Characterization of germ cell-specific expression of the orphan nuclear receptor, germ cell nuclear factor.

Nuclear receptors, such as those for androgens, estrogens, and progesterones, control many reproductive processes. Proteins with structures similar to these receptors, but for which ligands have not yet been identified, have been termed orphan nuclear receptors. One of these orphans, germ cell nuclear factor (GCNF), has been shown to be germ cell specific in the adult and, therefore, may also participate in the regulation of reproductive functions. In this paper, we examine more closely the expression patterns of GCNF in germ cells to begin to define spatio-temporal domains of its activity. In situ hybridization showed that GCNF messenger RNA (mRNA) is lacking in the testis of hypogonadal mutant mice, which lack developed spermatids, but is present in the wild-type testis. Thus, GCNF is, indeed, germ cell specific in the adult male. Quantitation of the specific in situ hybridization signal in wild-type testis reveals that GCNF mRNA is most abundant in stage VII round spermatids. Similarly, Northern analysis and specific in situ hybridization show that GCNF expression first occurs in testis of 20-day-old mice, when round spermatids first emerge. Therefore, in the male, GCNF expression occurs postmeiotically and may participate in the morphological changes of the maturing spermatids. In contrast, female expression of GCNF is shown in growing oocytes that have not completed the first meiotic division. Thus, GCNF in the female is expressed before the completion of meiosis. Finally, the nature of the two different mRNAs that hybridize to the GCNF complementary DNA was studied. Although both messages contain the DNA binding domain, only the larger message is recognized by a probe from the extreme 3' untranslated region. In situ hybridization with these differential probes demonstrates that both messages are present in growing oocytes. In addition, the coding region and portions of the 3' untranslated region of the GCNF complementary DNA are conserved in the rat.

A novel Ca2+/calmodulin-dependent protein kinase and a male germ cell-specific calmodulin-binding protein are derived from the same gene.

A cDNA representing a unique Ca2+/calmodulin-dependent protein kinase has been cloned and sequenced from a rat brain cDNA library. This enzyme, expressed in brain, testis, and spleen, is only 32% identical to the various isoforms of Ca2+/calmodulin-dependent protein kinase II. The sequence of the COOH-terminal 169 amino acids is identical to that of a previously described male germ cell-specific calmodulin-binding protein called calspermin (T. Ono, G.R. Slaughter, R.G. Cook, and A.R. Means, J. Biol. Chem. 264:2081-2087, 1989). This identity extends to the nucleic acid sequence and includes all but the first 130 nucleotides of the calspermin cDNA. Primer extension and sequence of a genomic fragment containing the unique calspermin sequence reveals that this mRNA is derived from the kinase transcription unit by germ cell-specific use of a unique exon. In situ hybridization was used to demonstrate that both kinase and calspermin mRNAs are expressed during spermatogenesis. The kinase mRNA is first detected in early meiotic cells and declines to a low level in haploid cells. Calspermin mRNA first appears in pachytene primary spermatocytes and continues to increase as cells complete meiosis and undergo terminal differentiation. These results show that differential utilization of a single gene during spermatogenesis is used to generate mRNAs that encode proteins with distinct functions.

Analysis of expression of multiple genes encoding calmodulin during spermatogenesis.

It has recently been determined that the intracellular calcium receptor, calmodulin, is encoded by a multigene family. At least three calmodulin genes that encode the identical protein are expressed in adult testis from a variety of mammalian species. The complementary techniques of RNA blot hybridization and in situ hybridization were used to assess the relative levels of calmodulin RNAs during rat testis cell differentiation. RNA isolated from highly purified populations of somatic or germinal cells and sections of fixed testis were analyzed for hybridization to specific probes corresponding to the three separate rat calmodulin genes. The level of each calmodulin RNA can be described by a unique developmental pattern during spermatogenesis. The steady state levels of transcripts corresponding to calmodulin gene I (CaM I) and III increase in early meiotic cells (leptotene-zygotene spermatocytes) and remain constant throughout meiosis. The level of the CaM II RNA increases between early and mid-pachytene spermatocytes, but is only transiently elevated. The level of this RNA decreases in round spermatids and is almost nondetectable in cytoplasts shed from elongating spermatids. The levels of CaM I RNAs are maintained in early round spermatids, but only RNA derived from the CaM III gene is still evident in late spermatids. The data indicate that an individual germ cell contains RNAs derived from multiple calmodulin genes and suggests that the divergent calmodulin genes may respond to different cellular regulatory signals.

