Mechanism of programmed cell death in follicular atresia / 生理学报

Apoptosis and autophagy of follicular granulosa cells play an important regulatory role in the process of ovarian follicular atresia in animals. Recent studies have shown that ferroptosis and pyroptosis are also involved in the process of ovarian follicular atresia. Ferroptosis is a form of cell death caused by iron-dependent lipid peroxidation and reactive oxygen species (ROS) accumulation. Studies have confirmed that autophagy- and apoptosis-mediated follicular atresia also have typical characteristics of ferroptosis. Pyroptosis is a pro-inflammatory cell death dependent on Gasdermin protein, which can regulate ovarian reproductive performance by regulating follicular granulosa cells. This article reviews the roles and mechanisms of several types of programmed cell death independently or interactively regulating follicular atresia, in order to expand the theoretical research on follicular atresia mechanism and provide the theoretical reference for the mechanism of programmed cell death-induced follicular atresia.

Manipulação de oócitos inclusos em folículos ovarianos pré-antrais e a importância dos antioxidantes ­ revisão de literatura / Manipulation of oocytes included in preantral ovarian follicles and the importance of antioxidants ­ literature review / Manipulación de ovocitos incluidos en folículos ováricos preantrales e importancia de los antioxidantes - revisión de la literatura

A manipulação de oócitos inclusos em folículos ovarianos pré-antrais (MOIFOPA) vem sendo estudada pensando na perspectiva futura de aplicação direta na reprodução humana, principalmente para mulheres que sofrem de doenças ou que precisam passar por tratamentos que interferem na função ovariana. Nesse contexto, o objetivo deste trabalho é revisar aspectos relacionados com a biotécnica de MOIFOPA e a importância dos antioxidantes no cultivo in vitro de folículos pré-antrais. Foi realizada uma pesquisa na base de dados PubMed, buscando artigos sobre a biotécnica, principalmente relacionados com a necessidade do uso de antioxidantes no cultivo. A grande maioria dos estudos sobre a biotécnica utilizam como modelo experimental os folículos ovarianos de diferentes espécies de animais. A MOIFOPA compreende o isolamento e o resgate de folículos ovarianos pré-antrais provenientes de ovários, seguido da conservação através da técnica de resfriamento ou congelação e o cultivo folicular in vitro, a fim de promover o crescimento, a maturação e a fecundação in vitro (FIV) dos oócitos inclusos nesses folículos, maximizando o potencial reprodutivo feminino e diminuindo a atresia folicular que acontece in vivo. Um aspecto que pode interferir no sucesso do cultivo in vitro de folículos ovarianos pré-antrais é a produção em excesso de espécies reativas de oxigênio (EROs). Os ácidos ascórbico e alfa lipóico vem demonstrando resultados interessantes para reduzir os efeitos que as EROs causam sobre os folículos ovarianos pré-antrais cultivados in vitro.

The manipulation of oocytes included in preantral ovarian follicles (MOEPF) has been studied considering the future perspective of direct application in human reproduction, especially for women who suffer from diseases or who need to undergo treatments that interfere with ovarian function. In this context, the objective of this paper is to review aspects related to the biotechnology of MOIFOPA and the importance of antioxidants. A search was carried out in the PubMed database, searching for articles on biotechnology, mainly related to the need to use antioxidants in cultivation. The vast majority of studies on biotechnology use ovarian follicles from different species of animals as an experimental model. MOIFOPA comprises the isolation and rescue of preantral ovarian follicles from ovaries, followed by conservation through the cooling or freezing technique and in vitro follicular cultivation, in order to promote growth, maturation and in vitro fertilization ( IVF) of the oocytes included in these follicles, maximizing the female reproductive potential and decreasing the follicular atresia that occurs in vivo. One aspect that may interfere with the success of in vitro culture of preantral ovarian follicles is the excess production of reactive oxygen species (ROS). Ascorbic and alpha lipoic acids have shown interesting results in reducing the effects that ROS cause on in vitro cultured preantral ovarian follicles.

