Asymmetry in previtellogenic and early vitellogenic oocytes, ultrastructure of follicular cells and egg envelope in the pigmented sterlet, Acipenser ruthenus L. 1758 (Chondrostei, Acipenseriformes).

Ovarian follicles of sterlets (Acipenser ruthenus) are composed of a single oocyte surrounded by follicular cells (FCs), basal lamina, and thecal cells. Previtellogenic oocytes are polarized. Homogeneous ooplasm (contains ribosomes) and granular ooplasm (comprises nuage aggregations of nuclear origin, rough endoplasmic reticulum (RER), Golgi complexes, ribosomes, and mitochondria) are distinguished. Granular ooplasm is initially located near the nucleus, contacts the plasma membrane of the oocyte (oolemma) and forms a thin layer underneath its entire perimeter. Next, a ring that surrounds the nucleus is formed and sends strands directed toward the oolemma. The lipid body composed of lipid droplets forms adjacent to this ring. Later, the granular ooplasm and strands enlarge toward the oolemma, lipid body disperses, and homogeneous ooplasm is no longer present. A thin cortical ooplasm is formed underneath the oolemma and does not contain any organelles. The oocyte nucleus moves to the center. The nucleoplasm contains lampbrush chromosomes, nuclear bodies, and multiple nucleoli. Early vitellogenic oocytes are polarized, too. Three regions in the ooplasm are distinguished: the perinuclear (contains lipid droplets near the nuclear envelope), the endoplasm (contains yolk platelets and lipid droplets), and the periplasm (contains yolk spheres, pigment granules, and microtubules). In all these regions the RER, Golgi complexes, nuage, and mitochondria are present. Micropinocytotic vesicles, Golgi vesicles and precursors of the internal layer of the egg envelope are in the cortical ooplasm. Some FCs delaminate from the follicular epithelium, degenerate and vesicles are released into the perioocytic space. They may contain precursors of egg envelope and may be involved in "cell-cell" communication. The egg envelope (zona radiata, zona pellucida) is made up of three layers: the vitelline envelope (inner layer), the middle layer, and the outer layer. In its deposition, both the oocyte and FCs are engaged.

Microscopic study of the primary growth ovarian follicles of the pike-perch Sander lucioperca (Linnaeus 1758) (Actinopterygii, Perciformes).

The ovaries of Sander lucioperca (Actinopterygii, Perciformes) are made up of the germinal epithelium and ovarian follicles, in which primary oocytes grow. Each follicle is composed of an oocyte surrounded by flattened follicular cells, the basal lamina, and thecal cells. The early stages of oocyte development (primary growth = previtellogenesis) are not fully explained in this species. The results of research with the use of stereoscopic, light, fluorescence, and transmission electron microscopes on ovarian follicles containing developing primary oocytes of S. lucioperca are presented. The polarization and ultrastructure of oocytes are described and discussed. The deposition of egg envelopes during the primary growth and the ultrastructure of the eggshell in maturing follicles of S. lucioperca are also presented. Nuclei in primary oocytes comprise lampbrush chromosomes, nuclear bodies, and nucleoli. Numerous additional nucleoli arise in the nucleoplasm during primary growth and locate close to the nuclear envelope. The Balbiani body in the cytoplasm of oocytes (ooplasm) is composed of nuage aggregations of nuclear origin and mitochondria, endoplasmic reticulum (ER), and Golgi apparatus. The presence of the Balbiani body was reported in oocytes of numerous species of Actinopterygii; however, its ultrastructure was investigated in a limited number of species. In primary oocytes of S. lucioperca, the Balbiani body is initially located in the perinuclear ooplasm on one side of the nucleus. Next, it surrounds the nucleus, expands toward the plasma membrane of oocytes (oolemma), and becomes fragmented. Within the Balbiani body, the granular nuage condenses in the form of threads, locates near the oolemma, at the vegetal oocyte pole, and then dissolves. Mitochondria and cisternae of the rough endoplasmic reticulum (RER) are present between the threads. During primary growth micropylar cells differentiate in the follicular epithelium. They contain cisternae and vesicles of the RER and Golgi apparatus as well as numerous dense vesicles suggesting high synthetic and secretory activity. During the final step of primary growth several follicular cells delaminate from the follicular epithelium, migrate toward the oocyte and submerge in the most external egg envelope. In the ooplasm, three regions are distinguished: perinuclear, endoplasm, and periplasm. Cortical alveoli arise in the perinuclear ooplasm and in the endoplasm as a result of the fusion of RER vesicles with Golgi ones. They are evenly distributed. Lamellar bodies in the periplasm store the plasma membrane and release it into a space between the follicular cells and the oocyte. The developing eggshell in this space is made up of two egg envelopes (the internal one and the external) that are pierced by canals formed around the microvilli of oocytes and the processes of follicular cells. In the deposition of egg envelopes the oocyte itself and follicular cells are engaged. In maturing ovarian follicles the eggshell is solid and the internal egg envelope is covered with protuberances.

