Genes in the Azoospermia Factor A Region of the Y Chromosome Show Sexual Dimorphism in Rat Brain Prior to Gonadal Sex Differentiation

The classical concept of brain sex differentiation suggests that steroid hormones released from the gonads program male and female brains differently. However, several studies indicate that steroid hormones are not the only determinant of brain sex differentiation and that genetic differences could also be involved. and E14. Analysis of genes expressed with the highest sex differences showed that Xist was highly expressed in females having XX genotype with an increasing ratio over time. Analysis of genes expressed with the highest male expression identied three main genes. At E12, two genes located in the azoospermia factor A (AZFa) region on the Y chromosome were highly expressed in males. These were Ddx3y (1552-fold higher in males) and Kdm6c (147-fold higher in males). The expression of Kdm6c, but not Ddx3y, remained high at both E13 and E14. In qRT-PCR analysis, these two genes were highly expressed in all the stages in male brain. In addition to these genes, one of the several copies of Sry in the rat genome, Sry4, showed a high expression in the male brains at all three time points. At all three time points several other genes were also found to show sex bias, but with lower differences in gene expression. The observed sex-specic expression of genes at early development suggests that the rat brain is sexually dimorphic prior to gonadal action on the brain and identies the AZFa region genes as a possible contributor to male brain development.


Introduction
Sexual dimorphism, including maternal care, sexual behavior, brain function, structure and susceptibility to neurological disorders are evident in humans as well as in nonhuman species. Earlier studies suggest that human male and female brains display differential connectome, methylome and transcriptome pro les (Ingalhalikar et al. 2014, Xu et al. 2014). Despite extensive advancement in neuroscience, the molecular regulation of these sex differences remains unclear. The classical model of brain sex differentiation that placed gonadal steroid hormones as the main drivers in establishing male and female neural networks was derived from earlier studies (Phoenix et al. 1959, Arnold 2009). This model states that the chromosomal constitution (XX or XY) determines the gonadal sex and that hormones secreted by these programs the brain neural network differently (Phoenix et al. 1959, Arnold 2009). However, a study on mice indicated that the developing whole head displayed differential gene expression prior to gonadal hormone action on the brain (Dewing et al. 2003). In that study, DEAD-box RNA helicase y (ddx3y; dby) and eukaryotic translation initiation factor 2 subunit 3 y (Eif2s3y) were identi ed as having the highest male biased gene expression at 10.5 dpc (days post coitum), while X-inactive speci c transcript (Xist) showed the highest female bias. Sex determining region Y (Sry) is the master regulator gene located on Y chromosome that determines testis fate (Koopman 2005). The expression of Sry can be detected at around 10.5 dpc in mouse gonads and peaks at 11.5 dpc to initiate testis differentiation (Sim et al. 2008).
A similar study in chicken also showed that male and female brains are sexually dimorphic prior to gonadal development (Lee et al. 2009). This indicates that there are genetic sex differences in developing brains prior to hormone action and suggests that these differences may be involved in the differential development of male and female brains.
Aromatase inhibitor treatment, that induced testicular tissue in the genetic female zebra nch, failed to masculinize the song system that remained feminine (Wade and Arnold 1996). Another study on zebra nch showed that androgen treatment of female birds did not fully masculinize the song center (Gahr and Metzdorf 1999). Furthermore, an involvement of genetic factors in brain sex development was suggested in a study of a gynandromorphic zebra nch that had one half of its brain and body genetically male and the other half genetically female (Arnold 2003). Despite the whole brain being under the in uence of the same gonadal hormones, the zebra nch still developed histologically identi able song centers in the male side of the brain while the female side of the brain remains feminine (Arnold 2003).
In the rat, it has been determined that Sry expression peaks on day 13, suggesting that this is the time when testis differentiation is initiated (Prokop et al. 2020). Therefore, we selected rat prenatal brains at E12, E13 and E14 to perform RNA sequencing to identify differentially expressed genes prior to the initiation of hormonal effects on the brain. At E12, prior to the initiation of testis development, we identi ed 2 genes located to the azoospermia factor A (AZFa) region on the Y chromosome, Ddx3y and N ϵ -methyl lysyl demethylase (Kdm6c), that may be involved in male brain sex differentiation. As Sry4 showed sex biased gene expression at all three developmental stages, locally expressed SRY cannot be excluded as a master regulator of gene expression in the brain prior to testis differentiation.

Materials And Methods
Sample processing, RNA sequencing and analysis Brain samples from Rattus norvegicus (Sprague Dawley) were purchased from Brain Bits USA. The samples were homogenized in Tri Reagent (Sigma) and RNA extraction was performed using Directzol RNA extraction kit (Zymo Research, USA). RNA was quanti ed using nano drop (Denovix, USA) and the quality was analyzed using RNA denaturing gel. RNA samples were sent to GATC Biotech/Euro ns for RNA sequencing. Sequencing was performed using Illumina platform with 30 million reads. RNA sequencing data was analyzed using Partek Flow software (Partek, USA). The raw data les were rst analyzed for sequence quality using pre-alignment QA/QC. The reads were aligned to rat genome (Rnor_6.0) using BWA-MEM alignment algorithm. The counts were normalized using counts per million reads method (CPM) and the differentially regulated genes were identi ed using gene speci c analysis (GSA). A p value of p<0.05 was used to identify signi cantly differentially expressed genes (DEGs). The regulated genes were then used for gene set enrichment analysis (GSEA) based on Gene Ontology (GO) annotations. GO enrichment analyses were performed with a p value threshold of 0.05.

