Species-specific differences in follicular antral sizes result from diffusion-based limitations on the thickness of the granulosa cell layer

The size of mature oocytes is similar across mammalian species, yet the size of ovarian follicles increases with species size, with some ovarian follicles reaching diameters more than 1000-fold the size of the enclosed oocyte. Here we show that the different follicular sizes can be explained with diffusion-based limitations on the thickness of the hormone-secreting granulosa layer. By analysing published data on human follicular growth and granulosa cell expansion during follicular maturation we find that the 4-fold increase of the antral follicle diameter is entirely driven by an increase in the follicular fluid volume, while the thickness of the surrounding granulosa layer remains constant at about 45+/-10 mkm. Based on the measured kinetic constants, the model reveals that the observed fall in the gonadotropin concentration from peripheral blood circulation to the follicular antrum is a result of sequestration in the granulosa. The model further shows that as a result of sequestration, an increased granulosa thickness cannot substantially increase estradiol production but rather deprives the oocyte from gonadotropins. Larger animals (with a larger blood volume) require more estradiol as produced by the ovaries to downregulate FSH-secretion in the pituitary. Larger follicle diameters result in larger follicle surface areas for constant granulosa layer thickness. The reported increase in follicular surface area in larger species indeed correlates linearly both with species mass and with the predicted increase in estradiol output. In summary, we propose a structural role for the antrum in that it determines the volume of the granulosa layer and thus the level of estrogen production.Comment: Mol Hum Repr 201

Follicle Structure Influences the Availability of Oxygen to the Oocyte in Antral Follicles

The ability of an oocyte to successfully mature is highly dependent on intrafollicular conditions, including the size and structure of the follicle. Here we present a mathematical model of oxygen transport in the antral follicle. We relate mean oxygen concentration in follicular fluid of bovine follicles to the concentration in the immediate vicinity of the cumulus-oocyte complex (COC). The model predicts that the oxygen levels within the antral follicle are dependent on the size and structure of the follicle and that the mean level of dissolved oxygen in follicular fluid does not necessarily correspond to that reaching the COC

Species-specific differences in follicular antral sizes result from diffusion-based limitations on the thickness of the granulosa cell layer

The size of mature oocytes is similar across mammalian species, yet the size of ovarian follicles increases with species size, with some ovarian follicles reaching diameters >1000-fold the size of the enclosed oocyte. Here we show that the different follicular sizes can be explained with diffusion-based limitations on the thickness of the hormone-secreting granulosa layer. By analysing published data on human follicular growth and granulosa cell expansion during follicular maturation we find that the 4-fold increase of the antral follicle diameter is entirely driven by an increase in the follicular fluid volume, while the thickness of the surrounding granulosa layer remains constant at ∼45 ± 10 µm. Based on the measured kinetic constants, the model reveals that the observed fall in the gonadotrophin concentration from peripheral blood circulation to the follicular antrum is a result of sequestration in the granulosa. The model further shows that as a result of sequestration, an increased granulosa thickness cannot substantially increase estradiol production but rather deprives the oocyte from gonadotrophins. Larger animals (with a larger blood volume) require more estradiol as produced by the ovaries to down-regulate follicle-stimulating hormone-secretion in the pituitary. Larger follicle diameters result in larger follicle surface areas for constant granulosa layer thickness. The reported increase in the follicular surface area in larger species indeed correlates linearly both with species mass and with the predicted increase in estradiol output. In summary, we propose a structural role for the antrum in that it determines the volume of the granulosa layer and thus the level of estrogen productio

An Agent-Based Model of Cryoprotectant Equilibration in Secondary Stage Preantral Ovarian Follicles

