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Morphometric and gene expression analyses of stromal expansion during development of the bovine fetal ovary
Abstract
During ovarian development stroma from the mesonephros penetrates and expands into the ovarian primordium and thus appears to be involved, at least physically, in the formation of ovigerous cords, follicles and surface epithelium. Cortical stromal development during gestation in bovine fetal ovaries (n = 27) was characterised by immunohistochemistry and by mRNA analyses. Stroma was identified by immunostaining of stromal matrix collagen type I and proliferating cells were identified by Ki67 expression. The cortical and medullar volume expanded across gestation, with the rate of cortical expansion slowing over time. During gestation, the proportion of stroma in the cortex and total volume in the cortex significantly increased (P < 0.05). The proliferation index and numerical density of proliferating cells in the stroma significantly decreased (P < 0.05), whereas the numerical density of cells in the stroma did not change (P > 0.05). The expression levels of 12 genes out of 18 examined, including osteoglycin (OGN) and lumican (LUM), were significantly increased later in development (P < 0.05) and the expression of many genes was positively correlated with other genes and with gestational age. Thus, the rate of cortical stromal expansion peaked in early gestation due to cell proliferation, whilst late in development expression of extracellular matrix genes increased.
... Introduction PCOS ovary or an over production of ovarian stroma when it first develops. Ovarian stroma first arises by migration or penetration from the mesonephros underlying the gonadal ridge [35]; this is a consistent process observed in ovine [36], bovine [35,37] and human [38,39] ovarian development. ...
... Non-laser capture micro-dissected fetal ovary samples were grouped into five groups based on their histological morphology; stage I: ovigerous cord formation (n = 7, 79 ± 6 days of gestation), stage II: ovigerous cord breakdown (n = 4, 127 ± 6 days), stage III: follicle formation (n = 3, 173 ± 12 days), stage IV: ovarian surface epithelium formation (n = 8, 234 ± 9 days) and stage V: tunica albuginea formation (n = 5, 264 ± 6 days) [37]. ...
... The other genes, C8H9orf3, RAB5B, ERBB4, YAP1, SUOX, RAD50, THADA, and KRR1 were expressed throughout development, however, their mRNA levels did not correlate with gestational age (Fig 4A-4H). We also classified the fetal ovary samples into 5 groups reflecting key stages in development as shown previously [37] and described in the Materials and Methods. We compared the mRNA levels at each stage and with those in the adult ovary. ...
Polycystic ovary syndrome (PCOS) affects around 10% of young women, with adverse consequences on fertility and cardiometabolic outcomes. PCOS appears to result from a genetic predisposition interacting with developmental events during fetal or perinatal life. We hypothesised that PCOS candidate genes might be expressed in the fetal ovary when the stroma develops; mechanistically linking the genetics, fetal origins and adult ovarian phenotype of PCOS. In bovine fetal ovaries (n = 37) of 18 PCOS candidate genes only SUMO1P1 was not expressed. Three patterns of expression were observed: early gestation (FBN3, GATA4, HMGA2, TOX3, DENND1A, LHCGR and FSHB), late gestation (INSR, FSHR, and LHCGR) and throughout gestation (THADA, ERBB4, RAD50, C8H9orf3, YAP1, RAB5B, SUOX and KRR1). A splice variant of FSHB exon 3 was also detected early in the bovine ovaries, but exon 2 was not detected. Three other genes, likely to be related to the PCOS aetiology (AMH, AR and TGFB1I1), were also expressed late in gestation. Significantly within each of the three gene groups, the mRNA levels of many genes were highly correlated with each other, despite, in some instances, being expressed in different cell types. TGFβ is a well-known stimulator of stromal cell replication and collagen synthesis and TGFβ treatment of cultured fetal ovarian stromal cells inhibited the expression of INSR, AR, C8H9orf3 and RAD50 and stimulated the expression of TGFB1I1. In human ovaries (n = 15, < 150 days gestation) many of the same genes as in bovine (FBN3, GATA4, HMGA2, FSHR, DENND1A and LHCGR but not TOX3 or FSHB) were expressed and correlated with each other. With so many relationships between PCOS candidate genes during development of the fetal ovary, including TGFβ and androgen signalling, we suggest that future studies should determine if perturbations of these genes in the fetal ovary can lead to PCOS in later life.
... This stroma, including its vasculature, penetrates the mass of GREL cells and PGCs/oogonia, branching as it does and so corralling the GREL and germ cells into forming the ovigerous cords [1]. Subsequently the continued expansion of the stroma [13] likely separates the ovigerous cords into smaller cords until the first primordial follicles are formed, consisting of one layer of flattened pre-granulosa cells and a meiotically-arrested oocyte [1,14,15]. In the mouse, two different populations of primordial follicles have been identified [16]. ...
... Whilst these have helped to make substantial gains on our knowledge, these studies generally have not fully assessed the behaviours of the different somatic cell types, such as GREL cells and fibroblasts, and the processes that these can undertake in unison at different stages of ovarian development. To address this, the current study first examined replication of non-stromal cells and the changes in their volume during gestation and compares this with changes in the stromal compartments [13]. The expression patterns of genes related to germ and stem cells, and GREL and granulosa cells in fetal ovaries across gestation were also examined and discussed in relation to changes occurring in the ovary at those times. ...
... Fixed ovaries, as used previously [13] were embedded in paraffin using a Leica EG 1140H (Leica Microsystems, Nussloch, Germany). Six-μm sections were cut using a CM1850 V2.2 Leica microtome (Leica Microsystems), mounted on Superfrost glass slides (HD Scientific Supplies, Wetherill Park, NSW, Australia) and stored at RT until used for haematoxylin-eosin (H&E) staining and immunohistochemistry. H&E-stained sections were used for sample grouping based on histological morphology: stage 1-ovigerous cord formation (n = 7, 79 ± 6 days of gestation), stage 2-ovigerous cord breakdown (n = 4, 127 ± 6 days), stage 3-follicle formation (n = 3, 173 ± 12 days), stage 4-ovarian surface epithelium formation (n = 8, 234 ± 9 days) and stage 5-tunica albuginea formation (n = 5, 264 ± 6 days)] [13]. ...
Cells on the surface of the mesonephros give rise to replicating Gonadal Ridge Epithelial-Like (GREL) cells, the first somatic cells of the gonadal ridge. Later germ cells associate with the GREL cells in the ovigerous cords, and the GREL cells subsequently give rise to the granulosa cells in follicles. To examine these events further, 27 bovine fetal ovaries of different gestational ages were collected and prepared for immunohistochemical localisation of collagen type I and Ki67 to identify regions of the ovary and cell proliferation, respectively. The non-stromal cortical areas (collagen-negative) containing GREL cells and germ cells and later in development, the follicles with oocytes and granulosa cells, were analysed morphometrically. Another set of ovaries (n = 17) were collected and the expression of genes associated with germ cell lineages and GREL/granulosa cells were quantitated by RT-PCR. The total volume of non-stromal areas in the cortex increased significantly and progressively with ovarian development, plateauing at the time the surface epithelium developed. However, the proportion of non-stromal areas in the cortex declined significantly and progressively throughout gestation, largely due to a cessation in growth of the non-stroma cells and the continued growth of stroma. The proliferation index in the non-stromal area was very high initially and then declined substantially at the time follicles formed. Thereafter, it remained low. The numerical density of the non-stromal cells was relatively constant throughout ovarian development. The expression levels of a number of genes across gestation either increased (AMH, FSHR, ESR1, INHBA), declined (CYP19A1, ESR2, ALDH1A1, DSG2, OCT4, LGR5) or showed no particular pattern (CCND2, CTNNB1, DAZL, FOXL2, GATA4, IGFBP3, KRT19, NR5A1, RARRES1, VASA, WNT2B). Many of the genes whose expression changed across gestation, were positively or negatively correlated with each other. The relationships between these genes may reflect their roles in the important events such as the transition of ovigerous cords to follicles, oogonia to oocytes or GREL cells to granulosa cells.
... The stroma spreads laterally below the surface thus partitioning some GRELs onto the surface of the ovary which then develop into a specialised epithelial layer. The expansion of cortical stroma is greatest in early gestation due to stromal cell proliferation [3]. ...
... Little is known about ovarian stroma, much less its development in the fetal ovary. In a morphometric study of the developing bovine ovary [3] it was found that during growth of cortex and medulla across gestation, the rate of cortical expansion slowed and the proliferation index and numerical density of proliferating cells in stroma significantly decreased. However, the proportion of stroma in the cortex significantly increased as the non stromal cells ceased dividing. ...
... They included the pluripotency marker OCT4, the stem cell marker LGR5, the desmosomal protein DSG2, genes connected with steroid synthesis, action or regulation, such as CYP19A1, ESR2, and LHCGR, extracellular matrix associated genes FBN3 and TGFB1, the cell cycle gene CCND2, and the transcription factors NR5A1 and GATA4 which are important for development and differentiation of cells in the ovary. During the first trimester when the cluster 1 genes are highly expressed the stroma is penetrating into the cortex from the medulla and is expanding at its fastest rate [3]. Both NR5A1 and FBN3 are expressed in the stromal cells [1,6]. ...
Movement and expansion of mesonephric-derived stroma appears to be very important in the development of the ovary. Here, we examined the expression of 24 genes associated with stroma in fetal ovaries during gestation (n = 17; days 58–274) from Bos taurus cattle. RNA was isolated from ovaries for quantitative RT-PCR. Expression of the majority of genes in TGFβ signalling, stromal transcription factors (NR2F2, AR), and some stromal matrix genes (COL1A1, COL3A1 and FBN1, but not FBN3) showed a positive linear increase with gestational age. Expression of genes associated with follicles (INSL3, CYP17A1, CYP11A1 and HSD3B1), was low until mid-gestation and then increased with gestational age. LHCGR showed an unusual bimodal pattern; high levels in the first and last trimesters. RARRES1 and IGFBP3 also increased with gestational age. To relate changes in gene expression in stromal cells with that in non stromal cells during development of the ovary we combined the data on the stromal genes with another 20 genes from non stromal cells published previously and then performed hierarchical clustering analysis. Three major clusters were identified. Cluster 1 genes (GATA4, FBN3, LHCGR, CYP19A1, ESR2, OCT4, DSG2, TGFB1, CCND2, LGR5, NR5A1) were characterised by high expression only in the first trimester. Cluster 2 genes (FSHR, INSL3, HSD3B1, CYP11A1, CYP17A1, AMH, IGFBP3, INHBA) were highly expressed in the third trimester and largely associated with follicle function. Cluster 3 (COL1A1, COL3A1, FBN1, TGFB2 TGFB3, TGFBR2, TGFBR3, LTBP2, LTBP3, LTBP4, TGFB1I1, ALDH1A1, AR, ESR1, NR2F2) had much low expression in the first trimester rising in the second trimester and remaining at that level during the third trimester. Cluster 3 contained members of two pathways, androgen and TGFβ signalling, including a common member of both pathways namely the androgen receptor cofactor TGFβ1 induced transcript 1 protein (TGFB1I1; hic5). GATA4, FBN3 and LHCGR, were highly correlated with each other and were expressed highly in the first trimester during stromal expansion before follicle formation, suggesting that this could be a critical phase in the development of the ovarian stroma.
... In contrast, the expression levels of genes regulating spermatocyte development after meiosis are upregulated in the heavier-testes group (> 5 g) [27][28][29][30]. On the other hand, the expression of all these genes regulating ovarian cell development was up-regulated in the heavier-ovaries group (> 3 g) [31][32][33][34]. Overall, these DEGs play an important role in gonadal development during the onset of puberty. ...
Background
Puberty onset signifies the beginning of sexual maturation and reproductive phase in poultry indeed, and plays an essential role in genetics and breeding. Studying gonadal development is one of the important approaches to exploring the genetic mechanism of puberty onset.
