Testicular Microenvironment Research Articles

Effect of Herbal Medicine on Fertility Potential in Experimental Animals - an Update Review.

Introduction:Sexual function greatly affects the quality of life. Any problem which affects satisfaction is considered sexual dysfunction.Aim:The aim of this study was to investigate how recently used of complementary and alternative medicines has increased in treating infertility.Methods:Study has descriptive character based on searched and analyzed facts from the published articles in scientific journals.Results:The majority of the studies done for the detection of the efficacy of medicinal plants in treating male infertility were done on experimental animals with few clinical studies. However, there is still a need for more studies to have certain results, as conflicting results were noted in different studies done on the effect of the same medicinal plant on spermatogenesis. It might be referred it to the absence of standardization among research models, the difference in extracts, administration route, doses and how long did the experiment last added to differences in the part of the plant used and plant collection. This review summarized the finding of many studies that studied the effect of different medicinal herbs on the testes, epididymis, sperm and prostate parameters, testosterone level and steroidogenesis, erection/ejaculation function and libido.Conclusion:The protective effect might be due to OLE competence to antagonize the oxidative stress and LPO. Londium suffruticosum I. suffruticosum leaf extract was administered to sub-fertile male rats, subfertility was induced by carbendazim, it resulted in increased spermatogenesis, increased sperm counts, lessened sperm agglutination by preserving normal pH in testicular microenvironment, increasing the testicular oxidative biomarkers, SOD, and CAT. It was found that the main active components of the extract are alkaloids, steroidal lactones and flavonoids.

Alginate oligosaccharides improve germ cell development and testicular microenvironment to rescue busulfan disrupted spermatogenesis.

Rationale: Busulfan is currently an indispensable anti-cancer drug, particularly for children, but the side effects on male reproduction are so serious that critical drug management is needed to minimize any negative impact. Meanwhile, alginate oligosaccharides (AOS) are natural products with many consequent advantages, that have attracted a great deal of pharmaceutical attention. In the current investigation, we performed single-cell RNA sequencing on murine testes treated with busulfan and/or AOS to define the mitigating effects of AOS on spermatogenesis at the single cell level.Methods: Testicular cells (in vivo) were examined by single cell RNA sequencing analysis, histopathological analysis, immunofluorescence staining, and Western blotting. Testes samples (ex vivo) underwent RNA sequencing analysis. Blood and testicular metabolomes were determined by liquid chromatography-mass spectrometry (LC/MS).Results: We found that AOS increased murine sperm concentration and motility, and rescued busulfan disrupted spermatogenesis through improving (i) the proportion of germ cells, (ii) gene expression important for spermatogenesis, and (iii) transcriptional factors in vivo. Furthermore, AOS promoted the ex vivo expression of genes important for spermatogenesis. Finally, our results showed that AOS improved blood and testis metabolomes as well as the gut microbiota to support the recovery of spermatogenesis.Conclusions: AOS could be used to improve fertility in patients undergoing chemotherapy and to combat other factors that induce infertility in humans.

Leptin Secreted from Testicular Microenvironment Modulates Hedgehog Signaling to Augment the Endogenous Function of Leydig Cells

Leptin Secreted from Testicular Microenvironment Modulates Hedgehog Signaling to Augment the Endogenous Function of Leydig Cells

Ефективність сумісного застосування метформіну та ліпофлавону за умов метаболічного синдрому в щурів

У чоловіків молодого та середнього віку, які страждають на метаболічний синдром (МС) та ожиріння, досить поширеними є випадки порушень статевої функції. Сучасні дані щодо ефективності застосування метформіну за порушень чоловічої репродуктивної функції є досить суперечливими. Таким чином, пошук нових мішеней для таргетної терапії патологій, пов’язаних з МС, є одним з найважливіших завдань сучасної фундаментальної та прикладної медицини. Мета дослідження – оцінка ефективності метформіну та його комбінації з ліпофлавоном за введення щурам-самцям з МС за основними критеріальними показниками гонадопротекторної дії. У дослідах використовували самців щурят з початковою масою тіла 50–70 г, віком 3 тижні. Тварин було розподілено на 4 групи по 12 у кожній: 1– інтактні; 2 – тварини, які замість питної води отримували 10 % розчин фруктози протягом 60 днів (модель МС); 3 – тварини з МС, яким внутрішньошлунково вводили метформін у дозі 266 мг/кг протягом останніх 30 днів споживання фруктози; 4 – сумісне введення на фоні МС метформіну та ліпофлавону (внутрішньоочеревинно) у дозі 3,1 мг/кг м. т. (за кверцетином) протягом останніх 30 днів споживання фруктози. Показано, що введення ліпофлавону до схеми лікування МС метформіном сприяло оптимізації гонадопротекторної дії за рахунок відновлення про/антиоксидантного статусу тестикулярного середовища, про що свідчить позитивна модуляція активності компонентів антиоксидантного захисту (активність СОД, вміст церулоплазміну та вміст відновленого глутатіону), зниження рівня ПОЛ і відновлення стероїдогенної активності сім’яників. Отримані результати свідчать на користь того, що сумісне введення метформіну та ліпофлавону виявилось більш ефективним для корекції наслідків МС на репродуктивну функцію, ніж окреме застосування базового препарату метформіну.

