Primordial Germ Cells In The Developing Chicken Gonads Biology Essay

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Understanding the avian germ line development and its manipulation provides a great potential for the basic science as well as practical applications. Production of pharmaceutical materials in the egg of transgenic chicken is the first target application, and success in this production could lead to the commencement of a new industry. Avian germ line, the primordial germ cells (PGC) or their precursors are localized in the central zone of the area pellucida (Tagami and Kagami, 1998; Yamamoto et al., 2007), on the ventral surface of the epiblast, at stage X (Roman numerals refer to the staging system of Eyal-Giladi and Kochav, 1976). These cells gradually translocate into the hypoblast during stages -XIV (Karagenc et al., 1996). Following the formation of the primitive streak, PGC are carried interiorly to the germinal crescent region (Ginsburg and Eyal-Giladi, 1986), from which they enter the embryonic circulation associated with formation of the blood vascular system. Subsequently, PGC actively leave capillary vessels close to the germinal epithelium and migrate along the dorsal mesenterium to the gonadal anlage (Nakamura et al., 1988; Yasuda et al., 1992; Kuwana and Rogulska, 1999 ). They settle in the gonads by day 6 of the 21 day incubation period, with more cells populating the left versus the right gonad (in both sexes) (Fujimoto et al., 1976).

Male germ cells do not proliferate significantly from the time of testicular organization (day 6 onwards), while female germ cells proliferate considerably, from at least as early as day 9 (Mendez et al., 2005 and Hughes, 1963). In the female, germ cell fate differs between the left and right gonads. In the left gonad, somatic cells develop a thickened outer cortex, where the synchronously proliferating germ cells nest and become apparent from day 10 onwards. It has been estimated that the total population of germ cells in the left ovarian cortex increases about twenty-five fold between day 9 and day 17 of incubation (Hughes, 1963). In contrast, the right gonad fails to proliferate in the cortex. Germ cells in the right gonad proliferate somewhat in the underlying medulla, but they later undergo apoptosis (Ukeshima and Fujimoto, 1991). Craig et al. (2008) reported that meiosis in the chicken embryo is initiated late in embryogenesis (day 15.5), relative to gonadal sex differentiation (from day 6). Meiotic germ cells are first detectable only in female gonads from day 15.5, correlating with the expression of the meiosis marker, SCP3.However the fact that weather all the PGCs enter to their meiotic status before the egg hatches are yet not reported. Similarly observation of the differentiating PGCs in male gonads till hatch and even soon after hatch are still needed to be probed.

Different approaches are currently used to study chicken PGCs. Originally they are using morphological criteria, such as a remarkably large size, large spherical nuclei, and the presence of refractive lipids in the cytoplasm (Tajima,(2002); Zhao and Kuwana, 2003), coupled with either a histochemical marker such as periodic acid-Schiff (PAS), which stains for glycogen (Meyer, 1960; Ishiguro et al. 2009) , or antibodies such as stage-specific embryonic antigen-1 (antiSSEA-1) and embryonic mouse antigen-1 (antiEMA-1), which recognize cell-surface carbohydrate antigens. However, PAS staining efficiently detects PGC only after stage 4 (Arabic numerals refer to the staging system of Hamburger and Hamilton, 1951). Moreover, the SSEA-1 epitope, galactose-N-acetyl glucosamine-fucose (Gooi et al., 1981), is expressed on inner cell mass, epiblastic cells, and migratory PGC in mice, suggesting that the antigen is not germline-specific. AntiEMA-1 recognizes fucosylated polylactosamine carbohydrate groups and was originally raised against mouse embryonic carcinoma cells (Hahnel and Eddy, 1986). AntiEMA-1 labels not only mouse PGC, but also chicken PGC. However, only one-third to one-fifth of PAS-positive cells are labeled by antiEMA-1 in the chicken (Urven et al., 1988), indicating that antiEMA-1 is not suitable to define germ cell lineage. Consequently, morphology, PAS staining and immunohistochemical staining, such as with antiSSEA-1 or antiEMA-1, are not specific enough to allow a direct investigation of germline segregation in the chicken.

Presently the gene vasa are considered a reliable molecular marker for tracing avian germ cell lineage (Ishiguro et al. 2009). The vasa gene was originally discovered in Drosophila (Lasko and Ashburner, 1988), and genes homologous to vasa have now been identified in various species, including Caenorhabditis elegans, Xenopus laevis, zebrafish, mice, humans, trout, and rat (Roussell and Bennett, 1993; Fujiwara et al., 1994; Komiya et al., 1994; Olsen et al., 1997; Yoon et al., 1997; Castrillon et al., 2000; Yoshizaki et al., 2000). Tsunekawa et al. (2000) isolated the chicken vasa homolog (CVH) gene and demonstrated germline-specific expression of CVH protein, mainly in sections of embryos. Their research showed that CVH could be used as one of the reliable molecular markers for investigating PGCs. Recently Craig et al. (2008) assessed the onset of meiosis in the chicken embryo histologically by haematoxylin and eosin (H & E) staining of serial sections through the developing gonads. The authors have also reported successful use of polyclonal rabbit SCP3 antibodies for observing meiotic cells in the developing chicken gonads.

