Does vitamin A (retinol, Rol) prevent round spermatid nuclear damage and increase the production of motile sperm during in vitro maturation of vitrified pre-pubertal mouse testicular tissue?
The supplementation of an in vitro culture of ~0.75 mm3 testicular explants from pre-pubertal mice with Rol enhances spermatogenesis progression during the first spermatogenic wave.
The production of functional spermatozoa in vitro has only been achieved in the mouse model and remains a rare event. Establishing an efficient culture medium for vitrified pre-pubertal testicular tissue is now a crucial step to improve the spermatic yield obtained in vitro. The role of Rol in promoting the differentiation of spermatogonia and their entry into meiosis is well established; however, it has been postulated that Rol is also required to support their full development into elongated spermatids.
A total of 60 testes from 6.5 days post-partum (dpp) mice were vitrified/warmed, cut into fragments and cultured for 30 days: 20 testes were used for light microscopy and histological analyses, 20 testes for DNA fragmentation assessment in round spermatids and 20 testes for induced sperm motility assessment. Overall, 16 testes of 6.5 dpp were used as in vitro fresh tissue controls and 12 testes of 36.5 dpp mice as in vivo controls. Testes were vitrified with the optimal solid surface vitrification procedure and cultured with an in vitro organ culture system until Day 30 (D30). Histological analysis, cell death, degenerating round spermatids, DNA fragmentation in round spermatids and induced sperm motility were assessed. Testosterone levels were measured in media throughout the culture by radioimmunoassay.
At D30, better tissue development together with higher differentiation of spermatogonial stem cells, and higher global cell division ability were observed for vitrified/warmed testicular fragments of ~0.75 mm3 with a culture medium supplemented with Rol compared to controls. During in vitro culture of vitrified pre-pubertal testicular tissue, Rol enhanced and maintained the entry of spermatogonia into meiosis and promoted a higher spermatic yield. Furthermore, decreased round spermatid nuclear alterations and DNA damage combined with induced sperm motility comparable to in vivo highlight the crucial role of Rol in the progression of spermatogenesis during the first wave.
Despite our promising results, the culture media will have to be further improved and adapted within the context of a human application.
The results have potential implications for the handling of human pre-pubertal testicular tissues cryopreserved for fertility preservation. However, because some alterations in round spermatids persist after in vitro culture with Rol, the procedure needs to be optimized before human application, bearing in mind that the murine and human spermatogenic processes differ in many respects.
This study was supported by a Ph.D. grant from the Normandy University and a financial support from ‘la Ligue nationale contre le cancer’ (both awarded to L.D.), funding from Rouen University Hospital, Institute for Research and Innovation in Biomedicine (IRIB) and Agence de la Biomédecine. The authors declare that there is no conflict of interest.
Does the rapid transit through the uterine environment modulate the sperm physiological state?
The uterosome-like vesicles (ULVs) secreted by endometrial epithelial cells (EECs) in vitro are able to fuse with human spermatozoa, prompting their fertilizing capacity.
Early studies suggest that sperm capacitation begins in the uterus and ends in the oviduct, and that a synergistic effect of both female organs may accelerate this process. Although it has been reported that co-incubation of human spermatozoa with endometrial cell-conditioned medium (CM) stimulates sperm capacitation, the mechanism mediating this communication is unknown.
Human ULVs secreted by EECs were characterized and their effect on human sperm physiology was analysed. Spermatozoa were incubated with EEC-derived CM or ULV, after which sperm capacitation was evaluated at different time points. In addition, the interaction of spermatozoa with ULV was analysed.
ULVs were isolated by ultracentrifugation and identified using electron microscopy and Western blotting to assess the presence of specific protein markers. Following seminal plasma removal, human spermatozoa were incubated CM or ULV, after which sperm capacitation was evaluated as the ability of the sperm to undergo the induced acrosome reaction and the level of protein tyrosine phosphorylation (PY) determined by Western blot and immunocytochemistry. The interaction of spermatozoa with labelled ULV was analysed by fluorescence microscopy. In all cases, at least three biological replicates from different sperm donors were performed for each set of experiments. Significant differences between mean values were determined by one-way ANOVA followed by Tukey's post hoc test. Differences between treatments were considered statistically significant at P ≤ 0.05.
