Nucleoside boranephosphonates as potential therapeutic agents / Nukleozydo boranofosfoniany jako potencjalne srodki terapeutyczne.

Nucleoside boranephosphonates are nucleotide analogues in which one of the non-bridging oxygen atom of the phosphate part has been replaced by a borane group (-BH3). This modification imparts a wide spectrum of biological activity, e.g., activation of ribonuclease H, resistance to endo- and exonucleases, and their respective triphosphates are good substrates for DNA and RNA polymerases. Nucleoside boranephosphonate derivatives are used in antisense therapy, silencing gene expression using siRNA strategies, and as potential antiviral and anti-cancer prodrugs. Boranephosphonates find also applications as aptamers and as substrates in a new method of DNA sequencing. This review briefly presents potential biological applications of nucleoside boranephosphonates.

Sperm DNA Fragmentation Index and Hyaluronan Binding Ability in Men from Infertile Couples and Men with Testicular Germ Cell Tumor.

Objective. To investigate sperm DNA fragmentation and sperm functional maturity in men from infertile couples (IC) and men with testicular germ cell tumor (TGCT). Materials and Methods. Semen samples were collected from 312 IC men and 23 men with TGCT before unilateral orchiectomy and oncological treatment. The sperm chromatin dispersion test was performed to determine DNA fragmentation index (DFI) and the ability of sperm to bind with hyaluronan (HA) was assessed. Results. In comparison with the IC men, the men with TGCT had a higher percentage of sperm with fragmented DNA (median 28% versus 21%; p < 0.01) and a lower percentage of HA-bound sperm (24% versus 66%; p < 0.001). Normal results of both analyses were observed in 24% of IC men and 4% of men with TGCT. Negative Spearman's correlations were found between DFI and the percentage of HA-bound sperm in the whole group and in IC subjects and those with TGCT analyzed separately. Conclusions. Approximately 76% of IC men and 96% with TGCT awaiting orchiectomy demonstrated DNA fragmentation and/or sperm immaturity. We therefore recommend sperm banking after unilateral orchiectomy, but before irradiation and chemotherapy; the use of such a deposit appears to be a better strategy to obtain functionally efficient sperms.

The risk of neoplasm associated with dysgenetic testes in prepubertal and pubertal/adult patients.

INTRODUCTION: In patients with Y-chromosome in the karyotype, partial gonadal dysgenesis and disorders of male reproductive sex organs development are usually resected in childhood because of the high risk of germ cell tumours (GCT). In patients with Y-chromosome, complete gonadal dysgenesis and female genitalia gonadectomy is performed markedly later. However, due to the relatively low number of adult patients with preserved dysgenetic gonads, the true risk of neoplasm is unknown. The aim of the study was to evaluate the prevalence of neoplasia in dysgenetic gonads of children and adults with Y-chromosome in a retrospective study. MATERIAL AND METHODS: A review of medical documentation of 94 patients with disorders of sex development (DSD), Y-chromosome and gonadal dysgenesis (GD), aged 1.2-32 years (47 prepubertal, 1.2-10 years; 47 pubertal/adult, 13-32 years), was conducted. Serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone were determined. Bilateral gonadectomy was performed in 73.4% of patients, and unilateral gonadectomy with biopsy of the contralateral gonad in 26.4%. All gonadal tissues were subjected to immunohistochemical evaluation with antibodies against PLAP and OCT3/4 (markers of malignant germ cells, but also foetal multipotent germ cells), while gonads of prepubertal patients were examined by c-KIT, as well. RESULTS: Streak gonads were identified on both sides (complete GD) in 30.8%, a streak gonad on one side and an underdeveloped testis on the other (asymmetric GD) in 38.3%, and underdeveloped testicular structure on both sides (partial GD) in 30.8% of cases. Germ cell neoplasia was found in 53.2% of patients (51.1% in children, 55.3% in pubertal/adults). Invasive GCT were identified in 11.7% of cases, of which 90.9% were in pubertal/adult patients. Other neoplastic lesions included gonadoblastoma (16% prevalence) and testicular carcinoma in situ (25.5%). In younger patients FSH serum levels were increased in 81% of cases (mean 2.82 ± 2.18 IU/L), while LH in 58% (mean 1.82 ± 1.69 IU/L). Hypergonadotropic hypogonadism was diagnosed in most of the pubertal/ /adult patients (mean FSH 54.2 ± 23.3 IU/L, mean LH 21.7 ± 12.1 IU/L, mean testosterone 5.5 ± 4.5 nmol/L). CONCLUSIONS: Dysgenetic gonads in patients with Y chromosome have a high risk of germ cell neoplasia (ca. 50%). If they are preserved until puberty/early adulthood, they may develop overt, invasive GCT. The gonads also have poor hormonal activity (hypergonadotropic hypogonadism) in most of the pubertal/adult patients. Each of these cases must be considered individually and a decision to remove the gonad or not should be based on the comprehensive analysis of the phenotype by a multidisciplinary team of specialists in consultation with the patient and the parents. If dysgenetic gonads are not resected in childhood, these patients need careful ongoing follow-up examination, including biopsy and histopathological evaluation.

