Growth hormone production and role in the reproductive system of female chicken.

General and Comparative Endocrinology xxx (2015) xxx–xxx

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Growth hormone production and role in the reproductive system of female chicken Anna Hrabia ⇑ Department of Animal Physiology and Endocrinology, University of Agriculture in Krakow, Al. Mickiewicza 24/28, 30-059 Krakow, Poland

a r t i c l e

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Article history: Received 27 September 2014 Revised 2 December 2014 Accepted 3 December 2014 Available online xxxx Keywords: GH Development Function Ovary Oviduct Chicken

a b s t r a c t The expression and role of growth hormone (GH) in the reproductive system of mammals is rather well established. In birds the limited information thus far available suggests that GH is an endocrine or paracrine/autocrine regulator of ovarian and oviductal functions too. GH and its receptors are expressed in all compartments of the ovary and oviduct and change accordingly to physiological state. The intraovarian role of GH likely includes the regulation of steroidogenesis, cell proliferation and apoptosis, the modulation of LH action and the synthesis of IGFs (insulin-like growth factors). In the oviduct, GH is also involved in the regulation of oviduct-specific protein expression. The present study provides a review of current knowledge on the presence and action of GH in the female reproduction, in which it is likely that act in endocrine, autocrine or paracrine mechanisms. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Growing evidence demonstrates that growth hormone (GH) is produced not only by pituitary gland but also by numerous extrapituitary tissues including neural (Arámburo et al., 2014), immunological (Luna et al., 2008, 2013) and reproductive ones (Harvey, 2010; Luna et al., 2014; Sirotkin, 2005) in which it can act through paracrine and/or autocrine pathways. In this brief review, based on available literature and the author’s own studies, up-to-date discoveries concerning the expression of GH and its receptors in the female reproductive system, mainly of chickens are presented. The possible participation of GH in the local regulation of the avian ovary and oviduct is also discussed. 2. Physiological evidence of growth hormone-controlled avian reproduction It is well established that pituitary GH is involved in a wide array of reproductive functions in mammals such as sexual differentiation, pubertal maturation, gonadal steroidogenesis, gametogenesis and ovulation as well as pregnancy and lactation (Codner and Cassorla, 2002; Harvey, 2010; Hull and Harvey, 2000, 2001; Sirotkin, 2005). In contrast much less is known about participation of GH in the regulation of reproductive processes in ⇑ Fax: +48 12 662 41 07.

female birds. Involvement of pituitary GH is, however, strongly suggested by the correlated elevation in blood plasma GH concentration in pullets at the onset of lay (Williams et al., 1986) and around the time of oviposition and ovulation in hens (Harvey et al., 1979). Attenuation of GH secretion in chickens, turkey and bantam hens with the cessation of egg laying is also observed (Bedrak et al., 1981; Scanes et al., 1979; Sharp et al., 1979). Moreover, a role for pituitary GH in the function of the ovary is also supported by an increase in the number of small ovarian follicles after GH administration to laying hens (Williams et al., 1992). Similarly, injections of recombinant chicken GH (cGH) to chickens prior to the onset of egg laying resulted in increased ovarian weight (13.5 ± 6.14 g vs 0.89 ± 0.14 g or 7.70 ± 4.17 g vs 1.10 ± 0.12 g, dependently on experiment) about one week before sexual maturity (Hrabia et al., 2011). Moreover, yellow hierarchical follicles were present in the ovaries of most GH treated hens but not in the control hens (Fig. 1), suggesting the involvement of GH in the selection of small prehierarchical follicles into hierarchy of yellow preovulatory follicles (Hrabia et al., 2011). In other experiments Donoghue et al. (1990) found that GH injections for 3 weeks to older egg laying White Leghorn hens did not affect egg production, but significantly improved eggshell quality. Within the follicle it is also possible that GH is involved in yolk deposition as in the synthesis of yolk proteins in the liver, which are primarily under control of ovarian estrogens (Stevens, 1996), but the liver is also GH responsive organ (Van As et al., 2001). In author’s experiment (unpublished data) cGH administration into

