Human Fetal Testicular Tissue Research Articles

Maintenance of Sertoli Cell Number and Function in Immature Human Testicular Tissues Exposed to Platinum-Based Chemotherapy-Implications for Fertility Restoration.

Background: Retrospective studies in adult survivors of childhood cancer show long-term impacts of exposure to alkylating chemotherapy on future fertility. We recently demonstrated germ cell loss in immature human testicular tissues following exposure to platinum-based chemotherapeutic drugs. This study investigated the effects of platinum-based chemotherapy exposure on the somatic Sertoli cell population in human fetal and pre-pubertal testicular tissues. Methods: Human fetal (n = 23; 14–22 gestational weeks) testicular tissue pieces were exposed to cisplatin (0.5 or 1.0 μg/ml) or vehicle for 24 h in vitro and analysed 24–240 h post-exposure or 12 weeks after xenografting. Human pre-pubertal (n = 10; 1–12 years) testicular tissue pieces were exposed to cisplatin (0.5 μg/ml), carboplatin (5 μg/ml) or vehicle for 24 h in vitro and analysed 24–240 h post-exposure; exposure to carboplatin at 10-times the concentration of cisplatin reflects the relative clinical doses given to patients. Immunohistochemistry was performed for SOX9 and anti-Müllerian hormone (AMH) expression and quantification was carried out to assess effects on Sertoli cell number and function respectively. AMH and inhibin B was measured in culture medium collected post-exposure to assess effects on Sertoli cell function. Results: Sertoli cell (SOX9+ve) number was maintained in cisplatin-exposed human fetal testicular tissues (7,647 ± 459 vs. 7,767 ± 498 cells/mm2; p > 0.05) at 240 h post-exposure. No effect on inhibin B (indicator of Sertoli cell function) production was observed at 96 h after cisplatin (0.5 and 1.0 μg/ml) exposure compared to control (21 ± 5 (0.5 μg/ml cisplatin) vs. 23 ± 7 (1.0 μg/ml cisplatin) vs. 25 ± 7 (control) ng/ml, p > 0.05). Xenografting of cisplatin-exposed (0.5 μg/ml) human fetal testicular tissues had no long-term effect on Sertoli cell number or function (percentage seminiferous area stained for SOX9 and AMH, respectively), compared with non-exposed tissues. Sertoli cell number was maintained in human pre-pubertal testicular tissues following exposure to either 0.5 μg/ml cisplatin (6,723 ± 1,647 cells/mm2) or 5 μg/ml carboplatin (7,502 ± 627 cells/mm2) compared to control (6,592 ± 1,545 cells/mm2). Conclusions: This study demonstrates maintenance of Sertoli cell number and function in immature human testicular tissues exposed to platinum-based chemotherapeutic agents. The maintenance of a functional Sertoli cell environment following chemotherapy exposure suggests that fertility restoration by spermatogonial stem cell (SSC) transplant may be possible in boys facing platinum-based cancer treatment.

The oncogene Gankyrin is expressed in testicular cancer and contributes to cisplatin sensitivity in embryonal carcinoma cells

