mRNAs Encoding a von Ebner's-like Protein and the Huntington Disease Protein Are Induced in Rat Male Germ Cells by Sertoli Cells*

Viqar SyedDagger , Edith Gomez§, and Norman B. Hecht

From the Center for Research on Reproduction and Women's Health and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, Pennsylvania 19104

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The success of spermatogenesis is dependent upon closely coordinated interactions between Sertoli cells and germ cells. To identify specific molecules that mediate interactions between somatic cells and germ cells in the rat testis, Sertoli cell-germ cell co-cultures and mRNA differential display were used. Two cDNAs, clone 1 (660 nucleotides) and clone 2 (390 nucleotides) were up-regulated when Sertoli cells were co-cultured with pachytene spermatocytes or round spermatids. Northern blot analyses confirmed the differential display expression patterns. Sequence analyses indicated that clone 1 was similar to a von Ebner's gland protein (87% at the nucleotide level and 80% at the amino acid level) and clone 2 was identical to a region of the Huntington disease protein. The von Ebner's-like protein mRNA was induced after 4 h of co-culture, while the Huntington disease protein required 18 h of co-culture for expression. The von Ebner's-like protein was induced in germ cells by a secreted Sertoli cell factor(s) smaller than 10 kDa that is sensitive to freezing and thawing or boiling. The Huntington disease protein was induced in germ cells by a Sertoli cell secreted factor(s) larger than 10 kDa which survives freezing and thawing, but is inactivated by boiling.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A prerequisite for normal spermatogeneis is the coordination of Sertoli cell function with the needs of germ cells in the seminiferous epithelium. Regulatory interactions between Sertoli cells and germ cells facilitate the sequential expression of genes needed for male germ cell differentiation (1-4). Disruption of testicular cell associations by heat, disease, or cytotoxic agents often affect the interactions between Sertoli cells and germ cells and lead to infertility (5-7).

Numerous experiments have utilized co-cultures of Sertoli cells and germ cells to investigate how Sertoli cell factors influence germ cell development. Sertoli cells stimulate germ cell RNA and DNA synthesis (8), induce the appearance of germ cell surface antigens (9), and maintain spermatogenic cell glutathione synthesis (10). Sertoli cell secretory products have been proposed to mediate regulatory factors such as insulin growth factor 1 (11-14) and transforming growth factors alpha  and beta  (15-17). Sertoli cells secrete transferrin (18, 19) and an acute-phase protein identical to rat haptoglobin (20), matrix metalloproteases, plasminogen activators, cyclic protein-2, and protease inhibitors such as tissue inhibitor of metalloproteases-2 and alpha 2-macroglobulin (21-24). The activities of Sertoli cell proteases and protease inhibitors display pronounced cyclic changes (25). The highest activities of plasminogen activator and cyclic protein-2 are observed at stages where germ cells translocate to the adluminal compartment, stages VII-VIII and VI-VII, respectively. At the same time, protease inhibitors such as cystatin (stages VII-VIII) and alpha 2-macroglobulin (stages VII-X) show low levels of activity (25). The number of germ cells in the testis is also regulated by Fas ligand from Sertoli cells, a molecule initiating apoptosis in germ cells expressing Fas (26).

Germ cells can also affect Sertoli cell functions. Germ cells secrete proteinacious inhibitors or stimulators that modulate Sertoli cell function (27-29). For instance, germ cell-conditioned medium stimulates the phosphorylation of proteins in Sertoli cells (30), stimulates gamma -glutamyl transpeptidase activity of Sertoli cells (31), and decreases RNA synthesis in Sertoli cells (32). Germ cells in co-cultures can influence Sertoli cell protein glycosylation (33), increase basal and follicle-stimulating hormone-induced androgen-binding protein production (34, 35), inhibit CP-2/cathepsin L mRNA expression (36), enhance inhibin and transferrin secretion and transferrin gene expression (37-39), inhibit Sertoli cell estradiol production (38), stimulate fibroblast growth factor receptor type-1 expression (40), stimulate alpha 2-macroglobulin expression (41), and N-cadherin expression (42). Testin, a glycoprotein secreted by Sertoli cells, shows a sharp increase when Sertoli-germ cell junctions are disturbed suggesting that germ cells may down-regulate testin gene expression (43-45). Nerve growth factor expressed in spermatocytes and early spermatids (46, 47) has also been proposed to be a germ cell paracrine factor for the nerve growth factor receptor in Sertoli cells (48). Differential regulation of Sertoli cells by germ cells has been studied by examining the effects pachytene spermatocytes or round spermatids have on Sertoli cells. A 24.5-kDa protein, homologous to phosphatidylethanolamine-binding protein, produced by cultured round spermatids stimulates the secretion of proteins from Sertoli cells, suggesting that the phosphatidylethanolamine-binding protein participates in the negative regulation of Sertoli cell secretory function during spermatogenesis (49). Soluble protein(s) from pachytene spermatocytes stimulate ceruloplasmin, transferrin, and sulfated glycoprotein 1 and 2 in Sertoli cells (50). Clearly, interactions between somatic and germ cells lead to the induction of factors that selectively up- or down-regulate gene expression in recipient cells.