Result
In our study, the phenotype data of the testes and ovaries of the 120-day-old Nonghua duck showed a large coefficient of variation, indicating that their gonads were in different developmental states. The CNV-based GWAS results for 358 Nonghua ducks showed two deleted-type CNVRs were associated with testicular weight (TW) and testicular percentage (TP), namely CNVR492 (Chr2: 59473501–59478500 bp) and CNVR494 (Chr2: 59514001–59517000 bp). Additionally, two both-type CNVRs were associated with ovarian weight (OW) and ovarian percentage (OP), namely CNVR557 (Chr2: 99951001–99956500 bp) and CNVR891 (Chr7: 39115001–39122500 bp). RNA-seq analysis showed 6228 and 1070 differentially expressed genes (DEGs) related to the TW and OW. These DEGs were mainly enriched in the MAPK signaling pathway, cytokine-cytokine receptor interaction, and focal adhesion, which were reported to affect gonadal development. Further, by joint analysis of CNV-based GWAS and RNA-seq data, 3 genes, including LOC106019197, CDH19 (LOC101793040), and TYW5 were identified as potential candidate genes for TW and OW. LOC106019197 and CDH19 were down-regulated in the heavier-testes group (> 5 g), while TYW5 was also down-regulated in the heavier-ovaries group (> 3 g). The qRT-PCR revealed that LOC106019197 and CDH19 exhibited higher expression levels in the wild/CN0 and CN0/CN0 genotypes compared to the wild/wild genotype. TYW5 showed the highest expression level in the wild/CN0 genotype and the lowest in the CN2/CN2 genotype. In addition, the expression levels of LOC106019197 and CDH19 were significantly higher at 0w than at 8w and 24w.
Conclusion
Our results revealed that LOC106019197 and CDH19 may act as inhibitors of duck testicular development. TYW5 may play a role in delaying ovarian development. These findings provide new insights into the mechanism of puberty onset in ducks.
... ELN was identified as a gene significantly related to chicken follicular development by transcriptome profiling analysis [46]. LUM has been reported to be involved in ovulation in Homo sapiens and bovine [47,48]. MFAP5 was related to ovarian hyperplasia and endometrial growth [49,50]. ...
Simple Summary
FBN1, ZFAT and FAM184B have been screened as candidate genes for the reproduction of sheep. Therefore, it is necessary to verify these genes in the population and determine the associated loci for litter size. The association of litter size with the genotypes of three candidate genes was analyzed using the fixed effects model in Xinggao mutton sheep. The results showed that the g.160338382 T > C in FBN1 was significantly associated with litter size in Xinggao mutton sheep and that this effect was independent of the FecB mutation. Overall, this study provides a useful genetic marker for improving sheep fecundity.
Abstract
Previous studies have screened key candidate genes for litter size in sheep, including fibrillin-1 (FBN1), family with sequence similarity 184 member B (FAM184B) and zinc finger and AT-hook domain containing (ZFAT). Therefore, it is necessary to verify these genes in the Xinggao mutton sheep population and determine the associated loci for litter size. In this study, three loci (FBN1 g.160338382 T > C, FAM184B g.398531673 C > T and ZFAT g.20150315 C > T) were firstly screened based on the population differentiation coefficient between the polytocous and monotocous sheep groups. Then, population genetic analysis and association analysis were performed on these loci. The results revealed that the g.160338382 T > C in FBN1 was significantly associated with the litter size of sheep. Moreover, there was no significant interaction effect between the g.160338382 T > C locus and FecB on litter size. Notably, g.160338382 T > C is adjacent to the anterior border of exon 58 and belongs to a splice polypyrimidine tract variant, which may lead to alternative splicing and ultimately cause changes in the structure and function of the protein. In summary, our results provided a potentially effective genetic marker for improving the litter size of sheep.
... The body weight gain in male fetus of Nili-Ravi buffalo is relatively slow from 96 th to 170 th days but a sharp increase is observed in weight from 180 th to 311 th days of life [4]. The development of the fetal ovary has been studied in various mammals, cattle [5], pigs [6], and rats [7]. The weight of the ovary increase steadily with the increase in number of germ cell from 50 th day post cotium in cattle [8] and sharp increase is observed in the 9 th month of pregnancy [9]. ...
This study aimed to evaluate the Nili-Ravi buffalo changes in fetal growth relationship with crown rump length (C-R) ovarian at fetal ages from 51 to 290 days
... The body weight gain in male fetus of Nili-Ravi buffalo is relatively slow from 96 th to 170 th days but a sharp increase is observed in weight from 180 th to 311 th days of life (Asma Ul, et al. [4]). The development of the fetal ovary has been studied in various mammals, cattle, pigs (Yuan, et al. [5]), and rats (Zhang, et al. [6]). The weight of the ovary increase steadily with the increase in number of germ cell from 50th day post cotium in cattle (Galindo [7]) and sharp increase is observed in the 9 th month of pregnancy (Mossa Ireland [8]). ...
This study aimed to evaluate the Nili-Ravi buffalo changes in fetal growth relationship with crown rump
length (C-R) ovarian at fetal ages from 51 to 290 days. In the methods, the weight, size, and other proximate
measurements were compared with the ovarian histology of female sex were externally differentiated up
to 56 days. In the results, a linear trend in increase between C-R length, age and body weight is observed.
The mean weight of prenatal ovary from 51–70-day-old fetus was 18.8 ± 2.33 mg which increased to
163.30±39.00 mg at 271-290 days of fetal age. Linear regression showed highly significant increase with
age in right ovarian weight (b=4.89, P <0.001), and left ovarian weight (b=4.79, P <0.001). In conclusion, the
novelty of the distinguished age for differentiating the fetus ovary could have critical information of altered
ovarian steroidogenesis in a way that might affect sexual maturation in Nili-Ravi Buffalo.
... AMH and WNT5A are associated with follicle development in cows (Biase et al., 2013;Monniaux et al., 2012;Rico et al., 2011;Souza et al., 2015). TGFB1I1 has been shown to have effect on ovarian development by affecting collagen synthesis in bovine ovarian cortex (Hartanti et al., 2019;Hatzirodos et al., 2019). NCOA2 has been reported to act as a transcription factor in the hypothalamus, and isinvolved in the response to progesterone and the protein-protein interactions of puberty regulation (Fortes et al., 2011). ...
China has diversified resources of indigenous cattle, which are classified into Northern, Central, and Southern groups according to their geographical distribution. Chaling cattle belong to Southern group. This breed is famous for the production of good quality meat with elite meat grades. To analyze the genetic diversity of Chaling cattle, 20 samples were sequenced using whole‐genome resequencing technology, along with 138 published whole‐genome sequencing data of Indian indicine cattle, Chinese indicine cattle, East Asian taurine cattle, Eurasian taurine cattle, and European taurine cattle as control. It was found that Chaling cattle originated from Chinese indicine cattle. The genetic diversity of Chaling cattle is higher than that of Indian indicine cattle, East Asian taurine cattle, Eurasian taurine cattle, and European taurine cattle, but lower than that of Chinese indicine cattle and Xiangxi cattle. Annotating the selection signals obtained by composite likelihood ratio, θπ, FST, π‐ratio, and XP‐EHH methods, several genes associated with immunity, heat tolerance, reproduction, growth, and meat quality showed strong selection signals. In general, this study provides a theoretical basis for analyzing the genetic mechanism of Chaling cattle with excellent adaptability, rough feeding tolerance, good immune performance, and good meat quality. This work lays a foundation for genetic breeding of Chaling cattle in future.
... Stereological analysis of relative frequency (%) was performed in each section (n = 15 sections per animal) by multipoint test system (400 points). The mean percentage of stroma (collagen, blood vessels), interstitial glands, and follicle population (primordial follicle to corpus luteum) were multiplied by total ovary weight, according to Hartanti et al. (2019), to determinate volume-density of each compartment. ...
The ovaries regulate fertility and hormonal control in females, and aging is a crucial factor in this process, when ovarian function is drastically impacted. Exogenous endocrine disruptors may accelerate this process, acting as the main agents in decreased female fertility and hormonal imbalance, since they impact different features related to reproduction. In the present study, we demonstrate the implications of exposure of adult mothers to the endocrine disruptor bisphenol A (BPA) during pregnancy and lactation on their ovarian function during the transition to later in life (aging). The follicle population of BPA exposed ovaries showed impairment in the development of follicles to the mature stages, with growing follicles being halted in the early stages. Atretic and early-atretic follicles were also enhanced. Expression of estrogen and androgen receptors in the follicle population demonstrated impairment in signaling function: ERβ was highly expressed in follicles from BPA exposed females, which also showed a higher incidence of early atresia of developed follicles. ERβ1 wild-type isoform was also enhanced in BPA-exposed ovaries, compared to its variant isoforms. In addition, steroidogenesis was targeted by BPA exposure: aromatase and 17-β-HSD were reduced, whereas 5-α reductase was enhanced. This modulation was reflected in serum levels of estradiol and testosterone, which decreased in BPA-exposed females. Imbalances in steroidogenesis impair the development of follicles and play an important role in follicular atresia. Our study demonstrated that BPA exposure in two windows of susceptibility - gestation and lactation - had implications during aging, enhancing perimenopausal and infertile features.
... Reproductive efficiency is an important indicator to measure the breeding value for the traits of economic importance. Ovary and uterus, being the "source" and "soil" of embryo formation, play pivotal role during gestation period (Hartanti et al., 2019). Bovine uterus supply the embryo with oxygen and nutrients, and it has to drive out the calf during parturition. ...
Reproductive efficiency of a heifer is directly dependent on the health of reproductive system especially ovary and uterus. Current experiment was carried out to screen candidate genes related to reproductive traits and detect molecular mechanisms of DEGs by using RNA-sequencing (RNAseq) and Gene Set Enrichment Analysis (GSEA) to understand the environmental adaptability, molecular breeding and analyze the reproductive performance. Furthermore, Illumina II high throughput sequncing technology was utilized in the current experiment to sequence transcriptomes and analysis related to pathway of different genes between uterus and ovary in Xiangxi cattle. The total number of ovary assembled was compared to the reference genome sequence, DEGs analysis, GO and KEGG enrichment analysis and candidate gene screening were performed. Based on the transcriptome data, seven cDNA libraries were constructed by utilizing total RNA. Out of seven contracted cDNA libraries, in every sequencing library, raw reads reached >133 Mb, and >94% clean reads remained. After comparing the Xiangxi cattle ovaries with the uterus, a total of 3635 mRNAs were found, in which 1608 were significantly up-regulated expressed and 2027 were down-regulated expression mRNAs. The mRNA expression of ADCY3, ADCY4, ADCY9, FSHR, BMP15, LHCGR, INSR, CYP11A1, CYP17A1, CYP19A1, STAR and SCARB1 in ovary of Xiangxi Cattle were significant different in ovarian and uterus tissues. Through GSEA and PPI network, we found the ovarian steroidogenesis pathway played significant role in signal regulation during the development of the ovarian tissue of the Xiangxi cattle
... We identified fibroblast-like cells in the ovarian stroma expressing many of the extracellular matrix protein known to play a role in these processes (Mara et al., 2020;Duffy et al., 2019). Another important player in ovulatory would repair is the ovarian surface epithelium (OSE), a simple mesothelial cell layer that covers the surface of the ovary and must dynamically expand to cover wound (Hartanti et al., 2019;Xu et al., 2011). The OSE cluster could be identified based on well-established markers such as keratin (Krt) 7, 8, and 18 (Kenngott et al., 2014) and was represented by only 3% of all cells in our dataset, which could be further subdivided in proliferative and non-proliferative states. ...
The estrous cycle is regulated by rhythmic endocrine interactions of the nervous and reproductive systems, which coordinate the hormonal and ovulatory functions of the ovary. Folliculogenesis and follicle progression require the orchestrated response of a variety of cell types to allow the maturation of the follicle and its sequela, ovulation, corpus luteum formation, and ovulatory wound repair. Little is known about the cell state dynamics of the ovary during the estrous cycle and the paracrine factors that help coordinate this process. Herein, we used single-cell RNA sequencing to evaluate the transcriptome of >34,000 cells of the adult mouse ovary and describe the transcriptional changes that occur across the normal estrous cycle and other reproductive states to build a comprehensive dynamic atlas of murine ovarian cell types and states.
... However, it was subsequently discovered that fibrillin 3 is expressed in bovine and human fetal ovaries and specifically in the developing stroma as it first penetrates from the mesonephros (Hatzirodos et al. 2011, Hummitzsch et al. 2013. This penetration of the stroma in this early phase is critical for the formation of the ovary and its ovigerous cords, surface epithelium and follicles (Hummitzsch et al. 2013, 2015, Hartanti et al. 2019, 2020a. Fibrillin 3 is also expressed in other fetal tissues but has not been examined in the fetal liver, muscle, endocrine pancreas or adipose tissue ) -all tissues likely involved in PCOS metabolic pathology. ...
