Global Infertility Solutions

Platelet Activating Factor Promotes Hyperactivated Sperm Motility In Fertile Males

Correspondence and request reprints to: Barry A. Ripps, MD, University of Florida- Pensacola, Florida, Department of Reproductive Endocrinology, 5147 North Ninth Avenue Suite # 315, Pensacola, Florida 32504, Office: 850-857-3733, Fax: 850-857-0670, Email: newfertility@aol.com

Heidi E. MacVittie, MD, University of Florida – Pensacola
Brijinder S. Minhas, Ph.D., Global Infertility Solutions – Kenilworth, Illinois

Keywords: Platelet Activating Factor, spermatozoa motility, hyperactivated sperm motility, IVF

Abstract

Objective
To determine whether or not exogenous platelet activating factor affects hyperactivated sperm motility.

Study Design
Human spermatozoa, washed with Ham’s F-10, were exposed to 10^-7 M and 10^-5 M of PAF

and compared to an untreated control. Each sample was then evaluated for hyperactivated

sperm motility using the Hamilton Thorn motility analyzer.

Results
The PAF exposed sperm exhibited decreased linearity, 37.5% to 30.0 % (P<0.05), and a significant increase in curvilinear velocity, 121.7 m/s up to 135.2m/s (P<0.05) without a change noted in the amplitude of lateral head displacement.

Conclusions
Spermatozoa exposed to PAF exhibited an increase in hyperactivated motility. This further substantiates the theory that PAF is intricately involved in spermatozoal function and may prove to be an effective first line therapeutic adjuvant in treating male factor infertility.

Introduction

Platelet Activating Factor (PAF) is the name assigned to a family of acetylated glycerophospholipids primarily because of their ability to release histamine from platelets. Central roles for PAF in reproductive processes have been documented over the years.  These functions include ovulation, fertilization, pre-implantation embryo development, implantation and parturition (Minhas,1992).  PAF has also been found in rabbit, mouse, pig, bovine and human spermatozoa. Our group reported the first study documenting the presence of PAF in human spermatozoa and it’s absence in seminal plasma (Minhas et al.,1991).

This finding of PAF in human spermatozoa by our group has been followed up by numerous studies from many groups documenting a key role for PAF in sperm function modification. Fertilization requires that the sperm is able to swim to the oocyte, attach and penetrate the oocyte followed by fusion of the gametes.  PAF has been implicated in several studies in many species to promote gamete interaction (Sengoku et al.,1993; Minhas,1993; Minhas and Ripps,1996; Naz and Minhas,1995; Angle et al., 1993; Huo and Roudebush, 2000; Hellstrom et al., 1991; Ricker et al., 1989).

Recent reports have documented the presence of spermatozoal   PAF receptor mRNA and of key interest is the finding that normal sperm have a distinct distribution of PAF receptors primarily at the proximal head and the midpiece regions (Roudebush et al., 2000, Reinhardt et al., 1999).  Reproductive insufficiency in men is heterogeneity of biochemical and molecular level disorders whose common and frequently only clinical expression is failure to sire offspring. Changes in the distribution of PAF receptors on spermatozoal surfaces may play a key role in modulating sperm function.

Binding of PAF to its membrane receptor protein, activates the adenylate cyclase system producing diacylglycerol and inositol triphosphate as secondary messengers, which in turn cause an increase in intracellular calcium. The fore-mentioned distribution of the PAF receptors in the midpiece of the sperm, which contains the mitochondria, in concert with calcium influxes upon exposure to exogenous PAF, are indicative of an important role for PAF in sperm motility. Several studies have indeed demonstrated increased motility in sperm upon exposure to PAF (Wang et al., 1994; Hellstrom et.al., 1991; Ricker et al., 1989).

This study supports these previous studies and further describes for the first time PAF promotion of spermatozoal hyperactivation, an essential predecessor of human fertilization. Mature spermatozoa of all mammalian species must complete a series of membrane and metabolic changes before they can fertilize an egg. This process has been termed capacitation and is functionally associated with the acrosome reaction and a distinctive type of motility termed hyperactivation.  Hyperactivation is considered to be a second functional marker of the completion of capacitation, along with the first maker of the acrosome reaction. During the short lived phase of hyperactivation spermatozoa display an increased curvilinear velocity and decreased linearity (Burkman, 1991).   Promotion of human sperm hyperactivation as noted in this paper is a key finding in the finely tuned autocoid regulation of spermatozoal function by PAF.

Methods

Specimen Collection
Fertile male donors were recruited to provide semen samples for treatment analysis with the approval of the Institution Review Board.  Nine subjects were recruited with an age range of 29-42 years.  All subjects had proven fertility with the last conception occurring within the preceding three years.

Experimental Design
The subjects were instructed to collect a semen sample by masturbation. The semen was then allowed to liquefy for thirty minutes and then washed twice with Ham’s F-10 (0.3% BSA – Bovine Serum Albumin) media. Sperm were allowed to swim up into overlaid fresh media and then divided into three siliconized glass tubes. One tube was the untreated control. The second tube contained 10^-7 M PAF and the third tube contained 10^-5 M PAF.  PAF (C16:0) (Nova Biochem) was kept as a 1x10^-6 M stock solution dissolved in chloroform/methanol (1:1) and stored at –20 C in a sterile glass container.  The treatment tubes were prepared by dehydrating a 1.5 ml aliquot of PAF under a stream of nitrogen that was then re-dissolved in Ham’s F-10 (0.3%BSA) The amount of fresh media used was adjusted to yield a final concentration of 10 million motile sperm per ml. This permitted dose response analysis of hyperactivated motility in paired samples of treated and control sperm.

