Demonstration of cytokeratin intermediate filaments in oocytes of the developing and adult human ovary

The intermediate filaments (IF) present in the various cells of human ovaries were studied by immunolocalization using antibodies to cytokeratins (CKs), vimentin, desmin and alpha-smooth muscle (-SM) actin. Oocytes revealed a single paranuclear aggregate, which reacted with antibodies to CKs 8, 18 and 19 both in adult and fetal ovaries. The existence of this aggregate was also documented by electron microscopy. Ovarian surface epithelium and granulosa cells consistently coexpressed CKs 8, 18, 19 and vimentin. During follicle maturation vimentin remained unchanged in the granulosa layer while CKs content decreased, showing variation in the amount and distribution of the different CK-types. Thecal cells of secondary and mature follicles showed -SM actin positivity. These contractile fibres increased in mature follicles. Ordinary fibrous stromal cells showed isolated cells which were desmin and -SM actin positive. A similar pattern of IF expression and distribution existed in all stages of development in fetal and embryonic ovaries. These results indicate that CKs are present in human oocytes and that the coexpression of vimentin and CKs can be regarded as a peculiar feature of all ovarian cell types except oocytes and ordinary stromal cells. Contractile properties have been documented associated with a modification in expression of IF proteins. This is likely to represent an integral part of folliculogenesis along with the functional hormone-dependent changes.     

Localization of an activin/activin receptor system in the porcine ovary.

The aim of this study was to locate a possible activin/activin receptor system within porcine ovaries containing functional corpora lutea. In situ hybridization was used to assess the gene expression of beta(A)- and beta(B)-activin subunits, and immunohistochemical studies were done to detect activin-A protein and activin receptor type II. mRNA expression of the beta(A)- and beta(B)-activin subunits was found in the granulosa from the unilaminar follicle stage onward, in the developing thecal layer of multilaminar and small antral follicles, in the theca interna of mid-sized antral follicles, in corpora lutea, and in the ovarian surface epithelium. Immunoreactive activin A protein could be detected at the same ovarian sites, but in thecal tissue of small antral follicles only. This protein was also demonstrated at the peripheral zone of oocytes from multilaminar and antral follicles. A positive immunoreaction for activin receptor was found in granulosa cells from multilaminar and older follicles and in oocytes from the earliest stages of follicular development onward. In late multilaminar follicles and in antral follicles, the oolemma was stained. Except for small antral follicles, a positive activin receptor immunoreaction was absent in the follicular theca. Activin receptor immunoreaction was furthermore present in corpora lutea and in the ovarian surface epithelium. It is concluded that, within porcine ovaries containing functional corpora lutea, an activin/activin receptor system is present in all intact follicles, the corpora lutea and the surface epithelium. Within follicles, granulosa and theca cells are the main sites of activin synthesis, while oocytes and granulosa cells are the main activin binding sites.     

Ontogeny and cellular localization of 125I-labeled basic fibroblast growth factor and 125I-labeled epidermal growth factor binding sites in ovaries from bovine fetuses and neonatal calves.

We have recently reported that although specific 125I-FSH receptors are present in granulosa cells from primary and secondary follicles, gonadotropin responsiveness is very low in ovaries from bovine fetuses, which consist mainly of preantral follicles with few early antral follicles. It is well established that a number of polypeptide growth factors show pronounced mitogenic effects on follicular cells. Therefore, we have compared autoradiographically the ontogeny and cellular localization of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) binding activities to assess their possible involvement in the regulation of early follicular growth in fetuses and neonatal calves. Follicular growth was initiated around Day 180 of gestation in fetuses. 125I-bFGF binding values were high in granulosa cells from preantral follicles (mean +/- SEM, 7.8 +/- 1.1-9.8 +/- 0.7 grains/cell, 0.05-0.15-mm diam.) but decreased in early antral follicles (0.16-3.0 mm) to a constant level (5.7 +/- 1.2 grains/cell). Specific 125I-EGF binding values were low in preantral follicles but showed a 2.5- and 5.0-fold increase in both granulosa cells and the theca interna from antral I (0.16-0.5 mm) and antral II follicles (0.6-3.0 mm), respectively. In atretic follicles, 125I-bFGF specific binding values were high (10.4 +/- 0.8 grains/cell), whereas 125I-EGF binding levels were significantly reduced or absent. None of the radioligands tested bound significantly to primordial follicles. There was no age-related difference in any ligand binding to follicles of comparable size. These results provide novel evidence that bFGF, a potent mitogen, is involved in the regulation of granulosa cell function as early as the preantral stage in cattle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of estrogen receptors alpha and beta in the baboon fetal ovary

