Lipoprotein receptor expression during luteinization of the ovarian follicle

Ovarian follicles luteinize after ovulation, requiring structural and molecular remodeling along with exponential increases in steroidogenesis. Cholesterol substrates for luteal steroidogenesis are imported via scavenger receptor-BI (SR-BI) and the low-density lipoprotein (LDL) receptor from circulating high-density lipoproteins and LDL. SR-BI mRNA is expressed in pig ovaries at all stages of folliculogenesis and in the corpus luteum (CL). An 82-kDa form of SR-BI predominates throughout, is weakly present in granulosa cells, and is robustly expressed in the CL, along with the less abundant 57-kDa form. Digestion of N-linked carbohydrates substantially reduced the SR-BI mass in luteal cells, indicating that differences between forms is attributable to glycosylation. Immunohistochemistry revealed SR-BI to be concentrated in the cytoplasm of follicular granulosa cells, although found mostly at the periphery of luteal cells. To examine receptor dynamics during gonadotropin-induced luteinization, pigs were treated with an ovulatory stimulus, and ovaries were collected at intervals to ovulation. SR-BI in granulosa cell cytoplasm increased through the periovulatory period, with migration to the cell periphery as the CL matured. In vitro culture of follicles with human chorionic gonadotropin induced time-dependent upregulation of 82-kDa SR-BI in granulosa cells. SR-BI and LDL receptor were reciprocally expressed, with the latter highest in follicular granulosa cells, declining precipitously with CL formation. We conclude that luteinization causes upregulation of SR-BI expression, its posttranslational maturation by glycosylation, and insertion into luteal cell membranes. Expression of the LDL receptor is extinguished during luteinization, indicating dynamic regulation of cholesterol importation to maintain elevated steroid output by the CL.     

Prolactin-binding activity of bovine granulosa cells on luteinization and exposure to somatotropin in vitro

The influence of luteinization and bovine somatotropin (ST, 5-50 ng/ml) during cultivation of bovine granulosa cells on their ability to bind [125I]-labeled bovine prolactin (PRL) was studied. On the second day of cultivation in serumfree medium, granulosa cells from immature antral follicles underwent spontaneous luteinization, in both the absence and presence of ST. The level of [125I]-PRL specific binding to cells increased after two days of cultivation, with a negative correlation being revealed between estradiol production by the cells and their PRL-binding activity. At the same time, the addition of ST to the culture medium had no effect on the level of [125I]-PRL specific binding to native and luteinizing granulosa cells. The findings suggest a stimulatory influence of the luteal differentiation process on the PRL-binding activity of bovine granulosa cells, this influence is independent of the action of ST.     

Expression of basigin, an inducer of matrix metalloproteinases, in the rat ovary

The extensive tissue remodeling that occurs during follicular development, ovulatory rupture, and the formation and regression of the corpus luteum (CL) requires local degradation of the extracellular environment by matrix metalloproteinases (MMPs). This report characterizes the expression pattern of basigin (Bsg), a putative regulator of MMP induction, in the rat ovary. An induced superovulation model (eCG/hCG) was used in immature rats to evaluate Bsg expression profiles in ovaries collected during the follicular phase, the preovulatory period, and the luteal lifespan. Levels of Bsg mRNA were unchanged through follicular growth (0-48 h post-eCG) and increased during postovulatory luteinization (24 and 48 h post-hCG; P < 0.01). Bsg expression persisted into pseudopregnancy (4-8 days post-hCG) and after functional luteal regression (12 days post-hCG). The profile of Bsg expression during regression of the CL was examined using a model of induced luteolysis. Both functional and structural regression was associated with a decline in Bsg expression levels. Bsg mRNA and protein localized to the theca of preovulatory follicles (12 h post-hCG) and formative and functional CL (24 h-8 days post-hCG). Bsg expression profiles in the induced ovulation and CL regression models were similar to observations made in naturally cycling mature rats. In the cycling ovary, Bsg signaling localized to newly forming CL, the theca of preovulatory follicles, and appeared to be lower in CL from previous estrous cycles. A putative regulatory mechanism of Bsg expression was identified using an in vitro model; treatment of cultured granulosa cells with hCG significantly augmented Bsg mRNA expression levels. The processes of ovulation and luteogenesis may be facilitated by Bsg expression and its induction or regulation of the MMPs.     

