A novel dominant B-cell epitope of FSHR identified by molecular docking induced specific immune response and suppressed fertility

The follicle stimulating hormone (FSH) is of great importance in reproduction modulation of both sexes. The extracellular domain (ECD) of its receptor (FSHR) is crucial for FSH binding and subsequent signal transduction; therefore, it is the potential target for fertility control. To avoid unwanted side-effect when used as immunocontraceptive agent, the ECD was analysed by online prediction combined with molecular docking to identify the candidate B-cell epitopes. Four potential B-cell epitopes were identified and synthesised in tandem with Pan DR epitope. Then the epitope-based peptides were used to boost adult male mice following rhFSHR protein priming, thus to determine their immune responses and fertility inhibition capacity. Three of the four peptides showed suppressed fertility accompanied with small testis and lower serum testosterone level, which was consistent with absolutely lower sperm quantity and poor quality. Among the four epitope peptides, Pep2 displayed the lowest fertility rate of 26.67%, which was similar to that of rhFSHR homologously prime/boost mice (23.30 and 25.00%). Thus, we identified a novel immunodominant B-cell epitope by molecular docking and protein prime/peptide boost strategy.     

Active immunization with follicle-stimulating hormone for fertility control: a 4 1/2-year study in male rhesus monkeys

Active immunization of four adult rhesus monkeys with highly purified ovine follicle-stimulating hormone (FSH) over 4 1/2 years resulted in the production of specific FSH antibodies. While luteinizing hormone and testosterone secretion were not affected, sperm counts were in most instances reduced to or below the lower normal range. On a few occasions, azoospermia or high sperm counts were observed. Although the antibodies produced neutralized the biologic activity of FSH throughout, spermatogenesis gradually returned, as evidenced by testicular histologic characteristics. However, the diameter of the seminiferous tubules remained decreased, and the germinal epithelium was depleted. No adverse side effects could be demonstrated in the immunized animals, e.g., immune complexes in either serum or tissues and resultant tissue damage. These results show that although active immunization with FSH may not result in an effective method of male fertility control, long-term immunization against a circulating hormone may not result in deleterious side effects.     

Chronic suppression of testicular function by constant infusion of gonadotropin-releasing hormone agonist and testosterone supplementation in the bonnet monkey (Macaca radiata).

OBJECTIVE: To study the efficacy of long-term buserelin acetate infusion to desensitize pituitary and block testicular function in adult male monkeys (Macaca radiata). ANIMALS: Proven fertile male monkeys exhibiting normal testicular function. PROTOCOL: Each of the control (n = 5) and experimental monkeys (n = 10) received a fresh miniosmotic pump every 21 days, whereas pumps in controls delivered vehicle of experimentals released 50 micrograms buserelin acetate every 24 hours. On day 170 (renewed every 60 days) a silastic capsule containing crystalline testosterone (T) was implanted in the experimental monkeys. At the end of 3 years, treatment was stopped, and recovery of testicular function and fertility monitored. RESULTS: (1) Treatment resulted in marked reduction of nocturnal but not basal serum T; (2) the pituitary remained desensitized to buserelin acetate throughout the 3-year period; (3) animals were largely azoospermic with occasional oligospermia exhibited by two monkeys; and (4) withdrawal of treatment restored testicular function, with 70% of animals regaining fertility. CONCLUSION: Long-term infertility (but restorable) can be induced in male monkeys by constant infusion of buserelin acetate and T.     

Oral immunization with sperm antigens: possible therapy for sperm antibodies?