Reproduction disrupts stem cell homeostasis in testes of aged male Drosophila via an induced microenvironment.

Stem cells rely on instructive cues from their environment. Alterations in microenvironments might contribute to tissue dysfunction and disease pathogenesis. Germline stem cells (GSCs) and cyst stem cells (CySC) in Drosophila testes are normally maintained in the apical area by the testicular hub. In this study, we found that reproduction leads to accumulation of early differentiating daughters of CySCs and GSCs in the testes of aged male flies, due to hyperactivation of Jun-N-terminal kinase (JNK) signaling to maintain self-renewal gene expression in the differentiating cyst cells. JNK activity is normally required to maintain CySCs in the apical niche. A muscle sheath surrounds the Drosophila testis to maintain its long coiled structure. Importantly, reproduction triggers accumulation of the tumor necrosis factor (TNF) Eiger in the testis muscle to activate JNK signaling via the TNF receptor Grindelwald in the cyst cells. Reducing Eiger activity in the testis muscle sheath suppressed reproduction-induced differentiation defects, but had little effect on testis homeostasis of unmated males. Our results reveal that reproduction in males provokes a dramatic shift in the testicular microenvironment, which impairs tissue homeostasis and spermatogenesis in the testes.

Xenotransplantation of canine spermatogonial stem cells (cSSCs) regulated by FSH promotes spermatogenesis in infertile mice

BackgroundXenotransplantation of spermatogonial stem cells (SSCs) has become a popular topic in various research fields because manipulating these cells can provide insights into the mechanisms associated with germ cell lines and the entire spermatogenesis process; moreover, these cells can be used in several biotechnology applications. To achieve successful xenotransplantation, the in vitro microenvironment in which SSCs are cultured should be an ideal microenvironment for self-renewal and similar to the in vivo testicular microenvironment. The age of the donor, the correct spermatogenesis cycle, and the quality of the donor tissue are also important. Although cell culture-related factors, such as the in vitro supplementation of hormonal factors, are known to promote successful xenotransplantation in mice, little is known about the influence of these factors on SSCs in vitro or in vivo in other mammalian species, such as dogs (Canis lupus familiaris). In this context, the goals of this study were to test the effect of follicle-stimulating hormone (FSH) on canine spermatogonial stem cell (cSSC) cultures since this hormone is related to the glial cell-derived neurotrophic factor (GDNF) signaling pathway, which is responsible for the self-renewal and maintenance of these cells in vivo, and to investigate the microenvironment of the SSC culture after FSH supplementation. Additionally, in vivo analyses of transplanted FSH-supplemented cSSCs in the testes of infertile mice were performed to assess the capacity of cSSCs to develop, maintain, and restore spermatogenesis.MethodsSSCs from canine prepubertal testes (aged 3 months) were cultured in vitro in the presence of FSH (10 IU L−1). GFRA1 transcript expression was detected to confirm the spermatogonia population in culture and the effect of FSH on these cells. The protein and transcript levels of late germ cell markers (GFRA1, DAZL, STRA8, PLZF, and CD49f) and a pluripotency marker (OCT4) were detected at 72 and 120 h to confirm the cSSC phenotype. In vivo experiments were performed by transplanting GFP+ cSSCs into infertile mice, and a 10-week follow-up was performed. Histological and immunofluorescence analyses were performed to confirm the repopulation capacity after cSSC xenotransplantation in the testis.ResultsSupplementation with FSH in cell culture increased the number of cSSCs positive for GFRA1. The cSSCs were also positive for the pluripotency and early germline marker OCT4 and the late germline markers PLZF, DAZL, C-kit, and GFRA-1. The in vivo experiments showed that the cSSCs xenotransplanted into infertile mouse testes were able to repopulate germline cells in the seminiferous tubules of mice.ConclusionsIn conclusion, our results showed for the first time that the treatment of cSSC cultures with FSH can promote in vitro self-renewal, increase the population of germline cells, and possibly influence the success of spermatogenesis in infertile mice in vivo.