Influence of Age and Breed on Bovine Ovarian Capillary Blood Supply, Ovarian Mitochondria and Telomere Length.

Worldwide, dairy cows of the type of high-producing cattle (HPC) suffer from health and fertility problems at a young age and therefore lose productivity after an average of only three lactations. It is still contentious whether these problems are primarily due to genetics, management, feeding or other factors. Vascularization plays a fundamental role in the cyclic processes of reproductive organs, as well as in the regeneration of tissues. In a previous study, HPC were shown to have a greater ovarian corpus luteum vascularization compared to dual-purpose breeds. We hypothesize that this activated angiogenesis could likely lead to an early exhaustion of HPC's regenerative capacity and thus to premature reproductive senescence. The objective of this study was to investigate if a HPC breed (Holstein-Friesian, HF) exhibits higher ovarian angiogenesis than a dual-purpose breed (Polish Red cow, PR) and if this is related to early ovarian aging and finally reproductive failure. For this purpose, we assessed the degree of vascularization by means of ovarian blood vessel characterization using light microscopy. As indicators for aging, we measured ovarian mitochondrial size and telomere length in peripheral leukocytes. We report in this study that in both breeds the distance between capillaries became smaller with increasing age and that the mean telomere length decreased with increasing age. The only difference between the two breeds was that PR developed larger capillaries than HF. Neither a relationship between telomere length, nor the morphology of the mitochondrial apparatus and nor angiogenesis in HF was proven. Although the data trends indicated that the proportion of shortened telomeres in HF was higher than in the PR, no significant difference between the two breeds was detected.

Mitochondrial content, activity, and morphology in prepubertal and adult human ovaries.

PURPOSE: To investigate whether mitochondrial content, activity, and morphology differ in prepubertal versus adult ovarian follicles. METHODS: Ovarian tissue was collected from 7 prepubertal girls (age 1-10 years) and 6 adult women (age 20-35 years). Primordial and primary follicles were isolated from frozen-thawed prepubertal and adult ovarian tissue and their viability was assessed. Mitochondrial content was investigated by TOMM20 immunostaining of prepubertal and adult ovarian tissue, while mitochondrial activity in isolated follicles was analyzed by MitoTracker CM-H2XRos and JC-1. Frozen-thawed ovarian tissue from the same patients was also evaluated by transmission electron microscopy to examine mitochondrial morphology. RESULTS: Higher TOMM20 staining was detected in prepubertal follicles compared to their adult counterparts, indicating the presence of more mitochondria in prepubertal follicles. Analysis of mitochondrial activity by MitoTracker showed higher fluorescence intensity in prepubertal follicles, suggesting that follicles in this group are more active than adult follicles. JC-1 analysis did not reveal any statistically significant difference in the inactive/active ratio between the two groups. Moreover, ultrastructural analysis by TEM detected morphological differences in the shape and cristae of prepubertal mitochondria, probably suggesting a mechanism of response to autophagy. CONCLUSION: Differences in the number, activity, and morphology of mitochondria were reported, suggesting that consequential modifications might occur during puberty, which could be the window of opportunity required by mitochondria to undergo changes needed to reach maturity, and hence the capacity for ovulation and fertilization.

