Immunocytochemical localization of angiotensin II receptor subtypes 1 and 2 in the porcine fetal, prepubertal and postpubertal ovary

Abstract

There is considerable evidence for a mammalian ovarian renin–angiotensin system, which may influence ovulation, angiogenesis and steroidogenesis via the autocrine and/or paracrine actions of the biologically active product of the cascade, angiotensin II (AngII). There are two characterized AngII receptors – type 1, AT₁ and type 2, AT₂. We report the localization of these receptor subtypes within porcine fetal, prepubertal and postpubertal ovaries. Positive staining for AT₁ and AT₂ receptors was observed in egg nests in all fetal ovaries studied, as well as in a defined two-cell layer at the ovarian periphery. In prepubertal tissue, positive AT₁ and AT₂ staining was localized to granulosa cells adjacent to the basement membrane of pre-antral and antral follicles, with no staining in the thecal layer. There was immunostaining for both receptors in prepubertal oocytes and zona pellucida. In postpubertal tissue, positive AT₁ and AT₂ immunostaining was localized to areas of putative neovascularization, the zona pellucida and the oocyte. Further AT₁ staining was located to the postpubertal antral follicle granulosa cells. The results indicate that there are higher densities of AT₁ receptors than AT₂ receptors in the porcine fetal, prepubertal and postpubertal ovary, and this has profound implications for the role of AngII in ovarian development.

Introduction

The renin–angiotensin system (RAS) is classically associated with the regulation of blood pressure and salt balance, particularly in the development of hypertension (Levy, 1998). However, there is an increasing body of evidence to link the RAS with physiological mechanisms in other tissues, including the reproductive tract and the mammalian ovary (for reviews see Robertson, 1993; Nielsen et al. 1995a; Vinson et al. 1997). Individual components of the RAS cascade have been identified within the porcine ovary. Both prorenin and renin have been shown to be present in porcine follicular fluid (Hagemann et al. 1992; Nielsen et al. 1995b), and follicular fluid renin levels decreased with increasing follicle size and animal maturity (Shuttleworth, 2000). However, the principal biologically active product of the RAS is the octapeptide angiotensin II (AngII). This molecule has been shown to inhibit the transcription of steroidogenic enzymes in porcine granulosa cells, which indicates a role for AngII in porcine ovarian function (Li et al. 1995). Additionally, AngII has been demonstrated to have a role in renal development (Grone et al. 1992), glomerular growth (Fogo & Ichikawa, 1991) and the long-term control of renal function (Woods & Rasch, 1998). As, embryologically, the ovary is formed from tissue that also develops to form the kidney, it may be hypothesized from these studies that the RAS has an active role in fetal ovarian development and function, and may have implications for subsequent ovarian function.

AngII acts through specific receptors of which two have been classified by the use of selective antagonists [subtype 1, AT₁ (Chiu et al. 1989; Whitebread et al. 1989), subtype 2, AT₂ (Timmermans et al. 1993)]. AT₁ receptors are principally involved in the regulation of vasoconstriction and salt and fluid balance (Chiu et al. 1989; Whitebread et al. 1989), but are also implicated in vascular smooth muscle proliferation, collagen deposition and angiogenesis (Inagami et al. 1999). AT₂ receptors, however, may play a role in growth regulation and apoptosis (Timmermans et al. 1993). There is a general paucity of information regarding AT₁ and AT₂ receptors within the mammalian ovary, and many reports were not able to differentiate between subtypes. In the rat, autoradiographical studies have demonstrated AngII receptor binding sites on granulosa and theca interna cells although these studies did not differentiate between the two characterized AngII receptor types (Husain et al. 1987; Daud et al. 1988). A study involving the preparation of a membrane fraction to examine ¹²⁵I-AngII binding demonstrated AT₂ receptors in cultured bovine thecal cells (Brunswig-Spickenheier & Mukhopadhyay, 1992). Also from the use of a membrane fraction AngII receptor assay, it has been suggested that there are AngII receptors present in postpubertal porcine follicle wall tissue, although these were not characterized or precisely localized to specific follicular cell types (Nielsen et al. 1995b). An autoradiographical study in our laboratory has indicated that there are both AT₁ and AT₂ receptors within the pre- and postpubertal porcine ovary, although the methodology did not allow the precise localization of these receptors (Shuttleworth et al. 2001).