Polycystic ovary syndrome (PCOS) is a common endocrine condition characterised by a range of reproductive, endocrine, metabolic and psychological abnormalities. Reports estimate that around 10% of women of reproductive age are affected by PCOS, representing a significant prevalence worldwide, which poses a high economic health burden. As the origin of PCOS remains largely unknown, there is neither a cure nor mechanism-based treatments leaving patient management suboptimal and focussed solely on symptomatic treatment. However, if the underlying mechanisms underpinning the development of PCOS were uncovered then this would pave the way for the development of new interventions for PCOS. Recently there have been significant advances in our understanding of the underlying pathways likely involved in PCOS pathogenesis. Key insights include the potential involvement of androgens, insulin, anti-Müllerian hormone (AMH) and transforming growth factor beta (TGFβ) in the development of PCOS. This review will summarise the significant scientific discoveries on these factors that have enhanced our knowledge of the mechanisms involved in the development of PCOS, and discuss the impact these insights may have in shaping the future development of effective strategies for women with PCOS.
... As one of the main reproductive organs of the female reproductive system, the growth and development of the ovary are extremely complex processes involving the participation of multiple regulatory mechanisms, regulatory factors and effect factors. Additionally, some studies have suggestion that mechanical factors are involved in normal ovarian development and the malignant process of ovarian cancer in mammals and play vital roles (1)(2)(3)(4)(5). Based on the role of biomechanical factors in the growth and development of various tissues and organs of the human body, their influence on the cell and tissue levels of bone (6), muscle (7), intestine (8), joints (9) and other organs has been studied in depth. ...
Biomechanics is a physical phenomenon which mainly related with deformation and movement of life forms. As a mechanical signal, it participates in the growth and development of many tissues and organs, including ovary. Mechanical signals not only participate in multiple processes in the ovary but also play a critical role in ovarian growth and normal physiological functions. Additionally, the involvement of mechanical signals has been found in ovarian cancer and other ovarian diseases, prompting us to focus on the roles of mechanical signals in the process of ovarian health to disease. This review mainly discusses the effects and signal transduction of biomechanics (including elastic force, shear force, compressive stress and tensile stress) in ovarian development as a regulatory signal, as well as in the pathological process of normal ovarian diseases and cancer. This review also aims to provide new research ideas for the further research and treatment of ovarian-related diseases.
... Fibulin-5 expression is up-regulated in endometrial decidual cells during the first trimester to regulate the invasion of extravillous trophoblast cells and the placentation process [67]. In the developing bovine fetal ovary, the expression of this protein is increased during the formation of surface epithelium and tunica albuginea, suggesting its role in ovary development [68]. A strong expression of fibulin-5 was found in rat lungs between the 18 th embryonic day and 17 th postnatal day, and an in situ hybridization technique revealed its expression in interstitial cells and pulmonary vessels [69]. ...
The extracellular matrix (ECM) plays an important role in the evolution of early metazoans, as it provides structural and biochemical support to the surrounding cells through the cell–cell and cell–matrix interactions. In multi-cellular organisms, ECM plays a pivotal role in the differentiation of tissues and in the development of organs. Fibulins are ECM glycoproteins, found in a variety of tissues associated with basement membranes, elastic fibers, proteoglycan aggregates, and fibronectin microfibrils. The expression profile of fibulins reveals their role in various developmental processes such as elastogenesis, development of organs during the embryonic stage, tissue remodeling, maintenance of the structural integrity of basement membrane, and elastic fibers, as well as other cellular processes. Apart from this, fibulins are also involved in the progression of human diseases such as cancer, cardiac diseases, congenital disorders, and chronic fibrotic disorders. Different isoforms of fibulins show a dual role of tumor-suppressive and tumor-promoting activities, depending on the cell type and cellular microenvironment in the body. Knockout animal models have provided deep insight into their role in development and diseases. The present review covers details of the structural and expression patterns, along with the role of fibulins in embryonic development and disease progression, with more emphasis on their involvement in the modulation of cancer diseases.
... As reported previously [35], ovaries were separated into five groups; stage I: ovigerous cord formation (n = 7), stage II: ovigerous cord breakdown (n = 4), stage III: follicle formation (n = 3), stage IV: ovarian surface epithelium formation (n = 8), and stage V: tunica albuginea formation (n = 5). ...
Polycystic ovary syndrome (PCOS) appears to have a genetic predisposition and a fetal origin. We compared the expression levels of 25 PCOS candidate genes from adult control and PCOS human ovaries (n = 16) using microarrays. Only one gene was potentially statistically different. Using qRT-PCR, expression of PCOS candidate genes was examined in bovine fetal ovaries from early stages when they first developed stroma through to completion of development (n = 27; 60–270 days of gestation). The levels of ERBB3 mRNA negatively correlated with gestational age but positively with HMGA2, FBN3, TOX3, GATA4 and DENND1A.X1,2,3,4, previously identified as correlated with each other and expressed early. PLGRKT and ZBTB16, and less so IRF1, were also correlated with AMH, FSHR, AR, INSR and TGFB1I1, previously identified as correlated with each other and expressed late. ARL14EP, FDFT1, NEIL2 and MAPRE1 were expressed across gestation and not correlated with gestational age as shown previously for THADA, ERBB4, RAD50, C8H9orf3, YAP1, RAB5B, SUOX and KRR1. LGCGR because of its unusual bimodal expression pattern had some unusual correlations with other genes. In human ovaries (n = 15, < 150 days of gestation), ERBB3.V1 and ERBB3.VS were expressed and correlated negatively with gestational age and positively with FBN3, HMGA2, DENND1A.V1,3,4, DENND1A.V1–7, GATA4 and FSHR, previously identified as correlated with each other and expressed early. Thus, the general lack of differential expression of candidate genes in adult ovaries contrasting with dynamic patterns of gene expression in fetal ovaries is consistent with a vulnerability to disturbance in the fetal ovary that may underpin development of PCOS.
... 30 This description is supported by the localization of stroma around the cords and the known substantial expansion that the stroma undergoes during early fetal development of the ovary. 19 During development, the GREL cells on the surface of the ovary began to epithelialize, as seen by the increased intensity of staining for CK19. This occurred once the stroma had penetrated to below the surface, and there was then a sub-epithelial basal lamina and epithelial-stromal interface. ...
When first formed, the ovary only has an established epithelium at its base or hilum. Later, an epithelium is established around the rest of the ovary. To examine this further, we conducted scanning electron microscopy of the surface of bovine fetal ovaries and immunohistochemistry of ovarian cross-sections. From the earliest time point, the cells on the surface of the base or hilum of the ovary were cuboidal. On the remainder of the ovary, the surface was more irregular. By mid-development, the surface was covered completely with either a stratified or simple epithelium of cuboidal cells. Clefts were observed in the surface and appeared to form due to the expansion of stroma surrounding each open ovigerous cord, elevating the areas surrounding each cord, while leaving the opening of the cord to form the base of each cleft. The continued expansion of the surrounding stroma below the surface appeared not only to close the ovigerous cords from the surface but to compress the clefts into the shape of a groove. Later, most of the ovarian surface was covered with a simple cuboidal epithelium. The changes to the ovarian surface during fetal development coincide with the remodeling of the stroma and cords below.
Polycystic ovary syndrome (PCOS) is an endocrine metabolic disorder that appears to have a genetic predisposition and a fetal origin. The fetal ovary has two major somatic cell types shown previously to be of different cellular origins and different morphologies and to differentially express 15 genes. In this study, we isolated the somatic gonadal ridge epithelial-like (GREL) cells ( n = 7) and ovarian fetal fibroblasts ( n = 6) by clonal expansion. Using qRT-PCR, we compared the gene expression levels of PCOS candidate genes with previous data on the expression levels in whole fetal ovaries across gestation. We also compared these levels with those in bovine adult ovarian cells including fibroblasts ( n = 4), granulosa cells ( n = 5) and surface epithelial cells ( n = 5). Adult cell types exhibited clear differences in the expression of most genes. In fetal ovarian cells, DENND1A and ERBB3 had significantly higher expression in GREL cells. HMGA2 and TGFB1I1 tended to have higher expression in fetal fibroblasts than GREL cells. The other 19 genes did not exhibit differences between GREL cells and fetal fibroblasts and FBN3 , FSHB , LHCGR , FSHR and ZBTB16 were very lowly expressed in GREL cells and fibroblasts. The culture of fetal fibroblasts in EGF-containing medium resulted in lower expression of NEIL2 but higher expression of MAPRE1 compared to culture in the absence of EGF. Thus, the two fetal ovarian somatic cell types mostly lacked differential expression of PCOS candidate genes.
Lay summary
Polycystic ovary syndrome (PCOS) is one of the most common reproductive problems. The cause is not known so there are no specific treatments or prevention strategies. We know it can be linked to issues that occur in the womb and that some people may be more likely to get PCOS due to their genetic makeup. Our recent studies showed that many of the genes linked to PCOS were found to be switched on in the fetal ovary and are likely to be involved in the development of the fetal ovary. In order to improve our understanding of PCOS, we need to identify the type of cells in the fetal ovary where these genes are switched on. In this study, we examined the PCOS genes in two types of cells that mature as the fetal ovary develops and found very little difference between them but bigger differences to their mature adult counterparts.
During ovarian development, gonadal ridge epithelial-like (GREL) cells arise from the epithelial cells of the ventral surface of the mesonephros. They ultimately develop into follicular granulosa cells or into ovarian surface epithelial cells. Stromal fibroblasts arise from the mesonephros and penetrate the ovary. We developed methods for isolating and culturing fetal ovarian GREL cells and ovarian fibroblasts by expansion of colonies without passage. In culture, these two cell types were morphologically different. We examined the expression profile of 34 genes by qRT-PCR, of which 24 genes had previously been studied in whole fetal ovaries. Expression of nine of the 10 newly-examined genes in fetal ovaries correlated with gestational age (MUC1, PKP2, CCNE1 and CCNE2 negatively; STAR, COL4A1, GJA1, LAMB2 and HSD17B1 positively). Comparison between GREL cells and fetal fibroblasts revealed higher expression of KRT19, PKP2, OCLN, MUC1, ESR1 and LGR5 and lower expression of GJA1, FOXL2, NR2F2, FBN1, COL1A1, NR5A1, CCND2, CCNE1 and ALDH1A1. Expression of CCND2, CCNE1, CCNE2, ESR2 and TGFBR1 was higher in the fetal fibroblasts than in adult fibroblasts; FBN1 was lower. Expression of OCLN, MUC1, LAMB2, NR5A1, ESR1, ESR2, and TGFBR3 was lower in GREL cells than ovarian surface epithelial cells. Expression of KRT19, DSG2, PKP2, OCLN, MUC1, FBN1, COL1A1, COL3A1, STAR and TGFBR2 was higher and GJA1, CTNNB1, LAMB2, NR5A1, CYP11A1, HSD3B1, CYP19A1, HSD17B1, FOXL2, ESR1, ESR2, TGFBR3 and CCND2 was lower in GREL cells compared to granulosa cells. TGFβ1 altered the expression of COL1A1, COL3A1 and FBN1 in fetal fibroblasts and epidermal growth factor altered the expression of FBN1 and COL1A1. In summary, the two major somatic cell types of the developing ovary have distinct gene expression profiles. They, especially GREL cells, also differ from the cells they ultimately differentiate in to. The regulation of cell fate determination, particularly of the bi-potential GREL cells, remains to be elucidated.
Study question:
Could changes in transforming growth factor β (TGFβ) signalling during foetal ovary development alter the expression of polycystic ovary syndrome (PCOS) candidate genes leading to a predisposition to PCOS?
Summary answer:
TGFβ signalling molecules are dynamically expressed during foetal ovary development and TGFβ1 inhibits expression of the androgen receptor (AR) and 7 (INSR, C8H9orf3, RAD50, ERBB3, NEIL2, IRF1 and ZBTB16) of the 25 PCOS candidate genes in foetal ovarian fibroblasts in vitro, whilst increasing expression of the AR cofactor TGFβ-induced transcript 1 (TGFB1I1 or Hic5).
What is known already:
The ovarian stroma arises from the mesonephros during foetal ovary development. Changes in the morphology of the ovarian stroma are cardinal features of PCOS. The ovary is more fibrous and has more tunica and cortical and subcortical stroma. It is not known why this is and when this arises. PCOS has a foetal origin and perhaps ovarian stroma development is altered during foetal life to determine the formation of a polycystic ovary later in life. PCOS also has a genetic origin with 19 loci containing 25 PCOS candidate genes. In many adult tissues, TGFβ is known to stimulate fibroblast replication and collagen deposition in stroma, though it has the opposite effect in the non-scaring foetal tissues. Our previous studies showed that TGFβ signalling molecules [TGFβs and their receptors, latent TGFβ binding proteins (LTBPs) and fibrillins, which are extracellular matrix proteins that bind LTBPs] are expressed in foetal ovaries. Also, we previously showed that TGFβ1 inhibited expression of AR and 3 PCOS candidate genes (INSR, C8H9orf3 and RAD50) and stimulated expression of TGFB1I1 in cultured foetal ovarian fibroblasts.
