Real-time quantitative monitoring of hiPSC-based model of macular degeneration on Electric Cell-substrate Impedance Sensing microelectrodes.

Age-related macular degeneration (AMD) is the leading cause of blindness in the developed world. Humanized disease models are required to develop new therapies for currently incurable forms of AMD. In this work, a tissue-on-a-chip approach was developed through combining human induced pluripotent stem cells, Electric Cell-substrate Impedance Sensing (ECIS) and reproducible electrical wounding assays to model and quantitatively study AMD. Retinal Pigment Epithelium (RPE) cells generated from a patient with an inherited macular degeneration and from an unaffected sibling were used to test the model platform on which a reproducible electrical wounding assay was conducted to model RPE damage. First, a robust and reproducible real-time quantitative monitoring over a 25-day period demonstrated the establishment and maturation of RPE layers on the microelectrode arrays. A spatially controlled RPE layer damage that mimicked cell loss in AMD disease was then initiated. Post recovery, significant differences (P < 0.01) in migration rates were found between case (8.6 ± 0.46 µm/h) and control cell lines (10.69 ± 0.21 µm/h). Quantitative data analysis suggested this was achieved due to lower cell-substrate adhesion in the control cell line. The ECIS cell-substrate adhesion parameter (α) was found to be 7.8 ± 0.28 Ω(1/2)cm for the case cell line and 6.5 ± 0.15 Ω(1/2)cm for the control. These findings were confirmed using cell adhesion biochemical assays. The developed disease model-on-a-chip is a powerful platform for translational studies with considerable potential to investigate novel therapies by enabling real-time, quantitative and reproducible patient-specific RPE cell repair studies.

Changes in cumulus-oocyte complexes of pregnant and non-pregnant camels (Camelus dromedarius) during maturation in vitro.

The aim of the present study was to examine the cumulus morphology and the oocyte chromatin quality of camel cumulus-oocyte complexes (COCs) at the time of recovery, and to monitor changes in oocyte chromatin configuration and apoptosis in cumulus cells from camel COCs during in vitro maturation (IVM) (0, 12, 24, 32, 36, 42, and 48 p.IVM) depending on pregnancy of donors. A total of 1023 COCs were isolated from sliced ovaries after slaughtering of 47 pregnant and 43 non-pregnant camels in an abattoir. The mean number of COCs per donor was 10.3 in pregnant and 12.5 in non-pregnant donors. The cumulus morphology of COCs was independent of the type of donor and was divided in COCs with compact (26.9 and 28%), dispersed (39.3 and 46%), corona radiata cumulus investment (27.9 and 21.7%) and without cumulus (6 and 4.2%), respectively for pregnant and non-pregnant donors. The highest proportion of COCs exhibited dispersed cumulus (P<0.05). Oocytes with meiotic stages of diplotene >50% were found only in compact (55 and 56.5%) and in dispersed COCs (58.4 and 60%), respectively for pregnant and non-pregnant donors. During IVM (0-48h) the first significant onset of specific meiotic stages were different in oocytes from pregnant donors: metaphase 1 (24-32h), metaphase 2 (36-42h), versus oocytes from non-pregnant donors: metaphase 1 (24h), metaphase 2 (32-48h) (P<0.05). The level of apoptotic cells in cumuli of matured COCs increased during IVM and was higher in matured COCs from non-pregnant donors for each time point during IVM (P<0.01). Camel oocytes meiosis during IVM is accompanied by a drastic increase of apoptosis in the surrounding cumulus cells 0-32 and 0-24h during IVM, respectively for pregnant and non-pregnant donors. The oocytes of pregnant camels require 36h of maturation to reach levels of >50% metaphase 2 stage in comparison to oocytes from non-pregnant donors where 32h are sufficient. The earlier onset of apoptosis in the COCs derived from non-pregnant donors possibly determines the faster progression of the oocytes through the final stages of meiosis.

Effect of ascorbic acid on health and morphology of bovine preantral follicles during long-term culture.