Young adult male CD-1 mice were given intraperitoneal injections (IP) of saline (controls) and pooled sperm or seminal plasma of two autoimmune infertile men and two nonautoimmune fertile men (n = 40 per treatment). Other mice received only an oral challenge with the same antigens (oral controls; n = 20 per treatment). Three weeks after the booster challenge (day 36), 20 mice in each group were orally immunized with the antigens, whereas the other 20 were not (IP controls). Cytotoxic antibody titers (immunoglobulin M) to human sperm were significantly higher in mice IP immunized with sperm or seminal plasma from autoimmune infertile men or orally immunized with autoimmune men's sperm, in contrast to the controls. Oral challenge with sperm or seminal plasma of autoimmune infertile men after the IP immunization with the same resulted in significantly decreased cytotoxic sperm antibody titers (P less than 0.001 versus oral or IP controls in sperm immunization; P less than 0.001 versus IP controls in seminal plasma immunization). Fertility was unaffected by any mode of immunization. It is concluded that, in mice, sperm and seminal plasma antigens from autoimmune infertile men are more immunogenic than those from nonautoimmune fertile men, and oral challenge with the former after an IP establishment of cytotoxic sperm immunity desensitizes the immune mice. These findings may have practical implications in the diagnosis and immunotherapy of infertile men with cytotoxic sperm antibodies.     

Variants in follicle-stimulating hormone receptor gene in infertile brazilian men and the correlation to FSH serum levels and sperm count

The aim of the study was to analyze the distribution of the follicle-stimulating hormone (FSH) receptor (FSHR) Ala307Thr and Asn680Ser polymorphisms in infertile Brazilian men and evaluate the possible role of these polymorphisms on the serum levels of FSH and in sperm count. A case-control study was performed comprising138 infertile men with nonobstructive azoospermia (n = 53) or severe oligozoospermia (n = 85), and 217 fertile men as controls. Genotyping of FSHR polymorphisms was performed by real-time polymerase chain reaction (PCR). The results were analyzed statistically and a P value <.05 was considered significant. According to the sperm count, relatively similar FSHR polymorphisms genotype and allele frequencies were found among the groups, and combined genotypes of 2 polymorphisms did not identify a haplotype associated with sperm count. Considering FSH serum level according to genotypes of the Ala307Thr and Asn680Ser polymorphisms individually, statistical analysis showed no difference among the groups. When the combined genotypes of the FSHR polymorphisms were compared to FSH serum levels, no difference was also found among the groups. In conclusion, the findings demonstrate that, in Brazilian population studied, genetic variations, Asn680Ser and Thr307Ala, of the FSHR gene are not correlated with serum FSH levels or sperm count in male infertility.     

Identification of the region 285-309 of follicle stimulating hormone receptor as a bioneutralizing epitope

Structure-function relationship studies of the follicle stimulating hormone and its receptor assume importance as this hormone is essential for folliculogenesis and spermatogenesis in females and males, respectively. The interaction between the hormone and the receptor is complex and not well understood. In vitro studies using synthetic peptides from the extracellular domain of the receptor and corresponding antipeptide antibodies have suggested that the 285-309 region is surface-oriented. Antipeptide antibodies to this region also inhibit hormone-receptor interaction in a dose-dependent manner and the mechanism of inhibition appears to be competitive in nature. To test this hypothesis in an animal model, antibodies to peptide 285-309 from rat follicle stimulating hormone receptor (FSHR) were developed and characterized. These antibodies were able to detect FSHR in rat ovaries by immunohistochemistry. Further, these antibodies were administered into adult female rats and their effect on fertility status was monitored. These antibodies were found to neutralize the biological activity of endogenous receptor, which resulted in the induction of infertility in the treated animals. Thus, bioneutralization of FSHR has been achieved by targeting its region 285-309 in an in vivo system.     

