scholarly journals Morphometric Estimation of the Numbers of Granulosa Cells in Preovulatory Follicles of the Ewe

1987 ◽  
Vol 40 (4) ◽  
pp. 451
Author(s):  
JD O'Shea ◽  
PJ Wright ◽  
KE Davis
Keyword(s):  
Light Microscopy ◽  
Corpus Luteum ◽  
Mitotic Index ◽  
Granulosa Cells ◽  
Prostaglandin Analogue ◽  
Previous Estimate ◽  
Luteal Cells ◽  
Preovulatory Follicles ◽  
Morphometric Methods ◽  
Lines Of Evidence

Several lines of evidence suggest that follicular granulosa cells give rise to the large luteal cells of the corpus luteum in the sheep. To further investigate this suggestion, numbers of granulosa cells in preovulatory follicles were estimated by morphometric methods for comparison with a previous estimate of numbers of large luteal cells (9.6 � 0.9 x 106). Preovulatory follicles from five Corriedale ewes were obtained after synchronization of the oestrous cycle with the prostaglandin analogue cloprostenol. Morphometry was undertaken using light microscopy of plastic-embedded tissue sectioned at Il-'m. Mitotic index in the membrana granulosa was 0.05 � s.e.m. 0.05"70. Mean follicular diameter was 6.25 � 0.25 mm and there were 7.68 � 0.53 x 106 granulosa cells per follicle. These results demonstrate a similarity between the number of granulosa cells per follicle and the number of large luteal cells per corpus luteum and thus support the hypothesis that large luteal cells are derived from granulosa cells.

scholarly journals Secreted Protein, Acidic and Rich in Cysteine (SPARC) and Thrombospondin in the Developing Follicle and Corpus Luteum of the Rat

1998 ◽  
Vol 46 (9) ◽  
pp. 1043-1049 ◽  
Author(s):  
P. Bagavandoss ◽  
E. Helene Sage ◽  
Robert B. Vernon
Keyword(s):  
Corpus Luteum ◽  
Granulosa Cells ◽  
Molecular Mechanisms ◽  
Basement Membranes ◽  
Oocyte Nucleus ◽  
Variable Degree ◽  
Matricellular Proteins ◽  
Luteal Cells ◽  
Postovulatory Follicle ◽  
Thecal Cells

In adult mammals, growth of new vasculature from extant blood vessels (angiogenesis) is rare in the absence of pathology. However, nonpathogenic angiogenesis occurs in the cycling ovary when the avascular postovulatory follicle transforms into a highly vascularized corpus luteum (CL). To improve our understanding of molecular mechanisms that regulate nonpathogenic vascular growth, we characterized the expression of two secreted matricellular proteins associated with angiogenesis, SPARC and thrombospondin (TSP), in postovulatory preluteal follicles and CL of hormone-primed immature rats. By indirect immunofluorescence with specific antibodies, we found SPARC in the cytoplasm of granulosa cells and thecal cells of preluteal follicles, in connective tissue cells of the ovarian interstitium, and in the oocyte nucleus. Administration of a luteinizing stimulus (chorionic gonadotropin) increased the expression of SPARC in granulosa cells. TSP was prominent in the basement membranes of growing follicles. Many cells in the early vascularizing CL expressed both SPARC and TSP. Neovascularization of CL was accompanied by expression of SPARC in nascent vessels and concentration of TSP in central avascular areas. In mature CL, steroidogenic luteal cells expressed both SPARC and TSP. Luteal cells of regressing CL retained SPARC to a variable degree but did not express TSP. The observed changes in expression of SPARC and TSP during development of the CL support distinct roles for these matricellular proteins in nonpathological morphogenesis and angiogenesis.

