Gonadotropins and Sex Steroid Hormones in Captive-Reared Small Yellow Croaker (Larimichthys polyactis) and Their Role in Female Reproductive Dysfunction
Round 1
Reviewer 1 Report
The authors conducted a series of experiments aimed at a better understanding of the reproductive endocrine system related to gonadotropins and sex steroids in the captive broodstock of small yellow croaker species. They analysed the mRNA expression levels of pituitary GtH subunits and the levels of steroids in the serum and gonads at different reproductive stages. They also analysed the effect of GnRHa on pituitary GtHs and evaluated the feedback regulation of sex steroids on pituitary GtHs. The authors suggested that the lower GtHs and steroids observed might explain the reproductive dysfunction in captive-reared females. This study is providing new insights about mechanisms underlying reproductive dysfunctions in captive-reared fish. However, I have several major concerns that should be attended.
Major comments.
Material methods
How many replicates were used for each experimental condition?
In the statistical analysis it’s said the results were analysed using a nonparametric one-way ANOVA in GraphPad Prims, followed Tukey’s post hoc test. However, it’s not possible use this test post hoc after nonparametric ANOVA. Could the author explain how they make it? Did nonparametric one-way ANOVA the only analytical methods use when data were compared? Why did they use two different GraphPad Prism software (9.4.1 and 9.3.1)?
Concerning the number of samples used in all experiments, it is not clear. Did they use only three samples (n=3), as noted in all the graphics or as described in sections 4.2.1 and 4.24, 10 samples were collected (lines 392 and 421)? I think 3 samples are not a good representative number for qPCR.
On the other hand, in section 4.2, it said that a portion of the gonad was fixed in 4% PFA for histology. Where is the section histology in this manuscript? Why histology of gonads was not included in the manuscript? To improve the manuscript the histology of gonads should be included.
Section 4.2.5 (line 438) when were the pituitaries collected at 6h, 12h and 24h or 3h, 6h and 12h? Please check results, material and methods, graphics and figure legends.
Results
Figure 1 shows significant differences in lhb and fshb mRNA expression in the gonads, with lower levels in females. I believe that there is something wrong with the statistical analysis. All analysis were performed with a small number of samples (n=3), and because there are some values too close, these do not seem to be statistically significant. Could the authors show the p-values analysis?
On the other hand, as mentioned above, the data analysis used in this study was performed using nonparametric one-way ANOVA. However, regarding the analysis results of figure 1, 2, 3, 6, and 7, two-way ANOVA should be used because we are studying the effect of two different factors: time and treatment. The analysis must be repeated accordingly.
Figure 5 shows the lower-case letters for males and the capital letters for females. Are the authors analysing both sexes together? If so, a two-way ANOVA should be used to determine significant differences (two Factors: sexes and gonadal developmental stages).
In the PCR analysis, the authors have only chosen one reference gene (β-actin gene) as internal control. Did the authors check the possibly differences in the β-actin transcripts in response to the treatments? However, even having performed a stability test, it is always better to include more reference genes to get more stable measurements.
Minor concerns
According to the rules for nomenclature of vertebrate’s genes and proteins, I would recommend use the following guidelines (https://www.gmb.org.br/geneprotein-nomenclature-guidelines).
· Mammalian (and other tetrapods); Proteins: All letter in capitals (GNRH).
· Mammalian (and other tetrapods); Genes and cDNA/RNA: All letters in capitals and italics (GNRH)
· Fish proteins: First letter in capitals and the remaining in lowercase (Gnrh)
· Fish genes and cDNA/RNA: all letters in lowercase and italics (gnrh)
What were the length and weight of animals?
4.2.3. Pituitary Samples from In vivo induction of GnRHa in SYC (Line 401)
Which was the criteria in relation to GnRHa dosage?
4.2.5. Pituitary Samples from In Vitro Cultures with E2 and MT (line 424)
What is cells density used in this assay? Did the authors perform a Time-course experiment? Was each treatment repeated at least twice?
