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Biology of Reproduction 59, 330-337 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Identification of Insulin-Like Growth Factor I in Bovine Seminal Plasma and Its Receptor on Spermatozoa: Influence on Sperm Motility1

Donald M. Henricksc,d,2, Andrew J. Koubae, Brett R. Lackeyc,d, William R. Booned,f, , and Sandra L. Grayc,d

c Endocrine Physiology Laboratory, d Animal and Veterinary Sciences Department, Clemson University, Clemson, South Carolina 29634 e Department of Obstetrics and Gynecology, College of Medicine, University of Florida, Gainesville, Florida 32610 f Andrology Laboratory, Reproductive Endocrinology and Infertility, Greenville Memorial Hospital, Greenville, South Carolina 29605


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Insulin-like growth factor I (IGF-I) has been identified in human seminal plasma. This study was conducted to determine whether IGF-I is present in bovine seminal plasma, whether sperm cells express the IGF-I receptor (IGF-IR), and whether IGF-I affects sperm motility. Semen samples were collected from bulls by electroejaculation and maintained at 37°C, and motility of sperm was assessed. After centrifugation to separate sperm cells from seminal plasma, the seminal plasma was submitted to a validated heterologous RIA for IGF-I. Significant concentrations of IGF-I (116.29 ± 40.83 ng/ml expressed as mean ± SD) were measured in bovine seminal plasma. Sperm cells were washed with buffer and subjected to either radioreceptor assay (RRA) or immunocytochemistry (IC). RRA revealed a single high affinity for the IGF-IR with a Kd of 0.83 nM as determined by the computer program LIGAND. IC, using three monoclonal antibodies, localized the IGF-IR to the acrosomal region of the sperm. Computer-assisted sperm-motion analysis was used to determine the effects of IGF-I and IGF-II on bovine sperm motility parameters. Both IGF-I and IGF-II increased sperm motility and straight-line velocity (p < 0.05) relative to the control. The presence of IGF-IR on sperm, the presence of IGF-I in semen, and the ability of IGF-I to stimulate sperm motility provide evidence that the IGF system may be involved in the fertilization process in the bovine species.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Insulin-like growth factor I (IGF-I) is a potent mitogenic, metabolic, and differentiating polypeptide that is becoming widely recognized as an important regulator of reproductive functions [1]. The biological actions of the IGF ligands (IGF-I and IGF-II) are mediated through cell surface receptors (type 1 and type 2) by a family of high-affinity IGF binding proteins (IGFBPs 1–6) and IGFBP proteases, which regulate ligand bioavailability. This multi-component network of molecules has been identified in the male testis.

In several species, IGF-I and IGF-II are involved in Leydig cell differentiation and function [2–5]. IGF-I and IGF-II stimulate steroidogenesis by increasing gonadotropin (LH/hCG) receptor density and expression of key steroidogenic enzymes [3–7]. IGF-I and IGF-II are also implicated as important factors in spermatogenesis [8–12]. Mice with an Igf1 null mutation were 30% smaller, had decreased reproductive organ weights, and had testosterone and sperm concentrations that were 18% of normal controls [2]. The production of IGF-I by cultures of porcine Sertoli and rat Leydig cells suggests that IGF-I may act as a developmental and differentiation factor for spermatogonia, spermatocytes, and spermatids [9–14]. The expression of the IGF-I and IGF-II ligands by Sertoli and Leydig cells and the identification of their receptors on Leydig and Sertoli cells, spermatogonia, spermatocytes, and spermatids indicate that IGF-I and -II are regulators of testicular function [4, 15–17].