Expression of RNAs for calmodulin, actins, and tubulins in rat testis cells.

Messenger RNAs encoding calmodulin, alpha- and beta-tubulins, and actins were analyzed in nucleic acid isolated from purified rat testis cell populations. Cell-specific patterns were discovered that suggest coordinate regulation of members of different gene families. Genes that are candidates for coordinate regulation include those encoding 1.4 kb calmodulin RNA and 1.8 kb alpha-tubulin RNAs; 1.6 kb calmodulin RNA and a 1.8 kb beta-tubulin RNA; and 1.6 kb actin and 2.1 kb alpha-tubulin RNAs. The steady state level of the 2.2 kb actin RNA(s) varies less than any other RNA analyzed. Actin RNAs of approximately 1.6 kb are observed in fractions enriched for A spermatogonia or round spermatids or cytoplasts shed from elongating spermatids. The level of 1.8 kb alpha-tubulin RNA increases in a fraction enriched for leptotene-zygotene spermatocytes and peaks in pachytene spermatocytes. As the level of this alpha-tubulin RNA species decreases in maturing spermatids a new 2.1 kb alpha-tubulin RNA increases. beta-Tubulin RNAs of 2.7 and 1.8 kb are present in somatic and premeiotic cell fractions. beta-Tubulin RNA transcripts of 1.8 kb that increase in pachytene spermatocytes are derived from a different gene than that expressed in premeiotic cells. The major RNA that hybridizes to the chicken calmodulin cDNA is 1.4 kb; it increases during the development of spermatocytes, reaching a maximum level in pachytene spermatocytes before it declines during spermiogenesis. Calmodulin RNAs of 1.6 and 1.0 kb are clearly evident in the round spermatid fraction. These data reveal that mechanisms exist for cell-selective elevation of mRNAs encoding cytoskeletal proteins during rat spermatogenesis.

Molecular cloning sequence and distribution of rat calspermin, a high affinity calmodulin-binding protein.

Calspermin is a heat-stable, acidic calmodulin-binding protein predominantly found in mammalian testis. The cDNA representing the rat form of this protein has been cloned from a rat testis lambda gt11 library. Sequence analysis of two overlapping clones revealed a 232-nucleotide 5'-nontranslated region, 510 nucleotides of open reading frame, a 148-nucleotide 3'-untranslated region, and a poly(A) tail. Authenticity of the clones was confirmed by comparison of a portion of the deduced amino acid sequence with the sequence of a tryptic peptide obtained from the rat testis protein. The lambda gt11 fusion protein was recognized by affinity purified antibodies to pig testis calspermin and bound 125I-calmodulin in a Ca2+-dependent manner. Calspermin cDNA encodes a 169-residue protein with a calculated Mr of 18,735. The putative calmodulin-binding domain is very close to the amino terminus of the protein. This region shows 46% identity with the calmodulin-binding region of rat brain Ca2+/calmodulin-dependent protein kinase II and 32% identity with the equivalent region of chicken smooth muscle myosin light chain kinase. The 5'-nontranslated region reveals significant homology with a portion of the catalytic region of the calmodulin-dependent protein kinase family. Calspermin contains a stretch of 17 contiguous glutamic acid residues in the central region of the molecule. Computer analysis predicts calspermin to be 81% alpha-helix and 14% random coil. Analysis of genomic DNA indicates calspermin to be the product of a unique gene. Northern blot analysis of rat testis RNA reveals a 1.1-kilobase mRNA. This RNA is restricted to testis among several rat tissues examined and could not be identified in total RNA isolated from testes of other mammals. Analysis of cells isolated from rat testis reveals calspermin mRNA to be predominantly expressed in postmeiotic cells indicating that it may be specific to haploid cells.

