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Biology of Reproduction 66, 346-353 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Gene Expression of the Tyrosine Kinase Receptor c-kit During Ovarian Development in the Brushtail Possum (Trichosurus vulpecula)1

Douglas C. Eckery2,a, Stephen B. Lawrencea, Jennifer L. Juengela, Penny Greenwooda, Kenneth P. McNattya, and Andrew E. Fidlera

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ovarian development and function have been extensively studied in eutherian species, with stem cell factor and its receptor, c-kit, having been shown to play key roles at various stages of these processes. In contrast, relatively little is known regarding ovarian development in marsupials. The aims of this study were, first, to establish the timing of key events during germ cell maturation and follicular development and, second, to determine the timing and cellular localization of gene expression for c-kit in the ovaries of a marsupial, the brushtail possum (Trichosurus vulpecula). For this study, ovaries were collected from possums ranging in age from Day 1 after birth to adult. Using stereology, the number of germ cells was found to increase rapidly during the first 60–100 days of life. This was followed by a sharp decline in number, wherein almost 90% of germ cells had disappeared by Day 180. From histological examinations, the time of initiation of meiosis, follicular formation, and follicular growth were determined to occur on Days 35, 50, and 60, respectively. Using in situ hybridization, c-kit gene expression was localized to germ cells and somatic cells during the first 15 days of life; however, after Day 30 and into adult life, c-kit expression was exclusive to germ cells. Results from this study suggest that the pattern of ovarian development is similar in marsupials to eutherians, and that c-kit may play a key role in germ cell development at various stages throughout life.

early development, follicle, gene regulation, oocyte development, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gonadal development and differentiation have been extensively studied in many eutherian species [1]. In contrast, relatively few accounts have been published of similar events in marsupial species. A major difference between eutherian species and marsupials is that, in marsupials, most gonadal development occurs after birth. Aspects of early gonadal development and follicular growth have been described in the tammar wallaby (Macropus eugenii) [2], bandicoots (Perameles nasuta and Isoodon macrourus) [3], gray short-tailed opossum (Monodelphis domestica) [4–6], and Virginia opossum (Didelphis virginiana) [7]. In the brushtail possum (Trichosurus vulpecula), various aspects of ovarian development have been reported [8–11]; however, these studies were limited by small numbers of animals and gaps in the ages studied. Therefore, one of the aims of the present study was to provide a more comprehensive account of key events during ovarian development in the brushtail possum.

Studies of mutant mice strains with phenotypes arising from aberrant migration of germ cells led to the discovery that stem cell factor (SCF) and its receptor, c-kit, are required for germ cell migration, survival, and proliferation [12, 13]. Subsequent studies have defined further roles for SCF and c-kit in early follicular growth [14–16] and ovarian somatic cell interactions [17]. The gene for SCF has been characterized for possums, and the predicted protein structure has been found to be similar to that in eutherian species, suggesting a similar role [18]. Gene expression was found in the ovary and testis, although specific cell types expressing SCF were not identified. Therefore, a second aim of the present study was to determine if c-kit is expressed in ovaries of possums and to identify the timing and cellular localization of gene expression for this receptor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Collection of Tissues

Experiments reported here were performed in accordance with the 1987 Animal Protection (Codes of Ethical Conduct) Regulations of New Zealand with approval from the Animal Ethics Committee of the Wallaceville Animal Research Centre. All animals used were wild-caught brushtail possums ranging in age from Day 1 postpartum to adult. Ages of pouch young (Days 1–200) were estimated from head length and/or crown-rump length based on the nomogram described by Lyne and Verhagen [19]. Adult possums and pouch young >70 days old were killed with an overdose of sodium pentobarbital. Pouch young <70 days old are unable to regulate body temperature; these animals were immersed in ice water to induce hypothermia and then killed by decapitation. Ovaries were collected immediately after the animals were killed and then put into fixative.

