Autocrine Regulation of Insulin Secretion in Human Ejaculated Spermatozoa
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内分泌学杂志 2005年第2期
Department of Pharmaco-Biology (Faculty of Pharmacy) (S.A., E.M.), Department of Cell Biology (M.G., S.A.), and Centro Sanitario (S.C.), University of Calabria, 87030 Arcavacata di Rende, Italy
Address all correspondence and requests for reprints to: Dr. Saveria Aquila , Centro Sanitario, University of Calabria, Arcavacata di Rende (CS) 87030, Italy. E-mail: sebastiano.ando@unical.it and aquisav@libero.it.
Abstract
A striking feature of insulin expression is its almost complete restriction to ?-cells of the pancreatic islet in normal mammals. Here we show that insulin is expressed in and secreted from human ejaculated spermatozoa. Both insulin transcript and protein were detected. In addition, the large differences in insulin secretion, assessed by RIA, between noncapacitated and capacitated sperm suggest a role for insulin in capacitation. Insulin had an oscillatory secretory pattern involving glucose dose-dependent increases and significant decreases during the blockage of an insulin autocrine effect. It appears that the effect of glucose on the fertilizing ability of sperm is mediated by glucose metabolism through the pentose phosphate pathway. Then we evaluated the autocrine effect of sperm insulin on glucose metabolism by studying the activity of glucose-6-phosphate dehydrogenase, the key rate-limiting enzyme in the pentose phosphate pathway. The simultaneous decrease in both insulin release and glucose-6-phosphate dehydrogenase activity induced by blocking the autocrine insulin effect with three different procedures (blockage of insulin release by nifedipine, immune neutralization of the released insulin by antiinsulin serum, and blockage of an insulin intracellular effector such as phosphotidylinositol 3-kinase by wortmannin) strongly suggests a physiological role of sperm insulin on these two events. Insulin secretion by spermatozoa may provide an autocrine regulation of glucose metabolism based on their energetic needs independent of systemic insulin. In conclusion, these data open a new area of study in male reproduction.
Introduction
INSULIN IS IMPORTANT for promoting and regulating growth, differentiation, and metabolism. During embryonic development, extrapancreatic insulin gene expression is detectable in the yolk sac (1) and brain (2) of rodents. In adult mammals, apart from the thymus, insulin is thought to be produced only in ?-cells in the pancreas (3). In addition, insulin has been shown to play a central role in the regulation of gonadal function. However, the significance of insulin in male fertility is not complete. It was reported that IGF-II and insulin as well as IGF-I promote spermatogonial differentiation into primary spermatocytes by binding to the IGF-I receptor (4). It was also shown that both the sperm plasma membrane and the acrosome represent cytological targets for insulin (5). In men affected by insulin-dependent diabetes, sperm have severe structural defects, significantly lower motility (6), and lower ability to penetrate hamster eggs (7). In this study we show that insulin is expressed in and secreted from human ejaculated spermatozoa.
Materials and Methods
Chemicals
The Total RNA Isolation System kit, enzymes, buffers, nucleotides, and 100-bp ladder used for RT-PCR were purchased from Promega Corp. (Milan, Italy). Moloney murine leukemia virus (M-MLV) was obtained from Invitrogen Life Technologies, Inc., Italia (Milan, Italy). Oligonucleotide primers were made by Invitrogen Life Technologies, Inc., Italia. BSA protein standard, Laemmli sample buffer, prestained molecular weight marker, Percoll (colloidal polyvinylpyrrolidone-coated silica for cell separation), sodium bicarbonate, dimethylsulfoxide, Earle’s balanced salt solution, triethanolamine buffer, MgCl2, glucose-6-phosphate, NADP+, and all other chemicals were purchased from Sigma-Aldrich Corp. (Milan, Italy). The ECL Plus Western blotting detection system, Hybond ECL, was purchased from Amersham Biosciences (Little Chalfont, UK). The human insulin RIA kit was purchased from Diagnostic Systems Laboratories ICN (Biogemina Sas, Catania, Italy). Antibodies (rabbit antiinsulin, peroxidase-coupled antirabbit, and fluorescein isothiocyanate-conjugated antirabbit immunoglobulin G) were obtained from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany).
Semen samples and spermatozoa preparations
Sperm samples with normal parameters of semen volume as well as sperm count, motility, vitality, and morphology, as specified in the WHO Laboratory Manual (8), were pooled and included in this study. In each experiment, four normal samples were pooled. Spermatozoa preparations were performed as previously described (9).
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 of the different treatments on human sperm viability (data not shown).
RNA isolation and RT-PCR
Total RNA was isolated from human ejaculated spermatozoa purified as previously described (10). PCR amplification of cDNA was performed using the following primer pair: forward, 5'-GCC TTT GTG AAC CAA CAC CTG-3'; and reverse, 5'-GTT GCA GTA GTT CTC CAG CTG-3'. The forward primer, located in exon II, and the reverse primer, located in exon III, produce a 261-bp cDNA. Because the primers span an intron, the genomic product is about 1200 bp.
The conditions for PCR were: denaturation at 95 C for 1 min, annealing at 62 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 the amplified product. As a negative control, an RT-PCR was performed without M-MLV reverse transcriptase.
Western blot analysis of sperm proteins
During Western blot analysis, sperm samples were processed as previously described (10). The negative control was performed using a sperm lysate that was immunodepleted of insulin [i.e. preincubate lysate with antiinsulin antibody (1 h at room temperature) and immunoprecipitate with protein A/G-agarose].
