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.

Representative 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 (Figure 6A) and all the eggs were fertilized and allowed for embryonic development (Figure 6B). Sham-operated males were also able to fertilize eggs after only 1-2 weeks. Two out of six partly-gonadectomized females reared with partly-gonadectomized males also showed oviposition with 100% of fertilized eggs after 2 months. In contrast, no oviposition in females or fertilization by males was observed in fully gonadectomized fish, even after 4 months.

When performed correctly, the body shape of the fish slightly changes (Figure 7A), and no piece of gonad should remain after the gonadectomy procedure (Figure 7B). Meanwhile, 4 weeks post-gonadectomy, the incision and suture completely disappeared (Figure 8), and after 4 months, all gonadectomized fish still showed healthy phenotype, and no gonadal tissue was found.

E2 blood concentrations in females (Table 1), measured with ELISA following the manufacturer's instructions, revealed that the E2 level in OVX fish is significantly lower than in sham-operated fish 24 hours after surgery (p < 0.00001). After 4 months, the E2 level in OVX fish is also significantly lower than in sham-operated fish (p < 0.00001) and shows no significant difference compared to that in 24 hours post-OVX fish (p > 0.05). Finally, partly OVX fish, where only 1/3 to 1/2 of the gonad was removed, show significantly lower E2 levels than sham-operated fish (p = 0.0437) and significantly higher E2 levels than fully OVX fish (p < 0.00001) (Figure 9A).

Similarly in males (Table 1), the 11-KT concentration in orchidectomized fish is significantly lower than in sham-operated fish 24 hours after surgery (p < 0.00001). The level of 11-KT in orchidectomized fish after 4 months is also significantly lower than in sham-operated fish (p < 0.00001) and shows no difference compared to 24 hours post-orchidectomized fish (p > 0.05). Finally, partly orchidectomized fish show significantly lower levels of 11-KT than sham-operated fish (p = 0.0428) and significantly higher levels of 11-KT than fully orchidectomized fish (p < 0.00001) (Figure 9B).

Figure 1. Instrument preparation. (A) Razor blade for gonadectomy, (B) glass needle for blood extraction, and (C) a plastic tube together with a lid with a hole for blood collection. Please click here to view a larger version of this figure.

Figure 2. Location of the incision area. A) Drawing of the incision area located between the ribs, between the pelvic and anal fins in females (left panel) and males (right panel); B) gonad removal in females (left panel) and males (right panel), white circles showing the joint part, white arrow showing the testis and black arrow showing the fat. Please click here to view a larger version of this figure.

Figure 3. The procedure of suture. 1) A hole is made on the right side of the incision using ultra-fine forceps. 2) The nylon thread is passed through the skin using the hole made in 1. 3) A hole is made in the left side of the incision. 4) The nylon thread is passed through the hole made in 3. 5) An overhand knot is made twice to close the incision. 6) Excess thread is cut. Please click here to view a larger version of this figure.

Figure 4. Fish position during blood sampling (A), the installation of glass needle with the silicone capillary (B). Please click here to view a larger version of this figure.

Figure 5. The suction area of blood sampling (A), drawn blood (B) and blood collection steps (C). Please click here to view a larger version of this figure.

Figure 6. Sham-operated fish shows oviposition of eggs pointed by white arrow (A) and fertilized eggs (B). Please click here to view a larger version of this figure.

Figure 7. Morphological (A) and anatomical (B) appearance of intact and gonadectomized fish. White arrows (top panels) show the surgery mark on gonadectomized fish. Black arrows (bottom panels) show gonads in intact fish. Please click here to view a larger version of this figure.

Figure 8. Surgery marks in male and female fish after 4 weeks. Please click here to view a larger version of this figure.

Figure 9. Blood levels of E2 in female (A) and 11-KT in male (B) Japanese medaka, 24 hours after sham operation (control), partly gonadectomy or gonadectomy, and 4 months after gonadectomy (OVX, ovariectomy in females; Cas, orchidectomy in males). The statistical analyses were performed using One Way ANOVA followed by Tukey Post Hoc test. Different letters (a-c) show significant differences (p-value < 0.05). Data in the graph are provided as mean + SD, n = 5. Please click here to view a larger version of this figure.

	E2 levels (Females)	11-KT levels (Males)
Sham-operated	4.15 ± 0.5 (n = 5)	10.38 ± 1.32 (n = 5)
Partly-gonadectomized	3.37 ± 0.6 (n = 5)	8.37 ± 1.92 (n = 5)
24h post-gonadectomy	0.36 ± 0.2 (n = 5)	0.4 ± 0.2 (n = 5)
4 months post-gonadectomy	0.54 ± 0.28 (n = 5)	0.74 ± 0.22 (n = 5)

Table 1. E2 and 11-KT levels (ng/mL) in females and males of sham-operated and gonadectomized and partly gonadectomized fish.

