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Biology of Reproduction 66, 1310-1317 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Ovarian Expression of Messenger RNA Encoding the Receptors for Luteinizing Hormone and Follicle-Stimulating Hormone in a Marsupial, the Brushtail Possum (Trichosurus vulpecula)1

Douglas C. Eckerya, Stan Luna, Brian P. Thomsona, Winny Ng Chiea, Lloyd G. Moorea, and Jennifer L. Juengel2,a

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Both LH and FSH play a central role in controlling ovarian function in mammals. However, little is known about the type of ovarian cells that are responsive to LH and FSH in marsupials. We determined, using in situ hybridization, the localization of mRNA encoding the receptors (R) for LH and FSH in ovaries of brushtail possums. The mRNA encoding FSH-R was observed in granulosa cells of healthy follicles containing at least two complete layers of cells. The mRNA encoding LH-R was first observed in granulosa cells at the time of antrum formation. Cells of the theca interna expressed LH-R mRNA but not FSH-R mRNA. Neither FSH-R nor LH-R mRNA was detected in atretic follicles. Both FSH-R and LH-R mRNAs were observed in luteal tissue, but only LH-R mRNA was observed in interstitial cells. Granulosa cells from follicles of various sizes (0.5 to >2 mm in diameter) responded to LH and FSH treatment with an increase in cAMP synthesis. In contrast, luteal tissue did not respond to either FSH or LH treatment. In conclusion, expression of FSH-R in the brushtail possum ovary was similar to that observed in many eutherian mammals. However, active LH-R was expressed in granulosa cells much earlier in follicular development than has been previously observed. In addition, although mRNAs for both FSH-R and LH-R were observed, neither FSH nor LH treatment stimulated cAMP synthesis in luteal tissue.

corpus luteum, corpus luteum function, follicle, follicle-stimulating hormone receptor, follicular development, ovary, receptor, theca cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The brushtail possum (Trichosurus vulpecula) is a marsupial that was introduced into New Zealand in the mid-1800s to establish a fur industry. Because of a lack of competitors and predators, the population of this introduced species is largely uncontrolled and threatens much of New Zealand's unique flora and fauna [1–3]. In addition, this animal is the main reservoir for bovine tuberculosis and thus represents an animal health threat to New Zealand cattle and deer [4]. Understanding essential reproductive processes in this marsupial may allow for the development of methods to control fertility to humanely manage the brushtail possum population and thus protect the unique flora and fauna of New Zealand as well as its pastoral agriculture industries.

The brushtail possum is a monovular, polyestrous seasonal breeder with an estrous cycle of around 26 days consisting of a 9- to 11-day follicular phase and a 16- to 18-day luteal phase [5]. Many aspects of ovarian function in the brushtail possum appear similar to those observed in many eutherian mammals. For example, follicles at all stages of development (primordial to antral) can be observed in the ovaries at any given time [6]. After ovulation, the corpus luteum develops from the ovulatory follicle and is a major source of progesterone during the luteal phase [7]. In addition, as is observed in many other mammalian species [7, 8], the possum ovary contains numerous steroidogenic-like interstitial cells [9]. However, the potential role of the interstitial tissue in the regulation of reproductive processes in the brushtail possum is unknown.

In eutherian mammals, the pituitary hormones FSH and LH are known to be central to ovarian function. The early stages of follicular development are thought to be facilitated by FSH, whereas the later stages of follicular development and ovulation are dependent on FSH and LH [10, 11]. Indeed, removal of the pituitary gland in eutherian mammals prevents the final stages of follicular development, ovulation, and normal luteal function. In most species, normal development and function of the corpus luteum requires LH [12]. Little is known about the potential role of FSH and LH in regulating ovarian function in the brushtail possum. Therefore, as a first step toward gaining a better understanding of these processes, the main aim of the present studies was to determine which cellular types within the ovary of the brushtail possum contain receptors for FSH and LH. Since receptors for both FSH and LH have been shown to be present in forms that do not activate the protein kinase A (PKA) second messenger system [13–15], a second aim was to determine if mRNAs encoding FSH-R and LH-R observed in granulosa and luteal cells encode a receptor capable of stimulating cAMP synthesis.


   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 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), Gibco BRL (Auckland, New Zealand), or Roche Diagnostics N.Z. Ltd. (Auckland, New Zealand).