The level of capacitated spermatozoa and those recruited by chemotaxis increased 3- to 4-fold when spermatozoa were incubated in the presence of CM for 4 h. Even a 15 min incubation of spermatozoa with CM was also enough to increase the level of capacitated cells 3- to 4-fold (P < 0.05). Furthermore, a short co-incubation of spermatozoa with ULV stimulates sperm capacitation, as determined by the increase in the level of induced acrosome reaction and the induction of PY. In addition, after the co-incubation of spermatozoa with fluorescent labelled ULV, the sperm cells acquired the fluorescent staining which indicates that ULV might be transferred to the sperm surface by a fusion mechanism.
This is an in vitro study performed with human biological material, spermatozoa and endometrial derived cells; the latter being a cell line originally isolated from a uterine adenocarcinoma.
The capability of spermatozoa to briefly interact with ULVs supports the hypothesis that any step of sperm transport may have physiological consequences, despite the interaction lasting for only a limited period of time. This way of communication of spermatozoa with cell products of uterine origin opens new frontiers of investigation (e.g. the signalling molecules involved), shedding light on the sperm processes that prepare the male gamete for fertilization, which might have implications for human infertility treatment.
The project was financially supported by SECyT-UNC. The authors declare no conflict of interest.
Can host fertility be rescued by grafting of a fragment of a healthy ovary soon after chemotherapy?
We found that grafting a green fluorescent protein (GFP)-positive fragment from a healthy isogenic ovary to the left ovary of a chemo-treated host rescued function and fertility of the grafted host ovary, and resulted in the production of host-derived offspring as late as the sixth litter after chemotherapy (CTx) treatment, whereas none of the ungrafted controls produced a second litter.
In women and girls undergoing chemotherapy, infertility and premature ovarian failure are frequent outcomes. There are accumulating reports of improved endocrine function after autotransplantation of an ovarian fragment, raising the possibility that the transplant is beneficial to the endogenous ovary.
We first established a CTx treatment regimen that resulted in the permanent loss of fertility in 100% of female mice of the FVB inbred strain. We grafted an isogenic ovary fragment from a healthy female homozygous for a GFP transgene to the left ovary of 100 CTx-treated hosts, and compared fertility to 39 ungrafted controls in 6 months of continuous matings, using GFP to distinguish offspring derived from the graft, and those derived from the host.
Immunofluoresece and western blot analysis of 39 treated ovaries during and 15 days after CTx treatment revealed elevated apoptosis, rapid loss of granulosa cells and an increased recruitment of growing follicles. Using immunofluorescence and confocal imaging, we tracked the outcome of the grafted tissue over 4 months and its effect on the adjacent and contralateral ovary of the host.
Fifty-three percent of grafted females produced a second litter whereas none of the ungrafted females produced a second litter. The likelihood that this could occur by chance is very low (P < 0.0001).
These results are shown only in mice, and whether or how they might apply to chemotherapy patients subjected to different CTx regimens is not yet clear.
Our experiments prove that rescue of a chemo-treated ovary is possible, and establish a system to investigate the mechanism of rescue and to identify the factors responsible with the long-term goal of developing therapies for preservation of ovarian endocrine function and fertility in women undergoing chemotherapy.
No large datasets were produced.
Duke University Medical Center Chancellor's Discovery Grant to BC; ESJ was supported by an NRSA 5F31CA165545; SK was supported by NIH RO1 GM08033; RWT was supported by the Duke University School of Medicine Ovarian Cancer Research Fellowship; XBM was supported by CONICYT. The authors have no conflicts of interest to declare.
Are follicular fluid (FF) sphingosine-1-phosphate (S1P) levels in patients at risk of developing ovarian hyperstimulation syndrome (OHSS) altered and in part responsible for the high vascular permeability observed in these patients.
FF S1P levels are lower in FF from patients at risk of OHSS and treatment with S1P may reduce vascular permeability in these patients.
Although advances have been made in the diagnosis, and management of OHSS and in basic knowledge of its development, complete prevention has proven difficult.
A total of 40 FF aspirates were collected from patients undergoing ART. The women (aged 25–39 years old) were classified into a control group (n = 20) or a group at risk of OHSS (n = 20). The EA.hy926 endothelial cell line was used to assess the efffects of FF from patients at risk of OHSS with or without the addition of S1P. An animal model that develops OHSS in immature Sprague-Dawley rats were also used.
