In vitro culture of reptile PGCS to preserve endangered species.

Primordial germ cells (PGCs), are the source of gametes in vertebrates. There are similarities in the development of PGCs of reptiles with avian and mammalian species PGCs development. PGCs culture has been performed for avian and mammalian species but there is no report for reptilian PGCs culture. In vitro culture of PGCs is needed to produce transgenic animals, preservation of endangered animals and for studies on cell behaviour and research on fertility. Reptiles are traded as exotic pets and a source of food and they are valuable for their skin and they are useful as model for medical research. Transgenic reptile has been suggested to be useful for pet industry and medical research. In this research different aspects of PGCs development was compared in three main classes of vertebrates including mammalian, avian and reptilian species. It is proposed that a discussion on similarities between reptilian PGCs development with avian and mammalian species helps to find clues for studies of reptilian PGCs development details and finding an efficient protocol for in vitro culture of reptilian PG.

Spermatogenesis regeneration by transfected spermatogonial stem cells in infertile roosters through testicular transplantation.

Investigations pertaining to spermatogonial stem cells (SSCs) have led to the use of these cells in a variety of fields including infertility treatments, production of transgenic animals, and genome editing. The aim of the present study was to investigate the plausibility of regenerating spermatogenesis in infertile roosters by transplanting transfected SSCs into testes. Spermatogonial stem cells were isolated and cultured for seven days. Afterward, pDB2, a plasmid vector carrying a reporter gene, GFP, was transfected into the SSCs. Transfected SSCs were transplanted into the left testis of infertile roosters. Tissue samples from the recipients' testes were obtained six weeks after the transplantation and transplanted SSCs were observed in the basement membrane. After eight weeks, GFP-positive spermatozoa were observed in collected semen from the recipient roosters and GFP gene in spermatozoa was confirmed using PCR. The recipient roosters were mated with hens. Hatchlings were visually checked and their tissue samples were tested by PCR to identify transgenesis but both of them were negative. Overall, it seems that regeneration of spermatogenesis in roosters via transfected SSCs is possible but more studies are need to produce recombinant proteins by this way.

Designing A Transgenic Chicken: Applying New Approaches toward A Promising Bioreactor.

Specific developmental characteristics of the chicken make it an attractive model for the generation of transgenic organisms. Chicken possess a strong potential for recombinant protein production and can be used as a powerful bioreactor to produce pharmaceutical and nutritional proteins. Several transgenic chickens have been generated during the last two decades via viral and non-viral transfection. Culturing chicken primordial germ cells (PGCs) and their ability for germline transmission ushered in a new stage in this regard. With the advent of CRISPR/Cas9 system, a new phase of studies for manipulating genomes has begun. It is feasible to integrate a desired gene in a predetermined position of the genome using CRISPR/Cas9 system. In this review, we discuss the new approaches and technologies that can be applied to generate a transgenic chicken with regards to recombinant protein productions.

The in vitro effect of chick embryo extract on mice pre-antral follicles.

Chick embryo extract (CEE) contains a variety of growth factors which may improve in vitro follicle growth. Therefore, the effect of CEE on mouse pre-antral follicle culture was evaluated. Different percentages of CEE (0, 0.50%, 1.00%, 5.00% and 10.00%) were added to culture medium. Hence, the osmolarity of media was measured. Pre-antral follicles with diameter of 120-150 µm were isolated from 12-14 days old mouse ovary and cultured for 12 days. After culture, the maturation rate was assessed. Granulosa cells viability was evaluated using MTT test and estradiol levels were evaluated using related radio-immunoassay (RIA). Genes expression (BMP15 and ALK6) was also evaluated. The osmolarity of media and granulosa cells viability were the same in all groups. Estradiol level in group with 10.00% CEE was significantly decreased compared to the control group. After 12 days culture, the percentage of antral follicles development was significantly higher in the group with 5.00% CEE compared to control group. The percentage of metaphase II and germinal vesicle breakdown oocytes was significantly higher in group 5.00% CEE compared to control group. The expression of BMP15 gene in antral follicles in 5.00% CEE and control groups was significantly lower compared to pre-antral follicles. However, the expression of ALK6 gene in antral follicles in 5.00% CEE and control groups was not significantly different compared to pre-antral follicles. The increasing effect of CEE on follicle viability with keeping normal gene expression indicates that addition of proper percentage of CEE to culture media improves culture conditions, making it a possible choice to be used as a follicular growth enhancer in infertility clinics.