Characterization of integrin expression in the mouse ovary

Integrin alpha:beta heterodimers mediate cell contacts to the extracellular matrix and initiate intracellular signaling cascades in response to a variety of factors. Integrins interact with many determinants of cellular phenotypes and play roles in controlling the development, structural integrity, and function of every type of tissue. Despite their importance, little is known about the regulation of integrin subunits in the mammalian ovary and how they function in folliculogenesis. To determine their relevance to ovarian physiology, we have studied the expression of integrin subunit mRNAs by Northern blot analysis and in situ hybridization in ovaries of wild-type, growth differentiation factor 9 (Gdf 9) knockout, FSHbeta (Fshb) knockout, and inhibin alpha (Inha) knockout mice. Integrin alpha6 mRNA is expressed in oocytes and granulosa cells of single-layer follicles and in oocytes and theca cells of multilayer follicles. Integrin alpha6 is highly expressed in Gdf 9 knockout ovaries, which are enriched in oocytes and primary (single layer) follicles because of a block at this stage of follicular development. Integrin alpha(v) mRNA is most highly expressed in the granulosa cells of multilayer growing follicles, and therefore only low levels of expression are detectable in the Gdf 9 knockout ovaries. Integrin beta1 mRNA exhibits a broad expression pattern in ovaries, including oocytes, granulosa cells, theca cells, and corpora lutea. Integrin beta3 mRNA is expressed in theca and interstitial cells and is upregulated in corpora lutea. It is nearly undetectable in ovaries of Fshb knockout mice, which develop preantral follicles but have no luteal cells. Integrin beta5 mRNA is predominantly expressed in granulosa cells of multilayer follicles. It is expressed at high levels in the Fshb knockout mice and in a compartmentalized manner in the granulosa cell/Sertoli cell tumors that develop in the Inha knockout mice. Specific integrins are associated with ovarian cellular phenotypes in mice, which raises intriguing possibilities as to integrin functions in oocyte competence, follicular development, luteinization, and granulosa cell proliferation.     

Lectin binding sites of the mouse ovary,intraovarian and ovulated ova

Summary Fluorescein isothiocyanate (FITC) labeled lectins were used to study the distribution of specific binding sites in histologic sections of mouse ovaries as well as ovulated ova. Four distinct patterns of reactivity of the components of the follicle (exclusive of the ovum) and the surrounding ovarian stroma were recognized: 1. uniform staining of granulosa cells, theca cells and surrounding stroma; 2. weak to moderate staining of the granulosa cells and strong staining of the theca cells and stromal cells; 3. no reactivity of the granulosa cells coupled with strong reactivity of the theca and stromal cells; 4. no reactivity with any component of thecumulus oophorus. Three lectins (from Triticum vulgare, Arachis hypogaea and Maclura pomifera) distinctly accentuated the basal lamina of thecumulus oophorus. The reaction of lectins with oocytes and zona pellucida revealed six distinct patterns: 1. no reactivity with either structure; 2. weak reactivity with the cytoplasm of the oocyte and no reactivity with the zona pellucida; 3. very strong reactivity with the cytoplasm of the oocyte and no reactivity with the zona pellucida; 4. moderate reactivity with both the oocyte and the zona pellucida; 5. moderate reactivity with the oocyte and very strong reactivity with the zona pellucida; 6. no reactivity with the oocyte and moderate reactivity with the zona pellucida. The same pattern of reactivity was seen in the ovulated ova in the oviduct except for the lectin from Arachis hypogaea, the reactivity of which changed upon ovulation and/or fertilization of the ovum. These data provide a list of lectin markers for distinct components of the mouse ovary.     