The long-term effects of FSH and triiodothyronine administration during the pubertal period on Connexin 43 expression and spermatogenesis efficiency in adult rats.

Follicle-stimulating hormone (FSH) and triiodothyronine (T3) are known regulatory factors of spermatogenesis initiation. Hyperstimulation of both hormones evokes regressional changes in connexin 43 expression and the seminiferous epithelium in young rats during testicular maturation. However, separate treatments with T3 reduce Sertoli cell number, which seems to be closely connected with the maturation of connexin 43 gap junctions. FSH elevates Sertoli cell number and function, but this effect may take place regardless of the presence of connexin 43-dependent intercellular communication. The aim of the study was to evaluate the later effects of such treatments. Newborn, male Wistar rats were divided randomly into experimental groups receiving daily subcutaneous injections of either 7.5 IU/animal FSH, or 100 mg/kg b.w. T3, or both substances or the same volume of vehicle (control group) until day 15 of life. The animals were sacrificed on day 50. Morphometric analysis and immunohistochemical reactions were performed using antibodies against Vimentin, Proliferating Cell Nuclear Antigen and Connexin 43 in the testis. Sertoli cell count, efficiency of spermatogenesis, and hormonal pattern were examined. Disturbances in the connexin 43 expression reduced the number of Sertoli cells, the efficiency of spermatogenesis and impaired endocrine function of testes in adult rats treated with FSH and T3 during puberty. Stimulation with FSH alone increased Sertoli cell number, but was associated with a negative effect on cell-to-cell connexin 43-dependent communication, with a consequential reduction of spermatogenesis efficiency. J. Exp. Zool. 323A: 256-265, 2015. © 2015 Wiley Periodicals, Inc.

Free and bioavailable fractions of sex steroids may influence bones in young men, depending on age and oestradiol level.

INTRODUCTION: Longitudinal bone growth ceases by the end of puberty, and it is thought to be a result, in both sexes, of increased pubertal oestrogen serum concentrations. Since peak bone mass is achieved by the third decade of life or later, the aim of this study was to relate sex steroid hormones and sex hormone binding globulin (SHBG) levels to bone quality in men during their third and fourth decades of life. MATERIAL AND METHODS: Eighty men, healthy volunteers aged between 18 and 39 years, were subjected to an interviewer-administered questionnaire, body mass index (BMI) measurement, blood sample and calcaneal quantitative ultrasound (QUS) (Hologic-SAHARA). Blood was assessed for testosterone (T), oestradiol (E2), dehydroepiandrosterone sulfate (DHEAS), SHBG, luteinising hormone (LH) and follicle stimulating hormone (FSH). Free and bioavailable T and E2 levels were calculated knowing SHBG and albumin levels. RESULTS: While T, E2, DHEAS, LH and FSH levels were not related, free and bioavailable fractions of T and E2 were positively associated with QUS readings. SHBG level was associated negatively. After dichotomisation for age, the associations remained significant only for younger subjects (18-30 years, n = 47). After adjustment for other co-variants, only SHBG in younger subjects retained its negative association with QUS. Older subjects (31-39 years, n = 33) revealed higher BMI and lower serum concentrations of total (-17 %), free (-18.5%) and bioavailable (-22.5%) levels of E2 than younger subjects. CONCLUSION: Free and bioavailable fractions of sex steroids may influence bones in young men, depending on age and E2 level.

