Ultrastructural Analysis of Large Japanese Field Mouse (Apodemus speciosus) Testes Exposed to Low-Dose-Rate (LDR) Radiation after the Fukushima Nuclear Power Plant Accident.

Since the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident, great attention has been paid to the impact of chronic low-dose-rate (LDR) radiation exposure on biological systems. The reproductive system is sensitive to radiation, with implications connected to infertility. We investigated the testis ultrastructure of the wild large Japanese field mouse (Apodemus speciosus) from three areas contaminated after the FDNPP accident, with different levels of LDR radiation (0.29 µSv/h, 5.11 µSv/h, and 11.80 µSv/h). Results showed good preservation of the seminiferous tubules, comparable to the unexposed animals (controls), except for some ultrastructural modifications. Increases in the numerical density of lipid droplet clusters in spermatogenic cells were found at high levels of LDR radiation, indicating an antioxidant activity rising due to radiation recovery. In all groups, wide intercellular spaces were found between spermatogenic cells, and cytoplasmic vacuolization increased at intermediate and high levels and vacuolated mitochondria at the high-level. However, these findings were also related to the physiological dynamics of spermatogenesis. In conclusion, the testes of A. speciosus exposed to LDR radiation associated with the FDNPP accident showed a normal spermatogenesis, with some ultrastructural changes. These outcomes may add information on the reproductive potential of mammals chronically exposed to LDR radiation.

Retinoic acid and proteotoxic stress induce AML cell death overcoming stromal cell protection.

BACKGROUND: Acute myeloid leukemia (AML) patients bearing the ITD mutation in the tyrosine kinase receptor FLT3 (FLT3-ITD) present a poor prognosis and a high risk of relapse. FLT3-ITD is retained in the endoplasmic reticulum (ER) and generates intrinsic proteotoxic stress. We devised a strategy based on proteotoxic stress, generated by the combination of low doses of the differentiating agent retinoic acid (R), the proteasome inhibitor bortezomib (B), and the oxidative stress inducer arsenic trioxide (A). METHODS: We treated FLT3-ITD+ AML cells with low doses of the aforementioned drugs, used alone or in combinations and we investigated the induction of ER and oxidative stress. We then performed the same experiments in an in vitro co-culture system of FLT3-ITD+ AML cells and bone marrow stromal cells (BMSCs) to assess the protective role of the niche on AML blasts. Eventually, we tested the combination of drugs in an orthotopic murine model of human AML. RESULTS: The combination RBA exerts strong cytotoxic activity on FLT3-ITD+ AML cell lines and primary blasts isolated from patients, due to ER homeostasis imbalance and generation of oxidative stress. AML cells become completely resistant to the combination RBA when treated in co-culture with BMSCs. Nonetheless, we could overcome such protective effects by using high doses of ascorbic acid (Vitamin C) as an adjuvant. Importantly, the combination RBA plus ascorbic acid significantly prolongs the life span of a murine model of human FLT3-ITD+ AML without toxic effects. Furthermore, we show for the first time that the cross-talk between AML and BMSCs upon treatment involves disruption of the actin cytoskeleton and the actin cap, increased thickness of the nuclei, and relocalization of the transcriptional co-regulator YAP in the cytosol of the BMSCs. CONCLUSIONS: Our findings strengthen our previous work indicating induction of proteotoxic stress as a possible strategy in FLT3-ITD+ AML therapy and open to the possibility of identifying new therapeutic targets in the crosstalk between AML and BMSCs, involving mechanotransduction and YAP signaling.

Sorcin promotes migration in cancer and regulates the EGF-dependent EGFR signaling pathways.

The epidermal growth factor receptor (EGFR) is one of the main tumor drivers and is an important therapeutic target for many cancers. Calcium is important in EGFR signaling pathways. Sorcin is one of the most important calcium sensor proteins, overexpressed in many tumors, that promotes cell proliferation, migration, invasion, epithelial-to-mesenchymal transition, malignant progression and resistance to chemotherapeutic drugs. The present work elucidates a functional mechanism that links calcium homeostasis to EGFR signaling in cancer. Sorcin and EGFR expression are significantly correlated and associated with reduced overall survival in cancer patients. Mechanistically, Sorcin directly binds EGFR protein in a calcium-dependent fashion and regulates calcium (dys)homeostasis linked to EGF-dependent EGFR signaling. Moreover, Sorcin controls EGFR proteostasis and signaling and increases its phosphorylation, leading to increased EGF-dependent migration and invasion. Of note, silencing of Sorcin cooperates with EGFR inhibitors in the regulation of migration, highlighting calcium signaling pathway as an exploitable target to enhance the effectiveness of EGFR-targeting therapies.

