Effect of Neurosteroids on Basal and Stress-Induced Oxytocin Secretion in Luteal-Phase and Pregnant Sheep

Round 1

Reviewer 1 Report

This paper by Mlotkowska et al focuses on a potential role of neurosteroids in modulating oxytocin mRNA levels in the PVN and SON as well as in the blood following allopregnanelone administration or an acute stressor, of the combination, in luteal phase ewes. In a second study, blood oxytocin concentrations are assessed in pregnant sheep following stress or finestaride administration, or the combination of both. The authors report that stress markedly elevates oxytocin release in luteal phase ewes, which can be largely blocked by allpregnanolone administration. In addition, hypothalamic area-specific changes are reported for oxytocin mRNA and PAM expression were noted as well as changes in oxytocin mRNA in the posterior pituitary.  The work builds on previous efforts by this group and addresses an interesting and timely topic, that of the role of neurosteroids in various reproductive processes. I would appreciate the authors responding to the following comments.

 

L136 – here, and elsewhere. Usually, in an experiment of this design, the individual treatments are conventionally presented first and the combined treatment last. 

L146 – Is there a reason for this interrupted treatment paradigm? Why not just a constant infusion?

L154 – I assume this was on day 3?

L174 – while finestaride may block the conversion to allopregnanelone, does is also block synthesis of other steroids? How specific is it for allopregnanolone?

L174 – the treatments here, from what I can see, are confounded by time. Is there a reason that the treatment order couldn’t be randomized or balanced across groups?

L183 – is there a rationale for this dose of finestaride?

Figure 2 – the superscript labeling of bars is confusing to me. If the LSD was applied, then it should make all comparisons and letters should denote differences. For instance, in Figure 1, top panel, the bar for A doesn’t have a letter designation. In the text, the statement is made that the values for A do not differ from controls, thus is should be labeled with an A superscript. In the bottom panel of that figure, the need to label AS and A with a “D” is confusing. If levels in AS and A are not different than controls, then they should also be labeled with an “A”. 

L243 – I would use “reduced” here instead of “down-regulated”.

Figure 2, top panel – I may have missed it, but is there any idea as to why A would reduce the impact of stress but increase OT in the SON when given by itself?

Figure 3 – what is the physiological relevance of measuring mRNA for OT in the posterior pituitary? My understanding is that this arises from transport from the hypothalamus, but the importance of that in the context of the paper is not inherently obvious.

Figure 4 – I would suggest reconsidering the coloring of the lines used in this and Figure 5. Some are hard to distinguish from another.

Figure 5 – one thing that seems obvious is that values in pregnant sheep are lower than in luteal phase ewes. Is that correct? Is that due to the presumably elevated allopregnanelone? Would that also offer an explanation for the much reduced response to an acute stressor?  Also, from the text, it seems that there are data in rodents about how finestaride impacts central allogpregnanelone levels, but the authors do not know its effectiveness in this paradigm. Is that correct? If so, that caveat needs to be mentioned in the discussion.

L326 – OT and PAM gene expression are not secretory parameters, just simply static mRNA levels. 

L339 – It’s not clear to me what the authors mean by the statement “To avoid additional and unexpected effects from estrogen” when there is obviously estrogen present during the luteal phase.

L347 – oxytocin content in the posterior pituitary was not looked at in this study, so this is speculative. Is there evidence that could be referenced to support this?

L349 – given the short time frame involved and the acute nature of the suppressor, is it likely that allopregnanelone is working through membrane receptors?

L386 – the authors reference a previous work where cortisol was examined. Would it be useful to assess it here relative to changes observed in oxytocin?

L429 – what are typical levels of allopregnanelone in a steroid-depleted animal?  The authors talk about it increasing during pregnancy, but in this study the levels in the pregnant animals seem to be similar to or even lower than luteal phase animals. Likewise, I don’t really qualify and increase from 2 to 5 ng/ml as pronounced, especially relative to what was observed in experiment 1 in response to stress.

