RESUMO
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.
Assuntos
Apoptose , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Núcleo Celular/metabolismo , Regulação para Baixo , Drosophila melanogaster/ultraestrutura , Retículo Endoplasmático/metabolismo , Feminino , Fertilidade , Mutação/genética , Oogênese , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Folículo Ovariano/ultraestrutura , FenótipoRESUMO
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.
Assuntos
Hormônio Antimülleriano , Folículo Ovariano/fisiologia , Animais , Feminino , Células da Granulosa/ultraestrutura , Microscopia Eletrônica , Folículo Ovariano/ultraestrutura , RatosRESUMO
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.
Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Doenças Ovarianas/metabolismo , Folículo Ovariano/metabolismo , Ovário/metabolismo , Puberdade/metabolismo , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Criopreservação , Feminino , Humanos , Lactente , Microscopia Eletrônica de Transmissão , Doenças Ovarianas/patologia , Folículo Ovariano/ultraestrutura , Ovário/ultraestrutura , Adulto JovemRESUMO
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.
Assuntos
Comunicação Celular/fisiologia , Conexina 43/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/ultraestrutura , Adulto , Conexina 43/genética , Criopreservação , Everolimo/farmacologia , Feminino , Células da Granulosa/metabolismo , Humanos , Oócitos/metabolismo , Técnicas de Cultura de Órgãos , RNA Mensageiro/genéticaRESUMO
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.
Assuntos
Criopreservação , Congelamento , Ovário , Vitrificação , Adulto , Animais , Biomarcadores , Contagem de Células , Proliferação de Células , Sobrevivência Celular , Criopreservação/métodos , Dano ao DNA , Feminino , Preservação da Fertilidade , Imunofluorescência , Xenoenxertos , Humanos , Camundongos , Neovascularização Fisiológica , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Folículo Ovariano/ultraestrutura , Ovário/citologia , Ovário/metabolismo , Ovário/transplante , Ovário/ultraestrutura , Adulto JovemRESUMO
The extracellular matrix (ECM) is a group of molecules that offer structural and biochemical support to cells and interact with them to regulate their function. Also, growth factors (GFs) stored in the ECM can be locally released during ECM remodeling. Here, we hypothesize that the balance between ECM components and remodelers is regulated according to the ovarian steroid milieu to which the oviduct is exposed during the periovulatory period. Follicular growth was manipulated to generate cows that ovulated small follicles (SF-small corpus luteum [SCL]; n = 20) or large follicles (LF-large corpus luteum [LCL]; n = 21) and possess corresponding Estradiol (E2) and Progesterone (P4) plasmatic concentrations. Ampulla and isthmus samples were collected on day 4 (day 0 = ovulation induction) and immediately frozen or fixed. The transcriptional profile (n = 3/group) was evaluated by RNA sequencing. MMP Antibody Array was used to quantify ECM remodelers' protein abundance and immunohistochemistry to quantify type I collagen. Transcriptome analysis revealed the over-representation of ECM organization and remodeling pathways in the LF-LCL group. Transcription of ECM components (collagens), remodelers (ADAMs and MMPs), and related GFs were upregulated in LF-LCL. Protein intensities for MMP3, MMP8, MMP9, MMP13, and TIMP4 were greater for the LF-LCL group. Type I collagen content in the mucosa was greater in SF-SCL group. In conclusion, that the earlier and more intense exposure to E2 and P4 during the periovulatory period in LF-LCL animals stimulates ECM remodeling. We speculate that differential ECM regulation may contribute to oviductal receptivity to the embryo.
