Changes in Sex Steroids and Ovarian Steroidogenic Enzyme mRNA Levels in Artificially Maturing Japanese Eel (Anguilla japonica) and Naturally Maturing New Zealand Longfin Eel (Anguilla dieffenbachii) during Vitellogenesis

Round 1

Reviewer 1 Report

General comment:

The manuscript entitled” Changes in mRNA Levels Encoding Steroidogenic Enzymes during Induced Vitellogenesis in the Japanese eel (Anguilla japonica) – is cyp19a Over-expressed?” by Matsubara et al. mainly described the comparisons of sex steroids (testosterone, T; estradiol-17b, E2) and ovarian steroidogenic enzyme mRNAs levels between artificially maturing Japanese eels and wild-caught, spontaneously maturing New Zealand longfin eels. So I would suggest the authors to change the title to “Changes in Sex Steroids and Ovarian Steroidogenic Enzyme mRNA Levels in the Induced Maturing Japanese Eel and Naturally Maturing New Zealand Longfin Eel during Vitellogenesis”. This study indicated that serum T levels in induced maturing Japanese eel changed with stage in a pattern that was comparable to that in longfin eels. Likewise, ovarian mRNA levels of most steroidogenic enzyme genes were not qualitatively dissimilar between both eel species when taking developmental stage into account. Moreover, sex steroid and target gene mRNA levels fluctuated drastically with each hormone injection. However, “aromatase (cyp19a) mRNA levels, together with serum E2 levels, rapidly increased in artificially maturing Japanese eels in mid-late stages of oogenesis” needed further revision, as I found that cyp19a mRNA levels in artificially maturing Japanese eels at the mid-vitellogenic stage (prior to SPH injections ) were not consistent with serum E2 levels from the results.

 

The manuscript is well English writing, and these results may be worth for publication, however, the manuscript should need some revisions before publication.

 

Specific comments:

L86: in artificially maturing “female” eels; L87: non-anguillid “female” fish L104-105: artificially maturing Japanese “eels” L141 & L146: delete “soon” and change to “2 days” L147: change between into “in both” L158: change to “Gonadosomatic index (GSI) of artificially maturing Japanese eels (black circles) and wild-caught longfin eels (white columns) during maturation” L164: change to “Serum levels of testosterone, T (A) and estradiol-17b, E2 (B) in artificially maturing Japanese eels (circles) and wild-caught longfin eels (white columns) during maturation” L174, L276: Japanese “eels” L213: change oil-seed into “cotton-seed” L243: “view point”→“viewpoint” L265: What is eel Ringer? Please add a reference paper. L262-L270: From the Fig. 2, I saw that "Steroid levels in Japanese eels were determined just prior to (open circles) or 2 days after (black circles) exogenous hormone treatment.", but why you did not mention that blood samples were also collected prior to exogenous hormone treatment here. Please describe the Materials and Method more precisely. L299-L309: add a table for the primers. L338-L339: change “during artificial maturation” into “in artificially maturing Japanese eels and wild-caught longfin eels” Please check the reference, some wrong format needed to be revised, especially for the abbreviation of author's name and the Italics of proper nouns and journal's name. Discussion is well-writing, but I suggested that the authors can add more discussion about the E2 levels (low, abnormal steroidogenesis) in artificially maturing Japanese eels at MV stage (prior to SPH injection). The mRNA levels of cyp19a (apparently elevated) seemed to be not consistent with E2 levels (non-apparently elevated) when comparing between EV and MV groups of artificially maturing Japanese eels. These results may imply that the induced spawning protocols for artificially maturing Japanese eels not only lead to the drastic fluctuations on steroidogenic enzyme gene expression and steroid levels, but also cause the abnormal steroidogenesis during vitellogenesis.

Author Response

Response to Reviewer 1 Comments

 

Point 1: Reviewer 1 suggested the authors to change the title to “Changes in Sex Steroids and Ovarian Steroidogenic Enzyme mRNA Levels in the Induced Maturing Japanese Eel and Naturally Maturing New Zealand Longfin Eel during Vitellogenesis”.

Response 1: Thank you for this suggestion. We have changed the title almost verbatim as suggested by Reviewer 1, as indicated in red font on the revised manuscript, see L2-L6:

 

“Changes in Sex Steroids and Ovarian Steroidogenic Enzyme mRNA Levels in Artificially Maturing Japanese Eel and Naturally Maturing New Zealand Longfin Eel during Vitellogenesis”

 

 

Point 2: Reviewer 1 suggested a number of editorial revisions, as outline below.

 

Response 2: We value the suggestions for our ms to be improved and have made amendments largely in keeping with the suggestions, as indicated in red front, below. Yellow highlight is used (below) in instances where editorial suggestions were not verbatim implemented.

