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Biology of Reproduction 67, 648-654 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Insulin-Like Growth Factor-I and Insulin-Like Growth Factor Binding Protein-2 and -5 in Equine Seminal Plasma: Association with Sperm Characteristics and Fertility1

M.L. Macpherson2, R.C.M. Simmena, F.A. Simmena, J. Hernandez, B.R. Sheerin, D.D. Varnerb, P. Loomisc, M.E. Cadario, C.D. Millerd, S.P. Brinskob, S. Rigbyb, and T.L. Blanchardb

a Departments of Large Animal Clinical Sciences and b Animal Sciences, University of Florida, Gainesville, Florida 32610 c Department of Large Animal Medicine and Surgery, Texas A&M University, College Station, Texas 77843 d Select Breeders Services Inc., Colora, Maryland 21917 e Equine Medical Center of Ocala, Ocala, Florida 34474


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objectives of this study were 1) to determine whether insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) were present in seminal plasma of stallions; 2) to compare semen parameters (IGF proteins, sperm numbers, morphology, and motility) from stallions at sexual rest (SR) and when sexually active (SA); 3) to compare semen parameters between stallions with high and low seminal plasma IGF-I concentrations; and 4) to examine the relationship between seminal plasma IGF-I concentrations and fertility parameters of stallions. Ejaculates were collected from stallions at SR (n = 51) and SA (n = 46). Concentrations of IGF-I and IGFBP-2 in seminal plasma samples were determined by radioimmunoassay. Presence of IGFBPs in equine seminal plasma was verified using immunoprecipitation and Western ligand blot procedures. IGF-I, IGFBP-2, and IGFBP-5 were present in equine seminal plasma. Concentrations of IGF-I, IGF-I/protein, total IGF-I, IGFBP-2, IGFBP-2/protein, and total IGFBP-2 were not significantly different (P >= 0.13) in seminal plasma between stallions at either SR or SA. At SR, stallions with higher seminal plasma IGF-I had more total IGFBP-2 per ejaculate (P < 0.01), more morphologically normal sperm (P = 0.05), and higher first-cycle pregnancy rates (P = 0.02). At SA, stallions with higher seminal plasma IGF-I had fewer cycles per pregnancy (P = 0.02). An association of seminal plasma IGF-I concentration with sperm motility, sperm morphology, and pregnancy rates in bred mares suggests that IGF-I may play a role in sperm function.

growth factors, insulin-like growth factor receptor, male reproductive tract, male sexual function, sperm


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Steroidogenesis and spermatogenesis, components of male reproductive function, are controlled by endocrine and local regulatory factors. Although endocrine control is integral to male reproductive function, some infertile males lack demonstrable endocrine abnormalities [1–3], so endocrine parameters alone do not always explain abnormalities in sperm production or infertility. Recent investigation of idiopathic infertility in humans, rodents, cattle, and horses has focused on local regulators of reproductive events including growth factors, differentiation factors, and cytokines [4–9].

Growth factors are polypeptides that function as paracrine, autocrine, and/or endocrine regulators of cell growth and differentiation. Specifically, insulin-like growth factor-I and -II (IGF-I and IGF-II) are ubiquitous peptide hormones that exert potent mitogenic, metabolic, and differentiating actions on cells throughout the body [10]. IGFs are produced in the liver and several other organs, and are frequently released in response to pulsatile secretion of growth hormone from the anterior pituitary [11]. However, biosynthesis of IGFs is not exclusively regulated by growth hormone, and both IGF-I and IGF-II have many actions that are independent of growth hormone. Signal transduction from IGFs occurs when a ligand binds to one of three specific receptor types: type I IGF receptor, type II IGF receptor, or the insulin receptor. IGF availability is modulated by a group of six high-affinity binding proteins (insulin-like growth factor binding proteins; designated IGFBP-1 through IGFBP-6) that are widely dispersed in the extracellular environment of many tissues [10]. Binding protein functions are regulated by interaction with the extracellular matrix and cell surface [12], as well as by specific proteases [13].

