Evidence that activin A directly modulates early human male germline differentiation status.

Disrupted fetal germline development underpins testicular germ cell neoplasia, which is increasing worldwide. The complex signaling milieu during normal testis development includes TGFß superfamily ligands; this study tests the hypothesis that, activin A, a TGFß superfamily member, can influence gonocyte development. The human seminoma-derived cell line, TCam-2, a model of fetal gonocytes, was cultured with activin A (1.25-25 ng/mL) for 48 h, or with 5 ng/mL activin A for short- (6, 24, and 48 h) and long-term (13 days) exposures, and downstream targets measured by qRT-PCR. Transcripts that exhibited significant dose-dependent responses to activin A included the early germ cell markers KIT, NODAL, and CRIPTO (NODALl co-receptor and activin inhibitor) which all increased and the differentiation marker DNMT3L which decreased. After 48 h, KIT, NODAL, and CRIPTO levels were significantly higher, while the differentiation marker NANOS2 was significantly lower. Interestingly, activin A exposure also significantly reduced both transcript and protein levels of the PIWI/piRNA pathway component DNMT3L. Because TCam-2 cells produce the activin inhibitor CRIPTO, CRIPTO was reduced using siRNA prior to activin A exposure. This selectively increased KIT in response to activin A. Other ligands present in the fetal testis (BMP4, FGF9, TGFß1, and TGFß2) induced distinct effects on germline marker expression. This study showed that activin A can directly modulate germline markers in this human gonocyte-like cell, promoting a less-differentiated phenotype. Additional findings indicate evidence of signaling crosstalk between activin A and NODAL, leading to target-specific effects on gonocyte differentiation.

TGF-ß superfamily signaling in testis formation and early male germline development.

The TGF-ß ligand superfamily contains at least 40 members, many of which are produced and act within the mammalian testis to facilitate formation of sperm. Their progressive expression at key stages and in specific cell types determines the fertility of adult males, influencing testis development and controlling germline differentiation. BMPs are essential for the interactive instructions between multiple cell types in the early embryo that drive initial specification of gamete precursors. In the nascent foetal testis, several ligands including Nodal, TGF-ßs, Activins and BMPs, serve as key masculinizing switches by regulating male germline pluripotency, somatic and germline proliferation, and testicular vascularization and architecture. In postnatal life, local production of these factors determine adult testis size by regulating Sertoli cell multiplication and differentiation, in addition to specifying germline differentiation and multiplication. Because TGF-ß superfamily signaling is integral to testis formation, it affects processes that underlie testicular pathologies, including testicular cancer, and its potential to contribute to subfertility is beginning to be understood.

Anatomical View of the Internal Carotid Artery Occlusion in Japanese Black Cattle.

The internal carotid artery (ICA) is a branch of the common carotid artery (CCA), along with the external carotid artery (ECA), which together provide the blood supply for the brain. The description of the ICA in cattle is vague, including denial of its existence or degeneration at an early stage after birth. This anatomical study investigated the internal carotid artery in Japanese black cattle. Sixty-five heads of Japanese black cattle aged from newborn to 13 years were dissected and injected with colored latex from the CCA after separating the head and body. Diameter measurements of the artery branches from the CCA on its bifurcation were conducted. Furthermore, a histological examination of the ICA wall's structures, which consist of the tunica intima, tunica media, and tunica externa, was performed. The ICA of Japanese black cattle is closed on the left side after age 3 years, except for a small lumen at 13 years, whereas the right ICA remains open at all ages. The location of occlusion of the left internal carotid artery (LICA) shows thickness of the tunica intima and an increased connective tissue layer area. The diameter of the ICA does not differ between the left and right sides, and there is no correlation with age. Therefore, further studies are needed, especially of ICA occlusion related to Japanese black cattle's physiology or cerebrospinal disease.

Prevalence of anatomic variants in coronary arteries of Japanese Black cattle.

Coronary artery anomalies that can cause sudden cardiac death have been described in some mammals. However, few studies have investigated coronary anomalies and coronary artery branching patterns in cattle. Therefore, understanding of bovine coronary arteries is incomplete. The aim of this study was to clarify anatomic variants in coronary arteries of Japanese Black cattle. The gross anatomy of the coronary arteries was examined in the hearts of 100 cattle. A total of 14 of 100 hearts exhibited confirmed coronary anomalies in the left main trunk. These 14 hearts were classified into 5 types potentially associated with cardiac ischemia. Regarding the coronary branches, the subsinuosal interventricular branch originated from the left circumflex branch (LCX) in 98 hearts and from the right coronary artery in 2 hearts. The origin of the sinoatrial nodal branch was classified into 3 types. In 99 hearts, the atrioventricular nodal branch originated from the LCX at the heart crux. This study characterized the prevalence of coronary anomalies, branching patterns of coronary arteries, and coronary artery anatomic variants in Japanese Black cattle.

