scholarly journals Three-dimensional organization of transzonal projections and other cytoplasmic extensions in mouse ovarian follicles

2018 ◽  
Author(s):  
Valentina Baena ◽  
Mark Terasaki
Keyword(s):  
Granulosa Cells ◽  
Ovarian Follicle ◽  
Cumulus Cell ◽  
Three Dimensional ◽  
Cumulus Cells ◽  
Ovarian Follicles ◽  
New Methods ◽  
Section Electron ◽  
Paracrine Interaction ◽  
Serial Section Electron Microscopy

AbstractEach mammalian oocyte is nurtured by its own multi-cellular structure, the ovarian follicle. We used new methods for serial section electron microscopy to examine entire cells and their projections in mouse antral ovarian follicles. It is already known that cumulus cells send towards the oocyte thin cytoplasmic projections called transzonal projections (TZPs), which are crucial for normal oocyte development. We found that most TZPs do not reach the oocyte, and that they often branch and make gap junctions with each other. Furthermore, the connected TZPs are usually contacted on their shaft by oocyte microvilli. Mural granulosa cells were found to possess randomly oriented cytoplasmic projections that are strikingly similar to free-ended TZPs. We propose that granulosa cells use cytoplasmic projections to search for the oocyte, and cumulus cell differentiation results from a contact-mediated paracrine interaction with the oocyte.

Alterations in the cell cycle of mouse cumulus granulosa cells during expansion and mucification in vivo and in vitro

1996 ◽  
Vol 8 (6) ◽  
pp. 935 ◽  
Author(s):  
AW Schuetz ◽  
DG Whittingham ◽  
R Snowden
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Granulosa Cells ◽  
Dna Content ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Ovarian Follicles ◽  
S Phase

The cell cycle characteristics of mouse cumulus granulosa cells were determined before, during and following their expansion and mucification in vivo and in vitro. Cumulus-oocyte complexes (COC) were recovered from ovarian follicles or oviducts of prepubertal mice previously injected with pregnant mare serum gonadotrophin (PMSG) or a mixture of PMSG and human chorionic gonadotrophin (PMSG+hCG) to synchronize follicle differentiation and ovulation. Cell cycle parameters were determined by monitoring DNA content of cumulus cell nuclei, collected under rigorously controlled conditions, by flow cytometry. The proportion of cumulus cells in three cell cycle-related populations (G0/G1; S; G2/M) was calculated before and after exposure to various experimental conditions in vivo or in vitro. About 30% of cumulus cells recovered from undifferentiated (compact) COC isolated 43-45 h after PMSG injections were in S phase and 63% were in G0/G1 (2C DNA content). Less than 10% of the cells were in the G2/M population. Cell cycle profiles of cumulus cells recovered from mucified COC (oviducal) after PMSG+hCG-induced ovulation varied markedly from those collected before hCG injection and were characterized by the relative absence of S-phase cells and an increased proportion of cells in G0/G1. Cell cycle profiles of cumulus cells collected from mucified COC recovered from mouse ovarian follicles before ovulation (9-10 h after hCG) were also characterized by loss of S-phase cells and an increased G0/G1 population. Results suggest that changes in cell cycle parameters in vivo are primarily mediated in response to physiological changes that occur in the intrafollicular environment initiated by the ovulatory stimulus. A similar lack of S-phase cells was observed in mucified cumulus cells collected 24 h after exposure in vitro of compact COC to dibutyryl cyclic adenosine monophosphate (DBcAMP), follicle-stimulating hormone or epidermal growth factor (EGF). Additionally, the proportion of cumulus cells in G2/M was enhanced in COC exposed to DBcAMP, suggesting that cell division was inhibited under these conditions. Thus, both the G1-->S-phase and G2-->M-phase transitions in the cell cycle appear to be amenable to physiological regulation. Time course studies revealed dose-dependent changes in morphology occurred within 6 h of exposure in vitro of COC to EGF or DBcAMP. Results suggest that the disappearance of the S-phase population is a consequence of a decline in the number of cells beginning DNA synthesis and exit of cells from the S phase following completion of DNA synthesis. Furthermore, loss of proliferative activity in cumulus cells appears to be closely associated with COC expansion and mucification, whether induced under physiological conditions in vivo or in response to a range of hormonal stimuli in vitro. The observations indicate that several signal-transducing pathways mediate changes in cell cycle parameters during cumulus cell differentiation.