Immunocytochemistry has been used to trace precisely the expression of AngII receptors to specific cell types in the ovine fetal adrenal gland throughout gestation (Wintour et al. 1999) and rat fetal and newborn kidney (Ozono et al. 1997). There was a large variation in AngII receptor expression observed by these authors throughout development, and so it is appropriate to study porcine ovaries at various stages of development: fetal, prepubertal and postpubertal. Therefore, the aims of this study were two-fold: to confirm and determine the presence of AT₁ and AT₂ receptors in the porcine ovary throughout development, and further to localize these receptor subtypes to specific cell types using immunocytochemistry.

Materials and methods

Tissue examined and preparation

Both ovaries from fetal (n = 3 from two pregnant sows), prepubertal (aged 12–15 weeks; n= 4) and postpubertal (n = 4) pigs (Large White hybrid) were collected from a local abattoir, and transported to the laboratory on ice within 1 h. Follicles within prepubertal ovaries were less than 5 mm in diameter. Postpubertal ovarian samples were selected as being in the follicular phase with several large follicles > 6 mm (Grant et al. 1989), and which therefore presented no active corpora lutea, although corpora albicantia from previous cycles were present. Ovaries were dissected free from surrounding tissue, fixed in neutral buffered formal saline for 72 h, processed and embedded in paraffin. Serial 4-µm-thick sections were cut and duplicate consecutive sections mounted on glass slides precoated with 2.5% (w/v) 3-aminopropyltriethoxysilane (APES – Sigma Chemical Co., Dorset, UK). Slides were stored at room temperature for less than 4 days prior to staining. Porcine adrenal gland was prepared as above for a positive control.

Immunocytochemistry

Immunocytochemistry was completed using the DAKO EnVision+® System (Dako, Cambs., UK). The protocol has been developed from the manufacturer's instructions with the addition of a 15-min 0.5% (v/v) hydrogen peroxide in methanol step to block endogenous peroxidases (Jackson & Blythe, 1993). Antibodies were diluted in 0.25% (w/v) BSA (Sigma Chemical Co.) in 0.05 m Tris buffered saline (pH 7.6). The monoclonal antibody to the AT₁ receptor [6313/G2, a kind gift from Prof G. P. Vinson (Barker et al. 1993)] was used at a dilution of 1 in 10, and sections then incubated with the antimouse IgG secondary antibody polymer received as part of the EnVision+® System kit. A polyclonal anti-AT₂[a kind gift from Dr W. T. Cheung (Yiu et al. 1997)] was used at 1 in 4000 dilution, and sections then incubated with antirabbit IgG secondary antibody polymer. Positive controls for both receptor subtypes comprised paraffin-embedded porcine adrenal gland. The AT₁ and AT₂ antibodies used in this study have been previously tested using absorbed antisera as a control and are specific for AngII (Harrison-Bernard et al. 1997; Cooper, 1999). Therefore, negative controls initially comprised sections of all tissue types (including adrenal gland) processed as above, with substitution of the primary antibody with mouse non-immune serum (anti-AT₁ negative control) or rabbit non-immune serum (anti-AT₂ negative control). As neither serum produced false positive immunoreactivity, all subsequent negative controls comprised Tris buffer containing 0.25% (w/v) BSA replacing receptor subtype antibody in the protocol.

The intensity of immunostaining in the tissue samples studied within the same set of slides examined was compared using a Leica DMRB microscope, and staining was scored from – (no immunostaining present) through to +++ (maximum immunostaining observed). This was verified independently by blind scoring by another person with assessment of between-observer agreement. Slides were taken using Fujichrome Provia colour film (100 ASA) using a MPS 48 photographic unit.

Results

All positive controls stained for either AT₁ or AT₂ receptors, respectively, in the adrenal medulla. All negative controls showed no positive immunostaining. A summary of staining intensity and number of structures observed is presented in Table 1.

Table 1.

Immunostaining in fetal (n = 3) prepubertal (n = 4) and postpubertal (n = 4) ovarian tissue where +++ indicates maximal staining and – represents no staining (NA – not applicable)

Ovarian type	Follicle status	Ovarian structure	No. analysed	AT1 receptors	AT2 receptors
Fetal	(NA)	Egg Nests	(NA)	++	+
	(NA)	Surface Epithelium	(NA)	+++	++
Prepubertal	All	Oocyte	10	+++	+/−
	All	Zona Pellucida	10	+	+
	Preantral	Granulosa Cells	20	+	+/−
	Preantral	Thecal Cells	20	−	−
	Antral	Granulosa Cells	20	+++	+/−
	Antral	Thecal Cells	20	−	−
	(NA)	Blood Vessels	(NA)	+++	−
Postpubertal	All	Oocyte	10	+	+/−
	All	Zona Pellucida	10	++	+
	Antral	Granulosa Cells	20	++	−
	Antral	Thecal Cells	20	−	−
	(NA)	Neovascularization	(NA)	+++	++
	External to follicle	Neovascularization	(NA)	++	++

