Insulin-like growth factor binding protein-3: its biological effect on bovine granulosa cells

This study was aimed at testing the hypothesis that insulin-like growth factor binding protein (IGFBP)-3 can modulate hormone-dependent differentiation of granulosa cells in vitro. Granulosa cells from small (1 to 5 mm) follicles were collected from cattle, cultured for 2 d in medium containing 10% fetal calf serum, washed, and then treated for an additional 2 d in serum-free medium with follicle-stimulating hormone (FSH) (50 ng/ml), recombinant human IGF-I (0, 1.3, 4.0, or 13.3 nM), or recombinant human IGFBP-3 (0 to 4.26 nM). In one series of experiments, IGFBP-3 (0.53 and 2.13 nM) inhibited (51% to 92% decreases; P < 0.05) progesterone and estradiol production induced by 1.3 nM of IGF-I, but did not influence (P > 0.10) granulosa cell numbers or steroidogenesis in the absence of IGF-I. Only 4.26 nM of IGFBP-3 inhibited (by 35%) the increase in granulosa cell numbers induced by 1.3 nM of IGF-I. In another series of experiments, 13.3 nM of IGF-I, but not 4.0 nM of IGF-I, was able to completely overcome the inhibitory effect of 4.26 nM of IGFBP-3 on estradiol production. The increase in cell numbers induced by 4.0 and 13.3 nM of IGF-I was attenuated (P < 0.001) by 4.26 nM of IGFBP-3. In a third series of experiments, IGFBP-3 inhibited 125I-IGF-I binding to granulosa cells. These results indicate that IGFBP-3 has a pronounced inhibitory effect on IGF-I action in cultured bovine granulosa cells, and that this inhibitory effect is likely attributable to IGFBP-3 binding/sequestering IGF-I. Thus, IGFBP-3 may play a significant role in regulating granulosa cell proliferation and steroidogenesis during follicular development in cattle.     

Seasonal differences in progesterone production by luteinized bovine thecal and granulosa cells

This study examined seasonal differences in progesterone (P4) production by granulosa cells (GC) and thecal cells (TC) that were luteinized in vitro during the winter or the summer; it also compared plasma P4 concentrations of lactating dairy cows in the two seasons. First-wave dominant follicles obtained from Holstein cows were dissected on day 6 of the cycle, GC and TC were separated, enzymatically dispersed, and cultured for 9 days in media containing 1% fetal calf serum, forskolin (10 micromol/mL) and insulin (2 microg/mL), to induce cell luteinization. All experimental procedures were identical and characteristics of the follicles were similar in the two seasons. During 9 days of culture, P4 production by luteinized GC was higher in winter than in summer, but the difference only tended to be significant. In contrast, luteinized TC produced three times as much P4 in winter as in summer (324 versus 100 ng/10(5)cells). In the in vivo experiment, P4 concentrations in plasma collected during entire estrous cycles in winter and summer were compared. The cows were, on average, at 70 days postpartum and yielded similar amounts of milk. Concentrations of progesterone in plasma were significantly higher in winter than in summer; during the mid-luteal phase the difference between the two seasons was 1.5 ng/mL. These results indicate that chronic effects of heat-stress are possibly carried over from an impaired follicle to an impaired corpus luteum (CL), and that luteinized TC are more susceptible to heat-stress than luteinized GC.     

Pregnancy-associated plasma protein-A and insulin-like growth factor binding protein mRNAs in granulosa cells of dominant and subordinate follicles of preovulatory cattle