L440 – The results in the pregnant ewes are intriguing. While the infusion of finestaride alone caused an increase in oxytocin, stress did as well. If allopregnanelone is inhibiting oxytocin release, then might you expect the oxytocin response in the stress+finestaride group to be higher than that of finestaride alone?

 

Author Response

Response to the Reviewer 1

The authors thank very much for the critical comments regarding the manuscript. Referring to them, we tried to improve the content of the manuscript as much as possible.

 

L136 – here, and elsewhere. Usually, in an experiment of this design, the individual treatments are conventionally presented first and the combined treatment last. 

• The description of the groups in the Materials and Methods section and their presentation in the figures have been changed as suggested

 

L146 – Is there a reason for this interrupted treatment paradigm? Why not just a constant infusion?

• Intermittent infusion of the active substance into the CNS was previously validated in our sheep model [25,26] as maintaining effective signaling without completely blocking the receptors and/or binding sites.(L148-150)

 

L154 – I assume this was on day 3?

• Yes, it was on the 3rd day – reworded (L158 and 190)

 

L174 – while finestaride may block the conversion to allopregnanolone, does is also block synthesis of other steroids? How specific is it for allopregnanolone?

• Finasteride, as a 5α-reductase inhibitor, blocks the production of neurosteroids, including allopregnanolone. It is not specific for allopregnanolone – L181 was reworded. All comments regarding the use of finasteride in pregnant sheep should refer to the effects of neurosteroids in general.

 

L174 – the treatments here, from what I can see, are confounded by time. Is there a reason that the treatment order couldn’t be randomized or balanced across groups?

• The use of pregnant sheep in the experiment was constrained by ethical considerations. In large animals, the active substance can be applied to the same animal after a previous control trial. In our design, we wanted to avoid exposing pregnant animals to a double dose of finasteride. This design was validated previously: Misztal et al. Theriogenology 2021; 174: 114-120.

 

L183 – is there a rationale for this dose of finestaride?

• The dose of finasteride was chosen on the basis of the rodent research literature [32] and our preliminary study (unpublished data, Grant No. 2015/19/B/NZ9/03706) – L188-189

 

Figure 2 – the superscript labeling of bars is confusing to me. If the LSD was applied, then it should make all comparisons and letters should denote differences. For instance, in Figure 1, top panel, the bar for A doesn’t have a letter designation. In the text, the statement is made that the values for A do not differ from controls, thus is should be labeled with an A superscript. In the bottom panel of that figure, the need to label AS and A with a “D” is confusing. If levels in AS and A are not different than controls, then they should also be labeled with an “A”. 

• Superscript labeling of bars in each figure has been improved

 

L243 – I would use “reduced” here instead of “down-regulated”.

• The sentence has been reworded – L248

 

Figure 2, top panel – I may have missed it, but is there any idea as to why A would reduce the impact of stress but increase OT in the SON when given by itself?

• The case of SON is interesting, but cannot be explained based on this study and literature data. The Discussion section dealing with these results has been reworded. (L351-359)

 

Figure 3 – what is the physiological relevance of measuring mRNA for OT in the posterior pituitary? My understanding is that this arises from transport from the hypothalamus, but the importance of that in the context of the paper is not inherently obvious.

• This finding suggested that, in addition to the mature hormone, significant amounts of OT mRNA were also translocated from both hypothalamic nuclei to nerve terminals in the PP. The phenomenon of axonal transport has been described previously for hypothalamic vasopressin neurons [44], as well as for GnRH neurons [45]. According to Kaplan et al. [46], peripheral protein biosynthesis does not occur during homeostasis milieu, but it is initiated in response to external stimuli. Therefore, the observed increase in axonal transport of OT mRNA in stressed animals could be particularly important in increasing the pool of OT transcript for hormone synthesis also in the vicinity of the release site. L364-372

 

Figure 4 – I would suggest reconsidering the coloring of the lines used in this and Figure 5. Some are hard to distinguish from another.

