Three dimensional culture of fresh and vitrified mouse pre-antral follicles in a hyaluronan-based hydrogel: a preliminary investigation of a novel biomaterial for in vitro follicle maturation

Background

Folliculogenesis within the ovary requires interaction between somatic cell components and the oocyte. Maintenance of 3-dimensional (3-D) architecture and granulosa-oocyte interaction may be critical for successful in vitro maturation of follicles. Testing of novel biomaterials for the 3-D culture of follicles may ultimately lead to a culture model that can support the longer in vitro culture intervals needed for in vitro maturation of human oocytes from ovarian tissue biopsies.

Methods

A novel tyramine-based hyaluronan (HA) hydrogel was tested for its biocompatibility with ovarian follicles. The HA was prepared at concentrations from 2 to 5?mg/ml. HA hydrogel was also formulated and tested with matrix proteins (ECM). Enzymatically isolated pre-antral follicles from the ovaries of 10?C12?day SJL pups were divided amongst control (CT) and HA treatments. The growth of both fresh and vitrified follicles was assessed after encapsulation in the hydrogel. The basal culture medium was MEM alpha supplemented with FSH, LH, ITS and 5% FBS. Maturation was triggered by addition of hCG and EGF after in vitro culture (IVC). Outcome parameters monitored were follicle morphology, survival after IVC, antrum formation, GVBD and MII formation. Differences between treatments were analyzed.

Results

HA and ECM-HA encapsulated follicles looked healthy and maintained their 3-D architecture during IVC. In control cultures, the follicles flattened and granulosa:oocyte connections appeared fragile. Estradiol secretion per follicle was significantly higher by Day 12 in ECM-HA compared to HA or CT (4119, 703 and 1080?pg/ml, respectively). HA and ECM-HA cultured follicles had similar survival rates (62% and 54%, respectively), percent GV breakdown (96?C97%), MII formation (47?C48%) and oocyte diameters at the end of IVC. Control cultures differed significantly in percent GVBD (85%) and MII formation (67%) . Vitrified-warmed follicles encapsulated in HA had an oocyte maturation rate to MII of 54% as compared to 57% in non-embedded follicles.

Conclusions

Initial testing of this new and unique HA-based hydrogel was quite promising. The ease of follicle encapsulation in HA, its optical transparency and ability to be molded combined with its support of follicle growth, estradiol secretion and resumption of meiosis make this HA-hydrogel particularly attractive as model for 3-D ovarian follicle culture.     

The roles of THY1 and integrin beta3 in cell adhesion during theca cell layer formation and the effect of follicle-stimulating hormone on THY1 and integrin beta3 localization in mouse ovarian follicles

The mechanism of theca cell layer formation in mammalian ovaries has not been elucidated. In the present study, we examined the roles of THY1 and integrin beta3 in theca cell layer formation during mouse folliculogenesis. The localization pattern of THY1 and integrin beta3 in adult mouse ovary was investigated immunohistochemically. The strongest THY1 signal was observed in theca cell layers from secondary to preantral follicles, at which time theca cells have begun to participate in follicle formation. Integrin beta3 also localized to the theca cell layer of secondary to preantral follicles and showed a localization pattern similar to that of THY1. Moreover, the role of THY1 in theca cell layer formation was examined using a follicle culture system. When anti-THY1 antibody was added to this culture, no theca cell layers were formed, and the granulosa cells were distanced from each other. Because a THY1 signal was not observed in ovaries at stages earlier than prepuberty, THY1 localization also appeared to be affected by mouse development. This possibility was examined by determining the effect of administering follicle-stimulating hormone, luteinizing hormone, and 17beta-estradiol to 7-day-old mice on THY1 localization in the ovary 3 days later. Only follicle-stimulating hormone induced a THY1 signal in 10-day-old mouse ovaries. No THY1 signal was observed in untreated 10-day-old ovaries. In conclusion, THY1 might play a role in cell adhesion via binding to integrin beta3 in mouse ovaries. The present results suggest that THY1 localization may be affected by follicle-stimulating hormone in mouse ovaries.     

