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
Animals ; Female ; Follicular Atresia ; Immunohistochemistry ; Inhibins* ; Mice* ; Ovary* ; RNA, Messenger

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.
Female ; Animals ; Follicular Atresia ; Apoptosis ; Cell Death ; Ferroptosis ; Pyroptosis

Extracellular vesicles (EVs) are membrane-bound particles actively released by cells. In prokaryotes and eukaryotes, EVs are effective bridges for communication between cells. EVs carry biological macromolecules, including proteins, lipids and nucleic acid, which affects different physiological functions of parent cells and recipient cells. Among them, the microRNA carried by EVs is the most reported and plays an important role in physiological function of organisms. During the development of follicles, only a few follicles can fully develop and ovulate, whereas most of them undergo atresia at different stages of development. In the whole process of follicular development, the changes at each stage and the regulation mechanism of follicular atresia are not completely understood. In this paper, we introduced the types, characteristics, isolation methods and uses of EVs, and emphasized how microRNA carried by EVs in follicular fluid regulated follicular atresia from the aspects of different cytokines and hormones. Additionally, the application prospect of microRNA carried by EVs in follicular fluid in reproductive regulation and reproductive disease diagnosis was discussed. This paper is significant for studying the regulation of follicular development and the effective utilization of oocytes.
Animals ; Extracellular Vesicles/metabolism* ; Female ; Follicular Atresia ; Follicular Fluid ; MicroRNAs/metabolism* ; Oocytes

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.
Apoptosis* ; DNA ; Female ; Follicular Atresia ; Granulosa Cells ; Humans* ; Hysterectomy ; Oocytes ; Ovarian Follicle* ; Ovary ; Pathology ; Pathology, Surgical ; Uterine Diseases

In mammal, lots of follicles start simultaneously their growth but only a few oocytes are ovulated in every sexual cycles. Most of matured and grown oocytes are destined to degenerate by atresia. However, the molecular and physiological mechanisms are not elucidated yet. The present study was designed to establish an induction method of follicular atresia with ketamine or pentobarbitone and evaluate the effect of these anesthetics on oocyte maturation and granulosa cell apoptosis of the mouse ovarian follicle. The percentages of degenerated oocyte and apoptotic granulosa cell in ketamine treated groups were significantly higher than that in controls (58.9% vs 33.5%, p<0.01, degeneration; 44.9% vs 26.6%, p<0.01, apotosis). Futhermore, it was revealed that the concentrations of progesterone in both groups were markedly higher than that in control. In conclusion, it is considered that ketamine induce an atresia as pentobarbitone, and may be useful for inducing follicular atresia.
Anesthetics ; Animals ; Apoptosis ; Female ; Follicular Atresia ; Granulosa Cells* ; Ketamine* ; Mammals ; Mice* ; Oocytes* ; Ovarian Follicle ; Ovary* ; Pentobarbital* ; Progesterone

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.
Animals ; Apoptosis ; Corpus Luteum ; Cytokines ; Female ; Follicular Atresia* ; Granulosa Cells ; Macrophages* ; Microscopy, Electron ; Oocytes ; Ovarian Follicle* ; Ovary ; Phagocytosis ; Rats* ; Theca Cells

Normal and abnormal follicular growth and steroidogenesis depend on gonadotropins as well as intraovarian peptides, which may mediate or potentiate gonadotropin action. Inhibin also affect follicular development and steroidogenesis and may play a role in dominant follicle selection and follicular atresia. Therefore, we studied the differences of serum inhibin, gonadotropin and androgen levels in the women with only the ultrasound findings and no disorder, and polycystic ovary (PCO) with ovulatory disturbance. We prospectively analysed forty-three women with PCO. The diagnosis of PCO was based on typical appearance of the ovaries on TVS. Twelve women with regular menstrual cycle and normal ovarian morphology were selected as control. Basal levels of inhibin, luteinizing hormone(LH), follicle stimulating hormone(FSH), estradiol(E2), testosterone(T), androstenedione(ADD), dehydroepiandrosterone-sulfate(DS), prolactin and TSH in serum were determined. There were significant differences in basal LH levels and LH/FSH ratio between the control and the women with PCO. The basal levels of inhibin and E2 in the oligo-amenorrheic PCO (N=34) were significantly higher than those in the control. There was higher negative correlation between the inhibin and T levels in the oligo-amenorrheic PCO, but, not in the regular cycling PCO. Also, there was higher positive correlation between the LH and T levels in the oligo-amenorrheic PCO, but not in the regular cycling PCO. These data presume that the initial event of PCO is elevated pituitary LH secretion. Elevated levels of LH may down-regulate LH receptors on granulosa cells and also cause hypertrophy of the thecal layer. High level of androgen secreted by the hypertrophied thermal layer may stimulate inhibin secretion from granulosa cells and can be converted to estrogen by extraovarian tissues and could serve to augment pituitary sensitivity to GnRH with a resultant secretion of more LH follicular development and dominant follicle selection resulted in ovulatory disturbance.
Diagnosis ; Estrogens ; Female ; Follicular Atresia ; Gonadotropin-Releasing Hormone ; Gonadotropins ; Granulosa Cells ; Humans ; Hypertrophy ; Inhibins* ; Lutein ; Menstrual Cycle ; Ovary* ; Peptides ; Prolactin ; Prospective Studies ; Receptors, LH ; Ultrasonography