In adult mammals, estrogen regulates ovarian function, and estrogen receptor (ER) is expressed in granulosa cells of antral follicles of the adult baboon ovary. Because the foundation of adult ovarian function is established in utero, the present study determined whether ERalpha and/or ERbeta were expressed in fetal ovaries obtained on Days 100 (n = 3) and 165-181 (n = 5) of baboon gestation (term = Day 184). On Day 100, ERalpha protein was detected by immunocytochemistry in surface epithelium and mesenchymal-epithelial cells but not oocytes in germ cell cords. ERbeta protein was also detected by immunocytochemistry on Day 100 of gestation and was abundantly expressed in mesenchymal-epithelial cells in germ cell cords, lightly expressed in the germ cells, but was not detected in the surface epithelium. On Days 165-180 of gestation, ERalpha expression was still intense in the surface epithelium, in mesenchymal-epithelial cells throughout the cortex, and in nests of cells between follicles. ERalpha expression was lighter in granulosa cells and was not observed in all granulosa cells, particularly in follicles close to the cortex. In contrast, ERbeta expression was most intense in granulosa cells, especially in flattened granulosa cells, was weaker in mesenchymal-epithelial cells and nests of cells between follicles, and was absent in the surface epithelium. Using an antibody to the carboxy terminal of human ERbeta, ERbeta protein was also detected by Western immunoblot with molecular sizes of 55 and 63 kDa on Day 100 and primarily 55 kDa on Day 180. The mRNAs for ERalpha and ERbeta were also detected by Northern blot analysis in the baboon fetal ovary. These results are the first to establish that the ERalpha and ERbeta mRNAs and proteins are expressed and exhibit changes in localization in the primate fetal ovary between mid and late gestation. Because placental estrogen production and secretion into the baboon fetus increases markedly during advancing pregnancy, we propose that estrogen plays an integral role in programming fetal ovarian development in the primate.     

Immunocytochemical study of anti-Müllerian hormone in sheep ovarian follicles during fetal and post-natal development

Anti-Müllerian hormone (AMH) was detected in perinatal and postnatal sheep ovaries, using avidin-biotin immunohistochemistry with a monoclonal antibody specific for ruminant AMH. Immunoreactivity was limited to granulosa cells, and was influenced both by the degree of follicular development, and by the age of the animal. In the fetus, only the most advanced follicles exhibited a faint immunoreactivity at 120 days gestation, and no reaction was observed in younger animals. Immediately before and after birth, primordial follicles were still negative, but a faint reaction was elicited in young growing follicles, increasing with follicle size. Strong immunoreactivity was visible in antral follicles, especially in the innermost granulosa cell layers, close to the oocyte and lining the antral cavity.     

Ontogeny and cellular localization of 125I-labeled insulin-like growth factor-I, 125I-labeled follicle-stimulating hormone, and 125I-labeled human chorionic gonadotropin binding sites in ovaries from bovine fetuses and neonatal calves.

