Regulation of Follicle Differentiation by Gonadotropins and Growth Factors

Regulation of Follicle Differentiation by Gonadotropins and Growth Factors A. L. JOHNSON Department of Animal Sciences, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903

1993 Poultry Science 72:867-873

INTRODUCTION Although the poultry industry is currently one of the more efficient animal production industries, there are several characteristics of the hen egg production cycle that remain suboptimal. These aspects include the decline in egg production in laying strains of chickens from a high of greater than 90% early in the production cycle to as low as 70 to 75% late in the production cycle. Broiler breeders have a comparatively shorter production cycle (average of 41 wk) and produce at an average rate of only 55%. Key components to the decrease in the rate of ovulation are the general decline in the number of small, growing follicles (from which follicles are recruited into the preovulatory hierarchy), and the increased rate of follicle atresia (Williams and Sharp, 1978; Palmer and Bahr, 1992). Recent work has focused on elucidating the physiological mechanisms that regulate the growth and development of small

Received for publication August 3, 1992. Accepted for publication December 31, 1992.

follicles and promote the final stages of differentiation following recruitment into the preovulatory hierarchy. Follicles can be classified into at least four stages of development; these include resting follicles, slow-growing white follicles, stage of follicle selection, and final differentiation (Figure 1). Virtually all follicles that undergo atresia do so during the slowgrowing stage (Gilbert et al, 1983), whereas the selection of a single follicle per day occurs from a pool of approximately 10 to 15, 6- to 8-mm follicles. At least two separate indicators of cellular activity provide evidence that granulosa cells are highly active at the time of follicle selection. First, plasminogen activator (PA) activity is greatest in 6- to 12-mm follicles and lowest in preovulatory follicles (Tilly et al, 1992). The type of PA activity detected within the granulosa layer is predominantly urokinase (u) PA (Armstrong et al, 1990), although tissue-type PA mRNA can be detected within granulosa cells by reverse transcriptase-polymerase chain reaction analysis (Johnson and Anthony, unpublished data). The presence of uPA activity has been linked to cellular reorganization

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ABSTRACT The rate of ovulation is determined by, among other things, the availability of small follicles that can be recruited into the follicular hierarchy. A decrease in the rate of lay with, for instance, aging has been attributed to both an increase in the rate of atresia and a decrease in the number of small, growing follicles that provide the pool from which follicles are selected into the final growth phase (the preovulatory hierarchy). Among the most important endocrine, paracrine, and autocrine factors that mediate follicle growth and differentiation are the gonadotropins and growth factors. A better understanding of gonadotropin-growth factor interactions that occur during follicle selection and differentiation should lead to the development of management practices or genetic manipulations (Mendelian or molecular) that will enhance the rate of lay at times during the production cycle when egg production is suboptimal. (Key words: follicle, growth factors, gonadotropins, steroids, reproduction)

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STEROIDOGENESIS IN SLOW-GROWING FOLLICLES It has been recognized for some time that slow-growing follicles (<10 mm in diameter) are steroidogenically active and are capable of producing steroids in vitro both in the absence and presence of luteinizing hormone (LH) (Robinson and Etches, 1986). However, this information was derived from incubation of whole follicles and was not designed to address either the question of relative contribution by the theca versus granulosa layer or the regulatory control of steroidogenic enzymes. Therefore, studies were initiated to evaluate the regulation of steroidogenesis in theca and granulosa cells in small, growing follicles.

Theca Tissue Similar to the theca layer from preovulatory follicles, theca cells from 1- to

8-mm follicles are steroidogenically active, and are capable of synthesizing progestins, androgens, and estrogens. For instance, Kowalski et al. (1991) have demonstrated that theca cells from 6- to 8-mm follicles express cytochrome P450 side-chain cleavage (P45oscc) mRNA and enzyme activity, and that these cells produce progesterone, androstenedione, and estradiol in vitro in the absence of granulosa cells. Moreover, theca cells from large white follicles express high levels of 3/3-hydroxysteroid dehydrogenase (3/3-HSD) activity (Davidson et al., 1979), cytochrome P450 17ahydroxylase mRNA (Li and Johnson, unpublished data), and aromatase immunoreactivity (Nitta et ah, 1991). Steroid production in theca cells from 6- to 8-mm, as well as preovulatory, follicles is thought to be primarily under the stimulatory control of LH (acting via the adenylyl cyclase pathway), because highly purified chicken follicle-stimulating hormone (FSH) and recombinant human FSH stimulate steroid production in vitro only at high doses (100 to 200 ng/mL) (Tilly and Johnson, 1989; Kowalski et al, 1991; Levorse, 1992). By contrast, activation of the protein kinase C pathway inhibits androgen and estrogen production, but these effects are apparently not mediated by the growth factors TGFa or EGF (Kowalski et al, 1991; Levorse, 1992). Protein kinase Cmediated inhibition of steroid production appears to be a mechanism by which steroidogenesis is modulated within theca tissue.