Use of platelet lysate for in-vitro embryo production and treatment of repeat breeding in cows.

Platelet-rich plasma (PRP) is a biological hemocomponent derived from blood after the complete removal of red blood cells and the partial or complete removal of white blood cells to concentrate platelets in an appropriate volume of plasma. Platelets have important growth factors, cytokines, and active metabolites that improve the endometrial environment and positively affect implantation. This study evaluated the effect of the addition of activated PRP (platelets lysate; PL) on in vitro bovine oocyte maturation and embryonic development and the effect of intrauterine (IU) infusion of autologous PL in repeat breeder (RB) cows. Experiment 1 examined the effects of allogeneic PL, fetal calf serum (FCS), mixed PL + FCS, or platelet-poor plasma (PPP) supplementations to in vitro maturation and development media on in vitro oocyte maturation and embryo development in good- and poor-quality oocytes of Japanese Black cows. Experiment 2 examined the IU infusion of autologous PL, 24 h post-insemination, in 21 RB Holstein-Friesian dairy cows. The cleavage rate of good-quality oocytes was higher in the PL group (85.93 ± 2.50%) than in the PPP group (67.16 ± 3.41%) (P < 0.05), while the cleavage rate of the poor-quality oocytes was higher in the PL alone (76.13 ± 4.04%) and mixed PL + FCS treated (73.59 ± 4.22%) groups than in the PPP group (54.64 ± 2.93%) (P < 0.05). The blastocyst rate of the good-quality oocytes was higher in the PL group (40.97 ± 3.03%) than in the FCS (27.97 ± 3.31%) and PPP (25.33 ± 2.15%) groups (P < 0.05). The blastocyst rate of poor-quality oocytes and the hatching rates of both good and poor-quality oocytes showed no significant differences among all groups. The conception rate in the autologous PL-treated group was 41.67% (5/12), while it was 11.11% (1/9) in the control group. The platelets' count in the pregnant PL-treated cows (n = 5; mean ± SEM, 1.07 ± 0.10 × 109/mL) was higher than in the non-pregnant ones (n = 7; 0.67 ± 0.10 × 109/mL) (P < 0.05). In conclusion, allogeneic PL was effective in stimulating the in vitro oocyte maturation and embryonic development in both good and poor-quality bovine oocytes, and post-insemination IU infusion of autologous PL derived from high platelets' count-PRP would be recommended for the treatment of RB cows.

Expression and localization of NO synthase isoenzymes (iNOS and eNOS) in development of the rabbit placenta.

Nitric oxide synthase (NOS) is a key regulator of angiogenesis and embryogenesis in the mammalian reproductive process. Here, we attempted to clarify the expression and localization of inducible and endothelial NOS (iNOS and eNOS) in the developing rabbit placenta. Real-time RT-PCR analysis indicated that iNOS mRNA was significantly upregulated till the complete development of the placenta (d18), and then significantly decreased at the end of fetal growth stage (d28) during successful pregnancy. The eNOS mRNA was also enhanced in the pregnant uteri and gradually decreased near the term of pregnancy. Western blot analysis also showed elevation of the iNOS and eNOS protein levels during the course of successful pregnancy till the functional maturation of the placenta (d18). Immunohistochemical study revealed distinct localizations of iNOS along the radial arteries and eNOS at the spiral arteries and arterial sinuses in the developing placenta. This may reflect that iNOS and eNOS participate in pregnancy success through placentation-specific vascular formation and by supporting adequate blood circulation in the rabbit placenta.

Insufficiency of CD205-positive cortical thymic epithelial cells in immature Japanese Black cattle with severe thymic abnormalities and poor prognosis.

