The Relevance of Neutral α-Glucosidase Activity in Andrology

Abstract

To assess the usefulness of routine determination of neutral α-glucosidase (NAG) in andrology, 216 ejaculates were analyzed for NAG activity and semen quality. A correlation between NAG activity and semen volume and sperm concentration was determined; however, no correlation was observed between NAG activity and sperm motility or sperm morphology. The number of azoospermic ejaculates that had NAG activity below acceptable levels was significantly higher than the number of non-azoospermic ejaculates with similarly low NAG levels. Routine determination of NAG activity is not practical; however, when an epididymal pathology leading to a physiological or anatomical functional alteration is suspected, the determination of NAG activity is a valuable tool in the diagnosis, and would also aid in the prognosis.

Keywords:

• azoospermia
• cryptozoospermia
• NAG
• neutral α-glucosidase
• normozoospermia

Abbreviations
NAG	=	Neutral α-glucosidase
PNPG	=	p-nitrophenyl glucopyranoside
PNP	=	p-nitrophenol

Introduction

The activity of neutral α-glucosidase (NAG) is significantly reduced when there is an occlusion distally to the epididymis [Cooper et al. 1990; Dohle 2003; Guerin et al. 1986; Kret et al. 1995; Mahmoud et al. 1998; Rivas et al. 1981; Sandoval et al. 1995; Schill and Henkel 1999]. Low NAG activity is also associated with functional deficiency of the epididymis in non-azoospermic patients [Comhaire et al. 1999; 2002].

Although the exact role played by α-glucosidase in sperm function is not well understood, some speculate that NAG is responsible for sperm maturation through protein modification [Decheux et al. 2003]. Others have reported a decrease in NAG activity as sperm abnormality increases [Casano et al. 1987; Pena et al. 2004; Rivas et al 1981; Viljoen et al. 1990]. Individuals with low NAG activity also exhibited poor ability to bind the zona pellucida, suggesting that NAG may have a role in sperm-egg binding [Ben Ali et al. 1994]. Still others have suggested that NAG aids in the diagnosis of infection of the reproductive tract [Loko et al. 1997; Wolff et al. 1991]. Given these findings, the possibility that decreased NAG activity leads to abnormal sperm quality cannot be overlooked.

The above considerations suggest that the determination of NAG activity is useful for the diagnosis of epididymal patency, and the diagnosis of sperm abnormality. However, one study evaluated 653 ejaculates [Krause and Bohring 1999] and concluded that the determination of NAG activity did not provide any additional relevant information as compared to data obtained from routine semen analysis results.

The objective of the present study was therefore to determine NAG activity in semen samples routinely evaluated as part of infertility workup, and assess the usefulness of NAG enzymatic activity as a marker for semen quality.

Results

Forty of the 216 ejaculates had a semen volume more than 1.4 ml, sperm concentration of more then 19.9×10⁶ ml⁻¹, sperm motility and normal sperm morphology of more than 39%, respectively. These ejaculates were considered normozoospermic (sperm concentration more than 20×10⁶ ml⁻¹ with sperm motility and normal morphology more than 40%, respectively). Twenty-seven of the 216 ejaculates were azoospermic (no spermatozoa in the ejaculate). Of these, 10 were cryptozoospermic (sperm concentration less than 0.1×10⁶ ml⁻¹) since spermatozoa were only identified in the centrifuged pellet. The remaining 149 ejaculates had an additional abnormality in the semen parameters analyzed.

The correlation coefficients of various semen parameters and NAG activity are listed in Table 1. The mean NAG activity values obtained for normozoospermic ejaculates was significantly higher (p<0.01) from that obtained for azoospermic and cryptozoospermic patients (Table 2). The 8.5 mU ml⁻¹ enzyme activity (mean—2 S.D.) obtained for normozoospermic ejaculates was considered the minimum acceptable NAG activity level for comparison with other samples.

Relationship between NAG Activity and Various Semen Parameters

Parameters	Number of ejaculates	Correlation coefficient	Student t test	P value
Volume	216	−0.219	−3.34	0.001
Sperm concentration	189	0.441	6.72	<0.0001
Sperm motility	189	0.066	0.92	0.36
Sperm morphology	189	0.037	0.50	0.61

Mean +/− S.D. of NAG Activity for Various Categories of Semen Quality

Ejaculate groups	Number of patients	Mean +/− S.D.
Normozoospermia	40	43.9 +/− 17.7*
Azoospermia/cryptozoospermia	27	26.2 +/− 23.7*
Abnormal semen	149	34.7 +/− 18.4

*P<0.01.

When the number of ejaculates that had either acceptable or below acceptable levels of NAG activity was compared with the number of azoospermic and cryptozoospermic ejaculates, a highly significant difference (p=0.0001) was noted (Table 3). Four of 7 azoospermic ejaculates and one of 5 cryptozoospermic ejaculates, low in NAG were post vasectomy samples.

Number of Ejaculates with or without Acceptable NAG Activity for Various Categories of Semen Quality

	Number of ejaculates
Ejaculate groups	NAG activity ≥ 8.5 mU ml^−1	NAG activity <8.5 mU ml^−1
Normozoospermia	38	2
Abnormal semen	147	2
Volume
≥1.5 ml	132	2
<1.5 ml	15	0
Sperm concentration
≥20×10^6 ml^−1	117	0
<20×10^6 ml^−1	30	2
Sperm motility
≥40%	137	2
<40%	10	0
Sperm morphology
≥40%	20	0
<40%	127	2
Azoospermia/cryptozoospermia*
Azoospermia	10	7
Cryptozoospermia	5	5

*Number of Azoospermia/Cryptozoospermia ejaculates with <8.5 mU ml⁻¹ NAG activity is significantly different from normozoospermic ejaculates and ejaculates with abnormal semen quality. Also, four of seven azoospermic and one of five cryptozoospermic ejaculate low in NAG activity were post vasectomy specimens.