Immunofluorescence assay
Sperm cells 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. Insulin staining was carried out after blocking with normal horse serum (10%) using a rabbit polyclonal antihuman insulin antibody (1:100) and an antirabbit immunoglobulin G fluorescein isothiocyanate-conjugated antibody (1:200). Sperm cells incubated without the primary antibodies were used as negative controls. The slides were examined under a fluorescence microscope (BX41, Olympus, Milan, Italy), and a minimum of 200 spermatozoa/slide were scored.
Measurement of insulin secreted by human ejaculated spermatozoa
A competitive RIA was applied to measure insulin in the sperm culture medium. Increasing numbers of spermatozoa were washed twice with unsupplemented Earle’s medium and incubated in the same medium for 1 h at 37 C in 5% CO2. In other experiments, sperm cultures were split into two series, then incubated under noncapacitating (unsupplemented Earle’s medium alone) or capacitating conditions (Earle’s balanced salt solution medium supplemented with 600 mg BSA/100 ml and 200 mg sodium bicarbonate/100 ml) for 1 h in a 37 C water bath at a final concentration of 10 x 106 sperm/500 μl. Each group was treated with 0.6, 8.3, and 16.7 mM glucose or with 3.3 or 10 nM insulin, antiinsulin (or normal) rabbit serum (1:100) plus 16.7 mM glucose, 10 μM wortmannin plus 16.7 mM glucose, or 25 μM nifedipine. The antiinsulin (or normal) rabbit serum and wortmannin were added 30 min before glucose stimulation. The antiinsulin serum dilution of 1:100 was empirically determined to neutralize 97% of the insulin released into the incubation medium from 10 x 106 sperm (data not shown). Nifedipine (11) and wortmannin (9) were used at concentrations tested by other researchers or used in our previous experiments. At the end of the sperm incubations, the culture media were recovered by centrifugation. Human insulin concentrations were determined in duplicate using an insulin RIA kit according to manufacturer’s instructions. Insulin standards ranged from 0–300 μIU/ml. The limit of sensitivity for the assay was 0.01 μIU/ml. Inter- and intraassay variations were 6.4% and 5.1%, respectively. Insulin results are presented as the original concentrations of the supernatants and are expressed as microinternational units per milliliter.
Glucose-6-phosphate dehydrogenase (G6PDH) activity
The conversion of NADP+ to NADPH, catalyzed by G6PDH, was measured by the increase in absorbance at 340 nm. Washed spermatozoa were incubated for 1 h at 37 C under noncapacitating or capacitating conditions as described above. Both noncapacitated and capacitated sperm were treated with 0.6, 8.3, and 16.7 mM glucose or with insulin (3.3 or 10 nM), antiinsulin (or normal) rabbit serum (1:100) plus 16.7 mM glucose, 10 μM wortmannin plus 16.7 mM glucose, or 25 μM nifedipine. The antibody and wortmannin were added 30 min before glucose stimulation. After treatment, 50 μl sperm extracts were loaded into individual cuvettes containing buffer (100 mM triethanolamine, 100 mM MgCl2, 10 mg/ml glucose-6-phosphate, and 10 mg/ml NADP+, pH 7.6) for spectrophotometric determination. The absorbance of samples was read at 340 nm every 20 sec for 1.5 min. Data are expressed as nanomoles per minute per 106 sperm. The enzymatic activity was determined with three control media: one without glucose-6-phosphate as substrate, one without the coenzyme (NADP+), and the third without either substrate or coenzyme (data not shown). Every experiment was performed six times, including three replicates within each experiment.
Statistical analysis
The experiments for RT-PCR were repeated on at least three independent occasions, whereas Western blot analysis was performed in at least 10 independent experiments. The data obtained from RIA were presented as the mean ± SEM, and 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).
Results
RT-PCR and Western blot
To determine whether mRNA for insulin is present in human ejaculated spermatozoa, RNA isolated from pooled Percoll-separated samples from normal men was analyzed by RT-PCR. The primer sequences were based on the human insulin gene sequence, and RT-PCR amplification revealed the expected PCR product size of 261 bp (Fig. 1A).
FIG. 1. Insulin expression in human ejaculated spermatozoa. A, RT-PCR analysis of human insulin gene in Percoll-separated human ejaculated spermatozoa (S1). N, Negative control (no M-MLV reverse transcriptase added); M, marker. The arrow indicates the expected size of the PCR product. B, Western blot of insulin protein: expression in three samples of ejaculated spermatozoa from normal men (S1, S2, and S3). The negative control (N), performed using sperm lysates, where insulin was previously removed by preincubation with the antibody to human insulin (1 h at room temperature) and immunoprecipitated with protein A/G-agarose, is represented in lane 2. The experiments were repeated more than four times, and this autoradiograph shows the results of one representative experiment.
The presence of insulin protein in human ejaculated spermatozoa was investigated by Western hybridization using an antibody raised against the carboxyl terminus of the mature human insulin protein. Our antibody principally revealed one immunoreactive band at 36 kDa in different lysates from noncapacitated sperm (Fig. 1B), whereas no bands were observed in the negative control. The 36-kDa protein corresponds to the hexameric insulin form, which is present within the storage granules.
Immunolocalization of insulin in human sperm
Using an immunofluorescence technique, we identified a positive signal for insulin in human spermatozoa (Fig. 2). Insulin immunoreactivity showed a different distribution between noncapacitated and capacitated sperm. It is worth noting that the signal was less intense in capacitated sperm.
FIG. 2. Immunolocalization of insulin in human ejaculated spermatozoa. Washed spermatozoa were incubated in unsupplemented Earle’s medium for 1 h at 37 C in 5% CO2 (NC) or in capacitating medium (CAP) at the same conditions. Sperm cells incubated without the primary antibody were used as the negative control (NEG). The pictures shown are representative examples of experiments that were performed at least three times with consistent results.
