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Growth and paracrine factors regulating follicular formation and cellular function
Molecular and Cellular Endocrinology 163 (2000) 11 – 20 www.elsevier.com/locate/mce
Growth and paracrine factors regulating follicular formation and cellular function K.P. McNatty *, A.E. Fidler, J.L. Juengel, L.D. Quirke, P.R. Smith, D.A. Heath, T. Lundy, A. O’Connell, D.J. Tisdall 1 AgResearch, Wallace6ille Animal Research Centre, PO Box 40063, Centre Ward Street, Upper Hutt, New Zealand Received 25 June 1999; accepted 20 October 1999
Abstract The purpose of this paper is to review, using fetal sheep as the animal model, aspects of ovarian development related to follicular formation and to report on the identity of growth and paracrine factors which might be involved in this process. Before follicular formation there is a massive and sustained colonisation of the fetal ovary by mesonephric cells, which become a precursor source of follicular cells. From within the ovarian medulla, somatic ‘cell-streams’ branch into the cortex around nests of oogonia and oocytes. These ‘cell-streams’, which contain elongated cells with either flattened or cuboidal shaped nuclei, express steroidogenic factor-1 (SF-1), steroid acute regulatory protein (StAR), 3b-hydroxysteroid dehydrogenase (3b-HSD), cytochrome P450scc, and P450aromatase mRNA and/or protein. Follicles form from the association of an oocyte with the ‘cell-stream’ with either a single layer of flattened cells (i.e. type 1 follicle) or with a mixture of flattened and cuboidal cells (i.e. type 1a follicle). These newly-formed follicles have between 3 and 57 somatic cells (i.e. granulosa cells) and contain oocytes which vary in diameter between 23 and 52 mm. Newly formed and early growing follicles have been identified with growth factors or growth factor receptors in either the oocytes or granulosa cells. Many of the growth factors are from the TGFb superfamily and are expressed in a cell- and stage-specific manner. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cell streams; SF-1; StAR; 3b-HSD
1. Introduction Development of the human ovary begins some 3–4 weeks after conception. Many key events in ovarian development such as germ cell migration, gonadal sex differentiation, germ cell mitosis and atresia, and follicular formation are initiated and completed during fetal life. Other key events such as germ cell meiosis and follicular growth and atresia, which occur throughout the prepubertal and reproductive years, are also initiated during fetal life. Whilst the timing of these events has been determined in the human, knowledge relating to function is largely derived from animal models such
* Corresponding author. Tel.: +64-4-5286089; fax: +64-45281380. E-mail address: [email protected] (K.P. McNatty). 1 Present address: National Centre for Disease Investigation, Ministry of Agriculture and Forestry, PO Box 40742, Upper Hutt, New Zealand.
as genetically-altered (mutant) rodents, rhesus monkeys and farm animals. The purpose of this paper is to review, using fetal sheep as the animal model, aspects of ovarian development related to follicular formation, and to report on the identity of growth and paracrine factors which might be involved in this process.
2. Gonadal development before follicular formation in fetal sheep (i.e. days 23–75 of fetal life)
2.1. Sequence of e6ents and morphology The gonad is first evident as a thickening of the coelomic epithelium on the medial aspect of the mesonephros at days 23–24 of fetal life (Fig. 1). As in other species, the gonad evolves from at least four different tissues or cell-types namely: (1) the coelomic epithelium; (2) the mesenchyme of the mesonephros; (3)
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Fig. 1. Photomicrographs showing aspects of early ovarian and follicular formation in fetal sheep. (a) The mesonephros (m) and site of gonadal formation, namely the coelomic epithelium ( ) at day 23 of gestation; (b) the prominent surface epithelium of the gonad (g) 2 – 3 cell layers thick ( ) with mesonephros (m) and glomerulii (gl) at day 28 of gestation; (c) the surface epithelium ( ) of the ovary (o) at day 40 of gestation. The mesonephros (m) is shown on the left; (d) evidence of the cell-continuum between the glomerulii (gl) of the mesonephros and ovary (o) at day 35 of gestation; (e) a ‘cell-stream’ ( ) between the ovarian medulla and cortical germ-cell nests ( ) at day 75 of gestation; (f) a higher magnification of the ‘cell-stream, showing elongated cells with flattened nuclei ( ) and cuboidal nuclei ( ) at day 75 of gestation; (g) a ‘cell-stream’ ( ) at follicular formation around oocytes ( ), and adjacent to some type 1 follicles () at day 75 of gestation; (h) ‘cell-streams’ ( ) and type 1 ( ) and type 1a follicles () at day 75 of gestation. Scale bars: a, b, c, 100 mm; d, 200 mm; e, 50 mm; f, h, 20 mm; g, 25 mm.
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cells from the mesonephric glomerulii and tubules (e.g. mesangial, epithelial); and (4) primordial germ cells (PGC) which migrate to the genital ridge from the dorsal endoderm of the yolk sac (Zamboni et al., 1979; Byskov, 1986) (Fig. 1). Between days 23 and 30, the coelomic epithelium of the developing gonad is a very prominent feature with columnar cells up to three layers in thickness (Fig. 1). Thereafter, the surface
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epithelium of the gonad is less prominent with a single layer of cuboidal or flattened elongated cells. In sheep most germ cells migrate into the gonad between days 23 and 35, and by day 40 are mainly in the cortex. A massive and sustained invasion of the gonad by mesonephric cells concomitant with the involution of the giant glomerulii occurs between days 23 and 55 of fetal life (Zamboni et al., 1979, see also Fig. 2). Many
Fig. 2. Contribution of mesonephric cells to ovarian and follicular formation in sheep from days 24 to 75 of fetal life. Germ cells are shown as cells with both cytoplasm and nuclei whereas somatic cells are shown only as cell nuclei.
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Table 1 Dimensions of cell nuclei in cell-streams and rete cell tubules or masses in fetal sheep ovaries. Values are means 9S.E.M. in mma Axis
Major Minor Mean of major and minor axis
Cell nuclei in cell streams
Cell nuclei in rete cell tubules or masses
Flattened
Cuboidal
11.6 9 0.4a 2.69 0.1a 7.19 0.3a
11.5 9 0.4a 5.5 90.4b 8.5 90.2b
8.0 9 0.5b 4.9 9 0.2b 6.5 90.2c
a For each row values with a different alphabetical superscript are different from one another (PB0.05; ANOVA with least significant differences). Values are means from three animals and the mean for each animal was obtained from the measurements of 50 cells.
of the cells in the continuum between the mesonephros and ovary are highly irregular in shape and have pseudopodia which are characteristic of migrating cells. A significant proportion of the early migrating mesonephric cells are of mesenchymal or mesangial lineage (Fig. 2). Later, cells in the continuum between the mesonephros and ovary include increasing proportions of cells from the walls of the glomerulii and tubules. Between days 40 and 75, somatic cells within the ovarian medulla form ‘cell-streams’ that branch out into the cortex and surround nests of oogonia or germ cells undergoing meiosis (Figs. 1 and 2). Cells within the ‘cell-streams’ all appear to be orientated in the same direction and are easily identifiable as they transverse in a seemingly orderly fashion from the medulla to the cortex. These mesonephric-derived ‘cell-streams’ contain elongated cells, which when examined, by optical sectioning, consist of two distinct types, one with a flattened cell nuclei and the other with cuboidal nuclei. The dimensions of the cell nuclei are shown in Table 1. The nuclei of the flattened cells were similar in length to the nuclei of the cuboidal cells but only half the thickness (i.e. the minor axis, Table 1). It remains to be determined whether cells with cuboidal and flattened nuclei are of different or common origin.
