The turtle leg (Pollicipes mitella), which belongs to the family Pollicipes, is widely distributed in the rock along the shore areas of the southern sea, Jeju island and Ulleungdo island of Korean Peninsula. Generally, the size, type, stripe pattern, and color of this species vary in accordance with water depth, turbidity, nutrition, growth period, water temperature, and other environmental aspects. Genomic DNAs were isolated from 21 individuals of three turtle leg (P. mitella) populations of Tongyeong, Yeosu and Manjaedo located in the southern sea of the Korean peninsula. After incubation, we added 300 l of 3 M NaCl, and gently pipetted for a few minutes. 600 l of chloroform was then added to the mixture and inverted (no phenol). The DNA pellets were then incubation-dried for more than 10 hours, maintained at -40℃ until analysis, then dissolved in the ultra-pure water. The concentration of the extracted genomic DNA was measured by its absorbance ratio at 260 nm, with a spectrophotometer (Beckman DU 600 series, UK). Seven primers were exposed to generate the unique loci to each population and number of shared loci by the three populations of turtle leg which could be clearly scored. The hierarchical clustering tree was analyzed by the similarity matrices to generate a dendrogram using pc-package program Systat version 10. As regards average bandsharing value (BS) results, the higher fragment sizes (>1,200 bp) are much more observed in the Yeosu population. The number of unique loci to each population and number of shared loci by the three populations generated by PCR using 7 decamer primers in the turtle leg (P. mitella) population of Tongyeong, Yeosu and Manjaedo. Genetic distances among different individuals of the Tongyeong population of the turtle leg (lane 01~07), Yeosu population of the turtle leg (lane 08~14) and Manjaedo population of the turtle leg (lane 15~21), respectively, were generated using the CLASSIFICATION option in Systat version 10 according to the bandsharing values and similarity matrix. Tongyeong population could be evidently discriminated with the other two Yeosu and Manjaedo populations among three populations. The longest genetic distance (0.305) was found to exist between individuals' no. 02 of the Tongyeong population and no. 13 of the Yeosu population. It seems to the author that this is a result of a high degree of inbreeding in narrow region for a long while. Three turtle leg (P. mitella) populations can be clearly distinguished, especially, by their morphological characters and PCR-based approach.

The mechanisms by which embryo exactly distributes mitochondria into the blastomeres during embryogenesis are one of the important issues in developmental biology. Although the mechanisms has been thought to be important for the proper embryonic development, our understanding has remained limited. In the present study, the distribution of mitochondria was examined in embryos of the ascidian, Halocynthia roretzi, by immunohistochemical staining with three-types of the mitochondria-specific antibodies and vital staining of mitochondria with a fluorescent probe, DiOC2(3). Results of the immunohistochemical staining coincided with that of vital staining, which is able to detect the distribution of mitochondria in cytoplasm of the embryo. Mitochondria was mainly segregated into the B4.1 posterior-vegetal blastomeres at the 8-cell stage. During the next stages, mitochondria was preferentially partitioned into cells of the B-line muscle and the A-line nerve cord precursor compared with each sister cell, endoderm in the 5th cleavage stage, and mesenchyme and notochord in the 6th cleavage stage. However, the mitochondria-rich cytoplasm is divided equally among the blastomeres of the animal hemisphere between the 8-cell and the 64-cell stages. When B6.2 blastomeres were isolated at the early 32-cell stage embryo and cultured in seawater, until control embryos reached the 64-cell stage, pattern of mitochondria distribution was similar to results of the coisolated B7.3 and B7.4 blastomeres from the 64-cell stage embryos. Therefore, it is likely that mitochondria are asymmetrically segregated into the marginal cells in the vegetal hemisphere of the ascidian embryo without cell-cell interaction.

