FIG 8 - uploaded by Michèle Weiss-Gayet
Content may be subject to copyright.
Immunocytochemical staining of the acrosomal granule and of BrdU-labeled nuclei in a coculture of PS and Sertoli cells. a) An RS positive for the MN7 antibody and negative for the anti-BrdU antibody. Counterstaining: Mayer's hematoxylin. Day 2 of coculture (X875). b) An RS positive with both antibodies. The acrosome granule is light brown as a result of the reaction with DAB. The nucleus is dark red as a result of the reaction with amino-3-ethyl-9-carbazole. Day 10 of coculture (x875).
Source publication
The present study was aimed at examining, by reverse transcription polymerase chain reaction, the expression of germ cell-specific genes in cocultures of Sertoli cells with either pachytene spermatocytes (PS) or round spermatids (RS). In situ hybridization studies showed that the mRNAs encoding phosphoprotein p19 and the testis-specific histone TH2...
Context in source publication
Context 1
... appearance of spermatids exhibiting a positive reaction with both anti-BrdU and anti-MN7 antibodies (see Materials and Methods) was monitored during the culture. While only spermatids not reacting with the anti-BrdU an- tibody could be visualized on Day I or Day 2, anti-BrdU- labeled RS could be observed from Day 4 until the end of the culture (Fig. 8). By contrast, the morphological ap- pearance of elutriated spermatids seeded on Sertoli cell monolayers became more and more impaired with time in culture so that, from Day 7 onward, it was rather difficult to identify these cells (data not ...
Similar publications
Objective: Microtubule (MT) stability in neurons is vital for brain development; instability is associated with neuropsychiatric disorders. The present study examined the effects of social defeat stress (SDS) on MT-regulating proteins and tubulin polymerization.
Methods: After 10 days of SDS, defeated mice were separated into susceptible (Sus) and...
Aptamers are chimerized with drug or antisense oligos or nanoparticles to generate targeted therapeutics for cancer. Aptamer chimerized siRNA rescues nonspecific delivery and, thereby, enhances the availability of siRNA to target cells. EpCAM RNA aptamer (EpApt or Ep) has potential for siRNA chimerization due to its secondary structure. Stathmin an...
Gastric cancer is the second common cause of cancer related death worldwide and lacks highly effective treatment for advanced disease. Nab-paclitaxel is a novel microtubule-inhibitory cytotoxic agent that has not been tested in gastric cancer as of yet. In this study, human gastric cancer cell lines AGS, NCI-N87 and SNU16 were studied. Nab-paclitax...
Tumor metastasis is the main cause of death in advanced colorectal cancer. Our previous research showed that upregulation of KIAA1199 predicted poorer outcomes, and promoted cell motility and tumor metastasis in colorectal cancer, with the mechanisms not being fully elucidated. Here, we demonstrate that silencing of KIAA1199 results in reduced tumo...
Recent studies have suggested a protective role of hsp27 against atherosclerosis and transplant graft vasculopathy. Here we have investigated the effects of over-expression of wild-type hsp27 and its phosphorylation mimics on proliferation of human endothelial cells (ECs) and smooth muscle cells (SMCs). ECs and SMCs cultured from human blood vessel...
Citations
... The germ cell-Sertoli cell coculture systems that we settled [8][9][10]; have been carefully validated from the physiological point of view, on many aspects, over the last decades. To our knowledge, there is no other system of culture of male germ cells and Sertoli cells which has been so carefully and extensively validated. ...
... In this study, porcine SSCs were successfully induced to differentiate into haploid spermatozoa in vitro. After 9 days of culture, spermatids with a single flagellum were formed (Gerton and Millette, 1984), indicating that a much shorter time span was required in-vitro compared with in-vivo spermatogenesis (Weiss et al., 1997). By adding RA, the differentiation efficiency of haploid cells was enhanced. ...