Involvement of insulin and growth hormone (GH) during follicular development in the bovine ovary

Insulin and growth hormone (GH) play critical roles in the process of follicular development and maturation. However, the involvement of insulin receptor (IR) and GH receptor (GHR) during follicular development is not well understood. The aim of this study was to investigate the expression of IR and GHR mRNAs in the granulosa cells (GCs) and theca tissues (TCs) of the follicle at different developmental stages (preovulatory dominant follicles, POFs; estrogen-active dominant follicles, EADs; estrogen-inactive dominant follicles, EIDs; and small follicles, SFs), and second, to examine the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the expression of IR and GHR genes in cultured bovine GCs. Although the concentration of insulin in follicular fluid (FF) was constant at all developmental stages, the GH concentration in FF was significantly increased in the EAD and POF compared with the EID. IR mRNA in GCs and TCs was significantly increased in the POF compared with other follicles. Regarding GHR expression, significant increases of mRNA expression were observed in GCs of EAD compared to those of SF, EID and POF. GHR mRNA in TCs was significantly decreased in the SF compared with other follicles. In cultured GCs, FSH, but not E2, stimulated the expression of IR and GHR genes. Our results suggest that the increase in the expression of GHR may be a turning point for follicles to enter the ovulatory phase during final follicular development and that the insulin system may support the maturation of preovulatory follicles.     

Mouse oocytes suppress cAMP-induced expression of LH receptor mRNA by granulosa cells in vitro

Mouse oocytes suppress follicle-stimulating hormone (FSH)–induced luteinizing hormone receptor (LHR) messenger ribonucleic acid (mRNA) expression in cultured granulosa cells. The objective of this study was to assess the mechanism by which oocytes suppress FSH-induced LHR expression. The effect of cumulus cell–denuded, germinal-vesicle-stage oocytes, isolated from antral follicles, on FSH-induced cyclic adenosine monophosphate (cAMP) production by cultured granulosa cells was determined by radioimmunoassays. In addition, the effect of oocytes on 8Br-cAMP–induced LHR mRNA steady-state expression by granulosa cells was assessed by RNase protection assays. Oocytes had no detectable effect on FSH-induced cAMP production. However, oocytes dramatically suppressed 8Br-cAMP–induced LHR mRNA steady-state expression by granulosa cells. It was concluded that the mechanism by which oocytes suppress FSH-induced steady-state expression of LHR mRNA is not by inactivating FSH, preventing functional interactions of FSH with its granulosa cell receptors, or by interfering with the signal-transduction mechanisms required for FSH-dependent cAMP production. In addition, since oocytes suppressed the 8Br-cAMP–induced increase in steady-state expression of mRNA for LHR, oocyte-derived factors probably suppress expression by acting downstream of FSH-induced elevation of granulosa cell cAMP. Mol. Reprod. Dev. 49:327–332, 1998. © 1998 Wiley-Liss, Inc.     

Expression of progesterone receptor (PR) A and B isoforms in mouse granulosa cells: stage-dependent PR-mediated regulation of apoptosis and cell proliferation

The intracellular progesterone receptor (PR) in the mammalian ovary is a part of the physiological pathway that facilitates ovulation. Two PR isoforms (A and B) exist, with different molecular and biological functions. Previous studies have revealed that the cellular ratio of the PR isoforms is important for progesterone-responsive tissues and is under developmental control in different species. However, the relative expression of PR isoforms in the ovary is unknown. In this study we have demonstrated first that the expression of both PR isoforms in mouse granulosa cells was rapidly up-regulated by hCG treatment and dramatically down-regulated when the granulosa cells were undergoing luteinization. The relative level of protein expression of the A and B forms was 2:1 and the highest total PR protein expression was found after hCG stimulation. Second, we demonstrated that the expression of PR protein was specific to granulosa cells of periovulatory follicles and was absent in undifferentiated granulosa cells of growing follicles. It was not detected in other cell types (i.e., corpora lutea or any stage of follicles with features of apoptosis). Third, we demonstrated that treatment with the PR antagonist RU 486 in vivo resulted in down-regulation of both isoforms in parallel with increased activation of caspase-3, a decreased level of proliferating cell nuclear antigen, and a reduced rate of ovulation. Fourth, we demonstrated, in vitro, that the PR antagonists RU 486 and Org 31710 increased internucleosomal DNA fragmentation parallel with a decrease in DNA synthesis in granulosa cells, which express PR. These results indicate that PR and its isoforms participate in regulation of ovulation, along with suppression of granulosa cell apoptosis and promotion of cell survival in the mouse ovary.     

