Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes)

Summary

The article describes a quick protocol to gonadectomize and sample blood from the small teleost fish, using Japanese medaka (Oryzias latipes) as a model, to investigate the role of sex steroids in animal physiology.

Abstract

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in all vertebrates by providing feedback to the brain and pituitary. Teleost fishes possess a higher degree of tissue plasticity and variation in reproductive strategies compared to mammals and appear to be useful models to investigate the role of sex steroids and the mechanisms by which they act. The removal of the main source of sex steroid production using gonadectomy together with blood sampling to measure steroid levels has been well-established and fairly feasible in bigger fish and is a powerful technique to investigate the role and effects of sex steroids. However, these techniques raise challenges when implemented in small size teleost models. Here, we describe the step-by-step procedures of gonadectomy in both males and female Japanese medaka followed by blood sampling. These protocols are shown to be highly feasible in medaka indicated by a high survival rate, safety for the life span and phenotype of the fish, and reproducibility in terms of sex steroid clearance. The use of these procedures combined with the other advantages of using this small teleost model will greatly improve the understanding of feedback mechanisms in the neuroendocrine control of reproduction and tissue plasticity provided by sex steroids in vertebrates.

Introduction

In vertebrates, sex steroids, which are mainly produced by the gonads, play important roles in the regulation of the Brain-Pituitary-Gonadal (BPG) axis through various feedback mechanisms^1,2,3,4,5. In addition, sex steroids affect the proliferation and activity of neurons in the brain^6,7,8 and endocrine cells, including gonadotropes, in the pituitary^9,10, and thus serve crucial roles in brain and pituitary plasticity. Despite relatively good knowledge in mammals, the mechanism of BPG axis regulation mediated by sex steroids is far from being understood in non-mammalian species, leading to poor understanding of evolutionary conserved principles¹¹. There is still a limited number of studies documenting the role of sex steroids on brain and pituitary plasticity, thus raising the need for further investigations of the role and effects of sex steroids on diverse vertebrate species.

Among vertebrates, teleosts have become powerful model animals in addressing numerous biological and physiological questions, including stress response^12,13, growth^14,15, nutritional physiology^16,17 and reproduction². Teleosts, in which sex steroids are mostly represented by estradiol (E2) in females and 11-ketotestosterone (11-KT) in males^18,19, have long been reliable experimental models for investigating the general principle of reproduction across species. Teleosts show uniqueness in their hypothalamic-pituitary connection^20,21 and distinct gonadotrope cells²², which are sometimes convenient for the elucidation of regulatory mechanisms. Moreover, due to their amenability to both laboratory and field experiments, teleosts offer many advantages compared to other organisms. They are relatively inexpensive to purchase and maintain^23,24. In particular, small teleost models such as zebrafish (Danio rerio) and the Japanese medaka (Oryzias latipes), are species with very high fecundity and a relatively short life cycle enabling rapid analysis of gene function and disease mechanisms²³, thus providing even greater advantages in addressing a plethora of biological and physiological questions, considering the numerous well-developed protocols and genetic toolkit available for these species²⁵.

In numerous studies, the removal of gonads (gonadectomy) along with blood sampling techniques have been used as a method for investigating many physiological questions, including its impact in vertebrate reproductive physiology in mammals^26,27,28, birds²⁹ and amphibians³⁰. Although the gonadectomy effect on reproductive physiology can be alternatively mimicked by sex steroid antagonists, such as tamoxifen and clomiphene, the effect of the drugs appears to be inconsistent due to bimodal effects^31,32. Chronic exposure to a sex steroid antagonist may lead to ovarian enlargement^33,34, which may disable observation of its effects for long-term purposes due to an unhealthy phenotype. In addition, it is impossible to perform a recovery experiment after sex steroid antagonist treatment, to warrant the specific effect of certain sex steroids. Along with those aforementioned points, other trade-offs of sex steroid antagonist use have been extensively reviewed^31,32. Therefore, gonadectomy still appears today as a powerful technique to investigate the role of sex steroids.

While gonadectomy and blood sampling techniques are relatively easy to perform in bigger species, such as European sea bass (Dicentrarchus labrax)³⁵, bluehead wrasse (Thalassoma bifasciatum)³⁶, dogfish (Scyliorhinus canicula)³⁷ and catfish (Heteropneustes fossilis and Clarias bathracus)^38,39, they raise challenges when applied in small fish as medaka. For instance, the use of Fish Anesthesia Delivery System (FADS)⁴⁰ is less feasible and appears to be prone to excessive physical damage for small fish. In addition, a gonadectomy procedure that is commonly used for bigger fish⁴⁰ is not suitable for small fish that requires high precision to avoid excessive damage. Finally, blood sampling is challenging due to the limited access to blood vessels and the small amount of blood in those animals. Therefore, a clear protocol demonstrating every step of gonadectomy and blood sampling in a small teleost is of importance.

