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Biology of Reproduction 63, 156-164 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular article

Expression of RUSH Transcription Factors in Developing and Adult Rabbit Gonads1

Alexia Rendona, Aveline Hewetsona, Beverly S. Chilton2,a, and Vaughan H. Leea

a Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

ABSTRACT

The RUSH transcription factors 1{alpha} and 1ß bind to the Rabbit Uteroglobin promoter and are members of the SWI/SNF complex that facilitates transcription by remodeling chromatin (Helicase). To characterize gonadal expression of RUSH, a cRNA probe that recognizes both isoforms was used for in situ hybridization studies. We found RUSH mRNA to be abundant in Sertoli cells from embryonic, neonatal, prepubertal, and pubertal rabbit testes. In adults, RUSH mRNA was detected in tubules with preleptotene spermatocytes and mature spermatids lining the lumen. However, RUSH was undetectable in tubules that contained leptotene spermatocytes and that lacked mature spermatids. In females, RUSH was expressed in presumptive granulosa cells of embryonic and neonatal ovaries before follicle organization. Abundant RUSH mRNA was detected in granulosa and theca cells surrounding preantral follicles of prepubertal and adult ovaries. Expression of RUSH remained high in granulosa cells of antral follicles in mature ovaries but was negligible in late-stage atretic follicles and in corpora lutea. Western blot analysis confirmed the RUSH-1{alpha} isoform predominated in both testicular and ovarian tissues. The expression pattern of RUSH indicates transcriptional activity in Sertoli cells and during multiple stages of differentiating granulosa cells, especially those of primordial follicles, which heretofore were considered to be dormant.

follicular development, granulosa cells, ovary, Sertoli cells, signal transduction, testes, theca cells

INTRODUCTION

Hormone-dependent activation of target genes requires receptors as activator proteins, coactivators, and basal transcription factors. The SWI/SNF complex, the first group of nuclear-receptor coactivators to be described [1], was originally identified in yeast as an ATP-dependent multiprotein complex that facilitated transcription by remodeling chromatin [2]. The SWI/SNF proteins were later described as a component of the yeast RNA polymerase II holoenzyme [3], which is considered to be responsible for most polymerase II transcription [4]. The highly conserved nature of the basic transcription machinery and chromatin structure in eukaryotic cells suggests that SWI/SNF-related proteins are also functionally conserved in higher eukaryotes. Recent experiments have shown that SWI/SNF interacts directly with nucleosomal DNA [5], and SWI/SNF binding to nucleosomes in vitro disrupts the DNA as it wraps around the histone octamer in the core particle. Additional in vitro evidence that SWI/SNF is an architectural protein comes from studies in which SWI/SNF preferentially bound synthetic, four-way junction DNAs that resemble the entry/exit point of the DNA in the nucleosome [6]. Thus, SWI/SNF binding might alter the helical twist of the DNA and destabilize the histone-DNA affiliation, resulting in either loss of the H2A-H2B histone dimer or eviction of the entire octamer.

Attention has recently focused on the RUSH family of SWI/SNF-related proteins [7]. The RUSH acronym identifies the key characteristics of the protein members of the family (i.e., RING-finger motif protein cloned in rabbit, binds the uteroglobin promoter, SWI/SNF-related, and Helicase-like). The RUSH cDNAs were isolated by recognition-site screening of {lambda}gt-11 cDNA expression libraries derived from rabbit endometrium. The RUSH-1{alpha} (1005 amino acids, 113 kDa) and RUSH-1ß (836 amino acids, 95 kDa) isoforms bind an 85-base pair (bp) (-170/-85) region of the rabbit uteroglobin (UG) gene, and their availability is regulated by a steroid-dependent, alternative splicing mechanism [8]. The RUSH-1{alpha} isoform is the progesterone-dependent splice variant whose expression correlates with maximal transcriptional activity of the uteroglobin gene, and the RUSH-1ß isoform is expressed when the uteroglobin gene is transcriptionally silent. Thus, the RUSH acronym also identifies the potential significance of the alternative splicing mechanism that allows an accelerated (i.e., a RUSHed) cellular response to selected physiological challenges.

