Sleep-wake patterns are altered with age, Prdm13 signaling in the DMH, and diet restriction in mice.

Old animals display significant alterations in sleep-wake patterns such as increases in sleep fragmentation and sleep propensity. Here, we demonstrated that PR-domain containing protein 13 (Prdm13)+ neurons in the dorsomedial hypothalamus (DMH) are activated during sleep deprivation (SD) in young mice but not in old mice. Chemogenetic inhibition of Prdm13+ neurons in the DMH in young mice promotes increase in sleep attempts during SD, suggesting its involvement in sleep control. Furthermore, DMH-specific Prdm13-knockout (DMH-Prdm13-KO) mice recapitulated age-associated sleep alterations such as sleep fragmentation and increased sleep attempts during SD. These phenotypes were further exacerbated during aging, with increased adiposity and decreased physical activity, resulting in shortened lifespan. Dietary restriction (DR), a well-known anti-aging intervention in diverse organisms, ameliorated age-associated sleep fragmentation and increased sleep attempts during SD, whereas these effects of DR were abrogated in DMH-Prdm13-KO mice. Moreover, overexpression of Prdm13 in the DMH ameliorated increased sleep attempts during SD in old mice. Therefore, maintaining Prdm13 signaling in the DMH might play an important role to control sleep-wake patterns during aging.

Sexual precocity in male microminipigs evaluated immunohistologically using spermatogonial stem cell markers.

Microminipigs are one of the smallest miniature pigs characterized as sexually precocious; the males achieve sexual maturity at around 3-4.5 months of age. However, the physiology of this sexual precocity is still unclear. To understand sexual precocity in male microminipigs, we analyzed their testes at five developmental stages: neonatal (<7 days), 30-day-old, 45-day-old, 80-day-old, and adult (>24 months) stages. We used 4 pigs in each of the stages. To analyze testicular development histologically, the seminiferous tubule diameter (SD) was measured, and the presence or absence of the seminiferous lumen was confirmed. Changes in the expression of pluripotency markers, DBA, UCHL1, ZBTB16, and vimentin, were evaluated immunohistologically. For the analyses, cells positive for DBA, UCHL1, and ZBTB16 per 150 round seminiferous tubules in cross sections from each testis were counted to evaluate the total number of positive cells. The number of positive cells per 100 Sertoli cells (DBA+/Sertoli, UCHL1+/Sertoli, and ZBTB16+/Sertoli) was calculated to compare the five developmental stages. Histologically, SDs became larger with piglet growth, and precocity was confirmed; seminiferous lumens were observed from the 30-day-old stage. Immunohistologically, the number of DBA+/Sertoli, which indicates the number of gonocytes, decreased rapidly to an undetectable level by the 45-day-old stage. In the same period, the number of UCHL1+/Sertoli, which indicates total SSCs, increased significantly, suggesting that the proliferation of SSCs was accelerated before 30 days of age. Consequently, our study clarified that differentiation of SSCs in microminipigs started during the fetal period, the differentiation of gonocytes and proliferation of SSCs was then accelerated before 30 days of age, and the early phase of spermatogenesis was finally completed at around 45 days after birth. Consequently, sexual precocity in male microminipigs was characterized by a shorter duration of the early phase of spermatogenesis.

Response to estrus induction with abortion treatment in microminipigs on different days after insemination.

In microminipigs, estrus induction with abortion treatment, which is typically performed between 25 and 40 days after mating, is not always successful. Thus, the authors hypothesized that it may be more difficult to induce estrus by treating microminipigs approximately 40 days after mating. Accordingly, in this study, estrus induction was performed with abortion treatment in four microminipigs as follows: 0.3 mg of cloprostenol, a prostaglandin F2-alpha analog, was administered (day 0); after 24 h, 0.15 mg of cloprostenol and 250 IU of equine chorionic gonadotrophin were administered intramuscularly and simultaneously (day 1); after 96 h, 120 IU of human chorionic gonadotropin was injected intramuscularly (day 4). These treatments were compared at two different stages of pregnancy: early treatment (26.5 ± 0.7 days) and late treatment (38.3 ± 0.8 days). In the early treatment, all four microminipigs exhibited estrus on day 5, whereas in the late treatment, estrus was observed clearly in only two pigs on day 6 and slightly in 1 pig on day 10, whereas it was unclear in 1 pig. These results suggest that it is difficult to induce estrus with abortion treatment in microminipigs at approximately 40 days after mating.

