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.     

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.     

Types I and IV collagenolytic and plasminogen activator activities in preovulatory ovarian follicles

During ovulation, enzymatic degradation of the extracellular matrix occurs within and around the graafian follicles. In this study, the activities of several different proteolytic enzymes were measured in the culture media of follicles taken from pregnant mare serum gonadotropin (PMSG)-primed immature rats. At 52 h after PMSG, the follicles were cultured for 2 to 15 h in media with or without human chorionic gonadotropin (hCG). Type I collagenase activity in hCG-stimulated follicles gradually increased within 6 h to 3.3-fold above that of the controls. Relatively pure populations of granulosa cells produced type I collagenase to a similar extent. Likewise, type IV collagenase increased 3.8-fold by 6 h after exposure of the follicles to hCG. In contrast, plasminogen activator activity increased by 3.9-fold at 2 h after hCG, but was negligible at 4, 6, and 15 h after incubation. These results suggest that plasminogen activator may activate both type I and type IV collagenase in hCG-stimulated ovulatory follicles.     

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.     

Effect of the preovulatory gonadotropin surge on matrix metalloproteinase (MMP)-14, MMP-2, and tissue inhibitor of metalloproteinases-2 expression within bovine periovulatory follicular and luteal tissue

The matrix metalloproteinases (MMPs) have been implicated in the ovulatory process, but the specific roles of individual MMPs are unclear. This study examined the effect of the preovulatory gonadotropin surge on localization and regulation of MMP-2, MMP-14, and tissue inhibitor of metalloproteinases-2 (TIMP-2) mRNA and MMP-2 and TIMP-2 activity in bovine preovulatory follicles and new corpora lutea (CL). Ovaries containing ovulatory follicles or new CL were collected at approximately 0, 6, 12, 18, 24, and 48 h (CL) after a GnRH-induced gonadotropin surge. Messenger RNA for TIMP-2 and MMP-14 increased within 6 and 24 h of the gonadotropin surge, respectively, whereas MMP-2 mRNA was constitutively expressed. Activity for MMP-2 in follicular fluid and follicle homogenates was not changed, but follicular fluid TIMP-2 activity increased in response to the gonadotropin surge. Messenger RNA for MMP-2 was localized to the thecal layer of bovine preovulatory follicles, whereas MMP-14 mRNA was localized primarily to the thecal layer and adjacent ovarian stroma. Expression of MMP-14 was also observed in the granulosal layer after the gonadotropin surge. In contrast, TIMP-2 mRNA was localized predominantly to the granulosal layer with intense expression in the antral portion of the granulosal layer in response to the gonadotropin surge. These data support the hypothesis that increased expression of MMP-14 and TIMP-2 may help regulate follicle rupture and/or the ovulatory follicle-CL transition in cattle.     

Gonadotropin surge-induced differential upregulation of collagenase-1 (MMP-1) and collagenase-3 (MMP-13) mRNA and protein in bovine preovulatory follicles

The ovulatory process is characterized by focalized extracellular matrix degradation at the apex of preovulatory follicles. Many studies have implicated the matrix metalloproteinases (MMPs) as potential mediators of follicle rupture. Objectives of this study were to determine localization and effect of the gonadotropin surge on temporal expression of MMP-1 and MMP-13 in bovine preovulatory follicles. Samples were collected at 0, 6, 12, 18, 24, and 48 h (corpora lutea) after GnRH injection (n = 5-6 per time point) and amounts of MMP-1 and MMP-13 mRNA and protein determined using dot blot or semiquantitative RT-PCR and Western blot analyses. Samples were also collected at 0 and 20 h after GnRH injection for immunohistochemical localization of MMP-1 and MMP-13. Results indicate that follicular expression of MMP-1 and MMP-13 increased following the gonadotropin surge. Abundance of MMP-1 mRNA increased at 6, 12, and 48 h post-GnRH injection. Immunoreactive MMP-1 was localized to granulosal and thecal layers of preovulatory follicles. Amounts of MMP-1 protein increased in both the apex and the base of preovulatory follicles. Abundance of MMP-13 mRNA increased at 6, 24, and 48 h post GnRH injection. Amounts of MMP-13 protein also increased in the follicular apex and base. Immunoreactive MMP-13 was localized to granulosal and thecal layers of preovulatory follicles. Results indicate MMP-1 and MMP-13 are increased in bovine preovulatory follicles following the gonadotropin surge but do not support a requirement for differential up-regulation of MMP-1 and MMP-13 (follicular apex vs. base) for the preovulatory collagenolysis required for follicle rupture.     