EDCs(endocrine disrupting chemicals)는 생물의 내분비계 작용기작에 비정상적으로 작용하여 호르몬 생산, 분비, 이동, 대사, 결합작용 및 배설을 간섭하는 외인성 물질이다. 특히, androgenic effector나 estrogenic effector로서 각각 다른 기작에 의해 생물의 생식관련 내분비계를 교란시켜 성의 표현이나 기능을 변화시킨다(Iguchi, 1998; Quinn et al., 2004). 전 세계적으로 EDCs로 약 67종의 화학물질을 선정하였으며, 이 물질들 가운데 중금속인 Cd, Pb, Hg, Zn이 EDCs 물질로 확인되었다(WWF, 2006; Ju et al., 2009). Zn은 수서생물에 생식에서 생식세포 발달을 억제하고 생식소 지수를 감소시키며(Timmermans et al., 1996), 배의 부화능력을 감소시킨다(Dave et al., 1987). 또한 Zn의 경우 고농도 또는 장시간 노출시 대복(Gomphina veneriformis)에서 웅성화를 일으킨다(Ju et al., 2009). 본 연구에서는 Zn에 노출된 꼬막의 성비와 intersexuality를 알아보고자 하였다. 본 연구에 사용된 꼬막, Tegillarca granosa은 남해안 여자만 장도에서 2013년 1월 초에 채집하였다. 대조구 하나와 Zn 노출구 9.5, 19.0, 28.5 ㎎/ℓ에 각각 200개체씩 2013년 1월부터 2월까지 5주간 노출시켰다. 그 후 생존개체는 채집지역에서 2013년 2월부터 2013년 5월까지 11주 동안 사육하였다. 조직표본 제작은 생식소가 포함된 내장낭을 적출하여 Bouin 용액에 고정하고 파라핀 절편법에 의해 4~6 ㎛ 두께로 연속 절편하였다. 제작된 표본은 Mayer's hematoxylin과 0.5% eosin(H-E) 비교염색을 실시하여 광학현미경으로 관찰하였다. 체내 아연 분석은 전처리한 시료를 ICP-MS(Perkin Elmer, NexION®300X)로 측정하였다. 꼬막의 성비는 대조구에서는 1:0.9로 나타났으며, Zn 노출구에서는 1:1.5로 나타났다. 노출구 가운데 19.0와 28.5 ㎎/ℓ에서 유의적으로 수컷의 비율이 높게 관찰되었다. Intersexuality는 대조구에서는 암컷에서만 3.2%로 나타났으며, Zn 노출구에서는 암컷(30.5%)에서 수컷(5.9%)보다 높게 관찰되었다. 노출구 가운데 28.5 ㎎/ℓ에서는 암컷에서 52.4%로 가장 높게 관찰되었다. 꼬막 체내 Zn 축적도는 28.5 ㎎/ℓ에서 대조구와 9.5 ㎎/ℓ에 유의적 차이가 나타났다.

해양에 유출된 중금속은 빠르게 퇴적물과 결합하며, 해수보다 퇴적물에 수천 배 이상 높은 농도로 분포한다(Martin & Whitifield, 1983; Lee & Kim, 2000). 이매패류는 주로 해양저질에 서식하며, 여과 섭식을 한다. 따라서, 오염된 퇴적물에 서식하는 생물은 중금속에 직,간접적으로 악영향을 받을 수 있다(Luoma & Fisher, 1997; Yoo et al., 2002). 본 연구에서는 베트남 Ha Long Bay의 저질중금속 농도와 이 지역에 서식하는 이매패류 두 종의 intersexuality를 확인하고자 하였다. 저질중금속 분석은 ICP-MS(Perkin Elmer, NexION®300X)를 이용하여 측정하였다. 이매패류는 백합목(Veneroida), 재첩과(Corbiculidae)에 속하는 Polymesoda erosa와 개량조개과(Mactridae)에 속하는 Lutraria lutraria를 이용하였다. 시료는 Bouin 용액에 24시간 동안 고정하여 파라핀 절편법으로 4~6 ㎛ 두께로 연속 절편하였다. 그 후 Mayer's hematoxylin과 0.5% eosin(H-E) 비교염색을 실시하여 광학현미경으로 관찰하였다. Ha Long Bay 5개 지점의 저질중금속 농도는 Table 1과 같으며, 모든 지점에서 Al 농도가 가장 높았고, Cd 농도가 가장 낮았다. Intersex는 난소에서 수컷의 생식세포 또는 정소에서 암컷의 생식세포가 관찰되는 것을 기준으로 하였다. 성비는 P. erosa에서 1:0.4로 암컷의 비율이 더 높게 나타났고, L. lutraria에서는 1:4.5로 수컷의 비율이 더 높았다. Intersexuality는 P. erosa에서 14.3%, L. lutraria에서 9.1%로 두 종 모두 암컷에서만 관찰되었다(Table 2).