2.2. C-kit and stem cell factor (SCF) Studies of mutant mice strains with a phenotype arising from aberrant migrations of germ cells led to the discovery that the ligand/receptor pair, SCF/c-kit plays a central role in the migration of germ cells to, and colonisation of, the developing genital ridge (Fleischman, 1993). It seems that concentration gradients of SCF from somatic cells interacting with c-kit on germ cells have a major influence on their directional migration (Keshet et al., 1991). In sheep at 23 to 26 days of fetal life, SCF mRNA and protein are present in somatic cells of the mesonephros and gonad. However, by day 30, SCF mRNA and protein become increasingly restricted to the cortical regions of the ovary where germ cells are also present as proliferating oogonia (Tisdall et al., 1999). By day 30 few, if any, of the mesonephric cells or those that are part of the contin-
uum of cells connected to the ovarian medulla expressed SCF mRNA or protein. In contrast to SCF, c-kit mRNA and protein were identifiable in both primordial germ cells and somatic cells of the gonad at 23 to 26 days of fetal life. Thereafter, from day 28 c-kit expression was confined largely to germ cells, most of which migrate to the cortical region of the ovary and adjacent to cells expressing SCF (Tisdall et al., 1999).
2.3. Wilms tumour-1 (WT-1) and steroidogenic factor-1 (SF-1) In WT-1 or SF-1 null mice, gonadal development is inhibited indicating that the expression of these two genes is critical for differentiation of the gonadal ridge (Kreidberg et al., 1993; Luo et al., 1994). In sheep, both SF-1 and WT-1 mRNA have been identified by in situ hybridisation or RT-PCR to be present in the gonad shortly after the thickening of the coelomic epithelium, and thereafter in both ovaries and testis throughout fetal life (Payen et al., 1996; McNatty et al., 1998). Between days 26 and 55, SF-1 mRNA and/or protein was identified in somatic cells in both the cortical and medullary regions of the gonad. It has been proposed that SF-1 protein regulates the production of gonadal aromatase and steroid hydroxylases (Ito et al., 1997). In fetal sheep, SF-1 expression in the gonad occurs some 10–15 days before the expression of steroidogenic regulatory genes such as steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage enzyme (P450scc), P450 17 alpha hydroxylase (P45017OH), and P450 aromatase (P450arom) (McNatty et al., 1998). In addition, SF-1 gene expression is more widespread in the gonad than has been noted for the genes involved in synthesis of steroid suggesting other roles for SF-1 in addition to the regulation of steroidogenesis.
2.4. Genes in6ol6ed in steroid synthesis In the sheep fetus, mRNA of genes involved in steroidogenesis (i.e. StAR, P450scc, P45017OH and P450arom) as well as the steroids themselves are present in the ovary around morphological sex differentiation,
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i.e. days 32–35 (Payen et al., 1996; Sweeny et al., 1997; McNatty et al., 1998; Lun et al., 1999): a punctuate pattern of gene expression was observed in ovarian somatic cells in the medulla and innermost regions of the cortex. Some of the somatic cells expressing the steroid regulatory genes are in close proximity to nests of oogonia and appear to be part of the ‘cell-streams’. Cells in these regions are likely to have migrated into the ovary after day 28 since they do not express SCF. In agreement with Zamboni et al. (1979) and Byskov et al. (1985), we consider these somatic cells to be mesonephric in origin and to include cells of mesenchymal and/or mesangial lineage. We hypothesise that these somatic cells synthesise steroids around the time of sexual differentiation of the gonad (i.e. some 12 days after gonadal formation) and during early follicular formation. Under in vitro conditions, mesonephrii and ovaries isolated and separated between days 35 and 55 each have a capacity to synthesise progesterone, androstenedione and oestradiol (Lun et al., 1999). Thus, the finding that the mesonephros is capable of synthesising steroid is consistent with morphological evidence that mesonephric cells are a potential source of follicular cells (see Grinsted, 1982). In some species it is considered that the surface epithelium is a contributor of follicular cells (see Peters, 1978; Byskov, 1986; Karl and Capel, 1998). However, in sheep, cells of the surface epithelium after day 30 of fetal life show no morphological evidence of invaginating or traversing down between the nests of oogonia either before or during follicular formation. Moreover, while expressing SCF mRNA, cells from the surface epithelium do not express StAR or any of the P450 steroid enzyme mRNA’s. Finally, the first oogonia to enter meiosis and the first follicles to form and grow are those where contact with the ‘cell-streams’ are first made in the innermost regions of the ovarian cortex as described for other species (Byskov, 1975). Thus, it seems unlikely that the surface epithelium is a significant contributor of follicular cells in sheep. Jost et al. (1973) have shown that ovarian steroids are not required for expression of the female phenotype. Nevertheless, the spatial and temporal pattern of steroidogenesis during gonadal development raises the possibility that intraovarian steroids are important for the developmental organisation and/or maturation of both the somatic and germ cells. It will be of interest to determine the location of steroid receptors in the developing ovary before follicular formation. 3. Gonadal events associated with follicular formation and growth in fetal sheep (i.e. days 75 – 135 of fetal life)
3.1. Sequence of e6ents and morphology By day 75 the mesonephros is at an advanced stage
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of involution and no longer contributes cells to the ovary (Zamboni et al. 1979). In the outer cortex large numbers of oogonia are present either in small nests or as individuals whereas in the mid-cortex numerous nests of oogonia are surrounded by mesonephricderived ‘cell-streams’ with some oogonia undergoing meiosis. Further into the cortex, large numbers of oocytes are in the process of follicular formation or in newly-formed follicles (Figs. 1 and 2). Some of the newly formed follicles consist of a single layer of flattened cells around the oocyte (i.e. type 1 follicles, Lundy et al., 1999) whereas others consist of a single layer of somatic cells which are a mixture of cells with either flattened or cuboidal nuclei (i.e. type 1a follicles; Lundy et al., 1999; Table 1): these two cell-types, namely those with flattened or cuboidal nuclei, are derived from the ‘cell-streams’ (Fig. 2). At day 75 a prominent mass of epithelial cells can be identified as part of a tubular system in the hilus region of the ovary but these are not a feature of the somatic cell population in the ovarian cortex. Zamboni et al. (1979); Fig. 2) suggest that these epithelial cells are from the capsule of the glomerulii, the walls of the capillaries and tubules and that they reach the ovary by mitosis as well as migration. To distinguish these mesonephric-derived epithelial cells from the ‘cellstreams’ we refer to the former as rete cell tubules or masses. When compared to cells of the ‘cell-streams’, the nuclei of the rete cell tubules or cell masses have distinctly different dimensions (Table 1), are more homogenous and as will be noted later develop different functional characteristics. By day 90 about 65% of the newly formed follicles are present as type 1 follicles and 35% as type 1a structures (unpublished data). By day 120 several type 2, 3 and 4 follicles are observed in the innermost regions of the cortex and by day 135 some follicles are noted at the type 5 stage of development (Table 2; McNatty et al., 1995; Lundy et al., 1999). Between days 90 and 135, somatic ‘cell-streams’ can be traced from the medulla traversing around follicles and nests of oocytes or oogonia right up to the surface epithelium. Also between day 90 and 105 a dramatic increase is noted in the extent and number of rete cell tubules or masses which have branched from the medulla into the cortex. Examination of individual 5 mm sections revealed that most growing follicles are in close proximity to the rete cell tubules or masses (Fig. 2). Hitherto, this close association was misinterpreted by Tisdall et al. (1999) and McNatty et al. (1999a) to mean that this composition of mesonephric-derived cells were a source of granulosa cells. However, with respect to the first forming follicles between days 75 and 90, it is clear that a different composition of mesonephric-derived cells (e.g. the ‘cell-streams’) precede the rete cell tubules or
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masses into the ovarian cortex; thus, the involvement of the latter in follicular formation remains obscure. One possibility is that the rete cell tubules or masses may contribute cells to the theca interna and interstitium as well as paracrine factors important for the initiation of follicular growth.