卵泡刺激素受体基因的点突变和单核苷酸多态性

卵泡刺激素受体(FSHR)是由FSHR基因编码的G蛋白耦联受体蛋白,由胞外区、跨膜区及胞内区3部分构成。胞外区与FSH特异性结合组成FSH/FSHR系统,在人类生殖过程中发挥着重要作用。FSHR基因上突变基因分为活性突变和失活突变2种,失活突变可能导致原发或继发性闭经、高促性腺激素性功能障碍、卵巢早衰及生精功能障碍等生殖疾病,活性突变主要与卵巢过度刺激综合征(OHSS)关系密切。FSHR基因的点突变出现概率非常小,大部分仅有1次报道,而大多数FSHR基因突变为单核苷酸多态性。卵巢和睾丸的正常发育及发挥功能均依赖于完整的FSHR介导,FSHR突变对两性生殖表型的影响存在着差异。     

FSH receptor gene polymorphisms in fertile and infertile Italian men

In women, single nucleotide polymorphisms (SNP) of the FSH receptor (FSHR) gene influence FSH concentrations and the sensitivity of the FSHR to FSH in vivo. In contrast, the significance of FSHR R gene SNP in the male is poorly understood. To this aim, the possible role of three FSHR SNP was evaluated in male infertility. SNP in exon 10 (codon 307 and 680) and in the core promoter region (at position -29) of the FSHR gene were analysed by polymerase chain reaction-restriction fragment length polymorphism technique in 150 men representative of the general population, 107 proven fathers, 92 normozoospermic controls, and 215 infertile patients classified according to sperm parameters (38 azoospermia, 53 severe oligozoospermia, 48 moderate oligozoospermia, and 76 slight oligozoospermia). Reproductive hormones were measured in infertile males and normozoospermic controls. No significant difference was found in allelic variants frequency and genotype distribution between each category of subjects when analysing the FSHR exon 10 SNP alone and in combination with the SNP at position -29. Serum FSH concentrations and other andrological parameters did not differ between subjects with different genotype within each group. The data showed that in the Italian population, FSHR genotypes have no influence on FSH concentrations both in normal and infertile males and do not associate with spermatogenetic impairment.     

Presence of immunoreactive beta-endorphin and calcitonin in human seminal plasma, and their relation to sperm physiology

Opioid peptides and calcitonin are found in high concentrations in the male reproductive tract. To further elucidate their role in sperm physiology, we studied semen samples from 49 infertile men and 25 men with proven fertility. beta-endorphin and calcitonin were measured in each sample by radioimmunoassay and then were correlated with seminal plasma testosterone (T) and dihydrotestosterone levels as well as sperm count, total motile sperm/milliliter, and percentage of penetrated hamster eggs. The levels of beta-endorphin (308 +/- 22 pg/ml) and calcitonin (331 +/- 32 pg/ml) in seminal plasma were 10 and 20 times higher than levels found in venous plasma (32 +/- 2 and 14.5 +/- 1.2 pg/ml, respectively) (P less than 0.001). There was no difference between the levels of beta-endorphin and calcitonin in seminal plasma of fertile and infertile men. However, seminal plasma T was significantly higher in fertile than infertile men (19.4 +/- 2 versus 11.5 +/- 1 ng/dl; P less than 0.05). No correlation could be demonstrated between either beta-endorphin or calcitonin and any of the parameters studied. In conclusion, beta-endorphin and calcitonin are produced locally in the male reproductive tract; however, their role in male reproduction remains to be elucidated.     

Establishment of the need for oestrogen during implantation in non-human primates

Deprivation of oestrogen during post-ovulatory mated cycles in proven fertile female bonnet monkeys by tamoxifen, aromatase inhibitor or oestrogen antiserum resulted in inhibition of pregnancy establishment in all three groups of animals. However, more than 85% of the animals became pregnant within three exposures to proven fertile males in the control group. These results suggest the requirement for oestrogen in pregnancy establishment in primates. Based on this conclusion, it is suggested that use of a suitable and potent anti-oestrogenic compound can be exploited as an alternative approach to contraception.     

DNA strand breaks in human spermatozoa: a possible factor, to be considered in couples suffering from unexplained infertility.