Young cancer patients have limited options for fertility treatment when facing gonadotoxic treatment. One promising fertility treatment for young cancer patients is the cryopreservation of immature ovarian follicles followed by maturation and subsequent reimplantation. However, preantral ovarian follicles currently have lower post-thaw success rates compared to mature oocytes and embryos. Previous research suggests that damage to vital intercellular connections, Transzonal Projections (TZPs), occurs during the cryopreservation process and may account for the observed lower post-thaw success rate in this tissue. It is likely that cryoprotective agent (CPA) equilibration is the cryopreservation step during which TZP damage occurs. Constructing a biologically relevant model of CPA equilibration and the associated damage may allow for improved protocols as measured by increased post-thaw success rates. Agent-based models are a promising technique to capture steps in the cryopreservation process, such as CPA equilibration. In this thesis, I conducted a series of experiments with typical CPAs and nonpermeating solutes at different temperatures using preantral ovarian follicles from a non-human primate (Rhesus monkeys) to measure TZP damage. In these experiments, I also estimated relevant permeability parameters within the tissue. I found that the majority of TZP damage was likely the result of mechanical forces that occurred during the cell volume reduction phase of CPA equilibration. Furthermore, through these experiments, I demonstrate that for this tissue type, parameters collected either during monolayer or single-cell experiments can be used to construct full tissue models. Using the derived experimental parameters and available literature values, I constructed and validated a 3-D agent-based model to capture CPA equilibration in preantral ovarian follicles. My agent-based model utilizes parallel computing on an average desktop computer and allows for the rapid design and testing of CPA equilibration protocols. The model I constructed can account for both mechanical and toxic damage. Importantly, my model accurately captures the experimental damage to TZPs in the majority of simulations. Lastly, I propose several theoretically improved cryopreservation protocols for preantral ovarian follicles. The research presented in this thesis demonstrates that agent-based models can be utilized to capture steps in the cryopreservation in silico and represents a non-invasive, less costly means to test and improve CPA equilibration protocols

Follicle-Stimulating Hormone Receptor: Advances and Remaining Challenges

International audienc

Protective effects of a SIRT1 inhibitor on primordial follicle activation and growth induced by cyclophosphamide: insights from a bovine in vitro folliculogenesis system

Purpose: Although oncological advances have improved survival rates of female cancer patients, they often suffer a reduced fertility due to treatment side effects. In the present study, we evaluated the potential fertoprotective effects of the specific inhibitor of SIRT1, EX-527, on the gonadotoxic action exerted by cyclophosphamide (CPM) on loss of primordial follicles (PFs). Methods: The effects of the CPM metabolite phosphoramide mustard (PM) on follicle activation, growth and viability and the protective action of EX-527 against PM effects were evaluated on bovine ovarian cortical strips in vitro cultured for 1 or 6 days. To understand whether PFs exposed to PM plus EX-527 were able to activate and grow to the secondary stage after suspension of the treatment, strips cultured for 3 days in PM plus EX-527 for 3 days were transferred to plain medium until day 6. Follicle growth and health were evaluated through histology and viability assay at a confocal microscope. In order to investigate the molecular pathways underlying the ovarian response to PM in the presence of EX-527, we analysed the protein level of SIRT1, HuR, PARP1 and SOD2 after 1 day of in vitro culture. Results: We found that (1) PM, the main CPM active metabolite, promotes PF activation; (2) the ovarian stress response induced by PM includes a SIRT1-dependent pathway; and (3) EX-527 reduces PF activation and growth induced by PM. Conclusion: SIRT1 can represent a candidate molecule to be targeted to protect ovarian follicles from alkylating agents and EX-527 could represent a potential fertoprotective agent for cancer patients

Synthetic human gonadal tissues for toxicology

Nishimura T., Takebe T.. Synthetic human gonadal tissues for toxicology. Reproductive Toxicology 126, 108598 (2024); https://doi.org/10.1016/j.reprotox.2024.108598.The process of mammalian reproduction involves the development of fertile germ cells in the testis and ovary, supported by the surrounders. Fertilization leads to embryo development and ultimately the birth of offspring inheriting parental genome information. Any disruption in this process can result in disorders such as infertility and cancer. Chemical toxicity affecting the reproductive system and embryogenesis can impact birth rates, overall health, and fertility, highlighting the need for animal toxicity studies during drug development. However, the translation of animal data to human health remains challenging due to interspecies differences. In vitro culture systems offer a promising solution to bridge this gap, allowing the study of mammalian cells in an environment that mimics the physiology of the human body. Current advances on in vitro culture systems, such as organoids, enable the development of biomaterials that recapitulate the physiological state of reproductive organs. Application of these technologies to human gonadal cells would provide effective tools for drug screening and toxicity testing, and these models would be a powerful tool to study reproductive biology and pathology. This review focuses on the 2D/3D culture systems of human primary testicular and ovarian cells, highlighting the novel approaches for in vitro study of human reproductive toxicology, specifically in the context of testis and ovary

Investigation of miRNAs enrichment and degradation in bovine granulosa cells during follicular development