To determine if (1) levels of pregnancy-associated plasma protein-A (PAPP-A) mRNA and insulin-like growth factor binding protein (IGFBP) (-2, -3, -4 and -5) mRNAs differ between the dominant and subordinate follicles during the follicular phase of an estrous cycle, and (2) these differences are associated with differences in follicular fluid (FFL) concentrations of steroids (estradiol, androstenedione, and progesterone), total and free IGF-I, or IGFBPs, estrous cycles of non-lactating Holstein dairy cows (n = 16) were synchronized with two injections of prostaglandin (PGF2 alpha) 11 days apart. Granulosa cells and FFL were collected either 24 h or 48 h after the second injection of PGF2 alpha. FFL from dominant follicles had lower concentrations of progesterone (P < 0.08) and higher concentrations of estradiol (P < 0.05), androstenedione (P < 0.0001), estradiol:progesterone ratio (P < 0.0001), free IGF-I (P < 0.0001), and calculated percentage free IGF-I (P < 0.01) than large subordinate follicles. Levels of IGFBP-2, -4, and -5 in FFL were 3.0- (P < 0.05), 2.4- (P < 0.06), and 3.4-fold (P < 0.05) greater, respectively, in subordinate than in dominant follicles. IGFBP-3, IGFBP-4 and PAPP-A mRNA expression and IGF-II concentration did not differ (P > 0.10) between dominant or subordinate follicles. Levels of IGFBP-2 and -5 mRNA were severalfold greater (P < 0.05) in subordinate than dominant follicles. IGFBP-5 mRNA in granulosa cells decreased (P < 0.05) 62% to 92%, between 24h and 48 h post-PGF2 alpha. We conclude that decreased levels of IGFBP-2 and -5 mRNA in granulosa cells may contribute to the decrease in FFL IGFBP-2 and -5 protein levels of preovulatory dominant follicles, and that changes in granulosa cell IGFBP-3 and -4 mRNA and PAPP-A mRNA levels do not occur during final preovulatory follicular development in cattle.     

Histological and immunohistochemical evaluation of granulosa cells during different stages of folliculogenesis in bovine ovaries

Bovine granulosa cells (GC) vary in their morphological aspect during different stages of folliculogenesis. In this study, 10 morphologically normal bovine ovaries were collected to study the structural aspects of different stages of GC using intermediate filament protein antibodies including cytokeratin AE1/AE3 (AE1/AE3), vimentin, nectin‐4 and desmin. Hormonal immunolocalization was assessed using the immunomarkers anti‐Müllerian hormone (AMH) and inhibin alpha. In addition, tumour markers and proliferation markers using c‐erbB‐2 oncoprotein and proliferating cell nuclear antigen, respectively, were investigated. The immunolabelling of AE1/AE3 in GC was strongest in the early follicle stage and gradually decreased when reaching the Graafian follicle stage. Its immunolabelling increased again as the stage progressed from stage I to stage III. The immunolabelling of inhibin alpha was inversely proportional to that of AE1/AE3 in the developing ovarian follicles as their immunolabelling is opposite to each other during folliculogenesis. AMH was immunopositive in almost all GC stages in different intensities and percentages, except for some negative staining in the atretic IV follicles. The atretic IV follicle is a unique type of atretic follicle that shows Call‐Exner body formation, which was mainly found in older cows in this study. The distinct patterns of immunoreactivity for various types of immunomarkers in the different GC stages will play an important role in diagnostic assistance of various follicle conditions, including cystic ovaries and GC tumours.     

Insulin-like growth factor I in sera, ovarian follicles and follicular fluid of cows with spontaneous or induced cystic ovarian disease

The objective of this research was to determine changes in IGF-I levels in serum and follicular fluid, and immunoreactivity of the follicle wall of cows with spontaneous (slaughter specimens) or ACTH-induced follicular cysts, and to compare results to normal cycling (control) cows after selection of the ovulatory follicle. Concentrations of IGF-I in serum did not differ between control and cystic animals (p=0.76). Fluid from the ovulatory follicle in control cows had 41% higher concentrations of IGF-I than that from cystic follicles collected at slaughter (spontaneous cysts; p<0.05) and 70% higher than that in induced follicular cysts (p<0.05). An intense positive immunostaining with anti-IGF-I was observed in granulosa cells (p<0.05) and in the theca interna (p<0.05) of secondary and tertiary follicles in all three groups of animals, but staining was less intense in cystic (p<0.05) and atretic follicles (p<0.05). This study provides evidence to suggest that cystic ovarian disease in cattle is associated with decreased concentrations of IGF-I in follicular fluid, but not in serum, and decreased production of IGF-I in the follicular wall. These data support the notion that IGF-I plays a role in the regulation of folliculogenesis, and may participate in the pathogenesis of cystic ovarian disease in cattle.     

