Impact of blood serum insulin-like growth factor I on
platelet-activating factor in bull spermatozoa
W.E. Roudebush
a,*, E.T. Purnell
b, M.E. Davis
ca
Reproductive Biology Associates, Atlanta, GA, USA b
Molecular Cell Biology & Pathology Program, Medical University of South Carolina, Charleston, SC, USA c
Department of Animal Sciences, The Ohio State University, Columbus, OH, USA
Received 22 April 2000; accepted 22 September 2000
Abstract
The objective of this study was to examine differences in platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF] in spermatozoa between two lines of Angus beef cattle divergently selected for blood serum insulin-like growth factor I (IGF-I) concentration. Endogenous lipids were extracted from the spermatozoa and endogenous PAF content was determined by radio-immunoassay. The amount of PAF detected in spermatozoa obtained from high IGF-I bulls (n5 8) ranged from 0.145 to 3.571 pM/106cells. The level of PAF extracted from spermatozoa obtained from low IGF-I- bulls (n5 5) ranged from 0.001 to 1.024 pM/106cells. Polynomial regression analysis revealed a significant cubic relationship (R250.374; F56.292; P,0.05) between spermatozoa PAF content and blood serum IGF-I concentration. Spermatozoa-derived PAF levels (mean6SEM) were significantly higher (P,0.05) in the high IGF-I group (1.90 60.39 pM/106cells) than in the low IGF-I group (0.5960.20 pM/106cells). High IGF-I bulls have a greater than three-fold higher PAF content in their spermatozoa than low IGF-I bulls. The data demonstrate that not only is PAF present in bull spermatozoa but that levels are significantly higher in individuals with high serum IGF-I concentrations. © 2001 Elsevier Science Inc. All rights reserved.
1. Introduction
Platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF] is a novel potent signaling phospholipid which has unique pleiotropic biologic properties in
* Corresponding author.
E-mail address: roudebush@rba-online.com (W.E. Roudebush).
addition to platelet activation [1,2]. PAF was first described by Benveniste et al. [3] when they documented rabbit platelet aggregation triggered by IgE stimulated basophils. But, in addition, PAF has a significant role in mammalian reproduction, influencing ovulation, fertilization, preimplantation embryo development, implantation and parturition [4].
This unique molecule is found in the spermatozoa of many laboratory and domestic species including the rabbit, mouse, and bovine [5,6,7]. PAF is not present in the ejaculatory plug or fluid. This finding is consistent with reports that PAF is localized on the spermatozoa and is not present in seminal plasma [5,8]. PAF is present in human spermatozoa [9] and has a positive relationship with pregnancy outcome [10]. We have recently reported the presence of PAF in spermatozoa in the squirrel monkey [11]. The levels are significantly higher during the breeding than during the nonbreeding season. Testosterone, estrogen and progesterone [12,13] affect PAF metabolism. Thus seasonal differences in hormone levels may affect spermatozoa-derived PAF levels.
PAF plays a significant role in the fertilization process, enhancing the in vitro fertilization rates of mouse and rabbit oocytes [14,15,16]. PAF antagonists inhibit fertilization [6,14]. Endogenous PAF of spermatozoa origin increases mouse in vitro fertilization rates [6]. Enhanced embryo development has also been reported in rabbit oocytes fertilized in vitro with PAF-treated spermatozoa [17]. However, there has been little research on relationships between PAF and male reproductive traits in cattle.
Insulin-like growth factor I (IGF-I) is a mitotic polypeptide that stimulates glucose and sulfate uptake. A relationship between IGF-I and PAF has been reported. IGF-I will attenuate the intracellular calcium response to PAF in cultured rat mesengial cells [18]. Additionally, PAF will induce production of IGF-I binding proteins in human adenocarcinoma cells [19]. Whereas IGF-I’s effects on female reproductive functions have been studied, little informa-tion is known concerning its effects on male reproductive funcinforma-tions. Scrotal circumference and percentage of normal sperm cells are related to blood serum IGF-I concentration in yearling Angus bulls [20]. IGF-I is an important factor for germ cell development and maturation of spermatozoa [21]. No information is available on the effect of blood serum IGF-I concentration on PAF content in spermatozoa.
