Transcriptome Alignment and Mapping
A total of 852,343,043 sequence reads were obtained from the hypothalamus and pituitary, with an average of 35,514,293 reads per sample (Supplemental Figure 1A). A significantly higher number of reads were obtained from the pituitary samples when compared to the hypothalamus samples, but no differences in the percentage of reads mapped to the turkey genome were seen. On average, 79.9% of reads mapped to the turkey reference genome (Ensembl Turkey_2.01). For each sample, read pairs were aligned with minimal discordant pairs or pairs with multiple alignments (average of 0.58% and 2.29% respectively) (Supplemental Figure 1B). The number of reads per sample, the number of mapped reads per sample, and the number of properly aligned pairs per sample did not differ significantly between egg production or ovulatory cycle groups in either of the tissue examined.
Overview of DEGs
A total of 1641 and 2778 DEGs were identified in the hypothalamus and pituitary, respectively. Analysis of the genes differentially expressed between LEPH and HEPH revealed a significantly higher number of DEGs in the hypothalamus during the PS and in the pituitary outside of the PS. In the hypothalamus, 248 DEGs were identified outside of the PS, whereas 1393 DEGs were identified during the PS (Figure 1A). The pituitary showed the opposite trend, with 2155 DEGs outside of the PS and 623 DEGs during the PS (Figure 1B). In the hypothalamus, equal numbers of genes were seen up-regulated in LEPH and HEPH outside of the PS, though a higher number of genes were up-regulated in LEPH during the PS. In the pituitary, both outside and during the PS, a higher number of genes were up-regulated in HEPH compared to LEPH. In the hypothalamus and pituitary, under both ovulatory cycle conditions, unannotated genes accounted for roughly 20-30% of the DEGs, indicating that further progress annotating the turkey genome may reveal additional genes involved in egg production rates or in triggering ovulation.
When comparing each hen group during the ovulatory cycle, LEPH displayed twice as many DEGs in the hypothalamus and pituitary between basal and PS conditions when compared to HEPH (Figure 1C and 1D). Of the genes differentially expressed in the hypothalamus during the ovulatory cycle, unannotated genes accounted for 26% of the DEGs unique to LEPH and 47% of the DEGs unique to HEPH. Lower fractions of unannotated DEGs were seen in the pituitary during the ovulatory cycle, with unannotated genes accounting for 21% of the DEGs unique to LEPH and 27% of the DEGs unique to HEPH. In total, LEPH and HEPH shared 64 genes in the hypothalamus and 210 genes in the pituitary that were differentially expressed during the ovulatory cycle. Roughly one-fourth of the common DEGs in the hypothalamus and pituitary were unannotated as well.
Of the DEGs common to both groups of hens during the ovulatory cycle, a majority showed similar expression patterns in LEPH and HEPH (73% of common DEGs in the hypothalamus and 93% of common DEGs in the pituitary) (Figure 1E and 1F). A larger percentage of the common DEGs showed down-regulation in both groups of hens in the hypothalamus and pituitary compared to the percentage of DEGs that showed up-regulation in both groups of hens. Among the genes in the hypothalamus showing similar expression patterns during the PS in both groups of hens was fatty acid 2-hydroxylase (FA2H) and somatostatin (SST). FA2H, which was up-regulated in both groups of hens during the PS, is involved in myelin production, which is essential for proper nerve conduction [8]. SST, which was also up-regulated in both groups of hens during the PS, is the main inhibitory hormone of the somatotropic axis but has been shown to inhibit GnRH neuron activity in mice [9]. Among the genes in the pituitary showing similar expression patterns during the PS in both groups of hens was Pre-MRNA processing factor 19 (PRPF19). PRPF19, which was down-regulated in both groups of hens during the PS, has been shown in mouse models to impact the splicing of gonadotropin subunits [10]. Common DEGs with similar expression patterns during the ovulatory cycle in both LEPH and HEPH could indicate a potential role for these genes in the regulation of ovulation.
A small percentage of the common DEGs showed inverse expression patterns in LEPH and HEPH (27% of common DEGs in the hypothalamus and 7% of common DEGs in the pituitary). Of the hypothalamic common DEGs showing inverse expression patterns between LEPH and HEPH, proteasome 26S subunit, non-ATPase 2 (PSMD2) displayed up-regulation in HEPH and down-regulation in LEPH during the PS. In mice, mutations in PSMD2 have been associated with decreased thyroid hormone production [11]. Of the pituitary common DEGs showing inverse expression patterns between LEPH and HEPH, NADH dehydrogenase 4 (ND4) and cyclooxygenase-2 (COX2) have been previously associated with reproductive functions [12, 13]. Both ND4 and COX2 showed up-regulation in HEPH and down-regulation in LEPH during the PS. Swine selected for high ovulation rates displayed higher pituitary ND4 gene expression when compared to control lines [12]. COX2 encodes the rate limiting enzyme in prostaglandin production, and deletion of COX2 in mice results in decreased ovulation [13]. Common DEGs during the ovulatory cycle with inverse expression patterns in LEPH and HEPH could signify a possible role in the regulation of egg production rates.