This protocol demonstrates the step-by-step procedures of gonadectomy followed by blood sampling in Japanese medaka, a small freshwater fish native to East Asia. Japanese medaka have a sequenced genome, several molecular and genetic tools available²⁵, and a genetic sex determination system allowing for investigation of sexual differences before secondary sexual characteristics or gonads are well developed⁴¹. Interestingly, Japanese medaka possess fused gonads contrary to many other teleost species⁴². These two techniques combined take only 8 minutes in total and will complete the list of video protocols already existing for this species that included labeling of blood vessels⁴³, patch-clamp on pituitary sections⁴⁴ and brain neurons⁴⁵, and primary cell culture⁴⁶. These techniques will allow the research community to investigate and better understand the roles of sex steroids in feedback mechanisms as well as brain and pituitary plasticity in the future.

Protocol

All experimentations and animal handling were conducted in accordance with the recommendations on the experimental animal welfare at Norwegian University of Life Sciences. Experiments using gonadectomy were approved by the Norwegian Food Safety Authority (FOTS ID 24305).

NOTE: The experiments were performed using adult male and female (6-7 months old, weight ca. 0.35 g, length ca. 2.7 cm) Japanese medaka. The sex was determined by distinguishing the secondary sexual characteristics, such as the size and shape of dorsal and anal fin, as described in^42,47.

1. Instruments and solutions preparation

1. Prepare anesthetic stock solution (0.6% Tricaine).
   1. Dilute 0.6 g of Tricaine (MS-222) in 100 mL of 10x Phosphate Buffer Saline (PBS).
   2. Distribute 1 mL of the Tricaine stock solution into several 1.5 mL plastic tubes and store at -20 °C until use.
2. Prepare recovery water (0.9% NaCl solution) by adding 18 g of NaCl into 2 L of aquarium water. Store the solution at room temperature until use.
3. Prepare the incision tools by breaking a razor diagonally to get a sharp point (Figure 1A).
4. Prepare blood anti-coagulant solution (0.05 U/µL of sodium heparin) by diluting 25 µL of sodium heparin into 500 µL of 1x PBS. Store the anti-coagulant solution at 4 °C until use.
5. Prepare two glass needles from a 90 mm long glass capillary by pulling a glass capillary with a needle puller (Figure 1B) following the instructions of the manufacturer.
   NOTE: The outer diameter of the glass needle is 1 mm, while the inner diameter is 0.6 mm.
6. Prepare a 1.5 mL plastic tube lid by cutting the lid and make a hole that fits with the needle outer diameter (Figure 1C). To make the hole, heat one end of the 9 mm glass capillary and stab the heated glass capillary through the lid. Alternatively, use a needle to stab through the lid until the diameter of the hole fits with the 9-mm glass capillary.

2. Gonadectomy procedure

1. Prepare 0.02% of anesthetic solution by diluting one tube of Tricaine stock (0.6%) in 30 mL of aquarium water.
2. Prepare dissection tools including one ultra-fine and two fine forceps (one with relatively wide tip), small scissors, nylon thread and razor as described in step 1.3.
3. Anesthetize the fish by putting it into the 0.02% anesthetic solution for 30-60 seconds.
   ​NOTE: The duration of the anesthesia depends on the size and weight of the fish and must be adapted. To ensure that the fish is fully anesthetized, the fish body can be pinched gently using forceps. If the fish does not react, the gonadectomy can be started.
4. Take out the fish from the anesthetic solution and place the fish horizontally on its side, out-of-water under a dissection microscope.
5. Ovariectomy (OVX) in females
   1. Remove oviposited eggs (eggs hanging outside the female body) if any and scrape the scales in the incision area (Figure 2A).
   2. Gently make an incision about 2-2.5 mm long between the ribs, between the pelvic and anal fins (Figure 2A), using the razor blade. Then, pinch gently the fish abdomen while taking out the ovaries little by little using fine forceps with wide tip.
   3. Cut the end of the ovaries using fine forceps and place the ovaries aside (Figure 2B).
      NOTE: Take care not to break the ovarian sac if possible. If the ovarian sac is broken, remove any gonad traces as completely as possible without leaving even any non-ovulated eggs.
6. Orchidectomy in males
   1. Gently make an incision between the ribs above the anus (Figure 2A), and open up the incision slowly using fine forceps.
   2. Gently grab the testes using the fine forceps and slowly take out the testes. Afterwards, cut the end of the testes to completely remove the testes (Figure 2B). For male orchidectomy, all preparations are similar to in females until the incision part. When grabbing the testes, sometimes the fat resembling the testes is obtained. However, after restoring the fat, it is possible to try to find the testes again (Figure 2B).
      NOTE: For both males and females, it is important to minimize the incision size in the abdomen to prevent excessive damage that can lead to mortality. Sometimes the intestines may also appear through the incision along with the gonads, so make sure they are properly returned inside the incision before closure. Prior knowledge on ovaries and testes location in medaka abdomen is essential.
7. Suture the incision similarly in males and females (Figure 3).
   1. Place the nylon thread beside the incision area and stab the skin from the right side of the incision through inner body cavity using ultra-fine forceps to take the thread in with fine forceps (Figure 3; 1-2).
   2. Stab the skin from the left side of the incision through outer body cavity to take out the thread ( Figure 3; 3-4).
   3. Close the incision opening and make two knots and cut the excessive thread (Figure 3; 4-6).
      NOTE: The suture must be adequately tight, and the remaining thread on the fish must be long enough to prevent loosening of the suture. The whole procedure from anesthesia until suturing commonly takes up to 6 minutes. Longer time may lead to mortality.
   4. Put the fish in the recovery water and leave them for at least 24 hours before transferring them to the aquarium system.
      ​NOTE: Gonadectomized fish usually show normal behavior after 1-2 hours in the recovery water. Therefore, depending on the experiment purpose, one can sample the fish after this time interval.