The effects of the anti-Müllerian hormone on folliculogenesis in rats: light and electron microscopic evaluation.

In this study, we evaluated the effects of anti-Müllerian hormone on follicle development and oocyte quality with light and electron microscopy. Twenty-four adult female rats were divided into four groups. After estrous cycle synchronization, on the first day, control group rats were injected with 0.5 ml saline, 2nd, 3rd, and 4th groups were injected 1 µgr, 2 µgr, and 5 µgr anti-Müllerian hormone, respectively. On the third day, intracardiac blood samples were taken for follicle-stimulating hormone, luteinizing hormone, estradiol, and progesterone serum level measurements. Ovaries were obtained for light and electron microscopic examinations. Secondary (antral) follicles were decreased while atretic follicles were increased in number parallel with an increased dose of anti-Müllerian hormone injection. Atresia of the follicles was demonstrated with apoptosis of granulosa cells characterized by apoptotic bodies and with paraptosis characterized by the vacuole formation in the cytoplasm, enlargement of granular endoplasmic reticulum cisternae and perinuclear cisternae in granulosa cells. Premature luteinization characterized by increased lipid droplets, mitochondria with tubular cristae, and smooth-surfaced endoplasmic reticulum in the cytoplasm of granulosa cells were detected in some growing follicles. In the anti-Müllerian hormone injected experimental groups, cystic follicles characterized by a large antrum, attenuated granulosa cell layer, and flattened granulosa cells that face the antrum were observed. Corpus luteum and stroma were similar in all groups. It was concluded that increasing doses of anti-Müllerian hormone caused increased atresia in developing follicles, premature luteinization of granulosa cells in some follicles, and cystic follicle formation in the further developing follicles.

5-Fluorouracil disrupts ovarian preantral follicles in young C57BL6J mice.

PURPOSE: 5-Fluorouracil (5-FU), an anti-cancer drug, has been used for hepatoblastoma (HB) chemotherapy in children, who may have impaired  ovarian follicle pool reserve with lasting effects to reproduction. Therefore, this study aimed to investigate 5-FU effects on survival, growth, and morphology of ovarian preantral follicles from C57BL6J young mice. METHODS: Experiments were carried-out both in vivo and in vitro. Mice were treated with 5-FU injection (450 mg/kg i.p) or saline and sacrificed 3 days after to obtain ovaries for histology and molecular biology. Ovaries for in vitro studies were obtained from unchallenged mice and cultured under basic culture medium (BCM) or BCM plus 5-FU (9.2, 46.1, 92.2 mM). Preantral follicles were classified according to developmental stages, and as normal or degenerated. To assess cell viability, caspase-3 immunostaining was performed. Transcriptional levels for apoptosis (Bax, Bcl2, p53, Bax/Bcl2) and Wnt pathway genes (Wnt2 and Wnt4) were also analyzed. Ultrastructural analyses were carried-out on non-cultured ovaries. In addition, ß-catenin immunofluorescence was assessed in mouse ovaries. RESULTS: The percentage of all-types normal follicles was significantly lower after 5-FU challenge. A total loss of secondary normal follicles was found in the 5-FU group. The highest 5-FU concentrations reduced the percentage of cultured normal primordial follicles. Large vacuoles were seen in granulosa cells and ooplasm of preantral follicles by electron microscopy. A significantly higher gene expression for Bax and Bax/Bcl2 ratio was seen after 5-FU treatment. A marked reduction in ß-catenin immunolabeling was seen in 5-FU-challenged preantral follicles. In the in vitro experiments, apoptotic and Wnt gene transcriptions were significantly altered. CONCLUSION: Altogether, our findings suggest that 5-FU can deleteriously affect the ovarian follicle reserve by reducing preantral follicles survival.

