Relaxin-like factor (RLF) mRNA expression in the fallow deer.

Employing RT- and RACE-PCR on RNA isolated from testicular tissue, we have cloned the coding cDNA sequence for the RLF, also known as Insl3, of the fallow deer. The RLF coding sequence consisted of 396 bp encoding a peptide of 131 amino acids and shared highest homology with bovine, sheep and goat RLF. Northern analysis revealed a single 0.9 kb transcript in the deer testis. There is only one RLF gene in the deer genome. Nonradioactive in situ hybridization revealed the Leydig cells to be the sole source for RLF mRNA in the deer testis. In the non-pregnant uterus, RLF transcripts were located in the luminal and glandular epithelium of the endometrium. Within the ovary of the pregnant doe, follicular theca interna cells and the corpus luteum expressed RLF transcripts. In uteroplacental tissues, luminal and glandular epithelium, fetal uninucleate and binucleate trophoblast cells (BNC) of the basic villous trophoblast layer expressed RLF mRNA. BNC located at the apical trophoblast layer or the tip of the fetal villus were devoid of RLF transcripts. Pseudostratified trophoblast cells at the base of fetal villi coexpressed RLF mRNA and immunoreactive MHC class Ib molecules.

Expression of proto-oncogenes in bovine preimplantation blastocysts.

Proto-oncogenes are involved in the regulation of gene expression, for example after ligand binding to growth factor receptors. Expression of the proto-oncogenes c-fos, c-jun, c-ha-ras and c-myc was studied in in vivo grown and in vitro cultured bovine preimplantation blastocysts employing RT-PCR, ribonuclease protection assay and immunohistochemistry. Thirteen- and 14- day-old preimplantation blastocysts, i.e. stages before and during trophoblast elongation, were used. In in vivo-grown blastocysts c-fos, c-jun and c-ha-ras transcripts as well as c-Fos, c-Jun and c-Myc proteins were detected in all stages studied. Cultured blastocysts were treated with 10 nM epidermal growth factor and 10 nM transforming growth factor-alpha simultaneously. Epidermal growth factor and transforming growth factor-alpha treatment induced c-fos mRNA and c-Myc protein expression. The induction of downstream targets of the epidermal growth factor receptor by epidermal growth factor and transforming growth factor-alpha indicates a functional epidermal growth factor signal transduction pathway in elongating bovine blastocysts.

Expression of connexin 43 in human testis.

In order to further characterize the Sertoli cell state of differentiation, we investigated the expression of connexin 43 (cx43) protein in the testis of adult men both with normal spermatogenesis and associated with spermatogenic impairment, since cx43 is first expressed during puberty. Cx43 protein was found as a single 43-kDa band on western blots of extracts of normal human testicular material. Cx43 immunoreactivity was generally present between Leydig cells. Within the normal seminiferous epithelium cx43 immunoreactivity was localized between adjacent Sertoli cells, except at stages II and III of the seminiferous epithelial cycle when primary spermatocytes cross from the basal to the adluminal compartment suggesting a stage-dependent Sertoli cell function. While testes with hypospermatogenesis and spermatogenic arrest at the level of round spermatids or spermatocytes revealed a staining pattern similar to that of normal adult testis, the seminiferous tubules showing spermatogenic arrest at the level of spermatogonia and Sertoli-cell-only syndrome were completely immunonegative. We therefore assume that severe spermatogenic impairment is associated with a population of Sertoli cells exhibiting a stage of differentiation deficiency.

Only a truncated epidermal growth factor receptor protein is present in porcine endometrium.

The epidermal growth factor receptor (EGF-R) is a 170-kDa transmembrane protein, of which a truncated 100-kDa form (trEGF-R) lacking the cytoplasmic and the transmembrane domains, and an oncogenic 68-kDa form (v-erb) lacking the extracellular domain have been described. The trEGF-R is secreted and not able to transmit signals into the cell. Growth factors of the EGF family have been shown in porcine uterine fluids and blastocyst. Employing differential immunohistochemistry, we found the extracellular domain, but not the cytoplasmic domain, of the EGF-R in porcine endometrium on Days 9-11 of pregnancy. Blastocysts from Days 9 to 11 post coitum (p.c.) were positive for both antibodies, indicating the presence of the full-size receptor. Western Blotting of endometrial protein resulted in a 100-kDa band, but no 170-kDa band. No evidence for a coexpression of the 170- or 68-kDa forms of the receptor was found. ELISA analysis demonstrated trEGF-R in porcine uterine flushings. We conclude that 1) the trEGF-R is the only EGF-R form present in porcine endometrium, and 2) growth factors of the EGF family in porcine uterine fluids can exert their function via the EGF-R only on blastocysts, not on the endometrium.

Epidermal growth factor receptor and ligands in elongating bovine blastocysts.

