In this study, in order to further understanding of function of Mullerian inhibiting substance (MIS) and the ontogeny of the production profile of biologically active MIS and MIS type II receptor (MISR II), the patterns of their localization according to the follicular development in 21 ovarian specimens from women in reproductive age were studied by immunohistochemical staining. The flattened granulosa cells in primordial follicles failed to stain for MIS and MISR II, but the first staining was detected in the cuboidal granulosa cells in primary follicles. MIS and MISR II were detected specifically and exclusively in the cytoplasm of granulosa cells. The granulosa cells of both single and multiple layered growing preantral follicles showed strong specific staining for MIS and MISR II. Among the growing follicles, large follicle stained more intensely than small one. Within the multiple layers of granulosa cells, the innermost cells, closer to the oocyte, stained more intensely for MIS than those near the basement membrane, but MISR II was evenly distributed. In antral follicles, expression of the MIS was only seen in the granulosa cells, but MISR II was seen in the granulosa cells and theca cells. In large antral follicles, cumulus cells and periantral granulosa cells stained more intensely for MIS than those in the periphery. MIS staining waned in the mature follicles just before ovulation and could not be found in atretic follicles, corpus luteum, and corpus albicans. The expression levels of MISR II in mature follicles was lower than those in growing follicles and were even further reduced, but still detectable, in corpus luteum. There was a decreased level of MISR II expression when follicles become atretic and eventually lost from atretic follicles. The MIS and MISR II staining were not found in primordial follicles, oocytes, interstitial cells, ovarian epithelium, and corpus albicans. It is concluded that actions of MIS via MISR II are autocrine and paracrine in nature. The pattern of MIS and MISR II expression according to the menstrual cycles and development suggest that MIS may act as an intraovarian regulator of follicle maturation, selection and ovulation during the adult reproductive cycle.
Adult ; Anti-Mullerian Hormone* ; Basement Membrane ; Corpus Luteum ; Cumulus Cells ; Cytoplasm ; Epithelium ; Female ; Granulosa Cells ; Humans* ; Menstrual Cycle* ; Oocytes ; Ovarian Follicle ; Ovary* ; Ovulation ; Theca Cells

In recent years,microRNAs(miRNAs)have been detected at different stages of follicular development and in different cells of follicles.Extracellular vesicle(EV)-derived miRNAs have also been detected in the follicular fluid of mature follicles.miRNAs participate in the regulation of normal follicular development,and the regulation disorder may lead to the occurrence of some ovarian diseases.In order to further systematically elucidate the regulatory mechanism of miRNAs on follicular development and find suitable EV-derived miRNAs that can predict oocyte development,we reviewed the functions of miRNAs in follicular development from the perspectives of granulosa cell development,oocyte development,and hormone synthesis.
Female ; Follicular Fluid ; Granulosa Cells ; Humans ; MicroRNAs/genetics* ; Oogenesis ; Ovarian Follicle

Human ovarian follicles reduce rapidly in number throughout fetal and adult life. Throughout the menstrual cycles, primordial follicles grow into mature follicles and then ovulate to form corpus luteum. Apoptosis has been implicated in several events that occur during the process of follicular growth, atresia and the regression of the corpus luteum. By the use of immunohistochemistry, we clarified the involvement of apoptosis in the human ovary during follicular growth, regression and atresia by investigating the expression of Fas, Fas-ligand, Bcl-2 and Bad in primordial follicles, primary follicles and mature follicles. Fas immunostaining was present in primordial oocytes, both oocytes and granulosa cells of primary follicles, preantral follicles and all follicular cells of mature follicles. Fas-ligand and Bad immunostaining patterns were similar to those of Fas except for theca cells. Bcl-2 immunostaining was present in both oocytes and granulosa cells of primary, preantral and mature follicles. In corpus luteum, Fas, Fas-ligand, Bcl-2 and Bad immunostaining were observed and decreased in the regressing corpus luteum. In postmenopausal ovary, Fas, Fas-ligand, Bcl-2 and Bad immunostaining were entirely negative. Bad immunostaining was observed but Bcl-2 was not in atretic follicle. These results suggest that Fas, Fas-ligand, Bcl-2 and Bad may play important roles in human ovary during follicular growth, regression and atresia simultaneously. Further studies should be required to elucidate the underlying mechanism and apoptosis of the disease associated with normal and abnormal ovarian aging.
Adult ; Aging ; Apoptosis ; Corpus Luteum ; Female ; Granulosa Cells ; Humans* ; Immunohistochemistry ; Menstrual Cycle ; Oocytes ; Ovarian Follicle* ; Ovary ; Theca Cells