Cloning of Possum FSH-R and LH-R cDNAs

Total cellular RNA was isolated from possum tissues using Trizol (Gibco BRL) according to the manufacturer's instructions. For the generation of FSH receptor (FSH-R) cDNA, first-strand cDNA was produced from 5 µg of ovarian total cellular RNA using the SuperScript preamplification system (Gibco BRL). The cDNA encoding possum FSH-R was generated with the primers 5'-GGAACCCAACTAGATGAGCTG-3' and 5'-CCCCATGATACTTCACATGG-3' corresponding to bases 648–668 and 1137–1157 of the published human sequence [16]. After denaturation at 94°C for 3 min, possum FSH-R cDNA was amplified using 40 cycles of denaturing for 1 min at 94°C, 1 min of annealing at 55°C, and extension at 72°C for 1 min. The product obtained from the ovarian cDNA was ligated into the pGEMT vector (Promega, Dade Diagnostics PTY Ltd., Auckland, New Zealand), and its nucleotide sequence was determined by automated sequence analysis (Waikato DNA Sequencing Facility, The University of Waikato, Hamilton, New Zealand). The obtained sequence was compared with known FSH-R sequences to confirm identity of the amplified product [17, 18]. The pGEMT plasmid containing possum FSH-R was linearized with NcoI and SalI for use in in situ hybridization analysis.

For generation of the LH receptor (LH-R) cDNA, first-strand cDNA was produced using 1.5 µg of testicular total cellular RNA using a primer specific for LH-R (5'-GGACTCCAGCCCATAACTAGG-3', corresponding to bases 776–796 of the porcine sequence [19]). The cDNA encoding a portion of possum LH-R was generated using the standard polymerase chain reaction (PCR) buffer from Roche Diagnostics N.Z. Ltd. with the forward primer 5'-CCCATCTCAAGCTTTCAGAGG-3' (corresponding to bases 256–276) and the reverse primer listed previously. The PCR conditions were identical to those listed for generation of the possum FSH-R cDNA except that the annealing step was performed at 53°C. The product obtained from the testicular cDNA was ligated into the pGEMTeasy vector (Promega), and its nucleotide sequence was determined by automated sequence analysis (Waikato DNA Sequencing Facility). The obtained sequence was compared with the known LH-R sequence to confirm identity of the amplified product [17, 18]. The pGEMTeasy possum LH-R construct was linearized with NcoI and SalI for use in in situ hybridization analysis.

In Situ Hybridization

Cellular localization of mRNAs for FSH-R and LH-R was determined using the in situ hybridization protocol described previously, with minor modifications [20]. Possums were killed by administration of an overdose of sodium pentobarbitone (1 ml, Pentobarb 500; Chemstock Animal Health, Christchurch, New Zealand) directly into the heart after sedation with Zoletil 100 (10 mg/kg of body weight i.m.; Virbac Laboratories Ltd., Auckland, New Zealand). Ovaries were collected from three juvenile and four adult animals (luteal phase; pregnant: n = 2; not pregnant: n = 2) and fixed in 4% (w/v) phosphate-buffered paraformaldehyde and embedded in paraffin wax. Sense and antisense RNA probes were generated from cDNA encoding FSH-R and LH-R (described previously) 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 50°C with 45 000 cpm/µl of 33P-labeled antisense RNA. Nonspecific hybridization of RNA was removed by RNase A digestion followed by stringent washes (2x saline-sodium citrate [SSC], 50% formamide, 65°C, and 0.2x SSC at 37°C). After the washes, sections were dehydrated, air-dried, and coated with autoradiographic emulsion (LM-1 emulsion; Amersham Pharmacia Biotech New Zealand, Auckland, New Zealand). Emulsion-coated slides were exposed at 4°C for 3–4 wk, developed, and fixed. Sections were stained with hematoxylin and viewed and photographed using both light- and dark-field illumination on an Olympus BH-2 microscope (Olympus New Zealand Limited, Lower Hutt, New Zealand). 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. No specific hybridization was observed for any section hybridized with the sense RNA for either gene (data not shown).