Formation of multiple-oocyte follicles in culture

Basement membranes are found in every organ of the body. They provide structure and a selective filter for molecules. The ovary is no different with the follicular basal lamina (FBL) separating the granulosa and theca cells, facilitating regulation of the changing follicular environment providing appropriate conditions for the developing oocyte. The FBL is modified in C1galt1 Mutant mice (C1galt1 ^FF:ZP3Cre) resulting from oocyte-specific deletion of C1galt1. Changes in the FBL lead to follicles joining to generate multiple-oocyte follicles (MOFs); where two or more oocytes are contained within a single follicle. This study aimed to determine if single-oocyte follicles could join in culture to become MOFs by co-culturing preantral follicles from Control or Mutant mice. Co-cultured follicles from both Control and Mutant follicles could superficially fuse (73% of Control follicle pairs; 84% of Mutant). Confocal microscopy revealed alterations in the organization of the space between follicles but was unable to discern MOFs. When co-cultured follicle pairs were embedded, sectioned and stained with haematoxylin, it was revealed that MOFs had formed from 50% of Mutant follicle pairs but none from Control follicle pairs. In conclusion, MOFs can form from C1galt1 Mutant follicles in culture and this model is a useful tool to elucidate the role of the oocyte in follicle development and the generation and function of the FBL. Furthermore, understanding the relationship between oocyte function and FBL generation will likely provide insight into optimizing conditions for follicle culture, which is important for fertility treatments and ART.     

Electron microscopy of cytodifferentiation and its subcellular steroidogenic sites in the theca cell of the human ovary

Summary The cytodifferentiation and subcellular steroidogenic sites in the theca cell of the human ovary during the follicular phase were investigated using the electron microscopic cytochemistry. Only fibroblast-like cells were seen around or near the primordial follicle. In the theca interna of the secondary and Graafian follicle however there were three different cell types: fibroblast-like cells, theca gland cells (steroid-secreting cells) and transitional cells (partially or incompletely differentiated theca cells). On the other hand the theca externa of these follicles consisted mainly of fibroblast-like cells. The hallmarks of the cytodifferentiation of the theca cells were: 1) the appearance of lipid droplets, 2) a structural change of the mitochondrial cristae from lamellar to tubular form and 3) the appearance and development of smooth endoplasmic reticulum. Reaction products of 3-hydroxysteroid ferricyanide reductase, indicating 3-hydroxysteroid dehydrogenase activity, were localized on tubular or lamellar cristae and inner membrane of the mitochondria, and on the membranes of smooth endoplasmic reticulum in the transitional cell as well as in the theca gland cell of the secondary and Graafian follicle. From these data, it is suggested that the transitional cell has a steroid-secreting activity and also plays an important role in follicular development in human reproduction.Supported by a grant from the Japanese Educational Ministry     

Comparison of the developmental potential of 2-week-old preantral follicles derived from vitrified ovarian tissue slices, vitrified whole ovaries and vitrified/transplanted newborn mouse ovaries using the metal surface method

Background

Cryopreservation of preantral follicles or ovarian tissues would enable the storage of large numbers of primordial follicles or preantral follicles and preserves the structural integrity of somatic and reproductive cells. In the present study, we compared the developmental potential of cryopreserved two-week-old mouse preantral follicles, ovarian tissue slices, two-week-old mouse ovaries and newborn mouse ovaries using a metal plate with a high cooling rate for cooling the droplet of vitrification solution.

Methods

Groups of 2 to 4 samples (including of 14-day old preantral follicles, ovarian tissue slices, whole ovaries, and whole newborn ovaries) were exposed to 4% ethylene glycol (EG) in DPBS + 10% FBS for 15 min and then rinsed in a vitrification solution composed of 6 M ethylene glycol and 0.4 M trehalose in DPBS + 10% FBS. Equilibration in room temperature was performed for 20–30 seconds for preantral follicle and 5 min equilibration was performed in an ice bath for ovaries. The samples were dropped onto the surface of metal plate around -180°C in the volume of 2 μl and 6 μl. After thawing, the ovarian tissue was mechanically isolated for collecting the preantral follicles. The thawed newborn ovaries were transplanted under the renal capsule of recipient male mice for 14 days. Preantral follicles collected from each groups were cultured individually in 20-μl droplets of α-MEM culture medium in culture dish for 12 days. On the day 12 of culture, the cumulus-oocyte complexes (COCs) were collected for IVM and IVF. Fertilization and embryo cleavage were scored.

