Intercellular junctions between the follicle cells and oocytes of Xenopus laevis.
Browne C L,Werner W
The Journal of experimental zoology
During development, the oocytes of Xenopus laevis establish junctional contact with the follicle cells enveloping them. These junctions have alternatively been described as desmosomes and as gap junctions. In this paper the morphology of these junctions has been examined in gonadotropin-stimulated and unstimulated animals at all stages of development. Contact between the oocyte and follicle cell plasma membranes is visible in stage I oocytes as thickenings in the membranes, separated by intercellular spaces of 20nm or greater. By stage III in unstimulated oocytes and stage II in gonadotropin-stimulated oocytes, intermembrane spaces at these junctional contacts are often reduced to 2 to 7 nm in width. These narrow intermembrane spaces persist through early stage IV, with greater frequency of occurrence in oocytes taken from hormonally stimulated animals. The closeness of these junctional contacts, and the permeability of the junctional spaces to intercellular tracer substances, supports the evidence that these are gap junctions.
10.1002/jez.1402300114
Ultrastructure of human preovulatory granulosa cells in follicular fluid aspirates.
Spanel-Borowski K,Sterzik K
Archives of gynecology
The ultrastructure of preovulatory granulosa cells may be distinct in follicles containing competent as opposed to non-competent oocytes. To test this assumption, granulosa cells were looked for in 36 follicular fluid aspirates from 8 patients taking part in an in vitro fertilization and embryo transfer program. Granulosa cells were absent from 16 aspirates and present in 20. Both aspirate types contained oocytes able to develop in culture. Granulosa cells were subdivided into three developmental stages. Stage 1 (5% of aspirates) showed proliferating cells, while stage 2 (60% of aspirates) and 3 (35% of aspirates) cells were in the preluteinization stage. These cells were recognizable by their number of lipid droplets and differentiated according to possession of a rough (stage 2) or smooth (stage 3) endoplasmic reticulum. Luteinization did not occur in these cells. All stages displayed desmosomes, gap junctions, and annular junctions. The structure of Call-Exner bodies and of fibrin deposits were unexpected findings. Our study indicates that there is no correlation between the previously used morphological parameters of granulosa cells and oocyte maturity.
10.1007/bf00207708
Expression of RNA from developmentally important genes in preimplantation bovine embryos produced in TCM supplemented with BSA.
Wrenzycki C,Herrmann D,Carnwath J W,Niemann H
Journal of reproduction and fertility
This study investigated the effects of a semi-defined culture system on the temporal pattern of expression of RNA from genes involved in compaction and cavitation: gap junction protein connexin43 (Cx43), desmosomal glycoproteins desmoglein 1 (Dg 1), desmocollins I, II and III (Dc I, Dc II, Dc III), desmosomal protein plakophilin (Plako); metabolism glucosetransporter-1 (Glut-1); RNA processing poly(A)polymerase (PolyA); heat shock protein 70.1 (HSP); and trophoblastic function trophoblast protein (TP) in bovine oocytes and embryos generated in vitro using TCM199 supplemented with BSA as the culture medium. Morulae and blastocysts derived in vivo were collected from superovulated heifers and also used for this study. Poly(A)+ RNA was extracted from pools of 20-50 oocytes or embryos, analysed by reverse transcription-polymerase chain reaction and the amplified fragments were verified by sequencing. Assays were repeated at least three times for each developmental stage and provided consistent results in all replicates. In bovine embryos produced in vitro, mRNA encoding Cx43 was detectable up to the morula stage, whereas blastocysts and hatched blastocysts did not express this gene. No transcripts were found for Dg 1 and Dc I throughout the tested preimplantation stages. Dc II and Dc III transcripts were found from 2-4-cell embryos up to the hatched blastocyst stage. mRNA encoding Plako was detected in immature and mature oocytes and zygotes, while no transcripts were seen in 2-4-cell and 8-16-cell embryos. The gene was expressed again from the morulae to the hatched blastocyst stage. Oocytes and bovine embryos produced in vitro showed transcripts for Glut-1, PolyA and HSP throughout preimplantation development up to the hatched blastocyst stage. The gene encoding TP was transcribed only in blastocysts and hatched blastocysts. Morulae and blastocysts produced in vivo showed the same expression as their in vitro counterparts, with one exception: the in vivo embryos transcribed Cx43. The results of this study reveal for the first time the transcriptional pattern of a set of 'marker' genes involved in various processes in early bovine embryonic development. Transferable morulae and blastocysts produced in vitro expressed most genes similar to their in vivo counterparts. These data contribute to the molecular characterization of this widely used in vitro culture system for bovine embryos and provide a major advance towards production of 'physiologically normal' embryos.