Assuntos
Matriz Extracelular/fisiologia , Hormônios Esteroides Gonadais/fisiologia , Oviductos/fisiologia , Proteínas ADAM/metabolismo , Animais , Bovinos , Colágeno Tipo I/biossíntese , Colágeno Tipo I/genética , Biologia Computacional , Estradiol/sangue , Matriz Extracelular/ultraestrutura , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Metaloproteinases da Matriz/metabolismo , Folículo Ovariano/fisiologia , Folículo Ovariano/ultraestrutura , Oviductos/ultraestrutura , Ovulação/fisiologia , Gravidez , Progesterona/sangueRESUMO
RESEARCH QUESTION: Does synchrotron X-ray fluorescence (XRF) provide novel chemical information for the evaluation of human ovarian tissue cryopreservation protocols? DESIGN: Tissues from five patients undergoing laparoscopic surgery for benign gynaecological conditions were fixed for microscopic analysis either immediately or after cryopreservation. After fixation, fresh and slowly frozen samples were selected by light microscopy and transmission electron microscopy, and subsequently analysed with synchrotron XRF microscopy at different incident energies. RESULTS: The distributions of elements detected at 7.3 keV (S, P, K, Cl, Fe, and Os) and 1.5 keV (Na and Mg) were related to the changes revealed by light microscopy and transmission electron microscopy analyses. The light elements showed highly informative findings. The S distribution was found to be an indicator of extracellular component changes in the stromal tissues of the freeze-stored samples, further revealed by the transmission electron microscopy analyses. Low-quality follicles, frequent in the freeze-thawed tissues, showed a high Na level in the ooplasm. On the contrary, good-quality follicles were detected by a homogeneous Cl distribution. The occurrence of vacuolated follicles increased after cryopreservation, and the XRF analyses showed that the vacuolar structures contained mainly Cl and Na. CONCLUSIONS: The study demonstrates that elemental imaging techniques, particularly revealing the distribution of light elements, could be useful in establishing new cryopreservation protocols.
Assuntos
Criopreservação/métodos , Preservação de Órgãos/métodos , Ovário/ultraestrutura , Feminino , Humanos , Microscopia Eletrônica de Transmissão , Folículo Ovariano/ultraestruturaRESUMO
Ovarian tissue cryopreservation is a promising technique for fertility maintenance. The aim of this study was to compare the morphology of domestic cat ovarian follicles after tissue cryopreservation with ethylene glycol (EG) and dimethyl sulfoxide (Me2SO). Ovaries from healthy adult cats undergoing elective ovariohysterectomy were used. Eight fragments were obtained from each pair of ovaries: two were used as fresh controls; three were submitted to fresh perfusion toxicity test and perfused with M199, 10% fetal calf serum and 0.4% sucrose containing Me2SO 1.5â¯M, EG 1.5â¯M or Me2SO 0.75 M + EG 0.75 M; and the remaining three fragments were perfused as described and submitted to slow freezing. After 45 days of cryopreservation, the samples were thawed, fixed and processed for light and transmission electron microscopy (TEM). The percentages of morphologically normal follicles identified by light microscopy were higher in the control group (94.45%) in comparison to the frozen groups (80.56% with EG, 78.7% with Me2SO and 75.87% with EG + Me2SO). The fresh perfused tissue showed no statistical difference compared to control or frozen samples. The TEM analysis showed less damage in the ultrastructure of follicles from the Me2SO group in comparison with the EG and Me2SO + EG groups. According to the morphological analysis, 1.5 M Me2SO is the best cryoprotectant for cryopreservation of domestic cat ovarian tissue regarding the morphology of preantral follicles after thawing. Further studies regarding the viability of these follicles should be performed.
Assuntos
Criopreservação/veterinária , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Etilenoglicol/farmacologia , Folículo Ovariano/ultraestrutura , Animais , Gatos , Feminino , Congelamento , Microscopia Eletrônica de TransmissãoRESUMO
The aim of this study was to define the population, morphological and ultrastructural characteristics of bitch preantral follicles (PAFs) and to compare the effects on the morphology of PAF of two cryopreservation techniques - slow freezing (SF) and vitrification (V) - of bitches' ovarian tissue. The average population (number per ovary) of PAFs was 48,541⯱â¯18,366, where 94.25% were primordial (45,145⯱â¯16,076). The average diameter of the primordial follicles was 27.5⯱â¯4.2⯵m. The overall percentage of morphologically normal PAFs was 93.66⯱â¯6.81% for the control group, 86.16⯱â¯11.05% after SF and 68.14⯱â¯12.75% after V. The percentage of normal primordial follicles was 96.69⯱â¯4.72% in control, 89.51⯱â¯10.39% in SF and 75.32⯱â¯9.23% in V. There was no significant difference in the overall percentage of normal PAFs among SF and the control. However, slow frozen follicles presented ultrastructural damage, while vitrified primordial and primary follicles were well preserved. In conclusion, although slow freezing seemed to be a good preserving method, vitrification was more effective than slow freezing in preserving the ultrastructure of primordial and primary follicles of bitches.
