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Effect of Transfer Primordial Germ Cells (PGCs) into Chick Gonad
Abstract
Primordial germ cells (PGCs) of white leghorn Maria line hens embryos as a donor were transferred white leghorn Laura line hens embryos as a recipient. During stage 12 to 15 of incubating fertilized eggs, donor PGCs which were taken out from blood vessels of donor embryos, were transferred into blood vessels of recipient embryos at the same stage. At 15 days of incubation, survival rate of the treated embryos was 35.7% when the PGCs were transferred into the same sex of donor and recipient embryos (homo-sexual) and 38.8% into antagonistic sex ones (hetero-sexual) respectively. Although the gonad of embryos which were transferred PCGs homo-sexually were developed normally, morphological and histological abnormalities were formed in the gonad derived with hetero-sexually transfer. The abnormalities of gonad were observed in 8 out of 33 samples transferred PCGs from male to female and 12 out of 49 from female to male respectively. Size of male right gonad was degenerated and flat sexual cords were observed. The gonads of germ line chimric chicken was detected donor derived DNA polymorphism to genetic diagnosis by LEI92 of microsatellite marker. These results suggest that hetero-sexual transfer of PGCs may influence gonadal development early-stage embryos.
... Numerous studies had reported the isolation and collection of PGCs from gonadal embryos (Nakamura et al., 2011;Nakajima et al., 2011;Nakajima et al., 2016). Moreover, germline chimeras had been produced in chicken by transferring PGCs or GGCs into the bloodstream at 2-day-old recipient embryos (Furuta et al., 2007(Furuta et al., , 2008 so that PGCs could be used as a genetic resource to produce germ line chimera (Furuta, 2012). Nakajima et al. (2012) reported that gonadal PGCs collected from 7-day-old chick embryos and incubated in PBS [-] can be used to produce germline chimeras. ...
Primordial germ cells (PGCs) are cells that will differentiate themselves into spermatogonia in the testis or oogonia in the ovary. Primordial germ cells arise from epiblast and circulate through the bloodstream and finally entering gonadal anlage. The aim of this study was to determine the number of gonadal PGCs of KUB chicken at different development stages. Sixty KUB chicken fertile eggs were divided into four groups (6, 7, 8, and 9 days incubation periods), and incubated at 38 oC with a humidity of 60%. Harvesting was synchronized to the embryonic development at 6-9 d. Gonads were collected using sharp tweezers, and were placed in Eppendorf tube 1.5 mL containing 500 µL PBS [-]. Gonadal PGCs were purified using PBS [-]. The results showed that the average number of gonadal PGCs at 6, 7, 8, and 9 d were 113.7; 143.5; 92.9; and 85.7 cells per embryo, respectively. Number of gonadal PGCs per embryo of KUB chicken were significantly affected by stage of embryonic development (P<0.05), which reached a peak at day 7 of incubation, so that the isolation and collection of PGCs from the gonads were recommended at day 7 of incubation. This information is useful in production of germline chimera of other Indonesian local chickens.
... Another application to control the amount of water loss is cutting windows with several diameters on the blunt end of the eggshell. Windowing on the eggshell is usually used for studying chicken chimeras (Furuta et al., 1999(Furuta et al., , 2001(Furuta et al., , 2007(Furuta et al., , 2008. It has been reported that windowing application on eggshell elevated water evaporation (Bednarczyk et al., 2000). ...