10.1530/jrf.0.1120387
Developmental, qualitative, and ultrastructural differences between ovine and bovine embryos produced in vivo or in vitro.
Rizos Dimitrios,Fair Trudee,Papadopoulos Serafeim,Boland Maurice P,Lonergan Patrick
Molecular reproduction and development
The objective of this study was to compare bovine and ovine oocytes in terms of (1) developmental rates following maturation, fertilization, and culture in vitro, (2) the quality of blastocysts produced in vitro, assessed in terms of their ability to undergo cryopreservation, and (3) the ultrastructural morphology of these blastocysts. In vitro blastocysts were produced following oocyte maturation/fertilization and culture of presumptive zygotes in synthetic oviduct fluid. In vivo blastocysts were used as a control from both species. In Experiment 1, the cleavage rate of bovine oocytes was significantly higher than that of ovine oocytes (78.3% vs. 58.0%, respectively, P < 0.001). The overall blastocyst yield was similar for both species (28.7% vs. 29.0%). However, when corrected for cleavage rate, significantly more ovine oocytes reached the blastocyst stage at all time-points (36.6% vs. 50.0% on day 8, for bovine and ovine, respectively, P < 0.001). Following vitrification, there was no difference in survival between in vivo produced bovine and ovine blastocysts (72 hr: 85.7% vs. 75.0%). However, IVP ovine blastocysts survived at significantly higher rates than IVP bovine blastocysts at all time points (72 hr: 47.4% vs. 18.1%, P < 0.001). At the ultrastructural level, compared with their in vivo counterparts, IVP blastocysts were characterized by a lack of desmosomal junctions, a reduction in the microvilli population, an increase in the average number of lipid droplets and increased debris in the perivitelline space and intercellular cavities. These differences were more marked in bovine IVP blastocysts, which also displayed electron-lucent mitochondria and large intercellular cavities. These observations may in part explain the species differences observed in terms of cryotolerance. In conclusion, the quality of ovine blastocysts was significantly higher than their bovine counterparts produced under identical in vitro conditions suggesting inherent species differences between these two groups affecting embryo quality.
10.1002/mrd.10138
Developmental potential of bovine oocytes cultured in different maturation and culture conditions.
Sagirkaya Hakan,Misirlioglu Muge,Kaya Abdullah,First Neal L,Parrish John J,Memili Erdogan
Animal reproduction science
Diverse groups of chemicals in culture media are needed for successful bovine oocyte maturation and embryo development during which dramatic cytoplasmic and nuclear reprogramming events take place. In vitro embryo production (IVP) procedures frequently include supplements such as serum and/or co-culture with various types of somatic cells. However, the presence of undefined serum in culture media introduces a variation from batch to batch, increases viral or prion contamination risk, and leads to problems during fetal development. The aim of the present study was to investigate the possibility of using chemically defined-synthetic serum substitute (SSS) in place of fetal calf serum (FCS) during maturation and long-term culture to stimulate in vitro maturation (IVM), fertilization (IVF) and subsequent embryo development. In Experiment I, the effect of the protein source on in vitro maturation was tested by maturing oocytes in culture media supplemented with 10% FCS (Control Group), 10% SSS (Group I) and 10% SSS+10 ng/ml epidermal growth factor (EGF) (Group II). In Experiment II, effects of SSS on both oocyte maturation and embryo development during in vitro culture (IVC) were tested by maturing oocytes in media supplemented with 10% FCS (FCS Group) or 10% SSS+10 ng/ml EGF (SSS Group), followed by IVF and IVC in SOF media supplemented with 10% FCS and 10% SSS on day 4 for FCS and SSS Groups, respectively. Even though rates for cleavage and development to blastocyst stage were not different, blastocyst cell numbers were higher in Group II containing SSS and EGF. The SSS supplementation group had higher apoptotic nuclei as compared to the FCS Group in Experiment II. Transcripts for heat shock protein 70 (Hsp70), interferon tau (IF-tau), DNA methyltransferase 3a (Dnmt3a), desmosomal glycoprotein desmocollin III (DcIII) and insulin-like growth factor II receptor (Igf-2r) were altered in different culture conditions in Experiment I. However, only glucose transporter-1 (Glut-1) mRNA was different in the SSS and FCS Groups in the second experiment. In summary, SSS and EGF in maturation medium and replacement of FCS with SSS alone in culture medium on day 4 of IVC support oocyte maturation and embryo development in vitro. However, significance of culture condition induced changes on the genome-wide abundance of messenger ribonucleic acid and the significance of the apoptotic nuclei during fetal development still remain to be determined.