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Please cite this article in press as: Hrabia, A. Growth hormone production and role in the reproductive system of female chicken. Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2014.12.022

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A. Hrabia / General and Comparative Endocrinology xxx (2015) xxx–xxx

the chicken during maturation did not change vitellogenin II and apoVLDL mRNAs expression in the liver as examined about one week before and at the time of maturity. Concomitantly, expression of estrogen receptor alpha mRNA was slightly, but not significantly elevated, and expression of estrogen receptor beta was significantly increased just after reaching maturity. Thus it seems possible that GH modulates estrogen-stimulated vitellogenesis in chickens as it was demonstrated in hypophysectomised pigeons (Harvey et al., 1978) and turtles of both sexes (Ho et al., 1982). Moreover, in in vitro culture of female eel hepatocytes, the time- and dose-dependent potentiating effect of GH on vitellogenin synthesis induced by estradiol was demonstrated, although, GH alone had no effect on vitellogenin synthesis or secretion (Peyon et al., 1996). On the other hand, in in vitro experiment in the frog Rana esculenta, the homologous pituitary homogenate stimulated vitellogenin synthesis in male and female livers, and no cooperation between pituitary homogenate and estradiol was found in the vitellogenin synthesis response (Carnevali and Mosconi, 1992).

3. Expression and localization of growth hormone in the ovary

Fig. 1. Effect of cGH administration on morphology of the chicken ovary. cGH (recombinant cGH prepared as described by Paczoska-Eliasiewicz et al. (2006) and purchased from Protein Laboratories Rehovot Ltd (Rehovot, Israel)) was injected three times a week at a dose of 200 lg per kg of body weight from 10 weeks of age. Hens were decapitated at 10 (a and f), 12 (b and g), 14 (c and h) and 16 (d and i) weeks of age and at 17 or 18 weeks (e and j) i.e., just after reaching maturity as evidenced by the first oviposition, and the ovary was weighed, photographed and characterized morphologically. It is of note, that at the age of 16 weeks, in the cGHtreated chickens, large yellow follicles (>8 mm diameter) were present but not in the control group. The mean number of these follicles was 0.8 ± 0.54, 1.0 ± 0.63, 1.8 ± 0.98 and 0.2 ± 0.17, respectively for follicles in diameter 8–12 mm, 12–18 mm, 18–24 mm and 24–30 mm. In other experiment it was 1.7 ± 0.47, 0.8 ± 0.48 and 0.8 ± 0.54, respectively for follicles 8–14 mm, 14–22 mm and 22–30 mm. For more detail see previous publication (Hrabia et al., 2011). Bar = 1 cm.

In addition to pituitary GH, numerous studies have suggested autocrine and/or paracrine actions of GH in reproductive functions, since the mammalian reproductive system has been shown to be an extrapituitary site of GH expression (Abir et al., 2007; Harvey, 2010; Izadyar et al., 1999; Schwarzler et al., 1997). Recent studies have also shown that the chicken ovary expresses the GH gene (Ahumada-Solórzano et al., 2012; Hrabia et al., 2008). Using RT-PCR, a full-length (690 bp) pituitary GH cDNA was, for the first time, detected in chicken ovarian stroma prior to and after the onset of lay, although GH expression was far less than that in the pituitary gland or hypothalamus. GH mRNA was also demonstrated in all classes of ovarian follicles (white, yellowish and yellow) after their ontogenetic appearance in the developing ovary. GH-immunoreactivity was similarly present in the tissues of the ovary and within the follicular wall, and it was more intense in the granulosa than in theca layer, and comparable to that in the epithelium with loose connective tissue. Development of the yellow preovulatory follicles was accompanied by increasing GH immunoreactivity in the granulosa layer, and by a significant reduction of GH staining in the theca layer (Hrabia et al., 2008). Furthermore, GH mRNA identical in nucleotide sequence to that in the pituitary gland and GH protein were detected in the ovarian compartments of laying hen in which stronger expression was in the granulosa cells and lesser in the theca cells (AhumadaSolórzano et al., 2012). Moreover, by Western blotting, nine moieties of GH proteins were detected in the follicular wall whose relative proportions changed with follicular development. The most abundant variant at all developmental stages of the follicles showed a molecular weight of 17 kDa. It has been proposed that difference in abundance of GH isoforms during development may provide a mechanism to modulate GH activity in the female reproductive system (Ahumada-Solórzano et al., 2012). Additionally, secretion of GH by the granulosa cells of the largest preovulatory ovarian follicles was also revealed (AhumadaSolórzano et al., 2012). Taken together, these results demonstrate that the ovary of the hen is thus an extrapituitary site of GH gene expression. Folliculogenesis, yolk deposition and ovulation may thus be dependent upon local GH production. The physiological importance of particular isoforms of GH protein in the avian ovary remains unknown.