3.2. Factors associated with follicular formation During the period of follicular formation in sheep (i.e. between days 75 and 135) SCF protein, but not mRNA, is present in somatic cells around oocytes. The SCF antibody used in this study detects both soluble and membrane forms of SCF (Tisdall et al., 1999). Thus, the presence of SCF protein but not RNA might be due to soluble SCF originating from a distant site, to a longer half-life of the protein relative to the message or differences in sensitivities between immunohistochemistry and in situ hybridisation. C-kit protein and mRNA are present in oocytes during follicular formation (Tisdall et al., 1999). Growth differentiating factor9 (GDF-9) mRNA is present in oocytes of newlyformed type 1 and 1a ovine follicles at day 135 of gestation although it remains to be established whether GDF-9 mRNA is present in newly-formed follicles between days 75 and 135 and/or in oocytes during follicular formation (Bodensteiner et al., 1999, personal communication). The ovary continues to be steroidogenically active
during the period of follicular formation since oestradiol was produced in measurable quantities (Smith et al., 1993). Moreover, under serum-free conditions, the ovary is capable of synthesising steroids in vitro (e.g. androstenedione and oestradiol; unpublished data). Immunohistochemical evidence reveals that at least some cells within cell-streams, rete cell tubules or masses and the membrana granulosa of newly formed follicles contain immunoreactive SF-1, StAR and 3b-HSD indicating that these cells have a potential to synthesise steroids (Fig. 3; see also Byskov et al., 1985). Studies with fetal sheep ovaries recovered between days 90 and 135 of gestation were unable to show the presence of follicle-stimulating hormone receptor (FSHR), luteinising hormone receptor (LH-R), follistatin or a, bA and bB inhibin/activin subunit mRNAs in ‘cellstreams’ adjacent to oocytes at follicular formation. However, follistatin and bB inhibin subunit mRNA were localised in most sections to the rete cell tubules or masses (Braw-Tal et al., 1994; Tisdall et al., 1995, unpublished data). The evidence suggests that the mesonephric-derived ‘cell-streams’ precede the rete cell tubules or masses as a precursor source of follicular cells during follicular formation. Cells in the ‘cell-streams’ and around the newly-formed follicles (i.e. granulosa cells) immunostain for SF-1, StAR and 3b-HSD supporting the notion that the former are the precursor follicular cells. The
Table 2 Characteristics of small ovine folliclesa Follicle (type)
Layers of granulosa cells
Primordial (type 1) 1 (all flattened) Transitory (type 1a) 1 (1 or more cuboidal cells) Primary (type 2) 1–B2 (all cuboidal cells) Small preantral 2–B4 (type 3) Large preantral 4–B6 (type 4) Small antral (type \5 5)
Total number of granulosa cells
Oocyte diameter in mm
Follicular diameter in mm
Presence of theca interna%
16b (3,52) 39c (9,136)
35b (23,52) 41c (27,53)
41b (21,61) 51c (37,64)
0 0
128d (30,520)
52d (31,80)
75d (50,119)
35
637e (127,2174)
73e (41,92)
129e (64,191)
100
2104f (1090,3404)
88f (77,100)
194f (164,256)
100
11649g (3425,51447)
119g (91,142)
327g (192,450)
100
a Data from Lundy et al. (1999). Values for granulosa cells, oocyte diameter and follicular diameter represent means and ranges. For each column separately, values with different alphabetical superscripts are different from one another (PB0.01).
Fig. 3. Immunostaining for steroidogenic factor-1 (SF-1), steroid acute regulatory protein (StAR) and 3b-hydroxysteroid dehydrogenase (HSD) during follicular formation and early follicular growth in the sheep ovary: (a) and (b) SF-1 localised to cells of the ‘cell-streams’ ( ) or granulosa cells of type 1, 1a or type 3 follicles (); (c) and (d) StAR immunolocalised to cells of the ‘cell-streams’ ( ) and oocytes () during follicular formation; (e) 3b-HSD localised to ‘cell-streams’ ( ) or granulosa cells of newly-formed follicles ( ) and; (f) 3b-HSD localised to granulosa cells ( ) of type 1 and type 2 follicles and also the oocytes. The SF-1 antibody (Catalogue No. 06-431) was obtained from Upstate Biotechnology, Lake Placid, New York, and used at a dilution of 0.5 mg/ml (w/v). The StAR antibody was generously supplied by Dr D. Stocco, Texas Tech University, Health Sciences Center, Texas and used at a dilution of 1:1000 (v/v). The 3b-HSD (5683) was generously supplied by Dr I. Mason, Department of Clinical Biochemistry, University of Edinburgh, Scotland and used at a dilution of 1:250 (v/v). The immunohistochemical procedures were those described in Tisdall et al., 1999. Scale bars for a, b, d, e and f, 43.5 mm; c, 87.0 mm.
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Fig. 3.
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Table 3 Expression of growth factors and receptors during early follicular growth in sheepa
a , mRNA, , protein, , protein observed only in zona pellucida. Act/inh, activin/inhibin. 1Observed in oocytes of all follicles types. 2At the type 1 and 1a stage respectively 23 and 36% of all follicles contain one or more granulosa cells immunostaining for bB act/inh whereas in type 2 to 4 follicles virtually all follicles immunostain for bB act/inh subunit. 3TGFb1,2,3 present in theca interna from follicle type 4 stage of growth. 4 LH-R mRNA present in theca interna of type 4 follicles. Modified from McNatty et al. (1999b) and Bodensteiner et al. (1999).5No data.
presence of SCF protein in the absence of SCF mRNA in the ‘cell-streams’ and in cells of newly-formed follicles suggests possible differences in sensitivities between the in situ hybridisation and immunohistochemical techniques, that the SCF protein degradation rate is very slow or that the soluble ligand has been derived from another cellular source. It seems unlikely however that the follicular cells are from the surface epithelium since the latter contains both SCF mRNA and protein. Moreover, cells of the surface epithelium do not show evidence that they are steroidogenic. The rete cell tubules or masses, which later become closely associated with follicles, also display characteristics of steroidogenic cells. However, the rete cell tubules or masses can be distinguished from the ‘cell-streams’ by the finding that both follistatin and bB inhibin/activin subunit mRNA have been identified in the rete cell tubules or masses.
