Granulosa Cells Of Healthy Follicles Research Articles

Whole-Transcriptome Analysis of LncRNAs Mediated ceRNA Regulation in Granulosa Cells Isolated From Healthy and Atresia Follicles of Chinese Buffalo.

Granulosa cells (GCs) are the main supporting cells in follicles and play an important role in the regulation of oocyte maturation and follicular atresia. Accumulating evidence indicates that non-coding RNAs participate in regulation of the physiological function of GCs. However, whole-transcriptome analysis for GCs of buffalo has yet to be reported. In this study, healthy follicles (HFs) and atretic follicles (AFs) were defined according to the apoptosis rate of GCs and the hormone level in follicular fluid. GCs were collected from HFs and AFs (n = 15, 5 < n < 8 mm) for whole-transcriptome analysis using second-generation high-throughput sequencing. A total of 1,861 and 1,075 mRNAs, 159 and 24 miRNAs, and 123 and 100 lncRNAs, were upregulated and downregulated between HFs and AFs, respectively. Enrichment of functions and signaling pathways of these differentially expressed (DE) genes showed that most of DEmRNAs and targets of DEmiRNAs were annotated to the categories of ECM–receptor interaction and focal adhesion, as well as PI3K-AKT, mTOR, TGF-beta, Rap1, and estrogen signaling pathways. The competing endogenous RNA (CeRNA) network was also constructed based on the ceRNA theory which further revealed regulatory roles of these DERNAs in GCs of buffalo follicles. Finally, we validated that lnc4040 regulated the expression of Hif1a as miR-709 sponge in a ceRNA mechanism, suggesting their critical functions in GCs of buffalo follicles. These results show that lncRNAs are dynamically expressed in GCs of HFs and AFs, and interacting with target genes in a ceRNA manner, suggesting their critical functions in buffalo follicular development and atresia.

Follicular fluid steroid profile in sows: relationship to follicle size and oocyte quality†.

Identification of reliable characteristics of follicle quality and developmental competence has been pursued in numerous studies, but with inconsistent outcomes. Here, we aimed to identify these characteristics by analysis of the follicular fluid (FF) steroid profile in relation to cumulus-oocyte complex (COC) morphology and follicle size, followed by molecular substantiation. Multiparous sows at weaning were used to facilitate analysis at the start of the follicular phase of the oestrus cycle. Sows with a higher average follicle size (≥5 mm vs. < 5 mm) had a higher follicular fluid β-estradiol concentration, but did not differ in other measured steroids. Sows with high compared to low percentage high-quality COCs (<70% vs. ≥70% high-quality) had follicular fluid with a higher concentration of β-estradiol, 19-norandrostenedione, progesterone, and α-testosterone, while the concentration of cortisol was lower. Transcriptome analysis of granulosa cells of healthy follicles of sows with a high percentage high-quality COCs showed higher abundance of transcripts involved in ovarian steroidogenesis (e.g., CYP19A2 and 3, POR, VEGFA) and growth (IGF1) and differential abundance of transcripts involved in granulosa cell apoptosis (e.g., GADD45A, INHBB). Differences in aromatase transcript abundance (CYP19A1, 2 and 3) were confirmed at the protein level. In addition, sows with a high percentage high-quality COCs lost less weight during lactation and had higher plasma IGF1 concentration at weaning, which may have affected COC quality. To the best of our knowledge, this study is also the first to report the relation between FF steroid profile and COC quality.

Polysialylation takes place in granulosa cells during apoptotic processes of atretic tertiary follicles.