The growth and development of follicles are regulated by genes,hormones and growth factors autocrined and paracrined from granulosa cells,theca cells,and oocytes.Products of glycolysis from granulosa cells such as pyruvate and lactate are one of the main energy sources,which play an important role during folliculogenesis and follicle maturity.Studies on the changes of the products and rate-limiting enzymes during granulosa cells' glycolysis help to clarify the molecular mechanism of energy demand in folliculogenesis and guide the clinical treatment of infertility due to abnormal follicular development.This article reviews recent research advances in the energy demand and regulatory mechanism in different states of folliculogenesis.
Energy Metabolism ; Female ; Glycolysis ; Granulosa Cells ; Humans ; Intercellular Signaling Peptides and Proteins ; Oocytes ; Ovarian Follicle ; growth & development ; Theca Cells

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

OBJECTIVES: This study was aimed to obtain information on the ontogeny of the production profile of MIS type II receptor (MISR II) and the pattern of its localization according to follicular development METHODS: Expression of MISR II were studied in 21 ovarian specimens from adult normal cycling women by RT-PCR and in situ hybridization of the MISR II mRNA and immunohistochemical staining of the MISR II. RESULTS: The first staining for MISR II and MISR II mRNA were detected in the granulosa cells in primary follicles. The granulosa cells of multiple layered growing follicles showed strong specific staining for MISR II and MISR II mRNA. Among the growing follicles, large follicle stained more intensely than small one. Expression of the MISR II and MISR II mRNA were also seen in the granulosa cells and theca cells of antral follicles. The expression levels of MISR II and MISR II mRNA in mature follicles were lower than those in growing follicles and were even further reduced, but still detectable, in corpus luteum. There was a decreased level of MISR II and MISR II mRNA expression when follicles become atretic. Both expressions were eventually lost from atretic follicles. And the MISR II and MISR II mRNA staining were not found in primordial follicles, oocytes, interstitial cells, ovarian epithelium, and corpus albicans. CONCLUSION: The production and localization of MISR II in granulosa cells, theca cells, and corpus luteum in normal reproductive ovary indicate that actions of MIS via MISR II are autocrine and paracrine in nature. The pattern of MISR II and MISR II mRNA expression according to follicular development indicate that MIS function in the ovary is turned on in primary follicles, increases to maximal levels in large growing follicles, and decreases just before ovulation. These experiments suggest that MIS may play an important role in follicle maturation and follicle selection during the adult reproductive cycle. And this study may yield important information to direct the development of newer contraceptive strategies.
Adult ; Anti-Mullerian Hormone* ; Corpus Luteum ; Epithelium ; Female ; Granulosa Cells ; Humans* ; In Situ Hybridization ; Oocytes ; Ovarian Follicle ; Ovary* ; Ovulation ; RNA, Messenger ; Theca Cells

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.
Adult ; Apoptosis ; Cytoplasm ; Female ; Follicular Atresia* ; Gap Junctions ; Granulosa Cells ; Humans ; Immunohistochemistry ; Macrophages* ; Microscopy ; Microscopy, Electron, Transmission ; Oocytes ; Ovarian Follicle ; Ovary* ; Phagocytosis ; Theca Cells ; Vacuoles ; Zona Pellucida

