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BOR - Papers in Press, published online ahead of print May 28, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.015446
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BIOLOGY OF REPRODUCTION 69, 947–958 (2003)
DOI: 10.1095/biolreprod.103.015446
© 2003 by the Society for the Study of Reproduction, Inc.

Ovary

Determination of Steroidogenic Potential of Ovarian Cells of the Brushtail Possum (Trichosurus vulpecula)1

Lisa J. Whale, Douglas C. Eckery, and Jennifer L. Juengel2

AgResearch, Wallaceville Animal Research Centre, Upper Hutt, New Zealand


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ovary of the brushtail possum (Trichosurus vulpecula) secretes steroids; however, little is known about the identity of the steroidogenic cells in the ovary. The aim of the present study was to determine the identity of the ovarian cell types expressing mRNAs encoding proteins important for steroidogenesis and determine at what stage of follicular development they are expressed. The genes examined were those for steroidogenic factor-1 (SF-1), steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage (P450scc), 3ß-hydroxysteroid dehydrogenase/{Delta}5,{Delta}4 isomerase (3ßHSD), cytochrome P45017{alpha}hydroxylase (P45017{alpha}OH), and P450 aromatase (P450arom). None of the genes examined were expressed in oocytes at any stage of follicular development. SF-1 was expressed in granulosa cells from the type 2 or the primary stage of development and thereafter to the preovulatory stage. In addition, the theca interna of small and medium-size antral but not preovulatory follicles and the interstitial glands and corpora lutea expressed SF-1 mRNA. Granulosa cells of preantral and small to medium-size antral follicles were not capable of synthesizing steroids from cholesterol because they did not contain P450scc mRNA. However, granulosa cells of many of the small to medium-size antral follicles expressed P450arom and 3ßHSD mRNA. The interstitial glands, theca interna, and corpus luteum expressed StAR, P450scc, 3ßHSD, and P45017{alpha}OH mRNA, suggesting that these tissues are capable of synthesizing progestins and androgens. The corpus luteum expressed P450arom, indicating that this tissue also has the potential to secrete estrogens in this species.

corpus luteum, follicle, interstitial cells, ovary, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Steroids produced by the ovary play a vital role in regulating the reproductive cycle because they are part of the complex communication system connecting the hypothalamus, pituitary gland, and ovary. Production of ovarian steroids and responsiveness of follicles to gonadotropins are key regulatory steps in the selection and/or maintenance of a single ovulatory follicle in many eutherian monovular mammals [1–4]. However, recent studies in the brushtail possum (Trichosurus vulpecula), a marsupial separated from eutherian mammals by 60–100 million years of evolution, have revealed that the regulation of LH receptor (LH-R) in granulosa cells does not play a similar role in the selection and/or maintenance of the dominant follicle in this marsupial [5]. This finding suggests that the mechanisms for selecting a dominant follicle in monovular marsupials may differ considerably from those observed in monovular eutherians. Because production of steroids from the ovary is also a key regulatory factor in the control of follicular growth, an understanding of the role that steroids play in this process in the brushtail possum may provide new insights into alternative methods of selection and maintenance of a single ovulatory follicle and may lead to new methods for controlling follicular development in a wide variety of mammalian species.

Key proteins regulating steroid synthesis include steroidogenic acute regulatory protein (StAR), which facilitates the rate-limiting step in steroidogenesis, i.e., the transport of cholesterol across the mitochondrial membranes to cytochrome P450 side chain cleavage (P450scc) [6]. P450scc converts cholesterol to pregnenolone and thus is essential for the synthesis of all steroid hormones. Synthesis of progesterone requires 3ß-hydroxysteroid dehydrogenase/{Delta}5,{Delta}4 isomerase (3ßHSD). Cytochrome P45017{alpha}hydroxylase (P45017{alpha}OH) is closely associated with androgen synthesis, whereas P450 aromatase (P450arom) converts androgens to estrogens [7]. In addition, transcription of most of these genes is controlled in part by steroidogenic factor 1 (SF-1) [8–13]. Because of the central role that steroids play in regulating reproductive events, the timing of onset of steroidogenesis during follicular development and the regulation of steroidogenesis in other ovarian cell types have been studied extensively in eutherian mammals (for reviews, see [1, 14–17]).