In a previous study we reported that ovaries from bovine fetuses, which consist mainly of preantral follicles with few antral follicles, are weakly responsive to gonadotropins (FSH and LH). Insulin-like growth factor-I (IGF-I) is known to enhance gonadotropin responsiveness in vitro, but there is a lack of consistent data on the involvement of IGF-I, FSH, and LH during early stages of folliculogenesis in cattle. In the study reported here, we assessed autoradiographically the ontogeny of 125I-gonadotropin and 125I-IGF-I binding activities during preantral and early antral stages in cattle. Follicular growth was initiated around Day 180 of gestation in fetuses. The density of 125I-FSH binding was high in granulosa cells from primary (mean +/- SEM 10.5 +/- 0.7 grains/cell, 0.05-mm diam.) and secondary follicles (10.8 +/- 0.8 to 13.6 +/- 1.2 grains/cell, 0.06-0.15 mm) but increased significantly (p < 0.05) in early antral follicles (18.2 +/- 1.1 grains/cell, 0.16-3.0 mm). Specific 125I-IGF-I binding levels were low in granulosa cells from preantral follicles, averaging 2.5 +/- 0.6-3.1 +/- 0.9 grains/cell. However, after antrum formation, the density of 125I-IGF-I binding increased significantly (p < 0.05) with follicular diameter in granulosa cells and was 5.7 +/- 0.7 and 9.1 +/- 0.6 grains/cell for antral I (0.16-0.5 mm) and antral II (0.6-3.0 mm) follicles, respectively. 125I-FSH and 125I-IGF-I binding densities were low in theca cells from preantral and early antral follicles as well as in the interstitial tissue and granulosa cells from atretic follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian prostaglandin endoperoxide synthase: cellular localization during the rat estrous cycle

The specific cellular localization of prostaglandin endoperoxide (PGH) synthase was studied throughout the rat estrous cycle. Animals were necropsied at 1300 h on each day of the 4-day cycle, and an additional group was necropsied at 2300 h on proestrus. Ovaries were removed and processed for cellular identification of PGH synthase by immunohistochemistry. At all stages of the cycle, intense immunostaining was observed in newly formed corpora lutea. Luteal cells were immunoreactive, but the connective tissue centrum was unstained. Interstitial tissue contained heavily labeled cells, whereas the germinal epithelium exhibited faint staining. During estrus, metestrus, and diestrus, thecal cells from preantral and antral follicles contained PGH synthase immunoreactivity, but granulosa cells were unstained. Faint staining of mural granulosa cells was observed first in 78% of preovulatory follicles (less than 400-microns diameter) in ovaries collected on the afternoon of proestrus. After the luteinizing hormone surge, 95% of the preovulatory follicles exhibited PGH synthase staining. The percentage of immunoreactive granulosa cells in these preovulatory follicles increased 4-fold in ovaries collected at 2300 h on proestrus. The presence of ovarian PGH synthase throughout the rat estrous cycle and the changes in cellular localization may reflect the potential role of PGs in follicular and luteal function.     

Immunocytochemical localization of aromatase in immature rat ovaries treated with PMSG and hCG, and in pregnant rat ovaries

Immunocytochemical localization of aromatase cytochrome P-450 was examined in immature rat ovaries treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), and in pregnant rat ovaries. It is well known that PMSG and hCG treatments induce ovulation about 12 h after hCG injection. At 24 h after hCG injection, many antral follicles were recognized in immature rat ovaries and only the granulosa cells in the antral follicles were stained weakly with the anti-aromatase antibody. At 0 to 9 h after hCG injection, in addition to the antral follicles, some large Graafian follicles could be observed in the rat ovaries, and the granulosa cells of these follicles were positively stained for aromatase. Each follicle was surrounded by the basal lamina which shows lineally distinct positive reaction against anti-laminin antibody. At 12 h after hCG injection, some large Graafian follicles without oocyte were weakly positive to the anti-aromatase antisera, and the outline of their basal lamina stained with anti-laminin antibody became irregular in shape and fragmentous. At 15 to 18 h after hCG injection, the luteinized cysts could be seen, and the granulosa-lutein cells of these cysts were almost negative for aromatase. Fragmentous reaction to the anti-laminin antibody was observed around the luteinized cysts. In the ovaries of day 4 in pregnancy, only the granulosa cells of the large antral follicles were weakly stained, but corpora lutea negatively reacted to the anti-aromatase antibody. At 7 to 19 days in gestation, both the granulosa cells of antral follicles and pregnant luteal cells were positively stained against aromatase antisera.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intercellular communication via connexin43 gap junctions is required for ovarian folliculogenesis in the mouse

