Osamu Takenaka

Comparative study of urinary reproductive hormones in great apes

Urinary estrone conjugates (E1C), pregnanediol-3-glucuronide (PdG), and follicle-stimulating hormone (FSH) were determined by enzyme immunoassays (EIAs) during the normal menstrual cycle in the orangutan, gorilla, chimpanzee, and bonobo. Furthermore, the data were compared to those levels in the human and long-tailed macaque. The results showed a typical preovulatory E1C surge and postovulatory increase in PdG in all species. The pattern of E1C during the menstrual cycle in the great apes more closely resembled the human than do the long-tailed macaque. A major difference of E1C pattern between these species appeared in the luteal phase. In the great apes and the human, E1C exhibited two peaks, the first peak detected at approximately mid cycle and the second peak detected during the luteal phase. On the other hand, in the long-tailed macaque, increase of E1C in the luteal phase was small or nonexistent. The gorilla, chimpanzee, and bonobo exhibited similar PdG trends. The orangutan excreted one tenth less PdG than these species during the luteal phase. The long-tailed macaque also excreted low levels of PdG. The patterns of FSH in orangutan, chimpanzee, bonobo and long-tailed macaque showed a marked mid-cycle rise and an early follicular phase rise, similar to those in the human. Comparing similar taxa, a large difference was found in FSH of gorilla; there were three peaks during the menstrual cycle. Thus, there is considerable species variation in the excretion of these hormones during the menstrual cycle and comparative studies could be approached with a single method. The methods and baseline data presented here provide the basis for a practical approach to evaluation and monitoring of ovarian events in the female great apes.

Wild chimpanzee infant urine and saliva sampled noninvasively usable for DNA analyses.

In many genetic studies on the great apes, fecal or hair samples have been used as sources of DNA. However, feces and hairs are difficult to collect from chimpanzee infants under 3xa0years of age. As alternative DNA sources, we investigated the efficiency of collecting urine samples from infants compared with fecal samples, as well as the validity of the DNA extracted from urine and saliva samples of well-habituated M group chimpanzees (Pan troglodytes schweinfurthii) in the Mahale Mountains National Park, Tanzania. We collected 40 urine and 3 fecal samples from 10 infants under 3xa0years. Compared with feces, the urine samples were relatively easy to collect. The saliva of infants, which remained on the twigs sucked by them, was collected using cotton swabs. The average amounts of DNA extracted from the 40 urine and 6 saliva samples were 3,920 and 458xa0pg/μl, respectively. The rate of positive PCR was low and the allelic dropout rate was high when using less than 25xa0pg of template DNA in the PCR mixtures. Based on the amounts of DNA, 50% of the urine samples and 100% of the saliva samples were judged usable for accurate microsatellite genotyping. For infant chimpanzees in particular, collecting urine and saliva as an alternative to fecal and hair samples can reduce the effort invested in collection in the field.

A prevalent POLG CAG microsatellite length allele in humans and African great apes.

The human nuclear gene for the catalytic subunit of mitochondrial DNA polymerase γ (POLG) contains within its coding region a CAG microsatellite encoding a polyglutamine repeat. Previous studies demonstrated an association between length variation at this repeat and male infertility, suggesting a mechanism whereby the prevalent (CAG)10 allele, which occurs at a frequency of >80% in different populations, could be maintained by selection. Sequence analysis of the POLG CAG microsatellite region of more than 1000 human chromosomes reveals that virtually all allelic variation at the locus is accounted for by length variation of the CAG repeat. Analysis of POLG from African great apes shows that a prevalent length allele is present in each species, although its exact length is species-specific. In common chimpanzee (Pan troglodytes) a number of different sequence variants contribute to the prevalent length allele, strongly supporting the idea that the length of the POLG microsatellite region, rather than its exact nucleotide or amino acid sequence, is what is maintained. Analysis of POLG in other primates indicates that the repeat has expanded from a shorter, glutamine-rich sequence, present in the common ancestor of Old and New World monkeys.