In marsupials, however, relatively little is known about when ovarian follicles become steroidogenically active or about the cellular origins of these steroids. In the brushtail possum, ovarian follicular development begins around Day 50 of pouch life, and antral follicles are common after Day 190 [18], which is shortly after the young has permanently left the pouch [19]. Numerous antral follicles are present throughout both the follicular phase (9–11 days duration) and luteal phase (16–18 days duration) of the estrous cycle (25–28 days duration) and during pregnancy (16–18 days duration, identical to the luteal phase of the estrous cycle). However, preovulatory-size follicles only develop during the follicular phase [5, 20] of the estrous cycle. Although numerous healthy and atretic antral follicles are present during the early follicular phase of the estrous cycle, typically only a single follicle is selected for ovulation at each cycle [21]. During the follicular phase, some of the developing antral follicles secrete estradiol, with concentrations appearing to increase as the follicles develop [22], and estradiol appears to be the dominant steroid during this phase of the reproductive cycle [21, 23]. Following ovulation, the corpus luteum develops from the ovulatory follicle and is a major source of progesterone during the last 8–10 days of the luteal phase [24, 25]. The possum ovary contains numerous interstitial cells scattered throughout the ovarian stroma that morphologically appear to be steroidogenically active [20]. Although steroidogenically active interstitial tissue is present in several species [25, 26], the steroidogenic characteristics of this tissue and its role in regulating reproductive activity in the brushtail possum is unknown. To provide fundamental information regarding the potential role of steroids in regulation of ovarian function in the brushtail possum, the aims of the present study were to determine in this marsupial the identity of steroidogenic cell types and the potential of the ovary to produce progestins, androgens, and estrogens.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All experiments were performed with the approval of the Animals Ethics Committee at Wallaceville Animal Research Centre and in accordance with the 1987 Animal Protection (Codes of Ethical Conduct) Regulations of New Zealand. Except where indicated, laboratory chemicals were obtained from BDH Chemicals New Zealand Ltd (Palmerston North, New Zealand), Invitrogen (Auckland, New Zealand), or Roche Diagnostics N.Z. Ltd. (Auckland, New Zealand).

Collection of Tissue Samples

All animals utilized in these experiments were captured within New Zealand. Following capture, animals were housed in groups of 6–10 possums/pen and were fed a mixed diet of fresh fruit, bread, and cereal-based pellets. All pens were furnished with fresh branches (predominately Pinus radiata) for browsing, and fresh water was freely available at all times [27]. Ovaries were collected from six juveniles, i.e., females that were sexually immature based on their body size and pouch development but were independent of their mothers; these animals were likely between 8 and 12 mo old (possums become independent from their mothers at approximately 8 mo of age and usually reach puberty between 12 and 24 mo of age). Ovaries were also collected from six adult animals in the luteal phase and three animals in the late follicular phase, as assessed by the presence of a large (approximately 5 mm) follicle and an enlarged vaginal cul-de-sac at the time of tissue collection; the follicle destined for ovulation is easily identifiable at this stage [21]. Gonads were recovered after administration of an overdose of sodium pentobarbitone (1 ml; Pentobarb 500 [500 mg/ml ]; Chemstock Animal Health, Christchurch, New Zealand) directly into the heart following anesthesia (10 mg per kg of body weight i.m. Zoletil 100; Virbac Laboratories, Auckland, New Zealand). Tissues were fixed in 4% (w/v) phosphate-buffered paraformaldehyde and embedded in paraffin wax.

Generation of cDNAs Encoding a Portion of the Coding Region of Genes of Interest

Total cellular RNA was isolated from possum tissues using TRIzol (Invitrogen) according to the manufacturer's instructions. These tissues were collected from untreated possums being killed for other approved experiments at Wallaceville Animal Research Centre. For the generation of the cDNAs of interest, first-strand cDNA was produced from 5 µg of total cellular RNA isolated from possum tissues using the SuperScript preamplification system (Invitrogen). Complementary DNAs encoding a portion of the coding sequence of genes of interest were isolated by polymerse chain reaction (PCR) using standard PCR buffer (Roche) under the following conditions: 1 cycle at 94°C for 1 min, 40 cycles of denaturing at 94°C for 1 min, annealing at gene-specific temperature for 1 min, and extension at 72°C for 2 min, and final extension at 72°C for 10 min. For individual cDNAs, tissue source of RNA, primer sequences, and annealing temperature are given in Table 1. Resulting PCR products were ligated into pGEMTeasy vector (Promega, Dade Diagnostics, Auckland, New Zealand), and the nucleotide sequence was determined by automated sequence analysis (Waikato DNA Sequencing Facility, University of Waikato, Hamilton, New Zealand). The obtained sequences were compared with known sequences to confirm identity using the GAP program (Wisconsin Package version 10.2; Genetics Computer Group, Madison, WI).


