Immunohistochemical and ultrastructural characterization of the initial post-hatching development of bovine embryos

The problems of sustaining placenta formation in embryos produced by nuclear transfer have emphasized the need for basic knowledge about epiblast formation and gastrulation in bovine embryos. The aims of this study were to define stages of bovine post-hatching embryonic development and to analyse functional mechanisms of germ-layer formation. Embryos developed in vivo were collected after slaughter from superovulated cows on days 9, 11, 14 and 21 after insemination and processed for transmission electron microscopy (n = 26) or immunohistochemistry (n = 27) for potential germ-layer characterization (cytokeratin 8 for potential ectoderm; alpha-1-fetoprotein for potential endoderm; and vimentin for potential mesoderm). On day 9, the embryos were devoid of zona pellucida and presented a well-defined inner cell mass (ICM), which was covered by a thin layer of trophoblast cells (the Rauber's layer). Formation of the hypoblast from the inside of the ICM was ongoing. On day 11, the Rauber's layer was focally interrupted and adjacent underlying ICM cells formed tight junctions. The hypoblast, which formed a thin confluent cell layer, was separated from the ICM and the tropho-blast by intercellular matrix. The embryos were ovoid to tubular and displayed a confluent hypoblast on day 14. The epiblast was inserted into the trophoblast epithelium and tight junctions and desmosomes were present between adjacent epiblast cells as well as between peripheral epiblast and trophoblast cells. In some embryos, the epiblast was more or less covered by foldings of trophoblast in the process of forming the amniotic cavity. Cytokeratin 8 was localized to the trophoblast and the hypoblast underlying the epiblast; alpha-1-fetoprotein was localized to most hypoblast cells underlying the trophoblast; and vimentin was localized to most epiblast cells. On day 21, the smallest embryos displayed a primitive streak and formation of the neural groove, whereas the largest embryos presented a neural tube, up to 14 somites and allantois development. These embryos depicted the gradual formation of the endoderm, mesoderm and ectoderm as well as differentiation of paraxial, intermediate and lateral plate mesoderm. Cytokeratin 8 was localized to the trophoblast, the hypoblast and the surface and neural ectoderm; and alpha-1-fetoprotein was localized to the hypoblast, but not the definitive endoderm, the intensity increasing with development. Vimentin was initially localized to some, but not all, cells positioned particularly in the ventral region of the primitive streak, to presumptive definitive endoderm cells inserted into the hypoblast, and to mesoderm. In conclusion, within 2 weeks of hatching, bovine embryos complete formation of the hypoblast and the epiblast, establishment of the amniotic cavity, ingression of epiblast cells for primitive streak formation, involution of cells through the node and the streak for endoderm and mesoderm fomation, neurulation and differentiation of the mesoderm. The recruitment of cells from the epiblast to form the primitive streak as well as the endoderm and mesoderm is associated with expression of the intermediate filament vimentin.     

葡萄果实发育过程中花色素还原酶的免疫组织化学定位

以赤霞珠葡萄果实为试材,采用免疫组织化学方法,对果实发育过程中花色素还原酶(ANR)的分布及其动态变化进行原位分析.结果表明,在果皮、果肉和种子中均可观察到ANR的存在;种子中的ANR主要定位于内珠被(in),果皮和果肉中的ANR则主要分布在维管束(VB)、果皮(BS)细胞壁和果肉(BP)细胞壁中,而且这种特异性分布并不随果实发育进程而发生改变.     

Production and localization of activins and activin type IIA and IIB receptors by the human endosalpinx

Fallopian tubes from ten premenopausal women were collected and examined for the presence of inhibin, activin and its type IIA and IIB receptors (ActRIIA and ActRIIB) in the endosalpinx. Immunocytochemistry demonstrated clear staining for the betaA, betaB subunits and ActRIIA and ActRIIB that increased in intensity from the isthmus to the ampulla. No staining for the alpha subunit was observed. Whilst the staining of the betaA subunit and ActRIIA was seen in almost every epithelial cell, staining for the betaB subunit and ActRIIB was more variable. In situ hybridization and RT-PCR confirmed the presence of mRNA for the betaA, betaB subunits and ActRIIA and ActRIIB. These results indicated that the epithelium of the uterine tube is able to synthesize activin but not inhibin and has receptors for activin. Activins may thus act as paracrine regulators of tubal epithelial cell function, and embryonic activity may also bind to epithelial receptor and initiate intracellular processes that alter epithelial cell secretions.     

