Insulin-like Growth Factor I and Associated Binding Proteins in Seminal Plasma of Stallions
ML Macpherson,¹ RCM Simmen,² FA Simmen,² BR Sheerin,¹
C D Miller,³ and P Loomis⁴
Departments of Large Animal Clinical Sciences¹ and Animal Science²
University of Florida, Gainesville, Florida USA
³Frank’s Farm, Ocala, Florida USA 
⁴Select Breeders Services Inc, Colora, Maryland USA

Introduction

Insulin-like growth factor I has been identified in seminal plasma and implicated as a post-testicular regulator of sperm physiology and function in other species.^1,2 The IGF system has not been studied in seminal plasma of the horse. Therefore, the objectives of this study were to determine if IGF-I and IGFBPs were present in seminal plasma of stallions and to determine the relationship of these proteins to sperm motility and fertility.  Our working hypotheses were that IGF-I and IGFBPS were present in seminal plasma of stallions. Furthermore, we hypothesized that IGF-I concentration in seminal plasma was positively associated with sperm motility and pregnancy rates in bred mares.

Method

Two ejaculates each were collected from 21 stallions during the period of February to July, 1999.  Samples were collected at sexual rest and daily sperm output. Daily sperm output (DSO) was defined as the number of sperm produced by an individual once the extra gonadal reserves were evacuated.³ Ejaculates were collected using a Missouri model artificial vagina and a live mare mount or a phantom mount. Semen was filtered using a nylon mesh filter and was collected into a sterile, prewarmed bag. Ejaculate volume was measured and recorded. Sperm concentration was determined using a densimeter (Animal Reproduction Systems, Pomona, CA) calibrated for use with equine semen.  Aliquots of semen were extended to a final concentration of 25 million sperm/ml with nonfat dried skim milk-glucose (NFDSM-G) and maintained at 20^oC in a light shielded environment until motility analysis.  Sperm motility was assessed using a Hamilton Thorne IVOS computerized sperm motility analyzer (Hamilton-Thorne, Beverly, MA).  An aliquot of sperm was preserved in buffered formal saline for morphologic evaluation.  Two hundred cells were assessed for morphologic abnormalities using a wet mount and differential interference contrast microscopy. The remaining semen was centrifuged at 2000 x g for 30 minutes. The seminal plasma supernatant was separated from the sperm pellet. Centrifuged seminal plasma was filtered, using  5.0 and 1.2 micron nylon syringe filters in tandem, to remove any remaining sperm.  Samples were stored in 500 ml aliquots at -80^oC until analyzed.

Concentration of IGF-I in equine seminal plasma samples was determined with a radioimmunoassay validated by Dr. R. Simmen using the method of Daughaday,et al.⁴ The inter and intra assay variation was 12% and 5.6%, respectively.  Samples were assayed for protein concentration (Bio-Rad, Hercules, CA). Samples from one horse on 6 different days were used to validate the ligand blotting procedure for IGFBP’s in equine seminal plasma by Dr. Susan Durham of Diagnostic Systems Laboratory. The samples were subjected to standard SDS-Page electrophoresis and ligand blotting techniques as described by Hossenlopp et al.⁵   The presence of IGFBPs were confirmed by immunoprecipitation using the method of Lee et al.⁶

Data were analyzed using Wilcoxon sign rank test to determine differences in IGF-I or IGF-I/protein concentration at sexual rest vs. daily sperm output.  Simple linear regression was used to compare changes in total and progressive sperm motility relative to IGF-I or IGF-I/protein. Pregnancy rates were calculated per cycle and per season for 6 stallions. Simple linear regression was used to compare pregnancy rates and concentration of IGF-I and IGF-I/protein.

Results and Discussion

Mean concentration (+SD) and median concentration (range) are reported for IGF-I (ng/ml) and IGF-I (ng/ml) per protein (mg/mg) in Table 1.

Table 1.  Comparison of IGF-I (ng/ml) and IGF-I/protein (ng/mg) by sampling periods.