Spermatogonial stem cells (SSCs) self-renew and contribute genetic information to the next generation. Pig is wildly used as a model animal for understanding reproduction mechanisms of human being. Inducing directional differentiation of porcine SSCs may be an important strategy in exploring the mechanisms of spermatogenesis and developing better treatment methods for male infertility. Here, we established an in-vitro culture model for porcine small seminiferous tubule segments, to induce SSCs to differentiate into single-tail haploid spermatozoa. The culture model subsequently enabled spermatozoa to express the sperm-specific protein acrosin and oocytes to develop to blastocyst stage after round spermatid injection. The addition of retinoic acid (RA) to the differentiation media promoted the efficiency of haploid differentiation. RT-PCR analysis indicated that RA stimulated the expression of stimulated by retinoic acid gene 8 (Stra8) but reduced the expression of NANOS2 in spermatogonia. Genes involved in post-meiotic development, transition protein 1 (Tnp1) and protamine 1 (Prm1), were up-regulated in the presence of RA. The addition of a retinoic acid receptor (RAR) inhibitor, BMS439, showed that RA enhanced the expression of cAMP responsive-element binding protein (CREB) through RAR and promoted the formation of round spermatids. We established an efficient culture system for in-vitro differentiation of pig SSCs. Our study represents a model for human testis disease and toxicology screening. Molecular regulators of SSC differentiation revealed in this study might provide a therapeutic strategy for male infertility.
... Different techniques of IVM of GCs have been studied in both animals and humans, namely 2D and 3D culture of testicular cells, organotypic culture of testicular tissue, and the latest approach based on organoids ( Galdon et al., 2016;Alves-Lopes and Stukenborg, 2017). In mice, since the first description of meiosis in vitro ( Weiss et al., 1997), the entire process of spermatogenesis has been reported successfully (Dumont et al., 2015) with generation of offspring both from fresh and cryopreserved murine ITT ( Sato et al., 2011;Yokonishi et al., 2014). In these reports, Knock-out Serum Replacement (KSR) 10% was used as culture medium, either with addition of FSH and testosterone (Sato et al., 2011;Yokonishi et al., 2014) or not ( Dumont et al., 2015). ...
While in mice various studies have described the completion of spermatogenesis in vitro using either organotypic culture of prepubertal testicular tissue or 3D culture of isolated cells, in humans it has not been possible to achieve germ cell differentiation from immature testicular tissue (ITT). In our study, we evaluated the ability of human ITT to differentiate via a long-term organotypic culture of frozen–thawed 1 mm3 testicular fragments from five prepubertal boys in two different culture media. Tissue and supernatants were analyzed at regular intervals up to day 139. Sertoli cell (SC) viability and maturation was evaluated using immunohistochemistry (IHC) for SOX9, GDNF, anti-Mullerian hormone (AMH) and androgen receptor (AR), and AMH concentration in supernatants. Spermatogonia (SG) and proliferating cells were identified by MAGE-A4 (for SG) and Ki67 (for proliferating cells) via immunohistochemistry (IHC). Apoptotic cells were studied by active caspase 3. To evaluate Leydig cell (LC) functionality testosterone was measured in the supernatants and steroidogenic acute regulatory protein (STAR) IHC was performed. Germ cell differentiation was evaluated on Hematoxylin-Eosin histological sections, via IHC for synaptonemal complex 3 (SYCP3) for spermatocytes, Protein boule-like (BOLL) for spermatocytes and round spermatids, angiotensin-converting enzyme (ACE), protamine 2 and transition protein 1 (for elongated spermatids) and via chromogenic in situ hybridization (CISH). We reported the generation of meiotic and postmeiotic cells after 16 days of culture, as shown by the histological analyses, the presence of differentiation markers and the increase of haploid germ cells. We showed SC viability and maturation by a decrease of AMH secretion in the supernatants (p ≤ 0.001) while the number of SOX9 positive cells did not show any variation. A decrease of spermatogonia (p ≤ 0.001) was observed. The number of apoptotic cells did not vary. LC functionality was shown by the increase in STAR expression (p ≤ 0.007) and a peak in testosterone secretion, followed by a reduction (p ≤ 0.001) with stabilization. According to our knowledge, this is the first report of generation of haploid cells in human ITT. Differentiating germ cells have to be further evaluated for their ability to complete differentiation, their fecundability and epigenetic characteristics.