Lectin binding sites of the mouse ovary, intraovarian and ovulated ova

Fluorescein isothiocyanate (FITC) labeled lectins were used to study the distribution of specific binding sites in histologic sections of mouse ovaries as well as ovulated ova. Four distinct patterns of reactivity of the components of the follicle (exclusive of the ovum) and the surrounding ovarian stroma were recognized: uniform staining of granulosa cells, theca cells and surrounding stroma; weak to moderate staining of the granulosa cells and strong staining of the theca cells and stromal cells; no reactivity of the granulosa cells coupled with strong reactivity of the theca and stromal cells; no reactivity with any component of the cumulus oophorus. Three lectins (from Triticum vulgare, Arachis hypogaea and Maclura pomifera) distinctly accentuated the basal lamina of the cumulus oophorus. The reaction of lectins with oocytes and zona pellucida revealed six distinct patterns: no reactivity with either structure; weak reactivity with the cytoplasm of the oocyte and no reactivity with the zona pellucida; very strong reactivity with the cytoplasm of the oocyte and no reactivity with the zona pellucida; moderate reactivity with both the oocyte and the zona pellucida; moderate reactivity with the oocyte and very strong reactivity with the zona pellucida; no reactivity with the oocyte and moderate reactivity with the zona pellucida. The same pattern of reactivity was seen in the ovulated ova in the oviduct except for the lectin from Arachis hypogaea, the reactivity of which changed upon ovulation and/or fertilization of the ovum. These data provide a list of lectin markers for distinct components of the mouse ovary.     

卵母细胞在哺乳动物卵巢排卵过程中的决定性作用

哺乳动物卵巢排卵是一个复杂的调控过程。卵泡成熟破裂后,卵母细胞从卵巢中排出。卵泡细胞感受排卵刺激,并诱导卵母细胞减数分裂的恢复及其随后的释放。卵母细胞及其周围颗粒细胞的旁分泌在对此起关键性作用,其中卵母细胞对其释放具有决定性作用。作者先前已经阐述过颗粒细胞在哺乳动物卵巢排卵过程中的调控作用,该文将从卵母细胞的发育及其调控角度重点阐明其在排卵过程中的决定作用,旨在进一步理解哺乳动物卵巢的排卵过程,同时为不孕不育等卵巢疾病的治疗提供重要的研究方向和理论基础。     

Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary

The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA) in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labeling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.     

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.     

The reproductive biology and estuarine movements of the gizzard shad, Nematalosa vlaminghi (Munro)

The microscopic development of the egg of the Perth herring, Nematalosa vlaminghi , has been related to the gross changes observed in the ovary. The patterns of change in the diameter of the oocyte, oocyte nucleus, yolk vesicles and yolk granules and the thickness of the theca, granulosa and zona radiata have been quantified. Three modes were present in frequency histograms of the diameter of oocytes in sexually-mature females, two of which corresponded to opaque and hyaline yolked-oocytes. The mean number of hyaline oocytes was 20430, which represented 35% of the mean number of all yolked oocytes (57610). Seasonal trends in gonadosomatic indices, together with the changes in the macroscopic and microscopic condition of the ovaries, demonstrated that spawning activity reached a peak in December-January. The place of capture of mature animals and 0+ recruits showed that breeding occurred near the top end of the upper estuary. During the ensuing weeks, the young fish dispersed throughout the upper and middle estuary while the older fish moved out to sea.     

TRAIL pathway components and their putative role in granulosa cell apoptosis in the human ovary

Extensive apoptotic oocyte reduction occurs during fetal ovarian development. The regulatory pathways responsible for oocyte selection to programmed cell death are, however, poorly understood. The aim of this study was to investigate the potential involvement of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 and decoy receptors TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in the apoptotic process characterizing human fetal and adult ovaries. For this purpose, in situ hybridization and immunohistochemistry were applied to human fetal and adult ovarian samples to study the mRNA and protein expression of TRAIL pathway components, and a human granulosa cell tumor-derived cell line (KGN) was used to elucidate functional effects of TRAIL on apoptosis. TRAIL was expressed in human fetal ovary from the 11th week until term. The pro-apoptotic TRAIL-R2/DR5 and the anti-apoptotic TRAIL-R4/DcR2 were also expressed in human ovaries throughout the fetal period. Among the different ovarian cell types, these TRAIL pathway components were mainly localized in the oocytes, and their expression increased towards term. Expression of TRAIL-R1/DR4 and TRAIL-R3/DcR1 was negligible in all of the fetal ovaries studied. Adult ovaries expressed TRAIL, TRAIL-R2/DR5, TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in granulosa cells and oocytes of small primary/secondary follicles as well as in granulosa and theca cells of more developed antral follicles. In KGN cells, TRAIL efficiently induced apoptosis in a dose-dependent manner, and this was blocked by a caspase inhibitor. The results indicate a role of the TRAIL pathway components in the regulation of granulosa cell apoptosis in in vitro and suggest that these factors may have a role in regulating ovarian apoptosis also in vivo.     