Determination of Responsiveness of Granulosa Cells and Luteal Tissue to FSH and LH

Experiment 1 A preliminary experiment was performed to determine the responsiveness of possum granulosa cells to purified possum (p) FSH and LH. Juvenile and adult possums were killed by inhalation of CO2, and ovaries were placed in sterile media (M199 with Earle salts, 20 mM Hepes, and 0.1% BSA) for transport to the laboratory, and all follicles between 1 and 2 mm in diameter were dissected from the ovaries. Granulosa cells were collected by scraping the cells from the follicular wall using a sterile steel loop in 1 ml of sterile media. Cells were pooled (n = 2 pools), washed in 10 ml of incubation media (Dulbecco PBS with 0.1% BSA and 0.2 mM 1-methyl-3-isobutylxanthine), and resuspended in incubation media at a concentration of 200 000 cells per milliliter. Incubation media (0.5 ml) containing 0–2000 ng/ml of pFSH or pLH was added to the cell suspension (0.5 ml) in an ice water bath. Purified preparations of pFSH and pLH (less than 0.05% cross-contamination) were isolated as described previously [21, 22]. After addition of the hormone, samples were incubated at 37°C for 1 h, and the reaction was stopped by incubation at 80°C for 20 min, followed by freezing at -20°C.

Experiment 2 The reproductive status of 14 adult female possums was monitored by vaginal cytology after removal of pouch young. The day after an observed cell rise and/or detection of sperm or leukocytes in the urine was considered to be Day 1 of the estrous cycle or pregnancy. Initially, fertile males were cohabitated with the females to obtain ovaries from pregnant animals. When approximately half of the possums had mated, the males were removed to allow for collection of ovaries from nonpregnant animals. Ovaries were collected from possums on Days 12–14 of the estrous cycle (luteal phase; n = 3) or pregnancy (n = 6) after the animals were killed with CO2. A blood sample was also collected from each animal by cardiac puncture for determination of progesterone concentrations (assay sensitivity, 0.2 ng/ml; intraassay coefficient of variation [CV], 9.5% [23]). An animal was classified as pregnant if an embryo was present in the uterus at the time of tissue collection. Ovaries were placed in sterile media for transport to the laboratory, and all follicles greater than 0.5 mm in diameter and the corpus luteum were dissected from the ovary. For each animal, follicles were pooled into three size groups (0.5–1.0 mm, 1.1–1.9 mm, and 2.0–2.5 mm), and granulosa cells were collected as described previously with the exception that cells were resuspended at concentrations ranging from 40 000 to 150 000 cells per milliliter. Although the preovulatory follicle reaches approximately 5 mm in diameter, no follicles >2.5 mm were present on the ovaries in this study. A small piece of luteal tissue was placed in 4% paraformaldehyde for use in in situ hybridization analysis. The remaining luteal tissue was divided into nine portions; each portion was weighed, placed into 0.5 ml of ice-cold incubation media, and finely chopped. Granulosa cells and luteal tissue were incubated in the presence or absence of pLH (2 ng/ml granulosa cells, 20 ng/ml luteal tissue) or pFSH (100 ng/ml) as described previously. Concentrations of cAMP were determined in samples using a previously validated RIA [24]. No samples were below the sensitivity of the assay (1 fmole per tube). The intraassay CV was 8.4%. The interassay CV was 7.5%.

Statistical Analyses

Data collected from the preliminary experiments examining the response of granulosa cells to LH and FSH were subjected to regression analysis. To determine if pLH and pFSH stimulated cAMP synthesis in granulosa cells of follicles of different sizes and in luteal tissue, data were examined by ANOVA and, where appropriate, differences between means were determined by least-significant differences. Data were normalized by log transformation before analysis. Pregnancy status did not influence responsiveness; therefore, data from all animals were combined for the final analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of Possum FSH-R and LH-R cDNAs

The nucleotide sequence of the cloned cDNA for the possum FSH-R (GenBank accession no. AF406604) was 74%–77% identical, whereas the deduced amino acid sequence was 66%–70% identical to the respective bear [25], pig [26], human [16], rat [27], and cow [28] sequences. The nucleotide and deduced amino acid sequences of the cDNA encoding possum LH-R (GenBank accession no. AF406605) was 83%–86% identical to the bear [25], pig [19], human [29], rat [30], and cow [31] sequences. A possum LH-R clone containing an in-frame 42-base pair insert between nucleotide 396 and 397 that encoded for an additional 14 amino acids was also isolated from the testis-derived PCR product (GenBank accession no. AF406606).

