Administration of testosterone inhibits initiation of seminal tubule growth and decreases Sertoli cell number in the earliest period of rat's postnatal development.

Sertoli cell (SC) number determines testes size and their capacity to produce spermatozoa. In the rat SC proliferate until 15th postnatal day (PND). Their proliferation is stimulated by FSH and inhibited by estradiol, but the role for androgens is uncertain. In this study we analyzed the effects of testosterone administration on testes growth and SC number in relation to timing of the treatment. Male rats were injected with 2.5 mg of testosterone propionate (TP) from birth until 5th PND and autopsied either on 6th PND [TP1-5(6)] or on 16th PND [TP1-5(16)] (transient administration). Other rats received TP from birth until 15th PND [TP1-15] or between 5th and 15th PND [TP5-15] continuously and were autopsied on day 16th. Control groups (C) received vehicle. In the Cs serum level of estradiol was 20-fold higher (

During seminiferous tubule maturation testosterone and synergistic action of FSH with estradiol support germ cell survival while estradiol alone has pro-apoptotic effect.

During establishment of spermatogenesis at the prepubertal age, an early germ cells apoptotic wave occurs likely aimed to remove abnormal germ cells and to maintain a proper cell number ratio between maturating germ cells and Sertoli cells. Here we assessed Sertoli and germ cell apoptosis in relation to morphological parameters of Sertoli cell maturation in neonatal rats under the influence of testosterone, estradiol and FSH given alone or in combinations. From postnatal day (PND) 5th to 15th male rats were daily injected with: 1) 2.5 mg of testosterone propionate (TP), or 2) 12.5 microg of 17beta-estradiol benzoate (EB), or 3) TP+EB, or 4) 7.5 IU of human purified FSH (hFSH), or 5) hFSH+EB or solvents (control-C). Autopsy was performed on PND 16th. Sertoli cell nuclei area and incidence of seminiferous tubule lumen formation (LF) were taken as markers of Sertoli cell maturation. Sertoli and germ cell apoptosis was assessed using TUNEL method. In comparison with C, the area of Sertoli cell nuclei was significantly reduced after EB (25.7+/-2.0 vs. 30.9+/-1.6 microm2 for C,

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

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

Features of impaired seminiferous tubule differentiation are associated with germ cell neoplasia in adult men surgically treated in childhood because of cryptorchidism.

Seminiferous tubule differentiation was related to the occurrence of germ cell neoplasia in 38 men, aged 17-47, treated surgically in childhood for cryptorchidism. Tissues from 46 testes obtained from biopsies taken as a neoplastic preventive procedure or whole testes removed because of GCT were evaluated quantitatively. Paraffin sections were treated with antibodies against placental like alkaline phosphatase (PLAP), a marker of germ cell neoplasia, and cytokeratin 18 (CK-18), a marker of immature Sertoli cells. Quality of spermatogenesis and number Leydig cells were assessed with a score count. Seminiferous tubules diameter, thickness of basal membrane and size of intertubular spaces were measured with image analysis software. In 17.4% of testes spermatogenesis was normal (9.9 points) (N) and neoplasia was not found there. In the other 38 specimens (83%) spermatogenesis was abnormal (A). When spermatogenesis was arrested or when germ cells were absent (3.7+/-1.8 points), neoplastic lesions were found in 13.1% of the specimens. In A group 5.1+/-7.1% of tubules contained immature Sertoli cells, while in N they were not found. Tubular diameter was significantly lower in A (161.5+/-31.8 microm) than in N (184.6+/-24.3 microm) and the percentage of seminiferous tubules with the thickening of tubular basal membrane was also greater in A. Intertubular spaces were significantly larger in A (49.9+/-18.6%) in comparison to N group (32.6+/-12.5%). Mean number of Leydig cells was similar in both groups. To conclude, in most of the formerly cryptorchid testes, despite surgical treatment, impaired seminiferous tubules differentiation is predominant. Germ cell neoplasia is present in testes with retarded seminiferous tubules differentiation. Retardation of seminiferous tubule differentiation consists of inhibited spermatogenesis, presence of tubules with immature Sertoli cells, decreased tubular diameter, increased thickness of basal membrane and enlarged intertubular spaces. Examination of testicular biopsy with respect to the state of seminiferous tubule differentiation may be helpful to predict the appearance of germ cell neoplasia in adult men with cryptorchidism in anamnesis. Orchiopexy of cryptorchid testes may not prevent the occurrence of features of testicular dysgenesis and the associated germ cell neoplasia

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.

During seminiferous tubule maturation testosterone and synergistic action of FSH with estradiol support germ cell survival while estradiol alone has pro-apoptotic effect.