Fetal ovaries

Positive immunostaining for AT₁ and AT₂ receptors was seen within ‘egg nests’ consisting of a primordial germ cell surrounded by a flat somatic pregranulosa cell component within the cortex of the ovary (Fig. 1a,c) Egg nests are also termed ‘germ cell cords’ (Byskov et al. 1986). Immunostaining was predominantly localized to the pregranulosa cells, and was also present in a clearly defined two-cell layer (the developing porcine ovary is stratified) at the surface epithelium of all ovaries studied (Fig. 1b,d). Staining for AT₁ was more intense than AT₂ in both areas for all ovaries studied.

Fig. 1. Immunocytochemical localization of AT₁ and AT₂ receptors in the porcine fetal (a–d), prepubertal (e–j) and postpubertal (k,l) ovary. Immunostaining for AT₁ is illustrated in parts a, b, e, g, i, k, m, o and against AT₂ in parts c, d, f, h, j, l, n, p. In the fetal ovary, staining is demonstrated at two magnifications per receptor subtype – low (a – AT₁, c – AT₂) and high (b – AT₁, d –AT₂). In prepubertal ovary examples, immunocytochemical localization of AT₁ and AT₂ receptors in antral follicles (e – AT₁, f – AT₂), blood vessels (g – AT₁, h – AT₂) and the oocyte and zona pellucida (i – AT₁, j – AT₂), is illustrated. In the postpubertal ovarian examples, immunocytochemical localization of AT₁ and AT₂ receptors antral follicle wall tissue (k – AT₁, l – AT₂), blood vessels at the edge of a degenerating follicle (m – AT₁, n – AT₂) and in the oocyte and zona pellucida (o – AT₁, p – AT₂) is illustrated. A, antral space; AF, antral space of atretic follicle; E, blood vessel endothelial cell layer; EN, egg nests; G, granulosa cells; M, medulla; O, oocyte; OP, ovary periphery; P, pericytes; S, stromal tissue; T, thecal cells; ZP, zona pellucida. Scale bars = 100 µm.

Prepubertal ovaries

Much denser AT₁ receptor immunostaining was observed in the granulosa cell layer of antral follicles (Fig. 1e) than pre-antral follicles (Fig. 1i,j). This was more intense adjacent to the basement membrane (allowing for variations in staining at the edge of the section and differences in cell sizes) and not seen within the thecal cell layer. AT₁ receptor, but not AT₂ receptor, staining was seen within ovarian blood vessels (Fig. 1g,h). Both AT₁ and AT₂ receptors were present in low levels in granulosa calls but not thecal cells of pre-antral follicles (Fig. 1i,j). Denser AT₁ receptor than AT₂ receptor staining was also observed within the oocyte and zona pellucida.

Postpubertal ovaries

AT₁ receptor staining was observed within granulosa cells in antral follicles (Fig. 1k), as had been seen in prepubertal tissue. Staining was dense and appeared to be slightly denser towards the granulosa thecal cell interface despite prominent edge staining at the internal face of the granulosa cell layer. However, AT₁ receptor immunostaining was seen within thecal cells of early luteinizing follicles (not shown), but not antral follicles. There was no AT₂ staining associated with follicular granulosa or thecal cells at any stage (Fig. 1l,p). Distinct AT₁ and AT₂ receptors were observed in discrete areas of neovascularization resulting from the degeneration of atretic follicles. This was comparable to a non-continuous area external to the follicles adjacent to the follicle edge in follicles displaying the early signs of atresia (sloughing away of the granulosa cell layer and poor cell–cell adhesion: Fig. 1m,n). This appeared to be an area of neovascularization due to the presence of pericytes. Both receptor subtypes were observed within the oocyte and zona pellucida (Fig. 1o,p).

Discussion

Clearly, both receptor subtypes were present within the fetal, prepubertal and postpubertal porcine ovary. AT₁ receptors were present at relatively higher densities than the AT₂ receptor in all tissues studied, and the receptor subtypes were localized to specific ovarian structures. As specific receptor subtype antagonists have only been available since the early 1990s, studies prior to this have reported non-specific AngII receptor types. The ability to examine the precise distribution of specific receptors throughout the tissues studied has implications for understanding the role of angiotensin in folliculogenesis, angiogenesis and steroidogenesis.