The objective of this study was to examine differences in spermatozoa PAF content between two lines of Angus beef cattle divergently selected for blood serum IGF-I concen-tration.
2. Materials and methods
Semen were collected by artificial vagina from yearling Angus bulls (Eastern Ohio Resource Development Center, Caldwell, Ohio) divergently selected for blood serum IGF-I concentration as previously described [20]. Briefly, IGF-I concentrations were measured by radioimmunoassay (R.C.M. Simmen, University of Florida, Gainesville, FL) on each bull calf at Day 28, 42 and 56 postweaning. The mean values for each calf were used for within line selection criterion.
and quick frozen in PAF assay buffer (0.1% sodium azide, 0.05% Tween 20 in 50 mM sodium citrate, pH 6.3) and transported on ice to the Medical University of South Carolina (Charleston, SC). Spermatozoa were thawed and endogenous lipids were extracted by the method of Bligh and Dyer [21] as previously reported [10]. Briefly, chloroform and methanol were added to the spermatozoa suspension (adjusted to 106 cells/ml) and mixed for 1 min every 10 min over a 1-hr period. Following extraction, the samples were centrifuged (2,000
3g, 5 min) and lipids obtained from the organic phase following the addition of chloroform and distilled water. The organic phase was evaporated to dryness under a gentle stream of nitrogen and redissolved in PAF assay buffer. Extracted PAF samples were stored at220°C until assayed. Endogenous PAF levels were determined by PAF specific radioimmunoassay [125I] according to manufacturer’s instructions (NEN Research Products, DuPont, Boston, MA). Briefly, primary antibodies were added to tubes containing the extracted spermatozoa samples or PAF assay buffer. Tubes were mixed and incubated for 15 min at room temperature. Secondary antibodies and tracer were added to samples, standards, blanks and total count tubes, mixed and incubated for 24 hr at room temperature. Following centrifu-gation (2,0003g; 30 min), the supernatant was decanted and the tubes blotted and counted. The standard curve was calculated by regression analysis (logit value of normalized percent bound versus log of ng PAF assayed).
PAF assay performance is as follows: sensitivity (detection limit), 0.1 ng/mL; intra-assay (determined by five replicates of samples measured in a single assay), 5.2760.15 ng/mL; inter-assay (determined by assaying same replicates in four separate assays), 5.62 6 0.40 ng/mL. Content of extracted spermatozoa PAF is expressed as pM/106 spermatozoa. Data were analyzed by using regression analysis and the Student’s t test.
3. Results
A total of 8 high IGF-I line (mean serum concentration 568.09619.78 ng/mL) and 5 low IGF-I line (mean serum concentration 511.34645.54 ng/mL) bull spermatozoa specimens were assayed for the presence of PAF as described. Mean serum IGF-I concentration for each bull and its associated selection line are provided in Table 1. PAF was measured in all bull spermatozoa samples assayed. The overall mean for PAF content in bull spermatozoa was 1.326 pM/106cells. The amount of PAF detected in spermatozoa obtained from high IGF-I line bulls ranged from 0.15 to 3.57 pM/106cells. The level of PAF extracted from sperma-tozoa obtained from low IGF-I line bulls ranged from 0.001 to 1.02 pM/106cells. Polynomial regression analysis revealed a significant cubic relationship (R2 50.374; F 56.292; P,
0.05) between PAF content and blood serum IGF concentration. The amount of PAF detected in high IGF-I line bull spermatozoa and low IGF-I line spermatozoa are presented in Table 2. Spermatozoa-derived PAF levels (mean6SEM) were significantly higher (P,0.05) in the high IGF-I line bulls (1.9060.39 pM/106cells) than in the low IGF-I line bull (0.596
4. Discussion
This study demonstrates the presence of PAF in the spermatozoa of the bull and that PAF levels appear to have a positive relationship with blood serum IGF-I concentration. Endog-enous PAF has been detected in human, rabbit, bull, mouse, rhesus macaque, squirrel monkey, and baboon spermatozoa [5,6,7,9,11,22,23]. PAF is localized to the spermatozoa and is not present in seminal fluid. The overall mean PAF content found in bull spermatozoa is comparable to that found in other species: mouse, 14.9 pM/106cells; and the rabbit, 3.5 pM/106 cells. However, the amounts contained are variable, and may be dependent on the source and method of preparation of the spermatozoa. There are no reports with regard to PAF content in bull spermatozoa and its relationship to fertility status nor components of the breeding soundness examination.