In the male reproductive tract, IGF-I has been identified in the testis [9, 14–17], where it is secreted by Leydig and Sertoli cells [14, 16, 18, 19]. Receptors for IGF-I have been identified on Sertoli cells [20], Leydig cells [16], secondary spermatocytes, spermatids [18, 21], and spermatozoa [7, 22]. Furthermore, IGF-I is believed to be involved in spermatogenesis [4, 23–25] and steroidogenesis [26]. An Igf1 gene null-mutant mouse model compellingly demonstrated the effect of IGF-I on rodent reproduction [27]. When male and female mice with an Igf1 null mutation were mated multiple times over the course of a year, only one litter was obtained. The offspring were dwarfs (~30% of normal size), and males had lower testosterone and sperm production (18% of that measured in mice without the mutation).

The role of IGF proteins as posttesticular regulators of reproductive function has also been examined. IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-4, and fragments of IGFBP-3 as well as IGFBP-3 protease activity have been identified in human seminal plasma [28–32]. Concentration of IGF-I in human seminal plasma was correlated (r = 0.748, P = 0.00001) to sperm concentration and morphology in one study [33]. The relationship of IGF proteins and sperm function in domestic species has been less clear. Henricks and coworkers [7] identified IGF-I in seminal plasma from bulls and IGF-I receptors on bovine sperm cells. Treatment of bovine sperm in vitro with IGF-I resulted in increased total and progressive sperm motility compared with untreated sperm. However, in bulls [8] and boars [34], seminal plasma IGF-I and IGF-II were not correlated with fertility nor with semen parameters in boars.

An effect of the IGF system on sperm physiology and function in some species has been demonstrated. However, the relationship between the IGF system and measures of fertility in stallions remains unclear [9]. The objectives of the present study were 1) to determine whether IGF-I and IGFBPs are present in seminal plasma of stallions; 2) to compare semen parameters (IGF proteins, sperm numbers, morphology, and motility) from stallions at sexual rest (SR) and when sexually active (SA); 3) to compare semen parameters between stallions with high and low seminal plasma IGF-I concentrations; and 4) to examine the relationship between seminal plasma IGF-I concentrations and fertility parameters of stallions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Ejaculates were collected from 51 light-breed, 3- to 20-yr-old stallions in Florida, Maryland, and Texas during January through August 2000. Horses were housed in box stalls and given daily exercise. They were fed concentrate and grass hay, and had ad libitum access to water. Experiments were approved by the University of Florida institutional animal care and use committee (permit A438).

Semen Collection Method and Analysis

One ejaculate was collected from each horse at SR, and a second ejaculate was collected after extragonadal sperm reserves were stabilized (i.e., SA) [35]. Extragonadal sperm reserves were stabilized by semen collection or natural breeding twice daily for three successive days (n = 6). A seventh semen sample was collected 20 h following the sixth sample and evaluated for analysis. Alternatively, horses ejaculating every other day for a minimum of 2 wk in a breeding program were considered SA.

Semen was collected using a Missouri (Nasco, Modesto, CA) or Colorado (Animal Reproduction Systems, Pomona, CA) model artificial vagina with each stallion mounted on an estrus mare or phantom breeding dummy. Ejaculates were collected into a warm, sterile plastic bag. The gel fraction was separated from the sperm-rich fraction using an in-line filter (Animal Reproduction Systems). A 2-ml aliquot of filtered semen was reserved in an individual tube to assess sperm concentration using a densimeter (Animal Reproduction Systems). A sample of filtered semen was mixed in nonfat dried skim milk glucose extender (Kenney extender; Hamilton-Thorne, Beverly, MA) to a final concentration of 25 million sperm/ml and maintained at 37°C in a light-shielded environment for approximately 30 min until motility analysis. Sperm motility was assessed using a negative phase contrast microscope (Labophot; Nikon, Melville, NY) fitted with a Sony XC-73 CCD (Sony Corp., New York, NY) camera connected to a video recorder. Sperm motility was analyzed live at the time of collection or videotaped and analyzed at a later date. Sperm motility analysis was performed using a Hamilton-Thorne IVOS computerized sperm motility analyzer system. The parameter settings for motility analysis were as follows: frames acquired, 40; frame rate, 60 Hz; minimum contrast, 70 units pixel brightness; minimum cell size, 4 pixels; straightness threshold (STR), 75%; low average path velocity (VAP), 20.0 µm/sec; medium VAP, 50.0 µm/sec; nonmotile head size, 4 pixels; nonmotile head intensity, 160; slow cells motile, no. A total of 400 cells were analyzed for motility. Measurements for VAP, straight line velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), STR, and linearity (LIN) were recorded for each ejaculate. An aliquot of sperm was preserved in 2% buffered formal saline for morphologic evaluation. One hundred cells were assessed for morphologic abnormalities using a wet mount and differential interference contrast microscopy (Axioskop II; Carl Zeiss Inc., Thornwood, NY). One operator performed all morphologic evaluations. The remaining semen was maintained on ice for a minimum of 15 min before further processing.