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  		TABLE 1. GenBank reference numbers used for primer design, tissue sources of RNA, primer sequences, annealing temperatures, and GenBank accession numbers for the resulting brushtail possum sequence for the various genes amplified

Specificity of generated cDNAs was tested with Northern blot analysis. Samples (50 µg total cellular RNA from corpus luteum [P450arom only], liver, adrenal, ovary, and kidney) were separated on a 1.5% agarose-formaldehyde-3 [N-morpholino] propanesulfonic acid gel. Integrity and concentrations of RNA samples loaded onto the gels were assessed by visual inspection of ethidium bromide-stained 18S and 28S ribosomal bands. Following electrophoresis, all samples had distinct 18S and 28S ribosomal bands and were judged to have been loaded in approximately equal concentrations. Total cellular RNA was transferred to a nylon filter (Hybond-NX; Amersham Pharmacia Biotech New Zealand, Auckland, New Zealand) by capillary action and cross-linked by ultraviolet light. Appropriate cDNAs were radiolabeled with 32P using a RadPrime kit (Invitrogen) according to the manufacturer's instructions. Filters were hybridized in ULTRAhyb (Ambion, Austin, TX) solution containing approximately 35–60 ng of 32P-labeled cDNA according to the manufacturer's instructions. The final wash was in 0.1x saline sodium citrate (SSC), 0.1% SDS for 15 min at 42°C (P45017{alpha}OH, P450arom, and SF-1 filters) or at 65°C (P450scc, StAR, and 3ßHSD filters). Filters were exposed to X-AR film (Eastman Kodak, Rochester, NY) for <24 h (17{alpha}OH, 3ßHSD, StAR, P450scc, P450arom) or 5 days (SF-1) at -70°C.

In Situ Hybridization

Cellular localization of mRNAs was determined using the in situ hybridization protocol described previously with minor modifications [28]. This protocol has been developed to maximize the sensitivity of detection of genes of interest in paraformaldehyde-fixed paraffin-embedded tissues and thus was not suitable for quantitative analysis because the signal in some cell types had reached saturation. Sense and antisense RNA probes were generated from cDNA encoding the gene of interest with T7 or SP6 RNA polymerase using the Riboprobe Gemini system (Promega). For all in situ hybridizations, 4- to 6-µm tissue sections were incubated overnight at 55°C with 45 000 cpm/µl (approximately 48 000 dpm/µl) of 33P-labeled antisense RNA. Nonspecific hybridization of RNA was removed by RNase A digestion followed by stringent washes (2x SSC and 50% formamide at 65°C and 0.2x SSC at 37°C). Following washing, sections were dehydrated, air dried, and coated with autoradiographic emulsion (LM-1 emulsion; Amersham). Emulsion-coated slides were exposed at 4°C for 3 wk and developed for 3.5 min in D19 developer (Kodak). Development was stopped using a 1-min incubation in 1% acetic acid, and slides were fixed with a 10-min incubation in Ilfofix II (Ilford, Cheshire, U.K.). Sections were stained with hematoxylin and then viewed and photographed using both light- and dark-field illumination with a BH-2 microscope (Olympus New Zealand, Lower Hutt, New Zealand). Specific hybridization was determined for each gene in at least three juvenile animals, four adult animals in the midluteal phase of the estrous cycle (two pregnant, two nonpregnant), and three animals in the follicular phase. In most instances, a single slide from each animal was hybridized with the antisense cRNA for each gene. For the juveniles and the adult animals in the luteal phase of the estrous cycle, further sections were examined if all follicular types were not present on the section chosen, so that all stages of follicular development described were represented in all animals examined for each gene for these groups. Nonspecific hybridization was monitored by hybridizing at least one tissue section from each group with approximately equal concentrations of the sense RNA for each gene. For all genes, hybridization of the sense RNA over the tissue section was similar to or lower in intensity than that observed on the areas of the slide not containing tissue of both the sense and antisense hybridized slides and thus was considered nonspecific.

