Author’s address (for correspondence): Patricia A. Johnson, Cornell University, Ithaca, NY, USA. E-mail: email@example.com
Follicle development in the highly efficient laying hen is characterized by a well-organized follicular hierarchy. This is not the case in other chickens such as the broiler breeder hen that has excessive follicle development and lower reproductive efficiency. Although management practices can optimize egg production in less productive breeds of chickens, the factors that contribute to this difference are not known. Interactions between the oocyte and surrounding somatic cells are believed to be involved in promoting follicle selection. Anti-Müllerian hormone (AMH) has been shown to have a role in regulating rate of follicle development in mammals. In hens, the expression of AMH is restricted to the growing population of follicles and, similar to mammals, is markedly decreased at around the time of follicle selection. The oocyte factors, growth and differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), have been identified in the hen, and their expression pattern has been characterized. Anti-Müllerian hormone expression in hens is decreased by a protein factor from the oocyte (not GDF9) and is also decreased by vitamin D. Associated with the decrease in AMH expression by vitamin D, follicle-stimulating hormone receptor mRNA is increased. These data suggest that information about AMH regulation may enhance our understanding of follicle selection, particularly in birds with aberrant follicle development.
Follicle selection is a widespread phenomenon in vertebrates and may result in the selection of several follicles from a cohort or may result in the selection of only one dominant follicle per ovulatory cycle. Examples of the first type are litter-bearing species such as rodents and pigs, while cattle and hens are typical of species in which only a single dominant follicle is usually selected. Growing follicles that are not selected to complete follicle development most often undergo atresia. The situation in the domestic hen is a bit different from other monovular species in that the hen ovary contains a hierarchy of growing follicles that progress gradually towards maturity (Fig. 1). In addition, in hens there is little atresia associated with the follicles as they advance towards ovulation, even though each follicle may be a number of ovulatory cycles away from ovulation. This is likely related to the energetically demanding process of yolk accumulation and the time required for growth to pre-ovulatory size. In spite of this difference between mammals and birds, there are many characteristics of maturing hen follicles that are similar to those occurring in mammals and that are also associated with development of ovulatory competence.
Optimal reproductive efficiency in domestic hens is achieved when follicle development proceeds through a well-distinguished follicular hierarchy. Generally, large pre-ovulatory follicles are selected from the pool of small growing follicles. These growing follicles are categorized by size (as 3–5 or 6–8 mm) or according to colour (as large white follicles or small yellow follicles). Follicle-stimulating hormone (FSH) appears to facilitate follicle selection and pregnant mare serum gonadotropin PMSG- or FSH-treatment can increase the number of follicles developing (Palmer and Bahr 1992). The granulosa layer of developing follicles expresses follicle-stimulating hormone receptors (FSHR) and the abundance of mRNA for these receptors changes with follicular maturation. Follicle-stimulating hormone receptors mRNA was shown to be highest in the granulosa layer of 6–8 and 9–12 mm follicles (You et al. 1996). In fact, one follicle in the pool of 6–8 mm follicles contains significantly more mRNA for FSHR than the other follicles in this size category, suggesting that this may be the follicle next selected to move into the larger group (Woods and Johnson 2005). As follicles progress through the hierarchy, the granulosa layer becomes capable of producing an increasing amount of progesterone (Robinson and Etches 1986), which eventually results in the luteinizing hormone surge and initiates ovulation.
Although this very orderly pattern of follicle development is characteristic of laying strains of hens, follicle development in commercial strains of broiler chickens is not so naturally organized. In broiler strains of hens permitted to feed ad libitum, follicle development is excessive and disorganized. As a result, ovulations are irregular or often multiple, resulting in fewer high-quality eggs that can be set. The tendency for ovarian overgrowth can be managed by limiting feed intake. Under these conditions, a distinct hierarchy can be maintained and egg production maximized. This underscores the importance of a well-organized follicular hierarchy for maintenance of high reproductive efficiency in hens. Some of the signalling pathways correlated with follicle selection in the domestic hen have been elucidated (Calvo and Bahr 1983; Woods and Johnson 2005), but the complete process is not yet understood. The vast majority of work has been with cells from the granulosa layer, in large part because pure preparations of these cells can be quite easily isolated and cultured. The theca layer has also been examined, but this is somewhat more difficult to study because there are multiple cell types in the layer. Recently, investigators have begun to study the role of the oocyte in follicle maturation and to consider that the oocyte is not simply a passive participant in development. This is particularly apparent in oviparous species because yolk accumulation and oocyte growth must be coordinated with steroidogenic competence and maturation of the somatic cell layers. The functions of the follicular cells cannot be viewed in isolation because the interactions between the oocyte and somatic cells together culminate in the production of a mature oocyte.