IGFs may also be post-testicular regulators of reproductive function, as Leydig, Sertoli, and peritubular cells secrete IGF-I [15–18]. Human seminal plasma contains IGF-I, IGF-II, IGFBP-2, IGFBP-3 (fragments), and IGFBP-4, as well as IGFBP-3 protease activity [19–22]. IGF-I in seminal plasma was shown to be primarily of testicular or epididymal origin, as vasectomized patients revealed a significant decrease in seminal plasma IGF-I concentrations [22–25]. IGFBP-2, IGFBP-3, and IGFBP-4 have been identified in human seminal plasma and may regulate the bioavailability of IGF-I in seminal plasma [26–28]. The serine protease, prostate-specific antigen, has been located in seminal plasma and is capable of cleaving IGFBP-3 from the IGF-I molecule [29]. The presence of IGF-I, IGFBP-2, IGFBP-3, and IGFBP-4, as well as prostate-specific antigen, in human seminal plasma suggests a complex interaction that may regulate sperm physiology and activity. In this study, we evaluate the presence of IGF-I in seminal plasma of a domestic species (bovine) and determine whether ejaculated spermatozoa express a receptor for IGF-I and whether IGF-I has a direct action on sperm motility.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Semen Collection

Seven sexually mature beef bulls ranging in age between 16 and 48 mo of age were the source of semen. Semen was collected by electroejaculation into a disposable collection cone and a conical centrifuge tube maintained at 37°C to prevent cold shock. This project was approved by the Animal Research Committee (ARC protocol #95–008).

Upon collection, the spermatozoa were centrifuged at 1000 x g for 10 min. Sperm were washed twice with Kreb's Ringer Buffer (KRB) [30] supplemented with 0.1% BSA. A special preparation of BSA (Sigma, St. Louis, MO; cat. no. A7030) was used because experiments conducted previously by Gray (unpublished results) showed that this BSA, unlike several others, contained no endogenous IGFs.

IGF-I Extraction and RIA Procedure

Seminal plasma was centrifuged at 1800 x g for 10 min. To 0.2 ml of seminal plasma was added 1.3 ml of 1% trifluoroacetic acid (TFA). Octadecyl C-18 columns (Waters Sep-Pak, Milford, MA) were activated by sequential addition and vacuum of acetonitrile, distilled water, and 0.1% TFA in water. The samples were added to the columns and vacuumed, and then 3 ml of 0.1% TFA was added and vacuumed for 3 min. One milliliter of 0.1% TFA-acetonitrile was added to the columns, and the eluent was vacuumed for approximately 45–60 sec into the test tubes. This step was repeated. The eluent was dried at 37°C under gentle air flow and reconstituted in 2 ml of RIA buffer [31]. IGF (human recombinant; Bachem, King of Prussia, PA) was assayed using a validated heterologous assay [31]. Using glass test tubes, a standard curve was created for each assay consisting of 10, 20, 40, 80, 160, 320, and 640 pg/100 µl. Tubes for total counts, nonspecific binding, and total bound also accompanied each assay. Ten microliters of seminal plasma sample was assayed in triplicate for IGF-I. The primary antiserum raised in rabbit (P.D. Gluckman, Auckland, New Zealand) was used at a 1:5000 dilution. After incubation overnight (> 16 h) at 4°C, 100 µl of a 1:30 dilution of goat anti-rabbit secondary antiserum (Linco, St. Charles, MO) was added and incubated at 4°C for 1 h; this was followed by addition of a 1:50 dilution of 100 µl of normal rabbit serum (Sigma) and incubation at 4°C for 1 h. One milliliter of RIA buffer was added, and all tubes except total counts were centrifuged at 1800 x g for 30 min. The tubes were decanted, inverted on paper towels, and counted for 1 min on the gamma counter (efficiency 75%) [31]. Cross-reactivity was less than 2% to other growth factors tested (IGF-II, epidermal growth factor [EGF], transforming growth factor alpha [TGF{alpha}]; data not shown). The level of quantitation of the assay was 0.1 ng/ml. Recovery rates of IGF-I were 91%. Extracted seminal plasma samples (5–100 µl) inhibited binding of 125I-IGF-I to antibody in a parallel manner when incubated with IGF-I standards (10–640 pg/100 µl).