Follicular Classification

The follicular classification system was based on that previously described for the brushtail possum [18]. Primordial follicles (type 1) were classified as oocytes surrounded by a layer of flattened or mixed flattened and cuboidal granulosa cells. Primary follicles (type 2) contain an oocyte surrounded by one or two layers of cuboidal granulosa cells. Secondary follicles were divided into small (type 3) secondary follicles containing two or three layers of granulosa cells, and large (type 4) secondary follicles containing four or more layers of granulosa cells without a formed antrum. Once an antrum was formed, follicles were classified as antral (type 5). Theca cells were first distinguishable in follicles forming an antrum. The largest nonatretic antral follicle (>4 mm) present on the ovaries of an animal in the follicular phase was classified as a preovulatory follicle. Atretic follicles, as assessed by the presence of several pyknotic nuclei in the granulosa layer, were excluded from the study.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of the Possum cDNAs

In all cases, the nucleotide and deduced amino acid sequences from the resulting cDNAs were compared to the corresponding regions of the human sequences (see Table 1 for GenBank accession numbers except for the SF-1 sequence, which was compared to sequence NM_004959). The size of the insert obtained, nucleotide identity, protein identity, protein similarity, and size and distribution of the band(s) observed following Northern blot analysis are summarized in Table 2. The obtained brushtail possum sequence for SF-1 was 94% and 96% identical to the corresponding region of the tammar wallaby nucleotide and amino acid sequence, respectively [29]. All cDNAs detected specific transcripts in steroidogenic tissues, and no hybridization was observed in nonsteroidogenic tissues (see Table 2).


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  		TABLE 2. Summary of the size of the resulting amplified PCR products, the homology between the brushtail possum sequence and the corresponding region of the human gene, the size of the transcript detected with Northern blot analysis, and tissue distribution of the mRNA for the various genes amplified

In Situ Hybridization

StAR Expression of StAR mRNA was observed in the granulosa cells of some type 3 and type 4 follicles and in preovulatory follicles but was not observed in granulosa cells of types 1, 2, or 5 follicles (Fig. 1 and Table 3). StAR mRNA was not observed in oocytes at any stage of follicular development examined (types 1–5). Expression was also observed in theca cells, interstitial glands, and the corpus luteum.



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  		FIG. 1. Corresponding brightfield (a, c, and e) and darkfield (b, d, and f) views of ovaries from adult brushtail possums following hybridization to StAR cRNA. Expression of StAR mRNA was not observed in type 1 and type 2 follicles (a and b; open ) but was observed in the granulosa cells of some type 3 and type 4 follicles (a and b; short ), in theca but not in granulosa cells of small and medium antral follicles (c and d; long *; follicle on left), and in granulosa and theca cells of the preovulatory follicle (c and d; long **; follicle on right). Hybridization was also observed in the interstitial glands (a–f; representative clusters denoted with *, with other clusters scattered through the ovarian stroma) and the corpus luteum (e and f; closed ). Expression was not observed in other stroma cells or the surface epithelium (a and b; {blacktriangleleft}). Bar = approximately 100 µm.


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  		TABLE 3. Summary of expressiona of mRNA encoding proteins important in steroidogenesis in the brushtail possum ovary

P450scc Expression of mRNA encoding P450scc was not observed in types 1–4 follicles and was limited to the theca cells of type 5 follicles (Fig. 2 and Table 3). The preovulatory follicles strongly expressed P450scc mRNA in both the theca and granulosa cells. The interstitial glands and corpus luteum also strongly expressed P450scc mRNA.