Three experiments were conducted to develop methods to control the amount of water loss and to evaluate the metabolic effects of water condition in the White Leghorn breeder eggs during incubation. One hundred twenty, 54, and 90 Julia strain White Leghorn breeder eggs were incubated at 37.8 degrees C, 60% RH in experiments 1, 2, and 3. In experiment 1, eggs were drilled with various bore diameters of 0, 0.5, 1, 2, 3, 4, and 5 mm on the blunt end of the eggshell. In experiment 2, 4 x 4 mm(2) windows were cut into the eggs or the eggs were drilled with 5 holes of bore diameter 2 mm on the blunt end of eggshell. In experiment 3, eggs were drilled with 1, 3, 5, and 7 holes of diameter 2 mm on the blunt end of eggshell. Eggs were treated on d 3 of each experiment and the amount of water loss was recorded on d 19 of incubation. Embryo growth was evaluated in experiments 2 and 3. In addition, the livers of embryos were collected in the 0-, 1-, 3-, and 5-hole treatment groups after weighing eggs to determine 3-hydroxy acyl coenzyme A dehydrogenase activity. In experiment 1, although higher water loss was observed in all windowed eggs than in control, there were no differences in amount of water loss among all bore diameters. Accordingly, that was not successful to control amount of water loss. In experiment 2, higher water loss was observed in drilled eggs at the same levels in windowed eggs as in control. Drilling holes was a more useful treatment to control amount of water loss on incubated eggs than windowing. In experiment 3, amount of water loss increased linearly with increasing number of holes on the blunt end of eggshell. Hepatic 3-hydroxy acyl coenzyme A dehydrogenase activity increased with increasing the number of drilled holes.
... It has been reported that exogenous genes could be introduced into the PGCs to produce a transgenic chicken (Naito et al. 1998;Inada et al. 1997;Eguma et al. 1999;Fujihara 2000, van de Lavoir et al. 2006). The transfer of PGCs from donor to recipient embryos is a commonly used technique to establish germline chimeric chickens (Naito et al. 1994;Ono et al. 1996;Yamaguchi et al. 2000;Furuta et al. 1999Furuta et al. , 2001Furuta et al. , 2007Furuta et al. , 2008. The chickens produced by the transfer of PGCs were shown to be chimeric by using a progeny test (Naito et al. 1994;Furuta et al. 2001). ...
Germline chimeric chickens were produced by the transfer of primordial germ cells (PGCs) or blastoderm cells. The hatchability of eggs produced by transfer of exogenous PGCs is usually low. The purpose of the present study was investigated to express (3-hydroxyacyl CoA dehydrogenase) 3HADH which is a limiting enzyme in the beta-oxidation of fatty acids for hatching energy. Manipulations of both donor and recipient eggshells were as follows. A window approximately 10 mm in diameter was opened at the pointed end of the eggs at stage 12-15 days incubation. Donor PGCs, taken from the blood vessels of donor embryos from fertilized eggs at the same stage of development, were injected into the blood vessels of recipient embryos. The muscles of chicks in the eggs with transferred PGCs were removed after 20 days of incubation. A cDNA was prepared from the total RNA. The expression of 3HADH in the manipulated embryos was investigated using real-time PCR analysis. Real-time PCR analysis showed that expression of 3HADH was reduced in the muscles of manipulated embryos.
Chicken primordial germ cells (PGCs) are used to restore and conserve genetic resources of some endangered birds or flocks of domestic chickens. When PGCs are transferred from a donor to a recipient, they can survive and gain sexual maturity in the recipient chicken. This indicates that PGCs retaining exogenous genes can proliferate and develop in recipient gonads, and proves that transferred PGCs are effective sources for establishing transgenic and germline chimeric chickens. The PGC transfer method has been used to produce germline chimeric chickens and may become popular in the field of reproductive science. Germline chimeric chickens have the potential to reconstruct and reproduce endangered birds, including both domestic and wild species.
It was proved that an exogenous gene was successfully transferred to chicken embryo and Primordial Germ Cells (PGCs) by the injection of the blastoderm. In this experiment, an exogenous gene, Lac Z constructs encoding Escherichia coli beta-galactosidase was introduced into chicken blastoderm (stage X) just ovoposition using lipofection reagent. After injection of gene the eggs were incubated in routine manner until developmental stage 12-15. Survival rate of the treated embryo was 35.6%. Lac Z expression was mosaic manner and low efficiency. However, it was obtained Lac Z specific band from chicken PGCs by PCR method.
