Expression and immunolocalisation of follicle-stimulating hormone receptors in gonads of newborn and adult female horses

2016 ◽  
Vol 28 (9) ◽  
pp. 1340 ◽  
Author(s):  
Dragos Scarlet ◽  
Ingrid Walter ◽  
Juraj Hlavaty ◽  
Christine Aurich
Keyword(s):  
Western Blot ◽  
Granulosa Cells ◽  
Hormone Receptors ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Cumulus Cells ◽  
Protein Levels ◽  
Fshr Gene ◽  
Luteal Tissue ◽  
Immunofluorescence Labelling

In mares, FSH and its receptor (FSHR) are essential for ovarian function. The objective of the present study was to analyse FSHR gene expression at the mRNA and protein levels in ovarian tissue from newborn and adult horses. Expression of mRNA was analysed by reverse transcription polymerase chain reaction, whereas FSHR protein was visualised by immunohistochemistry (IHC), immunofluorescence labelling (IF) and western blot. FSHR mRNA was detected in ovarian follicles and luteal tissue from adult mares, as well as in the ovaries of neonates. Follicular growth up to 4 mm in diameter was already present in neonates. Using IHC and IF, FSHR protein was detected in granulosa cells, cumulus cells and inconsistently in oocytes, independent of the animal’s age or the stage of folliculogenesis. A lower FSHR expression was observed in theca cells in comparison to granulosa cells. FSHR was abundant in the ovarian stroma cells of neonates but not of adults. Luteal cells stained positive for FSHR independent of the stage of corpus luteum development. The presence of FSHR protein in various cell populations of the ovary was confirmed by western blot. In conclusion, FSHR is present in horse ovaries consistently from birth onwards and expression remains constant during the oestrous cycle.

Maternal obesity is associated with ovarian inflammation and upregulation of early growth response factor 1

2016 ◽  
Vol 311 (1) ◽  
pp. E269-E277 ◽  
Author(s):  
Meghan Ruebel ◽  
Kartik Shankar ◽  
Dana Gaddy ◽  
Forrest Lindsey ◽  
Thomas Badger ◽  
...  
Keyword(s):  
Maternal Obesity ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Elevated Serum ◽  
Cumulus Cells ◽  
Gene Signature ◽  
Sprague Dawley ◽  
Protein Levels ◽  
Phosphorylated Akt ◽  
Nuclear Levels

Obesity impairs reproductive functions through multiple mechanisms, possibly through disruption of ovarian function. We hypothesized that increased adiposity will lead to a proinflammatory gene signature and upregulation of Egr-1 protein in ovaries from obese (OB; n = 7) compared with lean (LN; n = 10) female Sprague-Dawley rats during the peri-implantation period at 4.5 days postcoitus (dpc). Obesity was induced by overfeeding (40% excess calories for 28 days) via total enteral nutrition prior to mating. OB dams had higher body weight ( P < 0.001), greater fat mass ( P < 0.001), and reduced lean mass ( P < 0.05) and developed metabolic dysfunction with elevated serum lipids, insulin, leptin, and CCL2 ( P < 0.05) compared with LN dams. Microarray analyses identified 284 differentially expressed genes between ovaries from LN vs. OB dams (±1.3 fold, P < 0.05). RT-qPCR confirmed a decrease in expression of glucose transporters GLUT4 and GLUT9 and elevation of proinflammatory genes, including CCL2, CXCL10, CXCL11, CCR2, CXCR1, and TNFα in ovaries from OB compared with LN ( P < 0.05). Protein levels of PI3K and phosphorylated Akt were significantly decreased ( P < 0.05), whereas nuclear levels of Egr-1 ( P < 0.05) were increased in OB compared with LN ovaries. Moreover, Egr-1 was localized to granulosa cells, with the highest expression in cumulus cells of preovulatory follicles. mRNA expression of VCAN, AURKB, and PLAT ( P < 0.05) correlated with %visceral fat weight ( r = 0.51, −0.77, and −0.57, respectively, P ≤ 0.05), suggesting alterations in ovarian function with obesity. In summary, maternal obesity led to an upregulation of inflammatory genes and Egr-1 expression in peri-implantation ovarian tissue and a concurrent downregulation of GLUTs and Akt and PI3K protein levels.

scholarly journals Ovarian tissue freezing and activation after thawing: an update