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4. Expression and localization of growth hormone receptors in the ovary GH exerts pleiotropic functions in all vertebrates. The variety of GH actions are mostly as result of its direct effect on GH receptors (GHR) localized in the target cells which are single membranespanning glycoproteins that belong to the class I of the cytokine receptors family (Kelly et al., 1993; Leung et al., 1987; Pilecka et al., 2007). The expression of the GHR gene has been shown first in the non-compartmentalized ovaries of broiler chickens between 4 and 16 weeks of age, using RT-PCR (Heck et al., 2003). The expression and localization of GHR within the individual compartments of the chicken ovary was subsequently demonstrated during sexual maturation and at the time of maturity (Hrabia et al., 2008). The GHR mRNA and the corresponding immunoreactivity were comparable to GH detected in the stroma of developing ovary and in the wall of all follicles and were more intense in granulosa cells than in theca interna or theca externa. In the sexually mature hen ovary, GH-binding sites have also been shown, by radioreceptor assay, in the granulosa and theca layers of the 5 largest preovulatory follicles, in which the number of GH-binding sites falls in the theca while increasing in the granulosa during follicle enlargement (Lebedeva et al., 2004). A more recent study also revealed presence of GHR protein and GH in cultured granulosa cells which may indicate that the GHR may be activated by systemic or by GH produced locally (Ahumada-Solórzano et al., 2012). These results support the possibility that GH has paracrine and/or autocrine actions in ovarian functions of chickens. Molecular mechanisms of GH effects are, however, unclear.

5. Effect of growth hormone on ovarian steroidogenesis Subsequent studies have shown that the role of GH in the avian ovary is associated with the regulation of steroidogenesis. In the ovary of a laying hen the growing follicles are represented by numerous prehierarchical white and yellowish follicles and several yellow preovulatory ones arranged in a size hierarchy (F5-7–F1). Steroidogenic activity of follicles gradually changes during their development. In prehierarchical follicles the granulosa layer is steroidogenically inactive whereas the theca is the source of ovarian estradiol. In hierarchical follicles both theca and granulosa layer are steroidogenically active and theca is a source of estradiol while granulosa is a source of progesterone (Huang et al., 1979; Bahr et al., 1983; Robinson and Etches, 1986; Tilly et al., 1991a,b; Hrabia et al., 2004; Sechman, 2013). Experiments with cGH treatment have provided evidence for its involvement in the control of steroid production in the chicken ovary. Namely, administration of cGH in the pullets during maturation resulted in a significant increase in progesterone content in the ovary just before and after maturity (by 6.7-fold and 1.7-fold, respectively), as well as increase in estradiol content about one week before puberty (by 2.8-fold). Although it is unclear from this data whether injected cGH acted both centrally and/or peripherally to affect steroidogenesis, the widespread presence of GHR in the ovarian tissues and changes of its abundance in the wall of ovarian follicles along with follicle development support the notion of a direct effect of GH at the chicken ovary. Further in vitro studies revealed that cGH directly stimulates estradiol secretion by whole chicken prehierarchical ovarian follicles (Hrabia et al., 2012) whereas it inhibited release of estradiol and elevated secretion of progesterone by yellow hierarchical follicles (Hrabia et al., 2014a). Some effects of cGH on secretory activity of chicken ovarian follicles are illustrated in Fig. 2. It has been also reported that cGH at a dose of 0.1, 1 and 10 nM stimulated the synthesis of progesterone by the granulosa cells of