Comparison of androgen receptor CAG and GGN repeat length polymorphism in humans and apes

Two polymorphic trinucleotide repeats of human androgen receptor gene (hAR), CAG and GGN which encode glutamine and glycine, have been shown to be associated with human diseases. The number of repeats ranges from 8 to 35 for the CAG and from 10 to 30 for the GGN in human populations. Longer CAG repeats are associated with reduced hAR transcriptional activity, spinal bulbar muscular atrophy and lower cognitive function in older men, whereas shorter CAG repeats are associated with increased risk of prostate cancer and infertility in men. The functional roles of the CAG and GGN repeats have not been clarified. In order to compare the sequence of the CAG and GGN regions in apes, we analyzed 57 chimpanzees, 18 gorillas, 20 orangutans, 16 agile gibbons, and 17 siamangs by PCR and electrophoresis. Two bonobos and one long-tailed macaque were also sequenced and the sequences of all species were aligned, respectively, with one human registered sequence. Seventeen different alleles (4, 7, 8, 9, 12, 14, 15, and 17–26 repeats) and 11 alleles (11–14 and 16–22 repeats) were detected at the CAG and the GGN loci, respectively. Although the repeat tract was conserved among apes, chimpanzees had alleles with a wide range of repeat lengths: (CAG)14–26 and (GGN)14–22. Gorillas were less polymorphic with the (CAG)8 and (GGN)19 alleles being most common, and orangutans exhibited monomorphic (CAG)11 and (GGN)22 alleles. On the other hand, agile gibbons and siamangs had the shortest (CAG)4 allele, but showed variable length of GGN repeats (11–13 in agile gibbons and 16–21 in siamangs). In chimpanzees, frequent haplotypes consisting of short CAG repeats and long GGN repeats or vice versa was observed as in humans.

Molecular Evolution of the Periphilin Gene in Relation to Human Endogenous Retrovirus M Element

HERV-M (human endogenous retrovirus M), related to the super family of HERV-K, has a methionine (M) tRNA primer-binding site, and is located within the periphilin gene on human chromosome 12q12. HERV-M has been integrated into the periphilin gene as the truncated form, 5′LTR-gag-pol-3′LTR. Polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) approaches were conducted to investigate its evolutionary origins. Interestingly, the insertion of retroelements in a common ancestor genome can make different transcript variants in different species. In the case of the periphilin gene, human (10 variants) and mouse (2 variants) lineages show different transcript variants. Insertion of HERV-M (variant 1-3) could affect the protein-coding region. Also, Alusq/x (variant 4-9) and L1ME4a (mammalian-wide subfamilies of LINE-1) (variant 10) in humans and SINE (short interspersed repetitive element) and RLTR15 (the mouse putative long terminal repeat) (variant 2) in mice could be driving forces in transcript diversification of the periphilin gene during mammalian evolution. The HERV-M derived transcripts (variant 1-3) were expressed in different human tissues, whereas they were not detected in crab-eating monkey and squirrel monkey tissues by RT-PCR amplification. Taken together, HERV-M seems to have been integrated into our common ancestor genome after the divergence of simians and prosimians, and then was actively expressed during hominoid evolution.

Lineage-specific duplication and loss of pepsinogen genes in hominoid evolution.

Fourteen different pepsinogen-A cDNAs and one pepsinogen-C cDNA have been cloned from gastric mucosa of the orangutan, Pongo pygmaeus. Encoded pepsinogens A were classified into two groups, i.e., types A1 and A2, which are different in acidic character. The occurrence of 9 and 5 alleles of A1 and A2 genes (at least 5 and 3 loci), respectively was anticipated. Respective orthologous genes are present in the chimpanzee genome although their copy numbers are much smaller than those of the orangutan genes. Only A1 genes are present in the human probably due to the loss of the A2 gene. Molecular phylogenetic analyses showed that A1 and A2 genes diverged before the speciation of great hominoids. Further reduplications of respective genes occurred several times in the orangutan lineage, with much higher frequencies than those occurred in the chimpanzee and human lineages. The rates of non-synonymous substitutions were higher than those of synonymous ones in the lineage of A2 genes, implying the contribution of the positive selection on the encoded enzymes. Several sites of pepsin moieties were indeed found to be under positive selection, and most of them locate on the surface of the molecule, being involved in the conformational flexibility. Deduced from the known genomic structures of pepsinogen-A genes of primates and other mammals, the duplication/loss were frequent during their evolution. The extreme multiplication in the orangutan might be advantageous for digestion of herbaceous foods due to the increase in the level of enzymes in stomach and the diversification of enzyme specificity.

Species and sex identification of western lowland gorillas (Gorilla gorilla gorilla), eastern lowland gorillas (Gorilla beringei graueri ) and humans

Methods for the identification of the sex and species of individuals from samples non-invasively taken from humans and gorillas were established. Amplification of a segment of amelogenin (AMG), which is an X–Y homologous gene, using two pairs of primers from human AMG, revealed both X- and Y-specific bands. The possibility of sex identification was examined by typing the AMG gene using hair and fecal samples from captive western lowland gorillas (Gorilla gorilla gorilla) in Japan and hair samples from wild eastern lowland gorillas (Gorilla beringei graueri) in the Kahuzi-Biega National Park, Democratic Republic of Congo, which were sexed by direct observation. Species-specific bands of AMG in gorillas and humans were identified by restriction fragment length polymorphisms analysis. These tests could be used for sexing unidentified individuals of wild western and eastern lowland gorillas, and screening contamination of human DNA from non-invasively acquired samples.