107 Localization of Kisspeptin Immunoreactivity in the Cat Ovary on Different Reproductive Stages

2018 ◽  
Vol 30 (1) ◽  
pp. 193
Author(s):  
P. Tanyapanyachon ◽  
O. Amelkina ◽  
K. Chatdarong
Keyword(s):  
Corpus Luteum ◽  
Granulosa Cells ◽  
Ovarian Surface Epithelium ◽  
Domestic Cat ◽  
Primary Antibody ◽  
Surface Epithelium ◽  
Domestic Cats ◽  
Luteal Cells ◽  
Theca Cells ◽  
Steroidogenic Cells

Kisspeptin (Kp) is considered one of the main regulators of the reproductive axis, exerting its effects via stimulating GnRH expression in the hypothalamus. Apart from its central localization in the hypothalamus, the presence of Kp has been reported in the ovary, with possible local function. To date, very little is known about the ovarian Kp in the domestic cat. Therefore, our aim was to investigate the presence and localization of Kp at different reproductive stages in domestic cat ovaries. Twenty ovaries were collected from free-ranging domestic cats (body weight 2.7–4.5 kg) after routine ovariohysterectomy. Reproductive stages were classified by ovarian gross morphology, vaginal cytology, and blood progesterone level. Ovarian samples were grouped into inactive (n = 6), follicular (n = 8), and luteal stages (n = 6). Tissues were fixed in 4% paraformaldehyde and processed routinely. Immunohistochemistry was performed using polyclonal rabbit Kp-10 primary antibody (AB9754; Millipore, Billerica, MA, USA) at 1:500 at 4°C overnight. Immunoreactive cells were identified by avidin-biotin-peroxidase system. Rat hypothalamic tissue was used as a positive control. Primary antibody was substituted with PBS and normal rabbit IgG as the negative and isotypic negative controls, respectively. In addition, primary antibody was incubated with metastin overnight and applied for preabsorption test. Negative, isotypic negative, and preabsorption tests showed no staining. Immunoreactive Kp was detected in the ovaries of all reproductive stages with no obvious changes in localization or intensity of staining between stages. Kisspeptin was present in the cytoplasm of oocytes, granulosa cells, and theca cells of preantral (primordial, primary, and secondary) follicles and antral follicles. Interestingly, in most follicles, Kp staining was more prominent in theca cells and oocytes compared with granulosa cells. In corpus luteum, Kp was localised in the cytoplasm of luteal cells, with more intense staining on the periphery of corpus luteum compared with the middle in 3 luteal samples, whereas the rest of the samples demonstrated homogeneous staining distribution. Apart from oocytes and steroidogenic cells, Kp was also present in the cytoplasm of cells of the ovarian surface epithelium. Our study for the first time demonstrated the presence and localization of Kp in the ovary of the domestic cats. The localization of Kp in the cat oocyte is similar to previous reports on hamsters and dogs, indicating a possible function in oocyte development. The staining in steroidogenic cells, mainly theca cells and luteal cells, is in good agreement with studies on hamsters, rats, humans, and marmosets, suggesting the possible local involvement of Kp in steroidogenesis. In addition, Kp staining in the ovarian surface epithelium suggests a possible role in the ovarian remodeling after ovulatory defects, as reported in humans and marmosets. This research was funded by the RGJ PhD program PHD/01882556; RG 7/2559.

Immunocytochemical demonstration of oxytocin in bovine ovarian tissues

1985 ◽  
Vol 109 (4) ◽  
pp. 537-542 ◽  
Author(s):  
Th. A. M. Kruip ◽  
H. G. B. Vullings ◽  
D. Schams ◽  
J. Jonis ◽  
A. Klarenbeek
Keyword(s):  
Acetic Acid ◽  
Corpus Luteum ◽  
Granulosa Cells ◽  
Ovarian Tissue ◽  
Picric Acid ◽  
Corpora Lutea ◽  
Luteal Cells ◽  
Antral Follicles