Effect of intraovarian factors on porcine follicular cells: cumulus expansion, granulosa and cumulus cell progesterone production

The role of granulosa cell conditioned media (CM) containing luteinization stimulator (LS), and the role of EGF in the cumulus expansion of oocyte-cumulus complexes (OCC) isolated from large antral follicles was investigated. The CM were prepared by incubation of granulosa cells isolated from large antral follicles. After 24h incubation, more than 61 or 64% of OCC expanded to the +3 and +4 stage in the presence of CM (50%) or EGF (10ng/ml), respectively. The stimulatory effect of LS and EGF on the cumulus expansion was accompanied by the enhanced hyaluronic acid synthesis. Complete suppression of cumulus expansion stimulated by LS and EGF was observed in the presence of 10 micromol/l genistein (tyrosine kinase inhibitor), in the presence of 10mmol/l LiCl (the inhibitor of inositol 1,4,5-trisphosphate metabolism), and 100 micromol/l gallopamil, verapamil and norverapamil (calcium channel blockers). Stimulatory effect of EGF on the cumulus expansion of OCC isolated from large follicles was accompanied by the increased cumulus cell progesterone production. However, EGF did not affect the progesterone production by OCC isolated from small follicles. To determine whether EGF could modulate the granulosa cell steroidogenesis also, the effect of EGF on granulosa cells isolated from large (LGC) and small (SGC) follicles was compared. EGF (10ng/ml) failed to affect the progesterone synthesis during 72h culture of SGC but significantly enhanced the LGC progesterone production. Our results indicate that luteinization factor stimulates the cumulus expansion and hyaluronic acid synthesis by the OCC isolated from large antral follicles. The mechanism of LS- and EGF-induced cumulus expansion may involve tyrosine kinase activation and calcium mobilization. In addition, these results indicate the different response of porcine cumulus and granulosa cells originating from small and large follicles on the stimulatory effect of EGF.     

Relationship between progesterone and estradiol contents of follicular fluid and the morphological appearance of granulosa cells of follicles coexisting with corpora lutea in bovine ovaries

Bovine ovaries (n=149) bearing follicles (>5 mm) coexisting with mature corpora lutea (CL;>10 mm) were obtained at a local abattoir without regard for the reproductive status of the donor cows. Most corpora lutea were 21 to 25 mm in diameter, and nearly half of the largest follicles were 11 to 15 mm in diameter. When oocytes were aspirated from follicles 16 to 30 mm in diameter, approximately 60% of them proved to be degenerated. Concentrations of progesterone (P4) and estradiol-17beta (E2) in the follicular fluid of 23 follicles (>10 mm) were determined. Progesterone and estradiol-17beta were found to be the major hormone in 16 (69.6%) and 7 (30.4%) of the follicles, respectively. Light-microscope observations of the granulosa cells of the same 23 follicles showed that 7 were deficient in mural granulosa cells, and that 15 of the remaining 16 follicles were atretic or luteinizing. Ultrastructural observations of granulosa cells revealed many lipid droplets in the cytoplasm of follicles coexisting with mature CL, suggesting the initiation of luteinization. These results show that approximately 70% of the follicles were P4-dominant and that more than 95% of them were morphologically degenerated. Thus it is suggested that morphological signs of atresia precede changes in the concentrations of hormones in the follicular fluid of follicles coexisting with corpora lutea (>10 mm) during the middle of the estrous cycle.     

Possible involvement of transforming growth factor-beta 1 and transforming growth factor-beta receptor type II during luteinization in the marmoset ovary

The expression of transforming growth factor-beta 1 (TGF-beta 1), and transforming growth factor-beta receptor type II (T beta R-II), were evaluated in periovulatory marmoset ovaries. Histochemical methods were used, in particular double-labelling techniques, in order to correlate growth factor/receptor expression with proliferation (Ki 67), apoptosis (TUNEL method) and luteinization (3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)). The latter was used as a luteinization marker. Periovulatory ovaries are especially suited for studying all aspects since they typically consist of small non-luteinized follicles, large luteinizing follicles and corpora lutea accessoria (Clas), which have developed from large luteinizing follicles. TGF-beta 1 and T beta R-II expression was found in luteinizing theca cells of large periovulatory follicles and in all luteal cells of Clas. Non-luteinized theca cells, including those of small follicles were always devoid of any immunostaining. Granulosa cells of small follicles were immunopositive for T beta R-II. Large follicles with granulosa cell immunoreactivity of both antibodies coexisted with non-reactive follicles of comparable size. The highest activity of the luteal marker enzyme 3 beta-HSD was co-localized in the same cells that expressed TGF-beta 1 and T beta R-II. The double-labelling experiments revealed that TGF-beta 1 and T beta R-II expression is not correlated with proliferation or apoptosis of follicular cells. Our results indicate that TGF-beta 1 and T beta R-II participate in differentiation processes, i.e. luteinization, rather than proliferation. In particular, the dynamics of T beta R-II expression appear highly related to the process of luteinization.     