Classification of Germ Cells and Follicles

The following morphological classification of germ cells was used in this study (Fig. 1). Oogonia were identified as germ cells with an intact nuclear membrane. Histological distinction between oogonia and primordial germ cells is known to be very difficult [2, 10] and was not made in this study. Meiotic oocytes were identified as germ cells that had entered prophase of meiosis but were not surrounded by granulosa cells. Primordial follicles were defined as oocytes surrounded by a single layer of flattened granulosa cells or a combination of cuboidal and flattened cells. Primary follicles were identified as oocytes surrounded by at least one, and up to but not including two, complete layers of cuboidal granulosa cells. Secondary follicles were divided into type 3 (two to four complete layers of granulosa cells) and type 4 (more than four layers of granulosa cells) follicles. Tertiary follicles were classified as either early antral or mature antral follicles. Early antral follicles were those in which an antrum was just beginning to form but was not yet completed. Mature antral follicles had formed a complete antrum.



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  		FIG. 1. Germ cells and follicles at different stages of development in the brushtail possum. A) Section through the ovary of a Day 74 pouch young showing oogonia (open arrows), meiotic oocytes (asterisks), and primordial follicles (Pr). Bar = 25 µm. B–D) Sections through the ovaries of adult possums showing primary (P), secondary (S), early antral (EA), and mature antral (A) follicles. Note the typical configuration of granulosa cells (arrowheads) around the oocyte of a mature antral follicle. Bar = 25 µm (B) and 50 µm (C and D)

Morphometric Studies

Ovaries from pouch young (n = 75) ranging in age from Day 8 to Day 200 postpartum were fixed overnight in Bouin fixative, embedded in plastic (Technovit 7100; Kulzer & Co., GmBH, Wehrheim, Germany), serially sectioned at a thickness of 25–30 µm, and stained with hematoxylin and eosin. Ovarian volumes and germ cell numbers were estimated using stereological methods as previously described [20–23]. Briefly, ovarian volumes were estimated using the Cavalieri principle, in which the area of tissue was estimated by point-counting on projected images of every 3rd to 10th section, and the sum of the areas was multiplied by the distance between the sections to yield the volume. Germ cell numbers were estimated using the optical dissector method, which is based on a three-dimensional approach to quantifying cell numbers and a systematic, random-sampling procedure (e.g., sampling with a known and constant periodicity from a random start point). By focusing within the section through a known depth, generally between 15 and 20 µm, the germ cell nucleus or chromatin is counted as it comes into focus within an unbiased counting frame. The resulting number of germ cells represents the number in a volume derived from the area of the counting frame multiplied by the depth of the dissector (i.e., 15–20 µm). Germ cells counted were classified as oogonia, meiotic oocytes, and primordial follicles. The coefficient of error for the number of germ cells was <12%. The presence of primary to antral follicles was noted, but such follicles were not counted using this method because their frequency was too low, resulting in error estimates of >20%.

Diameters of randomly selected germ cells and their nuclei were measured at various stages of maturation from at least three different animals. Two measurements of each diameter were made at right angles and averaged using the public domain NIH Image program (v. 1.60; developed at the U.S. National Institutes of Health) from sections containing the nucleolus. No follicle of >2 mm was used to determine oocyte diameters, however, preovulatory follicles in possums reach 4.5–5 mm in diameter [24].

Statistical Analysis

Mean values for germ cell number, diameter, and ovarian volume were log transformed and analyzed by one-way ANOVA using SPSS statistical software (SPSS, Chicago, IL). However, for the ratio of total:volume and for germ cell diameters, tests for homogeneity of variance were not satisfied. Differences between means were determined by the Duncan multiple-comparison test.