Unexpected nuclear localization of Cdc25C in bovine oocytes, early embryos, and nuclear-transferred embryos

It is clear from a wide range of studies that the nuclear/cytoplasmic distribution of Cdc25C has important functional consequences for cell cycle control. It is now admitted that in somatic cells, the localization of Cdc25C in the cytoplasm is required to maintain the cell in an interphasic state and that Cdc25C has to translocate to the nucleus just before M-phase to induce mitotic events. We characterized the expression and localization of Cdc25C during oocyte maturation, the first embryo mitosis, and the first steps of somatic cell nuclear transfer (SCNT) in cattle. We demonstrated that Cdc25C was expressed throughout the maturation process and the early development. We clearly showed that Cdc25C was localized in the nucleus at the germinal vesicle stage and during the early development until the blastocyst stage. However, the signal change in blastocyst and Cdc25C became cytoplasmic as is the case in somatic cells. Thus, oocytes and early embryonic cells presented a specific nuclear Cdc25C localization different from the one observed in somatic cells, suggesting that Cdc25C could have a particular localization/regulation in undifferentiated cells. Following SCNT, Cdc25C became nuclear as soon as the nucleus swelled, and this localization persisted until the blastocyst stage, as is the case in in vitro fertilized embryos. The Cdc25C nuclear localization appeared to constitute a major change, which could be associated with the reorganization of the somatic nucleus upon nuclear transfer.     

Function and localization of oxytocin receptors in the reproductive tissue of rams

Oxytocin is present in the male reproductive tract and has been shown to increase contractility in the epididymis and to modulate steroidogenesis. This study investigated the effects of oxytocin in the testis in vivo, and the presence and cellular localization of oxytocin receptors in the reproductive tract of rams. During the breeding season, mature rams underwent efferent duct ligation before injection of either oxytocin (20 microg) or oxytocin plus an oxytocin antagonist (20 microg) into the testicular artery; the contralateral testicular artery received saline. Injection of oxytocin caused a significant increase (P < 0.05) in the concentration of spermatozoa collected from the rete testis. This effect was not observed after treatment with the oxytocin antagonist plus oxytocin. Western blot analysis performed using a specific oxytocin receptor antibody (020) identified a single immunoreactive band of 66 kDa in testicular and epididymal tissue. This band was present in uterine tissue but not in liver or muscle. Immunocytochemistry identified oxytocin receptors on Leydig and Sertoli cells of the testis, on epithelial cells throughout the epididymis, on peritubular smooth muscle cells in the cauda epididymidis, and on the epithelial cells and circular smooth muscle layer of the ductus deferens. These findings indicate that oxytocin can modulate sperm transport in the ram testis. A role for oxytocin in promoting sperm transit is supported by the localization of oxytocin receptors in the cauda epididymis and ductus deferens, and the presence of receptors on Leydig, Sertoli and epididymal epithelial cells provides further evidence that oxytocin may be involved in the local regulation of steroidogenesis.     

Immunohistochemical localization of active caspase-3 in the mouse ovary: growth and atresia of small follicles

Caspase-3 belongs to a family of highly conserved cysteine proteases that mediate the course of apoptotic cell suicide. It is recognized that ovarian follicular atresia is associated with apoptosis, a process that has been characterized mainly in larger antral follicles. The aims of this study were to investigate the expression of caspase-3 in the mouse ovary, and determine whether active caspase-3 is present within smaller follicles, which may constitute the resting pool. The inactive enzyme was expressed as a 32 kDa band on a western blot of tissue extracts, whereas the active form was localized immunohistochemically. Bromodeoxyuridine (BrdU) was administered to mice (n = 7) during a 12 h period and subsequently localized to identify potentially quiescent follicles. Measurements of BrdU-positive cells in the mouse ovary were extrapolated with data obtained by morphometric analyses of small follicles using the nucleator technique. BrdU was incorporated into the granulosa cells of follicles regardless of size and the number of cells they contained, but was absent in a large proportion (89%) of small, single layered follicles. Active caspase-3 was localized to both the oocyte and granulosa cells of follicles that were considered to be undergoing atresia, but was not localized to the granulosa cells of any small, single layered follicles. The results of this study indicate that, in small follicles, granulosa cell proliferation occurs independently of the size of follicles and the number of constituent cells, and that follicles of this type may be inherently less susceptible to the normal physiological factors that induce atresia.     

Immunohistochemical localization and expression of the hyaluronan receptor CD44 in the epithelium of the pig oviduct during oestrus

