Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
Summary:
Morgan et al. studied how paternal dietary alteration influenced testicular phenotype, placental and fetal growth using a mouse model of paternal low protein diet (LPD) or Western Diet (WD) feeding, with or without supplementation of methyl-donors and carriers (MD). They found diet- and sex-specific effects of paternal diet alteration. All experimental diets decreased paternal body weight and the number of spermatogonial stem cells, while fertility was unaffected. WD males (irrespective of MD) showed signs of adiposity and metabolic dysfunction, abnormal seminiferous tubules, and dysregulation of testicular genes related to chromatin homeostasis. Conversely, LPD induced abnormalities in the early placental cone, fetal growth restriction, and placental insufficiency, which were partly ameliorated by MD. The paternal diets changed the placental transcriptome in a sex-specific manner and led to a loss of sexual dimorphism in the placental transcriptome. These data provide a novel insight into how paternal health can affect the outcome of pregnancies, which is often overlooked in prenatal care.
Strengths:
The authors have performed a well-designed study using commonly used mouse models of paternal underfeeding (low protein) and overfeeding (Western diet). They performed comprehensive phenotyping at multiple timepoints, including the fathers, the early placenta, and the late gestation feto-placental unit. The inclusion of both testicular and placental morphological and transcriptomic analysis is a powerful, non-biased tool for such exploratory observational studies. The authors describe changes in testicular gene expression revolving around histone (methylation) pathways that are linked to altered offspring development (H3.3 and H3K4), which is in line with hypothesised paternal contributions to offspring health. The authors report sex differences in control placentas that mimic those in humans, providing potential for translatability of the findings. The exploration of sexual dimorphism (often overlooked) and its absence in response to dietary modification is novel and contributes to the evidence-base for the inclusion of both sexes in developmental studies.
Weaknesses:
The data are overall consistent with the conclusions of the authors. The paternal and pregnancy data are discussed separately, instead of linking the paternal phenotype to offspring outcomes. Some clarifications regarding the methods and the model would improve the interpretation of the findings.
(1) The authors insufficiently discuss their rationale for studying methyl-donors and carriers as micronutrient supplementation in their mouse model. The impact of the findings would be better disseminated if their role were explained in more detail.
We acknowledge the Reviewer’s comments regarding the amount of detail in support of the inclusion of methyl carriers and donors within our diet. Therefore, we will revise the manuscript to include more justification, especially within the Introduction section, for their inclusion. Please see lines 111-120.
(2) It is unclear from the methods exactly how long the male mice were kept on their respective diets at the time of mating and culling. Male mice were kept on the diet between 8 and 24 weeks before mating, which is a large window in which the males undergo a considerable change in body weight (Figure 1A). If males were mated at 8 weeks but phenotyped at 24 weeks, or if there were differences between groups, this complicates the interpretation of the findings and the extrapolation of the paternal phenotype to changes seen in the fetoplacental unit. The same applies to paternal age, which is an important known factor affecting male fertility and offspring outcomes.
We thank the Reviewer for their comments regarding the ages of the males analysed. As we had 5 treatment groups, and intended to generate a minimum of 8 litters of offspring per treatment group, this resulted in over 40 litters in total. In order to dissect these litters appropriately, and in a timely fashion, we had to stagger their generation over time. As such, this resulted in utilising our males at different ages/durations on the diet. However, in all our statistical analysis, we factored in the duration of time on the diet, which also acted as a proxy measure of paternal age. We also ensured that we staggered the generation of litters in each diet group so that any age effects were experienced across all paternal regimens.
We have revised the manuscript to acknowledge this fact and to highlight that the duration of time on any diet was factored into the statistical analysis.
(3) The male mice exhibited lower body weights when fed experimental diets compared to the control diet, even when placed on the hypercaloric Western Diet. As paternal body weight is an important contributor to offspring health, this is an important confounder that needs to be addressed. This may also have translational implications; in humans, consumption of a Western-style diet is often associated with weight gain. The cause of the weight discrepancy is also unaddressed. It is mentioned that the isocaloric LPD was fed ad libitum, while it is unclear whether the WD was also fed ad libitum, or whether males under- or over-ate on each experimental diet.
