sperm are able to secrete leptin. After the assay was validated for sperm, we demonstrated that the increase in leptin secretion was dependent on sperm concentration (Fig. 3A). The time course of leptin secretion from spermatozoa into the uncapacitating medium is shown in Fig. 3B. Leptin secretion from 10 x 106 sperm incubated in uncapacitating medium (range, 0.2–2 ng/ml) was significantly lower than that obtained from sperm incubated in capacitating conditions (range, 0.8–4.0 ng/ml) (Fig. 3C). We have attempted to measure the amount of leptin remaining in the sperm after secretion; however, the lysis buffer somehow interferes with the kit-system, altering the binding between antigen and antibody. Then we performed the Western blot analysis of sperm lysates. The time course of capacitating sperm showed a decrease of the hormone inside the sperm (Fig. 3D), according to the increased secretion during capacitation.
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The increased leptin secretion during capacitation suggests a possible role of the hormone in the process. Therefore, we investigated leptin effect on two different and representative aspects of capacitation: cholesterol efflux and protein tyrosine phosphorylation. The importance of the cholesterol efflux in inducing capacitation is historical known, and it has also been demonstrated that it initiates signaling events leading to tyrosine phosphorylation of sperm proteins (21, 22). Washed pooled sperm from normal samples were treated with increasing concentrations of leptin (0, 10, 50, and 100 ng/ml) and incubated under uncapacitating conditions. Then, samples were centrifuged, the upper phase was used to determinate the cholesterol levels, whereas the sperm were lysed to evaluate protein tyrosine phosphorylation. Our results showed a significant increase both in cholesterol efflux (Fig. 4A) and in protein tyrosine phosphorylation (Fig. 4B) upon leptin treatment, suggesting its involvement in the induction of capacitation. However, leptin induction of both processes was not dose-dependent; indeed, concentration as low as 10 ng/ml was able to sustain the greatest increase.
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The signaling events associated with capacitation and the changes in sperm energy metabolism are issues that remain to be resolved. In somatic cells, leptin and insulin play a central role in regulation of energy homeostasis. In several cell types, including muscle cells (23), adipocytes (24), and hypothalamic neurons (25), both insulin and leptin activate PI3K, which may account, at least in part, for similarities in the metabolic effects of these hormones. Our results showed that, in uncapacitated sperm, both leptin and insulin stimulation increased PI3K activity (Fig. 5A) as well as the phosphorylation of Akt and GSK3, two of the major metabolic intermediates downstream of PI3K (Fig. 5B). Intriguingly, GSK3 phosphorylation was abolished in capacitating sperm, suggesting that, during capacitation, there is a block in glycogen synthesis. Concomitantly, in capacitating sperm, we observed a reduction of both hormones inside the sperm (Fig. 5C).
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The above reported data led us to further investigate the mechanism underlying the regulation of glycogen synthesis in sperm. Therefore, we evaluated the action of the two hormones on GSA. In somatic cells, in vitro and in vivo evidence supports the hypothesis that leptin may mimic insulin action on glycogen synthesis (16). Our results showed that GSA was significantly higher in uncapacitated than in capacitated sperm (Figs. 6 and 7). Particularly, in uncapacitated sperm, GSA appears to depend on the hormones concentration: low concentrations (leptin, 10 ng/ml; insulin, 3.3 nM) stimulated the GSA, whereas high concentrations (leptin, 50 ng/ml; insulin, 10 nM) inhibited enzymatic activity. In capacitated sperm, the GSA was unable to discriminate between low and high hormone doses.
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) and antiinsulin rabbit serum (1:100) plus insulin (Fig. 7) significantly decreased GSA, compared with sperm incubated in the same conditions with normal rabbit serum (1:100) plus leptin or insulin. As further evidence of autocrine regulation of the hormones on glycogen metabolism, monensin (which blocks cell secretion) (26, 27) significantly decreased GSA. Besides, wortmannin plus leptin or wortmannin plus insulin disrupted the hormones signaling through PI3K (24, 25, 26). LiCl, an inhibitor of GSK3ß (28), was used as positive control of GSA enzymatic activity. In capacitating sperm, the decrease of GSA was not observed during the same experimental conditions. Finally, to evaluate whether, in sperm, the GSA was exclusively regulated by insulin and leptin, we used both antibodies simultaneously. Although we observed a significant decrease, the enzymatic activity still persists, suggesting that redundant actions of alternative pathways may exist in the sperm.
