Expression of Toll-Like Receptors in the Lung Tissue of Mouse Fetuses Generated by in vitro Embryo Culture and Embryo Transfer.

Mouse fetuses generated by in vitro embryo culture and embryo transfer exhibit impaired lung development, altered composition of pulmonary epithelial cells associated with downregulation of several genes involved in lung development and toll-like receptor (TLR) signaling pathway. The aims of the present study were to determine the expression of all TLRs and to examine if the expression of TLRs, along with genes involved in TLR signaling pathway, is altered in the lung tissue of mouse fetuses generated through embryo culture and embryo transfer. Two experimental (EGs) and one control (CG) group were included in the study. Embryos cultured at 5% CO2-95% air for 95 h or less than 24 h were transferred to pseudo-pregnant females to obtain fetuses comprising EGin vitro (n = 18) and EGin vivo (n = 18), respectively. Fetuses obtained from naturally ovulating females on day 18 of pregnancy served as the CG (n = 18). Western blot and immunohistochemistry were used to determine the expression of TLR proteins. The expression of transcripts encoding TLRs, and the genes involved in TLR signaling pathway (Lbp, Pik3r1, Pik3cb, Nfkbia, and Fos), was determined using qRT-PCR. While all TLRs were expressed by cells lining the bronchial/bronchiolar epithelium of lung tissues in all groups, some of the TLRs were expressed in a specific pattern. When compared to CG, the expression of transcripts encoding TLR-2, -3, -4, -5, -7, -8, -9, -12, -13, Lbp, Pik3r1, Pik3cb, Nfkbia, and Fos was significantly downregulated in both EGs. It appears that stress imposed on embryos at preimplantation stages of development is associated with downregulation of TLRs, along with some of the genes involved in TLR signaling pathway, in the lung tissue during the perinatal period. It remains to be determined if downregulation of TLRs, along with the genes involved in TLR signaling pathway, has any functional consequences in the adult lung tissue.

Altered expression of pulmonary epithelial cell markers in fetal and adult mice generated by in vitro embryo culture and embryo transfer.

Lung development is impaired in mice generated through transfer of in vitro-derived blastocysts. The main objective of the current study was to determine if the composition of epithelial cells in the fetal and adult lung tissue is altered in mice generated through transfer of in vitro-derived blastocysts. The study comprised of two experimental (EGs) and two control (CGs) groups. Fetuses (18.5 d.p.c.) and adult mice (8-week-old) of the EGs (EGfetus , n=18, EGadult, n=15) were produced by the transfer of day-5 F2 blastocysts to pseudo-pregnant females. F2 fetuses and adult mice derived from naturally-ovulating females served as the CGs (CGfetus, n=18, CGadult n=15). The expression of Tuba-1a (a marker of ciliated cells), Foxj-1 (a marker of motile ciliated cells), Uch-L1 (a marker of neuroendocrine cells), Cldn-10 (a marker of Club cells), Aqp-5 (a marker of Type I alveolar cells), and Sp-C (a marker of Type II alveolar cells) was determined using western blot, immunohistochemistry/immunofluorescence and qRT-PCR analyses. Weight of fetuses as well as adult mice is decreased in mice comprising the EGs. Impaired lung development observed in EGfetus was associated with altered expression of Tuba-1a, Foxj-1, Cldn10, Uch-L1, Sp-C and Aqp-5. Morphology of the adult lung tissue was similar between the groups except for a significant increase in the thickness of the epithelia in EGadult. The expression of Cldn-10 and Sp-C was also altered in EGadult. It remains to be determined whether altered expression of these genes has any long-term impact on epithelial cell functions in the adult lung tissue.

Transfer of mouse blastocysts exposed to ambient oxygen levels can lead to impaired lung development and redox balance.