In the neuronal system, polysialic acid (polySia) is known to be involved in several cellular processes such as the modulation of cell-cell interactions. This highly negatively-charged sugar moiety is mainly present as a post-translational modification of the neural cell adhesion molecule (NCAM). More than 20 years ago, differently glycosylated forms of NCAM were detected in the ovaries. However, the exact isoform of NCAM, as well as its biological function, remained unknown. Our analysis revealed that granulosa cells of feline tertiary follicles express the polysialylated form of NCAM-140. Unexpectedly, polySia was only expressed in the granulosa layers of atretic follicles and not of healthy follicles. By contrast, only the un-polysialylated form of NCAM was present on the membrane of granulosa cells of healthy follicles. To study a possible cellular function of polySia in feline follicles, a primary granulosa cell culture model was used. Interestingly, loss of polySia leads to a significant inhibition of apoptosis, demonstrating that polySia is involved during atretic processes in granulosa cells. Thus, polySia might not only directly influence regeneration processes as shown, for example, in the neuronal system, but also apoptosis.

Melatonin counteracts BMP-6 regulation of steroidogenesis by rat granulosa cells

The ovarian bone morphogenetic protein (BMP) system is a physiological inhibitor of luteinization in growing ovarian follicles. BMP-6, which is expressed in oocytes and granulosa cells of healthy follicles, specifically inhibits FSH actions by suppressing adenylate cyclase activity. In the present study, we studied the role of melatonin in ovarian steroidogenesis using rat primary granulosa cells of immature female rat ovaries by focusing on the interaction with BMP-6 activity. Treatment with melatonin had no direct effect on FSH-induced progesterone or estradiol production by granulosa cells, and the results were not affected by the presence of co-cultured oocytes. In addition, synthesis of cAMP by granulosa cells was not significantly altered by melatonin treatment. To elucidate the interaction between activities of melatonin and BMPs, the effect of melatonin treatment on suppression of progesterone synthesis by BMP-6 was investigated. Interestingly, the inhibitory effect of BMP-6 on FSH-induced progesterone production was impaired by co-treatment with melatonin. Granulosa cells express higher levels of MT1 than MT2, and BMP-6 had no significant effect on MT1 expression in granulosa cells. However, BMP-6-induced Smad1/5/8 phosphorylation and Id-1 transcription were suppressed by melatonin, suggesting that melatonin has an inhibitory effect on BMP receptor signaling in granulosa cells. Although the expression levels of ALK-2, -6, ActRII and BMPRII were not affected by melatonin, inhibitory Smad6, but not Smad7, expression was upregulated by melatonin. Thus, melatonin plays a role in the regulation of BMP-6 signal intensity for controlling progesterone production in the ovary. These findings suggest that the effect of melatonin on maintenance of ovarian function is, at least in part, due to the regulation of endogenous BMP activity in granulosa cells.

Apoptosis in Porcine Granulosa Cells During Follicular Atresia Is Regulated by Anti-Apoptotic cFLIP.

More than 99% of follicles are selectively eliminated via a degenerative process called gatresiah during follicular development and growth. Follicular atresia is indicated to begin with granulosa cell apoptosis. To reveal the molecular mechanisms of follicle selection, we examined changes in the expression levels of cellular FLICE-like inhibitory protein (cFLIP), which is a homologue of an intracellular apoptosis inducer (procaspase-8/FLICE), has two alternative splicing isoforms (cFLIP short and long form: cFLIPS and cFLIPL, respectively) and inhibits apoptosis initiated by death receptors, in porcine granulosa cells during atresia. In granulosa cells, we detected mRNA and protein of cFLIPL but no cFLIPS. mRNA and protein of cFLIPL were highly expressed in granulosa cells of healthy follicles and rapidly decreased during atresia. We examined changes in the localization of cFLIPL mRNA and protein in pig ovaries using in situ hybridization and immunohistochemistry, respectively, and found that they were abundant in granulosa cell layer of healthy follicles in comparison with that in those of atretic follicles. Moreover, when cFLIPL was stably expressed in granulosa cell-derived KGN cells following transfection of an expression vector, the cell death ligand/receptor-mediated apoptosis was inhibited. Suppression of cFLIPL by small interfering RNA (siRNA) spontaneously induced cell death in cultured KGN cells. We suggested that cFLIPL plays an anti-apoptotic role in the granulosa cells of healthy follicles of pig ovaries, and that cFLIPL is an essential pro-survival factor for granulosa cells and acts as a key determinant of follicular growth and/or atresia by regulating granulosa cell apoptosis.