4. Characteristics of newly formed follicles The characteristics of newly-formed and small follicles with respect to number of granulosa cells and diameter of oocyte etc are summarised in Table 2. For each follicular type, including type 1 and 1a follicles, the total populations of granulosa cells together with oocyte diameters are highly variable. It is possible that
the variability in number of granulosa cells may, in part, be related to both the diameter of the oocyte at follicular formation and the proliferative activity of the somatic cells when the follicle is forming (Hirshfield and De Santini, 1995). In fetal sheep, oogonia have diameters between 13 and 17 mm and isolated oocytes, diameters between 17 and 22 mm (Smith et al., 1997) whereas after follicular formation oocyte diameters are between 23 and 53 mm (Table 2). These results infer that growth of germ cells begins before and continues during as well as after follicular formation. It can be calculated from a knowledge of the total populations of granulosa cells in types 1, 1a, 2, 3, 4 and 5 follicles that types 1 and 1a follicles undergo 9 and 8 doublings of their populations of granulosa cells respectively to reach the numbers present at the type 5 stage of growth (Table 2): these numbers of doublings are similar to those estimated for the rat (Hirshfield, 1991) indicating consistency in follicular cell composition in small follicles across species. However, it has been suggested that the current follicular classification system for the sheep, cow, human and rat has limitations since follicular growth from types 1 to 2 or 2 to 3 or 3 to 4 etc each involves two doublings of the population of granulosa cells (McNatty et al., 1999b) and each successive doubling may be under some separate form of regulation. A summary of putative growth and paracrine factors present in sheep follicles with respect to stage of growth
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(follicle type) and cell type are shown in Table 3. The evidence from the sheep and other species, such as the cow and pig, show that oocytes in type 1 or 1a follicles immunostain for a number of growth factors or their receptors including c-kit, SCF, bA and bB activin/ inhibin subunit, follistatin, FGF-2, EGF and TGFb1, 2 and 3 (see McNatty et al., 1999b). However, of these factors, only c-kit mRNA together with GDF-9 mRNA have been unequivocally localised to oocytes whereas some of the other proteins (e.g. SCF, inhibins/ activins, follistatin) are probably synthesised by other cells. In type 2 follicles, SCF mRNA and protein together with the bB and bA activin/inhibin peptide subunits are localised to granulosa cells implicating the activins together with c-kit and GDF-9 in early follicular growth. At the type 2 – 3 stages of growth FSH-R, follistatin and a inhibin mRNA and protein are observed in granulosa cells implicating their involvement in early follicular growth. From the type 4 stage the TGFb mRNA’s and peptides together with LH-R and the cytochrome P450 mRNA’s for side-chain cleavage and 17a-hydroxylase are observed in the theca. These findings suggest that numerous growth factors and receptors together with the gonadotrophin receptors are expressed in early follicular growth in a stage and cell-specific manner.
5. Conclusions The mesonephros in sheep contributes a significant number of somatic cells to the developing ovary. The mesenchymal and mesangial cells of the mesonephros are considered to be a major source of follicular cells. These precursor follicular cells, which branch from the ovarian medulla as ‘cell-streams’, envelop germ cells in the cortex and express SF-1, StAR, 3b-HSD, P450scc and P450aromatase mRNA and/or protein. Most follicles form as either a type 1 or type 1a structure between days 75 and 135 of fetal life. The newly-formed and early growing follicles contain highly variable populations of granulosa cells and oocytes which vary considerably in diameter. An ever increasing number of growth factors and growth factor receptors are being identified in these small follicles and the evidence shows that they are expressed in a cell- and stage-specific manner.
References Bodensteiner, K.J., Clay, C.M., Moeller, C.L., Sawyer, H.R., 1999. Molecular cloning of the ovine growth/differentiating factor-9 gene and expression of growth/differentiating factor-9 in ovine and bovine ovaries. Biol. Reprod. 60, 351–386. Braw-Tal, R., Tisdall, D.J., Hudson, N.L., Smith, P., McNatty, K.P., 1994. Follistatin but not a or bA inhibin subunit mRNA is .
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expressed in ovine fetal ovaries in late gestation. J. Mol. Endocrinol. 13, 1 – 9. Byskov, A.G., 1975. The role of the rete ovarii in meiosis and follicle formation in the cat, mink and ferret. J. Reprod. Fert. 45, 201 – 209. Byskov, A.G., 1986. Differentiation of mammalian embryonic gonad. Physiol. Rev. 66, 71 – 117. Byskov, A.G., Hoyer, P.E., Westergaard, L., 1985. Origin and differentiation of the endocrine cells of the ovary. J. Reprod. Fert. 75, 299 – 306. Fleischman, R.A., 1993. From white spots to stem cells: the role of the kit receptor in mammalian development. Trends Genetics 9, 285 – 289. Grinsted, J., 1982. Influence of mesonephros on foetal and neonatal rabbit gonads. II. Sex-steroid release by the ovary in vitro. Acta Endocr. Copenh. 99, 281 – 287. Hirshfield, A.N., 1991. Development of follicles in the mammalian ovary. Int. Rev. Cytol. 124, 43 – 101. Hirshfield, A.N., De Santini, A.M., 1995. Patterns of ovarian cell proliferation in rats during the embryonic period and the first three weeks post partum. Bio. Reprod. 53, 1208 – 1221. Ito, M., Yu, R., Jameson, J.L., 1997. Dax-1 inhibits SF-1 mediated transactivation via a carboxy-terminal domain that is deleted in adrenal hypoplasia congenita. Mol. Cell. Biol. 17, 1476 –1483. Jost, A., Vigier, B., Prepin, J., Perchellet, J.P., 1973. Studies on sexual differentiation in mammals. Recent Prog. Horm. Res. 29, 1–41. Karl, J., Capel, B., 1998. Sertoli cells of the mouse testis originate from the coelomic epithelium. Dev. Biol. 15, 323 – 333. Keshet, E., Lyman, S.D., Williams, D.E., et al., 1991. Embryonic RNA expression patterns of the c-kit receptor and its cognate ligand suggest multiple functional roles in mouse development. EMBO J. 10, 2425 – 2435. Kreidberg, J., Sariola, H., Loring, J., et al., 1993. WT-1 is required for early kidney development. Cell 74, 679 – 691. Lun, S., Smith, P., Lundy, T., O’Connell, A., Hudson, N., McNatty, K.P., 1999. Steroid contents of and steroidogenesis in vitro by the developing gonad and mesonephros around sexual differentiation in fetal sheep. J. Reprod. Fertil. 114, 131 – 139. Lundy, T., Smith, P., O’Connell, A., Hudson, N.L., McNatty, K.P., 1999. Populations of granulosa cells in small follicles of the sheep ovary. J. Reprod. Fertil. 115, 251 – 262. Luo, X., Ikeda, Y., Parker, K., 1994. A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77, 481 – 490. McNatty, K.P., Smith, P., Hudson, N.L., Heath, D.A., Tisdall, D.J., O, W-S., Braw-Tal, R., 1995. Development of the sheep ovary during fetal and early neonatal life and the effect of fecundity genes. J Reprod Fertil 49, 123 – 135. McNatty, K.P., Quirke, L., Lun, S., Smith, P., Lundy, T., Tisdall, D., 1998. Ontogeny of steroidogenesis in the fetal sheep ovary around sexual differentiation. Proceedings of the Thirtyfirst Annual Meeting of the Society for the Study of Reproduction, Texas, Abstract 450. McNatty, K.P., Quirke, L.D., Fidler, A.E., Smith, P., Heath, D.A., Tisdall, D., 1999a. Ovarian development: fetus to puberty. In: Salamonsen, L.A. (Ed.) Hormones and Women’s Health. Gordon and Breach Science Publishers, Sydney, in press. McNatty, K.P., Heath, D.A., Lundy, T., Fidler, A.E., Quirke, L., O’Connell, A., Smith, P., Groome, N., Tisdall, D.J., 1999b. Control of early ovarian follicular development. J Reprod Fertil Suppl. 54, 3 – 16. Payen, E., Pailhoux, E., Merhi, R.A., et al., 1996. Characterization of ovine SRY transcript and developmental expression of genes involved in sexual differentiation. Int. J. Dev. Biol. 40, 567–575. Peters, H., 1978. Folliculogenesis in mammals. In: Jones, R.E. (Ed.), The Vertebrate Ovary. Plenum Press, New York, pp. 121–144.