3. Blood sampling procedure

1. Prepare the tools: a glass needle, a silicone capillary, a plastic tube with a hole, an empty 1.5 mL plastic tube, a minicentrifuge and tape.
2. Anesthetize the fish using 0.02% anesthetic solution as described in step 2.1 and place the fish under a dissection microscope in a vertical position (Figure 4A). Place the fish on a bright surface to ease visualization of the caudal puncture vein.
3. Install the blood drawer by attaching a glass needle to the silicone capillary (Figure 4B). Break the tip of the needle with wide tip forceps and coat the inside of the needle with anti-coagulant solution by suctioning and blowing.
   NOTE: The use of a sucker and a silicone capillary with at least 50 cm length are recommended for safety measures to avoid any direct contact of the blood when suctioning. In addition, make sure that the opening of the needle tip is sufficiently large to allow drawing the blood.
4. Direct the needle toward the peduncle area of the fish, aim at the caudal peduncle vein (Figure 5A) and draw the blood using mouth until at least one fourth the total volume of the needle is filled (Figure 5B).
   NOTE: It is important to stop suctioning before removing the needle from fish body.
5. Release the needle and put a piece of tape at the proximity of the sharp side of the needle. Place the lid with a hole on a collection tube and put the needle inside the tube through the hole with the needle tip on the outside (Figure 5C).
6. Put the fish in the recovery water and leave them for at least 24 hours before transferring them to the aquarium system.
   NOTE: To perform a second blood sampling from the same fish, sample the blood one week after the first blood sampling.
7. Flash spin down the collected blood for 1-2 seconds with 1,000 x g at room temperature to collect the blood in the tube.
8. Proceed directly to downstream applications or store the blood at -20 °C until use.
   NOTE: Refer to the previous study for sex steroid extraction from the whole blood⁴⁸.

Results

This protocol describes every step for performing gonadectomy and blood sampling in a small size model teleost, the Japanese medaka. The survival rate of the fish after ovariectomy (OVX) in females is 100% (10 out of 10 fish) while 94% (17 out of 18 fish) of the males survived after orchidectomy. Meanwhile, after the blood sampling procedure was performed, all (38 fish) fish survived.

Sham-operated females show oviposition (

Discussion

As reported in previous literature, gonadectomy and blood sampling have long been used in other model species to investigate questions related to the role of sex steroids in regulation of the BPG axis. However, these techniques seem to be amenable only for bigger animals. Considering the small size of the commonly used teleost model, Japanese medaka, we provide a detailed protocol for gonadectomy and blood sampling that is feasible for this species.

The fact that the survival rate of gonadecto...

Materials

Name	Company	Catalog Number	Comments
Glass capilary		GD1	Glass Capillary with Filament GD-1; Narishige
Heparin sodium salt		H4784-1G	Sigma-aldrich
Needle puller		P97	Flaming/Brown Micropipette puller Model P-97; Sutter Instrument
Nylon thread		N45VL	Polyamide suture, 0.2 metric; Crownjun
Plastic tube		T9661	Eppendorf Safe-lock microcentifuge tube 1.5 ml, Sigma-aldrich
Razor blade		-	Astra Superior Platinum Double Edge Razor Blades Green, salonwholesale.com
Silicone capillary		a16090800ux0403	Uxcell Silicone Tube 1 mm ID x 2 mm OD, amazon.com
Tricaine		WXBC9102V	Aldrich chemistry