Regulation of prohibitin expression during follicular development and atresia in the mammalian ovary

Prohibitin is a ubiquitous and highly conserved protein implicated as an important regulator in cell survival. Prohibitin content is inversely associated with cell proliferation, but it increases during granulosa cell differentiation as well as in earlier events of apoptosis in a temperature-sensitive granulosa cell line. In the present study, we have characterized the spatial expression patterns for prohibitin using established in vivo models for the induction of follicular development and atresia in the mammalian ovary. Comparative Western blot analyses of granulosa cell lysates from control ovaries and from ovaries primed with eCG or treated with eCG plus anti-eCG (gonadotropin withdrawal) were conducted. Prohibitin was immunolocalized in rat ovarian sections probed with antibodies against either proliferating cell nuclear antigen (PCNA) or cholesterol side-chain cleavage cytochrome P450 (P450(scc)) or in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeled sections. Additionally, porcine oocytes, zygotes, and blastocyts were also immunolocalized with prohibitin antibody. Immunolocalization revealed the presence of prohibitin in granulosa cells, theca-interstitial cells, and the oocyte. The results indicate that prohibitin protein expression in the gonadotropin-treated cells was upregulated. Immunoreactivity of prohibitin was inversely related to PCNA expression during follicular maturation and colocalized with P450(scc). Prohibitin appeared to be translocated from the cytoplasm to the nucleus in atretic follicles, germinal vesicle-stage oocytes, zygotes, and blastocysts. These results suggest that prohibitin has several functional regulatory roles in granulosa and theca-interstitial cells and in the ovum during follicular maturation and atresia. It is likely that prohibitin may play an important role in determining the fate of these cells and eventual follicular destiny.     

Gene expression during two alternative pathways of ovary development in Pisum sativum: fruit development and ovary senescence

Pea ovaries are induced to enter a fruit development pathway involving physiological and morphological changes by pollination or application of plant growth regulators. In the absence of these stimuli, overies stop growing and enter an alternative pathway of senesecence that leads to their degeneration. We have used two dimensional polyacrylamide gel electrophoresis in search of molecular changes underlying fruit development and ovary senescence at the level of total accumulated proteins, newly synthesized proteins, and translatable, RNA populations. We have found changes in gene expression during the processes of ovary formation and ovary senescence. Stimuli that induce fruit set do not appreciably alter the overall patterns of synthesized proteins or translatable RNAs, indicating that fruit development is apparently a natural continuation of ovary formation. However, ovary senescence is an alternative pathway that involves the presence of new RNA messengers and proteins as well as the disappearance of others. These changes were detected earlier than any morphological or structural changes could be observed in the ovary.     

Gene expression of matrix metalloproteinases and LH receptors in mare follicular development

The period from the emergence of a dominant follicle until its formation requires tissue remodeling. Enzymes promoting collagen lysis, such as matrix metalloproteinases (MMPs), are fundamental for the process of extracellular matrix remodeling, which allows changes in ovarian tissue architecture during follicular growth. It has been suggested that the production of these enzymes may be affected by the rise in circulating concentrations of LH, which acts on the ovarian surface epithelium (OSE). The aim of this study was to determine the expression of MMP-1, MMP-2, and LH receptor (LHR) in the ovulation fossa and in the central portion of the equine ovary during follicular deviation and dominance. Ovaries of 12 cyclic mares were selected and subsequently divided into two groups: development (DEV) group and dominant (DOM) group. The DEV group consisted of ovaries from six animals whose follicles were less than 28 mm in diameter (follicular deviation), and the DOM group consisted of ovaries from six animals whose follicles measured 28 mm or more in diameter (dominant follicles). The latter group was divided into two subgroups: the group of ovaries with a dominant follicle (DOM-D) and the group of contralateral ovaries (DOM-C). Our results showed that mRNA for MMP-1, MMP-2, and LHR was present in the equine ovary during follicle development, in the ovulation fossa, and in the central portion of the ovary. MMP-1 and LHR gene expression was greater (P < 0.05) for the DOM-D group compared with the DOM-C group. In the DOM-D group, MMP-1, MMP-2, and LHR gene expression was greater (P < 0.05) in the ovarian stroma compared with the ovulation fossa. Using immunohistochemistry, OSE from the DOM group showed increased expression compared with the DEV group (P < 0.05). In conclusion, we demonstrated that MMP-1 and MMP-2 might be fundamental for events related to tissue remodeling, which occurs during follicular development until the formation of the dominant follicle. We also demonstrated the relationship between the gene expression of MMPs and the gene and protein expression of LHR, suggesting that LHR in the OSE might be an important factor to initiate the signaling cascade that culminates with the production of MMPs.     

