Effect of sex chromosomes on mammalian behaviour: a case study in pygmy mice

Recent studies however suggest that gonadal hormones might not be the only biological factor involved in establishing sexual dimorphism, sex chromosomes might also play a role. The four core genotype (FCG) model and other similar systems allowing to decouple phenotypic and genotypic sex in mice have provided very convincing evidence of such a role (Gatewood et al. 2006; Arnold and Chen 2009; Arnold 2020a, 2020b). This however is a new field of research and the role of sex chromosomes in establishing sexually dimorphic behaviours has not been studied very much yet. Moreover, the FCG model has some limits. Sry, the male determinant gene on the mammalian Y chromosome might be involved in some sex differences in neuroanatomy, but Sry is always associated with maleness in the FCG model, and this potential role of Sry cannot be studied using this system.

Recent studies however suggest that gonadal hormones might not be the only biological factor involved in establishing sexual dimorphism, sex chromosomes might also play a role. The four core genotype (FCG) model and other similar systems allowing to decouple phenotypic and genotypic sex in mice have provided very convincing evidence of such a role (Gatewood et al. 2006;Arnold and Chen 2009;Arnold 2020aArnold , 2020b. This however is a new field of research and the role of sex chromosomes in establishing sexually dimorphic behaviours has not been studied very much yet. Moreover, the FCG model has some limits. Sry, the male determinant gene on the mammalian Y chromosome might be involved in some sex differences in neuroanatomy, but Sry is always associated with maleness in the FCG model, and this potential role of Sry cannot be studied using this system.
Heitzmann et al. have used a natural system to approach these questions. They worked on the African Pygmy mouse, Mus minutoides, in which a modified X chromosome called X* can feminize X*Y individuals, which offers a great opportunity for elegant experiments on the effects of sex chromosomes versus hormones on behaviour. They focused on maternal care and compared pup retrieval, nest quality, and mother-pup interactions in XX, X*X

Open Access
Published: 2022-10-31 Copyright: This work is licensed under the Creative Commons Attribution-NoDerivatives 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licen ses/by-nd/4.0/ and X*Y females. They found that X*Y females are significantly better at retrieving pups than other females. They are also much more aggressive towards the fathers than other females, preventing paternal care. They build nests of poorer quality but have similar interactions with pups compared to other females. Importantly, no significant differences were found between XX and X*X females for these traits, which points to an effect of the Y chromosome in explaining the differences between X*Y and other females (XX, X*X). Also, another work from the same group showed similar gonadal hormone levels in all the females (Veyrunes et al. 2022).

Heitzmann et al. made a number of predictions based on
what is known about the neuroanatomy of rodents which might explain such behaviours. Using cytology, they looked for differences in neuron numbers in the hypothalamus involved in the oxytocin, vasopressin and dopaminergic pathways in XX, X*X and X*Y females, but could not find any significant effects. However, this part of their work relied on very small sample sizes and they used virgin females instead of mothers for ethical reasons, which greatly limited the analysis.
Interestingly, X*Y females have a higher reproductive performance than XX and X*X ones, which compensate for the cost of producing unviable YY embryos and certainly contribute to maintaining a high frequency of X* in many African pygmy mice populations (Saunders et al. 2014(Saunders et al. , 2022. X*Y females are probably solitary mothers contrary to other females, and Heitzmann et al. have uncovered a divergent female strategy in this species. Their work points out the role of sex chromosomes in establishing sex differences in behaviours.  provided in the introduction -Following one comment of referee 3 (about discussion), I think showing a working model (figure?) of the effects of sex chromosomes on the studied phenotypes (with potential specific roles of XCI escapees -see referees 2 and 3, etc...) would be useful -I found referee 1 suggestion on analysing separately data from right and left parts of hypothalamus interesting, please consider it.

Reviewed by Marion Anne-Lise Picard, 26 May 2022 # SUMMARY OF THE MANUSCRIPT:
In this study, L. Heitzmann et al. investigate the role of the sex chromosome complement on maternal care, by combining behavioural and neurobiological aspects. The studied model, Mus minutoides, is characterized by three distinct female genotypes: XX, XX* and X*Y, all occurring naturally. Males are XY. This allow them not only to disentangle the role of gonadal hormone (phenotypic sex) vs sex chromosomes (genotypic sex); but also to discuss their results in the light of natural selection.
First, they performed an exhaustive behavioural assay, with a high number of replicates (from 75 to 105 females), and described as main results: (i) higher pup retrieval efficiency in X*Y females, (ii) no change in mother-pup interactions, (iv) poor nest quality in X*Y females, (v) no biparental care in X*Y females that are highly aggressive towards males.
Then, they focus on neural circuit known to underly such behaviors in model species, and report: (i) no significant differences in oxytocin expressing neurons number (ii) no significant differences in vasopressin expressing neurons number, but a tendancy for lower number in X*Y females (iii) no significant differences in the density of tyrosine hydroxylase expressing neurons, but a tendancy for greater density in X*Y females They conclude that the sex chromosome complement shapes sexually dimorphic behaviours, and that different combinations of sex chromosomes lead to alternative parental care strategies (more than the two expected under the sole effect of gonadal hormones). By providing the first description of the brain structure for the four genotypes of this species, they also open new perspectives for the study of the neural bases of such behaviours.

# MY OVERALL IMPRESSION:
That manuscript is straightforward and well written. It is attracting for a wide audience interested in reproductive biology, ranging from parental care, to the role of sex chromosomes in shaping phenotypic differences between sexes. I recommend it for three main reasons: 1. First, they shuffle the long standing thought that gonadal hormones are the main driver of sexually dimorphic behaviors; 2. their model, characterized by naturally occuring sex reversal, is original and relevant; 3. they propose complementary approaches.

# MINOR SUGGESTIONS:
1. The only important concern I could have is that their model do not allow them to perform the neurobiology assays on mothers, but on virgin females instead. Nonetheless, they adequately discuss this limitation (l390-l406) and their arguments are valuables. Even if I understand the reasoning behind the formulation "the neural basis of maternal behaviours", they maybe could find an alternative which better reflects the "potentiality"? 2. Sup. Fig. S5-S10: Why not comparing the "oxytocin neurons distribution" (S6) in the same way as vasopressin neurons (S5)? (It seems that at least that the regions #1 and #5 are also characterised by a tendancy for lower number of oxytocin neurons, aren't they?) Besides, did the autor studied the distribution of cell on left vs right side?? Indeed, there are growing evidences that the hypothalamus is functionally lateralized (Kiss et al. Brain Science, 2020 for review : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349050/). Such analysis may allow them to refine the signal?
3. Finally, did the authors previously assessed the level of gonadal hormones in the different female genotypes? Are they similar? # VERY MINOR SUGGESTIONS: -Fig2 : make slightly bigger panels A & B ? -Even if it is well explained in the text, maybe the authors could had a small chart of the studied neural circuit? # SOME TYPOS: l26 (keywords) : natural sex reversal and not "sex reversal, natural," ? l106 : remove the space before "Behavioural" l214 : remove space before "Higher" l352 : change the ref.

Reviewed by Caroline Hu, 19 Jun 2022
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