The ovarian follicle in mammals is a functional syncytium, with the oocyte being coupled with the surrounding cumulus granulosa cells, and the cumulus cells being coupled with each other and with the mural granulosa cells, via gap junctions. The gap junctions coupling granulosa cells in mature follicles contain several different connexins (gap junction channel proteins), including connexins 32, 43, and 45. Connexin43 immunoreactivity can be detected from the onset of folliculogenesis just after birth and persists through ovulation. In order to assess the importance of connexin43 gap junctions for postnatal folliculogenesis, we grafted ovaries from late gestation mouse fetuses or newborn pups lacking connexin43 (Gja1(-)/Gja1(-)) into the kidney capsules of adult females and allowed them to develop for up to 3 weeks (this was necessitated by the neonatal lethality caused by the mutation). By the end of the graft period, tertiary (antral) follicles had developed in grafted normal (wild-type or heterozygote) ovaries. Most follicles in Gja1(-)/Gja1(-) ovaries, however, failed to become multilaminar, with the severity of the effect depending on strain background. Dye transfer experiments indicated that intercellular coupling between granulosa cells is reduced, but not abolished, in the absence of connexin43, consistent with the presence of additional connexins. These results suggest that coupling between granulosa cells mediated specifically by connexin43 channels is required for continued follicular growth. Measurements of oocyte diameters revealed that oocyte growth in mutant follicles is retarded, but not arrested, despite the arrest of folliculogenesis. The mutant follicles are morphologically abnormal: the zona pellucida is poorly developed, the cytoplasm of both granulosa cells and oocytes is vacuolated, and cortical granules are absent from the oocytes. Correspondingly, the mutant oocytes obtained from 3-week grafts failed to undergo meiotic maturation and could not be fertilized, although half of the wild-type oocytes from 3-week grafted ovaries could be fertilized. We conclude that connexin43-containing gap junction channels are required for expansion of the granulosa cell population during the early stages of follicular development and that failure of the granulosa cell layers to develop properly has severe consequences for the oocyte.     

Occurrence of immunoreactivity for adipocyte-type fatty acid binding protein in degenerating granulosa cells in atretic antral follicles of mouse ovary

The localization of adipocyte-type fatty acid binding protein (A-FABP) in the mature mouse ovary was examined by immuno-light and electron microscopy. Solitary round cells showing the distinct immunoreactivity for A-FABP were detected in 1–6 antral follicles. In sets of two consecutive sections in a mirror alignment on slide glasses which were treated for immunoreactivity for A-FABP and TUNEL reaction separately, cells immunoreactive for A-FABP appeared in the same antral follicles as containing cells exhibiting TUNEL-reaction. In immunoelectron microscopy, A-FABP-immunopositive cells were found to contain highly electron-dense nuclei of round, irregular or crescent shapes together with cytoplasmic remnants without any features of macrophages or cells of extrinsic origin. Therefore the cells were identified as apoptotic granulosa cells. The apoptotic cells immunoreactive for A-FABP were often seen to be enclosed/engulfed in adjacent cells exhibiting normal ultrastructures without containing numerous lysosomes. The present findings suggest that A-FABP is involved in the apoptosis of ovarian granulosa cells, probably through its interaction with peroxisome proliferator activated receptors.     

Immunocytochemical analysis of the expression of gap junction protein connexin 43 in the rat ovary