We have previously shown that several genes including an isoform of casein kinase, an epidermal growth factor, a statin-related protein and an integral membrane glycoprotein are up-regulated and a basic fibroblast growth factor, fibronectin, an endoplasmic reticulum stress protein and a pro-alpha 2(XI) collagen are down-regulated when Sertoli cells are co-cultured with a mixed population of germ cells (51). Here we begin to define the molecular mechanisms regulating cellular interactions between Sertoli cells and specific populations of meiotic or post-meiotic male germ cells. We demonstrate that a von Ebner's-like protein and the Huntington disease protein are up-regulated in male germ cells by soluble factors secreted by Sertoli cells.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of Sertoli Cells-- Primary cultures of Sertoli cells were prepared from 20-day-old Sprague-Dawley rats (Charles River, Kingston, MA) by sequential enzymatic treatments as described previously (51-53). The testes were decapsulated and digested with trypsin (1 mg/ml, Sigma) for 30 min at 37 °C to remove interstitial cells and then with collagenase (1 mg/ml, Sigma) for 25 min at 37 °C. The cell suspensions were centrifuged at 800 rpm for 2 min. The supernatants, containing peritubular cells, were discarded. The pellets were washed twice with phosphate-buffered saline and incubated in phosphate-buffered saline containing hyaluronidase (1 mg/ml, Sigma) for 30 min at 37 °C. After incubation, the cell suspensions were centrifuged at 800 rpm and the pellets were washed twice with phosphate-buffered saline. The resulting Sertoli cells were plated at a density of 2 × 106/cm2 in polystyrene gamma -irradiated Petri dishes (Falcon, Oxnard, CA) in serum-free Ham's F-12 and Dulbecco's modified Eagle's medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10 µg/ml insulin (Sigma), 5 µg/ml transferrin (Sigma), and 4 µg/ml gentamycin (Life Technologies, Inc.). Cells were maintained in a humidified atmosphere of 95% air and 5% CO2 at 32 °C. To obtain Sertoli cells with a purity greater than 97%, cultures were hypotonically shocked with 20 mM Tris (pH 7.4) 48 h after plating to lyse contaminating germ cells, and then washed twice with culture medium. Twenty-four hours later, germ cells were added to the cultured Sertoli cells for different time periods as described.

Isolation of Germ Cells-- Germ cells were obtained from the testes of 60-day-old rats. The testes were decapsulated and incubated with collagenase (1 mg/ml) for 15 min at 37 °C. After the seminiferous tubules settled, interstitial cells were removed by decanting the supernatant. The seminiferous tubules were incubated with trypsin (1 mg/ml) for 20 min at 37 °C, pipetted up and down several times to obtain a single cell suspension, and filtered through 20-µm nylon mesh. Enriched populations of germ cells were obtained from adult rat testes by Staput sedimentation as described previously (53). Staput cell separations were performed in bovine serum albumin gradients (2-4%) in culture medium adjusted to pH 7.4. The cells were allowed to sediment for 3.5 h, fractions were collected, and cells were identified by microscopy.

Co-cultures of Sertoli Cells and Germ Cells-- Sertoli cells (2 × 106) from 20-day-old rats were co-cultured with pachytene spermatocytes (8 × 106) or round spermatids (8 × 106) for 24 h. Germ cells were cultured in medium supplemented with 2 mM sodium pyruvate and 6 mM DL-lactate at a density of 8 × 106/ml. At the end of culture, cells were scraped from the plates and RNA was extracted.