Large variation in steroid concentrations and insulin-like growth factor binding proteins exists among individual small antral follicles collected from within cows at random stages of the estrous cycle

Variation in the biochemical status of individual small (< or = 5 mm diameter) antral follicles within the ovaries of a cow at any given time likely influences the capacity for undergoing recruitment, selection, and establishing dominance. The objectives of this study were to provide insight into the magnitude of variation in follicular fluid concentrations of steroids and activities of IGFBP that exists among individual small antral follicles within and between cows, and to determine the relationships between follicular fluid IGFBP and steroid concentrations in these follicles. A total of 108 small antral follicles were collected from 6 cows at random stages of the estrous cycle, with 10 to 26 follicles/cow. Concentrations of steroids (ng/mL of follicular fluid) in the overall population of follicles ranged from 0.1 (lowest detectable limit) to 51 for estradiol (E2), 4 to 1,149 for progesterone (P4), and 5 to 504 for androstenedione (A4). Concentrations of E2 and A4 were associated positively (r = 0.2; P < 0.02), but E2 (r = -0.4) and A4 (r = -0.4) were associated negatively, with P4. The proportion of variation in steroid concentrations accounted for by differences among animals (P < 0.05) was small for E2 (12%), moderate for P4 (43%), and greatest for A4 (74%). Least differences between minimum and maximum concentrations of steroids observed in follicles from within a cow were 21-, 5.5-, and 3.5-fold for E2, P4, and A4, respectively, whereas the greatest differences between minimum and maximum concentrations were 505-, 108-, and 26-fold for E2, P4, and A4, respectively. Ranges of IGFBP concentrations (arbitrary densitometer units) detected in fluid from a sub-sample of 43 follicles were 1.18 to 4.50 for IGFBP-3, 0.54 to 4.68 for IGFBP-2, 0.07 to 2.56 for IGFBP-4, and 0.01 to 6.71 for IGFBP-5. Concentrations of E2 were correlated negatively with each IGFBP (r = -0.4 to -0.8; P < 0.05) except IGFBP-3. In contrast, concentrations of A4 were correlated positively with IGFBP-3 (r = 0.4; P < 0.05) but were not correlated with other IGFBP. Concentrations of P4 were correlated positively (r > 0.4; P < 0.05) with IGFBP-4 and -5. The results indicate that steroid concentrations and IGFBP activities vary substantially among small antral follicles collected from within and among individual animals and that increasing production of E2, the hallmark of a developing follicle, was associated with reduced activity of all IGFBP except IGFBP-3, thereby implicating these IGFBP in the regulation of follicular recruitment.     

Influence of polychlorinated biphenyls on the secretion of oxytocin from bovine luteal cells and from granulosa cells obtained from the follicles of different size

Effect of polychlorinated biphenyles (PCBs) on viability and secretory function of luteal and granulosa cells from mature cows was studied. Luteal cells from corpora lutea of different developmental stages and granulosa cells from follicles of >1 cm< in diameter were used. Neither individual congeners (PCB-126, -77, -153) nor mixture of PCBs Aroclor Ar) 1248 at the dose of 1, 10 or 100 ng/ml affected the viability of cells (P>0.05) compared to control after 72 h of incubation. PCBs markedly increased (P<0.05-0.001) oxytocin (OT) secretion from granulosa cells. This effect was the most evident when granulosa cells from follicles <1 cm diameter was treated with PCB-77 which is assumed to stimulate both arylhydrocarbon receptor (AhR) and estradiol (E2) receptor. Even the lowest dose of this compound (1 ng/ml) outranged the effect produced by cortisol (10(-5)M) used as positive control. There was marked effect (P<0.05-0.001) of PCBs on luteal cells from days 6-15 of the estrous cycle. However, influence of PCBs on OT secretion from luteal cells on day 1-5 and 16-18 of the estrous cycle was less evident. Again, PCB-77 was the most efficient stimulator of OT secretion. While the lowest effect was found after treatment of cells with PCB-126 which has dioxin-like properties. It can be assumed that diverse effect of PCBs on female reproduction largely results from the influence of these compounds on ovarian OT secretion. Since both synthesis and secretion of ovarian OT in bovine do not markedly depend on estradiol, some alternative cellular pathways of PCBs on ovary function are suggested.     