Luteinization of bovine granulosa cells and corpus luteum formation associated with loss of androgen-aromatizing ability.

The relative aromatizing ability of bovine luteinizing granulosa cells and dispersed luteal cells in tissue culture was studied. Luteinization of granulosa cells, as indicated by steadily increasing progesterone production (from 50 to 300 ng/10(5) cells/day over 4--5 days), was accompanied by a dramatic reduction in their capacity to aromatize exogenous androgen; oestradiol-17 beta production falling from 200 to less than 10 ng/10(5) cells/day over 4--5 days. Luteal cells also had only a very limited capacity to aromatize exogenous androgen, maximum oestradiol-17 beta production being less than 600 pg/10(5) cells/day. The loss in aromatizing capacity of granulosa cells during luteinization was also reflected in the relative endogenous steroid content of non-luteinized granulosa cells and luteal tissue, the former containing high levels of oestradiol-17 beta, less than or equal to 28 ng/mg protein, while the latter, although containing substantial amounts of testosterone, less than or equal to 5.7 ng/g tissue, contained very little oestradiol-17 beta, less than or equal to 0.35 ngG TISSUE. These findings suggest that luteinization of bovine granulosa cells and subsequent corpus luteum formation is associated with a loss in androgen aromatase activity.     

Low-density lipoprotein receptor-related protein 8 (LRP8) is upregulated in granulosa cells of bovine dominant follicle: molecular characterization and spatio-temporal expression studies

The low-density lipoprotein (LDL) receptor-related protein 8 (LRP8) is a member of the LDL receptor family that participates in endocytosis and signal transduction. We cloned the full-length bovine LRP8 cDNA in granulosa cells (GC) of the dominant follicle (DF) as well as several LRP8 mRNA splicing variants, including a variant that contains a proline-rich cytoplasmic insert (A759-K817) that is involved in intracellular signaling. Expression of the A759-K817 variant was analyzed in the GC of follicles at different developmental stages: the small follicle (SF; 2-4 mm), the DF at Day 5 (D5) of the estrus cycle, ovulatory follicles (OF) 24 h after hCG injection, and corpora lutea (CL) at D5. RT-PCR analysis showed that expression was predominant in the GC of DF compared to other follicles and CL (P<0.0001), whereas the expression of other related receptors, such as LDLR and VLDLR, did not show differences. Temporal analyses of follicular walls from the OF following hCG treatment revealed a decrease in LRP8 mRNA expression starting 12 h post-hCG treatment (P<0.0001). LRP8 protein was exclusively localized to the GC, with higher levels in the DF than in the SF (P<0.05). RELN mRNA, which encodes an LRP8 ligand, was highly expressed in the theca of the DF as compared to the OF (P<0.004), whereas MAPK8IP1 mRNA, which encodes an LRP8 intracellular interacting partner, is expressed in the GC of the DF. These results demonstrate the differential expression patterns of LRP8, RELN, and MAPK8IP1 mRNAs during final follicular growth and ovulation, and suggest that a RELN/LRP8/MAPK8IP1 paracrine interaction regulates follicular growth.     

Intracellular regulation of estradiol and progesterone production by cultured bovine granulosa cells.

