A spermatozoa-associated factor regulates proenkephalin gene expression in the rat epididymis

The gene encoding the opioid peptide precursor preproenkephalin is expressed at high levels in the initial segment of the adult rat epididymis. Expression is localized to principal cells, the secretory epithelial cells lining the epididymal duct. During development, epididymal proenkephalin mRNA levels show a pronounced increase at about 44 days of age, coincident with the initial entry of spermatozoa into the epididymal lumen. Hypophysectomy leads to a 60-fold decrease in epididymal proenkephalin mRNA levels. Testosterone replacement can prevent this decline in a manner consistent with an effect upon spermatogenesis. Castration studies demonstrate that a gonadal factor other than testosterone directly regulates epididymal proenkephalin expression, and the results of efferent duct ligation suggest that this factor must be supplied through an intact connection of the testis and epididymis. Proenkephalin mRNA levels in the epididymis correlate with the decline and reappearance of spermatozoa induced by the alkylating agent busulphan. Thus, the developmental profile of proenkephalin expression, coupled with the results of both surgical and pharmacological manipulations of the reproductive tract, indicate that spermatozoa, or a spermatozoa-associated factor, regulate proenkephalin gene expression in the epididymis.     

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.     

Quantitation of proenkephalin A messenger RNA in bovine brain, pituitary and adrenal medulla: correlation between mRNA and peptide levels.

Concentrations of mRNA coding for the opioid peptide precursor proenkephalin A were measured in bovine brain areas, pituitary and adrenal medulla. In all tissues, a single hybridizable species of 1400 bases in size was found by Northern blot analysis using as a probe a single-stranded (ss) cDNA complementary to bovine proenkephalin A mRNA. In solution hybridization experiments the distribution of the mRNA was quantified. Considerable differences were found for the abundance of proenkephalin A mRNA in the various tissues: from 0.023% in the adrenal medulla to 0.00002% in the adenohypophysis. Relative abundance in the various brain areas varied greater than 20-fold, being highest in the caudate nucleus (0.0025%) and lowest in the thalamus and substantia nigra (0.0001%). Comparison with immunoreactive peptide concentrations in these tissues showed a close correlation between the levels of proenkephalin A mRNA and the immunoreactive peptides.     

Changes in relaxin precursor mRNA levels in the rat ovary during pregnancy

Levels of preprorelaxin mRNA in the rat ovary during pregnancy were determined by cell-free translation and by hybridization analyses with cloned preprorelaxin cDNA. Translation of poly(A+) RNA from rat ovaries taken at different stages of pregnancy resulted in the incorporation of [35S]cysteine into two peptides, of Mr 17,500 and 20,500, that were specifically bound by anti-relaxin IgG. Both peptides also were demonstrated by translation of ovarian poly(A+) RNA that was hybrid-selected with cloned preprorelaxin cDNA, the sequence of which corresponds to the Mr 20,500 peptide. The origin of the Mr 17,500 putative precursor is not presently known. Preprorelaxin mRNA translational activities corresponded to previously reported concentrations of relaxin in rat ovaries during pregnancy. The results of hybridization analyses, both by Northern blotting of poly(A+) RNA and dot blotting of unfractionated RNA, agreed with those of translation assays. Preprorelaxin mRNA activity/concentration was low in early pregnancy, rose markedly and reached a plateau on days 15-20 (about 1-2% of total translation activity), and then fell to low levels again by day 23, the time of parturition. These findings indicate that the concentration of relaxin in the rat ovary is directly dependent on preprorelaxin mRNA levels.     

Cloning and characterization of a testis-specific thymosin beta 10 cDNA. Expression in post-meiotic male germ cells.

Thymosin beta 10 is one of a small family of proteins closely related in sequence to thymosin beta 4, recently identified as an actin-sequestering protein. A single molecular weight species of thymosin beta 10 mRNA is present in a number of rat tissues. In adult rat testis, an additional thymosin beta 10 mRNA of higher molecular weight was identified. Nucleotide sequencing of cDNA clones complementary to the testis-specific thymosin mRNA indicated that this mRNA differed from the ubiquitous thymosin beta 10 mRNA only in its 5'-untranslated region, beginning 14 nucleotides upstream of the translation initiation codon. These results, together with primer extension experiments, suggest that the two thymosin beta 10 mRNAs are transcribed from the same gene through a combination of differential promoter utilization and alternative splicing. The novel thymosin beta 10 mRNA could be detected only in RNA isolated from sexually mature rat testis. Both mRNAs were present in pachytene spermatocytes; only the testis-specific mRNA was detected in postmeiotic haploid spermatids. Immunoblot analysis using specific antibodies showed that the thymosin beta 10 protein synthesized in adult testis was identical in size to that synthesized in brain. Immunohistochemical analysis showed that the protein was present in differentiating spermatids, suggesting that the testis-specific thymosin beta 10 mRNA is translated in haploid male germ cells.     