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Li-fan Peng
Keyword(s):  
Granulosa Cells ◽  
Clinical Decision Making ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Clinical Decision ◽  
The Body ◽  
Endocrine Function ◽  
Ovarian Tissue Cryopreservation ◽  
Main Body ◽  
Tissue Cryopreservation

Abstract Background With the growth of women’s age, ovarian failure can be caused by various factors. For the women who need chemotherapy because of cancer factors, the preservation of fertility is more urgent. The treatment of cancer is also a process in which all tissues and organs of the body are severely damaged, especially in the reproductive system. Main body As a new fertility preservation technology, autologous ovarian tissue cryopreservation and transplantation is developing rapidly and showing great potentiality in preserving ovarian endocrine function of young cervical cancer patients. Vitrification and slow freezing are two common techniques applied for ovarian tissue cryopreservation. Thus, cryopreserved/thawed ovarian tissue and transplantation act as an important method to preserve ovarian function during radiotherapy and chemotherapy, and ovarian cryopreservation by vitrification is a very effective and extensively used method to cryopreserve ovaries. The morphology of oocytes and granulosa cells and the structure of organelles were observed under the microscope of histology; the hormone content in the stratified culture medium of granulosa cells with the diameter of follicle was used to evaluate the development potential of ovarian tissue, and finally the ovarian tissue stimulation was determined by the technique of ovarian tissue transplantation. Conclusions Although there are some limitations, the team members still carry out this review to provide some references and suggestions for clinical decision-making and further clinical research.

scholarly journals sRAGE Downregulates the VEGF Expression in PCOS Ovarian Granulosa Cells

2020 ◽  
Author(s):  
Jingyan Wang ◽  
Yichun Guan ◽  
Yi Liu ◽  
Liang Wang ◽  
Zhan Zhang ◽  
...  
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Granulosa Cells ◽  
Ovarian Tissue ◽  
Rt Pcr ◽  
Vegf Expression ◽  
Vitro Fertilization ◽  
Protein Levels ◽  
Ovarian Granulosa Cells

Abstract Objective High expression of VEGF in ovarian tissue, serum and follicular fluid of PCOS women is involved in the physiological and pathogenesis processes of PCOS. Our objective was to investigate the effect of sRAGE on VEGF expression and EGF-like growth factor in PCOS ovarian granulosa cells.Methods We collected ovarian granulosa cells of PCOS patients who underwent in vitro fertilization (IVF). Then treatment ovarian granulosa cells with different concentrations of sRAGE. Levels of VEGF, AREG, BTC and EREG mRNA were examined by quantitative RT-PCR. The protein levels of VEGF, AREG, BTC and EREG were measured by ELISA.Results Treatment with sRAGE decrease the production of VEGF, and the effects were dependent on the concentrations of sRAGE (P < 0.05). Simultaneously, the expression of the EGF-like growth factors AREG, BTC and EREG were decreased, and the expression were dependent on the concentrations of sRAGE (P < 0.05).Conclusions sRAGE may downregulate VEGF expression in PCOS ovarian granulosa cells,and EGF-like growth factor pathway may be involved in this process.

scholarly journals hUMSCs regulate the differentiation of ovarian stromal cells via  TGF-β1 /Smad3  signaling pathway to inhibit ovarian fibrosis to repair ovarian function in POI rats

2020 ◽  
Author(s):  
Linlu Cui ◽  
Hongchu Bao ◽  
Zhongfeng Liu ◽  
Xuejing Man ◽  
Hongyuan Liu ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Stromal Cells ◽  
Collagen Type ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Ovarian Insufficiency ◽  
Tissue Fibrosis ◽  
Function Recovery ◽  
Tgf Β1