The expression and fate of exogenous DNA (lacZ gene), introduced into the gonads of chimaeric embryos and chickens that had been produced by transfer of primordial germ cells (PGCs) transfected in vitro, were examined. PGCs obtained from embryonic blood were transfected in vitro by lipofection and transferred to the partially sterilized recipient embryos. Expression of the lacZ gene was observed in the gonads of chimaeric embryos incubated for 3 days after the PGC injection (71.2%, 37/52). Introduction of the lacZ gene into the gonads of chimaeric embryos was confirmed by PCR analysis. The percentage of embryos with gonads positive for the lacZ gene was 95% (38/40) after 3 days of incubation after the PGC injection. The lacZ gene, however, appeared to persist episomally but was gradually lost during embryonic development. After 17 days of incubation after the PGC injection, the lacZ gene was detected in only 14.3% (3/21) of the embryos examined. Although the lacZ gene was detected in the gonads of two hatched chicks (11.1%), it was not detected in the gonads of chimaeric chickens at sexual maturity. Offspring derived from the lipofected PGCs were obtained from the chimaeric chickens at frequencies of 12.1-69.9% in males and 71.6-97.6% in females. The technique developed in the present work could be used to test the expression of exogenous DNA in the gonads of early chicken embryos and should facilitate the production of transgenic chickens.
An attempt was made to improve gene transfer into chick embryos in order to produce transgenic chickens. The beta-actin-lacZ/MiwZ, a marker gene in transfection reagent, was injected into the blastodisc of either unincubated fertilized eggs (stage X) or eggs induced from the shell gland by treating the hens intravenously with oxytocin or arginine vasotocin (stages IV-VI). All the manipulated embryos were incubated to reach stage XIV, the period at which primordial germ cells (PGCs) migrate from the germinal crescent to the gonadal anlage via the blood stream. MiwZ was detected in the embryos, extraembryonic tissues and blood by the histochemical staining method of beta-galactosidase. The MiwZ DNA was detected in 57% (127/221) of the survival embryos and in 9% (12/127) of the embryonic tissues. The expression was observed mosaically in the epidermis, heart and neural tube. The PGCs in the blood collected from the vitelline artery or dorsal aorta also showed a positive histochemical staining. However, the expression of MiwZ using the soft shelled eggs was more intense in the extraembryonic tissues, although it did not emerge in the embryos. Thus, it is possible to introduce an exogenous gene into the embryonic tissues using incubated fertilized eggs without sacrificing the hens. This technique for successive genetic operations should facilitate the production of transgenic chickens.
Primordial germ cells (PGCs) are the precursors of sperm and eggs. In most animals, segregation of the germ line from the somatic lineages is one of the earliest events in development; in avian embryos, PGCs are first identified in an extra-embryonic region, the germinal crescent, after approximately 18 h of incubation. After 50-55 h of development, PGCs migrate to the gonad and subsequently produce functional sperm and oocytes. So far, cultures of PGCs that remain restricted to the germ line have not been reported in any species. Here we show that chicken PGCs can be isolated, cultured and genetically modified while maintaining their commitment to the germ line. Furthermore, we show that chicken PGCs can be induced in vitro to differentiate into embryonic germ cells that contribute to somatic tissues. Retention of the commitment of PGCs to the germ line after extended periods in culture and after genetic modification combined with their capacity to acquire somatic competence in vitro provides a new model for developmental biology. The utility of the model is enhanced by the accessibility of the avian embryo, which facilitates access to the earliest stages of development and supplies a facile route for the reintroduction of PGCs into the embryonic vasculature. In addition, these attributes create new opportunities to manipulate the genome of chickens for agricultural and pharmaceutical applications.
Primordial germ cells (PGCs) of White Leghorn chicken embryos as a donor were transferred to Rhode Island Red chicken embryos as a recipient. At 48-50 h (stage 13-15) of incubation of fertilized eggs. donor PGCs, which were taken out From blood vessels of donor embryos, were injected into blood vessels of recipient embryos. Sex of the treated embryos was determined after the transfer of PGCs using remaining blood samples. In the present experiments, survival rate of the treated embryos was 33.3% far homo-sexual and 35.4% for hetero-sexual transfers of PGCs, respectively, when determined at 17 days of incubation. In this study, most of the treated embryos could not survive more than 18 days of incubation, though the reason for that was not clarified in the present work. The gonads removed from embryos that died after 18 days of incubation and the organs from newly hatched chicks were examined for morphological and histological features. The gonads removed from the embryos with home-sexual transfer of PGCs showed normal development in appearance. On the contrary, some (35.3%) of the embryos with hetero-sexual transfer of PGCs possessed abnormal gonads similar to ovotestis by histological observation. In cases where the gonads developed to be normal organs (64.7%) the sex of embryos was the same as recipient ones. The present results suggest that hetero-sexual transfer of the PGCs may bring about the possibility of development of the embryos bearing sexually different gametes, spermatogonia or oogonia.
