Effect of calcitonin on adrenocorticotropic hormone secretion stimulated by corticotropin‐releasing hormone in the hen anterior pituitary

The presence of a receptor for calcitonin (CT) and the effect of chicken CT (cCT) on adrenocorticotropic hormone (ACTH) secretion stimulated by rat/human corticotropin‐releasing hormone (rhCRH) in the hen anterior pituitary were studied. The specific [¹²⁵I]cCT binding component was present in the plasma membrane of hen anterior pituitary and this binding component had properties of a receptor which has binding specificity to cCT, reversibility, saturable binding, high affinity and limited capacity. When anterior pituitary cells were incubated in vitro, cCT increased the maximal secretion of chicken ACTH stimulated by rhCRH. These results suggest that CT may act directly on the anterior pituitary via its receptor binding and enhances the ACTH secretion by CRH.     

Signature-tagged mutagenesis of Vibrio vulnificus

Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus.     

Piebald mutation on a C57BL/6J background

The classic piebald mutation in the endothelin receptor type B (Ednrb) gene was found on rolling Nagoya genetic background (PROD-s/s) mice with white coat spotting. To examine whether genetic background influenced the phenotype in the piebald mutant mice, we generated a congenic strain (B6.PROD-s/s), produced by repeated backcrosses to the C57BL/6J (B6) strain. Although B6.PROD-s/s mice showed white coat spotting, 7% of B6.PROD-s/s mice died between 2 and 5 weeks after birth due to megacolon. The PROD-s/s, s/s and Japanese fancy mouse 1 (JF1) strains, which also have piebald mutations on different genetic backgrounds with B6, showed only pigmentation defects without megacolon. In expression analyses, rectums of B6.PROD-s/s with megacolon mice showed ~5% of the level of Ednrb gene expression versus B6 mice. In histological analyses, aganglionosis was detected in the rectum of megacolon animals. The aganglionic rectum was thought to lead to severe constipation and intestinal blockage, resulting in megacolon. We also observed an abnormal intestinal flora, including a marked increase in Bacteroidaceae and Erysipelotrichaceae and a marked decrease in Lactobacillus and Clostridiales, likely inducing endotoxin production and a failure of the mucosal barrier system, leading ultimately to death. These results indicate that the genetic background plays a key role in the development of enteric ganglion neurons, controlled by the Ednrb gene, and that B6 has modifier gene (s) regarding aganglionosis.     

Pluripotent cell derivation from male germline cells by suppression of Dmrt1 and Trp53

Diploid germ cells are thought to have pluripotency potential. We recently described a method to derive pluripotent stem cells (PSCs) from cultured spermatogonial stem cells (SSCs) by depleting Trp53 and Dmrt1, both of which are known suppressors of teratomas. In this study, we used this technique to analyze the effect of this protocol in deriving PSCs from the male germline at different developmental stages. We collected primordial germ cells (PGCs), gonocytes and spermatogonia, and the cells were transduced with lentiviruses expressing short hairpin RNA against Dmrt1 and/or Trp53. We found that PGCs are highly susceptible to reprogramming induction and that only Trp53 depletion was sufficient to induce pluripotency. In contrast, gonocytes and spermatogonia were resistant to reprogramming by double knockdown of Dmrt1 and Trp53. PSCs derived from PGCs contributed to chimeras produced by blastocyst injection, but some of the embryos showed placenta-only phenotypes suggestive of epigenetic abnormalities of PGC-derived PSCs. These results show that PGCs and gonocytes/spermatogonia have distinct reprogramming potential and also suggest that fresh and cultured SSCs do not necessarily have the same properties.     

RNAi-mediated Knockdown of Xist Does Not Rescue the Impaired Development of Female Cloned Mouse Embryos

In mice, one of the major epigenetic errors associated with somatic cell nuclear transfer (SCNT) is ectopic expression of Xist during the preimplantation period in both sexes. We found that this aberrant Xist expression could be impeded by deletion of Xist from the putative active X chromosome in donor cells. In male clones, it was also found that prior injection of Xist-specific siRNA could significantly improve the postimplantation development of cloned embryos as a result of a significant repression of Xist at the morula stage. In this study, we examined whether the same knockdown strategy could work as well in female SCNT-derived embryos. Embryos were reconstructed with cumulus cell nuclei and injected with Xist-specific siRNA at 6–7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatment successfully repressed Xist RNA at the morula stage, as shown by the significant decrease in the number of cloud-type Xist signals in the blastomere nuclei. However, blastomeres with different sizes (from “pinpoint” to “cloud”) and numbers of Xist RNA signals remained within single embryos. After implantation, the dysregulated Xist expression was normalized autonomously, as in male clones, to a state of monoallelic expression in both embryonic and extraembryonic tissues. However, at term there was no significant improvement in the survival of the siRNA-injected cloned embryos. Thus, siRNA injection was largely effective in repressing the Xist overexpression in female cloned embryos but failed to rescue them, probably because of an inability to mimic consistent monoallelic Xist expression in these embryos. This could only be achieved in female embryos by applying a gene knockout strategy rather than an siRNA approach.     