Study design, size, duration:
We used Bos taurus for this study as we can ethically collect foetal ovaries from across the full 9-month gestational period. Foetal ovaries (62-276 days, n = 19) from across gestation were collected from pregnant B. taurus cows for RNA-sequencing (RNA-seq) analyses. Foetal ovaries from B. taurus cows were collected (160-198 days, n = 6) for culture of ovarian fibroblasts.
Participants/materials, setting, methods:
RNA-seq transcriptome profiling was performed on foetal ovaries and the data on genes involved in TGFβ signalling were extracted. Cells were dispersed from foetal ovaries and fibroblasts cultured and treated with TGFβ1. The effects of TGFβ regulation on the remaining eight PCOS candidate genes not previously studied (ERBB3, MAPRE1, FDFT1, NEIL2, ARL14EP, PLGRKT, IRF1 and ZBTB16) were examined.
Main results and the role of chance:
Many TGFβ signalling molecules are expressed in the foetal ovary, and for most, their expression levels increased accross gestation (LTBP1/2/3/4, FBN1, TGFB2/3, TGFBR2/3 and TGFB1I1), while a few decreased (FBN3, TGFBR3L, TGFBI and TGFB1) and others remained relatively constant (TGFBRAP1, TGFBR1 and FBN2). TGFβ1 significantly decreased expression of PCOS candidate genes ERBB3, NEIL2, IRF1 and ZBTB16 in cultured foetal ovarian fibroblasts.
Large scale data:
The FASTQ files, normalized data and experimental information have been deposited in the Gene Expression Omnibus (GEO) accessible by accession number GSE178450.
Limitations, reasons for caution:
Regulation of PCOS candidate genes by TGFβ was carried out in vitro and further studies in vivo are required. This study was carried out in bovine where foetal ovaries from across all of the 9-month gestational period were available, unlike in the human where it is not ethically possible to obtain ovaries from the second half of gestation.
Wider implications of the findings:
From our current and previous results we speculate that inhibition of TGFβ signalling in the foetal ovary is likely to (i) increase androgen sensitivity by enhancing expression of AR, (ii) increase stromal activity by stimulating expression of COL1A1 and COL3A1 and (iii) increase the expression of 7 of the 25 PCOS candidate genes. Thus inhibition of TGFβ signalling could be part of the aetiology of PCOS or at least the aetiology of polycystic ovaries.
Study funding/competing interest(s):
Funding was received from Adelaide University China Fee Scholarship (M.L.), Australian Research Training Program (R.A.) and the Faculty of Health and Medical Science Divisional Scholarship (R.A.), Adelaide Graduate Research Scholarships (R.A. and N.A.B.), Australia Awards Scholarship (M.D.H.), Robinson Research Institute Career Development Fellowship (K.H.) and Building On Ideas Grant (K.H.), National Health and Medical Research Council of Australia Centre for Research Excellence in the Evaluation, Management and Health Care Needs of Polycystic Ovary Syndrome (N.A.B., M.D.H. and R.J.R.; GTN1078444) and the Centre for Research Excellence on Women's Health in Reproductive life (R.A., R.J.R. and K.H.; GTN1171592) and the UK Medical Research Council (R.A.A.; grant no. G1100357). The funders did not play any role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. The authors of this manuscript have nothing to declare and no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
This chapter provides an overview of our present understanding of ovarian function in cattle, with emphasis on the dynamics and control of follicular and luteal gland development. It is arranged, more‐or‐less, in a chronological order, beginning with fetal ovarian development and follicle assembly, early folliculogenesis and oogenesis, antral follicle dynamics during the estrous cycle, ovulation, and development of the corpus luteum (CL). Ovulation involves a series of events that culminate in the evacuation of the antral contents of a follicle, including its mature cumulus‐oocyte complex, and the initiation of development of a CL. The chapter includes a description of ovarian dynamics during different reproductive states (i.e. pre‐and peripubertal periods, during pregnancy and the postpartum period, and during old age).
Fibrillins 1-3 are stromal extracellular matrix proteins that play important roles in regulating TGFβ activity, which stimulates fibroblasts to proliferate and synthesise collagen. In the developing ovary the action of stroma is initially necessary for formation of the ovigerous cords and subsequently for the formation of follicles and the surface epithelium of the ovary. FBN3 is highly expressed only in early ovarian development and then it declines. In contrast, FBN1 and 2 are up regulated in later ovarian development. We examined the expression of FBN1-3 in bovine and human fetal ovaries. We used cell dispersion and monolayer culture, cell passaging and tissue culture. Cells were treated with growth factors, hormones or inhibitors to assess the regulation of expression of FBN1-3. When bovine fetal ovarian tissue was cultured, FBN3 expression declined significantly. Treatment with TGFβ-1 increased FBN1 and FBN2 expression in bovine fibroblasts, but did not affect FBN3 expression. Additionally, in cultures of human fetal ovarian fibroblasts (9-17 weeks gestational age) the expression of FBN1 and FBN2 increased with passage whereas FBN3 dramatically decreased. Treatment with activin A and a TGFβ family signalling inhibitor, SB431542, differentially regulated expression of a range of modulators of TGFβ signalling and of other growth factors in cultured human fetal ovarian fibroblasts suggesting that TGFβ signalling is differentially involved in regulation of ovarian fibroblasts. Additionally since the changes in FBN1-3 expression that occur in vitro are those that occur with increasing gestational age in vivo, we suggest that the fetal ovarian fibroblasts mature in vitro.
Background
In society, there is a clear need to improve the success rate of techniques to restore fertility. Therefore a deeper knowledge of the dynamics of the complex molecular environment that regulates human gametogenesis and (early) folliculogenesis in vivo is necessary. Here, we have studied these processes focusing on the formation of the follicular basement membrane (BM) in vivo.
Results
The distribution of the main components of the extracellular matrix (ECM) collagen IV, laminin and fibronectin by week 10 of gestation (W10) in the ovarian cortex revealed the existence of ovarian cords and of a distinct mesenchymal compartment, resembling the organization in the male gonads. By W17, the first primordial follicles were assembled individually in that (cortical) mesenchymal compartment and were already encapsulated by a BM of collagen IV and laminin, but not fibronectin. In adults, in the primary and secondary follicles, collagen IV, laminin and to a lesser extent fibronectin were prominent in the follicular BM.
Conclusions
The ECM-molecular niche compartimentalizes the female gonads from the time of germ cell colonization until adulthood. This knowledge may contribute to improve methods to recreate the environment needed for successful folliculogenesis in vitro and that would benefit a large number of infertility patients.
Exploring stem cells in the mammalian ovary has unleashed a Pandora's box of new insights and questions. Recent evidence supports the existence of stem cells of a number of the different cell types within the ovary. The evidence for a stem cell model producing mural granulosa cells and cumulus cells is strong, despite a limited number of reports. The recent identification of a precursor granulosa cell, the gonadal ridge epithelial-like (GREL) cell, is exciting and novel. The identification of female germline (oogonial) stem cells is still very new and is currently limited to just a few species. Their origins and physiological roles, if any, are unknown and their potential to produce oocytes and contribute to follicle formation in vivo lacks robust evidence. The precursor of thecal cells remains elusive and more compelling data are needed. Similarly, claims of very small epithelial-like (VSEL) cells are also preliminary. Surface epithelial cells originating from GREL cells and from the mesonephric epithelium at the hilum of the ovary have also been proposed. Another important issue is the role of the stroma in guiding the formation of the ovary, ovigerous cords, follicles and surface epithelium. Immune cells may also play key roles in developmental patterning, given their critical roles in corpora lutea formation and regression. Thus, whilst the cellular biology of the ovary is extremely important for its major endocrine and fertility roles, there is much still to be discovered. This review draws together the current evidence and perspectives on this topic.
Protein restriction in early bovine gestation affects post-natal reproduction and production traits in progeny. This experiment evaluated the effects of dietary protein restriction during the peri-conception period and first trimester in yearling heifers on conceptus growth and development; this period of dietary intervention being earlier than any previous bovine fetal programming studies. Three-hundred and sixty primiparous 12-month-old Santa Gertrudis heifers were individually fed high [14% crude protein (CP)] or low (7% CP) diets for 60 days before conception. At 23 days post-conception (dpc), each high (HPERI) or low (LPERI) group was again split into high (HPOST) or low (LPOST) protein groups yielding four treatment groups in a 2 · 2 factorial design. From the end of the first trimester of gestation (98dpc), the pregnant heifers were individually fed a 12% CP diet until parturition. Forty-six fetuses were excised at 98dpc. Sixty-four heifers went on to calve. Conceptus development was assessed via transrectal ultrasound from 36dpc, fetal necropsy at 98dpc and live calf measures at term. At 36dpc, HPERI diet increased fetal crown–rump length (CRL) (P < 0.05) and at the 60dpc scan, biparietal diameter (BPD) tended to be increased by HPOST diet (P < 0.1) though the greater effect upon BPD was still the HPERI diet (P < 0.05). At 60dpc, BPD in the male fetus was affected by the peri-conception diet (P < 0.05), while in females, BPD was not different among nutritional groups. These ultrasound measures of fetal growth were validated by measures of the excised fetus at 98dpc. Fetal weight was heavier (P < 0.01) in those whose mothers were fed the HPOST diet than their LPOST counterparts. Males fetuses were heavier than female fetuses (P < 0.001). Fetal CRL was increased by HPERI diet (P < 0.05) and tended to be increased by HPOST diet (P < 0.1). Fetal BPD tended to be increased by HPERI diet (P < 0.1). In males, BPD tended to be increased in those fetuses whose mothers were fed HPERI (P < 0.1). For females, maternal nutrition during PERI or POST did not affect BPD at 98dpc (P > 0.1). At term, no dietary effect on birthweight was observed (P > 0.1) and males were not heavier than females (P > 0.1). These results suggest that maternal protein intake during the peri-conception (–60 to 23dpc) and first trimester (24–98dpc) may influence early conceptus growth and development in the bovine. The long-term effects on offspring metabolism and post-natal development of this dietary intervention are yet to be determined.
Prenatal and early postnatal ovarian development has assumed increasing importance over recent years. The full effects of perturbations to ovarian development on adult fertility, through environmental changes or genetic anomalies, is only now being truly appreciated. Mitigation of these perturbations requires an understanding of the processes involved in the development of the ovary. Here we review some recent findings from mouse, sheep and cow on the key events involved in ovarian development. We discuss the key process of germ cell migration, ovigerous cord formation, meiosis, follicle formation and follicle activation. We also review the key contribution of mesonephric cells to ovarian development and propose roles for these cells. Finally, we discuss polycystic ovary syndrome, premature ovarian failure and prenatal under nutrition; three key areas where perturbations to ovarian development appear to have major impacts on postnatal fertility.
Ultrasonographic imaging has afforded the ability to characterize dynamic physiologic events as they occur and has been intensively applied in the study of ovarian function, particularly in the bovine species. Distinct similarities exist between the bovine and human species in ovarian form and function, but ovarian dynamics and related endocrine events have not yet been critically characterized in women. Practical and ethical limitations prohibit gross, endocrine and histopathologic verification of ultrasonographic observations made in humans in vivo; consequently, meaningful diagnostic and therapeutic regimens have not been devised to precisely control ovarian function or ameliorate ovarian dysfunction. The bovine ultrasound model is the most well developed model with regard to characteristics of ovarian follicular and luteal dynamics, and preliminary study has revealed that physiologic characteristics of bovine ovarian follicles may be reflected by visual data analysis of ultrasound images. A research strategy has been designed, based on the bovine ultrasound model, to study ovarian function in women using the non-human primate as an intermediate step. It is anticipated that newly developed techniques of controlling folliculogenesis in cattle may be appropriately modified for diagnostic and interventional procedures in women to augment or suppress fertility.