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Smith, P., O, W-S., Hudson, N.L., Shaw, L., Heath, D.A., Condell, L., Phillips, D.J., McNatty, K.P., 1993. Effects of the Booroola gene (FecB) on body weight, ovarian development and hormone concentrations during fetal life. J Reprod Fertil 98, 41–54. Smith, P., O, W-S., Corrigan, K.A., Smith, T., Lundy, T., Davis, G.H., McNatty, K.P., 1997. Ovarian morphology and endocrine characteristics of female sheep fetuses that are heterozygous or homozygous for the Inverdale prolificacy gene (FecX1). Biol Reprod 57, 1183 – 1192. Sweeny, T., Saunders, P.T.K., Millar, M.R., Brooks, A.N., 1997. Ontogeny of anti-Mullerian hormone, 3b-hydroxysteroid dehy-
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drogenase and androgen receptor expression during ovine fetal gonadal development. J. Endocrinol. 153, 27 – 32. Tisdall, D.J., Watanabe, K., Hudson, N.L., Smith, P., McNatty, K.P., 1995. FSH receptor gene expression during ovarian follicle development in sheep. J. Mol. Endocrinol. 15, 273 – 281. Tisdall, D.J., Fidler, A.E., Smith, P., et al., 1999. Stem cell factor and c-kit gene expression and protein localisation in the sheep ovary during fetal development. J. Reprod. Fertil. 116, 277 – 291. Zamboni, L., Bezard, J., Mauleon, P., 1979. The role of the mesonephros in the development of the sheep fetal ovary. Ann. Biol. Anim. Biochim. Biophys. 19, 1153 – 1178.
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Growth and paracrine factors regulating follicular formation and cellular function K.P. McNatty *, A.E. Fidler, J.L. Juengel, L.D. Quirke, P.R. Smith, D.A. Heath, T. Lundy, A. O’Connell, D.J. Tisdall 1 AgResearch, Wallace6ille Animal Research Centre, PO Box 40063, Centre Ward Street, Upper Hutt, New Zealand Received 25 June 1999; accepted 20 October 1999
Abstract The purpose of this paper is to review, using fetal sheep as the animal model, aspects of ovarian development related to follicular formation and to report on the identity of growth and paracrine factors which might be involved in this process. Before follicular formation there is a massive and sustained colonisation of the fetal ovary by mesonephric cells, which become a precursor source of follicular cells. From within the ovarian medulla, somatic ‘cell-streams’ branch into the cortex around nests of oogonia and oocytes. These ‘cell-streams’, which contain elongated cells with either flattened or cuboidal shaped nuclei, express steroidogenic factor-1 (SF-1), steroid acute regulatory protein (StAR), 3b-hydroxysteroid dehydrogenase (3b-HSD), cytochrome P450scc, and P450aromatase mRNA and/or protein. Follicles form from the association of an oocyte with the ‘cell-stream’ with either a single layer of flattened cells (i.e. type 1 follicle) or with a mixture of flattened and cuboidal cells (i.e. type 1a follicle). These newly-formed follicles have between 3 and 57 somatic cells (i.e. granulosa cells) and contain oocytes which vary in diameter between 23 and 52 mm. Newly formed and early growing follicles have been identified with growth factors or growth factor receptors in either the oocytes or granulosa cells. Many of the growth factors are from the TGFb superfamily and are expressed in a cell- and stage-specific manner. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cell streams; SF-1; StAR; 3b-HSD
1. Introduction Development of the human ovary begins some 3–4 weeks after conception. Many key events in ovarian development such as germ cell migration, gonadal sex differentiation, germ cell mitosis and atresia, and follicular formation are initiated and completed during fetal life. Other key events such as germ cell meiosis and follicular growth and atresia, which occur throughout the prepubertal and reproductive years, are also initiated during fetal life. Whilst the timing of these events has been determined in the human, knowledge relating to function is largely derived from animal models such
* Corresponding author. Tel.: +64-4-5286089; fax: +64-45281380. E-mail address: [email protected] (K.P. McNatty). 1 Present address: National Centre for Disease Investigation, Ministry of Agriculture and Forestry, PO Box 40742, Upper Hutt, New Zealand.
as genetically-altered (mutant) rodents, rhesus monkeys and farm animals. The purpose of this paper is to review, using fetal sheep as the animal model, aspects of ovarian development related to follicular formation, and to report on the identity of growth and paracrine factors which might be involved in this process.
2. Gonadal development before follicular formation in fetal sheep (i.e. days 23–75 of fetal life)
2.1. Sequence of e6ents and morphology The gonad is first evident as a thickening of the coelomic epithelium on the medial aspect of the mesonephros at days 23–24 of fetal life (Fig. 1). As in other species, the gonad evolves from at least four different tissues or cell-types namely: (1) the coelomic epithelium; (2) the mesenchyme of the mesonephros; (3)
0303-7207/00/$ - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S0303-7207(99)00235-X
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Fig. 1. Photomicrographs showing aspects of early ovarian and follicular formation in fetal sheep. (a) The mesonephros (m) and site of gonadal formation, namely the coelomic epithelium ( ) at day 23 of gestation; (b) the prominent surface epithelium of the gonad (g) 2 – 3 cell layers thick ( ) with mesonephros (m) and glomerulii (gl) at day 28 of gestation; (c) the surface epithelium ( ) of the ovary (o) at day 40 of gestation. The mesonephros (m) is shown on the left; (d) evidence of the cell-continuum between the glomerulii (gl) of the mesonephros and ovary (o) at day 35 of gestation; (e) a ‘cell-stream’ ( ) between the ovarian medulla and cortical germ-cell nests ( ) at day 75 of gestation; (f) a higher magnification of the ‘cell-stream, showing elongated cells with flattened nuclei ( ) and cuboidal nuclei ( ) at day 75 of gestation; (g) a ‘cell-stream’ ( ) at follicular formation around oocytes ( ), and adjacent to some type 1 follicles () at day 75 of gestation; (h) ‘cell-streams’ ( ) and type 1 ( ) and type 1a follicles () at day 75 of gestation. Scale bars: a, b, c, 100 mm; d, 200 mm; e, 50 mm; f, h, 20 mm; g, 25 mm.