Abstract Objective: The basic pathological changes of primary ovarian insufficiency (POI) include ovarian tissue fibrosis and follicular development disorders. The human umbilical cord mesenchymal stem cells (hUMSCs) transplantation has been shown an effective method to improve the ovarian function in POI rat model; however, the exact mechanisms are still unclear. The purpose of this study is to investigate whether the recovery of ovarian function in POI rats is related to the inhibition of tissue fibrosis following hUMSCs transplantation. Furthermore, the transforming growth factor-β1 (TGF-β1 ) signaling pathway is explored to determine the mechanisms of ovarian function recovery through its inhibition of tissue fibrosis. Methods: The primary ovarian insufficiency (POI) rat model was established by intraperitoneal injection of chemotherapy drug cisplatin (CDDP) for 7 days, The levels of serum sex hormones were measured using enzyme-linked immunosorbent assay (ELISA). The tissue fibrosis in ovary was examined using masson staining and Sirus red staining. The collagen fibers in the ovarian tissues were detected by Western blot analysis. To investigate the mechanisms of ovarian function recovery following hUMSCs transplantation, ovarian stromal cells were isolated from the ovarian cortex of immature rats. The expression of Cytochrome P450 17A1 (Cyp17a1) and fibrosis marker of alpha smooth muscle Actin (α-SMA) in ovarian stromal cells were examined using immunofluorescence analysis. Also, the protein levels of Cyp17a1 and α-SMA in ovarian stromal cells were examined by western blot analysis. The expression of TGF-β1 and Smad3 signals were measured by Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis. Results: The results show that the function of ovary in POI rats were significantly improved after hUMSCs transplantation. The expression of fibrosis markers (α-SMA) and production of Collagen Type I (Collagen Ⅰ) and Collagen Type Ⅲ (Collagen Ⅲ) in POI rats were significantly inhibited in POI rats following hUMSCs transplantation. In the cultured ovarian stromal cells, the decrease of TGF-β1 and p-Smad3 protein expression was observed in hUMSCs treated POI rats. The treatment with TGF-β1 inhibitor of SB431542 further confirmed this signal pathway was involved in the process. Conclusion : Our study demonstrated that the TGF-β1 /Smad3 signaling pathway was involved in the inhibition of ovarian tissue fibrosis, which contributed to the restoration of ovarian function in POI rats following hUMSCs transplantation.

scholarly journals hUMSCs regulate the differentiation of ovarian stromal cells via TGF-β1/Smad3 signaling pathway to inhibit ovarian fibrosis to repair ovarian function in POI rats

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Linlu Cui ◽  
Hongchu Bao ◽  
Zhongfeng Liu ◽  
Xuejing Man ◽  
Hongyuan Liu ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Stromal Cells ◽  
Collagen Type ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Primary Ovarian Insufficiency ◽  
Ovarian Insufficiency ◽  
Tissue Fibrosis ◽  
Function Recovery

Abstract Objective The basic pathological changes of primary ovarian insufficiency (POI) include ovarian tissue fibrosis and follicular development disorders. The human umbilical cord mesenchymal stem cell (hUMSC) transplantation has been shown an effective method to improve the ovarian function in POI rat model; however, the exact mechanisms are still unclear. The purpose of this study is to investigate whether the recovery of ovarian function in POI rats is related to the inhibition of tissue fibrosis following hUMSC transplantation. Furthermore, the transforming growth factor-β1 (TGF-β1) signaling pathway is explored to determine the mechanisms of ovarian function recovery through its inhibition of tissue fibrosis. Methods The primary ovarian insufficiency (POI) rat model was established by intraperitoneal injection of chemotherapy drug cisplatin (CDDP) for 7 days. The levels of serum sex hormones were measured using enzyme-linked immunosorbent assay (ELISA). The tissue fibrosis in the ovary was examined using Masson staining and Sirius red staining. The collagen fibers in the ovarian tissues were detected by Western blot analysis. To investigate the mechanisms of ovarian function recovery following hUMSC transplantation, ovarian stromal cells were isolated from the ovarian cortex of immature rats. The expression of Cytochrome P450 17A1 (Cyp17a1) and fibrosis marker of alpha smooth muscle actin (α-SMA) in ovarian stromal cells was examined using immunofluorescence analysis. Also, the protein levels of Cyp17a1 and α-SMA in ovarian stromal cells were examined by Western blot analysis. The expression of TGF-β1 and Smad3 signals was measured by Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis. Results The results show that the function of the ovary in POI rats was significantly improved after hUMSC transplantation. The expression of fibrosis markers (α-SMA) and production of Collagen Type I (Collagen I) and Collagen Type III (Collagen III) in POI rats were significantly inhibited in POI rats following hUMSC transplantation. In the cultured ovarian stromal cells, the decrease of TGF-β1 and p-Smad3 protein expression was observed in hUMSC-treated POI rats. The treatment with TGF-β1 inhibitor of SB431542 further confirmed this signal pathway was involved in the process. Conclusion Our study demonstrated that the TGF-β1/Smad3 signaling pathway was involved in the inhibition of ovarian tissue fibrosis, which contributed to the restoration of ovarian function in POI rats following hUMSC transplantation.

scholarly journals Obese Women Exhibit Differences in Ovarian Metabolites, Hormones, and Gene Expression Compared with Moderate-Weight Women