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  		FIG. 2. Corresponding brightfield (a, c, and e) and darkfield (b, d, and f) views of ovaries from adult possums following hybridization to P450scc cRNA. Expression of P450scc mRNA was not observed in types 1–4 follicles (a–d; type 1 and type 2 follicles, open ; type 3 and type 4 follicles, short ). Theca but not granulosa cells of small and medium antral follicles (a–f; long *) expressed P450scc mRNA. The preovulatory follicle expressed P450scc in both granulosa and theca cells (c and d; long **; follicle on top). Hybridization was also observed in the interstitial glands (a–f; representative clusters denoted with *, with other clusters scattered through the ovarian stroma) and the corpus luteum (e and f; closed ). Expression was not observed in other stroma cells or the surface epithelium (a, b, e, and f, {blacktriangleleft}). Bar = approximately 100 µm

3ßHSD Granulosa cells of type 3 and larger follicles (including the preovulatory follicle) and theca cells expressed mRNA encoding 3ßHSD (Fig. 3 and Table 3). Expression was not observed in the oocytes of follicles at any stage examined (types 1–5). Strong signal for 3ßHSD was also observed in the interstitial glands and corpus luteum.



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  		FIG. 3. Corresponding brightfield (a, c, e, and g) and darkfield (b, d, f, and h) views of ovaries from adult possums following hybridization to 3ßHSD cRNA. Expression of 3ßHSD mRNA was not observed in type 1 and type 2 follicles (a and b; open ) but was observed in granulosa cells of type 3 and larger follicles (a-d; short ). Theca and granulosa cells of small and medium antral follicles (c and d; long *) expressed 3ßHSD mRNA. The preovulatory follicle expressed 3ßHSD in both granulosa and theca cells (e and f; long *). Hybridization was also observed in the interstitial glands (a–d, g, and h; representative clusters denoted with *, with other clusters scattered through the ovarian stroma) and the corpus luteum (g and h; closed ). Expression was not observed in other stroma cells or the surface epithelium (c and d, {blacktriangleleft}). Bars = approximately 50 µm (a and b), 100 µm (c, d, g, and h), and 25 µm (e and f)

P45017{alpha}OH In growing follicles (types 1–5), expression of P45017{alpha}OH was not observed until theca formation and was limited to theca cells (Fig. 4 and Table 3). However, the preovulatory follicle expressed P45017{alpha}OH mRNA in both the granulosa and theca layer. Strong expression was also observed in the interstitial glands and in the corpus luteum.



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  		FIG. 4. Corresponding brightfield (a, c, and e) and darkfield (b, d, and f) views of ovaries from juvenile (a and b) or adult (c–f) possums following hybridization to P45017{alpha}OH cRNA. Expression of P45017{alpha}OH mRNA was not observed in type 1–4 follicles (a–d; type 1 and type 2 follicles, open ; type 3 and type 4 follicles, short ). Theca but not granulosa cells of small and medium antral follicles (c–f; long *) expressed P45017{alpha}OH. The preovulatory follicle expressed P45017{alpha}OH in both granulosa and theca cells (c and d; long **; follicle on top). Hybridization was also observed in the interstitial glands (a, b, e, and f; representative cluster denoted with *, with other clusters scattered through the ovarian stroma) and the corpus luteum (e and f; closed ). Expression was not observed in other stroma cells or the surface epithelium (a and b, {blacktriangleleft}). Bar = approximately 100 µm.

P450arom Expression of P450arom was observed in the granulosa cells of many type 5 follicles of various sizes, including shortly after antrum formation until the preovulatory follicle developed (Fig. 5 and Table 3). Expression also was observed in the corpus luteum. The interstitial glands, theca cells, and oocytes did not express mRNA encoding P450arom.



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  		FIG. 5. Corresponding brightfield (a and c) and darkfield (b and d) views of ovaries from adult possums following hybridization to P450arom cRNA. Expression of P450arom mRNA was not observed in type 1–4 follicles (a–d; type 1 and type 2 follicles, open ; type 3 and type 4 follicles, short ). Granulosa but not theca cells of small and medium antral follicles (a and b; long *) expressed P450arom. The preovulatory follicle also expressed P450arom in granulosa but not theca cells (a and b; long **; follicle on top). Hybridization was not observed in the interstitial glands (c and d; *) but was present in the corpus luteum (c and d; closed ). Expression was not observed in other stroma cells or the surface epithelium (a and b; {blacktriangleleft}). Bar = approximately 100 µm

SF-1 The mRNA encoding SF-1 was first observed in primary follicles (Fig. 6 and Table 3). Expression was limited to the granulosa cells of these follicles. Granulosa cells continued to express SF-1 mRNA throughout follicular development. Expression of SF-1 mRNA was evident in theca cells from the time of antrum formation; however, expression was not evident in the theca of the preovulatory follicles. Expression of SF-1 mRNA was not observed in oocytes of any size of follicle examined (types 1–5). The mRNA encoding SF-1 was also present in interstitial glands and the corpus luteum (data not shown).