To study the time-dependent expression and cellular sites of the induced genes, Sertoli cells were co-cultured with pachytene spermatocytes or round spermatids for 2, 4, 8, 18, and 24 h. At the termination of each culture, the germ cells were removed from Sertoli cells by aggressive washing. Total RNAs were extracted from Sertoli cell-germ cell co-cultures and from separated Sertoli cells and germ cells with the RNAgents Kit (Promega, Madison, WI). Throughout the cultures, cell viability, monitored by trypan blue exclusion, revealed greater than 97% viability. To delineate whether secreted factors induced mRNAs, the germ cells and Sertoli cells were cultured separately for 24 h and the media were collected. The media collected from Sertoli cells were added to germ cells and the media from germ cells were added to Sertoli cells. The Sertoli and germ cells were then cultured for 24 h and collected for RNA extraction.

To start to characterize the factor(s) up-regulating genes in co-culture, conditioned media from germ cells or Sertoli cells were collected after 24 h of culture (35, 44, 45). The media were boiled for 10 min, frozen and thawed five times, or passed through Ultrafree-15 columns (Sigma) containing Biomax membranes with a molecular weight cut-off limit of 10 kDa. Germ cells were cultured with media prepared from Sertoli cells and Sertoli cells were cultured with media prepared from germ cells treated as described above. After 24 h, the cells were collected for RNA extraction.

Differential Display and Isolation of Clones-- Preparation of total RNA from Sertoli cells, from co-cultures of Sertoli cells with pachytene spermatocytes, or round spermatids, pachytene spermatocytes, and round spermatids were extracted as described previously (54). The mRNA differential display was performed as described earlier (51-53). Aliquots of total RNA (400 ng) were reverse-transcribed with 300 units of reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD) in a buffer containing 250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2, 10 µM dithiothreitol, 1 µM anchor primer (Operon, Alameda, CA), and 5 µM each of dATP, dCTP, dGTP, and dTTP for 60 min at 42 °C. After inactivating the reverse transcriptase, 2 µl of reverse-transcribed reaction mixtures were added to 18 µl of PCR1 buffer containing 100 mM Tris-HCl (pH 8.3), 500 mM KCl, 1.5 mM of MgCl2, 2 µM of each dATP, dCTP, dGTP and dTTP, 1 µl of anchor primer, 10 µCi of [35S]dATP, and 1 unit of Taq polymerase (Perkin Elmer, Norwalk, CT). The cycling parameters for PCR were 95 °C for 30 s, 42 °C for 2 min, and 72 °C for 30 s, followed by 72 °C for 5 min. The radiolabeled cDNAs were electrophoresed in 6% denaturing polyacrylamide gels and after drying, the gels were exposed to x-ray films.

Subcloning and Sequencing-- cDNA bands that were reproducibly detected in multiple preparations and in multiple differential displays were excised from the gels and extracted in a 0.3 M ammonium acetate solution containing 1% SDS and 5 µg of tRNA. The cDNAs were precipitated by the addition of 3 M sodium acetate (pH 5.2) and 1 ml of 100% ethanol, resuspended in 10 µl of water and amplified by PCR using the same reaction conditions as described above. The PCR amplified fragments were ligated into the PCR-II vector of the TA cloning kit according to the manufacturer's instructions. Plasmids were prepared by the QIAprep, Miniprep Kit (Qiagen, Santa Clarita, CA). Both strands of each cDNA were sequenced using M13 and T7 primers. The sequences of the isolated clones were compared with the GenBank and EMBL data bases.