Discussion

From the data presented, the routine determination of NAG enzyme activity may not provide any additional relevant information for semen analysis. For example, a significant correlation is observed between enzyme activity and semen volume as well as with sperm concentration (Table 1). This suggests that the semen volume and/or sperm concentration may reflect the level of NAG activity. However, these two semen parameters are dependent on the extent of sexual stimulation [Yamamoto et al. 2000] and the number of days of sexual abstinence. NAG activity was significantly lower after two to three days of abstinence when compared with both four to five days of abstinence, and six and seven days of abstinence, respectively [Elzanaty et al. 2005].

In addition, stability of the NAG is of concern. During storage at −20°C, the equine α-glycosidase activity was about 90% of the initial activity during the first week, and subsequently decayed approximately 10%, 30%, and 40% after 10, 20, and 30 days, respectively. After a 3-month storage period, about 30–40% of the activity remained [Dias et al. 2004]. Circannual changes in the NAG activity have also been documented [Henkel et al. 2006]. Considering these practical concerns as well as the poor correlation noted between the NAG activity and sperm motility or morphology (Table 1), routine assessment of NAG may not be advisable. This is in agreement with previous reports [Krause and Bohring 1999].

In specific instances, however, such as poor sperm egg binding [Ben Ali et al. 1994] or necrozoospermia (non-motile sperm in the ejaculate) in the presence of normal semen volume and sperm concentration, it may be advisable to determine NAG activity. For example, a patient following vasovasectomy had sperm concentration of more than 20 million, with no motility. His ejaculate repeatedly yielded NAG activity below the acceptable level (RSJ; personal communication). Although the cause of necrozoospermia could not be identified, low NAG activity nonetheless suggested epididymal involvement.

Should the ejaculate be azoospermic or cryptozoospermic (Table 2), then determination of enzyme activity is diagnostic, and may aid in the prognosis [Zopfgen et al. 2000]. Azoospermic ejaculates with low enzyme activity may suggest a complete post caput epididymal occlusion, since in humans most of the enzyme activity is found in or above this region [Pena et al. 2004]. Patients with low serum testosterone levels also have low levels of α-glucosidase activity [Gonzales 2002]. In the case of cryptozoospermia, however, a partial obstruction may cause this low enzyme activity; residual sperm following vasectomy may also result in this kind of low NAG activity.

Ten of the 27 (37%) azoospermic ejaculates were cryptozoospermic, as compared to about 20% reported previously [Jaffe et al. 1998]. The higher percent of cryptozoospermic ejaculates observed in the present study is probably due to 5 times the centrifugal force used (1,000 g for 10 min) as compared to the Jaffe study.

If a partial obstruction is suspected, then the examination of a second ejaculate collected within a half h to one h of the first ejaculate will reveal an increased number of sperm. In the 1980s, it was customary in some practices to request oligozoo spermic (sperm concentration less than 20×10⁶ ml⁻¹) partners of infertile couples to produce two consecutive ejaculates, which were then combined for insemination. When examining these two ejaculates, one occasionally notes a higher semen quality in the second ejaculate as compared to the first. For example, a patient following vasovasectomy had a sperm concentration of more than 20 million, but the sperm count declined to less than 5 million in 3 months. Two consecutive collections were combined for cryopreservation, revealing a sperm count of more than 30 million in the second ejaculate (RSJ; personal communication). In either case, sperm may be retrieved from the caput epididymal region.

Based upon the above discussion, under certain circumstances or conditions (such as when one suspects epididymal pathology leading to physiological or anatomical functional alteration), the determination of NAG activity proves a valuable tool for both diagnosis and prognosis in male infertility.

Materials and Methods

The investigation was undertaken to determine retrospectively, from records of routine semen analysis, the relevance of determining neural α-glucosidase activity as a part of routine semen analysis. No institutional review board approval was obtained as the data analyzed was obtained retrospectively from existing records.

Two-hundred and sixteen ejaculates obtained by self-masturbation were analyzed for semen quality as described previously [Jeyendran 2003]. NAG activity was determined by spectrophotometry [Cooper et al. 1990]. Briefly, NAG activity was determined by measuring the amount of p-nitrophenyl glucopyranoside (PNPG) converted to p-nitrophenol (PNP) by the enzyme. Seminal plasma was incubated for two hours in a culture tube containing PNPG in 1% sodium dodecyl sulfate solution, which inhibits the acid alpha glucosidase isoform so only the NAG activity is measured. One control sample also included castanospermine to provide the nonspecific enzyme blank value. After the incubation period, adding 0.1 M Na₂CO₃ to each reaction stopped the reaction. The absorbance of each sample was read at 405 nm; PNP concentration was determined by using the standard curve (prepared by measuring the absorbance of known concentrations of PNP at 405 nm). The results are expressed as mU ml⁻¹ enzyme activity.

All data are presented as the number of ejaculates or as the mean +/− standard deviation of the NAG activity. Pearson's coefficient of correlation was calculated when appropriate. To assess the significant differences in the means, data was subjected to paired t-test analysis.

Declaration of Interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

The corresponding author, Dr. R. S. Jeyendran, is the Director of Andrology Laboratory Services. Although the laboratory specializes in semen analysis, this investigation does not provide or will not provide any financial rewards to the laboratory.