In the Japanese hagfish Eptatretus burgeri, 16 chromosomes (eliminated [E]-chromosomes) have been lost in somatic cells (2n = 36), which is equivalent to approx. 21% of the genomic DNA in germ cells (2n = 52). At least seven of the 12 eliminated repetitive DNA families isolated in eight hagfish species were selectively amplified in the germline genome of this species. One of them, EEEb1 (eliminated element of E. burgeri 1) is exclusively localized on all E-chromosomes. Herein, we identified four novel eliminated repetitive DNA families (named EEEb3–6) through PCR amplification and suppressive subtractive hybridization (SSH) combined with Southern-blot hybridization. EEEb3 was mosaic for 5S rDNA and SINE elements. EEEb4 was GC-rich repeats and has one pair of direct and inverted repeats, whereas EEEb5 and EEEb6 were AT-rich repeats with one pair and two pairs of sub-repeats, respectively. Interestingly, all repeat classes except EEEb3 were transcribed in the testes, although no open reading frames (ORF) were identified. We conducted fluorescence in situ hybridization (FISH) to examine the chromosomal localizations of EEEb3–6 and EEEb2, which was previously isolated from the germline genome of E. burgeri. All sequences were only found on all EEEb1-positive E-chromosomes. Copy number estimation of the repeated elements by slot-blot hybridization revealed that (i) the EEEb1–6 family members occupied 39.9% of the total eliminated DNA, and (ii) a small number of repeats were retained in somatic cells, suggesting that there is incomplete elimination of the repeated elements. These results provide new insights into the mechanisms involved in the chromosome elimination and the evolution of E-chromosomes.

In the Japanese hagfish Eptatretus burgeri, 16 chromosomes (eliminated [E]-chromosomes) have been lost in somatic cells (2n = 36), which is equivalent to approx. 21% of the genomic DNA in germ cells (2n = 52). At least seven of the 12 eliminated repetitive DNA families isolated in eight hagfish species were selectively amplified in the germline genome of this species. One of them, EEEb1 (eliminated element of E. burgeri 1) is exclusively localized on all E-chromosomes. Herein, we identified four novel eliminated repetitive DNA families (named EEEb3–6) from this species through PCR amplification and suppressive subtractive hybridization (SSH) combined with Southern-blot hybridization. EEEb3 was mosaic for 5S rDNA and SINE element. EEEb4 was GC-rich repeats and had one pair of direct and inverted repeats, whereas EEEb5 and EEEb6 were AT-rich repeats and composed of several sub-repeats. Interestingly, all but EEEb3 were transcribed in the testes, although no functional open reading frame (ORF) was found. We conducted fluorescence in situ hybridization (FISH) to examine the chromosomal localizations of EEEb3–6 and EEEb2, which was initially isolated from the germline DNA of this species. All of them were completely colocalized on all EEEb1-positive E-chromosomes. The estimation of the copy number by slot-blot hybridization revealed that (i) EEEb1–6 family members occupied 39.9% of the total eliminated DNA, and (ii) a small number of repeats were retained in somatic cells, suggesting the incomplete elimination. These results provide new insights about the mechanisms involved in the chromosome elimination and the evolution of E-chromosomes.

Introduction Genitourinary syndrome of menopause (GSM) is a symptom complex that occurs secondary to vulvovaginal atrophy. It is generally accepted that GSM is more prevalent in postmenopausal women, and that it can affect their quality of life (QOL). However, there is little data regarding GSM in Japan. Objective The aim of this survey was (i) to determine the prevalence of vulvovaginal symptoms of GSM among Japanese peri- and post-menopausal women and (ii) to examine whether sexual activity affected the prevalence of GSM. Methods We conducted an online survey of 4,134 Japanese women aged 40 – 79 years in the general population. In this primary study, we focused on vulvovaginal symptoms of GSM. GSM was defined as a condition characterized by presenting at least one vaginal symptom such as vulva dryness, vulva hurting, dryness during sexual activity, and dyspareunia using the vulvovaginal symptoms questionnaire. The correlations between age, sexual activity, and GSM were analyzed. Results The prevalence of GSM was 11.6%. The respondents who reported GSM were more likely to be younger. The most common symptom was vulva dryness (6.0%). Only 22.0% of respondents were currently sexually active. Sexual activity was significantly correlated with GSM (P<0.01). The participants aged their 40s – 60s who had sexual activity showed an age-dependent increase in the prevalence of GSM such as vulva dryness and hurting. The respondents aged their 70s who were sexually active reported less dyspareunia than the other ages. Conclusions The vulvovaginal symptoms of GSM were not found with high frequency in Japanese peri- and post-menopausal women. This primary study was conducted in limited areas of GSM. We are planning to analyze the features of GSM including urinary symptoms and QOL more comprehensively with our whole database. Disclosure Work supported by industry: yes, by KOBAYASHI Pharmaceutical Company Limited. A consultant, employee (part time or full time) or shareholder is among the authors (KOBAYASHI Pharmaceutical Company Limited).