Results

After the vitrification of 14-day-old preantral follicles using 2 μl or 6 μl droplet onto surface of metal plate, the results indicated that no significant difference in survival rate, antral-like cavity formation, COCs collected, 2 cell embryo cleavage and blastocyst development was found in vitrification of the 2 μl and 6 μl droplet groups. As comparing 14-day old ovarian tissue (ovarian tissue slices and whole ovaries) and whole newborn ovaries vitrified in 6 μl droplet, lower success rates of antral-like cavity formation and COCs collection were found in the whole ovaries group.

Conclusion

Our results suggest that the metal plate surface vitrification method is an appropriate and convenient method for cryopreservation of mouse ovaries and preantral follicles. The droplet volume of vitrification solution in 2 μl and 6 μl can be an option.     

Stimulation of mouse vibrissal follicle growth by recombinant human fibroblast growth factor 20

Objectives

To explore potential effects of recombinant human fibroblast growth factor 20 (rhFGF20) in the growth of cultured mouse vibrissal follicles.

Results

The growth of cultured mouse vibrissal follicles was significantly induced by rhFGF20 in a dose dependent pattern in the in vitro vibrissal follicle organ culture model. However, too high concentration of rhFGF20 could inhibit the growth of vibrissal follicles. We further demonstrated that rhFGF20 stimulated the proliferation of hair matrix cells and activated Wnt/β-catenin signaling pathway.

Conclusions

The rhFGF20 might be a potential therapeutic agent to treat hair loss disorders.

     

Kit ligand actions on ovarian stromal cells: effects on theca cell recruitment and steroid production

Factors that control recruitment of theca cells from ovarian stromal-interstitial cells are important for early follicle development in the ovary. During recruitment, theca cells organize into distinct layers around early developing follicles and establish essential cell-cell interactions with granulosa cells. Recruitment of theca cells from ovarian stromal stem cells is proposed to involve cellular proliferation, as well as induction of theca cell-specific functional markers. Previously, the speculation was made that a granulosa cell-derived "theca cell organizer" is involved in theca cell recruitment. Granulosa cells have been shown to produce kit-ligand/stem cell factor (KL). KL is known to promote stem cell proliferation and differentiation in a number of tissues. Therefore, the hypothesis was tested in the current study that granulosa cell-derived KL may help recruit theca cells from undifferentiated stromal stem cells during early follicle development. The actions of KL were examined using adult bovine ovarian fragment organ culture and isolated ovarian stromal-interstitial cells. In organ culture KL significantly increased the number of theca cell layers around primary follicles. Experiments using purified stromal-interstitial cell cultures showed that KL stimulated ovarian stromal cell proliferation in a dose-dependent manner. Stromal cell differentiation into theca cells was analyzed by the induction of theca cell functional markers (i.e., androstenedione and progesterone production). Bovine ovarian stromal cells produced low levels of androstenedione (5-40 ng/microg DNA) and progesterone (5-30 ng/microg DNA) in vitro that were approximately 20-fold lower than theca cells under similar conditions. Treatment with KL did not affect ovarian stromal cell androstenedione or progesterone production. Interestingly, hormones such as estrogen and hCG did stimulate stromal cell steroid production. The results in this study suggest that granulosa cell-derived KL appears to promote the formation of theca cell layers around small (i.e., primary) ovarian follicles. KL directly stimulated ovarian stromal cell proliferation but alone did not induce functional differentiation (i.e., high steroid production). Therefore, KL is proposed to promote early follicle development by inducing proliferation and organization of stromal stem cells around small follicles. Observations suggest that KL may act as a granulosa-derived "theca cell organizer" to promote stem cell recruitment of ovarian stromal cells in a manner similar to the way that KL promotes hematopoietic and lymphoid stem cells in bone marrow and the thymus.     

