Testis Isoform Research Articles

Neprilysin II: A Putative Novel Metalloprotease and Its Isoforms in CNS and Testis

Metalloproteases of the M13 subfamily, comprising namely neprylisin (NEP) and endothelin-converting enzyme (ECE), are involved in the metabolism of various neuronal and hormonal peptides, and inhibitors thereof have already led to therapeutically useful agents. Using homology cloning, we have identified a new member of this family in rat tissues. It is a glycosylated, type II integral membrane protein of 774 amino acids, containing a zinc-binding consensus motif, highly homologous to NEP and, therefore, designated NEPII. We have characterized multiple splice variants of NEPII mRNA with distinct expression patterns in brain regions, pituitary and testis. In situ hybridization of testis, where levels of the NEPII gene transcript are the highest, reveals a localization within round spermatids. In brain, NEPII is expressed heterogeneously among several neuronal populations and according to a pattern grossly complementary to that of NEP.

Shedding of somatic angiotensin-converting enzyme (ACE) is inefficient compared with testis ACE despite cleavage at identical stalk sites

The somatic and testis isoforms of angiotensin-converting enzyme (ACE) are both C-terminally anchored ectoproteins that are shed by an unidentified secretase. Although testis and somatic ACE both share the same stalk and membrane domains the latter was reported to be shed inefficiently compared with testis ACE, and this was ascribed to cleavage at an alternative site [Beldent, Michaud, Bonnefoy, Chauvet and Corvol (1995) J. Biol. Chem. 270, 28962-28969]. These differences constitute a useful model system of the regulation and substrate preferences of the ACE secretase, and hence we investigated this further. In transfected Chinese hamster ovary cells, human somatic ACE (hsACE) was indeed shed less efficiently than human testis ACE, and shedding of somatic ACE responded poorly to phorbol ester activation. However, using several analytical techniques, we found no evidence that the somatic ACE cleavage site differed from that characterized in testis ACE. First, anti-peptide antibodies raised to specific sequences on either side of the reported cleavage site (Arg1137/Leu1138) clearly recognized soluble porcine somatic ACE, indicating that cleavage was C-terminal to Arg1137. Second, a competitive ELISA gave superimposable curves for porcine plasma ACE, secretase-cleaved porcine somatic ACE (eACE), and trypsin-cleaved ACE, suggesting similar C-terminal sequences. Third, mass-spectral analyses of digests of released soluble hsACE or of eACE enabled precise assignments of the C-termini, in each case to Arg1203. These data indicated that soluble human and porcine somatic ACE, whether generated in vivo or in vitro, have C-termini consistent with cleavage at a single site, the Arg1203/Ser1204 bond, identical with the Arg627/Ser628 site in testis ACE. In conclusion, the inefficient release of somatic ACE is not due to cleavage at an alternative stalk site, but instead supports the hypothesis that the testis ACE ectodomain contains a motif that activates shedding, which is occluded by the additional domain found in somatic ACE.

Shedding of somatic angiotensin-converting enzyme (ACE) is inefficient compared with testis ACE despite cleavage at identical stalk sites