... Different techniques of IVM of GCs have been studied in both animals and humans, namely co-culture with Vero cells, 3D culture and organotypic culture (Galdon et al., 2016). In humans, none have yet resulted in completion of the spermatogenic process in vitro, but in mice, since the first report of meiosis completion in vitro (Weiss et al., 1997), the entire process of spermatogenesis has been reproduced in an organotypic culture system, yielding fertile offspring (Sato et al., 2011;Yokonishi et al., 2013). Sato et al. (2011) used fetal bovine serum and AlbuMAX™, a lipidrich albumin fraction of bovine serum and transferrin which, according to the authors, is the principal component able to induce spermatogonial differentiation. ...
Study question:
Is an organotypic culture system able to provide the appropriate testicular microenvironment for in-vitro maturation of human immature testicular tissue (ITT)?
Summary answer:
Our organotypic culture system provided a microenvironment capable of preserving seminiferous tubule (ST) integrity and Leydig cell (LC) functionality and inducing Sertoli cell (SC) maturation.
What is known already:
Cryopreservation of human ITT is a well-established strategy to preserve fertility in prepubertal boys affected by cancer, with a view for obtaining sperm. While spermatogenesis in mice has been replicated in organotypic culture, yielding reproductively efficient spermatozoa, this process has not yet been achieved in humans.
Study design, size, duration:
The aim of this study was to in vitro mature frozen-thawed ITT. To this end, 1 mm(3) tissue fragments from three prepubertal patients aged 2 (P1), 11 (P2) and 12 (P3) years were placed in organotypic culture for 139 days. Culture media, supplemented with either testosterone or hCG, were compared.
Participants/materials, setting, methods:
ST integrity and tissue viability were assessed by histological score and lactate dehydrogenase (LDH) levels in supernatants. Spermatogonia (SG), proliferating cells and proliferating SG were identified by the use of MAGE-A4 and Ki67 immunohistochemical markers. Glial cell line-derived neurotrophic factor (GDNF) was used as a marker of SC functionality, while SC maturation was evaluated by androgen receptor (AR), anti-Müllerian hormone (AMH) immunohistochemistry (IHC) and AMH immunoenzymatic assay. LC functionality was determined by testosterone levels in supernatants and by 3β-hydroxysteroid dehydrogenase (3β-HSD) IHC. Apoptosis was studied by IHC with active caspases 3 and 8 and by TUNEL (terminal deoxynubocleotidyl transferase-mediated dUTP nick end labeling) analysis.
Main results and the role of chance:
Tissue viability was preserved, as demonstrated by the decrease in and stabilization of LDH release, and evolution of ST scoring, with the percentage of well-preserved STs showing no statistical differences during culture in either medium. GDNF was expressed until Day 139, demonstrating SC functionality. Moreover, a significant reduction in AMH expression and release indicated SC maturation. Testosterone concentrations in supernatants increased in both culture media, demonstrating LC functionality with paracrine interactions. SG were present up to Day 139, although the ratio between MAGE-A4-positive cells and well-preserved tubules was significantly reduced over the course of culture (P ≤ 0.001). SCs exhibited a decreased proliferation rate over time (P ≤ 0.05). The proliferation rate of SG remained stable until Day 64, but over the total culture period (139 days), it was found to have decreased (P ≤ 0.05). The number of apoptotic cells did not vary during culture, nor was any statistical difference observed between the two culture media for any of the studied parameters.
Large scale data:
N/A LIMITATIONS, REASONS FOR CAUTION: Loss of SG constitutes a limitation for evaluating full functionality of spermatogonial stem cells and warrants further investigation. The scarcity of human immature material is the reason for the limited amount of tissue available for experiments, precluding more comprehensive analysis.
Wider implications of the findings:
Our culture system, mimicking the peripubertal testicular microenvironment with SC maturation, LC functionality and preserved paracrine interactions, and the first to use human ITT, opens the door to a deeper understanding of niche and culture conditions to obtain sperm from cryostored ITT, with the ultimate goal of restoring fertility after gonadotoxic treatments.
Study funding/competing interests:
This project was supported by a grant from the Fond National de la Recherche Scientifique de Belgique (grant Télevie N° 7.4554.14F and N° 7.4512.15F) and the Fondation Salus Sanguinis. No conflict of interest is declared.