Changes in Expression and Localization of X-linked Inhibitor of Apoptosis Protein (XIAP) in Follicular Granulosa Cells During Atresia in Porcine Ovaries

Follicular selection predominantly depends on granulosa cell apoptosis in porcine ovaries, but the molecular mechanisms regulating the induction of apoptosis in granulosa cells during follicular selection remain incompletely understood. To determine the role of X-linked inhibitor of apoptosis protein (XIAP), which suppresses caspase-3, -7 and -9 activities and acts as an endogenous inhibitor of apoptotic cell death, in the regulation of granulosa cell apoptosis during follicular atresia, we examined the changes in the expression level and localization of XIAP mRNA and protein in granulosa cells during follicular atresia using reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, Western blotting and immunohistochemistry, respectively. High levels of XIAP mRNA and protein were noted in the granulosa cells of healthy follicles, and decreased levels were noted during follicular atresia. In situ hybridization and immunohistochemistry demonstrated that XIAP mRNA and protein were strongly expressed in the granulosa cells of healthy follicles, but negative/trace stainings were noted in those of atretic follicles. The present findings strongly indicate that XIAP is a candidate molecule which acts as an anti-apoptotic/pro-survival factor by inhibiting intracellular apoptosis signaling and is involved in the regulation of apoptosis in porcine granulosa cells.

The Role of Interleukin-6 in the Regulation of Granulosa Cell Apoptosis During Follicular Atresia in Pig Ovaries

More than 99% of follicles in mammalian ovaries undergo a degenerative process known as atresia, and only a few follicles actually ovulate during follicular growth and development. Follicular selection mostly depends on granulosa cell apoptosis, but the molecular mechanism behind the regulation of this selective atresia is still largely unknown. In the present study, to examine whether or not interleukin-6 (IL-6), a multifunctional cytokine, is involved in apoptosis during atresia in pig ovaries, the expression of IL-6 mRNA in granulosa cells was quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR). The level of mRNA decreased during atresia. Enzyme-linked immunosorbent assay (ELISA) showed that the level of IL-6 protein in follicular fluid also decreased during atresia. Moreover, recombinant human IL-6 upregulated the expression of an intracellular apoptosis inhibitor, cellular FLICE-like inhibitory protein long form (cFLIP(L)), in cultured cells derived from human granulosa cells. It is possible that IL-6 is produced in the granulosa cells of healthy follicles, that it increases the cFLIP(L) level, and cFLIP(L) then prevents apoptotic cell death.

Role and Regulation of Nodal/Activin Receptor-Like Kinase 7 Signaling Pathway in the Control of Ovarian Follicular Atresia

Although the role of the TGF beta superfamily members in the regulation of ovarian folliculogenesis has been extensively studied, their involvement in follicular atresia is not well understood. In the present study, we have demonstrated for the first time that Nodal, a member of the TGF beta superfamily, is involved in promoting follicular atresia as evidenced by the following: 1) colocalization of Nodal and its type I receptor Activin receptor-like kinase 7 (ALK7) proteins in the granulosa cells was only observed in atretic antral follicles, whereas they were present in theca cells and granulosa cells of healthy follicles, respectively; 2) addition of recombinant Nodal or overexpression of Nodal by adenoviral infection induced apoptosis of otherwise healthy granulosa cells; 3) constitutively active ALK7 (ALK7-ca) overexpression mimicked the function of Nodal in the induction of granulosa cell apoptosis. Furthermore, overexpression of Nodal or ALK7-ca increased phosphorylation and nuclear translocation of Smad2, decreased X-linked inhibitor of apoptotic proteins (Xiap) expression at both mRNA and protein level and phospho-Akt content, as well as triggered mitochondrial release of death proteins Smac/DIABLO, Omi/HtrA2, and cytochrome c in the granulosa cells. Dominant-negative Smad2 significantly attenuated ALK7-ca-induced down-regulation of Xiap and thus rescued granulosa cells from undergoing apoptosis. In addition, whereas up-regulation of Xiap significantly attenuated ALK7-ca-induced apoptosis, down-regulation of Xiap sensitized granulosa cells to ALK7-ca-induced apoptosis. Furthermore, ALK7-ca-induced apoptosis was significantly attenuated by forced expression of activated Akt, and Akt rescued granulosa cells from undergoing apoptosis via proteasome-mediated ALK7 degradation. Taken together, Nodal plays an atretogenic role in the ovary where it induces granulosa cell apoptosis through activation of Smad2, down-regulation of the key survival molecules Xiap and phospho-Akt, as well as the activation of mitochondrial death pathway.