The somatic and testis isoforms of angiotensin-converting enzyme (ACE) are both C-terminally anchored ectoproteins that are shed by an unidentified secretase. Although testis and somatic ACE both share the same stalk and membrane domains the latter was reported to be shed inefficiently compared with testis ACE, and this was ascribed to cleavage at an alternative site [Beldent, Michaud, Bonnefoy, Chauvet and Corvol (1995) J. Biol. Chem. 270, 28962-28969]. These differences constitute a useful model system of the regulation and substrate preferences of the ACE secretase, and hence we investigated this further. In transfected Chinese hamster ovary cells, human somatic ACE (hsACE) was indeed shed less efficiently than human testis ACE, and shedding of somatic ACE responded poorly to phorbol ester activation. However, using several analytical techniques, we found no evidence that the somatic ACE cleavage site differed from that characterized in testis ACE. First, anti-peptide antibodies raised to specific sequences on either side of the reported cleavage site (Arg(1137)/Leu(1138)) clearly recognized soluble porcine somatic ACE, indicating that cleavage was C-terminal to Arg(1137). Second, a competitive ELISA gave superimposable curves for porcine plasma ACE, secretase-cleaved porcine somatic ACE (eACE), and trypsin-cleaved ACE, suggesting similar C-terminal sequences. Third, mass-spectral analyses of digests of released soluble hsACE or of eACE enabled precise assignments of the C-termini, in each case to Arg(1203). These data indicated that soluble human and porcine somatic ACE, whether generated in vivo or in vitro, have C-termini consistent with cleavage at a single site, the Arg(1203)/Ser(1204) bond, identical with the Arg(627)/Ser(628) site in testis ACE. In conclusion, the inefficient release of somatic ACE is not due to cleavage at an alternative stalk site, but instead supports the hypothesis that the testis ACE ectodomain contains a motif that activates shedding, which is occluded by the additional domain found in somatic ACE.

Isolation of rat mitochondrial transcription factor A (r-Tfam) cDNA.

We have isolated rat mitochondrial transcription factor A (Tfam; formerly known as mtTFA) cDNA clones from a rat cerebellum cDNA library using human Tfam cDNA as a probe. The deduced amino acid sequence of r-Tfam shows 62% and 89% overall identity to human and mouse Tfam, respectively. We also show the presence of two r-Tfam isoforms in testis as for mouse. Our findings suggest that the mechanisms underlying transcription of mitochondrial genes are conserved among rat, mouse, and human.

Cbfa1, an essential transcription factor for bone formation, is expressed in testis from the same promoter used in bone.

Cbfa1 is an essential transcription factor for bone formation, but its transcripts have also been detected in thymus and testis. To elucidate the expression of Cbfa1 in testis, we isolated Cbfa1 cDNA from mouse testis. First we examined the length of the transcripts expressed in mouse testis by Northern hybridization. Using a cDNA probe that can detect both Type-I and Type-II Cbfa1 (Type-I: originally reported as Pebp2alphaA by Ogawa et al.; Type-II: originally reported as til-1 by Stewart et al.), 1.8-kb transcripts were detected in testis, and larger transcripts (6.3 kb and 7.4 kb) in T-cell lines, thymus and bone. Using a Type-II specific probe (bone isoform-specific probe), surprisingly, 1.8-kb testis transcript(s) were detected as well as those (6.3 kb) found in bone. In addition, the transcription start site in mouse testis coincides with one of three start sites identified in bone. These results suggest that the testis transcript is generated from the same promoter of Type-II Cbfa1, which is thought to be active only in osteoblasts and some chondrocytes. Next, to define the difference in length of transcripts, we isolated the Cbfa1 cDNA from mouse testis. Sequence analysis of the cDNA showed that alternative splicing around exon 2 and poly-adenylation within exon 8 occurred in the testis isoform, which produce the smaller transcripts. These data revealed that testis Cbfa1 mRNA is transcribed from same promoter used in bones, but the post-transcriptional regulation is different between testis and skeletal tissues.

Molecular cloning and characterization of a novel splicing variant of the Kir3.2 subunit predominantly expressed in mouse testis.

