Expression of G-Protein-Coupled Estrogen Receptor (GPER) in Whole Testicular Tissue and Laser-Capture Microdissected Testicular Compartments of Men with Normal and Aberrant Spermatogenesis.

In this study, we retrospectively investigated GPER expression in biopsies of azoospermic men with complete (obstructive azoospermia-OA) and aberrant spermatogenesis (nonobstructive azoospermia-NOA). Each biopsy was histologically evaluated with morphometry. The testicular GPER expression was analyzed by the immunohistochemistry and RT-PCR technique in the whole testicular tissue and in seminiferous tubules and Leydig cells after laser-capture microdissection. In laser-microdissected compartments, we also analyzed transcriptional expression of selected Leydig (CYP17A1, HSD17B3, StAR) and Sertoli cell (AMH, SCF, BMP4) function markers. Immunohistochemical staining revealed expression of GPER in the cytoplasm of Leydig and Sertoli cells. Its stronger intensity was observed in Sertoli cells of NOA biopsies. The RT-PCR analysis of the GPER mRNA level unequivocally showed its increased expression in seminiferous tubules (i.e., Sertoli cells), not Leydig cells in NOA biopsies. This increased expression correlated positively with the transcriptional level of AMH-a marker of Sertoli cell immaturity, as well as FSH serum level in NOA but not in the OA group. Our results clearly demonstrate altered GPER expression in testes with primary spermatogenic impairment that might be related to Sertoli cell maturity/function.

Risk of gonadal neoplasia in patients with disorders/differences of sex development.

BACKGROUND: Patients with disorders/differences of sex development (DSD), especially those possessing the Y chromosome, have a higher risk of gonadal germ-cell tumours (GCTs). We aimed to examine the incidence of different types of gonadal neoplasia and associated risk factors. METHODS: A total of 1040 DSD patients aged ≥16 years participated in a cross-sectional multicentre European study (dsd-LIFE). Data on medical history were gathered from the patients' archival medical documents. A web-based questionnaire was filled out individually by the participants. A physical examination was performed in all, while ultrasonography of gonads was carried out in 214 and semen analysis was performed for 53 patients. RESULTS: Germ-cell neoplasia was present in 12 % of patients with DSD and in 14 % of those with XY DSD. The highest risk (36 %) was observed in 46,XY patients with gonadal dysgenesis (GD): complete GD (33 %) and partial GD (23 %), but also in mixed GD (8 %) and complete androgen insensitivity syndrome (AIS) (6%). It was not reported in partial AIS, XX male, 46,XX DSD and congenital adrenal hyperplasia, Turner and Klinefelter syndromes, or in androgen biosynthesis defects. Benign sex cord-stromal tumours (Sertoli- and Leydig-cell tumours) were noted only in patients with complete AIS (3.1 %) and Klinefelter syndrome (14.3 %). A relationship between risk factors for GCT and gonadal neoplasia appearance, other than the Y chromosome, was not found. CONCLUSION: Adult patients with GD and the Y chromosome have the highest risk of GCT and should be kept under thorough medical control and receive special medical follow-up to prevent the development of gonadal tumours.

[High risk of atherosclerosis in men aged 20-39 from Lodz agglomeration]. / Wysokie ryzyko wystapienia miazdzycy u mezczyzn w wieku 20-39 lat z aglomeracji lódzkiej.