Abstract. The presence of oxytocin in ovarian tissue was examined immunocytochemically. Bovine antral follicles and corpora lutea were fixed with glutaraldehyde, picric acid and acetic acid fixative and immuno-stained by the indirect peroxidase-antiperoxydase (PAP) technique. Immunoreactive oxytocin was demonstrated in the granulosa cells of small and large follicles, in the granulosalutein cells of the young corpus luteum and in the large luteal cells of the mature corpus luteum. The regressing corpus luteum was not stainable. It is discussed that these findings additionally support the view that oxytocin is actually synthesized in ovarian tissues.

scholarly journals Analysis of TGFB1, CD105 and FSP1 expression in human granulosa cells during a 7-day primary in vitro culture

2020 ◽  
Vol 8 (4) ◽  
pp. 152-157
Author(s):  
Artur Bryja ◽  
Wojciech Pieńkowski ◽  
Katarzyna Stefańska ◽  
Błażej Chermuła ◽  
Rut Bryl ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Corpus Luteum ◽  
Expression Profile ◽  
Granulosa Cells ◽  
Luteal Cells ◽  
Human Granulosa Cells

AbstractThe human granulosa cells (GCs) surround the oocyte and form the ovarian follicle’s proper architecture.These sub-populations include mural granulosa cells, antral granulosa cells, and cumulus granulosa cells.Their main functions are to support the oocyte’s growth (cumulus granulosa cells) and estradiol production (mural granulosa cells). After ovulation, the granulosa cells transform into the luteal cells of the corpus luteum and produce progesterone.Our study investigated the expression profile of three genes: TGFB1, CD105, and FSP1 during a 7-day in vitro culture. The analysis was conducted using the RT-qPCR technique.Changes in the expression of CD105 and FSP1 could be observed during the 7-day in vitro culture. In the case of TGFB, the expression remained at a similar level, with no statistically significant differences observed.Running title: Expression of TGFB1, CD105 and FSP1 in granulosa cells

scholarly journals Localization and temporal regulation of tissue inhibitor of metalloproteinases-4 in mouse ovary

Reproduction ◽  
2006 ◽  
Vol 131 (6) ◽  
pp. 1099-1107
Author(s):  
Shumin Bu ◽  
Chenfu Cao ◽  
Yongjun Yang ◽  
Chenglin Miao ◽  
Zeng Hu ◽  
...  
Keyword(s):  
Protein Expression ◽  
Corpus Luteum ◽  
Granulosa Cells ◽  
Expression Profiles ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Mouse Ovary ◽  
Luteal Function ◽  
Temporal Regulation ◽  
Preovulatory Follicles

Tissue inhibitors of metalloproteinases (TIMPs) are potential regulators of tissue remodeling in the ovary. The aim of the present study was to examine the localization and temporal regulation of TIMP-4 protein in the mouse ovary. An induced superovulation model (eCG/hCG) was employed in immature mice to evaluate TIMP-4 protein expression profiles in ovaries collected during the follicular phase, the pre ovulatory period, and the luteal lifespan. Immunofluorescence results indicated that TIMP-4 protein was localized to theca of both antral and preovulatory follicles and adjacent ovarian stroma. After the initiation of luteinization with hCG, TIMP-4 was observed within the luteinizing granulosa cells and persisted throughout the lifespan of the corpus luteum. In the cycling ovary, TIMP-4 signaling localized to corpus luteum from previous estrous cycles, the theca of preovulatory follicles, and appeared to be lower in newly forming corpus luteum. Western analysis further showed that the levels of TIMP-4 increased significantly during the luteinization process of granulosa cells, but no significant change was found among all corpus luteum stages. A putative regulatory mechanism of TIMP-4 expression was identified utilizing an in vitro model. Treatment of cultured granulosa cells with hCG significantly augmented TIMP-4 protein expression levels. Together our data indicate that the luteinization process of granulosa cells is associated with up-regulation of TIMP-4 and that TIMP-4 might play an essential role in maintenance of the luteal function during the whole lifespan of corpus luteum.