Migration assays, confocal microscopy analysis of actin filaments, immunoblotting and quail chorioallantoic membrane (CAM) assays of in-vivo angiogenesis were performed and statistical comparisons between groups were made.
The S1P concentration was significantly lower in FF from patients at risk of OHSS (P = 0.03). The addition of S1P to this FF decreased cell migration (P < 0.05) and prevented VE-cadherin phosphorylation in endothelial cells (P < 0.05). S1P in the FF from patients at risk of OHSS increased the levels of VE-cadherin (P < 0.05), N-cadherin (P < 0.05) and β-catenin (P < 0.05), and partially reversed actin redistribution in endothelial cells. The addition of S1P in FF from patients at risk of OHSS also decreased the levels of vascular endothelial growth factor (VEGF121; P < 0.01) and S1P lyase (SPL; P < 0.05) and increased the levels of S1PR1 (P < 0.05) in endothelial cells. In CAMs incubated with FF from patients at risk of OHSS with S1P, the number of vessel branch points decreased while the periendothelial cell coverage increased. Additionally, in a rat OHSS model, we demonstrated that vascular permeability and VEGF121 and its receptor KDR expression were increased in the OHSS group compared to the control group and that S1P administration decreased these parameters.
The results of this study were generated from an in-vitro system. This model reflects the microvasculature in vivo. Even though the ideal model would be the use of human endothelial cells from the ovary, it is obviously not possible to carry out this kind of approach in ovaries of patients from ART. More studies will be necessary to delineate the effects of S1P in the pathogenesis of OHSS. Hence, clinical studies are needed in order to choose the most appropriate method of prevention and management.
The use of bioactive sphingolipid metabolites may contribute to finding better and safer therapeutic strategies for the treatment of OHSS and other human diseases that display aberrant vascular leakage.
This work was supported by grants ANPCyT (PICT 2012-897), CONICET (PIP 5471), Roemmers and Baron Foundation, Argentina. The authors declare no conflict of interest.
2016-12-07T08:35:53-08:00STUDY QUESTION Can supplementation of media with a glutathione (GSH) donor, glutathione ethyl ester (GEE), prior to vitrification protect the mouse oocyte from oxidative damage and critical changes in redox homeostasis, and thereby improve cryotolerance? SUMMARY ANSWER GEE supplementation supported redox regulation, rapid recovery of spindle and chromosome alignment after vitrification/warming and improved preimplantation development of mouse metaphase II (MII) oocytes. WHAT IS KNOWN ALREADY Cryopreservation may affect mitochondrial functionality, induce oxidative stress, and thereby affect spindle integrity, chromosome segregation and the quality of mammalian oocytes. GEE is a membrane permeable GSH donor that promoted fertilization and early embryonic development of macaque and bovine oocytes after IVM. STUDY DESIGN, SIZE, DURATION Two experimental groups consisted of (i) denuded mouse germinal vesicle (GV) oocytes that were matured in vitro in the presence or absence of 1 mM GEE (IVM group 1) and (ii) in vivo ovulated (IVO) MII oocytes that were isolated from the ampullae and exposed to 1 mM GEE for 1 h prior to vitrification (IVO group 2). Recovery of oocytes from both groups was followed after CryoTop vitrification/warming for up to 2 h and parthenogenetic activation. PARTICIPANTS/MATERIALS, SETTING, METHODS Reactive oxygen species (ROS), spindle morphology and chromosome alignment were analyzed by confocal laser scanning microscopy (CLSM) and polarization microscopy in control and GEE-supplemented MII oocytes. The relative overall intra-oocyte GSH content was assessed by analysis of monochlorobimane (MBC)-GSH adduct fluorescence in IVM MII oocytes. The GSH-dependent intra-mitochondrial redox potential (Em GSH) of IVM MII oocytes was determined after microinjection with specific mRNA at the GV stage to express a redox-sensitive probe within mitochondria (mito-Grx1-roGFP2). The absolute negative redox capacity (in millivolts) was determined by analysis of fluorescence of the oxidized versus the reduced form of sensor by CLSM and quantification according to Nernst equation. Proteome analysis was performed by quantitative 2D saturation gel electrophoresis (2D DIGE). Since microinjection and expression of redox sensor mRNA required removal of cumulus cells, and IVM of denuded mouse oocytes in group 1 induces zona hardening, the development to blastocysts was not assessed after IVF but instead after parthenogenetic activation of vitrified/warmed MII oocytes from both experimental groups. MAIN RESULTS AND ROLE OF CHANCE IVM of denuded mouse oocytes in the presence of 1 mM GEE significantly increased intra-oocyte GSH content. ROS was not increased by CryoTop vitrification but was significantly lower in the IVM GEE group compared to IVM without GEE before vitrification and