The present experiments were designed to examine whether exogenous DNA injected into the germinal crescent region (GCR) of early stage of developing embryos, which is considered to be the main place from which PGCs originate, can be transferred to recipient chicken embryos. In this experiment, Miw Z (DNA) dissolved in the transfection reagent (TR: Boehringer, Germany) was introduced into the GCR of donor embryos at stage 3-5 or 9-11, followed by continued incubation until the stage 13-15 of embryonic development. The PGCs collected from the embryonic blood vessels were examined for the incorporation of the injected DNA into the PGCs by the methods of X-gal staining and PCR analysis. As the results, the foreign DNA was successfully incorporated into the PGCs, leading to their transfer to the gonadal tissues. The present results, therefore, suggest that the early stage (3-5 or 9-11) of chicken embryonic development would be more successful than stage 13-15 in transferring exogenous genes to the recipient embryos, leading to the possibility of producing transgenic chicken medianting the PGCs.
In this experiment, an exogenous gene, green fluorescent protein (GFP), was introduced into chicken blastodermal cells (stage X) just after oviposition using two types of electrode, parallel and needle, comparing embryonic survivability and subsequent development of embryos. The needle type electrode was prepared with 27-gauge needle of syringe which was covered with an insulating paint to isolate electricity. Electricity employed in this study was 10v (needle type) and 200v (parallel type) of pressure, 10msec of pulse and 10 times of repeated intervals. The embryonic survivabilities were 65% for needle type and 53% for parallel one, respectively, though no significant (P>0.05) differ-ence was observed between two types of electrode. The expression rates of GFP in embryonic tissues were 48% for needle type and 7% for parallel one, respectively, showing significant (P<0.01) difference between two types of electrode. In all of these treated embryos, GFP expression was mosaic manner for both types of electrode, indicating especially positive expression in embryonic tissues for needle type and in vitelline membrane for parallel one. Therefore, the present results suggest that the needle type of electrode might be superior to parallel one for introducing foreign genes into chicken embryos when used for electroporation.
The cells in the center of the area pellucida of the blastoderm have been used as donors for production of chimeric chickens, but the developmental fate of the cells has not been analyzed in detail. Also, little is known regarding the molecular mechanism of germline development and differentiation in early chick embryos. To clarify the developmental fate precisely, donor cells fluorescently labeled by PKH26 were analyzed in chimeric embryos. The cells were obtained from the center of the area pellucida of the donor and injected into the center of the area pellucida of the recipient. The dynamic states of the donor cells were tracked during development of the chimeric embryos, thereby allowing analysis of the developmental fate of the cells of the center of the area pellucida. The manipulated embryos that were injected with marked donor cells from the center of the area pellucida were cultured. The presence of the marked donor cells was confirmed in many embryos, through observation of embryos at each development stage under a fluorescence microscope. The results suggest that the migration system and developmental fate of cells from the center of the area pellucida are identical to those of primordial germ cells (PGCs). It was concluded that many PGCs or their precursor cells are localized in the center of the area pellucida of the blastoderm region.
This experiment was designed to produce chimeric chicken by transferring primordial germ cells (PGCs) of Ehime native chicken embryos to White Leghorn ones. Fertilized eggs produced by both chicken breeds were incubated until stage 12-15 of embryonic development. The donor PGCs were taken out from blood vessel of Ehime native chicken embryos and injected into blood vessel of White Leghorn ones at the same stage. After the injection, the recipient eggs were incubated until hatching. Finally, three hatchlings including one male and two females were obtained. The birds survived to sexual maturity were used for progeny test to check chimerism. Of 29 offspring obtained by progeny test, 15 chicks showed feather color which was considered to be derived from Ehime native chicken. The present results suggest that the production of chimeric chicken by means of the transfer of PGCs may be able to restore genetic resources from endangered domestic chicken or some of the wild birds.