2009 ◽  
Vol 94 (5) ◽  
pp. 1533-1540 ◽  
Author(s):  
Rebecca L. Robker ◽  
Lisa K. Akison ◽  
Brenton D. Bennett ◽  
Penny N. Thrupp ◽  
Lindsay R. Chura ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Ovarian Function ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Activity Levels ◽  
Obese Women ◽  
C Reactive Protein ◽  
Specific Expression ◽  
Reactive Protein

Abstract Context: Obese women experience longer times to conception, even if they are young and cycling regularly, which is suggestive of alterations in ovarian function during the periconceptual period. Objective: This study sought to determine whether there are alterations in the preovulatory follicular environment that are likely to influence oocyte developmental competence. Design, Setting, and Participants: Women attending a private infertility clinic were categorized into body mass index (BMI) groups of moderate (n = 33; BMI 20–24.9 kg/m2), overweight (n = 31; BMI 25–29.9 kg/m2), and obese (n =32; BMI ≥30 kg/m2). Intervention: For each patient, follicular fluid was recovered from single follicles at oocyte retrieval, granulosa cells were pooled from multiple follicular aspirates and cumulus cells were pooled after separation from the oocytes. Main Outcome Measures: Follicle fluid was assayed for hormones and metabolites. Granulosa and cumulus cells were analyzed for mRNA expression of insulin signaling components (IRS-2 and Glut4), glucose-regulated genes (ChREBP, ACC, and FAS) and insulin-regulated genes (SREBP-1, CD36, and SR-BI) associated with obesity/insulin resistance. Results: Increasing BMI was associated with increased follicular fluid insulin (P &lt; 0.001), lactate (P = 0.01), triglycerides (P = 0.0003), and C-reactive protein (P &lt; 0.0001) as well as decreased SHBG (P = 0.001). IRS-2, Glut4, ChREBP, and SREBP exhibited cell-type-specific expression but were not affected by BMI. CD36 and SRBI mRNA were modestly altered in granulosa cells of obese compared with moderate-weight women. Conclusions: Obese women exhibit an altered ovarian follicular environment, particularly increased metabolite, C-reactive protein, and androgen activity levels, which may be associated with poorer reproductive outcomes typically observed in these patients.

scholarly journals Cryptotanshinone Regulates Androgen Synthesis through the ERK/c-Fos/CYP17 Pathway in Porcine Granulosa Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Danfeng Ye ◽  
Meifang Li ◽  
Yuehui Zhang ◽  
Xinhua Wang ◽  
Hua Liu ◽  
...  
Keyword(s):  
Western Blot ◽  
Mrna Expression ◽  
Molecular Mechanism ◽  
Granulosa Cells ◽  
Protein Level ◽  
Salvia Miltiorrhiza ◽  
Androgen Excess ◽  
Protein Levels ◽  
Androgen Synthesis ◽  
Porcine Granulosa Cells

The aim of the study is to investigate the molecular mechanism behind androgen reduction in porcine granulosa cells (pGCs) withSalvia miltiorrhizaBunge extract cryptotanshinone. PGCs were isolated from porcine ovaries and identified. Androgen excess model of the pGCs was induced with the MAPK inhibitor PD98059 and then treated with cryptotanshinone. The testosterone level was measured by radioimmunoassay in the culture media. The protein levels of P-ERK1/2, c-Fos, and CYP17 in the cells were measured by western blot. Cryptotanshinone decreased the concentration of testosterone and the protein level of CYP17 and increased the protein levels of P-ERK1/2 and c-Fos in the androgen excess mode. After the c-Fos gene was silenced by infection with c-Fos shRNA lentivirus, we measured the mRNA expression by quantitative RT-PCR and protein level by western blot of P-ERK1/2, c-Fos, and CYP17. This showed that the mRNA expression and protein level of P-ERK1/2 and c-Fos were significantly reduced in the shRNA–c-Fos group compared to the scrambled group, while those of CYP17 were significantly increased. So we concluded that cryptotanshinone can significantly reduce the androgen excess induced by PD98059 in pGCs. The possible molecular mechanism for this activity is regulating the ERK/c-Fos/CYP17 pathway.

scholarly journals hUMSCs Regulate the Differentiation of Ovarian Stromal Cells via TGF-β1/Smad3 Signaling Pathway to Inhibit Ovarian Fibrosis to Repair Ovarian Function in POI Rats