Fig. 2. Effect of cGH (10 ng/ml medium; purchased as described in Fig. 1) on estradiol (a and b) and progesterone (c) secretion in vitro by whole prehierarchical (a) and yellow hierarchical follicles (b and c) of the laying hens. 1–4 mm – white follicles, 4–8 mm – yellowish follicles, 8–30 mm – yellow follicles, nd – not detected. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001 – in comparison with the control, as determined by Student’s t-test. (Modified from Hrabia et al., 2012, 2014a).

the second largest follicle (F2) in a dose-dependent manner (1.5-, 2.9- and 5.4-fold, respectively). This action of exogenous GH was blocked by cGH specific antibody (Ahumada-Solórzano et al., 2012). This direct effect of cGH on progesterone secretion was due to a direct up-regulation of cholesterol side chain cleavage enzyme expression (cytochrome P450scc, a rate-limiting enzyme


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in progesterone synthesis pathway) but not 3b-hydroxysteroid dehydrogenase mRNA (3b-HSD; a constitutive enzyme). Locally produced GH was similarly able to stimulate progesterone production in cultured granulosa cells in dose-dependent fashion (Ahumada-Solórzano et al., 2012). On the other hand, Hrabia et al. (2001, 2014a) failed to find any effect of cGH on progesterone secretion in vitro (24 h incubation) by granulosa layer explants of the 3 largest preovulatory follicles (F3–F1). In turn, inhibitory (Hrabia et al., 2001) or lack (Hrabia et al., 2014a) of GH action on estradiol secretion by fragments of the theca layer of the F3–F1 follicles has been observed. Furthermore, GH lowered LH-stimulated secretion of estradiol by the theca layer of the 3 largest yellow follicles isolated 22 h but not 3 h before the F1 ovulation (Hrabia et al., 2014a). These observations highlight that the direct effect of GH on ovarian steroid production in the chicken depend on the stage of follicle maturation and the layer of follicular wall. GH may also temporarily modulate the actions of gonadotropins during the ovulatory cycle as suggested by Hull and Harvey (2000, 2002). Further experiments involving examination of interaction of GH with FSH and LH as well as with some growth factors will be very useful for better understanding of GH role in the ovarian steroidogenesis.

6. Effect of growth hormone on ovarian apoptosis and proliferation From among about 480,000 of oocytes present in the chicken ovary at the time of hatching only a relative few of these (200– 500) reach maturity and are ovulated. Most of follicles undergo atresia during maturation and reproductive life (Johnson, 2000). Follicular atresia is mediated via apoptosis and is initiated within the granulosa layer (Johnson, 2002). Apoptosis allows removal of aged, damaged, infected or unwanted cells, maintaining homeostasis and remodeling in organisms. Control of cell death is dependent on multiple signals (Elmore, 2007; Hawkins et al., 2013; McIlwain et al., 2013). The hormonal mechanisms determining the atresia or selection of chicken ovarian follicles into hierarchy are, however, not fully understood. Since anti-apoptotic, pro-survival and proliferating effects of GH are well known in mammalian ovaries (Harvey, 2010; Sirotkin, 2005) and increased weight of the ovary and number of ovarian follicles were observed in the chicken after cGH treatment, to study in more detail these phenomena the apoptotic (TUNEL-positive) and proliferating (PCNA-positive) cell number was examined in the chicken ovary during several weeks before and at the time of maturity (Hrabia et al., 2011). It was revealed that cGH caused a significant decrease in the number of

apoptotic cells in the ovarian stroma from 12 weeks of age by 17–27% and in the white follicles at 14 and 16 week of age, i.e. 3 and 1 week before maturity, by 35% and 18%, respectively. Concomitantly, cGH caused a significant increase in the number of proliferating cells, i.e. by 32–60% in the stroma, by 27–40% in the white follicles, by 26% in yellowish follicles just after maturity (Table 1). On the other hand, in the yellow follicles the number of apoptotic and proliferating cells was not affected by cGH treatment (Hrabia et al., 2011). This is consistent with the notion that within the chicken ovary only prehierarchical follicles are highly susceptible to atresia, whereas hierarchical ones, under normal physiological conditions, are resistant to becoming atretic (Johnson, 2000). Moreover, in the yellow hierarchical follicles proliferation is restricted only to germinal disc area (Tischkau et al., 1997; Yoshimura et al., 1996). Our results indicate that GH is involved in the regulation of cell apoptosis and proliferation in the chicken ovary during maturation (Hrabia et al., 2011). Nevertheless, further studies are necessary to elucidate the molecular mechanism of GH action in these processes.