Radiolabeling DNA Probes and Northern Blot Analysis-- Plasmid DNAs (10 µg) were digested with EcoRI and the cDNA inserts were separated from the vector by electrophoresis on 1% agarose gels. The insert bands were excised from the gels and purified with the Sephaglas Band Prep Kit (Pharmacia Biotech Inc., Piscataway, NJ). Approximately 25 ng of cDNAs were radiolabeled with 50 µCi of [alpha -32P]dCTP (NEN Life Science Products Inc.) and the random primer labeling system (Life Technologies Inc.). Unincorporated dNTPs were removed by centrifugation through Chroma-Spin columns (CLONTECH, Palo Alto, CA) and the probes were denatured at 100 °C for 5 min immediately before hybridization. Northern blot analysis was performed with GeneScreen membranes (NEN Life Science Products Inc.) as described earlier (51-54). Equal amounts of RNA (10 µg) were hybridized and after hybridization, the blots were stripped, and rehybridized with an actin cDNA to monitor for equal RNA loading and transfer.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To define genes whose expression is induced by cell-cell interactions of specific germ cells and Sertoli cells, we have co-cultured rat Sertoli cells and pachytene spermatocytes or round spermatids. Using mRNA differential display, two cDNAs were detected with RNAs isolated from co-cultures that were not detectable in equivalent RNA preparations from separate cultures of Sertoli cells, pachytene spermatocytes, or round spermatids. Both genes, up-regulated in germ cells, will be discussed in turn.

The mRNA of a von Ebner's-like Gene Is Up-regulated in Co-cultures-- A cDNA of 660 nucleotides (clone 1 in Fig. 1) was up-regulated in co-cultures of Sertoli cells with pachytene spermatocytes or round spermatids. It showed 87% similarity at the nucleotide level (from nucleotides 107 to 767) and a coding region amino acid similarity of 79.5% (from amino acid 18 to 178) to von Ebner's protein (Fig. 2) (55). The von Ebner's-like gene encodes a transcript of 1.2 kb in RNA from co-cultures or total testis, which is not detectable in RNA preparations from brain, lung, or liver confirming the selective differential display expression of this cDNA (Fig. 3A). Although von Ebner's protein is normally expressed in salivary glands, tear glands, and prostate, we did not detect hybridization with this 660-nucleotide von Ebner's-like protein cDNA to RNA from these tissues (Fig. 4).


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Fig. 1.   mRNA differential display profile of cDNAs expressed in co-cultures of Sertoli cells and germ cells. RNAs were isolated from 24-h cultures of Sertoli cells (SC), Sertoli cells co-cultured with pachytene spermatocytes (SC+P), pachytene spermatocytes (P), Sertoli cells co-cultured with round spermatids (SC+RS), and round spermatids (RS) and analyzed by reverse transcriptase-PCR. The primers used were OPA-12 (TCGGCGATAG) and T11GT. The arrowheads show two cDNAs (1 and 2) that are differentially expressed. Size markers are shown on the right. bp, base pair.


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Fig. 2.   Amino acid sequence of clone 1. Amino acid sequence comparison of clone 1 with rat von Ebner's gland protein (GenBank accession number P20289). 159 amino acids of open reading frame of clone 1 (lower sequence) share high extent of similarity (79.5%) with amino acids 18 to 178 of the von Ebner's protein (upper sequence). The shaded area indicates identity.


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Fig. 3.   Northern blot of RNAs differentially expressed in Sertoli cell-germ cell co-cultures. Total RNAs (10 µg) were extracted from 24-h cultures of Sertoli cells alone (SC), Sertoli cells co-cultured with pachytene spermatocytes (SC+P), Sertoli cells co-cultured with round spermatids (SC+RS), pachytene spermatocytes alone (P), round spermatids alone (RS) and from testis, brain, lung, and liver. RNAs were electrophoresed and hybridized with the cDNAs encoding the von Ebner's-like protein (A) and Huntington disease protein (B). The blots were rehybridized with an actin coding region cDNA.


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Fig. 4.   Northern blot showing selective expression of the von Ebner's-like gene. Total RNAs (10 µg) were extracted from testis, brain, prostate, lacrymal gland, salivary gland, and interstitial cells. RNAs were electrophoresed and hybridized with the cDNA encoding the von Ebner's-like protein. The blots were rehybridized with an actin coding region cDNA.

The von Ebner's-like Transcript Is Induced in Germ Cells by a Sertoli Cell Factor(s) after 4 h of Co-culture-- In order to study the time dependent induction and cellular site of expression of the von Ebner's-like gene, Sertoli cells were co-cultured with pachytene spermatocytes or round spermatids for 2, 4, 8, 18, and 24 h. At the termination of culture, germ cells and Sertoli cells were separated and RNA was prepared from each. The 1.2-kb transcript was first detected in RNA from co-cultured germ cells after 4 h co-culture, but not in individual cultures of Sertoli cells, pachytene spermatocytes, or round spermatids under identical culture conditions (Fig. 5A). Surprisingly, no mRNAs were detected in either Sertoli cells or germ cells following their disassociation after co-culture.