During ovarian folliculogenesis, ascorbic acid may be involved in collagen biosynthesis, steroidogenesis and apoptosis. The aims of this study were to determine the effects of ascorbic acid on bovine follicle development in vitro. Preantral follicles were cultured for 12 days in serum-free medium containing ascorbic acid (50 microg ml(-1)). Half of the medium was replaced every 2 days, and conditioned medium was analysed for oestradiol and matrix metalloproteinase 2 (MMP-2) and MMP-9 secretion. On day 12, cell death was assessed by TdT-mediated dUTP-biotin nick end labelling (TUNEL). In the absence of serum, there was significant (P < 0.05) follicle growth and oestradiol secretion over the 12 day culture period. Ascorbic acid had no effect on these parameters. The addition of serum from day 0 stimulated follicle growth (P < 0.05), but compromised follicle integrity. By day 12 of culture, a higher proportion of follicles remained intact in the presence of ascorbic acid in serum-free conditions (P < 0.05), and significantly (P < 0.01) less granulosa and theca cell death was observed in these follicles than in control follicles. Moreover, ascorbic acid significantly (P < 0.05) increased production of MMP-9, an enzyme involved in basement membrane remodelling. In conclusion, this culture system was capable of supporting follicle differentiation over the 12 day culture period. Furthermore, ascorbic acid maintains bovine follicle health and basement membrane remodelling in vitro.

Combined effect of follicle-follicle interactions and declining follicle-stimulating hormone on murine follicle health in vitro.

Follicle selection occurs throughout an adult female's reproductive life, with selected, dominant follicle(s) developing to the preovulatory stage whereas the remaining, subordinate follicles within the growing cohort instead undergo atresia and die. To date, most research into follicle dominance has concentrated on its endocrine regulation, although it seems likely that intraovarian mechanisms are also involved in its regulation. We demonstrate here that the response of singly cultured murine follicles to declining concentrations of FSH depends on their developmental stage, with follicles at an earlier stage of development being much more susceptible than mature follicles to a lowering of FSH levels. We then extrapolate this information to follicle cocultures, in which a large dominant follicle was grown with a small subordinate follicle in a manner that maintained a dominant/subordinate relationship, with follicle health assessed by a terminal transferase-mediated 2'-deoxyuracil 5'-triphosphate nick end-labeled reaction on whole-follicle mounts. Our investigations show a combined negative effect of coculture and FSH withdrawal on small subordinate follicles, such that subordinate follicles cocultured with dominant follicles and subjected to a lowering of FSH levels during the culture period exhibit a greatly increased incidence of apoptosis in the granulosa cells (750% increase) compared with that exhibited by the dominant follicles (97% increase). We suggest that a similar interaction between endocrine and intraovarian factors regulates follicular dominance in vivo, such that dominant follicles, in addition to bringing about a fall in FSH levels via the hypothalamic-pituitary axis, exert local, direct effects on subordinate follicles, with both of these influences combining to induce atresia in subordinate follicles.

Developmental regulation of effect of epidermal growth factor on porcine oocyte-cumulus cell complexes: nuclear maturation, expansion, and F-actin remodeling.

Epidermal growth factor (EGF) efficiently stimulates expansion of mouse and rat oocyte-cumulus complexes (OCC). Contradictory data have been published by several laboratories about the ability of EGF to stimulate expansion of porcine OCC. We assumed that these contradictions may have resulted from heterogeneous conditions used for isolation, culture, and assessment of OCC. The present experiments were designed to test the hypothesis that porcine OCC acquire the ability to synthesize hyaluronic acid (HA) and undergo expansion following EGF-stimulation gradually during the growth of follicles. For this reason, we isolated OCC from follicles of different sizes and assessed quantity of produced HA and proportions of expanding OCC after stimulation by EGF. In addition, we assessed in those OCC changes in morphology of cumulus cells and assembly of F-actin microfilaments, which are necessary for expansion to occur. Finally, nuclear maturation of EGF-stimulated OCC was assessed and its relationship with occurrence of expansion was evaluated. In all experiments, OCC stimulated with FSH were used as positive controls. The results showed that EGF did not stimulate production of HA, rearrangement of F-actin and expansion in OCC isolated from small follicles (<4 mm in diameter). OCC isolated from large preovulatory follicles (6-7 mm in diameter and PMSG-stimulated follicles) underwent efficient expansion when stimulated by EGF (93% and 100%, respectively). EGF dramatically stimulated total production of HA in these OCC and its retention in extracellular matrix of the expanding cumulus. Cumulus cells of the large OCC underwent essential changes of their morphology and extensive rearrangement of F-actin microfilaments following stimulation with EGF. Interestingly, EGF enhanced nuclear maturation of OCC isolated from both small and large follicles, which suggest diversity of signaling pathways controlling maturation and expansion. FSH caused cumulus expansion, F-actin remodeling, and enhancement of oocyte nuclear maturation in OCC originated from both small and large follicles. We conclude that EGF can stimulate expansion of porcine OCC in vitro; however, only of those isolated from large follicles. This indicates that EGF may have a physiological role in regulation of porcine cumulus expansion in preovulatory follicles, presumably as a mediator of signals elicited by the LH surge.