Measurement of insulin secretion
RIAs were used to evaluate whether sperm secrete insulin. After the assay was validated for spermatozoa, experiments showed that the increase in insulin secretion was dependent on the sperm concentration (Fig. 3A). Insulin production from 10 x 106 sperm incubated in noncapacitating medium (range, 0.1–0.73 μIU/ml) was significantly lower than that measured from sperm incubated under capacitating conditions (range, 4–12 μIU/ml). The time courses of insulin secretion from sperm incubated in noncapacitating and capacitating media are shown in Fig. 3, B and C, respectively.
FIG. 3. Insulin secretion from human ejaculated spermatozoa. Insulin secretion in culture medium from human ejaculated spermatozoa was measured by RIA. A, An increasing number of washed sperm were incubated in unsupplemented Earle’s balanced salt solution for 1 h at 37 C in 5% CO2. Linear regression analysis was performed, and the correlation coefficient (r) was calculated (r2= 0.97). B, Time course of changes in insulin secretion from noncapacitated sperm (NC) at the indicated times. C, Time course of changes in insulin secretion from capacitated sperm (CAP) at the indicated times. Values are the mean ± SEM of 10 replicates. , P < 0.05; , P < 0.01 (vs. control).
Glucose is the main physiological secretagogue of insulin in pancreatic ?-cells (12). To evaluate whether insulin secretion from sperm is also responsive to D-glucose, we generated a dose-response curve of exogenous glucose and evaluated insulin secretion from both noncapacitated and capacitated sperm. These experiments used 0.6, 8.3, or 16.7 mM glucose so that results could be compared with previously published reports. As shown in Fig. 4, glucose significantly stimulated insulin secretion in a dose-dependent fashion. Pretreatment with 10 μM wortmannin significantly decreased the amount of insulin released in response to 16.7 mM glucose. A similar decrease was observed in the presence of antiinsulin serum (1:100) or 25 μM nifedipine. These data suggest that insulin secretion is regulated in sperm.
FIG. 4. Effects of glucose, antiinsulin serum, wortmannin, and nifedipine on insulin secretion from human ejaculated spermatozoa. Washed sperm pooled from normal seminal samples was split into two samples and incubated under noncapacitating () or capacitating () conditions. Each group was treated with 0.6, 8.3, and 16.7 mM glucose (Gluc) or with antiinsulin [or normal (nrs)] rabbit serum (1:100) plus 16.7 mM glucose (Ab anti-ins + gluc), 10 μM wortmannin plus 16.7 mM glucose (W + gluc), or 25 μM nifedipine (Nif). Values are the mean ± SEM of 10 replicates. , P < 0.05; , P < 0.01; , P < 0.005 (vs. control).
Insulin effects on G6PDH activity
We evaluated possible autocrine regulation of G6PDH activity by sperm insulin, because the effect of glucose on the fertilizing ability of sperm appears to be mediated by the pentose phosphate pathway (PPP) (13, 14, 15). The production of NADPH from glucose-6-phosphate by sperm increased significantly when the concentration of exogenous insulin (3.3 and 10 nM) was increased and when the glucose concentration in the incubation medium was raised from 0.6 to 16.7 mM (Fig. 5). To prove that insulin secreted by sperm is acting on G6PDH activity, we stimulated insulin secretion with 16.7 mM glucose, but absorbed the secreted insulin with antiinsulin antibody. Antiinsulin rabbit serum (1:100) significantly decreased G6PDH activity in sperm lysates compared with sperm incubated under the same conditions, with normal rabbit serum (1:100). As additional evidence of autocrine regulation of insulin on glucose metabolism, nifedipine significantly decreased insulin secretion in sperm. This protocol has been shown to abolish the Ca2+ influx via L-type Ca2+ channels and to completely block insulin secretion (11). Finally, insulin signaling in sperm was disrupted using wortmannin, which inhibits the activity of phosphotidylinositol 3-kinase, the kinase principally involved in the insulin signaling cascade (11).
FIG. 5. Effects of insulin, glucose, antiinsulin serum, wortmannin, and nifedipine on G6PDH activity in human ejaculated spermatozoa. Washed spermatozoa were incubated for 1 h at 37 C under noncapacitating () or capacitating conditions () as described previously. Both groups were treated with 0.6, 8.3, and 16.7 mM glucose (Gluc) or with insulin (3.3 nM or 10 nM[SCAP];; Ins), antiinsulin [or normal (nrs)] rabbit serum (1:100) plus 16.7 mM glucose (Ab anti-ins + gluc), 10 μM wortmannin plus 16.7 mM glucose (W + gluc), or 25 μM nifedipine (Nif). Data are expressed as nanomoles per minute per 106 sperm. , P < 0.05; , P < 0.01; , P < 0.005 (vs. control).
Discussion
In the present study we reported the novel finding that insulin is expressed in human sperm, as demonstrated by evaluation of mRNA expression, protein expression, and protein immunolocalization. In addition, we detected insulin secretion by sperm and the existence of an autocrine feedback of insulin on its own secretion and on G6PDH activity.