Expression and Localization of Fas Ligand and Fas During Atresia in Porcine Ovarian Follicles

To reveal the mechanisms regulating the selective atresia of follicles in porcine ovaries, we examined the changes in the mRNA and protein levels of cell-death ligand, Fas/APO-1/CD95 ligand (FasL), and its receptor, Fas/APO-1/CD95 (Fas), and the localization of the proteins in granulosa cells during follicular atresia using the reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical techniques, respectively. Trace levels of FasL mRNA and protein were detected in the granulosa cells of healthy follicles; however, weak levels were detected in those of early atretic follicles, and the levels increased during atresia. Trace/weak levels of Fas mRNA and protein were detected in the granulosa cells of healthy follicles. Fas protein was located in the cytoplasmic area, not in cell membrane area, indicating that it has no activity in regard to inducing apoptosis. When apoptosis commences in granulosa cells, Fas moves from the cytoplasmic to cell membrane area. FasL and Fas mRNAs and proteins in granulosa cells were upregulated during follicular atresia. The FasL and Fas system may play a crucial role in the regulation of apoptosis in granulosa cells during selective follicular atresia in porcine ovaries.

Changes in expression of anti-apoptotic protein, cflip, in granulosa cells during follicular atresia in porcine ovaries

Follicular selection is performed in mammalian ovaries, as most follicles undergo atresia during follicular development and growth. Follicular regression is indicated to begin with granulosa cell apoptosis. To reveal the molecular mechanisms of the selection, we examined the changes in the levels of cellular-Flice like inhibitory protein (cFLIP) expression in porcine granulosa cells. cFLIP is the homologue of intracellular apoptosis inducer (procaspase-8/Flice), and has two alternative splicing isoforms: cFLIP short form (cFLIP(S)) and long form (cFLIP(L)). By competing with caspase-8, cFLIP inhibits apoptosis initiated by death receptors. The changes in the levels of cFLIP(S) and cFLIP(L) mRNA and protein expression in granulosa cells were determined by RT-PCR and Western blotting, respectively. cFLIP(L) mRNA and protein were highly expressed in granulosa cells of healthy follicles and decreased during atresia. cFLIP(S) mRNA levels in granulosa cells were low and showed no change among the stages of follicular development, and its protein level was extremely low. We examined the changes in the localization of cFLIP mRNAs in pig ovaries by in situ hybridization and found that cFLIP(L) is abundant in granulosa cells of healthy follicles in comparison with those of atretic follicles. Immunohistochemical analyses demonstrated that the cFLIP protein is highly expressed in the granulosa cell of healthy follicles but weakly expressed in that of atretic follicles. We presumed that cFLIP, especially cFLIP(L), plays an anti-apoptotic role in the granulosa cells of healthy follicles of pig ovaries, and that cFLIP could be a major survival factor that determines whether growth or atresia occurs in porcine follicles.