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Fig. 5.   Time-dependent expression and cellular localization of cDNAs that are up-regulated in co-cultures of Sertoli cells with pachytene spermatocytes or round spermatids. Total RNAs (10 µg) were isolated from 2-, 4-, 8-, 18-, and 24-h cultures of Sertoli cells with pachytene spermatocytes (SC+P), Sertoli cells co-cultured with either pachytene spermatocytes or round spermatids and then separated (SC), pachytene spermatocytes that had been co-cultured with Sertoli cells and then separated (P), Sertoli cells co-cultured with round spermatids and then separated (SC+RS), and round spermatids that had been co-cultured with Sertoli cells and then separated (RS). The three lanes underlined with C represent Sertoli cells (SC), pachytene spermatocytes (P), or round spermatids (RS) cultured alone for 24 h as controls. The blot was hybridized with cDNAs encoding the von Ebner's-like protein (A) and the Huntington disease gene (B). The blot was rehybridized with an actin coding region probe.

To determine whether germ cells or Sertoli cells or both express the von Ebner's-like protein mRNA, conditioned media were prepared from germ cells and Sertoli cells. The conditioned medium from Sertoli cells induced the 1.2-kb von Ebner's-like protein mRNA in germ cells, while the germ cell-conditioned medium did not induce the mRNA in Sertoli cells, demonstrating that the germ cells are transcribing the 1.2-kb mRNA (Fig. 6A). The Sertoli cell factor(s) that induces the 1.2-kb von Ebner's-like protein mRNA in germ cells is smaller than 10 kDa and is inactivated by freezing and thawing or boiling (Fig. 7A).


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Fig. 6.   Northern blot of RNAs from Sertoli cells cultured with germ cell conditioned medium or germ cells cultured with conditioned medium from Sertoli cells. RNAs (10 µg) were isolated from 24-h cultures of Sertoli cells and germ cells (SC+GC), Sertoli cells cultured with germ cell conditioned media (SC+GCM), or germ cells cultured with Sertoli cell conditioned media (GC+SCM). RNAs were electrophoresed and hybridized with cDNAs encoding the von Ebner's-like protein (A) and the Huntington disease gene (B). Blots were rehybridized with an actin coding region cDNA.


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Fig. 7.   Partial characterization of Sertoli cell and germ cell factor(s). Total RNAs (10 µg) were extracted from Sertoli cells co-cultured with germ cells for 24 h (SC+GC), Sertoli cells cultured with germ cell conditioned medium (SC+GCM), Sertoli cells cultured with germ cell conditioned medium that had been passed through a Millipore Ultrafree-15 column with a 10-kDa molecular mass cut-off limit (SC+GCM >10 kd), Sertoli cells cultured with germ cell medium that had been frozen and thawed 5 times (SC+GCM F & T), Sertoli cells cultured with germ cell medium previously boiled for 10 min (SC+GCM 100 C), germ cells cultured with Sertoli cell conditioned medium (GC+SCM) germ cells cultured with Sertoli cell medium that had been passed through a Millipore Ultrafree-15 column of 10-kDa molecular mass cut-off limit (GC+SCM >10 kd), germ cells cultured with Sertoli cell medium that had been frozen and thawed 5 times (GC+SCM F & T), and germ cells cultured with Sertoli cell medium previously boiled for 10 min (GC+SCM 100 C). RNAs were electrophoresed and hybridized individually with cDNAs encoding the von Ebner's-like protein (A) and the Huntington disease gene (B). The blots were rehybridized with an actin coding region cDNA.

The Huntington Disease Protein mRNA Is Up-regulated in Co-cultures-- A cDNA of 390 nucleotides (clone 2 in Fig. 1) was up-regulated when Sertoli cells were co-cultured with pachytene spermatocytes or round spermatids. Sequence analysis of the cDNA revealed 100% identity with nucleotides 5618 to 6008 of the Huntington disease gene. A transcript of about 10 kb was detected in RNA isolated from co-cultures of Sertoli cells and pachytene spermatocytes, from co-cultures of Sertoli cells and round spermatids or total testis, but not in RNA from Sertoli cells cultured alone, germ cells cultured alone, brain, lung, or liver (Fig. 3B).