A role for orphan nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) in primordial follicle activation.

Liver receptor homolog-1 (NR5A2) is expressed specifically in granulosa cells of developing ovarian follicles where it regulates the late stages of follicle development and ovulation. To establish its effects earlier in the trajectory of follicular development, NR5A2 was depleted from granulosa cells of murine primordial and primary follicles. Follicle populations were enumerated in neonates at postnatal day 4 (PND4) coinciding with the end of the formation of the primordial follicle pool. The frequency of primordial follicles in PND4 conditional knockout (cKO) ovaries was greater and primary follicles were substantially fewer relative to control (CON) counterparts. Ten-day in vitro culture of PND4 ovaries recapitulated in vivo findings and indicated that CON mice developed primary follicles in the ovarian medulla to a greater extent than did cKO animals. Two subsets of primordial follicles were observed in wildtype ovaries: one that expressed NR5A2 and the second in which the transcript was absent. Neither expressed the mitotic marker. KI-67, indicating their developmental quiescence. RNA sequencing on PND4 demonstrated that loss of NR5A2 induced changes in 432 transcripts, including quiescence markers, inhibitors of follicle activation, and regulators of cellular migration and epithelial-to-mesenchymal transition. These experiments suggest that NR5A2 expression poises primordial follicles for entry into the developing pool.

Comparison of methods for quantifying primordial follicles in the mouse ovary.

BACKGROUND: Accurate evaluation of primordial follicle numbers in mouse ovaries is an essential endpoint for studies investigating how endogenous and exogenous insults, such as maternal aging and chemotherapy, impact the ovarian reserve. In this study, we compared and contrasted two methods for counting healthy primordial follicles following exposure to cyclophosphamide (75 mg/kg), a well-established model of follicle depletion. The first was the fractionator/optical dissector technique, an unbiased, assumption-free stereological approach for quantification of primordial follicle numbers. While accurate, highly reproducible and sensitive, this method relies on specialist microscopy equipment and software, requires specific fixation, embedding and sectioning parameters to be followed, and is largely a manual process that is tedious and time-consuming. The second method was the more widely used serial section and direct count approach, which is relatively quick and easy. We also compared the impacts of different fixatives, embedding material and section thickness on the overall results for each method. RESULTS: Direct counts resulted in primordial follicle numbers that were significantly lower than those obtained by stereology, irrespective of fixation and embedding material. When applied to formalin fixed tissue, the direct count method did not detect differences in follicle numbers between saline and cyclophosphamide treated groups to the same degree of sensitivity as the gold standard stereology method (referred to as the Reference standard). However, when Bouin's fixative was used, direct counts and stereology were comparable in their ability to detect follicle depletion caused by cyclophosphamide. CONCLUSIONS: This work indicates that the direct count method can produce similar results to stereology when Bouin's fixative is used instead of formalin. The findings presented here will assist others to select the most appropriate experimental approach for accurate follicle enumeration, depending on whether the primary objective of the study is to determine absolute primordial follicle numbers or relative differences between groups.

Vps13 is required for timely removal of nurse cell corpses.

Programmed cell death and consecutive removal of cellular remnants is essential for development. During late stages of Drosophila melanogaster oogenesis, the small somatic follicle cells that surround the large nurse cells promote non-apoptotic nurse cell death, subsequently engulf them, and contribute to the timely removal of nurse cell corpses. Here, we identify a role for Vps13 in the timely removal of nurse cell corpses downstream of developmental programmed cell death. Vps13 is an evolutionarily conserved peripheral membrane protein associated with membrane contact sites and lipid transfer. It is expressed in late nurse cells, and persistent nurse cell remnants are observed when Vps13 is depleted from nurse cells but not from follicle cells. Microscopic analysis revealed enrichment of Vps13 in close proximity to the plasma membrane and the endoplasmic reticulum in nurse cells undergoing degradation. Ultrastructural analysis uncovered the presence of an underlying Vps13-dependent membranous structure in close association with the plasma membrane. The newly identified structure and function suggests the presence of a Vps13-dependent process required for complete degradation of bulky remnants of dying cells.