Semen Preparation for Laboratory Analysis

Aliquots of chilled semen were centrifuged at 2000 x g for 10 min at 20°C. Supernatant was separated from the sperm pellet and both components were maintained on ice. Chilled supernatant samples were centrifuged at 2000 x g for 30 min, and filtered using 5.0-µm and 1.2-µm nylon syringe filters (Cameo filter; MSI, Westboro, MA) in tandem to remove any remaining sperm. Filtered supernatant was stored in 500-µl aliquots at -80°C until analyzed. Sperm pellets were preserved at -80°C for additional analysis.

Protein Concentration

Protein concentration in filtered seminal plasma samples was determined using the Bio-Rad protein assay kit (500-0001; Bio-Rad Laboratories, Hercules, CA), according to the manufacturer's instructions.

IGF-I Radioimmunoassay

The radioimmunoassay for IGF-I was performed following previously published procedures [36] and validated for equine seminal plasma. Displacement curves produced by serial dilutions of acid ethanol extracts of seminal plasma (1–50 µl) were parallel with that of a standard curve using human IGF-I (Upstate Biotechnology, Lake Placid, NY); therefore, an assay dose of 20 µl was selected for this study. Percentage recovery of IGF-I for the assay was 85%.

Tubes for total counts, nonspecific binding, and total bound IGF-I accompanied each assay. Each sample was assayed in duplicate. The primary antiserum raised in rabbit against human IGF-I (UBK487; a generous gift of Dr. A.F. Parlow through the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, Torrance, CA) was used at a 1:18 000 dilution. Aliquots of 100 µl of primary antibody solution were added to each tube except total count and nonspecific binding tubes. One hundred microliters of 125I-IGF-I (~20 000 cpm) were added to all tubes. Samples were incubated for 16–20 h. Antigen-antibody complexes were precipitated with sheep anti-rabbit secondary antiserum (100 µl; Upstate Biotechnology) and normal rabbit serum (100 µl) for 3 h. One milliliter of radioimmunoassay buffer was added to all tubes (except total count), and the tubes were then vortexed and centrifuged for 30 min at 3000 x g at 4°C. The supernatant was aspirated and the pellet was counted using a gamma counter. Intraassay and interassay variations were 3.9% and 7.5%, respectively. Concentrations of IGF-I were expressed as nanograms per milliliter of seminal plasma and as nanograms of IGF-I per milligram of seminal plasma protein. Total IGF-I concentration in an ejaculate was determined by multiplying IGF-I concentration (ng/ml) with ejaculate volume (ml).

