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

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

OBJECTIVE: To understand the physiologic effects and secretion pattern of inhibin A and inhibin B during menstrual cycle and menopausal transition, inhibin A and inhibin B levels were measured. And to detect any changes in expression of inhibins in human ovary with age, we examined immunohistochemical staining of alpha, beta A, and beta B subunits of inhibin in ovarian tissues. This study was also designed to investigate whether or not inhibin is an early marker for menopausal transition. METHODS: Inhibin A and inhibin B levels were measured in 320 samples from normal reproductive women, in 60 from perimenopausal women, and in 20 from menopausal women by ELISA. And we examined the immunohistochemical staining of alpha, beta A, and beta B subunits of inhibin in ovarian tissues of 35 normal reproductive, 20 perimenopausal, and 5 menopausal women, respectively. RESULTS: In the normal reproductive women, inhibin A begins to increase in the late proliferative phase (16.53 +/- 1.57 pg/ml), reaches the peak in the mid-secretory phase (45.85 +/- 2.08 pg/ml), and subsequently decreases. Inhibin B begins to increase in the early proliferative phase (65.40 +/- 4.08 pg/ml), reaches the peak in the ovulatory phase (110.74 +/- 9.83 pg/ml), and thereafter declines rapidly. In the perimenopausal women, mean inhibin A serum concentration was 6.68 +/- 0.53 pg/ml during proliferative phase and 21.78 +/- 3.61 pg/ml during secretory phase, which were significantly lower than that of the same phase in the normal reproductive women (P<0.01). Mean inhibin B serum concentration was 52.16 +/- 7.46 pg/ml during proliferative phase and 22.41 +/- 6.73 pg/ml during secretory phase, which were significantly lower than that of the same phase in the normal reproductive women (P<0.01, P=0.025). In the menopausal women, both inhibin A and inhibin B were not detected. In the normal reproductive women, we observed strong immunostaining for alpha subunit in granulosa cells, theca cells, and corpus luteum. Immunostaining for beta A subunit was observed in corpus luteum, but not in growing follicles. Immunostaining for beta B subunit was observed in primary follicle, granulosa and theca cells of growing follicle, and mature follicle, but less strong than immunostaining for alpha subunit. No staining for beta B subunit was observed in the corpus luteum. In the perimenopausal women, immunostaining for inhibin subunits were observed in the same pattern as that of the normal reproductive women, but weaker. Stronger immunostaining was observed in theca cells than in granulosa cells. In the menopausal women, none of the immunostaining of inhibin subunits were observed. CONCLUSION: It is concluded that inhibin A is associated with the luteal function and inhibin B, the follicular function. The secretion of inhibins decreased rapidly in the perimenopausal transition period and were not detected in the menopausal period. Inhibin A and inhibin B are associated with the follicular maturation and development. It suggests that the inhibin A and inhibin B are good candidates as markers for perimenopausal transition.
Corpus Luteum ; Enzyme-Linked Immunosorbent Assay ; Female ; Granulosa Cells ; Humans ; Inhibins* ; Menstrual Cycle ; Ovary* ; 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

This study was undertaken to investigate the characterization and distribution patterns of MHC class II positive dendritic cells[DCs] and ED2 positive tissue macrophages throughout the estrous cycle and during pregnancy in the rat ovary. The immunohistochemical characterization of the cells was carried out using the monoclonal antibodies OX6 and ED2 in cryostat-cut sections. DCs were distributed in the theca cell layer of the growing and mature follicles,stroma and corpus luteum. Tissue macrophages were distributed in the theca externa of the growing and mature follicles, stroma and corpus luteum but they were smaller in number than DCs. None of DC and tissue macrophage was found in the ovum, granulosa layer and follicular cavity of the ovarian follicle. However, DCs and tissue macrophages were present in the granulosa layer and follicular cavity in the atretic follicles. Degenerating corpus luteum contained a vast number of OX6 positive cells. On the contrary, fewer tissue macrophages were founcl in the degenerating corpus luteum. More macrophages tended to be observed in the former follicular cavity and theca lutein cell layer than in the granulosa lutein cell layer of the corpus luteum. In stroma,DCs and tissue macrophages were more frequently found around the blood vessels than in the other region, however, DCs were relatively greater in number than tissue macrophages. There was no estrous cycle and pregnancy dependent variation in the numbers and distribution patterns of DCs and tissue macrophages. In conclusion, the rat ovary contains rich networks of MHC class II positive dendritic cells and ED2 positive tissue macrophages. These findings suggest the existence of a well-developed system of immunological surveillance in the rat ovary. The results of this study have potentially important implications for the understanding not only of the ovarian immune system and the pathogenesis of various ovarian diseases but also of various physiologic functions of the ovary.
Animals ; Antibodies, Monoclonal ; Blood Vessels ; Corpus Luteum ; Dendritic Cells* ; Estrous Cycle ; Female ; Immune System ; Immunologic Surveillance ; Luteal Cells ; Macrophages* ; Ovarian Diseases ; Ovarian Follicle ; Ovary* ; Ovum ; Pregnancy ; Rats* ; Theca Cells