In vitro culture under atmospheric oxygen puts embryos under oxidative stress and impairs preimplantation development. However, to what extent this process alters the redox balance in the perinatal period remains largely unknown. The aim of the present study was to examine if the redox balance is altered in the lung tissue of fetuses generated through transfer of mouse embryos exposed to atmospheric oxygen at different stages of development and to determine if this has any effect on lung morphogenesis and gene expression. Two experimental groups (EGs) were generated by transferring in vitro- and in vivo-derived blastocysts to pseudo-pregnant females. In vivo-developed fetuses served as control. Enzymatic/nonenzymatic antioxidants, malondialdehyde (MDA) levels, total antioxidant capacity, stage of lung development and gene expression were evaluated on day 18 of pregnancy. Weight of fetuses was significantly less in both experimental cohorts (ANOVA, P < 0.001 versus control), associated with delayed lung development, higher amounts of MDA (ANOVA, P < 0.001 versus control) and altered expression of several genes in oxidative stress/damage pathways. Evidence gathered in the present study indicates that pre-implantation stress caused by culture under atmospheric oxygen, even for a short period of time, leads to fetal growth restriction, impaired lung development and redox balance along with dysregulation of several genes in oxidative stress response. Absence of an EG in which in vitro embryo culture was performed at 5% oxygen and the use of genetically heterogeneous F2 fetuses are the limitations of the study. In any case, the long-term impact of such dramatic changes in the developmental programming of resulting fetuses warrants further investigations.

Changes on the Pancreas in Experimental Diabetes and the Effect of Lycopene on These Changes: Pdx-1, Ngn-3, and Nestin Expressions.

The aim of the present study was to investigate changes occurring in the number of beta cells, as well as the expressions of Ngn-3, nestin and Pdx-1 of pancreatic progenitor cells in the pancreas of experimentally-induced adult diabetic rats and to determine the effect of orally-administered lycopene on these changes. Following the administration of 50 mg/kg streptozotocin to rats, four groups of animals were established: control + corn oil, control + lycopene, diabetic + corn oil and diabetic + lycopene. The animals in the control + lycopene and diabetic + lycopene groups received 4 mg/kg lycopene for a period of four weeks. The expressions of insulin, Ngn-3, nestin, and Pdx-1 were determined through immunohistochemistry in sections taken from pancreas tissue samples at the end of the experiment. The number of insulin-positive cells was found to be significantly low in the diabetic groups compared to the control groups. In addition, the presence of Ngn-3 and nestin-positive cells within the exocrine pancreas surrounding the islands was noted in the diabetic groups. Lycopene, in general did not have any effect in any of the parameters analyzed in the present study. It is suggested that these cells would function as stem cells to replace the lost beta-cell population. It is also suggested that it is possible to demonstrate the antioxidant effects of lycopene in the pancreas of diabetic rats by increasing the dose and duration of lycopene administration. Anat Rec, 300:2200-2207, 2017. © 2017 Wiley Periodicals, Inc.

Changes in the Pancreas in Experimental Diabetes and the Effect of Lycopene on These Changes: Proliferating, Apoptotic, and Estrogen Receptor α Positive Cells.

This study aimed to investigate the changes occurring in estrogen receptor (ER) α-positive cells, proliferating cells, apoptotic cells and malondialdehyde (MDA) expression in the pancreas of experimentally induced adult diabetic rats and to determine the effect of orally administered lycopene on these changes. Experimental diabetes was induced using a single dose of 50 mg/kg streptozotocin (STZ). Following the administration of STZ, four groups of animals were established: Control + corn oil, control + lycopene, diabetic + corn oil and diabetic + lycopene. The expressions of ER α, Ki-67, and MDA were determined through immunohistochemistry in sections taken from pancreas tissue samples at the end of the experiment. Apoptotic cells were determined through the TUNEL method. In the diabetic groups, the densities of ER α expression in islets and ER α-positive cells in exocrine parts increased. Whereas the number of proliferating Ki-67 positive cells was higher in the diabetic groups, no significant difference was observed in terms of apoptotic cell number between the control and diabetic groups. Lycopene in general did not have any effect on any of the parameters analyzed in the study. The presence of ER α-positive cells around the islets was demonstrated for the first time in diabetic groups. Based on these observations, demonstrating the antioxidant effects of lycopene in the pancreas of diabetic rats may be possible by increasing the dose and/or the duration of lycopene. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:2000-2007, 2017. © 2017 Wiley Periodicals, Inc.

Contribution of cells derived from the area pellucida to extraembryonic mesodermal cell lineages in heterospecific quail chick blastodermal chimeras.