IGFBP-2 Radioimmunoassay

Seminal plasma concentrations of IGFBP-2 were determined by using a commercially available double-antibody radioimmunoassay system (Diagnostic Systems Laboratories, Webster, TX). Available radioimmunoassay systems for IGFBP-1, -3, -4, and -5 were not cross-reactive for equine IGFBPs, therefore, these assays were not used. The radioimmunoassay for IGFBP-2 was validated using equine seminal plasma following the procedures of Bang et al. [37]. Parallelism was established by comparing the IGFBP-2 reference curve and a curve generated with equine seminal plasma using four dilutions (1:2 to 1:16) from two horses. Recovery was 103% when the equine seminal plasma was incubated with unlabeled IGFBP-2. Cross-reactivity for IGFBP-2 was 100% and was not detected for the other binding proteins. The intraassay coefficient of variation was 6.2%, the interassay coefficient of variation was 7.1%, and the sensitivity was 0.5 ng/ml. Concentration of IGFBP-2 was expressed as ng/ml of seminal plasma and also relative to seminal plasma protein concentration (mg/ml) as nanograms of IGFBP-2 per milligrams of protein. Total IGFBP-2 concentration in an ejaculate was determined by multiplying IGFBP-2 concentration (ng/ml) with ejaculate volume (ml).

Western Ligand Blotting for IGF Binding Proteins

Equine seminal plasma and serum (control) samples were subjected to Western ligand blotting according to the method of Hossenlopp et al. [38]. Briefly, serum (2 µl) and seminal plasma (20-µl samples from five stallions at SR and SA with protein content in each sample as follows: horse 1, SR = 144 µg, SA = 120 µg; horse 2, SR = 94 µg, SA = 41 µg; horse 3, SR = 226 µg, SA = 220 µg; horse 4, SR = 170 µg, SA = 210 µg; horse 5, SR = 110 µg, SA = 54 µg) were diluted in nonreducing 4x sample buffer (250 mM Tris, 12% SDS, 40% glycerol, and 0.05% bromphenol blue) and denatured at 95°C for 5 min. Samples were then electrophoresed through a 5% stacking gel and 12.5% separating gel at 12 mA overnight in running buffer (25 mM Tris, 192 mM glycine, and 0.1% SDS pH 8.3) using a Bio-Rad Protean II vertical slab instrument. Prestained broad-range molecular weight standards were coelectrophoresed. Gels were electroblotted to nitrocellulose membrane (0.45 µm; Bio-Rad) according to the method of Towbin et al. [39]. Membranes were blocked in 100 ml of TBS-Tween-20 (TBS-T, 0.1%) and 0.5% BSA for 1 h at 20°C, washed with TBS-T, and incubated overnight at 4°C with approximately 1 x 106 cpm 125I-IGF-II (Diagnostic Systems) in 40 ml of TBS-T and 0.5% BSA. Membranes were then washed with TBS-T, and after drying were autoradiographed with Kodak-XAR (Eastman Kodak Company, Rochester, NY) film and exposed at -70°C for 7 days.

Immunoprecipitation

Ligand blot analysis of immunoprecipitated IGFBPs was performed as described by Lee et al. [40]. Briefly, IGFBPs were immunoprecipitated from a pooled sample of seminal plasma (six stallions, 20-µl samples with 112 µg of total protein/sample). Seminal plasma samples were incubated overnight at 4°C with each specie of anti-IGFBP (1–6; Upstate Biotechnology)) and nonimmune rabbit serum (Gibco, Gaithersburg, MD) in dilutions recommended by the manufacturer. Following incubation, prewashed Protein A-Sepharose (Amersham Pharmacia, Piscataway, NJ) was added and samples were incubated overnight at 4°C. The immunoprecipitated IGFBPs were centrifuged and washed with 50 mM Tris containing 0.5% Triton X-100 (Bio-Rad). The samples were solubilized in 4x sample buffer and visualized by Western ligand blotting as described above.

Fertility Parameters of Stallions

For each stallion included in the study, the following fertility data were obtained from historical breeding records (January–December 2000): seasonal pregnancy rate = total number of mares pregnant/total number of mares bred; first cycle pregnancy rate = mares pregnant on first cycle breeding/total numbers of mares bred; and cycles/pregnancy = overall number of mare cycles bred/total number of mares pregnant. Mares were bred naturally or by artificial insemination.

Statistical Analyses

IGF proteins, semen parameters, and fertility data were reported as means ± SEM. Data were analyzed using nonparametric tests because these study variables failed to meet assumptions for parametric testing (i.e., the data were not normally distributed).