Effect of Antiprogesterone RU486 on VEGF Expression and Blood Vessel Remodeling on Ovarian Follicles before Ovulation

Background

The success of ovarian follicle growth and ovulation is strictly related to the development of an adequate blood vessel network required to sustain the proliferative and endocrine functions of the follicular cells. Even if the Vascular Endothelial Growth Factor (VEGF) drives angiogenesis before ovulation, the local role exerted by Progesterone (P₄) remains to be clarified, in particular when its concentration rapidly increases before ovulation.

Aim

This in vivo study was designed to clarify the effect promoted by a P₄ receptor antagonist, RU486, on VEGF expression and follicular angiogenesis before ovulation, in particular, during the transition from pre to periovulatory follicles induced by human Chorionic Gonadotropins (hCG) administration.

Material and Methods

Preovulatory follicle growth and ovulation were pharmacologically induced in prepubertal gilts by combining equine Chorionic Gonadotropins (eCG) and hCG used in the presence or absence of RU486. The effects on VEGF expression were analyzed using biochemical and immunohistochemical studies, either on granulosa or on theca layers of follicles isolated few hours before ovulation. This angiogenic factor was also correlated to follicular morphology and to blood vessels architecture.

Results and Conclusions

VEGF production, blood vessel network and follicle remodeling were impaired by RU486 treatment, even if the cause-effect correlation remains to be clarified. The P₄ antagonist strongly down-regulated theca VEGF expression, thus, preventing most of the angiogenic follicle response induced by hCG. RU486-treated follicles displayed a reduced vascular area, a lower rate of endothelial cell proliferation and a reduced recruitment of perivascular mural cells. These data provide important insights on the biological role of RU486 and, indirectly, on steroid hormones during periovulatory follicular phase. In addition, an in vivo model is proposed to evaluate how periovulatory follicular angiogenesis may affect the functionality of the corpus luteum (CL) and the success of pregnancy.     

The ultrastructure of the layers enveloping yolk-forming oocytes from Triturus viridescens

Conclusion The peripheral membranous and extracellular layers of oocytes at the onset of yolk formation were studied by electron microscopy. It was shown that three cellular layers are present at this time. The outer or surface epithelium contains typical squamous cells. The middle or theca is the connective tissue layer which contains fibroblasts, blood vessels, and collagen fibers. The inner or follicular epithelium proper consists of compactly arrayed follicle cells that have distinct cell boundaries. Two extracellular layers were observed, a coarse granular homogeneous layer and a dense zona radiata. Macrovilli (0.2 in diameter), extensions from the follicle cells, project through the extracellular layers into the peripheral cytoplasm while more numerous microvilli (0.1 in diameter) project up to the dense matrix of the zona radiata. The plasmolemma separating the peripheral cytoplasm from the follicle cells is completely irregular; it forms microvilli. The relations of the enveloping layers as seen with both light and electron microscopes are discussed.This investigation was supported by a Public Health Service research grant (5803-C3) and research career program award (K-3-5356) from the Division of General Medical Sciences.     

Galactoglucomannan oligosaccharides are assumed to affect tracheary element formation via interaction with auxin in Zinnia xylogenic cell culture

Key message

Galactoglucomannan oligosaccharides seem to interact with auxin in xylogenic cell culture, thus influencing mainly metaxylem-like tracheary element differentiation depending on timing with hormones and the process kinetics.

Abstract

Complex mapping of Zinnia mesophyll cell transdifferentiation into tracheary elements with or without prior cell division was documented after palisade and spongy parenchyma cell immobilization during the first 4 days of culture. Here, we report a positive effect of galactoglucomannan oligosaccharides on cell viability and density and higher metaxylem-like tracheary element formation in xylogenic cell culture. The maximal positive effect was achieved by the simultaneous addition of the oligosaccharides and growth hormones (auxin, cytokinin) to the cell culture medium. Moreover, a large number of metaxylem-like tracheary elements were observed in a low-auxin medium supplemented with oligosaccharides, but not in a low-cytokinin medium, suggesting a close relationship between auxin and the oligosaccharides during tracheary element formation.     