The Huntington Disease Protein mRNA Is Induced in Germ Cells by a Sertoli Cell Factor(s) after 18 h of Co-culture-- When germ cells are separated from Sertoli cells after co-culture for 18 h or more, the transcript for the Huntington's disease gene was seen in RNA isolated from pachytene spermatocytes or round spermatids, but not from Sertoli cells (Fig. 5B). The 10-kb transcript was also induced in germ cells with Sertoli cell-conditioned media, but not in Sertoli cells with germ cell-conditioned media (Fig. 6B). The Sertoli cell factor(s) appears to be larger than 10 kDa and survives freezing and thawing, but is inactivated by boiling (Fig. 7B).

    DISCUSSION
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INTRODUCTION
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DISCUSSION
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Local secretory factors and cell to cell communication within the seminiferous tubule are essential to create and maintain microenvironments that allow the normal progression of spermatogenic events. Inappropriate or absent intratesticular signals from either germ cells or Sertoli cells may cause abnormal spermatogenesis leading to infertility. Many paracrine and autocrine factors mediate interaction between different testicular cell types (3, 4, 56, 57). The expression of inhibin, transferrin, testin, and prepro-enkephalin in Sertoli cells is under the influence of paracrine factors potentially of germ cell origin (37, 44, 45, 58, 59). Here we report that Sertoli cells in culture can induce meiotic or post-meiotic male germ cells to up-regulate a von Ebner's-like protein and the Huntington disease protein.

von Ebner's gland protein belongs to the lipocalin protein family, a large group of small proteins including bilin-binding protein, retinol-binding protein, retinoic acid-binding protein, beta -lactoglobulin, odorant-binding protein, apolipoprotein D, and murine beta -trace (60). Members of this family bind and transport small hydrophobic molecules such as retinoids, steroids, bilins, and lipids (61). Some members of this family such as the odorant-binding protein show little ligand specificity, while others, such as the retinol-binding protein, are highly specific. The lipocalins have been implicated in the modulation of immune responses and serve as carrier proteins helping to clear both endogenous and exogeneous compounds (60). In the tongue, von Ebner's protein is secreted from the lingual gland where it is believed to help clear bitter tasting compounds (55). Lipocalin from human von Ebner's gland contains three sequence motifs corresponding to the papain-binding domains of cysteine proteinase inhibitors. Since von Ebner's gland protein is able to inhibit papain activity to a similar extent as salivary cystatin, it may also function as an inhibitor of cysteine proteinases and have a role in the control of inflammatory processes in oral tissues (62).

Several lipocalin proteins including apolipoprotein D, murine beta  trace, lipocalin-type prostaglandin D synthase, and odorant receptors are expressed in the testis (63-65). Substantial levels of apolipoprotein D have been detected in rat and rabbit Sertoli cells (63, 64). Apolipoprotein D may serve as a transport protein carrying small hydrophobic molecules such as unesterified and esterified cholesterol and lecithin (66). Lipid metabolism is essential for steroid hormone biosynthesis in the testis and a role for apolipoprotein D in steroid hormone-binding and transport in the testis could be envisaged. Murine beta  trace has been localized to Leydig cells of postpubertal animals and has been proposed to function in transport of small hydrophobic molecules across the blood testis barrier (65). Lipocalin-type prostaglandin D synthase also is expressed in Leydig cells (67). The receptors of a subfamily of lipocalin proteins, odorant-binding proteins (68), have been detected in rat spermatids (69-72).

Our finding that induction of a von Ebner's-like protein in meiotic and post-meiotic germ cells suggests the presence of an additional lipocalin in the testis. A number of different transport proteins (73, 74), including ceruloplasmin (75, 76), retinoid-binding protein (77, 78), and clusterin (79, 80) have been detected in Sertoli cells. Many of these proteins have been implicated in the transport of nutritional components to germ cells. Our detection of a von Ebner's-like protein induced in germ cells suggests that a similar transport molecule could carry small molecules in germ cells or from germ cells to Sertoli cells. Although the von Ebner's-like protein induced in germ cells shows homology (Fig. 2) to a gene expressed in salivary glands (57), tear glands (68), and the prostate (81), under the stringent hybridization conditions we have used our rat testicular cDNA does not hybridize to RNAs from these tissues, suggesting it encodes a distinct isoform of this family of proteins.