Developmental regulation of calmodulin, actin, and tubulin RNAs during rat testis differentiation.

Postnatal testis differentiation involves transition through neonatal, pre-meiotic, meiotic, haploid, and mature stages. We have examined the qualitative and quantitative changes in rat testis RNAs that specifically hybridize to cDNAs encoding the cytoskeletal proteins, calmodulin, beta-actin, alpha- and beta-tubulin at ages corresponding to each of these developmental periods. We compared the species and relative levels of specific RNAs from testes of animals engaged in normal spermatogenesis with RNA from germ cell-depleted, Sertoli cell-enriched (SCE) testis. Distinct developmental patterns of expression of the specific RNAs were found with each of the cDNAs in the two animal models. A 2.2 kb (kilobase) actin RNA and a 2.7 kb beta-tubulin RNA are maximal at 5-10 days of age, suggesting these RNAs are required by somatic and germ cells in the postnatal phase prior to puberty. Between 19 and 29 days, when pachytene spermatocytes appear in significant numbers, there is a slight increase in the 2.2-kb actin RNA, but a 4- to 10-fold increase in RNAs hybridizing to cDNAs for calmodulin, alpha- and beta-tubulin. These changes are much less pronounced in the SCE testis than in the normal testis, indicating increases in these RNAs are related to germinal cell maturation. The germ cell-related increase in 1.8-kb beta-tubulin RNA appears to reflect a developmental "switch" in the gene from which the RNA is derived. This hypothesis is based on the observation that the ratio of hybridization of a chicken brain beta-tubulin cDNA versus a rat spleen beta-tubulin cDNA to the 1.8-kb RNA band increases more than 40-fold between 5 and 29 days of age in normal testis, but is constant in SCE testis. These data suggest that a specific beta-tubulin gene is activated in maturing germ cells. Analogously, a 2.1-kb alpha-tubulin RNA is found only in maturing normal testis and increases as spermatids are produced. A 2.0-kb beta-tubulin RNA, not found in normal testes, is maximal in maturing SCE testes, suggesting this RNA is of somatic cell origin. All of the RNA species studied, except the 2.0-kb beta-tubulin RNA, decrease between 5 and 19 days in SCE testes, as Sertoli cell mitotic activity wanes, indicating that their levels may be regulated by the developmental signals that influence mitosis.

Genetically engineered calmodulins differentially activate target enzymes.

Three mutant calmodulin (CaM) genes together with the normal chicken CaM cDNA have been expressed in bacteria for the purpose of determining structure/function relationships in CaM. The mutant CaM genes were generated by in vitro recombination between a chicken CaM cDNA and a processed pseudogene that encodes a full-length CaM but with 19 amino acid substitutions as compared to authentic vertebrate CaM. The calmodulin-like (CaML) proteins derived from the pseudogene are called CaML19, CaML16, and CaML3 and contain 19, 16, and 3 amino acid substitutions, respectively. CaML3 is functionally identical to CaM by all criteria tested. The functional characteristics of CaML16 and CaML19 are also indistinguishable yet quite different from normal CaM. CaML19 and CaML16 will maximally activate myosin light chain kinase but will only half-maximally activate calcineurin and CaM-dependent multiprotein kinase. In addition, CaML16 and CaML19 do not activate phosphorylase kinase. The differential activation of these enzymes does not result from the loss of Ca2+-binding sites, since CaML16 binds four Ca2+ with affinity similar to CaM or CaM23. It is more likely that the functional characteristics of the mutant proteins result from an altered tertiary structure, since the Ca2+-dependent enhancement of tyrosine fluorescence and limited proteolysis pattern of CaML16 are different from that of CaM. The data demonstrate that the nature of the interaction of CaM with myosin light chain kinase is different from its interaction with calcineurin, CaM-dependent multiprotein kinase, and phosphorylase kinase and may involve different functional domains in CaM.