Serum-dependent phosphorylation of human MAP4 at Ser696 in cultured mammalian cells.

In the previous paper (Ookata et al., (1997) Biochemistry, 36: 249-259), we identified two mitotic cdc2 kinase phosphorylation sites (Ser696 and Ser787) in the proline-rich region of human MAP4. One (Ser696) of them was also phosphorylated during interphase. A protein kinase responsible for interphase phosphorylation of Ser696 could necessarily be distinct from cdc2/cyclin B kinase. To get insights into a physiological role for Ser696 phosphorylation, we searched for a Ser696 kinase and for cellular conditions under which Ser696 is dephosphorylated. Because Ser696 conforms to the MAP kinase phosphorylation consensus motif (PXSP), MAP kinase was tested as a possible kinase phosphorylating Ser696. MAP kinase, in fact, did phosphorylate Ser696 in MTB3, the carboxy-terminal half of human MAP4 in vitro. Phosphorylation of Ser696 in HeLa cell extract was suppressed by a MAP kinase inhibitor, DBTM-0004. Also consistent with the notion that Ser696 is a MAP kinase site were the fact that serum-starvation induced dephosphorylation of Ser696 in HeLa cells, TIG-3 and MRC-5-30 human fibroblasts, while readdition of serum recovered Ser696 phosphorylation, albeit after a surprisingly long interval. Thus, phosphorylation of Ser696 of MAP4, most likely carried out by MAP kinase, may play a role in modulation of MAP4 activity in proliferating versus quiescent cells.

Effects of follicle-stimulating hormone, bovine somatotrophin and okadaic acid on cumulus expansion and nuclear maturation of blue fox (Alopex lagopus) oocytes in vitro.

The meiotic competence and meiosis resumption of Blue fox (Alopex lagopus) oocytes from anoestrous animals were followed. Oocyte-cumulus complexes (OCC) were cultured in modified TC 199 medium with or without FSH, recombinant bovine somatotrophin (bST) and okadaic acid (OA). The results showed that oocytes less than 100 microns in diameter did not achieve germinal vesicle breakdown (GFBD) by 72 h of culture, which indicates their meiotic incompetence. Oocytes larger than 100 microns in diameter underwent GVBD after 48 h of culture (27%) and reached metaphase II (MII) after 72 and 96 h (20% and 27%) in control medium. Both bST and OA accelerated resumption of meiosis (bST: 55% GVBD and 42% MII after 48 h; OA: 66% GVBD after 18 h). In contrast, FSH significantly reduced meiosis resumption (only 3% GVBD and MII after 72 h) and induced changes in the shape of cumulus granulosa (CG) cells and F-actin assembly typical for cumulus expansion. However, the innermost layers of CG cells (corona radiata) remained connected with the oocyte via gap junctions until the end of culture. Cumuli of oocytes cultured in control, bST-supplemented or OA-supplemented medium did not expand (changes in cell shape and F-actin redistribution did not occur). Moreover, especially in media with bST and OA an increased detachment and rapid disconnection of their gap junctions with the oocyte were observed. These results suggest that under in vitro conditions FSH stimulates expansion of the CG cells and the attached membrana granulosa cells but in contrast it secures heterologous gap junctions between cytoplasmic processes of the corona radiata cells and oolemma during 3 days of culture. Thus, in agreement with the in vivo situation in which Canidae oocytes are ovulated in the GV stage, the cumulus, mainly corona radiata cells, controls resumption of meiosis in Blue fox oocytes under in vitro conditions also.

Activation of bovine and baboon primordial follicles in vitro.