Changes in Expression and Localization of Connexin 43 mRNA and Protein in Porcine Ovary Granulosa Cells during Follicular Atresia

Gap junctions contain channels that connect neighboring cells by allowing the movement of molecules smaller than 1,200 Da. They are formed by connexins and may play a crucial role in the regulation of apoptotic cell death. To determine the role of connexin 43 (Cx43), which is dominantly expressed in granulosa cells, in the regulation of granulosa cell apoptosis during follicular atresia, we examined the changes in the expression and localization of Cx43 mRNA and protein in granulosa cells during atresia using the quantitative real-time revese transcription-polymerase chain reaction, in situ hybridization, Western blot, and immunohistochemistry. Stages of follicular atresia were assessed based on histochemical terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling (TUNEL) and/or the ratio of progesterone and 17beta-estradiol levels in follicular fluid measured by radioimmunoassay. Cx43 mRNA was detected in granulosa cells of secondary follicles and of healthy, early and progressed atretic tertiary follicles, but not in those of primordial or primary follicles. Both phosphorylated/activated and non-phosphorylated/native Cx43 proteins were detected in granulosa cells of secondary and tertiary follicles, but not in those of primordial or primary follicles. Moreover, in tertiary follicles, these Cx43 proteins were expressed most strongly in granulosa cells of healthy follicles, but only trace levels were noted in cells of early atretic and progressed atretic follicles, an indication that the expression levels of Cx43 protein decrease during follicular atresia. These findings indicate that Cx43 is involved in the apoptosis of granulosa cells during atresia in porcine ovaries.

222THE ROLE OF TUMOR NECROSIS FACTOR-RELATED APOPTOSIS INDUCING LIGAND DURING FOLLICULAR ATRESIA IN PIG OVARIES

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce cell death by binding to its receptors (DR4 and DR5). However, binding to DcR1 or DcR2 cannot induce apoptosis. DcRs compete with DRs. TRAIL has been reported to induce apoptosis in various tumor cells but not in normal cells. However, a recent study revealed that TRAIL induces apoptosis in normal hepatocytes of human but not in those of rat, mouse, or rhesus monkey, indicating that there are species-specific differences in TRAIL and receptor systems. In the present study, we demonstrated Immunohistochemical, Western immunoblotting, and reverse transcription-polymerase chain reaction analyses (RT-PCR) of TRAIL and DR4 in granulosa cells during follicular atresia in pig ovaries. For immunohistochemistry, pig ovaries obtained at a local slaughterhouse were fixed with 20% buffered formalin. For Western blotting and RT-PCR analysis, individual preovulatory antral follicles were dissected from the ovaries. Based on morphological and endocrinological criteria, the antral follicles were divided into three categories as follows: healthy, early stage of atresia, progressed stage of atresia. Significant increases were demonstrated in TRAIL protein and mRNA levels during atresia, but not in DR4 protein. Moreover in an in vitro apoptosis-inducing assay using cultured granulosa cells prepared from healthy follicles, we showed that more than 200ng/mL TRAIL could activate caspase-3 and induce apoptotic cell death in a dose-and time-dependent manner, but less than 100ng/mL of TRAIL could not induce apoptosis. When DcR1 was removed from the cell membrane of granulosa cells, a lower dose of TRAIL could induce apoptosis. The present findings suggested that the TRAIL can induce granulosa cell apoptosis, and that DcR1 blocks TRAIL-induced apoptosis in granulosa cells of healthy follicles in porcine ovaries.