We agree with the Reviewer that the general trend towards a lighter body weight for our experimental animals is unexpected. We can confirm that all diets were fed ad libitum. However, as males were group housed, we were unable to measure food consumption for individual males. We also observed that for males fed the high fat diets, they often shredded significant quantities of their diet, rather than eating it, so preventing accurate measurement of food intake.
We also agree with the Reviewer that body weight can be a significant confounder for many paternal and offspring parameters. However, while the experimental males did become lighter, there were no statistical differences between groups in mean body weight. As such, body weight was not included as a variable within our statistical analysis.
(4) The description and presentation of certain statistical analyses could be improved.
(i) It is unclear what statistical analysis has been performed on the time-course data in Figure 1A (if any). If one-way ANOVA was performed at each timepoint (as the methods and legend suggest), this is an inaccurate method to analyse time-course data.
(ii) It is unclear what methods were used to test the relative abundance of microbiome species at the family level (Figure 2L), whether correction was applied for multiple testing, and what the stars represent in the figure. 3) Mentioning whether siblings were used in any analyses would improve transparency, and if so, whether statistical correction needed to be applied to control for confounding by the father.
We apologies for the lack of clarity regarding the statistical analyses. Going forward, we will revise the manuscript and include a more detailed description of the different analyses, inclusion of siblings and correction for multiple testing.
Reviewer #1 (Public review):
Summary:
The authors investigated the effects of a low-protein diet (LPD) and a high sugar- and fat-rich diet (Western diet, WD) on paternal metabolic and reproductive parameters and fetoplacental development and gene expression. They did not observe significant effects on fertility; however, they reported gut microbiota dysbiosis, alterations in testicular morphology, and severe detrimental effects on spermatogenesis. In addition, they examined whether the adverse effects of these diets could be prevented by supplementation with methyl donors. Although LPD and WD showed limited negative effects on paternal reproductive health (with no impairment of reproductive success), the consequences on fetal and placental development were evident and, as reported in many previous studies, were sex-dependent.
Strengths:
This study is of high quality and addresses a research question of great global relevance, particularly in light of the growing concern regarding the exponential increase in metabolic disorders, such as obesity and diabetes, worldwide. The work highlights the importance of a balanced paternal diet in regulating the expression of metabolic genes in the offspring at both fetal and placental levels. The identification of genes involved in metabolic pathways that may influence offspring health after birth is highly valuable, strengthening the manuscript and emphasizing the need to further investigate long-term outcomes in adult offspring.
The histological analyses performed on paternal testes clearly demonstrate diet-induced damage. Moreover, although placental morphometric analyses and detailed histological assessments of the different placental zones did not reveal significant differences between groups, their inclusion is important. These results indicate that even in the absence of overt placental phenotypic changes, placental function may still be altered, with potential consequences for fetal programming.
Weaknesses:
Overall, this manuscript presents a rich and comprehensive dataset; however, this has resulted in the analysis of paternal gut dysbiosis remaining largely descriptive. While still valuable, this raises questions regarding why supplementation with methyl donors was unable to restore gut microbial balance in animals receiving the modified diets.
We thank the Reviewer for their considered thoughts on the gut dysbiosis induced in our models the minimal impact of the methyl donors and carriers. We will include additional text within the Discussion to acknowledge this. However, at this point in time, we are unsure as to why the methyl donors had minimal impact. It could be that the macronutrients (i.e. protein, fat, carbohydrates) have more of an influence on gut bacterial profiles than micronutrients. Alternatively, due to the prolonged nature of our feeding regimens, any initial influences of the methyl donors may become diluted out over time. We will amend the text to reflect these potential factors.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
The authors have done an immense amount of work, which should be commended. In addition to the public review, I have a few suggestions for improvement.
(1) To further explore the weight discrepancy between the males subjected to diet alteration and those on the control diet, further details about the intake and provision of the diets would be beneficial. Seeing as the fat mass was increased in males fed a WD, do you have information on where the weight 'loss' originated from?