7. Participation of growth hormone in oviduct function Most recent investigation showed that the chicken oviduct, like the ovary, is an extrapituitary site of GH production and a target organ for this hormone (Hrabia et al., 2013, 2014b; Luna et al., 2014). The avian oviduct is a unique organ divided morphologically and functionally into five parts. The infundibulum engulfs the ovum after ovulation. The magnum synthesizes and secretes majority of egg white proteins by the epithelial and tubular gland cells. In the isthmus shell membranes are formed and in the shell gland (uterus) the calcified eggshell is deposited. The vagina helps in egg expulsion. The expression of GH mRNA has been detected by RT-PCR in the infundibulum, and sequence of 690 bp cDNA fragment was 99.6% identical to that obtained from the pituitary (Luna et al., 2014). In other oviductal parts GH mRNA was also found, and the expression was higher in the lower segments (Fig. 3). Further, GH-immunoreactivity was determined by ELISA in proteins extracted from whole oviduct (Luna et al., 2014). Along with oviduct length the GH concentration was consistent, but it progressively declined from puberty to the late laying period. As determined by SDS– PAGE and Western blotting, immunoreactive GH was predominantly a 17 kDa variant in addition to other moieties (Luna et al., 2014). Like GH, expression of the GHR has also been found in the chicken oviduct. This was first shown by Ni et al. (2007) in the shell

Table 1 Effect of cGH on apoptotic and proliferating cell number in the ovarian stroma and prehierarchical follicles during sexual maturation. Apoptotic (TUNEL-positive) and proliferating (PCNA-positive) cells counted on 10–15 randomly chosen fields (50 lm 50 lm) of each stroma and follicular wall were averaged for each chicken for that microscopic field size and subsequently the mean value from 6 birds was calculated. Each value represents the mean ± SEM from 6 chickens. Means (in columns) marked with different letters are significantly different at P < 0.05 as determined by two-way ANOVA followed by Duncan’s test. C, control group; cGH, experimental group (cGH prepared and injected as in Fig. 1); –, lack of follicles; #, onset of egg lay. (Modified from Hrabia et al., 2011). Age [weeks]

10 12 14 16 17#

Group

C cGH C cGH C cGH C cGH C cGH

Apoptosis

Proliferation

Stroma

White follicles (1–4 mm)

32.3 ± 0.93abe 31.8 ± 0.85abe 36.0 ± 0.94a 30.3 ± 0.86bcd 36.7 ± 1.83ab 28.8 ± 1.27cde 35.6 ± 1.18a 25.9 ± 1.07c 32.8 ± 0.81ad 27.0 ± 1.7ce

– – – – 45.7 ± 2.42a 31.8 ± 1.05bc 36.5 ± 1.49b 29.9 ± 1.45c 35.8 ± 0.81bc 30.9 ± 1.43bc

Yellowish follicles (4–8 mm)

– – 33.3 ± 1.46a 33.3 ± 1.54a 33.2 ± 1.53a 31.2 ± 1.60a

Stroma

White follicles (1–4 mm)

Yellowish follicles (4–8 mm)

28.7 ± 2.59ac 29.8 ± 2.89ac 23.8 ± 0.89a 38.0 ± 1.53b 26.8 ± 0.96ad 33.5 ± 0.67bc 31.1 ± 0.72ce 32.0 ± 1.08cd 26.0 ± 1.56ae 34.3 ± 1.22bc

– – – – 25.3 ± 0.94a 35.4 ± 1.29b 30.3 ± 1.04ab 33.5 ± 2.89ab 26.9 ± 1.81a 34.3 ± 1.57b