To investigate the involvement of thymic function in the development of diseases with poor prognosis in calves, this study conducted a survey for the assessment of thymus cell composition in immature Japanese Black cattle with poor prognosis. Histopathological evaluation of 47 cattle showed signs of acute thymic involution in most cases. Less than half of the cases had a cortex predominant over the medulla in the thymic parenchyma, and a quarter of the cases indicated severe histological condition with an unclear boundary between the cortex and medulla. Correlation analysis revealed a close relationship between the corresponding stages of acute involution, cortical occupancy, and the expression of CD4, CD8B, and CD205. When cases were grouped by cortical occupancy, the expression of CD4 and CD8B expression was lower in the severe group with less than 25 % cortical occupancy, and the expression of CD205 was lower in the group with an unclear cortical-medullary boundary. Meanwhile, there was no difference in the expressions of IL7, CD80, FEZF2, and FOXN1 according to cortical occupancy. Immunohistochemistry has shown that cytokeratin-positive thymic epithelial cells are more densely populated in the severe thymus. UEA-I-binding medullary thymic epithelial cells were also present, but CD205-positive cortical thymic epithelial cells were rare in severe thymus. Moreover, there were significantly fewer Ki-67-positive cells in cattle with severe thymus. Therefore, these results indicate that thymic histological abnormalities frequently occur in immature cattle with a poor prognosis, and the presence of CD205-positive cortical thymic epithelial cells is associated with the severity of the abnormalities.

Feeding ecology of Swinhoe's tree lizard (Diploderma swinhonis (Günther, 1864)) in Hyuga City, Miyazaki Prefecture, Japan.

Swinhoe's tree lizard (Diploderma swinhonis: D. swinhonis) is an arboreal agamid that is native to Taiwan. In Taiwan, the lizard is considered to be a generalist that feeds primarily on ants and a diversity of small insect prey by employing an opportunistic sit-and-wait foraging strategy. In Japan, D. swinhonis is considered as an invasive alien species that was discovered in Hyuga city, Miyazaki Prefecture, in 2016. Despite concerns about the impact of D. swinhonis on native fauna, little information about the diet of this alien species has been published to date. This study, therefore, investigated the feeding ecology of D. swinhonis in Hyuga city to evaluate their potential impact on the ecosystem. Specifically, prey preference was investigated by examining the stomach contents of males, females, and juveniles captured from April to December 2020 and in March 2021. The results showed that the lizards in Hyuga preyed upon a wide variety of invertebrates as in Taiwan, while ants accounted for the largest proportion of the prey items consumed regardless of sex, age or changes in season. These findings indicated that D. swinhonis might cause a decrease in the abundance of the native insect fauna of Hyuga city or competition with native lizards for foods in Hyuga city. Since its impact is not currently apparent, it's necessary to monitor its effect in the future.

Embryo implantation is blocked by intraperitoneal injection with anti-LIF antibody in mice.

Leukemia inhibitory factor (LIF) is essential for embryo implantation in mice and plays an important role in other mammals including humans. Intraperitoneal (i.p.) injections with anti-LIF antibody (7.5 µg/g body weight, 3 times) between D3 (D1 = day of vaginal plug detection) and D4 effectively blocked embryo implantation; complete inhibition was achieved in C57BL/6J mice, and implantation was dramatically reduced in ICR mice (reduced to 27%). Normal rabbit IgG used as the control did not disturb embryo implantation. Anti-LIF antibody was localized not only in the stroma, but also in the luminal epithelium and the glandular lumen after i.p. injections. Growth-arrested blastocysts were recovered from the uterus without any implantation sites in both strains. Blastocysts made contact with the LE on the antimesometrial side; however, uterine stromal cells did not undergo secondary decidual reaction, and the uterine lumen was open, even at D7. Several regions of decidualization in ICR mice treated with anti-LIF antibody were smaller than those of the control, and development of blastocysts was delayed. The expression of LIF-regulated genes, such as immune-responsive gene-1 and insulin-like growth factor binding protein-3, was significantly decreased in C57BL/6J mice treated with anti-LIF antibody compared with the control, but not in ICR mice. The present study demonstrated that simple ip injections of an antibody are sufficient to block one of the important factors involved in embryo implantation in mice, and this method should also be easily applicable to the investigation of other factors involved in implantation.

Reproductive cycle and maturation of Swinhoe's tree lizard (Diploderma swinhonis (Günther, 1864)) in Hyuga City, Miyazaki Prefecture, Japan.

Swinhoe's tree lizard (Diploderma swinhonis) is an arboreal agamid that is native to Taiwan. The species has been introduced to some areas of Japan and is regarded as an invasive alien species. In 2016, a nonnative population of D. swinhonis was discovered in Hyuga City, Miyazaki Prefecture, Japan, but little information was available on the ecology of the population at the time. The main purpose of this study was therefore to investigate the reproductive cycle and maturation of this population. Field research was conducted from 2017 to 2019, and 764 lizards were collected. Euthanized lizards were dissected and the reproductive organs were examined to determine the reproductive period, clutch size, clutch frequency and size at sexual maturity. Females with oviductal eggs or vitellogenic ovarian follicles were observed from May to October. Clutch size ranged from 2 to 8, and clutch frequency was more than twice a year. In males, spermiogenesis started in early May and testicular regression was observed in September. Males with spermatozoa in the epididymides were found from May to November. Minimum snout-vent length at sexual maturity was 50.2 mm in females and 53.0 mm in males. Comparisons of the findings of this study and reports from Taiwan suggest that the nonnative population of D. swinhonis in Hyuga City has a higher fecundity than populations in Taiwan. It is therefore considered necessary to exterminate the population in Hyuga City before this species colonizes other areas.