Localization of mRNAs Encoding FSH-R and LH-R

The mRNA encoding FSH-R was first observed in the granulosa cells of follicles containing at least two complete layers of cuboidal granulosa cells (secondary follicles, Fig. 1, a and b) and continued to be expressed in granulosa cells of healthy antral follicles but was not observed in granulosa cells of atretic follicles (as assessed by the presence of pyknotic nuclei; Fig. 1, c and d). Corpora lutea of the two adult animals that were pregnant expressed mRNA encoding FSH-R (Fig. 2, e and f), whereas the other two adult animals, which were not pregnant, had little if any expression (Fig. 1, g and h). However, further analysis of luteal tissue collected from pregnant (n = 6) and nonpregnant (n = 3) possums showed variable low levels of expression of FSH-R mRNA in both pregnant and nonpregnant possums. Expression of FSH-R mRNA was not observed in the theca interna, interstitial cells, surface epithelium, or oocytes of any animal examined (Fig. 1, a–f).



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  		FIG. 1. Localization of mRNA for FSH-R in ovaries from juvenile (a, b) and adult (c–h) possums. Left, light-field photomicrographs; right, corresponding dark-field view. The mRNA for FSH-R was observed in granulosa cells of healthy secondary (a, b; ->) and antral (c, d; ->) follicles but not in primordial or primary (a, b; {twoheadrightarrow}) or atretic (c, d; {twoheadrightarrow}) follicles. Expression was observed in luteal tissue of many (e, f; ->) but not all (g, h; ->) possums. Expression was not observed in interstitial tissue (a, b; *), surface epithelium (a, b; {blacktriangleleft}), theca interna (c, d), or oocytes (a, b) of either healthy or atretic follicles. Bar = ~50 µm for all panels



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  		FIG. 2. Localization of mRNA for LH-R in ovaries from juvenile (a, b) and adult (c–j) possums. Left, light-field photomicrographs; right, corresponding dark-field view. In follicles, the mRNA for LH-R was first observed in granulosa cells (a–f; ->) and theca interna (c–d; {blacktriangleleft}) at the time of antrum formation and was not observed in less developed follicles (g, h; {twoheadrightarrow}). Expression continued in granulosa cells and theca interna of healthy antral follicles (e, f; ->) but was not present in either cell type of atretic follicles (e, f; {twoheadrightarrow}). Note the strong expression observed in the granulosa cells next to the oocyte (c, d; ->). All luteal tissue examined expressed mRNA encoding LH-R (i, j; ->). Hybridization was observed in some interstitial cells (g, h; *) but not in others, especially in juvenile possums (a, b; *). Expression of LH-R mRNA was not observed in surface epithelium (g, h; {blacktriangleleft}) or oocytes of follicles at any stage of development (a–d, g, h). Bar = ~50 µm (a, b, g–j) or ~25 µm (c–f)

Expression of LH-R mRNA was first observed in granulosa cells, cumulus cells, and theca cells at the time of antrum formation (Fig. 2, a–d) and continued to be expressed in healthy, but not atretic, antral follicles through the large antral stage (Fig. 2, e and f). The LH-R mRNA was also observed in corpora lutea of all adult animals examined (Fig. 2, g and h) and in some interstitial cells (Fig. 2, i and j). Interestingly, the interstitial tissue of two out of three juvenile animals did not contain detectable LH-R mRNA (Fig. 2, a and b). Moreover, expression of the LH-R gene was not observed in the surface epithelium or oocytes of any animal (Fig. 2, a–d, g, and h).

Determination of Responsiveness of Granulosa Cells and Luteal Tissue to FSH and LH

Both FSH and LH stimulated cAMP production from possum granulosa cells in a dose-dependent manner (Fig. 3). Although the absolute quantity of cAMP generated from each pool of granulosa cells in response to a given dose of FSH differed, the slopes of the dose response lines were identical. Granulosa cells from all sizes of follicles examined responded to both FSH and LH with increased production of cAMP (Table 1). Luteal tissue did not respond to FSH or LH. Average concentrations (geometric means and 95% confidence intervals) of cAMP (expressed in picomoles per 100 mg of tissue) in control and FSH- and LH-treated luteal tissue were 852 (596–1219), 841 (692–1022), and 836 (618–1131), respectively. The mRNAs encoding FSH-R and LH-R were detected by in situ hybridization analysis in all luteal samples (data not shown), and all animals had an active corpus luteum at the time of ovarian collection based on the presence of a serum progesterone concentration of greater than 7 ng/ml.