Relationship between sexual function, body mass index and levels of sex steroid hormones in young men.

INTRODUCTION: In older men, sexual disorders may be the result of a decrease in testosterone and an increase in sex hormone binding globulin (SHBG) serum levels. Although obesity may enhance the decline of testosterone, it is also the cause of metabolic disorders, which are additional risk factors of erectile dysfunction. The purpose of this study was to investigate whether elevated body weight is associated with decreased serum testosterone concentrations and reduced sexual function in young men. MATERIAL AND METHODS: Data on general health, medication, depressive symptomatology and sexual life was obtained from 136 men aged 20-49 years. Blood levels of LH, total testosterone (TT), dehydroepiandrosterone sulfate (DHEA-S), oestradiol, SHBG, total cholesterol, LDL- and HDL-cholesterol, and triglycerides were determined. Body mass index (BMI), waist to hip ratio (WHR) and free testosterone index (FTI) were calculated. RESULTS: A significantly reduced occurrence of sexual fantasies, morning erections and erectile function scores was observed in the oldest group compared to the youngest men with normal BMI, although orgasmic function was unchanged. A significant decrease in TT serum levels was observed in obese 30-year-olds compared to men with normal BMI, while in obese 40-year-olds decreased LH and SHBG levels were also found. No differences in the levels of lipids and sexual achievements were found among men with different BMI. However, erectile function and morning erections significantly negatively correlated with age, BMI and WHR, and positively with FTI, but not with other studied hormones and lipids. CONCLUSIONS: In young men, obesity can lead to a deterioration of erectile function as a result of lower testosterone levels as the only reason.

Estradiol and testosterone inhibit rat seminiferous tubule development in a hormone-specific way.

Follicle stimulating hormone (FSH), testosterone (T) and estradiol (E2) are known to regulate testis maturation, and changes in FSH secretion induced by sex steroid treatment may mediate the effects of sex hormones. The aim of this study was to compare the effects of T and E2 on the pre-meiotic steps of first spermatogenesis and FSH level in rats. Male rat pups were injected daily with 17ß-estradiol benzoate (EB; 12.5 µg) or testosterone propionate (TP; 2.5mg) with the use of one of the two administration modes: 1/transient mode; hormone injections on postnatal days (PND) 1-5 followed by daily vehicle injections until PND 15 (t-EB and t-TP, respectively) or 2/continuous mode; hormone injections on PND 1-15 (c-EB and c-TP, respectively). The control group was injected with vehicle alone. On PND 16, blood was taken for serum hormone measurement and testes were collected for analysis of seminiferous tubule morphometry as well as cell number, proliferation and apoptosis. Testis weight, tubule length, Sertoli and germ cell numbers were reduced, and cell apoptosis in seminiferous epithelium was increased after transient EB and TP treatments. Despite normal or increased FSH secretion, the c-EB treatment inhibited pre-meiotic germ cell development and augmented cell apoptosis, whereas the c-TP treatment reduced the spermatocyte number and inhibited the formation of seminiferous tubule lumen. In conclusion, transient administration of EB or TP during PND 1-5 inhibited testis growth, whereas continuous administration (PND 1-15) impaired pre-meiotic germ cell development in a hormone-specific way.

Semen analysis standardization: is there any problem in Polish laboratories?

The aim of the study was to determine the degree of compliance of Polish laboratories with World Health Organization (WHO) recommendations, with regard to semen analysis methodology. A survey requesting information about methods of semen analysis was distributed to employees of 55 laboratories. Respondents who had participated in external seminological workshops (31%) were termed certified respondents (CR), the remaining (69%)-non-certified respondents (NCR). Only one laboratory (6%) in the CR group and none in the NCR were compliant with WHO guidelines for methods and equipment used to evaluate seminal volume, sperm motility, concentration, vitality and morphology. Most problems were of volume measurement (weighing method was reported by 17% of CR and 10% of NCR) and staining method for sperm morphology (Papanicolau or Diff-Quik were found in 33% of CR and 23% of NCR). A three- or four-point grading of sperm motility was used by the majority of respondents; however, 17% of CR and 37% of NCR did not use a laboratory counter to tally spermatozoa. Although a haemocytometer method was used by 80% of laboratories in each group, the improved Neubauer chamber was used only by 42% of CR and 19% of NCR. In each group, 24% of laboratories did not perform a vitality test. Procedural errors and the interchangeable utilization of two or even three methods to analyse a given parameter was observed in both groups. The results indicate a need for standardisation of the methods and continuous, unified training in semen analysis in Polish laboratories.