New reports firmly establish the presence of mRNA in mammalian ejaculated spermatozoa. To date about 14 transcripts have been identified. 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 (16). Worthy new findings suggest that some of these transcripts code for proteins essential in early embryo development (17). In our study insulin expression has been evidenced by Western blot and immunofluorescence. In particular, immunocytochemical analysis of insulin showed a heterogeneous expression pattern, implying different degrees of energetic status among sperm. In a majority of the sperm in noncapacitated samples, insulin was located at the subacrosomial level, in the midpiece, and throughout the tail. Moreover, insulin is arranged within granules, similar to the insulin storage granules within pancreatic ?-cells. An overall decrease and uniform distribution in the signal intensity for insulin were observed in capacitated sperm. This result can be attributed to a major release of hormone in accordance with the high levels (RIA values) of insulin in the medium of capacitating sperm. In pancreatic ?-cells, changes in insulin release are accompanied by simultaneous changes in glucose metabolism. Changes in insulin release are also related to different physiological conditions, such as when sperm switch from noncapacitated to capacitated status. It is worth noting that insulin secretion from spermatozoa into the capacitating medium takes place immediately (at time zero), suggesting a possible involvement of insulin in the induction of capacitation.
Our time-course studies showed that insulin from sperm is released in a pulsatile manner that results in oscillatory concentrations in the incubation medium in both noncapacitated and capacitated sperm. Similarly, under basal as well as stimulated conditions, pancreatic ?-cells secrete insulin in a pulsatile manner, and the oscillatory pattern is believed to improve release control and to enhance hormonal action (18). Glucose is the main secretagogue of insulin in pancreatic ?-cells (12), and we now have shown that insulin secretion from sperm is also responsive to glucose.
Moreover, our results indicate that insulin exerts a physiological autocrine stimulatory effect on glucose-induced insulin release. Specifically, the blockage of insulin release, immune neutralization of the released insulin, or blockage of the activity of intracellular insulin messenger significantly decreased insulin secretion (50%) in both noncapacitated and capacitated sperm. These data address an autocrine regulation of insulin on its own secretion in sperm, which is in agreement with recent data in pancreatic ?-cells showing that secreted insulin may have a positive effect on insulin exocytosis (19).
In pancreatic islets, the release of insulin requires hexose metabolism mediated by glucokinase (20), which has also been identified in sperm (21). Insulin secretion is triggered by ATP generated in the course of glucose metabolism that depolarizes the ?-cell membrane and increases the cytosolic concentration of Ca2+ (22). Of note, these latter events are also important in the initiation of capacitation (23), such as when insulin efflux was detected. In addition, the IGF-I receptor has been found in human sperm (24), and spermatozoa have been reported to bind radioinsulin in a time- and concentration-dependent manner (5). These conditions create the potential for an autocrine insulin loop during the induction of capacitation.
The insulin secretion by spermatozoa suggests an autocrine regulation of glucose metabolism. Glucose is provided to sperm by seminal plasma, by female reproductive tract fluid in vivo (25), or by culture medium in vitro (26, 27), and several studies have indicated that stores of glycogen are endogenous sources of glucose in sperm, allowing sperm to accommodate glucose-free conditions (27). Hexokinase, in the initial step of glycolysis, generates glucose-6-phosphate from glucose, supplying this substrate to both glycolysis and the PPP. Although glycolysis is important for sperm functions such as hyperactivated motility, this metabolic pathway does not appear to be responsible for successful gamete fusion (28, 29). Instead, the beneficial effect of glucose on the acquisition of fertilizing ability as well as on gamete fusion is mediated by glucose metabolism through the PPP (13, 14, 15). We evaluated the possible autocrine insulin modulatory effect on G6PDH activity, because G6PDH is the key rate-limiting enzyme in the PPP, which has been shown to be functional in human spermatozoa (29). G6PDH regulates the production of NADPH by controlling the metabolism of glucose (14). Insulin induction of G6PDH activity has been studied both in vivo and in vitro (30). In our study the blockage of insulin release, immune neutralization of the released insulin, or blockage of intracellular insulin signaling significantly decreased G6PDH activity in sperm in both noncapacitated and capacitated sperm. A similar decrease in the effect of insulin on glucose metabolism was recently obtained in pancreatic islets (19).
Ejaculated spermatozoa require extratesticular maturation, termed capacitation, that allows them to become competent to fertilize an egg. Capacitation is a multifaceted process that has been shown to correlate with changes in spermatozoal metabolism, intracellular ion concentrations, plasma membrane fluidity, intracellular pH, intracellular cAMP concentration, and reactive oxygen species (23). Intriguingly, capacitation in vitro occurs spontaneously after the removal of seminal plasma and without a requirement for an exogenous mediator, suggesting autocrine induction (31) 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 as yet unknown. On the basis of our results, sperm-derived insulin might be considered a factor involved in the induction of capacitation. Upon achieving threshold concentrations, insulin may act on sperm receptors to induce the signal transduction and molecular changes of capacitation.
This study showed a new possible endogenous mediator of capacitation, because we found that human ejaculated spermatozoa are able to secrete insulin during this process. Using a variety of experimental approaches, we found abundant pancreatic ?-cell features, most notably insulin secretion. It is unexpected that insulin expression would be found in sperm, considering the limited cell types that produce insulin and considering the differences between sperm and pancreatic ?-cells. However, given the essential role in the propagation of life, it would be conceivable that metabolic and signaling pathways expressed in terminally differentiated cells might be subsumed into a sperm cell. The spermatozoon leaves the testis and moves through the female genital tract in the host body of the opposite gender, so sperm needs to be autonomous.
The secretion of insulin by spermatozoa may provide an autocrine regulation of glucose metabolism according to their energetic needs independent of systemic insulin. Given the simultaneous decrease in both insulin release and hexose metabolism by blocking the autocrine effect of sperm insulin, we have shown a modulatory effect of the hormone on these two events. Insulin, in addition to other candidates, may be implicated in control of the energy status in the various sperm compartments as well as during the different stages of fertilization.
In conclusion, the findings of insulin expression and secretion from human sperm open a new area of study in male reproduction.