The current study has two main objectives: first, to determine if cells derived from the area pellucida are able to populate extraembryonic membranes, and second, to determine if donor cells have the potential to differentiate to endothelial (EC) and hematopoietic cells (HC) in the yolk sac and allantois, the two extraembryonic membranes functioning as hematopoietic organs in the avian embryo. To this end, quail chick chimeras were constructed by transferring dissociated cells from the areae pellucidae of the stage X-XII (EG&K) quail embryo into the subgerminal cavity of the unincubated chick blastoderm. The distribution of quail cells in the allantois, yolk sac, amnion, and chorion of resulting putative chimeras was examined using quail cell-specific antibody against a perinuclear antigen (QCPN) after 6 days of incubation. The presence of EC, HC, and smooth muscle cells among the QCPN(+) donor cells was examined using QH-1, a quail-specific marker identifying HC and EC and an anti-α-smooth muscle actin antibody. Evidence gathered in the present study demonstrates that quail cells derived from the areae pellucidae are able to populate all of the extraembryonic membranes of resulting heterospecific quail chick chimeras and, most importantly, give rise to HC, EC, and smooth muscle cells, all of the three main mesodermal lineages derived from the posterior mesoderm both in the yolk sac and allantois.

Tissue distribution of cells derived from the area opaca in heterospecific quail-chick blastodermal chimeras.

The objective of the current study was to determine the tissue distribution of cells derived from the area opaca in heterospecific quail-chick blastodermal chimeras. Quail-chick chimeras were constructed by transferring dissociated cells from the area opaca of the stage X-XII (EG&K) quail embryo into the subgerminal cavity of the unincubated chick blastoderm. The distribution of quail cells in embryonic as well as extra-embryonic tissues of the recipient embryo were examined using the QCPN monoclonal antibody after 6 days of incubation in serial sections taken at 100-mum intervals. Data gathered in the present study demonstrated that, when introduced into the subgerminal cavity of a recipient embryo, cells of the area opaca are able to populate not only extra-embryonic structures such as the amnion and the yolk sac, but also various embryonic tissues derived from the ectoderm and less frequently the mesoderm. Ectodermal chimerism was confined mainly to the head region and was observed in tissues derived from the neural ectoderm and the surface ectoderm, including the optic cup, diencephalon and lens. Although the possibility of random incorporation of transplanted cells into these embryonic structures cannot be excluded, these results would suggest that area opaca, a peripheral ring of cells in the avian embryo destined to form the extra-embryonic ectoderm and endoderm of the yolk sac, might harbor cells that have the potential to give rise to various cell types in the recipient chick embryo, including those derived from the surface ectoderm and neural ectoderm.

Early cleavage morphology affects the quality and implantation potential of day 3 embryos.

OBJECTIVE: To assess the development and implantation potential of early-cleaved embryos displaying various morphological patterns. DESIGN: Retrospective analysis. SETTING: Private IVF center. PATIENT(S): Embryos obtained from 1,556 transfer cycles were assessed. Early-cleaved embryos were grouped according to their cleavage patterns as: even (1,490); uneven (3,238); and fragmented (768), or according to nuclear morphologies as: mononucleation (2,008) and other nuclear morphologies; nonmononucleation (3,488). Seven thousand four hundred forty-five embryos were late cleaved. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Embryo quality, pregnancy (PR), and implantation rates. RESULT(S): Day 3 embryo quality was highest in evenly early-cleaved embryos and in those displaying mononucleation. Early-cleaved embryos displaying fragmentation and late-cleaved embryos yielded the poorest day 3 quality. Early cleavage cycles displayed higher PR and implantation rate than late cleavage with the exception of other nuclear morphologies, in which similar outcome was obtained. Mononucleated early-cleaved embryos implanted at a higher frequency than early-cleaved embryos displaying other nuclear morphologies. CONCLUSION(S): The morphology of early cleavage correlates to day 3 embryo quality and implantation rate.

Effect of oxygen concentration during the incubation of embryos of women undergoing ICSI and embryo transfer: a prospective randomized study.

The hypothesis that ICSI outcome can be improved by culturing human embryos in an atmosphere of controlled O(2) concentration (5%) compared with 20% was tested in a prospective randomized study of 712 transfer cycles. The cycle characteristics and the embryology parameters were similar between groups. The embryo qualities were similar with day 2 transfers; however, they were better with day 3 transfers incubated in 5% O(2) than in 20% O(2). The clinical outcome parameters did not differ between groups according to the O(2) concentration. The results indicated that culture of embryos under atmospheric conditions of O(2) for the first 2 or 3 days did not alter the clinical outcome in ICSI cycles.

Use of both early cleavage and day 2 mononucleation to predict embryos with high implantation potential in intracytoplasmic sperm injection cycles.