BACKGROUND: The aim of this study was to assess the presence of DNA strand breaks and sperm cell morphology in men suffering from unexplained infertility, and to compare these results with normal fertile and oligozoospermic men. METHODS: One fresh sperm sample from proven fertile sperm donors (n=20) and from infertile men with oligozoospermia, (<20 x 10(6)/ml sperm cells) (n=74), and men suffering from unexplained infertility (>20 x 10(6)/ml sperm cells) (n=39) who delivered two sperm samples with 24 hours interval, were tested for the presence of DNA strand breaks in the spermatozoa by direct immunoperoxidase detection of digoxigenin-labeled genomic DNA. Correlations to other sperm parameters, sperm cell counts, motility, activation and Krüger's strict criteria were performed. RESULTS: DNA strand breaks in sperm cell nuclei were found significantly more often in sperm samples from men suffering from unexplained infertility compared to those from normal fertile men, and significantly more rarely compared with sperm samples from men with oligozoospermia. The percentages of normal spermatozoa (Krüger's strict criteria) were significantly lower in samples from men suffering from unexplained infertility compared to those of normal fertile men, but significantly higher compared to those of men with oligozoospermia. No difference was found between first and second day samples used for insemination, as regards DNA strand breaks, sperm cell morphology, total number of motile sperm cells, activation and motility degree. CONCLUSION: The present data suggest that a subgroup of men suffering from unexplained infertility have DNA strand breaks in their sperm cell DNA. This group might suffer from the same malfunction as many men with oligozoospermia, however, their apoptotic activated sites in the testis are different. Delivery of sperm samples with 24 hours interval does not affect any sperm cell counts, CASA, DNA strand breaks or morphology findings in sperm samples from men suffering from unexplained infertility.     

Comparison of gonadal function between fertile and infertile men with varicoceles

The high prevalence of men with varicoceles who by history are fertile has led some to question the suggested causal relationship between a varicocele and male infertility. However, testicular function in these fertile men has not previously been studied in detail. Fifty-five normal fertile men, 42 fertile men with varicoceles, and 24 infertile men with varicoceles and normal female partners were studied. Semen analyses were done, baseline serum testosterone and gonadotropin levels tested and the gonadotropin response to luteinizing hormone-releasing hormone (LH-RH) measured. The infertile men with varicoceles exhibited lower sperm counts, abnormal sperm morphologic features, increased baseline serum gonadotropins, and increased gonadotropin responses to LH-RH, compared with the normal fertile men. The fertile men with varicoceles showed similar abnormalities, although this was not statistically significant in all cases. That semen and hormone abnormalities were observed in both the fertile and infertile men with varicoceles suggests that the presence of a varicocele is associated with some degree of primary testicular dysfunction, regardless of present fertility status.     

A novel mutation in exon8 of the follicle-stimulating hormone receptor in a woman with primary amenorrhea

The FSH receptor (FSHR) gene mutation are rare, but recently have been detected in several cases with primary amenorrhea. We report a 25-year-old female who had primary amenorrhea, moderately developed secondary sex characteristics and normal sized ovaries with small antral follicles. Her ovaries reacted slightly to clomiphene citrate therapy; they did not react to the ordinary dose of human menopausal gonadotropin (hMG) (150 IU/day × 9 days), but did react to high-dose hMG (300 IU/day × 6 days) treatment. These ovarian responses to hMG stimulation suggested an FSHR dysfunction of her ovaries. We extracted genomic DNA and analysed FSHR gene sequence after we obtained the written informed consent from the patient under the approval by the Ethics Committee of Yamaguchi Grand Medical Centre and the Yamaguchi University School of Medicine. Direct sequencing revealed a heterozygous mutation 662T?>G resulting in the substitution of valine for glycine at position 221 in exon8 of the FSHR extracellular domain, which was also confirmed by the PCR-RFLP method. The sequencing results also showed two SNPs, 919G?>A (Ala307Thr) and 2039G?>A (Ser680Asn), in exon10. A novel mutation in exon8 of FSHR was identified in a woman with primary amenorrhea whose ovaries reacted to high-dose hMG treatment.     