Accuracy of follicle count and ovulation confirmation using magnetic resonance imaging in microminipigs with normal estrus cycles.

Magnetic resonance imaging (MRI) is suggested to be useful for counting follicles and confirming ovulation in microminipigs. However, its accuracy is unknown. We have compared the number of follicles counted by MRI to that of corpus hemorrhagicum confirmed directly by visual inspection. The follicles of 17 microminipigs were counted by using ovarian MRI on a 0.4 Tesla MRI System every 24 hr after estrus until follicle images disappeared. Then, we performed laparotomy to count their corpus hemorrhagicum. Significant correlation was observed between follicle counts obtained using MRI (5.18 ± 1.78 per head) and the numbers of corpus hemorrhagicum (5.47 ± 1.74 per head). In conclusion, follicle counts using 0.4-T MRI were reliable, and confirmed microminipig ovulation.

Characterization of domestic pig spermatogenesis using spermatogonial stem cell markers in the early months of life.

In pigs, spermatogonial stem cells (SSCs), which include gonocytes and undifferentiated spermatogonia, are classically defined as being present till up to 2 months of life. However, knowledge about this transition from gonocytes to undifferentiated spermatogonia in pigs is limited. In this study, we characterized pig SSCs in neonatal (n = 6), one-month-old (1-mo-old, n = 6), two-month-old (2-mo-old, n = 6), and adult testes (n = 6) anatomically, histologically, and immunohistologically. Anatomically, testicular circumference (TC) was measured at each development stage. Histologically, the presence or absence of seminiferous lumen was confirmed, and seminiferous tubule diameter (SD) was measured. Immunohistologically, changes in expression of pluripotent markers: DBA, UCHL1, ZBTB16, and POU5F1, were evaluated. For the analyses, cells positive for DBA, ZBTB16, UCHL1, and Vimentin per 150 round seminiferous tubules of cross sections from each testis were counted to evaluate the total number of positive cells, and then the positive cells per 100 Sertoli cells (UCHL1+/Sertoli, DBA+/Sertoli, and ZBTB16+/Sertoli) were calculated to compare the four developmental stages. Anatomically, piglet testes became larger with increasing age. Histologically, there was no seminiferous lumen in piglet testes, and only a single layer of cells was observed within the seminiferous tubules. Immunohistologically, the average number of DBA+/Sertoli, which indicates the number of gonocytes, was significant lowerthan that of UCHL1+/Sertoli (P < 0.05), which indicates the number of total SSCs in neonatal testes, suggesting that spermatogenesis had already started at birth. In 2-mo-old testes, although the average number of UCHL1+/Sertoli was the same as that in neonatal and 1-mo-old testes, the average number of DBA+/Sertoli decreased rapidly to an undetectable level. Moreover, the numbers of ZBTB16+/Sertoli and DBA+/Sertoli in neonatal testes were similar, and the number of ZBTB16+/Sertoli in 1-mo-old testes was significantly lower than that of DBA+/Sertoli (P < 0.05). Since ZBTB16 may be a marker for stable gonocytes or spermatogonia, the multiplication of the SSCs in neonatal testes may not be very vigorous, and this may be accelerated around one month of age. Consequently, our study clarified that differentiation of pig SSCs starts during the fetal period; then, transition of SSCs from gonocytes to undifferentiated spermatogonia is accelerated at one month of age and finally completed at two months of age.

Genetic diversity of the Yonaguni horse based on polymorphisms in microsatellites and mitochondrial DNA.