Three independent teams of investigators have cloned the human homologue of RUSH-1{alpha} by recognition-site screening of {lambda}gt-11 cDNA expression libraries derived from HeLa cells. HIP116 [9], hHLTF [10], and Zbu1 [11] colocalize to a single locus on human chromosome 3q25.1-q26.1 [12]. Their cDNAs encode identical 1009-amino-acid proteins (116 kDa). Activation of the PAI-1 gene in HeLa cells by hHLTF [10] and of the PAI-1 gene in 30A5 cells by P113, the mouse homologue of RUSH-1{alpha} [13], provides in vivo evidence that RUSH proteins are transcription factors. The suggestion that RUSH proteins mediate the ability of prolactin to increase progesterone-dependent transcription of the uteroglobin gene [14] is consistent with the hypothesis that RUSH proteins are targeted to an active promoter via an interaction with other transcriptional activators. All RUSH family members are characterized by domains that are typical of ATPases and DNA helicases [15]. Strong DNA-dependent ATPase activity has been demonstrated for HIP116, Zbu1, and P113, and this supports the suggestion that RUSH proteins use the energy of ATP hydrolysis to disrupt the chromatin structure.

Quantitative reverse transcriptase-polymerase chain reaction and the ion-pair, reversed-phase HPLC product purification and detection system were used to show that changes in the ratio of RUSH mRNA isoforms occur in a tissue-specific pattern [16]. Of all the reproductive tract tissues surveyed, the RUSH-1{alpha} splice variant was most abundant in the testis and ovary. As a result, the goal of this study was to use in situ hybridization to define cell-specific RUSH mRNA expression during gonadal differentiation and in adult organs.

MATERIALS AND METHODS

Reagents and Buffers

The following key reagents were used: Paraplast Plus (Sherwood Medical Labs, St. Louis, MO), Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA), EcoRI (Promega, Madison, WI), SeaPlaque GTG (BioWhittaker Molecular Applications, Rockland, ME), Gene Clean II Kit (Bio 101, La Jolla, CA), Biogel-P30 (Bio-Rad Laboratories, Hercules, CA), nitrocellulose transfer/immobilization membranes (Schleicher and Schuell, Keene, NH), Biotrans nylon membranes (0.2 µm; ICN, Costa Mesa, CA), and Renaissance Western Blot Chemiluminescence Kit (NEN Life Science Products, Inc., Boston, MA). NTB-2 emulsion, X-OMAT AR film, and D19 developer were purchased from Eastman Kodak Co. (New Haven, CT). Horseradish peroxidase (HRP)-conjugated donkey antirabbit immunoglobulin (Ig) G and the isotopes [{alpha}-32P]uridine triphosphate (UTP) (>3000 Ci/mmol) and [{alpha}-35S]UTP (>1000 Ci/mmol) were purchased from Amersham Pharmacia Biotech (Arlington Heights, IL).

Anti-RUSH antibodies [8] recognize RUSH-1{alpha} and -1ß, because they were made to a sequence (amino acids 370–387) common to both proteins that displayed strong antigenicity according to the PeptideStructure program (Genetics Computer Group Software, Madison, WI). Antibodies were generated in rabbits by the staff at Research Genetics (Huntsville, AL). Antibody titer was determined by ELISA with free MAP-peptide on the solid phase (1 µg/well), goat antirabbit IgG-HRP conjugate as the secondary antibody, and peroxidase dye. Antibodies were affinity purified to eliminate complications from nonspecific binding.

Stock solutions of PBS (20 mM sodium phosphate [pH 7.5] and 0.9% sodium chloride) and 20x SSC (3 M sodium chloride and 0.3 M sodium citrate [pH 7.0]) were prepared. For immunoblotting, a 10x stock solution of Tris-buffered saline (TBS) consisted of 1.5 M sodium chloride and 200 mM Tris (pH 7.6).

Animals and Tissue Collection

All studies were conducted in accord with the NIH Guidelines for the Care and Use of Laboratory Animals, as reviewed and approved by the Animal Care and Use Committee at Texas Tech University Health Sciences Center. Adult New Zealand white rabbits (age, 6 mo) were housed for 3 wk before experimentation. Tissues from prenatal and juvenile New Zealand white rabbits were harvested at the time of delivery.

Gonads were quickly removed from all killed animals. For RNA and protein isolation, tissues were frozen in liquid nitrogen and stored at -80°C. For in situ hybridization, tissues were fixed overnight in 4% paraformaldehyde in PBS and embedded in Paraplast Plus. Serial sections (5 µm) were mounted on Superfrost Plus slides, dried, placed in desiccated boxes, and stored at 4°C for later use.