Granulosa Tissue In contrast to theca tissue, granulosa cells from follicles <8 mm are incapable of producing progestins or androgens due to low levels of P45oscc mRNA and absence of enzyme. activity (Figure 2) (Tilly et al, 1991a,b). The fact that both P450scc mRNA and protein is present in such granulosa cells, albeit at extremely low levels, indicates that the lack of P45oscc enzyme activity at this stage of development may be due to the lack of cholesterol availability within the mitochondria. By contrast, granulosa cells from 6- to 8-mm follicles express 3/3-HSD activity, as evidenced by the ability to effectively convert pregnenolone to progesterone, in vitro (Tilly et al, 1991a).

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and tissue remodeling (Sappino et al, 1989). In addition, proliferation of granulosa cells (as indicated by 3 Hthymidine incorporation) is higher in 6- to 12-mm follicles than in follicles that have entered the preovulatory hierarchy (Tilly et al, 1992). Although there is considerable information about gonadotropin and growth factor control of steroidogenesis in large, preovulatory (F5 to Fj) follicles, there exists comparatively little information about the regulation of growth and differentiation in granulosa and theca cells from follicles that have yet to be recruited into the follicular hierarchy (e.g., follicles less than 8 m m in diameter). The purpose of the present review was to summarize information about interactions between the gonadotropins and the growth factors [primarily transforming growth factor a (TGFa) and epidermal growth factor (EGF)], as they relate to follicle development and differentiation. For the sake of brevity, emphasis has been placed upon data from granulosa and theca tissue of small, growing follicles. A more complete summary of the effects of gonadotropins and growth factors within preovulatory follicles can be found elsewhere (Etches, 1990; Johnson, 1990; Tilly and Johnson, 1990).

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FIGURE 2. The lack of steroidogenesis in granulosa (GR) cells less than 8 mm in diameter is the result of low cytochrome P450 side-chain cleavage (P450 ) mRNA levels and absence of enzyme activity. Panel A shows a Northern blot of P450<,cc mRNA in granulosa cells relative to stage of follicle maturation. Note that whereas P450scc mRNA is detectable in 1- to 8-mm follicles, levels are very low compared to those of 9- to 12-mm follicles, the third largest (F3) and largest (Fj) preovulatory follicles. The low levels of P450Scc m R N A are transcribed to protein as evidenced by the presence of P450scc protein found in 40 /xg crude mitochondrial protein (Lane b) and in immunoprecipitated protein (Lane a) as determined by Western blot analysis; Lane c represents bovine adrenal P45OSCC standard (Panel B). Nevertheless, enzyme activity is absent as determined by the inability of isolated mitochondrial preparations from granulosa cells of 6- to 8-mm follicles to convert 25-hydroxycholesterol to pregnenolone (Panel C). Km = Michaelis-Menten constant; Vmax = maximal rate of enzyme-catalyzed reaction, Values represent the mean ± SEM from three replicate experiments. Data adapted from Tilly et al. (1991b).

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FIGURE 1. Stages of follicular development in the hen's ovary. The vast majority of ovarian follicles are less than .5 mm in diameter and are considered to be in a resting stage. Once a follicle begins to grow, it does so over a period of weeks to months, and it is during this stage that atresia occurs most commonly (Gilbert et a\., 1983). Selection of a single follicle per day into the preovulatory hierarchy is proposed to take place at the 6- to 8-mm stage of development, after which final differentiation prior to ovulation (particularly in the granulosa layer) occurs over a 5- to 10-day period.