Our time course co-culture experiment (Fig. 5) reveals that the mRNA of the von Ebner's-like protein is induced after 4 h of co-culture and upon cell separation the turnover of its mRNA is very rapid as no RNA is detected in separated Sertoli cells or germ cells following co-culture (Fig. 6). Continued cellular contact does not seem to be essential for the stability of the von Ebner's-like protein mRNA, since a soluble factor(s) secreted by Sertoli cells induces the 1.2-kb mRNA in germ cells (Fig. 6A). More likely, a continued presence of the inducing factor is needed. Preliminary analyses indicate the factor(s) is less than 10 kDa, does not survive freezing and thawing, and is inactivated by boiling.

The Huntington disease protein gene is also up-regulated in pachytene spermatocytes or round spermatids co-cultured with Sertoli cells. Huntington disease is a late onset progressive lethal neurogenerative disorder of autosomal dominant inheritance. The Huntington disease gene (82, 83) contains repeating CAG triplets which are translated as a polyglutamine stretch near the amino terminus of the protein (84-87).

In rats and humans, Huntington disease is transcribed as two transcripts of 10 and 13.7 kb (88). Using Northern blot hybridization we have detected the 10-kb mRNA in co-cultures and in total testis (Fig. 3), but not in RNA preparations from brain, lungs, or liver. Our results are in agreement with previous studies (88, 89) that have detected the shorter transcript of the Huntington gene in testis, but not in non-neuronal tissues such as liver, lung, and kidney. It has recently been shown that the 10-kb transcript is due to a different polyadenylation signal (90). Although a 13-kb transcript has been reported in the rat brain (88), our inability to detect this brain transcript is likely due to differences in hybridization conditions.

Our detection of Huntington disease protein mRNA in pachytene spermatocytes and round spermatids is in agreement with the finding of Schmitt et al. (88) and Lin et al. (90) who have shown by in situ hybridization a stage-dependent expression in spermatocytes and spermatids. The presence of Huntington disease protein mRNA in total testis and separated germ cells following co-culture indicates it is induced in germ cells by Sertoli cells. Unlike the apparently unstable von Ebner's-like mRNA, both pachytene spermatocytes and round spermatids contain the 10-kb mRNA after the germ cells are separated from the inducing Sertoli cells (Fig. 5B). The Sertoli cell factor(s) inducing the Huntington disease gene appears larger than 10 kDa, survives freezing and thawing, but is inactivated by boiling, suggesting that it is a protein. The Huntington disease gene is localized in the cytoplasm of most cells including testicular germ cells. However, in some spermatocytes the Huntington disease protein has been detected in the nucleus, suggesting an additional but unknown function during spermatogenesis (91). The induction of Huntington disease protein mRNA in germ cells by a soluble factor from Sertoli cells suggests its regulation is under paracrine control in the testis.

In summary, we have detected two genes that are up-regulated in meiotic or post-meiotic germ cells by Sertoli cell factors. We believe these inductions are cell- and factor-specific, since no inductions are seen when Sertoli cells are co-cultured with a rat kidney cell line (NRK-5ZE) (data not shown). These studies demonstrate that information transfer between Sertoli cells and germ cells can be maintained through soluble factors and the von Ebner's-like gene and the Huntington disease gene provide specific markers for physiological studies of germ cells and Sertoli cells in culture.

    ACKNOWLEDGEMENT

We are indebted to Dr. Vargheese Chennathukuzhi for helpful discussions.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant HD-11878.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF 123454.

Dagger Supported by Grant T32HD07305 from NICHD, National Institutes of Health.

§ Present address: Dept. of Anatomy and Physiology, University of Dundee, Dundee, United Kingdom.

To whom correspondence should be addressed: Center for Research on Reproduction and Women's Health, 415 Curie Blvd., 752b CRB/6142 University of Pennsylvania, Philadelphia, PA 19104. Tel.: 215-898-0144; Fax: 215-573-5408; E-mail: nhecht{at}mail.med.upenn.edu.

    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; kb, kilobase(s).

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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REFERENCES
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