manipulación de ovocitos incluidos en folículos ováricos preantrales (MOIFOPA) se ha estudiado con la perspectiva futura de su aplicación directa en la reproducción humana, especialmente en mujeres que padecen enfermedades o que necesitan someterse a tratamientos que interfieren en la función ovárica. En este contexto, el objetivo de este trabajo es revisar los aspectos relacionados con la biotécnica de MOIFOPA y la importancia de los antioxidantes en el cultivo in vitro de los folículos pré-antrais. Se realizó una investigación en la base de datos PubMed, buscando artículos sobre la biotecnología, principalmente relacionados con la necesidad del uso de antioxidantes en el cultivo. La mayoría de los estudios sobre biotecnología utilizan como modelo experimental los folículos ováricos de diferentes especies de animales. El MOIFOPA incluye el aislamiento y rescate de los folículos ováricos preantrales de los ovarios, seguido de su conservación mediante la técnica de enfriamiento o congelación y el cultivo folicular in vitro, con el fin de promover el crecimiento, la maduración y la fecundación in vitro (FIV) de los ovocitos incluidos en estos folículos, maximizando el potencial reproductivo femenino y disminuyendo la atresia folicular que se produce in vivo. Un aspecto que puede interferir en el éxito del cultivo in vitro de folículos ováricos preantrales es la producción excesiva de especies reactivas de oxígeno (ROS). El ácido ascórbico y el ácido alfa lipoico han mostrado resultados interesantes para reducir los efectos que causan las ERO en los folículos ováricos preantrales cultivados in vitro.

Reproductive dynamics of Leporinus obtusidens captured downstream from an important hydropower Station in Brazil / Dinâmica reprodutiva de Leporinus obtusidens capturados a jusante de uma importante usina hidrelétrica no Brasil

This study evaluated the reproductive dynamics and gonadal morphology of piaparas (Leporinus obtusidens) captured downstream from the Funil Hydroelectric Power Station. The 107 piaparas evaluated were captured monthly from Sep 2006 to Aug 2007. The data were stratified among the seasons to evaluate gonad maturation and the reproductive period was determined by assessing the gonadosomatic index, hepatosomatic index, abdominal fat index (AFI), weight:length ratio, condition factor and sexual proportion. The reproductive period lasted from winter to summer (Jul-Mar) in females and from spring to autumn (Sept-Jun) in males. AFI was high during and after the reproductive period in females and only during the reproductive period in males. The highest HIS values for males and females were obtained in spring and summer (Sept-Mar). The females observed did not spawn and showed follicular atresia. These characteristics suggest that L. obtusidens does not use the study area as a reproductive site and probably migrates for this purpose.

Este estudo avaliou a dinâmica reprodutiva e a morfologia gonadal de piaparas (Leporinus obtusidens) capturados a jusante de uma usina hidrelétrica. Os 107 exemplares avaliados foram capturados mensalmente, de setembro de 2006 a agosto de 2007. Os dados foram estratificados entre as estações do ano para se avaliar a maturação gonadal, e o período reprodutivo foi determinado por meio da avaliação do índice gonadossomático, índice hepatossomático, índice de gordura abdominal (AFI), relação peso:comprimento, fator de condição e proporção sexual. O período reprodutivo foi observado do inverno para o verão, julho-março, para as fêmeas, e da primavera para o outono, setembro-junho, para os machos. AFI foi alto durante e após o período reprodutivo em fêmeas e apenas durante o período reprodutivo em machos. Os maiores valores para machos e fêmeas foram obtidos na primavera e no verão. As fêmeas observadas não desovaram e mostraram atresia folicular. Essas características sugerem que L. obtusidens não usa a área de estudo como sítio reprodutivo e, provavelmente, realiza migração.

Atresia Folicular en Ovarios de Prochilodus lineatus / Follicular Atresia in Ovaries of Prochilodus lineatus

Durante el período reproductivo, en el ovario de los teleósteos se forman cíclicamente folículos primarios, los que maduran y son liberados en el momento de la ovulación. Sin embargo, algunos de ellos desarrollan un proceso de atresia con degeneración y reabsorción, el que para ser considerado un evento fisiológico debería predominar al final del ciclo reproductivo. Las características morfológicas de ovarios de Prochilodus lineatus fueron analizadas para identificar la presencia de folículos atrésicos como indicadores de posibles modificaciones de su biología reproductiva que afecte la repoblación de la especie. En los ovarios de P. lineatus predominaron los cambios morfológicos que se corresponden con la denominada atresia tipo I, y los folículos atrésicos fueron observados en peces con índice gonadosomático bajo que se encontraban al final del ciclo reproductivo. La atresia folicular hallada no pudo vincularse con algún proceso patológico que provoque una disminución de la especie en el ecosistema del río Paraná.