Uterine natural killer cells in perforin and beta(2)-microglobulin deficient mice.

Uterine natural killer (uNK) cells have roles for immune responses at the feto-maternal interface in mice. We studied the effects of beta(2)-microglobulin (beta(2)m) and perforin on proliferation and differentiation of uNK cells in pregnancy, using beta(2)-microglobulin-deficient (beta(2)m(-/-)) mice and perforin-deficient (P(-/-)) mice. The cell population of uNK cells in the metrial gland of P(-/-) mice was tended to be higher than the control B6 mice. The cell population of uNK cells in the metrial gland of beta(2)m(-/-) mice was significantly increased at Day 12 of pregnancy comparing to B6 and P(-/-) mice. On the other hand, the cell population of uNK cells in the decidua basalis of beta(2)m(-/-) mice was tended to be lower than B6 and P(-/-) mice. These results indicate that beta(2)m may be involved in proliferation of uNK cells in the metrial gland, and that beta(2)m may affect the maturation of uNK cells in the decidua basalis.

Reproductive performance in diabetes mice with a special reference to uterine natural killer cells and placental growth factor.

To determine the effect of diabetes on reproductive performance, two kinds of diabetes mice, i.e., KK/TaJcl mice with Type-II diabetes and Streptozotocin-induced diabetes mice with Type-I diabetes, were used in this study. Particular attention was paid to uterine natural killer (uNK) cells and placental growth factor (PlGF). The number of fetuses, the fetal and placental weights in both diabetes mice were significantly decreased when compared to controls. Surprisingly, uNK cells in both diabetes mice persisted in the metrial gland even at the term of pregnancy. Although PlGF expression in both diabetes mice was significantly decreased, PlGF protein did not change. These results show that diabetes condition affects reproductive performance, particularly uNK cell behavior, but not PlGF production.

Effects of leukemia inhibitory factor on lectin-binding patterns in the uterine stromal vessels of mice.

Lectin histochemistry was performed on mouse uteri to determine what effects leukemia inhibitory factor (LIF) has on carbohydrate epitope expressions at the time of implantation. Twenty-two biotinylated lectins were used in this study. Following injection of LIF, specific binding to the apical surface of the uterine glandular epithelium (GE) was recognized by six lectins. Particularly, binding of the lectin from Griffonia (Bandeiraea) simplicifolia was specific to the glandular epithelium close to the luminal epithelium. Succinylated wheat germ agglutinin (WGA), which has specificity for oligosaccharides recognized by WGA without sialic acid residues, showed weaker binding to the uterine luminal epithelium (LE) and the stroma than WGA, suggesting that terminal residues of glyco-conjugates on these tissues may be modified by sialic acids. Lectin binding to the glandular and luminal epithelium was not influenced by LIF. However, three lectins including a lectin from Dolichos biflorus showed specificity for stromal vessels 6h after LIF injection. Since the lectin from D. biflorus binds to neo-vascular vessels, LIF may play a role in regulating maternal angiogenesis directly and/or indirectly during implantation.

The differentially DNA-methylated region responsible for expression of runt-related transcription factor 2.

Runt-related transcription factor 2 (Runx2) is essential for osteogenesis. This study aimes at identification of the genomic region differentially methylated in DNA for regulation of Runx2 expression. In the proximal promoter of mouse Runx2, DNA methylation was frequent at the region further than 3 kb relative to the transcription start site, in contrast to lower methylation status of the closer locus within 2 kb from the transcription start site. At the intermediate part, we identified a novel differentially methylated region in the Runx2 promoter region (Runx2-DMR): from -2.7 to -2.2 kb relative to the start site of Runx2 transcription in mice. In this region, the DNA methylation rate correlated negatively with Runx2 expression among mouse organs as well as among primary cultures of bone marrow from different dogs. Induction of mouse and dog mesenchymal-like cells into osteoblastic differentiation decreased the methylation rate of Runx2-DMR. Thus, in this study, we identified a novel genomic region in which DNA methylation status is related to Runx2 expression and detected demethylation of Runx2-DMR during osteoblastic differentiation in mouse and dog.