Oocyte growth is the most obvious characteristic of follicular development in oviparous species like birds, while accumulation of follicular fluid external to the oocyte characterizes mammalian follicular development. Growth of the chicken oocyte is due in large part to the accumulation of yolk, and this has been shown to be due to a receptor-mediated process (Stifani et al. 1990; Schneider 2009). The yolk proteins are synthesized in the liver through the action of oestrogen, and these proteins are taken into the oocyte by a specific lipoprotein receptor with 8 ligand binding domains (LR8; Stifani et al. 1990). The yolk proteins are delivered to the oocyte through the vascularized theca layer, traverse the basement membrane and then pass between the granulosa cells to access the oocyte (Schuster et al. 2004). Hens that are natural mutants for the LR8 receptor (termed ‘restricted ovulator hens’; Nimpf et al. 1989) are unable to transport adequate amounts of yolk into the growing oocyte and consequently ovulate few or no eggs (Elkin et al. 2006). This model was particularly useful in elucidating the critical nature of the oocyte-specific yolk receptor.
Yolk particles have been observed passing between the granulosa cells, implying another regulatory step in oocyte growth (Shen et al. 1993). In fact, Schuster et al. (2004) found that the protein occludin, which contributes to tight junctions between granulosa cells, was altered during follicle development. They showed that occludin was expressed at the apical region of granulosa cells in small white follicles, although no distinct localization of occludin was noted in larger follicles (Schuster et al. 2004). They also showed that occludin protein was increased in cultured granulosa cells from the F1 (largest) follicle by treatment with FSH or activin (Schuster et al. 2004). The authors had previously demonstrated by Western blot that occludin was not detectable in granulosa cells from the F1 follicle (Schuster et al. 2004) and granulosa cells from these follicles are not particularly FSH responsive (You et al. 1996). These results are somewhat difficult to reconcile with the increase in FSH responsiveness (Woods and Johnson 2005) and large increase in yolk accumulation associated with follicle selection in smaller follicles. If occludin is important in restricting yolk uptake, it would not be expected that FSH increases expression of this protein. Additional studies are needed on the hormonal regulation of yolk accumulation by the growing oocyte and the coordination with other events during follicle development.
In our recent studies of follicle development in the hen, we have been interested in the role of AMH because of its function to limit follicular progression in mice. Studies using mice null for the AMH gene revealed that follicular development was enhanced and the ovary became more rapidly depleted of oocytes in the absence of AMH (Durlinger et al. 1999). Additionally, AMH was shown to decrease FSH sensitivity and thereby, perhaps, regulate follicle selection (Durlinger et al. 2001). The well-organized avian hierarchy suggested that AMH might have a role in follicular progression in the hen. Anti-Müllerian hormone had been previously studied in birds in association with its known role in regressing the Müllerian duct in males (Tran and Josso 1977) and the right oviduct in females (Teng 1987; Teng et al. 1987). Similar to mammals, AMH is expressed predominantly in the granulosa layer of the ovary of mature female hens (Johnson et al. 2008). Quantitative PCR results showed that AMH mRNA is the highest in small follicles, prior to selection into the pre-ovulatory hierarchy. Studies in mammals have indicated that AMH expression is primarily restricted to the interval between primary and antral staged follicles (Visser and Themmen 2005). Our studies (incorporating both immunohistochemistry and quantitative PCR) suggested that follicles larger than approximately 50 μm and smaller than 6 mm showed the most AMH expression (Fig. 2), supporting the idea that AMH function was restricted to particular stages of follicle development in hens. Although mammalian work had indicated increased AMH expression in cumulus granulosa cells as compared to the mural granulosa cells (Baarends et al. 1995), we did not find a difference in AMH mRNA expression between granulosa cells adjacent to the germinal disc and those distal to the germinal disc (Johnson et al. 2008). The difference in proximity of granulosa cells to the oocyte in mammalian as compared to avian follicles may explain the difference in regional AMH expression.