Genotypic variation in the ability of wheat roots to penetrate wax layers

Background and aims

The role of the root system in mediating crop yields has recently been emphasised, resulting in several laboratory approaches for phenotyping root traits. We aimed to determine the existence of, and reasons for, genotypic variation in wheat (Triticum aestivum L.) root penetration of strong wax layers.

Methods

Three contrasting groups (UK elite lines, CIMMYT lines and near-isogenic lines of cv Mercia containing dwarfing and semi-dwarfing Rht alleles) comprising 18 different genotypes with contrasting phenologies were studied. We determined the ability of roots of these genotypes to penetrate strong wax layers and the angular spread of the root systems.

Results

There were no intrinsic differences in root system ability to penetrate strong wax layers (consistent with the similar root diameter of all lines) since greater root penetration was simply related to more root axes. Recording root penetration of concentric zones of the wax layer demonstrated that cv. Battalion had a root system with a smaller angular spread than cv. Robigus, which had the root system with the greatest angular spread.

Conclusions

There was limited genotypic variability in root penetration of strong layers within the wheat cultivars studied. A key challenge will be to determine the physiological and agronomic significance of the variation in root angular spread.     

Spatial variation in root system activity of tomato (Solanum lycopersicum L.) in response to short and long-term waterlogging as determined by 15N uptake

Background and aim

Root system activity is affected by abiotic stresses, which often creates spatial differences in root conditions. This is expected to influence plants ability to cope with suboptimal conditions.

Methods

Changes in root system activity were determined as ¹⁵N root uptake in top and bottom layers of potted tomato plants (Solanum lycopersicum L.), after waterlogging the bottom layer for 24 h or 5 d. The plants were grown in peat-based media; non-compacted or highly-compacted, resulting in differences in gas diffusion, air permeability and oxygen availability.

Results

The roots were affected by short-term waterlogging (24 h) by decreasing uptake in the bottom layer and increasing uptake in the pot top layer. Long-term waterlogging (5 d) decreased the ¹⁵N root uptake more in both layers. Root uptake recovered fast (within 6 h) after short-term waterlogging, whereas recovery of long-term waterlogged roots took more than 24 h, suggesting production of new root biomass. Despite affecting physical properties, medium compaction did not affect root uptake. Aboveground biomass was affected by waterlogging by increasing the dry matter percentage, decreasing nitrogen (N) percentage and increasing starch content.

Conclusions

The results confirmed that root uptake in different layers of small pots could be distinguished by the ¹⁵N technique, which was applicable under potentially denitrifying conditions. The results demonstrated that during short-term stress in part of the root system plants increased uptake from the non-affected parts of the root system, probably as compensation for suboptimal conditions.     

Capacity of biological soil crusts colonized by the lichen <Emphasis Type="Italic">Diploschistes</Emphasis> to metabolize simple phenols

Background and aims

Soil compaction strongly affects water uptake by roots. The aim of the work was to examine soil—plant interactions with focus on the impact of distribution of compacted soil layers on growth and water uptake by wheat roots.

Methods

The growth-chamber experiment was conducted on wheat growth in soil with compacted soil layers. The system for maintaining constant soil water potential and measurement of daily water uptake from variously compacted soil layers was used.

Results

Layered soil compaction differentiated vertical root distribution to higher extent for root length than root mass. The propagation rate of a water extraction front was the highest through layers of moderately compacted soil. The root water uptake rate was on average 67 % higher from moderately than heavily compacted soil layers. Correlations between water uptake and the length of thick roots were increasing with increasing level of soil compaction.

Conclusions

The study shows that root amount, water uptake, propagation of water extraction and shoot growth strongly depend on the existence of compacted layers within soil profile. The negative effects of heavily compacted subsoil layer on water uptake were partly compensated by increased uptake from looser top soil layers and significant contribution of thicker roots in water uptake.     