The objective of the present study was to investigate the implication of protein kinase A (PKA), protein kinase C (PKC), and receptor protein tyrosine kinase (R-PTK) pathways in the regulation of estradiol (E2) and progesterone (P4) production by bovine granulosa cells. Cells were harvested from bovine follicles (8-15 mm diameter) and cultured without serum for an initial 3 days (37 degrees C; 5% CO(2) in air; D1-D3). On the fourth day of culture (D4), E2 and P4 production were stimulated with FSH (1-6 ng/ml) or forskolin (FSK) in the presence or absence of intracellular effectors of PKA, PKC, and R-PTK. Culture medium was collected and replaced each day. Stimulation of granulosa cell adenylate cyclase activity with FSK (0.06-3.75 microM) mimicked FSH, inducing a quadratic increase (P < 0.001) of E2 production and a continuous elevation of P4 (P < 0.01). Inhibition of R-PTK activity with genistein (25-50 microM) increased the sensitivity of cells to FSH as demonstrated by a leftward shift in the dose response curve (P < 0.001). Treatment with transforming growth factor-alpha (TGFalpha; 0. 1 ng/ml) abolished the FSH-induced E2 production (P < 0.001) and this effect was not reversed (P < 0.001) by FSK or by genistein. Furthermore, the inhibitory effect of TGFalpha on FSH-induced E2 production was reproduced by phorbol 12-myristate 13-acetate (PMA; 1. 25-2.5 microM), a PKC activator (P < 0.001). Interestingly, genistein inhibited P4 production (P < 0.05). From these results, we conclude that E2 production by bovine granulosa cells is mediated by intracellular factors and can be stimulated downstream from the FSH receptor. The results also suggest that stimulation of R-PTK and/or PKC activities, as probably occurs with TGFalpha, negatively affects the PKA pathway, thus decreasing E2 production. Furthermore, inhibition of R-PTK leads to an increase production of E2 and may limit luteinization of bovine granulosa cells.     

Cyclic guanosine 5'-monophosphate-dependent protein kinase II is induced by luteinizing hormone and progesterone receptor-dependent mechanisms in granulosa cells and cumulus oocyte complexes of ovulating follicles

Cyclic GMP (cGMP)-dependent protein kinase II (Prkg2, cGK II) was identified as a potential target of the progesterone receptor (Nr3c3) in the mouse ovary based on microarray analyses. To document this further, the expression patterns of cGK II and other components of the cGMP signaling pathway were analyzed during follicular development and ovulation using the pregnant mare serum gonadotropin (PMSG)-human chorionic gonadotropin (hCG)-primed immature mice. Levels of cGK II mRNA were low in ovaries of immature mice, increased 4-fold in response to pregnant mare serum gonadotropin and 5-fold more within 12 h after hCG, the time of ovulation. In situ hybridization localized cGK II mRNA to granulosa cells and cumulus oocyte complexes of periovulatory follicles. In progesterone receptor (PR) null mice, cGK II mRNA was reduced significantly at 12 h after hCG in contrast to heterozygous littermates. In primary granulosa cell cultures, cGK II mRNA was induced by phorbol 12-myristate 13-acetate enhanced by adenoviral expression of PR-A and blocked by RU486 and trilostane. PR-A in the absence of phorbol 12-myristate 13-acetate was insufficient to induce cGK II. Expression of cGK I (Prkg1) was restricted to the residual tissue and not regulated by hormones. Guanylate cyclase-A (Npr1; GC-A) mRNA expression increased 6-fold by 4 h after hCG treatment in contrast to pregnant mare serum gonadotropin alone and was localized to granulosa cells of preovulatory follicles. Collectively, these data show for the first time that cGK II (not cGK I) and GC-A are selectively induced in granulosa cells of preovulatory follicles by LH- and PR-dependent mechanisms, thereby providing a pathway for cGMP function during ovulation.     

Induction of alpha-caveolin-1 (alphaCAV1) expression in bovine granulosa cells in response to an ovulatory dose of human chorionic gonadotropin

Caveolins are implicated in endocytosis, cholesterol trafficking and signal transduction. A cDNA fragment corresponding to caveolin-1 (CAV1) was identified in a mRNA profiling expression study in bovine granulosa cells (GC) following human chorionic gonadotropin (hCG)-induced ovulation. Thus, we have characterized CAV1 cDNA and studied its spatio-temporal expression pattern in bovine ovarian follicles. The full-length bovine alphaCAV1 cDNA was cloned and encodes a putative 22 kDa protein. Expression of alphaCAV1 was studied in bovine GC obtained from follicles at different developmental stages: small follicles (SF: 2-4 mm), dominant follicles (DF), ovulatory follicles (OF: 24 hr post-hCG), and corpus luteum (CL). Semiquantitative RT-PCR analysis showed a 6.5-fold increase in alphaCAV1 mRNA in GC of OF versus DF (P < 0.0001), whereas CAV2 mRNA was increased by only twofold (P < 0.0007). Temporal expression of alphaCAV1 mRNA from OF recovered at 0, 6, 12, 18, and 24 hr after hCG injection showed an 8.5-fold increase of alphaCAV1 mRNA after 24 hr compared to 0 hr (P < 0.0018) whereas no significant variation was detected for CAV2. Immunoblot demonstrated an initial increase in alphaCAV1 protein level 12 hr post-hCG, reaching a maximum at 24 hr. Immunohistochemical localization of CAV1 was observed in GC of OF isolated 18 and 24 hr after hCG injection, whereas no signal was detected in GC of DF and SF. The induction of alphaCAV1 in GC of OF suggests that alphaCAV1 likely contributes to control the increase in membrane signaling that occurs at the time of ovulation and luteinization.     