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Granulosa cells acquire the ability to pro- estrogens under the primary control of LH duce progesterone in response to FSH, but in the absence of progestin precursor from not LH, during the transition of follicles the granulosa layer. Such production can from 6- to 8-mm to 9- to 12-mm in diameter entirely account for the steroids produced (Figure 3), and this occurs coincident with following incubation of whole follicles, increased levels of P450 mRNA. The because coincubation of granulosa plus absence of LH responsiveness in granulosa theca cells fails to result in an additive effect cells during this period of development has (Tilly et al, 1991a). On the other hand, it is been attributed to a lack of coupling of the proposed that the granulosa layer from LH receptor to transmembrane G proteins follicles <8-mm is steroidogenically inor the adenylyl cyclase enzyme or both (see competent due, at least in part, to the tonic Tilly et al, 1991a). suppression of P45oscc mRNA and enzyme Dispersed granulosa cells from 6- to activity by TGFa and EGF. Increased P450 8-mm follicles do, however, respond to FSH mRNA levels and the initiation of enzyme (chicken and recombinant human), but not activity is under the stimulatory control of LH, with increased accumulation of cyclic FSH via the adenylyl cyclase pathway, and adenosine monophosphate (cAMP) (Tilly et occurs coincident with selection of the al, 1991a,b). Moreover, granulosa cells follicle into the preovulatory hierarchy. The cultured in serum-free medium with FSH mechanism(s) by which granulosa cells or the cAMP analog, 8-bromo-cAMP, re- escape the inhibitory effects of TGFa and spond within 8 h with the induction of EGF on P450SCC mRNA synthesis and enP450 enzyme activity and within 16 h with zyme activity in response to FSH at the time increased levels of P450 mRNA. These of follicle selection has yet to be established, data indicate that in a serum-free environ- but could be explained by: 1) a decrease in ment in vitro, granulosa cells from TGFa and EGF production by the follicle; 2) 6- to 8-mm follicles will begin to differenti- a decrease in EGF receptor expression; or 3) ate rapidly in response to an appropriate a change in the receptor signalling pathway signal (e.g., FSH), and lead to the question of the EGF receptor (see Chakravorty et al, why P450 mRNA and enzyme activity is 1992). Shortly after a follicle has been not fully expressed in vivo in granulosa cells selected into the preovulatory hierarchy, from 6- to 8-mm (or smaller) follicles. the granulosa layer acquires the ability to respond to LH (follicles > 12 mm; Tilly et al, Subsequent studies determined that FSH 1991a) and loses responsiveness to FSH and 8-bromo-cAMP failed to stimulate (Ritzhaupt and Bahr, 1987; Figure 3). Subseincreased levels of P450 mRNA or induce quent steroid production from granulosa enzyme activity in granulosa cells from cells of preovulatory follicles appears to be 6- to 8-mm follicles when cocultured with predominantly, if not exclusively, under the TGFa or EGF (Li and Johnson, 1991). By stimulatory control of LH, and results in the comparison, insulin-like growth factor-I formation of progesterone, which is re(IGF-I) neither enhanced nor inhibited FSH- quired for initiating or potentiating the induced P450 mRNA levels or enzyme preovulatory LH surge or both. activity. These results suggest that TGFa or EGF or both may act in an autocrine or Clearly, additional growth factors beparacrine fashion to tonically suppress the sides TGFa, EGF, and IGF-I modulate the expression of P45oscc mRNA and enzyme growth and differentiation of ovarian folliactivity in follicles that have not yet entered cles. One such newly identified growth the follicular hierarchy. Whereas the pres- factor is the ligand for the protooncogene cence and source (granulosa or theca cells) of kit receptor, stem cell factor (SCF) (Martin et TGFa or EGF has yet to be unequivocally al, 1990). established, it has recently been determined that granulosa cells from 1- to 8-mm i n C f t l T i r i r t A T I A k l />£? O T C f t l S*EI I follicles express EGF receptor mRNA 0ohnI U C N 1 inv/M 1 i v n v/r o i c m U E L I . son, unpublished data). FACTOR IN THE HEN OVARY The SCF (also called c-kit ligand or mast Taken collectively, it is concluded that the theca layer of growing follicles cell growth factor) is a 28- to 30-kDa produces progesterone, androgens, and glycoprotein growth factor that is synthe-

SYMPOSIUM: RECENT ADVANCES IN REPRODUCTION

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FIGURE 3. Differential effects of recombinant human (rh) follicle-stimulating hormone (FSH) (courtesy of A.J.W. Hsueh, Stanford University Medical Center, Stanford, CA 94305; potency was 1.7 ng ovine (o) FSH-16 = 1 mlU rhFSH) and luteinizing hormone (LH) (oLH-23) on progesterone production from granulosa cells at three stages of follicle development. Granulosa cells (4 x 10^) were incubated for 4 h, then the medium plus cells assayed for progesterone. Values represent the mean ± SEM from a minimum of three replicate experiments. *P < .05 versus control.