Hyaluronan is related to essential reproductive processes in pigs. Hyaluronan produced by cumulus cells builds, via specific cell surface receptors, an extracellular matrix responsible for cumulus cell cloud expansion during final oocyte maturation, a preparatory event for ovulation and fertilization. In addition, hyaluronan that has been localized in the pig oviduct both in the intraluminal fluid and on the surface of the lining epithelium of the preovulatory sperm reservoir, has proven beneficial during in vitro fertilization and embryo culture, thus indicating that it has a role in vivo. This study monitored the immunolocalization, protein determination and gene expression of the major cell surface hyaluronan receptor CD44 in the epithelial lining of the pig oviduct during selected stages of standing oestrus, in relation to spontaneous ovulation. The CD44 immunostaining in the lining epithelium was localized to the surface membrane and the supranuclear domain of mainly the secretory cells, particularly in the sperm reservoir of both treatment (inseminated) and control (non-inseminated) specimens. Up to four hyaluronan-binding protein (HABP) bands (60, 90, 100 and 200 kDa) were detected in the tubal epithelium, and the 200 kDa band was determined as CD44 by immunoblotting. The expression of CD44 mRNA was higher before than after ovulation (P < 0.05), most conspicuously in the uterotubal junction (UTJ). In addition, CD44 expression in the preovulatory UTJ and the ampullary-isthmic junction (AIJ) of control animals was higher than in those that were inseminated (P < 0.05 and P < 0.01 for UTJ and AIJ, respectively). The results demonstrate for the first time that the specific hyaluronan receptor CD44 is expressed by the oviduct epithelial cells during spontaneous oestrus, and is particularly abundant in the sperm reservoir before ovulation. Presence of spermatozoa in this segment seemed to downregulate the receptor. The variation in the expression of CD44 in relation to spontaneous ovulation and the presence of spermatozoa indicate that the hyaluronan CD44-signalling pathway may play a role in oviduct function during sperm storage and fertilization in pigs.     

DNA methylation pattern in human zygotes and developing embryos

We report on observations of the global methylation/demethylation pattern of both pronuclei in human zygotes and in early embryos up to the blastocyst stage. Our results demonstrate that in about half of the zygotes examined the paternal chromatin was less methylated than the maternal chromatin. In the other half, both pronuclei exhibited the same intensity of labeling. The nuclei in developing embryos were intensively labeled for up to the four-cell stage; thereafter, a decline of labeling intensity was detected. Remethylation in some nuclei starts in late morulae. Surprisingly, and unlike the mouse, at the blastocyst stage the inner cell mass showed a weaker intensity of labeling than the trophectodermal cells.     

Expression and localization of inhibin alpha,inhibin/activin betaA and betaB and the activin type II and inhibin beta-glycan receptors in the developing human testis

Inhibins and activins have roles in the regulation of cell proliferation and differentiation in a variety of tissues. This study investigated the distribution of the three inhibin/activin subunits (alpha, betaA and betaB) and their receptors in the human testis between week 13 and week 19 of gestation using RT-PCR and immunohistochemistry. mRNA for all three subunits and for the activin type II receptors ActRIIA and ActRIIB was detected at all stages of gestation examined. Sertoli cells showed intense immunostaining for the alpha subunit and some staining for the betaB subunit, whereas only the betaB subunit was detected in gonocytes. No betaA subunit staining was detected within the tubules. All three subunits were localized to interstitial Leydig cells. Cells of the rete testis and the epididymal epithelium also showed immunostaining for betaB; however, staining for the other subunits was weak or absent. Peritubular cells showed intense immunostaining for the beta-glycan inhibin receptor, which was also localized to interstitial cells, but was not detected within the tubular compartment, rete testis or epididymal epithelium. ActRIIA was detected in gonocytes and in interstitial cells; ActRIIB was distributed widely. These data indicate that fetal Leydig and Sertoli cells have the potential to produce both activins and inhibins, whereas gonocytes may produce only activin B. The distribution of activin and inhibin receptors implies that the intratubular compartment and developing duct system are sites of action of activin B but not inhibin at this stage of development, whereas both activins and inhibins may be involved in the development and function of the peritubular and interstitial cells.     

Differential hormonal regulation of estrogen receptors ERalpha and ERbeta and androgen receptor expression in rat efferent ductules

Estrogen receptors, in addition to the androgen receptor (AR), are expressed at high levels in efferent ductules of the male reproductive tract and it is now well recognized that estrogen receptor (ER) alpha is required for the maintenance of normal structure and function of the ductules. However, little is known regarding the hormonal regulation of the receptors themselves in the male. In the present study, efferent ductule ligation and castration, followed by replacement with testosterone, dihydro-testosterone (DHT) or estradiol was used to investigate the relative importance of circulating and luminal sources of steroid for the modulation of ERalpha, ERbeta and AR in rat efferent ductules. Uni- or bilateral castration and ligation did not affect the expression of ERalpha and ERbeta, but bilateral castration caused down-regulation of AR. Replacement with DHT and testosterone alone or in combination with estradiol caused the recovery of AR expression to control levels. A slight recovery of AR was also observed after estrogen replacement. ERalpha expression was decreased to nearly undetectable levels after estrogen replacement. On the other hand, ERbeta did not show evident effects following any of the treatments, suggesting a constitutive expression of this receptor. This differential modulation of the steroid hormone receptors highlights the importance of maintaining a physiological androgen-estrogen balance to regulate the structure and function of efferent ductules in the male.     