• Various line patterns were used

 

Figure 5 – one thing that seems obvious is that values in pregnant sheep are lower than in luteal phase ewes. Is that correct? Is that due to the presumably elevated allopregnanolone? Would that also offer an explanation for the much reduced response to an acute stressor?  Also, from the text, it seems that there are data in rodents about how finestaride impacts central allopregnanolone levels, but the authors do not know its effectiveness in this paradigm. Is that correct? If so, that caveat needs to be mentioned in the discussion.

• The intriguingly low plasma OT concentrations observed in our pregnant sheep confirmed several early observations. OT levels were shown to remain low throughout pregnancy until term, at concentrations even below as during the luteal phase of the cycle [40,68]. However, episodic increases in OT concentration were found during several days after mating [69]. L 435-439

and

• Moreover, lowered OT concentration and reduced response to stress in the pregnant sheep, compared to the luteal-phase sheep, may confirm a strong inhibitory influence of brain neurosteroids in this species. L454-456

 

L326 – OT and PAM gene expression are not secretory parameters, just simply static mRNA levels. 

• The sentence has been reworded: L327-329

 

L339 – It’s not clear to me what the authors mean by the statement “To avoid additional and unexpected effects from estrogen” when there is obviously estrogen present during the luteal phase.

• The statement has been reworded: To avoid fluctuations in estrogen levels in cycling animals, our study used AL in late luteal-phase sheep, whose reproductive status was previously described by Młotkowska et al. [39]. Early studies in sheep showed that circulating OT concentrations were similar to those of progesterone during the estrous cycle and reached low levels during the period of luteal regression [40,41]. L340-344

 

L347 – oxytocin content in the posterior pituitary was not looked at in this study, so this is speculative. Is there evidence that could be referenced to support this?

• The paragraph has been reworded without this speculation. L345-359

 

L349 – given the short time frame involved and the acute nature of the suppressor, is it likely that allopregnanolone is working through membrane receptors?

• The authors are aware of the action of AL through membrane progesterone receptors, but rather in the anterior pituitary in relation to prolactin secretion.Due to the short frame of action, this is rather ruled out and, above all, not explored in the posterior pituitary.The discussion on this topic in this manuscript could be based on speculation.

 

L386 – the authors reference a previous work where cortisol was examined. Would it be useful to assess it here relative to changes observed in oxytocin?

• References No 47, 48 and 49 refer to oxytocin as well.

 

L429 – what are typical levels of allopregnanolone in a steroid-depleted animal?  The authors talk about it increasing during pregnancy, but in this study the levels in the pregnant animals seem to be similar to or even lower than luteal phase animals. Likewise, I don’t really qualify and increase from 2 to 5 ng/ml as pronounced, especially relative to what was observed in experiment 1 in response to stress.

• The authors have removed the mention of unpublisheddata on oxytocin in pregnant sheep and also some questionable claims. Instead, a citation from earlier studies in sheep has been added. L435-439

 

L440 – The results in the pregnant ewes are intriguing. While the infusion of finestaride alone caused an increase in oxytocin, stress did as well. If allopregnanelone is inhibiting oxytocin release, then might you expect the oxytocin response in the stress+finestaride group to be higher than that of finestaride alone?

• Increasing the strength of the stimulus, it does not always lead to an increased secretory response. It depends on many factors, includingthe accumulated pool of hormone at a given moment and the rate of synthesis of new molecules. In this case, we are dealing with a rapid response, which is largely dependent on the dose of finasteride used. In our study, the stressed sheep had significantly higher levels of oxytocin after using finasteride than without.

Some changes can be found, according to the suggestions of the Reviewer 2

Reviewer 2 Report

The manuscript titled "Effect of neurosteroids on basal and stress-induced oxytocin secretion in luteal-phase and pregnant sheep" uses both in vivo and neuroanatomical approaches to examine a central mechanism of stress on the oxytocin secretion in female sheep (both luteal phase and pregnant animals).  A considerable amount of work has been conducted and with some minor edits and additional information should be acceptable for publication.  See below for comments.