In continuation with the above findings present study will be conducted for qualitative and quantitative analysis of differentiating PGCs in the developing chicken gonads

Materials and Methods

Experimental Birds Experimental flock of two layer breeds, the White Leg Horn(WLH) and Rhode Island Red(RIR)comprising 30 female and 10 male of each breed will be maintained at the poultry barn of Agriculture and Forestry Research Centre, University of Tsukuba. All the birds will be kept, maintained and treated adherence to accepted standards for the humane treatment of animals. After attaining their mature age the semen will be collected from male cocks and will be injected into the vaginal orifice of the females from the respective breed,@0.2ml per bird using tuberculin syringe. The eggs produced 2-3 days after artificial insemination (AI) till one week are usually fertile. After having the confirmed fertility, the eggs will be set in the incubator maintained at 37.8 °C and 60 % humidity.

Sampling strategies and project duration Sampling will be continued till clear pictures of reasonable number of sections carrying differentiating PGCs are obtained. It is planned to start the project from April 2010 and hoped to be completed by the end of October. Results compilation, data analysis and write up will be completed in the remaining period of the fellowship and the research paper will be ready to submission for publication in the first week of December 2010

Collection of Gonads Embryos from 10 to 20 days of incubation will be collected for the present project. After harvesting the embryo proper will be isolated from the egg yolk and placed in phosphate buffered saline (PBS) filled petri dish. The egg yolk and the embryonic membranes will be removed. The embryo will be fixed through needles in a rubber pad having petri dish filled with PBS. The gonads be will carefully removed under dissecting microscope and will be placed in bouin`s fluid maintained in sample glass bottle, properly labeled with the required information.

Fixation and Dehydration Each sample will be Fixed in 10 ml Bouin's fluid {Bouin's fluid is mixture of 15 parts saturated picric acid in distilled water, 5 parts neutralized formalin (Neutralized formalin is saturated calcium carbonate in formalin) and 1 part acetic acid} form over night to 24 hours with the vial rocking, stored at 4°C. After fixing the samples will be dehydrated through six serial washes using 100% ethanol with an interval of 30 minutes between the washes. Thereafter the samples will be stored at 4°C till further use

Clearing, Paraffin Infiltration The samples will be stained red through adding 3 ml coloring solution (made by dissolving red food coloring in 100% ethanol) for 5 minutes at room temperature. Excessive staining will be washed through 5ml ethanol 100 % for 10 minutes at room temperature. Then the sample will be cleaned in Xylene 5cc for three times with an interval of 30 minutes between each wash at room temperature. On third wash the Xylene will be washed and liquid parffine will be added to the sample bottle@ 5ml per sample at 65°F in hot air oven.( Xylene is volatile and harmful chemical, and therefore this work will need draft facilities)

Embedding Three washs with paraffin at 30 minutes interval in temperature of 65 °F will be applied to each sample. Efforts will be made to completely melt the paraffin even if the time is passed. After third wash the sample will be shifted to metal mold (labeled with sample identity) containing paraffin using Tweezer. The samples will shifted to the centre through Tweezer. Then the metal mold will be immersed in water and will be maintained at -20°C. Thereafter the sample containing mold will be separated from metal plate.

Trimming of the blocks The paraffin mold will be trimmed using shaving blade in such manner that some part is left for attachment with wooden block. After the block is ready it will be attached on the wooden block using heated spatula

Sectioning The block containing sample will be sectioned with microtome. Serial sections of all the samples will be prepared and will be divided on three slides for H&E staining, and immunohistochemistry using Vasa and SCP3 antibodies simultaneously. The slides will be dried on hot plate for overnight keeping at 45 °F.

H& E Staining

Name Reagents

Treatment timing

Name Reagents

Treatment timing

1 Xylene 1

10 min

11 Distilled Water

1 min

2 Xylene 2

10 min

12 Eosin Solution

2 min

3 Xylene 3

10 min

13 Distilled Water

1 min

4 100% Ethanol

10 min

14 50% Ethanol

1 min

5 100% Ethanol

10 min

15 70% Ethanol

1 min

6 70% Ethanol

1 min

16 100% Ethanol

5 min

7 50% Ethanol

1 min

17 100% Ethanol

5 min

8 Distilled Water

1 min

18 Xylene 1

5 min

9 Mayer's haematoxylin

5 min

19 Xylene 2

5 min

10 Running Water

5 min

20 Xylene 3

5 min

Immuno-staining of section s with chicken vasa homolog (CVH)

Reagents

PBS-T

Phosphate buffered saline containing 0.05% Tween 20

Blocking Solution

PBS-T containing 0.2% Blocking One (03953-95, Nacalai Tesque)

Primary Antibody

Mixture of 100 parts Blocking Solution and 1 part anti-CVH antibody

Secondary Antibody

Mixture of 200 parts Blocking Solution and 1 part alkaline phosphatase conjugated Goat Anti-Rabbit IgG, IgM, IgA (H&L) antibody (SAB1005, Open Biosystems)

BCIP/NBT (5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium)

BCIP/NBT Substrate System (K0598, Dako)

Aqueous Mounting Medium

Mount-Quick Aqueous

Procedure All the steps will be performed at room temperature

S.No

Name Reagents

Time

Purpose

1

Xylene 1

10 min

Deparaffinization

2

Xylene 2

10 min

3

Xylene 3

10 min

4

100% Ethanol

10 min

Rehydration

5

100% Ethanol

10 min

6

70% Ethanol

1 min

7

50% Ethanol

1 min

8

Distilled Water

1 min

9

PBS(-)

1 min

Proper quantity, Rinse

10

Blocking Solution

1 h.