The null hypotheses that semen parameters (paired seminal plasma concentrations of IGF-I and IGFBP-2, sperm numbers, morphology, and motility) are not significantly different in stallions at SR and SA were tested using the Wilcoxon signed rank nonparametric test [41].

Because IGF-I data were not normally distributed, stallions were assigned into one of two groups with low and high seminal plasma IGF-I concentrations on the basis of their frequency distribution (median). The median of a sample is the middle number in an array of data when the number of observations is odd, or the arithmetic mean of the two middle numbers in the array when the number of observations is even. For example, at SR, 25 stallions were identified as having low IGF-I (3.4–9.4 ng/ml) and 26 stallions with high IGF-I concentrations (9.5–31.5 ng/ml). The null hypotheses that semen parameters (seminal IGF proteins, motility, and morphology) are not significantly different between stallions with low and high IGF-I concentrations at SR and SA were tested using the Mann-Whitney nonparametric test [42].

The null hypotheses that seasonal pregnancy rates, first-cycle pregnancy rates, and number of cycles per pregnancy are not significantly different between stallions with low and high IGF-I concentrations were compared using the Mann-Whitney nonparametric test [42] and ANOVA (rank transformed data) [43]. Additional covariates (age, artificial insemination, total IGF-I, total IGFBP-2, progressive motility, total motility, and normal morphology) were included in the final ANOVA to address possible confounding effects that these factors may have had on fertility. All tests were performed using Statistix Analytic Software (version 7, Tallahassee, FL) and values of P <= 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IGF-I and IGFBP-2 Concentrations in Seminal Plasma

At SR, seminal plasma concentrations (mean ± SEM) of IGF-I and IGFBP-2 were 10.4 ± 0.7 ng/ml and 35.5 ± 3.0 ng/ml, respectively. At SA, seminal plasma concentrations of IGF-I and IGFBP-2 were 10.2 ± 1.0 ng/ml and 32.7 ± 2.8 ng/ml, respectively (Table 1). Mean and median values for IGF-I in seminal plasma at SR and SA are depicted in Figure 1.


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  		TABLE 1. Semen characteristics (mean ± SEM) in stallions at sexual rest and when sexually active



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  		FIG. 1. Box and whiskers plot with mean (dashed line) and median (solid line) values of seminal plasma IGF-I concentrations from stallions at SR and SA

IGFBP Identification

The presence of IGFBP-2 in equine seminal plasma measured by radioimmunoassay was verified using ligand blot analysis. Seminal plasma IGFBP-5 was detected using ligand blot analysis before and after immunoprecipitation. Ligand blot analysis (without immunoprecipitation) of seminal plasma samples revealed protein bands with molecular masses of {approx}30–32 and 35 kDa, respectively (Fig. 2). These proteins would be consistent with IGFBP-5 and IGFBP-2, respectively. Bands from seminal plasma samples of different horses showed varying degrees of intensity, possibly reflecting varying amounts of protein between horses (horse 1, SR = 144 µg, SA = 120 µg; horse 2, SR = 94 µg, SA = 41 µg; horse 3, SR = 226 µg, SA = 220 µg; horse 4, SR = 170 µg, SA = 210 µg; horse 5, SR = 110 µg, SA = 54 µg). A doublet band was detected in human serum (and faintly in equine serum) at 42–44 kDa, consistent with IGFBP-3. Proteins in the range of 32–35 kDa were detected in both human and equine serum. A protein band at 180 kDa was detected in equine serum, and this was likely a soluble Type II IGF receptor, albeit of a lower molecular weight than that reported (240 kDa) in rat and monkey serum [44]. Immunoprecipitation combined with ligand blotting was used to enhance detection of IGFBPs from a pooled seminal plasma sample. A band was detected at 32 kDa after the sample was immunoprecipitated with anti-IGFBP-5 (Fig. 3). This molecular weight is consistent with IGFBP-5. A very weak band was detected at 35 kDa (not readily visible in Fig. 3) following immunoprecipitation with anti-IGFBP-2. This band would be compatible with IGFBP-2. Proteins with molecular weights of 42–44 kDa (IGFBP-3) and 32 kDa (IGFBP-5) were present in human and equine serum in this assay. A faint band was visible in equine serum at 35 kDa (IGFBP-2), and a strong band of 180 kDa was again detectable.