Our studies with AMH led us to hypothesize that aberrant AMH expression was somehow involved in the excessive follicle selection in broiler strains of hens as compared to laying strains. In mice, inactivation of the AMH gene resulted in accelerated follicle development (Durlinger et al. 1999), implicating AMH in the regulation of follicle recruitment and development. We hypothesized that increased follicle selection might be associated with a lower level of AMH in broiler breeder hens. Interestingly, we found just the opposite in that broiler breeder hens showed an increased expression of AMH mRNA (Johnson et al. 2009) in the granulosa layer of growing follicles as compared to expression in the granulosa layer from laying hens. Commercial strains of broiler breeder hens are routinely feed-restricted to enhance follicle development. This practice improves egg production, but follicle development is still not as precise as that in laying hens. Broiler breeder hens that were fed a restricted diet (87% of ad libitum) had lower expression of AMH compared to broiler breeder hens fed ad libitum (Johnson et al. 2009). The increased expression of AMH associated with excessive growth of follicles in hens resembles polycystic ovary syndrome (PCOS) in women, which is also associated with increased serum AMH (Cook et al. 2002). Ovaries in women with PCOS contain many large antral follicles although ovulation is rare in these women with resulting infertility (Visser et al. 2006). In the full-fed broiler breeder hens, ovulation does occur, although often there are multiple ovulations, with the result of poor egg production (Yu et al. 1992).
As noted earlier, AMH reduces the rate of follicle development and reduces FSH sensitivity in rodents (Durlinger et al. 1999, 2001). Previous studies have indicated that mammalian AMH is not biologically active in birds, although avian AMH has demonstrated bioactivity in a mammalian system (Tran and Josso 1977; di Clemente et al. 1992). In the absence of purified avian AMH, we have utilized medium conditioned by embryonic chick testes (Teng et al. 1987) as a source of biologically active chicken AMH. We called this testis-conditioned medium (TCM) and demonstrated by Western blot that it contains immunologically active AMH. Furthermore, the TCM stimulated a dose-related increase in granulosa cell proliferation, which could be inhibited by pre-incubation of the TCM with AMH antibody (Johnson et al. 2009). We also assessed FSHR expression in granulosa cells in response to TCM and found that it decreased FSHR expression, in a dose-related way. This response, however, could not be blocked by pre-incubation of the TCM with AMH antibody (unpublished results). These results further demonstrate the difficulties of working with a crude hormone preparation, and we are currently working on producing chicken AMH by recombinant means. Our data may indicate that the AMH signal must be reduced to promote FSH sensitivity in developing follicles, but that reduction of AMH alone is not sufficient.
There is limited information available on the regulation of AMH in female mammals (Salmon et al. 2004, 2005; Taieb et al. 2011). We have studied the regulation of AMH in granulosa cells of the hen. We cultured hen granulosa cells from 6 to 8 mm follicles with estradiol and progesterone, with both hormones tested at a variety of doses, and found no effect on AMH mRNA expression (Johnson et al. 2008). The expression of AMH mRNA in granulosa cells cultured in a range of doses of epidermal growth factor (EGF) and insulin-like growth factor I was also examined, and no effects were observed (unpublished results). The findings from broiler breeder hens (Johnson et al. 2009) suggested that dietary factors may influence expression of AMH, because full-fed hens had higher levels of AMH mRNA compared to restricted-fed hens. As a result, we cultured granulosa cells with a range of doses of insulin and glucose and found no effect on AMH mRNA expression. Studies in a human prostate cancer cell line had suggested that vitamin D regulates AMH (Krishnan et al. 2007) and these investigators identified a functional vitamin D-response element in the human AMH promoter (Malloy et al. 2009). Vitamin D caused a decrease in the expression of AMH mRNA in granulosa cells of the hen from 3- to 5-mm and 6- to 8-mm follicles. Interestingly, FSHR was increased in these same samples at the higher dose of vitamin D utilized, and cell proliferation was increased at both dosages (Wojtusik and Johnson 2012). In these experiments, the decrease in AMH expression occurred at a lower dose in the granulosa cells from 6- to 8-mm follicles as compared to the increase in FSHR after treatment with vitamin D. This could indicate that vitamin D directly affects AMH expression and has a secondary effect on FSHR.