Ovarian follicular granulosa cells surround and nurture oocytes, and produce sex steroid hormones. It is believed that during development the ovarian surface epithelial cells penetrate into the ovary and develop into granulosa cells when associating with oogonia to form follicles. Using bovine fetal ovaries (n = 80) we identified a novel cell type, termed GREL for Gonadal Ridge Epithelial-Like. Using 26 markers for GREL and other cells and extracellular matrix we conducted immunohistochemistry and electron microscopy and chronologically tracked all somatic cell types during development. Before 70 days of gestation the gonadal ridge/ovarian primordium is formed by proliferation of GREL cells at the surface epithelium of the mesonephros. Primordial germ cells (PGCs) migrate into the ovarian primordium. After 70 days, stroma from the underlying mesonephros begins to penetrate the primordium, partitioning the developing ovary into irregularly-shaped ovigerous cords composed of GREL cells and PGCs/oogonia. Importantly we identified that the cords are always separated from the stroma by a basal lamina. Around 130 days of gestation the stroma expands laterally below the outermost layers of GREL cells forming a sub-epithelial basal lamina and establishing an epithelial-stromal interface. It is at this stage that a mature surface epithelium develops from the GREL cells on the surface of the ovary primordium. Expansion of the stroma continues to partition the ovigerous cords into smaller groups of cells eventually forming follicles containing an oogonium/oocyte surrounded by GREL cells, which become granulosa cells, all enclosed by a basal lamina. Thus in contrast to the prevailing theory, the ovarian surface epithelial cells do not penetrate into the ovary to form the granulosa cells of follicles, instead ovarian surface epithelial cells and granulosa cells have a common precursor, the GREL cell.
We present the qgraph package for R, which provides an interface to visualize data through network modeling techniques. For instance, a correlation matrix can be rep-resented as a network in which each variable is a node and each correlation an edge; by varying the width of the edges according to the magnitude of the correlation, the structure of the correlation matrix can be visualized. A wide variety of matrices that are used in statistics can be represented in this fashion, for example matrices that contain (implied) covariances, factor loadings, regression parameters and p values. qgraph can also be used as a psychometric tool, as it performs exploratory and confirmatory factor analysis, using sem and lavaan; the output of these packages is automatically visualized in qgraph, which may aid the interpretation of results. In this article, we introduce qgraph by applying the package functions to data from the NEO-PI-R, a widely used personality questionnaire.
Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.
The development of a normal ovary during foetal life is essential for the production and ovulation of a high-quality oocyte in adult life. Early in embryogenesis, the primordial germ cells (PGCs) migrate to and colonise the genital ridges. Once the PGCs reach the bipotential gonad, the absence of the sex-determining region on the Y chromosome (SRY) gene and the presence of female-specific genes ensure that the indifferent gonad takes the female pathway and an ovary forms. PGCs enter into meiosis, transform into oogonia and ultimately give rise to oocytes that are later surrounded by granulosa cells to form primordial follicles. Various genes and signals are implicated in germ and somatic cell development, leading to successful follicle formation and normal ovarian development. This review focuses on the differentiation events, cellular processes and molecular mechanisms essential for foetal ovarian development in the mice and humans. A better understanding of these early cellular and morphological events will facilitate further study into the regulation of oocyte development, manifestation of ovarian disease and basis of female infertility.
Although not often discussed, the ovaries of women with polycystic ovary syndrome (PCOS) show all the hallmarks of increased TGF-β activity, with increased amounts of fibrous tissue and collagen in the ovarian capsule or tunica albuginea and ovarian stroma. Recent studies suggest that PCOS could have fetal origins. Genetic studies of PCOS have also found linkage with a microsatellite located in intron 55 of the extracellular matrix protein fibrillin 3. Fibrillins regulate TGF-β bioactivity in tissues by binding latent TGF-β binding proteins. We therefore examined expression of fibrillins 1-3, latent TGF-β binding proteins 1-4, and TGF-β 1-3 in bovine and human fetal ovaries at different stages of gestation and in adult ovaries. We also immunolocalized fibrillins 1 and 3. The results indicate that TGF-β pathways operate during ovarian fetal development, but most important, we show fibrillin 3 is present in the stromal compartments of fetal ovaries and is highly expressed at a critical stage early in developing human and bovine fetal ovaries when stroma is expanding and follicles are forming. These changes in expression of fibrillin 3 in the fetal ovary could lead to a predisposition to develop PCOS in later life.
To verify the feasibility of ovarian stromal evaluation and correlate ovarian parameteres (echogenicity and volume) with hyperandrogenism, and both cardiovascular and metabolic risk factors in PCOS.
Twenty four young PCOS patients and twelve age-matched control women were enrolled. Diagnosis of PCOS was based on the Rotterdam criteria. Ultrasound ovarian study included ovarian volume, stromal volume, stromal area and stromal area/total ovarian area ratio (S/A). Concerning hormones, insulin, LH, FSH, estradiol, androstenedione, testosterone, DHEAS, 17-hydroxy-progesterone, and SHBG were measured during the early follicular phase (days 2-5). Cardiovascular risk factors were represented by fasting plasma levels of glucose, lipids (total and HDL-cholesterol), plasminogen activator inhibitor 1 (PAI-1), von-Willebrand factor (vWF), and adiponectin. Carotid intima-media thickness (C-IMT) was measured as a parameter of cardiovascular risk.
A positive correlation between the S/A ratio and plasma levels of testosterone (p < 0.05) and androstenedione (p < 0.05) was found. The stromal volume, stromal area and S/A ratio were also significantly and positively correlated with PAI-1, and vWF levels, and with IMT in PCOS women (P < 0.05).
This study shows that the ultrasound measurement of ovarian stroma is a predicting factor of hyperandrogenism degree, prothrombotic factors and cardiovascular risk in patients with PCOS.
Ovarian function is dependent on the establishment and continual remodelling of a complex vascular system. This enables the follicle and/or corpus luteum (CL) to receive the required supply of nutrients, oxygen and hormonal support as well as facilitating the release of steroids. Moreover, the inhibition of angiogenesis results in the attenuation of follicular growth, disruption of ovulation and drastic effects on the development and function of the CL. It appears that the production and action of vascular endothelial growth factor A (VEGFA) is necessary at all these stages of development. However, the expression of fibroblast growth factor 2 (FGF2) in the cow is more dynamic than that of VEGFA with a dramatic upregulation during the follicular-luteal transition. This upregulation is then likely to initiate intense angiogenesis in the presence of high VEGFA levels. Recently, we have developed a novel ovarian physiological angiogenesis culture system in which highly organised and intricate endothelial cell networks are formed. This system will enable us to elucidate the complex inter-play between FGF2 and VEGFA as well as other angiogenic factors in the regulation of luteal angiogenesis. Furthermore, recent evidence indicates that pericytes might play an active role in driving angiogenesis and highlights the importance of pericyte-endothelial interactions in this process. Finally, the targeted promotion of angiogenesis may lead to the development of novel strategies to alleviate luteal inadequacy and infertility.
The interactions of the ECM (extracellular matrix) protein asporin with ECM components have previously not been investigated. Here, we show that asporin binds collagen type I. This binding is inhibited by recombinant asporin fragment LRR (leucine-rich repeat) 10-12 and by full-length decorin, but not by biglycan. We demonstrate that the polyaspartate domain binds calcium and regulates hydroxyapatite formation in vitro. In the presence of asporin, the number of collagen nodules, and mRNA of osteoblastic markers Osterix and Runx2, were increased. Moreover, decorin or the collagen-binding asporin fragment LRR 10-12 inhibited the pro-osteoblastic activity of full-length asporin. Our results suggest that asporin and decorin compete for binding to collagen and that the polyaspartate in asporin directly regulates collagen mineralization. Therefore asporin has a role in osteoblast-driven collagen biomineralization activity. We also show that asporin can be expressed in Escherichia coli (Rosetta-gami) with correctly positioned cysteine bridges, and a similar system can possibly be used for the expression of other SLRPs (small LRR proteoglycans/proteins).
Lumican and fibromodulin compete for collagen type I binding in vitro, and fibromodulin-deficient mice have 4-fold more lumican in tendons. These observations indicate that homologous sequences in lumican and fibromodulin bind to collagen type I. Here, we demonstrate that lumican binding to collagen type I is mediated mainly by Asp-213 in leucine-rich repeat (LRR) 7. The mutation D213N in lumican impairs interaction with collagen, and the lumican fragment spanning LRRs 5-7 is an efficient inhibitor of collagen binding. Also, the lumican LRR 7 sequence-based synthetic peptide CYLDNNKC inhibits the binding to collagen. Homologous collagen-binding site in fibromodulin, located in LRRs 5-7, inhibits the binding of lumican to collagen, and the mutation E251Q in this fibromodulin fragment does not inhibit the lumican-collagen binding. Lumican, but not the D213N mutation, lowers the melting point and affects the packing of collagen fibrils.
Factors that control the onset of primordial follicle growth are unknown. We have tested the hypothesis that primordial follicles from fetal calves can survive and initiate growth in vitro in serum-free conditions. Superficial pieces of ovarian cortex, containing mostly primordial follicles, were isolated from bovine fetuses 6-8 mo old and cultured for 0, 2, 4, or 7 days in Waymouth MB 752/1 medium supplemented with insulin, transferrin, selenium, linoleic acid, and BSA (ITS+). Histological examination of cortical pieces after 2, 4, and 7 days in culture showed that the number of healthy primordial follicles had decreased by 88%, 90%, and 94%, respectively (p < 0.01), whereas the number of healthy primary follicles had increased to 260%, 209%, and 197%, respectively, of the number present on Day 0 (p < 0.05). The percentage of follicles that showed signs of atresia did not change with time in culture and was about 28% and 50% for primordial and primary follicles, respectively. After 7 days in culture, the mean diameter of the few remaining healthy primordial follicles was 1.2 times the average diameter of primordial follicles present on Day 0 (p < 0.01). In contrast, after 2, 4, and 7 days in culture, primary follicles were 1.2, 1.3, and 1.4 times larger in diameter, respectively, relative to Day 0 (p < 0.01). There was little change in the diameter of oocytes in primordial follicles during culture, whereas in primary follicles an increase in oocyte diameter became apparent after 4 and 7 days (1.1 and 1.2 times, respectively, p < 0.01). That follicle growth was initiated in vitro was further confirmed by immunolocalization of proliferating cell nuclear antigen (PCNA), a marker for cell growth and proliferation, in cultured and freshly isolated pieces of ovarian cortex. In freshly isolated tissue, PCNA staining was absent from pre-granulosa cells and oocytes of the quiescent primordial follicles but was intense in granulosa cells and oocytes of the few growing primary follicles. After 2, 4, and 7 days in culture, PCNA was expressed intensely in the oocyte and many granulosa cells of newly activated primary follicles. These results demonstrate that bovine primordial follicles can enter the growth phase in vitro and that PCNA expression by granulosa cells and oocytes is closely associated with the onset of primordial follicle growth. The fact that a high percentage of primordial follicles initiated growth in vitro suggests that the ovarian stroma exerts inhibitory control over the initiation of primordial follicle growth in vivo. The culture system we describe may provide the means to test this hypothesis and others.
Type VI collagen filaments are found associated with interstitial collagen fibers, around cells, and in contact with endothelial basement membranes. To identify type VI collagen binding proteins, the amino-terminal domains of the alpha1(VI) and alpha2(VI) chains and a part of the carboxyl-terminal domain of the alpha3(VI) chain were used as bait in a yeast two-hybrid system to screen a human placenta library. Eight persistently positive clones were identified, two coding the known matrix proteins fibronectin and basement membrane type IV collagen and the rest coding new proteins. The amino-terminal domain of alpha1(VI) was shown to interact with the carboxyl-terminal globular domain of type IV collagen. The specificity of this interaction was further studied using the yeast two-hybrid system in a one-on-one format and confirmed by using isolated protein domains in immunoprecipitation, affinity blots, and enzyme-linked immunosorbent assay-based binding studies. Co-distribution of type VI and type IV collagens in human muscle was demonstrated using double labeling immunofluorescent microscopy and immunoelectron microscopy. The strong interaction of type VI collagen filaments with basement membrane collagen provided a possible molecular pathogenesis for the heritable disorder Bethlem myopathy.
In the ovary, differentiation of germinal cells into primordial follicles, functional ovulatory follicles and corpus luteum, all take place in a connective tissue matrix. We postulated that extracellular matrix (ECM) of the ovary participates actively in ovarian functions. To test this, the mRNA levels for several ECM components were determined in the mouse ovary at six distinct stages of the 4-day oestrous cycle. Northern analysis revealed statistically significant cyclic expression patterns for the mRNAs coding for type III, IV and VI collagens as well as for the small proteoglycan, biglycan, and for syndecan-1 and osteonectin. The cyclic changes observed in the mRNAs for these structural components exceeded those for matrix metalloproteinases (MMP)-2, -9 and -13, and for tissue inhibitors of matrix metalloproteinases (TIMP)-1, -2 and -3, where the changes were not statistically significant, despite their apparent role in ECM remodelling in the ovary. These observations support the hypothesis that cyclic changes in the production and degradation of ECM are part of normal ovarian function connected with follicular maturation, rupture and corpus luteum formation.