A New Strong-Acid Free Route to Produce Xanthan Gum-PANI Composite Scaffold Supporting Bioelectricity.

Conductive hybrid xanthan gum (XG)-polyaniline (PANI) biocomposites forming 3D structures able to mimic electrical biological functions are synthesized by a strong-acid free medium. In situ aniline oxidative chemical polymerizations are performed in XG water dispersions to produce stable XG-PANI pseudoplastic fluids. XG-PANI composites with 3D architectures are obtained by subsequent freeze-drying processes. The morphological investigation highlights the formation of porous structures; UV-vis and Raman spectroscopy characterizations assess the chemical structure of the produced composites. I-V measurements evidence electrical conductivity of the samples, while electrochemical analyses point out their capability to respond to electric stimuli with electron and ion exchanges in physiological-like environment. Trial tests on prostate cancer cells evaluate biocompatibility of the XG-PANI composite. Obtained results demonstrate that a strong acid-free route produces an electrically conductive and electrochemically active XG-PANI polymer composite. The investigation of charge transport and transfer, as well as of biocompatibility properties of composite materials produced in aqueous environments, brings new perspective for exploitation of such materials in biomedical applications. In particular, the developed strategy can be used to realize biomaterials working as scaffolds that require electrical stimulations for inducing cell growth and communication or for biosignals monitoring and analysis.

RIPK4 regulates cell-cell adhesion in epidermal development and homeostasis.

Epidermal development and maintenance are finely regulated events requiring a strict balance between proliferation and differentiation. Alterations in these processes give rise to human disorders such as cancer or syndromes with skin and annexes defects, known as ectodermal dysplasias (EDs). Here, we studied the functional effects of two novel receptor-interacting protein kinase 4 (RIPK4) missense mutations identified in siblings with an autosomal recessive ED with cutaneous syndactyly, palmoplantar hyperkeratosis and orofacial synechiae. Clinical overlap with distinct EDs caused by mutations in transcription factors (i.e. p63 and interferon regulatory factor 6, IRF6) or nectin adhesion molecules was noticed. Impaired activity of the RIPK4 kinase resulted both in altered epithelial differentiation and defective cell adhesion. We showed that mutant RIPK4 resulted in loss of PVRL4/nectin-4 expression in patient epidermis and primary keratinocytes, and demonstrated that PVRL4 is transcriptionally regulated by IRF6, a RIPK4 phosphorylation target. In addition, defective RIPK4 altered desmosome morphology through modulation of plakophilin-1 and desmoplakin. In conclusion, this work implicates RIPK4 kinase function in the p63-IRF6 regulatory loop that controls the proliferation/differentiation switch and cell adhesion, with implications in ectodermal development and cancer.

Repeated hyperstimulation affects the ultrastructure of mouse fallopian tube epithelium.

Controlled ovarian hyperstimulation (COH) is routinary used in assisted reproductive technologies (ARTs) to increase the yields of mature oocytes. The possibility that patients with a history of failures or poor-responders may develop side-effects following these treatments is still debated. Epidemiological studies reported controversial results about pregnancy outcome and the risk of developing gynecological cancers. By using a mouse model, here we compared the ultrastructural features of fallopian tubes (FTs) obtained from mice undergoing or not (control, CTR) four (4R) and eight (8R) rounds of gonadotropin stimulation. Although the morphological characteristics of oviductal layers seemed unaffected by repeated treatments, dose-response ultrastructural alterations in the ampulla appeared in the 4R group and even more in the 8R group. The targets were oviductal ciliated (CCs) and non-ciliated (NCCs) cells, which showed damaged mitochondria and glycogen accumulations in the cytoplasm. The drastic reduction of CCs, evident after 4R, was supported by the absence of cilia. After 8R, glycogen granules were significantly reduced and massive degeneration of mitochondria, which appeared swollen and/or vacuolated, occurred in NCCs. Moreover, disintegrated mitochondria were found at the periphery of mitophagic vacuoles with evident signs of cristolysis. The morphometric analysis evidenced a significant increase in the density and frequency of damaged mitochondria after 4R and 8R. The absence of cilia, necessary to sustain oviductal transport of oocytes, spermatozoa and embryos, may originate from either mitochondrial dysfunction or glycogen consumption. These results suggest that repeated COH treatments could induce alterations impairing fertilization and embryo transport toward the uterus.

