Yuji Takagi

Identification of pig primordial germ cells by immunocytochemistry and lectin binding

Monoclonal antibodies anti‐SSEA‐1 and EMA‐1, and the lectins DBA and LTA, bound to the surface of large, round cells randomly distributed in the 26‐day pig genital ridge. Other antibodies, SSEA‐3, SSEA‐4, TRA‐1‐60, and TRA‐1‐81, did not react with any cells in the pig genital ridge. SSEA‐1‐positive cells displayed pseudopods and appeared to migrate from the dorsal mesentery of the hindgut (18‐day) to the primordium of the gonad (day 23) and entered the genital ridge by 26 days. The number of SSEA‐1‐positive cells associated with the dorsal mesentery and genital ridge markedly increased from the 18‐day to the 26‐day pig embryo. It was concluded that the SSEA‐1‐positive cells were primordial germ cells (PGCs). Using these markers and alkaline phosphatase histochemistry, pig PGCs derived from the 26‐day genital ridge showed no proliferation when grown in STO co‐culture in the presence of human LIF, bFGF and SCF. Mol. Reprod. Dev. 46:567–580, 1997. Published 1997 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Development of early bovine embryos to the blastocyst stage in serum-free conditioned medium from bovine granulosa cells

SummaryBovine granulosa cell — conditioned medium (BGC-CM) was prepared in a serum-free medium consisting of TCM 199, 5µg/ml insulin, and 0.5µg/ml aprotinin (TCM 199 IAP). Granulosa cells surrounded with embryos were denuded 24 to 30 h after in vitro fertilization. The proportion of denuded granulosa cell-free embryos that developed to the blastocyst stage in BGC-CM (43/219; 20%) as well as in the co-culture system (43/178; 24%) was significantly greater (P<0.001) than in fresh TCM 199 IAP medium (FM: 10/191; 5%), whereas the proportion of embryos that developed to the eight-cell stage was similar (P>0.05) in all three culture systems (95/178; 53% in co-culture, 111/219; 51% in BGC-CM, and 86/191; 45% in FM, respectively). Higher rates of hatching and hatched blastocysts 8.5 days after in vitro fertilization were observed in co-culture (13/44; 29.5%) and in conditioned medium (8/39; 20.5%). On the other hand, no hatching or hatched blastocysts were obtained in the fresh medium (0.7; 0%). Cell numbers per blastocyst in BGC-CM (178.3 cells/blastocyst) were approximately two-fold higher than those in FM (97.1 cells/blastocyst). However, higher cell numbers (249.3 cells/blastocyst) were observed in co-culture with BGC than in BGC-CM. The embryotrophic activity in BGC-CM was stable upon freezing and thawing, lyophilization, and heating at 56° C whereas activity was reduced by dilution in fresh medium, dialysis, pronase digestion, and heating at 80° C. These results suggest that BGC cultured in a serum-free medium can synthesize and secrete an embryotrophic factor(s) that supports blastocyst formation in vitro beyond the 8- to 16-cell stage.

Development of bovine oocytes matured, fertilized and cultured in a serum-free chemically defined medium

Abstract This experiment was conducted to determine if bovine embryos derived from in vitro maturation, fertilization and culture could develop in serum-free medium. Oocytes were matured and cultured in TCM199 supplemented with or without fetal calf serum (FCS), and in TCM199 supplemented with growth factors (GF-TCM199), namely epidermal growth factor, insulin and transferrin. The proliferation of cumulus cells co-cultured with embryos was also examined. The highest rate of embryo cleavage (48%; 2-cell/total) and blastocyst formation (30%; blastocyst/2-cell) was obtained in serum-supplemented medium, and the extensive cumulus cells proliferation formed a monolayer within 3 d of culture. In TCM199 alone, no cleaved embryos developed to the blastocyst stage, and very limited cell proliferation was observed. In GF-TCM199, 3% of cleaved embryos developed to the blastocyst stage. The collagen-coated dish improved cumulus cell growth, and the rate of blastocyst formation was 8%. The viability of these embryos was judged by transfer, with one of the three recipients becoming pregnant and delivering one calf. In conclusion, the results indicated that collagen-coating and growth factors-supplementation can support embryonic development in serum-free TCM199; however, development in vitro was significantly less extensiver than that in serum-supplemented TCM199.

