Benha Veterinarymedicaljournal
Total Page：16
File Type：pdf, Size：1020Kb
BENHA VETERINARY MEDICAL JOURNAL, VOL. 24, NO. 1, JUNE 2013:343-358 BENHA UNIVERSITY BENHA VETERINARY MEDICAL JOURNAL FACULTY OF VETERINARY MEDICINE BIOCHEMICAL EFFECT OF LIPOPROTEIN MODULATORS IN INFERTILE PATIENTS (POLYMORPHISM OF ESTROGEN RECEPTOR BETA) Ahmed A. Sayed a, Afaf A. Desoky b, Hussein A. Abd Elmaksoud b, Mohamed H. Motawea c a Clinical Pathology Dept (Biochemistry), Faculty of Medicine, Al-Azhar University, Egypt. bBiochemistry Dept., Faculty of Vet., Banha University, Egypt. cBiochemistry Dept., Faculty of science, Tanta University, Egypt. A B S T R A C T Male infertility is a multifactorial condition with a strong genetic component. In the last decade a large number of investigations focused on the identification of gene variants affecting spermatogenesis in human. Polymorphisms of the estrogen receptor (ER) genes, have been implicated in male infertility, however, comprehensive data are lacking. We investigated the association between the ER-β gene (ESR2) RsaI and Alul polymorphisms and the idiopathic male infertility in Egyptian males. Allel specific PCR method and restriction fragment length polymorphism (RFLP) were used to detect the ER-β gene polymorphisms in 24 infertile men and 34 age-matched healthy controls. Reproductive hormones were measured and at least two semen analyses were performed in each subject. Compared with the controls, the frequency of the heterozygous Rsa1 AG genotype was almost three times higher in infertile men (16.6 vs. 5.8 %; P = 0.17). The heterozygous Rsa1 AG genotype was associated with an reduction in LH concentration compared with the wild-typ1e Rsa1 GG genotype in both controls and infertile men. Our results further suggest a possible role of ER-β polymorphisms on male infertility. Further studies are needed to replicate our findings as well as to better elucidate the biological mechanisms of the modulation of ER-β on male infertility. Keywords: polymorphism, idiopathic infertility, estrogen receptor beta, genotype, homozygous, heterozygous. (BVMJ-24(1): 343-358, 2013) 1. INTRODUCTION cytochrome P450. Some studies have shown that aromatase deficiencies cause progressive In almost 50% of infertile couples, the infertility in adult mice, resulting in reduced problem is related to the male; the cause has sperm production and motility (11, 12, 48). not been determined in at least 30% of these However, although many studies have cases, and are thus considered as idiopathic demonstrated a strong association between infertility (43). Estrogen biology is ER inactivation and abnormal exceedingly complex and important in the spermatogenesis, the specific role of ER gene development and function of numerous mutations or polymorphisms in male tissues and physiological phenomena (34, 36, infertility is still novel. It has been 41). There is much evidence to suggest that hypothesized that changes in the androgen- estrogen and estrogen receptors (ERs) play estrogen balance in utero could lead to very important roles during male impairment of embryonic programming reproduction (17, 27, 37, 42). In the male including the development of male testes, estrogens are synthesized from reproductive organs during fetal life. The testosterone via the action of aromatase hormonal balance could, for example, be 343 Lipoprotein modulators in infertile patients disturbed by increased exposure to estrogenic 65, 68,). Estrogen signaling in the cell is or antiandrogenic endocrine disruptors (55, mediated by estrogen receptors (ERs), of 58). Any alteration in the genes involved in which at least two subtypes exist, ERα and androgen–estrogen action or metabolism may ERβ. Two silent polymorphisms in ERα have disturb the androgen–estrogen balance. It is been associated with azoospermia or severe well known that estrogenic activity is oligozoospermia (30, 64). Recently several mediated by at least two functional isoforms sequence variants of the ERβ gene have been of the ER, ERα and ERβ (24, 41). Animal described (49) including two silent G⇒ A models revealed that knockout (ERαKO) and polymorphisms, RsaI and AluI. Both double knockout (ERα/βKO) mice are polymorphisms have been overrepresented in infertile from puberty, and presented atrophy ovulatory dysfunctions (63). However, of the testes and seminiferous tubule studies on genetic variants of ERβ with dysmorphogenesis, which could lead to respect to male infertility are still lacking. decreased spermatogenesis and sperm Such information might add to our motility (23). Other studies have identified knowledge regarding the role of estrogens in silent polymorphisms in the human ERα, the physiology and pathophysiology of male which may be associated with male infertility reproductive systems. .xbaI polymorphism (rs9340799) was associated with either azoospermia or 2. PATIENTS AND METHODS idiopathic severe oligospermia in men (30). 