Porcine (Sus scrota) Cellular FLICE-like Inhibitory Protein (cFLIP): Molecular Cloning and Comparison with the Human and Murine cFLIP

To reveal the molecular regulation mechanism of selective follicular atresia in porcine ovaries, we isolated the porcine cDNA encoding cellular FLICE-like inhibitory protein (cFLIP), which inhibits death receptor-mediated apoptosis signal transduction. Two alternative splicing isoforms of cFLIP, porcine cellular FLIP-short form (pcFLIPS, 642 bp and 214-aa) and -long form (pcFLIPL, 1446 bp and 482-aa), were identified from a cDNA library prepared from follicular granulosa cells of pig ovaries. pcFLIPS and pcFLIPL indicated high identities with human and murine cFLIP, and both of them contain two tandem specific amino acid regions (death effector domain: DED) in their N-terminal, suggesting that pcFLIPS and pcFLIPL inhibit the death receptor-mediated apoptosis signal by binding to other pro-apoptotic factors mediated by DED. pcFLIPS contains a short C-terminal region, while pcFLIPL has a caspase-like domain in the C-terminal region. The reverse transcription-polymerase chain reaction analysis revealed that both pcFLIPS and pcFLIPL mRNAs were highly expressed in granulosa cells of healthy follicles, suggesting that these cFLIPs play important roles in the regulation mechanism of apoptosis in ovarian follicular granulosa cells. The present data will contribute to understanding of the physiological roles of cFLIPs in the apoptosis regulation in porcine tissues.

Expression and activity of Apaf1 and caspase-9 in granulosa cells during follicular atresia in pig ovaries.

Apoptosis in granulosa cells plays a crucial role in ovarian follicular atresia, but the intracellular regulating mechanism, especially the mitochondrion-dependent apoptosis signalling pathway, is still largely unknown. This study examined whether the mitochondrial pathway is associated with granulosa cell apoptosis during atresia in pig ovaries. Both mRNAs of caspase-9 and apoptotic protease-activating factor 1 (Apaf1), which are major signal transducing components in the mitochondrial pathway, were detected in granulosa cells in healthy, early atretic and progressed atretic follicles by RT-PCR. No changes in the expression of Apaf1 mRNA were seen during follicular atresia, but the expression of caspase-9 mRNA increased during atresia. Apaf1 protein was steadily detected in granulosa cells prepared from healthy, early atretic and progressed atretic follicles by western blot analysis, but high expression of the precursor of caspase-9 (procaspase-9) was detected only in granulosa cells of healthy follicles. Decreased procaspase-9 protein was demonstrated during follicular atresia. Proteolytic activity of caspase-9 increased during atresia, in agreement with the diminution of procaspase-9 protein. Intensive expression of caspase-9 mRNA was demonstrated in the granulosa cells of early atretic and progressed atretic follicles but not in those of healthy follicles. These results indicate that the mitochondrial signalling pathway, which is mediated by Apaf1 and caspase-9, plays a crucial role in determining the fate of granulosa cells during atresia in pig ovaries.

Expression and activity of Apaf1 and caspase-9 in granulosa cells during follicular atresia in pig ovaries

Apoptosis in granulosa cells plays a crucial role in ovarian follicular atresia, but the intracellular regulating mechanism, especially the mitochondrion-dependent apoptosis signalling pathway, is still largely unknown. This study examined whether the mitochondrial pathway is associated with granulosa cell apoptosis during atresia in pig ovaries. Both mRNAs of caspase-9 and apoptotic protease-activating factor 1 (Apaf1), which are major signal transducing components in the mitochondrial pathway, were detected in granulosa cells in healthy, early atretic and progressed atretic follicles by RT-PCR. No changes in the expression of Apaf1 mRNA were seen during follicular atresia, but the expression of caspase-9 mRNA increased during atresia. Apaf1 protein was steadily detected in granulosa cells prepared from healthy, early atretic and progressed atretic follicles by western blot analysis, but high expression of the precursor of caspase-9 (procaspase-9) was detected only in granulosa cells of healthy follicles. Decreased procaspase-9 protein was demonstrated during follicular atresia. Proteolytic activity of caspase-9 increased during atresia, in agreement with the diminution of procaspase-9 protein. Intensive expression of caspase-9 mRNA was demonstrated in the granulosa cells of early atretic and progressed atretic follicles but not in those of healthy follicles. These results indicate that the mitochondrial signalling pathway, which is mediated by Apaf1 and caspase-9, plays a crucial role in determining the fate of granulosa cells during atresia in pig ovaries.