Assuntos
Criopreservação/veterinária , Cães , Folículo Ovariano/ultraestrutura , Vitrificação , Animais , Crioprotetores/farmacologia , Feminino , Congelamento , Folículo Ovariano/efeitos dos fármacosRESUMO
PURPOSE: The aim of this study is to optimize fibrin matrix composition in order to mimic human ovarian tissue architecture for human ovarian follicle encapsulation and grafting. METHODS: Ultrastructure of fresh human ovarian cortex in age-related women (n = 3) and different fibrin formulations (F12.5/T1, F30/T50, F50/T50, F75/T75), rheology of fibrin matrices and histology of isolated and encapsulated human ovarian follicles in these matrices. RESULTS: Fresh human ovarian cortex showed a highly fibrous and structurally inhomogeneous architecture in three age-related patients, but the mean ± SD of fiber thickness (61.3 to 72.4 nm) was comparable between patients. When the fiber thickness of four different fibrin formulations was compared with human ovarian cortex, F50/T50 and F75/T75 showed similar fiber diameters to native tissue, while F12.5/T1 was significantly different (p value < 0.01). In addition, increased concentrations of fibrin exhibited enhanced storage modulus with F50/T50, resembling physiological ovarian rigidity. Excluding F12.5/T1 from further analysis, only three remaining fibrin matrices (F30/T50, F50/T50, F75/T75) were histologically investigated. For this, frozen-thawed fragments of human ovarian tissue collected from 22 patients were used to isolate ovarian follicles and encapsulate them in the three fibrin formulations. All three yielded similar follicle recovery and loss rates soon after encapsulation. Therefore, based on fiber thickness, porosity, and rigidity, we selected F50/T50 as the fibrin formulation that best mimics native tissue. CONCLUSIONS: Of all the different fibrin matrix concentrations tested, F50/T50 emerged as the combination of choice in terms of ultrastructure and rigidity, most closely resembling human ovarian cortex.
Assuntos
Órgãos Artificiais , Fibrina/química , Ovário , Materiais Biomiméticos/química , Composição de Medicamentos , Elasticidade , Feminino , Dureza , Humanos , Fenômenos Mecânicos , Folículo Ovariano/transplante , Folículo Ovariano/ultraestrutura , Ovário/química , Ovário/citologia , Ovário/ultraestruturaRESUMO
Most protocols to cryopreserve ovarian tissue utilize the permeable cryoprotective agents (CPAs) in 1.5 M concentration. However the issues related to the ability to use higher concentrations of CPAs have remained open. The research aim was to assess the efficiency of media containing osmotically active sugars (sucrose, mannitol) at stepwise adding/removing of 3 M dimethylsulphoxide (DMSO) and propanediol (PROH) on ovarian tissue integrity. After the CPAs adding/removing the ovarian tissue injury was histologically examined, as well as the oocyte volume in tissue structure was assessed. It has been found, that after adding/removing of PROH and DMSO solutions the maximum amount of normal follicles made 67-93% when using Dulbecco's Modified Eagle Medium (DMEM) with 200 mM sucrose. Assessment of tissue damage after adding/removing of CPAs has demonstrated that the percentage of normal follicles was 83-87% using DMSO in presence of both sucrose and mannitol as the dilution media components. While removing PROH the level of follicles preservation was 2.5× higher when using mannitol compared with sucrose. Our results indicate that the ovarian tissue injury was minimal during adding 3 M CPAs using DMEM, containing sucrose and following application of mannitol at removing both DMSO and PROH.
Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Manitol/farmacologia , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Animais , Dimetil Sulfóxido/metabolismo , Dimetil Sulfóxido/farmacologia , Feminino , Pressão Osmótica/efeitos dos fármacos , Folículo Ovariano/metabolismo , Folículo Ovariano/ultraestrutura , Propilenoglicóis/metabolismo , Propilenoglicóis/farmacologia , Ratos WistarRESUMO
The presence of follicular cellular processes (FCP) that cross the zona pellucida, has been recorded in the ovarian follicles of Callorhinchus callorhynchus. This constitutes the first report describing the presence of these structures in a species of the Holocephali. Considering that FCPs have only previously been reported in the Selachii, these findings suggest that FCPs could have been lost by the Batoidea after their divergence, around 280 M B.P.
