Aberrant association of chromatin with nuclear periphery induced by Rif1 leads to mitotic defect.

The architecture and nuclear location of chromosomes affect chromatin events. Rif1, a crucial regulator of replication timing, recognizes G-quadruplex and inhibits origin firing over the 50-100-kb segment in fission yeast, Schizosaccharomyces pombe, leading us to postulate that Rif1 may generate chromatin higher order structures inhibitory for initiation. However, the effects of Rif1 on chromatin localization in nuclei have not been known. We show here that Rif1 overexpression causes growth inhibition and eventually, cell death in fission yeast. Chromatin-binding activity of Rif1, but not recruitment of phosphatase PP1, is required for growth inhibition. Overexpression of a PP1-binding site mutant of Rif1 does not delay the S-phase, but still causes cell death, indicating that cell death is caused not by S-phase problems but by issues in other phases of the cell cycle, most likely the M-phase. Indeed, Rif1 overexpression generates cells with unequally segregated chromosomes. Rif1 overexpression relocates chromatin near nuclear periphery in a manner dependent on its chromatin-binding ability, and this correlates with growth inhibition. Thus, coordinated progression of S- and M-phases may require regulated Rif1-mediated chromatin association with the nuclear periphery.

Functional Sperm of the Yellowtail (Seriola quinqueradiata) Were Produced in the Small-Bodied Surrogate, Jack Mackerel (Trachurus japonicus).

Production of xenogeneic gametes from large-bodied, commercially important marine species in closely related smaller surrogates with short generation times may enable rapid domestication of the targeted species. In this study, we aimed to produce gametes of Japanese yellowtail (Seriola quinqueradiata) using jack mackerel (Trachurus japonicus) as a surrogate with a smaller body size and shorter maturation period. Donor spermatogonia were collected from the testes of yellowtail males and transferred into the peritoneal cavity of 10- and 12-day-old jack mackerel larvae. Twenty days later, 59.5% of the recipients survived of which 88.2% had donor-derived germ cells in their gonads. One year later, genomic DNA templates were prepared from the semen of 96 male recipients and subjected to polymerase chain reaction (PCR) analyses using primers specific for the yellowtail vasa sequence, resulting in the detection of positive signals in semen from two recipients. The milt collected from the recipients was used for fertilization with yellowtail eggs. Of eight hatchlings obtained from the crosses, two were confirmed to be derived from donor yellowtail by DNA markers, although the others were gynogenetic diploids. These findings indicate that it is possible to produce donor-derived sperm in xenogeneic recipients with a smaller body size and shorter generation time by transplanting spermatogonia. Thus, the xenogeneic transplantation of spermatogonia might be a potential tool to produce gametes of large-bodied, commercially important fish, although the efficiency of the method requires further improvement. This is the first report demonstrating that donor-derived sperm could be produced in xenogeneic recipient via spermatogonial transplantation in carangid fishes.

Production of donor-derived offspring by allogeneic transplantation of spermatogonia in the yellowtail (Seriola quinqueradiata).

Although the yellowtail (Seriola quinqueradiata) is the fish most commonly farmed in Japan, breeding of this species has not yet started. This is primarily due to the lack of sufficiently sophisticated methods for manipulating gametogenesis, which makes it difficult to collect gametes from specific dams and sires. If it were possible to produce large numbers of surrogate fish by transplanting germ cells isolated from donor individuals harboring desirable genetic traits, then the probability of acquiring gametes carrying the donor-derived haplotype would increase, and breeding programs involving this species might increase as a result. As a first step, we established a method for the allogeneic transplantation of yellowtail spermatogonia and the production of donor-derived offspring. Donor cells were collected from immature (10-month-old) yellowtail males with testes containing abundant type A spermatogonia, labeled with PKH26 fluorescent dye, and transferred into the peritoneal cavities of 8-day-old larvae. Fluorescence observation at 28 days post-transplantation revealed that PKH26-labeled cells were incorporated into recipients' gonads. To assess whether donor-derived spermatogonia could differentiate into functional gametes in the allogeneic recipient gonads, gametes collected from nine male and four female adult recipients were fertilized with wild-type eggs and milt. Analysis of microsatellite DNA markers confirmed that some of the first filial (F(1)) offspring were derived from donor fish, with the average contribution of donor-derived F(1) offspring being 66% and the maximum reaching 99%. These findings confirmed that our method was effective for transplanting yellowtail spermatogonia into allogeneic larvae to produce donor-derived offspring.