I. Introduction II. Insulin and Insulin Receptor A. Structures of insulin and insulin receptor B. Presence of insulin and insulin receptor in the ovary C. Insulin action and the ovary D. Summary III. IGFs and Their Receptors A. IGF peptides and receptors B. Expression of IGFs and IGF receptors in the ovary C. Role of IGFs in ovulatory function and steroidogenesis D. Summary IV. IGF-Binding Proteins (IGFBPs) and Proteases A. Structural relationships among IGFBPs B. IGFBP expression in the ovary C. IGFBP proteases in the ovary D. IGFBP actions in the ovary E. Role of IGFBPs in follicular development and atresia F. Summary V. Polycystic Ovary Syndrome (PCOS) A. Clinical features B. Theories of pathogenesis C. Insulin resistance in PCOS D. Alterations of IGFs and IGFBPs in PCOS E. Summary VI. The Insulin-Related Ovarian Regulatory System: Implications for Therapy A. Treatment of PCOS B. Therapeutic use of IGF-I and IGF-II C. Use of GH in ovulation induction VII. Summary and Conclusions

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The insulin-related ovarian regulatory system in health and disease.

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Selection of the dominant follicle in cattle

The ovarian insulin and insulin-like growth factor system with an emphasis on domestic animals

Regulation of ovarian activity is an integrated process encompassing both extraovarian signals and intrafollicular factors. Initiation of primordial follicle growth and the early stages of folliculogenesis can occur without gonadotropins, but FSH may affect the rate of preantral follicle growth. Antral follicle development from 1 to 4 mm in sheep and cattle is completely gonadotropin dependent. These recruited follicles express a range of mRNA encoding steroidogenic enzymes, gonadotropin receptors, and local regulatory factors and their receptors. As follicles continue to mature, there is a transfer of dependency from FSH to LH, which may be part of the mechanism involved in selection of follicles for continued growth. Locally produced growth factors, such as the IGF and members of the transforming growth factor-beta superfamily, work in concert with gonadotropins throughout the follicular growth continuum and can have significant effects on follicle selection. Environmental influences, such as changes in nutrition, also have an effect on ovarian activity. This can occur without significant variation in circulating gonadotropin concentrations and can be correlated with changes in circulating concentrations of metabolic hormones, including insulin, IGF-I, GH, and leptin. Nutrition can also affect the expression of mRNA encoding components of the ovarian IGF system to regulate the sensitivity/response of follicles toward gonadotropins. Hence, the roles of growth factors in follicular development and survival depend on gonadotropin status and differentiation state of the follicle. In conclusion, it is the integration of these extraovarian signals and intrafollicular factors that determine whether a follicle will continue to develop or be diverted into atretic pathways.

Control of follicular growth: local interactions and nutritional influences.

The objectives of this study were to develop a serum-free bovine granulosa cell culture system in which FSH-responsive estradiol production could be induced and maintained, and to use this system to evaluate the effects of FSH, insulin, and IGF-I on steroidogenesis and proliferation of bovine granulosa cells from different follicle size categories ( 8-mm diameter). In the presence of FSH, granulosa cells from small follicles differentiated in vitro, and estradiol secretion increased with time (p < 0.01) so that by the end of the culture period it was similar to that of cells from large follicles. Granulosa cells from medium and large follicles secreted estradiol throughout the culture period. Cells cultured in plasma-coated culture wells had an increased proliferative response but had lower estradiol production compared to cells cultured under serum-free conditions (p < 0.01). Insulin promoted proliferation and estradiol production by granulosa cells from the three follicle-size categories (p < 0.01). Physiological concentrations of FSH induced proliferation and estradiol secretion (p < 0.01) by granulosa cells in a dose-responsive manner. The inclusion of IGF-I in the culture system enhanced proliferation and estradiol production (p < 0.01), even in the absence of gonadotropic support, demonstrating the gonadotropic characteristics of this growth factor. These results demonstrate the development of a relevant physiological culture system for bovine granulosa cells. This system will permit the detailed study of the key factors controlling the differentiation and proliferation of bovine granulosa cells.

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Development of a long-term bovine granulosa cell culture system: induction and maintenance of estradiol production, response to follicle-stimulating hormone, and morphological characteristics.