Assuntos
Peixes/anatomia & histologia , Folículo Ovariano/ultraestrutura , Zona Pelúcida/ultraestrutura , Animais , Feminino , OvárioRESUMO
BACKGROUND: Ovarian failure is diagnosed by ovarian destruction and reducing sex hormonal levels. Platelet-rich plasma (PRP) contains several growth factors that induce tissue repair and may induce folliculogenesis. OBJECTIVE: This study evaluates the effect of PRP on ovarian structures and function in cyclophosphamide (Cy)-induced ovarian failure in female rats by a stereological method. METHODS: Thirty-two adult female rats were divided into four groups (Control, Cy, Cy + PRP, and PRP). Female infertility was induced by Cy (75 mg/kg, single dose, intraperitoneally). Animals were treated by PRP which was obtained from age-matched male rats (200 µL, single dose, intraperitoneally). Blood samples were collected for measurement of hormones. The animals were dissected and the right ovaries were removed, fixed, sectioned, and stained by H&E. Stereological methods were used to estimate cortex and medulla volume, and the number and diameter of follicles, follicular cell, and oocyte using light microscopy. RESULTS: Cyclophosphamide had the maximum effect in decreasing on cortex volume, the pre-antral follicles number, a diameter of follicular cells and oocyte diameter in the antral follicle and the reduction of estradiol and progesterone levels compared with the control group. PRP had a dominant positive effect on the ovarian cortex volume, pre-antral follicles number and antral follicle diameter relative to the control group. PRP also decreased oocyte diameter in pre-antral follicle in infertile animals (p < 0.001). CONCLUSION: It seems that PRP has a protective effect on ovarian failure in the infertile female rat model.
Assuntos
Ciclofosfamida/toxicidade , Oócitos/efeitos dos fármacos , Doenças Ovarianas/induzido quimicamente , Folículo Ovariano/efeitos dos fármacos , Plasma Rico em Plaquetas , Animais , Estradiol/sangue , Feminino , Oócitos/ultraestrutura , Doenças Ovarianas/sangue , Doenças Ovarianas/patologia , Folículo Ovariano/ultraestrutura , Progesterona/sangue , Ratos Sprague-Dawley , Reprodução/efeitos dos fármacosRESUMO
Hyperthermia or heat stress (HS) occurs when heat dissipation mechanisms are overwhelmed by external and internal heat production. Hyperthermia negatively affects reproduction and potentially compromises oocyte integrity and reduces developmental competence of ensuing embryos. Autophagy is the process by which cells recycle energy through the reutilization of cellular components and is activated by a variety of stressors. Study objectives were to characterize autophagy-related proteins in the ovary following cyclical HS during the follicular phase. Twelve gilts were synchronized and subjected to cyclical HS (n = 6) or thermal neutral (n = 6) conditions for 5 days during the follicular phase. Ovarian protein abundance of Beclin 1 and microtubule associated protein light chain 3 beta II were each elevated as a result of HS (P = 0.001 and 0.003, respectively). The abundance of the autophagy related (ATG)12-ATG5 complex was decreased as a result of HS (P = 0.002). Regulation of autophagy and apoptosis occurs in tight coordination, and B-cell lymphoma (BCL)2 and BCL2L1 are involved in regulating both processes. BCL2L1 protein abundance, as detected via immunofluorescence, was increased in both the oocyte (â¼1.6-fold; P < 0.01) and granulosa cells of primary follicles (â¼1.4-fold P < 0.05) of HS ovaries. These results suggest that ovarian autophagy induction occurs in response to HS during the follicular phase, and that HS increases anti-apoptotic signaling in oocytes and early follicles. These data contribute to the biological understanding of how HS acts as an environmental stress to affect follicular development and negatively impact reproduction.