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  		FIG. 6. Corresponding brightfield (a, c, and e) and darkfield (b, d, and f) views of ovaries from juvenile (a and b) or adult (c–f) possums following hybridization to SF-1 cRNA. Expression of SF-1 mRNA was not observed in type 1 follicles (a and b; open ) but was observed in type 2 (e and f; short ) and larger follicles (a–f; type 3 and type 4 follicles, short ; small and medium antral follicles, long *). Once theca cells were identifiable in the follicles, expression was observed in both granulosa and theca cells. Faint signal for SF-1 mRNA was observed in the granulosa cells of the preovulatory follicle, but no signal was detected in the theca cells of these follicles (e and f; long **; follicle on bottom). Hybridization was also observed in the interstitial glands (a–f; representative cluster denoted with *, with other clusters scattered through the ovarian stroma). Expression was not observed in other stroma cells or the surface epithelium (e and f; {blacktriangleleft}). Bar = approximately 100 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The nucleotide and deduced amino acid sequences of the isolated brushtail possum cDNAs corresponded closely with published human sequences. For SF-1, where the sequence was known for another marsupial, tammar wallaby [29], conservation was very high. Based on Northern blot analysis with total cellular RNA, abundant expression of the genes examined was observed in steroidogenic tissues (i.e., ovary and adrenal gland) but was not observed in nonsteroidogenic tissues (i.e., kidney and liver). With the exception of P45017{alpha}OH, the sizes of the transcripts observed were similar to those previously reported for humans (3ßHSD and P450scc [30]) or other mammalian species (SF-1, tammar wallaby [29]; StAR, sheep [31]; P450arom, pig [32]). The cDNA encoding a portion of brushtail possum P45017{alpha}OH detected a dominant transcript of approximately 3.4 kilobases (kb), which is larger than the approximately 1.9-kb transcript reported in other mammalian species [30, 33]. Potential reasons for the increase in size in this transcript were not examined in this study, but wide variation in size of transcripts for the same gene in different species is not uncommon, e.g., the dominant transcript detected for StAR is 1.6 kb in humans [34] and 2.8 kb in sheep [31] and the dominant P450arom transcript is approximately 6 kb in cattle [35] and approximately 3 kb in humans [30].

During antral follicular development in eutherian mammals with low ovulation rates (e.g., cow, sheep, and human), expression of mRNAs encoding proteins important for steroidogenesis in the granulosa cells is not observed until the later stages of follicular development, i.e., around the time that follicles become dependent on gonadotropins for continued growth [1, 36, 37]. Similarly, in possums expression of P450scc, which is required for synthesis of any steroid from cholesterol, was not observed in the granulosa cells of any follicle except in the presumptive preovulatory follicle. However, the mRNA encoding 3ßHSD was present in granulosa cells of most follicles from the type 3 stage of development, and aromatase mRNA was observed in the granulosa cells of some follicles as early as shortly after antrum formation. Thus, granulosa cells of small antral follicles may be able to synthesize progesterone and estradiol from precursors that could be provided by thecal or interstitial tissue. In this respect, steroidogenic potential of granulosa cells in the brushtail possum differs significantly from that of other eutherian mammals with low ovulation rates. In both sheep and cattle, expression of 3ßHSD occurs relatively late during antral follicular development and coincides with or occurs after the expression of LH-R [1, 37]. In cattle, expression of LH-R and 3ßHSD mRNA is only observed in dominant follicles that would be capable of ovulating if provided with the correct endocrine signal [1]. Similarly, expression of steroidogenic enzymes in the granulosa layer in humans is predominately restricted to a single presumptively preovulatory follicle [36]. We have previously shown that functionally active LH-Rs are present in possum granulosa cells of all healthy follicles shortly after antrum formation [5]. These data in the brushtail possum do not support the hypothesis that expression of LH-R and potentially synthesis of steroids in granulosa cells is a critical determinant for the selection of a single follicle for ovulation, as has been proposed for eutherian mammals [1–4].