We thank the Reviewer for their insight into the changes in male body weight. We agree that the differences in total body weight verses the amount of adipose tissue, is intriguing. Unfortunately, we were unable to monitor the food intake of our animals for two main reasons. The first was that for animal welfare considerations, all our males were initially group housed prior to mating. This meant that typically, males were housed in groups of 4 during the initial feeding (pre-mating) period. Males were only housed singly upon them being used for mating. As such, it was not possible to obtain food consumption data for individual males.
A second limitation arose due to the high extend of males who were fed the Western Diet effectively shredding the diet. This meant that it was not possible to weight the food to obtain a crude idea of how much they were consuming. The reason for this shredding is not clear to us. All mice received environmental enrichment, as we did not observed this behaviour for our control or low protein diet fed males.
With regards to the weight of the other organs, we did not observe and significant overall changes in organ weight, or weight relative to body weight. Unfortunately, we did not have access to, or conduct any whole body scanning, such as DEXA, which would have given more insight into the body composition of our mice.
(2) The testicular abnormalities and gene expression findings are linked nicely to the offspring's story. This is not as compelling for other findings, including the gut microbiome changes, which are not discussed in the context of the fetoplacental outcomes. More discussion of the potential impact of paternal changes on fetal outcomes would strengthen claims that these findings are impactful.
We thank the Reviewer for their comments and suggestion. Our caution with connecting the gut microbiota to offspring development is that, to the best of our understanding, there is little data with regards to its effect on post-fertilisation development. While there is data showing that the microbiome can produce compounds and metabolites that can affect sperm quality and metabolism, lipid composition and testicular morphology, the connection with post-fertilisation development is limited. Additionally, as we saw no difference in fundamental fertility, as measured by changes in litter size, we propose that there no overall changes in the ability of the sperm from our experimental males to reach, fertilise and support development.
However, we acknowledge the Reviewers comments on strengthening the manuscript and so have included some additional text within the Discussion to highlight the links between the microbiome and male reproductive fitness. Please see lines 337-348.
(3) It is clarified in the methods that n=8 males were used in the study, but different nnumbers are shown for some parameters. It would improve transparency for the reader if it were clarified whether these differences result from missing data or from the removal of statistical outliers.
The Reviewer is correct that while 8 males were initially placed on their respective diets, for some of the analyses, the n-number is less than 8. In some instances, for example the analysis of total body fat (Fig. 1D), data was unfortunately not collected during an initial round of dissections. As such, the n number here is only 6 in each group. Additionally, due to the high cost associated with sequencing the microbiome for 5 groups, we decided to only sequence 6 samples per group. However, we do not feel that this impacts significantly on the overall focus of the data presented.
(4) Despite this, you may have been underpowered to detect differences in some parameters, for example, the placental stereology. Alternative approaches, such as immunostaining with whole-section quantification, may be more sensitive to detect subtle changes. Alternatively, have you considered using smaller grids for improved sensitivity of the stereological analysis?
We thank the Reviewer for their insight into the data and their suggestion for immunostaining. We agree with the Reviewers that a greater number of samples would have strengthened our analyses. However, we are not in the possession of further samples which have been processed in the correct manner for additional stereological analysis. We are hoping to conduct further placental analyses based on our RNA-Seq data, but this will require the generation of new samples.
(5) It would be easier to interpret the figures if it were clear which datasets were analysed using non-parametric tests. Were Figure 2F, 2G, 6A, 6E, and 6I are shown differently for that reason, perhaps? It would improve transparency if non-normally distributed data are shown as medians, as that's what's being compared in a non-parametric test.
We apologies for any confusion regarding the analysis of our data. The Reviewer is correct that the data in 2F and 2G were analysed using a non-parametric test. We have now made this clearer in the legend to the figure highlighting which data sets were analysed by ANOVA or Kruskal–Wallis test. We have also done this for the other figure legends where appropriate. With regard to Figure 6, the data presented in Panels A, E and I were intended to show the range of data extending above and below the 90th and 10th centiles of the CD fetuses. As such, we felt that violin plots were the most appropriate way to display these data.