Assuntos
Autofagia , Transtornos de Estresse por Calor/patologia , Folículo Ovariano/patologia , Ovário/patologia , Animais , Apoptose/genética , Feminino , Febre/patologia , Genes bcl-2/genética , Células da Granulosa/metabolismo , Temperatura Alta , Infertilidade Feminina/etiologia , Infertilidade Feminina/fisiopatologia , Folículo Ovariano/ultraestrutura , Ovário/ultraestrutura , Gravidez , Transdução de Sinais/genética , Sus scrofa , Suínos , Vacúolos/ultraestrutura , Proteína bcl-X/genéticaRESUMO
The aim of the present study was to characterise the ovarian preantral follicle (PF) population and to establish a solid surface vitrification (SSV) process using dimethyl sulfoxide (DMSO) as a cryoprotectant for preservation of ovarian tissue from yellow-toothed cavies (Galea spixii). Ovaries were fixed for PF population analysis or were subjected to the SSV process. The mean (± s.e.m.) PF population per ovarian pair was estimated to be 416.0±342.8. There were 140.0±56.0 (63.4%) and 125.0±58.0 (64.0%) primary follicles on the right and left ovaries, respectively. The proportion of this follicle category was significantly greater than that of other follicle categories (P<0.05). The diameter of follicles (123.7±18.3µm), oocytes (50.1±5.0µm) and nuclei (14.27±2.01µm) was larger for secondary ones when compared with other PFs categories. Most PFs were morphologically normal (94.6%), with light microscopy identifying only a few atretic follicles (5.4%). After SSV, there was a reduction in the proportion of morphologically normal PFs compared with the non-vitrified group (69.5% vs 91.2%, respectively). Transmission electron microscopy revealed preservation of oocytes and granulosa cell membranes and the morphological aspect of follicles; the primary change observed in some vitrified PFs was the presence of vacuoles in the oocytes and granulosa cells cytoplasm and turgid mitochondria. In conclusion, the present study provides an estimative and characterization for the PF population in ovaries of G. spixii. Moreover, we report its PFs cryopreservation using an SSV process.
Assuntos
Criopreservação , Folículo Ovariano/anatomia & histologia , Ovário/anatomia & histologia , Vitrificação , Animais , Feminino , Microscopia Eletrônica de Transmissão , Folículo Ovariano/ultraestrutura , Ovário/ultraestrutura , RoedoresRESUMO
Epithelial morphogenesis contributes greatly to the development and homeostasis of the organs and body parts. Here, we analysed the consequences of impaired ecdysone receptor (EcR) signalling in the Drosophila follicular epithelium. Besides governing cell growth, the three EcR isoforms act redundantly in controlling follicle cell positioning. Flattening of the microvilli and an aberrant actin cytoskeleton arise from defective EcR signalling in follicle cells, and these defects impact on the organisation of the oocyte membrane. We found that this signalling governs a complex molecular network since its impairment affects key molecules as atypical protein kinase C and activated Moesin. Interestingly, the activity of the transcription factor Tramtrack69 isoform is required for microvilli and their actin core morphogenesis as well as for follicle cell positioning. In conclusion, our findings provide evidence of novel roles for EcR signalling and Tramtrack69 transcription factor in controlling stage-specific differentiation events that take place in the follicular epithelium.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Microvilosidades/metabolismo , Folículo Ovariano/citologia , Folículo Ovariano/crescimento & desenvolvimento , Receptores de Esteroides/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Animais , Drosophila/metabolismo , Drosophila/ultraestrutura , Feminino , Microvilosidades/ultraestrutura , Oócitos/citologia , Oócitos/metabolismo , Oócitos/ultraestrutura , Oogênese , Folículo Ovariano/ultraestruturaRESUMO
The present study provides the first ultrastructural data of the vitellogenesis in a cestode species of the cyclophyllidean family Paruterinidae, aiming to expand the limited data on the vitellogenesis in cyclophyllidean cestodes and to explore the potential of ultrastructural characters associated with vitellogenesis for phylogenetic and taxonomic studies of this order. The process of vitellocyte formation in Dictyterina cholodkowskii follows the general pattern observed in other tapeworms but exhibits several specific differences in the ultrastructure of vitelline cells. The vitellarium contains vitellocytes at various stages of maturation. The periphery of the vitellarium and the space between maturing vitellocytes are occupied by interstitial cells. Differentiation into mature vitellocytes is characterized by high secretory activity, which involves the development of granular endoplasmic reticulum, Golgi complexes, mitochondria and vitelline globules of various sizes. During vitellogenesis, the progressive fusion of these globules results in the formation of two large membrane-limited vitelline vesicles that eventually fuse into a single large vesicle. Mature vitellocytes are composed of a single vitelline vesicle, a high content of cytoplasmic organelles and have no nucleus. No traces of lipid droplets and glycogen granules are detected in the cytoplasm of mature vitellocytes, which might be related to biological peculiarities of this family, i.e. the release of eggs into environment within the tissues of the paruterine organ, which may serve as a source of nutrients for embryos.
