During the process of zygotic gene activation (ZGA), which occurs in the fertilized oocyte, genomic function is regulated by dynamic structural changes in the nucleus. Nakaya et al. developed an easy-to-use chamber device (EASI-FISH chamber) for three-dimensional fluorescence in situ hybridization (3D-FISH) in the embryos and succeeded in visualizing the intra nuclear spatial arrangements in the fertilized eggs (Nakaya M, et al., Visualization of the spatial arrangement of nuclear organization using threedimensional fluorescence in situ hybridization in early mouse embryos: A new “EASI-FISH chamber glass” for mammalian embryos. pp. 167–174). Pericentromeric regions which comprise heterochromatin clusters called chromocenters in somatic cells decondensed during the 1- to 2-cell stages and changed to the chromocenter-like structure thereafter. In the early embryos, the ZGA-related Zscan4 gene loci were located peripherally in the CTs but relocated interiorly in somatic cells. In addition, the CTs in the early embryos were enlarged and had deformed shapes, which were quite different from those in somatic cells. This convenient and reproducible 3D-FISH technique for mammalian embryos is a valuable tool that will provide insights into the nuclear dynamics during development.
Cell-secreted vesicles, such as exosomes, have been recognized as mediators of cell communication. In recent studies, exosomes were shown to be involved in the regulation of ovarian function including in the induction of cumulus expansion. To date, exosome function has not been investigated in pigs. Matsuno et al. reported the existence of exosome-like vesicles in porcine follicular fluid and examined the effects of these vesicles on cumulus expansion in porcine cumulus-oocyte complexes (Matsuno et al., Effects of exosome-like vesicles on cumulus expansion in pigs in vitro, pp. 51–58). The existence of exosome-like vesicles was confirmed by transmission electron microscopic observation (top left: negative stain method image; top right: cryo-TEM method image). The vesicles labeled with fluorescent tags were incorporated into cumulus and mural granulosa cells in vitro (bottom left and right: vesicles shown in green with white arrowhead). The vesicles had no significant effects on the expansion of porcine cumulus-oocyte complexes in vitro. Therefore, although exosome-like vesicles exist in ovarian follicles, they are not efficient in inducing cumulus expansion in pigs in vitro.
Meiosis-specific α-kleisin subunits (RAD21L and REC8) play roles in the formation of axial elements, assembly of the synaptonemal complex, recombination of homologous chromosomes, and cohesion of sister chromatids. However, the exact functions of the individual α-kleisin subunits remain to be elucidated. Using 3D-SIM, Rong et al. found that RAD21L and REC8 were localized at the connection sites between lateral elements and transverse filaments of pachynema with RAD21L locating interior to the REC8 sites (Rong et al.: Meiotic cohesin subunits RAD21L and REC8 are positioned at distinct regions between lateral elements and transverse filaments in the synaptonemal complex of mouse spermatocytes. pp. 623−630). Intriguingly, some bridge-like signals of RAD21L, but not REC8, were observed between unsynapsed regions of axial elements of zygonema. Furthermore, the signals of recombination intermediates overlapped with those of RAD21L to a greater degree than with the signals of REC8. These results highlight the different properties of the two meiotic α-kleisin subunits, and support the view that RAD21L is an atypical cohesin that establishes the association between homologous chromosomes rather than sister chromatids.
Appeltant et al. reviewed the involvement of cAMP in maturation regulation, the function of oocyte-secreted factors (OSFs), and the interaction of both in the bidirectional regulatory loop between porcine oocytes and their cumulus cells. (Appeltant et al. Porcine oocyte maturation in vitro: role of cAMP and oocyte-secreted factors—A practical approach, pp. 439–449). In vivo, follicle-stimulating hormone induces luteinizing hormone (LH) receptors and epidermal growth factor (EGF) receptors (EGFR). LH can stimulate the LH receptor, while EGFR can be stimulated by amphiregulin, epiregulin, and betacellulin. In vitro, EGF cannot activate the EGFR and extracellular signal regulated kinase (ERK1/2) signaling in cumulus cells of porcine cumulus oocyte complexes derived from small antral follicles (< 4 mm), leading to a low developmental competence of these oocytes. The co-operative action of dibutyryl cAMP sodium salt and OSFs in an amphiregulin-stimulated in vitro maturation system promotes the EGFR/ERK1/2 signaling pathway, consequently improving the developmental potential of the oocytes.