The inflammatory response to infections, such as bacteria and viruses is mediated by multiple host factors. The tumor related-genes are the important cytokines in mammals. However, a number of tumor related-genes are not identified in the rock bream. Here, we have reported the identification and molecular characterization of the tumor related genes. The LPS-induced TNF-α factor 1 and 2 (LITAF1, LITAF2), tumor necrosis factor superfamily member 14 (TNFSF14), tumor necrosis factor receptor superfamily member 14 (TNFRSF14) and translationally controlled tumor protein (TCTP), programmed cell death 10 (PCD10) from rock bream are used for the under investigations. The LITAF1 and LITAF2 consist of 138 and 163 amino acids with a conserved LITAF domain. TNFSF14 and TNFRSF14 comprise 266 and 181 amino acid, respectively. TCTP encompasses of 170 amino acid containing two conserved TCTP signatures. Furthermore PCD10 consists of 210 amino acids. Using quantitative real-time PCR, we have obtained expression analysis results of LITAF1 and 2, TNFSF14, TNFRSF14, TCTP, PCD10 in the various tissue. Compared to the control, the tumor related genes mRNA is detected at a high levels in gill (LITAF1, TCTP), intestine (LITAF2), liver (PCD10), spleen (TNFSF14) and RBC (TNFRSF14). We have also performed gene expression analysis in the kidney, spleen, liver and gill after challenging with Streptococcus iniae, Edwardsiella tarda and Red seabream iridovirus. We have acquired the dynamic regulated mRNA expression to each of pathogen according to the tissue. Expression of tumor related-genes mRNA are significantly increased by infected with pathogens in most of the tissue. But oddly, PCD10 mRNA is expressed significantly decreased by S. iniae infection in all of tissues. Our results reveal that rock bream tumor relatived-genes may be involved in rock bream immune responses to pathogen infections, as well as, they also act like potential biomarkers for innate immunity.

Gonadotropin-inhibitory hormone (GnIH) has been found to inhibit the synthesis and release of gonadotropin (GTH) in avian and mammalian species. It was originally identified in the brain of a quail as a novel hypothalamic neuropeptide with a C-terminal Arg-Phe-NH2 motif (RFamide peptide). Homologs of this peptide have been identified in a couple of model fish species such as goldfish (Carassius auratus) and zebrafish (Danio rerio). Understanding GnIH system could be particularly useful in some aquaculture species with problems of frequent reproduction and/or precautious sexual maturation. However, GnIH system in such species has not been studied yet. In this study, we have identified a pupative GnIH gene in the Nile tilapia (Oreochromis niloticus). We also investigated the role of GnIH in the reproduction of this species. The full length sequence of putative tilapia GnIH gene coded for a protein (197 amino acids) containing two modified RFamides (MPLRF and LSQRF) and a LPQRF cDNA sequence of 594 bp. This putative GnIH gene showed high homology with the GnIH genes of Takifugu rubripes (72%) and Tetraodon nigroviridis (74%). PCR analysis showed that expression of this gene was ubiquitously distributed in the whole brain, ovary and testis as well as in the peripheral tissues examined in this study (liver, kidney, intestine, spleen, muscle and gill) except heart and eye. Expression level of this gene in sexually inactive group was significantly higher than the expression level in first gonadal development and sexually active groups (P<0.05). On the contrary, the expression level of LH-β gene was low in sexually inactive group but significantly higher in first gonadal development and sexually active groups (P<0.05). However, no significant difference was observed in the level of FSH-β gene expression between different reproductive phases in this species. In vitro studies revealed an inhibitory effect of GnIH on LH-β mRNA and FSH-β mRNA levels. No significant difference between GnIH and GnIH with LHRH-a treatments on LH-β and FSH-β mRNA expression in female tilapia pituitary cells.