Expression of SF-1 was first observed in the granulosa cells of some type 2 follicles. Because SF-1 mRNA regulates expression of 3ßHSD and StAR mRNA, expression of SF-1 may be a prerequisite to expression of 3ßHSD and StAR mRNA in type 3 follicles. SF-1 mRNA continued to be expressed in developing small and medium antral follicles; however, very faint expression of SF-1 mRNA was observed in the granulosa cells of the preovulatory follicle, and no expression was detected in the theca cells. This expression pattern was similar to that observed in another marsupial, the tammar wallaby, where expression of SF-1 mRNA was observed in the granulosa and theca cells of small to medium antral follicles but was not detected in large antral follicles [29]. In contrast, expression of SF-1 mRNA remains strong in preovulatory follicles of the ewe and may be important for the expression of steroidogenic capability [37]. However, the preovulatory follicle of the brushtail possum is steroidogenically active; it strongly expresses mRNAs encoding proteins important for steroidogenesis and secretes estradiol [22, 23]. The precise timing of downregulation of SF-1 expression and upregulation of the steroidogenic enzymes during antral follicular development in the brushtail possum is not known because follicles do not grow larger than approximately 2.5 mm during the luteal phase [5, 20]. Decreased levels of SF-1 mRNA may not occur until after expression of the steroidogenic enzymes has been initiated. Alternatively, a related transcription factor, NR5A2, which also stimulates transcription of the steroidogenic enzymes [38], is also expressed in the ovary of humans [38] and specifically in granulosa cells of preovulatory follicles of horses [39]. Expression of NR5A2 has been proposed to play a key role in the regulation of steroidogenic enzymes in the preovulatory follicle during luteinization in the horse [39] and could also regulate expression of steroidogenic enzymes in brushtail possums.

As observed in sheep, cattle, and humans [1, 36, 37], expression of steroidogenic enzymes is first observed in the theca interna around the time of antrum formation in the brushtail possum. Thus, as observed in eutherian mammals, the theca interna of the brushtail possum might be capable of providing precursor steroids for the synthesis of estradiol by the granulosa cells. These cells also expressed mRNA encoding SF-1, a potential regulator of steroidogenic enzymes. In sheep, expression of SF-1 precedes expression of the steroidogenic enzymes as the theca interna progresses from a nonsteroidogenic tissue to a tissue capable of producing high levels of steroids [37]. In contrast, in the brushtail possum the theca interna is not identifiable until antrum formation, and at this time it already has a steroidogenic phenotype. In addition, the theca interna is not as prominent in brushtail possum follicles as it is in eutherian species. Small patches of theca cells are intermittently observed around the follicle throughout growth of antral follicles in the brushtail possum compared with the continuous layer of cells observed in most eutherian mammals.

In possums, clusters of interstitial cells are often observed in a concentric circle just at the perimeter of the follicle [20]. These interstitial cells appear capable of making progestins and androgens because they contain abundant StAR, P450scc, 3ßHSD, and P45017{alpha}OH mRNA. However, whether these steroids could provide a precursor source for steroid production by granulosa cells is not known. In addition, various clusters of interstitial cells are scattered throughout the stroma of the ovary. These interstitial glands also have a steroidogenic phenotype and strongly express mRNA for proteins important in steroidogenesis. The potential role of these cells in the regulation of ovarian function and the interactions between the ovary and the pituitary are not well understood [20], but they might be capable of synthesizing progestins and androgens that could regulate ovarian function directly or indirectly through regulation of pituitary function. The pattern of expression of mRNAs encoding steroidogenic enzymes was not different between the juvenile and adult animals or between the luteal and follicular phases of the estrous cycle. However, whether the amount of a particular steroid produced from these cells might be different at different ages or with changing reproductive status was not determined in this study. Little information is available on which hormones might control expression of steroid synthesis in this tissue. Previously, we demonstrated that the mRNA for LH-R but not FSH receptor was present in some interstitial cells [5], indicating a potential role for LH in regulating steroidogenesis in these cells, as has been shown for other species [26, 40]. However, LH-R mRNA expression was weak or absent in many interstitial cells. Because all interstitial glands strongly expressed the steroidogenic enzymes, it seems unlikely that LH would be a primary regulator of steroidogenesis in this tissue. In contrast to the weak expression of LH-R mRNA in interstitial tissue, preliminary studies have shown that prolactin receptor mRNA is strongly expressed ubiquitously in interstitial cells, raising the possibility that this pituitary hormone may be involved in regulation of steroidogenesis in these cells [20]. Expression of SF-1 by the interstitial glands may be important in maintaining their steroidogenic phenotype; however, because all interstitial glands examined expressed mRNAs encoding steroidogenic enzymes, the relative importance of expression of SF-1 to obtaining a steroidogenic phenotype could not be assessed. In the brushtail possum, the interstitial glands are thought to be primarily derived from the medullary cords [20], which are a prominent feature of the developing ovary. During the later stages of ovarian formation, these medullary cells appear to differentiate into interstitial cells with a steroidogenic phenotype. Thereafter, they are present throughout the animal's life. The pattern of expression of SF-1 in the medullary cells during this differentiation is currently unknown but may lend insight to the development of this tissue.