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  		FIG. 2. Ligand blot analysis (125I-IGF-II) of equine seminal plasma samples collected from five horses at SR and SA (sample dose = 20 µl; protein concentrations per lane: horse 1, SR = 144 µg, SA = 120 µg; horse 2, SR = 94 µg, SA = 41 µg; horse 3, SR = 226 µg, SA = 220 µg; horse 4, SR = 170 µg, SA = 210 µg; horse 5, SR = 110 µg, SA = 54 µg). Proteins with molecular weights of 32 and 35 kDa are visible in seminal plasma samples, consistent with IGFBP-5 and IGFBP-2, respectively. A doublet band was detected in human serum (and faintly in equine serum) at 42–44 kDa, consistent with IGFBP-3. Proteins in the range of 32–35 kDa were detected in both human and equine serum (IGFBP-5, IGFBP-2, or both). A protein band at 180 kDa was detected in equine serum, and this is possibly a soluble IGF receptor



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  		FIG. 3. Ligand blotting (125I-IGF-II) of six pooled equine seminal plasma samples after immunoprecipitation with anti-IGFBPs 1–6. Each lane contains 112 µg protein (20 µl). The control sample is nonimmunoprecipitated seminal plasma. A band is visible at 32 kDa, consistent with IGFBP-5. A very weak band was detected at 35 kDa but is not visible in this figure. This protein would be compatible with IGFBP-2. Proteins with molecular weights of 42–44 kDa (IGFBP-3) and 32 kDa (IGFBP-5) are evident in samples of human and equine serum. A faint band was visible in equine serum at 35 kDa (IGFBP-2), and a strong band was again detectable at 180 kDa (a possible soluble IGF receptor)

Comparison of Semen Parameters in Stallions at Sexual Rest and when Sexually Active

Paired seminal plasma concentrations of IGF-I, protein, IGF-I/protein, total IGF-I, IGFBP-2, IGBBP-2/protein, and total IGFBP-2 were not significantly different (P >= 0.13) from stallions at SR and SA (Table 1).

Comparisons of Semen Parameters Between Stallions with Low and High Seminal Plasma IGF-I Concentrations at SR and SA

At SR, quantity of total IGFBP-2 in seminal plasma and percentage of morphologically normal sperm were greater (P = 0.05) from stallions with high seminal plasma IGF-I concentrations compared with those with low IGF-I concentrations (Table 2). When horses were sexually active, total sperm motility tended to be higher (P = 0.08) from stallions with high IGF-I vs. low IGF-I concentrations (Table 3). Sexually active horses with high seminal plasma IGF-I concentrations were younger (P < 0.01) than horses with low IGF-I concentrations. Motility parameters VSL, VCL, ALH, STR, and LIN were not different (P > 0.05) from stallions with high or low IGF-I levels at either SR or SA.


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  		TABLE 2. Comparison of age and semen characteristics (mean ± SEM) at sexual rest in stallions with high or low seminal plasma IGF-I concentrations (n = 46)


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  		TABLE 3. Comparison of age and semen characteristics (mean ± SEM) in sexually active stallions with high or low seminal plasma IGF-I concentrations (n = 46)

Comparisons of Seasonal Pregnancy Rates, First-Cycle Pregnancy Rates, and Number of Cycles per Pregnancy Between Stallions with Low and High Concentrations of Seminal Plasma IGF-I

Fertility data were obtained from 46 stallions that were bred to between 2 and 109 mares. At SR, the mean number of mares bred per stallion was not significantly different (P = 0.26) between stallions with high and low IGF-I concentrations (24 and 24 mares bred/stallion, respectively; Table 4). Mean first-cycle pregnancy rate was significantly higher (P = 0.02) in stallions with high IGF-I (76%) compared with stallions with low IGF-I (55%). In the multivariable analysis, the observed difference remained significant after adjustment for progressive sperm motility. Mean pregnancy rate per season tended to be higher (P = 0.06) in stallions with high IGF-I (89%) compared with stallions with low IGF-I (73%).