Bacterial expression and characterization of proteins derived from the chicken calmodulin cDNA and a calmodulin processed gene.

Both normal chicken calmodulin (CaM) and a CaM-like mutant protein have been expressed in bacteria, isolated and evaluated with respect to several physical and biological properties. The mutant CaM is derived from a CaM-like gene that lacks intervening sequences and probably evolved from a CaM-processed gene (Stein, J. P., Munjaal, R. P., Lagacé, L., Lai, E. C., O'Malley, B. W., and Means, A. R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 6485-6489). The mutant CaM protein contains 16 of the 19 amino acids encoded by the CaM-like gene. Normal chicken CaM produced in bacteria is identical to rat CaM by all criteria tested except that it is not trimethylated. The protein product of the CaM-like gene has been termed CaML and exhibits properties which are very similar to CaM despite the presence of 16 amino acid substitutions. CaML binds Ca2+ as evidenced by Ca2+-dependent binding to phenothiazine- and phenyl-Sepharose affinity resins and a Ca2+-dependent electrophoretic mobility shift which is similar to but distinct from CaM. CaML cross-reacts with a monospecific CaM antibody and has an immunodilution curve which is identical to bacterially synthesized CaM. Finally, CaML can maximally activate rat brain phosphodiesterase but with altered kinetic parameters as compared to CaM. These data suggest that the nucleotide substitutions in the putative CaM processed gene are not random but are selected to retain CaM-like functions in the encoded protein. Such a mechanism may exist for other processed genes.

Quantitation and significance of 125I-calmodulin binding to myosin light chain kinase and phosphorylase distributed on polyacrylamide gels.

Glycogen phosphorylase (a or b) binds 125I-calmodulin in a Ca2+-dependent manner, in the 125I-calmodulin overlay technique. This binding is quantitatively identical to 125I-calmodulin binding to myosin light chain kinase. In an in vitro assay, calmodulin stimulates phosphorylase activity at limiting concentrations of either glucose-1-phosphate or glycogen, but the Ka is 1000 fold higher than for the kinase, and is not Ca2+-dependent. Activation of phosphorylase, but not myosin light chain kinase, by calmodulin can be mimicked by troponin C or bovine serum albumin. These results demonstrate that the properties of calmodulin interaction with proteins can vary between the 125I-calmodulin technique and a functional assay of calmodulin effect on the same protein.

Follicle-stimulating hormone activation of glycogen phosphorylase in the Sertoli cell-enriched rat testis.

The potential role of glycogen phosphorylase in providing energy for the Sertoli cell-enriched testis has been investigated. This enzyme is detectable in testes from rats 6-54 days of age. Glycogen phosphorylase in isolated Sertoli cell-enriched testes is specifically stimulated by FSH. Maximal activation (2-fold) is obtained within 10 min after adding 0.5 micrograms FSH/ml to isolated immature testes (16 days old). There is only a 1.1-fold activation by FSH in testes from mature (34 days old) animals. The sensitivity to the gonadotropin can be restored by adding 1-methyl-3-isobutylxanthine, a phosphodiesterase inhibitor, with the FSH. Phosphorylase can be activated by effectors that mimic the actions of the two proposed mediators of FSH action, cAMP and Ca+2. Phosphorylase from testis of either age is maximally activated by an analog of cAMP, 8-bromo-cAMP. While phosphorylase is rapidly activated 1.4-fold by incubating isolated testis for 2 min with A23187, a Ca+2 ionophore, the age, time, and dose dependence of FSH activation are consistent with conversion mediated by cAMP. Phosphorylase was localized in cultured Sertoli cells by indirect immunofluorescence microscopy. Affinity-purified antiphosphorylase decorated cytoskeletal structures that resemble stress fibers, suggesting that phosphorylase may function in Sertoli cells to provide energy for cytoskeletal motility.