Immunohistochemical localization of chorionic gonadotrophin on baboon placenta, dispersed trophoblast cells and those derived from blastocysts grown in vitro

An immunohistochemical technique using a high specificity antiserum against baboon CG was used to demonstrate the presence of a CG-like material on: (1) fixed baboon placental sections collected between 31 and 39 days of gestation, (2) trophoblast monolayers derived from hatched embryos grown in vitro for 15 days and (3) trophoblast cells derived from cells dispersed from placentae collected between Days 31 and 39 of pregnancy. A specific radioimmunoassay was used to detect concentrations of baboon CG in daily spent medium. Immunohistochemical studies showed that material cross-reacting with CG was present on all the three sources of trophoblast. The embryos secreted CG from attachment onwards and immunoactive CG was measurable in daily spent medium collected from placenta-derived trophoblast cultures. It is concluded that baboon CG is localized in the syncytiotrophoblast of fixed placental sections and cellular trophoblast derived from cultured embryos and placental cells.     

Purification and properties of baboon chorionic gonadotrophin

Baboon CG from the urine and placenta (Days 33-45 of gestation) was purified by ammonium acetate/alcohol extraction followed by ionic exchange, gel filtration and affinity chromatography. The CG activity was monitored using a double-antibody radioimmunoassay utilizing anti-baboon CG and hCG both as the labelled ligand and standard. Biological activity was measured by the rat luteal cell radioreceptor assay. The purified preparation exhibited heterogeneity in terms of its behaviour during ionic exchange chromatography and isoelectric focussing. Like hCG, baboon CG was made up of two non-covalently linked subunits: the Stokes' radii were 36.5, 29.0 and 22.0 X 10(-10) m for native CG, the beta subunit and the alpha subunit. The material focussed between pH 4.2 and 5.5. Relative to the second international hCG standard the biological potency of the purified urinary baboon CG was 4058 i.u. whilst the immunological activity was 4364 i.u. It is concluded that baboon and human CG are very similar with respect to their physicochemical properties and that baboon CG can be purified by the methods that have been developed for hCG.     

Human inhibin genes. Genomic characterisation and sequencing

Inhibin is a gonadal hormone involved in the non-steroidal regulation of follicle stimulating hormone (FSH) secretion. Using the cDNAs coding for bovine inhibin A and B subunits we have identified inhibin genes within the human genome using Southern blot hybridisation techniques. The genes are likely to be present as single copies. Cloning and sequencing inhibin genes obtained from lambda libraries of human genomic DNA provide structural and sequence data on the human A and B genes. Comparison of the known inhibin gene sequences showed, in particular, that the B subunits have identical sequences in man, pigs and cattle thus demonstrating a remarkable evolutionary conservation in these genes.     

Highly repeated DNA of the baboon: organization of sequences homologous to highly repeated DNA of the African green monkey.

In the African green monkey genome, 20% of the total DNA consists of a highly reiterated DNA sequence that occurs largely in long tandem arrays of a repeat unit that is 172 base-pairs in length. The DNA of the baboon contains sequences homologous to this repeat unit. However, in the baboon genome, these sequences comprise roughly 6% of the total DNA and alternate in a regular fashion with a DNA segment that may be distantly related to the monkey repeat unit. The sequences in the baboon that are homologous to the monkey repeat unit are contained within a 340 base-pair repeat unit of the highly repeated DNA fraction of the baboon. The extent of nucleotide divergence of the homologous repeated sequences between the two species is estimated to be about 10%.     

Regulation of placental villous angiopoietin-1 and -2 expression by estrogen during baboon pregnancy

We recently showed an increase in vascular endothelial growth factor (VEGF), decrease in angiopoietin-1 (Ang-1) and unaltered Ang-2 expression by the villous placenta with advancing baboon pregnancy. Moreover, placental VEGF expression was increased by estrogen in early pregnancy. In the present study, we determined whether placental Ang-1 and Ang-2 are regulated by estrogen. Ang-1 and Ang-2 mRNA and protein were determined by RT-PCR and immunocytochemistry in the placenta of baboons on Day 60 of gestation (term is 184 days) after administration of estrogen precursor androstenedione on Days 25-59 or on Day 54 after acute estradiol administration. Chronic androstenedione treatment increased serum estradiol levels three-fold (P < 0.001) and decreased (P < 0.05) villous cytotrophoblast Ang-1 mRNA to a level (0.36 +/- 0.08 relative to 18S rRNA) that was one-third of that in untreated animals (0.98 +/- 0.26). Within 2 hr of estradiol administration, cytotrophoblast Ang-1 mRNA was decreased to a level (0.24 +/- 0.05) one-fifth (P < 0.05) of that in untreated animals (1.14 +/- 0.23). However, Ang-2 mRNA levels were unaltered. Ang-1, Ang-2 and estrogen receptors alpha and beta protein were localized within villous cytotrophoblasts providing a mechanism for estrogen action at this site. In summary, estrogen increased VEGF, decreased Ang-1, and had no effect on Ang-2 expression within placental cytotrophoblasts during early baboon pregnancy. We propose that the estrogen-dependent differential regulation of these angioregulatory factors underpins the unique pattern of neovascularization established within the villous placenta during primate pregnancy.     