Radiolabeled Probes

For Northern blot analysis and in situ hybridization, the original 1509-bp RUSH cDNA clone [8] in pBluescript II SK(+) was used to synthesize cRNAs (sense and antisense) [17]. All labeled probes were separated from unincorporated radionucleotides on Biogel-P30 spin columns.

Northern Blot Analysis

Total RNA samples (20 µg) from ovarian and testicular tissue were fractionated by electrophoresis through formaldehyde containing agarose (1.5%) gels, transferred to Biotrans nylon membranes by capillary elution in 20x SSC, and baked at 80°C for 2 h under vacuum [17]. Membranes were prehybridized (2–4 h at room temperature) and then hybridized overnight (16–22 h at 50°C) in prehybridization solution containing 32P-labeled RUSH cRNA and 10% dextran sulfate. After hybridization, membranes were washed in 0.1x SSC at 75°C for 1 h. Autoradiographic exposure was performed at -80°C using XAR-5 x-ray film with an intensifying screen.

In Situ Hybridization

In situ hybridization was performed according to method described by Lee et al. [17]. Briefly, sections were deparaffinized, rehydrated, and treated with proteinase K. After treatment with acetic anhydride, slides were prehybridized for 2–4 h and then hybridized at 50°C overnight with either a sense or an antisense probe. Slides were treated with RNase A to remove unbound riboprobes and were washed in 0.1x SSC for 2 h at 65°C. After dehydration in ethanol plus 300 mM ammonium acetate, the slides were dried and exposed to film for 3 days to estimate the strength of the hybridization signal. Slides were then dipped in Kodak NTB-2 emulsion and exposed for 7–14 days at 4°C. Slides were developed with Kodak D19 developer, counterstained with Mayer hematoxylin, and viewed with an Olympus BX50 microscope (Leeds Instruments, Inc., Irving, TX) under light- and dark-field optics.

Western Blot Analysis

Proteins (each sample, 50 µg) were resolved on 12% SDS-PAGE (minigels) and transferred to nitrocellulose for 3 h at 75 V in 25 mM Tris-HCl (pH 8.3), 192 mM glycine, 20% methanol, and 0.1% SDS. To verify the molecular weight of the proteins, standards were separated by electrophoresis and transferred with the samples. Transfer efficiency was verified by staining membranes with Ponceau S (2% Ponceau S, 3% trichloroacetic acid, and 3% sulfosalicylic acid) according to the method described by Sambrook et al. [18]. Membranes were dried for 60 min and incubated in blocking buffer (1x TBS and 5% Carnation nonfat dry milk) for 60 min. Membranes were then incubated with affinity-purified RUSH antibodies (1:100 dilution) in antibody dilution buffer (1x TBS and 1% Carnation nonfat dry milk) overnight at 4°C. Membranes were washed twice for 30 min each time in 1x TBS and then incubated in HRP-conjugated donkey antirabbit IgG (1:5000 dilution) in antibody dilution buffer for 30 min at room temperature. Subsequently, membranes were washed twice for 30 min each time and once for 60 min in 1x TBS. Specific signals were detected by chemiluminescence.

RESULTS

In situ hybridization was used to examine the temporal and spatial expression of RUSH-1{alpha} and -1ß mRNA in developing and adult rabbit gonads. Antisense and sense riboprobes were characterized using total RNA isolated from adult rabbit ovaries (Fig. 1). The RUSH antisense riboprobe hybridized to a single 5.2-kilobase (kb) mRNA, whereas the sense riboprobe produced no hybridization signal. In situ hybridization of rabbit gonads collected from fetuses at 18 and 28 days postcoitus showed abundant RUSH mRNA in both testes and ovaries (Fig. 2). Expression of RUSH mRNA in testes predominated in the developing seminiferous tubules of the medullary region, although some scattered cells in the surface epithelium were also positive (Fig. 2B). In contrast, RUSH mRNA, although distributed in both the medullary and the cortical regions (Fig. 2E) of the ovary, was most abundant in the cortical area. Only micrographs from fetuses on Day 18 of gestation are presented here, because no major differences between the expression patterns in testes or ovaries from Days 18 and 28 of gestation were observed.



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  		FIG. 1. Northern blot characterization of antisense and sense riboprobes. A) RUSH antisense riboprobes (-) recognized the 5.2-kb mRNA transcripts of RUSH in total RNA isolated from adult ovaries. No mRNAs were detected with the RUSH sense riboprobes (+). B) Ethidium bromide–stained gel of total RNA samples that were transferred and probed in A.