Ovarian aging is related to the reduction of oocyte quality and ovarian follicles reservation leading to infertility. Vitamin C is a natural antioxidant which may counteract with adverse effects of aging in the ovary. The aim of this study was to evaluate the possible effect of vitamin C on NMRI mice ovarian aging according to the stereological study. In this experimental study, 36 adult female mice (25–30 g) were divided into two groups: control and vitamin C. Vitamin C (150 mg/kg/day) were administered by oral gavage for 33 weeks. Six animals of each group were sacrificed on week 8, 12, and 33, and right ovary samples were extracted for stereology analysis. Our data showed that the total volume of ovary, cortex, medulla and corpus luteum were significantly increased in vitamin C group in comparison to the control groups (P≤0.05). In addition, the total number of primordial, primary, secondary, and antral follicles as well as granulosa cells were improved in vitamin C group in compared to the control groups (P≤0.05). No significant difference was observed in total volume of oocytes in antral follicles between control and vitamin C groups. Our data showed that vitamin C could notably compensate undesirable effects of ovarian aging in a mouse model.
Adult ; Aging ; Animals ; Ascorbic Acid ; Corpus Luteum ; Female ; Granulosa Cells ; Humans ; Infertility ; Mice ; Oocytes ; Ovarian Follicle ; Ovary ; Vitamins

OBJECTIVE: This study was aimed to obtain information on normal MIS serum levels according to menstrual cycles of adult normal cycling women . It was also designed to obtain information on the ontogeny of the production profile of MIS and the pattern of its localization in ovary from adult normal cycling women. METHODS: Between January 1998 and January 1999, normal MIS serum levels were measured according to menstrual cycles using 160 serum samples from adult normal cycling women by ELISA. The ontogeny of the production profile of MIS and the pattern of its localization were also studied by immunohistochemical staining using the rabbit polyclonal antibody against human recombinant MIS in 35 ovarian specimens from adult normal cycling women. RESULT: The MIS levels were gradually increased through the follicular phase, reaching at its maximum at the ovulatory phase(4.2+/-2.6 ng/ml), and sharply decreased at the beginning of the luteal phase being minimized at the premenstrual phase(0.5+/-0.2 ng/ml). In average, the MIS levels of the follicular phase(3.7+/-1.9 ng/ml) were significantly higher than those of the luteal phase(1.8+/-2.4 ng/ml)(P<0.05). The MIS levels of the preovulatory and ovulatory phase were significantly higher than those of the other cycle days(P<0.05). Even the early follicular phase(2.9+/-1.6 ng/ml) showed higher MIS levels than the advanced luteal phase(0.9+/-0.7 ng/ml) and the premenstrual phase(0.5+/-0.2 ng/ml)(P<0.05 and P<0.05, respectively). The first staining for MIS was detected in the cytoplasm of granulosa cells when the flattened granulosa cells changed to the cuboidal cells in primordial follicles. The granulosa cells of both single and multiple layered growing follicles showed strong specific staining for MIS. but the MIS staining was not found not in the mature follicle just before ovulation, atretic follicles, corpus luteum, and corpus albicans. MIS staining waned in the mature follicles just before ovulation. CONCLUSION: These experiments demonstrate that the MIS is produced by ovarian granulosa cells in normal reproductive females. The MIS may play an important role as a hormone of follicular development and oocyte maturation through interactions with female steroid hormones, gonadotropins, and growth factors during the adult reproductive cycle.
Adult ; Anti-Mullerian Hormone* ; Corpus Luteum ; Cytoplasm ; Enzyme-Linked Immunosorbent Assay ; Female ; Follicular Phase ; Gonadotropins ; Granulosa Cells ; Humans* ; Intercellular Signaling Peptides and Proteins ; Luteal Phase ; Menstrual Cycle* ; Oocytes ; Ovarian Follicle ; Ovary* ; Ovulation

To understand the role of IGF2 gene in development of human ovary, IGF2 expression was detected by monoclonal antibody for IGF2 to its producted protein with immunohistochemical technique on human ovarian tissues. The results was as follows. IGF2 was highly expressed in ovum of mature follicle, IGF2 expression, however, was not high in granulosa and the cells. IGF2 was not highly expressed in ovum of primary follicle. Highly expressed IGF2 was found on corpus luteum and no expression of IGF2 was found in stroma and epithelial cells. These results suggest that IGF2 is important role in ovulation and in production of progesterone. Abnormal IGF2 expression may be concerned to carcinogenesis of ovarian tumor because most of all tumor from ovary is originated from epithelium.
Carcinogenesis ; Corpus Luteum ; Epithelial Cells ; Epithelium ; Female ; Humans* ; Ovary* ; Ovulation ; Ovum ; Progesterone ; Theca Cells

OBJECTIVES: The purpose of this study is to investigate the expression of CDK (Cyclin dependent kinase) inhibitor, p57(kip2) in human ovarian corpus luteum, benign and malignant ovarian tumors. METHODS: 46 women undergoing laparoscopic surgery or laparotomy for ovarian tumors were enrolled. Total 46 formalin-fixed, paraffin-embedded sections of corpus luteum, benign and malignant ovarian tumors were stained by immunohistochemistry for expression of p57(kip2). RESULTS: p57(kip2) was stained in theca cell of growing follicle but not induced in human corpus luteum. There was the expression of p57(kip2) in mature teratoma, immature teratoma and endometrioma but not in epithelial ovarian tumors. CONCLUSIONS: These results showed that p57(kip2) expression may be not important in luteinization of the ovary and seemed not to play a role in development of epithelial ovarian tumors. However, it may involve pathogenesis of mature teratoma, immature teratoma and endometrioma.
Corpus Luteum ; Endometriosis ; Female ; Humans ; Immunohistochemistry ; Laparoscopy ; Laparotomy ; Lutein ; Luteinization ; Ovary ; Teratoma ; Theca Cells