Impaired testicular signaling of vitamin A and vitamin K contributes to the aberrant composition of the extracellular matrix in idiopathic germ cell aplasia

To study pathogenic features of the somatic testicular microenvironment associated with idiopathic germ cell aplasia. Cross-sectional study. Tertiary referral center for reproductive medicine. Testicular specimens from men with idiopathic nonobstructive azoospermia (iNOA) prospectively submitted to microdissection testicular sperm extraction. Of 20 specimens used for histology, 10 were also available for proteomic analysis. Primary Sertoli cells with normal karyotype and phenotype were also used. Patients with iNOA were dichotomized according to a positive versus negative sperm retrieval at microdissection testicular sperm extraction, and on the isolated extracellular matrix (ECM) the proteomic analysis was performed. Proteomic analysis of the ECM from testicular specimens with positive versus negative sperm retrieval. Gene ontology enrichment was used to identify upstream regulators based on the 11 deregulated ECM proteins, which were validated by immunohistochemistry and quantitative polymerase chain reaction. Continuous variables were expressed as medians and interquartile range. Germ cell aplasia was characterized by an increased signaling of the retinoic acid in Sertoli cells and associated with decreased expression of the basal membrane markers nidogen-2 and heparan sulfate proteoglycan-2. Decreased levels of the interstitial matrisome-associated factor IX and its regulator VKORC1 were, instead, coupled with decreased signaling of vitamin K in Leydig cells. An altered expression of a further eight ECM proteins was also found, including laminin-4 and laminin-5. Peripheral levels of the two vitamins were within the reference range in the two cohorts of iNOA men. We identified the pathogenetic signature of the somatic human testicular microenvironment, providing two vitamin-related mechanistic insights related to the molecular determinants of the idiopathic germ cell aplasia.

The Impact of the Fine Ambient Particle on Infertile Male's Sperm Quality

Introduction: Infertility has been a major problem for several years. Previously, infertility was often attributed to female factors. Recently, however, male factors have been found to contribute to 50% of the infertility cases overall. Male infertility is a multifactorial issue. A genetic disorder, abnormal endocrine system, structural anomaly, and environmental causes could lead to male infertility. Studies have revealed a link between fine air particles and decreased fertility. The aim of this study was to discover the effect of particulate matter 2.5 (PM2.5), representing environmental fine particles, on male infertility. Materials and Methods: We retrospectively collected data from patients diagnosed as having infertility and visited National Taiwan University Hospital from January 2015 to October 2017. We recorded each patient's body weight, height, basic blood test, sperm analysis, and sex hormone profile. Air quality data, especially PM2.5 concentration, were acquired from the Environmental Protection Administration of Taiwan. A total of 78 monitoring stations throughout Taiwan record PM2.5 concentrations (μg/m3) each hour automatically. Multivariate linear regression was used to detect independent factors affecting sperm count, concentration, motility, and morphology and sex hormone profile. Results:We observed regional and seasonal differences in the distributions of PM2.5 concentrations. In nearly all cities in Taiwan, the PM2.5 concentrations increased during early winter and continued to increase until next spring, with a peak around January and February. In particular, regional differences were observed in winter. The peak PM2.5 concentrations in southern and northern cities in Taiwan ranged between 35 and 40 μg/m3 and between 18 and 23 μg/m3, respectively. PM2.5 in central Taiwan reached as high as 30 μL/m3. By contrast, the eastern part of Taiwan had the lowest peak PM2.5, which was mostly <15 μg/m3. The average 24-month PM2.5 concentration had a negative effect on sperm count, but the result was nonsignificant. Additionally, the effect of PM2.5 on sperm motility and morphology was minimal. Conclusion: In infertile Taiwanese men, there is a trend of a negative association between exposure to PM2.5 and sperm concentration and total sperm count. Exposure to ambient fine particles, especially PM2.5, might have a negative correlation with sperm quality, sex hormone balance, and the testicular microenvironment through different mechanisms.