The granulosa cells in the mammalian ovarian follicle respond to gonadotropin signalling and are involved in the processes of folliculogenesis and oocyte maturation. Although, several studies have been done on spatio temporal expression of genes during follicular development, little is known about the post-transcriptional regulation of those genes. This study unravelled the basic knowledge on bovine miRNA prevalence and expression pattern during the early luteal phase of the bovine estrous cycle. For this, miRNAs enriched total RNA isolated from granulosa cells of subordinate follicles (SF) and dominant follicles (DF) obtained from heifers slaughtered at day 3 and day 7 of the estrous cycle and were subjected for miRNAs deep sequencing. The data analysis revealed that 291 and 318 mature miRNAs were detected in granulosa cells of SF and DF, respectively at day 3 of estrous cycle, while 314 and 316 were detected in granulosa cells of SF and DF, respectively, at day 7 of estrous cycle. A total of 244 detected miRNAs were common to all follicle groups, of which 15 miRNAs including bta-miR-10b, bta-miR-26a, let-7 families, bta-miR-92a, bta-miR-191, bta-miR-125a, bta-miR-148 and bta-miR-30a-5p, were highly abundant (≥3000 reads) in both SF and DF at both days of the estrous cycle. At day 3 of the estrous cycle, 16 miRNAs including bta-miR-449a, bta-miR-449c, bta-miR-212, bta-miR-21-3p, bta-miR-183 and bta-mir-34c were differentially expressed (DE) in granulosa cell of subordinate follicle groups. Similarly, at day 7 of the estrous cycle, a total of 108 miRNAs including bta-mir-409a, bta-miR-2446, and bta-mir-383 were altered in granulosa cells of SF compared to DF. Nine miRNAs including bta-miR-21-3p, bta-miR-708, and bta-miR-335 were commonly DE between SF and DF at day 3 and day 7 of the estrous cycle. In addition to known miRNAs, a total of 21 novel miRNAs were identified and detected in granulosa cells of SF and/or DF at day 3 and day 7 of the estrous cycle. The majority of the DE miRNAs were found to be involved in regulation of programmed cell death and regulation of cell proliferation. In addition, the DE miRNAs were found to be involved in Wnt signaling, TGF-beta signaling, oocyte meiosis, MAPK signaling, focal adhesion, axon guidance and gap junction. Therefore, our findings suggest that temporal variation in the abundance of mature miRNAs during bovine follicular development in SF and DF of granulosa cells, which may be associated with recruitment, selection and development of bovine follicles.Untersuchung der miRNA Anreicherung und Abbau in bovine Granulosazellen während der Follikelreifung Die Granulosazellen aus ovarialen Säugertierfollikeln reagieren auf Gonadotropin-Signale und sind an den Prozessen der Follikulogenese und Eizellenreifung beteiligt. Obwohl bereits mehrere Studien über die spatio-temporale Genexpression während der follikulären Entwicklung erfolgten, ist bisher wenig über die post-transkriptionelle Regulierung dieser Gene bekannt. Daher befasst sich diese Studie mit der Untersuchung von der bovine miRNA Prävalenz und ihrer Expressionsmuster während der frühen Lutealphase des bovinen Östruszyklus. Dafür wurde miRNA angereicherte Gesamt-RNA aus Granulosazellen von untergeordneten Follikeln (SF) und dominanten Follikeln (DF) am Tag 3 und Tag 7 des Östruszyklus von geschlachteten Färsen isoliert und mittels Deep Sequenzierung analysiert. Durch die Datenanalyse konnten jeweils 291 und 317 miRNAs in Granulosazellen von SF und DF am Tag 3 des Östruszyklus ermittelt werden. Für Tag 7 des Östruszyklus gewonnene Granulosazellen konnten 314 und 316 miRNAs identifizierten werden. In allen Follikelgruppen wurden insgesamt 244 miRNAs detektiert, wobei 15 miRNAs einschließlich bta-miR-10b, bta-miR-26a, let-7 families, bta-miR-92a, bta-miR-191, bta-miR-125a, bta-miR-148 und bta-miR-30a-5p in beiden SF und DF und auch an beiden Tagen des Östruszyklus hoch reguliert (≥3000 reads) waren. Am Tag 3 des Östruszyklus waren 16 miRNAs einschließlich bta-miR-449a, bta-miR-449c, bta-miR-212, bta-miR-21-3p, bta-miR-183 und bta-mir-34c unterschiedlich in Granulosazellen der untergeordneten Follikelgruppe exprimiert (DE). Genauso zeigten am Tag 7 des Östruszyklus insgesamt 108 miRNAs einschließlich bta-mir-409a, bta-miR-2446, und bta-mir-383 in SF Granulosazellen im Vergleich zu DF eine unterschiedliche Expression. Neun miRNAs, u.a. bta-miR-21-3p, bta-miR-708 und bta-miR-335 waren sowohl am Tag 3 als auch am Tag 7 DE zwischen SF und DF des Östruszyklus. Insgesamt wurden 21 neue miRNAs zusätzlich zu den bekannten miRNAs in den Granulosazellen von SF und/oder DF am Tag 3 und 7 des Östruszyklus identifiziert und detektiert. Die Mehrheit der DE miRNAs sind an der Regulierung des programmierten Zelltods und der Regulierung der Zellproliferation beteiligt. Gleichwohl waren diese DE miRNAs auch an den Signalwegen Wnt, TGF-beta, Meiose der Eizelle, MAPK, fokal Adhäsion, Axon Guidance und Gap Junction involviert. Deshalb lassen unsere Ergebnisse darauf schließen, dass temporale Variationen in der Anreicherung von miRNAs während der bovinen Follikelentwicklung in SF und DF aus Granulosazellen, welche mit der Rekrutierung, Selektion und Entwicklung boviner Follikel assoziiert werden, vorkommen können