The luteal tissue examined in this study was collected during the time of maximum progesterone production, and as expected this tissue strongly expressed mRNAs encoding proteins important in steroidogenesis. As observed in other species [29, 41, 42], this tissue also expressed mRNA encoding SF-1, indicating a potential role for this transcription factor in regulation of steroidogenic enzyme expression in the corpus luteum of the brushtail possum. As observed in some species [36, 39], the corpus luteum of the brushtail possum expressed enzymes necessary for the synthesis of estrogens. In humans, expression of estrogens by the corpus luteum suppresses FSH synthesis, thus preventing the development of large antral follicles during the luteal phase [2]. In the tammar wallaby, a macropodid marsupial, follicular development is also suppressed by the corpus luteum, and administration of estradiol can mimic this suppression [43]. However, the corpus luteum of the tammar wallaby did not have detectable aromatase activity [44], and the potential role of estradiol in the observed luteal suppression of follicular growth in this species is unclear. In the brushtail possum, a similar suppression of development of large antral follicles occurs during the luteal phase [5, 20], and because the corpus luteum clearly has the ability to synthesize estradiol, a role for luteal estradiol in this suppression seems likely. However, further studies showing that the suppression of follicular growth during the luteal phase is due to the presence of the corpus luteum and that this suppression can be mimicked by estradiol treatment are needed to confirm the role of luteal estrogens in the suppression of follicular growth in the brushtail possum.

We isolated cDNAs encoding portions of the StAR, P450scc, 3ßHSD, P45017{alpha}OH, P450arom, and SF-1 genes from a marsupial, the brushtail possum. Based on expression of these genes in ovarian tissue, shortly after antrum formation some follicles are capable of synthesizing estrogens, with the theca/interstitial tissue supplying androgen precursors for aromatization by the granulosa cells. Granulosa cells of preantral and antral follicles express 3ßHSD. However, granulosa cells are not capable of synthesizing steroids from cholesterol until they have reached the later stages of antral development. Interstitial tissue would be capable of synthesizing androgens but does not have the ability to convert androgens to estrogens. The corpus luteum expressed all the mRNAs examined, indicating the potential for luteal tissue to synthesize progestins, androgens, and estrogens.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Ken McNatty for his helpful comments regarding the manuscript, Dr. Bernie McLeod for providing adult possum ovaries for the study, Brian Thomson, Lara Colbourne, Heath Liddington, and Bert Gwilliam for animal care and tissue collection, Lee-Ann Still and Lynn O'Donovan for preparation of histological material, Alan Barkus for preparation of the figures, and Sue Swaney for secretarial assistance.


   FOOTNOTES
 
1 This research was supported by the New Zealand Foundation for Research, Science and Technology. Back

2 Correspondence: Jennifer Juengel, Wallaceville Animal Research Centre, Ward St., P.O. Box 40063, Upper Hutt, New Zealand. FAX: 64 4 922 1380; jenny.juengel{at}agresearch.co.nz Back

Received: 15 January 2003.

First decision: 3 February 2003.

Accepted: 21 April 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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