Proteoglycans (PGs) consist of a core protein and attached glycosaminoglycans (GAGs) and have diverse roles in cell and tissue biology. In follicles PGs have been detected only in follicular fluid and in cultured granulosa cells, and the composition of their GAGs has been determined. To identify PGs in whole ovarian follicles, not just in follicular fluid and granulosa cells, small (1-3-mm) bovine follicles were harvested. A proportion of these was incubated with (35)SO(4) for 24 h to incorporate radiolabel into the GAGs. The freshly harvested and cultured follicles were sequentially extracted with 6 M urea buffer, the same buffer with 0.1% Triton X-100 and then with 0.1 M NaOH. Proteoglycans were subjected to ion-exchange and size-exclusion chromatography. The GAGs were analyzed by chemical and enzymic digestion, and on the basis of their composition, we chose a list of known PGs to measure by ELISA analyses. Versican, perlecan, decorin, but not aggrecan or biglycan, were identified. These, excluding decorin for technical reasons, as well as a basal lamina glycoprotein, nidogen/entactin, were immunolocalized. Versican was localized to the thecal layers, including externa and the interna particularly in an area adjacent to the follicular basal lamina. Perlecan and nidogen were localized to the follicular basal lamina of antral follicles, both healthy and atretic, but not to that of preantral follicles. Both were localized to subendothelial basal laminas, but the former was not readily detected in arteriole smooth muscle layers. This study has confirmed the presence of versican and perlecan, but not the latter as a component of follicular fluid, and identified decorin and nidogen in ovarian antral follicles.
Fibronectin is one of the main components of the extracellular matrix and associates with a variety of other matrix molecules including collagens. We demonstrate that the absence of secreted type VI collagen in cultured primary fibroblasts affects the arrangement of fibronectin in the extracellular matrix. We observed a fine network of collagen VI filaments and fibronectin fibrils in the extracellular matrix of normal murine and human fibroblasts. The two microfibrillar systems did not colocalize, but were interconnected at some discrete sites which could be revealed by immunoelectron microscopy. Direct interaction between collagen VI and fibronectin was also demonstrated by far western assay. When primary fibroblasts from Col6a1 null mutant mice were cultured, collagen VI was not detected in the extracellular matrix and a different pattern of fibronectin organization was observed, with fibrils running parallel to the long axis of the cells. Similarly, an abnormal fibronectin deposition was observed in fibroblasts from a patient affected by Bethlem myopathy, where collagen VI secretion was drastically reduced. The same pattern was also observed in normal fibroblasts after in vivo perturbation of collagen VI-fibronectin interaction with the 3C4 anti-collagen VI monoclonal antibody. Competition experiments with soluble peptides indicated that the organization of fibronectin in the extracellular matrix was impaired by added soluble collagen VI, but not by its triple helical (pepsin-resistant) fragments. These results indicate that collagen VI mediates the three-dimensional organization of fibronectin in the extracellular matrix of cultured fibroblasts.
Relaxin-like factor (RLF) is a new member of the insulin-relaxin gene family known to be expressed in the ovarian follicular thecal cells of ruminants. To investigate the pattern of RLF expression in development and atresia of bovine follicles, antisera were raised in rats and rabbits to recombinantly expressed bovine pro-RLF and to chemically synthesized ovine RLF B chain, respectively. On dot blotting analysis, the rat antiserum bound to pro-RLF and less strongly to a synthetic mature ovine RLF lacking the C-domain, whereas the rabbit antiserum bound the mature form of ovine RLF. These antisera were used to immunostain bovine ovarian follicles of differing sizes and stages of health and atresia. 3beta-Hydroxysteroid dehydrogenase was colocalized with pro-RLF (n = 86 follicles), and cholesterol side-chain cleavage cytochrome P450 was localized in another section of many of the same follicles (n = 66). Not all follicles expressed pro-RLF in the theca interna, so the results are presented as the proportion of follicles expressing pro-RLF. Both mature and pro-RLF were immunolocalized to steroidogenic thecal cells of healthy follicles. As follicles enlarged to >5 mm, the proportion expressing pro-RLF declined (19/19 for <5 mm and 18/26 for >6 mm). Atresia was divided into antral (antral granulosa cells dying first) or basal (basal cells dying first) and further divided into early, middle, and late. For antral atresia of small follicles (2-5 mm), no decline in the proportion expressing pro-RLF was observed (early 6/6, middle 2/2) until the late stages (1/4). For basal atresia, which only occurs in small follicles (2-5 mm), the proportion expressing pro-RLF declined in the middle (2/5) and late (0/8) stages. In larger follicles (>6 to <10 mm), the proportion expressing pro-RLF also declined with atresia (1/13). These declines in RLF expression with atresia or increasing size were not accompanied by a decline in the expression of steroidogenic enzymes in the theca interna. A significant (P < 0.001) inverse relationship in the expression of pro-RLF and 3beta-hydroxysteroid dehydrogenase in the membrana granulosa was observed. We conclude that the expression of pro-RLF in the theca interna is switched off as follicles enlarge or enter atresia, whereas the expression of steroidogenic enzymes is maintained in the theca interna.
The objective of the study was to demonstrate the mRNA expression of estrogen receptor a (ERa), ERbeta, and progesterone receptor (PR) by block reverse transcription-polymerase chain reaction(RT-PCR) and real-time RT-PCR (LightCycler) in bovine ovarian follicles and in corpus luteum during the estrous cycle and pregnancy. The mRNA expression of ERalpha and ERbeta mRNA in theca interna tissue (TI) (lower pg/microg RNA) increased continuously and significantly during final growth of follicles, with much higher levels for ERalpha. The mRNA expression of ERalpha and ERbeta in granulosa cells (GC) (fg/microg RNA) increased continuously during follicle growth but without any significant change. The expression of mRNA for PR in follicles (lower fg/microg RNA) increased continuously to maximum level in preovulatory follicles with a significant change only in TI. The highest mRNA expression for ERalpha (fg/microg RNA) was detected in corpus luteum (CL) during the early luteal phase, following by a significant decrease of expression during the mid, late, and regression phases. In contrast, ERbeta mRNA expression is relatively high during the early stage, decreased during the late early and mid luteal phase, and increased significantly again during the late luteal phase and after CL regression. During pregnancy (>3 mo), low levels of ERalpha and ERbeta mRNA expression (<25 fg/microg RNA) with no significant changes were measured. No significant change in PR mRNA expression (levels <13 fg/microg RNA) during the estrous cycle and pregnancy in bovine CL were found. The results suggest an autocrine/paracrine role of steroid receptors in the regulation of final follicle growth and corpus luteum formation and function.
Small leucine-rich proteoglycans (SLRPs) are extracellular molecules that bind to TGFbetas and collagens and other matrix molecules. In vitro, SLRPs were shown to regulate collagen fibrillogenesis, a process essential in development, tissue repair, and metastasis. To better understand their functions in vivo, mice deficient in one or two of the four most prominent and widely expressed SLRPs (biglycan, decorin, fibromodulin, and lumican) were recently generated. All four SLRP deficiencies result in the formation of abnormal collagen fibrils. Taken together, the collagen phenotypes demonstrate a cooperative, sequential, timely orchestrated action of the SLRPs that altogether shape the architecture and mechanical properties of the collagen matrix. In addition, SLRP-deficient mice develop a wide array of diseases (osteoporosis, osteoarthritis, muscular dystrophy, Ehlers-Danlos syndrome, and corneal diseases), most of them resulting primarily from an abnormal collagen fibrillogenesis. The development of these diseases by SLRP-deficient mice suggests that mutations in SLRPs may be part of undiagnosed predisposing genetic factors for these diseases. Although the distinct phenotypes developed by the different singly deficient mice point to distinct in vivo function for each SLRP, the analysis of the double-deficient mice also demonstrates the existence of rescuing/compensation mechanisms, indicating some functional overlap within the SLRP family.
The mammalian bone morphogenetic protein-1 (BMP-1)/Tolloid-related metalloproteinases play key roles in regulating formation of the extracellular matrix (ECM) via biosynthetic processing of various precursor proteins into mature functional enzymes, structural proteins, and proteins involved in initiating the mineralization of hard tissue ECMs. They also have been shown to activate several members of the transforming growth factor-beta superfamily, and may serve to coordinate such activation with formation of the ECM in morphogenetic events. Osteoglycin (OGN), a small leucine-rich proteoglycan with unclear functions, is found in cornea, bone, and other tissues, and appears to undergo proteolytic processing in vivo. Here we have successfully generated recombinant OGN and have employed it to demonstrate that a pro-form of OGN is processed to varying extents by all four mammalian BMP-1/Tolloid-like proteinases, to generate a 27-kDa species that corresponds to the major form of OGN found in cornea. Moreover, whereas wild-type mouse embryo fibroblasts (MEFs) produce primarily the processed, mature form of OGN, MEFs homozygous null for genes encoding three of the four mammalian BMP-1/Tolloid-related proteinases produce only unprocessed pro-OGN. Thus, all detectable pro-OGN processing activity in MEFs is accounted for by products of these genes. We also demonstrate that both pro- and mature OGN can regulate type I collagen fibrillogenesis, and that processing of the prodomain by BMP-1 potentiates the ability of OGN to modulate the formation of collagen fibrils.
Designing PCR and sequencing primers are essential activities for molecular biologists around the world. This chapter assumes acquaintance with the principles and practice of PCR, as outlined in, for example, refs. 1, 2, 3, 4.
This study was aimed at testing the hypothesis that different forms of fibronectin (FN), produced as a consequence of the alternative splicing of the precursor messenger RNA, play specific roles during development of the ovarian follicle. In particular, we were interested in determining the effect of the ED-I (also termed ED-A) type III repeat, which is absent in the plasma form. Analysis of FN levels in follicular fluids corresponding to different stages of development of bovine follicles revealed marked changes in the concentrations of ED-I+FN, whereas total FN levels remained relatively constant. ED-I+FN levels were higher in small follicles, corresponding to the phase of granulosa cell proliferation. The hypothesis of a physiological role for ED-I+FN was further supported by the finding of a regulation of the alternative splicing of FN in primary cultures of bovine granulosa cells by factors known to control ovarian follicular development. cAMP produced a 10-fold decrease in the relative proportion of...
Collagen VI represents a remarkable extracellular matrix molecule, and in the past few years, studies of this molecule have revealed its involvement in a wide range of tissues and pathological conditions. In addition to its complex multi-step pathway of biosynthesis and assembly that leads to the formation of a characteristic and distinctive network of beaded microfilaments in the extracellular matrix, collagen VI exerts several key roles in different tissues. These range from unique biomechanical roles to cytoprotective functions in different cells, including myofibers, chondrocytes, neurons, fibroblasts and cardiomyocytes. Indeed, collagen VI has been shown to exert a surprisingly broad range of cytoprotective effects, which include counteracting apoptosis and oxidative damage, favoring tumor growth and progression, regulating autophagy and cell differentiation, and even contributing to the maintenance of stemness. In this Cell Science at a Glance article and the accompanying poster, we present the current knowledge of collagen VI, and in particular, discuss its relevance in stemness and in preserving the mechanical properties of tissues, as well as its links with human disorders.
The extracellular matrix (ECM), constituting the follicular basal lamina and present also between follicular cells and in the follicular fluid, is believed to regulate granulosa cell (GC) function during follicular development. Ovine GCs isolated from small (1‐3 mm in diameter) or large (4‐ 7 mm in diameter) antral follicles were cultured on various pure ECM components (type I collagen, fibronectin, laminin), synthetic substrata enhancing (RGD peptides) or impairing (poly 2-hydroxyethylmethacrylate (poly-hema)) cell adhesion, or in the presence of heparin. The effects of these factors, used alone or in combination with IGF-I and/or FSH, were evaluated in terms of GC spread, survival, proliferation and steroidogenesis. When grown on type I collagen (CI) gel, poly-hema or heparin, GCs from both large and small follicles exhibited a round shape and a low proliferation rate. Compared with non-coated plastic substratum as a control, these ECM or synthetic compounds enhanced estradiol secretion and reduced progesterone secretion by large-follicle GCs. In contrast, GCs from both large and small follicles spread extensively on CI coating, fibronectin, laminin and RGD peptides. Fibronectin and laminin dramatically increased the proliferation rate and enhanced survival of GCs from both origins. Moreover, fibronectin, laminin and RGD peptides reduced estradiol secretion by large-follicle GCs. Unexpectedly, CI coating increased estradiol secretion and reduced progesterone secretion by large-follicle GCs, suggesting that type I collagen was able to maintain estradiol secretion independently of GC shape. Finally, GC responsiveness to IGF-I and FSH, in terms of proliferation and steroidogenesis, was generally maintained when cells were grown on ECM components, RGD peptides and in the presence of heparin. However, when large-follicle GCs were grown as non-adherent clusters (as observed on poly-hema) basal and IGF-I- and/or FSH-stimulated progesterone secretions were totally abolished. Overall, this study shows that GC shape, survival, proliferation and steroidogenesis can be modulated in vitro by pure ECM components in a specific and coordinated manner. It is suggested that, in vivo, fibronectin and laminin would sustain follicular development by enhancing the survival and proliferation of GCs, whereas type I collagen might participate in the maintenance of estradiol secretion in large antral follicles. Journal of Endocrinology (2001) 169, 347–360
In the ovary, differentiation of germinal cells into primordial follicles, functional ovulatory follicles and corpus luteum, all take place in a connective tissue matrix. We postulated that extracellular matrix (ECM) of the ovary participates actively in ovarian functions. To test this, the mRNA levels for several ECM components were determined in the mouse ovary at six distinct stages of the 4-day oestrous cycle. Northern analysis revealed statistically significant cyclic expression patterns for the mRNAs coding for type III, IV and VI collagens as well as for the small proteoglycan, biglycan, and for syndecan-1 and osteonectin. The cyclic changes observed in the mRNAs for these structural components exceeded those for matrix metalloproteinases (MMP)-2, -9 and -13, and for tissue inhibitors of matrix metalloproteinases (TIMP)-1, -2 and -3, where the changes were not statistically significant, despite their apparent role in ECM remodelling in the ovary. These observations support the hypothesis that cyclic changes in the production and degradation of ECM are part of normal ovarian function connected with follicular maturation, rupture and corpus luteum formation.