Assuntos
Cestoides/ultraestrutura , Filogenia , Vitelogênese/fisiologia , Animais , Cestoides/fisiologia , Citoplasma/ultraestrutura , Feminino , Folículo Ovariano/fisiologia , Folículo Ovariano/ultraestrutura , Membrana Vitelina/fisiologiaRESUMO
Cytological responses in different organs of sentinel organisms have proven to be useful tools for characterizing the health status of those organisms and assessing the impact of environmental contaminants. Our study shows that nickel (II) accumulated in both germ cells (oogonia and developing oocytes) and somatic cells (muscle cells, follicle cells) in the Astacus leptodactylus ovary. Muscle cells from ovarian wall show disorganization and the disruption of cytoplasmic microtubules and pyknosis of the cell nucleus. Follicle cells, both those that surround the developing oocytes and also those that are not associated with the oocytes contained within the cytoplasm vacuoles of different sizes, degenerated mitochondria, myelin bodies, disorganized microtubules, and pyknotic nuclei. The most evident pathological phenomenon was the alteration and disorganization of the basal matrix, which separates the ovarian interstitium from ovarian follicles compartment. Exposure to nickel induces cytoplasmic vacuolation in oogonia and developing oocytes, structural alteration of the developing yolk granules and condensation of the nucleoli. Ultrastructural autometallography has shown grains of silver-enhanced nickel inside the cytoplasm of the muscle cells with altered morphology, including the cytoplasm, nucleus, and basal matrix of the follicle cells, and in intracisternal granules and developing yolk granules of the oocytes.
Assuntos
Astacoidea/efeitos dos fármacos , Técnicas Citológicas/métodos , Eletroforese/métodos , Níquel/toxicidade , Ovário/efeitos dos fármacos , Ovário/diagnóstico por imagem , Ovário/ultraestrutura , Coloração e Rotulagem/métodos , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Citoplasma/efeitos dos fármacos , Citoplasma/ultraestrutura , Feminino , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Células Musculares/efeitos dos fármacos , Células Musculares/ultraestrutura , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/ultraestrutura , Oócitos/efeitos dos fármacos , Oócitos/ultraestrutura , Oogônios/efeitos dos fármacos , Oogônios/ultraestrutura , Folículo Ovariano/diagnóstico por imagem , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/ultraestrutura , VacúolosRESUMO
Quantitative analysis of tissues and organs can reveal large-scale patterning as well as the impact of perturbations and aging on biological architecture. Here we develop tools for imaging of single cells in intact organs and computational approaches to assess spatial relationships in 3D. In the mouse ovary, we use nuclear volume of the oocyte to read out quiescence or growth of oocyte-somatic cell units known as follicles. This in-ovary quantification of non-growing follicle dynamics from neonate to adult fits a mathematical function, which corroborates the model of fixed oocyte reserve. Mapping approaches show that radial organization of folliculogenesis established in the newborn ovary is preserved through adulthood. By contrast, inter-follicle clustering increases during aging with different dynamics depending on size. These broadly applicable tools can reveal high dimensional phenotypes and age-related architectural changes in other organs. In the adult mouse pancreas, we find stochastic radial organization of the islets of Langerhans but evidence for localized interactions among the smallest islets.
Assuntos
Imageamento Tridimensional/métodos , Ilhotas Pancreáticas/fisiologia , Oócitos/fisiologia , Folículo Ovariano/fisiologia , Análise de Célula Única/métodos , Envelhecimento , Algoritmos , Animais , Feminino , Ilhotas Pancreáticas/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/ultraestrutura , Folículo Ovariano/ultraestruturaRESUMO
Mobilization of endogenous retrotransposons can destabilize the genome, an imminent danger during epigenetic reprogramming of cells in the germline. The P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway is known to silence retrotransposons in the mouse testes. Several piRNA pathway components localize to the unique, germline structure known as the nuage. In this study, we surveyed mouse ovaries and found, for the first time, transient appearance of nuage-like structures in oocytes of primordial follicles. Mouse vasa homolog (MVH), Piwi-like 2 (PIWIL2/MILI) and tudor domain-containing 9 (TDRD9) are present in these structures, whereas aggregates of germ cell protein with ankyrin repeats, sterile alpha motif and leucine zipper (GASZ) localize separately in the cytoplasm. Retrotransposons are silenced in primordial ovarian follicles, and de-repressed upon reduction of piRNA expression in Mvh, Mili or Gasz mutants. However, these null-mutant females, unlike their male counterparts, are fertile, uncoupling retrotransposon activation from sterility.