Comments:

1) Some reorganization may help the reader.  Lines 71-79 would best be placed in the middle of Line 88.  Move the description of Finasteride from lines 177-178 to Line 174.  Move section on RTPCR to immediately after section 2.3 as that luteal tissue is then used for qPCR.  That will remove confusion as to what tissue was used for the RTPCR analysis.  Finally, my preference would be to show the in vivo data before the mRNA data.  At the very least the central drug delivery for luteal animals could be displayed followed by the mRNA in PVN, SON, and PostPit.  Then ending the results with the pregnant animal experiment.  

2) More information on AL in the introduction is warranted.  

3) Include a rational for using luteal phase ewes in the introduction.

4) Clearly describe your "stressful stimuli".  Indicate how you know this is a stressful stimuli and draw on that when you get into the discussion as this is the positive control (increase in oxytocin) needed to make comparisons throughout.  

5) Provide a reference to the previous use and validation of the Oxytocin RIA kit in sheep.  

6) Figure 4 clarification:  Is the line graph averages of all animals in each group or representatives from each group.  If the former, SEM is needed in the graph.  A representative would be sufficient given the mean values that are in the top panel of that graph.  The authors might consider moving the line graph to the top and the bar graph to the bottom.  

7) Minor grammatical corrections:  Line 8: start the simple summary with "Oxytocin is a hypothalamic..."; Line 22:  Start the Abstract with "The" instead of "A"; Line 40: replace "nonapeptide" with "nanopeptide" or maybe "neuropeptide" would be more appropriate; Line 50:  replace "At" with "In"; italicize the use of in vitro and in vivo throughout.  Line 100:  replace "representing" with "which displays"; Delete the final sentence of section 2.2 and modify the previous sentence to read "...after slaughter with data herein from only animals with correct cannula placement."

 

Author Response

Response to the Reviewer 2

The authors thank very much for the critical comments regarding the manuscript. Referring to them, we tried to improve the content of the manuscript as much as possible.

• Lines 71-79 would best be placed in the middle of Line 88 – In the opinion of the authors, moving the paragraphs will disturb the concept of the Introduction

Move the description of Finasteride from lines 177-178 to Line 174 – done (L178). 

Move section on RTPCR to immediately after section 2.3 as that luteal tissue is then used for qPCR – The authors prefer the standard article scheme: experimental design - analytics - statistics

Finally, my preference would be to show the in vivo data before the mRNA data. At the very least the central drug delivery for luteal animals could be displayed followed by the mRNA in PVN, SON, and PostPit – The proposed change in the scheme would force huge changes in the ongoing Discussion, which the authors prefer to avoid.

• More information on AL in the introduction is warranted
• Information on allopregnanolone has been added (L80-86).

 

3) Include a rational for using luteal phase ewes in the introduction.

• An explanation has been added in the Discussion section: “To avoid fluctuations in estrogen levels in cycling animals, our study used AL in late luteal-phase sheep, whose reproductive status was previously described by Młotkowska et al. [39]. Early studies in sheep showed that circulating OT concentrations were similar to those of progesterone during the estrous cycle and reached low levels during the period of luteal regression [40,41]”. L340-344.

 

 

4) Clearly describe your "stressful stimuli". 

• Sheep is a herd animal. Isolation from the herd is a strong psychological stimulus that has been described many times in sheep: “The combination of both stressors was validated previously in cycling, pregnant and lactating sheep [25,30,31], demonstrating a clear response of the HPA axis at the level of all its components.” L155-157.

 

5) Provide a reference to the previous use and validation of the Oxytocin RIA kit in sheep.

• A reference has been added (L225).

 

6) Figure 4 and 5 clarification:  Is the line graph averages of all animals in each group or representatives from each group.  If the former, SEM is needed in the graph.  A representative would be sufficient given the mean values that are in the top panel of that graph.  The authors might consider moving the line graph to the top and the bar graph to the bottom.  

• Individual representative sheep from each group have been shown on the bottom graphs.

 

7) Minor grammatical corrections: 

• The suggested correction has been carried out. The manuscript has been linguistically checked.

 

Several changes in the present version have been made according to the suggestion of the Reviewer 1.