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  		FIG. 2. In situ hybridization of RUSH mRNA in 18-day-postcoitus rabbit testis (A–C) and ovary (D–F). A and D) Photomicrographs of hematoxylin-and-eosin-stained sections. B and E) Dark-field photomicrographs of sections adjacent to A and D, respectively, probed with antisense RUSH riboprobes. C and F) Dark-field photomicrographs of sections adjacent to B and E, respectively, probed with sense RUSH riboprobes. A and B) Abundant expression of RUSH in seminiferous tubules in the medullary region (arrows) of the fetal testis. D and E) Abundant expression of RUSH in cells of the gonadal epithelium (arrowheads) and in the medullary region (arrows) of the fetal ovary. C and F) Negative controls. Bars = 36 µm

After birth, abundant RUSH mRNA was detected in the somatic Sertoli cells of the developing seminiferous tubules of testes from 5-day-old neonatal rabbits (Fig. 3). However, no transcripts were detected in the prespermatogenic germ cells that also populated the seminiferous tubules, which are little more than anastomosing cords at this stage. The RUSH transcripts were also undetectable in cells of the interstitial tissue and the tunica albuginea.



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  		FIG. 3. In situ hybridization of RUSH mRNA in a 5-day-old rabbit testis. A) Photomicrograph of hematoxylin-and-eosin-stained testicular section. B and C) Bright- and dark-field photomicrographs of a section adjacent to A probed with antisense RUSH riboprobes. D and E) Bright- and dark-field photomicrographs of a section adjacent to B probed with sense RUSH riboprobes. A–C) Abundant expression of RUSH in Sertoli cells (large arrows). The RUSH mRNA was undetectable in prespermatogenic germ cells (arrowheads) and in interstitial cells (small arrows). The conclusion that RUSH mRNA transcripts are undetectable in these areas is based on the observation that the density of silver grains over these cell types is the same as that in the negative controls (D and E). Bars = 36 µm

The same pattern of RUSH expression persisted among somatic Sertoli cells in the testes of 6-wk-old juvenile rabbits (Fig. 4, A–C). Although RUSH was confined to the seminiferous tubules, transcripts were not detected in the prespermatogenic germ cells that typify this stage of development. In addition, RUSH was undetectable in the differentiated Leydig cells, either located around the borders of the seminiferous tubules or grouped into perivascular locations. As a positive control (data not shown), steroidogenic acute regulatory gene expression was used to confirm the identity of Leydig cells on similar tissue sections [19]. Although not included in the illustrations, RUSH transcripts were observed in the cells of the testicular rete. The signal, however, was less intense than that found in Sertoli cells.



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  		FIG. 4. In situ hybridization of RUSH mRNA in rabbit testes. A, D, and G) Photomicrographs of hematoxylin-and-eosin-stained sections. B, C, E, F, H, and I) Bright- and dark-field photomicrographs of sections adjacent to A, D, and G probed with antisense RUSH riboprobes. A–C) sections from a 6-wk-old rabbit testes illustrating RUSH expression in Sertoli cells of developing seminiferous tubules (large arrows). The RUSH mRNA was undetectable in prespermatogenic germ cells (arrowheads) and in interstitial cells (small arrows). D–F) Sections (x10) of an adult testis illustrating RUSH mRNA expression in stage-specific tubules (large arrows). G–I) Sections (x40) of an adult testis illustrating detectable RUSH mRNA expression in tubules containing preleptotene spermatocytes and mature spermatids lining the lumen (large arrows) but undetectable RUSH mRNA expression in tubules containing leptotene spermatocytes but lacking mature spermatids (arrowheads). The RUSH mRNA was undetectable in interstitial cells (small arrows). The conclusion that RUSH mRNA transcripts are undetectable in these areas is based on the observation that the density of silver grains over these cell types is equal to that in sections probed with sense riboprobes. Bars = 36 µm in A–C and G–I and 144 µm in D–F.

A pattern of Sertoli cell-stage specificity emerged when the seminiferous epithelial cycle was analyzed for RUSH expression in adult testes (Fig. 4, D–I). The RUSH mRNA was detectable in somatic Sertoli cells of seminiferous tubules with preleptotene spermatocytes and mature spermatids lining the lumen. However, RUSH transcripts were undetectable in the Sertoli cells of seminiferous tubules with leptotene spermatocytes and no mature spermatids.