The aim of this study was to evaluate the influence of two vitrification techniques on the extra cellular matrix (ECM) and ovarian follicular development. The ovarian cortex was fragmented (9 mm(3) ) and divided into six groups, viz. fresh control, cultured control, vitrified by the Ovarian Tissue Cryosystem (OTC) method, conventional solid surface vitrification (SSV) method, OTC/cultured and SSV/cultured. Follicles from all the fragments were analysed for morphology, development and viability. The ECM was evaluated based on the condition of collagen and reticular fibres and the immunolocalization of type I collagen and fibronectin. After 7 days of culture, the tissue vitrified by OTC revealed a higher percentage (p < 0.05) of morphologically normal (30.66%) and viable (60.00%) follicles when compared with those vitrified using the SSV technique (21.33% and 23.00%). In all the fragments cultured, regardless of the vitrification method, a significantly higher percentage of developing follicles was observed when compared with the non-cultured tissue. Analysis of the type I collagen showed increased immunostaining after the in vitro culture in the vitrified fragments. In conclusion, the OTC is better for preserving the follicular viability and morphology and maintaining the integrity of the extracellular matrix components of the ovine ovary.
© 2014 Blackwell Verlag GmbH.
The aim of this research was to perform in situ quantification, morphometry evaluation, and apoptosis analysis of ovarian follicular wall cells in mechanically isolated follicles obtained from ovaries of bovine fetuses (Bos taurus indicus) between 3 and 9 months of age. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The number of isolated follicles increased from 3 months onward (102.5 ± 141.1, mean ± SEM), peaked at 6 months (12855.0 ± 9030.1), and then decreased by 7 months (3208.7 ± 3249.5), consistent with atresia occurring at these stages. Follicular density was greatest at 4 months, consistent with a sudden boost in follicular activity independent of a corresponding increase in ovarian size. Antral follicles were first observed at 5 months. As fetal age increased, there was a tendency for the percentage of primordial and primary follicles to decrease, and the percentage of secondary follicles to increase. However, the high variability (P < 0.05) for all follicle populations up to 5 months of age precluded further interpretation of these results. Oocyte diameter increased from the primordial (23.6 ± 4.4 μm) to the secondary follicular stages (38.0 ± 14.9 μm). Apoptosis was observed in ovaries from all fetal ages analyzed. We concluded that preantral follicles could be isolated from bovine fetuses by 3 months of age, with apoptosis affecting ovarian follicular dynamics throughout fetal life.
Different fibronectin (FN) isoforms are generated by the alternative splicing of 3 regions (ED-A, ED-B and IIICS) of the primary transcript. The FN isoform containing the ED-B sequence, a complete type-III-homology repeat, while having extremely restricted distribution in normal adult tissues, reveals high expression in fetal and tumor tissues. Using the monoclonal antibody (MAb) BC-1, specific for the FN isoform containing the ED-B sequence (B+·FN), we demonstrate here, using immunohistochemical techniques, that while this FN isoform is undetectable in mature vessels, it is highly expressed during angiogenesis both in neoplastic and in normal tissues, as in the case of the functional layer of endometrium during the proliferative phase. B+·FN is thus a marker for the formation of new vessels, and the BC-I MAb may be a useful reagent for evaluating the level of the angiogenetic process in different neoplasms.
During the growth of bovine follicles, one emerges from a wave as the largest and dominant follicle. What regulates dominance is not known but candidates include oestradiol, transforming growth factor beta beta1 (TGFB1), and recently CYP11AI (cholesterol side-chain cleavage) and focal intra-epithelial matrix (focimatrix). To examine this, pairs of bovine ovaries with 2 or more follicles of equal size (>5mm) and hence in a wave before deviation, were collected at an abattoir (6.7+/-SEM 0.1mm diameter; n=14 animals, 35 follicles in total). These follicles were dissected and follicular fluid collected to measure progesterone and oestradiol concentrations. A portion of the follicle wall was processed for histological classification of health or atresia and granulosa cells were harvested for quantitative RT-PCR of focimatrix components [COL4A1 (collagen type IV alpha1), LAMB2 (laminin beta2) and HSPG2 (perlecan)], steroidogenic enzymes [CYP11A1 and CYP19A1] and TGFB1. For statistical analyses follicles within each animal were grouped into either the highest (oestradiol, CYP11A1) or lowest (TGFB1) expression (n=14) for comparison with the remaining follicles (n=21). When grouped on oestradiol no other parameters differed significantly, and when grouped on TGFB1 some parameters were different however the levels were also lower, and not higher as expected. When grouped on CYP11A1 other parameters were significantly elevated in the high CYP11A1 group (COL4A1P<0.05; LAMB2P<0.01; HSPG2P<0.01 and CYP19A1P<0.001). This suggests that steroidogenesis and focimatrix might be important in a follicle attaining dominance.
The microsatellite D19S884, located in intron 55 of fibrillin-3 (FBN3) gene, associates with polycystic ovary syndrome (PCOS) in familial studies. The family of fibrillin proteins (FBN1-3), which includes latent TGF-beta binding proteins (LTBP-1 to -4), are extracellular matrix proteins. We localized and examined the expression of these proteins in the adult bovine ovaries (n=7-10 per group, average age 681 days) born to mothers fed high (13% protein per total dry weight) or a low protein diet (5%) in each of the first and second trimesters of pregnancy (n=4 groups). FBN1 and LTBP-1 and -2 were the major members expressed in the mature ovary. Each protein had a unique localization pattern but all were associated with stromal tissue including the tunica albuginea (FBN1 and LTBP-2 near surface, and FBN1 and LTBP-1 deeper in the tunica), cortical stroma (FBN1 and LTBP-1) and follicular thecal layers (FBN1 in theca interna, LTBP-1 in the inner regions of the theca externa, and LTBP-2 in the outer regions of the theca externa). No significant (P>0.05) effects of maternal diet were observed on either the localization or the levels of mRNA of any of these proteins in the tunica. Expression levels of all three FBNs were positively correlated with each other, and FBN1 and 2 were positively correlated with LTBP-2, suggesting some level of co-ordinate regulation. This is the first study to investigate the expression and localization of these genes affecting TGFbeta bioavailability in the ovary.
Ovariectomy and ovarian tissue cryopreservation has the potential to preserve the natural fertility of cancer patients prior to sterilizing chemo- and radiotherapies. Ovarian tissue cryopreservation with the conventional slow-freezing method has yielded limited success, partly because of oocyte loss during freeze-thaw and subsequent transplant. Based on the high-efficiency vitrification Cryotop method, a practical vitrification procedure for murine, bovine and human ovarian tissue was developed. A Cryotissue method was designed for cryopreservation of ovarian tissue, and vitrification experiments were performed in a bovine animal model with ovarian size and structure similar to the human. There was no difference in oocyte viability (>89%) between fresh and vitrified ovarian cortical tissue in either bovine or human samples. Ovarian tissue was successfully autotransplanted to six cattle. Autotransplantation of vitrified-warmed tissue back to the cattle resulted in no loss of oocyte viability. In addition, human ovarian tissue from cancer patients, and from ovary transplant donors was also vitrified by the Cryotissue method. After warming, high oocyte survival in human tissue (similar to bovine tissue) was obtained. These results indicate that an ultra-rapid cooling vitrification method has the potential for clinical use in human ovarian tissue cryopreservation.
Type VI collagen is a component of 100 nm long periodic filaments with a widespread distribution around collagen fibers and on the surface of cells. It is an unusual collagen constituted by three distinct chains, one of which (alpha 3) is much larger than the others and is encoded by a 9-kb mRNA. The amino acid sequence of the alpha 3(VI) deduced from the present cDNA clones specifies for a multidomain protein of at least 2648 residues made of a short collagenous sequence (336 residues), flanked at the N-terminus by nine 200 residue long repeating motifs and at the C-terminus by two similar motifs that share extensive identities with the collagen-binding type A repeats of von Willebrand factor. Type VI collagen and alpha 3(VI) fusion proteins bound to insolubilized type I collagen in a specific, time-dependent, and saturable manner. The alpha 3(VI) chain has three Arg-Gly-Asp sequences in the collagenous domain, and cell attachment was stimulated by the triple helix of type VI collagen and by alpha 3(VI) fusion proteins containing Arg-Gly-Asp sequences. This function was specifically inhibited by the Arg-Gly-Asp-Ser synthetic peptide. The type I collagen-binding and the cell-attachment properties of the alpha 3(VI) chain provide direct information for the role of type VI collagen in connective tissues.
The distribution of interstitial collagen type I and III was studied immunocytochemically and ultrastructurally in the fetal rat testis and ovary from the undifferentiated stage (day 12) until birth. The results suggest that there is a correlation between the differentiation, organization, and abundance of the mesenchyme and the differentiation of the testicular vs. ovarian cords. Type III collagen was already present in the undifferentiated gonadal mesenchyme, and it appeared at an early stage around the organizing gonadal cords. Type I collagen appeared later in a similar mesenchymal distribution as type III collagen. Fragmentation of the subepithelial basement membrane in the gonads starting morphogenesis was considered to indicate that the surface epithelium participates in the gonadal cord formation. The expression of type III collagen at first on the surface of the developing testicular cords and later around the ovarian cords suggests that the mesenchymal premyoid cells are actively involved in the male cord formation. Focal discontinuities were found in the basement membrane of the ovarian cords, which in part were separated from each other by a ramified and relatively sparse mesenchyme. A complex linkage between the cytoskeleton and the extracellular matrix is illustrated both in the cord forming Sertoli and granulosa cells, and in the adjacent mesenchymal cells. Depletion of the mesenchyme and the basement membrane around the germ cell-granulosa cell associations of the wide ovarian medullary cords may be causal for their subsequent degeneration.
A study of 34 full-thickness Stein-Leventhal ovarian wedges and 30 age-matched control ovaries allowed comparison of the entire ovarian cross-sections and quantification of their features. As compared with controls, Stein-Leventhal ovaries showed on average (i) double the cross-sectional area, (II) the same number of primordial follicles, (iii) double the number of ripening and subsequent atretic follicles from the earliest stages, (iv) a tunica increased by 50 per cent and more collagenized, (v) cortical stromal thickness increased by a third, (vi) subcortical stroma, whether deep cortical or medullary in site, increased five times, (vii) ovarian hilus cell nests four times as frequently. The increased subcortical stroma was derived partly from the regressive conversion into stroma of the over-numerous older follicles, so augmenting steadily with duration, and partly from a concurrent stromal hyperplasia. Stromal smooth muscle and lutein cell nests were each found in four-fifths of cases. So-called "hyperthecosis," in which such nests are combined with marked stromal increase, is arguably just a late stage of Stein-Leventhal morphology. The whole picture may result directly or indirectly from the raised LH output, although androgens possibly promote early follicle ripening.