Mammalian ovaries contain a large pool of non-growing, primordial follicles. The ability to initiate growth of this pool of resting follicles in vitro and to maintain follicular growth to a stage when the oocyte could be matured and fertilized would increase the reproductive potential of valuable domestic animals, endangered species and infertile women. This paper summarizes our progress to date in activating primordial follicles of cattle and baboons. Pieces of ovarian cortex, rich in primordial follicles, were obtained from fetal bovine and baboon ovaries during late gestation. Pieces were maintained in organ culture in serum-free medium containing ITS+ (insulin-transferrin-selenium-linoleic acid-BSA) for up to 20 days and at various times during culture some pieces were fixed for histological morphometry. As early as 2 days of culture, the number of primordial follicles had decreased by 88% or 55%, whereas the number of primary follicles had increased 2.5- or 5-fold, compared to tissue freshly isolated from bovine or baboon ovaries, respectively (P < 0.01). In baboon cortical pieces a small number of secondary follicles developed during a 20-day culture period. The development of primary and secondary follicles was accompanied by an increase in diameter of both the granulosa cell layer and the oocyte. The addition of FSH (1, 10, or 100 ng/ml) had no effect on the development of follicles in bovine cortical pieces after 7 or 14 days of culture, relative to control cultures without FSH. These results show that a high percentage of primordial follicles from cattle and baboons can be activated to grow in serum-free medium in the absence of gonadotropins. Conditions that will support further growth in vitro of follicles from these species remain to be elucidated. The culture system we have developed could be used to develop such conditions and to explore factors that regulate the movement of primordial follicles into the pool of growing follicles.

Initiation of growth of baboon primordial follicles in vitro.

Factors that cause some primordial follicles to enter the growth phase while the others remain quiescent are unknown. The hypothesis was tested that primate primordial follicles can survive and initiate growth in vitro in serum-free medium. Superficial pieces of ovarian cortex, containing mostly primordial follicles, were obtained from baboon fetuses during late gestation and cultured for 0, 2, 4, 7, 10 or 20 days in Waymouth MB 752/1 medium supplemented with insulin, transferrin, selenium, linoleic acid, and bovine serum albumin (ITS +). Histological examination of cortical pieces revealed that after 2 and 4 days in culture, the total number of primordial follicles had decreased by 55 and 76% (P < 0.01) respectively, relative to day 0 of culture. This was associated with a sustained, 5- to 8-fold increase in total primary follicles (P < 0.01) beginning on day 2 of culture. There was also a gradual increase in the total number of early secondary and secondary follicles. The average diameter of follicles and oocytes increased gradually throughout culture for all follicular categories (P < 0.01), except secondary follicles and oocytes. Immunohistochemical localization of proliferating cell nuclear antigen (PCNA), a marker for cell proliferation and growth, showed that PCNA was generally absent in primordial follicles on day 0, but was observed after 2 or 4 days in culture in both granulosa cells and oocytes of most growing follicles. Comparison of cortical pieces cultured for 10 or 20 days with ITS + versus 10% fetal bovine serum (FBS) showed a more pronounced decrease in the numbers of primordial follicles and more primary, early secondary and secondary follicles in ITS + compared to FBS-treated cortical pieces (P < 0.01 at 20 days). These results show that primordial follicles from non-human primates can survive and develop to the secondary stage in vitro in serum-free conditions.

Establishment of the block against sperm penetration in parthenogenetically activated bovine oocytes matured in vitro.

The ability of a single electric pulse to mimic a block against sperm penetration in bovine oocytes matured in vitro was investigated. Confocal laser scanning microscopy detected a global loss of spots, presumed to be cortical granules, stained with Lens culinaris agglutinin, in pulsed oocytes. Transmission electron microscopy revealed that cortical granule exocytosis occurred within 1 min of stimulation and the number of remaining cortical granules was significantly reduced in all pulsed oocytes. The ability of pulsed oocytes to undergo fertilization in vitro was also affected, as only 31% of the pulsed oocytes were penetrated compared with 87% in the control group. Since incidences of penetration in pulsed oocytes (31%), and of polyspermy in control oocytes (18%) did not differ and were highly correlated (P = 0.009) among trials (n = 15), the induced block is considered to be comparable with the natural block triggered by a spermatozoon. The increased resistance of the zona pellucida to pronase E observed in pulsed oocytes suggests that the induced block depends, at least partly, on modifications of zona pellucida glycoproteins. Finally, the majority (66%) of pulsed, penetrated oocytes did not form male pronuclei, probably as a consequence of asynchrony between the formation of female pronucleus and sperm penetration. The reduced ability of the cytoplasm to induce the formation of a male pronucleus was accompanied by a fall in histone H1 kinase activity to basal values by 3 h after stimulation. These results demonstrate that a single electric pulse can induce a block against sperm penetration similar to that of the spermatozoon itself.

