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Preview: Molecular Human Reproduction - current issue

MHR: Basic science of reproductive medicine Current Issue





Published: Mon, 02 Apr 2018 00:00:00 GMT

Last Build Date: Wed, 18 Apr 2018 07:49:50 GMT

 



Thank you to our reviewers

Mon, 02 Apr 2018 00:00:00 GMT







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

Mon, 12 Mar 2018 00:00:00 GMT

AbstractSTUDY QUESTIONHow 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?SUMMARY ANSWERThe 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.WHAT IS KNOWN ALREADYAlthough 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.STUDY DESIGN, SIZE, DURATIONHistological 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.PARTICIPANTS/MATERIALS, SETTING, METHODSTesticular 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.MAIN RESULTS AND THE ROLE OF CHANCEImmunohistochemical 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.LARGE SCALE DATAN/A.LIMITATIONS REASONS FOR CAUTIONAccessibility 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 imp[...]



Characterization of migratory primordial germ cells in the aorta-gonad-mesonephros of a 4.5-week-old human embryo: a toolbox to evaluate in vitro early gametogenesis

Thu, 08 Mar 2018 00:00:00 GMT

AbstractSTUDY QUESTIONWhich set of antibodies can be used to identify migratory and early post-migratory human primordial germ cells (hPGCs)?STUDY FINDINGWe validated the specificity of 33 antibodies for 31 markers, including POU5F1, NANOG, PRDM1 and TFAP2C as specific markers of hPGCs at 4.5 weeks of development of Carnegie stage (CS12–13), whereas KIT and SOX17 also marked the intra-aortic hematopoietic stem cell cluster in the aorta-gonad-mesonephros (AGM).WHAT IS KNOWN ALREADYThe dynamics of gene expression during germ cell development in mice is well characterized and this knowledge has proved crucial to allow the development of protocols for the in vitro derivation of functional gametes. Although there is a great interest in generating human gametes in vitro, it is still unclear which markers are expressed during the early stages of hPGC development and many studies use markers described in mouse to benchmark differentiation of human PGC-like cells (hPGCLCs). Early post-implantation development differs significantly between mice and humans, and so some germ cells markers, including SOX2, SOX17, IFITM3 and ITGA6 may not identify mPGCs and hPGCs equally well.STUDY DESIGN, SIZE, DURATIONThis immunofluorescence study investigated the expression of putative hPGC markers in the caudal part of a single human embryo at 4.5 weeks of development.PARTICIPANTS/MATERIALS, SETTING, METHODSWe have investigated by immunofluorescence the expression of a set of 33 antibodies for 31 markers, including pluripotency, germ cell, adhesion, migration, surface, mesenchymal and epigenetic markers on paraffin sections of the caudal part, including the AGM region, of a single human embryo (CS12–13). The human material used was anonymously donated with informed consent from elective abortions without medical indication.MAIN RESULTS AND THE ROLE OF CHANCEWe observed germ cell specific expression of NANOG, TFAP2C and PRDM1 in POU5F1+ hPGCs in the AGM. The epigenetic markers H3K27me3 and 5mC were sufficient to distinguish hPGCs from the surrounding somatic cells. Some mPGC-markers were not detected in hPGCs, but marked other tissues; whereas other markers, such as ALPL, SOX17, KIT, TUBB3, ITGA6 marked both POU5F1+ hPGCs and other cells in the AGM. We used a combination of multiple markers, immunostaining different cellular compartments when feasible, to decrease the chance of misidentifying hPGCs.LARGE SCALE DATANon-applicable.LIMITATIONS REASONS FOR CAUTIONMaterial to study early human development is unique and very rare thus restricting the sample size. We have used a combination of antibodies limited by the number of paraffin sections available.WIDER IMPLICATIONS OF THE FINDINGSMost of our knowledge on early gametogenesis has been obtained from model organisms such as mice and is extrapolated to humans. However, since there is a dedicated effort to produce human artificial gametes in vitro, it is of great importance to determine the expression and specificity of human-specific germ cell markers. We provide a systematic analysis of the expression of 31 different markers in paraffin sections of a CS12–13 embryo. Our results will help to set up a toolbox of markers to evaluate protocols to induce hPGCLCs in vitro.STUDY FUNDING AND COMPETING INTEREST(S)M.G.F. was funded by Fundação para a Ciência e Tecnologia (FCT) [SFRH/BD/78689/2011] and S.M.C.S.L. was funded by the Interuniversity Attraction Poles (IAP, P7/07) and the European Research Council Consolidator (ERC-CoG-725722-OVOGROWTH). The authors declare no conflict of interest.[...]



Mimicking physiological O2 tension in the female reproductive tract improves assisted reproduction outcomes in pig