Cloning of Possum c-kit cDNA

A range of PCR primers were designed based on evolutionarily conserved regions identified by alignment and comparison of published eutherian mammalian c-kit cDNA sequences (i.e., human [25], mouse [26], rat [27], and bovine [28]). Products were amplified by Taq DNA polymerase-catalyzed polymerase chain reaction (PCR) from total RNA isolated from possum tissues (ovary, testes, brain), and amplification products of the anticipated size were cloned into the TA-cloning vector pCR2.1 (Invitrogen, Carlsbad, CA) and sequenced to determined their identity. The primer pair (forward primer, 5'-GTTGAAT(A/T)(C/T)GAGGCIT(A/T)(C/T)CCIAAACC-3'; reverse primer, 5'-CAICT(C/T)TG(C/T)TCAGTTCCIGG-3'), amplified from adult possum ovarian total RNA, yielded an approximately 0.35-kilobase (kb) product that was identified as being a portion of the possum c-kit homologue. This amplified sequence, corresponding to nucleotides 1081–1427 of the generated possum c-kit cDNA sequence (GenBank accession no. AF131209), was used as the basis for obtaining the remainder of the possum c-kit coding region. The c-kit sequence was extended by PCR amplification from pouch young testicular total RNA using a forward primer within the established possum c-kit sequence (5'-GACTCATGAATGGCCTGCTCCAGTG-3') and a reverse primer that was designed on the basis of further sequence comparisons among eutherian c-kit sequences (5'-AACTCAGCCTGTTICTGGGAAACTCCA-3'). The sequence amplified by these two primers corresponds to nucleotides 1337–1837 of the completed sequence (AF131209). Using this sequence, two further (nested) primers were designed to obtain the 5' sequence of the c-kit cDNA (5' RACE kit; Gibco BRL, Auckland, New Zealand): primer 1, 5'-GTTTCTGGAAATACTTGTAGGTTAG-3' and primer 2: 5'-CTGCTGCTACCACAAAGCCAATC-3'. The amplification product produced with these primers yielded sequence data 1–1666 (AF131209). Obtaining further sequence using 3' RACE proved to be difficult, and this was attributed to a putative long 3'-untranslated region that is present in other mammalian c-kit sequences and is thought likely to be present in the possum c-kit sequence also. Therefore, the remaining portion of the c-kit coding region was amplified using a forward primer designed to the 3' end of the known sequence (5'-GGAAGAGATAAATGGGAACAACTATG-3') and a reverse primer (5'-GCTGCTGCCIACIGA(C/T)TTGAIIC(A/C)CAC-3') designed from further mammalian c-kit sequence comparisons located near the C-terminus of the coding region. The amplification product produced with these primers yielded nucleotides 1746–2924 of the completed sequence.

For all PCR reactions, the following conditions were used: PCR Supermix (Gibco BRL), one cycle of 94°C for 1 min, 35 cycles of 94°C for 30 sec, 55°C for 1 min, and 72°C for 2 min, and one cycle of 72°C for 7 min.

Sequencing Procedure

The PCR amplification products were cloned into the TA-cloning vectors pCR2.1 (Invitrogen) for the initial c-kit cDNA cloning (e.g., nucleotides 1081–1427 of the AF131209 sequence) or pGemT-easy (Promega, Madison, WI) for all subsequent cloning. Plasmids having inserts were first identified by restriction enzyme digestion, purified for DNA sequencing by alkaline lysis (Boehringer Mannheim, Mannheim, Germany), and sequenced using an ABI 373 DNA sequencer (Applied Biosystems, Foster City, CA). All enzymes were used according to the manufacturer's specifications (Boehringer Mannheim). Sequence changes due to Taq DNA polymerase-catalyzed replication errors were identified by comparison of sequences from a minimum of three independent PCRs. Nucleotide sequences common to at least two clones from independent PCRs were considered to be correct because of the low probability of Taq DNA polymerase introducing identical alterations at the same nucleotide position in two independent PCR amplifications.