scholarly journals Human Granulosa Cells—Stemness Properties, Molecular Cross-Talk and Follicular Angiogenesis

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1396
Author(s):  
Claudia Dompe ◽  
Magdalena Kulus ◽  
Katarzyna Stefańska ◽  
Wiesława Kranc ◽  
Błażej Chermuła ◽  
...  
Keyword(s):  
Stem Cells ◽  
Granulosa Cells ◽  
Polycystic Ovary ◽  
Ovarian Follicle ◽  
Cumulus Cells ◽  
Ovary Syndrome ◽  
Different Types ◽  
Metabolite Exchange ◽  
Reproductive Techniques ◽  
New Strategies

The ovarian follicle is the basic functional unit of the ovary, comprising theca cells and granulosa cells (GCs). Two different types of GCs, mural GCs and cumulus cells (CCs), serve different functions during folliculogenesis. Mural GCs produce oestrogen during the follicular phase and progesterone after ovulation, while CCs surround the oocyte tightly and form the cumulus oophurus and corona radiata inner cell layer. CCs are also engaged in bi-directional metabolite exchange with the oocyte, as they form gap-junctions, which are crucial for both the oocyte’s proper maturation and GC proliferation. However, the function of both GCs and CCs is dependent on proper follicular angiogenesis. Aside from participating in complex molecular interplay with the oocyte, the ovarian follicular cells exhibit stem-like properties, characteristic of mesenchymal stem cells (MSCs). Both GCs and CCs remain under the influence of various miRNAs, and some of them may contribute to polycystic ovary syndrome (PCOS) or premature ovarian insufficiency (POI) occurrence. Considering increasing female fertility problems worldwide, it is of interest to develop new strategies enhancing assisted reproductive techniques. Therefore, it is important to carefully consider GCs as ovarian stem cells in terms of the cellular features and molecular pathways involved in their development and interactions as well as outline their possible application in translational medicine.

scholarly journals Serum deprivation affects secretory activity of cultured porcine ovarian follicles and granulosa cells and their response to hormones

2009 ◽  
Vol 54 (No. 10) ◽  
pp. 455-460
Author(s):  
A.V. Sirotkin
Keyword(s):  
Granulosa Cells ◽  
Ovarian Follicle ◽  
Secretory Activity ◽  
Ovarian Follicles ◽  
Serum Deprivation ◽  
Ovarian Cells ◽  
Igf I ◽  
Granulose Cells ◽  
Vitro Model

The aim of the present study is to understand the hormonal mechanisms of the effect of malnutrition on ovarian follicle functions. For this purpose, we examined the effect of malnutrition/serum deprivation, addition of metabolic hormones and gonadotropin (IGF-I, leptin and FSH) and their combination on the release of progesterone (P<sub>4</sub>), testosterone (T), estradiol (E<sub>2</sub>) and insulin-like growth factor I (IGF-I) by cultured whole ovarian follicles and on P<sub>4</sub> and IGF-I output by cultured granulosa cells isolated from porcine ovaries. It was observed that in ovarian follicles cultured with nutrients/serum addition of IGF-I reduced release of P<sub>4</sub>, but not of T or E<sub>2</sub>. Exogenous leptin reduced output of E<sub>2</sub>, but not of P<sub>4</sub> or T, and increased IGF-I output. No significant effect of FSH on release of steroid hormones by isolated follicles was found. Serum deprivation did not affect release of P<sub>4</sub>, but reduced output of T and E<sub>2</sub>, and promoted IGF-I release by cultured ovarian follicles. Addition of hormones failed to prevent the effect of malnutrition on the secretory activity of cultured ovarian follicles. In cultured granulose cells, all the tested hormones promoted release of both P<sub>4</sub> and IGF-I. Food restriction/serum deprivation reduced both P<sub>4</sub> and IGF-I output. Additions of either IGF-I, leptin and FSH prevented the inhibitory action of malnutrition on both P<sub>4</sub> and IGF-I release. The present observations (1) confirm the involvement of the hormones IGF-I, leptin and FSH in the control of secretory activity of ovarian cells, (2) demonstrate, that both isolated ovarian granulosa cells and whole follicles cultured in the absence of serum nutrients could be an adequate in-vitro model for studying the effect of malnutrition on ovarian secretory functions, and (3) suggest, that malnutrition could affect ovarian functions through changes in the release of ovarian hormones.