The mechanism of bovine sperm functional regulation in vivo is still largely unknown. By using spermatozoa from elite bulls (top left), Umezu et al. have proposed a new regulation model of bovine sperm functions mediated by the neurotensin (NT) ligand-receptor system (Umezu et al.: Exogenous neurotensin modulates sperm function in Japanese Black cattle. pp. 409–414). They confirmed the expression of neurotensin receptor 1 (NTR1) in the bovine sperm neck region (top right) and the secretion of NT in the bovine uterus and oviduct. They also examined the effects of exogenous NT on bovine sperm functions such as hyperactivation (middle right). Exogenous NT enhanced protein tyrosine phosphorylation in bovine sperm and acrosomal disappearance rates in a dose-dependent manner in vitro (middle left). These results suggest that NT acts as a facilitator of sperm capacitation and acrosome reaction in the female reproductive tract in cattle, thus highlighting the importance of NT-mediated signaling to regulate sperm functions (bottom).
Data on pituitary transcription factors have demonstrated the regulatory mechanisms of the three pituitary glycoprotein hormone subunit genes (Fshβ. Lhβ, and Cga) constituting two types of gonadotropins. However, the functional differences between the LIM-homeobox transcription factors LHX2 and LHX3 in the regulation of Fshβ and Cga remain unclear. Yoshida et al. reported the DNA binding properties and transcriptional activities of Cga and Fshβ between LHX2 and LHX3 as well as their localization in the pituitary gonadotrope of rat (Yoshida et al., Transcription of Follicle-Stimulating Hormone Subunit Genes is Modulated By Porcine LIM Homeobox Transcription Factors, LHX2 and LHX3. pp. 241–248). In particular, immunohistochemical analysis demonstrated a rare population of LHX2 in contrast to LHX3 in the gonadotrope. The present study showed that LHX2 and LHX3 differentially regulate Fshβ and Cga.
Miyahara et al. revealed that the membrane-bound form of chicken stem cell factor (chSCF2) was highly beneficial for the in vitro culture of chicken primordial germ cells (PGCs) and that it exerted its growth-promoting effects by cooperating with fibroblast growth factor 2 (Miyahara et al., Chicken stem cell factor enhances primordial germ cell proliferation cooperatively with fibroblast growth factor 2, pp. 143–149). The cultured PGCs were maintained as large spheres (differential interference image) on a feeder layer stably expressing chSCF2. These cultured PGCs expressed the germ cell marker CVH (green) and the undifferentiated cell marker SSEA-1 (red). The cultured PGCs were isolated from a white donor chicken and then transplanted into a recipient embryo from a black chicken, generating a germline chimeric chicken expected to possess both donor- and recipient-derived sperm. The presence of a white chick among the offspring of this chimeric chicken demonstrates that these cultured PGCs were able to undergo normal gametogenesis. These results suggest that chSCF2 induces hyperproliferation of chicken PGCs while retaining their germline competency.
In vitro growth of immature oocytes provides the opportunity to increase gametic resources and to understand the mechanisms underlying oocyte development. Eppig et al. demonstrated that non-growing oocytes in primordial follicles can develop into functional oocytes in vitro; however, no one has been able to reproduce such excellent data in the last 20 years. Morohaku et al. established a novel and robust protocol for producing mature oocytes from non-growing oocytes in vitro (pp. 1–5). The addition of fetal bovine serum and polyvinylpyrrolidone to the medium is the primary difference between the protocols established by Morohaku et al. and that of Eppig et al. The resulting oocytes can develop into fertile offsprings after in vitro fertilization and embryo transfer. The birth rate, by this method, greatly exceeds that of the previous study. Although there is still scope for further improvement, Morohaku’s report will facilitate the in vitro growth of mammalian oocytes for practical applications.
Artificial insemination (AI)-subfertile bulls have occasionally been found in the sire candidates of high-performance Japanese Black cattle. Kishida and Sakase et al. reported that it is valid to perform exact examinations of acrosomal conditions of cryopreserved spermatozoa for exclusion of these subfertile bulls from the AI program for Japanese Black cattle (Kishida and Sakase et al. Effects of acrosomal conditions of frozen-thawed spermatozoa on the results of artificial insemination in Japanese Black cattle, pp. 519–524). The acrosomal conditions were assessed by peanut agglutinin-lectin staining and immunostaining of acrosomal tyrosine-phosphorylated proteins. The percentages of cryopreserved spermatozoa with normal acrosomal conditions were significantly correlated with the conception rates of routine AI, rates of transferable embryos in superovulation/AI-embryo collection tests and in vitro fertilization rates. These results indicate that low conception rates in AI using cryopreserved spermatozoa with poor acrosomal conditions result from reproductive dysfunctions in the processes between sperm insemination into females and early embryo development, probably from failed fertilization of oocytes with cryopreserved spermatozoa.