Up-regulation of adipogenin, an adipocyte plasma transmembrane protein, during adipogenesis

Until now, the various proteins highly expressed in adipose tissues have been identified and characterized by traditional gene cloning techniques. However, methods of computer analysis have been developed to compare the levels of expression among various tissues, and genes whose expression levels differ significantly between tissues have been found. Among these genes, we report on the possible function of a new adipose-specific gene, showed higher expression in adipose tissue through ‘Search Expression’ on Genome Institute of Norvartis Research Foundation (GNF) SymAtlas v0.8.0. This database has generated and analyzed gene expression of each gene in diverse samples of normal tissues, organs, and cell lines. This newly discovered gene product was named adipogenin because of its role in stimulating adipocyte differentiation and development. Adipogenin mRNA was highly expressed in four different fat depots, and exclusively expressed in adipocytes isolated from adipose tissues. The level of adipogenin mRNA was up-regulated in the subcutaneous and visceral adipose tissues of mice fed a high-fat diet compared to those on the control diet. The expression of adipogenin mRNA is dramatically elevated during adipocyte differentiation of 3T3-L1 cells. Troglitazone, which up-regulated peroxisome proliferators-activated receptor γ2 (PPAR-γ2) expression, increased adipogenin mRNA expression, although this gene was down-regulated by retinoic acid. Confocal image analyses of green-fluorescent protein-adipogenin (pEGFP-adipogenin) transiently expressed in 3T3-L1 adipocytes showed that adipogenin was strictly localized to membranes and was absent from the cytosol. Moreover, small interfering RNA (siRNA) mediated a reduction of adipogenin mRNA in 3T3-L1 cells and blocked the process of adipocyte differentiation. These results indicate that adipogenin, an adipocyte-specific membrane protein, may be involved with adipogenesis, as one of the regulators of adipose tissue development.

Multipotential ability of primitive germ cells from neonatal pig testis cultured in vitro

Gonocytes are progenitor-type germ cells that arise from primordial germ cells and differentiate further into spermatogonia, thereby initiating spermatogenesis. In the present study, freshly isolated gonocytes were found to have either weak or no expression of pluripotency determining transcription factors, such as POU5F1, SOX2 and C-MYC. Interestingly, the expression of these transcription factors, as well as other vital transcription factors, such as NANOG, KLF4 and DAZL, were markedly upregulated in cultured cells. Cells in primary cultures expressed specific germ cell and pluripotency markers, such as lectin Dolichos biflorus agglutinin (DBA), KIT, ZBTB16, stage-specific embryonic antigen (SSEA-1), NANOG and POU5F1. Using a monoclonal antibody to specifically identify porcine germ cells, the stem cell potential of fresh and cultured cells was determined with a testis xenotransplantation assay. Colonised porcine germ cells were detected only in mouse testes that were either transplanted with fresh testicular cells or with cells from primary cultures. Interestingly, testes transplanted with cells from primary cultures showed colonisation of germ cells in the interstitial space, reflecting their tumourigenic nature. The formation of teratomas with tissues originating from the three germinal layers following the subcutaneous injection of cells into nude mice from primary cultures confirmed their multipotency. The results of the present study may provide useful information for the establishment of multipotent germ stem cell lines from neonatal pig testis.

Involvement of Transient Receptor Potential Vanilloid (TRPV) 4 in mouse sperm thermotaxis

Transient Receptor Potential Vanilloid (TRPV) 4 is one of the temperature-sensitive ion channels involved in temperature receptors, and it is known to be activated from 35 to 40ºC. Here we analyzed sperm motility function of Trpv4 knockout (KO) mouse in temperature-gradient conditions to elucidate the thermotaxis of mouse sperm and the involvement of TRPV4 in thermotaxis. The sperm were introduced at the vertical column end of a T-shaped chamber filled with medium in a plastic dish, and we measured the number of sperm that arrived at both ends of the wide column where we had established a temperature gradient of approx. 2ºC, and we evaluated the sperm’s thermotaxis. Large numbers of wild-type (WT) mouse sperm migrated into the high level of the temperature gradient that was set in the wide column, and thermotaxis was confirmed. The ratio of migrated sperm at the high temperature level of the T-shaped chamber was decreased in the KO sperm and Ruthenium red (a TRPV antagonist) treated sperm compared with the WT sperm. The thermotaxis of the mouse sperm was confirmed, and the involvement of TRPV4 in this thermotaxis was suggested.