Evolution of Personality-Related Genes in Primates

Human dopamine receptor D4 (DRD4) and serotonin transporter (5-HTT) genes are polymorphic in terms of the repeat numbers of the GC-rich sequences located in the exon and promoter region, respectively. These genes have attracted special interest as genes related to personality traits. To assess the diversities of these genes in the evolutionary context, we examined the polymorphic regions of these genes in nonhuman primates. Simian primates (apes and monkeys) had four or more repeated sequences in the DRD4 gene polymorphic region, whereas pro simians had one or two repeat units of the 48-bp sequence. These observations imply that the frequency of long-repeated alleles tended to increase during primate evolution. On the other hand, apes and monkeys had relatively longer 5-HTT promoter sequences with an increased number of tandem repeats than human sequences, suggesting that the frequency of short-repeated alleles tended to increase during the process of hominization.

Diet-induced atherosclerosis in cynomolgus monkey aorta and regression by the sixth-month observation

Pathomorphologic analysis was employed to evaluate diet-induced athero sclerosis in cynomolgus monkey aorta and regression by administration of a hypolipidemic agent for six months after the atherogenic ration. Twenty-seven male cynomolgus monkeys were divided into three groups. Group A was fed individually with a high-fat diet containing 0.3% cholesterol under identical conditions for six months. Group B was fed with normal monkey chow for six months after the same atherogenic ration. Group C was fed with normal mon key chow and administered a hypolipidemic agent 1% of 4-[2-(4-isopropylben zamido)ethoxy] benzonic acid for six months after the same atherogenic ration. Each thoracic and abdominal aorta of animal models was separately analyzed. Lipid composition analysis and esterified cholesterol (CE) in aortic wall, ratio of free cholesterol to phospholipid, surface involvement, and atherosclerotic index after Sudan IV staining were studied for evaluation of progression and regres sion. The configurations of atherosclerotic involvement were histologically eval uated among each group. These observed lesions, features specific to cynomol gus lesions, mainly consisted of lipid-rich foam cells, lipid debris, and prolifer ated extracellular matrix. No different lesion composition was noted between the thoracic and abdominal aorta. This may suggest that some local factors play an important role for development of atherosclerosis after the initial event. Group C had remarkable reduction of foam cells and of CE accumulation in both the thoracic and abdominal aortic wall. Accelerated regression in group C as compared with group B was demonstrated both biochemically and pathohis tologically. These results suggest that substantial regression of atherosclerosis in both the thoracic and abdominal aorta can be expected. This hypolipidemic agent exerts notable antiatherosclerotic activity, along with a lowering effect on plasma total cholesterol levels.

Comparative analysis of estrogen receptor gene polymorphisms in apes.

Polymorphic microsatellite repeats in the promoter region of estrogen receptor α gene (ESRα and the intron 6 region of estrogen receptor β gene (ESRβ) have been reported in human populations. To examine the evolutional state of both repeats, we surveyed the corresponding regions in DNA sequences from the following great apes and gibbons: 56 chimpanzees, 3 bonobos, 16 gorillas, 20 orangutans and 60 gibbons (four species: 17 of Hylobates agilis, 11 of H. lar, 15 of H. muelleri, and 17 of H. syndactylus). In the corresponding region of the TA repeat of human ESRα, chimpanzees and bonobos had two motifs in the repeat tract, (TA)7–9 and (CA)4–6. Gorillas had the (TA)9–10 repeat tracts and orangutans had monomorphic (TA)7 repeats. Although all great apes maintained the TA expansion, all gibbon sequences contained (TA)2, implying that the CA dinucleotide expansion arose in the ancestor of chimpanzees and bonobos. The nucleotide sequences of ESRβ showed a very complex repeat pattern in apes. The human sequences had a non-variable preceding sequence at (CA)n, (GA)2(TA)8(CA)4(TA). In apes that region included {(TA)n(CA)n}n. Gibbon sequences included (TATG)n and (TATC)n and no regular construction was observed. A deletion event in the reverse primer site seems to have occurred in the orangutan lineage. In addition, a great diversity of allele length was detected in each gibbon species.