Two types of apoptotic cell death of rat central nervous system-derived neuroblastoma B50 and B104 cells: apoptosis induced during proliferation and after differentiation.

We describe here two types of apoptotic cell death observed in the rat CNS-derived neuroblastoma B50 and B104 cells. One type was induced by dibutyryl cyclic AMP (DBcAMP) after differentiation, and the other was induced by treatment of proliferating cells with cycloheximide. When B50 and B104 cells were treated with 1 mM DBcAMP in the presence of 0.5% fetal calf serum, they began to extend neurites within 12 h and differentiated into neurons at 24 h, as reported previously. However, further cultivation with DBcAMP for up to 72 h led to flotation and, finally, death. Death was by apoptosis as shown by chromatin condensation and DNA fragmentation. Addition of a protein kinase A inhibitor or removal of DBcAMP after differentiation suppressed apoptosis, indicating the involvement of cyclic AMP and protein kinase A in apoptotic cell death. Cell death was also induced in proliferating cells without neurite outgrowth by treatment with cycloheximide. The death was also judged to be by apoptosis based on chromatin condensation and apoptotic body formation, although DNA fragmentation into small sizes was not detected. Both types of cell death showed similar responses to inhibitors for protein kinases and protein phosphatases.

Initiation in vitro of growth of bovine primordial follicles.

Factors that control the onset of primordial follicle growth are unknown. We have tested the hypothesis that primordial follicles from fetal calves can survive and initiate growth in vitro in serum-free conditions. Superficial pieces of ovarian cortex, containing mostly primordial follicles, were isolated from bovine fetuses 6-8 mo old and cultured for 0, 2, 4, or 7 days in Waymouth MB 752/1 medium supplemented with insulin, transferrin, selenium, linoleic acid, and BSA (ITS+). Histological examination of cortical pieces after 2, 4, and 7 days in culture showed that the number of healthy primordial follicles had decreased by 88%, 90%, and 94%, respectively (p < 0.01), whereas the number of healthy primary follicles had increased to 260%, 209%, and 197%, respectively, of the number present on Day 0 (p < 0.05). The percentage of follicles that showed signs of atresia did not change with time in culture and was about 28% and 50% for primordial and primary follicles, respectively. After 7 days in culture, the mean diameter of the few remaining healthy primordial follicles was 1.2 times the average diameter of primordial follicles present on Day 0 (p < 0.01). In contrast, after 2, 4, and 7 days in culture, primary follicles were 1.2, 1.3, and 1.4 times larger in diameter, respectively, relative to Day 0 (p < 0.01). There was little change in the diameter of oocytes in primordial follicles during culture, whereas in primary follicles an increase in oocyte diameter became apparent after 4 and 7 days (1.1 and 1.2 times, respectively, p < 0.01). That follicle growth was initiated in vitro was further confirmed by immunolocalization of proliferating cell nuclear antigen (PCNA), a marker for cell growth and proliferation, in cultured and freshly isolated pieces of ovarian cortex. In freshly isolated tissue, PCNA staining was absent from pre-granulosa cells and oocytes of the quiescent primordial follicles but was intense in granulosa cells and oocytes of the few growing primary follicles. After 2, 4, and 7 days in culture, PCNA was expressed intensely in the oocyte and many granulosa cells of newly activated primary follicles. These results demonstrate that bovine primordial follicles can enter the growth phase in vitro and that PCNA expression by granulosa cells and oocytes is closely associated with the onset of primordial follicle growth. The fact that a high percentage of primordial follicles initiated growth in vitro suggests that the ovarian stroma exerts inhibitory control over the initiation of primordial follicle growth in vivo. The culture system we describe may provide the means to test this hypothesis and others.

Absence of BoLA class I antigens on bovine spermatozoa.

Forty AI bulls were tested for BoLA class I antigens by means of eight specific polyclonal reagents. By means of immobilization and sperm penetration tests these antigens were not detected on sperm cells. Isoimmunization studies with the use of sperm as antigenic stimuli and insemination of frozen spermatozoa diluted in specific reagents did not prove the presence of BoLA class I antigens on bovine spermatozoa. The cytotoxic tests used in this investigation were not reliable.