Regulation of Bovine Corpus Luteum Function

1995 ◽  
Author(s):  
Rina Meidan ◽  
Robert Milvae
Keyword(s):  
Arachidonic Acid ◽  
Corpus Luteum ◽  
Granulosa Cells ◽  
Luteal Cells ◽  
Bovine Corpus Luteum ◽  
Pure Cell ◽  
Igf Binding ◽  
Gonadotropin Surge ◽  
Follicular Fluids

The main goal of this research plan was to elucidate regulatory mechanisms controlling the development, function of the bovine corpus luteum (CL). The CL contains two different sterodigenic cell types and therefore it was necessary to obtain pure cell population. A system was developed in which granulosa and theca interna cells, isolated from a preovulatory follicle, acquired characteristics typical of large (LL) and small (SL) luteal cells, respectively, as judged by several biochemical and morphological criteria. Experiments were conducted to determine the effects of granulosa cells removal on subsequent CL function, the results obtained support the concept that granulosa cells make a substaintial contribution to the output of progesterone by the cyclic CL but may have a limited role in determining the functional lifespan of the CL. This experimental model was also used to better understand the contribution of follicular granulosa cells to subsequent luteal SCC mRNA expression. The mitochondrial cytochrome side-chain cleavage enzyme (SCC), which converts cholesterol to pregnenolone, is the first and rate-limiting enzyme of the steroidogenic pathway. Experiments were conducted to characterize the gene expression of P450scc in bovine CL. Levels of P450scc mRNA were higher during mid-luteal phase than in either the early or late luteal phases. PGF 2a injection decreased luteal P450scc mRNA in a time-dependent manner; levels were significantly reduced by 2h after treatment. CLs obtained from heifers on day 8 of the estrous cycle which had granulosa cells removed had a 45% reduction in the levels of mRNA for SCC enzymes as well as a 78% reduction in the numbers of LL cells. To characterize SCC expression in each steroidogenic cell type we utilized pure cell populations. Upon luteinization, LL expressed 2-3 fold higher amounts of both SCC enzymes mRNAs than SL. Moreover, eight days after stimulant removal, LL retained their P4 production capacity, expressed P450scc mRNA and contained this protein. In our attempts to establish the in vitro luteinization model, we had to select the prevulatory and pre-gonadotropin surge follicles. The ratio of estradiol:P4 which is often used was unreliable since P4 levels are high in atretic follicles and also in preovulatory post-gonadotropin follicles. We have therefore examined whether oxytocin (OT) levels in follicular fluids could enhance our ability to correctly and easily define follicular status. Based on E2 and OT concentrations in follicular fluids we could more accurately identify follicles that are preovulatory and post gonadotropin surge. Next we studied OT biosynthesis in granulosa cells, cells which were incubated with forskolin contained stores of the precursor indicating that forskolin (which mimics gonadotropin action) is an effective stimulator of OT biosynthesis and release. While studying in vitro luteinization, we noticed that IGF-I induced effects were not identical to those induced by insulin despite the fact that megadoses of insulin were used. This was the first indication that the cells may secrete IGF binding protein(s) which regonize IGFs and not insulin. In a detailed study involving several techniques, we characterized the species of IGF binding proteins secreted by luteal cells. The effects of exogenous polyunsaturated fatty acids and arachidonic acid on the production of P4 and prostanoids by dispersed bovine luteal cells was examined. The addition of eicosapentaenoic acid and arachidonic acid resulted in a dose-dependent reduction in basal and LH-stimulated biosynthesis of P4 and PGI2 and an increase in production of PGF 2a and 5-HETE production. Indomethacin, an inhibitor of arachidonic acid metabolism via the production of 5-HETE was unaffected. Results of these experiments suggest that the inhibitory effect of arachidonic acid on the biosynthesis of luteal P4 is due to either a direct action of arachidonic acid, or its conversion to 5-HETE via the lipoxgenase pathway of metabolism. The detailed and important information gained by the two labs elucidated the mode of action of factors crucially important to the function of the bovine CL. The data indicate that follicular granulosa cells make a major contribution to numbers of large luteal cells, OT and basal P4 production, as well as the content of cytochrome P450 scc. Granulosa-derived large luteal cells have distinct features: when luteinized, the cell no longer possesses LH receptors, its cAMP response is diminished yet P4 synthesis is sustained. This may imply that maintenance of P4 (even in the absence of a Luteotropic signal) during critical periods such as pregnancy recognition, is dependent on the proper luteinization and function of the large luteal cell.

