Acrosin Activity in Patients with Idiopathic Infertility

ACROSIN ACTIVITY IN PATIENTS WITH IDIOPATHIC INFERTILITY

A. AGARWAL and K. R. LOUGHLIN

Acrosin is a sperm acrosomal enzyme that is involved in the acrosorne reaction, the binding of spermatozoa to the zona pellucida, and fertilization. This study was designed to determine whether sperm acrosin measurements can identify subpopulations of infertile or subfertile patients that are not recog- nized by routine semen analyses. We measured the total acrosin activity of ejaculates in a group of 19 men (15 suspected subfertile patients and 4 fertile donors). The acrosin activity was measured in liquefied semen specimens using the methodology described by Kennedy et al. ((1989) J Urol 10:221- 231). Ten patients in the suspected subfertile group had a mean acrosin value of 7.8 pIU acrosin/million sperm, which is clearly in the abnormal range (<14 pIU/106 sperm). Three patients had a mean acrosin value of 20.1 pIU/106 sperm, which is in the indeterminate range. Two other patients and four proven fertile donors had acrosin values in the normal range (>25 pIU/106 sperm) (Agarwal A, Loughlin KR (1990): 2d International Meeting of Andrology, Como, Italy; Abstr 22). The normal fertile controls had a mean acrosin value of 32.5 pIU/106 sperm.

Key Words: Acrosin; Fertilization; Male infertility.

INTRODUCTION The mechanism of successful fertilization remains poorly understood and as a result many men who are treated for infertility have no identifiable cause for their problem. Acrosin is a sperm-specific acrosomal serine proteinase whose activity is essential for fertilization. Evi- dence obtained in nonhuman species suggests that acrosin enables sperm penetration through For personal use only. the zona pellucida of the oocyte [3]. In addition, possible roles for acrosin include causing or facilitating the acrosome reaction [4], being involved in the zona reaction [5], facilitating sperm passage through the vitelline membrane [6],enhancing sperm migration in the female reproductive tract by the release of kinins from kininogens [7], and degrading sperm prot- amines and causing the dispersion of sperm chromatin in the egg cytoplasm [8]. Traditional methods for the evaluation of male infertility have consisted of taking a history, physical examination, and repeated semen analyses with special attention to sperm count, motility, and morphology. Semen analysis, however, fails to provide any information relative to the ability of sperm to fertile the ovum. In cases of unexplained infertility where a man is judged normal by clinical evaluation and semen analysis and yet continues to remain infertile,

Syst Biol Reprod Med Downloaded from informahealthcare.com by Cleveland Clinic on 12/06/12 the fertilizing capacity of spermatozoa can be tested by assaying acrosin, an enzyme involved

From the Division of Urology, Department of Surgery, Brigharn and Women’s Hospital, Harvard Medical School, Boston, MA, USA. Address correspondence to: Kevin R. Loughlin, MD, Associate Professor in Urologic Surgery, Brigham and Women’s Hospital, Harvard Medical School, 45 Francis Street, Boston, MA 02115, USA.

ARCHIVES OF ANDROLOGY 27:97-101 (1991) Copyright 0 1991 by Hemisphere Publishing Corporation 97 98 A. Agarwal and K. R. Loughlin