During the reproductive period, primary follicles are formed cyclically in the ovary of teleost, which mature and are released at the ovulation time. However, some of them develop a process of atresia with degeneration and resorption, which to be considered a physiological event should prevail at the end of the reproductive cycle. Morphological characteristics of ovaries of Prochilodus lineatus were analyzed for the presence of atresic follicles as indicators of possible changes in their reproductive biology affecting the repopulation of the species. In the ovaries of P. lineatus dominated the morphological changes that correspond to the so-called type I atresia, and atresic follicles were observed in fish with low gonadosomatic index who were at the end of the reproductive cycle. Detected follicular atresia could not be linked to a pathological process that causes a decline of the species in the Paraná river ecosystem.

Histological and immunohistochemical study of apoptosis in granulosa cells of atretic ovarian follicles of adult albino rat

It is well known that during ovarian follicular development, the majority of follicles undergo atresia at various stages of their development. Recent studies have reported that the degenerative changes associated with atresia appear initially in the granulosa cell layer, and follicular atresia occurs by apoptosis, which is an essential physiological process. The present study aimed to examine granulosa cell apoptosis using different, histological, immunohistochemical, and biochemical methods. Fifteen adult female albino rats were used in this study. The rats were sacrificed and bilateral oophorectomy was carried out; the ovaries of rats were processed and paraffin sections were prepared for staining with H and E and also to study immunohistochemistry for apoptotic proteins such as caspase-3 and antiapoptotic proteins such as B-cell lymphoma-2 [Bcl-2]. Some ovarian specimens were processed by electrophoresis for DNA separation. Apoptosis in granulosa cell showed morphological characteristics, including nuclei with marginated chromatin, a single condensed pyknotic nucleus, multiple nuclear fragments, and apoptotic bodies containing variable amounts of chromatin. Moreover, apoptotic changes were detected by immunohistochemical staining in the form of increased staining intensity to caspase-3 [apoptotic protein] and decreased staining intensity to Bcl-2 [antiapoptotic protein], and also increased fragmentation of DNA, which was indicated by a ladder step appearance that was detected using DNA electrophoresis

Correlation between function-morphology of effect of the stress and flunitrazepam administered during gestation in adult mouse ovary / Correlación entre la función y la morfología de los efectos de estrés y flunitrazepam administrado durante la gestación en ovario de ratón adulto

Previously we report long lasting effects on ovary of mice prenatally exposed to flunitrazepam (FNZ), a benzodiazepine with tranquilized action. In this work we find that the FNZ don't prevent the effects on ovary prenatally exposure to stress in mice. We studied adult females born from mothers that had been stressed by immobilization on day 6 ofgestation (GD-6) or group S, and from mothers stressed also by immobilization at GD-6, but which received a single oral dose of FNZ immediately after the stress group FNZS. The control groups were the SS that received the GD-6 saline solution and the group NT non-treated. Their ovaries were extracted for histology studies and to observe the activity of 3b hydroxysteroid dehydrogenase/isomerase (3 b-HSD). The histological analysis revealed high staining affinity ovarian cell of S and FNZS. Double oocytes and apoptotic bodies were found in the secondary atretic follicles, as well as abnormal primordial, primary and secondary follicle populations, as compared to SS and NT groups. The primordial, primary, and secondary follicles were significantly reduced in the experimental groups. But the primary and secondary atretic follicles were higher in both groups, and the number of corpora lutea was lower in both groups. The activity of 3 b-HSD was abnormally increased in both FNZS- and S-groups. These findings suggest that FNZ did not counteract the impairing effects of prenatal stress on adult offspring ovarian follicles, and could rather be responsible for long lasting changes occurring during embryonic programming.