Timing of Puberty and Its Relationship with Body Growth and Season in Male Raccoons (Procyon lotor) in Hokkaido

The raccoon (Procyon lotor), indigenous to North America, has naturalized in Japan as an invasive alien species, having been introduced into the country in the 1970s. In Hokkaido, the northernmost island of Japan, feral raccoons have been increasing in number and spreading throughout the island. The age at the onset of puberty for raccoons is important for estimating individual lifetime reproductive success and population growth. The present study investigated the timing of and potential factors affecting the onset of puberty in male raccoons in Hokkaido. External characteristics and histology of testes were studied in 151 male feral raccoons and in 1 captive juvenile. For the majority of feral yearling raccoons, prepubertal development began in May, and spermatozoa production began in October prior to their second mating season. However, some larger juveniles attained puberty during the juvenile period. The captive juvenile, which was fed throughout the winter, attained puberty only 11 months after birth. These results suggest that if male raccoons can achieve enough body growth before the first mating season, puberty can be attained early. In both juveniles and yearlings, spermatozoa production was only observed after autumn. This timing coincided with the recrudescence of seasonally active spermatogenesis in adult males. Therefore, attaining puberty in male raccoons appears to require both adequate body nutrient development and several environmental factors that control seasonal testicular changes.     

Low Incidence of an Altered Endometrial Epidermal Growth Factor (EGF) Profile in Repeat Breeder Holstein Heifers and Differential Effect of Parity on the EGF Profile Between Fertile Holstein (Dairy) and Japanese Black (Beef) Cattle

A high incidence (about 70%) of alteration in endometrial epidermal growth factor (EGF) profile, i.e., loss of 2 peaks on days 2–4 and 13–14, has been linked to a reduced fertility in multiparous repeat breeder Holstein cows. However, the EGF profile in Holstein heifers and other breeds (types) of cattle has not been investigated. In study 1, EGF concentrations were determined using endometrial tissues obtained by biopsy on days 3, 7 and 14 from 84 fertile Holstein heifers to obtain a normal range and 53 repeat breeder Holstein heifers to estimate incidence of alterations in the EGF profile. In repeat breeder heifers, EGF concentrations were similar to fertile controls on 3 days and five animals (9.4%) had an altered EGF profile with EGF concentrations below the normal range on days 3 and 14. In study 2, EGF concentrations on day 3 were repeatedly examined from the nulliparous period to the third postpartum period in 28 Holstein (dairy) and 47 Japanese Black (beef) cattle. The effect of parity on EGF concentrations on day 3 was different between Holstein and Japanese Black cattle. In Japanese Black cows, the EGF concentrations were consistently high throughout the study period, while in Holstein cows, the EGF concentrations decreased after the second calving. In conclusion, unlike multiparous repeat breeder Holstein cows, an altered EGF profile may not be a major cause of repeat breeding in Holstein heifers, and the peak EGF concentrations around day 3 may decrease even in fertile populations of multiparous dairy cows, but not in beef cows.     

Motility and fertility of boar semen after liquid preservation at 5°C for more than 2 weeks

This study investigated the effects of skim milk on the quality and fertility of boar spermatozoa under long‐term chilled preservation. Semen samples were stored in Modena solution supplemented with 0 (control) to 50 mg/mL skim milk at 5°C for 4 weeks; spermatozoa stored with 7.5 and 15 mg/mL of skim milk (7.5‐SM and 15‐SM groups, respectively) exhibited significantly higher motility indices than those of the control group up to 3 weeks (P < 0.05), and the 7.5‐SM group showed improved motility indices even after 4 weeks (P < 0.05). In vitro fertilization using spermatozoa in the 7.5‐SM and 15‐SM groups stored at 5°C for 2 weeks showed significantly higher fertilization rates of spermatozoa and the development rates to blastocyst than the control group (P < 0.05), and the 7.5‐SM group showed similar rates of fertilization and blastocyst formation in the fresh non‐stored spermatozoa group. After artificial insemination using spermatozoa stored for 2 weeks in the 7.5‐SM group, healthy piglets were obtained. Boar spermatozoa can be stored at 5°C in a Modena solution containing skim milk. Supplementation of 7.5 mg/mL skim milk improves boar spermatozoa motility and fertility even after liquid preservation at 5°C for 2 weeks.