RNA sequencing confirmation
A total of 8 genes per tissue were confirmed through RT-qPCR. Confirmation genes were equally distributed to have one of four expression profiles: genes showing up-regulation in HEPH compared to LEPH (both outside and during the PS), genes showing up-regulation in LEPH compared to HEPH (both outside and during the PS), genes showing up-regulation in one hen group outside of the PS and up-regulation in the other hen group during the PS, and genes showing no changes in expression between hen groups (both outside and during the PS). Each of the confirmation genes examined in the hypothalamus (Figure 2A) and pituitary (Figure 2B) showed expression profiles similar to those obtained through RNA sequencing.
Overview of network analysis
All DEGs between LEPH and HEPH with an absolute fold change greater than 1.5 and a P-value less than 0.05 were submitted for Ingenuity® Pathway Analysis (IPA) (RPKM>0.2). Hypothalamic transcriptome differences between LEPH and HEPH included 160 genes outside of the PS and 305 genes during the PS. Pituitary transcriptome differences between LEPH and HEPH included 1626 genes outside of the PS and 438 genes during the PS. IPA analysis of the DEGs revealed two common themes in the hypothalamus and pituitary: up-regulation of the HPG axis and estradiol signaling in HEPH and up-regulation of the HPT axis in LEPH.
The HPG axis
In the hypothalamus during the PS, LEPH displayed up-regulation of genes associated with ovulation inhibition as well as an abnormal up-regulation of ovulation stimulation genes when compared to HEPH (Figure 3A). LEPH exhibited up-regulation of neuropeptide VF precursor (NPVF), which encodes avian gonadotropin inhibitory hormone (GNIH) and of gonadotropin releasing hormone 1 (GNRH1). GnIH negatively regulates the HPG axis to decrease gonadotropin production in the pituitary [14]. Up-regulation of NPVF may play a role in reduced ovulation rates seen in LEPH. GNRH1 mRNA levels were previously shown to decrease during the PS in hens with average egg production, whereas in the present study, LEPH showed increased expression relative to HEPH [15]. In the same study, no expression changes in NPVF were seen during the PS in average egg producing hens, whereas in the present study, LEPH showed up-regulation of NPVF [15]. Up-regulation of GNRH1 during the PS may prevent hormone levels from returning to basal levels, prolonging the interval between ovulations.
When comparing HEPH outside and during the PS, HEPH showed up-regulation of estrogen related receptor beta (ESRRB) and down-regulation of follicle stimulating hormone (FSH) and LH during the PS (Figure 3B). Estrogen related receptors are ligand-dependent transcription factors capable of estradiol binding. Though the function of estrogen related receptors in avian reproduction have not been characterized, function analysis of estrogen related receptors in knock-out mice and zebrafish models indicate that estrogen related receptors are essential for female reproduction [16]. Decreased LH during the PS is consistent with decreased mRNA levels for the beta-subunit of LH (LHB) seen in average egg producing hens during the PS [7]. Additionally, in this network, casein kinase 2 alpha 2 (CSNK2A2) is down-regulated in the pituitary of HEPH during the PS. CSNK2A2 encodes an uncharacterized protein in avian species but this protein was shown to be decreased in laying geese pituitaries when compared to non-laying geese, indicating a possible role in egg production or ovulation [17].
Examination of the expression changes of DEGs related to the HPG axis revealed differential regulation of the HPG axis during the ovulatory cycle in LEPH and HEPH (Table 1). Outside of the PS, LEPH showed up-regulation of genes involved in prolactin signaling and androgen signaling. During the PS, LEPH showed up-regulation of genes involved in stimulatory and inhibitory HPG axis signaling, whereas HEPH showed up-regulation of estradiol and prolactin signaling. When LEPH and HEPH were compared individually outside and during the PS, LEPH displayed further increased expression of HPG axis inhibition and prolactin signaling (Table S1). HEPH displayed decreased expression of HPG axis stimulatory genes and increased expression of androgen and prolactin signaling. Prolactin signaling showed inverse trends in LEPH and HEPH and was impacted by the PS. Prolactin signaling has been shown to impact LH release in mammals and was up-regulated in HEPH during the PS, indicating a possible role in the shortened ovulation intervals seen in HEPH [18]. Both LEPH and HEPH showed down-regulation of gonadotropin releasing hormone receptor (GNRHR) during the PS, which was also seen in average egg producing turkey hens during the PS [7]. Generally, HEPH displayed down-regulation of the HPG axis during the PS, whereas LEPH displayed up-regulation of both genes that stimulate and inhibit the HPG axis during the PS, presumably leading to a longer ovulation interval in LEPH.