Germ cell apoptosis and survival in testicular inflammation.

Male infertility is due to genetics, hormonal or environmental causes, or is idiopathic. Azoospermia is linked to local testicular microenvironment deregulation, with inflammatory cells present in the 15% of testicular biopsies of infertile patients. As widely reported, spermatogenesis and steroidogenesis are controlled by local immunoregulatory agents produced by immune and nonimmune cells. Moreover IL-6R, TNFR1, Fas and IL-1R are expressed on germ cells, indicating a direct action of pro-inflammatory agents on these cells. Beyond the known function of cytokines and nitric oxide on testicular function at the stable levels present in the normal testis, this review focalises on the effect of pro-inflammatory factors on germ cell survival and death when inflammatory conditions are established in the testis. As no cure for male infertility has been found up to the present, intracytoplasmic sperm injection is the therapeutic option for azoospermic patients who wish to achieve genetic parenthood. Therapies with antioxidant and anti-inflammatory agents in experimental models of testicular damage have been successful. However, clinical implementation is uncertain in cases with a prolonged inflammatory state of the testis. Therapies offering multiple approaches to treat infertility by restoring the spermatogonial stem cell niche and protecting germ cells from apoptosis should be considered.

Microenvironmental signals govern the cellular identity of testicular macrophages.

Testicular macrophages (TM) comprise the largest immune cell population in the mammalian testis. They are characterized by a subdued proinflammatory response upon adequate stimulation, and a polarization toward the immunoregulatory and immunotolerant M2 phenotype. This enables them to play a relevant role in supporting the archetypical functions of the testis, namely spermatogenesis and steroidogenesis. During infection, the characteristic blunted immune response of TM reflects the need for a delicate balance between a sufficiently strong reaction to counteract invading pathogens, and the prevention of excessive proinflammatory cytokine levels with the potential to disturb or destroy spermatogenesis. Local microenvironmental factors that determine the special phenotype of TM have just begun to be unraveled, and are discussed in this review.

C-MET receptor as potential biomarker and target molecule for malignant testicular germ cell tumors.

Type II testicular germ cell tumors (TGCTs) represent the most frequent malignancy in Caucasian males (20–40 years). Even if diagnosed with disseminated disease, >80% of patients are cured; however, a small percentage of cases progress and result in death. It is commonly accepted that these cancers arise from a disturbed testicular embryonic niche that leads to the block of gonocyte differentiation. The subsequent development of the invasive seminomas and non-seminomas is due to a combination of genetic, epigenetic and microenvironment-based alterations (genvironment). Hepatocyte growth factor (HGF) is present in the testicular microenvironment, together with its receptor c-MET, from early embryonic development to an adult stage. In addition, c-MET is a well-known proto-oncogene involved in the onset and progression of various human cancers. Herein, we have investigated the expression and availability of HGF and c-MET in TCam-2, NCCIT and NT2D1 cells, which are type II (T)GCT representative cell lines, and the effect of c-MET activation/repression on the regulation of cancerous biological processes. We found that NT2D1 cells increase their proliferation, polarized migration, and invasion in response to HGF administration. NCCIT cells respond to HGF stimulation only partially, whereas TCam-2 cells do not respond to HGF, at least according to the investigated parameters. Interestingly, the immunohistochemical study of c-MET distribution in TGCTs confirm its presence in both seminoma and non-seminoma lesions with different patterns. Notably, we found the highest c-MET immunoreactivity in the epithelial elements of the various components of TGCTs: teratoma, yolk sac tumor and choriocarcinoma.

A young testicular microenvironment protects Leydig cells against age-related dysfunction in a mouse model of premature aging.