Oxygen concentration alters mitochondrial structure and function in in vitro fertilized preimplantation mouse embryos.

STUDY QUESTION: Does the oxygen concentration in the culture medium [either physiologic (5%) or atmospheric (20%)] affect mitochondrial ultrastructure and function in preimplantation mouse embryos generated by IVF? SUMMARY ANSWER: Embryos cultured in 20% oxygen show increased mitochondrial abnormalities compared to embryos cultured in 5% oxygen. WHAT IS KNOWN ALREADY: ART are widely used and have resulted in the birth of more than 8 million children. A variety of media and oxygen concentrations are used to culture embryos. Embryos cultured under physiological O2 tension (5%) reach the blastocyst stage faster and have fewer alterations in gene expression when compared with embryos cultured under atmospheric oxygen conditions (20%). The mechanisms by which oxygen tension affects preimplantation development remain unclear, but mitochondria are believed to play an important role. The aim of this study was to evaluate how mitochondrial ultrastructure and function in IVF embryos were affected by culture under physiologic (5%) or atmospheric (20%) oxygen concentrations. STUDY DESIGN, SIZE, DURATION: Zygotes, 2-cell, 4-cell, morula and blastocyst were flushed out of the uterus after natural fertilization and used as controls. IVF was performed in CF1 x B6D2F1 mice and embryos were cultured in Potassium simplex optimized medium (KSOM) with amino acids (KAA) under 5% and 20% O2 until the blastocyst stage. Embryo development with the addition of antioxidants was also tested. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mitochondrial function was assessed by measuring mitochondrial membrane potential, reactive oxygen species (ROS) production, ATP levels, and the expression of selected genes involved in mitochondrial function. Mitochondria ultrastructure was evaluated by transmission electron microscopy (TEM). MAIN RESULTS AND THE ROLE OF CHANCE: Embryos cultured under 20% O2 had fewer mitochondria and more vacuoles and hooded (abnormal) mitochondria compared to the other groups (P < 0.05). At the blastocyst stage the mitochondria of IVF embryos cultured in 20% O2 had lower mtDNA copy number, a denser matrix and more lamellar cristae than controls. Overall IVF-generated blastocysts had lower mitochondrial membrane potential, higher ROS levels, together with changes in the expression of selected mitochondrial genes (P < 0.05). ATP levels were significantly lower than controls only under 5% O2, with the 20% O2 IVF group having intermediate levels. Unexpectedly, adding antioxidant to the culture medium did not improve development. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Findings in mice embryos might be different from human embryos. WIDER IMPLICATIONS OF THE FINDINGS: This study suggests that changes in the mitochondria may be part of the mechanism by which lower oxygen concentration leads to better embryo development and further emphasize the importance of mitochondria as a locus of reprogramming. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by R01 HD 082039 to PFR, the Department of Life, Health and Environmental Sciences, University of L'Aquila, Italy (RIA 2016-2018) and the Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, La Sapienza University of Rome, Italy (University grants 2016-2017). The authors declare no competing interests.

Mancozeb impairs the ultrastructure of mouse granulosa cells in a dose-dependent manner.

Mancozeb, an ethylene bis-dithiocarbamate, is widely used as a fungicide and exerts reproductive toxicity in vivo and in vitro in mouse oocytes by altering spindle morphology and impairing the ability to fertilize. Mancozeb also induces a premalignant status in mouse granulosa cells (GCs) cultured in vitro, as indicated by decreased p53 expression and tenuous oxidative stress. However, the presence and extent of ultrastructural alterations induced by mancozeb on GCs in vitro have not yet been reported. Using an in vitro model of reproductive toxicity, comprising parietal GCs from mouse antral follicles cultured with increasing concentrations of mancozeb (0.001-1 µg/ml), we sought to ascertain the in vitro ultrastructural cell toxicity by means of transmission (TEM) and scanning (SEM) electron microscopy. The results showed a dose-dependent toxicity of mancozeb on mouse GCs. Ultrastructural data showed intercellular contact alterations, nuclear membrane irregularities, and chromatin marginalization at lower concentrations, and showed chromatin condensation, membrane blebbing, and cytoplasmic vacuolization at higher concentrations. Morphometric analysis evidenced a reduction of mitochondrial length in GCs exposed to mancozeb 0.01-1 µg/ml and a dose-dependent increase of vacuole dimension. In conclusion, mancozeb induced dose-dependent toxicity against GCs in vitro, including ultrastructural signs of cell degeneration compatible with apoptosis, likely due to the toxic breakdown product ethylenethiourea. These alterations may represent a major cause of reduced/delayed/missed oocyte maturation in cases of infertility associated with exposure to pesticides.