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cells from the mesonephric glomerulii and tubules (e.g. mesangial, epithelial); and (4) primordial germ cells (PGC) which migrate to the genital ridge from the dorsal endoderm of the yolk sac (Zamboni et al., 1979; Byskov, 1986) (Fig. 1). Between days 23 and 30, the coelomic epithelium of the developing gonad is a very prominent feature with columnar cells up to three layers in thickness (Fig. 1). Thereafter, the surface
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epithelium of the gonad is less prominent with a single layer of cuboidal or flattened elongated cells. In sheep most germ cells migrate into the gonad between days 23 and 35, and by day 40 are mainly in the cortex. A massive and sustained invasion of the gonad by mesonephric cells concomitant with the involution of the giant glomerulii occurs between days 23 and 55 of fetal life (Zamboni et al., 1979, see also Fig. 2). Many
Fig. 2. Contribution of mesonephric cells to ovarian and follicular formation in sheep from days 24 to 75 of fetal life. Germ cells are shown as cells with both cytoplasm and nuclei whereas somatic cells are shown only as cell nuclei.
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Table 1 Dimensions of cell nuclei in cell-streams and rete cell tubules or masses in fetal sheep ovaries. Values are means 9S.E.M. in mma Axis
Major Minor Mean of major and minor axis
Cell nuclei in cell streams
Cell nuclei in rete cell tubules or masses
Flattened
Cuboidal
11.6 9 0.4a 2.69 0.1a 7.19 0.3a
11.5 9 0.4a 5.5 90.4b 8.5 90.2b
8.0 9 0.5b 4.9 9 0.2b 6.5 90.2c
a For each row values with a different alphabetical superscript are different from one another (PB0.05; ANOVA with least significant differences). Values are means from three animals and the mean for each animal was obtained from the measurements of 50 cells.
of the cells in the continuum between the mesonephros and ovary are highly irregular in shape and have pseudopodia which are characteristic of migrating cells. A significant proportion of the early migrating mesonephric cells are of mesenchymal or mesangial lineage (Fig. 2). Later, cells in the continuum between the mesonephros and ovary include increasing proportions of cells from the walls of the glomerulii and tubules. Between days 40 and 75, somatic cells within the ovarian medulla form ‘cell-streams’ that branch out into the cortex and surround nests of oogonia or germ cells undergoing meiosis (Figs. 1 and 2). Cells within the ‘cell-streams’ all appear to be orientated in the same direction and are easily identifiable as they transverse in a seemingly orderly fashion from the medulla to the cortex. These mesonephric-derived ‘cell-streams’ contain elongated cells, which when examined, by optical sectioning, consist of two distinct types, one with a flattened cell nuclei and the other with cuboidal nuclei. The dimensions of the cell nuclei are shown in Table 1. The nuclei of the flattened cells were similar in length to the nuclei of the cuboidal cells but only half the thickness (i.e. the minor axis, Table 1). It remains to be determined whether cells with cuboidal and flattened nuclei are of different or common origin.
2.2. C-kit and stem cell factor (SCF) Studies of mutant mice strains with a phenotype arising from aberrant migrations of germ cells led to the discovery that the ligand/receptor pair, SCF/c-kit plays a central role in the migration of germ cells to, and colonisation of, the developing genital ridge (Fleischman, 1993). It seems that concentration gradients of SCF from somatic cells interacting with c-kit on germ cells have a major influence on their directional migration (Keshet et al., 1991). In sheep at 23 to 26 days of fetal life, SCF mRNA and protein are present in somatic cells of the mesonephros and gonad. However, by day 30, SCF mRNA and protein become increasingly restricted to the cortical regions of the ovary where germ cells are also present as proliferating oogonia (Tisdall et al., 1999). By day 30 few, if any, of the mesonephric cells or those that are part of the contin-
uum of cells connected to the ovarian medulla expressed SCF mRNA or protein. In contrast to SCF, c-kit mRNA and protein were identifiable in both primordial germ cells and somatic cells of the gonad at 23 to 26 days of fetal life. Thereafter, from day 28 c-kit expression was confined largely to germ cells, most of which migrate to the cortical region of the ovary and adjacent to cells expressing SCF (Tisdall et al., 1999).
2.3. Wilms tumour-1 (WT-1) and steroidogenic factor-1 (SF-1) In WT-1 or SF-1 null mice, gonadal development is inhibited indicating that the expression of these two genes is critical for differentiation of the gonadal ridge (Kreidberg et al., 1993; Luo et al., 1994). In sheep, both SF-1 and WT-1 mRNA have been identified by in situ hybridisation or RT-PCR to be present in the gonad shortly after the thickening of the coelomic epithelium, and thereafter in both ovaries and testis throughout fetal life (Payen et al., 1996; McNatty et al., 1998). Between days 26 and 55, SF-1 mRNA and/or protein was identified in somatic cells in both the cortical and medullary regions of the gonad. It has been proposed that SF-1 protein regulates the production of gonadal aromatase and steroid hydroxylases (Ito et al., 1997). In fetal sheep, SF-1 expression in the gonad occurs some 10–15 days before the expression of steroidogenic regulatory genes such as steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage enzyme (P450scc), P450 17 alpha hydroxylase (P45017OH), and P450 aromatase (P450arom) (McNatty et al., 1998). In addition, SF-1 gene expression is more widespread in the gonad than has been noted for the genes involved in synthesis of steroid suggesting other roles for SF-1 in addition to the regulation of steroidogenesis.