Biochemical Characterization of the IGF-I Receptor

In order to characterize the biochemical properties of the spermatozoal IGF-I receptor, a radioreceptor assay was validated. Assay conditions were optimized for 125I-IGF-I, sperm cell concentration, time, and temperature. Whole sperm cell suspensions were utilized for each assay, and all assays were run in quadruplicate. Binding reactions were stopped by addition of 3 ml of KRB supplemented with 25% polyethylene glycol (8000 Mr). Bound ligand was separated by centrifugation at 1800 x g for 30 min at 4°C. The supernatant was decanted, and the radioactivity bound in the sperm pellet was counted on a gamma counter. Polypropylene tubes (12 x 75 mm) were precoated with 1% BSA-KRB to minimize nonspecific binding. In brief, varying quantities of 125I-IGF-I were added to a fixed quantity of sperm cells (1 x 107) and incubated for 1 h at 37°C. The optimum amount of radiolabeled ligand was determined to be 0.06 pmol of 125I-IGF-I. Using this fixed concentration of radiolabeled ligand, binding to increasing amounts of sperm cells was examined (5 x 105-4 x 107 cells). To determine saturation kinetics of the IGF-I receptor, binding was examined at various times (15, 30, 60, 90, 120, and 240 min). The dissociation rate of prebound 125I-IGF-I from its receptor was analyzed by allowing 125I-IGF-I to bind to sperm cells under optimum conditions (0.06 pmol 125I-IGF-I, 1 x 107 sperm cells, 37°C for 50 min). A 2000-fold molar excess of unlabeled IGF-I in KRB or KRB alone was then added to the assay, and dissociation of 125I-IGF-I was observed at various times (5, 10, 15, 20, 30, 40, and 50 min). Competitive inhibition with IGF-I, IGF-II, and insulin was examined by adding a 1000-fold molar excess of each hormone at the outset of the binding experiment under optimum conditions. Scatchard data, obtained by adding varying amounts of unlabeled IGF-I (0.01–24 pmol) at the beginning of the assay, were analyzed using the computer program LIGAND [32], which provides information on the affinity of the receptor for the ligand, the association constant, the molar concentration of receptor sites per tube, and nonspecific binding.

Immunocytochemistry. Monoclonal Antibody Staining of the IGF-I Receptor

Two Angus bulls, D184 and D764 (36–48 mo of age), were used for the immunostaining study. Fifty microliters of sperm cells (2 x 107 cells/ml) were incubated with 10.7 µg goat-anti-mouse IgG for 10 min to minimize background fluorescence [33]. The monoclonal antibodies 1H7 and 3B7 (Santa Cruz Biotechnology, Santa Cruz, CA) were generated by immunoprecipitation of the precursor form of the IGF-I receptor (IGF-IR) and do not cross-react with the insulin-receptor. Monoclonal antibody Ab-1 (Oncogene Research Products, Cambridge, MA), a clone of alpha-IR3, recognizes an epitope on the alpha subunit near the binding site. Five micrograms of the control mouse IgG and the mouse monoclonal primary antibodies to the IGF-IR were added and incubated for 15 min. Four milliliters of 0.01% PBS were added to the cells and then vortexed and centrifuged at 1200 x g for 3 min. The supernatant was discarded, and 50 µl of the fluorescein isothiocyanate (FITC)-antimouse-Ab (Sigma) was added (1:50 dilution). The cells were washed with 4 ml of 0.01% PBS, vortexed, and centrifuged at 1200 x g for 3 min. The supernatant was discarded, and the precipitate was resuspended in 0.01% PBS, fixed with formaldehyde, analyzed with fluorescence microscopy, and photographed at 40x using a Nikon (Garden City, NY) Optiphot 2 equipped with an epifluorescence filter with an excitation wavelength of 450–490 nm.