Testicular Leydig cells (LCs) are the primary source of circulating androgen in men. As men age, circulating androgen levels decline. However, whether reduced LC steroidogenesis results from specific effects of aging within LCs or reflects degenerative alterations to the wider supporting microenvironment is unclear; inability to separate intrinsic LC aging from that of the testicular microenvironment in vivo has made this question difficult to address. To resolve this, we generated novel mouse models of premature aging, driven by CDGSH iron sulfur domain 2 (Cisd2) deletion, to separate the effects of cell intrinsic aging from extrinsic effects of aging on LC function. At 6 mo of age, constitutive Cisd2-deficient mice display signs of premature aging, including testicular atrophy, reduced LC and Sertoli cell (SC) number, decreased circulating testosterone, increased luteinizing hormone/testosterone ratio, and decreased expression of steroidogenic mRNAs, appropriately modeling primary testicular dysfunction observed in aging men. However, mice with Cisd2 deletion (and thus premature aging) restricted to either LCs or SCs were protected against testicular degeneration, demonstrating that age-related LCs dysfunction cannot be explained by intrinsic aging within either the LC or SC lineages alone. We conclude that age-related LC dysfunction is largely driven by aging of the supporting testicular microenvironment.—Curley, M., Milne, L., Smith, S., Jørgensen, A., Frederiksen, H., Hadoke, P., Potter, P., Smith, L. B. A Young testicular microenvironment protects Leydig cells against age-related dysfunction in a mouse model of premature aging.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin promotes inflammation in mouse testes: The critical role of Klotho in Sertoli cells

Increasing evidence shows that 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) enhances inflammation, and inflammation has a significant negative impact on fertility. Therefore, the aim of this study was to investigate the effects of TCDD on testis inflammation. Pregnant mice and primary Sertoli cells were treated with TCDD, and male offspring and Sertoli cells were treated with lipopolysaccharides(LPS). We then measured testis apoptotic cells, proinflammatory cytokines, and observed the Klotho/PDLIM2/p65 pathway. In vivo results revealed that TCDD further enhanced LPS-increased testis apoptotic cells and concentrations of testicular proinflammatory cytokines (IL1β, IL18, and IL12) (p < 0.05). An in vitro investigation showed the levels of proinflammatory cytokines were increased in TCDD + LPS-treated cells compared with LPS-treated cells (p < 0.05). Compared with the LPS-treated cells, expression of Klotho and PDLIM2 was significantly decreased in TCDD + LPS-treated cells (p < 0.05), while expression of p65 and NLRP3 were significantly increased in the cotreatment cells (p < 0.05). However, the addition of Klotho to the TCDD + LPS-cotreated cells significantly increased PDLIM2 and decreased p65 activation and NLRP3 (p < 0.05). Meanwhile, mRNA levels and the secretion of proinflammatory cytokines were both suppressed by exogenous Klotho (p < 0.05). Administration of Klotho decreased TCDD + LPS-induced cytokines and apoptosis in mice (p < 0.05). Taken together, TCDD may increase testicular inflammation by affecting the secretion of proinflammatory cytokines in Sertoli cells via the Klotho/PDLIM2/p65 pathway, which influences the testicular microenvironment and induces germ cell apoptosis.

Testicular microbiome in azoospermic men-first evidence of the impact of an altered microenvironment.

STUDY QUESTIONGiven the relevant role of the extracellular microenvironment in regulating tissue homeostasis, is testicular bacterial microbiome (BM) associated with germ cell aplasia in idiopathic non-obstructive azoospermia (iNOA)?SUMMARY ANSWERA steady increase of dysbiosis was observed among testis with normal spermatogenesis vs. iNOA with positive sperm retrieval and iNOA with complete germ cell aplasia.WHAT IS KNOWN ALREADYTissue-associated BM has been reported to be a biologically important extracellular microenvironment component for numerous body habitats, but not yet for the human testis.STUDY DESIGN, SIZE, DURATIONCross-sectional study, investigating tissue-associated BM in the testis of (i) five men with iNOA and negative sperm retrieval at microdissection testicular sperm extraction (microTESE); (ii) five men with iNOA and positive sperm retrieval at microTESE; and (iii) five normozoospermic men upon orchiectomy. Every testicular specimen was histologically classified and analyzed in terms of bacterial community.PARTICIPANTS/MATERIALS, SETTING, METHODSMassive ultra-deep pyrosequencing was applied to investigate testis microbiome. Metagenome was analyzed using Quantitative Insights Into Microbial Ecology (QIIME). Tissue-associated bacterial load was quantified by digital droplet PCR.MAIN RESULTS AND THE ROLE OF CHANCENormozoospermic men showed small amounts of bacteria in the testis, with Actinobacteria, Bacteroidetes, Firmicutes Proteobacteria as the dominating phyla; iNOA individuals had increased amounts of bacterial DNA (P = 0.02), associated with decreased taxa richness due to the lack of Bacteroidetes and Proteobacteria (P = 2 × 10−5). Specimens with negative sperm retrieval at microTESE depicted complete germ cell aplasia and a further decrease in terms of Firmicutes and Clostridia (P < 0.05), a complete lack of Peptoniphilus asaccharolyticus, but increased amount of Actinobacteria.LIMITATIONS, REASONS FOR CAUTIONThe limited number of specimens analyzed in this preliminary study deserves external validation. The paraneoplastic microenvironment could have an impact on the residential bacterial flora.WIDER IMPLICATION OF THE FINDINGSHuman testicular microenvironment is not microbiologically sterile, containing low amounts of Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. A dysbiotic bacterial community was associated with iNOA and complete germ cell aplasia. Novel findings on testicular BM could support future translational therapies of male-factor infertility.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by URI-Urological Research Institute free funds. Authors declared no conflict of interest.TRIAL REGISTRATION NUMBERN/A.