In the mammalian ovary, follicular atresia occurs through apoptosis. Apoptosis is known as the physiological cell death, which is regulated by bcl-2 gene family. In the bcl-2 gene family, bcl-2/bcl-xLong is known as an inhibitor of apoptosis, whereas bax/bcl-xShort is known as an inducer of apoptosis. We thought that these genes are associated with follicular development and atresia. Therefore, the present study used a in situ hybridization to examine the expression of bcl-2 and bax mRNAs during ovarian follicular development and atresia induced by PMSG (15 IU) treatment in the immature rat ovary. Morphological changes were occurred with the manner of five days periodicity after PMSG treatment. One or two days after PMSG treatment, ovaries had growing follicles with antrum and healthy granulosa cells. Three days after treatment, some degenerating follicles which had thinner granulosa cell layers than growing follicles were observed. At day four and five after treatment, degenerating follicles which have pyknotic nuclei and thin distorted granulosa cell layers appeared. These atretic follicles showed positive reaction with in situ DNA end labelling which indicates apoptotic changes. This study showed that bcl-2 mRNA was expressed in the theca and interstitial cells. Growing follicles of one or two days have showed stronger bcl-2 mRNA signals than atretic follicles of four or five days in these cells. Bax mRNA was expressed in the theca cells, interstitial cells, and granulosa cells. Atretic follicles of four or five days showed stronger bax mRNA signals than growing follicles of one or two days in these cells. Expression of bcl-2 mRNA was increased in growing follicles while decreased in atretic follicles. In contrast, expression of bax mRNA was increased in atretic follicles while decreased in growing follicles. Therefore, we confirmed that follicular development and atresia were affected by the change in the ratio of bcl-2 and bax mRNAs. According to these data, we proposed that these two genes are associated with follicular development and atresia.
Animals ; Apoptosis ; Cell Death ; DNA ; Female ; Follicular Atresia ; Genes, bcl-2 ; Granulosa Cells ; Humans ; In Situ Hybridization ; Ovarian Follicle ; Ovary* ; Periodicity ; Rats* ; RNA, Messenger* ; Theca Cells

Mullerian inhibiting substance(MIS) has been known as a non-steroidal testicular Sertolicell product responsible for the regression of Mullerian duct in male embryos. More recently MIS was also found to be present in an bioactive form in the bovine and rat ovaries but the function of MIS in the ovary has not been fully delineated. In this study, in order to understand its function in the ovary the ontogeny of the production profile of MIS and the pattern of its localization in ovaries from adult normal cycling women were studied by immunohistochemical staining using the rabbit polyclonal antibody against human recombinant MIS that almost completely blocks its biological activity. MIS was detected specifically and exclusively in the cytoplasm of granulosa cells. The flattened granulosa cells in primordial follicles failed to stain for MIS, but the cuboidal cells of growing follicles stained intensely. The granulosa cells of both single and multiple layered growing follicles showed strong specific staining for MIS. Within the multiple layers of granulosa cells, closer to the oocyte, stained more intensely than those near the basement membrane. Similarly, in antral follicles, cumulus cells and periantral granulosa cells stained more intensely than those in the periphery. MIS staining waned in the mature follicles just before ovulation and could not be found in atretic follicles, corpus albicans. In conclusion, this specific localization suggest that MIS may act as an intraovarian regulator of follicular development and oocyte maturation during the adult reproductive cycle.
Adult ; Animals ; Anti-Mullerian Hormone* ; Basement Membrane ; Cumulus Cells ; Cytoplasm ; Embryonic Structures ; Female ; Granulosa Cells ; Humans* ; Male ; Oocytes ; Ovarian Follicle ; Ovary* ; Ovulation ; Rats

OBJECTIVE: This study was performed to explore the possibility that each oocyte and its surrounding cumulus cells might have different genetic expression patterns that could affect human reproduction. METHODS: Differential gene expression analysis was performed for 10 clusters of cumulus cells obtained from 10 cumulus-oocyte complexes from 10 patients. Same procedures related to oocyte maturation, microinjection, and microarray analyses were performed for each group of cumulus cells. Two differential gene expression analyses were performed: one for the outcome of clinical pregnancy and one for the outcome of live birth. RESULTS: Significant genes resulting from these analyses were selected and the top 20 affected pathways in each group were analyzed. Circadian entrainment is determined to be the most affected pathway for clinical pregnancy, and proteoglycans in cancer pathway is the most affected pathway for live birth. Circadian entrainment is also amongst the 12 pathways that are found to be in top 20 affected pathways for both outcomes, and has both lowest p-value and highest number of times found count. CONCLUSION: Although further confirmatory studies are necessary, findings of this study suggest that these pathways, especially circadian entrainment in cumulus cells, may be essential for embryo development and pregnancy.
Circadian Clocks ; Cumulus Cells ; Embryonic Development ; Female ; Gene Expression ; Granulosa Cells ; Humans ; Infertility ; Live Birth ; Microarray Analysis ; Microinjections ; Oocytes ; Ovarian Follicle ; Pregnancy ; Proteoglycans ; Reproduction ; Reproductive Techniques, Assisted