– – – – – – 29.4 ± 0.83ab 30.8 ± 1.56ab 26.3 ± 1.77a 33.1 ± 0.43b



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8. IGFs as a mediators of growth hormone action

Fig. 3. Expression of GH mRNA in the oviduct of the laying chicken as demonstrated by Real-time PCR. Oviducts were isolated from hens 2 h after oviposition. Expression level of investigated gene was normalized with a 18S rRNA reference gene. The data were analyzed by the 2 DDCT method using the expression in the infundibulum as the calibrator and presented as RQ ± SEM from 5 birds. Values marked with different letters differ significantly at P < 0.05, as determined by oneway ANOVA followed by Duncan’s multiple range test.

gland, but Hrabia et al. (2013) later demonstrated differential mRNA expression of GHR in the infundibulum, magnum, isthmus and shell gland of laying hens. Recently GHR mRNA was also found in the vagina (Luna et al., 2014). Expression of GHR was lower in the infundibulum than in the magnum, isthmus and shell gland. As these segments of the oviduct are characterized by high metabolic activity related to formation of the egg components (25– 30 g of white and 5–6 g of shell) the results may indicate that GH participates in the regulation of egg formation in them. It is of interest that GHR immunoreactivity within the wall of the oviduct was particularly present in the mucosa but not in the stroma composed of muscles and connective tissue. It additionally suggests the involvement of GH in the production of egg constituents, as protein of egg white, shell membranes and egg shell, are produced by the mucosa of the magnum, isthmus and shell gland, respectively. A subsequent study provided the evidence which supports this hypothesis and extends the proposal of Donoghue et al. (1990) that GH is involved in eggshell formation. Namely, it has been revealed that treatment of chickens with cGH during puberty (from 10 to 16 weeks of age) resulted in an increase (by 211%) in mRNA expression of ovalbumin, major egg-white protein synthesized in the magnum as well as ovocalyxins 32 and 36 (by 592% and 303%, respectively), eggshell matrix proteins, produced in the shell gland (Hrabia et al., 2014b). The action of GH in the avian oviduct appears to be also related to the regulation of apoptotic cell death, since injections of cGH for several weeks during maturation in chickens caused inhibition of cell apoptosis as identified by TUNEL assay, and reduced expression and activity of caspases 2 and 3, markers of apoptosis, in the magnum (Hrabia et al., 2014b). Simultaneously, cGH did not affected expression of anti-apoptotic genes as Bcl-2 protein (Hrabia et al., 2014b) or survivin (Hrabia, unpublished data). Since the ratio of proliferating to apoptotic cells was increased, it cannot be, however, excluded that GH acts as a survival factor in the chicken oviduct. It should be noted that these results may demonstrate the endocrine effects of GH on chicken oviduct development and function as well as paracrine and/or autocrine actions, as GHR were co-localized with GH in the chicken oviduct (Luna et al., 2014).

Some biological effects of GH are mediated by insulin-like growth factors (IGF-1, IGF-2) which are produced in many tissues in response to GH (Yoshimura et al., 1994). Thus, in chicken reproductive system GH may exert some effects at least in part by IGFs as was proposed by Onagbesan et al. (1999, 2009). In the chicken ovary all members of the IGFs system have been identified and the intra-ovarian functions reported for the IGFs include regulation of steroidogenesis, cell proliferation and differentiation, as well as follicle selection (Onagbesan et al., 1999, 2009). In vitro, additive effect of cGH and IGF-1 on estradiol secretion by whole prehierarchical follicles was not observed, although separately both hormones stimulated estradiol secretion by these follicles (Hrabia et al., 2012). In turn, exogenous GH induced IGFs secretion from the granulosa cells of the largest preovulatory follicles (Onagbesan et al., 1999) which stimulate progesterone production and decrease estradiol secretion by these follicles (Onagbesan et al., 1999). In contrary, in author’s study injections of cGH in

Fig. 4. Expression of IGF-1 (a) and IGF-2 (b) mRNA in the chicken ovary after subcutaneous cGH injections (200 lg/kg of b.w.; 3 a week from 10 weeks of age; purchased as described in Fig. 1) during sexual maturation determined by semiquantitative RT-PCR at the time of maturity. STR – ovarian stroma, 1–4 mm – white follicles, 4–8 mm – yellowish follicles, 8–30 mm – yellow follicles. Each value represents the mean ± SEM from 5 or 6 determinations of IGFs that were normalized with respect to 18S rRNA mRNA expression. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄ P < 0.001 – in comparison with control, as determined by Student’s t-test.