Maturational changes in connexin 43 expression in the seminiferous tubules may depend on thyroid hormone action.

INTRODUCTION: Connexin 43 (Cx43) mediates the effect of thyroid hormone on Sertoli cell maturation in vitro. We investigated the influence of triiodothyronine (T3) administration on Cx43 expression in relation to the progress in seminiferous tubule maturation. MATERIAL AND METHODS: Male rats were daily injected with 100 µg T3/kg body weight from birth until postnatal day (pnd) 5 (transient treatment - tT3) or until pnd 15 (continuous treatment - cT3) or solvent - control (C). On pnd 16 serum hormone levels, body and testes weight, seminiferous tubule morphometry, Cx43 immunostaining and germ cell degeneration were investigated. Cx43 expression was also assessed in six 50-day-old adult untreated rats. RESULT: tT3 increased 2.6-fold serum level of T3, testes weight, and seminiferous tubule diameter, and induced maturation-like dislocation of Cx43 expression from the apical to the peripheral region of Sertoli cell cytoplasm. In addition, incidence of Cx43-positive tubules declined from 86% in C to 46% after tT3, being similar to the adult value (30% of tubules Cx43-positive). In turn, cT3 increased serum T3 level 12-fold, and decreased body weight. Seminiferous tubules became shortened and distended, Sertoli cell cytoplasm vacuolated, Cx43 expression had minimal intensity and germ cell degeneration increased. CONCLUSIONS: Cx43 might intermediate a short and transient stimulatory effect of T3 on seminiferous tubule maturation that disappeared together with exposure to the toxic effect of a continuously high level of the hormone.

Estrogen receptor alpha localization in the testes of men with normal spermatogenesis.

It is known that estrogens act on the male reproductive tract by binding to estrogen receptors (ER) α and ß. However, studies on ER localization in the human testis are discordant. The aim of this study was to investigate the localization of ERα in the testes of adult men with normal spermatogenesis. Semen analysis of ten adult men revealed azoospermia. FSH, LH and testosterone serum concentrations were within normal values, and the volume of the testes was normal, hence obstructive azoospermia was suspected. The tissues from testicular surgical biopsies were fixed in Bouin's fluid and embedded in paraffin. Assessments of the seminiferous epithelium (scoring 10 to -1), the number of Leydig cells (scoring 1 to 5), the areal fraction of intertubular space (IS), measurements of seminiferous tubule diameter, and the thickness of the tubular wall, were performed on microscopic sections. Immunohistochemical staining was applied with monoclonal antibodies against ERα. The mean spermatogenesis score was 10 points; IS - 30.6 ± 8.1%; seminiferous tubule diameter - 193.9 ± 19.4 µm; thickness of tubular wall - 7.44 ± 1.1 µm; number of Leydig cells - 1.6 ± 1.1 points. Immunohistochemical staining showed the localization of ERα to be in the Sertoli and Leydig cell cytoplasm, while ERα was absent in germ cells. The results of testicular tissue analysis confirmed its normal structure and normal, full spermatogenesis. The presence of ERα in Sertoli and Leydig cells in normal human testis demonstrated in this study suggests that estrogens may affect testicular function.

Role of FSH and triiodothyronine in Sertoli cell development expressed by formation of connexin 43-based gap junctions.