Follicle growth in the ovary of the rabbit after ovulation-inducing application of human chorionic gonadotropin

Summary The growth of tertiary follicles, i.e., the proliferation of cells in the stratum granulosum and in the capillary network of the theca interna, after injection of ovulation-inducing human chorionic gonadotropin (HCG), was investigated in the rabbit by means of autoradiographic and morphometric methods.Based on the frequency distribution of follicles with different sizes and on the labeling index (LI) of granulosa cells as a function of follicle size and of time prior to and after HCG stimulation, two groups of tertiary tollicles can be distinguished: growing (250–900 m in diameter) and mature (>900 m in diameter) elements. The growth of both groups is influenced by the release of gonadotropins.After HCG stimulation, follicles belonging to the first group grow rapidly. During, and a short time after ovulation, almost all non-ruptured follicles larger than 600 m in diameter become atretic. Within 35–50 h the ruptured and atretic mature follicles (>900 m in diameter) are replaced by follicles out of the group of growing follicles.From these results the following concept for regulation of follicle growth is derived: In principle, all growing follicles possess the potential to develop into mature follicles. When a sufficient number of mature follicles is generated, these mature follicles determine the number of succeeding growing follicles. Follicles that are not required for providing mature follicles become atretic as soon as they reach a diameter of 700 m. When the majority of mature follicles is lost during ovulation (by rupture or atresia), this inhibition regulated by mature follicles is abolished, and all of the growing follicles again are capable to develop into mature follicles.The relative amount of capillaries in the theca interna of growing and mature follicles remains constant with increasing follicle size. This means that the capillary network grows parallel to the increasing size of follicles. No differences are found between intact and atretic follicles; advanced atretic follicles were excluded from this study.The labeling index (LI) of granulosa cells in the stratum granulosum and of endothelial cells in the theca interna, as a function of follicle size and of time after HCG stimulation, are closely correlated. A change in the LI of granulosa cells is usually followed with a certain delay by a similar alteration of the LI of endothelial cells in the theca interna. This suggests that granulosa cells have a certain regulatory function on capillary growth.     

Speckle reducing bilateral filter for cattle follicle segmentation

Background

Ultrasound imaging technology has wide applications in cattle reproduction and has been used to monitor individual follicles and determine the patterns of follicular development. However, the speckles in ultrasound images affect the post-processing, such as follicle segmentation and finally affect the measurement of the follicles. In order to reduce the effect of speckles, a bilateral filter is developed in this paper.

Results

We develop a new bilateral filter for speckle reduction in ultrasound images for follicle segmentation and measurement. Different from the previous bilateral filters, the proposed bilateral filter uses normalized difference in the computation of the Gaussian intensity difference. We also present the results of follicle segmentation after speckle reduction. Experimental results on both synthetic images and real ultrasound images demonstrate the effectiveness of the proposed filter.

Conclusions

Compared with the previous bilateral filters, the proposed bilateral filter can reduce speckles in both high-intensity regions and low intensity regions in ultrasound images. The segmentation of the follicles in the speckle reduced images by the proposed method has higher performance than the segmentation in the original ultrasound image, and the images filtered by Gaussian filter, the conventional bilateral filter respectively.

     

Catecholamine content of the preovulatory follicles of the domestic hen

The role of catecholamines in ovarian function of the domestic hen has not been examined extensively. The aim of this study was first to determine the location of catecholamines in the preovulatory follicle of the domestic hen. Second, norepinephrine (NE), epinephrine (EPI) and dopamine (DA) were measured in the isolated theca layer of the five largest preovulatory follicles at specific times during the ovulatory cycle and changes in catecholamine content were correlated with ovarian events. The five largest preovulatory follicles were removed from chickens at 24, 18, 12, 6 and 2 h before ovulation of the largest (F1) follicle. Theca and granulosa layers were isolated, frozen, weighed and prepared for measurements of catecholamines by the double isotope radio-enzymatic assay. Catecholamines were localized primarily in the theca layer with only small amounts present in the granulosa layer. Norepinephrine was present in the theca layer in concentrations 6- and 30-fold those of EPI and DA, respectively. The content of NE and EPI in the theca layer of the F1 follicle was significantly (p less than 0.01) higher at 6 h before ovulation than at other times for the F1 follicle. In contrast, NE and EPI content of the theca layer of second (F2) and third (F3) largest follicles did not change during the ovulatory cycle. The content of DA was elevated (p less than 0.05) at 12 h before ovulation in F1 and F2 follicles. There was a significant reduction in NE in the theca layer of the fifth largest (F5) follicle between 24 and 18 h before ovulation of the F1 follicle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of cellular and vascular changes in equine follicles during hCG-induced ovulation