scholarly journals E- and N-cadherin expression and distribution during luteinization in the rat ovary

Reproduction ◽  
2003 ◽  
pp. 791-800 ◽  
Author(s):  
NH Machell ◽  
R Farookhi
Keyword(s):  
Western Blot ◽  
Corpus Luteum ◽  
Expression Pattern ◽  
Granulosa Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Adult Rats ◽  
Preovulatory Follicles ◽  
E Cadherin

Cadherins, a family of Ca(2+)-dependent cell adhesion molecules, play an important role in ovarian tissue remodelling processes. The aim of this study was to examine the expression pattern of E- and N-cadherin in rat preovulatory follicles, luteinizing follicles and corpora lutea. Immature female rats were treated with equine chorionic gonadotrophin (eCG) to promote preovulatory follicle development. At 48 h after eCG treatment, the rats were injected with an ovulatory dose of hCG. Ovaries were analysed by western blot analysis and immunofluorescence for E- and N-cadherin expression at 48 h after eCG injection, and at 24 and 72 h after hCG injection. Ovaries of cyclic adult rats were examined to assess whether the changes in the expression pattern of cadherin were in agreement with those of the gonadotrophin-treated rats. Finally, expression of E-cadherin in luteinizing granulosa cells in vitro was assessed by RT-PCR and western blot analysis. Immunofluorescence results indicate that E-cadherin is expressed in the theca-interstial cells surrounding preovulatory follicles. N-cadherin expression is prominent in the membrana granulosa of these follicles. The initiation of luteinization with hCG leads to a decreased expression of N-cadherin in the membrana granulosa, whereas expression of E-cadherin starts within the luteinizing follicle. Both cadherins are prominently expressed in the fully formed corpus luteum at 72 h after hCG treatment. Immunofluorescence results revealed that the patterns of E- and N-cadherin expression in the gonadotrophin-treated rats were similar to those of the cyclic adult rats. Western blot analysis reflected similar changes for N-cadherin in the ovaries of both the cyclic adults and gonadotrophin-treated rats; however, they were different in E-cadherin expression. The expression of E-cadherin mRNA and protein was induced in vitro in luteinized granulosa cells. These results support the hypothesis that modulation of cadherin expression is an integral component of remodelling processes, including corpus luteum formation, in the ovary. The results also indicate that expression of E- and N-cadherin in granulosa-lutein cells appear to be under hormonal control.

HISTOCHEMICAL OBSERVATIONS ON THE GRANULOSA AND THECA INTERNA DURING FOLLICULAR DEVELOPMENT AND CORPUS LUTEUM FORMATION AND REGRESSION IN THE AMERICAN OPOSSUM

1968 ◽  
Vol 40 (2) ◽  
pp. 237-241 ◽  
Author(s):  
S. S. GURAYA
Keyword(s):  
Corpus Luteum ◽  
Granulosa Cells ◽  
Follicular Development ◽  
Follicle Cell ◽  
Luteal Cell ◽  
Histochemical Change ◽  
Luteal Cells ◽  
Lipid Granules ◽  
American Opossum ◽  
Theca Interna