In situ hybridization was also used to examine RUSH expression in the ovaries of neonatal rabbits during the formation of primordial follicles and the initiation of follicular growth. Abundant RUSH mRNA was detected in the presumptive granulosa cells of 5-day-old neonatal rabbits (Fig. 5, A–E). The RUSH transcripts were also detectable in the ovarian surface epithelium and the ovarian rete of ovaries from neonatal rabbits, which are structures that share a mesodermal origin with the granulosa cells (data not shown). In 4-wk-old juvenile rabbits, RUSH was expressed in the squamous layer of the granulosa cells of primordial follicles and the cuboidal layer of the granulosa cells of primary follicles (Fig. 5, F–J). The RUSH mRNA was abundant in the granulosa cells of secondary follicles and detectable in the early organizational stages of the theca folliculi in the ovaries of 8-wk-old juvenile rabbits (Fig. 5, K–O). However, with progressive folliculogenesis, RUSH was consistently absent from primary oocytes and extrafollicular stroma. With approaching follicle maturity, RUSH transcripts were abundant in both the granulosa cells and the theca interna of the antral follicles of adult ovaries (Fig. 6, A–C). Whereas RUSH expression persisted in these regions of early atretic follicles (Fig. 6, D–F), expression was undetectable in regions of late atretic follicles and in luteinized granulosa cells (Fig. 6, G–I). The RUSH transcripts were not observed in the ovarian surface epithelium of adult ovaries.



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  		FIG. 5. In situ hybridization of RUSH mRNA in rabbit ovaries. A, F, and K) Photomicrographs of hematoxylin-and-eosin-stained sections. B, G, L, C, H, and M) Bright- and dark-field photomicrographs of sections probed with antisense RUSH riboprobes. D, I, N, E, J, and O) Bright- and dark-field photomicrographs of sections probed with sense RUSH riboprobes as negative controls. A–C) Sections of a 5-day-old ovary illustrating expression of RUSH mRNA in presumptive granulosa cells (arrows). D and E) Negative controls for B and C, respectively. F–H) Sections of a 4-wk-old ovary illustrating RUSH mRNA expression in granulosa cells of primordial follicles (arrowheads). I and J) Negative controls for G and H, respectively. K–M) Sections of an 8-wk-old ovary illustrating RUSH mRNA expression in granulosa cells of primary and secondary follicles (arrows). Labeling for RUSH mRNA was also detectable in presumptive theca cells surrounding these preantral follicles (arrowheads). N and O) Negative controls for L and M, respectively. Bars = 36 µm



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  		FIG. 6. In situ hybridization of RUSH mRNA in adult rabbit ovarian sections. A, D, and G) Photomicrographs of hematoxylin-and-eosin-stained sections. B, C, E, F, H, and I) Bright- and dark-field photomicrographs of sections probed with antisense RUSH riboprobes. A–C) Sections of adult virgin ovary illustrating expression of RUSH mRNA in granulosa cells (arrows) and theca cells (arrowheads). D–F) Ovarian sections illustrating RUSH mRNA expression in granulosa cells (arrows) and theca cells (arrowheads) of an early atretic follicle. G–I) Ovarian sections demonstrating undetectable labeling for RUSH mRNA in a corpus luteum. Bars = 36 µm

Western blot analysis of RUSH proteins was performed with affinity-purified, antipeptide antibodies prepared against amino acids 370–387 of RUSH-1{alpha} and -1ß. Immunoreactive proteins were identified in whole-tissue extracts of immature and mature ovaries and testes (Fig. 7). With an estimated molecular mass of 113 kDa, RUSH-1{alpha} was the predominant isoform. With an estimated molecular mass of 95-kDa, RUSH-1ß was negligible.



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  		FIG. 7. Expression of RUSH proteins in rabbit gonadal tissue. Affinity-purified, antipeptide antibodies recognize RUSH-1{alpha} (113-kDa) protein expression in immature (6-wk-old) and adult (Ad) ovaries and testes (large arrow). The RUSH-1ß (95-kDa) protein expression was negligible in these tissues (small arrow). The 105-kDa molecular weight marker is indicated (MW)

DISCUSSION

These results indicate that the abundant expression of RUSH mRNA in rabbit gonads is localized in the Sertoli cells of developing testes and in the granulosa and theca interna cells of ovaries. Expression of RUSH in these cell types during critical stages of differentiation and development suggests an important role for this transcription factor in gonadal function. Because the posttranscriptional regulation of active versus inactive RUSH in uterine cells involves steroid hormones, this system may be associated with steroid hormone regulation of gonadal development and function.