THE AIM OF THE STUDY: To estimate the incidence of atherosclerosis risk factors in young men of Lodz city because of the highest in Poland fatality rate of circulatory system diseases. MATERIAL AND METHODS: Anamnestic data on actual diseases, smoking, alcohol drinking and physical activity were achieved from 80 men, volunteers aged 20-39 years. Body weight and height, waist and hip circumference and arterial blood pressure were measured. Blood levels of lipids: total cholesterol (TCh), its fractions LDL, and HDL (LDL-Ch, HDL-Ch) ,and triglicerydes (TG), glucose, albumins, sex hormone binding globulin (SHBG), FSH, LH, total testosterone, dehydroepiandrosterone sulphate (DHEA-S) and estradiol were determined. Calculated were body mass index (BMI), waist to hip ratio (WHR), free testosterone index (FTI), free and bioactive testosterone. RESULTS: At least 3 atherosclerosis risk factors were simultaneously found in 33.7% of men, of which 22.7% were 20-29-year-old and 47.2% 30-39-year-old subjects. Elevated values of TG were found in 16.2% of men, TCh in 13.7%, LDL-Ch in 7.5% and decreased values of HDL-Ch in 6.2%. Positive significant correlations were found between WHR and TCh (R = 0.39; p = 0.01), LDL-Ch (R = 0.38; p = 0.02), TG (R = 0.41; p = 0.009). WHR negatively correlated with HDL-Ch (R = -0.31; p = 0.04). 50% of men had the excessive body weight. Obese men had abdominal type of obesity in 90%. As many as 62% of subjects had excessive systolic and 21% excessive diastolic arterial blood pressure. Blood pressure positively correlated with body weight (R = 0.51; p < 0.001), BMI (R = 0.51; p < 0.001), waist circumference (R = 0.55; p < 0.001) and WHR (R = 0.44; p < 0.001). In the whole group 35% of subjects led sitting life style and did not report any other physical activity. 57.5% of men were present or past smokers. 44% of men consumed alcohol everyday or almost everyday. FTI diminished with the advancing age, what was connected with the increase in SHBG blood concentration. There were no changes in total, free or bioactive testosterone, or LH and FSH concentrations with the age. Correlations between androgens and lipid profiles were not found. Estradiol blood levels negatively correlated with TG (R = -0.35; p = 0.03) and was significantly lower in 30-39-year-old men than in younger (20-29). CONCLUSION: The results indicate considerably higher incidence of atherosclerosis risk factors in young men, citizens of Lodz agglomeration, than it was found before for other regions of Poland. This phenomenon increases with the advancing age already between 20 and 39 years. Implementation of intensive prophylactic actions may prevent it.

Endoscopic extraperitoneal radical prostatectomy: An initial report following the first 30 cases.

INTRODUCTION: To present initial observations after the first 30 cases of endoscopic extraperitoneal radical prostatectomy carried out at our department, which so far has had no experience with this surgical procedure. MATERIAL AND METHODS: In the period of 15 months a group of 30 patients with organ confined prostate cancer, underwent endoscopic extraperitoneal radical prostatectomy using Montsouris technique. All procedures were performed by the same team of two urologists and one resident. RESULTS: The mean age of the patients was 65.3 years (43-73 years), the mean preoperative prostate specific antigen (PSA) was 7.2 ng/ml (4-9.8 ng/ml), the mean prostate volume measured in TRUS was 41 cm³ (25-80 cm³). The mean operative time was 3 h 55 min (3 h 15 min - 5 h 30 min). The negative margin was achieved in 26 patients (86%). In seven patients (23%) blood transfusion was required. Three patients had intraoperative rectal injury. In two cases trauma was supplied laparoscopically, and in one case it was decided to perform diverting colostomy. The majority of patients (65%) were discharged home on the fifth day after surgery. Two months postoperatively 13 patients (43%) were continent, 16 (35%) presented moderate stress incontinence with occasional urine leakage during normal activity and 1 patient (3%) presented severe stress incontinence. CONCLUSIONS: Endoscopic extraperitoneal radical prostatectomy during the early phase of learning is technically difficult, requiring from the operator the laparoscopic skills, determination and a thorough knowledge of the theoretical basis of the subsequent stages of the procedure. Urologists who start performing this procedures must be aware of possible intra as well as postoperative complications.

Undescended testis - current trends and guidelines: a review of the literature.

The best mode of undescended testis (UDT) treatment remains controversial. However, knowledge gained from randomized controlled studies and meta-analyses allowed different groups of researchers to set out guidelines on management of patients with UDT. The authors reviewed recent literature and came to the following conclusions: (1) Hormonal treatment is not recommended, considering both the immediate results (only 15-20% of retained testes descend) and the possible long-term adverse effects on spermatogenesis. (2) Surgery is the treatment of choice; orchiopexy is successful in about 95% of UDT, with a low rate of complications (about 1%). (3) Orchiopexy should be performed between 12 and 18 months of age, or at first contact if diagnosed later.

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.

[Important functions of estrogens in men--breakthrough in contemporary medicine]. / Wazne funkcje estrogenów u mezczyzn--przelom we wspóczesnej medycynie.