OBJECTIVE: To evaluate the value of early cleavage and day 2 mononucleation as combined parameters in predicting the implantation potential of embryos. DESIGN: Prospectively designed retrospective cohort analysis. SETTING: Private IVF center. PATIENT(S): Two hundred eighty-seven ICSI cycles were evaluated in four groups according to the presence of early cleavage and mononucleation in all (ECM), some (pECM, ECpM), or none (noECnoM) of the transfer embryos. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Embryo quality and pregnancy and implantation rates. RESULT(S): The cycle characteristics, embryo quality, and pregnancy rates were similar when all (ECM) or some (pECM and ECpM) transfer embryos displayed these parameters. The poorest cycle characteristics and outcome were observed in the absence of both parameters (noECnoM). When early cleavage was present, a higher implantation rate was observed when all transfer embryos displayed mononucleation at day 2 (ECM) than when this was the case only in some (ECpM). CONCLUSION(S): Combination of early cleavage and day 2 mononucleation improves selection of embryos with high implantation potential in ICSI cycles with good prognosis.

Outcomes in couples undergoing ICSI: comparison between fresh and frozen-thawed surgically retrieved spermatozoa.

We retrospectively evaluated the impact of cryopreservation on spermatozoa obtained from patients with azoospermia and used for intracytoplasmic sperm injection (ICSI). Frozen-thawed epididymal spermatozoa (FTEPS) was used in 34 couples, whereas frozen-thawed testicular spermatozoa (FTTS) was used in 50 couples for ICSI during assisted conception, and these results were compared with results using fresh spermatozoa for ICSI in the same individuals. The fertilization rate (FR) was significantly lower for FTTS (65.8%) but not for FTEPS (73.1%) compared with the FR using fresh spermatozoa (72.3% and 73.2% respectively). In contrast, neither the implantation nor the pregnancy rate was altered when FTEPS or FTTS was used. In conclusion, our results indicate that surgically retrieved spermatozoa can be efficiently used for ICSI after freezing and thawing without compromising the outcome.

Granulocyte-macrophage colony-stimulating factor stimulates mouse blastocyst inner cell mass development only when media lack human serum albumin.

The aim of the current study was to examine the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the development and differentiation of preimplantation mouse embryos from different strains and under different culture conditions. Embryos from F1 hybrid mice were cultured in a modified G1 medium lacking amino acids and EDTA (simple G1), human tubal fluid medium (HTF) or in G1/G2 sequential media, supplemented with GM-CSF (0, 2, 4, 8, and 16 ng/ml). Embryos from CF1 mice were subsequently cultured in G1/G2 with (5 mg/ml) or without HSA, in the absence or presence of GM-CSF (2 ng/ml). GM-CSF had no effect at any concentration on F1 embryo development and blastocyst cell numbers, irrespective of the culture media used. Similarly, GM-CSF had no effect on CF1 blastocyst development. However, a stimulatory effect of GM-CSF was evident on total blastocyst cell number and ICM development when CF1 embryos were cultured in the absence of HSA. When HSA was present in the media the beneficial effect of GM-CSF was negated. There was no difference in the number of apoptotic cells in CF1 blastocysts when G1/G2 were supplemented with GM-CSF with or without HSA. These data indicate that there is no beneficial effect of supplementing either simple (simple G1 or HTF) or more complete (G1/G2) media with GM-CSF when protein is present in the medium. However, when culture conditions are suboptimal and non-physiological, i.e. the absence of protein, GM-CSF stimulates development of both total cell numbers and ICM development of CF1 blastocysts.

Impact of assisted hatching on ART outcome in women with endometriosis.

BACKGROUND: Assisted hatching can improve the implantation rate in cycles with poor outcome. The impact of assisted hatching in embryos from women with endometriosis is not known. Therefore, the hypothesis that the implantation potential of embryos obtained from women with endometriosis can be improved with assisted hatching was tested. METHODS: In a prospective randomized study, transfer embryos obtained from 60 women with endometriosis were hatched using a laser system and compared to embryos obtained from patients with the same diagnosis which were left intact (n = 30). RESULTS: The characteristics of cycles were similar between groups. The pregnancy (40% zona intact, 28.3% assisted hatching), and implantation rates (19.4% zona intact, 17.8% assisted hatching) did not differ in endometriosis cycles regardless of assisted hatching. CONCLUSION: Assisted hatching does not improve outcome in women with endometriosis undergoing assisted reproduction.