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  		TABLE 4. Fertility parameters (mean ± SEM) between stallions with high or low seminal plasma IGF-I concentrations (n = 46)

At SA, the mean number of mares bred per stallion was also not significantly different (P = 0.64) between stallions with high and low IGF-I (22 and 29 mares bred/stallion, respectively; Table 4). Mean number of cycles per pregnancy was significantly different (P = 0.02) between stallions with high IGF-I (1.3 cycles/pregnancy) compared with stallions with low IGF-I (1.8 cycles/pregnancy). In the multivariable analysis, the observed difference remained significant after adjustment for age (stallions 3–10 yr old vs. stallions 11–20 yr old). Mean pregnancy rate per season tended to be higher (P = 0.07) in stallions with high IGF-I (87%) compared with stallions with low IGF-I (83%). Age, artificial insemination, total IGF-I, total IGFBP-2, progressive motility, total motility, and normal morphology were not significantly associated (P > 0.05) with pregnancy rate per season and were not considered in the ANOVA analysis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Results of this study demonstrated that IGF-I, IGFBP-2, and IGFBP-5 were present in equine seminal plasma. Frazer and Bucci [45] detected 14 proteins in equine seminal plasma with molecular weights ranging between 14 kDa and 120 kDa. Two proteins identified in samples from most horses had molecular weights of 32 and 34 kDa, respectively. In the present study, proteins with molecular weights of 32 and 35 kDa were detected in equine seminal plasma using ligand blotting, and were positively identified as IGFBP-2 (35 kDa) and IGFBP-5 (32 kDa) using immunoprecipitation, ligand blotting, and radioimmunoassay techniques.

IGF-I and IGFBP-2 (measured per seminal plasma volume) were quantified in equine seminal plasma samples. Concentrations of IGF-I and IGFBP-2 were not affected by sexual activity. Frequency of ejaculation in stallions has been shown to affect semen parameters such as sperm production [46]. Sexual activity was considered in this study to determine if ejaculation frequency, sperm production, or both influenced the quantity of IGF-I in seminal plasma. Differences in IGF-I concentrations were not detected in samples from the individual stallions at different levels of sexual activity. However, substantial variation in IGF-I concentrations occurred between stallions. Therefore, concentrations of IGF-I and IGFBP-2 were also analyzed per milligram of protein and as total IGF-I or IGFBP-2 per ejaculate. Differences were not detected in IGF-I or IGFBP-2 at SR and SA after accounting for these variables, and variation between stallions remained high. Factors influencing production or concentration of IGF-I, IGFBP-2, or both in equine seminal plasma could not be determined from these data. Possible sources of seminal plasma IGF proteins in horses include the testis [9], epididymis, and accessory genitalia. The source of seminal plasma IGF-I production in men was determined by examining samples before and after vasectomy [33]. The authors postulated that high IGF-I concentrations from samples after vasectomy would indicate production from the prostate, whereas low concentrations would suggest testicular/epididymal production. Following vasectomy, seminal plasma IGF-I concentrations dropped to approximately one-third of presurgical levels, leading the authors to conclude that IGF-I was primarily testicular/epididymal in origin. However, presence of IGF-I in seminal plasma after vasectomy suggests that accessory glands also produce IGF-I in men. A similar scenario may hold true for horses; however, specific work has not been performed to determine the origin of seminal plasma IGF-I in this species. The accessory genitalia of stallions contribute a significant fluid fraction to the final ejaculate [47], therefore, secretions from these organs likely could include IGF-I. Examination of IGF-I concentrations in ejaculate fractions and epididymal compartments with analysis of epididymal IGF-I gene expression by Northern blot analysis would be useful for further defining the source of this protein in stallion seminal plasma.