2.4. Genes in6ol6ed in steroid synthesis In the sheep fetus, mRNA of genes involved in steroidogenesis (i.e. StAR, P450scc, P45017OH and P450arom) as well as the steroids themselves are present in the ovary around morphological sex differentiation,
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i.e. days 32–35 (Payen et al., 1996; Sweeny et al., 1997; McNatty et al., 1998; Lun et al., 1999): a punctuate pattern of gene expression was observed in ovarian somatic cells in the medulla and innermost regions of the cortex. Some of the somatic cells expressing the steroid regulatory genes are in close proximity to nests of oogonia and appear to be part of the ‘cell-streams’. Cells in these regions are likely to have migrated into the ovary after day 28 since they do not express SCF. In agreement with Zamboni et al. (1979) and Byskov et al. (1985), we consider these somatic cells to be mesonephric in origin and to include cells of mesenchymal and/or mesangial lineage. We hypothesise that these somatic cells synthesise steroids around the time of sexual differentiation of the gonad (i.e. some 12 days after gonadal formation) and during early follicular formation. Under in vitro conditions, mesonephrii and ovaries isolated and separated between days 35 and 55 each have a capacity to synthesise progesterone, androstenedione and oestradiol (Lun et al., 1999). Thus, the finding that the mesonephros is capable of synthesising steroid is consistent with morphological evidence that mesonephric cells are a potential source of follicular cells (see Grinsted, 1982). In some species it is considered that the surface epithelium is a contributor of follicular cells (see Peters, 1978; Byskov, 1986; Karl and Capel, 1998). However, in sheep, cells of the surface epithelium after day 30 of fetal life show no morphological evidence of invaginating or traversing down between the nests of oogonia either before or during follicular formation. Moreover, while expressing SCF mRNA, cells from the surface epithelium do not express StAR or any of the P450 steroid enzyme mRNA’s. Finally, the first oogonia to enter meiosis and the first follicles to form and grow are those where contact with the ‘cell-streams’ are first made in the innermost regions of the ovarian cortex as described for other species (Byskov, 1975). Thus, it seems unlikely that the surface epithelium is a significant contributor of follicular cells in sheep. Jost et al. (1973) have shown that ovarian steroids are not required for expression of the female phenotype. Nevertheless, the spatial and temporal pattern of steroidogenesis during gonadal development raises the possibility that intraovarian steroids are important for the developmental organisation and/or maturation of both the somatic and germ cells. It will be of interest to determine the location of steroid receptors in the developing ovary before follicular formation. 3. Gonadal events associated with follicular formation and growth in fetal sheep (i.e. days 75 – 135 of fetal life)
3.1. Sequence of e6ents and morphology By day 75 the mesonephros is at an advanced stage
15
of involution and no longer contributes cells to the ovary (Zamboni et al. 1979). In the outer cortex large numbers of oogonia are present either in small nests or as individuals whereas in the mid-cortex numerous nests of oogonia are surrounded by mesonephricderived ‘cell-streams’ with some oogonia undergoing meiosis. Further into the cortex, large numbers of oocytes are in the process of follicular formation or in newly-formed follicles (Figs. 1 and 2). Some of the newly formed follicles consist of a single layer of flattened cells around the oocyte (i.e. type 1 follicles, Lundy et al., 1999) whereas others consist of a single layer of somatic cells which are a mixture of cells with either flattened or cuboidal nuclei (i.e. type 1a follicles; Lundy et al., 1999; Table 1): these two cell-types, namely those with flattened or cuboidal nuclei, are derived from the ‘cell-streams’ (Fig. 2). At day 75 a prominent mass of epithelial cells can be identified as part of a tubular system in the hilus region of the ovary but these are not a feature of the somatic cell population in the ovarian cortex. Zamboni et al. (1979); Fig. 2) suggest that these epithelial cells are from the capsule of the glomerulii, the walls of the capillaries and tubules and that they reach the ovary by mitosis as well as migration. To distinguish these mesonephric-derived epithelial cells from the ‘cellstreams’ we refer to the former as rete cell tubules or masses. When compared to cells of the ‘cell-streams’, the nuclei of the rete cell tubules or cell masses have distinctly different dimensions (Table 1), are more homogenous and as will be noted later develop different functional characteristics. By day 90 about 65% of the newly formed follicles are present as type 1 follicles and 35% as type 1a structures (unpublished data). By day 120 several type 2, 3 and 4 follicles are observed in the innermost regions of the cortex and by day 135 some follicles are noted at the type 5 stage of development (Table 2; McNatty et al., 1995; Lundy et al., 1999). Between days 90 and 135, somatic ‘cell-streams’ can be traced from the medulla traversing around follicles and nests of oocytes or oogonia right up to the surface epithelium. Also between day 90 and 105 a dramatic increase is noted in the extent and number of rete cell tubules or masses which have branched from the medulla into the cortex. Examination of individual 5 mm sections revealed that most growing follicles are in close proximity to the rete cell tubules or masses (Fig. 2). Hitherto, this close association was misinterpreted by Tisdall et al. (1999) and McNatty et al. (1999a) to mean that this composition of mesonephric-derived cells were a source of granulosa cells. However, with respect to the first forming follicles between days 75 and 90, it is clear that a different composition of mesonephric-derived cells (e.g. the ‘cell-streams’) precede the rete cell tubules or
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masses into the ovarian cortex; thus, the involvement of the latter in follicular formation remains obscure. One possibility is that the rete cell tubules or masses may contribute cells to the theca interna and interstitium as well as paracrine factors important for the initiation of follicular growth.
3.2. Factors associated with follicular formation During the period of follicular formation in sheep (i.e. between days 75 and 135) SCF protein, but not mRNA, is present in somatic cells around oocytes. The SCF antibody used in this study detects both soluble and membrane forms of SCF (Tisdall et al., 1999). Thus, the presence of SCF protein but not RNA might be due to soluble SCF originating from a distant site, to a longer half-life of the protein relative to the message or differences in sensitivities between immunohistochemistry and in situ hybridisation. C-kit protein and mRNA are present in oocytes during follicular formation (Tisdall et al., 1999). Growth differentiating factor9 (GDF-9) mRNA is present in oocytes of newlyformed type 1 and 1a ovine follicles at day 135 of gestation although it remains to be established whether GDF-9 mRNA is present in newly-formed follicles between days 75 and 135 and/or in oocytes during follicular formation (Bodensteiner et al., 1999, personal communication). The ovary continues to be steroidogenically active
during the period of follicular formation since oestradiol was produced in measurable quantities (Smith et al., 1993). Moreover, under serum-free conditions, the ovary is capable of synthesising steroids in vitro (e.g. androstenedione and oestradiol; unpublished data). Immunohistochemical evidence reveals that at least some cells within cell-streams, rete cell tubules or masses and the membrana granulosa of newly formed follicles contain immunoreactive SF-1, StAR and 3b-HSD indicating that these cells have a potential to synthesise steroids (Fig. 3; see also Byskov et al., 1985). Studies with fetal sheep ovaries recovered between days 90 and 135 of gestation were unable to show the presence of follicle-stimulating hormone receptor (FSHR), luteinising hormone receptor (LH-R), follistatin or a, bA and bB inhibin/activin subunit mRNAs in ‘cellstreams’ adjacent to oocytes at follicular formation. However, follistatin and bB inhibin subunit mRNA were localised in most sections to the rete cell tubules or masses (Braw-Tal et al., 1994; Tisdall et al., 1995, unpublished data). The evidence suggests that the mesonephric-derived ‘cell-streams’ precede the rete cell tubules or masses as a precursor source of follicular cells during follicular formation. Cells in the ‘cell-streams’ and around the newly-formed follicles (i.e. granulosa cells) immunostain for SF-1, StAR and 3b-HSD supporting the notion that the former are the precursor follicular cells. The
Table 2 Characteristics of small ovine folliclesa Follicle (type)
Layers of granulosa cells
Primordial (type 1) 1 (all flattened) Transitory (type 1a) 1 (1 or more cuboidal cells) Primary (type 2) 1–B2 (all cuboidal cells) Small preantral 2–B4 (type 3) Large preantral 4–B6 (type 4) Small antral (type \5 5)
Total number of granulosa cells
Oocyte diameter in mm
Follicular diameter in mm
Presence of theca interna%
16b (3,52) 39c (9,136)
35b (23,52) 41c (27,53)
41b (21,61) 51c (37,64)
0 0
128d (30,520)
52d (31,80)
75d (50,119)
35
637e (127,2174)
73e (41,92)
129e (64,191)
100
2104f (1090,3404)
88f (77,100)
194f (164,256)
100
11649g (3425,51447)
119g (91,142)
327g (192,450)
100
a Data from Lundy et al. (1999). Values for granulosa cells, oocyte diameter and follicular diameter represent means and ranges. For each column separately, values with different alphabetical superscripts are different from one another (PB0.01).