The present immunocytochemical study examines in the rat ovary the pattern of expression of connexin 43 (Cx43), a subunit of gap junctions. Using a well-characterized specific antiserum against rat Cx43, immunoreactivity was not detected in the fetal ovary, i.e., prior to follicular formation. However, in the ovary of 20-day-old, 35-day-old, and adult rats, strong Cx43-immunore-activity was associated with the cell borders of the follicular epithelium/granulosa cells of all developmental stages (primordial follicles, preantral and antral secondary follicles). In general, immunoreactivity of the granulosa cells of large antral follicles appeared more intense than the one of smaller follicles. Staining was also seen in oocytes (cytoplasmic staining). Theca cells of large antral follicles, but not of small follicles were immunoreactive. Immunoreactive interstitial cells were not seen in ovaries of 20- and 35-day-old animals, but staining in these cells was present in adult rats. In large follicles with signs of atresia, granulosa cells lacked Cx43-immunoreactivity, whereas Cx43-immunoreactivity in their theca interna strikingly increased. Corpora lutea in the cyclic adult rats were heterogeneously stained, with either no detectable immunoreactivity, staining of cell borders of most luteal cells, or with conspicuous staining of only a few cells. In the pregnant animals on gestation days (GD) 12, 14, and 17, all luteal cells stained strongly for Cx43 at the cell surface. Shortly before delivery (GD 21), however, the staining pattern vanished and only few, presumably luteal cells remained immunoreactive. In Western blots (using homogenates of whole ovaries), the Cx43 antiserum recognized a major band of approximate Mr 43 × 10³, together with minor bands, which may reflect the presence of several differently phosphorylated Cx43 forms. This is indicated by treatment with alkaline phosphatase, which reduced the banding pattern to one single band. In summary, the gap junction molecule Cx43 is abundantly expressed in all endocrine compartments of the rat ovary. The staining pattern obtained in the present study indicates that Cx43 and presumably gap-junctional communication are associated with follicular development, atresia, and the development of the interstitial gland, as well as with the development and regression of the corpus luteum. The heterogeneous staining within the ovary furthermore hints to a contribution of the local intraovarian factors in the regulation of Cx43 expression. © 1995 Wiley-Liss, Inc.     

Immunocytochemical localization of aromatase in immature rat ovaries treated with PMSG and hCG,and in pregnant rat ovaries

Summary Immunocytochemical localization of aromatase cytochrome P-450 was examined in immature rat ovaries treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), and in pregnant rat ovaries. It is well known that PMSG and hCG treatments induce ovulation about 12 h after hCG injection.At 24 h after hCG injection, many antral follicles were recognized in immature rat ovaries and only the granulosa cells in the antral follicles were stained weakly with the anti-aromatase antibody. At 0 to 9 h after hCG injection, in addition to the antral follicles, some large Graafian follicles could be observed in the rat ovaries, and the granulosa cells of these follicles were positively stained for aromatase. Each follicle was surrounded by the basal lamina which shows lineally distinct positive reaction against anti-laminin antibody. At 12 h after hCG injection, some large Graafian follicles without oocyte were weakly positive to the anti-aromatase antisera, and the outline of their basal lamina stained with anti-laminin antibody became irregular in shape and fragmentous. At 15 to 18 h after hCG injection, the luteinized cysts could be seen, and the granulosa-lutein cells of these cysts were almost negative for aromatase. Fragmentous reaction to the anti-laminin antibody was observed around the luteinized cysts.In the ovaries of day 4 in pregnancy, only the granulosa cells of the large antral follicles were weakly stained, but corpora lutea negatively reacted to the anti-aromatase antibody. At 7 to 19 days in gestation, both the granulosa cells of antral follicles and pregnant luteal cells were positively stained against aromatase antisera. The luteal cells were increased in size during pregnancy. And weakly positive reaction was detected on day 7 of pregnancy, then the immunoreaction became stronger in the corpora lutea on day 15 and 19 of pregnancy.The localization of aromatase was immunocytochemically examined in immature rat ovaries treated with PMSG and hCG injection, and the reaction of the granulosa cells of the antral follicles against anti-aromatase antibody became strongly positive about 12 h before ovulation and the became very weak suddenly after ovulation. In rat-ovaries, the pregnant corpora lutea was positively stained for aromatase after day 7 of pregnancy.This study was supported by Grants from the Ministry of Education, Science and Culture, Japan, and from USPHS Research Grants HD04945, USA     

Spatiotemporal expression of epidermal growth factor receptor messenger RNA and protein in the hamster ovary: follicle stage-specific differential modulation by follicle-stimulating hormone,luteinizing hormone,estradiol, and progesterone