Sperm Motility Analysis

Sperm motility parameters were analyzed using a Hamilton-Thorne Integrated Visual Optical System (IVOS; Hamilton-Thorne Research, Beverly, MA). The parameter settings for motility analysis were as follows: frame rate—60 Hz; frames acquired—25; minimum contrast—30; minimum cell size—9; threshold straightness—80; medium average path velocity (VAP) cut-off—75; low VAP cut-off—25; nonmotile head size—5; nonmotile head intensity—55; slow cells motile—no; LO/HI size gates—0.70 to 2.75; and LO/HI intensity gates—0.30–2.0 [34, 35]. These parameters were selected to maximize the accuracy of the motility analysis by distinguishing motile sperm from detritus, debris, and nonmotile sperm. Slow cells were not counted as motile to prevent collision-induced artifacts from being recorded as motile. Semen was collected by electroejaculation from two sexually mature Angus bulls (D759 and D764; 36–48 mo of age) during a 3-wk period. A total of three collections for each bull were made with a minimum of 48 h between collections. The semen was collected into an Ag-Tek cone (Sioux Falls, SD) and Falcon 2095 conical centrifuge tube (VWR, Atlanta, GA) inside an examination glove containing 37°C water. The semen was diluted 1:2 into 37°C modified Tyrode's medium (MTM) for sperm-motion analysis [30]. MTM was used to dilute any sperm motility inhibitors in the seminal plasma [36]. The diluted semen samples were wrapped in cloth, surrounded by three Deltaphase Isothermal Pads (Braintree Scientific, Braintree, MA), which maintained the temperature at 37°C, and placed in a cooler for transport. The samples were centrifuged at approximately 400 x g for 5 min, the supernatant was discarded, and the sperm were resuspended in MTM to determine the proper dilution for computer-assisted sperm-motion analysis (CASA), which was 20–60 million sperm/ml [35, 37]. Test solutions consisted of MTM, MTM with 100 ng/ml IGF-I (MTM-1), and MTM with 250 ng/ml IGF-II (MTM-2). The plasticware to be used in the motility studies underwent mouse embryo toxicity testing to ensure that the sperm motility studies were performed in an environment free of toxic substances [38].

Sperm were incubated in a 37°C incubator with 5% CO2. At each time point (0, 90, 180, and 360 min), samples were removed and placed in a 37°C warming block for processing. MicroCell 20-µm slides (Conception Technologies, La Jolla, CA), were warmed on the heating block for 3 min before samples were dispensed. The samples were vortexed briefly, and 5 µl of the test solution was loaded onto prewarmed MicroCell slides and allowed to settle in the chamber for 30 sec, and excess solution was removed. The slide was placed in IVOS and analyzed [39]. Five to seven fields from a stage position of 0.0–7.9 mm and a minimum of 200 sperm cells were analyzed for each replicate [34, 38]. At each time point, three samples from each treatment group were analyzed.

Statistical Analysis and Methods

The design of this study was a randomized block design consisting of three treatments and repeated measures. The bulls and sample collections were blocks. Treatments consisted of MTM-1, MTM-2, and MTM control. The incubation time points (90, 180, and 360 min) were the repeated measures. The data were analyzed by two-way ANOVA. Specific comparisons of treatment and time were performed using least-squares means and Student's t-tests. Significant differences were reported when the p value from the t-test was less than alpha (p < 0.05). Statistical analysis was performed using the SAS System for Windows, Release 6.11 (SAS Institute, Cary, NC) [40].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Seminal Plasma IGF-I Concentrations

Seminal plasma IGF-I concentrations varied between bulls and often between consecutive ejaculates. The mean IGF-I concentration (± SD) ranged from 87.37 ± 30.23 to 179.39 ± 0.88 ng/ml. The mean seminal plasma concentrations for each bull are shown in Table 1. The overall mean concentration of IGF-I in seminal plasma from the six bulls was 116.29 ± 40.83 ng/ml.


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  		TABLE 1. Concentrations of IGF-1 in seminal plasma of six bulls.