1. One of the features of weaver mutant mice is male infertility, which suggests that Kir3.2, a G-protein-gated inwardly rectifying K+ channel subunit, may be involved in spermatogenesis. Therefore, we have characterized the Kir3.2 isoform in mouse testis using immunological, molecular biological and electrophysiological techniques. 2. Testicular membrane contained a protein that was recognized by the antibody specific to the C-terminus of Kir3.2c (aG2C-3). Its molecular mass was approximately 45 kDa, which was smaller than that of Kir3.2c ( approximately 48 kDa). The immunoprecipitant obtained from testis with aG2C-3 contained a single band of the 45 kDa protein, which could not be detected by the antibody to the N-terminus common to the known Kir3.2 isoforms (aG2N-2). 3. A novel alternative splicing variant of Kir3.2, designated Kir3.2d, was isolated from a mouse testis cDNA library. The cDNA had an open reading frame encoding 407 amino acids, whose molecular mass was calculated to be approximately 45 kDa. Kir3.2d was 18 amino acids shorter than Kir3.2c at its N-terminal end, which was the only difference between the two clones. The 18 amino acid region possesses the epitope for aG2N-2. 4. In heterologous expression systems of both Xenopus oocytes and mammalian cells (HEK 293T), Kir3.2d either alone or with Kir3.1 exhibited G-protein-gated inwardly rectifying K+ channel activity. 5. Prominent Kir3.2d immunoreactivity in the testis was detected exclusively in the acrosomal vesicles of spermatids, while Kir3.1 immunoreactivity was diffuse in the spermatogonia and spermatocytes. These results indicate the possibility that the testicular variant of Kir3.2, Kir3. 2d, may assemble to form a homomultimeric G-protein-gated K+ channel and be involved in the development of the acrosome during spermiogenesis.

Expression and localization of protein kinase C θ isoform in mouse testis

Protein kinase C (PKC) is encoded by a complex of a gene family, and its multiple isoforms are expressed in various mammalian tissues. The objective of this study was to investigate the expression and localization of a PKC θ isoform in mouse testis. PKC θ displays the highest homology to PKC δ, lacks the Ca 2+-binding C2 domain and, thus, belongs to the subfamily of Ca 2+-independent PKC enzymes which also includes the δ, ε, ζ and η isoforms. We analyzed the PKC θ mKNA and protein by Northern blotting, in situ hybridization, and immunohistochemistry. In testes of normal mice, signals of PKC θ isoform expression were detected specifically in the interstitial cells of testes. The expression of PKC θ isoform was also detected in testes of germ cell-deficient W W′ mice. These results suggest that PKC θ isoform has the specific biological functions in the interstitial cells of testis.

Alternative splicing of hMSH4: two isoforms in testis and abnormal transcripts in somatic tissues.

Alternative splicing of hMSH4: two isoforms in testis and abnormal transcripts in somatic tissues.

Review:Pdha-2, past and present

Pyruvate dehydrogenase (PDH) is a multiunit enzymatic complex essential for the process of generating cellular energy. One of the most important of its subunits is the E1 alpha subunit. Perturbations in the expression of this subunit lead to reduced or lost function of the PDH complex as a whole, resulting in a loss of ATP production. The consequence of such perturbations can lead to neurological abnormalities, lactic acidosis, and in males, death. Pdha-2 codes for the mouse testis isoform of the E1 alpha subunit and maps to chromosome 19 (chromosome 4 in humans). This is a fortuitous evolutionary development because the somatic isoform of the E1 alpha subunit is linked to the X-chromosome, which is not only inactivated early in spermatogenesis but is represented in only half of the haploid spermatid population. Consequently, activation of the testis-specific E1 alpha subunit is essential for the progression of spermatogenesis. Despite its importance, the molecular mechanisms governing the tight tissue- and temporal-specific regulation of Pdha-2 have, until recently, remained poorly understood. In this review, we describe our current understanding of the transcriptional regulation of Pdha-2 and propose potential mechanisms that may play a role in this process.

Review: Pdha2, past and present

Pyruvate dehydrogenase (PDH) is a multiunit enzymatic complex essential for the process of generating cellular energy. One of the most important of its subunits is the E1α subunit. Perturbations in the expression of this subunit lead to reduced or lost function of the PDH complex as a whole, resulting in a loss of ATP production. The consequence of such perturbations can lead to neurological abnormalities, lactic acidosis, and in males, death. Pdha-2 codes for the mouse testis isoform of the E1α subunit and maps to chromosome 19 (chromosome 4 in humans). This is a fortuitous evolutionary development because the somatic isoform of the E1α subunit is linked to the X-chromosome, which is not only inactivated early in spermatogenesis but is represented in only half of the haploid spermatid population. Consequently, activation of the testis-specific E1α subunit is essential for the progression of spermatogenesis. Despite its importance, the molecular mechanisms governing the tight tissue- and temporal-specific regulation of Pdha-2 have, until recently, remained poorly understood. In this review, we describe our current understanding of the transcriptional regulation of Pdha-2 and propose potential mechanisms that may play a role in this process. J. Exp. Zool. 282:231–238, 1998. © 1998 Wiley-Liss, Inc.