... These steps are regulated by pituitary hormones (mainly FSH and LH) and by soluble and membrane bound factors produced by both the somatic cells and the germ cells of the testis [1,2]. During the past twenty years, we have developed two culture systems of rat germ cells together with Sertoli cells, the somatic cells of the seminiferous tubules supporting the germ cells [3,4]. We were the first to show that the whole meiotic process could occur in-vitro/ex-vivo in a mammal, the rat [5]. ...
Until now, complete ex-vivo spermatogenesis has been reported only in the mouse. In this species the duration of spermatogenesis is 35 days, whereas it is 54 days in the rat and 74 days in the human. We performed long term (until 60 days) cultures of fresh or frozen rat or human seminiferous tubule segments in a bioreactor, made of a hollow cylinder of chitosan hydrogel. Testicular tissues were obtained from 8 or 20 day old male rats, or from adult human subjects having undergone hormonal treatments leading to a near complete regression of their spermatogenesis, before bilateral orchiectomy for gender reassignment. The progression of spermatogenesis was assessed by cytological analyses of the cultures; it was related with a dramatic increase in the levels of mRNAs specifically expressed by round spermatids: Transition protein 1, Transition protein 2 and Protamine 3 in rat cultures. Two to 3.8% of cells were found haploid cells by FISH analysis of human cultures. In this bioreactor, long term cultures of seminiferous tubule segments from (pre)-pubertal rats, or from adult men, allowed the completion of the spermatogenic process, leading to morphologically mature spermatozoa. Further studies will have to address the way of optimizing the yield of every step of spermatogenesis, by adjusting the composition of the culture medium, the geometry and material properties of the chitosan hydrogel bioreactors. Another essential requirement is to assess the quality of the gametes produced ex-vivo by showing their ability to produce normal offspring (rat) or their biochemical normality (human).
... During mammalian spermatogenesis, diploid spermatogonia divide mitotically to provide spermatocytes that proceed through meiosis to haploid spermatids. This process depends on a specific environment provided by the somatic cells of the testis and requires endocrine and auto/paracrine regulation, as well as direct cell-cell interactions [8]. Sertoli cells (SCs) in the mammalian seminiferous epithelium are involved in the regulation of germ cell development by providing nutrients and hormonal signals needed for spermatogenesis [9]. ...
Background
Microenvironment signals play a critical role in directing the differentiation of stem cells. Sertoli cells (SCs) provide a unique microenvironment that is essential for germ cell differentiation.Methods
Our previous study has demonstrated that human umbilical cord Wharton¿s jelly-derived mesenchymal stem cells (HUMSCs) could differentiate towards male germ cells in vitro, but HUMSC-derived germ-like cells expressed only few germ cell markers. The aim of this study was to investigate the effect of SCs on the differentiation of HUMSCs towards male germ cells using a co-culture system that mimicked the in vivo male germ cell microenvironment.ResultsHUMSCs formed clump-like features on SC monolayers after seeding for 3 weeks. Differentiated cells formed round colonies that share the morphological features of spermatogonial colonies. RT-PCR, immunofluorescence, confocal microscopy, and Western blot analyses revealed the expression of early germ cell markers STELLA and VASA and male germ cell-specific marker DAZL in differentiated HUMSCs, confirming the presence of cells with characteristics of male germ cells.Conclusion
The HUMSC-SC co-culture system mimics a native microenvironment for germ cell colonization without any in vitro artificial manipulation and can be used to explore the mechanisms controlling the differentiation of male germ cells from HUMSCs. Male germ cells derived from HUMSCs may be used in the therapy for male infertility.
... MEFs were isolated from E12.5 embryos as previously described. Sertoli cells were isolated from ∼10to 14-day-old mice and cultured as described previously (Weiss et al., 1997). The mouse TM4 Sertoli and human BOSC cell lines were obtained from the American Tissue Culture Collection (ATCC). ...