Insulin-like growth factor binding proteins and mitogenic activity of partially fractionated sheep amniotic fluid

Amniotic fluid collected from ewes on various days of gestation was examined for the presence of insulin-like growth factor (IGF) binding proteins. IGF-binding proteins with a molecular mass of 40-45 kDa appeared at day 41 of gestation. The level of these major IGF-binding proteins increased during pregnancy and reached a maximum at day 106. Smaller IGF-binding molecules with an approximate molecular mass of 35 kDa and 25 kDa appeared at day 90, also reaching a concentration peak at day 106. The mitogenic activity of sheep amniotic fluid after chromatography on Sephadex G-50 was separated into two peaks. The peak having lower molecular mass corresponded to an elution profile of 125I-IGF-I. The first peak, having higher molecular mass, was eluted immediately after the void volume of column. Electrophoresis and ligand blotting showed that proteins in the first peak had similar properties as IGF-binding proteins.     

TRAIL-decoy receptor 1 plays inhibitory role in apoptosis of granulosa cells from pig ovarian follicles

Previously, we histochemically examined the localization of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its receptors in porcine ovarian follicles, and demonstrated a marked reduction in the expression of TRAIL-decoy receptor-1 (DcRI) in granulosa cells of atretic follicles. In the present study, to confirm the inhibitory activity of DcR1 in granulosa cells, granulosa cells prepared from healthy follicles were treated with phosphatidylinositol-specific phospholipase C (PI-PLC) to cleave glycophospholipid anchor of DcR1 and to remove DcR1 from the cell surface, and then incubated with TRAIL. PI-PLC treatment increased the number of apoptotic cells induced by TRAIL. The present finding indicated the possibility that TRAIL and its receptors were involved in induction of apoptosis in granulosa cells during atresia, and that DcR1 plays an inhibitory role in granulosa cell apoptosis.     

Anovulation in postpartum suckled beef cows. II. Associations among binding of 125I-labeled gonadotropins to granulosa and thecal cells, and concentrations of steroids in serum and various sized ovarian follicles

To determine if specific binding of 125I-labeled gonadotropins to granulosa and thecal cells, or concentrations of steroids in ovarian follicles change during the postpartum anovulatory period, 21 suckled beef cows were slaughtered on d 7, 14, 28, 42 or 56 after parturition (n = 4 to 6 per d). After slaughter, 10 to 15 follicles were dissected from each pair of ovaries and categorized by diameter: small (1.0 to 3.9 mm), medium (4.0 to 7.9 mm) or large (greater than or equal to 8 mm). Progesterone (221 to 612 ng/ml), androstenedione (48 to 94 ng/ml) and estradiol (2.7 to 23.9 ng/ml) did not change (P greater than .10) in fluid of small or medium follicles from d 7 to 42 to 56 after parturition. Similarly, specific binding of human chorionic gonadotropin (125I-hCG) or follicle stimulating hormone (125I-oFSH) to homogenates of small, medium or large follicles did not change (P greater than .05). In contrast, progesterone in fluid of large follicles increased (P less than .05) 3.4-fold between d 7 and 14, but decreased (P less than .05) 55% between d 14 and 28. Concentrations of androstenedione in fluid of large follicles did not change (P greater than .10) from d 7 to 42 to 56. Concentrations of estradiol in fluid of large follicles remained constant between d 7 and 14, but increased (P less than .05) 4.2-fold between d 14 and 28. We conclude that during the postpartum anovulatory period, there is no change in steroidogenic capabilities of small or medium follicles, both of which predominantly produce progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kisspeptin在牛卵巢颗粒细胞的表达及功能研究