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.
Adenosine Triphosphate* ; Annexin A5 ; Apoptosis* ; Calcium ; Chorionic Gonadotropin ; Female ; Fertilization in Vitro ; Flow Cytometry ; Follicular Atresia ; Fura-2 ; Granulosa Cells* ; Humans* ; Lutein* ; Microscopy, Confocal ; Mitochondria ; Ovum ; Receptors, Purinergic

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.
Animals ; Apoptosis ; Chromatin ; Female ; Follicular Atresia* ; Granulosa Cells ; Immunohistochemistry ; In Situ Nick-End Labeling ; Macrophages* ; Microscopy, Electron, Transmission ; Ovarian Follicle ; Radiation, Ionizing ; Rats

There have been many reports to date regarding the role of GnRH as a local regulatory factor of ovarian function as studies of human and rat ovaries revealed GnRH and its receptor. In recent studies it has been shown that GnRH directly causes apoptosis in the granulosa cells of the rat ovary, and such results leads to the suggestion that the use of GnRH agonist for more stable long term ovarian hyperstimulation in human IVF-ET programs causes granulosa cell apoptosis which may lead to follicular atresia. Therefore this study attempts to determine if granulosa-luteal cell apoptosis occurs in patients during IVF-ET programs in which GnRH agonist is employed for ovarian hyperstimulation. The quality of oocyte-cumulus complexes obtained during ovum pickup procedures were assessed morphologically and then the fertilization rate and developmental rate was determined. Apoptotic cells among the granulosa-luteal cells obtained during the same procedure were observed after staining with Hematoxylin-rosin. The fragmentation degree of DNA extracted from granulosa-luteal cells was determined and comparatively analyzed. There was no difference in the average age of the patients, the number of oocytes retrieved, and fertilization and developmental rates between the FSH/hMG group and GnRH-long group. There was also no difference in the apoptosis rate and pyknosis rate in the granulosa-luteal cells between the two groups. However, when the oocyte-cumulus complexes were morphoogically divided into the healthy group and atretic group without regard for the method of hyperstimulation, the results showed that the number of oocytes obtained averaged 11.09+/-8.75 and 10.33+/-4.53 per cycle, respectively, showing no significant difference, but the fertilization rate (77.05%, 56.99%, respectively, p<0.01) and developmental ,ate (65.96%, 41.51%, respectively, p<0.01) was significantly increased in the healthy group when compared to the atretic group. The degree of apoptosis in the granulosa-luteal cells showed that in the healthy group it was 2.25% which was not significantly different from the atretic group (2.77%), but the pyknosis rate in the atretic group (27.81%) was significantly higher compared to the healthy group (11.35%, p<0.01). The quantity of DNA fragmentation in the FSH/hMG group was 32.22%, while in the GnRH-long group it was 34.27%, showing no significant difference. On the other hand the degree of DNA fragmentation was 39.05% and 11.83% in the healthy group and atretic group, respectively, showing significantly higher increase in the atretic group (p<0.01). The above results suggest that death of granulosa-luteal cells according to the state of the oocyte-cumulus complex is more related to pyknosis rather than apoptosis. Also, the GnRH agonist used in ovarian hyperstimulation does not seem to directly affect the apoptosis of retrieved oocytes and granulosa-luteal cells, and which is thought to be due to the suppression of the apoptogenic effect of GnRH agonist as a result of the high doses of FSH administered.
Animals ; Apoptosis* ; DNA ; DNA Fragmentation ; Female ; Fertilization ; Follicular Atresia ; Gonadotropin-Releasing Hormone* ; Granulosa Cells ; Hand ; Humans* ; Luteal Cells ; Oocytes ; Ovary ; Ovum ; Rats