Primordial follicle activation and follicular development in the juvenile rabbit ovary

Of all the stages of mammalian folliculogenesis, the primordial to primary follicle transition is the least understood. In order to gain new insights into this process, we have conducted a comprehensive morphological, morphometric and molecular study of ovarian organisation and early follicle development in the rabbit. The structure of ovaries collected from rabbits aged from 2–12 weeks (a period encompassing primordial follicle formation, activation and the first wave of folliculogenesis in this species) has been analysed by light microscopy and the follicles present have been measured and scored for their developmental stage. To establish useful molecular markers of activation, we have further classified follicles according to their expression of the proliferative marker, proliferating cell nuclear antigen, and the zona pellucida protein, ZPB. The activation of primordial follicles is initiated immediately following their formation in the rabbit ovary and is characterised by oocyte growth, granulosa cell morphogenesis and increased granulosa cell mitosis. Enhanced ZPB protein expression at the oolemma is also associated with follicle activation and development. Few primordial follicles in the juvenile rabbit ovary are lost by atresia, as assessed by the TUNEL assay. The appearance of apoptotic granulosa cells is however coincident with the development of antral follicles. This study thus describes the temporal and spatial regulation of early follicular development in the post-natal rabbit ovary and, for the first time, shows that the primordial to primary transition in the juvenile rabbit is a highly ordered process occurring within quantifiable parameters.K.J.H. was supported by the Pest Animal Control CRC and Post Graduate scholarships from the Australian National University.     

Collagenase and gelatinase messenger ribonucleic acid expression and activity during follicular development in the rat ovary.

Metalloproteinases are members of a family of proteinases that remodel the extracellular matrix throughout the body. To test the hypothesis that metalloproteinases are regulated by gonadotropin-induced changes during follicular growth, rats were injected with eCG (20 IU, s.c.), and ovaries and serum were collected at the time of eCG administration (0 h) and at 6, 12, 24, 36, or 48 h later for analysis of metalloproteinase mRNA expression, metalloproteinase activity, and steroidogenesis. Serum estradiol levels increased from 18.9 pg/ml at 0 h to 503.8 pg/ml at 48 h. Analysis of mRNA expression was performed for collagenase-3, 72-kDa gelatinase, and 92-kDa gelatinase (n = 3-4). For collagenase-3, eCG stimulated a 32-fold increase in collagenase-3 mRNA at 48 h after eCG injection as compared to that in ovaries collected at the time of eCG administration (i.e., 0-h control). The mRNA levels for 72-kDa gelatinase were 2.8-fold compared to 0 h at 36 h after eCG treatment and returned to control levels by 48 h after gonadotropin treatment. Levels of the 92-kDa mRNA expression peaked at 24 h (4. 2-fold compared to 0 h) and returned to control levels by 36 h. Gel zymography revealed 3 gelatinolytic bands corresponding to the gelatinases of approximately 72 kDa, 92 kDa, and 105 kDa. Analysis of metalloproteinase activity as the degradation of collagen or gelatin per ovary showed an increase in gelatinolytic and collagenolytic activity between 12 and 48 h after eCG treatment. In summary, these findings demonstrate that the gonadotropin induction of folliculogenesis results in changes in the metalloproteinases that may be responsible for extracellular matrix remodeling associated with follicular growth.     

Estrogen receptor alpha and beta expression in the porcine ovary

In order to investigate the expression of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in the porcine ovary, in situ hybridization was applied. Specific ovine ERalpha and bovine ERbeta cDNA probes were labeled with [-32S]dCTP. In the porcine ovary, positive signals for ERbeta were found in both granulosa and theca cells of all types of antral follicles as well as in the corpora lutea at all stages of regression. ERalpha mRNA was limited exclusively to the granulosa cells of preovulatory follicles and was present in a few cells of early corpora lutea. Thus, we showed differential expression of ERalpha and ERbeta at the mRNA level. Large antral follicles and early corpora lutea are the site, where both forms of estrogen receptor are expressed.     