Previamente comprobamos efectos de larga duración sobre el ovario de ratones expuestos prenatalmente a flunitrazepam (FNZ), una benzodiazepina con acción tranquilizante. En este trabajo encontramos que el FNZ, no revierte los efectos producidos por la exposición prenatal a estrés. Estudiamos hembras adultas nacidas de madres que se estresaron por inmovilización el día 6 de la gestación (DG-6) o grupo S, y de madres estresadas también por inmovilización el DG-6, las que recibieron una sola dosis de FNZ inmediatamente después del estrés (grupo FNZS). Los grupos de control fueron el SS al que se le administró solución salina y el NT no tratado. Se extrajeron sus ovarios para su estudio histológico y para observar la actividad de delta 3b-deshidroxiesteroide dehidrogenasa/isomerasa (3 b-HSD). El análisis histológico reveló una gran afinidad tintoreal en los ovarios de los grupos S y FNZS. En los ovarios de los ratones del grupo FNZS se encontraron en los folículos secundarios atrésicos ovocitos dobles y cuerpos apoptóticos así como una población mayor de folículos anormales primordiales, primarios y secundarios en comparación con los grupos SS y NT. Los folículos primarios y secundarios tuvieron una reducción significativa en los grupos experimentales pero los folículos atrésicos primarios y secundarios fueron más en ambos grupos y el número de cuerpos lúteos fue menor en ambos grupos. La actividad de 3 b-HSD aumentó de manera anormal tanto en los grupos FNZ y S. Estos hallazgos sugieren que el FNZ no contrarresta los efectos negativos del estrés prenatal sobre los folículos ováricos de las crías adultas, y podría ser responsable de los cambios largo plazo que ocurren a durante la programación embrionaria.

Falência ovariana prematura: aspectos atuais / Premature ovarian failure: present aspects

A falência ovariana prematura (FOP) acomete aproximadamente 1:1000 mulheres antes dos 30 anos, 1:250 em torno dos 35 anos e de 1:100 aos 40 anos. Manifesta-se como amenorréia primária ou amenorréia secundária, não podendo ser considerada definitiva em todas as pacientes, uma vez que a concepção espontânea pode ocorrer em até 5-10 por cento das FOP. Na maioria dos casos apresenta-se na forma esporádica, pois apenas 5 por cento apresentam história familial. Entre as causas conhecidas estão as alterações cromossômicas, dos genes ligados ao cromossomo X e cromossomos autossômicos, doenças autoimunes, alterações tóxicas e iatrogênicas. Com relativa freqüência, a causa etiológica não é obtida, sendo então denominada de idiopática. O diagnóstico da FOP é feito baseado na história clínica e níveis elevados do hormônio folículo estimulante (FSH), sendo posteriormente investigadas as causas mais específicas. O manejo clínico visa o suporte emocional, o tratamento hormonal com estrogênios e progestogênios, a abordagem da infertilidade e a prevenção de co-morbidades como a osteoporose e potencial maior risco cardiovascular.

Premature ovarian failure occurs in approximately 1:1000 women before 30 years, 1:250 by 35 years and 1:100 by the age of 40. It is characterized by primary or secondary amenorrhea and cannot be considered as definitive because spontaneous conception may occur in 5 to 10 percent of cases. In 95 percent of cases, premature ovarian failure is sporadic. The known causes of premature ovarian failure include chromosomal defects, autoimmune diseases, exposure to radiation or chemotherapy, surgical procedures, and certain drugs. Frequently, however, the etiology is not clear and these cases are considered to be idiopathic. Premature ovarian failure is defined by gonadal failure and high serum follicle-stimulating hormone (FSH) levels. Clinical approach includes emotional support, hormonal therapy with estrogens and progesterone or progestogens, infertility treatment, and prevention of osteoporosis and potential cardiovascular risk.

Low-dose Radiation-induced Hormetic Effect in the Rat Ovarian Follicle / 체질인류학회지

Hormesis is the generally-favorable biological responses to low exposures to toxins and other stressors. Radiation hormesis is the theory that ionizing radiation is benign at low levels of exposure, and that doses at the level of natural background radiation can be beneficial. The purpose of this study is to reveal the hormetic effect of low-dose radiation of ionizing radiation on the ovarian follicles of 4-week old female mice. Mice were grouped into control group, 2 cGy irradiated group, 2 cGy and 2 Gy irradiated group (2 cGY pre-exposure group), and 2 Gy irradited group. Mice were sacrificed by cervical dislocation 24 hours after irradiation, removed ovaries, fixed in neutral formaldehyde solution for 24 hours, embedded with paraffin, stained with hematoxylin and eosin and TUNEL immunohistochemically, and observed light microscopically the atretic follicles and normal follicles in various follicular developmental stages. In this experiment, the ratrio of atretic follicles to entire follicles in an ovary increased significantly in 2 Gyirradiated group compared with 2 cGY pre-exposure group, and the ratio of normal follicles to the entire follicles in an ovary in all the developmental stages were increased significantly in the 2 cGY pre-exposure group compared with 2 Gy-irradiated group. These results mean that low-dose radiation pre-exposure can induce the hormetic effect in the developing ovarian follicle.