Comparative effects of TiO2 and ZnO nanoparticles on growth and ultrastructure of ovarian antral follicles.

Titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NP) have been demonstrated to reach the ovary. However, the potential detrimental effects of these metal-based NP on ovarian antral follicles and whether they can be directly taken up by follicular cells are unknown. The aim of this study was to evaluate whether TiO2 and ZnO NP internalize into the antral follicle, and further compared any potential detrimental effects of either NP on growth, ultrastructure and viability of antral follicles. It has been described that TiO2 and ZnO NP induce oxidative stress, thus this study indirectly assessed whether oxidative stress was involved. Antral follicles were cultured with TiO2 (5, 25 and 50 µg/mL) or ZnO (5, 15 and 25 µg/mL) NP for 96 h. TiO2 NP were internalized and agglomerated into cells, increased follicle diameter and disrupted the cytoskeleton arrangement, effects that were partially prevented by a co-exposure with trolox. Moreover, ZnO NP partially dissolved into culture media, decreased follicle diameter, and disrupted cytoskeletal arrangement, and these effects were not prevented by trolox. Ultrastructural alterations induced by exposure to both NP were evidenced by impaired transzonal projections and swelling mitochondria. Oxidative stress mediates TiO2 NP-induced effects but not those from ZnO NP in antral follicle development. Our results suggest that both NP induced ovarian follicle toxicity through different toxic mechanisms, possibly due to a stimulation of ZnO NP solubility and agglomeration of TiO2 NP into the follicular cells.

Follicle populations and vascularization in ovarian tissue of pediatric patients before and after long-term grafting.

OBJECTIVE: To characterize ovarian tissue from pediatric patients by evaluating development and vascularization in follicle populations and comparing it with adult tissue after xenografting. DESIGN: Prospective experimental study. SETTING: Academic research center. PATIENT(S): Five children (median age 3 years) and seven women (median age 28 years). INTERVENTION(S): Hematoxylin and eosin staining, immunofluorescence, and transmission electron microscopy (TEM) evaluation before and after grafting. MAIN OUTCOME MEASURE(S): Follicle density, morphology, classification, and size, ovarian tissue vascularization, follicle ultrastructure. RESULT(S): Frozen-thawed ovarian tissue was divided into three fragments: nongrafted controls, TEM, and xenografting for 20 weeks. Follicle density was statistically significantly higher in pediatric than adult patients; even though it decreased in both groups after transplantation, it remained higher in pediatric patients. In the pediatric group, quiescent-stage follicles were the majority of the follicle pool before and after grafting, while growing follicles statistically significantly increased in both groups after grafting. Abnormal and atretic follicles were also observed in pediatric tissue and declined with age and after grafting. Pediatric ovarian tissue contained more and larger immature vessels, while mature vessels were larger in adults. The TEM analysis of abnormal pediatric follicles showed loss of shape and vacuolization of the cytoplasm without organelle damage. CONCLUSION(S): Statistically significant differences in follicle density were observed between pediatric and adult patients, but the follicle proportions were similar before and after grafting, with the exception of atretic and abnormal follicles. Pediatric tissue contains more and larger immature vessels than adult tissue, and the posttransplantation revascularization process is accelerated in this group.

The Attenuating Effect of the Intraovarian Bone Morphogenetic Protein 4 on Age-Related Endoplasmic Reticulum Stress in Chicken Follicular Cells.