비대칭 세포분열은 분열장치, 세포 표층의 구조, 세포골격의 역동성, 세포골격 관련 단백질 등이 종합적으로 관련된 현상이다. 비대칭 세포분열은 세포질 결정인자 및 세포 내 특정 인자의 분포에 영향을 미쳐 발생을 조절하며, 크기가 서로 다른 딸세포의 형성을 통하여 형태형성을 뒷받침한다. 본 연구는 척삭동물의 기본형으로 알려진 멍게 배에서 미토콘드리아의 비대칭 분포와 이에 영향을 미치는 신호전달에 대하여 조사하였다. 척삭동물에 속하는 멍게는 모자이크 발생을 하는 대표적 동물로 알려져 있으며 초기 발생에서 개체에 따라 난할 패턴이나 할구의 발생운명에 차이를 보이지 않는 특징을 갖는다. 멍게 64세포기 배아를 구성하는 대부분의 할구는 하나의 조직을 형성하도록 발생운명이 결정된다. 멍게 유생의 주요한 중배엽 조직은 척삭, 근육 및 간충직이다. 멍게의 미토콘드리아를 특이적으로 인식하는 단일클론성 항체를 이용하여 발생 과정에서 미토콘드리아의 분포를 조사하였다. A6.2 세포가 분열하여 형성된 A7.3 척삭 전구세포는 언제나 A7.4 신경삭 전구세포에 비하여 세포 크기가 크며, 미토콘드리아가 적게 분포하였다. 또한, B6.2 세포로부터 형성되는 B7.3 간충직 전구세포도 B7.4 근육전구세포에 비하여 적은 미토콘드리아가 분포하였다. 즉, 멍게 초기 배에서 미토콘드리아는 신경삭과 척삭 및 근육과 간충직 전구세포 사이에서 비대칭적인 분포를 보였다. 이러한 미토콘드리아의 비대칭 분포가 어떻게 조절되는지 연구하기 위하여 MEK 신호전달을 억제한 배아에서 미토콘드리아의 분포를 조사하였다. 척삭과 간충직 세포는 내배엽 세포로부터 방출되는 FGF/Ras/MEK 신호전달에 의해 32세포기부터 64세포기 사이에 유도된다. MEK 신호전달이 억제된 배아에서 간충직과 근육 전구세포 사이의 비대칭적인 미토콘드리아의 분포는 정상 배아와 유사하였다. 그러나 척삭과 신경삭 전구세포 사이의 비대칭적인 미토콘드리아의 분포는 방해를 받아 대칭적인 분포를 나타내었다. 다른 동물이나 세포에서 미토콘드리아의 비대칭적인 분포가 어떤 신호에 의하여 조절되는지 현재 명확하지 않다. 특히 발생 과정에서 미토콘드리아의 비대칭적인 수송에 대하여 거의 알려진 바가 없다. 앞으로 멍게에서 이 과정이 어떠한 신호전달에 의하여 조절되는지 매우 흥미로운 연구주제로 사료된다.

Na+/K+-ATPase, an energy-transducing ion pump, is responsible for maintenance of relatively high concentrations of potassium ions but low concentrations of sodium ions in the cell by transport of these ions across the plasma membrane and participates in transport of various nutrients including glucose, amino acids, and ions. In addition, Na+/K+-ATPase is also involved in regulation of intracellular calcium ion concentration by coupling with Na+/Ca+ exchanger expressed at the maternal-fetal interface in pigs. Na+/K+-ATPase consists of α, β, and FXYD subunits, but only α and β subunits are required for primary functions. FXYD subunit is an auxiliary protein for αβ complex of Na+/K+-ATPase. However, it has not been determined that subunits of Na+/K+-ATPase are expressed in the uterine endometrium during the estrous cycle and pregnancy in pigs. In this study, we determined expression of alpha (ATP1A1-4), beta (ATP1B1-3), and FXYD (FXYD1-7) subunits of Na+/K+-ATPase in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that all alpha, beta, and FXYD subunits, except ATP1A3, were expressed in the uterine endometrium during the estrous cycle and pregnancy in a pregnancy status- and stage-specific fashion. In situ hybridization analysis exhibited that ATP1A1, ATP1A4, and ATP1B1 were localized to luminal (LE) and glandular epithelium (GE) during the estrous cycle and early pregnancy, and during mid to term pregnancy. ATP1A1 mRNA was localized to LE, GE, and areolae of the chorioallantois, especially at high levels to LE in areolae regions. ATP1B3 mRNA was detected only in LE during the estrous cycle and pregnancy with highest levels on day (D) 12 of pregnancy. Transcripts of all subtypes of FXYD subunit were primarily localized to LE and GE during the estrous cycle and early pregnancy and to chorionic membrane (CM) during mid to term pregnancy. RT-PCR analysis showed that all subtypes of Na+/K+-ATPase subunits, except ATP1A2, ATP1A3, and ATP1B2 mRNAs, were expressed in conceptuses on D12 and D15 of pregnancy. These results indicate that Na+/K+-ATPase subunits are expressed in the uterine endometrium and conceptuses during the estrous cycle and pregnancy in a pregnancy status- and stage-specific manner. These suggest that Na+/K+-ATPase subunits may be involved in the establishment and maintenance of pregnancy by coordinate regulation of absorption and secretion of nutrients such as glucose, amino acids, and ions at the maternal-fetal interface in pigs.