Genotyping
For genotyping, tissue sample from the body was taken and genomic DNA was extracted using DNA isolation kit (Zymo Research, USA). DNA was quanti ed using nano drop (Denovix, USA) and PCR was performed for the Sry4 gene. Beta actin was used as positive control. Primer sequences are listed in Table  S1. The PCR reaction conditions were as follows: 95°C for 5 mins followed by 35 cycles of 95°C for 10 secs, 55°C for 15 sec and 72°C for 1 min. The PCR product was run on 1% agarose gel.

Quantitative real-time PCR (qRT-PCR) validation
Equal amounts of total RNA, from male and female brains at E12 to E14, were converted to cDNA using qScript cDNA synthesis kit (Quanta Biosciences, USA) according to the manufacturer's instructions. qRT-PCR was performed on a CFX96 Real-Time PCR Detection System using SsoAdvanced SYBR Green (BioRad, USA). Thermocycling conditions for SYBR Green qRT-PCR consisted of an initial denaturation at 95°C for 2 mins, followed by 40 cycles of 95°C for 2 secs and 60°C for 30 secs. Gapdh was used as the normalizing control. For primer speci city, melting curves were analyzed and the PCR product was run on agarose gel. Quantitative data analysis of relative gene expression was performed using the ΔΔCt method (Schmittgen 2008). Primer sequences are listed in Table S1.

Statistical analysis
For the transcriptomics data, differentially expressed genes were considered statistically signi cant if p values were ≤ 0.05. Pathway enrichment analysis and gene set enrichment analysis were performed using differentially genes and were considered statistically signi cant if p values were ≤ 0.05.
Student's t-test was performed for qRT-PCR data using the GraphPad Prism 8 software (GraphPad software). The differences were considered signi cant when the p value was <0.05 ( * p < 0.05; * * p < 0.01).

Results
Gene expression patterns during rat brain development The three developmental stages, from E12 to E14, were compared to identify genes that could be involved in sex differentiation of the brain. Principal component analysis (PCA) showed that the overall gene expression in male and female brains from the three different stages clustered according to embryonic stages rather than sex ( Figure 1A). The PC1 explained 43.19% of the variability, while PC2 and PC3 explained 12.67% and 8.2% of the variability, respectively.
Within each developmental stage, sexual dimorphic gene expression patterns were identi ed. In E12 brains, the expression of 13,440 genes was identi ed in male and female brains. Of these, 91 genes were upregulated in males, whereas 91 genes were upregulated in females ( Figure 1B). In the E13 brains, 13,979 genes were expressed in male and female brains ( Figure 1C). Of these, 80 genes were upregulated in male brain, whereas 349 genes were upregulated in female brains. In the E14 brains, the expression of 14043 genes was identi ed in male and female brains, of which 144 genes were upregulated in males and 368 genes were upregulated in females ( Figure 1D). The increased number of differentially expressed genes indicates that the sex differences in the brain increased with developmental time.

Differentially expressed genes in rat brains
Analysis of the sex-speci c gene expression patterns revealed that a substantial number of genes were upregulated in male brain already at E12 (Figure 2A, Supplementary  Con rmation of RNA seq data by qPCR qRT-PCR analysis was performed on Sry4, Ddx3x, Ddx3y, Xist, Kdm6c, Polysaccharide Biosynthesis Domain Containing 1 (Pbdc1), and Eif2s3 to con rm RNA seq data ( Figure 3). The qRT-PCR analysis con rmed that there were sex speci c differences in gene expression of these genes, in agreement with the data obtained from the RNA sequencing.

Pathway Analysis of Differentially Expressed Genes and Gene Ontology
Following identi cation of differentially expressed genes, we performed a pathway enrichment analysis to determine the main affected signaling pathways (Table S2). In E12 brains, three pathways were signi cantly enriched in males, whereas six were signi cantly enriched in females. Of these the Rap1 signaling pathway was affected in both males and females at E12 and continued to be enriched in females at E13. The PI3K-AKT signaling pathway showed enrichment in female rat brains at both E12 and E13 as well as in males at E12. At E14, the insulin signaling pathway showed the strongest enrichment in the male brain, while the Ribosome pathway was highly enriched in female brains.
Analysis of GO enrichment data revealed that 467 and 535 biological processes were altered in the E12 male and female brains, respectively ( Figure S1). At E13, 474 and 2076 biological processes were altered in male and female brains, respectively. At E14, 495 and 592 biological processes were altered in male and female brains, respectively. At E12, the main GO function was associated with intracellular membrane-bound organelles, including the nucleus. In females at E12 and in both males and females at E13 and E14 the results are more complex with several GO functions being altered as listed in Figure S1, Supplementary Tables 4, 5, 6).