Molecular cloning and tissue-specific expression of the mouse homologue of the rat brain 14-3-3 θ protein: Characterization of its cellular and developmental pattern of expression in the male germ line

The highly conserved 14-3-3 family of proteins, originally reported as brain-specific and then found in various somatic cells and oocytes, interacts with several important signal transduction kinases so that actually the 14-3-3 proteins are considered as modulators of multiple signal transduction pathways. Here we show that a 14-3-3 protein is also expressed in the male germ cells, thus extending the protein cellular distribution to a cell line never reported to express 14-3-3 proteins. Screening of a mouse spermatogenic cells λgt11 cDNA library with affinity-purified polyclonal antibodies to the tyrosine kinase SP42 allowed the isolation of several positive clones. Sequencing of a positive cDNA clone revealed a 735-nucleotide open reading frame encoding a protein of 245 amino acids (27,778 Da). The predicted protein was found to be identical to the most recently discovered 14-3-3 isoform, the θ subtype from a rat brain. Here we demonstrate that 14-3-3 θ mRNA is highly expressed in testis and brain only. Western immunoblot analyses confirm the Northern blot data. Developmental Northern and Western blot analyses are consistent with an expression and translation of the 14-3-3 θ gene throughout spermatogenesis. However, analysis of RNA from purified populations of spermatogenic cells at different developmental stages and immunohistochemistry on adult testis sections reveal that within the testis the 14-3-3 θ gene products are most abundant in meiotic prophase spermatocytes, and, above all, in differentiating spermatids. Both testicular and epididymal spermatozoa are negative. The present study is the first report on the presence and molecular characterization of the 14-3-3 θ gene product in the male germ line. Our observations suggest that this specific member of the 14-3-3 protein family could play distinct modulatory roles in the complex development of the mammalian male germ cell lineage. Mol. Reprod. Dev. 47:370–379, 1997. © 1997 Wiley-Liss, Inc.     

Novel identification of peripheral dopaminergic D2 receptor in male germ cells

Dopamine is a recognized modulator in the central nervous system (CNS) and peripheral organ functions. The presence of peripheral dopamine receptors outside the CNS has suggested an intriguing interaction between the nervous system and other functional systems, such as the reproductive system. In the present study we analyzed the expression of D2R receptors in rat testis, rat spermatogenic cells and spermatozoa, in different mammals. The RT-PCR analysis of rat testis mRNA showed specific bands corresponding to the two dopamine receptor D2R (L and S) isoforms previously described in the brain. Using Western blot analysis, we confirmed that the protein is present in rat testis, isolated spermatogenic cells and also in spermatozoa of a range of different mammals, such as rat, mouse, bull, and human. The immunohistochemistry analysis of rat adult testis showed that the receptor was expressed in all germ cells (pre- and post-meiotic phase) of the tubule with staining predominant in spermatogonia. Confocal analysis by indirect immunofluorescence revealed that in non-capacitated spermatozoa of rat, mouse, bull, and human, D2R is mainly localized in the flagellum, and is also observed in the acrosomal region of the sperm head (except in human spermatozoa). Our findings demonstrate that the two D2 receptor isoforms are expressed in rat testis and that the receptor protein is present in different mammalian spermatozoa. The presence of D2R receptors in male germ cells implies new and unsuspected roles for dopamine signaling in testicular and sperm physiology.     

Murine Myak, a member of a family of yeast YAK1-related genes, is highly expressed in hormonally modulated epithelia in the reproductive system and in the embryonic central nervous system

We have cloned a mouse homologue (designated Myak) of the yeast protein kinase YAK1. The 1210 aa open reading frame contains a putative protein kinase domain, nuclear localization sequences and PEST sequences. Myak appears to be a member of a growing family of YAK1-related genes that include Drosophila and human Minibrain as well as a recently identified rat gene ANPK that encode a steroid hormone receptor interacting protein. RNA blot analysis revealed that Myak is expressed at low levels ubiquitously but at high levels in reproductive tissues, including testis, epididymis, ovary, uterus, and mammary gland, as well as in brain and kidney. In situ hybridization analysis on selected tissues revealed that Myak is particularly abundant in the hormonally modulated epithelia of the epididymis, mammary gland, and uterus, in round spermatids in the testis, and in the corpora lutea in the ovary. Myak is also highly expressed in the aqueduct of the adult brain and in the brain and spinal cord of day 12.5 embryos.