The implantation process in pigs is initiated when the conceptus begins secretion of estrogen, the signal for maternal recognition of pregnancy, and cytokines including interleukin-1β(IL1B), interferon delta (IFND) and interferon gamma (IFNG). Our previous study showed that IFNG receptors, IFNGR1 and IFNGR2, were expressed in the uterine endometrium during the estrous cycle and early pregnancy. However, the molecular and cellular mechanism of IFNG in the uterine endometrium in pigs is poorly understood. To determine the role of IFNG on the uterine endometrium during the implantation period, we took advantage of RNA-Seq analysis using explant tissues treated with IFNG in the presence of estrogen and progesterone, and found that many genes including CXCL9, CXCL10, CXCL11, IDO1, IL15, IL15RA, TNFSF10 (TRAIL), and WARS were up-regulated by IFNG. Additional analysis in the uterine endometrial tissues from day (D) 12 and D15 of the estrous cycle and from D12, D15, D30, D60, D90 and D114 of pregnancy determined the expression of these IFNG-regulated genes in pigs by quantitative real-time PCR Results showed that expression of CXCL9, CXCL10, and IDO1 dramatically increased on D15 of pregnancy, and expression of CXCL11 and TNFSF10 was high during mid- to term pregnancy. These results indicate that IFNG regulates immune-associated genes in the uterine endometrium in a stage-specific fashion during pregnancy, and may play a critical role to support the establishment and maintenance of pregnancy at the fetomaternal interface in pigs.

Cleft palates with or without cleft lip is one of the most common congenital craniofacial defects in dogs. It has been reported that monogenic autosomal recessive inheritance caused this defect in this species. However, here, we aimed to report cleft palate in a cloned dog. A fibroblast cell line was established from skin tissues of an eight-year-old German shepherd dog. Blood was collected from oocyte donor dogs, and serum progesterone concentration was measured by chemiluminescence enzyme immunoassay method. Ovulation was determined when serum progesterone results reached 5-10 ng/ml, and in vivo matured oocytes were collected surgically about 72 hr after ovulation. Donor cells were cultured with Dulbecco’s modified Eagle medium supplemented with 10% (v/v) fetal bovine serum until confluence. An in vivo matured oocyte was enucleated, and a donor cell was injected into the perivitelline space. The oocyte-cell couplet was electrically fused, and chemically activated. Reconstructed embryos were transferred to an oviduct of a recipient. Pregnancy diagnosis was performed 27 days after the embryo transfer, and ultrasonography of fetal heart beat, and rectal temperature and serum progesterone value of recipient was monitored until the day of delivery. Microsatellite analysis was performed using genomic DNA of cell donor, clones, and oocyte donors. As results, a total of 74 cloned embryos were transferred to five recipients, and one recipient diagnosed as pregnant with two fetuses by ultrasonography and radiology. Caesarean section was performed on day 58 after embryo transfer due to a decreased heart beat of a fetus, which was lower than 180. Two cloned puppies with 640g and 320g of birth weight were delivered safety, but the small one was born with a cleft palate. Microsatellite analysis results of both clones were identical with the cell donor. Cleft palate of the clone was surgically corrected on day 40 after birth. To our knowledge, there has been no report about cleft palate in cloned dogs, and also, no report about clones with different phenotype of cleft palate in dogs. Therefore, this study can give a clue of cleft palate in dogs, which might not be a genetic cause. Further studies about aberrant epigenetic reprogramming in those clones are needed.