Estradiol (E2) is traditionally recognised as the female sex hormone. Since discovery of estrogens in the early forties of XX century it has been believed, that these hormones caused impairment of the gonadal function in men or didn't exert any influence. New studies are contradictory, but indicate also a possible involvement of estrogens in the pathogenesis of some systemic diseases of men. The main source of E2 in men is adipose tissue and the brain. E2 is also produced in adrenals, liver, mammary glands, hair and in male gonads. Daily production and blood level of E2 in men are higher than those in postmenopausal women. In 1988 we were the first to demonstrate that E2 is an important hormonal signal for initiation of spermatogenesis. The traditional view about unimportant or inhibitory role of E2 in male physiology was finally refuted thanks to discovery of the estrogen receptors in males. In the middle 90ties transgenic mice with the lack of estrogen receptor (ER knockout) or enzyme aromatase, that enable the conversion of testosterone into E2, were produced. Observations of men with inherited mutations of these genes, considerably extended our knowledge about stimulatory role of E2 in men in the formation of bone stroma, inhibition of their linear growth, lipids metabolism and sexual maturation, the effects that were attributed to testosterone action until today. New data indicate role of estrogens and ER in the function of the cardio-vascular system. Their link with development of arteriosclerosis seems, however, to be bipolar. In single reported cases of men with the inactivating mutations of ERalpha or aromatase genes, a precocious arteriosclerosis is noted. From the other site, men homozygous for the most common variant of ERalpha gene (ESR1c.454-397cc) have a significantly increased risk of myocardial infraction. Estrogens are the risk factors in prostatic cancer and their local tissue increase in autoimmune diseases is connected with aggravation of the proliferative complications of these disorders.

[Physiological significance of estrogens in men--breakthrough in endocrinology]. / Znaczenie fizjologiczne estrogenów u mezczyzn--przelom w endokrynologii.

Estradiol (E2) is traditionally recognised as the female sex hormone. It has been believed for 40 years, that E2 didn't exert any influence or caused impairment of the gonadal function in men. The main source of E2 in men is adipose tissue and the brain. E2 is also produced in adrenals, liver, mammary glands, hair and in male gonad. Daily production and the level of E2 in the blood in men are higher than those in postmenopausal women. At the end of the 80-ties we were first reporting that during sexual maturation E2 can be the important hormonal signal for the initiation of spermatogenesis. The traditional view about unimportant or inhibitory role of E2 in male physiology was finally refuted thanks to discovering estrogen receptors in males. In the 90-ties, transgenic mice with the lack of estrogen receptor (ER) or gene encoding enzyme aromatase, that enable the conversion of testosterone into E2, were also produced. Observations of men with inherited mutations of these genes, considerably extended our knowledge about E2 in men in stroma bones formation, inhibition of their growing, lipids metabolism and sexual maturation, the effects that were attributed to testosterone action until today. New data also points at important role of estrogens and ER in the function of the cardio-vascular system and in counteracting coronary disease in men.

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.

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.

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.

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.

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.

Triiodothyronine modulates initiation of spermatogenesis in rats depending on treatment timing and blood level of the hormone.

Triiodothyronine (T3) stimulates spermatogenic onset but the influence of T3 on spermatogonia development is unknown. The aim of the study was to investigate the role of T3 for both processes simultaneously. Male rats were given daily injections of 100 µg T3/kg body weight or vehicle from birth until postnatal day (pnd) 5 and euthanized on pnd 6 (short T3-sT3). Other rats, euthanized on pnd 16, were treated either transiently with T3 (tT3) during the initial 5 days or continuously until pnd 15 (cT3). sT3 was found to increase gonocyte differentiation, spermatogonia number, cell degeneration and proliferation. tT3 increased serum T3 level and spermatogonial development to adult values precociously, but cell degeneration or proliferation were not affected. cT3 increased serum T3 together with cell degeneration and proliferation, but cell number was not affected. In conclusion, T3 may modulate spermatogonial development quantitatively depending on treatment timing and blood level of the hormone.

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.

Semen quality in men from subfertile couples from the region of Lódz (Poland) according to the new reference values recommended by WHO 2010.

The semen analysis is the main diagnostic tool for evaluating the male fertility potential. The standard semen analysis includes evaluation of the sperm concentration, motility, and their morphology. The most important question is whether the results from semen analysis may be accurate markers for male fertility. Therefore, we retrospectively studied sperm quality among men attending the infertility clinic due to reproductive problems consistent with the WHO manual from 1999, which were reassessed according to the manual from 2010. Semen results from 571 males from couples undergoing fertility investigation were analyzed. All subjects included in the study had no abnormalities during examination. In 64 samples (11.2%), a leukocyte count above 1 x 10(6)/ml was found and their semen volume (median 3.2 ml) was significantly lower in comparison with the group without leukocytes (3.6 ml; p <0.001). Normal semen parameters were found in 290 subjects (50.8%) according to the 1999 manual and in 362 men (63.4%) according to the 2010 manual. The normozoospermia group, according to the 2010 manual, had a significantly lower percentage of sperm with progressive motility, motile sperm concentration, and total number of motile sperm in comparison with the normozoospermia group according to the manual from 1999. It seems that routine semen analysis is not sufficient to estimate male fertility potential and some men with normal semen parameters may be subfertile. Further investigations are needed.

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.