Biol Open. 2017 Oct 15;6(10):1493-1501. doi: 10.1242/bio.024844.

How does oestradiol influence the AVT/IT system in female round gobies during different reproductive phases?

Hanna Kalamarz-Kubiak, Magdalena Gozdowska, Tatiana Guellard, Ewa Kulczykowska

Department of Genetics and Marine Biotechnology, Institute of Oceanology Polish 18, Academy of Sciences, Powstańców Warszawy 55, 81-712 Sopot, Poland.

 

Abstract

In this in vitro gradient perfusion study, we determined whether there is a functional relationship between oestradiol and the arginine vasotocin/isotocin (AVT/IT) system in the female round goby (Neogobius melanostomus). Brain explants were perfused in medium supplemented with 17β-oestradiol (E2) at doses mimicking the plasma levels of this hormone in nature during the spawning-capable phase and regressing phase. We aimed to establish which pathway, genomic or non-genomic, is involved in this mechanism in different reproductive phases. For this purpose, brain explants were perfused in medium supplemented with Fulvestrant (ICI 182.780) or Actinomycin D (Act D) separately or in combination with E2. The contents of AVT and IT in the perfusion media were determined using high-performance liquid chromatography (HPLC) with fluorescence and UV detection. During the spawning-capable phase, the effect of E2 on AVT release is mediated through oestrogen receptors (ERs) via both genomic and non-genomic pathways, while IT release is mediated through ERs via a genomic pathway only. In the regressing phase, release of both nonapeptides is mediated through ERs via a genomic pathway. This is the first study to present a feasible mechanism of oestradiol action on the AVT/IT system in female fish during different phases of the reproductive cycle.

KEY WORDS: Oestradiol, AVT, IT, In vitro gradient perfusion, Genomic and non-genomic pathways, Female round goby

 

Supplement 

In vitro models are widely and even routinely applied in the physiological, pharmacological, toxicological and biomedical research as they simplified the test systems to a minimum whilst aligning with the principles of reduction in animal use.

Organ culture techniques can bridge the methodological gap between in vivo and in vitro studies. Compared to the monolayer, three-dimensional (3D) cultures are associated with the higher cell densities. In fact, conventional two dimensional (2D) static culture systems can not sufficiently mimic this physiological in vivo situation. Moreover, static systems have disadvantages in the mass transport of nutrients and oxygen and the elimination of metabolic waste products. As a result, they are not comparable to the situation within in an organ anymore because cells and tissues lose their morphological, physiological and biochemical features, and their interactions as a cell to cell or cell to the matrix, by de-differentiation in this stagnant environment of static culture. However, appropriate conditions can be archived by using an automated medium replacement in perfusion culture system, thereby guaranteeing a stable concentration of nutrients an pH level, and reduced risk of contamination. The development of an innovative system for organ perfusion by Minuth in the early 1990s was driven by the idea to create under in vitro conditions an environment resembling as near as possible the situation of specialized tissues found within the organism. This MINUCELLS and MINUTISSUE tissue engineering technique meets the requirements for studies of retina, blood-retina and blood-brain barrier, regeneration of blood vassals, nervous tissue, skin renewal, bone and muscular tissue in mammals 1. To the authors’ knowledge, organ perfusion methods have not often been used in fish for lack of suitable techniques. It is known that in fish, simple organ perfusion systems were applied in pituitary 2,3,4,5 and pineal gland 6,7,8. For the first time, this tissue engineering technique has been adapted for perfusion of fish brain tissue and pituitary explants by Kalamarz-Kubiak and co-workers 9. It should be mentioned that the gradient perfusion technique (3D) allows monitoring the dynamic hormone secretion and registering even small and short-term fluctuations in their release while ensuring optimal culture conditions. Trying to overcome the problems with “dead space” and gas bubbles, tissue carriers are therefore used in combination with the gradient perfusion culture container. Tissues are placed on the membrane between rings of tissue carriers inside the gradient container. A specific construction of this container facilitates the uniform supply of medium to the luminal and basal sides. The flowing medium could be aerated inside the gas exchange modules by an air pump (0.3% CO2) or a gas mixture (95% O2 and 5% CO2) at an appropriate pressure. In procedures presented by Kalamarz-Kubiak and co-workers 9, three perfusion sets can be applied depending on the method of medium transport into gradient container: one medium supplied from the top of gradient container without aeration (set 1), or with aeration (set 2) and one or two media supplied from the top and bottom of gradient container, simultaneously, with aeration (set 3). Set 1 is recommended only for short-term studies. Set 2, where the medium is aerated with a mixture of 95% O2 and 5% CO2 at a pressure of 127.51 mmHg, is necessary for a long-term research. Set 3 is also recommended for a long-term research but required aeration with a mixture of 95% O2 and 5% CO2 at a pressure of 315.03 mmHg. Moreover, it should be noted that the presented procedures can serve many other purposes after only minor modifications 9,10,11.

In the presented study, we determine whether there is a functional relationship between circulating oestradiol and arginine vasotocin (AVT), and isotocin (IT) in female fish during different phases of the reproductive cycle. For this purpose, the brain explants of female round goby (Neogobius melanostomus) were perfused in medium supplemented with E2 at doses mimicking the plasma levels of this hormone in nature during the spawning capable phase and the regressing phase. In the perfusion of brain explants, we used E2 separately or in combination with Fulvestrant—oestrogen receptors (ERs) antagonist or Actinomycin D—transcription inhibitor. Perfusion of brain explants was carried out using set 3 where medium was supplied from the top and bottom of gradient container, simultaneously and was aerated inside the gas exchange modules by the gas mixture (95% O2 and 5% CO2) at a pressure of 315.03 mm Hg. The set used in the experiments consisted of storage medium bottles, a peristaltic pump, two gas exchange modules, a gradient perfusion container and plastic vials for the sampling medium after perfusion (Figure 1).

In conclusion, it should be noted that fish may be a perfect model for studying the mechanisms whereby hormones modulate the sexual behaviours in both humans and non-humans vertebrates.

 

Figure 1. The scheme of experimental design. Components of perfusion culture sets: 1. Storage medium bottles, 2. Peristaltic pumps, 3. Connecting fittings, 4. Gas exchange modules, 5. Gradient culture container, 6. Sampling vials.

 

Acknowledgement

This study was supported by the National Science Centre (Narodowe Centrum Nauki) [2012/05/B/NZ9/01024 to H.Kalamarz-Kubiak].

 

Hanna Kalamarz-Kubiak, PhD, DSc, Eng.

Associate Professor 

Genetic & Marine Biotechnology Department,

Institute of Oceanology Polish Academy of Sciences,,

Powstańców Warszawy 55,

81-712 Sopot, Poland

e-mail: hkalamarz@iopan.gda.pl

Tel.: +48 58 7311766

Fax: +48 58 5512130

 

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