Ovarian expression of messenger RNA encoding the receptors for luteinizing hormone and follicle-stimulating hormone in a marsupial, the brushtail possum (Trichosurus vulpecula).

Both LH and FSH play a central role in controlling ovarian function in mammals. However, little is known about the type of ovarian cells that are responsive to LH and FSH in marsupials. We determined, using in situ hybridization, the localization of mRNA encoding the receptors (R) for LH and FSH in ovaries of brushtail possums. The mRNA encoding FSH-R was observed in granulosa cells of healthy follicles containing at least two complete layers of cells. The mRNA encoding LH-R was first observed in granulosa cells at the time of antrum formation. Cells of the theca interna expressed LH-R mRNA but not FSH-R mRNA. Neither FSH-R nor LH-R mRNA was detected in atretic follicles. Both FSH-R and LH-R mRNAs were observed in luteal tissue, but only LH-R mRNA was observed in interstitial cells. Granulosa cells from follicles of various sizes (0.5 to >2 mm in diameter) responded to LH and FSH treatment with an increase in cAMP synthesis. In contrast, luteal tissue did not respond to either FSH or LH treatment. In conclusion, expression of FSH-R in the brushtail possum ovary was similar to that observed in many eutherian mammals. However, active LH-R was expressed in granulosa cells much earlier in follicular development than has been previously observed. In addition, although mRNAs for both FSH-R and LH-R were observed, neither FSH nor LH treatment stimulated cAMP synthesis in luteal tissue.

Expression and localization of RAP250 mRNA in rat ovary: possible implications in follicular development and ovulation.

The expression levels of nuclear receptor coregulators in specific tissue compartments and cells are thought to influence the expression of hormone-responsive genes involved in metabolism, development, and reproduction. RAP250 is a novel nuclear receptor coactivator highly expressed in brain and reproductive organs. To investigate the possible involvement of RAP250 in tissue-specific regulation of ovarian function, untreated immature, pregnant mare's serum gonadotropin luteinizing hormone (PMSG-LH)-primed, cycling, and pregnant rat models were used to study the localization and expression of RAP250 mRNA in rat ovary by in situ hybridization (ISH) and reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that RAP250 mRNA was primarily localized to granulosa cells of healthy follicles in immature, cycling, and pregnant rats and increased during PMSG-induced follicular development. In the preovulatory and ovulatory follicles from the LH-primed rats of 48-h post-PMSG administration, the signals for RAP250 mRNA increased further and remained high until early luteal formation. Only a subset of corpora lutea during diestrus 1, diestrus 2, and initiation of pregnancy was weakly positive, and atretic follicles were largely negative. The RT-PCR results confirmed the presence of RAP250 mRNA in the rat ovary and strengthen the data from ISH. These findings suggest that RAP250 may play potential roles in follicular development and ovulation.

Expression of the bovine oestrogen receptor-β (bERβ) messenger ribonucleic acid (mRNA) during the first ovarian follicular wave and lack of change in the expression of bERβ mRNA of second wave follicles after LH infusion into cows