Spatiotemporal expression, endocrine regulation, and activation of epidermal growth factor receptor (EGFR) in the hamster ovary were evaluated by immunofluorescence and in situ hybridization localization. Whereas granulosa cells (GC) of primordial through large preantral (stage 6, 7-8 layers GC) follicles had low immunoreactivity, granulosa cells of antral follicles, theca, and interstitial cells had intense EGFR immunoreactivity. EGFR expression in GC of primordial and small preantral follicles increased progressively from estrous through proestrous, but a significant increase occurred in mural GC of antral follicles following the gonadotropin surge. Interstitial cells around small preantral follicles had strong immunofluorescence, and the intensity increased significantly in fully differentiated thecal cells. Distinct EGFR protein was localized in the nucleus of the oocytes and granulosa cells. FSH significantly stimulated EGFR expression in the GC, especially the mural GC, theca, and interstitial cells in hypophysectomized hamster. Estrogen stimulated EGFR expression in preantral GC as well as in interstitial cells. Progesterone and hCG effect was limited to theca and interstitial cells. EGFR expression correlated well with EGFR activation following endogenous or exogenous gonadotropin exposure. Receptor mRNA expression closely followed the protein expression, with increased mRNA expression in mural GC of antral follicles. These results suggest that low levels of EGF signal as a consequence of low levels of receptors in preantral GC may be critical for cell proliferation, but higher receptor density may evoke increased signal intensity due to activation of other intracellular signal pathways, which activate cellular processes related to granulosa, theca, and interstitial cell differentiation. The spatiotemporal cell type and follicle stage-specific expression of receptor mRNA and protein and EGFR activation is critically regulated by gonadotropins and ovarian steroids, primarily estradiol.     

The structure of the spiny mouse (Acomys cahirinus) ovary during development

The study presents the structure of the ovaries of the spiny mouse (Acomys cahirinus) during the first months of life. The ovaries in neonate females exhibit a large number of primordial and primary follicles, sometimes clustered in nests. The growing follicles were also observed within the ovary at that period. The first, early antral follicles appeared in the ovary during the second week of life. In the group of 60-day old females, the structure of the ovaries was characterized by a significant increase in the connective tissue elements. Moreover, ovarian follicles at various stages of development were observed, except for the antral ones with cumulus oophorus. The first mature follicles were identified in 3-month old females. In the ovarian follicles, apoptosis occurs at all stages of follicle development, especially in the early antral follicles. In the atretic follicles, apoptotic cells were identified in the layer of granulosa cells.     

Unilaminar follicular cells transiently express galectin-3 during ovarian folliculogenesis in pigs

The localization of galectin-3, a β-galactoside-binding animal lectin, was immunohistochemically studied in the ovaries of pigs to determine its expression in ovarian folliculogenesis. Various stages of ovarian follicles were identified in the ovaries of adult pigs. Galectin-3 was immunostained in the squamous follicular cells surrounding oocytes in primordial follicles and in the unilaminar granulosa cells of primary follicles, but not in oocytes of multilaminar follicles (including primary, secondary, and tertiary Graafian follicles). As in adult ovaries, galectin-3 immunoreactivity was prominent in the unilaminar follicles in neonatal ovaries. Galectin-3 was also immunolocalized in the luteal cells in the corpus luteum and granulosa cells of atretic follicles as well as in interstitial macrophages in porcine ovaries. Collectively, these results suggest that galectin-3 is transiently expressed in follicular cells in the unilaminar ovarian follicles (primordial and primary) but not in multilaminar ovarian follicles (primary to tertiary), implying that galectin-3 is embryologically involved in ovum generation.     

Differential expression of estrogen receptor-alpha and -beta and androgen receptor in the ovaries of marmosets and humans

Estrogens and androgens are essential for the maturation of the ovarian follicle and normal fertility in the female. We have used antibodies specific for both forms of estrogen receptor (alpha [ERalpha] and beta [ERbeta]) and androgen receptor (AR) to investigate the pattern of receptor expression in ovaries obtained from women and from a New World primate, the Common marmoset (Callthrix jacchus). On Western blots, three antibodies directed against different peptides within human ERbeta all recognized recombinant human (h) ERbeta but did not bind to recombinant hERalpha. The ERbeta protein was extracted from human ovary and prostate and marmoset ovary. In marmoset and human ovaries, ERbeta protein was detected in the nuclei of granulosa cells in all sizes of follicle (both before and after formation of the antrum), and it was also detected in thecal cells, corpora lutea, surface epithelium, and stroma. In contrast, ERalpha protein was not detected in the nuclei of granulosa cells in preantral follicles, was low/absent from stromal and thecal cells, but was expressed in granulosa cells of antral follicles and in the surface epithelium. The pattern of expression of AR protein more closely resembled that of ERbeta than ERalpha. In conclusion, three independent antibodies have demonstrated convincingly that ERbeta is expressed in a wide range of cells in the primate ovary. Granulosa cells in preantral follicles could contain ERbeta:beta dimers. In antral follicles, however, ERalpha is also expressed, and the formation of homo- or heterodimers containing ERalpha may influence the pattern of gene activation within these cells.     