The results in fetal tissue offer a novel and unequivocal mechanism to identify precisely the location of discrete egg nests (Mauleon & Mariana, 1977; Christenson et al. 1985). Egg nests consist of a primordial germ cell surrounded by a relatively smaller, flatter granulosa cell component (Byskov et al. 1986). The ability to identify the germ cell which is surrounded by intensely stained pregranulosa cells aids in localization of egg nests within the ovary. An alternative method of identifying germ cells – alkaline phosphatase cytoplasmic staining by the azodic technique – is unsatisfactory due to the asymmetrical distribution of cytoplasmic activity (Mauleon & Mariana, 1977). The use of AngII receptor staining therefore represents a significant advance in the identification of fetal ovarian components.

Differences in the intensity of immunoreactivity for AT₁ and AT₂ were evident in fetal ovaries from animals at different stages of development. The fetuses studied had crown-rump lengths of 10.0 cm, 15.5 cm and 19.5 cm, corresponding to a fetal age of approximately 50, 65 and 75 days, respectively (term at 114 days). There was less intense immunostaining in ovaries from younger fetuses, and the egg nests appeared more clustered. This clustering is expected as germ cells are contained in egg nests until 60 days post conception (p.c.) when primordial follicles begin to form and the percentage of egg nests then progressively decreases (Mauleon & Mariana, 1977; Christenson et al. 1985). A later study more precisely indicates that this occurs at day 50 p.c. and continues until day 76 when the first primordial follicles are formed (Byskov et al. 1986). This period is reflected in the samples that we have studied. Byskov et al. (1986) demonstrate that break-up occurs in the most centrally placed cords and does not extend to those in the cortical periphery where we primarily observe AngII receptors to be localized. Therefore, if AngII were involved in early folliculogenesis, it is not implicated solely in the break-up of the cords as staining was spread throughout the cortical region, but may be unregulated in these areas prior to cord break-up. The increasing intensity of AngII receptor immunostaining with fetal age suggests a role in embryonic ovarian development, such as in tissue remodelling and collagen deposition required during cord break-up with the onset of meiosis, or primordial follicle formation. Further, in the current study, a defined two-cell layer at the periphery of the fetal ovary immunostained for both AT₁ and AT₂ receptors. The ovary grows rapidly during gestation, when the increase in porcine fetal mass is not under gonadotrophin control (Christenson et al. 1985). In situ hybridization studies demonstrated that the AT₂ receptor was expressed in the fetal and newborn rat kidney (Ozono et al. 1997), and, as the fetal kidney and ovary have the same embryological origin, it may be expected that AngII receptors would be present within the fetal ovary. This has been clearly demonstrated in the current study, although both receptor subtypes were present, and it was the AT₁ receptor that dominated. AT₂ receptors have previously been identified in porcine fetal membranes (Nielsen et al. 1996) and other RAS components have been demonstrated in the circulation of mid-term pig fetuses (Broughton Pipkin et al. 1986). This supports a role for the RAS in fetal development as a whole and indicates a specific function in porcine ovarian fetal development.

It appears that the majority of receptors within the porcine ovaries studied from non-fetal animals were localized to follicular granulosa cells and were subtype 1. There was most AT₂ staining in the granulosa cell layer of a subpopulation of follicles (n = 3, data not shown) that had an irregular undulating granulosa cell layer and a number of pyknotic nuclei (∼25%). The granulosa cell layer was sloughing away, implying that these follicles were in the early stages of atresia. AT₂ receptors have also been shown to mediate the progression of follicle atresia through granulosa cell apoptosis by inhibiting FSH actions in immature rats treated with equine chorionic gonadotrophin (Kotani et al. 1999). However, in healthy postpubertal antral follicles there were only AT₁ receptors within granulosa cells, and no staining in thecal cells. Closer examination revealed that the basement membrane appeared to be breaking down which is typically observed after an LH surge and indicates that these specific follicles may be in the early stages of luteinization (Hunter et al. 1989).