为明确kisspeptin在牛卵巢颗粒细胞的表达和功能,本试验首先利用免疫荧光技术研究了kisspeptin在牛卵巢颗粒细胞的表达,随后利用MTT和ELISA技术研究了不同浓度的kisspeptin-10(Kp10)对牛卵巢颗粒细胞增殖和孕酮分泌的影响。结果表明,kisspeptin在牛卵巢颗粒细胞有表达,10、100、1 000nmol/L的Kp10处理24h,均可以极显著提高牛卵巢颗粒细胞活力(P0.01);100nmol/L的Kp10处理24、72h,可极显著提高牛卵巢颗粒细胞的活力(P0.01),但处理48h未能显著提高细胞活力(P0.05)。100nmol/L的Kp10处理24h,可显著提高牛卵巢颗粒细胞孕酮的分泌水平(P0.05),而10、1 000nmol/L的Kp10对孕酮分泌无显著影响(P0.05)。研究结果提示kisspeptin可促进牛卵巢颗粒细胞增殖和孕酮分泌。     

Insulin-like growth factors and IGF-binding proteins in bovine seminal plasma.

Insulin-like growth factor-I (IGF-I) is an important factor for germ cell development and maturation of spermatozoa. Actions of IGFs are modulated by IGF-binding proteins (IGFBPs) that may, depending on their concentration and site of expression, inhibit or enhance effects of IGF-I. We characterized IGFs and IGFBPs in seminal plasma from bulls routinely used for artificial insemination (AI) and from bulls producing poor-quality semen (low mass and individual motility of spermatozoa). IGFs were measured by specific radioimmunoassay in 22 samples of seminal plasma from nine different AI bulls with high (> 76.8%), average (72.8-73.4%), or low (< 69.5%) nonreturn rate (NRR). IGF-I and IGF-II levels were 144 +/- 9 ng/ml (mean +/- SE; range, 79-238 ng/ml) and 144 +/- 10 ng/ml (range, 55-221 ng/ml), respectively, and did not correlate with NRRs. IGF-I concentrations in seminal plasma from bulls producing poor-quality semen (n = 10) were significantly (P < 0.05) greater (194 +/- 26 ng/ml; range, 94-370 ng/ml), whereas IGF-II levels were significantly (P < 0.05) lower (93 +/- 17 ng/ml; range, 38-183 ng/ml) than in AI bulls. Ligand blot analysis of seminal plasma for IGFBPs revealed the presence of a 38-/45-kDa doublet band and a 30-kDa IGFBP. These IGFBPs were identified as IGFBP-3 and IGFBP-5, respectively, by immunoprecipitation using specific antibodies. In addition, a low amount of IGFBP-4 was detected in bovine seminal plasma by immunoprecipitation. There was a marked difference in the activity of IGFBPs between individual bulls, with a relatively small within-bull variance. The differences in IGFBP activities did not correlate with the fertilization capacity of the bulls in vivo or in vitro nor with immunoreactive IGF-I and IGF-II levels in seminal plasma. Our results demonstrate the presence of IGFBPs in bovine seminal plasma. In contrast to human seminal plasma, high activity of IGFBP-3 was detected in seminal plasma of some bulls, suggesting species-specific regulation of IGFBP activity by proteases.     

Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland

We have demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) production by mammary epithelial cells increases dramatically during forced involution of the mammary gland in rats, mice and pigs. We proposed that growth hormone (GH) increases the survival factor IGF-I, whilst prolactin (PRL) enhances the effects of GH by decreasing the concentration of IGFBP-5, which would otherwise inhibit the actions of IGFs. To demonstrate a causal relationship between IGFBP-5 and cell death, we created transgenic mice expressing IGFBP-5, specifically, in the mammary gland. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. The concentrations of the pro-apoptotic molecule caspase-3 was increased in transgenic animals whilst the concentrations of two pro-survival molecules Bcl-2 and Bcl-x were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I, we examined IGF receptor- and Akt-phoshorylation and showed that both were inhibited. These studies also indicated that the effects of IGFBP-5 could be mediated in part by IGF-independent effects involving potential interactions with components of the extracellular matrix involved in tissue remodeling, such as components of the plasminogen system, and the matrix metallo-proteinases (MMPs). Mammary development was normalised in transgenic mice by R3-IGF-I, an analogue of IGF-I which binds weakly to IGFBPs, although milk production was only partially restored. In contrast, treatment with prolactin was able to inhibit early involutionary processes in normal mice but was unable to prevent this in mice over-expressing IGFBP-5, although it was able to inhibit activation of MMPs. Thus, IGFBP-5 can simultaneously inhibit IGF action and activate the plasminogen system thereby coordinating cell death and tissue remodeling processes. The ability to separate these properties, using mutant IGFBPs, is currently under investigation.     

Co-culture model reveals the characteristics of theca cells and the effect of granulosa cells on theca cells at different stages of follicular development

Theca cells (TCs) play an important role in follicular development, which cannot be separated from granulosa cells (GCs). However, compared with mammals, the TCs and the effects of GCs on TCs at different follicular development stages (FDSs) have specific characteristics in avian species, but none of them have been clearly defined. In this study, we established an in vitro co-culture (with GC at the corresponding stage) model of goose TCs at different FDSs (pre-hierarchical, hierarchical and F1) by using a transwell system. The properties of TCs in co-culture at the three FDSs, including cell morphology, activity and intracellular lipid content, as well as the expression of key genes involved in de novo lipogenesis, steroidogenesis, proliferation and apoptosis, were examined and defined. We further compared the mono-culture and co-culture groups. After co-culture, the activity of TCs showed significant (p < .01) increases in all stages; moreover, in pre-hierarchical TCs, the expression levels of FAS, SREBP, 3β-HSD and CCND1 were promoted, and PPARγ, CYP19, BCL2 and CAS3 were inhibited (p < .05); in the hierarchical TCs, the expression levels of PPARγ, FAS, CYP19, CCND1 and BCL2 were promoted, and SREBP, STAR, 3β-HSD and CAS3 were inhibited (p < .05), whereas in the F1 TCs, the expression levels of PPARγ, FAS, 3β-HSD, CYP19 and CCND1 were promoted, and STAR and CAS3 were inhibited (p < .05). These results suggested that GCs at the three FDSs have dynamic and complex influences on the physiological characteristics of TCs, and the influences on TCs at the three FDSs were varied.     

Production of insulin-like growth factor-I by granulosa cells but not thecal cells is hormonally responsive in cattle