Measurement of Motility Parameters
Sperm motility parameters were measured with the Hamilton Thorn motility analyzer (Hamilton Thorn Research, Beverly, MA. Model HTM-S 2030, Version 7), with the following settings: Analysis time = 0.67 sec (20 frames), minimum contrast = 7, minimum size = 6, low size gate = 0.4, high size gate = 1.6, low intensity gate = 0.4, high intensity gate = 1.6, medium average path velocity = 25, low VAP = 5m/s, slow cell count  = yes, threshold straightness = 80%, magnification = 2.13. A SORT function identified cell populations with defined hyperactivated motility.  Hyperactivated motility is defined as linearity (LIN) £ 65, curvilinear velocity (Vcl) ³ 100m/s and amplitude of lateral head displacement (ALH) ³ 7.5m/s.  Measurements were performed in Microcell Ò chambers 20 m depth (MicrocellÒ Spectrum Technologies, Berkeley, CA) and obtained at time 0 in the swim up preparation and at 30, 60, and 120 minutes after PAF exposure.

Statistical Analysis
The control and treatment groups were compared at each time interval using a two-tailed paired t test. A P value of < 0.05 was considered significant.

Results

The proportion of motile sperm exhibiting hyperactivated motility in the control, 10^-7 M PAF (treatment group 1) and 10^-5 M (treatment group 2) at time 0 were 8.7%, 10.4%, 12.8% respectively. These proportions did not change appreciably during the interval of study. The PAF exposed sperm exhibited decreased linearity when compared to controls (Figure 1). The linearity of controls was 37.5% while the PAF exposed sperm was 30.2% (p<0.05) and 30.0% (p<0.02) respectively by sixty minutes after treatment. Curvilinear velocity (Vcl) increased significantly within the same time interval (Figure 2), with control = 121.7m/s, PAF 10-7 = 130.0 m/s (p< 0.05), PAF 10-5= 135.2 m/s (P<0.05). Amplitude of lateral head displacement did not significantly change. Analysis at two hours from exposure failed to reveal persistent differences between control and treated pairs.

Discussion

Exogenous PAF treatment of human spermatozoa has been shown to enhance motility, cervical mucous penetration, hamster oocyte penetration, and pregnancy rates in patients undergoing intrauterine insemination (Wild and Roudebush 2001; Roudebush et al., 1990; Minhas, 1993; Minhas et al., 1994).  The amount of endogenous PAF present (Reinhardt et al., 1999; Sanwick et al., 1992) in human spermatozoa is inversely correlated with normalcy (Roudebush et al., 2000, Roudebush and Purnell, 2000). Furthermore, human spermatozoa posses PAF receptors and abnormal spermatozoa have an altered receptor distribution. This altered distribution of PAF receptors in abnormal spermatozoa has been speculated to be due to defective gene expression (Roudebush et al., 2000).

Exogenous PAF treatment of spermatozoa has  a stimulatory effect on spermatozoal motility which may lead to enhanced fertilization rates and subsequently higher  pregnancy rates(Ricker et.al.,1989, Minhas, 1992).

In the normal process of fertilization, spermatozoa must undergo capacitation, acquire hyperactivated motility, and undergo an acrosomal reaction prior to penetrating the oocyte. Hyperactivated motility is thought to be essential to allowing the spermatozoa to move through the complex system of folds and furrows of the oviductal mucosa. Previous studies have shown that exogenous PAF treatment of spermatozoa is effective therapeutically for patients undergoing intrauterine insemination (Wild and Roudebush, 2001). Our study is the first to provide direct evidence of hyperactivated sperm motility as a result of exogenous PAF treatment.

We demonstrate that spermatozoal exposure to PAF at 10^-7 M and 10^-5 M results in a significant increase in curvilinear velocity and decreases linearity by sixty minutes after exposure.  However, two hours after exposure these differences do not persist. This short – lived response is consistent with calcium mobilization seen with sperm treated with progesterone and 17 hydroxyprogesterone (Blackmore et al., 1990). Intracellular calcium has been shown to increase, in both human and rabbit spermatozoa, when exposed to PAF and measured by flow cytometry (Minhas 1996). Studies on spermatozoal  PAF exposure  consistently show  that  in both animals and humans, PAF improves in vitro fertilization rates, intrauterine insemination fertility rates and fertilization potential (Minhas 1993, Angle et al., 1993; Wild and Roudebush 2001; Roudebush et al., 1990; Minhas et al., 1994; Fukuda et al., 1994). The hyperactivation of sperm motility seen in this study along with the enhancement of the acrosome reaction previously reported (Angle et al., 1993, Minhas et al., 1994) may be a mechanistic reason for the improvement of fertilization seen with PAF treated spermatozoa.

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Figure 1

Linearity, %

Figure 2