Acknowledgments
Our special thanks to Dr. Vincenzo Cunsulo (Biogemina SAS, Catania, Italy). We also thank D. Sturino (Faculty of Pharmacy, University of Calabria, Calabria, Italy) and Dr. M. Young assistance with reviewing the manuscript text.
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Address all correspondence and requests for reprints to: Dr. Saveria Aquila , Centro Sanitario, University of Calabria, Arcavacata di Rende (CS) 87030, Italy. E-mail: sebastiano.ando@unical.it and aquisav@libero.it.
Abstract
A striking feature of insulin expression is its almost complete restriction to ?-cells of the pancreatic islet in normal mammals. Here we show that insulin is expressed in and secreted from human ejaculated spermatozoa. Both insulin transcript and protein were detected. In addition, the large differences in insulin secretion, assessed by RIA, between noncapacitated and capacitated sperm suggest a role for insulin in capacitation. Insulin had an oscillatory secretory pattern involving glucose dose-dependent increases and significant decreases during the blockage of an insulin autocrine effect. It appears that the effect of glucose on the fertilizing ability of sperm is mediated by glucose metabolism through the pentose phosphate pathway. Then we evaluated the autocrine effect of sperm insulin on glucose metabolism by studying the activity of glucose-6-phosphate dehydrogenase, the key rate-limiting enzyme in the pentose phosphate pathway. The simultaneous decrease in both insulin release and glucose-6-phosphate dehydrogenase activity induced by blocking the autocrine insulin effect with three different procedures (blockage of insulin release by nifedipine, immune neutralization of the released insulin by antiinsulin serum, and blockage of an insulin intracellular effector such as phosphotidylinositol 3-kinase by wortmannin) strongly suggests a physiological role of sperm insulin on these two events. Insulin secretion by spermatozoa may provide an autocrine regulation of glucose metabolism based on their energetic needs independent of systemic insulin. In conclusion, these data open a new area of study in male reproduction.
Introduction
INSULIN IS IMPORTANT for promoting and regulating growth, differentiation, and metabolism. During embryonic development, extrapancreatic insulin gene expression is detectable in the yolk sac (1) and brain (2) of rodents. In adult mammals, apart from the thymus, insulin is thought to be produced only in ?-cells in the pancreas (3). In addition, insulin has been shown to play a central role in the regulation of gonadal function. However, the significance of insulin in male fertility is not complete. It was reported that IGF-II and insulin as well as IGF-I promote spermatogonial differentiation into primary spermatocytes by binding to the IGF-I receptor (4). It was also shown that both the sperm plasma membrane and the acrosome represent cytological targets for insulin (5). In men affected by insulin-dependent diabetes, sperm have severe structural defects, significantly lower motility (6), and lower ability to penetrate hamster eggs (7). In this study we show that insulin is expressed in and secreted from human ejaculated spermatozoa.
Materials and Methods
Chemicals
The Total RNA Isolation System kit, enzymes, buffers, nucleotides, and 100-bp ladder used for RT-PCR were purchased from Promega Corp. (Milan, Italy). Moloney murine leukemia virus (M-MLV) was obtained from Invitrogen Life Technologies, Inc., Italia (Milan, Italy). Oligonucleotide primers were made by Invitrogen Life Technologies, Inc., Italia. BSA protein standard, Laemmli sample buffer, prestained molecular weight marker, Percoll (colloidal polyvinylpyrrolidone-coated silica for cell separation), sodium bicarbonate, dimethylsulfoxide, Earle’s balanced salt solution, triethanolamine buffer, MgCl2, glucose-6-phosphate, NADP+, and all other chemicals were purchased from Sigma-Aldrich Corp. (Milan, Italy). The ECL Plus Western blotting detection system, Hybond ECL, was purchased from Amersham Biosciences (Little Chalfont, UK). The human insulin RIA kit was purchased from Diagnostic Systems Laboratories ICN (Biogemina Sas, Catania, Italy). Antibodies (rabbit antiinsulin, peroxidase-coupled antirabbit, and fluorescein isothiocyanate-conjugated antirabbit immunoglobulin G) were obtained from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany).
Semen samples and spermatozoa preparations
Sperm samples with normal parameters of semen volume as well as sperm count, motility, vitality, and morphology, as specified in the WHO Laboratory Manual (8), were pooled and included in this study. In each experiment, four normal samples were pooled. Spermatozoa preparations were performed as previously described (9).
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 of the different treatments on human sperm viability (data not shown).
RNA isolation and RT-PCR
Total RNA was isolated from human ejaculated spermatozoa purified as previously described (10). PCR amplification of cDNA was performed using the following primer pair: forward, 5'-GCC TTT GTG AAC CAA CAC CTG-3'; and reverse, 5'-GTT GCA GTA GTT CTC CAG CTG-3'. The forward primer, located in exon II, and the reverse primer, located in exon III, produce a 261-bp cDNA. Because the primers span an intron, the genomic product is about 1200 bp.
The conditions for PCR were: denaturation at 95 C for 1 min, annealing at 62 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 the amplified product. As a negative control, an RT-PCR was performed without M-MLV reverse transcriptase.
Western blot analysis of sperm proteins
During Western blot analysis, sperm samples were processed as previously described (10). The negative control was performed using a sperm lysate that was immunodepleted of insulin [i.e. preincubate lysate with antiinsulin antibody (1 h at room temperature) and immunoprecipitate with protein A/G-agarose].
Immunofluorescence assay
Sperm cells 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. Insulin staining was carried out after blocking with normal horse serum (10%) using a rabbit polyclonal antihuman insulin antibody (1:100) and an antirabbit immunoglobulin G fluorescein isothiocyanate-conjugated antibody (1:200). Sperm cells incubated without the primary antibodies were used as negative controls. The slides were examined under a fluorescence microscope (BX41, Olympus, Milan, Italy), and a minimum of 200 spermatozoa/slide were scored.