Expression of glutathione transferase isoenzymes in the porcine ovary in relationship to follicular maturation and luteinization

The expression of different isoenzymes of glutathione transferase (GST), i.e. the cytosolic subunits GSTA1/A2, A3, A4, A5, M1/2, M2 and P1, T2, and the microsomal GST in follicles of different sizes and in corpora lutea from porcine ovary, was investigated by Western blotting. No immunoreactivity was obtained with anti-rat GSTT2 or anti-rat microsomal GST polyclonal antibodies. In contrast, GSTA1/A2, A3, A4, A5, M1/2, M2 and P1 are all expressed in the cytosol from porcine ovaries. In general, the highest levels of these GST isoenzymes were present in the cytosol from corpora lutea, in agreement with measurements of activity towards 1-chloro-2,4-dinitrobenzene. Immunoreactivity with anti-rat GSTP1 was only obtained with follicles. The cytosolic GSTs from follicles and corpora lutea were affinity purified on glutathione-Sepharose and separated by reversed-phase high-performance liquid chromatography in order to quantitate the different subunits. A peak corresponding to the class pi subunit was present in follicles. This peak was also seen with corpora lutea, although at very low level. There were four peaks containing class mu subunits. The remaining peaks were concluded to contain the class alpha subunits, except for two peaks which are suggested to contain proteins other than GSTs. The levels of the different subunits were quantitated on the basis of the areas under the peaks and the relative amounts in follicles of different sizes and in corpora lutea corresponded well with the Western blot analysis.     

Gene expression profiling in porcine fetal thymus

To obtain an initial overview of gene diversity and expression pattern in porcine thymus, 11,712 ESTs (Expressed Sequence Tags) from 100-day-old porcine thymus (FTY) were sequenced and 7,071 cleaned ESTs were used for gene expression analysis. Clustered by the PHRAP program, 959 contigs and 3,074 singlets were obtained. Blast search showed that 806 contigs and 1,669 singlets (totally 5,442 ESTs) had homologues in GenBank and 1,629 ESTs were novel. According to the Gene Ontology classification, 36.99% ESTs were cataloged into the gene expression group, indicating that although the functional gene (18.78% in defense group) of thymus is expressed in a certain degree, the 100-day-old porcine thymus still exists in a developmental stage. Comparative analysis showed that the gene expression pattern of the 100-day-old porcine thymus is similar to that of the human infant thymus.     

Presence of endothelin-1 in porcine spinal cord: isolation and sequence determination

We investigated the molecular forms of endothelin (ET) related peptides in porcine spinal cord by high performance liquid chromatography coupled with radioimmunoassays using three antisera raised against ET-1 and C-terminal fragments of ET-1 and big ET-1. ET-1 and its oxidized form were isolated as major immunoreactive peptides and sequenced. Furthermore, immunoreactivities like ET-3 and big ET-1(22-39) (contents: less than 8% and less than 1% of ET-1, respectively) were detected based on their chromatographic retention times and characteristics of immunoreactivity to the antisera. Big ET-1 was only scarcely detected. Immunohistochemical study showed the presence of ET-1-like immunoreactivity in motoneurons, dorsal horn neurons and dot- and fiber-like structures in the dorsal horn of lumbar spinal cord. These results indicate that ET-1 is present not only in endothelial cells but also in spinal cord, and that big ET-1 is converted into ET-1 in spinal cord by specific processing between Trp21-Val22. The data also indicate that ET-1 may act as a neuropeptide in the central nervous system.     

Identification of calcitonin expression in the chicken ovary: influence of follicular maturation and ovarian steroids

Calcitonin (CALCA), a hormone primarily known for its role in calcium homeostasis, has recently been linked to reproduction, specifically as a marker for embryo implantation in the uterus. Although CALCA expression has been documented in several tissues, there has been no report of production of CALCA in the ovary of any vertebrate species. We hypothesized that the Calca gene is expressed in the chicken ovary, and its expression will be altered by follicular maturation or gonadal steroid administration. Using RT-PCR, we detected Calca mRNA and the calcitonin receptor (Calcr) mRNA in the granulosa and theca layers of preovulatory and prehierarchial follicles. Both CALCA and Calca mRNA were localized in granulosa and thecal cells by confocal microscopy. Using quantitative PCR analysis, F1 follicle granulosa layer was found to contain significantly greater Calca mRNA and Calcr mRNA levels compared with those of any other preovulatory or prehierarchial follicle. The granulosa layer contained relatively greater Calca and Calcr mRNA levels compared with the thecal layer in both prehierarchial and preovulatory follicles. Progesterone (P(4)) treatment of sexually immature chickens resulted in a significantly greater abundance of ovarian Calca mRNA, whereas estradiol (E(2)) or P(4) + E(2) treatment significantly reduced ovarian Calca mRNA quantity. Treatment of prehierarchial follicular granulosa cells in vitro with CALCA significantly decreased FSH-stimulated cellular viability. Collectively, our results indicate that follicular maturation and gonadal steroids influence Calca and Calcr gene expression in the chicken ovary. We conclude that ovarian CALCA is possibly involved in regulating follicular maturation in the chicken ovary.     