A calcium dependent phospholipase A2 is present in human, mouse, hamster, guinea pig and ram spermatozoa [24]. Phospholipase A2catalyzes the formation of lyso-PAF [1-alkyl-2-lyso-sn-glycero-3-phosphocholine] from alkyl-acyl-glycerophosphocholine (an inert struc-tural cellular membrane component). The lyso-PAF can either be acetylated by lyso-PAF-acetyltransferase (with acetyl-CoA as the acetate donor) to form PAF or acetylated by CoA-independent arachidonyltransacylase to form alky-acyl-glycerophosphocholine. PAF-acetylhydrolase is responsible for removal of the acetate group from the PAF molecule, resulting in reformation of the biologically inactive lyso-PAF. Lyso-PAF-acetyltransferase
Table 1
Blood serum and PAF concentrations of angus bulls divergently selected for IGF-I
Bull number (IGF-I line) IGF-I [ng/ml]* PAF [pM/106
spermatozoa]*
PAF levels in spermatozoa derived from angus bulls divergently selected for blood serum IGF-I concentration IGF-I status (mean6SEM) PAF concentration [pMol/106
spermatozoa] High(568.09619.78) 1.90 (60.39)*
and PAF-acetylhydrolase are both present in spermatozoa [8]. PAF acetylhydrolase activity present in bovine semen [25] is primarily secreted by the seminal vesicles [26]. Thus, the enzymes necessary for PAF activation and deactivation are certainly present in spermatozoa. However, there are no reports on the presence of lyso-acetyltransferase or PAF-acetylhydrolase in bull spermatozoa. PAF metabolism is affected by the endocrine system (e.g., testosterone; 12). PAF-acetylhydrolase is also affected by estrogen and progesterone [13]. Additional studies are warranted to compare hormone levels with spermatozoa-derived PAF and PAF-acetylhydrolase.
The PAF-acetylhydrolase present in seminal plasma is believed to have a role as a decapacitation factor [27]. Removal of acetylhydrolase during the capacitation process may promote PAF synthesis, which, in turn, would allow for the increase in spermatozoa motility [28,29]. The PAF stimulated capacitation and acrosome reaction is calcium dependent [31]. PAF plays a significant role in the fertilization process, enhancing the in vitro fertilization rates of mouse and rabbit oocytes [15,16]. Enhanced embryo development has also been reported in rabbit oocytes fertilized in vitro with PAF exposed spermatozoa [17].
PAF-antagonists inhibit the motility, acrosome reaction and hamster oocyte penetration in exposed spermatozoa [6,14,16]. Additionally, these antagonists inhibit fertilization [15,16]. These data indicate the presence of a PAF-specific receptor in spermatozoa. Immunofluo-rescent studies have verified that spermatozoa possess a receptor for PAF [23,30]. Therefore, PAF would appear to bind to cell surface receptors on spermatozoa, initiating the formation of inositol triphosphate and diacylglycerol, and increasing intracellular calcium [31]. As a secondary messenger, calcium may regulate spermatozoa function by modulating the activity of molecules that transduce intracellular signals, which in turn influence spermatozoa motility. The PAF-receptor appears in higher concentrations at the neck and midpiece regions on spermatozoa. The midpiece is the location of the mitochondria, essential for spermatozoa motility. The neck region is the location of the proximal centriole, which plays a critical role in preimplantation embryo development [32]. PAF may have a stimulatory effect on centriole-intact spermatozoa, enhancing their fertilization success, resulting in improved development rates. Enhanced embryo development has also been reported in rabbit oocytes fertilized in vitro with PAF-treated spermatozoa [17]. Additional studies are war-ranted to elucidate the role of PAF in spermatozoa selection in the fertilization process and PAF’s effect on postfertilization development.
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