Specific comments:

L86: in artificially maturing “female” eels (L93 in revised manuscript = R1)

L87: non-anguillid “female” fish (L94 in R1)

L104-105: artificially maturing Japanese “eels” (L112 in R1)

L141 & L146: delete “soon” and change to “two days” (L150 and L155 in R1)

L147: change between into “in both” (L156 in R1)

L158: change to “Gonadosomatic index (GSI) of artificially maturing Japanese eels (black circles) and wild-caught longfin eels (white columns) during vitellogenesis” (L171 in R1)

L164: change to “Serum levels of testosterone, T (A) and estradiol-17b, E2 (B) in artificially maturing Japanese eels (circles) and wild-caught longfin eels (white columns) during vitellogenesis” (L178 in R1)

L174, L276: Japanese “eels” (L188 and L299 in R1)

L213: change oil-seed into “cotton-seed” (L225 in R1)

L243: “view point”→“viewpoint” (L258 in R1)

L265: What is eel Ringer? Please add a reference paper→“[41] ” (L281 in R1)

L262-L270: From the Fig. 2, I saw that "Steroid levels in Japanese eels were determined just prior to (open circles) or 2 days after (black circles) exogenous hormone treatment.", but why you did not mention that blood samples were also collected prior to exogenous hormone treatment here. Please describe the Materials and Method more precisely→ a subclause (“Blood was sampled by syringe from the caudal vasculature ….”) was added to the sentence that describes sample collection (L285 in R1)

L299-L309: add a table for the primers. → Table 1 was added, as requested (L326 in R1)

L338-L339: change “during artificial maturation” into “in artificially maturing Japanese eels and wild-caught longfin eels” (L360 in R1)

 

 

Point 3: Reviewer 1 pointed out that

this study indicated that serum T levels in induced maturing Japanese eel changed with stage in a pattern that was comparable to that in longfin eels. Likewise, ovarian mRNA levels of most steroidogenic enzyme genes were not qualitatively dissimilar between both eel species when taking developmental stage into account. Moreover, sex steroid and target gene mRNA levels fluctuated drastically with each hormone injection. However, “aromatase (cyp19a) mRNA levels, together with serum E2 levels, rapidly increased in artificially maturing Japanese eels in mid-late stages of oogenesis” needed further revision, as I found that cyp19amRNA levels in artificially maturing Japanese eels at the mid-vitellogenic stage (prior to SPH injections ) were not consistent with serum E2 levels from the results. Discussion is well-writing, but I suggested that the authors can add more discussion about the E2 levels (low, abnormal steroidogenesis) in artificially maturing Japanese eels at MV stage (prior to SPH injection). The mRNA levels of cyp19a(apparently elevated) seemed to be not consistent with E2 levels (non-apparently elevated) when comparing between EV and MV groups of artificially maturing Japanese eels. These results may imply that the induced spawning protocols for artificially maturing Japanese eels not only lead to the drastic fluctuations on steroidogenic enzyme gene expression and steroid levels, but also cause the abnormal steroidogenesis during vitellogenesis.  Please check the reference, some wrong format needed to be revised, especially for the abbreviation of author's name and the Italics of proper nouns and journal's name.

Response 3: We appreciate the suggestion of the Reviewer to add further detail on the mismatch between cyp19 mRNA levels and E2 levels. In keeping with this recommendation, we checked the reference and added the following passage (Lines 250-260 in R1):

 

“We propose that these acute changes in steroid levels are a reflection of the cyclical presence of injected GTHs which are cleared rapidly from circulation after administration [38]; these GTHs, in turn, are likely to increase the expression of the gene encoding steroidogenic acute regulatory protein (star), a protein that is regulating cholesterol transport for subsequent use in steroidogenesis [46]. Indeed, given the strong regulation of the star gene by tropic stimulation in vertebrates [46], including the eel [37], a cyclical SPH-dependent pattern in star expression can be expected to result in the observed rise and fall in levels of steroid substrates. Accordingly, the mismatch between cyp19a mRNA and plasma E2 levels that were seen in Japanese eels sampled just before and those sampled two days post-SPH injection conceivably result from the changes in availability of T rather than that of the abundance of the enzyme aromatase or its transcript.”

 

We further added (L261 in R1):

“….and indicative of abnormal steroidogenesis?”

Author Response File: Author Response.docx

Reviewer 2 Report

The only concern this reviewer has is that at MV before SPH injection, high cyp19a expression  (4-5 fold higher than the level of EV, Fig 3e) did not increase serum E2 level (when compare it with E2 level at EV, Fig.2b). The authors may need to explain and discuss it. 

Author Response

Response to Reviewer 2 Comments

 

Point 1: Reviewer 2pointed out that at MV before SPH injection, high cyp19a expression  (4-5 fold higher than the level of EV, Fig 3e) did not increase serum E2 level (when compare it with E2 level at EV, Fig.2b). The authors may need to explain and discuss it. 

 

Response 1: We appreciate the suggestion of the Reviewer to add further detail on the mismatch between cyp19 mRNA levels and E2 levels. In keeping with this recommendation, we checked the reference and added the following passage (Lines 250-260 in R1):

 

“We propose that these acute changes in steroid levels are a reflection of the cyclical presence of injected GTHs which are cleared rapidly from circulation after administration [38]; these GTHs, in turn, are likely to increase the expression of the gene encoding steroidogenic acute regulatory protein (star), a protein that is regulating cholesterol transport for subsequent use in steroidogenesis [46]. Indeed, given the strong regulation of the star gene by tropic stimulation in vertebrates [46], including the eel [37], a cyclical SPH-dependent pattern in star expression can be expected to result in the observed rise and fall in levels of steroid substrates. Accordingly, the mismatch between cyp19a mRNA and plasma E2 levels that were seen in Japanese eels sampled just before and those sampled two days post-SPH injection conceivably result from the changes in availability of T rather than that of the abundance of the enzyme aromatase or its transcript.”

 

We further added (L261 in R1):

“….and indicative of abnormal steroidogenesis?”

 

Author Response File: Author Response.docx