Biochemical Measurements of the IGF-IR

The binding of a constant amount of 125I-IGF-I (0.06 pmol), representing 120 000 cpm, was found to be linear with increasing amounts of sperm cells from 5–40 x 106 (Fig. 1). Saturable binding was observed, which reached a plateau 20–25 min after incubation with 0.06 pmol of 125I-IGF-I at 37°C with 1 x 107 sperm cells (Fig. 2). Binding remained fairly constant for 4 h and then began to decline (data not shown). Prebound 125I-IGF-I was disassociated from the receptor by a 2000-fold molar excess of unlabeled IGF-I within 50 min (Fig. 3). KRB alone did not affect the disassociation rate of prebound 125I-IGF-I. This indicated that the ligand-receptor complex was not internalized, nor was there a spontaneous loss of 125I-IGF-I due to incubation conditions. Binding of 125I-IGF-I was competitively inhibited by a 1000 molar excess of IGF-I and IGF-II, but not insulin (Fig. 4). This indicated that IGF-I binding was not occurring through an insulin receptor and also supported the hypothesis that it was an IGF-I receptor, as IGF-II binds the receptor with an equal or slightly lower affinity depending on the cell type. IGF-I minimally binds to the IGF-II receptor. Scatchard analysis of 125I-IGF-I revealed a curvilinear relationship (Fig. 5). Regression analysis by the program LIGAND revealed a significant (r2 = 0.93) single high-affinity binding site (Fig. 5, insert).



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  		FIG. 1. Dose-response curve of 125I-IGF-I to increasing numbers of spermatozoa.



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  		FIG. 2. Time course plot exhibiting saturation kinetics.



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  		FIG. 3. Disassociation curve of prebound 125I-IGF-I upon addition of 2000-fold molar excess of IGF-I.



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  		FIG. 4. Competitive inhibition of 125I-IGF-I by KRB and 1000-fold molar excess of IGF-I, IGF-II, and insulin.



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  		FIG. 5. Scatchard and LIGAND plots of radioreceptor assay data. Scatchard plot of 125I-IGF-I binding. Specific binding was expressed as the ratio bound/free (B/F) IGF-I and plotted as a function of IGF-I bound. The data are from a representative experiment. Insert: Scatchard data fitted by the LIGAND program. Nonspecific binding was removed from B/F and bound. The x-intercept and slope yield the concentration of receptor sites (R) and the disassociation constant (KD), respectively.

Immunocytochemical Localization of the IGF-IR

Three mouse monoclonal antibodies—Ab-1, 3B7, and 1H7—were used to screen bovine spermatozoa for immunocytochemical localization of the IGF-IR. No background or nonspecific fluorescence resulted when sperm were incubated with only FITC-labeled secondary antibody (data not shown) [32]. Mouse IgG controls (Fig. 6a, insert) also did not exhibit fluorescence, as seen in Figure 6a. However, sperm incubated with monoclonal antibodies Ab-1, 3B7, and 1H7 displayed intense fluorescence of the acrosomal region of the sperm head, pictured in Figure 6, b, c, and d, respectively.



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  		FIG. 6. Immunocytochemical identification of the IGF-IR. a) Control (mouse IgG). Sperm incubated with monoclonal antibody Ab-1 (b), 3B7 (c), and 1H7 (d). Bar equals 20 µm. Pictures are from a representative experiment.

Effect of IGF-I and -II on Sperm Motility

Our RIA results revealed a mean IGF-I concentration of 116.29 ± 40.83 ng/ml in seminal plasma. Using physiological concentrations of IGF-I and IGF-II (100 and 250 ng/ml [9, 25, 41], respectively), two motility parameters were measured: percentage of motile sperm and straight-line velocity (VSL). Treatment with IGF-I and IGF-II increased (p < 0.05) sperm motility at all time points when compared to the control (90, 180, and 360 min; Fig. 7, a and b). However, only treatment with IGF-I resulted in significant increases in VSL when compared to the control (180 and 360 min; Fig. 7, c and d). Although time-dependent declines in both motility and VSL occurred, sperm incubated with IGF-I and IGF-II were better able to maintain these parameters.



