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.
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.
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 mechanisms1,2,3,4,5. In addition, sex steroids affect the proliferation and activity of neurons in the brain6,7,8 and endocrine cells, including gonadotropes, in the pituitary9,10, 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 response12,13, growth14,15, nutritional physiology16,17 and reproduction2. Teleosts, in which sex steroids are mostly represented by estradiol (E2) in females and 11-ketotestosterone (11-KT) in males18,19, have long been reliable experimental models for investigating the general principle of reproduction across species. Teleosts show uniqueness in their hypothalamic-pituitary connection20,21 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 maintain23,24. 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 mammals26,27,28, 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 effects31,32. Chronic exposure to a sex steroid antagonist may lead to ovarian enlargement33,34, 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 reviewed31,32. 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.
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 in42,47.
1. Instruments and solutions preparation
2. Gonadectomy procedure
3. Blood sampling procedure
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.
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 zebrafish49,50,51, 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 plasma53,54. 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 teleosts59,60, 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.
The authors have nothing to disclose.
The authors thank Ms Lourdes Carreon G Tan for her assistance in the fish husbandry. This work was funded by NMBU, Grants-in-Aid from Japan Society for the Promotion of Science (JSPS) (Grant number 18H04881 and 18K19323), and grant for Basic Science Research Projects from Sumitomo Foundation to S.K.
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 |