2.1. Infertile men: Other ERα mutations may interfere with human spermatogenesis (25, 64). ERα is Twenty-four Egyptian men from infertile known to be strongly expressed in the couples were included in the study. All men epididymis, efferent ductules and Leydig presented with sperm count lower than 2 cells, whereas ERβ is predominantly million in all (at least two) ejaculates, expressed in germ cells, particularly in the delivered for examination. Although the primary spermatocytes and round spermatids fertility of their female partners was not of the human testis (18, 45, 51, 52). These explored. Men with known genetic causes of data suggest that ERβ may also play an infertility. As well as those with a history of important role in male infertility. Noticeably, cryptorchidism were excluded. the male-infertility rate has significantly 2.2. Controls. increased in our reproductive center over the past decade, as well as in other countries (8). Normal controls served a group of thirty four A DNA polymorphism is defined when a Egyptian men without genital abnormalities single nucleotide—A, T, C, or G—in the and with sperm count higher than 20 millions/ genome differs between members of a ml. biological species, these single nucleotide 2.3. Hormone analysis polymorphisms (SNPs( may occurs within the coding sequences of genes ,noncoding Circulating levels of FSH, LH, testosterone, regions of genes ,or in the intergenic regions . and estradiol were measured by the Changes in the regulatory parts of the gene electrochemiluminescence immunoassay could affect the degree of expression of the (ELICIA) which is intended for use on Elcsys gene ,and thus the levels of the protein )29). and cobas immunoassay analyzer. Polymorphic variants of both ERα and ERβ 2.4. Allele-specific PCR genes have been identified in recent years, In all subjects, allele-specific PCR and studied for possible association with was performed to detect the RsaI and AluI reproductive outcomes (15, 20, 25, 33, 50, 61, 344 Sayed et al. (2013) variants of ERβ. For each polymorphism two AluI (nucleotide numbering according to reactions per subject were run, using a GenBank accession no. AB006590). specific primer for either the polymorphic A In both positions a G nucleotide was variant or for the wild-type G variant, considered the wild-type sequence and was together with an upstream and a downstream not digestible by RsaI or AluI. RsaI digestion primer. PCR conditions were established to produced one uncleaved band of 409 bp in generate both a control fragment and a subjects with the homozygous wild-type GG shorter, allele-specific band in the presence of genotype, two bands of 110 and 299 bp in the variant and only the control fragment in homozygous polymorphic AA subjects, and its absence. all three bands in heterozygous AG carriers. Allele-specific PCR of the RsaI AluI digestion yielded one band of 405 bp in polymorphism was performed in a total the uncleaved homozygous wild-type GG volume of 25 μl containing 25 ng genomic polymorphism, two bands of 163 and 242 bp DNA, 45 mmol/liter KCl, 10 mmol/liter Tris in the homozygous polymorphic AA HCl (pH 9.1), 0.1% Tween 20, 0.2 mmol/liter polymorphism, and all three bands in deoxynucleotide triphosphate, 1.5 mmol/liter heterozygous AG subjects. We did not MgCl2, 1 U Dynazyme Taq polymerase compare the hormone levels between the two (Qiagen, USA), and 0.5 μmol/liter of each of groups but instead compared the hormone the primers RsaI forward (Fw), RsaI reverse concentrations between the genotypes within (Rev), and either RsaI RevA or RsaI RevG. each group. Primer sequences are presented in Table 1. 2.6. Semen analysis Amplification was performed for 35 cycles; each cycle including denaturation for 1 min at The ejaculate was obtained by masturbation 96 C, primer annealing for 30 sec at 58 C, and after a minimum 48 h of sexual abstinence. primer extension for 3 min at 72 C, with an Semen analysis includes physical initial denaturation step for 3 min at 96 C, and examination (color, PH and viscosity) and a final extension step for 7 min at 72 C. For microscopic examination (sperm count, the AluI polymorphism, an annealing motility and morphology). temperature of 54 C for 30 sec was used. 2.7. Statistical analysis Other conditions were the same as for the RsaI reaction. The sequences of the primers The distributions of ERβ polymorphisms are presented in Table 1. were compared between the patient groups and controls using Fisher’s exact test. Median 2.5. Analysis of restriction fragment length and mean for hormone parameter in control polymorphisms and infertile men and also for hormone levels Both the RsaI and AluI polymorphisms are in the different genotypes, in two groups, restriction fragment length polymorphisms, separately, were compared by Mann-Whitney and digestion with the respective restriction U test applying the SPSS statistical software enzymes was performed according to the (version 11). P < 0.05 was considered manufacturer (real biogene , Canada) to statistically significant. verify the results from the allele-specific PCR. In the RsaI polymorphism, a G to A 3. RESULTS nucleotide exchange at nucleotide 1082 in exon 5 created a recognition site for RsaI, and The RsaI AA genotype was not found in in the AluI polymorphism an exchange of G control or infertile men subject.