79 microRNA EXPRESSION IN BOVINE CUMULUS CELLS

2015 ◽  
Vol 27 (1) ◽  
pp. 133
Author(s):  
K. Uhde ◽  
L. T. A. van Tol ◽  
T. A. E. Stout ◽  
B. A. J. Roelen
Keyword(s):  
Mirna Expression ◽  
Reference Genes ◽  
Noncoding Rna ◽  
Ovarian Follicle ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Small Samples ◽  
Variable Expression

A mammalian oocyte within an ovarian follicle is surrounded by cumulus cells, together this structure is known as the cumulus-oocyte complex (COC). Cumulus cells are important for the development of the oocyte, they support the maturation process of the oocyte within the ovary and aid in sperm recognition. Because it is known that a Dicer knockout leads to infertility, microRNAs (miRNA) are focused to have an important role in oocyte development. MiRNAs are small noncoding RNA sequences that act as transcriptional regulators. Little is known about the expression of miRNA in cumulus cells or how cumulus-derived miRNA may regulate or be used to indicate the developmental competence of the maturing oocyte. Our aim was to investigate miRNA expression in oocytes and to identify and establish how specific miRNA influence the acquisition of developmental competence by bovine oocytes. Normalization of qPCR data requires stable reference genes. To this end, we tested the expression of various miRNA with respect to their ability to be used as reference miRNA for bovine cumulus cells; these included miR-103, miR-93, miR-26, let-7a, miR-191, and the small noncoding nuclear RNA U6. Cumulus-oocyte complexes were recovered from the ovaries of slaughtered cows and matured in vitro. Small samples of cumulus cells were collected from these COC before and after maturation. From the cumulus cell groups recovered at different stages, small RNA were extracted and cDNA was synthesised, followed by qRT-PCR. To identify the optimal combination of reference genes, the geNorm algorithm was used. MiR-26a and let-7a were identified as the most stably expressed miRNAs, whereas U6 showed the most variable expression levels. Future investigations are planned to identify miRNA in cumulus cells that can be used as markers for oocyte developmental competence. Using a single oocyte-embryo culture system will enable us to retrospectively relate cumulus miRNA expression to the developmental capacity of the oocyte.This work was supported by EU FP7 EpiHealthNet (N°317146).

037. IMMUNE-LIKE MECHANISMS ASSOCIATED WITH OVULATION

2009 ◽  
Vol 21 (9) ◽  
pp. 10
Author(s):  
J. Richards
Keyword(s):  
Granulosa Cells ◽  
Immune Cell ◽  
Expression Patterns ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Mature Oocyte ◽  
Innate Immune ◽  
Innate Immune Cell ◽  
Oocyte Meiosis ◽  
Mitogen Activated Protein

Ovulation is the unique biological process by which a mature oocyte and surrounding somatic cells, the cumulus cell-oocyte complex (COC), are released from the surface of the ovary into the oviduct for transport and fertilization. Ovulation is similar to an inflammatory response: the follicles become hyperemic, produce prostaglandins and synthesize a hyaluronan-rich extracellular matrix. However, this view of ovulation may be too restrictive and need to be broadened to encompass the innate immune cell surveillance response system. This hypothesis is being proposed because ovarian granulosa cells and cumulus cells express and respond to innate immune cell related surveillance proteins (Toll-like receptors 2 and 4) and cytokines such as interleukin 6 (IL6) during ovulation. In addition, recent studies indicate that the ovulation process that is set in motion by the surge of luteinizing hormone (LH) is mediated, in large part, by the EGF-like factors (Amphiregulin, epiregulin and betacellulin) and their critical activation of RAS, most probably KRAS that is expressed at high levels in granulosa cells, and the mitogen activated protein kinases, MAP3/1 (ERK1/2). Mice in which granulosa cells are depleted of ERK1/2 fail to ovulate, oocyte meiosis does not resume, COC expansion is impaired and luteinization is blocked. Thus the global molecular reprogramming of granulosa cell gene expression patterns is completely derailed. Supported, in part by NIH-HD-16229, -16272 and -07495 (SCCPIR).