in the process of fertilization. Acrosin can be present on spermatozoa in both an active (nonzy- mogen) and inactive (zymogen) form. The latter is called proacrosin and represents approximately 90% of the acrosin on human spermatozoa [9]. Proacrosin is thought to be converted into its active form, acrosin, during the acrosome reaction and sperm penetration through the zona pellucida. It is of clinical importance to measure the total amount of active acrosin that can be generated by spermatozoa, which is the sum of the active acrosin already associated with the spermatozoa plus the active acrosin that is produced by conversion of proacrosin. The total acrosin content is probably less useful as a fertility market than the total acrosin activity because it is conceivable that proacrosin conversion to acrosin cannot take place leaving intact the total acrosin content but greatly decreasing the amount of active acrosin available to the spermatozoa for function. To measure the total acrosin activity available for sperm function, the assay has to incorporate proacrosin conversion to acrosin before the activity can be measured. Many assays are available to measure sperm acrosin levels. Most are either complicated, time-consuming, not sufficiently sensitive (such as gelatin plate assay), or measure acrosin content rather than activity (such as immunological techniques). A simple, sensitive clinical assay that measures the total available acrosin activity of human spermatozoa based on the benzamidine-sensitive amidase activity of the enzyme was recently developed by Kennedy et al. [ 11. We are reporting our results on sperm acrosin levels in a group of infertile men with idiopathic infertility using the above method. MATERIALSMETHODS The acrosin assay [l] consists of three main steps. First, the spermatozoa were washed free oi seminal plasma by centrifugation over Ficoll to remove the soluble proteinase inhibitors in human semen1 that can interfere with acrosin activity. The sperm pellet was subsequently suspended in buffer that has, (1) a detergent that facilitates disruption of the acrosomes and releases the acrosomal enzymes, (2) a1 basic pH that allows activation of proacrosin into enzymatically active acrosin, and (3) a synthetic For personal use only. arginine amide substrate that, when hydrolyzed, releases a chromophoric product. Finally, the total. amount of color developed after a 3-h incubation period is measured spectrophotometrically. Benzamidine hydrochloride, Ficoll (type 400), N-benzoyl-DL-arginine p-nitroanilide hydrochloride: (BAPNA), dimethylsulfoxide (DMSO) , Hepes (N-2-hydroxyethylpiperazine-n’ -2-ethanesulfonicacid) ., and Triton X-100 were obtained from Sigma Chemical Company (St. Louis, MO). Semen samples were: obtained from apparently normal healthy men (n = 4) and from suspected subfertile men (n = 15). After complete liquefaction of the ejaculate, a small aliquot was removed to determine sperm motility and sperm concentration on a Cell-Soft 2000 computerized semen analyzer (Cry0 Resources, Ltd, New York). As a standard procedure, an aliquot of the ejaculate containing between 2 and 10 x lo6 sperm was carefully layered over 0.5 mL 11% Ficoll (in 0.12 M NaCl, 0.025 M Hepes, pH 7.5) in a plastic conical centrifuge tube. One control and two tests were run simultaneously for each ejaculate. The tubes Syst Biol Reprod Med Downloaded from informahealthcare.com by Cleveland Clinic on 12/06/12 were centrifuged at lOOOg for 30 min in a tabletop centrifuge. The Ficoll and seminal plasma were plasma were removed from the sperm pellets with a thin stemmed Pasteur pipette leaving approximately 100 pL of Ficoll and the sperm pellet behind. To the control tube, 100 pL of 500 mM benzamidinc: hydrochloride in water was added. The sperm pellets in the control and test samples were thoroughly suspended in 1.0 mL of detergent substrate solution obtained by mixing 22.5 mL of detergent buffer (0.1 % Triton X-100,0.055M NaCl in 100 mL distilled deionized water adjusted to pH 8.0) with 2.5 mL substrate (25 mg BAPNA in 2.5 mL DMSO, prepared fresh). The mixtures were incubated for 3 h at room temperature after which 100 pL of 500 mM benzamidine in distilled water was added to all tubes except for the control. The samples were then centrifuged at lOOOg for 30 min and the supernatants were collected. An LKB spectrophotometer (Ultrospec 11) was adjusted to an absorbance reading of 0.0 at 410 Acrosinhfertility 99

nm with the substrate-detergent mixture. Subsequently, the absorbance of each supernatant solution was recorded at 410 nm. The acrosin activity of the ejaculate was calculated according to the following formula:

pIU acrosin/million sperm = [(Mean OD test) - OD control] x 10 1485 x number of sperm (in millions layered over Ficoll mL)

RESULTS AND DISCUSSION Acrosin content of all of our four proven fertile donors was in the normal range (>25 pIU/ lo6 spermatozoa) [I]. However, only 2 out of 15 of our infertile patients demonstrated acrosin levels in the normal range, whereas 10 of the 15 infertile men exhibited acrosin levels in the subfertile range (< 14 pIU/106 spermatozoa). The remaining 3 patients with clinical infertility had acrosin levels in the indeterminate range (> 14, < 25). Another interesting observation was that 5 patients (Table 1, patients 1, 3, 5, 9, and 14) exhibited low acrosin activity despite normal semen parameters.