4.3.1. RNA Extraction and cDNA Syntheses. (Line 441)
Did the RNA samples check for quality and purity? Describe how.
cDNA synthesis: Please include amount of RNA used (ng or ug) to carry out cDNA
Figure 6 and 7, Treatments for 3h, 6h, 12h or 6h, 12h, 24h?
Figure 3, 6 and 7, What do those asterisks represent exactly?
Figure 6 (line 198) and 7 (line 219), there are not letters on those bar graphs.
Figure 3, 6 and 7, What does IC mean? Please include it in the figure legends
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
The article titled "Gonadotropins and Sex Steroid Hormones in Captive-Reared Small Yellow Croaker and Their Role in Female Reproductive Dysfunction" deals with the gene expression of gonadotropins and the evaluation of some steroid hormones in the Small Yellow Croaker (SYC) in order to identify the potential causes of reproductive dysfunction in captive-reared SYCs.
This article deals with a topic that from an economic point of view is of particular importance in the economy of China, Korea and Japan. In fact, due to overfishing, this fish is captivity-reared, and problems related to reproductive functionality often occur. The experimental work was very laborious, and the results obtained were numerous, but it is necessary to clarify some points reported below.
L. 366: you write: "1 °C at each 7-day interval". It means that you, in broodstock tank, increase 1 °C per week, so you need 7 weeks to reach 18 °C and commence the breeding. Do you confirm?
L. 414: Was GnRHa injected intramuscularly.?
In general, I would suggest paying attention to acronyms, quoting the full form only in the first quotation.
I noticed that also in a previous work you used methyltestosterone in in vitro experimentation: could you explain the reasons for this choice?
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
Comments about the manuscript:
“Gonadotropins and Sex Steroid Hormones in Captive-Reared Small Yellow Croaker and Their Role in Female Reproductive Dysfunction”
In captivity, egg production of small yellow croakers Larimichthys polyactis, is limited by endocrine dysfunction. The work presented here aims to understand this type of dysfunction. For this, the authors identified gonadotropins (FSH, LH and GP) and sex steroids (17β-estradiol; testosterone; progesterone) using qRT-PCR, ELISA, in individuals and cultures in vitro, in males, females and during development. The results show that gonadotropins are essential for final gonadal maturation and that steroids promote negative feedback from their regulation. Low levels of these different hormones could result in reproductive dysfunctions in females of small yellow croakers bred in captivity.
This work provides useful elements for the understanding of endocrine dysfunction in captive animals. Before considering its publication, however, it is necessary to make some improvements to the manuscript.
Title: add the scientific name (Larimichthys polyactis) to the usual name (small yellow croaker).
Page 3, line 99, 2.1. Distribution of FSHβ, LHβ and GPα in Different Organs. Indicate the number of individuals used for the study. How many males, females were used for the study of each organ?
Page 3, figure 1. INT (intestine?) has been forgotten in the legend.
Page 4, line 115, 2.2. How many individuals of each sex (if the latter can be determined) were used for each stage of development studied?
Page 5, line 131, 2.3. How many males, females were used for each detection?
Page 6, line 152, 2.4. How many males, females were used for each detection?
Page 6, line 167, 2.5. How many males, females were used for each each stage? For each hormone ?
Page 8, figure 6. What do the numbers 3, 6, 12 under the graphs mean? If this is the duration of the treatment, change to 6, 12, 24 specifying in the legend.
Page 9, figure 7. Same remark as for Figure 6 above.
Page 12, line 370, 4.2. For each experiment, specify the number of males, females, stages of development.
Page 12, line 397. Briefly describe the histological method used: fixation, dehydration, embedding, staining, mounting...
Page 13, line 425. “Pituitaries of SYCs from both sexes”: Specify how many male and female individuals were used for in vitro cultures.
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Thank you to the authors the paper has been improved accordingly.