In a previous study, the ERβ cDNA protein-coding region was utilised to clone bovine ERβ. The objectives in this study were to examine (1) ERβ mRNA expression in ovarian follicles throughout the bovine first follicular wave, and (2) effect of LH infusion into cows on bERβ mRNA expression during the second follicular wave. In experiment 1, heifers (4–5 per time point) were ovariectomized at 12, 24, 36, 48, 60, 72, 84, 96, 144, or 216 h after emergence of the first follicular wave after oestrus. In experiment 2, saline or LH was pulsed hourly (computer-controlled syringe pump) into cows ( n=31; 5–6 per treatment) at wave emergence for 2 or 4 days: wave 1-saline (W1S), wave 2-saline (W2S), or wave 2-LH (25 μg/h; W2LH). Ovaries were removed on day 2 or day 4 after wave emergence. Follicles, 2–19 mm in size, were dissected, frozen, and stored at −80°C for in situ hybridisation with two bERβ cRNA probes. Expression of bERβ mRNA was localised in granulosa cells of healthy follicles. In experiment 1, bERβ mRNA expression did not change with time points of the wave showing no association of bERβ mRNA expression with follicular selection and dominance. However, bERβ mRNA expression decreased with increase in size of all follicles. Expression of bERβ mRNA was greater in very small follicles (2–4 mm) than in large (≥9 mm) follicles. In experiment 2, expression of bERβ mRNA in follicles did not differ either between W1S and W2S or between W2S and W2LH. In summary, bERβ mRNA expression decreased with increasing follicular size. However, neither stage of the wave (selection or dominance), nor pulsatile infusion of LH influenced bERβ mRNA expression.

Clusterin Protects Granulosa Cells from Apoptotic Cell Death during Follicular Atresia

Clusterin expression is associated with programmed cell death (apoptosis) in many cell types but its exact role has not yet been defined. This study was carried out to determine the cellular localization of clusterin in the ovary and its functional role in the apoptotic cell death of ovarian follicles. A homogenous population of healthy and atretic follicles was obtained by treating immature rats with pregnant mare serum gonadotropin (PMSG). Apoptotic cell death was evaluated by TUNEL. Clusterin expression in the healthy and atretic follicles was examined by immunohistochemical and Western blot analyses, and gene expression was examined by Northen blot analysis. Clusterin protein and its mRNA are only expressed in granulosa cells of atretic follicles obtained from PMSG-treated rats on day 5 of the treatment. Healthy follicles from PMSG-treated rats on day 2 of the treatment do not express clusterin. Theca and stroma cells of both healthy and atretic follicles showed no signs of apoptosis and did not express clusterin. Withdrawal of trophic support from granulosa cells in cultures to induce apoptosis resulted in a dramatic increase in the levels of clusterin and its mRNA compared to cells cultured in serum-supplemented medium. In an attempt to establish the functional role of clusterin in the apoptotic cell death of ovarian follicles, the biosynthesis of clusterin in granulosa cells of healthy follicles was blocked by treatment of cells with antisense oligonucleotide to its cDNA. Treatment of granulosa cells with the antisense oligonucleotide resulted in an increase in the apoptotic cell death compared to the control. These findings indicate that depletion of clusterin can lead to the programmed cell death in ovary, suggesting a functional role for this protein in follicular atresia.

Differential distribution of oestrogen receptor-alpha and -beta mRNAs in the female reproductive organ of rats as revealed by in situ hybridization.

The cellular distribution of two oestrogen receptor (ER) subtypes, ERalpha and ERbeta mRNAs, was studied in the entire female reproductive organ of the rat using in situ hybridization. Expression of ERalpha and ERbeta mRNAs was predominant in the reproductive tract and ovary respectively. ERalpha mRNA had the most pronounced expression in epithelial cells and subepithelial stromal cells from the oviduct to the vagina, while in the ovary it was moderately detected in only the theca folliculi and interstitial glands. The oviduct showed a region-dependent expression of ERalpha mRNA: the isthmus had the most intense signals while the infundibulum revealed a low intensity of expression. Signals for ERbeta mRNA in the ovary were most intense in the granulosa cells of healthy follicles, whereas degenerating follicles lacked any significant expression. Less intense signals for ERbeta mRNA were localized in the theca folliculi and corpus luteum. Detectable levels of ERbeta mRNA were observed in the subepithelial stromal cells from the oviduct to the vagina. This study shows that the two ER subtypes are differentially expressed in cells and compartments of the reproductive organ, suggesting that the mediation of oestrogen action in these tissues may be accomplished through the respective predominant receptor.