The pesticide Lindane induces dose-dependent damage to granulosa cells in an in vitro culture.

Lindane, which is one of the most persistent organochlorine pesticide contaminating the Aral Sea region, is associated with numerous pathologies of the female reproductive system, including infertility, due to its gap junction blocker activity. By using an in vitro model of reproductive toxicity consisting of mouse parietal granulosa cells (GCs) exposed to increasing concentrations of Lindane ranging from 1 to 100µM (L1; L10; L100), we aimed to ascertain the Lindane toxicity by evaluating the ultrastructure and expression of the cell death protein p53. GCs exposed to L1 showed an early sign of degeneration as chromatin marginalization and initial reduction of cell-to-cell contacts. Such effects increased at L10 with nuclear membrane invagination, cytoplasmic blebbing, reduction of microvilli and intercellular connections. L100 induced evident cellular damages with an extensive presence of vacuoles, cytoplasmic fragments, nuclear membrane vesiculation and abundant cellular debris. A dose-dependent increase of p53 expression was evident in the L1 and L10 groups but not in L100. These data provide evidence for a dose-dependent reproductive toxicity of the gap junction blocker Lindane, as seen in mouse GCs cultured in vitro by ultrastructural damage compatible with apoptosis. Since gap junctions may play a critical role in FSH-stimulated progesterone production, the ultrastructural damage here evidenced could explain the increase in the prevalence of reproductive pathologies and infertility in exposed women. Finally, this study provided a useful and repeatable model of reproductive toxicity in vitro, which is applicable to evaluate the detrimental effects of toxicants or the reversing effect of protective substances.

Vitrification of human immature oocytes before and after in vitro maturation: a review.

The use of immature oocytes subjected to in vitro maturation (IVM) opens interesting perspectives for fertility preservation where ovarian reserves are damaged by pathologies or therapies, as in PCO/PCOS and cancer patients. Human oocyte cryopreservation may offer some advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation and postponing childbirth. It also eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In addition, a successful oocyte cryopreservation program could eliminate the need for donor and recipient menstrual cycle synchronization. Recent advances in vitrification technology have markedly improved the oocyte survival rate after warming, with fertilization and implantation rates comparable with those of fresh oocytes. Healthy live births can be achieved from the combination of IVM and vitrification, even if vitrification of in vivo matured oocytes is still more effective. Recently, attention is given to highlight whether vitrification procedures are more successful when performed before or after IVM, on immature GV-stage oocytes, or on in vitro matured MII-stage oocytes. In this review, we emphasize that, even if there are no differences in survival rates between oocytes vitrified prior to or post-IVM, reduced maturation rates of immature oocytes vitrified prior to IVM can be, at least in part, explained by underlying ultrastructural and biomolecular alterations.

In-Vitro Application of Pentoxifylline Preserved Ultrastructure of Spermatozoa After Vitrification in Asthenozoospermic Patients.

PURPOSE: To evaluate the effect of in vitro application of pentoxifylline (PX) on sperm parameters and ultrastructure after vitrification in asthenozoospermic patients. MATERIALS AND METHODS: A total of 30 asthenozoospermic semen samples (aged 25-45 years) were divided into four groups before vitrification, after vitrification, control (without PX) and experimental (with PX). In experimental group, each sample was exposed for 30 min to 3.6mmol/l PX and the control group without any treatment apposing in 370C for 30 min. After incubation, the samples were washed and analyzed again. Vitrification was done according to straw method. Eosin-nigrosin and Papanicolaou staining were applied for assessment of sperm viability and morphology, respectively. The samples without PX and post treatment with PX were assessed by transmission electron microscopy (TEM). RESULTS: A significant decrease in sperm motility (P ≤ .001), morphology (11.47 ± 2.9 versus 6.73 ± 2.01) and viability (73.37 ± 6.26 versus 54.67 ± 6.73) was observed post vitrification, but sperm motility (19.85 ± 4.75 versus 32.07 ± 5.58, P ≤ .001) was increased significantly following application of PX. This drug had no significant (P >.05) detrimental neither negative effect on ultrastructure acrosome, plasma membrane and coiled tail statues of spermatozoa. CONCLUSION: Vitrification had detrimental effects on sperm parameters, but PX reversed detrimental effects on sperm motility. However, PX had no alteration on ultrastructure morphology of human spermatozoa after vitrification.