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  		FIG. 7. Effects of IGF-I and IGF-II on CASA parameters. a) Overall motility for treatment groups. b) Time effects on motility for treatment groups. c) Overall VSL for treatment groups. d) Time effects on VSL for treatment groups.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study provides evidence that immunoreactive IGF-I is found in bovine seminal plasma and that an IGF-I receptor is present on ejaculated bovine sperm. The concentration of IGF-I in bovine seminal plasma is similar to that reported for IGF-I in human seminal plasma [25]. Concentrations of IGF-I are 15–20 times lower than the concentrations of IGF-II in human seminal plasma [20]. Other growth factors that have been reported in human seminal plasma include EGF [42] and TGF{alpha} [43]. The presence of these growth factors, and of as yet unidentified growth factors in seminal plasma, suggests a regulatory role of growth factors in sperm physiology or reproductive tract maintenance. The exact location of the IGF-I production in human seminal plasma is still unknown. Vasectomized patients revealed a much lower IGF-I concentration than did intact patients [22–25], indicating that seminal plasma IGF-I was primarily of testicular or epididymal origin. IGF-I is necessary for development of normal germ cells, and seminal IGF-I is associated with normal morphology [25]. Mice lacking the IGF-I gene have decreased organ weights and 80% lower sperm concentrations [2]. Also of testicular origin are several IGFBPs capable of regulating the bioavailability of IGF-I [16, 44]. It is of interest to note that a prostate-specific antigen capable of cleaving IGF-I from various binding proteins [29] is found within accessory sex gland fluid. This suggests that IGF-I bound to binding proteins is unavailable until immediately before or after ejaculation. IGF-I in seminal plasma may have a regulatory function in pre- and post-ejaculated sperm affecting motility and capacitation.

Four major proteins of bovine seminal plasma—BSP-A1, -A2, -A3, and -30-kDa (collectively called the BSP proteins)—have been shown to bind IGF-II [45]. These BSP proteins are not related to the IGFBPs found in human seminal plasma [46]. The BSP proteins have been shown to bind to sperm cells by interacting with choline phospholipids on the sperm plasma membrane [47]. It is possible that these BSP proteins, which act as sperm cell surface binding sites for IGF-II, might also bind IGF-I in a similar fashion. IGFBPs along with BSP proteins may serve to modulate IGF-I in bovine seminal plasma. The present study, however, shows that IGF-I can interact directly with a spermatozoal IGF-I receptor.

Characterization of the bovine sperm cell IGF-IR revealed biochemical properties similar to those previously reported [48]. Under optimum assay conditions for intact bovine sperm cells, 125I-IGF-I binding was found to be specific, saturable, and competitively inhibited by both IGF-I and IGF-II in an equal fashion. A dose-response test was not done for IGF-I and IGF-II inhibition of binding, but this presumably would have shown slight differences in affinity for the receptor. The binding affinity (Ka = 1.2 x 109 M-1) for IGF-I bound to sperm cells is similar to those reported for IGF-I binding to rat Leydig cells [49, 50]. By using the molar concentration of receptor sites per tube, it was possible to calculate the number of IGF-I receptors per sperm cell, which was on average 1000 receptors per cell. The presence of an IGF-I ligand in seminal plasma and its receptor on sperm cells suggests that this complex might be involved in metabolic or differentiating activities within the sperm cell.

The immunocytochemistry experiment using three distinct monoclonal antibodies and the radioreceptor experiment were mutually supportive and agreed with other studies that have localized the IGF-IR to differentiating germinal cells [9, 11–17, 51]. The physiological status of the sperm cells may explain why some cells fluoresce and others do not. Receptors for progesterone and mannose identify a subset of sperm that are acrosome-inducible [52, 53]. Perhaps sperm that are functionally viable express the IGF-I receptor. Future work needs to be done to identify the subset of sperm expressing the IGF-I receptor. Our results indicate that the IGF-I receptor is primarily localized to the acrosomal region on the plasma membrane of differentiated, uncapacitated sperm. This region is involved in capacitation and the acrosome reaction. It is hypothesized that growth factor signaling may be important in these membrane reorganizational events. Indeed, an EGF receptor has been identified on mature sperm cells from humans, mice, rabbits, and rats [54]. This EGF receptor was functionally active as shown by increased tyrosine kinase activity and phosphorylation. Future work needs to be done to determine the signal transduction pathway of the IGF-I receptor on bovine sperm cells. EGF was shown to stimulate human and mouse capacitation [55], but it had no effect on fertilization [56]. Furuya's group [55] reported no effect of EGF on motility; however, they suggest that to correctly evaluate EGF effects on motility, CASA would need to be examined.