In Situ Hybridization

Ovaries were collected from possums ranging in age from Day 1 postpartum to adult (Days 1–15, n = 9; Days 30–40, n = 2; Days 75–115, n = 6; Days 150–190, n = 5; prepubertal, n = 2; and adult, n = 2), fixed overnight in 4% (w/v) phosphate-buffered paraformaldehyde, and embedded in paraffin. Cellular localization of mRNAs was determined using an in situ hybridization protocol described previously [29] with minor modifications. Sense and antisense RNA probes were generated from cDNA encoding c-kit (described above) with T7 or SP6 RNA polymerase using the Riboprobe Gemini system (Promega). For all in situ hybridizations, 4- to 6-µm sections of tissue were incubated overnight at 55°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 SSC [single strength: 0.15 M sodium chloride and 0.015 M sodium citrate] and 50% [v/v] 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 Pharmacia Biotech New Zealand, Auckland). The emulsion-coated slides were exposed at 4°C for 3 wk. Slides were then developed and fixed. Sections were stained with hematoxylin and then viewed and photographed using both light- and dark-field illumination on an Olympus BH-2 microscope (Tokyo, Japan). Nonspecific hybridization was monitored by hybridizing at least one tissue section from each group with approximately equal concentrations of the sense mRNA. No specific hybridization was observed for any section hybridized with the sense mRNA (data not shown).

Sexing of Pouch Young

To ensure that data were collected only for ovarian tissue, the sex of pouch young <7 days old was determined from either tissue sections [29, 30] or tail tips recovered at the time of ovary collection by PCR using possum SRY gene-specific primers (GenBank accession no. AF103878). Briefly, paraffin wax-embedded tissue sections or tail tips were incubated at 55°C with shaking for 2 h in lysis buffer (10 mM Tris-HCl [pH 7.5], 50 mM KCl, 2.5 mM MgCl, 0.45% [v/v] Tween 20, and 0.1 mg/ml of proteinase K [Boehringer Mannheim]). Tubes containing tail tips were vortexed every 30 min. Aliquots (10 µl) of the tissue extract were used as templates for amplification of the possum SRY sequence using 2.5 U of Taq DNA polymerase (Boehringer Mannheim) in a total volume of 50 µl containing 20 pmol of forward (5'-TCCGTGAGAAGTGGATCAAGCAGTACA-3', corresponding to nucleotides 1–27 of the possum SRY cDNA sequence) and reverse (5'-GGGTATTCTTCTCTGTGTTTAGCACGC-3', corresponding to nucleotides 204–230 of the possum SRY cDNA sequence) primers and the manufacturer's recommended buffer. As a control to ensure that DNA was present in all samples, forward (5'-ATGGCAAACAGAGCCTACCTTGAGCAG-3') and reverse (5'-AGCGTACCACTGCACGGTCACATTCCA-3') primers for possum GnRH receptor (GenBank accession no. AF032379) were also added to the reaction mixture. Reactions were conducted using a thermal cycler (Perkin-Elmer Cetus, Norwalk, CT) with the following conditions: one cycle of 94°C for 3 min, 60°C for 3 min, and 72°C for 5 min, 30 cycles of 94°C for 30 sec, 64°C for 1 min, and 72°C for 2 min, and a final extension of 72°C for 10 min. The amplification products for SRY (0.23 kb) and GnRH receptor (0.33 kb) were visualized in an ethidium bromide-stained, 2% (w/v) agarose gel. Negative control reactions, in which deionized-water replaced the crude DNA preparation, were used to control for possible contamination of reagents (Fig. 2).



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  		FIG. 2. Example of the use of PCR to determine the genetic sex of newborn possum pouch young. A single product (0.33 kb, derived from the GnRH-receptor gene) was amplified from the DNA of female pouch young (lanes 5, 6, and 8), whereas two products (0.33 kb, derived from the GnRH-receptor gene; and 0.23 kb, derived from the SRY gene) were amplified from the DNA of male pouch young (lanes 4 and 7). Negative controls were performed using both primer pairs together (lane 1), the GnRH-receptor primer pair (lane 2), and the SRY primer pair (lane 3)