The HPT axis
DEGs up-regulated in LEPH compared to HEPH were associated with HPT axis expression in each tissue and condition examined (Figure 4, S3, and S4). In the hypothalamus during the PS, LEPH displayed increased expression of thyroid stimulating hormone receptor (TSHR) and solute carrier organic anion transporter family member 1C1 (SLCO1C1) relative to HEPH (Figure 4A). In the pituitary during the PS, LEPH displayed increased expression of the beta-subunit of thyroid stimulating hormone (TSHB) in contrast to HEPH (Figure 4B). TSHR expression in the hypothalamus is related to short loop feedback control on thyrotropin releasing hormone signaling [19]. Retrograde regulation of TSHB on the hypothalamus has also been implicated in increased GnRH production in response to a changing photoperiod in seasonally reproductive birds [20]. It is plausible that retrograde TSHB feedback on the hypothalamus could also be involved in the timing of ovulation, due to the role of TSHB in GnRH signaling initiation coupled with the finding that molecular clockwork components impact TSHB pituitary expression in several mammalian species [21]. SLCO1C1 is a thyroid hormone transporter that participates in transporting thyroid hormone across the blood-brain barrier [22]. Up-regulation of SLCO1C1 in LEPH during the PS would allow greater thyroid hormone concentrations in the hypothalamus, which could ultimately have genomic effects on ovulation rates [23].
Additionally, in the hypothalamus during the PS, LEPH showed up-regulation of solute carrier family 16 member 12 (SLC16A12) and integrin (encoded by ITGAV and ITGB3) relative to HEPH (Figure S3A and S3B). SLC16A12 encodes a thyroid hormone transporter similar to SLCO1C1, allowing greater transport of thyroid hormone past the blood brain barrier in LEPH rather than HEPH [22]. Integrin is a plasma membrane receptor capable of binding thyroid hormones to elicit non-genomic actions of thyroid hormone, such as protein translocation and phosphorylation [23]. Up-regulation of integrin in the hypothalamus of LEPH relative to HEPH during the PS, infers possible non-genomic implications of thyroid hormone in the hypothalamus of LEPH [24].
In the pituitary during the PS, HEPH showed up-regulation of iodothyronine deiodinase 1 (DIO1) relative to LEPH (Figure S4A). DIO1 is capable of converting thyroid hormone to the biologically active form but is also capable for thyroid hormone deactivation [25]. Increased thyroid hormone deactivation could mitigate the effect of thyroid hormone on the tissues of the HPG axis in HEPH. When comparing HEPH outside and during the PS, HEPH showed down-regulation of TSHB in the pituitary during the PS (Figure S4B). Thyroid stimulating hormone (TSH) acts on the thyroid gland to promote the synthesis of thyroid hormones [26]. Down-regulation of TSHB during the PS in HEPH could indicate lower circulating levels of TSH, ultimately impacting circulating thyroid hormones.
Examination of the expression changes of DEGs related to HPT axis revealed that LEPH exhibited up-regulation of a majority of the key genes of the HPT axis when compared to HEPH (Table 2). Outside and during the PS, LEPH displayed higher expression of genes related to HPT axis signaling, thyroid hormone receptors, thyroid hormone transporters, thyroid hormone metabolism, and thyroid hormone synthesis when compared to HEPH. During of the PS, LEPH displayed further increased expression thyroid related genes. When LEPH and HEPH were compared individually outside and during the PS, LEPH displayed increased expression of HPT axis genes during the PS, whereas HEPH displayed decreased expression of HPT axis genes during the PS (Table S2). HEPH during the PS showed down regulation of thyroid hormone transporters and genes involved in HPT axis signaling. Generally, LEPH displayed higher expression of HPT axis genes both outside and during the PS compared to HEPH and displayed further up-regulation of the HPT axis during the PS when compared to levels outside of the PS. HEPH, on the other hand, displayed down-regulation of the HPT axis during the PS and lowered HPT axis expression both outside and during the PS when compared to LEPH.
Upstream analysis
Analysis of the predicted upstream regulators for each comparison made also showed a common theme: the involvement of beta-estradiol. While the calculated Z-score varied for the comparisons examined, beta-estradiol was the only upstream regulator common to all of the comparisons (Figure 5). Additionally, beta-estradiol was among the top five upstream regulators in the pituitary both outside and during the PS (Table 3). The predicted involvement of beta-estradiol across all conditions examined with target genes involved in the HPG and HPT axes supports the hypothesis that beta-estradiol feedback on the hypothalamus and pituitary impacts the ovulatory process and possibly egg production rates.