Effects of methoxychlor on IGF-I signaling pathway in rat ovary

Follicular development and other ovarian functions are regulated by growth factors that can be affected by exogenous agents. Methoxychlor (MXC) is an organochloride pesticide that causes female infertility. We investigated how MXC affects the distribution of developing ovarian follicles in adult rats after treatment between embryonic day (E) 18 and postnatal day (PND) 7. We also measured insulin-like growth factor-I (IGF-I) and its receptor, IGF-IR, expressions in ovarian follicles and investigated whether MXC changed the levels of IGF-I and IGF-IR in the ovary. Using immunohistochemical (IHC) staining, we detected IGF-I expression in oocytes and granulosa cells of the follicles, luteal cells, interstitial cells, theca externa and theca interna, and the smooth muscle of ovarian vessels. IGF-IR was co-localized with IGF-I in the ovary except for the theca externa. IGF-I expression was decreased in granulosa cells of preantral and antral follicles after treatment with MXC compared to granulosa cells of preantral and antral follicles of the control group. We also observed that oocytes of secondary follicles and granulosa cells of secondary and preantral follicles of the MXC treated groups showed increased IGF-IR expression compared to oocytes of secondary follicles and granulosa cells of secondary and preantral follicles of the control group. We also detected more secondary and preantral follicles, and fewer primordial and antral follicles after MXC administration compared to controls. Therefore, the IGF signaling pathway may participate in MXC induced ovary dysfunction and female infertility.     

Morphological development and characterization of aromatase and estrogen receptors alpha and beta in fetal ovaries of cattle from days 110 to 250

To better understand the role of estradiol-17β in fetal ovarian development, presence and localization of cytochrome P450 aromatase (P450arom) and estrogen receptors alpha (ERα) and beta (ERβ) proteins were characterized in fetal ovaries of cattle using immunohistochemistry. Fetal cattle ovaries were collected from an abattoir and sorted into fetal age groups (days 110, 130, 150, 170, 190, 210, 230, 250+) based on crown-rump length. In addition to immunohistochemistry, morphological analysis of ovarian and follicular formation was made. Ovaries appeared lobular at day 110, but by the end of gestation (day 250+) ovaries were oval-shaped similar to those found in adult animals. Ovarian structures within different lobes appeared to be at different developmental stages. At day 110, oocytes and pre-granulosa cells were observed in ovigerous cords that were still open to the surface epithelium. Most ovigerous cords appeared to be closed to the surface epithelium on day 130, all closed by day 150 and were no longer present at day 210. Ovarian follicles were classified as follows: Type 1(primordial): single layer of flattened granulosa cells, Type 1a (transitory): single layer of mixed flattened and cuboidal granulosa cells, Type 2 (primary): at least one but less than two layers of cuboidal granulosa cells, Type 3 (small preantral): two to three layers of granulosa cells, Type 4 (large preantral): four to six layers of granulosa, and the theca layer is forming around the follicle, Type 5 (antral): contain greater than six layers of granulosa cells, several layers of theca cells and the antrum has formed. Type 1 follicles were observed in day 110 ovaries. Follicle Types 1a and 2 were first observed on day 130. Type 3 follicles were first observed on day 150 and Types 4 and 5 were first observed on day 170. P450arom protein was localized in granulosa cells of follicle Types 2–5 and cells of rete tubules throughout the experimental period. There was punctate expression within stroma and rete masses. There was ERα protein localization in pre-granulosa cells and germ cells of ovigerous cords and all surface epithelial cells. There was also localization in granulosa cells and oocytes of all follicle types and cells of rete tubules. There was punctate ERα protein expression in stroma and rete masses. ERβ protein was localized in pre-granulosa cells and germ cells of ovigerous cords. Expression was also localized to granulosa cells of all follicle types and cells of rete tubules. ERβ protein was punctate in oocytes of follicles, surface epithelial cells, stroma and rete masses. Thus, the fetal ovary of cattle has the steroidogenic enzyme (P450arom) to convert androgens to estradiol-17β, and estrogen receptors α and β to facilitate an estrogen response within the fetal ovary.     

Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells <Emphasis Type="Italic">in vitro</Emphasis>

Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2–6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0–10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids.     

Notch pathway genes are expressed in mammalian ovarian follicles.

Folliculogenesis is the process of development of ovarian follicles that ultimately results in the release of fertilizable oocytes at ovulation. This is a complex program that involves the proliferation and differentiation of granulosa cells. Granulosa cells are necessary for follicle growth and support the oocyte during folliculogenesis. Genes that regulate the proliferation and differentiation of granulosa cells are beginning to be elucidated. In this study, the expression patterns of Notch receptor genes and their ligands, which have been shown to regulate cell-fate decisions in many systems during development, were examined in the mammalian ovary. In situ hybridization data showed that Notch2, Notch3, and Jagged2 were expressed in an overlapping pattern in the granulosa cells of developing follicles. Jagged1 was expressed in oocytes exclusively. Downstream target genes of Notch also were expressed in granulosa cells. These data implicate the Notch signaling pathway in the regulation of mammalian folliculogenesis.     

Ultrastructural observations of previtellogenic ovarian follicles of dove

Dove ovarian follicle is a complex structure composed of oocyte surrounded by a somatic compartment consisting of theca externa, theca interna and granulosa. The structure of ovarian follicle (1 and 2 mm) of dove was studied by electron microscopy. The granulosa was pseudostratified in the 1-mm-diameter follicles and stratified with two or three irregular rows of cells in the 2-mm-diameter follicles. In the larger follicle indentations between oocyte and granulosa cells become more numerous and the microvilli of granulosa cell elongated to form a zona radiata with similarly elongated oocyte microvilli. Lining bodies were present at the tips of granulosa microvilli and in the cortical region of the oocyte. In the oocyte cortex were observed coated pits, coated vesicles, dense tubules, multivesicular bodies and primordial yolk spheres. Primordial yolk spheres may contain lining bodies and were observed fused with dense tubules and multivesicular bodies or associated with smooth cisternae.     

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime’s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.     

Tumor necrosis factor alpha enhances oocyte/follicle apoptosis in the neonatal rat ovary.

Tumor necrosis factor alpha (TNFalpha) is a multifunctional cytokine present in oocytes and macrophages in the neonatal rat ovary. The presence of both TNFalpha and its receptors in the neonatal rat ovary suggests a potential role for it in follicle assembly or oocyte atresia. Previous studies have provided support for effects of TNFalpha on isolated granulosa and theca cells and intact follicles; however, to our knowledge, this is the first study to investigate the effects of TNFalpha on the earliest stages of follicular development. Effects of TNFalpha on oocyte/follicle number and apoptosis were investigated using an ovarian organ-culture system that supported assembly of primordial follicles in vitro. Ovaries were collected on the day of birth and treated with TNFalpha (0, 0.1, 1.0, 10, or 50 ng/ml), a function-blocking TNFalpha antibody (5 microg/ml), or control immunoglobulin (Ig) G. At 1 ng/ml, TNFalpha decreased follicle and oocyte numbers during 3 days of culture, whereas higher (10 and 50 ng/ml) or lower (0.1 ng/ml) doses had no effect. Treatment with TNFalpha antibodies increased the number of oocytes and follicles compared to nonspecific IgG control. To determine whether the decreased oocyte/follicle numbers were due to an apoptotic effect of TNFalpha, apoptosis was examined by DNA laddering. At 1 ng/ml, TNFalpha increased apoptotic DNA laddering twofold, with no significant effect from lower or higher doses. The cells undergoing apoptosis, as determined by in situ end-labeling, were oocytes, interstitial cells, and granulosa cells. These findings suggest that TNFalpha may be involved in oocyte atresia that normally occurs during the perinatal period.