Identification of novel candidate genes for follicle selection in the broiler breeder ovary

Sexually mature hens are capable of ovulating almost once per day for the duration of their period of lay. However, although modern breeding programmes consider many more traits[1], in early breeding programmes, commercial broiler breeders that were selected primarily for rapid growth and high meat yield produced fewer eggs[2, 3]. The result of that selection pressure is that when fed ad libitum, broiler breeders produce multiple ovulations due to the development of a numerically large ovarian hierarchy[4]. While many ova from multiple ovulations are lost into the body cavity, some result in the production of double- or multiple-yolked eggs, or eggs with defective shells[3]. Additionally ad-libitum fed broiler breeders will become very heavy and develop various weight-related problems. Consequently, although juvenile broiler breeders are fed ad libitum, they are required to be limited to at least 40% of their natural nutritional intake as they reach sexual maturity to alleviate these problems[5]. However, this leaves the birds in a permanently hungry state. Feed restriction does however reduce follicle numbers in the ovarian hierarchy to a normal level[5], and consequently decreases the chance of multiple ovulation. Despite the benefits to the health of the birds, the degree of hunger experienced by the birds has raised concerns in some quarters as to the resulting impact on the birds’ welfare[3, 6, 7].

The mechanism by which feed restriction regulates follicle number has not been completely elucidated, though it is known to influence Gonadotrophin Releasing Hormone (GnRH) secretion within the hypothalamo-pituitary-gonadal (HPG) axis[8, 9]. It is likely that food restriction exploits a natural mechanism. In wild birds, clutch size is affected by availability of food and the energy required to obtain it[10]. It is clear that food intake and related growth can influence follicular selection in the ovary of birds and the result of selection for these traits has affected the regulatory mechanisms that govern follicle selection within the ovary[11, 12]. Modifying existing breeding programmes to select against multiple ovulation in commercial flocks is a potential route for reducing the requirement for feed restriction of broiler breeders.

Despite obvious differences in post-ovulatory mechanisms and specific hormone role reversals, the chicken ovulatory cycle is generally comparable with that of mammalian species[13–15]. The follicle is comprised of a series of membranes and highly vascularised cell layers that surround, support, and protect the oocyte[16]. Follicles within the avian ovary are ordered progressively by size from many follicles <1 mm in diameter to the pre-ovulatory or F1 follicle at around 40 mm[17]. Follicles can be grouped into 2 basic types, white follicles, which range from 1–7 mm in diameter, and yellow follicles, from approximately 8–40 mm, which are distinguishable by the deposition of yolk, that gives them their colour, and the increased vascularisation needed to support their growth[17]. Most follicles will not grow sufficiently to undergo ovulation and the majority <8 mm will become atretic. Normally under appropriate endocrine conditions following ovulation a follicle from the 6–8 mm pool is recruited into the pre-ovulatory hierarchy[18, 19]. Once recruited and unless gonadotrophin support is removed these follicles are highly likely to mature and ovulate[17]. In pre-hierarchical development, Follicle Stimulating Hormone Receptor (FSHR) is the predominant gonadotrophin receptor[20]. However, once follicles are drafted into the hierarchy, this predominance shifts towards the Luteinising Hormone Receptor (LHR)[20].

Atresia in mammals predominantly occurs immediately prior to follicle recruitment[14, 15, 21, 22]. Studies have shown that this occurs even in the absence of the pituitary-suggesting that regulation of pre-recruitment development is intra-ovarian once it has been initiated[14, 15]. Pre-hierarchical development would therefore constitute the ‘normal’ lifespan of follicles, with progression to the hierarchy and subsequent ovulation being triggered externally[14, 15, 23].