DNA strand breaks in human sperm cells: a comparison between men with normal and oligozoospermic sperm samples

BACKGROUND: The aim of this report was to compare the degree of DNA strand breaks in known normal fertile men to those with oligozoospermia, and evaluate the presence of DNA strand breaks in normal raw sperm, after Percoll and swim-up and following conventional cryopreservation, as all these preparation methods might differ in selection of healthy sperm cells. METHODS: Sperm samples from proven fertile sperm donors (n=20) and infertile men with oligozoospermia (n=33) were tested for the presence of DNA strand breaks in the spermatozoa, by direct immunoperoxidase detection of digoxigenin-labeled genomic DNA. A correlation to other sperm parameters, sperm counts, motility and Krüger's strict criteria was performed. RESULTS: DNA strand breaks were found significantly more often in sperm samples from men with oligozoospermia compared to sperm samples of normal fertile men. The degree of spermatozoa with DNA strand breaks was correlated proportional with the degree of morphological pathological sperm cells judged by Krüger's strict criteria. The percentage of spermatozoa with DNA strand breaks in the samples was not influenced by procedures such as the swim-up technique, Percoll gradients or cryopreservation and thawing. CONCLUSION: DNA strand breaks were found significantly more often in men with oligozoospermia compared to normospermic men. The DNA strand breaks might play an important role for the maturation process of the spermatozoa in the same way as apoptosis is controlling the number of early meiotic germ cells in the testis, and hereby play an important role in advanced fertility treatments (ICSI).     

不育症患者精子X,Y及18染色体的荧光原位杂交分析

目的:分析不育症患者精子X,Y,18染色体的荧光原位杂交情况。方法:在男性不育症组中,2例无精子症患者从附睾抽吸获取精子、3例从睾丸获取精子、2例严重少精症患者从射出精液中找到精子。选择5例正常射出精液者作为对照组。应用荧光原位杂交法(FISH)检查精子X,Y以及18号染色体,比较两组精子染色体非整倍体的发生率。结果:不育症组睾丸精子、附睾精子的非整倍体率无差别,但不育症组精子与正常男性组精子比较,非整倍体总发生率、性染色体二体性率及缺对染色体率明显增高(2.8％vs0.58％,0.81％vs 0.19％,2.1％vs0.37％),P<0.001。结论:无精子症与严重少精子症患者的精子比正常精子具有更高的染色体非整倍体率,需要进行大样本的研究,为不育症患者的治疗和遗传咨询提供有效的证据。     

A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence

A new and practical test for evaluating the fertility of a male subject has been developed. Twenty-eight donors whose semen had induced at least one pregnancy resulting in a normal delivery and 61 patients attending our infertility clinic were studied. Semen smears stained with acridine orange were read on a fluorescence microscope; sperm heads appeared either green (fertile) or red ( nonfertile ). We introduce the concept of an "effective sperm count" which is obtained by multiplying the percentage of green-fluorescing sperm by the actual sperm count. Of our fertile subjects, 27 of 28 (96.4%) exhibited an effective sperm count of greater than or equal to 50 million/ml, while 60 of 61 (98.3%) infertile patients fell below this value. The percent green correlates with neither actual sperm count nor motility, indicating that this test measures a new parameter of male fertility.     

Effect of intranasal administration of norethisterone and progesterone on pituitary and gonadal function in adult male and female bonnet monkeys (Macaca radiata)

The effect of intranasal spray of norethisterone (NET) and progesterone (P) on serum testosterone (T) and total sperm count in adult male bonnet monkeys and the effect of NET spray on the menstrual cycle in female monkeys has been studied. Whereas NET spray resulted in a drastic decrease in serum T levels and sperm count, P spray caused a decrease in total sperm count only. Intranasal spray of NET in adult cycling female monkeys between days 5 and 14 resulted in shortening of the menstrual cycle; this could be ascribed to a decrease in serum follicle-stimulating hormone, luteinizing hormone, 17 beta-estradiol, and P levels. Nasal spray of only solvent in male or female monkeys, however, had no effect on any of the parameters tested.     