Relationships between seminal plasma IGF-I and sperm parameters (sperm numbers, morphology, and motility) were identified in this study, but not consistently. Sperm morphology and motility were greater in some samples having high IGF-I. Other workers [48–50] have investigated the relationship of IGF-I to sperm morphology and motility in growth hormone-deficient dwarf (dw/dw) rats. Growth hormone-deficient dw/dw rats were selected for study because they had lower reproductive parameters (i.e., sperm motility and morphology) as well as lower concentrations of IGF-I in blood, seminal vesicle fluid, and epididymal fluid [48–50]. Treating growth hormone-deficient rats with exogenous growth hormone resulted in improved sperm motility and higher concentrations of IGF-I in blood plasma and seminal vesicle fluid compared with non-dwarf control rats [48]. When growth hormone-deficient rats were treated with IGF-I, sperm morphology and motility improved, and concentrations of IGF-I in blood plasma and seminal vesicle fluid were higher [50]. From these studies, the authors concluded that growth hormone may stimulate the production of IGF-I, but that IGF-I had more of a functional effect at a local (testicular/seminal vesicle) level. The authors postulated that the relationship of IGF-I and sperm morphology originated in the testis. Furthermore, they believed that the effects of seminal plasma IGF-I on sperm morphology and motility were mediated via secretion of IGF-I in the seminal vesicles and binding to the IGF-I receptor on developing sperm cells as the cells were mixed with vesicular fluid [50]. In support of this theory, when bull sperm were treated in vitro with IGF-I or IGF-II, sperm motility and VSL were significantly higher than untreated sperm [7]. In the same study, IGF-I receptors were identified on sperm cells [7]. With treated sperm, the response to IGF-I or IGF-II may reflect a physiologic effect of IGF-I or IGF-II through ligand-receptor binding.

The role of IGF-I of male origin in pregnancy-related events is supported by data from this study. Pregnancy rates were higher in horses with high seminal plasma IGF-I, which is suggestive of IGF-I effects on sperm function. Several proteins that affect sperm function have recently been identified in bovine seminal plasma (BSP) [51–53]. Specifically, BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa proteins are produced by the seminal vesicles and ampullae [51, 52, 54, 55] and bind to sperm at ejaculation [56]. The proteins have been shown to accelerate capacitation of bovine epididymal sperm in the presence of heparin and high-density lipoprotein [57]. In addition, heparin-binding proteins found in bovine seminal plasma have been linked to fertility of bulls [58]. Heparin-binding proteins are secreted by the accessory sex glands and modulate heparin-sperm activity [59]. Although the role of the seminal plasma IGF proteins on sperm function of humans or domestic species has not been fully elucidated, work from this study and others suggests that these proteins are also linked to sperm function. Examination of IGF regulation of events such as capacitation and the acrosome reaction may help define the physiological relevance of IGF proteins in seminal plasma.

In conclusion, IGF-I, IGFBP-2, and IGFBP-5 have been identified in seminal plasma from a large population of stallions. A positive but variable relationship between IGF-I and sperm morphology and motility was noted. Stallions with high levels of IGF-I in seminal plasma achieved higher pregnancy rates in mares. Further work is necessary to localize IGF receptors on equine sperm and to determine at which juncture IGF-I and IGFBPs affect sperm function.


   ACKNOWLEDGMENTS
 
We are grateful to staff at the University of Florida Horse Teaching Center and Horse Research Center, Texas A&M Horse Center; the Texas Department of Corrections; Select Breeders Services; and Franks Farm for obtaining samples and breeding records for this study. In addition, we thank Dr. Susan Durham of Diagnostic Systems Laboratories for her validation procedures for the Western ligand blot, Mr. Frank Michel for his expertise with the IGF-I radioimmunassay, Mr. Eric Reller for his assistance with IGF-I and IGFBP assays, and Ms. Lisa Hinder for validating the IGFBP-2 radioimmunassay.


   FOOTNOTES
 
First decision: 7 December 2001.

1 This work was funded by a grant from the Grayson-Jockey Club Research Foundation, Inc. and published as Journal Series R- from the Florida Agricultural Experiment Station. Back

2 Correspondence. FAX: 352 392 8289; macphersonm{at}mail.vetmed.ufl.edu Back

Accepted: March 18, 2002.

Received: November 16, 2001.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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