Pyruvate dehydrogenase E1 α isoform in rat testis: cDNA cloning, characterization, and biochemical comparison of the recombinant testis and liver enzymes

Previous data indicated a tissue-specific regulation of mitochondrial pyruvate dehydrogenase (PDH) complex, especially in the brain and testis. The lack of biochemical data on the rat testis PDH limits comparative analysis between testis and liver enzymes. Therefore, we have isolated a cDNA clone encoding rat testis PDH E1 α isoform, determined its nucleotide sequence, studied the tissue-specific expression, and characterized the recombinant protein produced in bacteria, compared to the liver counterpart. Our cDNA clone (2.2 kb) contained the identical open reading frame (from nt 974 to 2149) with that previously reported (Cullingford et al., 1993 Biochim Biophys Acta 1216:149–153) but contained a long 5′ untranslated region, which has little identity to the other clone. Northern blot confirmed testis-specific expression of this isoform. Genomic DNA analyses by PCR amplification suggested this clone is a gene product distinct from its X-linked somatic counterpart. Our biochemical and kinetic analyses revealed that the purified recombinant rat testis PDH E1 (containing both E1 α and E1 β subunits) was enzymatically active and phosphorylated in vitro by purified PDH-kinase p48 or p45, similar to the recombinant human liver enzyme. Our current data thus indicate that the differential regulation of testis PDH observed in the animal model may result from differential modulation of PDH-kinase or -phosphatase in this tissue rather than the presence of functionally different PDH E1 subunit.

Evidence for four cytoplasmic dynein heavy chain isoforms in rat testis.

Recent studies have revealed the expression of multiple putative cytoplasmic dynein heavy chain (DHC) genes in several organisms, with each gene encoding a separate protein isoform. This finding is consistent with the hypothesis that different isoforms do different things, as is the case for the axonemal dyneins. Furthermore, the large number of tasks ascribed to cytoplasmic dynein suggests that there may be additional isoforms not yet identified. Two of the mammalian cytoplasmic dynein heavy chains are DHC1a and DHC1b. DHC1a is conventional cytoplasmic dynein and is found in all organisms examined. DHC1b is expressed in organisms that have multiple dyneins, and has been implicated in the intracellular trafficking of molecules in unciliated and ciliated cells. In the present study, we examined the DHC1b protein from rat testis. Testis cytoplasmic dynein contains a large amount of dynein heavy chain reactive with an antibody raised against a peptide sequence of rat DHC1b. The testis anti-DHC1b immunoreactive protein is slightly smaller than testis DHC1a, as assessed by SDS-PAGE. In Northern blots, the DHC1b mRNA is smaller than the DHC1a mRNA. In sucrose gradients made in low ionic strength, DHC1a sedimented at approximately 20S, and the anti-1b immunoreactive heavy chains sedimented in a broad band centered at approximately 14S. The V1-photolysis reaction of individual sucrose gradient fractions revealed three distinct patterns of photolysis, suggesting that there are at least three separate 1b-like heavy chain isoforms in testis. Using a high-stringency Western blotting protocol, the anti-1b antibody and the anti-DHC2 antibody recognized the same heavy chain and specifically bound to one of the three 1b-like heavy chains. We conclude that rat testis contains three 1b-like dynein heavy chains, and one of these is the product of the DHC1b/DHC2 gene previously identified.

Characterization of cytoplasmic dynein light-intermediate chain isoforms in rat testis.