Occurrence and significance of ovarian and follicular regression in cachara Pseudoplatystoma fasciatum (Linnaeus, 1766): a histology approach

The processes of ovarian regression and follicular atresia which reproduction was not induced by hormone in confined cachara, Pseudoplatystoma fasciatum, were investigated. The macro and microscopic characteristics (oocytes diameter and histology) of the ovaries were described every 20 days, in four stages: initial regression (Rg I = first 20 days), intermediate regression (Rg II = from 21st to 40th day), final regression (Rg III = from 41st to 80th day) and the recovering stage, called resting II (R II = from 81st to 150th day). The experiment was conducted from late January (summer - longer days) to May (autumn - shorter days). In the beginning, A0 samples showed oocyte diameters ranging from 437.5 to 1,187.5mm, suggesting that oocytes were in perinucleolar, at final maturation and atretic phases. After 150 days, the diameters reached the lowest values and a ruptured zona radiata, as well as the nearly complete reabsorption of the yolk could be visualized. At the same time, a sharp decrease in the mean values of the gonadosomatic index (GSI), water temperature, photophase and rainfall was observed. The gradual involution of this long process was dynamic and complex, affecting the spawning success (fertilization, eclosion and larvae survival rates) and, consequently, the whole productive system.

Estudaram-se os processos de regressão ovariana e atresia folicular em cachara, Pseudoplatystoma fasciatum, mantida em cativeiro, na reprodução não induzida por hormônios. As características macro e microscópicas (diâmetro dos ovócitos e histologia) dos ovários foram descritas a cada 20 dias, em quatro estádios: na regressão inicial (Rg I - os primeiros 20 dias), na regressão intermediária (Rg II - do 21° ao 40° dia), na regressão final (Rg III - do 41° ao 80° dia) e na fase de recuperação ou de repouso II (R II - do 81° ao 150° dia). O experimento foi realizado do final de janeiro (verão-dias longos) a maio (outono-dias curtos). No início do experimento, as amostras apresentaram ovócitos com diâmetros que variaram de 437,5 a 1.187,5mm, sugerindo encontrarem-se nas fases perinucleolar, de maturação final e atrésicos. Aos 150 dias, os diâmetros atingiram os menores valores e pôde-se visualizar a zona radiata rompida e o vitelo reabsorvido. Concomitantemente, houve diminuição abrupta dos valores médios do índice gonadossomático, da temperatura da água, das horas de luz e de chuva. A involução gradual do longo processo foi dinâmica e complexa, afetando o êxito da desova (taxas de fertilização, de eclosão e de sobrevivência de larvas) e, conseqüentemente, o sistema produtivo.

The expression of BH3-only Bcl-2 family members in mouse ovary and influence of gonadotropin on Nix expression in granulosa cells / 대한산부인과학회지

OBJECTIVE: In the ovary apoptosis eliminates granulosa cells (GC) during follicle atresia. While we identified many cell death regulatory molecules not yet characterized in the ovary, of particular interest were members of the Bcl-2 family which contain only the Bcl-2 homology (BH)-3 domain. The objectives of this study was to investigate and compare the expression patterns of BH3-only Bcl-2 family members in various organs and evaluate their function in ovarian granulosa cells. METHODS: Total RNA was extracted from GC, ovaries, uteri, hearts (low rate of cell turnover) and livers (high rate of cell turnover) of prepubertal female mice. BH3-only Bcl-2 family members were cloned to make riboprobe. The expression patterns in the tissues evaluated by Northern blot analysis. Nix mRNA expression in ovarian granulosa cells after gonadotropin treatment also compared by Northern blot analysis. RESULTS: Bad, Bid, Bim, Bmf, Map-1 and Nix were expressed in granulosa cells. Nix was most abundantly expressed in GC. In contrast, Blk was expressed in the ovary, liver, heart and uterus, but not in GC. Bmf, a sensor or microfilament disassembly, was expressed in GC, ovary and uterus, with limited to no expression in non-reproductive tissues. Nix mRNA expression was not regulated by gonadotropin after 42 hour. CONCLUSION: These studies will help to complete a molecular blueprint of the regulatory network that controls GC death during follicular atresia. In addition, these data, which show a tissue/cell-selective profile of BH3-only expression, may also explain the known variation in the in vivo apoptotic response of different tissues/cells to generic stimuli that should be globally lethal.