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  		FIG. 3. Concentrations of cAMP after exposure of granulosa cells collected from 1- to 2-mm follicles to increasing doses of FSH (A) and LH (B). Both hormones stimulated a dose-dependent increase in cAMP concentrations


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  		TABLE 1. Concentrations of cAMP after treatment with FSH and LH of granulosa cells collected from various-sized follicles.a


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the brushtail possum, as reported in eutherian mammals [32], expression of FSH-R was observed in granulosa cells of follicles shortly after they had begun to grow. Moreover, in follicles, expression of FSH-R was limited to granulosa cells. Although we were unable to collect granulosa cells of preantral follicles, the FSH-R was active in granulosa cells of follicles shortly after antrum formation. As in eutherian mammals, in the brushtail possum, multiple ovulatory follicles can be induced to develop by administration of exogenous FSH [33], suggesting a key role for this hormone in regulation of follicular growth. However, in eutherian mammals [32, 34] and in the marsupial tammar wallaby [35, 36], early follicular growth is not dependent on gonadotropins, indicating that FSH acts in a facilitatory rather than an obligatory role until the final stages of follicular maturation. The pattern of FSH-R expression in follicles and the demonstrated activity of the receptor in granulosa cells suggest that the role of FSH during follicular growth in the brushtail possum is similar to that in other species.

Expression of LH-R in theca interna was first observed at the time of antrum formation in the brushtail possum, which is similar to the pattern present in many eutherian mammals [32, 34]. In eutherian mammals, expression of LH-R in theca interna corresponds with the onset of steroidogenic capability and is limited to the theca interna until the later stages of antral follicular growth, when expression is also observed in the granulosa cells [32, 34]. In fact, in eutherian mammals, expression of the LH-R in granulosa cells is considered a hallmark in follicular growth and signifies the development of a follicle capable of ovulation if provided with the correct endocrine milieu (e.g., a dominant follicle). In monovular species, expression of LH-R in granulosa cells is thought to be one of the key mechanisms by which a follicle maintains dominance, allowing the follicle to continue to grow as circulating concentrations of FSH decrease [11, 12, 34]. In contrast, in the brushtail possum, all healthy follicles express active LH-R in the granulosa cells beginning at the time of antrum formation. Thus, assuming that the preceding hypothesis is correct, selection and maintenance of a single dominant follicle in the brushtail possum must occur by a different mechanism than that observed in monovular eutherian mammals.

The mRNAs encoding both FSH-R and LH-R were observed in the corpus luteum of the brushtail possum. Likewise, expression of FSH-R has been observed in luteal tissue of some eutherian mammals, and the corpus luteum is known to express LH-R in most all species studied to date [12], including the possum [37]. However, neither FSH nor LH was able to stimulate generation of cAMP by luteal tissue. Both FSH-R and LH-R are known to exist in luteal tissue in forms that are not linked to the PKA second messenger system [13–15], and it would appear that this is the case for these receptors in possum luteal tissue. In cattle, luteinization of granulosa cells causes a loss of the full length FSH-R transcript; however, expression of transcripts encoding the extracellular domain, which would bind FSH-R without stimulating PKA, persists in the formed corpus luteum [38]. In rabbits, the corpus luteum contains LH-R that are not able to increase concentrations of cAMP or progesterone [39]. Similarly, in ewes, large luteal cells are able to bind LH without causing an increase in cAMP production [12]. Interestingly, in ewes, large luteal cells are thought to be derived from granulosa cells [12], and the possum corpus luteum is thought to be derived solely from granulosa cells of the ovulatory follicle [7]. In possums and other marsupials such as Macropus eugenii (tammar wallaby), secretion of progesterone from the corpus luteum is thought to be independent of LH and FSH, as hypophysectomy does not decrease secretion of progesterone once ovulation has occurred [7]. In fact, in the tammar wallaby the pituitary gland suppresses normal luteal function during embryonic diapause as evidenced by reactivation of the corpus luteum after hypophysectomy. Prolactin has been identified as the pituitary hormone responsible for this inhibition of the corpus luteum [7]. However, it is important to note that luteal function in the tammar wallaby is very different from that in the brushtail possum; in particular, possums do not undergo embryonic diapause, and luteal tissue of the tammar wallaby does not contain LH-R [37].