Measurement of insulin secreted by human ejaculated spermatozoa
A competitive RIA was applied to measure insulin in the sperm culture medium. Increasing numbers of spermatozoa were washed twice with unsupplemented Earle’s medium and incubated in the same medium for 1 h at 37 C in 5% CO2. In other experiments, sperm cultures were split into two series, then incubated under noncapacitating (unsupplemented Earle’s medium alone) or capacitating conditions (Earle’s balanced salt solution medium supplemented with 600 mg BSA/100 ml and 200 mg sodium bicarbonate/100 ml) for 1 h in a 37 C water bath at a final concentration of 10 x 106 sperm/500 μl. Each group was treated with 0.6, 8.3, and 16.7 mM glucose or with 3.3 or 10 nM insulin, antiinsulin (or normal) rabbit serum (1:100) plus 16.7 mM glucose, 10 μM wortmannin plus 16.7 mM glucose, or 25 μM nifedipine. The antiinsulin (or normal) rabbit serum and wortmannin were added 30 min before glucose stimulation. The antiinsulin serum dilution of 1:100 was empirically determined to neutralize 97% of the insulin released into the incubation medium from 10 x 106 sperm (data not shown). Nifedipine (11) and wortmannin (9) were used at concentrations tested by other researchers or used in our previous experiments. At the end of the sperm incubations, the culture media were recovered by centrifugation. Human insulin concentrations were determined in duplicate using an insulin RIA kit according to manufacturer’s instructions. Insulin standards ranged from 0–300 μIU/ml. The limit of sensitivity for the assay was 0.01 μIU/ml. Inter- and intraassay variations were 6.4% and 5.1%, respectively. Insulin results are presented as the original concentrations of the supernatants and are expressed as microinternational units per milliliter.
Glucose-6-phosphate dehydrogenase (G6PDH) activity
The conversion of NADP+ to NADPH, catalyzed by G6PDH, was measured by the increase in absorbance at 340 nm. Washed spermatozoa were incubated for 1 h at 37 C under noncapacitating or capacitating conditions as described above. Both noncapacitated and capacitated sperm were treated with 0.6, 8.3, and 16.7 mM glucose or with insulin (3.3 or 10 nM), antiinsulin (or normal) rabbit serum (1:100) plus 16.7 mM glucose, 10 μM wortmannin plus 16.7 mM glucose, or 25 μM nifedipine. The antibody and wortmannin were added 30 min before glucose stimulation. After treatment, 50 μl sperm extracts were loaded into individual cuvettes containing buffer (100 mM triethanolamine, 100 mM MgCl2, 10 mg/ml glucose-6-phosphate, and 10 mg/ml NADP+, pH 7.6) for spectrophotometric determination. The absorbance of samples was read at 340 nm every 20 sec for 1.5 min. Data are expressed as nanomoles per minute per 106 sperm. The enzymatic activity was determined with three control media: one without glucose-6-phosphate as substrate, one without the coenzyme (NADP+), and the third without either substrate or coenzyme (data not shown). Every experiment was performed six times, including three replicates within each experiment.
Statistical analysis
The experiments for RT-PCR were repeated on at least three independent occasions, whereas Western blot analysis was performed in at least 10 independent experiments. The data obtained from RIA were presented as the mean ± SEM, and 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).
Results
RT-PCR and Western blot
To determine whether mRNA for insulin is present in human ejaculated spermatozoa, RNA isolated from pooled Percoll-separated samples from normal men was analyzed by RT-PCR. The primer sequences were based on the human insulin gene sequence, and RT-PCR amplification revealed the expected PCR product size of 261 bp (Fig. 1A).
FIG. 1. Insulin expression in human ejaculated spermatozoa. A, RT-PCR analysis of human insulin gene in Percoll-separated human ejaculated spermatozoa (S1). N, Negative control (no M-MLV reverse transcriptase added); M, marker. The arrow indicates the expected size of the PCR product. B, Western blot of insulin protein: expression in three samples of ejaculated spermatozoa from normal men (S1, S2, and S3). The negative control (N), performed using sperm lysates, where insulin was previously removed by preincubation with the antibody to human insulin (1 h at room temperature) and immunoprecipitated with protein A/G-agarose, is represented in lane 2. The experiments were repeated more than four times, and this autoradiograph shows the results of one representative experiment.
The presence of insulin protein in human ejaculated spermatozoa was investigated by Western hybridization using an antibody raised against the carboxyl terminus of the mature human insulin protein. Our antibody principally revealed one immunoreactive band at 36 kDa in different lysates from noncapacitated sperm (Fig. 1B), whereas no bands were observed in the negative control. The 36-kDa protein corresponds to the hexameric insulin form, which is present within the storage granules.
Immunolocalization of insulin in human sperm
Using an immunofluorescence technique, we identified a positive signal for insulin in human spermatozoa (Fig. 2). Insulin immunoreactivity showed a different distribution between noncapacitated and capacitated sperm. It is worth noting that the signal was less intense in capacitated sperm.
FIG. 2. Immunolocalization of insulin in human ejaculated spermatozoa. Washed spermatozoa were incubated in unsupplemented Earle’s medium for 1 h at 37 C in 5% CO2 (NC) or in capacitating medium (CAP) at the same conditions. Sperm cells incubated without the primary antibody were used as the negative control (NEG). The pictures shown are representative examples of experiments that were performed at least three times with consistent results.