TABLE 1 Semen Analysis and Acrosin Activity

Sperm Concentration Layered Sperm Concentration over Ficoll mL Ejaculate Acrosin Value Patient # (lo6 per mL) Motility (%) (lo6 per mL) Volume (m~) (~Iu/~o~sperm)

Fertile Donors 80 74 10.00/0.15 1.o 40 For personal use only. 63 72 15.OO/0.25 1.5 26 72 55 08.50/0.10 3.1 28 240 88 12 .OO/0.05 3.5 36 Idiopathic Infertile Patients 1 188 93 09.40/0.05 1.8 29.7 2 22 49 11 .OO/0.50 3.0 11.1 3 193 67 19.00/0.10 3.0 25 .O 4 41 23 12.00/0.30 1.2 8.0 5 50 52 12.0010.25 0.9 9.6 6 21 23 05 .OO/0.25 2.0 5.6 7 20 28 10 .OO/O. 50 2.5 8.7

Syst Biol Reprod Med Downloaded from informahealthcare.com by Cleveland Clinic on 12/06/12 8 15 38 07.40/0.50 6.5 10.6 9 65 59 13.00/0.20 2.5 10.9 10 19 61 09.50/0.50 3.5 21.4 11 18 36 09 .00/0.50 4.6 1.o 12 195 96 19.00/0.10 2.5 21.4 13 19 24 04.50/0.25 2.5 3.7 14 121 47 12.00/0.10 0.75 7.1 15 74 49 1 1 .00/0. 15 2.0 12.6 16 149 66 15.00/0.10 2.0 9.5 17 38 42 07.0010.20 2.0 1.o 18 21 28 06.00/0.30 4.5 15.0 100 A. Agarwal and K. R. Loughlin

During the past few years, acrosin activity has been shown to be an important marker of sperm fertilizing potential because the acrosin system affects the acrosome reaction [lo, 111, as well as sperm binding to the zona pellucida, and penetration ability of the zona pellucida and vitelline membrane [12, 131. In addition, infertile men as a group have lower sperm acrosin activity than fertile men. Spermatozoa from apparently normal, asymptomatic men possess approximately three times the acrosin as those from subfertile men [ 14, 151. Cryopres- ervation, which is known to decrease sperm fertilizing ability has been associated with de- creased acrosin levels [ 16, 171, Also, acrosomeless spermatozoa with almost undetectable levels of acrosin are infertile even if they are motile and possess an intact plasma membrane [ 181. Finally, recent preliminary results show a correlation between sperm acrosin activity and the fertilization rate in vitro [l]. The presence of a hydrolytic enzyme, acrosin, is a prerequisite for fertilization. Men with low sperm acrosin activity may be infertile because of the lower enzyme level. The acrosin activity has been shown to vary independently of the physical sperm parameters [19] and can identify a subpopulation of infertile men not identified by routine semen parameters [20]. In the present study 87% of our total cohort of men with unexplained infertility had sperm acrosin levels below the range of values for normal men. Among normal fertile controls and infertile patients, however, sperm acrosin activity does not correlate with cell density, percent motility, or forward progression measured in the initial semen sample. Sperm acrosin activity is lower among infertile men than fertile men [14, 151 and lower in cells obtained from the second portion of a split ejaculate [ 151. However, the mechanism(s) of the lower sperm acrosin in some infertile men remain unknown. Potential causes for altered acrosin levels include failure of initial synthesis or packaging of the enzyme within the acroso- ma1 space during spermatid formation, loss of acrosin activity during sperm transit through the epididymis and loss of acrosin activity from “fragile sperm” following ejaculation. Although

For personal use only. the epididymis is important for sperm to achieve normal motility characteristics and fertilizing capacity [21], it is not clear whether the acrosin system is normally altered during epididymal transit. Lower sperm acrosin activity of some infertile men probably reflects changes in the acrosome status before ejaculation [22]. Acrosin appears to play a crucial role in the fertilization process. Patients with normal routine semen parameters may have low acrosin levels which can explain the cause of their infertility. Further studies of factors influencing acrosin activity as essential to achieve a better understanding of the factors contributing to successful fertilization.

REFERENCES

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