Localization and developmental expression of the activin signal transduction proteins Smads 2, 3, and 4 in the baboon fetal ovary

We recently demonstrated that the reduction in the number of primordial follicles in ovaries of near-term baboon fetuses deprived of estrogen in utero was associated with increased expression of alpha-inhibin, but not activin betaA and betaB or the activin receptors. Therefore, we proposed that estrogen regulates fetal ovarian follicular development by controlling the intraovarian inhibin:activin ratio. As a prelude to conducting experiments to test this hypothesis, in the current study we determined whether the primate fetal ovary expressed Smads 2/3 and 4 and whether expression of these activin-signaling proteins was altered in fetal ovaries of baboons in which estrogen production was suppressed. Western blot analyses demonstrated that the 59 kDa Smad 2, 54 kDa Smad 3, and 64 kDa Smad 4 proteins were expressed in fetal ovaries of untreated baboons at both mid and late gestation and that the level of expression was not significantly altered in late gestation by in vivo treatment with CGS 20267 or CGS 20267 and estrogen. Immunocytochemistry localized Smads 2/3 and 4 to cytoplasm of oocytes and pregranulosa cells at midgestation and oocytes and granulosa cells of primordial follicles in late gestation. Smad 4 was also detected in granulosa cell nuclei in late gestation, and nuclear expression appeared to be decreased in fetal ovaries of baboons deprived of estrogen. The site of localization of Smads correlated with localization of the activin receptors IA and IIB, which we previously showed were abundantly expressed in oocytes and (pre)granulosa cells at both mid and late gestation and unaltered by estrogen deprivation. In summary, the results of the current study are the first to show that the intracellular signaling molecules required to transduce an activin signal are expressed in the baboon fetal ovary and that expression was not altered by estrogen deprivation in utero. These findings, coupled with our previous observations showing that estrogen deprivation reduced follicle numbers and upregulated/induced expression of inhibin but not activin or the activin receptors, lend further support to the hypothesis that estrogen regulates fetal ovarian folliculogenesis by controlling the intraovarian activin:inhibin ratio.     

Assembly of amino-terminally deleted desmin in vimentin-free cells

To study the role of the amino-terminal domain of the desmin subunit in intermediate filament (IF) formation, several deletions in the sequence encoding this domain were made. The deleted hamster desmin genes were fused to the RSV promoter. Expression of such constructs in vimentin- free MCF-7 cells as well as in vimentin-containing HeLa cells, resulted in the synthesis of mutant proteins of the expected size. Single- and double-label immunofluorescence assays of transfected cells showed that in the absence of vimentin, desmin subunits missing amino acids 4-13 are still capable of filament formation, although in addition to filaments large numbers of desmin dots are present. Mutant desmin subunits missing larger portions of their amino terminus cannot form filaments on their own. It may be concluded that the amino-terminal region comprising amino acids 7-17 contains residues indispensable for desmin filament formation in vivo. Furthermore it was shown that the endogenous vimentin IF network in HeLa cells masks the effects of mutant desmin on IF assembly. Intact and mutant desmin colocalized completely with endogenous vimentin in HeLa cells. Surprisingly, in these cells endogenous keratin also seemed to colocalize with endogenous vimentin, even if the endogenous vimentin filaments were disturbed after expression of some of the mutant desmin proteins. In MCF-7 cells some overlap between endogenous keratin and intact exogenous desmin filaments was also observed, but mutant desmin proteins did not affect the keratin IF structures. In the absence of vimentin networks (MCF-7 cells), the initiation of desmin filament formation seems to start on the preexisting keratin filaments. However, in the presence of vimentin (HeLa cells) a gradual integration of desmin in the preexisting vimentin filaments apparently takes place.