In the poultry, only less than 5% primordial follicles in the ovary can develop into the prehierarchical follicles (PHFs) leading to progressive development, ovulation, and egg formation. This low rate of recruitment indicates a huge potential for improvement of the laying performance. A great reduction in egg production is caused by aging with extensive follicular atresia. In this study, age-related changes in the laying performance and ovarian status were compared between the peak-lay (D280) and aged (D580) chickens. Subsequently, a cross coculture of PHFs and granulosa cells (GCs) from D280 or D580 hens was adopted to reveal the mechanism of declined follicle development. Results showed that persistent endoplasmic reticulum (ER) stress in GCs of the aged hens was accompanied with intensified apoptosis. Bone morphogenetic protein 4 (BMP4) secreted by GCs of PHFs in D280 hens was capable of relieving ER stress and improving follicular dominance for selection in D580 hens. During this action, BMP4 reduced free calreticulin (CALR, an ER marker) content and attenuated cell apoptosis in PHFs of D580 hens via the PERK-CHOP-BCL2/caspase3 or CALR-Ca2+-BCL2-caspase12 pathway. Furthermore, BMP4 prevented follicular atresia by promoting production of steroid hormones to improve survival of GCs in PHFs from the aged hens. In conclusion, intensified ER stress and apoptosis occurred in GCs of PHFs in aged chickens, while BMP4 secreted by GCs was capable of improving follicular viability by alleviating ER stress to promote follicular development.

Asymmetry in the cytoplasm of oocytes of largescale yellowfish Labeobarbus marequensis Smith 1841 (Teleostei: Cypriniformes: Cyprinidae).

The ovaries of the largescale yellowfish, Labeobarbus marequensis (Teleostei: Cypriniformes: Cyprinidae), are made up of the germinal epithelium, nests of late chromatin nucleolus stage oocytes, and ovarian follicles. Each follicle is composed of a single oocyte, which is surrounded by somatic follicular cells and a basal lamina covered by thecal cells. We describe polarization and ultrastructure of oocytes during the primary growth stage. The oocyte nucleus contains lampbrush chromosomes, nuclear bodies and fibrillar material in which multiple nucleoli arise. Nuage aggregations composed of material of a nuclear origin are present in the perinuclear cytoplasm. The Balbiani body (Bb) contains aggregations of nuage, rough endoplasmic reticulum, individual mitochondria and complexes of mitochondria with nuage (cement). Some mitochondria in the Bb come into close contact with endoplasmic reticulum cisternae and vesicles that contain granular material. At the start of primary growth, the Bb is present in the cytoplasm close to the nucleus. Next, it expands towards the oocyte plasma membrane. In these oocytes, a spherical structure, the so-called yolk nucleus, arises in the Bb. It consists of granular nuage in which mitochondria and vesicles containing granular material are immersed. Later, the Bb becomes fragmented and a fully grown yolk nucleus is present in the vegetal region. It contains numerous threads composed of granular nuage, mitochondria, lysosome-like organelles and autophagosomes. We discuss the formation of autophagosomes in the cytoplasm of primary growth oocytes. During the final step of primary growth, the cortical alveoli arise in the cytoplasm and are distributed evenly. The eggshell is deposited on the external surface of the oocyte plasma membrane and is made up of two egg envelopes that are pierced by numerous pore canals. The external egg envelope is covered in protuberances. During primary growth no lipid droplets are synthesized or stored in the oocytes.

Cellular Heterogeneity of the Luteinizing Hormone Receptor and Its Significance for Cyclic GMP Signaling in Mouse Preovulatory Follicles.