In vitro formation of the blood-testis barrier during long-term organotypic culture of human prepubertal tissue: comparison with a large cohort of pre/peripubertal boys.

How does the formation of the blood-testis barrier (BTB), as reflected by the expression of connexin 43 and claudin 11 proteins during the pubertal transition period, take place in vitro compared to samples from a large cohort of pre/peripubertal boys? The BTB connexin 43 and claudin 11 expression patterns appeared to be partially achieved in organotypic culture when compared to that in samples from 71 pre/peripubertal patients. Although alterations in the protein expression patterns of the BTB, whose main components are connexin 43 and claudin 11, are known to be associated with impaired spermatogenesis in mice and adult men, there is a lack of knowledge on its formation in pre-peripubertal human tissue both in vitro and in vivo. Moreover, despite Sertoli cell (SC) maturation during long-term organotypic culture of immature testicular tissue (ITT), initiation of spermatogenesis has not yet been achieved. Histological sections from 71 pre-peripubertal patients were evaluated for the formation of the BTB acting as in vivo controls according to age, SC maturation, clinical signs of puberty and germ cell differentiation. Testicular tissue fragments retrieved from three prepubertal boys were cultured in a long-term organotypic system to analyze the BTB formation and expression pattern in correlation with SC maturation. Testicular histological sections from 71 patients aged 0-16 years who underwent a biopsy between 2005 and 2014 to preserve their fertility before gonadotoxic treatment were examined. Immunohistochemistry (IHC) results for connexin 43 and claudin 11 as BTB markers, using a semi-quantitative score for their expression, and for Anti-Mullerian hormone (AMH), as SC maturation marker, were analyzed. Germ cell differentiation was evaluated on Hematoxylin-Eosin sections. Tanner stages at the time of biopsy were recorded from medical files. A longitudinal analysis of connexin 43, claudin 11 and AMH expressions on immunohistological sections of organotypic cultured testicular tissue from three prepubertal boys who underwent a biopsy for fertility preservation was performed. Immunostaining was evaluated at culture Days 0, 1, 3, 10, 16, 27, 32, 53, 64 and 139 for two different types of culture media. Immunohistochemical control sections showed progressive maturation of SCs, as shown by the decrease in AMH expression, with increasing age (P ≤ 0.01) and the AMH expression was negatively correlated with the expression of connexin 43 and claudin 11 (P ≤ 0.01 for both proteins). Androgen receptor (AR) expression increased with age (P ≤ 0.01) and was significantly correlated with the expression of connexin 43 (P = 0.002) and claudin 11 (P = 0.03). A statistical correlation was also found between the reduction of AMH expression and both the advancement of Tanner stages (P ≤ 0.01) and the differentiation of germ cells (P ≤ 0.01). Furthermore, positive correlations between BTB formation (using connexin 43 and claudin 11 expression) and age (P ≤ 0.01 for both the proteins), higher Tanner stages (P ≤ 0.001 and P ≤ 0.01 for connexin 43 and claudin 11, respectively), and presence of more advanced germ cells (P ≤ 0.001 for both proteins) were observed. In the subanalysis on organotypic cultured ITT, where a significant decrease in AMH expression as a marker of SC maturation was already reported, we showed the onset of expression of connexin 43 at Day 16 (P ≤ 0.001) and a constant expression of claudin 11 from Days 0 to 139, for all three patients, without differences between the two types of culture media. N/A. Accessibility of prepubertal human testicular tissue is a major limiting factor to the analysis of cultured tissue samples from a wide number of patients, as would be needed to assess the in vitro development of the BTB according to the age. The impossibility of performing longitudinal studies on in vivo BTB formation in the same patient prevents a comparison of the time needed to achieve effective BTB formation and protein expression patterns in vivo and in vitro. To the best of our knowledge, this is the first report describing the expression of two BTB proteins in samples from a cohort of prepubertal and peripubertal boys, for the in vivo pattern, and in cultured ITT from a few prepubertal boys, for the in vitro evaluation. Since the formation of this barrier is essential for spermatogenesis and because little is known about its protein expression patterns and development in humans, a deeper understanding of the testicular microenvironment is essential to improve ITT in vitro culture conditions. The final aim is to restore fertility by acheiving in vitro differentiation of spermatogonial stem cells, using cryopreserved ITT collected before gonadotoxic therapies. Funding was received from Fonds National de la Recherche Scientifique de Belgique (Grant Télevie Nos. 7.4554.14F and 7.6511.16) and Fondation Salus Sanguinis. No conflict of interest has to be disclosed.