SUMMARY A study has been made of the histochemical changes which occur during follicular growth and formation and regression of the corpus luteum in the ovary of the American opossum. The granulosa cells show abundant cytoplasmic RNA. Some lipid bodies consisting of phospholipids are sparsely distributed among the granulosa cells. After ovulation, the granulosa cells undergo 'luteinization' to form the large luteal cells. The most striking histochemical change involved in the differentiation (or luteinization) of the granulosa follicle cell into a luteal cell is the development of abundant diffuse lipoproteins throughout the cytoplasm. Fine lipid granules consisting of phospholipids are also formed in the cytoplasm of luteal cells. The stromal elements of the theca interna, which contain some sparsely scattered phospholipid granules, do not show any histochemical change during corpus luteum formation. With the regression of luteal cells, coarse lipid granules consisting of cholesterol and cholesterol esters, triglycerides and some phospholipids accumulate abundantly in the cytoplasm. Some of these regressing luteal cells continue to persist in the ovarian stroma for some time.

scholarly journals Expression and Localization of Serum/Glucocorticoid-Induced Kinase in the Rat Ovary: Relation to Follicular Growth and Differentiation1

Endocrinology ◽  
2000 ◽  
Vol 141 (1) ◽  
pp. 385-395 ◽  
Author(s):  
Tamara N. Alliston ◽  
Ignacio J. Gonzalez-Robayna ◽  
Patricia Buse ◽  
Gary L. Firestone ◽  
JoAnne S. Richards
Keyword(s):  
In Situ Hybridization ◽  
Subcellular Localization ◽  
Granulosa Cells ◽  
Corpora Lutea ◽  
Pregnant Rats ◽  
Western Blots ◽  
Stromal Compartment ◽  
Luteal Cells ◽  
Preovulatory Follicles

Abstract Expression of serum/glucocorticoid-inducible kinase (Sgk), one member of an inducible serine/threonine kinase family, is induced by FSH/cAMP in rat granulosa cells cultured in defined medium. The FSH-stimulated pattern of sgk expression is biphasic, and transcriptional activation of the sgk gene depends on an intact Sp1/Sp3 binding site within the proximal promoter. To determine whether sgk was expressed in a hormone-dependent and physiologically relevant manner in vivo, the cellular levels of sgk messenger RNA (mRNA) and protein as well as the subcellular localization of this kinase were analyzed in ovaries containing follicles and corpora lutea at specific stages of differentiation. To stimulate follicular development and luteinization, hypophysectomized (H) rats were treated with estradiol (E; HE) and FSH (FSH; HEF) followed by hCG (hCG; HEF/hCG). To analyze Sgk in functional corpora lutea, PRL was administered to HEF/hCG rats, or ovaries of pregnant rats were obtained on day 7, 15, or 22 of gestation. In situ hybridization indicated that sgk mRNA was low/undetectable in granulosa cells of H and HE rats. An acute injection (iv) of FSH to HE rats rapidly increased sgk mRNA at 2 and 8 h. Sgk mRNA was also elevated in granulosa cells of preovulatory follicles of HEF rats and in luteal cells of HEF/hCG and pregnant rats. Northern blots and Western blots confirmed the in situ hybridization data, indicating that the amount and cellular localization Sgk protein were related to that of sgk mRNA. When the subcellular localization of this kinase was analyzed by immunohistochemistry, Sgk protein was nuclear in granulosa cells and some thecal cells of large preovulatory follicles. In contrast, Sgk protein was cytoplasmic in luteal cells as well as some cells within the stromal compartment. Intense immunostaining was also observed in oocytes present in primordial follicles, but not in growing follicles. Collectively, these results show that FSH and LH stimulate marked increases in the cellular content of Sgk, as well as dramatic changes in the subcellular distribution of this kinase. The specific nuclear vs. cytoplasmic compartmentalization of Sgk in granulosa cells and luteal cells, respectively, indicates that Sgk controls distinct functions in proliferative vs. terminally differentiated granulosa cells.

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