BackgroundTesticular germ cell cancer (TGCC) develops from pre-malignant germ neoplasia in situ (GCNIS) cells. GCNIS originates from fetal gonocytes (POU5F1+/MAGE-A4−), which fail to differentiate to pre-spermatogonia (POU5F1−/MAGE-A4+) and undergo malignant transformation. Gankyrin is an oncogene which has been shown to prevent POU5F1 degradation and specifically interact with MAGE-A4 in hepatocellular carcinoma (HCC) cells. We aimed to investigate the role of Gankyrin in progression from gonocyte to pre-invasive GCNIS and subsequent invasive TGCC.MethodsWe determined Gankyrin expression in human fetal testicular tissue (gestational weeks 9–20; n = 38), human adult testicular tissue with active spermatogenesis (n = 9), human testicular tissue with germ cell maturation delay (n = 4), testicular tissue from patients with pre-invasive GCNIS (n = 6), and invasive TGCC including seminoma (n = 6) and teratoma (n = 7). Functional analysis was performed in-vitro by siRNA knock-down of Gankyrin in the NTera2 cells (derived from embryonal carcinoma).ResultsGerm cell expression of Gankyrin was restricted to a sub-population of prespermatogonia in human fetal testes. Nuclear Gankyrin was also expressed in GCNIS cells of childhood and adult pre-invasive TGCC patients, and in GCNIS from seminoma and non-seminoma patients. Cytoplasmic expression was observed in seminoma tumour cells and NTera2 cells. Gankyrin knock-down in NTera2 cells resulted in an increase in apoptosis mediated via the TP53 pathway, whilst POU5F1 expression was unaffected. Furthermore, Gankyrin knock-down in NTera2 cells increased cisplatin sensitivity with an increase in cell death (13%, p < 0.05) following Gankyrin knock-down, when compared to cisplatin treatment alone, likely via BAX and FAS. Our results demonstrate that Gankyrin expression changes in germ cells during normal transition from gonocyte to prespermatogonia. In addition, changes in Gankyrin localisation are associated with progression of pre-invasive GCNIS to invasive TGCC. Furthermore, we found that Gankyrin is involved in the regulation of NTera2 cell survival and that a reduction in Gankyrin expression can modulate cisplatin sensitivity.ConclusionsThese results suggest that manipulation of Gankyrin expression may reduce the cisplatin dose required for the treatment of TGCC, with benefits in reducing dose-dependent side effects of chemotherapy. Further studies are required in order to assess the effects of modulating Gankyrin on GCNIS/TGCC using in vivo models.

This Month in Investigative Urology

No AccessJournal of UrologyThis Month in Investigative Urology1 Aug 2015This Month in Investigative Urology Karl-Erik Andersson Karl-Erik AnderssonKarl-Erik Andersson More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.05.031AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "This Month in Investigative Urology." The Journal of Urology, 194(2), pp. 276–277 References 1 : Bone metastasis in renal cell carcinoma is preprogrammed in the primary tumor and caused by AKT and integrin α5 signaling. J Urol2015; 194: 539. Link, Google Scholar 2 : Synergy of histone-deacetylase inhibitor AR-42 with cisplatin in bladder cancer. J Urol2015; 194: 547. Link, Google Scholar 3 : Human fetal testicular tissue xenotransplantation: a platform to study the effect of gonadotropins on human germ cell development in utero. J Urol2015; 194: 585. Link, Google Scholar 4 : Abnormalities in expression of structural, barrier and differentiation related proteins, and chondroitin sulfate in feline and human interstitial cystitis. J Urol2015; 194: 571. Link, Google Scholar © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 194Issue 2August 2015Page: 276-277 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Karl-Erik Andersson More articles by this author Expand All Advertisement PDF downloadLoading ...

Human Fetal Testicular Tissue Xenotransplantation: A Platform to Study the Effect of Gonadotropins on Human Germ Cell Development In Utero

We examined the effects of long-term hCG stimulation on germ cell maturation, and Sertoli and Leydig cell function in a xenotransplantation model of the human fetal testis. A total of 20 human fetal testes were ectopically xenografted on 20 castrated NCr male nude mice. Grafts were collected for analysis 24 weeks later. Mice were treated with saline as the control or with hCG beginning 4 weeks after the grafts were transplanted. Of the grafts 65% survived at 24 weeks. In contrast to untreated pregrafted samples, hCG stimulated xenografts showed significantly increased density of seminiferous tubule formation with Sertoli cell migration to the basement membrane. Germ cell proliferation and differentiation from gonocytes (M2A(+)) to prespermatogonia (MAGE-4A(+)) were observed in graft samples recovered from the hCG and nonhCG treated groups at 24 weeks of treatment. Leydig cells in hCG treated grafts produced significantly more testosterone than nonhCG treated grafts. Although further studies are required to investigate the potential for further differentiation and maturation of xenografted human fetal testes, normal in utero testicular development was reproduced under long-term hCG stimulation. This model represents a means to study long-term effects of gonadotoxins or hormonal stimulation on the maturation of human fetal testes.