Thirty-two microsatellites and a mitochondrial DNA haplotypes of endangered Yonaguni horses were analyzed to establish a pedigree registration system and to understand their genetic diversity for planning effective conservation. Blood samples were collected from 78 of the 130 horses in existence, and DNA was extracted and genotyped. There were two major findings. One is that it is possible to use microsatellites for Yonaguni horse pedigree registration in the future because the power of exclusion of parentage testing is reliable at 0.999998. The second is the clarification of the current genetic diversity of Yonaguni horses. The average number of alleles, observed heterozygosity, expected heterozygosity and fixation index were 4.4, 0.591, 0.601 and 0.016, respectively, for the analyzed horses. The probability of a genetic bottleneck, under the assumptions of the stepwise mutation model, was 0.432, suggesting that the genetic structure of the horses was not influenced by a recent bottleneck. Genetic distance between individuals was visualized by a phylogenetic tree based on the proportion of shared alleles. Structure analysis based on Bayesian clustering revealed the possibility that Yonaguni horses comprise four or five subpopulations. Consequently, although only two haplotypes were identified in the mitochondrial analysis, genetic diversity of Yonaguni horses was not particularly low in comparison with that of other breeds that are at risk of extinction.

Genetic characterization of the Miyako horse based on polymorphisms of microsatellites and mitochondrial DNA.

To help plan conservation of the endangered Miyako horse, a biological resource of the Miyako Islands in Japan, we characterized the genetics of the breed by genotyping 32 microsatellites and identifying mitochondrial DNA haplotypes. We also calculated genetic distances between individuals based on the proportion of shared alleles and visualized the genetic relationships with a phylogenetic tree. Two important results were obtained. One is that accurate pedigree registration of the horse by using microsatellites is possible, as the exclusion power of parentage testing is 0.999998. Another is that the current genetic diversity of the horses was clarified. The average number of alleles, observed heterozygosity and expected heterozygosity were 4.2, 0.701 and 0.649, respectively, for the 35 analyzed horses. The probability values for bottleneck models (infinite allele model: 0.00000; stepwise mutation model: 0.00026; and two-phase model: 0.00000) suggested that Miyako horses have experienced a recent genetic bottleneck. Only one mitochondrial haplotype was identified. Consequently, genetic diversity within the population is relatively well-maintained despite a very small population size (41 at the time of the study), and the first priority in conservation of the Miyako horse is to increase the population size.

Cryopreservation of lar gibbon semen collected by manual stimulation.

We confirmed ejaculation as a result of manual stimulation in a lar gibbon, and attempted to cryopreserve the semen using TES-Tris-egg yolk-based (TTE) extender. After measuring the amount of semen (g), we first diluted the semen with TTE extender, and calculated sperm concentration (sperm/ml), total sperm count (sperm), and progressive sperm motility (%). Then, we cooled diluted semen slowly to 4 °C over 2 h, and added an equal volume of secondary extender containing glycerol over 30 min. Finally, we flash-froze the semen solution by plunging into liquid nitrogen. In addition, we freeze-thawed the solution to determine the recovery rate of the motile sperm. Collection of semen was successful on four of the five occasions. The median (min-max) quantity of ejaculate was 0.19 g (0.09-0.26 g), the median sperm concentration was 1.38 × 10(9) sperm/ml (1.20-1.53 × 10(9) sperm/ml), and the median total sperm count was 0.26 × 10(9) sperm (0.11-0.40 × 10(9) sperm). Moreover, the median sperm motility immediately after ejaculation was 65 % (60-75 %), the median sperm motility after freeze-thawing was 30 % (25-35 %), and the median recovery rate was 42.3 % (40.0-58.3 %). We were able to (1) collect semen from a lar gibbon by manual stimulation, (2) reveal andrological findings regarding semen characteristics, and (3) preserve the genetic resource using TTE cryopreservation.

Magnetic Resonance Imaging of Ovarian Activity in Microminipigs Showing Normal Estrous Cycles.

We investigated whether magnetic resonance imaging (MRI) might be applicable to evaluation of the ovarian activity of microminipigs. Firstly, using three mature microminipigs, we confirmed ovarian position and morphology by laparotomy or laparoscopy, and then acquired MRI images in various patterns to determine the most suitable condition for the acquisition. Next, using four microminipigs, we performed daily MRI, starting 10 days after ovulation and ending 10 days after the subsequent ovulation, as the starting day of standing estrus was taken as day 0. While the ovarian structure could not be discriminated on T1-weighted imaging, it was possible to confirm the follicles during estrus as hyperintense regions on T2-weighted imaging. With chronological MRI, 3-5 follicles were visible on T2-weighted imaging during the interval from day -2 to day 1, and their size immediately prior to ovulation was 3-5 mm. However, confirmation of the presence of small follicles and the corpus luteum was difficult.