We obtained three kinds of novel cytoplasmic dynein light-intermediate chain (LIC) isoforms from rat testis and brain by reverse transcription polymerase chain reaction (RT-PCR). The primers for RT-PCR were designed according to LIC-2 (LIC 53/55) (15). In one novel isoform, the 42 bp specific sequence named TDL was inserted between 1,106 and 1,107 nucleotides (nts) of LIC-2, whereas the 57 bp sequence corresponding to LIC-2 1,339-1,395 nts (BDL) was absent. The TDL and BDL regions were specifically digested with restriction enzymatic treatment and followed by subcloning of non-digested cDNA band in testis and brain, producing an isoform without TDL and BDL regions. BDL specific RT-PCR of testis cDNA followed by sequencing produced an isoform with two specific regions. By Northern blot hybridization using TDL and BDL specific antisense oligo DNA probe, 4.4, 3.5, and 2.0 kb of signals were detected. With both TDL and BDL probes, the 2.0 kb signal was intensely detected in testis, while the 4.4 kb was defected in brain. This indicates that TDL and BDL are derived from the same size of mRNAs. In situ hybridization method using these probes showed that all seminiferous epithelial cells, especially late pachytene spermatocytes, were positive, indicating that LIC 53/55 isoforms were coexpressed in these cells. These findings indicate that LIC 53/55 isoforms provide a variety of dynein subunits, and thus may regulate the dynein-dependent intracellular transport system.

CP β3, a Novel Isoform of an Actin-Binding Protein, Is a Component of the Cytoskeletal Calyx of the Mammalian Sperm Head

In the mammalian sperm head, the nucleus is tightly associated with the calyx, a cell type-specific cytoskeletal structure. Previously, we have identified and characterized some basic proteins such as calicin and cylicins I and II as major calyx components of bovine and human spermatids and spermatozoa. Surprisingly we have now discovered another calyx constituent which by amino acid sequencing and cDNA cloning was recognized as a novel isoform of the widespread β subunit of the heterodimeric actin-binding “capping protein” (CP). This polypeptide, CP β3, of sperm calices, is identical with the β2 subunit present in diverse somatic cell types, except that it shows an amino-terminal extension of 29 amino acids and its mRNA is detected only in testis and, albeit in trace amounts, brain. This CP β3 mRNA contains the additional sequence, encoded by exon 1 of the gene, which is missing in β2 mRNAs. Antibodies specific for the β3 amino-terminal addition have been used to identify the protein by immunoblotting and to localize it to the calyx structure by immunofluorescence microscopy. We conclude that in spermiogenesis the transcription of the gene encoding the β1, β2, and β3 CP subunits is regulated specifically to include exon 1 and to give rise to the testis isoform CP β3, which is integrated into the calyx structure of the forming sperm head. This surprising finding of an actin-binding protein isoform in an insoluble cytoskeletal structure is discussed in relation to the demonstrated roles of actin and certain actin-binding proteins, such asLimulusα-scruin, in spermiogenesis and spermatozoa.

The Testis Isoform of the Phosphorylase Kinase Catalytic Subunit (PhK-γT) Plays a Critical Role in Regulation of Glycogen Mobilization in Developing Lung

In order to identify the form of phosphorylase kinase catalytic subunit expressed in developing lung, degenerate polymerase chain reaction primers were designed based on conserved domains of the two known catalytic subunits, expressed primarily in muscle and testis. Amplification of cDNA from day 19 fetal rat lung followed by cloning and sequence analyses indicated that only the testis isoform of phosphorylase kinase (PhK-gammaT) was detectable in fetal lung. In situ hybridization analyses indicated that expression of PhK-gammaT RNA in developing lung tissue was widespread and not restricted to Type II epithelial cells; PhK-gammaT protein expression was temporally and spatially correlated with expression of PhK-gammaT RNA. PhK-gammaT RNA and protein expression was also characterized in the PhK-deficient glycogen storage disease (gsd) rat. PhK-gammaT RNA levels were similar in Type II cells isolated from wild type and gsd/gsd fetuses; in contrast, PhK-gammaT protein was virtually undetectable in gsd/ gsd Type II cells and enzyme activity was very low. These results suggest that PhK-gammaT plays a critical role in mobilization of glycogen during fetal lung development and that failure to catabolize glycogen in the gsd/gsd rat is related to an untranslatable PhK-gammaT RNA or unstable protein.