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Discussion |
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Expression of leptin in the male gamete is a novel finding. In our study, we have demonstrated the presence of leptin in human sperm at different levels: mRNA expression, protein expression and immunolocalization. New reports firmly establish the presence of messenger RNAs in mammalian ejaculated spermatozoa. Originally, it was hypothesized that these transcripts were carried over from earlier stages of spermatogenesis, but the analysis and significance of mRNA in these cells are currently under investigation (31). Worthy new findings suggest that some of these transcripts code for proteins essential in early embryo development (32). In our study, leptin expression has also been evidenced by Western blot and by immunofluorescence. Particularly, immunocytochemical analysis showed that the hormone is specifically compartmentalized in uncapacitated sample at the equatorial segment and the midpiece, whereas capacitated sperm showed an overall decrease and a uniform distribution in the signal intensity. These data fit well with the RIA values that showed a significant increase of leptin secretion from capacitating sperm, suggesting an involvement of the hormone in capacitation.
Ejaculated spermatozoa require an extratesticular maturation termed ‘capacitation’ (14) that in vivo occurs within the female reproductive tract. It consists of several molecular events that involve different aspects of sperm physiology whose final aim is making the ejaculated sperm competent to fertilize an egg. Intriguingly, capacitation in vitro occurs spontaneously, after the removal of seminal plasma, without a requirement for exogenous mediator, suggesting an autocrine induction of the process (14) by endogenous sperm-derived factors. Despite the importance of capacitation, the biochemical and molecular events of the phenomenon are still poorly understood, and the identities of factors that trigger gamete activation are still unknown.
The efflux of cholesterol is historically known to be one of the first steps of capacitation, and most work has been concentrated on it (Ref. 21 and references therein). Our results showed a significant increase in cholesterol efflux upon leptin treatment in uncapacitated sperm. Moreover, leptin increases the sperm proteins tyrosine phosphorylation, which is an event tightly related to the capacitation and resulting downstream cholesterol efflux (21). However, leptin action on these two events was not dose dependent. These findings may be in context with the hypothesis of a possibly double role of leptin in the male gonads. So far, most studies indicated both positive and negative effects of leptin in gonadal functions (13, 33). Besides, seminal plasma leptin levels were significantly lower in patients with normal spermiogram parameters, compared with pathological semen samples, and showed a negative correlation with the motility of human spermatozoa (9).
It has long been recognized that capacitated sperm display an increased metabolic rate, presumably to affect the changes in sperm signaling and function during capacitation (34). The relationship between the signaling events associated with capacitation and changes in sperm energy metabolism is poorly understood. Recently, we have demonstrated that insulin is expressed in and secreted from human ejaculated spermatozoa (26). In somatic cells, both leptin and insulin play a central role in regulation of energy homeostasis, acting on PI3K/Akt pathway, which principally mediates their metabolic effects (16). Similarly, in uncapacitated sperm, both insulin and leptin increased PI3K activity, Akt S473 and GSK-3 S9 phosphorylations, leading us to hypothesize a similar action of the two hormones in modulating sperm energetic substrates availability during capacitation. Interestingly, during capacitation, when the insulin and leptin secretions are maximal, we observed a strong decrease of GSK-3 S9 phosphorylation, suggesting a potential role of these hormones in modulating sperm glycogen synthesis. In somatic cells, the regulation of glycogen metabolism involves GSK-3 activity, which, in turn, is regulated by tyrosine and serine/threonine phosphorylations, the latter mediated by PI3K and Akt (35). Akt phosphorylates GSK3 on serine residue 9 and then deactivates this enzyme, which, in turn, reduces the phosphorylation and thus enhances the activity of glycogen synthase (36, 37). The conversion of UDP-glucose to glycogen by glycogen synthase is the rate-limiting step in glycogen synthesis (38); and in somatic cells, both leptin and insulin are central in the regulation of GSA.
Worthy, in our study, was the observation that GSA was significantly higher in uncapacitated than in capacitated sperm. Particularly, in uncapacitated sperm, the GSA was stimulated at low levels of both leptin and insulin, whereas it was inhibited at high concentrations. The outcome of signaling activation can depend on differences in ligand concentration (40, 41). Besides, recently it was hypothesized that the net effect of leptin upon male reproductive function may depend on the circulating level of the molecule. Thus, predominant stimulatory effects are observed at leptin levels above a minimal threshold; in contrast, direct inhibitory actions may take place in the presence of a significantly elevated leptin concentration (2, 13).