Evaluation of texture features for analysis of ovarian follicular development

Ovarian follicles in women are fluid-filled structures in the ovary that contain oocytes (eggs). A dominant follicle is physiologically selected and ovulates during the menstrual cycle. We examined the echotexture in ultrasonographic images of the follicle wall of dominant ovulatory follicles in women during natural menstrual cycles and dominant anovulatory follicles which developed in women using oral contraceptives (OC). Texture features of follicle wall regions of both ovulatory and anovulatory dominant follicles were evaluated over a period of seven days before ovulation (natural cycles) or peak estradiol concentrations (OC cycles). Differences in echotexture between the two classes of follicles were found for two co-occurrence matrix derived texture features and two edge-frequency based texture features. Co-occurrence energy and homogeneity were significantly lower for ovulatory follicles while edge density and edge contrast were higher for ovulatory follicles. In the each feature space, the two classes of follicle were adequately separable.This thesis employed several statistical approaches to analyses of texture features, such as plotting method and the Mann-Kendall method. A distinct change of feature trend was detected 3 or 4 days before the day of ovulation for ovulatory follicles in the two co-occurrence matrix derived texture features and two edge-frequency-based texture features. Anovulatory follicles, exhibited the biggest variation of the feature value 3 or 4 days before the day on which dominant follicles developed to maximum size. This discovery is believed to correspond to the ovarian follicles responding to system hormonal changes leading to presumptive ovulation

Comparative Sequence and Structural Analyses of G-Protein-Coupled Receptor Crystal Structures and Implications for Molecular Models

BACKGROUND:Up until recently the only available experimental (high resolution) structure of a G-protein-coupled receptor (GPCR) was that of bovine rhodopsin. In the past few years the determination of GPCR structures has accelerated with three new receptors, as well as squid rhodopsin, being successfully crystallized. All share a common molecular architecture of seven transmembrane helices and can therefore serve as templates for building molecular models of homologous GPCRs. However, despite the common general architecture of these structures key differences do exist between them. The choice of which experimental GPCR structure(s) to use for building a comparative model of a particular GPCR is unclear and without detailed structural and sequence analyses, could be arbitrary. The aim of this study is therefore to perform a systematic and detailed analysis of sequence-structure relationships of known GPCR structures. METHODOLOGY:We analyzed in detail conserved and unique sequence motifs and structural features in experimentally-determined GPCR structures. Deeper insight into specific and important structural features of GPCRs as well as valuable information for template selection has been gained. Using key features a workflow has been formulated for identifying the most appropriate template(s) for building homology models of GPCRs of unknown structure. This workflow was applied to a set of 14 human family A GPCRs suggesting for each the most appropriate template(s) for building a comparative molecular model. CONCLUSIONS:The available crystal structures represent only a subset of all possible structural variation in family A GPCRs. Some GPCRs have structural features that are distributed over different crystal structures or which are not present in the templates suggesting that homology models should be built using multiple templates. This study provides a systematic analysis of GPCR crystal structures and a consistent method for identifying suitable templates for GPCR homology modelling that will help to produce more reliable three-dimensional models