Expression and regulation of oestrogen receptors in the human corpus luteum

The molecular mechanisms underlying the control of corpus luteum lifespan in women are not fully understood. Oestradiol has various luteolytic, or luteotrophic, functions in some species, and as it is synthesised within the human corpus luteum, it is an excellent candidate molecule to be a paracrine regulator of luteal function. This study aimed to comprehensively investigate the expression, regulation and effects of oestrogen receptors (ER) in human luteal cells. Genomic oestrogen receptors ERalpha, ERbeta1 and ERbeta2 were immunolocalised in human corpora lutea from throughout the luteal phase. mRNA expression was investigated throughout the luteal phase and after luteal rescue with exogenous human chorionic gonadotrophin (hCG). The regulation of ER expression and oestradiol action was investigated in cultures of luteinised granulosa cells. ER subtypes ERbeta1 and ERbeta2 were localised throughout the luteal phase to steroidogenic cells in the human corpus luteum and cells of the surrounding stroma. Unlike follicular granulosa cells, steroidogenic cells in the corpus luteum showed minimal ERalpha immunostaining. The presence of endothelial cells in the granulosa cell layer with ERbeta1 and ERbeta2 positive nuclei was noted. ERbeta1 and ERbeta2 were differentially regulated across the luteal phase with ERbeta1 maximally expressed in the mid-luteal phase, while ERbeta2 expression was maximal in the early luteal phase. In vivo and in vitro, hCG had no long-term effect on ER expression, although in vitro hCG and oestradiol acutely down-regulated ERs. Treatment with oestradiol in vitro down-regulated 11beta-hydroxysteroid dehydrogenase type 1 and inhibin betaA subunit confirming a functional oestradiol response. These data highlight functional and differentially regulated oestradiol reception in human luteal cells.     

Quantitative analysis of human ras localization and function in the fission yeast Schizosaccharomyces pombe

Ras signalling is central to fundamental and diverse cellular processes. In higher eukaryotes ras signalling is highly complex, involving multiple isoforms, regulatory proteins and effectors. As a consequence, the study of ras activity in mammalian systems presents a number of technical challenges. The model organism Schizosaccharomyces pombe has previously proved a key system for the study of human signalling components and provides an ideal model for the study of ras, as it contains just one ras protein (Ras1p), which is non‐essential and controls a number of downstream processes. Here we present data demonstrating the quantitative analysis of three distinct Ras1‐related signalling outputs, utilizing the three most abundant human ras isoforms, H‐Ras, N‐Ras and K‐Ras4B, in Sz. pombe. Further, we have characterized the localization of these three human ras isoforms in Sz. pombe, utilizing quantitative image analysis techniques. These data indicate that all three human ras isoforms are functional in fission yeast, displaying differing localization patterns which correlate strongly with function in the regulation of pheromone response and cell shape. These data demonstrate that such yeast strains could provide powerful tools for the investigation of ras biology, and potentially in the development of cancer therapies. Copyright © 2013 John Wiley & Sons, Ltd.     

The effect of human chorionic gonadotrophin on the expression of progesterone receptors in human luteal cells in vivo and in vitro

The human corpus luteum expresses genomic progesterone receptors (PRs) suggesting that progesterone may have an autocrine or paracrine role in luteal function. We hypothesised that the reduction in luteal PR reported in the late-luteal phase augmented progesterone withdrawal and had a role in luteolysis. We therefore tested the hypothesis that luteal rescue with human chorionic gonadotrophin (hCG) would maintain PR expression. PR was immunolocalised to different cell types in human corpora lutea (n = 35) from different stages of the luteal phase and after luteal rescue with exogenous hCG. There was no change in the staining intensity of theca-lutein cell or stromal cell PR throughout the luteal phase or after luteal rescue. In the late-luteal phase, granulosa-lutein cell PR immunostaining was reduced (P < 0.05) but the trend to reduction was also seen after luteal rescue with hCG (P = 0.055). To further investigate the effect of hCG on granulosa-lutein cell PR expression, an in vitro model system of cultured human luteinised granulosa cells was studied. Cells were cultured for 12-13 days exposed to different patterns of hCG and aminoglutethamide to manipulate progesterone secretion (P < 0.0001). Expression of PR A/B and PR B isoforms was examined by quantitative real-time RT-PCR. PR A/B mRNA was lower (P < 0.05) after 11-13 days of culture than after 7 days of culture. This reduction could not be prevented by hCG in the presence (P < 0.05) or absence (P < 0.05) of stimulated progesterone secretion. The expression of PR B mRNA showed a similar pattern (P = 0.054). Simulated early pregnancy in vivo and hCG treatment of luteinised granulosa cells in vitro did not appear to prevent the down-regulation of PR seen during luteolysis.