10.1016/j.anireprosci.2006.09.016
Characterization and significance of adhesion and junction-related proteins in mouse ovarian follicles.
Mora Jocelyn M,Fenwick Mark A,Castle Laura,Baithun Marianne,Ryder Timothy A,Mobberley Margaret,Carzaniga Raffaella,Franks Stephen,Hardy Kate
Biology of reproduction
In the ovary, initiation of follicle growth is marked by cuboidalization of flattened granulosa cells (GCs). The regulation and cell biology of this shape change remains poorly understood. We propose that characterization of intercellular junctions and associated proteins is key to identifying as yet unknown regulators of this important transition. As GCs are conventionally described as epithelial cells, this study used mouse ovaries and isolated follicles to investigate epithelial junctional complexes (tight junctions [TJ], adherens junctions [AJ], and desmosomes) and associated molecules, as well as classic epithelial markers, by quantitative PCR and immunofluorescence. These junctions were further characterized using ultrastructural, calcium depletion and biotin tracer studies. Junctions observed by transmission electron microscopy between GCs and between GCs and oocyte were identified as AJs by expression of N-cadherin and nectin 2 and by the lack of TJ and desmosome-associated proteins. Follicles were also permeable to biotin, confirming a lack of functional TJs. Surprisingly, GCs lacked all epithelial markers analyzed, including E-cadherin, cytokeratin 8, and zonula occludens (ZO)-1alpha+. Furthermore, vimentin was expressed by GCs, suggesting a more mesenchymal phenotype. Under calcium-free conditions, small follicles maintained oocyte-GC contact, confirming the importance of calcium-independent nectin at this stage. However, in primary and multilayered follicles, lack of calcium resulted in loss of contact between GCs and oocyte, showing that nectin alone cannot maintain attachment between these two cell types. Lack of classic markers suggests that GCs are not epithelial. Identification of AJs during GC cuboidalization highlights the importance of AJs in regulating initiation of follicle growth.
10.1095/biolreprod.111.096156
A correlative microscopical analysis of differentiating ovarian follicles of mammals.
Anderson E,Wilkinson R F,Lee G,Meller S
Journal of morphology
The mammalian ovary has been studied by optical microscopy and by scanning and transmission electron microscopy with the purpose of presenting an integrated view of the differentiating mammalian follicle. During follicular development, changes in the granulosa cells are particularly noteworthy and include dramatic modifications in cell shape coincident with antrum formation. The cytoplasmic processes of those granulosa cells immediately surrounding the oocyte, as well as the more peripheral granulosa cells comprising a second and third layer, traverse the zona pellucida, infrequently interdigitate with the microvilli of the egg, and make both desmosomal and gap junction contacts with the oocyte. The zona pellucida is thus distinguished by numerous fenestrations of varying diameters. The membrana limitans (basal lamina) is a bipartite structure composed of (a) a homogenous stratum upon which the peripheral layer of granulosa cells rests, and (b) an outer region of collagen-like fibers. The specific advantages and limitations of the different methodologies utilized to study folliculo-genesis are discussed.
10.1002/jmor.1051560303
Breakdown of starfish ovarian follicle induced by maturation-promoting factor.
Kishimoto T,Usui N,Kanatani H
Developmental biology
Immature starfish oocytes are surrounded by envelopes consisting of follicular cells. These cells adhere to each other and to the oocyte, immobilizing the latter within the ovary. When isolated oocytes in their follicles are treated with 1-methyladenine (1-MeAde), germinal vesicle breakdown (GVBD) and follicular envelope breakdown (FEBD) occur simultaneously. The 1-MeAde acts on the oocyte surface to produce a maturation-promoting factor (MPF) in the cytoplasm, which brings about GVBD. In the present study, MPF was found to induce FEBD as well as GVBD when injected into immature oocytes with their follicles in Asterina pectinifera. Although GVBD was induced by MPF in the presence of cytochalasin D, this drug prevented MPF-induced FEBD, and each follicular cell remained in situ on the surface of the oocyte. However, desmosomes connecting the processes of the follicle cell with the oocyte surface were disrupted following MPF injection even in the presence of cytochalasin D, and the processes became detached from the oocyte. FEBD occurred in these oocytes when cytochalasin D was removed, resulting in the formation of a small follicular clump by microfilament-mediated contraction of the follicle cells. These results show that FEBD is not brought about by the direct action of 1-MeAde but by the action of MPF. Therefore, in starfish, spawning as well as oocyte maturation is directly triggered by MPF produced under the influence of 1-MeAde.