scholarly journals In vitro ovarian follicle growth: a comprehensive analysis of key protocol variables†

2020 ◽  
Vol 103 (3) ◽  
pp. 455-470
Author(s):  
Leah E Simon ◽  
T Rajendra Kumar ◽  
Francesca E Duncan
Keyword(s):  
Wildlife Conservation ◽  
Culture Media ◽  
Ovarian Follicle ◽  
Three Dimensional ◽  
Ovarian Follicles ◽  
Culture Methods ◽  
Growth And Survival ◽  
Follicle Culture ◽  
Culture Techniques

Abstract Folliculogenesis is a complex process that requires integration of autocrine, paracrine, and endocrine factors together with tightly regulated interactions between granulosa cells and oocytes for the growth and survival of healthy follicles. Culture of ovarian follicles is a powerful approach for investigating folliculogenesis and oogenesis in a tightly controlled environment. This method has not only enabled unprecedented insight into the fundamental biology of follicle development but also has far-reaching translational applications, including in fertility preservation for women whose ovarian follicles may be damaged by disease or its treatment or in wildlife conservation. Two- and three-dimensional follicle culture systems have been developed and are rapidly evolving. It is clear from a review of the literature on isolated follicle culture methods published over the past two decades (1980–2018) that protocols vary with respect to species examined, follicle isolation methods, culture techniques, culture media and nutrient and hormone supplementation, and experimental endpoints. Here we review the heterogeneity among these major variables of follicle culture protocols.

scholarly journals Heparan Sulfate Proteoglycans Regulate Responses to Oocyte Paracrine Signals in Ovarian Follicle Morphogenesis

Endocrinology ◽  
2012 ◽  
Vol 153 (9) ◽  
pp. 4544-4555 ◽  
Author(s):  
Laura N. Watson ◽  
David G. Mottershead ◽  
Kylie R. Dunning ◽  
Rebecca L. Robker ◽  
Robert B. Gilchrist ◽  
...  
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Cell Function ◽  
Ovarian Follicle ◽  
Heparan Sulphate ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Distinct Cell ◽  
Heparan Sulphate Proteoglycans

In the ovarian follicle, oocyte-secreted factors induce cumulus-specific genes and repress mural granulosa cell specific genes to establish these functionally distinct cell lineages. The mechanism establishing this precise morphogenic pattern of oocyte signaling within the follicle is unknown. The present study investigated a role for heparan sulphate proteoglycans (HSPG) as coreceptors mediating oocyte secreted factor signaling. In vitro maturation of cumulus oocyte complexes in the presence of exogenous heparin, which antagonizes HSPG signaling, prevented cumulus expansion and blocked the induction of cumulus-specific matrix genes, Has2 and Tnfaip6, whereas conversely, the mural granulosa-specific genes, Lhcgr and Cyp11a1, were strongly up-regulated. Heparin also blocked phosphorylation of SMAD2. Exogenous growth differentiation factor (GDF)-9 reversed these heparin effects; furthermore, GDF9 strongly bound to heparin sepharose. These observations indicate that heparin binds endogenous GDF9 and disrupts interaction with heparan sulphate proteoglycan coreceptor(s), important for GDF9 signaling. The expression of candidate HSPG coreceptors, Syndecan 1–4, Glypican 1–6, and Betaglycan, was examined. An ovulatory dose of human chorionic gonadotropin down-regulated Betaglycan in cumulus cells, and this regulation required GDF9 activity; conversely, Betaglycan was significantly increased in luteinizing mural granulosa cells. Human chorionic gonadotropin caused very strong induction of Syndecan 1 and Syndecan 4 in mural granulosa as well as cumulus cells. Glypican 1 was selectively induced in cumulus cells, and this expression appeared dependent on GDF9 action. These data suggest that HSPG play an essential role in GDF9 signaling and are involved in the patterning of oocyte signaling and cumulus cell function in the periovulatory follicle.

scholarly journals Centriolar remodeling underlies basal body maturation during ciliogenesis in Caenorhabditis elegans