Membrane type 1-matrix metalloproteinase (MMP)-associated MMP-2 activation increases in the rat ovary in response to an ovulatory dose of human chorionic gonadotropin

Gonadotropins stimulate ovarian proteolytic enzyme activity that is believed to be important for the remodeling of the follicular extracellular matrix. Membrane type 1-matrix metalloproteinase (MT1-MMP) has been identified in vitro as an activator of pro-MMP-2 by forming a complex with tissue inhibitors of metalloproteinase-2 (TIMP-2). In the present study, the expression pattern of MT1-MMP mRNA and the role of MT1-MMP were examined in the ovary using the gonadotropin-treated immature rat model. Ovaries were collected at selected times after eCG or hCG. RNase protection assays revealed a transient increase in MT1-MMP mRNA beginning 4 h after hCG. High expression of MT1-MMP mRNA was localized to the theca-interstitial layer of developing and preovulatory follicles, while low expression was observed in the granulosa cell layer of developing follicles by in situ hybridization. The localization pattern of MT1-MMP mRNA was compared with TIMP-2 mRNA. Both MMP-2 and TIMP-2 mRNA were expressed in the theca layer of preovulatory follicles, showing a similarity to MT1-MMP mRNA expression. To further determine whether MT1-MMP activates pro-MMP-2 in the ovary, crude plasma membrane fractions from preovulatory ovaries were analyzed by gelatin zymography. In plasma membrane fractions, pro-MMP-2 increased around the time of ovulation. Upon incubation, pro-MMP-2 was activated with the highest levels of activation at 12 h post-hCG. The addition of MT1-MMP antibody or excess TIMP-2 to membrane fractions inhibited pro-MMP-2 activation. The increase in MT1-MMP mRNA may be an important part of the mechanism necessary for the efficient generation of active MMP-2 during the ovulatory process.     

Group IVA phospholipase A2 regulates testosterone biosynthesis by murine Leydig cells and is required for timely sexual maturation

In the present paper, we report that PLA2G4A (Group IVA phospholipase A2) is important in the development and function of rodent testes. Interstitial cells of rat testes had high PLA2 (phospholipase A2) activity that was very sensitive to the PLA2G4A-preferential inhibitor AACOCF3 (arachidonyl trifluoromethyl ketone). PLA2G4A protein was expressed primarily in the interstitial cells of wild-type mouse testes throughout maturation. Although Pla2g4a knockout (Pla2g4a-/-) male mice are fertile, their sexual maturation was delayed, as indicated by cauda epididymal sperm count and seminal vesicle development. Delayed function of Pla2g4a-/- mice testes was associated with histological abnormalities including disorganized architecture, swollen appearance and fewer interstitial cells. Basal secretion of testosterone was attenuated significantly and steroidogenic response to hCG (human chorionic gonadotropin) treatment was reduced in Pla2g4a-/- mice compared with their Pla2g4a+/+ littermates during the sexual maturation period. Chemical inhibition of PLA2G4A activity by AACOCF3 or pyrrophenone significantly reduced hCG-stimulated testosterone production in cultured rat interstitial cells. AACOCF3 inhibited forskolin- and cAMP analogue-stimulated testosterone production. These results provide the first evidence that PLA2G4A plays a role in male testes physiology and development. These results may have implications for the potential clinical use of PLA2G4A inhibitors.     

Inverse relationship between the expression of messenger ribonucleic acid for peroxisome proliferator-activated receptor gamma and P450 side chain cleavage in the rat ovary

Messenger RNA for peroxisome proliferator-activated receptor gamma (PPARgamma) has been found in granulosa cells, and its expression decreases after the LH surge. We determined which developmental stage of ovarian follicle expresses mRNA for PPARgamma and evaluated the impact of PPARgamma agonists on steroidogenesis. Ovaries were collected from immature eCG/hCG-treated rats at 0 (no eCG), 24, and 48 h post-eCG and 4 and 24 h post-hCG. Ovarian tissue was serially sectioned and processed for in situ hybridization to localize mRNA corresponding to PPARgamma, aromatase, and the LH receptor, and P450 side chain cleavage (P450SCC) and to determine whether apoptotic cells were present. During follicular development, there was no correlation between the expression of mRNAs for PPARgamma and aromatase or the presence of apoptotic cells, but a general inverse correlation was observed between the expression of PPARgamma mRNA and LH receptor mRNA. At 4 h post-hCG, follicles expressing P450SCC mRNA had lost expression of PPARgamma mRNA. This inverse pattern of expression between PPARgamma and P450SCC mRNAs was also observed 24 h post-hCG, with developing luteal tissue expressing high levels of P450SCC mRNA but little or no PPARgamma mRNA. To determine the impact of PPARgamma on steroidogenesis, granulosa cells were collected from ovaries 24 h post-eCG and cultured alone, with FSH alone, or with FSH in combination with the PPARgamma agonists ciglitazone or 15-deoxy-delta 12,14-prostaglandin J2 (PGJ2). Treatment of granulosa cells with PGJ2 stimulated basal progesterone secretion, whereas ciglitazone or PGJ2 had no significant effect on FSH-stimulated steroid production. These findings suggest that 1) PPARgamma may regulate genes involved with follicular differentiation and 2) the decline in PPARgamma in response to LH is important for ovulation and/or luteinization.     