Pluripotent cells are categorized as either "naive" or "primed" based upon their pluripotent status. According to previous studies, embryonic stem cells and embryonic germ cells are identified as naive pluripotent stem cells and epiblast stem cells are identified as primed pluripotent stem cells. In a permissive species such as the mouse, naive and primed pluripotent stem cells can be derived from embryos without genetic manipulations. In non-permissive species such as humans and pigs, primed pluripotent cells are only established from embryos. However, previous studies have shown that the embryonic germ cells of non-permissive species share similar morphology and features with naive pluripotent cells. For these reasons porcine embryonic germ cells (pEGCs) may provide a useful cell source for comparative studies on naive pluripotent cells in non-permissive species. In this study, we attempted to establish and characterize porcine embryonic germ cells. Consequently, an embryonic germ cell line was derived from the genital ridges of a porcine dpc 30 fetus in media containing bFGF. This cell line displayed a dome-shaped colony morphology. The cell line was maintained in a stable condition over an extended time period and was able to differentiate into the three germ layers in vitro. Pluripotency markers such as OCT4, SOX2, NANOG and SSEA4 were expressed in these pEGCs. Similar with pESCs, Mek/Erk signaling pathway were activated by bFGF in the cultured pEGCs. In conclusion, we were able to successfully derive embryonic germ cells from genital ridges of a porcine fetus. Unlikely naive pluripotent cells such as mESCs, pluripotency of pEGCs were regulated by Mek/Erk signaling pathway activated by bFGF. This cell line could potentially be used as naive pluripotent cell source for comparative study with porcine embryonic stem cells and other pluripotent cell lines. As porcine pluripotent cells, pEGCs could be useful candidates for preliminary studies of human disease as well as a source for generating transgenic animals.

The present study determined the differences in the morphometric characteristics of truss and classical dimensions between diploid and triploid Far Eastern catfish, Silurus asotus and provided methods for separating diploid and triploid Far Eastern catfish based on morphometric observations. Significant variables were the direct distance between the anterior edge of the lower lip and the anterior insertion of the dorsal fin (DALAD), the horizontal distance between the anterior edge of the lower lip and the anterior insertion of the ventral fin (HALAV), the direct distance between the anterior edge of the upper lip and the first nostril (DAUF), the direct distance between the anterior edge of the upper lip and the second nostril (DAUS), the interorbital width (IW), and the mandible barbel length (ManBL). The more significant variables were DALAD, HALAV, DAUF, DAUS, and IW. The most useful combination of variables for separating the two groups was DALAD, IW, and DAUF, which correctly classified 85% of the catfish as triploid or diploid, the maximum degree of separation obtained (P<0.05). The triploid catfish had high head region and body depth growth rates during the first year after hatching. Triploid catfish had smaller heads and shorter mandible barbels than diploid catfish.

The influence of triploidization on histological characteristics of retina, trunk kidney, liver and midgut tissue, and comparative analysis of cell cycle and expression of cell cycle protein from tail fin and gill tissue in Far Eastern catfish, Silurus asotus were analyzed. In the infertile triploid fish, the nucleus and/or cell size of secondary proximal tubule cells of trunk kidney, hepatocyte and midgut epithelium are much larger than those of the corresponding cells in the diploid fish (P<0.05). However, triploid tissue showed fewer number of outer nuclear layer in retina and nuclei in secondary proximal tubule of trunk kidney than those for diploid tissue. The mean percentages of the G1-, the S- and the G2+M-phase fractions were 92.5%, 3.2% and 4.3% in tail fin tissue of diploid, and 93.4%, 2.6% and 4.0% in those of triploid, respectively. There were no significant differences in the percentages of each cell cycle fraction between diploid and triploid. The differences of cell cycle between tail fin tissue and gill tissue were statistically significant in diploid and triploid (P<0.05). Also, the differences between diploid and triploid were statistically significant in tail fin tissue and gill tissue (P<0.05). Results from this study suggest that some characteristics in the triploid exhibiting larger cell and nucleus size with fewer number of cell than diploid can be used as an indicator in the identification of triploidization and ploidy level in Far Eastern catfish.

In sexually reproducing animals, primordial germ cells (PGCs) are often set aside early in embryogenesis, a strategy which minimizes the risk of genomic damage associated with replication and mitosis during the cell cycle. Here, we have used germ line markers (piwi, vasa and nanos) and micro-injected cell lineage tracers to show that PGC specification in the leech genus Helobdella follows a different scenario: in this hermaphrodite, the male and female PGCs segregate from somatic lineages only after more than 20 rounds of zygotic mitosis; the male and female PGCs share the same (mesodermal) cell lineage for 19 rounds of zygotic mitosis. Moreover, while all three markers are expressed in both male and female reproductive tissues of the adult, they are expressed differentially between the male and female PGCs of the developing embryo: piwi and vasa are expressed preferentially in female PGCs at a time when nanos is expressed preferentially in male PGCs. A priori, the delayed segregation of male and female PGCs from somatic tissues and from one another increases the probability of mutations affecting both male and female PGCs of a given individual. We speculate that this suite of features, combined with a capacity for selffertilization, may contribute to the dramatically rearranged genome of Helobdella robusta relative to various other animals.