Testicular organoids: a new model to study the testicular microenvironment in vitro?

In recent decades, a broad range of strategies have been applied to model the testicular microenvironment in vitro. These models have been utilized to study testicular physiology and development. However, a system that allows investigations into testicular organogenesis and its impact in the spermatogonial stem-cell (SSC) niche in vitro has not been developed yet. Recently, the creation of tissue-specific organ-like structures called organoids has resurged, helping researchers to answer scientific questions that previous in vitro models could not help to elucidate. So far, a small number of publications have concerned the generation of testicular organoids and their application in the field of reproductive medicine and biology. Here, we aim to elucidate whether testicular organoids might be useful in answering current scientific questions about the regulation and function of the SSC niche as well as germ cell proliferation and differentiation, and whether or not the existing in vitro models are already sufficient to address them. Moreover, we would like to discuss how an organoid system can be a better solution to address these prominent scientific problems in our field, by the creation of a rationale parallel to those in other areas where organoid systems have been successfully utilized. We comprehensively reviewed publications regarding testicular organoids and the methods that most closely led to the formation of these organ-like structures in vitro by searching for the following terms in both PubMed and the Web of Science database: testicular organoid, seminiferous tubule 3D culture, Sertoli cell 3D culture, testicular cord formation in vitro, testicular morphogenesis in vitro, germ cell 3D culture, in vitro spermatogenesis, testicular de novo morphogenesis, seminiferous tubule de novo morphogenesis, seminiferous tubule-like structures, testicular in vitro model and male germ cell niche in vitro, with no restrictions to any publishing year. The inclusion criteria were based on the relation with the main topic (i.e. testicular organoids, testicular- and seminiferous-like structures as in vitro models), methodology applied (i.e. in vitro culture, culture dimensions (2D, 3D), testicular cell suspension or fragments) and outcome of interest (i.e. organization in vitro). Publications about grafting of testicular tissue, germ-cell transplantation and female germ-cell culture were excluded. The application of organoid systems is making its first steps in the field of reproductive medicine and biology. A restricted number of publications have reported and characterized testicular organoids and even fewer have denominated such structures by this method. However, we detected that a clear improvement in testicular cell reorganization is recognized when 3D culture conditions are utilized instead of 2D conditions. Depending on the scientific question, testicular organoids might offer a more appropriate in vitro model to investigate testicular development and physiology because of the easy manipulation of cell suspensions (inclusion or exclusion of a specific cell population), the fast reorganization of these structures and the controlled in vitro conditions, to the same extent as with other organoid strategies reported in other fields. By way of appropriate research questions, we might use testicular organoids to deepen our basic understanding of testicular development and the SSC niche, leading to new methodologies for male infertility treatment.