In the Japanese hagfish Eptatretus burgeri, 16 chromosomes (eliminated [E]-chromosomes) have been lost in somatic cells (2n=36), which is equivalent to approx. 21% of the genomic DNA in germ cells (2n=52). At least seven of the 12 eliminated repetitive DNA families isolated in eight hagfish species were selectively amplified in the germline genome of this species. One of them, EEEb1 (eliminated element of E. burgeri 1) is exclusively localized on all E-chromosomes. Herein, we identified four novel eliminated repetitive DNA families (named EEEb3–6) from this species through polymerase chain reaction (PCR) and suppressive subtractive hybridization (SSH) combined with Southern-blot hybridization. EEEb3 was mosaic for 5S rDNA and SINE element. EEEb4 was GC-rich repeats and had one pair of direct and inverted repeats, whereas EEEb5 and EEEb6 were AT-rich repeats and composed of several sub-repeats. Interestingly, all but EEEb3 were transcribed in the testes, although no functional open reading frame (ORF) was found. We conducted fluorescence in situ hybridization (FISH) to examine the chromosomal localizations of EEEb3–6 and EEEb2, which was initially isolated from the germline DNA of this species. All of them were completely colocalized on all EEEb1-positive E-chromosomes. The estimation of the copy number by slot-blot hybridization revealed that (i) EEEb1–6 family members occupied 39.9% of the total eliminated DNA, and (ii) a small number of repeats were retained in somatic cells, suggesting the incomplete elimination. These results provide new insights about the mechanisms involved in the chromosome elimination and the evolution of E-chromosomes.

In the Japanese hagfish, Eptatretus burgeri, approximately 21% of the genomic DNA in germ cells (2n=52) consists of 16 chromosomes (eliminated [E]-chromosomes) that are eliminated from presumptive somatic cells (2n=36). To uncover the eliminated genome (E-genome), we have identified 16 eliminated repetitive DNA families from eight hagfish species, with 11 of these repeats being selectively amplified in the germline genome of E. burgeri. Furthermore, we have demonstrated that six of these sequences, namely EEEb1–6, are exclusively localized on all 16 E-chromosomes. This has led to the hypothesis that the eight pairs of E-chromosomes are derived from one pair of ancestral chromosomes via multiple duplication events over a prolonged evolutionary period. NGS analysis has recently facilitated the re-assembly of two distinct draft genomes of E. burgeri, derived from the testis and liver. This advancement allows for the prediction of not only nonrepetitive eliminated sequences but also over 100 repetitive and eliminated sequences, accomplished through K-mer-based analysis. In this study, we report four novel eliminated repetitive DNA sequences (designated as EEEb7–10) and confirm the relative chromosomal localization of all eliminated repeats (EEEb1–10) by fluorescence in situhybridization (FISH). With the exception of EEEb10, all sequences were exclusively detected on EEEb1-positive chromosomes. Surprisingly, EEEb10 was detected as an intense signal on EEEb1-positive chromosomes and as a scattered signal on other chromosomes in germ cells. The study further divided the eight pairs of E-chromosomes into six groups based on the signal distribution of each DNA family, and fiber-FISH experiments showed that the EEEb2–10 family was dispersed in the EEEb1-positive extended chromatin fiber. These findings provide new insights into the mechanisms underlying chromosome elimination and the evolution of E-chromosomes, supporting our previous hypothesis.