Evaluation of Enkephalin-Degrading Enzymes in Sperm from Heroin-Addicted Men

Infertility is one of the most serious social problems that has an effect on a huge percentage of couples. In general, the basic cause of infertility refers to the male partner in approximately 40-45% of cases (1). In some national data and available sources, the number of drug abusers in Iran is estimated to be between 1,200,000 and 2,000,000 people (2-4) with a mean age of 33 years (5). This means that they are generally young in reproductive age. Heroin, from the opioid group, is morphine O-acetylated at position 3 and 6 (diacetylmorphine) (6). Heroin addiction has been linked to some degenerative diseases including aging, abscesses, arthritis, other rheumatologic disorders, and immunological disorders (7).

The activity of the opioid system is mediated by endogenous opioid peptides (EOPs). EOPs or opiate alkaloids carry out their function through three types of opioid receptors [the delta-opioid receptor (DOR), the mu opioid receptor (MOR) and the kappa opioid receptor (KOR)] on membranes (8, 9). EOPs control male reproductive function at different levels (1): i. At the hypothalamus, they inhibit the secretion of gonadotropinreleasing hormone (GnRH) and suppress the rate of luteinizing hormone (LH) from the pituitary gland (decrease the levels oftestosterone), ii. At the testes, leydig cells produce EOPs through LH affect, and these peptides perform an inhibitory effect on sertoli cells [inhibiting the production of Androgen-binding protein (ABP)] and iii. In germ cells, somatic cells of the testes and iv. In sperm cells (regulates sperm motility). As a result, EOPs may be involved in human reproductive function by a direct effect on sperm. However, EOP levels are controlled by enzymatic degradation by aminopeptidase N (APN) and endopeptidase neutral N (NEP). These enzymes are present in both sperm and seminal fractions (10), and their activity in semen is particularly high compared with other body tissues (11). Interestingly, APN activity levels were found to be altered in semen from sub-fertile patients, suggesting that this enzyme may play an important role in male fertility (12).

The effect of opiates on spermatozoa in some aspects is still unknown or controversial. Some studies have been performed on the effect of certain drug abuse on the human and mouse sperm parameters (13-15). In our previous study, we showed the deleterious effects of kerack, (unlike to crack cocaine), consumption in Iran on testis structure, sperm parameters, and particularly sperm morphology in the adult mouse. Also, It down-regulated the expression of CatSper genes, resulting in depression of sperm motility (16). Nazmara et al. (17) reported that heroin is strongly associated with abnormalities in histone-to-protamine transition and with human semen quality, particularly sperm morphology and motility.

Sperm motility is considered a key functional parameter that controls reproduction (18) and is widely used as an indicator of semen quality (19) since sperm must move to reach the oocyte and then penetrate it using sperm movement. More reports on heroin (13) and nicotine (20) consumption in animals and opioids in human (14, 17) showed decreasing in sperm parameters.

Since decreased motility (asthenozoospermia) is a common abnormality among opiate drug addicts (17), it is likely that the opioid system involves the sperm movement. Previous studies have suggested expression of enkephalin-degrading enzymesin human spermand semen (1, 10, 12, 21). Subiran et al. (10) reported Enkephalindegrading enzymes were expressed in human sperm including messenger RNA of both enzymes and APN and NEP, (in a small number of sperms), were detected in spermatozoa at the protein level. In addition, the activity of the enkephalin-metabolizing enzyme aminopeptidase N was measured in various fractions of human semen from normal and subfertile patients by Irazusta et al. (12). They reported that the activity of aminopeptidase N was lower in males with asthenozoospermia as compared with normal semen. It has been described that the inhibition of some of those enzymes can significantly improve sperm motility (21). In the present study, we investigated the expression of two enkephalin-degrading enzymes APN and NEP in heroin-users’ sperm cells to assess any correlation between expression of abovementioned and sperm motility.

The present study showed that enkephalin-degrading enzymes (NEP and APN) and sperm viability and motility were reduced in men addicted to heroin. We also showed that there was a significant negative correlation between NEP and APN gene expression levels with the duration of heroin dependence.

Infertility is one of the most important problems of human societies throughout the world. Considering the role of recreational heroin consumption as an idiopathic etiology of male infertility and increasing consumption of illicit drugs, especially among young people of reproductive age, socio-medical studies on this issue has been done less yet. The living conditions, lack of cooperation, simultaneous use of various drugs and legal and ethical problems in sampling in addicted people make the research difficult and complicated in this area. Therefore, research in this area can be very valuable. Our findings suggest a remarkable association among heroin addiction, asthenozoospermia and decreased APN and NEP mRNA levels. In addition, the duration of drug dependence is one of the main factors contributing to the detrimental effects of heroin on impaired male fertility.