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Morphometric Studies

Mean numbers of germ cells and ovarian volumes are shown in Table 1. During the first 30–35 days of pouch life, all germ cells in developing ovaries were present as either primordial germ cells or oogonia. From Days 1–10, germ cells were found scattered throughout the ovaries but were increasingly found in small clusters or nests with age. By Day 12 of pouch life, germ cells were mostly located in nests in the outer cortex, and a clear delineation was seen between the cortex and medulla. Many germ cells were seen in mitosis, reflected by the rapid increase in germ cell numbers during the first 90 days of life. The first oogonia to enter prophase of meiosis (oocytes) were found in the innermost regions of the cortex at approximately Day 35. Thereafter, oocytes were observed until Day 190; however, at this time, they were found only occasionally in small, isolated nests. Primordial follicles began to form at approximately Day 50 in the inner cortex. The number and proportion of primordial follicles increased steadily with age (Fig. 3). The maximum number of germ cells was reached between Days 60 and 100 of pouch life; however, when normalized to ovarian volume, the maximum was reached between Days 40 and 60. The highest number of germ cells in an individual animal (n = 861 242) was recorded in a Day 67 pouch young. Ovarian volume steadily increased with age. After the maximum number of germ cells was reached, a sharp decline was observed in total number due to atresia, and by Day 180, this number was reduced by almost 90%. The biggest decrease was noted between Days 100 and 119 (Fig. 3). The first growing (primary) follicles were observed at approximately Day 65; however, very few primary follicles were seen until Days 90–100, when they became much more prevalent. Secondary follicles were first evident at approximately Day 105 but, like primary follicles did not become prevalent for another 35–40 days. The first signs of antrum formation began at approximately Day 140, but antral follicles were not common until after Day 190. Polyovular follicles were often observed from Days 90–140. Oocytes in mature antral follicles are typically surrounded by a single layer of granulosa cells except in one place, where a cluster of granulosa cells protruding into the antrum is found (Fig. 1). A steady and significant increase in germ cell diameter was found with stage of maturation (Table 2). Diameters of germ cell nuclei also steadily increased up to the early antral stage.


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  		TABLE 1. Mean (±SEM) number of germ cells and ovarian volumes in possum pouch young after birth



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  		FIG. 3. Bar graph showing the mean number of germ cells and the proportion of each germ cell type with age


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  		TABLE 2. Mean (±SEM) diameters of germ cells and their nuclei at different stages of maturation in possums

Possum c-kit Gene

The cDNA sequence of possum c-kit was determined (GenBank accession no. AF131209). Because of the cloning procedure used, the sequence encoding the final C-terminal region (estimated as 21 amino acids) was not obtained; however, possum c-kit protein likely is approximately the same length as its eutherian homologues (~976 amino acids). Hydrophobicity plots (not shown) indicated that the broad structure of the protein resembles its eutherian homologues, having a 22-amino acid signal sequence, a 493-residue extracellular domain, a 23-residue hydrophobic transmembrane region, and an intracellular domain of at least 410 amino acids. In the extracellular domain are 10 Asn residues that are potential N-linked glycosylation sites (Asn 32, 64, 146, 282, 292, 299, 319, 351, 461, and 485). Of these sites, 8 appear to be evolutionarily conserved and present in the majority of other eutherian c-kit sequences. Twelve evolutionarily conserved cysteine residues in the extracellular domain and nine residues in the intracellular domain of eutherians are also present in the possum c-kit sequence.

The localization of c-kit mRNA during ovarian development is shown in Figures 4 and 5. At the time of birth, c-kit gene expression was observed in germ cells and somatic cells within the ovary and in tubules of the mesonephros, particularly just under the coelomic epithelium. By Day 5, expression in the ovary was much stronger and associated primarily with clusters of germ cells in the ovarian cortex. At Day 13, in addition to a strong signal in germ cells, expression was observed in tubules of the rete ovarii. These tubules were located primarily in the region between the mesonephros and ovary, although isolated tubules expressing c-kit were also seen with the ovarian medulla. Oocytes that had entered prophase of meiosis did not express c-kit, however, expression was evident again when meiosis was arrested at the diplotene stage. After Day 30, c-kit gene expression was specific to germ cells and localized to oogonia and oocytes of primordial to antral follicles. No apparent effect of animal age on the expression of c-kit in oocytes was observed.