The dynamics of sex chromosomes during spermatogenesis have been unclear in vivo. Otaka et al. showed the localization of sex chromosomes before and after meiosis in mouse testis sections by fluorescence in situ hybridization (FISH) using specific probes for the X and Y chromosomes (Otaka et al. Distribution of the sex chromosome during mouse spermatogenesis in testis tissue sections. pp. 375–381). The positions of the X chromosomes (red) and Y chromosomes (green) were detected in spermatogenic cells (nucleus; blue), and the dynamics of sex chromosomes during spermatogenesis were demonstrated.
Although Rb1 is reportedly essential for self-renewal of spermatogonial stem cells (SSCs), its mechanism has remained unknown. Tanaka et al. examined the phenotype of Rb1-deficient SSCs in detail using cultured SSCs and conditional knock-out mice and found that Rb1 is critical for protection from genomic damage, which induces cell cycle arrest and apoptosis (Tanaka et al. The CDKN1B-RB1-E2F1 pathway protects mouse spermatogonial stem cells from genomic damage. pp.305–316). This picture shows increased expression of γH2AX (a double-strand break marker, red) in GFRA1(green)-expressing undifferentiated spermatogonia in 10-day-old Ddx4-Cre Rb1flox/– mice, in which Rb1 is specifically inactivated in germ cells (blue: Hoechst 33342).
Monomeric Plum, a far-red fluorescent protein with photostability and photopermeability, is a potentially suitable cell marker for a variety of animal experiments. Watanabe et al. created a transgenic cloned pig systemically expressing Plum (Watanabe et al. Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum. pp. 169–177). Expression of Plum commenced at the early embryonic stage and was confirmed in all of the tissues/organs examined, including blood cells. Cells expressing Plum could be clearly distinguished in culture or by flow cytometry from those expressing other fluorescent proteins with shorter wavelengths, such as green fluorescent protein and Kusabira-Orange. These results suggested that the cells, tissues, and organs of pigs expressing Plum could be a useful tool in biomedical research.
Rejuvenation research provides the latest information on the molecular and cellular mechanisms necessary for most effective therapeutic approaches for human welfare. Collagen is known to be one of the elements necessary for rejuvenation. Chung et al. investigated whether treatment with collagen complexes had beneficial effects on the rejuvenation or reprogramming of adult mouse-derived fibroblasts (Chang et al. Collagen complexes increase the efficiency of iPS cells generated using fibroblasts from adult mice. pp. 145–153). They found that adult-derived fibroblasts cultured with collagen complexes showed a more youthful state, expanded at a higher rate, and exhibited reduced spontaneous cell death than those cultured without collagen complexes. Further, the efficiency of reprogramming of fibroblasts to become induced pluripotent stem (iPS) cells was significantly higher in adult-derived fibroblasts cultured with collagen complexes than in adult-derived fibroblasts cultured alone. As shown in the cover photo, these iPS cells derived from fibroblasts cultured with collagen complexes showed positive staining for stemness markers such as c-myc, Nanog, Oct4, Sox3, and SSEA-1.
It is important to identify which pluripotent stem cells (PSCs) are suitable for human medical application; however, it is unclear how the qualitative differences between different types of PSCs should be evaluated. To provide a human ES/iPS cell model, Honsho et al. converted rabbit ES/iPS cells into a naïve-like state and differentiated them in vitro into a neural lineage cells—oligodendrocytes (Honsho et al. Naïve-like conversion enhances the difference in innate in vitro differentiation capacity between rabbit ES cells and iPS cells, pp. 13–19). It is thought that naïve PSCs have much better differentiating ability than primed PSCs. Naïve-like converted ESCs differentiated much more effectively than primed-state ESCs and naïve-like iPSCs. Also, this study demonstrated that naïve-like conversion can increase the slight qualitative differences of primed-state PSCs and can be used as a valuable strategy for examining the innate capacity of human PSCs for therapeutic use.