To determine whether the hormonal regulation of IGF-I production differs between granulosa and thecal cells in cattle, granulosa and thecal cells from bovine follicles were collected, cultured for 2 d in medium containing 10% fetal calf serum, washed, and then treated for an additional 24 h in serum-free medium with various hormones. In Exp. 1, granulosa cells were treated with 0 or 100 ng/mL of insulin and(or) 50 ng/mL of follicle-stimulating hormone (FSH), insulin plus 10 ng/mL of epidermal growth factor, or insulin plus 10 ng/mL of basic fibroblast growth factor. In Exp. 2, thecal cells were treated as described in Exp. 1 except that 100 ng/mL of luteinizing hormone (LH) was used instead of 50 ng/mL of FSH. In Exp. 3, granulosa and thecal cells were treated with 0 or 30 ng/mL of cortisol with or without 100 ng/mL of insulin, 300 pg/mL of glucagon, or glucagon plus insulin. In Exp. 4, granulosa and thecal cells were treated with 0 or 300 ng/mL of estradiol with or without 100 ng/mL of insulin and(or) 100 ng/mL of LH. At the end of treatment, medium was collected, concentrated with Centricon-3 concentrators, and assayed for IGF-I by radioimmunoassay. Cell numbers were determined by Coulter counting at the end of culture. Thecal cells produced low amounts of IGFI (0.48 +/- 0.04, 0.63 +/- 0.03, and 0.82 +/- 0.03 ng per 100,000 cells per 24 h in Exp. 2, 3, and 4, respectively), and this production was not influenced (P > 0.05) by the various treatments. In contrast, IGF-I production by granulosa cells (2.0 to 6.2 ng per 100,000 cells per 24 h) was influenced by treatment in Exp. 1, 3, and 4 and was greater than IGF-I production by thecal cells (Exp. 2, 3, and 4). Alone, insulin, FSH, LH, and cortisol (but not estradiol) each decreased (P < 0.05) granulosa-cell IGF-I production by 20 to 57%; combined treatments of insulin plus FSH or insulin plus cortisol decreased IGF-I production to levels seen with insulin alone. Glucagon had no effect (P > 0.10) on IGF-I production in the absence or presence of insulin. In the presence of insulin, epidermal growth factor, basic fibroblast growth factor, and estradiol decreased (P < 0.05) IGF-I production below that observed for insulin alone. These results indicate that, during follicular development in cattle, changes in intrafollicular levels of IGF-I may be due to hormonally-induced changes in granulosa-cell, but not thecal-cell, IGF-I production.     

Microvascular distribution and vascular endothelial growth factor expression in bovine cystic follicles

The aim of this study was to examine the distribution of microvessels in the theca and the expression of vascular endothelial growth factor (VEGF) in the theca and granulosa of cystic follicles. Paraffin sections of cystic follicles were stained with Bandeiraea simplicifolia-I (BS-I) to visualize the endothelial cells of microvessels. The other sections were immunostained with anti-VEGF antibody. The mRNA expression of VEGF in the theca interna of cystic and healthy follicle was determined by RT-PCR. In the theca interna, cystic follicles with granulosa cells had significantly greater microvessel number density (the number of microvessels per given field) and area (area occupied by microvessels per given area) than healthy follicles in various sizes (<3, 4–8, >9 mm). Loss of granulosa cells from cystic follicles resulted in a similar number density, but significantly smaller area of microvessels in the theca interna. There was no significant difference in the microvessel number density and area of the theca externa between the types of follicle. VEGF protein was expressed in the granulosa and theca interna of healthy and cystic follicles. These results demonstrate that cystic follicles have a highly developed vasculature network in the theca interna, especially in cystic follicles containing granulosa cells. It is also suggested that VEGF is highly expressed in the cystic follicle as well as healthy follicle, which may be associated with advanced vasculature and the accumulation of follicular fluid in cystic follicles.     

Insulin-like growth factors and binding proteins in ruminants and their nutritional regulation.

Insulin-like growth factors (IGF) are important mediators of growth, lactation, reproduction, and health. Considerable information on their role in ruminant animals has been learned in the past several years, but the precise mechanisms of their action are not known. The exact biological response of target cells is undoubtedly determined by the developmental state of the cell and synergism with other growth factors. Overall, somatomedins and their binding proteins seem to be major links between cellular developmental processes and nutrient supply. The mechanism by which nutrients control biological actions of somatomedins is not known but clearly involves the synthesis of IGF, as well as their binding proteins and receptors. In ruminants, severe feed restriction decreases circulating concentrations of IGF-I, whereas subtle alterations typical of those that occur in production systems have minimal effect. However, the responses of IGF to somatotropin are affected by modest alterations in nutritional status, including differences in nutritional status that are typically encountered in animal production systems.     