GLUT3 glucose transporter isoform in rat testis: localization, effect of diabetes mellitus, and comparison to human testis

Facilitative hexose transporter expression was compared in rat and human testes. In rat testis, only GLUT1 and GLUT3 proteins were expressed. By contrast, human testis expressed GLUT1 and GLUT3 in addition to GLUT5. Immunocytochemical studies showed that GLUT3 was expressed in all cells of the seminiferous epithelium of rat testis, including sperm. In human testis, GLUT3 was expressed exclusively in cells juxtaposed to the lumen of the seminiferous tubule and ejaculate sperm, a pattern of expression that was identical to that of GLUT5. Induction of insulinopenic diabetes mellitus in the rat did not alter the levels or the distribution of GLUT3 protein or mRNA in the testis. Moreover insulin treatment of the diabetic rats did not produce changes in GLUT3 mRNA or protein levels. The results show that rat and human testis express the high-affinity glucose transporter GLUT3, which allows for the efficient uptake of glucose. In addition, the testis may be protected from changes in glucose transporter expression in experimental diabetes.

Interchangeable associations of calcineurin regulatory subunit isoforms with mammalian and fungal catalytic subunits.

Two mammalian genes for the Ca(2+)-binding regulatory subunit of the calmodulin-dependent protein phosphatase (calcineurin) were identified recently, suggesting unique associations with tissue-specific catalytic subunits. The murine brain (beta 1) and testis (beta 2) isoforms of the regulatory subunit were expressed with poly-histidine carboxyl termini and purified by Ni(2+)-chelate chromatography, each exhibiting high affinity Ca2+ binding on nitrocellulose overlays. Using chromatographic methods to assess complex formation, the beta 1 and beta 2 isoforms appeared indistinguishable in their binding to bacterially expressed forms of the murine brain (alpha 1) or testis (alpha 3) catalytic subunits; this suggests that multiple heterodimeric forms may be present in some tissues. Furthermore, both beta 1 and beta 2 formed complexes with the recombinant catalytic subunit from Neurospora crassa. For this catalytically active fungal enzyme, stoichiometric amounts of mammalian regulatory subunit increased calmodulin-dependent activity without affecting that stimulated by Mn2+ alone. Maximal hydrolysis of rho-nitrophenyl phosphate by the N. crassa catalytic subunit was stimulated 80-120% with the beta 1 isoform and 30-50% by beta 2. Significantly, the incubation time at 4 degrees C required for optimal activation (6-8 h) was much greater than that for association (1 h), indicating that the catalytic subunit undergoes a slow transition to an activated conformation after binding the regulatory subunit. The production of functional heterodimers of mammalian and fungal proteins implies highly conserved interaction domains on the catalytic and regulatory subunits of this phosphatase.

Structure and expression of two isoforms of the murine calmodulin-dependent protein phosphatase regulatory subunit (calcineurin B)

Murine cDNAs representing distinct genes for the regulatory subunits of calmodulin-dependent protein phosphatase (CaM-PrP) were cloned from a testis library, using probes prepared by PCR amplification of brain and testis mRNA. The cDNA sequence of the brain-specific isoform (beta 1) encodes a 170 amino acid protein (M(r) approximately 19.3 kDa), whereas that for the testis isoform (beta 2) contains 179 residues (M(r) approximately 20.7 kDa); these two sequences show approximately 80% amino acid identity. An oligonucleotide probe for the brain isoform hybridized to a single mRNA of 3.6 kilobases (kb) in many tissues, whereas using the beta 2 probe, two mRNAs of 1.8 and 0.8 kb were detected only in testis. The mRNA for the testis-specific isoform increases markedly during development, its pattern being virtually identical to that of mRNA for a testicular form of the catalytic subunit (alpha 3). These data are consistent with the biological co-regulation of catalytic and regulatory subunits of a testis-specific isoenzyme during germ cell maturation.