Fig. 3. Immunostaining for steroidogenic factor-1 (SF-1), steroid acute regulatory protein (StAR) and 3b-hydroxysteroid dehydrogenase (HSD) during follicular formation and early follicular growth in the sheep ovary: (a) and (b) SF-1 localised to cells of the ‘cell-streams’ ( ) or granulosa cells of type 1, 1a or type 3 follicles (); (c) and (d) StAR immunolocalised to cells of the ‘cell-streams’ ( ) and oocytes () during follicular formation; (e) 3b-HSD localised to ‘cell-streams’ ( ) or granulosa cells of newly-formed follicles ( ) and; (f) 3b-HSD localised to granulosa cells ( ) of type 1 and type 2 follicles and also the oocytes. The SF-1 antibody (Catalogue No. 06-431) was obtained from Upstate Biotechnology, Lake Placid, New York, and used at a dilution of 0.5 mg/ml (w/v). The StAR antibody was generously supplied by Dr D. Stocco, Texas Tech University, Health Sciences Center, Texas and used at a dilution of 1:1000 (v/v). The 3b-HSD (5683) was generously supplied by Dr I. Mason, Department of Clinical Biochemistry, University of Edinburgh, Scotland and used at a dilution of 1:250 (v/v). The immunohistochemical procedures were those described in Tisdall et al., 1999. Scale bars for a, b, d, e and f, 43.5 mm; c, 87.0 mm.
K.P. McNatty et al. / Molecular and Cellular Endocrinology 163 (2000) 11–20
Fig. 3.
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Table 3 Expression of growth factors and receptors during early follicular growth in sheepa
a , mRNA, , protein, , protein observed only in zona pellucida. Act/inh, activin/inhibin. 1Observed in oocytes of all follicles types. 2At the type 1 and 1a stage respectively 23 and 36% of all follicles contain one or more granulosa cells immunostaining for bB act/inh whereas in type 2 to 4 follicles virtually all follicles immunostain for bB act/inh subunit. 3TGFb1,2,3 present in theca interna from follicle type 4 stage of growth. 4 LH-R mRNA present in theca interna of type 4 follicles. Modified from McNatty et al. (1999b) and Bodensteiner et al. (1999).5No data.
presence of SCF protein in the absence of SCF mRNA in the ‘cell-streams’ and in cells of newly-formed follicles suggests possible differences in sensitivities between the in situ hybridisation and immunohistochemical techniques, that the SCF protein degradation rate is very slow or that the soluble ligand has been derived from another cellular source. It seems unlikely however that the follicular cells are from the surface epithelium since the latter contains both SCF mRNA and protein. Moreover, cells of the surface epithelium do not show evidence that they are steroidogenic. The rete cell tubules or masses, which later become closely associated with follicles, also display characteristics of steroidogenic cells. However, the rete cell tubules or masses can be distinguished from the ‘cell-streams’ by the finding that both follistatin and bB inhibin/activin subunit mRNA have been identified in the rete cell tubules or masses.
4. Characteristics of newly formed follicles The characteristics of newly-formed and small follicles with respect to number of granulosa cells and diameter of oocyte etc are summarised in Table 2. For each follicular type, including type 1 and 1a follicles, the total populations of granulosa cells together with oocyte diameters are highly variable. It is possible that
the variability in number of granulosa cells may, in part, be related to both the diameter of the oocyte at follicular formation and the proliferative activity of the somatic cells when the follicle is forming (Hirshfield and De Santini, 1995). In fetal sheep, oogonia have diameters between 13 and 17 mm and isolated oocytes, diameters between 17 and 22 mm (Smith et al., 1997) whereas after follicular formation oocyte diameters are between 23 and 53 mm (Table 2). These results infer that growth of germ cells begins before and continues during as well as after follicular formation. It can be calculated from a knowledge of the total populations of granulosa cells in types 1, 1a, 2, 3, 4 and 5 follicles that types 1 and 1a follicles undergo 9 and 8 doublings of their populations of granulosa cells respectively to reach the numbers present at the type 5 stage of growth (Table 2): these numbers of doublings are similar to those estimated for the rat (Hirshfield, 1991) indicating consistency in follicular cell composition in small follicles across species. However, it has been suggested that the current follicular classification system for the sheep, cow, human and rat has limitations since follicular growth from types 1 to 2 or 2 to 3 or 3 to 4 etc each involves two doublings of the population of granulosa cells (McNatty et al., 1999b) and each successive doubling may be under some separate form of regulation. A summary of putative growth and paracrine factors present in sheep follicles with respect to stage of growth
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(follicle type) and cell type are shown in Table 3. The evidence from the sheep and other species, such as the cow and pig, show that oocytes in type 1 or 1a follicles immunostain for a number of growth factors or their receptors including c-kit, SCF, bA and bB activin/ inhibin subunit, follistatin, FGF-2, EGF and TGFb1, 2 and 3 (see McNatty et al., 1999b). However, of these factors, only c-kit mRNA together with GDF-9 mRNA have been unequivocally localised to oocytes whereas some of the other proteins (e.g. SCF, inhibins/ activins, follistatin) are probably synthesised by other cells. In type 2 follicles, SCF mRNA and protein together with the bB and bA activin/inhibin peptide subunits are localised to granulosa cells implicating the activins together with c-kit and GDF-9 in early follicular growth. At the type 2 – 3 stages of growth FSH-R, follistatin and a inhibin mRNA and protein are observed in granulosa cells implicating their involvement in early follicular growth. From the type 4 stage the TGFb mRNA’s and peptides together with LH-R and the cytochrome P450 mRNA’s for side-chain cleavage and 17a-hydroxylase are observed in the theca. These findings suggest that numerous growth factors and receptors together with the gonadotrophin receptors are expressed in early follicular growth in a stage and cell-specific manner.
5. Conclusions The mesonephros in sheep contributes a significant number of somatic cells to the developing ovary. The mesenchymal and mesangial cells of the mesonephros are considered to be a major source of follicular cells. These precursor follicular cells, which branch from the ovarian medulla as ‘cell-streams’, envelop germ cells in the cortex and express SF-1, StAR, 3b-HSD, P450scc and P450aromatase mRNA and/or protein. Most follicles form as either a type 1 or type 1a structure between days 75 and 135 of fetal life. The newly-formed and early growing follicles contain highly variable populations of granulosa cells and oocytes which vary considerably in diameter. An ever increasing number of growth factors and growth factor receptors are being identified in these small follicles and the evidence shows that they are expressed in a cell- and stage-specific manner.
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