Preimplantation development depends on multiple interactions between mother and embryo. The Epidermal Growth Factor Receptor (EGF-R) and its ligands are potential components of the embryo-maternal cross-talk: Employing RT-PCR, in situ hybridization, and immunohistochemistry, we investigated on mRNA and protein level the expression of EGF-R, Epidermal Growth Factor (EGF), Transforming Growth Factor alpha (TGF-alpha), and Heparin-binding EGF-like Growth Factor (HB-EGF) in spherical and elongating bovine blastocysts between day 13 and day 16 of gestation, and in endometrium at day 13 of gestation. EGF-R mRNA and protein were detected in trophoblast and endoderm cells of all blastocyst stages that were studied, and in luminal and some glandular epithelial cells of the endometrium at day 13. EGF protein was detected in both blastocysts and endometrial epithelium. TGF-alpha transcripts and protein were present in blastocysts prior to and after elongation and in uterine glandular and luminal epithelium at day 13 of gestation. HB-EGF mRNA and protein was shown in the endoderm, and the protein also was detected immunohistochemically in about 45% of the blastocysts. This presence of the EGF receptor-ligand system in the endometrium and the preimplantation embryo at the time of blastocyst elongation suggests an important role for these growth factors during bovine preimplantation development.

Molecular remodeling of members of the relaxin family during primate evolution.

Employing comparative analysis of the cDNA-coding sequences of the unique preprorelaxin of the Afro-lorisiform Galago crassicaudatus and the Malagasy lemur Varecia variegata and the relaxin-like factor (RLF) of G. crassicaudatus, we demonstrated distinct differences in the dynamics of molecular remodeling of both hormones during primate evolution. The lorisiform and lemuriform preprorelaxin sequences encoded identical hormones, providing the first endocrinological evidence for the monophyletic origin of all Strepsirrhini. Structural analysis revealed the lemuriform members of the relaxin family to be potentially bioactive single-gene products. In contrast to the "two-prong" relaxin receptor-binding motif (RELVR) present within the B-domains of other primate relaxins, strepsirrhine relaxin contained a unique "three-prong" motif (RRLIR) with highest sequence homology to the receptor-binding motif of the evolutionarily much older skate relaxin. In contrast to relaxin, the RLF molecule was highly conserved during primate evolution and contained within its B-domain the putative relaxin receptor-binding motif and a pentameric sequence implicated in binding to specific RLF receptors. Mutually exclusive expression of strepsirrhine preprorelaxin and RLF were observed in the fetal villous trophoblast cells of the strepsirrhine placenta and postpubertal testicular Leydig cells, respectively, reflecting distinct functional roles for both hormones within the reproductive tract of Strepsirrhini.

Increased expression of NADH-ubiquinone oxidoreductase chain 2 (ND2) in preimplantation rabbit embryos cultured with 20% oxygen concentration.

In vitro culture of mammalian preimplantation embryos is associated with various developmental disorders such as retardation in development and cell damage. The molecular mechanisms underlying these processes are poorly understood. One of the possible reasons may be the unphysiologically high oxygen concentration used for culture. Four-day-old rabbit blastocysts were cultured with 5% O2 (physiologic oxygen concentration) or 20% O2 (usually used for in vitro culture) for 4 hr. Differences in gene expression were analysed by differential display reverse-transcriptase polymerase chain reaction (DD RT-PCR). Thirty-two differentially expressed RNA bands were found. Two of them revealed a high sequence homology to the equine NADH-ubiquinone oxidoreductase chain 2 (ND2), a subunit of complex I of the respiratory chain. mRNA expression of ND2 was increased in blastocysts cultured with the higher oxygen concentration. Increased expression of ND2 was confirmed by semiquantitative and semiquantitative competitive RT-PCR.

Localization of fibroblast growth factor 2 (FGF-2) protein and the receptors FGFR 1-4 in normal human seminiferous epithelium.

Fibroblast growth factor 2 (FGF-2), which occurs in various isoforms both species and tissue specifically, regulates cell proliferation and differentiation via a dual receptor system consisting of heparan sulphate proteoglycans and receptor tyrosine kinases (FGFRs). This study demonstrates for the first time the distribution pattern of FGF-2 and the receptors FGFR 1-4 in the normal seminiferous epithelium of adult men. In western blot analyses, the polyclonal antibody, anti-FGF-2, shows two immunoreactive bands at 18 and 24 kDa. On paraffin sections, positive immunoreaction occurs within the cytoplasm of spermatogonia. The distribution pattern of the polyclonal anti-FGFR 1-4 antibodies is as follows: anti-FGFR-1 (one 68-kDa band) stains nuclei and cytoplasm of spermatogonia; anti-FGFR-3 (five bands at 68, 78, 105, 125 and 145 kDa) stains the nuclei of all germ cells except those of elongated spermatids; and anti-FGFR-4 (one 48-kDa band) stains the cytoplasm of primary pachytene spermatocytes. We were unable to demonstrate FGFR-2 immunoreactivity either in western blot analysis or on paraffin sections. This distribution pattern suggests that FGF-2 in spermatogonia is involved in the autocrine and paracrine regulation of the proliferation and differentiation of spermatogonia and spermatocytes via the receptors FGFR-1, FGFR-3 and FGFR-4.

Molecular cloning and localization of caprine relaxin-like factor (RLF) mRNA within the goat testis.