Removal of the pituitary gland or corpus luteum prevents parturition in both the brushtail possum and the tammar wallaby [5, 7, 40], providing evidence that both the pituitary gland and the corpus luteum are necessary for normal parturition. The roles of the pituitary gland and the corpus luteum have been most extensively studied in the tammar wallaby. At parturition, peaks of the oxytocic peptide mesotocin from the pituitary gland, the prostaglandin F2{alpha} (PGF2{alpha}) metabolite 13,14-dihydro-15-oxo-prostaglandin F2{alpha}, and prolactin are observed, and a precipitous decline in progesterone occurs [41–44]. In addition, relaxin concentrations in the corpus luteum are increased after Day 22 of pregnancy [45]. The rapid decline in progesterone or surge of prolactin is not necessary for normal parturition, although survival of the young is dependent on prolactin production [42, 43]. Both mesotocin and PGF2{alpha} appear to be central to regulation of parturition as inhibition of PGF2{alpha} synthesis prevents normal parturition [41], and treatment with an oxytocin/mesotocin antagonist delays parturition [46]. Currently, the role of luteal-derived relaxin is unknown. Again, it is important to note that it is unknown if similar mechanisms controlling parturition exist in the brushtail possum. However, several notable differences are known to exist during the time of parturition in brushtail possums. Although the brushtail possum corpus luteum is known to contain relaxin activity [47], it also contains mesotocin [48], which the corpus luteum of the tammar wallaby does not [49]. In addition, in the brushtail possum no precipitous decline in progesterone is observed at the time of birth [5, 7, 42]. Finally, in the tammar wallaby, the female enters estrus shortly after birth of her young, and if the oocyte is fertilized, embryonic diapause occurs [7]. No such events are observed in the brushtail possum [7]. Thus, additional information regarding the potential roles of the pituitary gland and the corpus luteum and their interactions in controlling parturition in the brushtail possum is needed.

As is observed in many eutherian mammalian species [50, 51], the possum ovary contains numerous potentially steroidogenically active interstitial cells [9]. These cells are presumed to be steroidogenically active in the brushtail possum based on their morphologic appearance and the localization of 3ß-hydroxysteroid dehydrogenase enzyme activity in these cells [9, 52]. However, very little is known about their function and regulation. We could find no evidence that FSH would be able to regulate interstitial cells in the possum. In other species, LH is considered a primary regulator of the interstitial tissue [50, 51], and it is possible that LH does play a role in the regulation of interstitial cells in the brushtail possum. We were not able to isolate interstitial cells without some contaminating granulosa cells and therefore were unable to test whether the expressed LH-R in the interstitial cells was capable of generating cAMP. However, expression of LH-R was weak or even absent in many interstitial cells and, in particular, was not observed in the interstitial cells of some juvenile animals, yet these interstitial cells had the morphologic appearance of being steroidogenically active. Thus, it seems likely that steroidogenesis may not be primarily regulated by LH in interstitial cells of the brushtail possum.

In conclusion, we have cloned cDNAs encoding a portion of the FSH-R and LH-R in a marsupial, the brushtail possum. Although the corpus luteum expressed mRNAs encoding both FSH-R and LH-R, neither FSH nor LH was able to stimulate cAMP formation in luteal tissue. The pattern of gene expression for FSH-R during follicular development in the brushtail possum was very similar to that observed in other eutherian mammals: gene expression was first observed in granulosa cells of small growing preantral follicles. In contrast, expression of LH-R in granulosa cells of brushtail possums occurred much earlier in follicular development than that in eutherian mammalian species, suggesting that LH may play a very different role in regulation of follicular development in the brushtail possum. Thus, both FSH and LH are likely to be key regulators of follicular development in the brushtail possum, and regulation of secretion of FSH and LH may be a viable mechanism to control reproductive function in these marsupials.


   ACKNOWLEDGMENTS
 
The authors would like to thank Lara Colbourne, Heath Liddington, and Bert Gwilliam for animal care and tissue collection; Tim Manley for determination of progesterone concentrations in possum sera; Lee-Ann Still and Lynn O'Donovan for preparation of histologic material; Lillian Morrison for assistance with statistical analysis; Alan Barkus for preparation of the figures; Sue Swaney and Vera Bent for secretarial assistance; and Ken McNatty for helpful discussion of the experiments.


   FOOTNOTES
 
First decision: 18 September 2001.

1 Supported by 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

Accepted: November 27, 2001.

Received: August 15, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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L. J. Whale, D. C. Eckery, and J. L. Juengel
Determination of Steroidogenic Potential of Ovarian Cells of the Brushtail Possum (Trichosurus vulpecula)
Biol Reprod, September 1, 2003; 69(3): 947 - 958.
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