For the comparisons between LEPH and HEPH, beta-estradiol was significantly more active in HEPH in the hypothalamus (z-score = 2.011) and pituitary (z-score = 2.079) outside of the PS. Differentially expressed target genes of beta-estradiol in the hypothalamus outside of the PS included thyroid releasing hormone receptor (TRHR), TSHB, transthyretin (TTR), prolactin (PRL), hydroxysteroid 17 beta dehydrogenase 2 (HSD17B2), and aromatase (CYP19A1), while differentially expressed target genes of beta-estradiol in the pituitary outside of the PS included the androgen receptor (AR), glycoprotein hormones alpha subunit (CGA), steroidogenic acute regulatory protein (STAR), and solute carrier family 7 member 5 (SLC7A5) (Table S3). Beta-estradiol tended to be more active in HEPH in the pituitary during the PS (z-score = 1.75), though not significantly.
For the comparisons during the ovulatory cycle for each hen group, in the pituitary beta-estradiol was significantly more active during the PS for LEPH (z-score = 2.014) and significantly more active outside of the PS for HEPH (z-score = -2.079). Differentially expressed target genes of beta-estradiol in the pituitary of LEPH included albumin (ALB), prolactin receptor (PRLR), STAR, and TTR, whereas differentially expressed target genes of beta-estradiol in the pituitary of HEPH included CGA and TSHB (Table S4).
Effect of thyroid hormone and estradiol on pituitary gonadotropin production
To further examine the impact of thyroid hormone and estradiol on HPG axis function, gonadotropin subunit mRNA levels were measured in pituitary cells from LEPH and HEPH after thyroid hormone pretreatment (T3) or estradiol pretreatment (E2) combined with GnRH treatment. Pituitary cells from LEPH and HEPH responded differently to each pretreatment in terms of gonadotropin subunit mRNA levels, indicating functional differences in the response of the HPG axis to thyroid hormone and estradiol that could be related to differences seen in egg production levels between the two groups of hens (Figure 6). The in vitro effects of T3 and E2 were seen both with and without GnRH treatment, indicating that both hormones could be capable of pituitary gonadotropin regulation outside and during the PS.
T3 decreased LHB, follicle stimulating hormone beta subunit (FSHB), and CGA mRNA levels compared to no pretreatment in HEPH pituitary cells, regardless of GnRH treatment concentration. T3 also decreased LHB, FSHB, and CGA mRNA levels in LEPH pituitary cells, but only at 10-9 M GnRH for LHB, 0 M and 10-9 M GnRH for FSHB, and 0 M and 10-8 M GnRH for CGA. T3 negatively impacted LHB, FSHB, and CGA mRNA levels in cells from LEPH and HEPH, however the effect was more prominent in HEPH cells. Negative regulation of LHB, FSHB, and CGA by thyroid hormone treatment was also reported in male rats [27, 28]. One possible mechanism for response differences to T3 between LEPH and HEPH is desensitization or down-regulation of thyroid hormone receptors in LEPH due to the general up-regulation of the HPT axis seen in the hypothalamus and pituitary of LEPH. Thyroid hormone receptor desensitization in the liver has been documented after thyroid hormone injections in mice and in vitro thyroid hormone treatment decreased thyroid hormone receptor expression in rat pituitary cells [29, 30]. Generally, T3 negatively regulated gonadotropin production, independent of GnRH treatment concentration, with a higher negative response from HEPH. These findings suggest that HEPH are more sensitive to the effect of T3 on gonadotropin production, whereas LEPH are more resistant to the effects of T3.
E2 decreased LHB mRNA levels in HEPH pituitary cells compared to no pretreatment at 10-8 M GnRH. E2 also decreased FSHB mRNA levels in HEPH pituitary cells relative to no pretreatment at 0 M GnRH and increased FSHB mRNA levels in LEPH pituitary cells at 10-9 M GnRH. E2 in HEPH pituitary cells decreased FSHB mRNA levels at lower GnRH treatment concentrations but decreased LHB mRNA levels at higher GnRH treatment concentrations. Previous studies in chickens have shown estradiol to inhibit pituitary LH production [31]. In contrast, E2 upregulated FSHB in pituitary cells from LEPH at 10-9 M GnRH. The effect of estradiol on FSHB mRNA levels has not been examined in avian species but estradiol injections in quail did not impact FSH plasma levels, which is consistent with the mRNA levels seen in HEPH [32]. Overall, E2 had varied impacts on gonadotropin production, depending on the rate of egg production of the hens.