The relationship between ovarian follicular steroidogenesis and insulin-like growth factor binding protein (IGFBP) activity was evaluated during the follicular phase of the bovine estrous cycle. In experiment 1, follicles were collected from cyclic cows (n = 11) slaughtered at 48 h after administration of prostaglandin F2 alpha (PGF; 35 mg i.m.). In experiment 2, cows were injected twice daily with saline (control) or FSH (FSH cows; total dosage = 42 mg) from Day 2 to Day 6 (estrus = Day 0) and with PGF (35 mg i.m.) on Day 7; follicles were collected from control cows (n = 20) slaughtered at 0, 24, 48, or 72 h and from FSH cows (n = 8) at 0 and 48 h after PGF. Follicular fluid was assayed for estradiol (E2), androstenedione (A4), progesterone (P4), and insulin-like growth factor-I (IGF-I) by RIA and for IGFBP activity by ligand blotting and densitometry. Intensities of the 34-kDa (IGFBP-2), 29-27-kDa, and 22-kDa IGFBP bands in follicular fluid were nondetectable or were lower (p or = 8 mm) E-active (E-A; E2 > 50 ng/ml and > P4) follicles than in large E-inactive (E-I), medium (5-7 mm), or small (< 5 mm) follicles. IGFBP-3 (44-40-kDa doublet) was unaffected by follicle stage in experiment 1, but IGFBP-3 was lower (p < 0.01) in follicular fluid of E-A vs. E-I large follicles in experiment 2. Profiles of IGFBP activity were similar in follicular fluid of small, medium, and E-I large follicles. In experiment 2, E2 concentrations in large E-A follicles increased (p < 0.01) from 0 to 48 h after the PGF injection for control cows but decreased (p < 0.01) for FSH cows, whereas follicular fluid IGFBP-2 binding activity decreased from 0 to 48 h after PGF in controls and increased in FSH cows (treatment x time, p < 0.05). IGFBP-3 binding was unaffected by FSH treatment or time after administration of PGF. Profiles of IGFBP activity in homogenates of granulosa or theca cells were similar to follicular fluid profiles except for the absence of IGFBP-3 binding activity. The disappearance of binding activities for IGFBP-2 and smaller-molecular-mass IGFBPs in E-A follicles suggests a possible regulatory role for IGFBPs in follicular maturation and on aromatase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

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Relationships among concentrations of steroids, insulin-like growth factor-I, and insulin-like growth factor binding proteins in ovarian follicular fluid of beef cattle.

The expression of four major steroidogenic enzymes in porcine theca and granulosa cell layers of preovulatory follicles was related to the levels of steroids in follicular fluid and gonadotropin concentrations in peripheral serum at slaughter. Ovaries were collected during proestrus, early estrus, and late estrus as evidenced by behavioral signs. Follicles were dissected from the ovaries, and theca, granulosa, and follicular fluids were pooled for each of 24 sows. Cytochromes P450 17 alpha-hydroxylase/17-20 lyase (P450c17), aromatase (P450arom) and side-chain cleavage (P450scc), as well as 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), were subjected to Northern and Western immunoblot analyses. The concentrations of estradiol-17 beta, testosterone, androstenedione, and progesterone were determined in follicular fluid, and peripheral serum was assayed for estradiol-17 beta, LH, and FSH. Stages of preovulatory development were verified by plasma levels of LH, FSH, and estradiol-17 beta. Thecca expressed P450c17, P450arom, P450scc, and 3 beta HSD whereas granulosa expressed only P450arom and low levels of P450scc. Thecal P450c17, thecal P450arom, and granulosa P450arom expression decreased coincidentally as serum estradiol-17 beta and follicular fluid estradiol-17 beta, testosterone, and androstenedione levels declined after the presumed gonadotropin surge. Unlike P450c17 and P450arom P450scc and 3 beta HSD remained relatively constant in theca and granulosa. From these data, we suggest that the theca interna may be the primary steroidogenic compartment of the porcine follicle during its final stages of preovulatory development. Moreover, preovulatory estrogen secretion appears to be controlled by the coordinated expression of a triad of enzymes in the porcine follicle that includes theca P450c17, theca P450arom and granulosa P450arom.

Steroidogenesis in the preovulatory porcine follicle.

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)

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

Two experiments were conducted to examine responses of porcine granulosa cells to insulin-like growth factor-1 (IGF-1) or IGF-1 analogs (des [1-3] and Long R3) that have reduced affinity for IGF-binding proteins (IGFBP). Both experiments evaluated estradiol and IGFBP production by granulosa cells after separation of cells into subpopulations that maintain long-term estradiol production in vitro (tightly bound) and those that do not (weakly associated). Granulosa cells were obtained from medium-sized follicles (4-6 mm) at random stages of the estrous cycle in experiment 1 and from the 10 largest follicles per ovary at 0 or 48 h after weaning in experiment 2. Follicle diameter and follicular fluid estradiol concentrations increased with time after weaning (p < 0.05). Tightly bound cells produced more estradiol than weakly associated cells at 24-120 h of culture in experiment 1 and from 0 to 48 h in experiment 2 (p < 0.05). In tightly bound but not weakly associated cells, IGF-1 stimulated estradiol production. The IGF analogs were more potent stimulators than IGF-1 in experiment 1 (p < 0.05); and in experiment 2, this response was restricted to cells collected at 48 h after weaning. Conversely, tightly bound cells obtained at 0 h after weaning responded similarly to IGF-1 and des (1-3). During the final 48 h of culture, weakly associated cells produced greater quantities of 28-30-kDa IGFBP than did tightly bound cells in response to IGF-1 or analogs (both experiments; p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

H J Howard

Papers

Relationships among concentrations of steroids, insulin-like growth factor-I, and insulin-like growth factor binding proteins in ovarian follicular fluid of beef cattle.

Steroidogenesis in the preovulatory porcine follicle.

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

Differential steroidogenic response of subpopulations of porcine granulosa cells to insulin-like growth factor-1 (IGF-1) or IGF-1 analogs.