Syndecan-4 expression is associated with follicular atresia in mouse ovary

Ovarian granulosa cells synthesize heparan sulfate proteoglycans (HSPGs), that have anticoagulant properties. Moreover, HSPGs greatly increase in the granulosa cells during follicular atresia. However, the species of ovarian HSPGs have not yet been identified. Syndecan-4 (ryudocan, amphiglycan) is a membrane-spanning HSPG and a member of the syndecan family. Herein, we demonstrate that syndecan-4 is expressed in the granulosa cells of type 4-5b follicles and, most intensely, in those of the atretic follicles in the mouse ovary, as revealed by in situ hybridization. There is no relationship between syndecan-4 expression and age or sexual cycle stage. Compared with syndecan-4 expression, syndecan-1 and -3 are expressed more abundantly in postovulatory follicles and the corpora lutea, but less in the type 4-5b follicles and much less in the atretic follicles. Immunohistochemistry also demonstrates syndecan-4 expression in atretic follicles with apoptosis. The present study has revealed the distinct modes of expression of the syndecan family members, and the association of syndecan-4 expression and apoptosis in ovarian atretic follicles.     

Retrograde transfer of testosterone to the porcine ovary in follicular and luteal phase of the estrous cycle in vivo

This study was designed to determine the efficiency and rate of testosterone (T) retrograde transfer during the follicular and luteal phase of the estrous cycle in gilts (n=27). The efficiency and the rate of the retrograde transfer of T from the ovarian effluent into blood supplying the ovary were determined for the first time under in vivo conditions. Ovarian arterial blood concentration of T was higher than that in systemic blood during both, the follicular phase (p<0.01) and luteal phase (p<0.0001). The efficiency of the retrograde transfer of T into ovarian arterial blood was not dependent on concentration of testosterone in the ovarian venous blood. However, the efficiency and the rate of the retrograde transfer of T differed between phases of the estrous cycle. The presented results suggest that local retrograde transfer enriched the porcine ovary with an additional amount of T which may affect the ovarian secretory function.     

Endothelin-3 is a novel neuropeptide: isolation and sequence determination of endothelin-1 and endothelin-3 in porcine brain

The molecular forms of endothelin (ET) related peptides were investigated in porcine brain by using high performance liquid chromatography coupled with three specific radioimmunoassays. ET-1 and its oxidized form were isolated and sequenced as in the case of porcine spinal cord. A very small amount of big ET-1 (1-39) and its C-terminal fragment (big ET-1 (22-39] were also detected. Furthermore, immunoreactive (ir)-ET-3 was isolated and sequenced; its partial primary structure was identical to that of human (rat) ET-3. The concentrations of ir-ET-1 and ir-ET-3 in porcine brain were 140 fmol/g tissue and 5 fmol/g tissue, respectively. These results indicate that besides ET-1, ET-3 is a novel neuropeptide in the central nervous system.     

Follicular microvasculature in the porcine ovary

The microvasculature of porcine ovaries, with special regard to the follicles in the interstitial-stromal tissue, was studied by scanning electron microscopy (SEM) of vascular corrosion casts. Porcine ovaries displayed several coiled arteries in the hilus and many branches with small diameters and a tightly spiraling configuration in the cortical areas. However, small arterioles became straight before entering vascular complexes of follicles and finally divided into capillaries. Vascular baskets of various sizes (150-9,900 micro m in diameter) and architecture related to follicles in various developmental stages were observed in the ovarian cortex. Small follicles (150-300 micro m in diameter) began with a polygonal meshwork of a few large capillary meshes and developed to an obvious spherical microvascular network with a thin single layer of capillaries when reaching 500-700 micro m in diameter. The microvascular architecture of follicles 1,000-2,000 micro m in diameter developed further and had a three-layer vascular plexus. With a diameter of more than 2,000 micro m, the microvasculature of antral follicles was arranged as an inner vascular plexus of about 25 micro m, a middle plexus of about 100 micro m, and an outer capillary plexus of about 30 micro m in thickness. The present observations indicate that follicular vascular baskets of diverse sizes and architecture in various developmental stages support the gradual increase of follicular blood flow during follicle growth in the pig.     