200μl at each slide glasses Blocking for ndogenous alkaline phosphatase and permeabilization

11

PBS(-)

1 min

Proper quantity

12

First Antibody

30 min

200μl at each slide glasses

13

0.05% PBS-T

5 min

Proper quantity, Rinse

14

0.05% PBS-T

5 min

15

0.05% PBS-T

5 min

16

Secondary Antibody

30 min

200μl at each slide glasses

17

0.05% PBS-T

5 min

Proper quantity, Rinse

18

0.05% PBS-T

5 min

19

0.05% PBS-T

5 min

20

0.05% PBS-T

5 min

21

0.05% PBS-T

5 min

22

BCIP/BCT

a few minutes

2 or 3 drops, For colorimetric detection of alkaline phosphatase

color development will be observed under microscope

23

Distilled Water

1min

Rinse

24

Distilled Water

1min

25

Distilled Water

1min

26

Mayer's Haematoxylin

5 min

Counterstaining

S.No

Name Reagents

Time

Purpose

27

Running Water

5 min

Rinse

28

Distilled Water

1 min

Rinse

29

The slide glass will be using aqueous mounting medium

SCP3 antibodies for observing meiotic cells

The procedure adopted will be almost similar to Vasa but the reagents used will be different. The protocol for the use of this marker will be requested from the supplier company and will be proceeded accordingly.

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Embryo sexing

Sex identification prior to the appearance of morphological variation is a prerequisite for for identification of sex-related differences during embryonic development. Recently the polymerase chain reaction (PCR) has been used successfully to identify embryonic sex in chickens (Clinton, 1994; Petitte and Kegelmeyer, 1995). This method is reliable and accurate and has been used to sex a large number of chick embryos in a short time, thereby permitting subsequent manipulation of embryos of known sex. Unlike mammals, in birds, the female is the heterogametic sex (ZW). The W chromosome in Gallinaceous birds, such as the chicken, pheasant, and turkey, consists of a significant amount of repetitive DNA sequences (Tone et al., 1982, 1984;). The sex-specificity of the W chromosome repeats (XhoI) has enabled its use in sexing chicks and chick embryos by PCR and DNA-DNA hybridization (Kodama et al., 1987; Petitte and Kegelmeyer, 1995).

Sex determination in present study will be required to work out the difference in the differentiation pattern of stem cells in male as well as females. PCR -based sexing will be carried out according to the method described by Petitte and Kegelmeyer at al. (1995) using skin samples of the embryos. A 276- bp fragment of the 717 -bp W -chromosome specific Xh0I repetitive sequence will be amplified in a reaction mixture containing Takara ExTaq polymerase, dNTPs,10X Taq buffer and primers 5`-CGTGAGAAAAGTGGTAGTT-3`and`3-CTCTGTCCACCATAAAAACC-5`. Each primer (25 microM in a 5 micro M volume) will be mixed with one micol of dialuted DNA sample 4 microl dNTP mixture,5microl of 10X Taq buffer, 0.25 microl Taq polymerase ( 5unit per micro l and 29.75 microl of sterilized water).

The PCR will be carried out with an initial denaturation step at 94 oc for 2 min, followed by 20 cycles at 94 oc for 30 seconds and 72 0c 30 sec and a final extension step at 72 oc for 5 minutes in a Takara PCR Thermal Cycler. The PCR products will be separated by electroperosis on a 1.5% agrose gel and will be visualized under UV light after staining with ethidium bromide. Only samples from female will express a 276-bp PCR product.

Observations: The numbers of germ cells will be estimated in both gonads of embryos belonging to either sex removed between 10th and twentieth days of incubation. The sections will be examined under high magnification power of microscope. The germ cells situated in the ovarian cortex and medulla will be counted in every fortieth section (Gillian & Hhughes, 1963). In order to estimate the total number of germ cells, the counts will be simply multiplied by 40 (Green & Zuckerman, 1954). The sections will be systematically moved on the mechanical stage so that the whole of the cortical area is scanned once; an eye-piece graticule will be used as a guide. Care will be taken to focus at all levels through the thickness of the section, and only those cells whose nucleus is largely included in the section will be considered. The germ cells will be classified as stem and meiotic cells and will be counted separately. It is presumed that Vasa staining will provide us the whole cells count and SCP3 staining will enable us to observe and count the meiotic cells. After knowing the number of all cells and meiotic cells in a section we can clearly predict the number of stem cell present in the section.

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