Differences in the Kinetic of the First Meiotic Division and in Active Mitochondrial Distribution between Prepubertal and Adult Oocytes Mirror Differences in their Developmental Competence in a Sheep Model.

Our aim is to verify if oocyte developmental potential is related to the timing of meiotic progression and to mitochondrial distribution and activity using prepubertal and adult oocytes as models of low and high developmental capacity respectively. Prepubertal and adult oocytes were incorporated in an in vitro maturation system to determine meiotic and developmental competence and to assess at different time points kinetic of meiotic maturation, 2D protein electrophoresis patterns, ATP content and mitochondria distribution. Maturation and fertilization rates did not differ between prepubertal and adult oocytes (95.1% vs 96.7% and 66.73% vs 70.62% respectively for prepubertal and adult oocytes). Compared to adults, prepubertal oocytes showed higher parthenogenesis (17.38% vs 2.08% respectively in prepubertals and adults; P<0.01) and polispermy (14.30% vs 2.21% respectively in prepubertals and adults; P<0.01), lower cleavage rates (60.00% vs 67.08% respectively in prepubertals and adults; P<0.05) and blastocyst output (11.94% vs 34.% respectively in prepubertals and adults; P<0.01). Prepubertal oocytes reached MI stage 1 hr later than adults and this delay grows as the first meiotic division proceeds. Simultaneously, the protein pattern was altered since in prepubertal oocytes it fluctuates, dropping and rising to levels similar to adults only at 24 hrs. In prepubertal oocytes ATP rise is delayed and did not reach levels comparable to adult ones. CLSM observations revealed that at MII, in the majority of prepubertal oocytes, the active mitochondria are homogenously distributed, while in adults they are aggregated in big clusters. Our work demonstrates that mitochondria and their functional aggregation during maturation play an active role to provide energy in terms of ATP. The oocyte ATP content determines the timing of the meiotic cycle and the acquisition of developmental competence. Taken together our data suggest that oocytes with low developmental competence have a slowed down energetic metabolism which delays later development.

Repeated ovarian stimulation does not affect the expression level of proteins involved in cell cycle control in mouse ovaries and fallopian tubes.

PURPOSE: To understand if repeated cycles (2-4 rounds) of gonadotropin stimulation could affect intracellular localization/content of proteins controlling cell cycle progression in mouse fallopian tubes (FT) and ovaries. METHODS: FT and ovaries of estrous mice (control) and of stimulated mice were analyzed to detect Oct-3/4, Sox-2, p53, ß-catenin, pAKT and cyclin D1 localization/content. Spindles and chromosome alignment were analyzed in ovulated oocytes. RESULTS: After round 4, FT and ovaries of control and stimulated groups showed no differences in Oct-3/4, Sox-2 and ß-catenin localization nor in Oct-3/4, Sox-2, p53, ß-catenin and pAKT contents. Cyclin D1 level increased significantly in FT of treated mice. Oocytes number decreased meanwhile frequency of abnormal meiotic spindles increased with treatments. CONCLUSIONS: Repetitive stimulations affected oocyte spindle morphology but did not induce changes in a set of proteins involved in cell cycle progression, usually altered in ovarian cancer. The significant increase of cyclin D1 in the FT requires further investigation.

Restoration of corpus luteum angiogenesis in immature hypothyroid rdw rats after thyroxine treatment: morphologic and molecular evidence.