Localization of relaxin during formation of the porcine corpus luteum

The presence and localization of relaxin (RLX) in luteal tissue during the estrous cycle of the pig have been studied using the avidin-biotin immunoperoxidase method and homologous antisera to purified RLX. Prepubertal gilts were induced to ovulate by treatment with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Ovaries were obtained at laparotomy during the periovulatory period and at specified times through Day 19 post-ovulation. Emphasis was placed on obtaining ovarian tissue at 12- and 24-h intervals up to 96 h after ovulation. RLX immunostaining was evident in theca interna (TI) cells before and at 6 h after ovulation. At 18 h after ovulation, RLX immunostaining comparable to that seen in TI cells was observed for the first time in luteinizing granulosa (G) cells. As luteinization progressed, it became difficult to identify the origin of the RLX immunostaining cells. However, the intensity of RLX immunostaining increased with corpus luteum (CL) development, with the staining becoming localized in the large luteal cells. By Day 19 after ovulation, RLX immunostaining was undetectable. These results indicate RLX is present in the CL during its formation and functional lifespan. Also, it would appear that the presence of RLX in G cells post-ovulation is associated with cell luteinization.     

Steroid production and hCG binding by ram-induced ovarian follicles in seasonally anoestrous ewes

The introduction of rams to a group of previously isolated anoestrous ewes has been shown to stimulate ovarian follicular development and ovulation. The present experiment was carried out to determine the ability of follicles arising from this ram stimulus to produce steroids and bind hCG. Seasonally anoestrous Southdown ewes were exposed to rams for 24 h, 40 h, 3 days, 10 days or 20 days before ovariectomy. Steroid production and the concentration of hCG binding sites in follicles dissected from the ovaries were measured in vitro. The presence of a ram caused ovulation and enhanced oestradiol production by follicles, but had little effect on total androgen production or the number of hCG binding sites present in the follicles when compared to follicles from anoestrous ewes. The oestradiol concentrations in large follicles were not as high as in preovulatory follicles from cyclic ewes reported in other studies. Follicles continued to develop through the ram contact period and when incubated after 40 h and 10 days of ram contact produced high levels of progesterone, indicating partial luteinization, although the corpora lutea (CL) resulting from the induced ovulations regressed prematurely. We suggest that the lack of hCG binding sites in ram-induced follicles may be the cause of poor luteinization and suboptimal development of luteal tissue after induced ovulation in ewes during seasonal anoestrus.     

Analysis of gene expression in granulosa cells of ovine antral growing follicles using suppressive subtractive hybridization

Follicular growth, development and ovulation are highly ordered processes that involve the expression of many genes under precise temporal and spatial regulation. However, information on stage-specific gene expression during the antral follicle phase in sheep is not well understood. In the present study, suppressive subtractive hybridization (SSH) was performed to screen genes that were differentially expressed in the granulosa cells between large follicles (LF, >5 mm) and small follicles (SF, 3–5 mm), and subtractive cDNA library was constructed. Furthermore, with dot-blot analysis, a total of 90 clones randomly selected from the library were proven to be differentially expressed in the granulosa cells. Among these, 38 exhibited high homology to known genes, 14 sequences were corresponding to novel expressed sequence tags (ESTs). Four ESTs, LAPTM4A, SERPINE2, GSTA1, and INHBA, were further examined the reproducibility of the SSH data by the real-time quantitative PCR. Results confirmed an increase expression of respective mRNA in granulosa cells of large follicles compared with that of small follicles. It is concluded that we have identified several genes (known or unknown) that may effect follicular growth, dominance or ovulation in ewes.