All members of the RUSH family of transcription factors are characterized by sequence motifs that are suggestive of function. These include a nuclear localization signal, domains that are typical of ATPases and DNA helicases, and a consensus C3HC4 zinc finger (RING) in the C-terminal region. The RING finger binds two Zn2+ atoms, and each molecule is ligated tetrahedrally by four cysteines, or by three cysteines and one histidine, in a unique cross-brace scaffold. This cysteine-rich motif is characteristic of the first subclass of zinc-finger proteins [20, 21], and it is common among genes that regulate development and cell differentiation. The second subclass is characterized by replacement of the fourth cysteine with a histidine. Proteins in this class are involved in protein-protein and/or protein-membrane interactions. The third subclass is the so-called RING-B-Box-coiled-coil (RBCC) group that is characterized by one or two cysteine-rich B-boxes and a coiled-coil domain on the C-terminal end of the protein. Members of the RBCC group are proto-oncoproteins. The fourth subclass is the so-called TRAF subclass, which has a RING motif followed by a large cysteine/histidine-rich region, a coiled-coil domain, and a tumor necrosis factor receptor-associated factor domain. Members of this group are involved in signal transduction pathways.

As members of the first subclass of zinc-finger proteins associated with events of cellular differentiation, RUSH transcripts are most abundantly expressed in male and female gonads [16]. The preferential expression of the RUSH-1{alpha} isoform in the ovary compared with the RUSH-1ß isoform in the endometrium of rabbits in estrous indicates that RUSH pre-mRNAs are alternatively spliced in a tissue-specific manner. McKeown [22] showed that changes in the ratio of the steady-state levels of the mRNA products varied in a direct linear fashion with the splice rate constant. Thus, the 40-fold difference in the ratio of RUSH-1{alpha} to -1ß mRNA isoforms for ovary compared with endometrium reflects the same difference in the relative rates of splicing between the two tissues [16]. These results support the suggestion that RUSH transcription factors may be important regulators of gene expression in the gonad.

The rabbit is an ideal animal system in which to characterize expression of RUSH, because complete development and maturation of the gonads into ovaries and testes occurs primarily after birth. Studies evaluating germ cell kinetics in the testes of most mammalian species have indicated that spermatogenesis begins at puberty, and that oogenesis occurs during the fetal or neonatal period. In the rabbit, spermatogenesis begins in 7- to 8-wk-old juvenile rabbits [23, 24], and oogenesis is initiated and completed during the first 3 wk of neonatal life [25]. Therefore, rabbit gonads can easily be evaluated before and after the onset of either spermatogenesis or oogenesis.

The prenatal rabbit gonad contains primordial germ cells embedded in the urogenital ridge in association with cells derived from the mesonephros and gonadal surface epithelium [26]. On Day 18 of gestation, the fetal rabbit testis and ovary can be distinguished histologically by the presence of seminiferous tubules in the former and the absence of seminiferous tubules in the later [27]. As the testis differentiates, the primordial germ cells are enclosed within tubules along with the Sertoli cells, leaving the steroid-producing Leydig cells outside the compartment [26]. Expression of RUSH mRNA is confined to the Sertoli cells, which are the major supporters of germ cell development throughout postnatal differentiation of the testis. Moreover, RUSH expression by Sertoli cells precedes the formation of mature Leydig cells and the onset of spermatogonial mitoses [28]. This early stage of gonadal differentiation is believed to occur independent of stimulation by gonadotropins or steroid hormones [26]. However, fetal rabbit testes and ovaries synthesize androgens and estrogens, respectively, during this time [29]. If these steroids play a critical role during early gonadal development, RUSH transcription factors may mediate their effects.

In contrast to the well-organized tubules of the testis, the female primordial germ cells remain dispersed in the mesenchymal tissue that is closely associated with the ovarian surface epithelium and the invading mesonephric cells. Unlike the testes, RUSH mRNA is abundantly expressed in the cortical area of the developing ovary and, to a lesser degree, in scattered cells of the medullary region. Thus, even at this early stage of gonadal development, the pattern of RUSH expression reflects the morphological differentiation and compartmentalization of Sertoli cells as well as the delayed differentiation and separation of ovarian medullary and cortical compartments. This pattern is commensurate with a dual origin of Sertoli cells and granulosa cells from the mesonephros and the gonadal surface epithelium [26].