In our study, we evaluated the effects of IGF-I and IGF-II on bovine sperm motility using CASA. We showed that physiological concentrations of IGF-I (100 ng/ml) and IGF-II (250 ng/ml) were capable of maintaining motility significantly above that of the control, at all time points examined. This is the first report to our knowledge that shows a direct effect of this growth factor on sperm motility. The IGF concentration used in this study approximates the average concentration of IGF found in oviductal fluid [57]. The VSL of spermatozoa has been correlated with fertilization rates, and differences between the VSL of sperm from fertile and subfertile males has been reported [58–60]. IGF-I and IGF-II appear to increase the velocity of sperm relative to that of the control, which would therefore classify the IGFs as chemokinetic factors, factors that are involved in regulation of a cell's movement.

One possible way that IGF-I maintains motility is through energy metabolism. IGFs have been shown to increase glucose uptake, lactate production, pyruvate dehydrogenase activity, and conversion to glucose-6-phosphate [61]. Another possibility is the antioxidant effect of IGFs. Lipid peroxidation and reactive oxygen species can severely damage metabolic machinery, resulting in loss of sperm motility and function [53]. IGFs in neuronal cells prevent mitochondrial dysfunction when exposed to glutathione-depleting agents, maintain calcium homeostasis, and increase cell survival [62–64]. If IGFs act as antioxidants, this would then result in increased sperm viability [58–60]. Indeed, the greater motility and velocity of sperm cells treated with IGFs at the later time-points may reflect this antioxidant activity.

The effects of IGF-I on capacitation have not yet been evaluated. Capacitation of sperm reflects a change in the kinematics of sperm motility called hyperactivation. Results from our lab have shown that IGF-I may also affect sperm motion characteristics that are typical of hyperactivated sperm (data not shown). IGFs have been shown to increase ion transport [65], which may result in increased intracellular calcium levels. Hyperactivation is known to require extracellular calcium [66] and may therefore be partially regulated by growth factors. Our data showing that IGF-I can significantly increase the VSL of sperm suggest increased flagellar motion and thrust. These sperm-motion characteristics have been shown to be important in penetration of the zona pellucida [67] and in transport through oviductal mucus [68]. IGF-I has been shown to be produced by the oviductal epithelial cells and secreted into the oviductal lumen of pigs [57]. The prefertilization events suggested by Suarez [69] and Smith [70] may in part be regulated by an interaction between oviductal IGF-I and the sperm cell IGF-I receptor.

In summary, data presented here demonstrate that IGF-I present in bovine seminal plasma can interact with a specific IGF-I receptor on the acrosomal region of ejaculated sperm. This interaction of ligand and receptor can increase sperm motility and VSL. Further investigations are needed to understand IGF-I involvement in capacitation, hyperactivation, and the acrosome reaction. The presence of IGF-I in both the male and female reproductive tract as well as a receptor on sperm cells suggests a possible regulatory role in spermatozoal prefertilization events.


   ACKNOWLEDGMENTS
 
We wish to thank Dr. John Whitesides, Dr. Ronald Thurston, and Ms. Nancy Korn for assistance with immunocytochemistry, and Dr. Richard Hilderman for assistance with the radioreceptor analysis. We thank Mr. Gary Burns and Mr. Scott Hicks for assistance with semen collection, and Ms. Jane Johnson and Ms. Sherri Lee for CASA expertise. Appreciation is extended to Dr. William C. Bridges, Jr., for guidance and statistical proficiency.


   FOOTNOTES
 
1 This work was supported by the Animal Biotechnology Initiative (#1513), Clemson University, and a Pilot Grant from the Greenville Hospital System/Clemson University Biomedical Cooperative. Technical contribution No. 4415 of the South Carolina Agric. Experimental Station. Back

2 Correspondence: Donald M. Henricks, AVS Department, Clemson University, Poole Ag., Building, Box 340361, Clemson, SC 29634. FAX: (864) 656–3131; dhnrcks{at}clemson.edu Back

Accepted: March 24, 1998.

Received: December 29, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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