Tetraploid Embryonic Stem Cells Maintain Pluripotency and Differentiation Potency into Three Germ Layers.

Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.

Multiple Acyl-CoA Dehydrogenation Deficiency (Glutaric Aciduria Type II) with a Novel Mutation of Electron Transfer Flavoprotein-Dehydrogenase in a Cat.

Multiple acyl-CoA dehydrogenation deficiency (MADD; also known as glutaric aciduria type II) is a human autosomal recessive disease classified as one of the mitochondrial fatty-acid oxidation disorders. MADD is caused by a defect in the electron transfer flavoprotein (ETF) or ETF dehydrogenase (ETFDH) molecule, but as yet, inherited MADD has not been reported in animals. Here we present the first report of MADD in a cat. The affected animal presented with symptoms characteristic of MADD including hypoglycemia, hyperammonemia, vomiting, diagnostic organic aciduria, and accumulation of medium- and long-chain fatty acids in plasma. Treatment with riboflavin and L-carnitine ameliorated the symptoms. To detect the gene mutation responsible for MADD in this case, we determined the complete cDNA sequences of feline ETFα, ETFß, and ETFDH. Finally, we identified the feline patient-specific mutation, c.692T>G (p.F231C) in ETFDH. The affected animal only carries mutant alleles of ETFDH. p.F231 in feline ETFDH is completely conserved in eukaryotes, and is located on the apical surface of ETFDH, receiving electrons from ETF. This study thus identified the mutation strongly suspected to have been the cause of MADD in this cat.

Influence of atopic dermatitis on reproduction and uterine natural killer cells.

The causal relationship between severe allergic conditions and successful pregnancy remains unclear. We aimed to evaluate reproductive performance in an experimental mouse model of atopic disease (AD), and the appearance of uterine natural killer (uNK) cells that have crucial roles in placental formation was examined. In the NC/Nga pregnant mice with moderate skin allergic lesions and an 8.6-fold elevation of plasma IgE, significant differences were not detected in the reproductive indices of the number of normal fetuses, abortion rate and placental size. There were few uNK cells in the placenta of AD mice, and they showed a significant decrease regarding the immature subtype as compared with controls. These findings revealed that AD disturbs uNK cell differentiation and provides disadvantageous effects on placental formation, although it does not arrest the pregnancy process. It may be possible that specific immunological conditions behind AD operate favorably to recover the reproductive performance.

Quantitative expression and immunohistochemical detection of glucose transporters, GLUT1 and GLUT3 in the rabbit placenta during successful pregnancy.

Glucose is essential for the development of the fetus. We address here the quantitative expression and immunohistochemical localization of glucose transporter (GLUT1 and GLUT3) in the rabbit placenta during successful pregnancy. Blood glucose level showed a significant decrease at the gestation period in comparison with non-pregnancy. Maternal serum glucose was gradually increased according to fetal development. Quantitative RT-PCR results showed that expression of GLUT1 was significantly increased from day 13 to day 18, while GLUT3 mRNA level was significantly decreased during the same periods. Western blot analysis demonstrated that GLUT1 protein did not change significantly in the placenta during pregnancy when compared to non-pregnant uteri. Immunohistochemistry indicated that distribution of GLUT1 was observed mainly to the surface of the outer trophoblasts, whereas GLUT3 mainly localized to the basal site of the inner trophoblasts and fetal blood vessels. These results suggest that glucose is transported through GLUT1 from the maternal blood stream for use as a placental fuel and for further transport through GLUT3 to the fetal circulation, thus signifying the distinct anatomical localization of GLUT1 and GLUT3 in the rabbit placenta during successful pregnancy.

Differentiation of uterine natural killer cells in pregnant SCID (scid/scid) mice.

To determine whether functional T- and B-cells can affect differentiation and/or proliferation of uterine natural killer (uNK) cells, their numbers in SCID mice (genotype, C.B.-17/Icr-scid/scid) were compared with those of control mice (genotype, C.B.-17/Icr-+/+) on days 8, 12 and 16 of pregnancy. Using biotinylated-Dolichos biflorus agglutinin (DBA) lectin staining, uNK cells can be readily classified into 4 subtypes, I to IV, from immature to mature types. The number of uNK cells was significantly lower in the decidua basalis of SCID mice than in that of control mice on day 8 of pregnancy. Particularly, the number of uNK cells of immature subtype II was significantly lower in SCID mice than in the control mice. By day 12, however, the uNK cell number in the SCID mice reached the same level as that of the control mice. It is likely that uNK cell differentiation in SCID mice was delayed during the early placentation period due to a lack of functional T and B cells.