Can testosterone make your chick masculine?

Rachael Curley, Zac Martin, Michael Mendez

Milersville University

Purpose

To determine if the sex of chicken embryos will be influenced by the injections of hormones, particularly testosterone. Testosterone is a cholesterol-derived hormone that is found at higher concentrations in males than females. The desired results following the testosterone injections are to obtain all male, female and transgender containing ovary and testis, chick embryos. After the dissection of these chicks, the gonads will be stained to help distinguish the organism between male and female as described in our previous study (chick mini-lab).

Background

Chicken embryos begin to develop their sex organs around 7 days following fertilization. During this crucial developmental stage the left gonad of both male and female increase in volume. The difference is greater in females than in males, constituting early evidence of some sex differentiation of the gonads. Additionally, volume and surface epithelium thickness are greater in the left gonad in both sexes. These differences disappear in males, whereas in females only the left ovary and left Müllerian ducts fully develop and become functional (Hoshino, 2005). It has been previously hypothesized that sex hormones, when increased in concentration, may influence the development of gonads and in fact cause reveral sex development. Theelin, theelol, and an extract of male human urine (containing some estrogenic substances) brought about, in a number of cases, embryos in which the left gonad is an ovotestis and the right one entirely testicular (Willier, 1937).

Chicken embryos have distinct differences between their reproductive organs. Males have two testis and two ovaries form in the female, but then only one continues to grow and develop. It is very hard to distinguish between both, when just viewing the gonads under a microscope. Therefore, a staining technique using alkaline phosphatase substrate is implemented since germ cells can be stained based on their high levels of the enzyme alkaline phosphatase. Staining the primordial germ cells (PGCs) would allow you to distinguish between ovaries and testis in younger chicks because they still have two ovaries and the shapes are more similar. Alkaline phosphatase activity was first noted with consistency in PGCs as early as 2 days of incubation. PGCs found in the extra embryonic circulation at this stage of development exhibited a positive reaction for alkaline phosphatase activity in the form of a globular deposit(s) in the cytoplasm near the nucleus. (Swartz, 1982) According to Gilbert (2009), PGCs are arranged differently in testis and ovaries. In the testis the germ cells migrate to the periphery of the seminiferous tubules where they begin to differentiate into sperm and then are transported through the rete testis. In the females the germ cells become the ova and the surrounding cortical somatic cells will differentiate into granulose cells. From there the mesenchyme cells from the ovary differentiate into the thecal cells.

Materials:

24-two week old embryos

Howard’s Ringers Solution

Dissection tools (2x)

Petri-Dishes (1 per 2 Chicks)

Scanning Microscope

Alkaline Phosphatase Substrate (Western Blue, Fisher)

Alkaline Phosphatase Substrate Buffer (APSB)

1x PBS (Phosphate Buffered Saline) Stain Stop

4% PFA (PARAFORMALDEHYDE) in PBS

Capped Tubes (8 tubes)

Eye droppers (2x)

Testosterone Cypionate

1mL Syringe

Vegetable Oil

Beak nose tweezers

Scotch Tape

Procedure:

Making Solutions

APSB

(100nN NaCl, 100mM Tris, pH 9.5, 50mMMgCl₂)

5 M NaCl 2ml

2 M Tris, pH 9.5 5ml

4.9 M MgCl₂ 1ml

dH₂O (ultrapure) to 100ml

1x PBS

10X PBS

NaCl 175.3g

KH₂PO₄ 7.7g

K₂HPO₄ (anhydrous) 25g

dH₂O to 2L

100mL of this solution was added to 900mL of H2O to create 1L of 1xPBS

APS

Western Blue

Cat # S3841 Lot # 28564204

4% PFA

PBS – Warm to 65° (about 1 hour)

Add 4g/100ml Paraformaldehyde

Add 1 ml 1M NaOH, stir till dissolves

Neutralize with 1 ml of 1M HCl

Testosterone Cypionate/Vegetable Oil

The Testosterone Cypionate (TC) bottle recommended 400mg TC per 100kg body weight. This was converted to 50µg TC 10g body weight. A random sample of five eggs were weighed and averaged to be 66.576g. The weight was rounded up to 70g so the calculated dosage would be 350µg of TC per 70g of body weight. The TC bottle contained 10mL of solution with a dosage of 100mg/mL. We calculated that each egg should receive 3.5µL of TC. The final solution contained 105µL of TC solution mixed with 2.9mL of vegetable oil to give us a total of 3mL. This is the solution used for injection.

Testosterone Injection

Allow the eggs to sit up right for 5 min in order to ensure that the chick embryo is at the top of the egg. Using the beak nose tweezers, poke a small hole in the shell of the egg allowing enough room to see where you are going to stick the needle. Keep in mind you are not to poke the embryo itself with the needle. Using a 1mL syringe, measure out 100µL of Testosterone Cypionate/Vegetable Oil solution. Inject this into the yolk of the egg through the hole. Immediately after injecting the Testosterone Cypionate/Vegetable Oil solution into the yolk, place a piece of scotch tape over the hole to prevent the embryo from drying out.