10.1016/0012-1606(84)90113-1
Communications of Oocyte-Granulosa Cells in the Chum Salmon Ovary Detected by Transmission Electron Microscopy: (oocyte/granulosa cell/intercellular junction/salmonid ovary/teleost).
Development, growth & differentiation
The communications between oocytes and granulosa cells in the ovary of the chum salmon, Oncorhynchus keta, were examined in follicles in which the oocytes were at the stage of germinal vesicle migration. Microvilli were seen extending through the radial canals of the egg envelope and terminating in either the subfollicular space or at the surface of granulosa cells. Cytoplasmic processes extending from granulosa cells toward oocytes were also observed; most of the processes appeared to end in the radial canals, but some passed through the canals and terminated as bulbs in slight depressions or indentations of the oolemma. Various types of junctional structures that participated in intimate association between these cells were seen. The granulosa cells were found to be firmly fixed on the surface of the intrafollicular oocyte by means of desmosomes or desmosome-like junctions. It is speculated that intrafollicular oocytes communicate with surrounding granulosa cells directly through gap junctions and indirectly by endocytosis of material released from the granulosa cells.
10.1111/j.1440-169X.1985.00553.x
Electron microscope observations on LH-induced oocyte maturation in Japanese quail (Coturnix coturnix japonica).
Yoshimura Y,Okamoto T,Tamura T
Journal of reproduction and fertility
The aim of this study was to describe the temporal sequence of ultrastructural changes in the boundary between the preovulatory oocyte and its surrounding follicular wall during maturation induced by injection of LH. Female Japanese quail were injected with ovine LH (20 micrograms per bird) 10-12 h before the expected time of ovulation. The largest and second largest follicles were excised before or 1, 2, 4 or 6 h after injection. The oocyte and the surrounding follicular wall were processed for observations using light and electron microscopy. Before injection of LH, cytoplasmic projections of granulosa cells interdigitated with microvilli on the surface of the oocyte and formed spot desmosomes and gap junctions with the oolemma. Two hours after injection of LH, the germinal vesicles in the largest but not in the second largest follicles began to break down and membrane-bound vesicles increased in number and size in the surrounding germinal disc. The junctions between the oocyte surface and the granulosa cell projections started to dissociate and a perivitelline space began to develop, possibly as the result of an accumulation of fluids transported from the capillary sinus in the theca interna. The first maturation spindle was formed 4 h after injection of LH, whereas the first polar body and the second maturation spindle were formed 6 h after LH stimulation. These observations suggest that the dissociation of connections between the oocyte and granulosa cells 2 h after exposure to increased concentration of LH is the first process of oocyte maturation.(ABSTRACT TRUNCATED AT 250 WORDS)
From oogonia to mature oocytes: inactivation of the maternal centrosome in humans.
Sathananthan A Henry,Selvaraj Kamala,Girijashankar M Lakshmi,Ganesh Vijaya,Selvaraj Priya,Trounson Alan O
Microscopy research and technique
The fine structure of human oogonia and growing oocytes has been reviewed in fetal and adult ovaries. Preovulatory maturation and the ultrastructure of stimulated oocytes from the germinal vesicle (GV) stage to metaphase II (MII) stage are also documented. Oogonia have large nuclei, scanty cytoplasm with complex mitochondria. During folliculogenesis, follicle cell processes establish desmosomes and deep gap junctions at the surface of growing oocytes, which are retracted during the final stages of maturation. The zona pellucida is secreted in secondary follicles. Growing oocytes have mitochondria, Golgi, rough endoplasmic reticulum (RER), ribosomes, lysosomes, and lipofuscin bodies, often associated with Balbiani bodies and have nuclei with reticulated nucleoli. Oocytes from antral follicles show numerous surface microvilli and cortical granules (CGs) separated from the oolemma by a band of microfilaments. The CGs are evidently secreted by Golgi membranes. The GV oocytes have peripheral Golgi complexes associated with a single layer of CGs close to the oolemma. They have many lysosomes, and nuclei with dense compact nucleoli. GV breakdown occurs by disorganization of the nuclear envelope and the oocyte enters a transient metaphase I followed by MII, when it is arrested and ovulated. Maturation of oocytes in vitro follows the same pattern of meiosis seen in preovulatory oocytes. The general organization of the human oocyte conforms to that of most other mammals but has some unique features. The MII oocyte has the basic cellular organelles such as mitochondria, smooth endoplasmic reticulum, microfilaments, and microtubules, while Golgi, RER, lysosomes, multivesicular, residual and lipofuscin bodies are very rare. It neither has yolk nor lipid inclusions. Its surface has few microvilli, and 1-3 layers of CGs, aligned beneath the oolemma. Special reference has been made to the reduction and inactivation of the maternal centrosome during oogenesis. The MII spindle, often oriented perpendicular to the oocyte surface, is barrel-shaped, anastral and lacks centrioles. Osmiophilic centrosomes are not demonstrable in human eggs, since the maternal centrosome is nonfunctional. However, oogonia and growing oocytes have typical centrioles, similar to those of somatic cells. The sperm centrosome activates the egg and organizes the sperm aster and mitotic spindles of the embryo, after fertilization.