Discussion
Differential patterning of male and female brains has been suggested to be regulated by steroid hormones released by the gonads. However, studies on zebra nch (Arnold 2003), mouse (Dewing et al. 2003) and chicken (Lee et al. 2009) suggests an involvement of genetic signals in the development of male and female brain neuronal networks.
In the present study, we have used isolated brain samples to avoid any signal from other head tissues.
We selected three embryonic stages to determine sex differences in genetic signaling in the brain, prior to gonadal activation (E12), at the time of gonadal activation (E13), and following gonadal activation (E14). RNA sequencing analysis indicated that the rat brain shows sexually dimorphic gene expression already at the rst studied stage, at E12 which is prior to the initiation of gonadal differentiation that occur at around stage E13 (Schulz et al. 2019). The rat gonadal differentiation process initiates with a bipotential gonad, and after E13 it further differentiates either into testis or ovary (Val et al. 2003).
Sry is the master switch in determining mammalian gonadal sex. Expression of Sry in mice testis begins at 10.5 dpc and peaks at 11.5 dpc and by 12. . Hence, it can be assumed that steroid secretion at E13 is still not initiated from the testis. Thus, it can be concluded that the differential gene expression pro les observed for rat brain at E12 occur prior to hormone action on the brain.
Of the many rat Sry genes, Sry4 is the gene that most closely resembles Sry4A (Prokop et al. 2013). The main difference between these two genes is a P83S amino acid replacement in Sry4 and a ten Q insertion after amino acid 154 in Sry4A (Prokop et al. 2013). While Sry4A was the top candidate for sex determination in rat testis, Sry4 was not expressed in testis (Prokop et al. 2020). In the present study, we observed sex differences in brain gene expression of Sry4 at all three developmental stages, while Sry4A was not differentially expressed. As Sry4 was the only Sry gene expressed in the developing brain, this suggests that Sry4 may be involved in regulating brain sex differentiation.
In the mouse study by Dewing and co-worker, microarray was performed on whole head of 10.5 dpc and 51 differentially regulated genes were identi ed (Dewing et al. 2003). Of these, the two genes with the highest fold differences were Dby (Ddx3y) and Eif2s3y, both located on the Y chromosome. While the remaining genes indicated to be differentially expressed were not located to the Y chromosome, one was located to the X chromosome. In the present study, we identi ed a larger number of genes as male biased at all three timepoints. The 3 genes showing the highest male bias at E12 were located on the Ychromosome. Two of these genes, Ddx3y and Kdm6c, are located in the AZFa region of the Ychromosome. This region also contains the Usp9y gene that was not identi ed in the present study. The AZFa, b and c regions on the Y-chromosome have been identi ed to be required for normal spermatogenesis (Vog et al. 1996). In the AZFa region it has been suggested that Ddx3y is the main gene responsible for infertility (Foresta et al. 2000). In male gonads, Ddx3y is expressed in spermatogonia before meiosis and Ddx3x is expressed in spermatids (Rauschendorf et al. 2014). Deletion of Ddx3y disrupts germ cell development and leads to infertility in males (Ramathal et al. 2015). The peak expression of Ddx3y at E12 in rat brain indicate that this gene may also have key functions in male brain development.
The second gene that showed high expression in male rat brains was Kdm6c. The X chromosome homologue Kdm6a/Utx has been shown to be an active demethylase, acting on Lys 27 of histone H3 (H3K27). Kdm6a/Utx was also differentially regulated in mouse whole head (Dewing et al. 2003

Perspective and Signi cance
In the present study, we show that the top candidate genes, Ddx3y and Kdm6c, that are involved in male brain development are expressed in a sexually dimorphic pattern at stage E12, prior to the initiation of gonadal differentiation that occurs at E13 in rat (Clement et al. 2007, Prokop et al. 2020). Here we show that the expression of Kdm6c remain high at E13 and E14, while Ddx3y peak at E12. Thus, it is possible that Ddx3y may play a role in the initiation of brain sex differentiation prior to the initiation of gonadal differentiation. Previous studies have shown that genes encoded by the Y chromosome can result in differences in behavior and brain phenotypes in mice (van Abeelen 1988, van Abeelen et al. 1989). The expression of Sry at all three timepoints suggests that locally expressed Sry may be involved in male brain differentiation. Furthermore, two candidate genes, Ddx3y and Kdm6c in the AZFa region of the Y chromosome, were also identi ed as possible regulators of dimorphic rat brain differentiation prior to gonadal sex differentiation.

Declarations
Ethics approval and consent to participate Not applicable Consent for publication Not applicable  Validation of gene expression patterns using qRT-PCR. In order to con rm the differential expression patterns a qRT-PCR analysis was performed on a selection of genes with different expression patterns. This analysis con rmed the the expression patterns of the analysed genes (Sry, Ddx3y, Xist, Kdm6c, Pbdc1, and Eif2s3) as observed in the RNA sequencing data.

Supplementary Files
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