In contrast to other species, the histology of the equine follicle during ovulation has not been described. Preovulatory follicles were isolated during oestrus at 0, 12, 24, 30, 33, 36 and 39 h (n = 5-6 follicles per time point) after an ovulatory dose of hCG to characterize the cellular and vascular changes associated with ovulation in mares. Pieces of follicle wall were formalin-fixed and processed for light microscopy to evaluate the general follicular morphology and quantify selected parameters. Marked changes were observed in the histology of equine follicles in the hours before ovulation. The thickness of the granulosa cell layer doubled between 0 and 39 h after hCG (77.8 +/- 4.8 versus 158.8 +/- 4.8 microns, respectively; P < 0.01). This expansion was caused primarily by a pronounced accumulation of acid mucosubstances between granulosa cells, which was first detected at 12 h after hCG and peaked at 36-39 h. In contrast, a significant thinning of the theca interna was observed after hCG treatment. Fewer cell layers were present; theca interna cells appeared smaller than before hCG; and the presence of occasional pyknotic cells was noted at 36 and 39 h after hCG. In addition, the theca layers were invaded by numerous eosinophils. No eosinophils were observed in preovulatory follicles isolated between 0 and 24 h after hCG, but the number increased to 14.0 +/- 0.8 and 5.6 +/- 0.3 eosinophils per field (x 400) in theca interna and theca externa, respectively, 39 h after hCG treatment (P < 0.01). Severe oedema, hyperaemia and haemorrhages, and significant increases in the number of blood vessels in theca interna and externa were observed at 33, 36 and 39 h after hCG. This study provides the first in-depth characterization of the sequential cellular and vascular changes that occur in equine follicles before ovulation.     

Lacker's model: control of follicular growth and ovulation in domestic species

Lacker (1981) and Lacker & Akin (1988) developed a mathematical model of follicular maturation and ovulation; this model of only four parameters accounts for a large number of results obtained over the past decade or more on the control of follicular growth and ovulation in mammals. It establishes a single law of maturation for each follicle which describes the interactions between growing follicles. The function put forward is sufficient to explain the constancy of the number of ovulations or large follicles in a female as well as the variability of this number among strains or species and for either induced or spontaneous ovulators. According to the model, the number of ovulations or large follicles lies between two limits that are themselves simple functions of two parameters of the model. Moreover, Lacker's model exhibits interesting characteristics in agreement with results obtained by physiologists: in particular, it predicts that the number of ovulating or large follicles is independent of:

1. the total number of maturing follicles,
2. the process of recruitment of newly maturing follicles towards the terminal maturation (Poisson or other),
3. the form of the LH or FSH secretion curves as functions of the systemic level of oestradiol. The model further predicts that
4. selection and dominance of follicles result from the feedback between the ovary and the hypophysis through the interactions between follicles; these interactions are expressed by the maturation function of the model.
5. recovery from atresia is possible for a follicle: from decreasing, the rate of secretion of oestradiol may increase.
6. the revised model suggests a renewal of follicles during the sexual cycle, as “waves of follicular growth”.

Lacker's model is a model of strict dominance; it maintains a hierarchy of the follicles as soon as they start their final maturation to the ovulations as that is observed in bird or reptile ovary. Such a strict hierarchy is possible but it is probably not a general situation in all mammals. We therefore modified the maturing function of the follicle in order to make it compatible with the observations of physiologists: follicles always interact as in the initial model but they individually become old, the hierarchy of follicles can be modified with time and the largest follicles do not indefinitely grow as in the initial model.     