Furthermore, we have showed an autocrine regulation of GSA by both insulin and leptin in sperm. Particularly, the autocrine blockage significantly decreased GSA in uncapacitated sperm; whereas in capacitating sperm, it was not observed in the same experimental conditions. Besides, because, during capacitation, hormones efflux and multiple changes of the membrane structure rapidly occur, the autocrine blockage may be not appreciable. Our study suggests that, in uncapacitated sperm, the GSK3 is tightly blocked; whereas during capacitation, there is an activation of the enzyme, which, in turn, blocks the GSA. This effect may have physiological relevance in sperm because, during capacitation, energy demand increases, and then sperm mobilizes the glycogen reserves rather than produce it. Our results, together with the presence of leptin receptor in human ejaculated spermatozoa, create the condition for an autocrine leptin loop at this level. Upon achieving threshold concentrations, leptin may act on sperm receptors to induce signal transduction and molecular changes of capacitation.
It also has to be mentioned that mammalian spermatozoa have a fully functional glycogen metabolism, resulting in the presence of glycogen deposits and of GSK3 in the head and in the midpiece (17). Our results regarding the immunolocalization of leptin in uncapacitated sperm fit well with these findings, given that leptin works in the same sites. Glucose is needed for spermatozoa during zona pellucida penetration and sperm-oocyte fusion and to ensure that tyrosine phosphorylation occurs during capacitation (42, 43). Glucose is provided to sperm by the female reproductive tract fluid in vivo or by the culture medium in vitro; besides, several studies have indicated that stores of glycogen are endogenous sources of glucose in sperm allowing sperm to accommodate glucose-free conditions (39). It may be hypothesized that leptin in uncapacitated sperm is involved in the accumulation of energy substrates, which would be spent during capacitation.
This study shows a new possible endogenous mediator of capacitation, because we found that human ejaculated spermatozoa secrete leptin able to affect some events tightly related to this process. Leptin secretion suggests that the sperm has the ability to modulate its metabolism, according to its energy needs, independently by systemic leptin. In other words, sperm is an alternative site of leptin expression that may represent a protective mechanism in male reproduction to guarantee the accumulation of energy substrates to maintain the gamete fertilizing capability.
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Acknowledgments |
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Footnotes |
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First Published Online June 8, 2005
Abbreviations: CHOD, Cholesterol-oxidase; GSA, glycogen synthase activity; GSK, glycogen synthase kinase; M-MLV, Moloney murine leukemia virus; PI3K, phosphatidyl-inositol-kinase-3; POD, peroxidase; UDP, uridine diphosphate.
Received November 16, 2004.
Accepted May 26, 2005.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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and ERß are both expressed in human ejaculated spermatozoa: evidence of their direct interaction with phosphatidylinositol-3-OH kinase/Akt pathway. J Clin Endocrinol Metab 89:1443–1451This article has been cited by other articles:
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  S. Aquila, V. Rago, C. Guido, I. Casaburi, S. Zupo, and A. Carpino Leptin and leptin receptor in pig spermatozoa: evidence of their involvement in sperm capacitation and survival Reproduction, July 1, 2008; 136(1): 23 - 32. [Abstract] [Full Text] [PDF]
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 S. Aquila, E. Middea, S. Catalano, S. Marsico, M. Lanzino, I. Casaburi, I. Barone, R. Bruno, S. Zupo, and S. Ando Human sperm express a functional androgen receptor: effects on PI3K/AKT pathway Hum. Reprod., October 1, 2007; 22(10): 2594 - 2605. [Abstract] [Full Text] [PDF]
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I M Aparicio, M J Bragado, M C Gil, M Garcia-Herreros, L Gonzalez-Fernandez, J A Tapia, and L J Garcia-Marin Porcine sperm motility is regulated by serine phosphorylation of the glycogen synthase kinase-3{alpha} Reproduction, September 1, 2007; 134(3): 435 - 444. [Abstract] [Full Text] [PDF]
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, uncapacitated sperm (NC); 
, capacitated sperm (CAP).