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In summary, it has become clear that there are a myriad of growth factors and growth factor-gonadotropin interactions, only a few of which have been discussed here, which orchestrate growth and differentiation of hen ovarian follicles. Clearly, a major challenge for the coming years will be to identify those interactions that are most susceptible to genetic and molecular manipulation in an effort to enhance egg production, particularly in broiler breeder strains of birds and in laying hens during the latter stages of production. ACKNOWLEDGMENTS

The author is indebted to J. L. Tilly, J. A. Armstrong, C. Brown, K. Kowalski, J. Levorse, and Z. Li for conducting studies described herein. This work was supported by USDA Grants 88-37242-4013 and 90-37240-5510. REFERENCES Anderson, D. M., S. D. Lyman, A. Baird, J. M. Wignall, J. Eisenman, C. Rauch, C. J. March, H. S. Boswell, S. D. Gimpel, D. Cosman, and D. E. Williams, 1990. Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms. Cell 63:235-243. Armstrong, J. A., J. L. Tilly, and A. L. Johnson, 1990. Evidence for the presence of both a urokinase and tissue-type plasminogen activator (PA) in preovulatory follicles from the domestic hen. Biol. Reprod. 42 (Suppl. l):158.(Abstr.) Chakravorty, A., M. I. Joslyn, and J. S. Davis, 1992. Defective epidermal growth factor (EGF) receptor in bovine luteal cells: Lack of receptor tyrosine kinase activity. Proceedings DC Ovarian Workshop, Chapel Hill, NC. Abstract No. ll.(Abstr.) Davidson, M. F., A. B. Gilbert, and J. W. Wells, 1979. Activity of ovarian A5-3|3-hydroxysteroid dehydrogenase in the domestic fowl (Gallus domesticus) with respect to age. J. Reprod. Fertil. 57: 61-64. Dolci, S., D. E. Williams, M. K. Ernst, J. L. Resnick, C. I. Brannan, L. F. Lock, S. D. Lyman, H. S. Boswell, and P. J. Donovan, 1990. Requirement for mast cell growth factor for primordial germ cell survival in culture. Nature 352:809-811. Etches, R. J., 1990. The ovulatory cycle of the hen. CRC Crit. Rev. Poult. Biol. 2:293-318. Gilbert, A. B., M. M. Perry, D. Waddington, and M. A. Hardie, 1983. Role of atresia in establishing the follicular hierarchy in the ovary of the domestic hen (Gallus domesticus). J. Reprod. Fertil. 69:221-227.

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sized as a transmembrane protein; this protein can be proteolytically cleaved at the cell surface to produce a soluble SCF. To date, SCF has been studied primarily with respect to its potent ability to stimulate colony formation from immature hematopoietic progenitors (Anderson et al, 1990; Migliaccio et al, 1991). Recently, however, evidence has accumulated to indicate a role for SCF in promoting the viability, proliferation, or migration of mammalian primordial germ cells during embryonic development (Dolci et al, 1990; Godin et al, 1990). Moreover, the SCF receptor, c-kit, has been identified not only in embryonic germ cells (Manova and Bachvarova, 1991) but also in theca cells and oocytes from the mature mouse ovary (Manova et al, 1990). There is little information from any species about the source of SCF in the adult ovary, thus studies were initiated to evaluate the potential presence of this growth factor in the hen ovary. A chicken SCF partial cDNA (501 bp from the extracellular domain) was cloned and sequenced, and has been utilized as a probe to detect SCF mRNA in ovarian tissues (Johnson et al, 1992). The nucleotide sequence of chicken SCF is approximately 65% homologous to the rat, human, and ovine SCF moieties, and the deduced amino acid sequence contains four cysteine residues that are completely conserved with respect to the mammalian SCF amino acid sequences. Northern blot analysis of both poly A+-enriched and total cellular RNA shows a single 6.5-kb transcript that is present in both granulosa and theca tissues from preovulatory (Fi) and 6- to 8-mm follicles. Although the presence or biological activity of the SCF protein has yet to be evaluated within the hen ovary, these initial data indicate that yet another growth factor, SCF, may play a role in modulating the development of follicular tissues or viability of the oocyte in the adult, as well as embryonic, ovary via a paracrine or autocrine mechanism. Consistent with this proposal is the recent preliminary report that SCF regulates germinal vesicle breakdown within the rat oocyte during the periovulatory period (Ismail and Vanderhyden, 1992).

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