During establishment of spermatogenesis at the prepubertal age, an early germ cells apoptotic wave occurs likely aimed to remove abnormal germ cells and to maintain a proper cell number ratio between maturating germ cells and Sertoli cells. Here we assessed Sertoli and germ cell apoptosis in relation to morphological parameters of Sertoli cell maturation in neonatal rats under the influence of testosterone, estradiol and FSH given alone or in combinations. From postnatal day (PND) 5th to 15th male rats were daily injected with: 1) 2.5 mg of testosterone propionate (TP), or 2) 12.5 microg of 17beta-estradiol benzoate (EB), or 3) TP+EB, or 4) 7.5 IU of human purified FSH (hFSH), or 5) hFSH+EB or solvents (control-C). Autopsy was performed on PND 16th. Sertoli cell nuclei area and incidence of seminiferous tubule lumen formation (LF) were taken as markers of Sertoli cell maturation. Sertoli and germ cell apoptosis was assessed using TUNEL method. In comparison with C, the area of Sertoli cell nuclei was significantly reduced after EB (25.7+/-2.0 vs. 30.9+/-1.6 microm2 for C, p<0.001) and increased after hFSH+EB (33.1+/-2.3 microm2, p<0.05). Incidence of LF was completely arrested by steroid hormone treatments given separately, significantly inhibited after TP+EB (median: 0.0%, vs. 2.0% for C p<0.05) and significantly enhanced after hFSH+EB (median: 51.0%, p<0.001). hFSH alone did not influence LF. Incidence of TUNEL positive Sertoli cells significantly increased after EB (median: 2.9% vs. 0.5% for C, p<0.05) or TP+EB (median: 2.2%, p<0.01) and was not affected by other treatments. Incidence of TUNEL positive germ cells increased significantly after EB alone (median: 4.4% vs. 2.5%, for C, p<0.01 ) and was significantly decreased by hFSH+EB (median: 0.5%, p<0.01). CONCLUSIONS: 1) Administration of testosterone or estradiol to immature rats inhibits Sertoli cell maturation. 2) Estradiol stimulates Sertoli and germ cell apoptosis while testosterone has no effect. 3) Testosterone eliminates estradiol--induced germ cell apoptosis when both hormones act in concert. 4) FSH in concert with estradiol, but neither one of the hormone alone, accelerate Sertoli cell differentiation and effectively inhibit germ cell apoptosis. 5) During seminiferous tubule maturation testosterone and the synergistic action of FSH with estradiol support germ cell survival while estradiol alone has an inhibitory, pro-apoptotic effect

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

Features of impaired seminiferous tubule differentiation are associated with germ cell neoplasia in adult men surgically treated in childhood because of cryptorchidism.

Seminiferous tubule differentiation was related to the occurrence of germ cell neoplasia in 38 men, aged 17-47, treated surgically in childhood for cryptorchidism. Tissues from 46 testes obtained from biopsies taken as a neoplastic preventive procedure or whole testes removed because of GCT were evaluated quantitatively. Paraffin sections were treated with antibodies against placental like alkaline phosphatase (PLAP), a marker of germ cell neoplasia, and cytokeratin 18 (CK-18), a marker of immature Sertoli cells. Quality of spermatogenesis and number Leydig cells were assessed with a score count. Seminiferous tubules diameter, thickness of basal membrane and size of intertubular spaces were measured with image analysis software. In 17.4% of testes spermatogenesis was normal (9.9 points) (N) and neoplasia was not found there. In the other 38 specimens (83%) spermatogenesis was abnormal (A). When spermatogenesis was arrested or when germ cells were absent (3.7+/-1.8 points), neoplastic lesions were found in 13.1% of the specimens. In A group 5.1+/-7.1% of tubules contained immature Sertoli cells, while in N they were not found. Tubular diameter was significantly lower in A (161.5+/-31.8 microm) than in N (184.6+/-24.3 microm) and the percentage of seminiferous tubules with the thickening of tubular basal membrane was also greater in A. Intertubular spaces were significantly larger in A (49.9+/-18.6%) in comparison to N group (32.6+/-12.5%). Mean number of Leydig cells was similar in both groups. To conclude, in most of the formerly cryptorchid testes, despite surgical treatment, impaired seminiferous tubules differentiation is predominant. Germ cell neoplasia is present in testes with retarded seminiferous tubules differentiation. Retardation of seminiferous tubule differentiation consists of inhibited spermatogenesis, presence of tubules with immature Sertoli cells, decreased tubular diameter, increased thickness of basal membrane and enlarged intertubular spaces. Examination of testicular biopsy with respect to the state of seminiferous tubule differentiation may be helpful to predict the appearance of germ cell neoplasia in adult men with cryptorchidism in anamnesis. Orchiopexy of cryptorchid testes may not prevent the occurrence of features of testicular dysgenesis and the associated germ cell neoplasia