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  		FIG. 4. Localization of mRNA for c-kit in ovaries of possum pouch young during the first 15 days after birth. Light-field photomicrographs are shown on the left and corresponding dark-field photomicrographs on the right. A and B) Day 5, showing c-kit expression in germ cells within the ovary (o) and in tubules of the mesonephros (m). The inset (upper right), taken from the area indicated by a box, shows c-kit expression in a single germ cell. Bar = 100 µm (A and B) and 10 µm (inset). C and D) Day 13, showing c-kit expression in oogonia (open arrows) primarily located in the ovarian cortex. Expression was also found in cells of the rete ovarii (arrowheads). Bar = 50 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study defines the timing of key events during ovarian development in the brushtail possum and provides, to our knowledge, the most comprehensive account for this species to date. The general pattern of ovarian development in possums is similar to that in other marsupial and eutherian species [1, 11, 31]. A major exception is that most events occur after birth in marsupials, as opposed to during fetal life in eutherians. In possums, morphological sexual differentiation of the gonads occurs around the perinatal period, and testis cords are recognizable within the first few days after birth. As in eutherians, differentiation of the testis precedes that of the ovary. The first histological signs of sexual differentiation, however, can be observed before birth by the presence of either scrotal or mammary primordia [32]. Germ cells originate extragonadally and start to migrate to the developing gonads during fetal life, and they continue to migrate in the 1–2 days after birth [8]. Once in the gonads, germ cells form clusters or nests and begin a rapid proliferation. Maximum numbers of germ cells were observed between Days 60 and 100. This wide range of ages reflects the high degree of variation in the number of germ cells between animals and the method of estimating the ages of pouch young. Similar variation in the number of germ cells has been reported for possums [9] and other species [22, 33]. The degree of variation was greatly reduced, however, when total germ cell numbers were normalized to ovarian volume. As in other species, soon after the maximum number of germ cells was reached, a sharp decline in number was observed due to atresia, wherein numbers were reduced almost 90% by Day 180, with the most dramatic decrease being observed in the number of oogonia between Days 100 and 119.

In possums, as in other species, the first germ cells to progress to the next stage of maturation are those at the innermost regions of the ovarian cortex. In eutherian species, the rete ovarii, which is located in the medulla, is thought to produce substances (e.g., meiosis-activating substance) that induce the maturation process in those germ cells located at the corticomedullary interface [34]. Rete ovarii, although present in the possum ovary, are not thought to play a major role in ovarian development in marsupials [6, 8]. In possums as well as in other marsupials, the medulla is occupied primarily by structures known as medullary cords. These structures might perform a role similar to that of rete ovarii in marsupials [8, 11].

Meiosis was initiated at approximately Day 35 of pouch life in possums. Follicles were first formed at approximately Day 50, and evidence of growth was observed at Days 65, 105, and 140 for primary, secondary, and tertiary follicles, respectively. However, a period of 35–40 days occurred between the time that any type of growing follicle was first observed and the time at which that type became prevalent. Data for oocyte diameters indicated that the oocyte doubles in size in the transition from primary to secondary (type 3) and from secondary to tertiary (early antral) follicles. The relatively long periods of transition between follicle types may be due to the amount of growth needed for the oocyte between those stages. Diameters of oogonia and oocytes of primordial and tertiary follicles were similar to data previously reported [10, 35]. To our knowledge, this is the first study to report the growth of oocytes through the primary and secondary stages. As expected, continual oocyte growth was observed through all stages of follicular growth. The diameters of oocyte nuclei also increased with stage of maturation. Nuclear size in the growing mouse oocyte has been shown to be positively correlated to RNA synthesis and RNA polymerase activity [36, 37]. Also of interest was the configuration of granulosa cells surrounding the oocytes of antral follicles. A typical cumulus oophorus, as seen in eutherian species, is not formed. Instead, granulosa cells form only a single layer around the oocyte, except in one place, where there appears to be a large cluster of granulosa cells. The significance of this configuration for granulosa cell function or communication with the oocyte is not known.

Two major events occurred in the ovary at approximately Day 100 after birth in possums: a major reduction in the number of germ cells, and a greater prevalence of growing follicles. Day 100 marks a time of significant changes in the growth of possum pouch young as well [38]. Around this time, fur begins to grow, eyes open, and growth rate accelerates. Moreover, a dramatic change is seen in milk composition at this time. Although no direct evidence is available, these events occurring in the ovaries are likely associated with the overall metabolic changes.

