Levent Karagenc

Restoration of Fertility by Germ Cell Transplantation Requires Effective Recipient Preparation

Abstract 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.

Impact of oxygen concentration on embryonic development of mouse zygotes

The aim of the present study was to examine the effect of culture under 5 and 20% oxygen on the development, differentiation and viability of zygotes and in-vivo-produced embryos at the 2-cell and 8-cell stages of development. First, zygotes collected in a common pool were cultured in 20% O2 for 0, 23, 46 and 95 h. Zygotes and in-vivo-produced embryos at the 2-cell and 8-cell stages of development were then cultured in 5 or 20% O2. The proportion of embryos reaching the compaction and blastocyst stages of development did not differ between groups regardless of the period of time embryos were cultured in 20% O2 or the stage at beginning of culture. Duration of culture under 20% O2 had a significant effect on total number of blastocyst cells. A stage-specific effect was observed on total and trophectoderm cell numbers in blastocysts resulting from the culture of zygotes and in-vivo-produced embryos under 20% O2. ICM and percent ICM development was significantly decreased by culture in 20% O2 at all stages examined. Oxygen concentration had no effect on implantation rate and fetal weights upon embryo transfer. However, transfer of zygotes grown to the blastocyst stage in 20% O2 resulted in a dramatic decrease in fetal development per blastocyst and fetal development per implantation. These results demonstrate that culture of F1 mouse zygotes in 20% O2 compromises the developmental potential of resultant blastocysts, which appear to be normal on morphological assessment.

Origin of primordial germ cells in the prestreak chick embryo

The temporal and spatial pattern of segregation of the avian germline from the formation of the area pellucida to the beginning of primitive streak formation (stages VII-XIV, EG&K) was investigated using the culture of whole embryos and central and peripheral embryo fragments on vitelline membranes at stages VII-IX, immunohistological analysis of whole mount embryos and sections with monoclonal antibodies MC-480 against stage-specific embryonic antigen-1 (SSEA-1) and EMA-1, and with the culture of dispersed blastoderms at stages IX-XIV with and without on STO feeder layer. Whole embryos at intrauterine stages developed up to the formation of the primitive streak despite the absence of area pellucida expansion. Primordial germ cells (PGCs) appeared in the cultures of whole embryos and only in central fragments containing a partially formed area pellucida at stages VII-IX. When individual stage IX-XIV embryos were dispersed and cultured without a feeder layer, 25-45 PGCs/embryo were detected only with stage X-XIV, but not with stage IX blastoderms. However, the culture of dispersed cells from the area pellucida of stages IX-XIII on STO feeder layers yielded about 150 PGCs/embryo. The carbohydrate epitopes recognized by anti-SSEA-1 and EMA-1 first appeared at stage X on cells in association with polyingressing cells on the ventral surface of the epiblast and later on the dorsal surface of the hypoblast. The SSEA-1-positive hypoblast cells gave rise to chicken PGCs when cultured on a feeder layer of quail blastodermal cells. From these observations, we propose that the segregation and development of avian germline is a gradual, epigenetic process associated with the translocation of SSEA-1/EMA-1-positive cells from the ventral surface of the area pellucida at stage X to the dorsal side of the hypoblast at stages XI-XIV.

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.

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.

Sexing of newly-hatched chicks using DNA isolated from chorio-allantoic membrane samples by polymerase chain reaction in Denizli chicken

1. The aim of the present study was to determine the sex of newly-hatched chicks of Denizli chicken, a local Turkish breed, by polymerase chain reaction (PCR) using DNA extracted from the chorioallantoic membrane (CAM). 2. Fertilised eggs were incubated individually and a total of 20 CAM samples were collected following the hatching process. DNA was isolated from the CAM samples and PCR was performed using W-repeat (W) and 18 S ribosomal gene (R) primers. 3. Screening of the PCR products by agarose gel electrophoresis revealed that males have a single band (256 bp) and females have an extra second band (415 bp) as expected. 4. The present study describes a reliable, rapid, and simple multiplex PCR protocol that can be put into use to sex local breeds of chicken in which phenotypic sexing is impossible, using DNA isolated from the CAM that is discarded and remains attached to the egg shell following the hatching process.

Oestradiol, cyclodextrin-encapsulated 17β-oestradiol and the oestradiol solubilizer 2-hydroxypropyl-β-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 mares 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.

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‐μm 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.

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.