Tue, 27 Feb 2018 00:00:00 GMT

AbstractSTUDY QUESTIONIs O2 tension in the pig oviduct and uterus affected by the estrous cycle stage and the animal’s age, and can the outcome of in vitro embryo development be improved by mimicking these physiological values?SUMMARY ANSWERO2 tension within the pig reproductive organs is affected by the animal’s age, and values close to those measured in vivo have a positive impact on embryo development and quality when used during IVF and embryo culture (EC).WHAT IS KNOWN ALREADYTo obtain a healthy embryo in vitro, it is necessary to adopt a culture microenvironment that approximates physiological conditions. Despite advances in surgical procedures and sensitive probes that allow accurate assessment of in vivo O2 tension, few such studies have been conducted recently in mammals. In addition, no reference values of physiological O2 tension in the reproductive tract exist for large animal models such as pig, and the effect of O2 tension on ART outcomes is unknown.STUDY DESIGN, SIZE, DURATIONThis study was conducted in pigs. We measured oviductal and uterine O2 tension (n = 29 and 13, respectively) and then examined how the use of the physiological values in pig IVF and EC affected pig ART output (n = 1447 oocytes).PARTICIPANTS/MATERIALS, SETTING, METHODSThe oviductal and uterine O2 tension at the different stages of the estrous cycle was monitored using a laparo-endoscopic single-site surgery (LESS) assisted approach along with a flexible and thin miniaturized luminescent probe. Two groups of pigs, Large-white × Landrace breed, were used: for the first group, 16 pre-pubertal gilts (5 months old and 95 kg) were induced to ovulate with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG); in the second group 13 mature sows (24–48 months and 185 kg) were used. IVF and EC were performed at two different O2 tensions: Atmospheric O2 (20%) and the mean in vivo value measured (7%). At 18–20 h post-insemination (hpi), a small sample of presumptive zygotes were fixed, stained and examined under epifluorescence microscopy to assess the fertilization rates. At 48 hpi, cleavage was evaluated under the stereomicroscope. Finally, at 180 hpi, development to the blastocyst stage was quantified, blastocyst morphology was assessed, and embryos were fixed and stained to count the mean cell number per blastocyst.MAIN RESULTS AND THE ROLE OF CHANCEThe mean O2 content within the pig oviduct and uterus was always lower than in ambient air. The average O2 percentage was higher in gilts (10.0%) than in sows (7.6%) (P < 0.0001). The cleavage rate of porcine in vitro fertilized embryos maintained under 7% O2 during IVF and EC was significantly higher (60.0 ± 2.3) compared with those cultured under 20% O2 (32.0 ± 2.2) (P < 0.05). An increase in the number of cells in embryos cultured under the low O2 concentration (88.9 ± 5.9) was observed compared to those cultured under 20% O2 (59.0 ± 5.0) (P < 0.05).LARGE SCALE DATANone.LIMITATIONS, REASONS FOR CAUTIONAlthough minimally invasive surgery was used the effect of anesthesia and manipulations on O2 tension within the organs are unknown.WIDER IMPLICATIONS OF THE FINDINGSUsing physiological oxygen concentrations in IVF/EC could improve ART outcomes.STUDY FUNDING AND COMPETING INTEREST(S)This study was funded by Spanish Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER). Grants AGL2012-40180-C03-01 and AGL2015-66341-R. The authors declare no conflict of interest.[...]



A germline-specific role for the mTORC2 component Rictor in maintaining spermatogonial differentiation and intercellular adhesion in mouse testis

Fri, 09 Feb 2018 00:00:00 GMT

Abstract
STUDY QUESTION
What is the physiological role of Rictor in spermatogenic cells?
SUMMARY ANSWER
Germline expression of Rictor regulates spermatogonial differentiation and has an essential role in coordinating germ cells and Sertoli cells in maintaining intact cell–cell adhesion dynamics and cytoskeleton-based architecture in the seminiferous epithelium.
WHAT IS KNOWN ALREADY
The mechanistic target of rapamycin (mTOR) resides in its functions as the catalytic subunits of the structurally and functionally distinct mTORC1 and mTORC2 complexes. In the mammalian testis, mTORC1 regulates spermatogonial stem cell self-renewal and differentiation, whereas mTORC2 is required for Sertoli cell function. In contrast to mTORC1, mTORC2 has been much less well studied. Rictor is a distinct component of the mTORC2 complex.
STUDY DESIGN, SIZE, DURATION
We investigated the effects of germ cell-specific ablation of Rictor on testicular development by using a mouse model of germline-specific ablation of Rictor.
PARTICIPANTS/MATERIALS, SETTING, METHODS
We analyzed the in-vivo functions of Rictor through different methods including histology, immunofluorescent staining, chromosome spreads, blood–testis barrier (BTB) integrity assays and RNA sequencing.
MAIN RESULTS AND THE ROLE OF CHANCE
Mutant mice did not show a defect in meiotic synapsis or recombination, but exhibited compromised spermatogonial differentiation potential, disorganized cell–cell junctions, impaired BTB dynamics and defective spermiogenesis. Concomitantly, RNA-seq profiling revealed that many genes involved in adhesion and migration were expressed inappropriately.
LARGE SCALE DATA
RNA-seq data are published in the SRA database (PRJNA419273).
LIMITATIONS REASONS FOR CAUTION
A detailed analysis of the mechanisms underlying the phenotype needs further investigations.
WIDER IMPLICATIONS OF THE FINDINGS
Our work provides previously unidentified in-vivo evidence that germline expression of Rictor plays a role in maintaining spermatogonial differentiation and cell–cell adhesion. These findings are important for understanding the regulation of spermatogenesis and have clinical implications for the effect of mTOR inhibitors on human fertility.
STUDY FUNDING AND COMPETING INTEREST(S)
This study was supported by National Key R&D Program of China (2016YFA0500902), National Natural Science Foundation of China (31471228 and 31771653), Jiangsu Science Foundation for Distinguished Young Scholars (BK20150047), and Natural Science Foundation of Jiangsu Province (BK20140897, 14KJA180005 and 14KJB310004) to K.Z. The authors declare no competing or financial interests.