A cDNA sequence for the tyrosine kinase-receptor c-kit was determined from possum ovary and testis and represents the only known sequence of c-kit in a marsupial. When aligned with protein sequences for seven eutherian species—namely, human [25], mouse [26], rat [27], bovine [28], cat [39], horse (unpublished results, GenBank accession no. AF055037), and goat [40]—the predicted possum c-kit full-length protein shows a mean of 75.6% amino acid identity (range, 72.9%–77.2%). However, this identity is not evenly distributed through the protein with the extracellular domain showing significantly lower sequence identity (mean, 63.7%; range, 60.8%–65.9%) than the intracellular domain (mean, 90.2%; range, 87.9%–91.3%). Presumably, this regional difference in conservation reflects the differing evolutionary constraints on the two sections of the receptor, with the extracellular domain able to coevolve with the SCF ligand whereas the intracellular domain must preserve its tyrosine kinase activity and its interaction with components of the intracellular signaling pathway [41].

In neonatal possums (Days 1–15), c-kit mRNA was localized in both germ cells and somatic cells within the ovaries and in tubules of the mesonephros. Similar results have been reported in fetal sheep [29] and mice [42–44]. After Day 30, expression was specific to germ cells being positive in oogonia, absent during prophase of meiosis, and present again in oocytes of primordial to antral follicles. The absence of signal during meiosis has also been reported in sheep [29, 45] and mice [44] and been attributed to structural changes affecting chromatin during the meiotic process. Expression of c-kit in the oocytes of antral follicles appeared to be reduced compared to that in oocytes of primordial and primary follicles. Whether this is the result of a decreased transcription rate or a "dilution" of mRNA due to the increased size of the oocyte is not known. To our knowledge, possums are the only species reported to date in which c-kit expression within the ovaries is exclusive to germ cells during adult life. Thus, it does not appear that c-kit has a role in somatic cell interactions as it does in other species. In mice, c-kit is also expressed in theca interna and interstitial tissue [46, 47]. In sheep, expression is found in granulosa cells and corpus luteum [48, 49]. In humans, c-kit is expressed in granulosa cells [50], and in cows, expression is found in theca interna [17]. Species differences are not uncommon, and even with these differences, it seems clear that, in all mammalian species, c-kit is associated with germ cell development in a stage-specific manner from fetal to adult life.

In summary, we have identified the timing of key events during ovarian development in the brushtail possum. Moreover, we have shown that c-kit gene expression is associated with germ cells at nearly all stages of development, suggesting an important role for the SCF/c-kit signaling pathway during ovarian development in this species.



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  		FIG. 5. Localization of mRNA for c-kit in ovaries of possum pouch young and adults. Light-field photomicrographs are shown on the left and corresponding dark-field photomicrographs on the right. A and B) Day 78 pouch young showing expression of c-kit in oogonia (open arrows) and oocytes of primordial follicles (Pr). No expression was seen in oocytes undergoing meiosis (asterisks). Bar = 50 µm. C and D) Adult possum showing expression of c-kit in oocytes of primordial to tertiary follicles: primordial (Pr), primary (P), secondary (S), and mature antral (A). Bar = 100 µm


   ACKNOWLEDGMENTS
 
The authors would like to thank the following for their assistance: Lee-Ann Still for preparation of tissues for histology, Lisa Whale for sexing of pouch young, the technical staffs at AgResearch, Ltd., and Landcare Research for collection of pouch young, and Alan Barkus for preparation of figures.


   FOOTNOTES
 
First decision: 25 July 2001.

1 Supported by grants from the New Zealand Foundation for Research, Science and Technology. Back

2 Correspondence: Douglas C. Eckery, AgResearch, Wallaceville Animal Research Centre, P.O. Box 40063, Upper Hutt, New Zealand. FAX: 64 4 922 1380;doug.eckery{at}agresearch.co.nz Back

Accepted: September 11, 2001.

Received: June 22, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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