The relaxin-like factor (RLF) is one of the insulin-like molecules, which also includes insulin, insulin-like growth factor I and II, placentin, and relaxin. Employing RT- and RACE-PCR on RNA isolated from goat testicular tissue, we report the cloning and nucleic acid sequence of goat RLF. The caprine RLF cDNA coding sequence consisted of 396 base pairs encoding a peptide of 131 amino acids. Caprine RLF showed the highest homology in nucleic acid and amino acid sequence with bovine and sheep RLF, suggesting conservation of the RLF gene among ruminants. Northern blot analysis revealed a single 0.9 kb RLF transcript expressed in the goat testis but not in the epididymis, liver, or muscle tissue. Only a single goat RLF gene is present in the goat genome as determined by Southern blot analysis. Employing nonradioactive in situ hybridization for goat RLF mRNA and immunohistochemistry for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17alpha-hydroxylase, we identified the Leydig cells as the sole source of RLF mRNA in the goat testis.

Induction of arylhydrocarbon receptor expression in embryoblast cells of rabbit preimplantation blastocysts upon degeneration of Rauber's polar trophoblast.

The arylhydrocarbon receptor (AhR) is a ligand-activated transcription factor and mediates carcinogenic, teratogenic, and toxic effects of xenobiotics such as dioxin and coplanar polychlorinated biphenyls. The AhR nuclear translocator (ARNT) is involved in AhR signal transduction. We have analyzed the expression of AhR and ARNT mRNA and AhR protein in Day 3 pc (postcoitum) rabbit morulae and Days 4 and 6 pc blastocysts using RT-PCR, nested PCR, whole mount in situ hybridization, and whole mount immunohistochemistry with subsequent confocal laser scanning analysis. AhR and ARNT transcripts were detected in all stages investigated, indicating coexpression of both transcription factors. AhR protein was localized in the cytoplasm. It was detected in Day 3 pc morulae and in blastocysts. In Day 4 pc blastocysts, only trophoblast cells but not embryoblast cells were immunopositive. However, at Day 6 pc, the embryoblast cells also expressed AhR protein and this expression was correlated with the degeneration of Rauber's trophoblast layer.

Epidermal growth factor-like ligands and erbB genes in the peri-implantation rabbit uterus and blastocyst.

Molecular cloning of the partial cDNA coding sequences of the four erbB receptors and the epidermal growth factor (EGF)-like ligands EGF, transforming growth factor alpha (TGF), and heparin-binding EGF (HB-EGF) has provided the basis for a comprehensive analysis of the spatiotemporal expression pattern of the EGF receptor/ligand system during the peri-implantation period in the rabbit. Employing nonradioactive in situ hybridization and immunolocalization, we observed differential expression of erbB1-erbB3 within the trophectoderm of the blastocyst. ErbB1 was strongly expressed in the cytotrophoblast but was downregulated upon syncytium formation. ErbB3 was a product of both the cyto- and syncytiotrophoblast. Despite the expression of erbB2 mRNA, the trophectoderm was devoid of immunoreactive ErbB2. ErbB4 gene activity was exclusively detected in the trophoblast at midpregnancy. The luminal and glandular epithelium and stroma of the nonpregnant, pseudopregnant, and pregnant rabbit uterus at Day 6 of gestation also expressed ErbB1-ErbB3. In the peri-implantation period, gene activities of erbB1-erbB3 were upregulated upon decidualization. At the site of implantation, uterine luminal epithelial cells apposing the preimplantation blastocyst displayed a distinct membrane immunolocalization of ErbB2, identifying the uterine epithelium as target for EGF, TGFalpha, and HB-EGF derived from both the embryonic trophectoderm and the uterine epithelium. In the luminal epithelium at the antimesometrial uterine site, HB-EGF gene activity was upregulated at the time of blastocyst attachment, but this upregulation was not reflected in an increase in immunoreactive HB-EGF. The detection of tyrosine phosphorylated ErbB2 in the rabbit placenta indicated the presence of a functional ErbB/EGF-like system in the pregnant rabbit uterus. This study provides strong evidence for a role of the ErbB/EGF-like system in embryo/maternal interactions during the peri-implantation period in the rabbit.

Characterization of the Antirrhinum floral homeotic MADS-box gene deficiens: evidence for DNA binding and autoregulation of its persistent expression throughout flower development.

We have determined the structure of the floral homeotic deficiens (defA) gene whose mutants display sepaloid petals and carpelloid stamens, and have analysed its spatial and temporal expression pattern. In addition, several mutant alleles (morphoalleles) were studied. The results of these analyses define three functional domains of the DEF A protein and identify in the deficiens promoter a possible cis-acting binding site for a transcription factor which specifically upregulates expression of deficiens in petals and stamens. In vitro DNA binding studies show that DEF A binds to specific DNA motifs as a heterodimer, together with the protein product of the floral homeotic globosa gene, thus demonstrating that the protein encoded by deficiens is a DNA binding protein. Furthermore, Northern analysis of a temperature sensitive allele at permissive and non-permissive temperatures provides evidence for autoregulation of the persistent expression of deficiens throughout flower development. A possible mechanism of autoregulation is discussed.