Expression of kit ligand and insulin‐like growth factor binding protein 3 during in vivo or in vitro development of ovarian follicles in sheep

Expression of Kit ligand (KL) and insulin‐like growth factor binding protein (IGFBP3) genes was studied at different in vivo and corresponding in vitro stages of development of the ovarian follicles in sheep. The expression of both KL and IGFBP3 was significantly higher in the primordial follicles relative to any other stage of development. Compared to the other stages, the KL expression in the cumulus cells from in vivo grown large antral follicles and that of IGFBP3 in COCs’ isolated from large antral follicles matured in vitro for 24 hr were significantly higher. In the oocytes from in vivo grown ovarian follicles, the expression of KL was the same at all the stages of development. Insulin‐like growth factor binding protein 3 expression was also the same in the oocytes at all the stages of the development except for a significantly lower expression in those from antral follicles. The expression of KL in the cumulus cells decreased significantly in the in vitro grown early antral follicles but did not change further as the development progressed. The expression of IGFBP3 in the cumulus cells from in vitro grown ovarian follicles appeared to increase as the development progressed although the increase was not significant between any two consecutive stages of development. In the oocytes in in vitro grown ovarian follicles, the expression levels of KL and IGFBP3 genes did not change with development. It is concluded that (i) KL and IGFBP3 genes follow specific patterns of expression during ovarian folliculogenesis and (ii) in vitro culture of preantral follicles compromises the development potential through alterations in the stage‐specific patterns of expression of these and other developmentally important genes.     

Proteolysis of insulin-like growth factor binding proteins during preovulatory follicular development in cattle

The present study was conducted to evaluate changes in follicular fluid (FF) insulin-like growth factor binding protein (IGFBP) proteolytic activity and levels of steroids and IGFBP during follicular development in cattle. Estrous cycles of cows were synchronized with two injections of prostaglandin F2alpha (PGF) 11 d apart and follicular growth monitored via daily rectal ultrasonography in order to identify the dominant follicle. All cows were ovariectomized 48 hr after the second injection of PGF. Follicular fluid was collected individually for all follicles > 5 mm and pooled for small (1 to 5 mm) follicles. Follicular fluid estradiol and androstenedione levels were greater (P < 0.05) and progesterone and IGFBP-3 levels not different (P > 0.10) in large dominant than in small (1 to 5 mm) or large (>5 mm) subordinate follicles, whereas IGFBP-2, -4 and -5 levels were less (P < 0.05) in large dominant than in small or large subordinate follicles. To evaluate proteolysis of IGFBPs, FF was incubated with recombinant human (125) I-labeled IGFBP-2, -3, -4, and -5 and proteins separated by 12% SDS-PAGE. Follicular fluid caused little or no proteolysis of (125)I-lableled IGFBP-2 or -3. However, cleavage of (125)I-labeled IGFBP-4 and -5 by FF from large dominant follicles was greater (P < 0.05) than by FF from small or large subordinate follicles indicating that a protease to IGFBP-4 and -5 exists in estrogen dominant follicles. We conclude that lower levels of IGFBP-2 in estrogen dominant follicles of cattle are not due to increased proteolysis, whereas decreases in IGFBP-4 and -5 levels are likely due, in part, to increased protease activity. Changes in IGFBP may alter levels of bioavailable IGFs that stimulate steroidogenesis and mitogenesis in developing bovine follicles.     

玻璃化冷冻预处理方法对牛卵巢颗粒细胞的影响

为了研究不同玻璃化冷冻预处理对牛卵巢颗粒细胞培养的影响,试验采用4种不同的玻璃化冷冻预处理方法(①将冷冻保存管直接投入液氮罐中;②将冷冻保存管先置于4℃冰箱15 min,然后投入液氮罐中;③将冷冻保存管先置于-20℃冰箱10 min,然后投入液氮罐中;④将冷冻保存管先置于4℃冰箱15 min,再置于-20℃冰箱10 min,然后投入液氮罐中)处理牛卵巢颗粒细胞,解冻后检测细胞活力和传代时间。结果表明:解冻后4种方法的细胞活力分别为72%、74%、79%和86%;传代时间分别为76 h、73 h、67 h和62 h;方法④的细胞活力和传代时间与其他组比较差异显著(P<0.05)。说明玻璃化冷冻预处理方法有利于复苏后的牛卵巢颗粒细胞的进一步培养。