Studies in mice had indicated the importance of oocyte-derived factors in folliculogenesis (Eppig et al. 2002). Specifically, GDF9 and BMP15 have been shown to play critical roles in follicle development and ovulation (Dong et al. 1996; Aaltonen et al. 1999). Growth and differentiation factor 9 has been identified in the ovary of hens (Johnson et al. 2005), and the full-length transcript was found to be approximately 65% similar to mammalian cDNA sequences. Immunohistochemical analysis showed that GDF9 was primarily located in the oocyte (Fig. 3). Purified chicken GDF9 was not available, and therefore, we utilized oocyte-conditioned medium (OCM; media collected after 2–3 day culture of approximately 1 mm follicles) to examine GDF9 function. Oocyte-conditioned medium was collected and shown (by Western blot) to contain GDF9 (Johnson et al. 2005). Culture of granulosa cells with this OCM caused a significant dose-related increase in granulosa cell proliferation (Johnson et al. 2005), which could be inhibited by pre-incubation of the conditioned medium with GDF9 antiserum. This same medium was used to examine a potential effect of the oocyte on AMH expression. Previous studies (Salmon et al. 2004, 2005) had suggested that the oocyte was involved in AMH expression in mice. Oocyte-conditioned medium caused a dose-related decrease in AMH mRNA expression. Although the effect was not blocked by GDF9 antiserum, it was blocked by prior heat treatment of the OCM (Johnson et al. 2008). From these experiments, we concluded that the bioactive factor in the OCM responsible for the effect on AMH was likely a protein factor but not GDF9.
Another factor that is thought to be oocyte related is BMP15. Alteration in function of BMP15 has dramatic effects on mammalian fertility (Galloway et al. 2000), although the effects of this factor are believed to be quite species-specific (Hashimoto et al. 2005). Bone morphogenetic protein 15 in the hen has been shown to be primarily localized to the ovary by PCR analysis, and in situ hybridization has indicated that it is present in the oocyte (Elis et al. 2007). Recombinant human bone morphogenetic protein 15 (hBMP15) decreased progesterone production from granulosa cells removed from the F1 follicle, and Smad1, a downstream mediator of BMP15, was phosphorylated in cultured granulosa cells in response to BMP15, suggesting bioactivity of the human hormone (Elis et al. 2007). We have also observed biological effects of hBMP15 and found that BMP15 decreased proliferation and increased the expression of FSHR in cultured granulosa cells from 3 to 5 and 6 to 8 mm follicles (unpublished results). In spite of these effects, there was no effect of BMP15 (at a range of doses) on AMH expression.
Although vitamin D and a protein factor in OCM were found to decrease the expression of AMH, we have not yet found a substance that acts in a regulatory way to increase mRNA for AMH. This is interesting because AMH in the hen is maximally expressed in growing follicles up to approximately 6–8 mm and then dramatically reduced around the time of follicle selection. This suggests an important role for AMH in regulating the pre-selection pool. Understanding the regulatory factors for AMH may give important clues about follicle selection.
Another system that has been implicated in the regulatory loop between the oocyte and surrounding somatic cells in the hen is the c-Kit/Kit ligand system. It is believed that granulosa-derived kit ligand (KL) binds to the membrane-anchored c-Kit receptor on the oocyte. Studies in mammals have indicated that signalling between KL and c-Kit is important in follicular activation and/or follicular survival (Reddy et al. 2005; John et al. 2009). A previous study on hen ovarian tissue showed that heparin-binding EGF-like growth factor (HB-EGF) decreased mRNA expression of granulosa cell KL in a dose-related manner (Wang et al. 2007). We studied the expression of the mRNA for KL in the granulosa layer of different sized follicles. We found that there was a decreased expression of KL with increasing follicle size. Kit ligand was expressed at significantly higher levels in the granulosa layer of 3-mm follicles as compared to expression in the granulosa layer of 6- to 12-mm follicles (unpublished results). Western blot analysis showed that c-Kit is not expressed in the granulosa cells but is present in the theca layer and in protein extract from the whole ovary (unpublished results). These findings support the mammalian model suggesting that granulosa-derived KL interacts with c-Kit on the oocyte and theca layer.
In conclusion, we are working to characterize the factors involved in follicle selection and responsible for the well-defined hierarchy in strains of laying hens. Although hampered by a paucity of homologous reagents, progress is being made in defining some of the interactions between the oocyte and somatic cells ultimately leading to follicular maturation. This knowledge may be important in understanding the aberrant patterns of follicle development common in broiler breeder strains of chickens and other species.
I am grateful to Dr Jim Giles, Ms Jessye Wojtusik, Ms Mila Kundu, Dr Lindsey Trevino and Dr Mary Ellen Urick for their contributions to this work. This work was supported by National Research Initiative competitive grant 2008-35203-19097 from the U.S. Department of Agriculture National Institute of Food and Agriculture.
Conflicts of interest
The author has no conflicts of interest to declare.