(6) Supplemental Figure 1 seems to be missing.
We apologise sincerely for the lack of inclusion of Supplemental Figure 1. We will ensure that it is included in our resubmission
(7) Line 523 states that samples with RIN < 7 were used for microarray analysis. Do the authors mean RIN > 7?
We thank the Reviewer for identifying our mistake. The Reviewer is correct that this should have been a RIN >7. We have now corrected this.
(8) It is mentioned in lines 603-604 that paraffin shrinkage was accounted for. It could be useful to describe how this was done.
We have revised the text within the Materials and Methods to provide additional clarity on how we compensated for the shrinkage due to the paraffin processing.
In the revised Methods we have added a brief “Shrinkage correction” subsection describing how paraffin-embedding shrinkage was quantified for each placenta individually. Specifically, we now state that post-embedding placental volume was estimated using the Cavalieri Principle on systematic and uniformly-random sampled H&E sections, and a per-placenta volume shrinkage coefficient (kV = Vpost/Vpre) was calculated.
We have also added the equations showing how this coefficient was used to correct compartment volumes and the derived surface area estimates (surface area calculated from Sv and the corresponding shrinkage-corrected placenta volume). Please see lines 618-644.
(9) This may be due to the generation of the reviewer PDF, but Figure 4E and 4H are illegible in our version of the manuscript.
We apologies for the lower resolution with these figures and the difficulty in seeing the information presented. We have created revised versions of these figures which we hope are of higher quality and clarity.
(10) What do the stars represent in Figure 6A, E, I - compared to what, controls?
The Reviewer is correct that the asterisks in Figures 6A, E and I represent differences in the proportion of fetuses either above or below the 90th and 10th centile of the CD fetuses respectively. As such, in panel A, for both the LPD and MD-LPD groups, there are significantly more fetuses who are below the 10th centile of the CD group. Similarly, in panel E, there are significantly more placentas in the LPD group that have a weight above the 90th centile of the CD group. We have revised the graphs to make these differences, and their comparisons clearer.
Reviewer #2 (Recommendations for the authors):
Some Recommendations for improving the writing and presentation, and minor corrections to the text and figures:
(1) Please describe Wnt signaling in the Abstract.
The Abstract has been amended to provide some additional text regarding Wnt signalling. Please see lines 60-63.
(2) Page 6, line 134: A brief explanation of why measuring the inhibin beta-A chain should be included.
The text within this section has been amended to include a brief description of the role of Inhibin β-A chain on testicular function. Please see lines 135-139.
(3) The methodology used for Tnf determination is missing and should be described.
We apologies for the lack of detail regarding our analysis of serum Tnf in our males. This has now been included. Please see lines 479-480.
(4) It is important to mention that free fatty acid levels in the MD-WD group were similar to those in the CD group, although they remained comparable to the WD group.
We agree with the Reviewer and have amended the text to indicate that there was no difference in the FFA profile of the MD-WD males to either the CD or WD males. Please see lines 147-148.
(5) Figure 2 presents both metabolic parameters and bacterial profile analyses. Although the authors appear to relate these outcomes, clarity would be improved by presenting them in separate figures.
As requested, we have now presented these data as two separate Figures
(6) Figure 3H: The data suggest that the decrease in the number of spermatogonia (PLZF⁺) observed in the LPD and WD groups was prevented when the diets were supplemented with methyl donors.
(7) However, the description and interpretation of this result (or of a neutral effect) are missing.
We agree with the Reviewer in their interpretation of the PLZF+ data. We have indicated this in the text within the Results and Discussion sections. Please see lines 177-178 and lines.
(8) Line 284: Please check the abbreviation for MD-LPD.
We thank the Reviewer for identifying this typographical mistake. This has now been corrected to state MD-LPD and not MDL.
(9) Line 285: Please check the lettering in the text and in Figure 6H-K.
We thank the Reviewer for identifying this typographical mistake. This has now been corrected to state the panels are Figure 9H-K, as we have split the original Figure 2 into two figures.