Chorionic gonadotropin has a recent origin within primates and an evolutionary history of selection

Chorionic gonadotropin (CG) is a critical signal in establishing pregnancy in humans and some other primates, but this placentally expressed hormone has not been found in other mammalian orders. The gene for one of its two subunits (CG beta subunit [CGbeta]) arose by duplication from the luteinizing hormone beta subunit gene (LHbeta), present in all mammals tested. In this study, 14 primate and related mammalian species were examined by Southern blotting and DNA sequencing to determine where in mammalian phylogeny the CGbeta gene originated. Bats (order Chiroptera), flying lemur (order Dermoptera), strepsirrhine primates, and tarsiers do not have a CGbeta gene, although they possess one copy of the LHbeta gene. The CGbeta gene first arose in the common ancestor of the anthropoid primates (New World monkeys, Old World monkeys, apes, and humans), after the anthropoids diverged from tarsiers. At least two subsequent duplication events occurred in the catarrhine primates, all of which possess multiple CGbeta copies. The LHbeta-CGbeta family of genes has undergone frequent gene conversion among the catarrhines, as well as periods of strong positive selection in the New World monkeys (platyrrhines). In addition, newly generated DNA sequences from the promoter of the CG alpha subunit gene indicate that platyrrhine monkeys use a different mechanism of alpha gene expression control than that found in catarrhines.     

Number and organization of actin-related sequences in the mouse genome

Recombinant plasmids containing cDNA sequences complementary to the two mouse striated-muscle actin messenger RNAs (pAF81, pAM91) and to a non-muscle actin mRNA (pAL41) have been used to examine the number and organization of actin-related sequences in the mouse genome. A large number (greater than 20) of actin-related sequences are detected on Southern blots of restricted mouse DNA, the majority of which hybridize to both the 5' and 3' ends of the actin-coding sequence, even under conditions revealing only sequences greater than 80% homologous to the actin cDNA probes. More stringent washing of these blots indicates that the two striated muscle actins are each encoded by single genes, and that a non-muscle (beta or gamma) actin cDNA detects one homologous and two closely related sequences in mouse DNA. The segregation of the two striated-muscle actin genes in recombinant inbred mouse strains shows that these genes are not closely linked (greater than 1 centimorgan), and that the skeletal muscle actin gene is not linked to a non-muscle actin gene. Screening a bank of mouse genomic DNA, cloned in Charon 4A, indicates that the number of actin-related sequences in the mouse genome is much higher than 20. In particular, five phages have been isolated representing part of a sub-family of 20 to 50 similar but non-identical sequences, only weakly homologous to actin cDNA probes (probably a family of actin pseudogenes), which are the result of a recent amplification of a greater than 17 X 10(3) base region of mouse DNA.     

Toward a complete linkage map of the human X chromosome: regional assignment of 16 cloned single-copy DNA sequences employing a panel of somatic cell hybrids.

Closely linked restriction fragment length polymorphisms (RFLPs) are potentially useful as diagnostic markers of genetic defects, and, in principle, RFLPs can be employed to construct a complete linkage map of the human genome. On the X chromosome, linkage studies are particularly rewarding because in man more than 120 X-linked genes are known. Thus, it is probable that each X-specific RFLP will be of use as a genetic marker of one or several X-linked disorders. To facilitate the search for closely linked RFLPs, we have regionally assigned 16 cloned DNA sequences to various portions of the human X chromosome, employing a large panel of somatic cell hybrids. These probes have been used to correlate genetic and physical distances on Xp, and it can be extrapolated from these data that the number and distribution of available Xq sequences will also suffice to span the long arm of the X chromosome.