Decreased in heroin users BMI may be caused by caloric malnutrition (25), inhibition of androgen production (26), or disorders of the gastrointestinal tract (27). Although our finding appears to be consistent with a number of studies (16, 26, 27), Diamond et al. (25) showed drug and alcohol abuse did not change the BMI in adolescent males. The average BMI may affect spermatogenesis. However, multivariate regression analysis showed that duration of heroin consumption can be more effective than BMI in semen parameters.

In this study, reduced sperm motility was observed in addicted men. Our finding is in line with other studies who mentioned that opioids such as heroin, kerack, and morphine can impair sperm parameters in mice and human. They also showed those alterations were dose-dependent (8, 13, 14, 16, 17). The opioid system likely influences reproductive function by the central nervous system (28), the pituitary gland, and the testis (29), exerting a direct action on the spermatozoa (30).

Reduced total and progressive sperm motility may be caused directly by heroin because of alteration of the encephalin-degrading enzymes. Researchers scrutinized the presence of APN/CD13 in the sperm head, neck and along the tail (19, 31). APN/CD13 was acknowledged to play a critical role in the sperm binding to oocyte due to being in the sperm head and control its motility by existence along the tail and in the neck. In the fact that opioid levels in semen are in charge of degrading enzymes like APN/ CD13, alteration of these enzymes could regulate sperm motility. Indeed, an adequate level of enkephalin as a delta opioid agonist is essential to sperm motility, but this effect depends on opiate concentrations (10). We proposed that heroin directly affects sperm motility by two mechanisms; first, a higher concentration of mu opioid agonists not only bind mu opioid receptors but also have an affinity to delta-opioid receptors. Heroin bind to the receptors occupies the enkephalin-binding site on the sperm and could deactivate the receptors and cause to accumulate opioids in semen. Second, Reducing the APN gene and subsequently, its protein in heroin consumers may result in opioid accumulation in the semen microenvironment. Indeed, the occupation of receptors and degrading enzymes deficiency or inactivity may affect the sperm quality and male fertility. Agirregoitia et al. (8) reported the inhibitory effect of the delta-antagonist naltrindole on sperm motility.

Notwithstanding the level of NEP gene expression was statistically different between groups, its protein amount was similar in addicted and healthy men. In addition, a significant negative correlation between the NEP gene level and BMI was observed. Our data was proved by previous studies (10, 32, 33). Given that just a few cells after capacitation express NEP protein on sperm membranes (32), could justify the same protein amount between groups. This similarity may be due to the fact that NEP act by a mechanism that does not involve the opioid system which was suggested by Subiran et al. (10) who revealed naloxone does not affect sperm motility when incubated with thiorphan an encephalin degradation inhibitor for NEP. Our data showed that the amount of NEP gene and protein do not relate to each other. We hypothesize that this may be due to: i. Epigenetic regulators or the presence of NEP mRNAs in the profile of RNAs’ mature spermatozoa that interfere with embryogenesis; or ii. Since spermatozoa have a complex RNA package (34, 35), sperm cells could retain NEP messenger RNA for carrying epigenetic information in the zygote.

Based on the literature, NEP is the main peptidase that can hydrolyze tachykinins, which are present in spermatozoa and interfere in the regulation of sperm motility (12, 36, 37). Thus, alterations of NEP can cause slowly developing changes in sperm motility. Opioids and tachykinins, as bioactive peptides, could operate as signal molecules between spermatozoa and their environment, acting in an autocrine and/or paracrine fashion (38).

In addition, heroin can impair semen quality and alter sperm microenvironment by semen acidification and leukocytospermia (17), which probably affects the structure and function of these surface-expressed enzymes and influences semen parameters.

The main limitation in the present study were: i. Sampling of heroin-addicted men was complicated because (a) heroin consumers usually take various addiction and narcotic drugs like opiate, kerack or methamphetamines, (b) the majority of addicted men had no trends to provide semen. Hence, we selected persons who used only heroin for at least 12 months -without using other drugs, ii. In addition; cigarette smoking was a major interference variable, which was controlled by partial correlation, iii. Scio-economic and family backgrounds were different among the participants. For example, diet may affect sperm parameters, and iv. We could not follow up the fertility status after treatment.

We conclude that semen quality and enkephalindegrading enzymes were decreased in heroin-addicted men and there is a significant negative correlation between NEP and APN gene expression levels with the duration of heroin dependence. This study may increase our understanding of the effects of drugs and toxins on male infertility.