Furuta, H., Kim, K.B. and Fujihara, N. 2000. Gene transfer to chicken blastoderm by lipofection or electroporation. J. Appl. Anim. Res., 17: 209–216.It was proved that an exogenous gene was successfully transferred to chicken embryos by the injection of the gene into blastoderm of freshly oviposited fertilized eggs. In this study, green fluorescent protein (GFP) was used as a marker gene. Tlie GFP dissolved in DOTAP Liposomal Transfection Reagent was introduced into the blastoderm by the method of lipofection or electroporation. After the injection of gene, the eggs were incubated in routine manner until developmental stage 11 to 15. Survival rate of the treated embryos at the stage 11 was 46 per cent (27/59) for the lipofection and 49 per cent (81/166) for the electroporation, respectively. Expression percentages of the GFP for the treated eggs were 74 per cent (20/27) for the lipofection and 5 per cent (4/81) for the eleclroporation, respectively. The GFP was observed in several sites of embryonic tissues and in some of the primordial germ cells (PGCs) in the former method only. Thus lipofection method was considered, slightly superior to the electroporation method for the introduction of exogenous gene to chicken embryos.
Primoridal germ cells (PGCs) from the circulating blood in 2-day chick embryos were observed by histochemical techniques in smear preparation and by phase contrast microscopy in fresh samples. In the blood smear, PGCs were readily distinguished from blood cells by their large size (15–20 μm in diameter) and by the round and large nuclei (8–10 μm in diameter) occupying eccentrically the greater parts of the cells. Histochemically, they were demonstrated to contain abundant glycogen, a lesser amount of yolk granules (by the PAS reaction), and a large amount of lipid droplets (by the Oil red O and Sudan black B stains) in their cytoplasm. Alkaline phosphatase activity was proved slightly in their cytoplasm by the simultaneous diazo coupling method. In phase contrast microscopy, living PGCs in the blood and from the primitive gonad of 3-day embryos in parts of the examinations were observed to change their shape in somewhat amoeboid fashion, suggesting their capability for locomotion as a possible mechanism of the migration of PGCs in vivo.
Chicken primordial germ cells (PGCs) collected from thecirculating blood in embryonic vessels at stage 13-15 were inter-embryonically, homo- or hetero-sexually,transferred to the blood vessels of recipient embryosat the same stage of development. Approximately 30%of the embryos treated with hetero-sexual transfer of PGCs had abnormal gonads, showing ovotestis likeorgans. In this case, some of these reversed gonadswere considered to be dependent upon the ratio of thenumber of PGCs from donor to recipient embryos. Oneof the treated embryos possessed completely reversedorgans. Therefore, the introduction of exogenousembryonic vessels was thought to be also useful forproducing transgened gonads.
FORTY-FIVE FIGURES The preparation of a series of normal stages of the chick embryo does not need justification at a time when chick ernbryos are not only widely used in descriptive and experimental embryology but are proving to be increasingly valuable in medical research, as in work on viruses and cancer. The present series was planned in connection with the preparation of a new edition of Lillie’s DeueZopmerzt of the Chick by the junior author. It is being published separately to make it accessible immediately to a large group of workers. Ever since Aristotle “discovered” the chick embryo as the ideal, object for embryological studies, the embryos have been described in terms of the length of time of incubation, and this arbitrary method is still in general use, except for the first three days of incubation during which more detailed characteristics such as the numbers of somites are applied. The shortcomings of a classification based on chronological age are obvious to every worker in this field, for enormous variations may occur in embryos even though all eggs in a setting are plmaced in the incubator at the same time. Many factors are responsible for the lack of correlation between chronological and structural age. Among these are : genetic differences in the rate of development of different breccls (eg., the embryo of the White Leghorn breed develops more 49
We have demonstrated that quail PGCs possess a characteristic heterochromatin nuclear marker demonstrable already at stage 6 H & H with Feulgen staining. Chimaeras of stage-XIII E. G & K chick epiblast and quail hypoblast and vice versa have been made. The chimaeras were incubated up to stage 7-8 H & H and checked histologically with Feulgen staining for the presence of the heterochromatin marker in every detectable PGC. It was found that the PGCs are of epiblastic origin, contrary to previous data and speculations concerning avian PGCs.