Conversion of big endothelin-1 to endothelin-1 by two-types of metalloproteinases of cultured porcine vascular smooth muscle cells

Incubation of big endothelin-1 (big ET-1, 1-39) with the membrane fraction obtained from cultured vascular smooth muscle cells (VSMCs) resulted in an increase in immunoreactive-ET (IR-ET), which was inhibited by EDTA but not by phosphoramidon, a metalloproteinase inhibitor. When the incubation was performed in the presence of N-ethylmaleimide (NEM), the generation of IR-ET was markedly augmented and this augmentation was abolished by phosphoramidon. The pH profile for IR-ET generation in the presence of NEM was apparently distinct from that observed in the absence of NEM. Reverse-phase HPLC of the incubation mixture with or without NEM revealed one major IR-ET component corresponding to the elution position of synthetic ET-1 (1-21). When the cultured VSMCs were incubated with big ET-1, a conversion to the mature ET-1 was observed. This ET-1 generation from exogenously applied big ET-1 was markedly inhibited by the addition of phosphoramidon, although the inhibitor did not influence the basal secretion of ET-1-like materials. These results suggest the presence of two types of metalloproteinases, which can generate ET-1, in VSMCs. The possibility that ET-1 functions in an autocrine manner to control the cardiovascular system warrants further attention.     

miR-26b promotes granulosa cell apoptosis by targeting ATM during follicular atresia in porcine ovary

More than 99% of ovarian follicles undergo atresia in mammals, but the mechanism of follicular atresia remains to be elucidated. In this study, we explored microRNA (miRNA) regulation of follicular atresia in porcine ovary. A miRNA expression profile was constructed for healthy, early atretic, and progressively atretic follicles, and the differentially expressed miRNAs were selected and analyzed. We found that miR-26b, which was upregulated during follicular atresia, increased the number of DNA breaks and promoted granulosa cell apoptosis by targeting the ataxia telangiectasia mutated gene directly in vitro.     

Conversion of big endothelin-1 to endothelin-1 by two types of metalloproteinases derived from porcine aortic endothelial cells

Incubation of big endothelin-1 (big ET-1(1-39] with either the cytosolic or membrane fraction obtained from cultured endothelial cells, resulted in an increase in immunoreactive-endothelin (IR-ET), which was markedly inhibited by metal chelators. Phosphoramidon, a metalloproteinase inhibitor, specifically suppressed the membrane fraction-induced increase in IR-ET, whereas the increase in IR-ET observed with the cytosolic fraction was not influenced by phosphoramidon. Reverse-phase (RP)-HPLC of the incubation mixture of big ET-1 with the cytosolic or membrane fraction revealed one major IR-ET component corresponding to the elution position of synthetic ET-1(1-21). Simultaneously, immunoreactivities like the C-terminal fragment (CTF22-39) of big ET-1 were present, as deduced from the RP-HPLC coupled with the radioimmunoassay for CTF. Our results indicate the presence of two types of metalloproteinases, which convert big ET-1 to ET-1 via a single cleavage between Trp21 and Val22, in vascular endothelial cells.     

Differential effects of different statins on endothelin-1 gene expression and endothelial NOS phosphorylation in porcine aortic endothelial cells

It has been reported that 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitors (statins) produce a variety of cardiovascular protective effects independent of their ability to lower total and low-density lipoprotein cholesterol. Recent studies have also reported that statins produce pleiotropic effects through improved endothelial function, enhanced fibrinolysis, and antithrombotic actions. In the present study, we examined the effects of pitavastatin, pravastatin, atorvastatin, and cerivastatin on endothelin (ET)-1 production in cultured porcine aortic endothelial cells (PAECs). Treatment with cerivastatin but not pitavastatin, pravastatin, or atorvastatin decreased basal and TNF-alpha-stimulated ET-1 release from PAECs in a dose-dependent manner (1-10 microM). Northern blot analysis showed that cerivastatin markedly suppressed prepro ET-1 mRNA expression in both conditions. In addition, these inhibitory effects of cerivastatin on ET-1 release and prepro ET-1 mRNA expression were completely abolished by simultaneous treatment with 200 microM mevalonate. Furthermore, cerivastatin did not have any effects on endothelial nitric oxide synthase (eNOS) protein levels, but induced eNOS phosphorylation at Ser1177. From these findings, it is most likely that cerivastatin suppresses ET-1 production, possibly through an increase in eNOS activity and the subsequent nitric oxide production in PAECs. These findings also suggest that cerivastatin may have beneficial effects on ET-1-related diseases.