Review of recent experimental evidence: phthalates and male reproductive endpoints

The antiandrogenic properties of phthalates and their characterization as testicular developmental toxicants have been derived from a series of experimental studies in the rat. Effects of developmental exposure in the rat include decreased anogenital distance, hypospermatogenesis, underdeveloped reproductive organs, cryptorchidism and hypospadias, and formation of multinucleated germ cells (MNG) in the testis. The underlying mechanism is related to the suppression of testosterone production in Leydig cells in the rat testis. The mouse, however, did not show suppression of steriodogenesis and reproductive organ malformations, but did show MNG formation. Also in marmosets, phthalates did not affect reproductive organ development, nor did testicular histopathology reveal abnormalities. The human testicular developmental response to phthalates has been difficult to address experimentally for ethical reasons and due to the absence of appropriate alternative models. Two independent recent studies have used xenotransplants of human fetal testis in immunologically compromised rodents to study the effects of phthalate exposure in man. Both studies found no effects on steroidogenesis whereas MNG were induced in both models in human fetal testis implants, confirming adequate internal exposure of implanted target tissue. Moreover, in the same model, steroidogenesis in mouse implants was unaffected as well, although MNG were observed. Rat implants showed suppression of steroidogenesis as well as MNG formation, confirming the general sensitivity of the model as in the in vivo situation. These results strongly suggest that human fetal testicular tissue is not sensitive to the endocrine disrupting effects of phthalates.

Methods of cryopreservation of testicular tissue with viable spermatogonia in pre-pubertal boys undergoing gonadotoxic cancer treatment

Banking of testicular tissue from pre-pubertal boys before gonadotoxic treatment is a crucial step in fertility preservation. We wanted to find optimal methods for cryopreservation of testicular tissue from pre-pubertal boys, modifying techniques developed for fetal and adult human testicular tissue cryopreservation. Testicular tissue was collected from five pre-pubertal boys undergoing gonadotoxic treatment in a clinical programme. Two freezing protocols, originally developed for fetal and adult human testicular tissue, were applied for pre-pubertal testicular tissue cryopreservation. In both methods, 5% dimethyl sulphoxide (DMSO) was used as a cryoprotectant. The integrity of the tissue was investigated in non-frozen tissue cultured for 24 h and in cryopreserved-thawed tissue, using two different programmes. We also analysed frozen-thawed samples cultured for 24 h in comparison with untreated fresh fixed control tissue. Immunohistochemical analysis using anti-MAGE-A4, vimentin and CD34 monoclonal antibodies was performed in order to visualize and characterize the cryodamage of the different testicular cells and compartments. The structure of the tissue was evaluated using light microscopy. Qualitative control analysis was performed using transmission electron microscopy. No clear structural changes were observed in the fresh, fresh cultured and cryopreserved testicular tissue after using the protocol developed for adult testicular tissue. The programme earlier successfully used for human fetal testicular tissue cryopreservation caused more tissue damage. Pre-pubertal testicular tissue from boys facing gonadotoxic treatment survives cryopreservation, can be cryobanked and hopefully used for fertility preservation. Slow programmed freezing with DMSO as a cryoprotectant is efficient in maintaining the spermatogonia, Sertoli cells and stromal compartment during freezing, thawing and tissue culture.

Prospects for clinical transplantation of the testis as an organ and as a tissue

In our century the transplantation of testes as replacement therapy and the method of rejuvenation of the organism have elicited great interest. The history of the procedure started with the transplantation of testes from adult men; then came transplantation of fetal testes (with the description of various new surgical methods), followed by experiments in the transplantation of human fetal testicular tissue, including tissue cultures, and treatment with lyophilizates from animal testes. The transplantation of the fetal testis as a tissue and as cell cultures and treatment with testicular tissue extracts may effectively stimulate the regeneration of the testis proper in hypogonadism of various etiology, including incurable hypergonadotropic forms.

A Monoclonal Antibody Against Human Testicular Anti‐Müllerian Hormone

Using immunochromatography on a polyclonal antibody, testicular anti-Müllerian hormone (AMH) was purified from homogenates of human fetal testicular tissue and used as an antigen for hybridoma production. Two IgM clones were obtained. Both recognized AMH on biopsies of human testicular tissue and one blocked its anti-Müllerian activity. This monoclonal antibody (MAb) exhibited a relatively high affinity for AMH, and was studied further. It is interspecific, recognizing AMH in other mammalian species. The study of this MAb in relation to an IgM MAb raised against bovine AMH (bAMH) indicates that both MAbs recognize different but related epitopes on bAMH.