10.1002/jemt.20299
Oocyte follicle cells association during development of human ovarian follicle. A study by high resolution scanning and transmission electron microscopy.
Motta P M,Makabe S,Naguro T,Correr S
Archives of histology and cytology
Morphodynamics of oocyte follicle cells association during the development of human ovarian follicles were studied by transmission electron microscopy and high resolution scanning electron microscopy including the ODO method. For this study primordial, primary, growing preantral and antral follicles were systematically analysed in a total of 20 adult and fetal (3-8 months and at term) ovaries. In early stages of follicle development (primordial and primary stages) the flattened and/or polyhedral cells, closely associated with the growing oocyte, project an increasing number of microvillous processes. These are in apposition with the oolemma, and form bulbous terminals presenting attachment zones such as zonula adherens, desmosomes and communicating junctions (gap junctions). "Focal contacts" between oolemma, and lateral microvillous extensions of follicle cells were also present. Unusual forms of contact between follicle cell microvilli and oocytes in the early stages of growing primordial and primary follicles were also observed. These consist of long, thin extensions penetrating into the oocyte through deep invaginations of the oolemma. The aid of high resolution SEM of specimens subjected to the ODO method clearly reveals their 3-D arrangement within the ooplasm. They appear as long tortuous microvilli coming very close to the nucleus, and in their course are closely associated with a variety of organelles such as Golgi vesicles, endoplasmic reticulum membranes and nascent forms of smooth endoplasmic reticulum. Using integrated observations by TEM and SEM, there may be as many as 3-5 "intraooplasmic processes" even in only one plane of fracture of an oocyte. Therefore, if the total volume of the oocyte and associated cells is considered, their amounts appear to be higher than previously reported. Thus, they have to be considered as normal devices of deep contact between the ooplasm and associated follicle cell extensions. The presence of such structures within the ooplasm in early developing follicles well coincides with the great increase in volume of the oocyte. Although it is commonly believed that the activation of the growing oocyte may depend on the numerous contacts between the oolemma and follicle cells (mostly via gap junctions), the finding of these additional intraoocytic extensions suggests that they may in someway contribute to the initiation of growth in the human. In fact, these microvilli penetrate deep into the ooplasm, much like a sword in its sheath.(ABSTRACT TRUNCATED AT 400 WORDS)
10.1679/aohc.57.369
Oocyte morphology from primordial to early tertiary follicles of yak.
Yu S J,Yong Y H,Cui Y
Reproduction in domestic animals = Zuchthygiene
The objective of this study was to investigate the developmental morphology of yak oocytes from the primordial follicle to the tertiary follicle. Yak oocytes from resting primordial (n = 6), activated primordial (n = 12), primary (n = 9), secondary (n = 7) and early tertiary (n = 5) follicles were processed and analysed by light and transmission electron microscopy. The resting primordial follicular oocyte was characterized by relatively smooth surface on the oolemma, the accumulation of free and organelle-related smooth (SER) and rough endoplasmic reticulum (RER), round or oval mitochondria, and polyribosomes on the surface of the RER and throughout the ooplasm. The activated primordial follicular oocyte was dominated by numerous coated pits and coated vesicles on the oolemma, and round mitochondria. Up to the secondary follicular stage the oocyte displayed an increase in the number of microvilli, polyribosome, Golgi complexes and mitochondria with distinct cristae. During the secondary follicular stage, formation of the zona pellucida, development of a desmosome-like connection between the oocyte and the granulosa cells, formation of the cortical granules in the oocyte and elongated mitochondria in nearly all oocytes were seen. In the early tertiary follicular oocyte, the perivitelline space was present and a decrease in free SER and RER in the ooplasm occurred; finally, the nucleus migrated from an eccentric to a peripheral location. In conclusion, the growth of the yak oocyte is associated with the relocation and modulation of a number of cytoplasmic organelles as well as the development of oocyte-specific structures such as the zona pellucida, desmosome-like connection and cortical granules.
10.1111/j.1439-0531.2009.01347.x