External regulation of the avian ovary is managed through the HPG axis, with input signals being generated in the hypothalamus and relayed to the pituitary where hormone secretion is initiated[24]. The hypothalamus co-ordinates reproductive development and activity in response both to physiological and environmental signals, such as ovarian steroids[24] and photoperiod[25]. GnRH is released in pulses into the pituitary and the frequency and strength of these pulses modulate reproductive development[24] by inducing the production and release of LH and FSH from the pituitary[17, 20, 26–30]. While gonadotrophin signalling leads to cell growth and proliferation, as well as intrafollicular steroidogenesis and ovulation[13, 17, 29, 31–33], mapping of the downstream pathways is less complete. Due to difficulties in isolating FSH from LH, avian FSH has proved challenging to study, and is not as well understood as the mechanisms involved in LH regulation[24]. Work has been carried out to investigate the roles of Bone Morphogenetic Proteins (BMPs) and Transforming Growth Factor B (TGFB) superfamily members in intrafollicular signalling, both in early and post-recruitment development[17] but less so in the intervening stages, notably the period of follicle selection and recruitment to the hierarchy.

The aim of this study was to investigate intraovarian regulation of follicle number in chickens by identifying gene candidates which have a potential role in either intrafollicular signalling or feedback mechanisms that affect the HPG axis. Ultimately this information will increase our understanding of the mechanisms by which dysfunction in broiler breeder hen ovaries occurs and may lead to genetic or alternative strategies to reduce dependence on food restriction.

This research was based on the premise that, as broilers and layers have been genetically selected for different traits, the fact that adult broilers exhibit contrasting ovarian phenotypes under non-restricted conditions from layers is a result of genetic selection and there is ample evidence that this is the case[5]. Consequently, the effect of this genetic selection on expression of genes suspected of being important for reproductive development was undertaken, as regulation of their expression is likely to be the cause, directly or indirectly, of the phenotypic differences observed in broilers and layers. These are obvious targets for research to explain these differences but are also of fundamental interest in reproductive biology in a wider context. The study therefore used results from a range of experimental and analytical approaches to identify candidate genes for regulation of follicle selection and recruitment in the broiler breeder ovary. Two different approaches were used in complementary fashion to analyse the large dataset produced by the microarray. BioLayout Express, the more novel component of our analytical approach, has many built-in features that complement its use as a basic analytical tool. The most fundamental is the ability to visualise the data as a 3D network of datapoints that can be subsequently analysed by the internal clustering algorithm. The results can also be filtered by any available annotation or expression profile across samples to facilitate identification of possible associations between them. This makes it much easier to ask different questions of a single dataset and was an essential asset in switching focus between developmental stages and effect of nutrition. Using these different approaches to analyse the microarray data, as well as using information from different patterns of follicular development a number of genes that show changes in expression that would be consistent with a role in follicle selection have been identified. Three of these candidates have also been shown to exhibit altered expression levels as a result of ad libitum feeding.

From the 37 prioritised candidates considered for multi-level QPCR profiling, 10 candidates (including FSHR and TGFBR1 for validation of the approach) had sufficient experimental and/or literature-sourced evidence for basic hypothesis generation as to their role in follicle selection and recruitment. All of these candidates are associated with regulation of apoptosis[43–48], cell growth[20, 49, 50] and survival[51] or steroidogenesis[52] where there is any documented function. While these processes are all prerequisite to follicle survival, there is insufficient information at present to create a single model system incorporating all of our candidates, although FSHR, TGFBR1 and SMAD3, with the added possibility of MOSPD1, do interact with common signalling pathways. However, PDGFRL is by far the most intriguing novel candidate.

PDGFRL produces a product which is homologous with the functional domains of Platelet-derived Growth Factor Receptors that are involved in intrafollicular cell signalling associated with steroidogenesis in mice[52]. QPCR profiling in layers clearly shows significant (P <0.001) and substantial peaks in expression at the 5–6 mm and F2 stages, i.e. immediately prior to selection and ovulation respectively. This evidence would support a function in regulatory feedback mechanisms. The BioLayout Express profile from the broiler breeder microarray data suggests that the gene is downregulated in 5–7 mm follicles relative to the stroma and F1. This is consistent with the broiler breeder QPCR expression pattern for feed restricted birds. In contrast, the expression levels across tissues in ad libitum fed birds remain relatively constant. In ad libitum fed birds, where hierarchical follicle number is increased, the upregulation of PDGFRL expression, relative to feed restricted birds, suggests that it is likely to be in activation or upregulation of positive feedback signalling to the HPG axis. The observed downregulation in 5–7 mm follicles from broiler breeders is in marked contrast to the layers, which would support the hypothesis of potential dysregulation of part of the steroid-based feedback mechanisms, given what is already known of the PDGFR family in other species.