Meiotic arrest and resumption in mammalian oocytes are regulated by 2 opposing signaling proteins in the cells of the surrounding follicle: the guanylyl cyclase natriuretic peptide receptor 2 (NPR2), and the luteinizing hormone receptor (LHR). NPR2 maintains a meiosis-inhibitory level of cyclic guanosine 5'-monophosphate (cGMP) until LHR signaling causes dephosphorylation of NPR2, reducing NPR2 activity, lowering cGMP to a level that releases meiotic arrest. However, the signaling pathway between LHR activation and NPR2 dephosphorylation remains incompletely understood, due in part to imprecise information about the cellular localization of these 2 proteins. To investigate their localization, we generated mouse lines in which hemagglutinin epitope tags were added to the endogenous LHR and NPR2 proteins, and used immunofluorescence and immunogold microscopy to localize these proteins with high resolution. The results showed that the LHR protein is absent from the cumulus cells and inner mural granulosa cells, and is present in only 13% to 48% of the outer mural granulosa cells. In contrast, NPR2 is present throughout the follicle, and is more concentrated in the cumulus cells. Less than 20% of the NPR2 is in the same cells that express the LHR. These results suggest that to account for the LH-induced inactivation of NPR2, LHR-expressing cells send a signal that inactivates NPR2 in neighboring cells that do not express the LHR. An inhibitor of gap junction permeability attenuates the LH-induced cGMP decrease in the outer mural granulosa cells, consistent with this mechanism contributing to how NPR2 is inactivated in cells that do not express the LHR.

Morphogenesis of the ovarian follicular epithelium during initial stages of embryogenesis of the viviparous earwig, Hemimerus talpoides.

Representatives of the highly specialized earwig family Hemimeridae are epizoic and viviparous. Their embryos develop inside terminal ovarian follicles (termed also embryonic follicles) and rely solely on nutrients transferred from mother tissues. In this report, we present results of ultrastructural and histochemical studies of the initial stage of Hemimerus talpoides development. Our results show that the follicular cells surrounding fully grown oocyte of Hemimerus do not degenerate after initiation of embryogenesis, but transform and gradually form the wall of the incubation chamber in which the embryo develops. We also show that amniotic cells of the early embryo remain in direct contact with transformed follicular cells. In the region of contact, short outgrowths of the amniotic cells associate with irregular surface specializations of the transformed follicular cells. We suggest that extended "postfertilization" activity of hemimerid follicular cells represents an adaptation to viviparity and matrotrophy in this insect lineage.

Ultrastructure and intercellular contact-mediated communication in cultured human early stage follicles exposed to mTORC1 inhibitor.

The reproductive lifespan of a woman is determined by the gradual recruitment of quiescent follicles into the growing pool. In humans, ovarian tissue removal from its in vivo environment induces spontaneous activation of resting follicles. Similarly, pharmacological activation of the PI3K/Akt pathway leads to accelerated follicle recruitment, but has been associated with follicular damage. Recent findings demonstrate that everolimus (EVE), an mTORC1 inhibitor, limits primordial follicle activation. However, its potential benefit regarding growing follicle integrity remains unexplored. Ovarian cortical fragments were exposed to ± EVE for 24 h and cultured for an additional 5 days. After 0, 1 and 6 days of culture, fragments were either processed for ultrastructural analysis or subjected to follicular isolation for gene expression and immunofluorescence assessments. Data from transmission electron microscopy showed that growing follicles displayed similar ultrastructural features irrespective of the conditions and maintained close contacts between germinal and stromal compartments. Establishment of intra-follicular communication was confirmed by detection of a gap junction component, Cx43, in both groups throughout culture, whereas transzonal projections, which physically link granulosa cells to oocyte, formed later in EVE-treated follicles. Importantly, levels of GJA1 mRNA, encoding for the Cx43 protein, significantly increased from Day 0 to Day 1 in the EVE group, but not in the control group. Given that EVE-treated follicles were smaller than controls, these findings suggest that EVE might facilitate the establishment of appropriate intercellular communications without impairing follicle ultrastructure. Therefore, mTORC1 inhibitors might represent an attractive tool to delay the culture-induced primordial follicle activation while maintaining follicles in a functionally integrated state.

Comparison between Slow Freezing and Vitrification for Human Ovarian Tissue Cryopreservation and Xenotransplantation.