after recovery from vitrification/warming (P < 0.001). Vitrification alone significantly increased the GSH-dependent intra-mitochondrial redox capacity after warming (Em GSH, P < 0.001) in IVM oocytes, presumably by diffusion/uptake of cytoplasmic GSH into mitochondria. The presence of 1 mM GEE during IVM increased the redox capacity before vitrification and there was no further increase after vitrification/warming. None of the reproducibly detected 1492 spots of 2D DIGE separated proteins were significantly altered by vitrification or GEE supplementation. However, IVM of denuded oocytes significantly affected spindle integrity and chromosome alignment right after warming from vitrification (0 h) in group 1 and spindle integrity in group 2 (P < 0.05). GEE improved recovery in IVM group as numbers of oocytes with unaligned chromosomes and aberrant spindles was not significantly increased compared to unvitrified controls. The supplementation with GEE for 1 h before vitrification also supported more rapid recovery of spindle birefringence. GEE improved significantly development to the 2-cell stage for [...]
2016-12-07T08:35:53-08:00STUDY QUESTION Does the gene expression profile of cumulus cells (CC) accompanying oocytes with different degrees of chromatin compaction within the germinal vesicle (GV) reflect the oocyte's quality and response in culture during in-vitro embryo production (IVP). SUMMARY ANSWER The transcriptomic profile of the CC is related to oocyte competence, setting the stage for the development of customized pre-maturation strategies to improve IVP. WHAT IS KNOWN ALREADY Oocytes complete the acquisition of their competence during antral follicle development. During this period, the chromatin configuration within the GV changes dynamically and is indicative of oocyte's developmental potential. The interactions between somatic and germ cells modulate chromatin morphology and function and are critical for acquisition of oocyte competence. STUDY DESIGN, SIZE, DURATION Bovine cumulus–oocyte complexes (COC) were isolated from 0.5 to 6 mm antral follicles. Surrounding CC were separated from the oocyte and classified as GV0, GV1, GV2 and GV3 according to the degree of the oocyte's chromatin compaction. PARTICIPANTS/MATERIALS, SETTING, METHOD RNA extracted from CC of each group was amplified and hybridized on a bovine embryo-specific 44 K Agilent slide. The CC_GV1, CC_GV2 and CC_GV3 classes were each hybridized against the CC_GV0 class, representing an early oocyte differentiation stage with poor development competence. The data were normalized and fold changes of the differentially expressed genes were determined. Microarray data were validated using quantitative RT-PCR on selected targets. Microarray data were further analyzed through: (i) between-group analysis (BGA), which classifies the samples according to their transcriptomic profiles; (ii) cluster analysis according to the expression profile of each gene; and (iii) Ingenuity Pathway Analysis (IPA) to study gene regulation patterns and predicted functions. Furthermore, CC of each GV group were cultured and apoptotic cells were assessed after 3 h by caspase analysis. Finally, based on the analysis of CC transcriptomic profiles and the relationship between morphological features of the COC and the oocyte chromatin configuration, a customized, stage-dependent oocyte pre-maturation (pre-IVM) system was used to improve oocyte developmental potential before IVM. For this, the blastocyst rate and quality were assessed after in-vitro maturation and fertilization of pre-matured oocytes. MAIN RESULTS AND THE ROLE OF CHANCE Overall, quantitative RT-PCR results of a subset of five selected genes were consistent with the microarray data. Clustering analysis generated 16 clusters representing the main profiles of transcription modulation. Of the 5571 significantly differentially expressed probes, the majority (25.49%) best fitted with cluster #6 (downregulation between CC_GV0 and CC_GV1 and stable low levels in successive groups). IPA identified the most relevant functions associated with each cluster. Genes included in cluster #1 were mostly related to biological processes such as ‘cell cycle’ and ‘cell death and survival’, whereas genes included in cluster #5 were mostly related to ‘gene expression’. Interestingly, ‘lipid metabolism’ was the most significant function identified in clusters #6, #9 and #12. IPA of gene lists obtained from each contrast (i.e., CC_GV0 vs. CC_GV1; CC_GV0 vs. CC_GV2; CC_GV0 vs. CC_GV3) revealed that the main affected function in each contrast was ‘cell death and survival’. Importantly, apoptosis was predicted to be inhibited in CC_GV1 and CC_GV2, but activated in CC_GV3. Caspase analysis indicated that a low percentage of CC_GV0 was prone to undergo apoptosis but apoptosis increased significantly in CC from oocytes with condensed chromatin, reaching a peak in CC_GV3 (P < 0.05). Finally, the tailored oocyte pre-maturation strategy, based on morpholog[...]