Expression of Inhibin in the Whole-body gamma-irradiated Mouse Ovary / 대한불임학회지

OBJECTIVE: The purposes of the present study were to investigate the effect of gamma-radiation on the expression of inhibin-alpha proteins and genes for inhibin alpha, betaA, and betain the ovary. METHODS: Immature mice were whole-body gamma-irradiated with 25% of a lethal dose. At time 0, 3, 6, 12, and 24 hours after the irradiation,the ovaries were collected and used for immunohistochemistry for inhibin-alpha, and RT_PCR for inhibin-alpha, betaA, and betaB. RESULTS: The expression of the immunoreactive inhibins-alpha was maintained at 12 hours post-irradiation and reduced thereafter. The expression of inhibin-alpha mRNA was significantly increased with the time after the irradiation. However there were no significant changes in the expression of betaA and betaB mRNAs. CONCLUSION: It might be thought that inhibin acts as one of the regulatory factors in the gamma-radiation-induced follicular atresia in mice

TNFalpha and TNFR2 Immunohistochemistry During Ovarian Follicular Development and Atresia in the Rat / 대한해부학회지

Tumor necrosis factor alpha (TNFalpha) is an intraovarian cytokine that may play a role in ovarian development and function. Identification of ovarian TNFalpha receptors provides support for establishing a role of TNFalpha in ovarian development and function. TNFalpha exerts its effects by binding to either TNF receptor 1 or 2 (TNFR1 or TNFR2). When TNFalpha binds with TNFR2, expression of survival genes is up-regulated, resulting in proliferation of granulosa cells. In the present study, the authors identified the changes in localization of TNFalpha and the expression of TNFR2 in granulosa cells during follicular atresia in rat ovaries. In healthy follicles, intense signals for TNFalpha and TNFR2 were found in the outer surface of the granulosa layer, where many proliferating cells and no apoptotic cells were observed. In atretic follicles, decreased expression of TNFalpha and TNFR2 was observed in the granulosa layer, where many apoptotic cells were seen. These findings suggested that TNFalpha acts as a survival factor in granulosa cells during follicular atresia in rat ovaries.

Distribution and Role of Ovarian Follicle Macrophage in Rat Ovarian Follicular Atresia / 대한산부인과학회지

OBJECTIVE: Ovarian follicular atresia is initiated from ovarian granulosa cell apoptosis and macrophages exert their effects directly and/or indirectly on follicular atresia by phagocytosis of apoptotic bodies and secretion of various cytokines. In spite of the abundant data on ovarian macrophages, the presence of these cells within the follicles (i.e., among granulosa cells) remains controversial and the elimination methods of apoptotic bodies of atretic follicles, and the time and methods of penetration of macrophages into the follicles are not known completely. The aim of the present study is to demonstrate the presence of macrophage within the ovary as related to follicular atresia and the process of elimination of apoptotic granulosa cells by light and electron microscopy. METHODS: Using rat ovaries, immunohistochemical studies with rat macrophage monoclonal antibody ED1 for macrophages, and light and transmission electron microscopic observations were performed. RESULTS: In the rat, follicular atresia was initiated by the granulosa cell apoptosis which occured randomly within the all granulosa layers. Macrophages were observed within normal follicles, in antrum, granulosa and theca cell layers of atretic follicels, in interstium and in corpus luteum. Ultrastructurally, apoptotic granulosa cells showed characteristics, pyknotic nucleus and apoptotic body formation. Apoptotic bodies were eliminated by intact neighboring granulosa cells and macrophages. Intact granulosa cells ingested apoptotic bodies transiently, soon after they fell into the apoptosis. Finally, apoptotic bodies and degenerating oocytes were phagocytosed by macrophages. Macrophages entered the ovarian follicle at the time of initiation of granulosa cell apoptosis, and migrated with the progression of apoptosis. By elimination of theca cells, macrophages contributed the completion of follicular atresia. CONCLUSION: This study demonstrates both intact neighboring granulosa cells and macrophages in the elimination of apoptotic bodies in atretic follicles of the rat ovary. Macrophages are present within normal follicles, in atretic follicles such as antrum, granulosa and theca cell layers and in corpus luteum but are in different appearances according to their location in ovary. A number of macrophages appearing in atretic follicles and in corpora lutea suggest a role for macrophages in follicular atresia and corpus luteum differentiation. The function of macrophage according to their location in follicular development should be further studied.