Effects of relaxin and IGF-I on capacitation, acrosome reaction, cholesterol efflux and utilization of labeled and unlabeled glucose in porcine spermatozoa

AimRelaxin and insulin-like growth factor (IGF)-I have pronounced effects on the male and female reproductive tracts. The aim of this study was to investigate the effects of relaxin and IGF-I on the motility, capacitation, acrosome reaction, cholesterol efflux and utilization of glucose in porcine spermatozoa.MethodsSwim-up separated spermatozoa that had been washed twice were incubated at 37°C for 1 or 4 h in modified Tyrode’s albumin lactate pyruvate (mTALP) medium supplemented without (control) or with relaxin (20 ng/mL) or IGF-I (20 ng/mL) or both (10 + 10 ng/mL).ResultsProgressive motility and the induction rate of capacitation and acrosome reaction were increased (P < 0.05) by relaxin and IGF-I alone or in combination, especially after 4 h of incubation. Relaxin alone or combined with IGF-I enhanced (P < 0.05) the cholesterol efflux after 4 h, whereas IGF-I alone did not show any significant effect on the cholesterol efflux compared with the control at any time point. The utilization rates of labeled and unlabeled glucose increased (P < 0.05) in spermatozoa incubated with relaxin or IGF-I alone or in combination compared with the control.ConclusionThus, supplementation of relaxin alone or combined with IGF-I into the medium possibly plays a beneficial role in porcine spermatozoal prefertilization events in vitro.

Growth requirements and long-term subcultivation of bovine granulosa cells cultured in a serum-free medium

SummaryWe have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells. The cells on collagen-coated culture plates proliferated extensively in a nutrient medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2), lipoprotein, and bovine serum albumin (BSA). The cell doubling time at logarithmic phase and final cell density at confluent cultures were equal to those of cultures grown in the presence of medium supplemented with optimal concentration (10%) of fetal bovine serum (FBS). Whereas HBGF-2 or insulin alone had a small mitogenic effect of granulosa cells, lipoprotein or BSA did not. When lipoprotein, BSA, or insulin was added together with HBGF-2, synergistic cell proliferation was observed in all combinations. Insulin or lipoprotein had an additive mitogenic stimulation of these cells in the presence of BSA. After granulosa cells were subcultivated in a serum-containing medium until three generations [8.5 cumulative population doubling level (CPDL)], subsequent subcultivation of the cells in a complete serum-free medium could be achieved up to six generations (14.4 CPDL). These results demonstrate that this serum-free medium can support the optimal cell growth and long-term subcultivation of bovine granulosa cells.

Fatty acid composition of lipids in day 7–13 blastocysts, serum and uterine fluid of rabbits

PurposeThe fatty acid composition of rabbit blastocysts, blood serum and uterine fluids were analyzed to study embryonic lipid metabolism.MethodsEmbryos were collected from Japanese white rabbits and fatty acids were analyzed by gas chromatograph.ResultsTotal amount of fatty acids in blastocysts was higher than that in serum and uterine fluid. The amount of fatty acids in blastocysts markedly decreased during days 7–13 of pregnancy, and in serum had hovered, but in uterine fluid on day 13 was nine times higher than that on day 7 of pregnancy. Palmitic acid predominates in blastocysts, serum and uterine fluid during this period.ConclusionPalmitic acid is the most abundant fatty acid in the blastocysts, serum and uterine fluids of rabbit during days 7–13 of pregnancy.

Growth and Function of Bovine Granulosa Cells Cultured in a Serum-Free Medium

The granulosa cell has an important physiological role in supporting the formation of ovarian follicles and the maturation of oocytes and early embryos. We have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells (BGC). BGC seeded on collagen-coated culture plates proliferated progressively in a serum-free medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2), lipoprotein (LP), and BSA. The cell doubling time at logarithmic phase and final cell density were equal to those in serum-containing medium. Extensive cell proliferation of BGC was primarlily important to induce the early bovine embryo development in vitro. When embryos were co-cultured with BGC in a defined medium, insulin and HBGF-2 stimulated embryonic development to the blastocyst stage as effective as serum did. Denuded embryos (without granulosa cells) could not enter into the blastocyst stage even in the presence of insulin and HBGF-2. These results suggest that insulin and HBGF-2 stimulate early bovine embryonic development by activating BCC function.