Measurement of insulin secretion
RIAs were used to evaluate whether sperm secrete insulin. After the assay was validated for spermatozoa, experiments showed that the increase in insulin secretion was dependent on the sperm concentration (Fig. 3A). Insulin production from 10 x 106 sperm incubated in noncapacitating medium (range, 0.1–0.73 μIU/ml) was significantly lower than that measured from sperm incubated under capacitating conditions (range, 4–12 μIU/ml). The time courses of insulin secretion from sperm incubated in noncapacitating and capacitating media are shown in Fig. 3, B and C, respectively.
FIG. 3. Insulin secretion from human ejaculated spermatozoa. Insulin secretion in culture medium from human ejaculated spermatozoa was measured by RIA. A, An increasing number of washed sperm were incubated in unsupplemented Earle’s balanced salt solution for 1 h at 37 C in 5% CO2. Linear regression analysis was performed, and the correlation coefficient (r) was calculated (r2= 0.97). B, Time course of changes in insulin secretion from noncapacitated sperm (NC) at the indicated times. C, Time course of changes in insulin secretion from capacitated sperm (CAP) at the indicated times. Values are the mean ± SEM of 10 replicates. , P < 0.05; , P < 0.01 (vs. control).
Glucose is the main physiological secretagogue of insulin in pancreatic ?-cells (12). To evaluate whether insulin secretion from sperm is also responsive to D-glucose, we generated a dose-response curve of exogenous glucose and evaluated insulin secretion from both noncapacitated and capacitated sperm. These experiments used 0.6, 8.3, or 16.7 mM glucose so that results could be compared with previously published reports. As shown in Fig. 4, glucose significantly stimulated insulin secretion in a dose-dependent fashion. Pretreatment with 10 μM wortmannin significantly decreased the amount of insulin released in response to 16.7 mM glucose. A similar decrease was observed in the presence of antiinsulin serum (1:100) or 25 μM nifedipine. These data suggest that insulin secretion is regulated in sperm.
FIG. 4. Effects of glucose, antiinsulin serum, wortmannin, and nifedipine on insulin secretion from human ejaculated spermatozoa. Washed sperm pooled from normal seminal samples was split into two samples and incubated under noncapacitating () or capacitating () conditions. Each group was treated with 0.6, 8.3, and 16.7 mM glucose (Gluc) or with antiinsulin [or normal (nrs)] rabbit serum (1:100) plus 16.7 mM glucose (Ab anti-ins + gluc), 10 μM wortmannin plus 16.7 mM glucose (W + gluc), or 25 μM nifedipine (Nif). Values are the mean ± SEM of 10 replicates. , P < 0.05; , P < 0.01; , P < 0.005 (vs. control).
Insulin effects on G6PDH activity
We evaluated possible autocrine regulation of G6PDH activity by sperm insulin, because the effect of glucose on the fertilizing ability of sperm appears to be mediated by the pentose phosphate pathway (PPP) (13, 14, 15). The production of NADPH from glucose-6-phosphate by sperm increased significantly when the concentration of exogenous insulin (3.3 and 10 nM) was increased and when the glucose concentration in the incubation medium was raised from 0.6 to 16.7 mM (Fig. 5). To prove that insulin secreted by sperm is acting on G6PDH activity, we stimulated insulin secretion with 16.7 mM glucose, but absorbed the secreted insulin with antiinsulin antibody. Antiinsulin rabbit serum (1:100) significantly decreased G6PDH activity in sperm lysates compared with sperm incubated under the same conditions, with normal rabbit serum (1:100). As additional evidence of autocrine regulation of insulin on glucose metabolism, nifedipine significantly decreased insulin secretion in sperm. This protocol has been shown to abolish the Ca2+ influx via L-type Ca2+ channels and to completely block insulin secretion (11). Finally, insulin signaling in sperm was disrupted using wortmannin, which inhibits the activity of phosphotidylinositol 3-kinase, the kinase principally involved in the insulin signaling cascade (11).
FIG. 5. Effects of insulin, glucose, antiinsulin serum, wortmannin, and nifedipine on G6PDH activity in human ejaculated spermatozoa. Washed spermatozoa were incubated for 1 h at 37 C under noncapacitating () or capacitating conditions () as described previously. Both groups were treated with 0.6, 8.3, and 16.7 mM glucose (Gluc) or with insulin (3.3 nM or 10 nM[SCAP];; Ins), antiinsulin [or normal (nrs)] rabbit serum (1:100) plus 16.7 mM glucose (Ab anti-ins + gluc), 10 μM wortmannin plus 16.7 mM glucose (W + gluc), or 25 μM nifedipine (Nif). Data are expressed as nanomoles per minute per 106 sperm. , P < 0.05; , P < 0.01; , P < 0.005 (vs. control).
Discussion
In the present study we reported the novel finding that insulin is expressed in human sperm, as demonstrated by evaluation of mRNA expression, protein expression, and protein immunolocalization. In addition, we detected insulin secretion by sperm and the existence of an autocrine feedback of insulin on its own secretion and on G6PDH activity.
New reports firmly establish the presence of mRNA in mammalian ejaculated spermatozoa. To date about 14 transcripts have been identified. 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 (16). Worthy new findings suggest that some of these transcripts code for proteins essential in early embryo development (17). In our study insulin expression has been evidenced by Western blot and immunofluorescence. In particular, immunocytochemical analysis of insulin showed a heterogeneous expression pattern, implying different degrees of energetic status among sperm. In a majority of the sperm in noncapacitated samples, insulin was located at the subacrosomial level, in the midpiece, and throughout the tail. Moreover, insulin is arranged within granules, similar to the insulin storage granules within pancreatic ?-cells. An overall decrease and uniform distribution in the signal intensity for insulin were observed in capacitated sperm. This result can be attributed to a major release of hormone in accordance with the high levels (RIA values) of insulin in the medium of capacitating sperm. In pancreatic ?-cells, changes in insulin release are accompanied by simultaneous changes in glucose metabolism. Changes in insulin release are also related to different physiological conditions, such as when sperm switch from noncapacitated to capacitated status. It is worth noting that insulin secretion from spermatozoa into the capacitating medium takes place immediately (at time zero), suggesting a possible involvement of insulin in the induction of capacitation.