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 gonadectomized fish reached almost 100% indicates that the gonadectomy procedure is feasible to be applied on medaka. Similarly, the procedure of blood sampling does not affect the survivability of the fish as shown by the 100% survival rate after undergoing this procedure. In addition, sham-operated females reared together with sham-operated males show oviposition and 100% fertilized eggs, indicating that the incision and suture procedure do not affect the reproduction of the fish. In other words, they were healthy enough to spawn. Furthermore, partly gonadectomized fish show comparable concentrations of sex steroids to sham-operated fish, and oviposition in some females as well as fertilization of eggs by males were observed in those partly gonadectomized fish. These results suggest that the procedure of gonadectomy should be performed with high precision, meaning that the ovaries or testes should be completely removed.

As shown in Figure 8, the incision and suture mark on the fish completely disappeared 4 weeks post-gonadectomy, and the fish are still alive and look healthy 4 months after surgery. These indicate that the operation procedure is safe for the fish for long term purpose and does not affect the life span of the fish. In addition, after 4 months, no gonads were observed. This is confirmed by the low levels of E2 and 11-KT that are still similar to those found in gonadectomized fish after 24 hours.

The levels of E2 and 11-KT in gonadectomized fish are significantly lower than sham-operated fish, already after 24 hours post-gonadectomy and remain lower in fish sampled 4 months after gonadectomy. The significantly lower sex steroid levels in gonadectomized fish compared to control have been observed in previous studies in dogfish³⁷, catfish³⁹ and medaka⁴⁸. These consistent evidences suggest that the gonadectomy procedure described in the protocol is a reliable technique to clear circulating sex steroids.

Since this procedure does not rely on FADS as demonstrated in⁴⁰, the gonadectomy should be carried out as quickly as possible to prevent mortality during surgery. Indeed, the use of FADS enables to maintain the rhythm of operation since this tool allows continuous anesthetic condition to the fish despite being exposed to the air. Nonetheless, due to its lower feasibility in the small teleost as medaka, the use of FADS cannot be performed with that size of fish. In addition, unlike the previous gonadectomy protocol in bigger fish that enables wide incision to reach the gonad, the protocol described in this manuscript does not allow wide incision to avoid excessive damage to the small fish. Therefore, one should be very careful when trying to access the gonad using forceps to prevent damage in other tissues inside the fish body cavity.

The protocol relies on a quick and clean procedure. Training is thus highly recommended until reaching a high success rate, indicated by a high survival rate of the fish after gonadectomy as well as the complete removal of the gonads (see the difference of morphological and anatomical appearance of the fish before and after successful gonadectomy in Figure 7). In fact, many factors can affect the success rate of the procedure, including the anesthesia period, the wideness of incision, the accuracy and tidiness of the suture and fish handling during the procedure. Another important point is that one should prepare healthy fish by maintaining the fish optimally prior to performing the protocol.

With respect to blood sampling procedure, the previous studies have attempted to sample the blood from medaka⁴⁸ and zebrafish^49,50,51, but the procedure does not allow repeated blood sampling in the same fish since the blood is taken after euthanizing the fish. Repeated blood sampling has been demonstrated using zebrafish in another study⁵², but we report this type of protocol for the first time in medaka.

The evaluation of sex steroid concentrations is commonly carried out using an enzyme-linked immunosorbent assay (ELISA) kit, and there have been many ELISA kits commercially available for different types of sex steroids. Due to the low amount of blood collected during blood sampling, the downstream assays are intended for the whole blood. Previous studies have shown that there is a difference in the measured level of circulating steroid levels extracted from whole blood and plasma^53,54. Therefore, the difference in the sex steroid levels from whole blood and plasma needs to be validated prior to performing the real experiment using the protocol.

As documented in previous studies with different animal models, the protocol described here will allow to investigate questions related to reproductive physiology using a small size teleost as a model. In fact, these techniques have already contributed to answer questions concerning the regulation of the BPG axis and its feedback mechanisms, such as the involvement of kiss1 (kisspeptin gene type 1) expressing neurons in positive feedback loops⁵⁵, estrogen-mediated regulation of kiss1 expressing neurons in nucleus ventralis tuberis (NVT), and kiss2 (kisspeptin gene type 2) expressing neurons in preoptic area (POA)^56,57, the possible involvement of estrogen receptor β1 (Esr2a) in down-regulating fsh expression level in female Japanese medaka⁵⁸ as well as the profile of the circadian rhythm of E2 in female fish⁴⁸. Furthermore, since previous studies demonstrated that sex steroids also affect the proliferation of gonadotrope cells in the pituitary of teleosts^59,60, it is intriguing to investigate the effects of sex steroid clearance after gonadectomy on pituitary plasticity.

The blood sampling technique not only can be used for sex steroid analysis, but also for other blood content analysis, including blood glucose levels. Indeed, the protocol can also be applied for blood glucose measurements as demonstrated in zebrafish⁵² and medaka⁶¹. Therefore, this technique may be expanded to address research questions in other fields of physiology.

Finally, the protocols described here are intended and optimized for adult Japanese medaka, and the outcomes due to different size of fish and materials used during the procedures may vary. Moreover, as medaka left and right ovaries/ testes are fused, which might provide an important advantage for gonadectomy, this protocol might need several adaptations before being used in other species where this is not the case such as in zebrafish. Thus, an optimization according to the choice of laboratory equipment and fish size should be taken into account before testing these protocols.

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