Two methods for the cryopreservation of human ovarian tissue were compared using a xenotransplantation model to establish a safe and effective cryopreservation method. Ovarian tissues were obtained from women who underwent benign ovarian surgery in the gynecology research unit of a university hospital. The tissues were transplanted into 112 ovariectomized female severe combined immunodeficient mice 4 weeks after slow freezing or vitrification cryopreservation. Tissues were retrieved 4 weeks later. Primordial follicular counts decreased after cryopreservation and xenotransplantation, and were significantly higher in the slow freezing group than in the vitrification group (p < 0.001). Immunohistochemistry and TUNEL assay showed that the Ki-67 and CD31 markers of follicular proliferation and angiogenesis were higher in the slow freezing group (p < 0.001 and p = 0.006, respectively) and DNA damage was greater in the vitrification group (p < 0.001). Western blotting showed that vitrification increased cellular apoptosis. Anti-Müllerian hormone expression was low in transplanted samples subjected to both cryopreservation techniques. Electron microscopy revealed primordial follicle deformation in the vitrification group. Slow freezing for ovarian tissue cryopreservation is superior to vitrification in terms of follicle survival and growth after xenotransplantation. These results will be useful for fertility preservation in female cancer patients.

Three-dimensional organization of transzonal projections and other cytoplasmic extensions in the mouse ovarian follicle.

Each 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 cumulus and mural granulosa cells and their projections in mouse antral ovarian follicles. Transzonal projections (TZPs) are thin cytoplasmic projections that connect cumulus cells to the oocyte and are crucial for normal oocyte development. We studied these projections in detail and found that most TZPs do not reach the oocyte, and that they often branch and make gap junctions with each other. Furthermore, the TZPs that connect to the oocyte 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 the 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.

Electrospun patterned porous scaffolds for the support of ovarian follicles growth: a feasibility study.

Recently, the interest of the scientific community is focused on the application of tissue engineering approach for the fertility restoration. In this paper innovative patterned electrospun fibrous scaffolds were fabricated and used as 3D system for porcine follicles culture. The obtained scaffolds demonstrated to be a suitable support which did not alter or interfere with the typical spherical follicles morphology. The fibrillar structure of the scaffolds mimics the morphology of the healthy native tissue. The use of porcine follicles implied many advantages respect to the use of mouse model. Relevant results showed that more than the scaffold pattern and struts dimension, the selection of proper biomaterials improve the follicles adhesion and development.

Ultrastructural and histochemical study of previtellogenic oogenesis in the desert lizard Scincus mitranus (Squamata, Sauropsida).

The structure of the granulosa in reptilian sauropsids varies between groups. We investigated the follicle development in the desert lizard Scincus mitranus. In the germinal bed, oogonia, and primary oocytes were identified and found to be interspersed between the epithelial cells. Previtellogenesis was divided into three stages: early, transitional, and late previtellogenic stages. During the early previtellogenic stage (diplotene), the oocyte is invested by small epithelia cells that formed a complete single layer, which may be considered as a young follicle. The transitional previtellogenic stage was marked by proliferation and differentiation of the granulosa layer from a homogenous layer consisting of only small cells to a heterogeneous layer containing three cell types: small, intermediate, and large cells. The late previtellogenic stage was marked by high-synthetic activity of large cells and the initiation of cytoplasmic bridges between large granulosa cells and the oocyte. Small cells were the only type of granulosa cells that underwent division. Thus, these cells may be stem cells for the granulosa cell population and may develop into intermediate and subsequently large cells. The intermediate cells may be precursors of large cells, as suggested by their ultrastructure. The ultrastructure of the large granulosa was indicative of their high synthetic activity. Histochemical analysis indicated the presence of cholesterol and phospholipids in the cytoplasm of large cells, the zona pellucida, among the microvilli, in the bridges region, and in the cortical region of the oocyte cytoplasm. These materials may be transferred from large cells into the oocyte through cytoplasmic bridges and provide nutritive function to large cells rather than functioning in steroidogenesis or vitellogenesis.