3β-Hydroxysteroid dehydrogenase/Δ 5→4-isomerase expression in rat and characterization of the testis isoform

The isolation, cloning and expression of a DNA insert complementary to mRNA encoding rat testis 3β-hydroxysteroid dehydrogenase/Δ 5→4-isomerase (3β-HSD) is reported. The insert contains an open reading frame encoding a protein of 373 amino acids, which exhibits 73% and 78% identity to the cDNA encoding the human placental form at the amino acid and nucleotide levels respectively. Northern blot analysis of total RNA of rat tissues using as probe a specific radiolabeled cDNA insert encoding rat testis 3β-HSD demonstrated high levels of 1.6 kb mRNA species in ovary, adrenal and Leydig tumor, with lower but detectable message in testis and adult male liver, while the probe also hybridized to a 2.1 kb mRNA species in liver. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to 3β-HSD present in H540 Leydig tumor cell homogenate and human placental microsomal 3β-HSD, as detected by immunoblot analysis, and catalyzed the conversion of pregnenolone to progesterone, 17α-hydroxypregnenolone to 17α-hydroxyprogesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD +, but not NADP +, converted pregnenolone to progesterone and dehydroepiandrosterone to androstenedione with apparent K m values of 0.13 and 0.09 μM, respectively. Immunoblot analysis of various rat tissues using a polyclonal antibody directed against human placental 3β-HSD, in addition to immunoreactivity in the adrenal and testis, demonstrated immunoreactive 3β-HSD protein in adult male liver, but not in adult female or fetal liver. We conclude that while one gene product is highly expressed in testicular Leydig cells, and probably adrenal and ovary, accounting for their 3β-HSD content, a 3β-HSD is also expressed in liver in a sex-specific manner.

Two Drosophila beta tubulin isoforms are not functionally equivalent.

We have tested the functional capacity of different beta tubulin isoforms in vivo by expressing beta 3-tubulin either in place of or in addition to beta 2-tubulin in the male germ line of Drosophila melanogaster. The testes-specific isoform, beta 2, is conserved relative to major metazoan beta tubulins, while the developmentally regulated isoform, beta 3, is considerably divergent in sequence. beta 3-tubulin is normally expressed in discrete subsets of cells at specific times during development, but is not expressed in the male germ line. beta 2-Tubulin is normally expressed only in the postmitotic germ cells of the testis, and is required for all microtubule-based functions in these cells. The normal functions of beta 2-tubulin include assembly of meiotic spindles, axonemes, and at least two classes of cytoplasmic microtubules, including those associated with the differentiating mitochondrial derivatives. A hybrid gene was constructed in which 5' sequences from the beta 2 gene were joined to protein coding and 3' sequences of the beta 3 gene. Drosophila transformed with the hybrid gene express beta 3-tubulin in the postmitotic male germ cells. When expressed in the absence of the normal testis isoform, beta 3-tubulin supports assembly of one class of functional cytoplasmic microtubules. In such males the microtubules associated with the membranes of the mitochondrial derivatives are assembled and normal mitochondrial derivative elongation occurs, but axoneme assembly and other microtubule-mediated processes, including meiosis and nuclear shaping, do not occur. These data show that beta 3 tubulin can support only a subset of the multiple functions normally performed by beta 2, and also suggest that the microtubules associated with the mitochondrial derivatives mediate their elongation. When beta 3 is coexpressed in the male germ line with beta 2, at any level, spindles and all classes of cytoplasmic microtubules are assembled and function normally. However, when beta 3-tubulin exceeds 20% of the total testis beta tubulin pool, it acts in a dominant way to disrupt normal axoneme assembly. In the axonemes assembled in such males, the doublet tubules acquire some of the morphological characteristics of the singlet microtubules of the central pair and accessory tubules. These data therefore unambiguously demonstrate that the Drosophila beta tubulin isoforms beta 2 and beta 3 are not equivalent in intrinsic functional capacity, and furthermore show that assembly of the doublet tubules of the axoneme imposes different constraints on beta tubulin function than does assembly of singlet microtubules.