Concentrations of nitric oxide in equine preovulatory follicles before and after administration of human chorionic gonadotropin

In the present study, follicular fluids of estrous mares treated with saline solution (Control) or nitric oxide synthase (NOS) inhibitors were analyzed for nitric oxide (NO), estradiol-17beta (E2) and progesterone (P4) concentrations before and 36h after administration of human chorionic gonadotropin (hCG). Follicular fluids obtained before (0h) hCG administration from control mares had lower concentrations of NO than those obtained 36h after administration of hCG (58.3+/-17.8 micromol versus 340.4+/-57.7 micromol; P<0.05). A similar pattern was also noted for intrafollicular P4 in control mares, which had lower concentrations of intrafollicular P4 before hCG than 36h post-hCG administration (P<0.05). As expected, E2 concentrations of control follicles sampled before hCG administration were higher than those sampled 36h post-hCG administration (P<0.05). However, the E2 concentrations in follicles of mares treated with the NOS inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine (AG) did not decrease after hCG administration, unlike those in control mares (P>0.10). In addition, mares treated with NOS inhibitors had lower intrafollicular concentrations of NO and P4 than control mares, both before and after hCG administration (P<0.05). Increased intrafollicular concentrations of NO in control, hCG-stimulated mares provide evidence for the presence of an NO-generating system in the equine preovulatory follicle that is likely upregulated following administration of hCG.     

Involvement of cytosolic phospholipase A2 in the ovulatory process in gonadotropin-primed immature rats

The preovulatory LH surge induces a remarkable increase in ovarian prostaglandins (PGs) which help to mediate the ovulatory process. We investigated whether cytosolic phospholipase A2 (cPLA2) has a role in this PG production in PMSG/hCG-primed immature rats. The immunoreactive signal for cPLA2 was localized in both thecal and granulosa layers of mature follicles and became evident in response to gonadotropins. The PLA2 activity in the whole ovarian cytosol rose slightly after PMSG stimulation, persisted relatively constant until 24 h after hCG injection and thereafter increased gradually. Intra-ovarian bursal injection of arachidonyl trifluoromethyl ketone, a specific inhibitor for cPLA2 ( 1.0-3.0 mg/ovary), significantly reduced ovarian PGE2 content and the ovulation rate. These results suggest that cPLA2 exists in periovulatory follicles and functions in PG production related to the ovulation process.     

Relationships among concentrations of steroids, inhibin, insulin-like growth factor-1 (IGF-1), and IGF-binding proteins during follicular development in weaned sows.

The experimental objective was to determine how insulin-like growth factor binding proteins (IGFBP), as examined by Western ligand blot procedures, related to porcine follicular steroidogenesis. Weaned sows were ovariectomized at various times after litter removal in three experiments. In experiments 1 and 2, sows were ovariectomized at 48-120 h after weaning. In experiment 1, pools of all small (1-3 mm), medium (greater than 3-6 mm), or large (greater than 6-9 mm) follicles were made for each sow; in experiment 2, fluid was collected individually from the 10 largest follicles per ovary. A third experiment was conducted to examine changes after an ovulatory dose of hCG, but prior to ovulation. In this experiment, sows were treated with eCG at weaning, given hCG 72 h later, and ovariectomized 0-36 h after the ovulatory dose of hCG. Follicular fluid was collected from the 10 largest follicles per sow. In experiments 1 and 2, IGFBP-3 in follicular fluid remained constant over follicle diameters and stage sof development, and IGFBP-2 decreased with advancing follicular development as concentrations of estradiol, androstenedione, and progesterone increased. In experiment 1, after the presumed LH surge when the concentration of all steroids was low, there was a sharp increase in band intensity for IGFBP-2. Similarly, estradiol and androstenedione were low in preovulatory sows in experiment 2, though progesterone increased and IGFBP-2 decreased with follicle diameter. In experiment 3, progesterone remained elevated from 0 to 36 h after hCG, even though IGFBP-2 did not increase until after 24 h post-hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of follicular fluid in the porcine oviduct and its contribution to the acrosome reaction

Two experiments were conducted to measure the quantity of follicular fluid entering the porcine oviduct following ovulation and to establish its influence on the sperm acrosome reaction in vivo. Prepubertal gilts treated with pregnant mare serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG) were used in both experiments. In experiment 1, each of 64 gilts was assigned at random to one of four treatment groups (n = 16 per group): I (preovulatory), surgery 38 hr post-hCG; II (ovulatory), (surgery 42 hr post-hCG; III (postovulatory), surgery 46 hr post-hCG; IV (ovulation blocked), surgery 46 hr post-hCG but also treated with indomethacin (INDO) at 24 hr. At surgery, both follicular and oviductal fluid were collected for determination of volume and progesterone (P4) concentration. In experiment 2, sperm were recovered surgically from the uterine horn, isthmus, and ampulla of gilts at 46 hr post-hCG either 1) inseminated and non-INDO-treated controls (n = 5) or 2) inseminated and INDO-treated at 24 hr (n = 4). Using P4 as a marker, it was calculated that only 0.51% +/- 0.10% of the available follicular fluid was present in the oviduct near the time of ovulation and that this amount had decreased 10-12-fold 4 hr later. Mean sperm concentration at 46 hr post-hCG was higher in the uterine horn than in the other two regions (P less than 0.05) but the percentage of acrosome-reacted sperm was greater in the ampulla (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)