2020 ◽  
Author(s):  
Linlu Cui ◽  
Hongchu Bao ◽  
Zhongfeng Liu ◽  
Xuejing Man ◽  
Hongyuan Liu ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Stromal Cells ◽  
Collagen Type ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Primary Ovarian Insufficiency ◽  
Ovarian Insufficiency ◽  
Tissue Fibrosis ◽  
Function Recovery

Abstract Background: The basic pathological changes of primary ovarian insufficiency (POI) include ovarian tissue fibrosis and follicular development disorders. The human umbilical cord mesenchymal stem cells (hUMSCs) transplantation has been shown an effective method to improve the ovarian function in POI rat model; however, the exact mechanisms are still unclear. The purpose of this study is to investigate whether the recovery of ovarian function in POI rats are related to the inhibition of tissue fibrosis following hUMSCs transplantation. Furthermore, the transforming growth factor-β1 (TGF-β1) signaling pathway is explored to determine the mechanisms of ovarian function recovery through its inhibition of tissue fibrosis.Methods: The primary ovarian insufficiency (POI) rat model was established by intraperitoneal injection of chemotherapy drug cisplatin (CDDP) for 7 days, The levels of serum sex hormones were measured using enzyme-linked immunosorbent assay (ELISA). The tissue fibrosis in ovary was examined using masson staining and Sirus red staining. The collagen fibers in the ovarian tissues were detected by Western blot analysis. To investigate the mechanisms of ovarian function recovery following hUMSCs transplantation, ovarian stromal cells were isolated from the ovarian cortex of immature rats. The expression of Cytochrome P450 17A1 (Cyp17a1) and fibrosis marker of alpha smooth muscle Actin (α-SMA) in ovarian stromal cells were examined using immunofluorescence analysis. Also, the protein levels of Cyp17a1 and α-SMA in ovarian stromal cells were examined by western blot analysis. The expression of TGF-β1 and Smade3 signals were measured by Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis.Results: The results show that the function of ovary in POI rats were significantly improved after hUMSCs transplantation. The expression of fibrosis markers (α-SMA) and production of Collagen Type I (Collagen Ⅰ) and Collagen Type Ⅲ (Collagen Ⅲ) in POI rats was significantly inhibited in POI rats following hUMSCs transplantation. In the cultured ovarian stromal cells, the decrease of TGF-β1 and p-Smade3 protein expression was observed in hUMSCs treated POI rats. The treatment with TGF-β1 inhibitor of SB431542 further confirmed this signal pathway was involved in the process. Conclusion: Our study demonstrated that the TGF-β1/Smad3 signaling pathway was involved in the inhibition of ovarian tissue fibrosis, which contributed to the restoration of ovarian function in POI rats following hUMSCs transplantation.

99 GLUCOCORTICOID RECEPTORS ARE EXPRESSED IN OVARIES OF NEWBORN AND ADULT FEMALE HORSES

2015 ◽  
Vol 27 (1) ◽  
pp. 142
Author(s):  
D. Scarlet ◽  
I. Walter ◽  
C. Aurich
Keyword(s):  
Oocyte Maturation ◽  
Granulosa Cells ◽  
Glucocorticoid Receptors ◽  
Ovarian Tissue ◽  
Rna Extraction ◽  
Cumulus Cells ◽  
Oestrous Cycle ◽  
Luteal Function ◽  
Luteal Cells ◽  
Theca Cells