Follicular Atresia and Macrophages in the Porcine Ovary; Immunohistochemical and Transmission Electron Microscopic Study / 대한해부학회지

Apoptosis of granulosa cells leads follicular atresia and macrophages have an important role during the apoptotic process. However, the propagation of apoptosis within the follicle, the ways of elimination of apoptotic bodies and degenerated oocyte, and the completion of follicular atresia are still controversial and unidentified clearly. Using adult porcine (Yorkshire-breed) ovary, in this morphological study, transmission electron microscopic observation and immunohistochemical study with pig macrophage monoclonal antibody 4E9 were performed. In light microscopy, the follicular atresia initiated with apoptosis of granulosa cells, followed by degeneration of oocyte and apoptosis of theca interna cells. Apoptosis occured in random fashion among the granulosa cells and propagated multidirectionally, and finally to the granulosa cells surrounding zona pellucida of degenerating oocyte. Pyknosis of granulosa cells was the first sign of apoptosis. In immunohistochemistry, macrophages were found only in the granulosa layer at the stage of beginning of apoptosis. With progression of apoptosis, they were proliferated greatly in number enough to eliminate all the apoptotic bodies, and found within the follicular antrum. In advanced stage of atresia, macrophages surrounded the zona pellucida of degenerating oocyte, and found also in the theca interna. In transmission electron microscopy, phagocytic granulosa cells maintained characteristic gap junctions with neighboring granulosa cells and contained several apoptotic bodies and lipid droplets within their cytoplasm. Macrophages kept many apoptotic bodies, vacuoles and autophagosomes in their cytoplasm. Apoptotic granulosa cells were ingested by intact granulosa cells and macrophages initially, but lately, all the apoptotic granulosa cells and degenerated oocyte were eliminated by macrophages. Ovarian follicular atresia completed with phagocytosis of apoptotic theca interna cells by macrophages, and the remnants of the atretic follicle became ovarian stroma. It is well known that macrophages may play an important role during follicular atresia, such as elimination of apoptotic granulosa cells, theca interna cells and degenerated oocytes, but, the valid action mechanisms of macrophages on the initiation of granulosa cell apoptosis and on the completion of atresia through the secretion of paracrine factors and autocrine factors still unclear.

Morphological Study on the Radiation-induced Follicular Atresia and the Follicular Macrophage / 대한산부인과학회잡지

OBJECTIVE: It is well known that X-ray induces follicular atresia, but the exact mechanism of atresia is not still unveiled completely. In addition, the role of macrophage related with clean-up the dead granulosa cells and other functions within the ovarian follicle is emphasized recently. The aim of this study is to assess the radiation-induced morphological changes of ovarian follicles and follicular macrophages. METHODS: 8 Gy X-ray irradiated on the 3-week old rats (Sprague-Dawley strain), sacrificed at 6, 12, and 24 hours after irradiation, and performed morphological studies with light and transmission electron microscopy, TUNEL, and macrophage immunohistochemistry. RESULTS: Follicular atresia increased significantly (p<0.01) at 6 hours after X-irradiation, and it was decreased significantly (p<0.01) at 12 and 24 hours after irradiation. X-ray induced chromatin condensation in the nucleus and nuclear fragmentation of granulosa cells, which were the typical features of apoptosis. Apoptotic granulosa cells were phagocytosed by the neighboring normal granulosa cells and the macrophages. During atresia of follicles, radioresistant granulosa cells were found in some follicles, which showed similar features morphologically with the granulosa cells of normal follicles. Macrophages were found both within the antrum and at the follicular granulosa layer. CONCLUSION: X-radiation induced follicular atresia by means of granulosa cell apoptosis, and radioresistant granulosa cells which have similar features morphologically with the granulosa cells of normal follicles were observed in some follicles. And the macrophages which phagocytose the apoptotic granulosa cells were located within the follicular antrum and at the follicular granulosa layer.

Study of atresia in the ovary of bagrus bayad during the reproduction cycle by histological and electron microscopic investigation

Atretic follicles in ovary of the Nile teleost fish, Bagrus bayad, were followed through out the spawning period. They have been examined under light microscope. Six types of atresia have been distinguished, Two of them have been subjected to the electron microscope examination. In general, atretic follicles have been found in mature and tertiary yolk oocytes stage. They were measured to be larger than 400 micro in diameter. The Observed sequences for atretic follicles were as follows: erosion of thecal layer, hypertrophy of granulosa, and finally dissolution of ooplasm. In ultra structure examination erosion of thecal layer appeared as ill-defined cell outline with obvious separation between granulosa and thecal layer. Beside, junction of complex vanished. In addition, the zona radiata ruptured to small fragments. In conclusion atresia in Bagrus bayad is a functional activity of oocyte development which might be influenced by hormonal mechanism that regulate oocyte growth and recruitment