Germline chimeric chickens were produced by transfer of primordial germ cells from White Leghorn to Barred Plymouth Rock, and vice versa. Blood was collected from stage 13-15 embryos and primordial germ cells were concentrated by Ficoll density gradient centrifugation. Approximately 200 primordial germ cells were injected into the bloodstream through the dorsal aorta of stage 14-15 recipient embryos from which blood had been drawn via the dorsal aorta prior to the injection. Intact embryos were also prepared as recipients for White Leghorns only. The manipulated embryos were cultured in recipient eggshells until hatching. Germline chimerism of the chickens reaching maturity was examined by mating them with Barred Plymouth Rocks and donor-derived offspring were identified based on their feather color. The efficiency of production of germline chimeras was 95% (19/20). When primordial germ cells were transferred from White Leghorn to Barred Plymouth Rock, the average frequency of donor-derived offspring was 81% for three male chimeras (96% for one female chimera), and it was approximately 3.5 times higher for transfer in the opposite direction (23% for 6 male chimeras). Removing blood from recipient embryos prior to primordial germ cell injection enhanced the frequency of donor-derived offspring by 10% in resulting male chimeras. Male chimeras produced donor-derived offspring more frequently (approximately 3.8 times) than female chimeras. Increases, decreases, or no changes were observed in the frequency of donor-derived offspring from the germline chimeras with increasing age.(ABSTRACT TRUNCATED AT 250 WORDS)
Blood from an individual quail embryo at stages 13-16, when primordial germ cells (PGCs) were in circulation, was taken from its marginal vein and transfused into the marginal vein of a chick embryo at stages 13-16. Both donor and recipient embryos were cultured in vitro until day 8 of development and their sex was determined by morphological and histological observations of the gonads. Sections of recipient gonads were stained immunohistochemically with QCR1 monoclonal antibody positive for quail PGCs but negative for chick PGCs. Donor and recipient embryos were sexed in 17 pairs which included all four sex combinations. Transferred PGCs, either female-derived ZW type or male-derived ZZ type, were observed in the gonads of both sexes of 15 recipient embryos. The population of donor PGCs ranged from 20 to over 2500. In all four sex combinations, there was a higher population in the left than the right gonad of the embryos.
In avian species, the developmental fate of different-sex germ cells in the gonads is unclear. The present study attempted to confirm whether genetically female germ cells can differentiate into spermatozoa in male gonads using male germline chimeric chickens produced by the transfer of primordial germ cells (PGC), and employing molecular biological methods. As a result of Southern hybridization, specific sequences of the W chromosome (the female specific sex chromosome in birds) were detected in the genomic DNA extracted from one out of four male germline chimeric chickens. When two-color in situ hybridization was conducted on the spermatozoa of this germline chimera, 0.33% (average) of the nuclei of each semen sample showed the fluorescent signal indicating the presence of the W chromosome. The present study shows that female PGC can differentiate into spermatozoa in male gonads in the chicken. However, the ratio of produced W chromosome-bearing (W-bearing) spermatozoa fell substantially below expectations. It is therefore concluded that most of the W-bearing PGC could not differentiate into spermatozoa because of restricted spermatogenesis.
In vitro transfer of foreign DNA into germ cells (PGCs) of embryos
- K Eguma
- T Soh
- M A Hattori
- N Fujihara
Eguma K, Soh T, Hattori MA and Fujihara N. In vitro
transfer of foreign DNA into germ cells (PGCs) of
embryos. Asian-Australasian Journal of Animal Science,
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