Evaluation of three-dimensional SonoAVC ultrasound for antral follicle count in infertile women: its agreement with conventional two-dimensional ultrasound and serum levels of anti-Müllerian hormone

Background

Several studies have reported a correlation between antral follicle count by conventional 2D transvaginal sonography and serum anti-Müllerian hormone levels. However, few studies have investigated the effectiveness of 3D SonoAVC transvaginal ultrasound technology, particularly in infertile women. Therefore, this study aims to evaluate the usefulness of three-dimensional (3D) SonoAVC transvaginal ultrasound technology for antral follicle count and its correlation to conventional two-dimensional (2D) transvaginal ultrasound and serum levels of anti-Müllerian hormone in infertile women.

Methods

This cross-sectional study included 42 infertile women with age lower than 40 years that underwent treatment at a private fertility clinic between June and December 2015. Patient data included age, body mass index and cause of infertility. On cycle day 3 the following hormone levels were measured: serum levels of anti-Müllerian hormone, follicle-stimulating hormone, cancer antigen 125, prolactin, thyroid-stimulating hormone and oestradiol; the number of antral follicles was counted as well. The scanning were performed through 2D and 3D technology transvaginal ultrasound.

Results

Using a Bland-Altman test we demonstrated that both technologies are quite equivalent. However, antral follicle count is higher using 3D ultrasound technology compared to 2D technology (p?<?0.001; Wilcoxon test), this finding is mainly remarkable in ovaries with more than 20 antral follicles. Moreover, the mean time required for manual 2D ultrasound and 3D SonoAVC measurements were 275?±?109 and 103?±?57 s, respectively (p?<?0.001). Serum AMH concentration correlated to the total number of early antral follicles (correlation coefficients?=?0.678 and 0.612; p?<?0.001 by 2D ultrasound and 3D SonoAVC, respectively; Spearman’s correlation test).

Conclusions

Antral follicle count is better estimated using 3D ultrasound compared to 2D technology. A great advantage of 3D SonoAVC was less time required for an examination and the visual advantage when it need to count more than 20 follicles.

Trial registration

CAAE: 35141114.4.0000.5327. Registered 10 June 2015.

     

Growth and antrum formation of bovine preantral follicles in long-term culture in vitro

Culture of preantral follicles has important biotechnological implications through its potential to produce large quantities of oocytes for embryo production and transfer. A long-term culture system for bovine preantral follicles is described. Bovine preantral follicles (166 +/- 2.15 micrometer), surrounded by theca cells, were isolated from ovarian cortical slices. Follicles were cultured under conditions known to maintain granulosa cell viability in vitro. The effects of epidermal growth factor (EGF), insulin-like growth factor (IGF)-I, FSH, and coculture with bovine granulosa cells on preantral follicle growth were analyzed. Follicle and oocyte diameter increased significantly (P < 0.05) with time in culture. FSH, IGF-I, and EGF stimulated (P < 0.05) follicle growth rate but had no effect on oocyte growth. Coculture with granulosa cells inhibited FSH/IGF-I-stimulated growth. Most follicles maintained their morphology throughout culture, with the presence of a thecal layer and basement membrane surrounding the granulosa cells. Antrum formation, confirmed by confocal microscopy, occurred between Days 10 and 28 of culture. The probability of follicles reaching antrum development was 0.19 for control follicles. The addition of growth factors or FSH increased (P < 0.05) the probability of antrum development to 0.55. Follicular growth appeared to be halted by slower growth of the basement membrane, as growing follicles occasionally burst the basement membrane, extruding their granulosa cells. In conclusion, a preantral follicle culture system in which follicle morphology can be maintained for up to 28 days has been developed. In this system, FSH, EGF, and IGF-I stimulated follicle growth and enhanced antrum formation. This culture system may provide a valuable approach for studying the regulation of early follicular development and for production of oocytes for nuclear/embryo transfer, but further work is required.