In contrast to other domestic animal species, in vitro maturation (IVM) of oocytes in the horse is still not successful. Oocytes for IVM are obtained either from slaughterhouse ovaries or via ovum pick-up from living mares. Both situations may be associated with a stress-induced glucocorticoid release. So far, neither an involvement of glucocorticoids in follicle and oocyte maturation nor the presence of glucocorticoid receptors (GCR) in ovarian tissue has been investigated in the horse. We hypothesised that GCR are expressed in equine ovarian tissue independent of the animal's age and stage of the oestrous cycle. Ovaries (n = 40) were collected from killed newborn female foals (n = 10) and killed or slaughtered adult mares (n = 10). For assessment of GCR mRNA expression, ovarian samples were fixed in Tissue-Tek O.C.T. Compound (Sakura Finetek, Zoeterwoude, the Netherlands) and stored at –80°C. Various cell populations were isolated using laser capture microdissection on cryosections. After RNA extraction, samples were analysed by qualitative RT-PCR and real time-PCR. For analysis of GCR protein, tissue was fixed in Bouin's solution and histological slides immunostained using a monoclonal antibody for GCR (Ab2768, Abcam, Cambridge, UK), followed by visualisation with diaminobenzidine. One tertiary follicle per slide (40×; light microscopy) was analysed and percentages of cells staining positive for GCR calculated. Statistical analysis was done with the SPSS Statistics 21 software (SPSS Inc., Chicago, IL, USA). Expression of mRNA for GCR was detected in oocytes, cumulus cells, granulosa, and theca cells, independent of age and stage of the oestrous cycle. In both neonates and adults, nuclei of the oocytes and cumulus cells stained positive for GCR regardless of stage of folliculogenesis. Also, GCR were constantly expressed in granulosa cells from both preantral and antral follicles. Percentage of granulosa cells staining positive for GCR (adult: 73.6 ± 3.2, fillies: 72.4 ± 1.9%) was higher (P < 0.001) than of theca cells (adult: 56.8 ± 3.9, fillies: 57.2 ± 1.9%), but not affected by age. GCR were lacking in ovarian stroma of adults but not of neonates. In periovulatory follicles from adult mares, GCR were abundant in developing luteal cells. GCR were also detected in the nuclei of luteal cells in corpora haemorrhagica and corpora lutea. Follicular atresia was associated with a decrease of GCR independent of cell type and age. This study describes for the first time the expression of GCR in horse ovaries, which are present independent of age of the animal, stage of folliculogenesis, and oestrous cycle stage. Results suggest that glucocorticoids are involved in follicular and oocyte maturation, ovulation, and luteal function in the horse. Presence of GCR in the ovaries of newborn horses suggests a role of glucocorticoids in ovarian tissue maturation. Nevertheless, detrimental effects of excess glucocorticoid secretion due to stress on follicular development, oocyte maturation, and luteal function cannot be excluded in the mare.

scholarly journals 219.Analysis of ovarian macrophage populations using macrophage-specific green fluorescent protein (GFP) transgenic mice

2004 ◽  
Vol 16 (9) ◽  
pp. 219
Author(s):  
C. E. Haynes ◽  
R. J. Norman ◽  
R. L. Robker
Keyword(s):  
Transgenic Mice ◽  
Hormone Receptors ◽  
Ovarian Function ◽  
Fluorescent Protein ◽  
Immune Cell ◽  
Ovarian Tissue ◽  
Gene Promoter ◽  
Peritoneal Macrophages ◽  
Reproductive Tissues ◽  
Green Fluorescent

Macrophages represent a major immune cell type in reproductive tissues and are thought to regulate multiple aspects of reproduction, including ovarian function. We have previously shown distinctive phenotypes and functions of ovarian macrophages such that many immunological mediators, such as cytokines and hormone receptors, are uniquely regulated within these cells across the oestrus cycle. In order to isolate macrophages from ovarian tissue by fluorescence activated cell sorting (FACS), we acquired transgenic mice (from DA Hume, Institute for Molecular Bioscience, University of Queensland) which express GFP exclusively in macrophages (1). In these mice GFP is expressed under direction of the c-fms gene promoter, which encodes the receptor for colony-stimulating factor-1 (CSF-1R), a major macrophage growth factor. Using flow cytometry we confirmed that 95% of peritoneal macrophages express GFP and 88% co-express GFP and the classical macrophage marker F4/80. The distribution of GFP+ macrophages in tissues was co-localized with macrophage markers F4/80 and major histocompatibility complex class II (MHCII) by immunohistochemistry using phycoerythrin (PE)-labelled antibodies. The liver, uterus and oviduct exhibited many GFP+ cells in characteristic macrophage distributions. Furthermore, GFP fluorescence was tightly co-localized with PE fluorescence of either F4/80 or MHCII, indicating that CSF-1R is expressed in the macrophages of these tissues. In contrast, macrophages in the ovary were positive for F4/80 and MHCII, but rarely expressed GFP. Thus unlike macrophages of other reproductive tissues, ovarian macrophages do not consistently express CSF-1R. In ovaries from gonadotrophin-primed immature females, GFP was not expressed in macrophages (F4/80+/MHCII+) surrounding follicles but was detected in macrophages within the regressing corpus luteum. Thus CSF-1R is a hormonally regulated gene, expressed only in specific subsets of ovarian macrophages suggesting that CSF-1 controls functional activities of ovarian macrophages at specific stages of the ovarian cycle. (1) Sasmono, R. T., et al. (2003) Blood 101, 1155–1163.

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