Follicle-stimulating hormone (FSH) and triiodothyronine (T3) are known regulatory factors of spermatogenesis initiation. Connexin 43 (Cx43) is the most ubiquitous constitutive protein of gap junctions in the testis. This study evaluates the effects of the hyperstimulation of FSH and T3 during testicular maturation on Cx43 expression in the testis. The newborn, male Wistar rats were divided randomly into four experimental groups: FSH group-daily injections of FSH 7.5 IU/animal; T3 group-100 µg T3/kg body weight; FSH+T3 group-both substances; A control group-received vehicles in the same volume. Proliferating cell nuclear antigen immunohistochemistry and toluidine blue staining were used to determine the germ cell proliferation and degeneration. Cx43 immunolocalization was evaluated to find Cx43 maturational changes. Under FSH treatment, the proliferation rate was high so the total number of Sertoli cells increased with a low level of degeneration and lumen formation. T3 stimulation evoked a reduction in the proliferation rate and a decrease in Sertoli cell number but with intensive formation of lumen. T3+FSH inhibited the proliferation rate and stimulated lumen formation together with degeneration, which negatively influenced the number of germ cells in the seminiferous epithelium. We conclude that T3 action seems to be particularly connected with the maturation of Cx43 gap junctions. FSH stimulates maturation of Sertoli cell function, but this effect may take place regardless of the presence of Cx43-dependent intercellular communication. The hyperstimulation of both FSH and T3 damages Cx43 connections and hence evokes regressional changes in the seminiferous epithelium.

Di(n-butyl) phthalate has no effect on the rat prepubertal testis despite its estrogenic activity in vitro.

The aim of this study was to assess the impact of di(n-butyl) phthalate (DBP) on the rat's prepubertal testis. Male Wistar rats were given daily subcutaneous injections with DBP (20 or 200 µg) or a vehicle from the 5th to the 15th postnatal day (pd). On the 16(th) pd, the rats were euthanized, and the testes were dissected, weighed, and paraffin embedded. The blood was collected to determine the serum levels of testosterone (T), estradiol (E) and FSH. The following parameters were assessed in the testis sections: diameter and length of seminiferous tubules (st), numbers of spermatogonia A + intermediate + B (A/In/B), preleptotene spermatocytes (PL), leptotene + zygotene + pachytene spermatocytes (L/Z/PA) and Sertoli cells per testis, percentage of st containing gonocytes or pachytene spermatocytes or lumen. An estrogenicity in vitro test was performed by means of a transgenic yeast strain expressing human estrogen receptor alpha. At both doses, DBP had no influence on testis and seminal vesicle weight, st diameter and length, number of germ and Sertoli cells per testis, percentage of st containing gonocytes or pachytene spermatocytes or lumen. DBP did not change E, T or FSH serum levels. The in vitro yeast screen showed that DBP was a weak estrogenic compound, approximately six to seven orders of magnitude less potent than 17ß-estradiol. In conclusion, exposure of a rat to DBP in doses 100 or 1,000-fold higher than a Tolerable Daily Intake for humans had no effect on its testicular development.

Xenoestrogens diethylstilbestrol and zearalenone negatively influence pubertal rat's testis.

UNLABELLED: The aim of this study was to assess the impact of xenoestrogens: diethylstilbestrol (DES) and zearalenone (ZEA) on rat's pubertal testis and to compare it with the effect of natural estrogen - 17beta-estradiol (E). Male Wistar rats were daily, subcutaneously injected at 5th-15th postnatal days (p.d.) with E (1.25 or 12.5 mug) or DES (1.25 or 12.5 mug) or ZEA (4 or 40 mug) or vehicle. At 16th p.d. testes were dissected, weighted, and paraffin embedded. Following parameters were assessed: diameter and length of seminiferous tubule, numbers of spermatogonia A+intermediate+B (A/In/B), preleptotene spermatocytes (PL), leptotene+zygotene+pachytene spermatocytes (L/Z/PA) and Sertoli cells per testis. Testes weight, seminiferous tubule diameter and length were decreased by both doses of E, DES and ZEA. DES effect was the strongest, but its influence on testis weight and seminiferous tubule length, on the contrary to E and ZEA, was not dose-dependent. Similarly, DES in both doses had the most severe negative impact on the number of germ and Sertoli cells. The negative influence of E on germ cells was less pronounced. The negative effect of ZEA was seen only after administration of the higher dose on spermatogonia number, while DES and E decreased A/In/B number more evidently. Sertoli cell number were decreased after both doses of E. ZEA40 decreased Sertoli cell number while ZEA4 had no effect. CONCLUSION: exposure of prepubertal male rat to DES has the strongest detrimental effect on the developing testis in comparison to E and ZEA. Both, E and DES, decreased number of germ and Sertoli cells, diminished seminiferous tubule diameter, length and testis weight. ZEA had much more weaker effect than the potent estrogens.