Thyroxine (T4) plus gonadotropins might stimulate ovarian follicular angiogenesis in immature infertile hypothyroid rdw rats by upregulating mRNA expression of major angiogenic factors. Development of growing corpus luteum (CL) is strongly related to angiogenesis and to morphofunctional development of microcirculation. Our aim was to investigate if T4 is involved in CL angiogenesis and in the activation of capillary cells and angiogenic factors after ovulation in a spontaneous model of hypothyroidism, the rdw rat. Rdw rats were treated with T4 plus gonadotropins (equine chorionic gonadotropin plus human chorionic gonadotropin; eCG+hCG) or gonadotropins alone in order to evaluate the effects of T4 on early luteal angiogenesis, on microvascular cells and on expression of major growth factors which are involved in the regulation of angiogenesis. Wistar-Imamichi rats treated with gonadotropins were used as controls. The ovaries were collected 4 days after hCG administration and analyzed using morphologic and molecular approaches. Thyroxine plus gonadotropins stimulated the growth of CLs and follicles as in controls, differently from rdw rats treated only with gonadotropins, in which CLs were not found and only small follicles, often atretic, could be recognized. In T4 plus gonadotropin-treated rdw rats CLs showed increased microvasculature, numerous activated capillaries characterized by sprouting and other angiogenic figures, and associated pericytes. Quantitative analysis revealed that the number of pericytes in T4 plus gonadotropin-treated rdw rats was comparable with that found in control rats and was significantly higher than that found in gonadotropin-treated rdw rats. The mRNA expression of vascular endothelial growth factor and basic fibroblast growth factor was significantly higher in control rats and in T4 plus gonadotropin-treated rdw rats than in gonadotropin-treated rdw rats. mRNA expression of tumor necrosis factor α, transforming growth factor ß, and epidermal growth factor did not show significant changes. Our data originally demonstrated that T4 promoted the growth of an active microcirculation in developing CLs of gonadotropin-primed hypothyroid rdw rats, mainly by inducing sprouting angiogenesis, pericyte recruitment, and upregulation of mRNA expression of vascular endothelial growth factor and basic fibroblast growth factor. In conclusion, we suggest that T4 plays a key role in restoring luteal angiogenesis in ovaries of immature hypothyroid rdw rats.

Blood vessel remodeling in pig ovarian follicles during the periovulatory period: an immunohistochemistry and SEM-corrosion casting study.

BACKGROUND: The present research aims to describe the process of vascular readjustment occurring in pig ovary during the periovulatory phase (from LH surge to ovulation) that drives the transformation of the follicle, a limited blood supplied structure, into the corpus luteum, a highly vascularised endocrine gland required to maintain high levels of progesterone in pregnancy. The swine model was chosen because it is characterized by a long periovulatory window (about 40-44 hrs-similar to human) that permits to recover follicles at a precise endocrinological timing. METHODS: By validated hormonal protocol (eCG+hCG), able to mimic the physiologic gonadotropin stimulation, preovulatory follicles (PreOFs, 60 h-eCG), follicles in the middle (early periovulatory follicles, EPerOFs, 18 h-hCG) or late (LPerOFs, 36 h-hCG) periovulatory phase were isolated from prepubertal gilts. To understand the angiogenic process, morphological/morphometrical analyses were performed by combining immunohistochemistry (IHC) and SEM of vascular corrosion casts (VCC) techniques. RESULTS: PreOFs showed a vascular plexus with proliferating endothelial cells (EPI). This plexus was characterized by a dense inner capillary network, with angiogenic figures, connected to the outer network by anastomotic vessels (arterioles and venules of the middle network). EPerOFs decreased their EPI, blood vessel extension in the outer network, and evidenced a reduced compactness of blood vessels. In LPerOFs, a rapid neovascularization was associated to an intensive tissue remodeling: the follicle acquired an undulated aspect presenting arterioles/venules near the basal membrane, increased vascular extension by EPI, sprouting and non-sprouting angiogenesis.The analysis of vascular geometric relations and branching angles evidenced similar values at all stages. CONCLUSION: These data allow us to hypothesize that EPerOFs are in a quiescent status. LPerOFs represent the "metamorphic" follicles that rapidly turn-on angiogenesis to sustain a successful corpus luteum formation. Particularly, it is interesting to underlie that the non-sprouting angiogenesis, typical of structures in rapid neovascularization, occurred only in the LPerOFs. Moreover, vascular geometric relations showed as blood vessel remodeling occurs with the "maximum output and the minimum energetic expense".This knowledge will allow to better understand the mechanisms regulating the reproductive success and to clarify the complex physiological angiogenic process in adult tissues.