Analysis of aneuploidy in mini-percoll gradient centrifuged human sperm for intracytoplasmic sperm injection (ICSI) using fluorescence in situ hybridization

AIM: To study the aneuploidy rates of chromosomes 13, 18, 21, X and Y in Percoll gradient centrifuged sperm from infertile patients with male infertility factor treated by intracytoplasmic sperm injection (CSI) compared with healthy fertile donors and infertile patients with normal semen parameters. METHODS: This case-controlled study was conducted in a university hospital. Semen samples were obtained from three healthy fertile donors, eight infertile patients with normal semen parameters, and 18 infertile patients with male infertility factor. All samples were subjected to mini-Percoll gradient centrifugation before being processed through fluorescent in situ hybridization. The incidences of aneuploidy were compared using Chi-squared test. RESULTS AND CONCLUSIONS: A total of 64949 spermatozoa were analyzed. The disomy rates for chromosomes 13, 18, 21, and X or Y of sperm from patients with male infertility factor were 0.21%, 0.37%, 0.36% and 0.63%, respectively, whereas the diploidy rate was 0.17-0.23%. These incidences were higher than those from men with normal semen parameters. The result suggested that the embryos of patients with male infertility factor treated by ICSI are at increased risk of chromosome abnormalities.     

Varicocele is associated with abnormal retention of cytoplasmic droplets by human spermatozoa

OBJECTIVE: To determine whether varicocele is associated with retention of sperm cytoplasmic droplets in infertile men. DESIGN: Retrospective study.Setting: University infertility clinic. PATIENT(S): Nonazoospermic men with idiopathic (n = 69) and varicocele-associated infertility (n = 73), and 20 fertile controls presenting for vasectomy. INTERVENTION(S): None. MAIN OUTCOME MEASURES(S): Standard semen parameters and percentage of spermatozoa with cytoplasmic droplets on Papanicolaou smears. RESULT(S): No statistically significant differences were found between the fertile and infertile groups with respect to semen volume. Fertile controls had significantly greater mean percent sperm motility and normal morphology than infertile men. The mean percentage of sperm with residual cytoplasm was statistically significantly different in all three groups. Infertile men with varicocele had the highest percentage of sperm with cytoplasmic droplets, the next highest level being in men with idiopathic infertility and the lowest level in fertile controls (11.7 +/- 1.0, 8.1 +/- 0.9 and 3.2 +/- 0.4%, respectively, P<.0001). CONCLUSION(S): Our data show that idiopathic and even moreso, varicocele-related male infertility are conditions associated with impaired disposal of residual sperm cytoplasm by the testis and/or epididymis. These data provide a possible mechanism for the observed semen abnormalities and reduced fertility potential associated with varicocele and idiopathic male infertility.     

Immunological consequences of vasectomy and vasovasostomy in rhesus monkeys

The effects of circulating antibodies on fertility after vasovasostomy was studied in rhesus monkeys. After vasectomy there was rapid rise in antibody levels against spermatozoa; these reached a peak after 2 weeks, rather more rapidly than has been reported in man. Antibody levels declined after this period. Monkeys with high antibodies were also more likely to have granulomas and fistulas. After vasovasostomy several monkeys became fertile again with sperm counts in ejaculate reaching a high level at 3 months, although levels were lower than before vasectomy. Some males retained a high and sustained level of sperm immobilizing antibodies in plasma, sometimes for 18 months or more. These males produced fewer spermatozoa and were more likely to be subfertile or infertile after vasectomy. Almost all the monkeys without antibodies were fertile. Presence of antibodies at vasovasostomy was not a definite indication of subsequent infertility but did provide an indication of likelihood of continuing sterility. These are similar to observations of men undergoing vasectomy and vasosostomy.