Molecular Pathogenesis of Testicular Germ Cell Tumors

Testicular germ cell tumors (TGCT) are the most frequently diagnosed solid tumors in young men and their incidence has been increasing over the past decades. Several factors may combine and play a role in the TGCT etiology, including environmental factors and genetic predispositions at multiple genomic loci that affect both testicular germ cells and stromal cells, and their interactions within the testicular microenvironment. The pathogenesis of TGCT starts prenatally with primordial germ cell arrest, and further proceeds postnatally, giving rise to in situ germ cell neoplasia and, finally, to invasive TGCT with the characteristic 12p chromosome amplification. Apart from the genes localized here, further molecular mechanisms have been linked to TGCT pathogenesis, such as the activation of the KIT/KITL signaling pathway, and aberrations in genes involved in DNA reparation, regulation of cellular differentiation, proliferation, and survival. Despite the relatively good prognosis and known etiopathogenesis of these tumors, neither targeted therapy nor molecular prognostic/predictive factors have yet been implemented in the management of TGCT, because there is not enough information about the molecular pathways or molecules involved in TGCT development that could be used for patient stratification and treatment. Current high-throughput technologies, such as next generation sequencing at the exome or transcriptome level could provide this missing information on genetic predispositions and other factors influencing the clinical course of the disease and treatment response in TGCT. In this review, we summarize the main molecular characteristics of TGCT and the probable mechanisms participating in tumor initiation and progression.Key words: testicular germ cell tumors - signaling pathways - molecular aberrations - predictive factors - prognostic factors The work was supported by the Czech Ministry of Education, Youth and Sports NPU I nr.LO 1604. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 20. 3. 2017Accepted: 23. 7. 2017.

Fertility restoration with spermatogonial stem cells.

This review evaluates the state of the art in terms of challenges and strategies used to restore fertility with spermatogonial stem cells retrieved from prepubertal boys affected by cancer. Although these boys do not yet produce spermatozoa, the only option to preserve their fertility is cryopreservation of spermatogonial stem cells in the form of testicular cell suspensions or whole tissue pieces. Different techniques have been described to achieve completion of spermatogenesis from human, spermatogonial stem cells but none is yet ready for clinical application. A crucial point to address is gaining a full understanding of spermatogonial stem cell niche pathophysiology, where germ cells undergo proliferation and differentiation. Various fertility restoration approaches will be presented depending on the presence of an intact niche, dissociated niche, or reconstituted niche. Testicular organoids open the way to providing further insights into the niche. They can recreate the three-dimensional architecture of the testicular microenvironment in vitro, allowing a large number of applications, from physiology to drug toxicity investigations. In addition to the full elucidation of the niche microenvironment, achieving fertility restoration from cryopreserved human spermatogonial stem cells implies overcoming other important challenges. Testicular organoids might prove to be essential tools to progress in this field.

Testicular organoid generation by a novel invitro three-layer gradient system.

A system that models the testicular microenvironment and spermatogonial stem-cell (SSC) niche invitro has not been produced yet. Here, we developed and characterized a novel three-dimensional multilayer model, the Three-Layer Gradient System (3-LGS), which permits the generation of rat testicular organoids with a functional blood-testis barrier (BTB) and germ cell establishment and proliferation. The model is unique as regards the formation of cellular organizations that more closely represent the invivo germ-to-somatic cell associations invitro. Moreover, we also verified the roles of retinoic acid (RA), IL-1α, TNFα and RA inhibitors in germ cell maintenance and BTB organization invitro. Treatment with RA was beneficial for germ cell maintenance, while IL-1α and TNFα were observed to impair the formation of testicular organoids and germ cell maintenance. Taking in account our characterization and validation results, we propose the 3-LGS as a new platform to investigate the SSC niche invitro and to search for novel unknown factors involved in germ cell proliferation and differentiation. Moreover, we suggest that this model can be used in other scientific fields to study organogenesis and development by the generation of organoids.

Primary Human Testicular Cells Self-Organize into Organoids with Testicular Properties.

SummarySo far, successful de novo formation of testicular tissue followed by complete spermatogenesis in vitro has been achieved only in rodents. Our findings reveal that primary human testicular cells are able to self-organize into human testicular organoids (TOs), i.e., multi-cellular tissue surrogates, either with or without support of a biological scaffold. Despite lacking testis-specific topography, these mini-tissues harbored spermatogonia and their important niche cells, which retained specific functionalities during long-term culture. These observations indicate the posibility of in vitro re-engineering of a human testicular microenvironment from primary cells. Human TOs might help in the development of a biomimetic testicular model that would exert a tremendous impact on research and development, clinical treatment of infertility, and screening in connection with drug discovery and toxicology.