Oestradiol, cyclodextrin-encapsulated 17beta-oestradiol and the oestradiol solubilizer 2-hydroxypropyl-beta-cyclodextrin all impair preimplantation mouse embryo development.

The aim of this study was to examine the effects of 2-hydroxypropyl-beta-cyclodextrin (HbetaC) used as a solubilizer for oestradiol, 17beta-oestradiol (ethanol soluble) and HbetaC-encapsulated-17beta-oestradiol on mouse embryo development in vitro. HbetaC had no effect on day 3 development. In contrast, blastocyst development and blastocyst cell number were significantly reduced in the presence of 10(-4) mol/l solubilizer equivalent, but not at lower concentrations. The proportion of compacted embryos was significantly reduced with 10(-4) mol/l 17beta-oestradiol. No blastocysts were formed at 10(-4) mol/l concentration of 17beta-oestradiol, although the rate of blastocyst formation did not differ at lower concentrations. Blastocyst cell number was significantly decreased compared with controls at 10(-5) mol/l 17beta-oestradiol. The dose-response using HbetaC-encapsulated-17beta-oestradiol revealed that at 17beta-oestradiol concentrations of 10(-4) and 10(-5) mol/l, blastocyst development was significantly reduced. Blastocyst cell number was significantly reduced compared with controls for all concentrations of HbetaC-encapsulated-17beta-oestradiol. Exposure of embryos to 17beta-oestradiol (10(-4) mol/l) reduced blastocyst development on days 4 and 5 significantly in cultures initiated at the zygote, 2-cell and 8-cell, but not the morulae, stages of development. Trophectoderm, ICM and blastocyst cell numbers as well as percentage ICM development were reduced significantly, regardless of the stage of development. Therefore, 17beta-oestradiol does compromise embryo development.

Administration of increasing amounts of gonadotrophin compromises preimplantation development of parthenogenetic mouse embryos.

The aim of the present study was to examine the effect of ovarian stimulation with increasing amounts of pregnant mare's serum gonadotrophin (PMSG) on preimplantation development of diploid parthenogenetic embryos in vitro. Administration of 5, 10 and 20 IU PMSG significantly increased the number of oocytes obtained per mouse in a dose-dependent manner. The amount of PMSG administered did not alter the proportion of degenerate oocytes. However, there was a significant decrease in the proportion of 8-cell/compacted embryos after 53 h of culture with the administration of increasing amounts of PMSG. Proportion of embryos reaching at the blastocyst stage after 79 h of culture was reduced significantly in both the 10 and 20 IU PMSG groups. Reduced blastocyst development after 96 h of culture, however, was significant only in the 20 IU PMSG group. Total blastocyst, trophectoderm and inner cell mass numbers were also reduced significantly with the administration of 20 IU PMSG. It is concluded on the basis of these observations that preimplantation development of diploid parthenogenetic oocytes, which depends virtually entirely on maternal molecules accumulated during oogenesis along with gene products derived from the maternal genome, is compromised with the administration of increasing amounts of PMSG.

Restoration of fertility by germ cell transplantation requires effective recipient preparation.

Spermatogonial transplantation provides access to the mammalian germline and has been used in experimental animal models to study stem cell/niche biology and germline development, to restore fertility, and to produce transgenic models. The potential to manipulate and/or transplant the germline has numerous practical applications that transcend species boundaries. To make the transplantation technology more broadly accessible, it is necessary to develop practical recipient preparation protocols. In the current study, mouse recipients for spermatogonial transplantation were prepared by treating pregnant females with the chemotherapeutic agent busulfan at different times during gestation. Donor germ cells were introduced into the testes of male progeny between 5 and 12 days postpartum. Analysis of recipient animals revealed that busulfan treatment of pregnant females on 12.5 days postcoitum was the most effective; male progeny transplanted with donor germ cells became fertile and passed the donor genotype to 25% of progeny. This approach was effective because 1) the cytoablative treatment reduced (but did not abolish) endogenous spermatogenesis, creating space for colonization by donor stem cells, 2) residual endogenous germ cells contributed to a healthy testicular environment that supported robust donor and recipient spermatogenesis, and 3) fetal busulfan-treated males could be transplanted as pups, which have been established as better recipients than adults. Laboratory mice provide a valuable experimental model for developing the technology that now can be applied and evaluated in other species.