Expression of RUSH by Sertoli cells precedes the formation of mature Leydig cells and the onset of spermatogonial mitoses [28]. In the adult testes, the pattern of expression for RUSH mRNA is reminiscent of that of stage-specific Sertoli cell expression [30] for such gene products as transferrin and sulfated glycoprotein-2 [31]. Initially, it appeared that RUSH shared a common expression pattern in the testes with the Wilms' tumor susceptibility gene (WT1), which encodes WT1, a RING-finger protein that has been implicated in normal gonadal development [32]. Expression of WT1 is restricted to Sertoli cells and their precursors, the embryonic tunica albuginea, and the rete testis of the male gonad [33]. However, because RUSH mRNA was not detected in the tunica albuginea, its expression pattern was not identical to that of WT1. Additional RING-finger proteins that may be involved in sperm differentiation, include sperizin, a murine RING-finger protein that is exclusively expressed in the round spermatids, and mouse ret finger protein, which is expressed at the highest levels in pachytene spermatocytes and round spermatids [34]. Gong et al. [11] used immunohistochemical analysis to localize Zbu1, the human homologue of RUSH, to mature sperm and spermatids. Although RUSH transcripts were not detected in mature sperm or spermatids, RUSH expression in spermatocytes cannot be ruled out by the methods used in the present studies. Immunocytochemical analyses are required to determine whether spermatocytes transiently express RUSH proteins during different stages of spermatogenesis.

Oogenesis in the rabbit begins on the first day after birth [35]. The transformation of oogonia into oocytes occurs between Days 1–10, and the process of oogenesis is complete at the end of the second week of postnatal life [25]. The RUSH transcription factor is expressed in presumptive granulosa cells before follicle assembly. These presumptive granulosa cells are arranged close to the stromal wall of the oocyte clusters as well as in association with single oocytes. This cellular array in rabbits is similar to that described for forming primordial follicles in neonatal rat ovaries [36]. By 4 wk of age, the rabbit ovary contains a large population of primordial and primary follicles [37, 38]. Once the primordial follicles were assembled, RUSH mRNA was present during all stages of follicular development, including mature antral follicles. Expression of RUSH in primordial follicles is particularly intriguing, because these follicles are often considered to be dormant or inactive. Expression of transcription factors such as RUSH and the slow proliferation rate of granulosa cells in some primordial follicles [39, 40] indicate that active processes are occurring in these "dormant" follicles.

In summary, RUSH encodes a putative transcription factor implicated in hormone-dependent gene expression in the endometrium. In the developing gonad, RUSH mRNA expression is restricted to Sertoli cells during spermatogenesis/spermiogenesis and is linked to the different stages of granulosa cell development in females. Identification of RUSH in both Sertoli and granulosa cells is not surprising, because Sertoli cells are considered to be the male homologue of granulosa cells [41]. That the developing ovarian surface epithelium and ovarian rete express RUSH is also not surprising, because the NIH-OVCAR3 ovarian carcinoma cell line derived from surface epithelium expresses Zbu1 mRNA [11], which is the human homologue of RUSH-1{alpha}. These observations support the suggestion that RUSH transcription factors are involved in cell-specific gene expression in developing rabbit gonads, and they provide the groundwork for future studies to better understand the differentiation and development of mammalian gonads.

FOOTNOTES

First decision: 27 January 2000.

1 Supported in part by a Howard Hughes Medical Institute grant through the Undergraduate Biological Sciences Education Program to Texas Tech University and by NIH grants HD29457 (B.S.C.) and HD34457 (V.H.L.). Back

2 Correspondence: Beverly S. Chilton, Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430. FAX: 806 743 2990; beverly.chilton{at}ttmc.ttuhsc.edu Back

Accepted: February 22, 2000.

Received: January 6, 2000.

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A. Hewetson, E. C. Hendrix, M. Mansharamani, V. H. Lee, and B. S. Chilton
Identification of the RUSH Consensus-Binding Site by Cyclic Amplification and Selection of Targets: Demonstration that RUSH Mediates the Ability of Prolactin to Augment Progesterone-Dependent Gene Expression
Mol. Endocrinol., September 1, 2002; 16(9): 2101 - 2112.
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