The four related mammalian MEX-3 RNA-binding proteins are evolutionarily conserved molecules for which the in vivo functions have not yet been fully characterized. Here, we report that male mice deficient for the gene encoding Mex3b are subfertile. Seminiferous tubules of Mex3b-deficient mice are obstructed as a consequence of the disrupted phagocytic capacity of somatic Sertoli cells. In addition, both the formation and the integrity of the blood-testis barrier are compromised owing to mislocalization of N-cadherin and connexin 43 at the surface of Sertoli cells. We further establish that Mex3b acts to regulate the cortical level of activated Rap1, a small G protein controlling phagocytosis and cell-cell interaction, through the activation and transport of Rap1GAP. The active form of Rap1 (Rap1-GTP) is abnormally increased at the membrane cortex and chemically restoring Rap1-GTP to physiological levels rescues the phagocytic and adhesion abilities of Sertoli cells. Overall, these findings implicate Mex3b in the spatial organization of the Rap1 pathway that orchestrates Sertoli cell functions.
... Some of the first attempts to culture mammalian male germ cells were carried out by Durand and colleagues over the past decade [27][28][29][30][31]. Over this time they have developed a co-culture technique that allows meiotic progression of rat spermatocytes. ...
... Over this time they have developed a co-culture technique that allows meiotic progression of rat spermatocytes. Briefly, it consists of co-culture of prepuberal rat spermatocytes on a monolayer of Sertoli cells in a bicameral chamber [31]. In their experiments, meiotic progression was followed by different approaches, including electron microscopy ultrastructural analysis of the cultured cells, analysis of the DNA content by fluorescence activated cell sorting (FACS) and expression of some postmeiotic specific genes. ...
The study of meiosis is limited because of the intrinsic nature of gametogenesis in mammals. One way to overcome these limitations would be the use of culture systems that would allow meiotic progression in vitro. There have been some attempts to culture mammalian meiocytes in recent years. In this review we will summarize all the efforts to-date in order to culture mammalian sperm and oocyte precursor cells.
... To isolate Spga and Sertoli cells, testes of 45 rats at 9 dpp and 20 rats at 22 dpp were excised and decapsulated. The procedure of cell purification is depicted in Additional file 2, Figure S2 and was adapted from previous publications [84][85][86]. Seminiferous tubules were first isolated from surrounding interstitial cells by collagenase dispase treatement. After three sedimentation steps, the tubules were separated in two fractions. ...
Stem cells and their niches are studied in many systems, but mammalian germ stem cells (GSC) and their niches are still poorly understood. In rat testis, spermatogonia and undifferentiated Sertoli cells proliferate before puberty, but at puberty most spermatogonia enter spermatogenesis, and Sertoli cells differentiate to support this program. Thus, pre-pubertal spermatogonia might possess GSC potential and pre-pubertal Sertoli cells niche functions. We hypothesized that the different stem cell pools at pre-puberty and maturity provide a model for the identification of stem cell and niche-specific genes. We compared the transcript profiles of spermatogonia and Sertoli cells from pre-pubertal and pubertal rats and examined how these related to genes expressed in testicular cancers, which might originate from inappropriate communication between GSCs and Sertoli cells.
The pre-pubertal spermatogonia-specific gene set comprised known stem cell and spermatogonial stem cell (SSC) markers. Similarly, the pre-pubertal Sertoli cell-specific gene set comprised known niche gene transcripts. A large fraction of these specifically enriched transcripts encoded trans-membrane, extra-cellular, and secreted proteins highlighting stem cell to niche communication. Comparing selective gene sets established in this study with published gene expression data of testicular cancers and their stroma, we identified sets expressed genes shared between testicular tumors and pre-pubertal spermatogonia, and tumor stroma and pre-pubertal Sertoli cells with statistic significance.
Our data suggest that SSC and their niche specifically express complementary factors for cell communication and that the same factors might be implicated in the communication between tumor cells and their micro-environment in testicular cancer.
... Depuis plus de 12 ans, notre équipe a développé des systèmes de coculture des cellules germinales de rats mâles avec des cellules de Sertoli, dans un milieu de culture sans sérum, permettant d'étudier ex vivo différentes étapes de la spermatogenèse (la phase mitotique, la totalité de l'étape méiotique ainsi que le début de la spermiogenèse) sur une durée allant jusqu'à quatre semaines [6][7][8]. En effet, ces cocultures, réalisées en chambres de culture bicamérales, permettent la (re-)formation et/ou le maintien de la barrière hématotesticulaire, qui est essentielle au bon déroulement de la spermatogenèse. La fonctionnalité de la barrière hématotesticulaire ex vivo est attestée par plusieurs critères : ...