HCG attenuates ATP induced apoptosis in cultured human luteinized granulosa cells (hLGCs) / 대한산부인과학회잡지

OBJECTIVE: To determine the effects of hCG on extracellular ATP induced apoptosis in cultured human luteinized granulosa cells (hLGCs) METHODS: The addition of various concentrations of ATP (0, 0.1, 0.25, 0.5, 0.75 mM) and 5 IU hCG to luteinized granulosa cells obtained during in vitro fertilization ovum pickup procedures. After culture for 24 hours, purinoceptor activity and functional changes in mitochondria were measured by patch clamp, flow cytometry, and confocal microscopy. RESULTS: Calcium imaging with fura-2 revealed that ATP elevated [Ca2+]i by mobilizing intracellularly stored Ca2+. A patch clamp study showed that ATP exerted its effect by initially binding to the P2Y type purinoceptor, as evidenced by the ATP-evoked outward Ca2+-activated K+ current. Probing mitochondria with JC-1, a mitochondrial transmembrane potential-sensitive dye, revealed that ATP induced mitochondrial depolarization in a concentration-dependent manner. A quantitative flow cytometric analysis with Annexin V showed that apoptotic cells were increased in number in proportion to the concentration of ATP, having 18.57% of apoptotic cell populations in the presence of 0.75 mM ATP compared to 7.88% in the control. Moreover, treatments with human chorionic gonadotropin (hCG) at 5 IU reversed both the ATP-induced mitochondrial depolarization and apoptosis (18.57 vs 6.32%). CONCLUSION: Taken together, these results indicate, first, extracellular ATP recognized by the P2Y type purinoceptor on h-LGCs increases the intracellular Ca2+. Second, the increased intracellular Ca2+ triggers the apoptotic cascade by acting at least, in part, on mitochondria. Third, hCG reverses the ATP-induced apoptosis, raising a possible clinical implication of hCG in the treatment of degeneration of granulosa cells such as follicular atresia.

Effect of angiotensin II on follicular atresia in mouse / 生理学报

The effect of angiotensin II (Ang II) on the follicular development was studied by using an animal model of follicular atresia induced by pregnant mare s serum gonadotropin (PMSG). The results showed that: (1) a large number of atretic follicles were found in the ovary of 24-day-old mouse after 6-day treatment of PMSG. Deoxyribonucleic acid (DNA) extracted from granulosa cells clearly showed a ladder band under agarose gel electrophoresis analysis. (2) the contents of Ang II in the ovary extremely increased with the development of follicular atresia. (3) Ang II significantly antagonized the stimulating effect of the follicle-stimulating hormone (FSH) on estradiol (E(2)) generation of granulosa cells. It is suggested that Ang II may be involved in the regulation of follicular atresia in mouse.

The Immunologic Expression of Apoptosis Related Proteins and Apoptotic Cells in Human Ovarian Follicles / 대한불임학회지

OBJECTIVE: To investigate the expression of apoptosis related proteins and apoptotic cells on the human ovarian follicles. MATERIALS AND METHODS: Thirty five Formalin-fixed paraffin-embedded human ovarian tissue blocks were selected from the surgical pathology files of the department of pathology, College of Medicine, Yonsei University, for the period from 1996 to 1998. All specimen were from premenopausal women aged from 32~45. Ovarian tissues were collected from the patients performing hysterectomy for benign uterine diseases. Immunohistochemical staining was performed for the detection of DNA fragmented cell, Bcl-2, Bax, Fas and Fas-ligand. RESULTS: Bcl-2 and bax were not expressed on the surrounding cells and oocyte of the primary, primordial and preantral follicles. Fas and Fas-ligand (Fas-L) were not expressed on the surrounding cells on the primordial and primary follicles. But expressed on the surrounding granulosa cells and oocyte in the primordial and primary follicles. In the healthy follicles, Bcl-2 was expressed on the granulosa cells, however, Bax was not expressed. DNA fragmented cells were expressed on the inner granulosa cell layer of atretic follicles. CONCLUSION: Fas, Fas-ligand, and Bax may be responsible for the follicular atresia and Bcl-2 may be involved in the follicular survival in the human ovary.