^a,b Differences between superscripts are significant, P < 0.05

Median IGF-I and IGF-I/protein seminal plasma concentrations were significantly higher ( P < 0.05) at sexual rest when compared to DSO. A positive correlation was determined between samples at sexual rest and daily sperm output for both IGF-I (r = 0.70, P < 0.05) and IGF-I/protein (r = 0 .67, P < 0.05).

Concentrations of seminal plasma IGF-I and IGF-I/protein from 21 horses at DSO were compared to total and progressive motility of sperm from the same ejaculates. There was a tendency for both total (r = 0.43, P = 0.06) and progressive motility (r = 0.38, P = 0.08) to be positively associated with concentration of IGF-I/protein.

Concentrations of seminal plasma IGF-I and IGF-I/protein from 6 stallions at DSO were compared to pregnancy rate per first cycle and pregnancy rate per season. Both first cycle pregnancy rates (r = 0.88, P = 0.02) and seasonal pregnancy rates ( r = 0.87, P = 0.02) were positively associated with seminal plasma IGF-I concentration.

Blots were incubated with ¹²⁵I-IGF-I and ¹²⁵I-IGF-II to determine which ligand was more sensitive for binding proteins in equine seminal plasma. The affinity of the binding proteins in equine seminal plasma was less with IGF-I than IGF-II.  Human IGFBP-3 (control) was detectable at 38-43 kDa. A faint band was detectable at approximately 180kDa in equine serum.  A consistent band from all equine seminal plasma samples was apparent at approximately 43-45kDa.  A large, less defined band appeared in all seminal plasma samples at 28-32kDa. Immunoprecipitation confirmed the presence of IGFBP2 in equine seminal plasma.

Results from these preliminary studies verify that IGF-I and IGFBP2 are present in equine seminal plasma. These data suggest a positive relationship between sperm motility and concentration of IGF-I in seminal plasma.  Furthermore, in a small sample population, a positive relationship has been identified between IGF-I concentration in seminal plasma and pregnancy rates in bred mares. A study involving a larger population of stallions with known fertility data is underway to confirm the relationship of IGF-I in seminal plasma to sperm characteristics and fertility.

Results of a ligand blot using equine seminal plasma are shown in Figure 1.

Figure 1.  Ligand blot of equine seminal plasma using ¹²⁵I-IGF-I and ¹²⁵I-IGF-II.

            We have also confirmed the presence of IGFBP2 in equine seminal plasma. Current work is directed toward determining the relationship of IGFBP2 to IGF-I in seminal plasma as well as to sperm characteristics and fertility.   The regulatory role of the binding proteins in seminal plasma has not been well defined in any species. Identification of specific binding proteins in equine seminal plasma will be the first step toward characterizing the activity of these proteins in regulation of IGF-I in this body fluid.

References

1.   Henricks D, Kouba A, Lackey B, et al. 1998. Identification of insulin-like growth factor I in bovine seminal plasma and its receptor on spermatozoa: influence on sperm motility.  Biology of Reproduction 59: 330-337.

2.  Glander HJ, Kratzsch J, Weisbrich Ch et al. 1996. Insulin-like growth factor-I and "2- macroglobulin in seminal plasma correlate with semen quality. Human Reproduction 11(11): 2454-2460.

3.  Gebauer MR et al.1974. Reproductive physiology of the stallion: daily sperm output and testicular measurements. Journal of the American Veterinary Medical Association 165: 711-713.

4.  Daughaday WH, Mariz IK, Blethen SL 1980. Inhibition of access of bound somatomedin to membrane receptor and immunobinding sites: a comparison of radioreptor and radioimmunoassay of somatomedin in native and acid-ehtanol-extracted serum.  Journal of Clinical Endocrinology and Metabolism 51(4): 781-788.

5.  Hossenlopp P, Seurin D, Segovia-Quinson B et al. 1986. Analysis of serum insulin-like growth factor binding proteins using western blotting: use of the method for titration of the binding proteins and competitive binding sites.  Analalytical Biochemistry 154(1): 138-143.

6.  Lee CY, Bazer FW, Etherton TD et al.  1991. Ontogeny of insulin-like growth factors (IGF-I and IGF-II) and IGF-binding proteins in porcine serum during fetal and postnatal development. Endocrinology 128: 2336-2344.