Staining Process

Fertilized chicken eggs were obtained from Millersville University of Pennsylvania where they were incubated at 37°C. To insure the eggs were actually fertilized or developing, each egg was held up against a table light for the presence of blood vessels lining the interior of the egg. The eggs that did not fertilize or develop correctly were discarded. The eggs however that did develop normally were cracked open. Using forceps the chick embryos were pulled out and placed into a Petri dish containing 50 mL of Howards Ringers Solution. After the chicks were decapitated and then placed on its back, the ventral side was cut open and the stomach, intestinal tract (white), and the liver (green/brown) were removed or pushed aside. Once removed, the kidneys (yellow) were visible. The gonads were located towards the medial-anterior ends of the kidneys, depending on how far along the developing female chick embryos’ reproductive system was. The gonads were carefully teased away from the kidneys and then were placed into a 15 mL capped tube filled with 4% paraformalaldehyde (PFA). From there, the gonads were washed three times at 5 minute increments with another 15 ml of 4% PFA and then transferred into another capped tube containing alkaline phosphatase substrate buffer (APSB) for 30 minutes on a rocker. After 30 minutes, the gonads were then transferred to another capped tube filled with 5 mL APSB and 5 mL APB in a 1:1 ratio, respectively. The gonads were removed from the stain once the inside of the gonads turned purple and from there, were placed in 15 mL of 1% phosphate buffered saline (PBS) for 5 minutes to stop the precipitation. Lastly, the gonads were stored in the 15 mL of fixative. The differences between the male and female gonads were analyzed using a dissecting microscope.

Results

Following the injection of testosterone into the albumin of 42 individual chick eggs albumin and the incubation at 37°C for 7 days, the gonads were extracted. Each individual was observed using a dissecting microscope and then stained. After the staining of the primordial cells located inside the chick embryos gonads, a purple precipitate was deposited. The purple precipitate enabled us to view the make-up of the gonads. The female gonads (Figure 1) did not have a particular pattern to them, but seemed to have random spots of purple scattered all over the gonad(s). Female gonads are also much larger and most of them were more round in shape. The male gonads (Figure 2) had a pattern of purple to them, very similar to the human male’s seminiferous tubules pattern. Male gonads also tended to be longer and much thinner than the ovaries. Table 1 represent the percentage of males vs. females found in the total eggs and used as the control. A total of 36 eggs were obtained and 23 of these eggs were actually used for gonad extraction. Out of the eggs, 47.83% of them were males and 52.17% of them females. Table 2 presents the gender of the chicken embryos following testosterone injections. Only 18 chicken embryos survived the injection of testosterone and of these 61.1% were males and 38.9% transgender. There were no females distinguished using the dissecting microscope and staining technique. As you can see from the control to the testosterone injected embryos there was a significant decrease in the females developed and an increase in the males developed. From the testosterone injected embryos there were transgenders that developed containing both an ovary and testis. Of the 42 chicken embryos injected only 18 developed successfully (42.85%). The high rate of non-developing embryos following injection could be related to infection after cracking open the eggs or from accidentally injecting the testosterone directly into the embryos. Lastly, figure 3 represents a transgender chick embryo following testosterone solution injection of 1 ml.

Figure 1: The ovary of a 14-day-old chick embryo that has been stained with alkaline phosphatase substrate (5 mL) and alkaline phosphatase substrate buffer (5 mL) in a 1:1 ratio, respectively.

Figure 2: The testis of a 14 day old chick embryo that has been stained with alkaline phosphatase substrate (5 mL) and alkaline phosphatase substrate buffer (5 mL) in a 1:1 ratio, respectively.

	Total	# Male	% Male	# Female	% Female
Exp. 1	15	8	.53	7	.47
Exp. 2	8	3	.38	5	.62
Total	23	11	.48	12	.52

Table 1: The total number of eggs that were used to extract the embryo gonads are represented here. The total is the amount of these eggs that were developed. The number of male and female embryos was recorded and the percentage of each gender calculated.

Figure 3: The gonads of a transgender are shown. The larger pink medial object is an ovary. The smaller pink medial object below the ovary is a testis.

	Total	# Male	% Male	# Female	% Female	# Transgender	% Transgender
1	11	8	.727	0	0	3	.272
2	7	3	.429	0	0	4	.571
Total	18	11	.611	0	0	7	.389

Table 2: The total number of eggs that were used to extract the embryo gonads are represented here. The total is the amount of these eggs that developed following testosterone injections. The number of male, female, and transgender were recorded and the percentage of each gender calculated.

Discussion

This experiment achieved the ability to influence the sex of 7-day-old developing chick embryos by injecting higher amounts of testosterone into the albumin of the egg. This created a higher concentration of testosterone than normally present. The gonads of the chick embryo were removed at 14 days and observed. We also learned that alkaline substrate phosphatase, when used in the correct concentrations, can stain the PGCs of the gonads as well as other structures. We were able to successfully isolate the gonads of both genders as well as stain the PGCs present in the gonads of chick embryos to assist in the distinguishing of their gender. All of the surviving chicks were either male or transgender following testosterone injections. No females were present after the injection of testosterone. This experiment shows that the sex of developing chick embryos can be influenced using testosterone. Male or transgender chicks may be obtained by simply injecting the eggs with testosterone. This may be used in a practical sense on farms or other chicken producing industries. If a higher frequency of males is needed for reproduction of chickens then the handler of the chickens can perform the injection of testosterone to achieve his desired frequency. A way to expand on this experiment would be to inject chicken embryos with Estrogen, the female sex hormone. The same steps can be taken to see if the sex of chickens can be influenced to be female. This would allow for a wider range of achieving desired sex ratios. The staining process can be used to determine the structure or organs containing PGCs and also can be used to follow the migration patterns of PGCs through stages of development.

Literature Cited

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