The volumetric composition of the human ovary during the compartmentalization stage has been investigated using current stereological methods. Eight left ovaries removed from three fetuses (developmental age 20-25 weeks), four neonates, and one 8-month-old child all with a 46,XX karyotype, free from malformations of the genital apparatus, were completely cut obtaining serial sections and one 1 micron-thick section every 1,000 microns was examined. Ovarian volume was 30 mm3 at the 20th week of development, 36 mm3 at the 25th week, 129 mm3 at birth, and 287 mm3 at the eighth postnatal month. The primitive cortical tissue was the largest component of the fetal ovaries (17 mm3, corresponding to 60.2% of the organ). The second component was the interstitium (21% of the organ), followed by the medulla (11.8% of the organ). The primordial follicles occupied a small part of the organs: 1.8 mm3 at 20 weeks and 3.4 mm3 at 25 weeks (respectively 6.7% and 5.4% of the volumes of the relevant ovaries). At birth, most of the organ was composed of interstitial tissue (57 mm3, 44.2% of the volume) followed by the medulla (25 mm3, 20.3% of the volume). The germinal tissue occupied 46 mm3, mainly primitive cortical tissue (14.9% of the ovary) and primordial follicles (16.3% of the ovary), with a minor contribution from the antral follicles (about 3% of the ovary).(ABSTRACT TRUNCATED AT 250 WORDS)
The expression patterns of transforming growth factor (TGF)-beta receptor type I (TbetaRI) and -II (TbetaRII) in pre- and post-menopausal human ovaries, and the in-vitro effects of follicle stimulating hormone (FSH), epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1) on receptor expression in preantral follicles were evaluated using immunohistochemistry and immunoblotting. Both types of receptor were present in granulosa, theca and interstitial cells; however, more mural than antral granulosa cells were TbetaRII positive. Overall, more cells expressed TbetaRI than TbetaRII. TbetaRI and TbetaRII expressions were detected in the membrane as well as in the soluble fractions. However, marked increases in TbetaRII and TbetaRI expression in the soluble fraction and corresponding decreases in the membrane fraction were noted in the post-menopausal ovary. Preantral follicles in class 1 and 2 responded in vitro to FSH and EGF with increased growth and a corresponding increase in TbetaRII expression in granulosa cells; however, TGF-beta did not influence follicular growth or receptor expression. There was no apparent change in follicular TbetaRI immunoreactivity regardless of test factors or follicular growth status. These results suggest that TbetaRI and TbetaRII are expressed differentially in human ovarian cells, particularly in the granulosa cells. The shift in TbetaR-I and -II from transmembrane to soluble fraction after menopause indicates that the endocrine milieu provided by the granulosa cells positively influences receptor induction in the membrane and, hence, the biological action of TGF-beta. FSH- and EGF-induced preantral follicular development is associated with a selective induction of TbetaRII and may indicate a mechanism whereby gonadotrophins and growth factor(s) regulate preantral folliculogenesis.
The direct interaction of hyaluronic acid (HA) and heavy chain (HC) of the inter-alpha-trypsin inhibitor (IalphaI) family plays a critical role in the organization and stabilization of the extracellular matrix. The aim of the present investigation was to elucidate the distribution of the IalphaI HC and HA in adult mouse tissues. An immunohistochemical method using a rabbit polyclonal antibody raised against mouse IalphaI heavy-chain peptide and a specific probe for HA (biotinylated HA-binding protein) was used to demonstrate an immunolocalization of IalphaI HC and HA. Distribution and localization of HA was of three types, namely, colocalization with IalphaI HC itself (cartilaginous tissue and ovary), localization around IalphaI HC immunostaining (lung, intestine and skeletal muscle), and localization at a small distance from IalphaI HC or a different distribution pattern (brain, liver, skin and kidney). These results indicate that IalphaI HC could function as an HA-rich matrix stabilizer on the cells of cartilage and maturing ovary, in which IalphaI HC shows colocalization with its predominant ligand, HA.
To identify type VI collagen expression in human ovarian follicles during follicular growth.
In vitro experiment.
Department of Obstetrics and Gynecology, Wakayama Medical College, Japan.
Regularly cycling women who underwent adnexectomy.
Immunohistochemistry and in situ hybridization for human type VI collagen.
Expression of type VI collagen.
Expression of type VI collagen was observed in the theca cell layers during folliculogenesis, whereas no expression of type VI collagen was observed in the granulosa cell layers at the mRNA and protein levels. As the follicles grew, immunostaining for type VI collagen became intense in the theca cell layers, especially the theca externa. In preovulatory follicles, however, weak, fragmented, or discontinuous immunostaining of the theca cell layers was observed. This fragmented or discontinuous immunostaining was evident predominantly in the apical area of preovulatory follicles rather than in the basal area.
Type VI collagen is present in the theca cell layers of follicles during folliculogenesis and plays an important role in interactions between the theca cells and extracellular matrix. These interactions may lead to changes in the shape, proliferation, migration, or differentiation of follicular cells during follicular development, maturation, and ovulation.
Fibronectin is an extracellular matrix glycoprotein. Alternative splicing of fibronectin mRNA within three specific regions, the extra domains (ED) A and B and the variable (V) or IIICS region, result in the production of different isoforms of fibronectin. These isoforms differentially regulate tissue developmental processes, such as those occurring during follicular and luteal development. The specific isoforms of fibronectin present in follicles and corpora lutea have not been identified. To identify these, primers for reverse transcription polymerase chain reaction (RT-PCR) were designed to the known bovine amino acid sequence of exons flanking the ED-A, ED-B and V regions. PCR products from bovine fetal liver cDNA were determined to be bovine fibronectin by the correct product size and DNA sequence homology to other species, and to the known bovine amino acid sequence. Bovine ovarian follicles (0.5-9 mm diameter) and corpora lutea (cyclic, early to late mid-luteal phase) were shown to express ED-A+, ED-A-, ED-B+, ED-B-, V+ and V fibronectin isoforms, similar to the liver, lung and kidney of fetuses, but generally not of adult animals. Thus follicles and corpora lutea express isoforms of fibronectin usually expressed in developing tissues.
The extracellular matrix (ECM), constituting the follicular basal lamina and present also between follicular cells and in the follicular fluid, is believed to regulate granulosa cell (GC) function during follicular development. Ovine GCs isolated from small (1-3 mm in diameter) or large (4-7 mm in diameter) antral follicles were cultured on various pure ECM components (type I collagen, fibronectin, laminin), synthetic substrata enhancing (RGD peptides) or impairing (poly 2-hydroxyethylmethacrylate (poly-hema)) cell adhesion, or in the presence of heparin. The effects of these factors, used alone or in combination with IGF-I and/or FSH, were evaluated in terms of GC spread, survival, proliferation and steroidogenesis. When grown on type I collagen (CI) gel, poly-hema or heparin, GCs from both large and small follicles exhibited a round shape and a low proliferation rate. Compared with non-coated plastic substratum as a control, these ECM or synthetic compounds enhanced estradiol secretion and reduced progesterone secretion by large-follicle GCs. In contrast, GCs from both large and small follicles spread extensively on CI coating, fibronectin, laminin and RGD peptides. Fibronectin and laminin dramatically increased the proliferation rate and enhanced survival of GCs from both origins. Moreover, fibronectin, laminin and RGD peptides reduced estradiol secretion by large-follicle GCs. Unexpectedly, CI coating increased estradiol secretion and reduced progesterone secretion by large-follicle GCs, suggesting that type I collagen was able to maintain estradiol secretion independently of GC shape. Finally, GC responsiveness to IGF-I and FSH, in terms of proliferation and steroidogenesis, was generally maintained when cells were grown on ECM components, RGD peptides and in the presence of heparin. However, when large-follicle GCs were grown as non-adherent clusters (as observed on poly-hema) basal and IGF-I- and/or FSH-stimulated progesterone secretions were totally abolished. Overall, this study shows that GC shape, survival, proliferation and steroidogenesis can be modulated in vitro by pure ECM components in a specific and coordinated manner. It is suggested that, in vivo, fibronectin and laminin would sustain follicular development by enhancing the survival and proliferation of GCs, whereas type I collagen might participate in the maintenance of estradiol secretion in large antral follicles.
To evaluate whether some ultrasound parameters of ovarian morphology can discriminate between control women and patients with polycystic ovary syndrome (PCOS).
Retrospective data analysis.
Volunteers women in an academic research environment.
Eighty amenorrheic or oligomenorrheic women and 30 normal ovulatory control participants.
None.
We evaluated ovarian volume, area, stroma, and the stroma/total area (S/A) ratio by use of transvaginal pelvic ultrasound; and we assayed serum levels of gonadotropin, androgen, and estradiol during the early follicular phase (days 2 to 5) of the menstrual cycle in regularly cycling controls and on a random day in amenorrheic patients.
Patients with PCOS showed significantly higher ovarian volume, area, stroma, and mean S/A ratio when compared to multifollicular and control groups. Cut-off values have been defined for ovarian volume (13.21 mL), area (7.00 cm2), stroma (1.95 cm2), and S/A ratio (0.34). The sensitivity for PCOS diagnosis was 21%, 4%, 62%, and 100%, respectively. The S/A ratio showed the most significant correlation with the androgen levels.
The evaluation of the S/A ratio can differentiate between PCOS and control or multifollicular women with both a sensitivity and a specificity of 100%. Furthermore, this ultrasound parameter is strictly related to hormonal milieu and to anthropometric characteristics.
Stroke-prone spontaneously hypertensive rats (SHRSP) are a well-characterized, genetic model for stroke. We showed earlier that the structure and function of the tight junctions in SHRSP blood-brain barrier endothelial cells is disturbed prior to stroke. To investigate the molecular events leading to endothelial dysfunction in SHRSP cerebral capillaries, we carried out suppression subtractive hybridization (SSH) in combination with a cDNA filter screening step. We identified two cDNA fragments that were upregulated in SHRSP, compared to stroke-resistant spontaneously hypertensive rats (SHR), and found open reading frames of 133 and 138 amino acids, respectively. These peptides did not match any known proteins in public databases. A third upregulated SHRSP cDNA fragment was identified as the rat sulfonylurea receptor 2B (SUR2B). We also isolated and cloned the cDNA of the rat homologue for the mouse G-protein signaling 5 (RGS5) regulator. This regulator was downregulated in SHRSP. We used in situ hybridization to show that rat RGS5 is expressed in the brain capillary endothelium and in the choroid plexus. Our findings may lead to the identification of new stroke-related genes.
All blood capillaries consist of endothelial tubes surrounded by mural cells referred to as pericytes. The origin, recruitment, and function of the pericytes is poorly understood, but the importance of these cells is underscored by the severe cardiovascular defects in mice genetically devoid of factors regulating pericyte recruitment to embryonic vessels, and by the association between pericyte loss and microangiopathy in diabetes mellitus. A general problem in the study of pericytes is the shortage of markers for these cells. To identify new markers for pericytes, we have taken advantage of the platelet-derived growth factor (PDGF)-B knockout mouse model, in which developing blood vessels in the central nervous system are almost completely devoid of pericytes. Using cDNA microarrays, we analyzed the gene expression in PDGF-B null embryos in comparison with corresponding wild-type embryos and searched for down-regulated genes. The most down-regulated gene present on our microarray was RGS5, a member of the RGS family of GTPase-activating proteins for G proteins. In situ hybridization identified RGS5 expression in brain pericytes, and in pericytes and vascular smooth muscle cells in certain other, but not all, locations. Absence of RGS5 expression in PDGF-B and PDGFR beta-null embryos correlated with pericyte loss in these mice. Residual RGS5 expression in rare pericytes suggested that RGS5 is a pericyte marker expressed independently of PDGF-B/R beta signaling. With RGS5 as a proof-of-principle, our data demonstrate the usefulness of microarray analysis of mouse models for abnormal pericyte development in the identification of new pericyte-specific markers.
We identified regulator of G-protein signaling-5 (RGS-5) as an angiogenic pericyte marker at sites of physiologic and pathologic angiogenesis. In a mouse model of pancreatic islet cell carcinogenesis, RGS-5 is specifically induced in the vasculature of premalignant lesions during the "angiogenic switch" and further elevated in tumor vessels. Similarly, RGS-5 is overexpressed in highly angiogenic astrocytomas but not in hypoxia-inducible factor-1alpha (HIF-1alpha)-deficient tumors, which grow along preexisting brain capillaries without inducing neovessels. Elevated levels of RGS-5 in pericytes are also observed during wound healing and ovulation indicating a strong correlation between RGS-5 expression and active vessel remodeling beyond tumor angiogenesis. Moreover, antitumor therapy, which reverses tumor vasculature to an almost normal morphology, results in down-regulation of RGS-5 transcription. Taken together, these data demonstrate for the first time a factor that is specific for "activated" pericytes. This further supports the notion that pericytes, like endothelial cells, undergo molecular changes during neovascularization that makes them a novel target for antiangiogenic therapy.