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chickens during maturation (from 10 to 17 weeks of age) resulted in significant decrease in IGF-1 mRNA expression in the wall of yellowish follicles by 38% and yellow follicles (>18–30 mm) by 48–49% as measured at the time of maturity. Similarly, cGH decreased IGF-2 mRNA expression by ovarian stroma and yellow follicles larger than 12 mm by 32–49% (Fig. 4). Simultaneously, IGF expression was strongly up-regulated in the liver (author’s unpublished data), what indicates the tissue-specific response to GH. The discrepancy between the effect of GH on IGF protein secretion and IGF mRNA expression by ovarian follicles may be a result of different experimental conditions. Namely, IGF secretion by the granulosa cells of preovulatory follicles in response to GH was measured after 72 h of cell culture, whereas mRNA expression was examined in whole wall of follicles after cGH treatments in vivo, where many molecules may interact with GH in regulation of IGF expression in the ovary. Different regulation of IGF synthesis and secretion by GH cannot be also excluded. From the current results, it is suggested that participation of GH in development and activity of the chicken ovary occurs partially via the regulation of IGFs gene expression. Further studies will be required to elucidate the effect of GH on IGF expression on mRNA and protein levels by ovarian follicles at different developmental stages and different physiological conditions. Mediation of some GH actions by IGFs in the avian oviduct is also very likely since IGF-1, IGF receptor and IGF binding protein2 have been revealed and paracrine and/or autocrine actions of IGF-1 in the quail oviduct during its development was proposed (Fu et al., 2001; Kida et al., 1994; Ni et al., 2007). Moreover, in primary cultures of quail oviduct cells, elevation of ovalbumin synthesis by IGF-1 in cooperation with estrogen was shown (Kida et al., 1995).

9. Summary In summary, the results so far clearly indicate that GH is involved in general mechanism responsible for activation and functioning of avian ovary and oviduct. The action of GH may reflect the effect of pituitary GH, but evidence that GH is produced in the reproductive system and parallel localized with its receptors strongly suggests that GH may also act as paracrine and/or autocrine growth factor. In order to clarify the mechanisms of GH action in avian reproductive system further studies are needed. Acknowledgments The author is grateful to Prof. Steve Harvey (Department of Physiology, University of Alberta, Edmonton, Canada) for valuable comments on the manuscript and to following collaborators, J. Rza˛sa, A. Gertler, H.E. Paczoska-Eliasiewicz, A. Sechman, M. Mika, A.K. Grzegorzewska, A. Lesńiak-Walentyn. This work was supported by Grants: P06D 034 28 from the Ministry of Education and Science and 2011/01/B/NZ4/03665 from the National Science Centre, Poland to A.H., and DS-3243/KFiEZ. References Abir, R., Garor, R., Felz, C., Nitke, S., Krissi, H., Fisch, B., 2007. Growth hormone and its receptor in human ovaries from fetuses and adults. Fertil. Steril. 90, 1333– 1339. Ahumada-Solórzano, M.S., Carranza, M.E., Pedernera, E., Rodríguez-Méndez, A.J., Luna, M., Arámburo, C., 2012. Local expression and distribution of growth hormone and growth hormone receptor in the chicken ovary: effect of GH on steroidogenesis in cultured follicular granulosa cells. Gen. Comp. Endocrinol. 175, 297–310. Arámburo, C., Alba-Betancourt, C., Luna, M., Harvey, S., 2014. Expression and function of growth hormone in the nervous system: a brief review. Gen. Comp. Endocrinol. 203, 35–42.

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