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Inna V Nechipurenko ◽  
Cristina Berciu ◽  
Piali Sengupta ◽  
Daniela Nicastro
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Sensory Neurons ◽  
Basal Body ◽  
Three Dimensional ◽  
C Elegans ◽  
Mother Centriole ◽  
Section Electron ◽  
Serial Section Electron Microscopy ◽  
Section Electron Microscopy

The primary cilium is nucleated by the mother centriole-derived basal body (BB) via as yet poorly characterized mechanisms. BBs have been reported to degenerate following ciliogenesis in the C. elegans embryo, although neither BB architecture nor early ciliogenesis steps have been described in this organism. In a previous study (Doroquez et al., 2014), we described the three-dimensional morphologies of sensory neuron cilia in adult C. elegans hermaphrodites at high resolution. Here, we use serial section electron microscopy and tomography of staged C. elegans embryos to demonstrate that BBs remodel to support ciliogenesis in a subset of sensory neurons. We show that centriolar singlet microtubules are converted into BB doublets which subsequently grow asynchronously to template the ciliary axoneme, visualize degeneration of the centriole core, and define the developmental stage at which the transition zone is established. Our work provides a framework for future investigations into the mechanisms underlying BB remodeling.

scholarly journals Astrocytes refine cortical connectivity at dendritic spines

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
W Christopher Risher ◽  
Sagar Patel ◽  
Il Hwan Kim ◽  
Akiyoshi Uezu ◽  
Srishti Bhagat ◽  
...  
Keyword(s):  
Dendritic Spines ◽  
Cortical Neurons ◽  
Three Dimensional ◽  
Early Postnatal Development ◽  
Mouse Cortex ◽  
Section Electron ◽  
Refinement Process ◽  
Serial Section Electron Microscopy ◽  
Cortical Inputs ◽  
The Brain

During cortical synaptic development, thalamic axons must establish synaptic connections despite the presence of the more abundant intracortical projections. How thalamocortical synapses are formed and maintained in this competitive environment is unknown. Here, we show that astrocyte-secreted protein hevin is required for normal thalamocortical synaptic connectivity in the mouse cortex. Absence of hevin results in a profound, long-lasting reduction in thalamocortical synapses accompanied by a transient increase in intracortical excitatory connections. Three-dimensional reconstructions of cortical neurons from serial section electron microscopy (ssEM) revealed that, during early postnatal development, dendritic spines often receive multiple excitatory inputs. Immuno-EM and confocal analyses revealed that majority of the spines with multiple excitatory contacts (SMECs) receive simultaneous thalamic and cortical inputs. Proportion of SMECs diminishes as the brain develops, but SMECs remain abundant in Hevin-null mice. These findings reveal that, through secretion of hevin, astrocytes control an important developmental synaptic refinement process at dendritic spines.

scholarly journals High-Throughput Analysis of Ovarian Granulosa Cell Transcriptome

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Ewa Chronowska
Keyword(s):  
Reproductive Biology ◽  
High Throughput ◽  
Granulosa Cells ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Molecular Techniques ◽  
Embryo Morphology ◽  
Embryo Viability ◽  
High Throughput Analysis ◽  
Developmental Potential

The quality of follicular oocytes depends on interactions with surrounding granulosa cells. Development of molecular techniques and methods enables better understanding of processes underlying mammalian reproduction on cellular level. The success in reproductive biology and medicine in different species depends on reliable assessment of oocyte and embryo viability which presently mainly bases on embryo morphology. Although successful pregnancies have been achieved using this approach, its precision still should be improved and completed with other, more objective, and accurate assessment strategies. Global profiling of gene expression in follicular cumulus cells using microarrays is continuously leading to the establishment of new biomarkers which can be used to select oocytes with highest developmental potential. Even more potential applications and greater precision could be achieved using next generation sequencing (NGS) of granulosa and cumulus cell RNA (RNA-seq). However, due to the high cost, this method is not used as frequently as microarrays at the moment. In any case, high-throughput technologies offer the possibilities and advantages in ovarian somatic cell analysis on scale that has not been noted so far. The aim of this work is to present current directions and examples of global molecular profiling of granulosa cells and underline its impact on reproductive biology and medicine.

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