Regulation of human fetal testicular secretion of testosterone: Low-density lipoprotein-cholesterol and cholesterol synthesized de novo as steroid precursor

The results of the present investigation support the conclusion that low-density lipoprotein (LDL)-cholesterol facilitates androgen synthesis in human chorionic gonadotropin (hCG)-treated human fetal testicular tissue in vitro. Moreover, the number of LDL receptors and the rate of de novo synthesis of cholesterol are high during the period of active fetal testicular steroidogenesis and fall with advancing gestational age, suggestive of regulation by hCG.

Human chorionic gonadotropin binding to human fetal testes as a function of gestational age.

The characteristics of binding of hCG to testicular tissue obtained from human abortuses of 10--24 weeks gestational age were studied. Specific, saturable binding of [125I]hCG was demonstrated using homogenates of human fetal testicular tissue. The equilibrium dissociation constant ranged from 0.4 x 10(-10) M to 5.5 x 10(-10) M, a finding that is indicative of a high affinity receptor. The capacity to bind hCG was low, but varied strikingly with gestational age. The binding capacity for hCG of tissues from abortuses of gestational age less than 15 weeks and greater than 22 weeks was consistently less than 10.0 pg x mg-1 tissue (2.2 fmol x mg-1 tissue). The binding capacity for hCG of tissues from abortuses of gestational age between 15--20 weeks ranged from 2.4--29.8 pg x mg-1 tissue (0.5--6.5 fmol x mg-1 tissue) with the majority of values being greater than 10 pg x mg-1 tissue (2.2 fmol x mg-1 tissue). On the other hand, receptors for hCG in human fetal ovarian tissue were undetectable, irrespective of gestational age. It is concluded that specific high affinity binding sites for hCG are present in human fetal testes and that the binding capacity is maximum between gestational ages of 15--20 weeks. This increase in binding capacity parallels the surge in testosterone production known to occur during the same period of development. These results suggest that the increase in fetal plasma levels of testosterone during this time in gestation is the result of an increase in the sensitivity of the fetal testis to hCG caused by an increase in the number of hCG receptors, and that hCG most likely is responsible for stimulation of fetal testicular steroidogenesis in utero at this time of gestation.

Müllerian-inhibiting activity of human fetal testicular tissue deprived of germ cells by in vitro irradiation.

Extract: Fragments of testicular tissue from seven human fetuses were explanted in three organ-culture dishes for each fetus. One remained unirradiated while the others were submitted to, respectively, 500 and 700 rads of 6 0Co γ-rays. After an interval of 1–2 weeks, the mullerian-inhibiting activity of the explants was tested using the 14 0.5-day-old rat miillerian duct as end organ and the number of germ cells present in 50 orthogonal sections of seminiferous tubules was determined on a minimum of 10 histologic sections. In control explants, only 12.9% of the original number of germ cells were normal after 1–2 weeks in vitro. The mortality of germ cells was increased by γ-rays; only 7% of their original number surviving 500 rads, and 3% 700 rads. Mullerian-inhibiting activity of both control and irradiated explants was normal. Speculation: Mullerian-inhibiting activity of human fetal testicular explants is not affected either by in vitro survival or by γ-irradiation, although both these procedures significantly deplete the germ cell population of the seminiferous tubules, only 3% surviving exposure to 700 rads. If germ cells were involved in the secretion of the antimullerian hormone, such a drastic reduction of their number would certainly be reflected by an impairment of the mullerian-inhibiting activity of the irradiated explants. As this is not the case, and as it has been demonstrated previously that the antimullerian hormone is synthesized by seminiferous tubules and not by interstitial tissue, we speculate that Sertoli cells are the source of the hormone.

Studies on human sexual development. I. Fetal gonadal and adrenal sex steroids.

Testosterone and estradiol concentrations were measured by isotope-displacement assays in the gonads and adrenals of 54 human fetuses (33 male, 21 female) delivered by hysterotomy. Results were correlated with crownrump (CR) length which ranged from 5 to 24 cm, corresponding to 10–25 weeks fetal age. Testosterone concentration averaged 1400 pg/mg in the testes but was negligible (<90 pg/mg) in the ovaries and in the adrenals of both sexes. Testosterone was found in appreciable amounts in the testes from the youngest specimens, reached a peak at 7–10 cm CR-length and declined by 13–14 cm CR-length. Little or no estradiol was detected in fetal testes, ovaries and adrenals and there were no sex differences in these levels. These data provide evidence that human fetal testicular tissue accumulates testosterone in a pattern suggesting higher androgen production at the time of male genital differentiation.