2016-12-07T08:35:53-08:00STUDY QUESTION Does the changing molecular profile of the endometrium during menstruation correlate with the histological profile of menstruation. SUMMARY ANSWER We identified several genes not previously associated with menstruation; on Day 2 of menstruation (early-menstruation), processes related to inflammation are predominantly up-regulated and on Day 4 (late-menstruation), the endometrium is predominantly repairing and regenerating. WHAT IS KNOWN ALREADY Menstruation is induced by progesterone withdrawal at the end of the menstrual cycle and involves endometrial tissue breakdown, regeneration and repair. Perturbations in the regulation of menstruation may result in menstrual disorders including abnormal uterine bleeding. STUDY DESIGN, SIZE DURATION Endometrial samples were collected by Pipelle biopsy on Days 2 (n = 9), 3 (n = 9) or 4 (n = 6) of menstruation. PARTICIPANTS/MATERIALS, SETTING, METHODS RNA was extracted from endometrial biopsies and analysed by genome wide expression Illumina Sentrix Human HT12 arrays. Data were analysed using ‘Remove Unwanted Variation-inverse (RUV-inv)’. Ingenuity pathway analysis (IPA) and the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 were used to identify canonical pathways, upstream regulators and functional gene clusters enriched between Days 2, 3 and 4 of menstruation. Selected individual genes were validated by quantitative PCR. MAIN RESULTS AND THE ROLE OF CHANCE Overall, 1753 genes were differentially expressed in one or more comparisons. Significant canonical pathways, gene clusters and upstream regulators enriched during menstrual bleeding included those associated with immune cell trafficking, inflammation, cell cycle regulation, extracellular remodelling and the complement and coagulation cascade. We provide the first evidence for a role for glutathione-mediated detoxification (glutathione-S-transferase mu 1 and 2; GSTM1 and GSTM2) during menstruation. The largest number of differentially expressed genes was between Days 2 and 4 of menstruation (n = 1176). We identified several genes not previously associated with menstruation including lipopolysaccharide binding protein, serpin peptidase inhibitor, clade B (ovalbumin), member 3 (SERPINB3) and -4 (SERPINB4), interleukin-17C (IL17C), V-set domain containing T-cell activation inhibitor 1 (VTCN1), proliferating cell nuclear antigen factor (KIAA0101/PAF), trefoil factor 3 (TFF3), laminin alpha 2 (LAMA2) and serine peptidase inhibitor, Kazal type 1 (SPINK1). Genes related to inflammatory processes were up-regulated on Day 2 (early-menstruation), and those associated with endometrial repair and regeneration were up-regulated on Day 4 (late-menstruation). LIMITATIONS, REASONS FOR CAUTION Participants presented with a variety of endometrial pathologies related to bleeding status and other menstrual characteristics. These variations may also have influenced the menstrual process. WIDER IMPLICATIONS OF THE FINDINGS The temporal molecular profile of menstruation presented in this study identifies a number of genes not previously associated with the menstrual process. Our findings provide valuable insight into the menstrual process and may present novel targets for therapeutic intervention in cases of endometrial dysfunction. LARGE SCALE DATA All microarray data have been deposited in the public data repository Gene Expression Omnibus (GSE86003). STUDY FUNDING AND COMPETING INTEREST(S) Funding for this work was provided by a National Health and Medical Research Council of Australia (NHMRC) Project Grant APP1008553 to M.H., P.R. and J.G. M.H. is supported by an NHMRC Practitioner Fellowship. P.P. is supported by a NHMRC Early Career Fellowship. The authors have no conflict of interest to declare. [...]