Granulosa cells closest to the basement membrane stained more intensely and this could indicate two roles for AngII. Those receptors adjacent to the basement membrane may be involved in fluid transport into the developing follicle aiding antrum formation and/or follicle rupture. AngII has been classically associated with the transport of sodium and water across epithelial tissue (Robertson, 1993) and has also been associated with an increase in the transport of macromolecules across tumour vessels (Netti et al. 1999) so it is possible that uptake of follicular fluid components is stimulated by local production of angiotensin in the porcine ovary. Alternatively, the presence of these receptors indicates a role in steroidogenesis as it has also been shown that granulosa cell steroidogenic activity is most intense adjacent to the basement membrane (Ford & Lunstra, 1992). AngII has been shown to stimulate oestrogen production in the rat (Husain et al. 1987; Pucell et al. 1987; Speth & Husain, 1988), rabbit (Feral et al. 1995; Yoshimura et al. 1996) and cow (Acosta et al. 1999). However, AngII did not affect progesterone production in the rat (Cannon et al. 1997), although in humans it resulted in a dose-dependent increase (Morris et al. 1994). Further, in rats, oestradiol and progesterone themselves did not influence granulosa cell AngII receptor expression (Pucell et al. 1988). This indicates a marked species difference in the function of AngII in steroid synthesis. In cultured porcine granulosa cells, AngII reduced LH-induced progesterone accumulation and abrogated LH-induced increases in 3β-hydroxysteroid dehydrogenase (3β-HSD) protein and mRNA (Li et al. 1995). From the current study, AngII is bound to receptors in the granulosa cell layer of animals in the follicular phase and therefore potentially affects oestradiol synthesis in the steroid cascade. A recent study, however, demonstrated that AngII stimulated progesterone secretion by porcine pre-antral follicles with a theca cell layer cultured in vitro (Shuttleworth et al. 2002). Clearly the role of AngII in steroidogenesis is complicated and varies between species.

The ability to produce steroids differentiates between healthy and atretic follicles. Atretic follicles in the rat possess AngII receptors, although whether this is causative or a result of atresia is unclear (Husain et al. 1987; Daud et al. 1988). This could imply that follicles in the current study expressing AngII receptors were atretic, but this was not the case because receptors were present in follicles with a healthy morphology. In cattle, high levels of AngII receptors were also not confined to atretic follicles, but were localized to the thecal cell layer and corpora lutea (Nielsen et al. 1995b; Hayashi et al. 2000). Studies in rats have associated AngII, acting via AT₁ receptors, with tissue remodelling (Lefroy et al. 1996; Makino et al. 1996), such as associated with the degradation of atretic follicles or in corpora lutea formation. Results from the current study do not disprove a role for AngII in the luteal phase, or in regulation of atresia, but immunostaining in prepubertal tissue suggests an alternative or non-cyclical role as well. Further, both receptor subtypes were strongly associated with the fetal ovaries, so, although there is some atresia associated with fetal ovaries, it is unlikely that regulation of atresia is the sole function of AngII within the porcine ovary. Moreover, the predominant receptor subtype observed – AT₁– is not usually associated with apoptosis.

Additionally, staining localized to oocytes demonstrates AT₁ and AT₂ receptor expression varies with animal maturity. AngII has been linked to oocyte maturation and ovulation in rats (Peterson et al. 1993) and rabbits (Yoshimura et al. 1996). Both receptor subtypes were located to fetal egg nests and may be involved in deposition of the zona pellucida and germ cell development.

Both receptor subtypes positively stained blood vessels of postpubertal ovaries within regions of neovascularization, the pericytes of new blood vessels and also areas of neovascularization seen at the periphery of some antral follicles following the follicle edge (although they did not extend all the way round). AngII has been shown to play a role in neovascularization (Fernandez et al. 1985) and both receptors have been observed in the rat aorta during development (Viswanathan et al. 1991), indicating a role for AngII in adult vascular growth, which may be dependent on their parallel stimulation. A second area of vascularization may be associated corpora fibrosa, which result from the degeneration of atretic follicles and the temporary formation of blood vessels as granulosa cells are broken down. It has also been suggested that ovarian blood vessel AngII receptors mediate the effect of AngII on blood flow and thereby determine which follicles become atretic or dominant (Speth & Husain, 1988). Therefore, AngII may act as an angiogenic stimulus for neovascularization of the thecal layer in developing follicles, or transformation of follicles into corpora lutea.

In summary, there was a dominance of AT₁ receptors in the porcine fetal, pre- and postpubertal ovary. AT₂ receptors were present at lower concentrations. The study has localized receptor subtypes specifically in the oocyte and zona pellucida, granulosa cell layers, blood vessels and putative areas of neovascularization. The results indicate that AngII has many roles in the ovary, including folliculogenesis, angiogenesis and steroidogenesis.