Comparative study on accuracy of mucosal estradiol-17β, testosterone and 11-ketotestosterone

This

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study

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was designed

a

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as a

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comprehensive non-invasive procedure towards

sex

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identification

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and

maturity

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status that might be applied for other teleost species as well. Herein, E2,

T

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, 11

-KT

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,

VTG

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and Ca of

mucus

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were tracked, clarified and

then

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compared to

plasma

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, and gonad analyses in goldfish. In the present

study

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, mucosal androgens considering for

sex

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identification

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, and even contributing

in

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to

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males

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' reproductive status as an indicator.

T

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had an acceptable trend

resulted

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that resulted

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in

sex

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identification

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, its accuracy was 89%, at the same time, had the lowest

level

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(33%) of

identification

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efficacy in

maturity

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status. Regarding different roles of

T

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playing in spermatogenesis (Schulz et al., 2010; Basak et al., 2016), synthesizing of 11

-KT

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in spermiation evaluation

besides

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males

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' sexual behaviour hormone (Barry et al., 1990), apparently can track in

mucus

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in order to its

further

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potential. Measurement of

T

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levels

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in

water

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the water

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maintaining Dentex dentex and in

plasma

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, related to the number of

females

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stocked in the tank (Pavlidis et al., 2004).

Nevertheless

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,

T

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level

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in

females

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’

skin

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mucus

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has been currently lower than

males

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. Florida gar (Lepisosteus

platyrhincus

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platyrrhines

), group-synchronous teleost spawned,

sex

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steroids in

plasma

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showed an upward trend as it comes to reproduction season (Orlando et al., 2007). Already there is a group-synchronous pattern herein, but the border of stages is apparently distinguishable across both goldfish genders. Only 11

-KT

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of

plasma

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,

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apply

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seemingly hit a peak in spring over upcoming warm. Indeed both mucosal androgens were fairly stable in pre-reproduction and reproduction seasons. Schultz et al., (2005) reported 13-fold higher for a range of mucosal 11

-KT

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in

males

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than

females

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of Koi (Cyprinus carpio), which was not far from our results (

mucus

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content was 7-ply compared with

plasma

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). What is more, considering Barkowski and Haukenes (2014) indicating a direct relationship between 11

-KT

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of

mucus

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and

plasma

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, strengthening of mucosal hormone is hands-on.

Sex

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identification

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in white bass (Morone

chysops

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chops

) was correctly determined by 90% in the following, and a low error rate by 11

-KT

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in

mucus

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had been seen

as a result

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.

Similarly

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, the 11

-KT

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accuracy has been observed at

maturity

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stage detection (83%) and

sex

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identification

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(89%). Inadequate knowledge on fish

skin

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mucus

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components leads to hardly find out of how much

sex

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steroids exist within,

in particular

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E2 and

T

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,

However

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, there are several studies in mammals that proved the diffusion procedure and tissues' origin of

mucus

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(Friedl et al., 2013; Wang & Shi 2017; Lock et al., 2018). The mucosal layer might play passive and active roles during the diffusion process relied on the shape and size of

skin

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and

mucus

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(Dünnhaupt et al., 2015), and fish

sex

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hormones differentiation over seasonal and

sex

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alteration might directly affect

skin

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structure as a consequence (Pickering & Pottinger, 1985; Pottinger, 1987). Based on our

study

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, E2

levels

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in

mucus

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primarily were high at initial and late-vitellogenesis, even rather than

plasma

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. In

further

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, at vitellogenesis, protein

levels

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of

plasma

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were on top more than

mucus

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. The reason why these two matrices have a significant difference.

Although

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there is no data about fish mucosal E2, investigation of

estrogens

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encoding a protein in

mucus

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and epithelial cells have proved

hypothesis

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the hypothesis

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of controlling

mucus

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might be aligned with

estrogens

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, a pivotal hormone in reproduction (Massart et al., 2004). In

this

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survey, appearing of E2 in male’s

mucus

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would not be debatable since it seems rational predicted the

mucus

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VTG

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level

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or

vtg

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gene

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expression

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in the

skin

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. The ova-testis hypothesis and environmental

estrogens

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disruptors’ effects were failed by monitoring testis histological slides. There is a potent tendency for inducing a gender reverse in cyprinids (Saoshiro et al., 2013; Hassell et al., 2016), and

somales'

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sales

behaviour would probably

affectfemales

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affect females

or vice versa. Apart from androgens, E2 plays a critical role in mammals’ spermatogenesis process (Carreau & Galeraud-Denis, 2008), or even estrogen can take a burden as mediator testicular function and spermatogenesis in teleosts (Miura et al., 1999; Kobayashi et al., 2011; Laing et al., 2018). Pottinger et al., (2005) reported that

plasma

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E2 could not be a suitable discriminator of

sex

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in sea trout (Salmo trutta) and Atlantic salmon (Salmo salar). Even though, a high

level

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of E2 in

plasma

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of mature

females

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is indicated in Atlantic cod (Gadus morhua) (Dahle et al., 2003), goldfish (Kagawa et al., 1983; Kobayashi et al., 1988) and Florida gar (Orlando et al., 2007). Turning primary-oocytes into mature ones through E2 affected ovary, or controlling the

gene

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expression

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in different tissues like the

liver

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(Marlatt et al., 2010) and E2 circulation in the blood all are key stimulator to

VTG

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synthesis in the

liver

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. Normally,

VTG

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is synthesized and detectable only in matured

females

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(Denslow et al., 1999; Tian et al., 2009). Yet, the hypothesis of physiological interaction in

mucus

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, the

first

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line of body components, and

then

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direct interface with the peripheral aqueous medium (Fletcher et al., 1997; Sanahuja et al., 2019) leading to

combination

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a combination

the combination

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of environmental

estrogens

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. In the present

study

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,

mucus

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VTG

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was identically at a low

level

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compared to

plasma

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, but it was apparently detectable and acceptable.

Low

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The low

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expression

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level

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of the

vtg

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in

males

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'

skin

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or/and even the

level

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of E2 in

males

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' body confirm their

VTG

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secretion.

VTG

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can be transmitted from

plasma

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to

mucus

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by a diffusion mechanism or another regulated mechanism unknown (Kishida et al., 1992), and since

VTG

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is the yolk protein produced in the

liver

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by

estrogens

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activity; as a consequence, it is transferred to the ovary for oocyte as a final destination (Meucci & Arukwe 2005; Tian et al., 2009; Reading et al., 2017). High

levels

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of

vtg

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gene

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expression

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in the

liver

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,

therefore

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, would be expected inconsistent with our results.

Similarly

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,

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vtg

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the vtg

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gene

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expression

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of ovaries was more than

liver

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in white cloud mountain minnow (Tanichthys

albonubes

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bonuses

) (Wang et al., 2010) and sterlet sturgeon (Acipenser ruthenus) (Akhavan et al., 2016).

Sex

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identification

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has been more distinguished by

skin

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mucus

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Ca (73%) and

VTG

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(61%) than E2 (44%). Certainly, because blood Ca is an indirect tool for detection of

VTG

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during

maturity

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progress, Ca content can be assayed as a biomarker of

estrogens

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recirculation in

females

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at once

VTG

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levels

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(Svoboda et al., 2001; Linares-Casenave et al., 2003; Stahl et al., 2009).

Although

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the Ca

level

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of

plasma

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was higher than

mucus

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, there is

sustainable

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a sustainable

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amount of Ca observed in the

mucus

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particularly during vitellogenesis.