Growth and paracrine factors regulating follicular formation and cellular function.

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), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450(scc), and P450(aromatase) 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 microm. 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 TGFbeta superfamily and are expressed in a cell- and stage-specific manner.

Populations of granulosa cells in small follicles of the sheep ovary.

The aim of this study in sheep ovaries was to determine the total number of granulosa cells in primordial follicles and at subsequent stages of growth to early antrum formation. The second aim was to examine the interrelationships among the total number of granulosa cells in the follicles, the number of granulosa cells in the section through the oocyte nucleolus, and the diameter of the oocyte. A third aim was to examine whether proliferating cell nuclear antigen labelling occurred in flattened granulosa cells in primordial follicles or was confined to follicles containing cuboidal granulosa cells. The follicles were classified using the section through the oocyte nucleolus by the configuration of granulosa cells around the oocyte as type 1 (primordial), type 1a (transitory), type 2 (primary), type 3 (small preantral), type 4 (large preantral), and type 5 (small antral). In type 1 follicles, the number of granulosa cells and oocyte diameter were highly variable in both fetal and adult ovaries. Each type of follicle was significantly different from the others (all P < 0.05) with respect to oocyte diameter, number of granulosa cells in the section through the oocyte nucleolus and total number of granulosa cells. Follicles classified as type 2, 3, 4 or 5 each corresponded to two doublings of the total granulosa cell population. The relationships between oocyte diameter and the number of granulosa cells (that is, in the section through the oocyte nucleous or total population per follicle) could all be described by the regression equation loge chi = a + b loge gamma with the correlation coefficients R always > 0.93. For each pair of variables the slopes (b) for each type of follicle were not different from the overall slope for all types of follicle pooled. Immunostaining for proliferating cell nuclear antigen was observed in granulosa cells in type 1 follicles, as well as in the other types of follicle. These findings indicate that 'flattened' granulosa cells in type 1 follicles express an essential nuclear protein involved in cell proliferation before assuming the cuboidal shape. Thus, when considering factors that regulate specific phases of early follicular growth, it is important to consider: (i) the follicle classification system used; (ii) the animal model studied; (iii) whether type 1 follicles are all quiescent; and (iv) the likelihood that each follicle type represents more than one doubling of the population of granulosa cells.

Control of early ovarian follicular development.

Early follicular growth refers to the development of an ovarian follicle from the primordial to early antral phase. In sheep and cows these phases of growth can be classified by the configuration of granulosal cells in the largest cross-section of the follicle as types 1 (primordial), 1a (transitory) 2 (primary), 3 and 4 (preantral) and 5 (early antral). Follicles classified as type 1 may be highly variable within each species with respect to number of granulosal cells and diameter of oocyte. Much of the variation in granulosal cell composition of type 1 follicles may occur at formation and this may account for the variability in granulosal cell composition throughout subsequent stages of growth. There appear to be important differences among species (for example sheep and cattle) in the number and function of granulosal cells relative to the diameter of the oocyte during the initiation of follicular growth. There is evidence that most, if not all, of the growth phases from types 1 to 5 are gonadotrophin-independent and that follicles develop in a hierarchical manner. In sheep, cows and pigs, numerous growth factor, growth factor receptor and gonadotrophin receptor mRNAs and peptides (for example c-kit, stem cell factor, GDF-9, beta B and beta A activin/inhibin subunit, alpha inhibin subunit, follistatin, FGF-2, EGF, EGF-R, TGF beta 1,2 and 3 FSH-R and LH-R) are expressed in a phase of growth (for example types 1-5)-specific and cell-specific manner. However, the roles of many of these factors remain to be determined.

Steroid contents of and steroidogenesis in vitro by the developing gonad and mesonephros around sexual differentiation in fetal sheep.

The aim of the present study was to establish whether the steroids, progesterone, androstenedione, testosterone and oestradiol, were present in the mesonephric-gonadal complex of female and male sheep fetuses around sexual differentiation (that is, from day 28 to day 45 of gestation, with sexual differentiation occurring at approximately day 32). A second aim was to test whether the mesonephric-gonadal complex, mesonephros (days 35-45 only) and gonad (days 35-45 only) were capable of steroid synthesis in vitro. The steroid contents in the mesonephric-gonadal complex were not detectable before sexual differentiation. However, from day 35 of gestation onwards, the mesonephric-ovarian complex contained mainly oestradiol and the mesonephric-testicular complex contained mainly testosterone: from day 35 until day 45 the increase in content of these two steroids exceeded the increase in the mass of tissue by more than fivefold. From day 40 to day 45 of gestation, the contents of the other steroids in the pathways to oestradiol increased progressively in both sexes but more in parallel with the increase in tissue mass. In contrast to the steroid contents in the tissue at recovery, the mesonephric-gonadal tissue from both sexes in tissue culture was able to synthesize most steroids before and after sexual differentiation and also to metabolise supplementary androstenedione to oestradiol. These findings suggest that many, if not all, of the steroidogenic enzymes in the pathway from cholesterol to oestradiol are present before sexual differentiation. Most of the aforementioned steroids were present in detectable amounts in isolated mesonephros and gonad of both sexes after sexual differentiation. Moreover, for both the isolated mesonephros and gonad, there were increases in the mean contents of most steroids after culture relative to the contents in the tissues at recovery. These data suggest that the mesonephros, as well as the gonad, in both sexes is capable of synthesizing steroid. It is concluded that, in the sheep fetus, the female and male gonads are steroidogenically active after sexual differentiation, that the steroidogenic enzymes develop before sexual differentiation, and that the mesonephros is a site of steroid synthesis.

Ovarian morphology and endocrine characteristics of female sheep fetuses that are heterozygous or homozygous for the inverdale prolificacy gene (fecX1).

The Inverdale gene (fecX1), located on the X chromosome, is a major gene affecting the ovulation rate of sheep. At each ovulation, ewes heterozygous (I+) for the fecX1 gene ovulate, on average, one more egg than noncarriers (++), whereas ewes that are homozygous (II) for this gene are infertile and have "streak" ovaries. Since formation of the ovary occurs in fetal life, it is possible that the fecX1 gene influences ovarian development before birth. The aims of this study were to examine the effects of the fecX1 gene on germ cell development, follicular formation and growth, and plasma gonadotropin concentrations at 5 different days of gestation (i.e., Days 40, 90, 105, 120, and 135) and also in adult life. The results suggest that one copy of the X-linked mutation in female fetuses leads to a retardation of germ cell development at Days 40 and 90 of gestation. However, from Day 105 of gestation, follicular formation and growth appear normal. By contrast, in females with two copies of the X-linked mutation, germ cell development and follicular formation appear normal, but thereafter follicular growth from the primary stage of development is impaired. During fetal life the plasma concentrations of FSH and LH, although not measurable at Day 40, were similar between all the genotypes at Day 105, 120, and 135 of gestation. The only exception was for LH at Day 90 in the I+ and II animals: in ewes with these genotypes the plasma concentrations of LH were similar but significantly lower (p < 0.01) than in the ++ genotype. In adult animals the plasma concentrations of FSH and LH were not different between the ++ and I+ genotypes, reflecting similar levels of ovarian follicular activity. However, in adult II animals, the plasma concentrations of FSH and LH were significantly higher (both p < 0.01) than in the ++ and I+ genotypes, reflecting the absence of normal secondary and antral follicles. In summary, these data show that the fecX1 gene affects ovarian development before birth and that the nature of the effect is influenced by whether the female fetus is a homozygous or heterozygous carrier of the X-linked mutation.