The QPCR results from experiment 2 show that FSHR and TGFBR1, genes known to be involved in follicle growth, peak in their expression during early prehierarchical development. This agrees with previous results[20, 49]. Interestingly, both FSHR and TGFBR1 show a prominent peak at 8–10 mm, indicating that follicles immediately post-recruitment may have a heightened sensitivity to the ligands of these receptors at this stage. As both receptors activate pathways leading to cell growth, proliferation and differentiation, higher expression at those stages is not surprising. SMAD3, a known downstream signal mediator of TGFB family receptors demonstrates a very similar pattern of expression to TGFBR1. However, TGFBR1 and SMAD 3 did not show a significant effect in response to ad libitum feeding, whereas FSHR was significantly downregulated in the F1 follicle under ad libitum feeding. It is most likely that, as the lower FSHR expression shown in ad libitum fed broilers is more comparable with previous studies[53], the increased expression in feed restricted birds leads to negative feedback resulting from steroidogenic factors. Little functional significance has been placed on FSH control in the F1 and further investigation is warranted to explore the potential roles for FSHR in this follicle in light of the results reported here. MOSPD1 has been implicated in mesenchymal cell differentiation[50] and is upregulated in ovarian cancer[54]. BioLayout Express analysis showed it clustered with FSHR in broiler breeders and QPCR profiling in layers corroborated this. MOSPD1 is a membrane-associated protein[55] and may be involved in supporting or mediating signal transduction from the FSH receptor but further work will be required to determine this.

GDF9 was not identified through the original microarray analysis but was included due to its location near the putative QTL for follicle number in the chicken and because studies in sheep report an association between mutations in GDF9 and increased ovarian follicle number and ovulation rate[56, 57]. Despite showing little change in expression between tissues in the initial screen in layers, GDF9 does show significant downregulation in response to ad libitum feeding in broiler breeders in the stroma and in 6–8 mm follicles (P = 0.005). In conjunction with results from mamalian studies this result would imply an inhibitory effect on follicle number. It is interesting to note that the expression profile for GDF9 in broiler breeders, regardless of diet, is comparable with other species[58–61], indicating a high level of inter-species conservation for this gene. Reported inter-species sequence conservation from the UCSC Chicken Genome Browser supports this, with sheep being most comparable in terms of exon coverage. Further investigation is underway to determine if there are mutations in the chicken, as there are in sheep, that might be associated with multiple ovulation.

SLIT3 and VDAC1 have both been shown to be involved in pro-apoptotic signalling[43–46] and are located in the putative QTL for follicle number on chromosome 13. SLIT3 is also involved in ovary and follicle development in sheep[62] and its expression profile in layers is consistent with phases of increased apoptosis. Expression profiling of SLIT3 and VDAC1 in BioLayout Express for broiler breeders is consistent with the layer QPCR profiling. However, there was no significant differential expression either between tissues or dietary regimes in the broiler breeder QPCR validation for either candidate.

YAP1 is believed to be involved in cell survival signalling through regulation of the p53 signalling pathway[51]. The BioLayout Express profile of the broiler breeders suggests that it is upregulated in late prehierarchical follicles. However, profiling in layers shows higher expression in early prehierarchical follicles and the F5 follicle, where pro-survival signals would be expected to occur more prominently. Upregulation in ad libitum fed birds could not be validated by QPCR. This does not negate a role for YAP1 in follicular development, however it is unlikely to be responsible for multiple ovulation in broiler breeders.

GULP1 is expressed in macrophages and is involved in engulfment of apoptotic cells[47, 48]. Profiling in layers is consistent with this activity. While this is not likely to be a candidate for follicle selection, it does highlight the transitional stages of the follicle as it progresses through development.

RIGG1740, KRT75 and SPTY2D1 were also investigated for a potential role in follicle development, however their expression profiles in layers, in conjunction with the level of available evidence (summarised in Table5) did not indicate a central role in follicle recruitment.