Our time-course studies showed that insulin from sperm is released in a pulsatile manner that results in oscillatory concentrations in the incubation medium in both noncapacitated and capacitated sperm. Similarly, under basal as well as stimulated conditions, pancreatic ?-cells secrete insulin in a pulsatile manner, and the oscillatory pattern is believed to improve release control and to enhance hormonal action (18). Glucose is the main secretagogue of insulin in pancreatic ?-cells (12), and we now have shown that insulin secretion from sperm is also responsive to glucose.
Moreover, our results indicate that insulin exerts a physiological autocrine stimulatory effect on glucose-induced insulin release. Specifically, the blockage of insulin release, immune neutralization of the released insulin, or blockage of the activity of intracellular insulin messenger significantly decreased insulin secretion (50%) in both noncapacitated and capacitated sperm. These data address an autocrine regulation of insulin on its own secretion in sperm, which is in agreement with recent data in pancreatic ?-cells showing that secreted insulin may have a positive effect on insulin exocytosis (19).
In pancreatic islets, the release of insulin requires hexose metabolism mediated by glucokinase (20), which has also been identified in sperm (21). Insulin secretion is triggered by ATP generated in the course of glucose metabolism that depolarizes the ?-cell membrane and increases the cytosolic concentration of Ca2+ (22). Of note, these latter events are also important in the initiation of capacitation (23), such as when insulin efflux was detected. In addition, the IGF-I receptor has been found in human sperm (24), and spermatozoa have been reported to bind radioinsulin in a time- and concentration-dependent manner (5). These conditions create the potential for an autocrine insulin loop during the induction of capacitation.
The insulin secretion by spermatozoa suggests an autocrine regulation of glucose metabolism. Glucose is provided to sperm by seminal plasma, by female reproductive tract fluid in vivo (25), or by culture medium in vitro (26, 27), and several studies have indicated that stores of glycogen are endogenous sources of glucose in sperm, allowing sperm to accommodate glucose-free conditions (27). Hexokinase, in the initial step of glycolysis, generates glucose-6-phosphate from glucose, supplying this substrate to both glycolysis and the PPP. Although glycolysis is important for sperm functions such as hyperactivated motility, this metabolic pathway does not appear to be responsible for successful gamete fusion (28, 29). Instead, the beneficial effect of glucose on the acquisition of fertilizing ability as well as on gamete fusion is mediated by glucose metabolism through the PPP (13, 14, 15). We evaluated the possible autocrine insulin modulatory effect on G6PDH activity, because G6PDH is the key rate-limiting enzyme in the PPP, which has been shown to be functional in human spermatozoa (29). G6PDH regulates the production of NADPH by controlling the metabolism of glucose (14). Insulin induction of G6PDH activity has been studied both in vivo and in vitro (30). In our study the blockage of insulin release, immune neutralization of the released insulin, or blockage of intracellular insulin signaling significantly decreased G6PDH activity in sperm in both noncapacitated and capacitated sperm. A similar decrease in the effect of insulin on glucose metabolism was recently obtained in pancreatic islets (19).
Ejaculated spermatozoa require extratesticular maturation, termed capacitation, that allows them to become competent to fertilize an egg. Capacitation is a multifaceted process that has been shown to correlate with changes in spermatozoal metabolism, intracellular ion concentrations, plasma membrane fluidity, intracellular pH, intracellular cAMP concentration, and reactive oxygen species (23). Intriguingly, capacitation in vitro occurs spontaneously after the removal of seminal plasma and without a requirement for an exogenous mediator, suggesting autocrine induction (31) 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 as yet unknown. On the basis of our results, sperm-derived insulin might be considered a factor involved in the induction of capacitation. Upon achieving threshold concentrations, insulin may act on sperm receptors to induce the signal transduction and molecular changes of capacitation.
This study showed a new possible endogenous mediator of capacitation, because we found that human ejaculated spermatozoa are able to secrete insulin during this process. Using a variety of experimental approaches, we found abundant pancreatic ?-cell features, most notably insulin secretion. It is unexpected that insulin expression would be found in sperm, considering the limited cell types that produce insulin and considering the differences between sperm and pancreatic ?-cells. However, given the essential role in the propagation of life, it would be conceivable that metabolic and signaling pathways expressed in terminally differentiated cells might be subsumed into a sperm cell. The spermatozoon leaves the testis and moves through the female genital tract in the host body of the opposite gender, so sperm needs to be autonomous.
The secretion of insulin by spermatozoa may provide an autocrine regulation of glucose metabolism according to their energetic needs independent of systemic insulin. Given the simultaneous decrease in both insulin release and hexose metabolism by blocking the autocrine effect of sperm insulin, we have shown a modulatory effect of the hormone on these two events. Insulin, in addition to other candidates, may be implicated in control of the energy status in the various sperm compartments as well as during the different stages of fertilization.
In conclusion, the findings of insulin expression and secretion from human sperm open a new area of study in male reproduction.
Acknowledgments
Our special thanks to Dr. Vincenzo Cunsulo (Biogemina SAS, Catania, Italy). We also thank D. Sturino (Faculty of Pharmacy, University of Calabria, Calabria, Italy) and Dr. M. Young assistance with reviewing the manuscript text.
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