James M. Hammond

Comparative effects of insulin and insulin-like growth factors on DNA synthesis and differentiation of porcine granulosa cells.

The effects of insulin-like growth factors (IGF-I and II) were compared with those of insulin in porcine granulosa cells in vitro with regard to stimulation of thymidine incorporation, facilitation of FSH-induced progesterone secretion, and displacement of [125I] IGF-I from specific receptors. In all three assays, the order of potency was IGF-I from specific receptors. In all three assays, the order of potency was IGF-I greater than IGF-II greater than insulin. However, the difference in potency was marginally significant in the mitogenic assay. These data are consistent with the presence of specific IGF-I receptors on these cells and involvement of these binding sites in regulation of granulosa cell replication and differentiation.

Ovarian epidermal growth factor-like activity. Concentrations in porcine follicular fluid during follicular enlargement.

Numerous data indicate that epidermal growth factor has important effects on cultured granulosa cells. However, most of the few attempts to detect epidermal growth factor in ovarian tissue have been unrevealing. In this study, ovarian epidermal growth factor-like activity was easily detected by a radioreceptor assay based on the A431 cell line but not by an immunoassay for mouse epidermal growth factor. The concentration of this activity in follicular fluid from small porcine ovarian follicles was higher than that in fluid from medium or large follicles or serum (p less than 0.01), but lower than that in salivary gland extracts. Receptor-active epidermal growth factor-like peptides could function as local ovarian regulators.

The ovarian insulin-like growth factors, a local amplification mechanism for steroidogenesis and hormone action

The importance of the ovarian insulin-like growth factors (IGFs) has been suggested by data from numerous laboratories and several approaches in the last several years. In the aggregate, these data indicate that this system could function as an important local amplification mechanism for steroidogenesis and gonadotropin action. Studies supporting this hypothesis have described several interacting components of this autocrine/paracrine system. First, the several types of ovarian cells possess an IGF-response system, which includes receptors for IGFs and an effective intracellular transduction system. The IGFs can promote growth and/or differentiation of ovarian cells, and their predominant actions depend on the nature of the cells and the presence of additional modulating factors. The biochemical events leading to enhanced steroidogenesis are now understood in considerable detail and include induction of several steps in the cAMP-dependent steroidogenic cascade. The second component of the ovarian IGF system comprises hormone-responsive local production of IGFs. Both IGF-I and IGF-II may be secreted; gonadotropins, gonadal steroids and locally produced growth factors can regulate the IGF system at this level. Finally, ovarian cells secrete a heterogeneous and complex family of IGF-binding proteins (IGFBPs). These proteins can impact on multiple ovarian functions in a manner which is generally opposite to that of the IGFs themselves. As is the case for the IGFs, the secretion of these proteins by ovarian cells is regulated by gonadotropins and locally produced ovarian factors. Collectively, these several components provide an integrated, synergistically cooperative local network to promote gonadotropin-dependent growth and differentiation in the ovary.

Expression of Mouse Ovarian Insulin Growth Factor System Components During Follicular Development and Atresia1

Insulin growth factor I (IGF-I) appears necessary for the completion of follicular development in mice. However, little is known about changes in the IGF system components during follicular development and luteinization. This study determined the relation between gene expression of specific IGF system components and follicular growth, survival, or atresia in mice. Immature mice from three different strains (129, C57, and MF1), with or without gonadotropin treatment (2.5 IU PMSG/2.5 IU human CG (hCG)], were used. The strains were similar in all parameters measured. Apoptosis, as detected by in situ labeling of nicked DNA, preceded the appearance of morphological signs of atresia. In healthy follicles, IGF-I transcripts were low during the primary follicular stage but increased to a maximum in the late preantral and early antral stages (P < 0.001) irrespective of hormone treatment. Occasionally, IGF-I transcripts were also detected in apoptotic follicles but decreased (P < 0.05) as a function of atresia as assessed by morphological criteria. IGF binding protein-4 (IGFBP-4) messenger RNA (mRNA) expression in granulosa cells was restricted to apoptotic and atretic follicles (P < 0.001). IGFBP-5 transcript levels, on the other hand, were elevated in granulosa cells of healthy primary and secondary follicles but decreased in subsequent follicular stages and in atretic follicles (P < 0.001). Conversely, IGFBP-2 mRNA was constitutively expressed in granulosa cells. PMSG/hCG treatment induced the appearance of IGFBP-2 transcripts in the ovarian interstitium. Following PMSG/hCG-induced ovulation, IGFBP-2 and -4 and IGF type-I receptor mRNAs were strongly expressed in virtually all luteal cells, whereas IGFBP-3 and -5 transcripts were selectively localized to some cell types in the corpus luteum. Conversely, IGF-I mRNA was essentially undetectable in the corpus luteum. This study represents the most comprehensive and detailed analysis of the physiology and anatomy of the mouse ovarian IGF system, and shows that 1) IGFBP-5-is linked to the survival of the slow growing and immature preantral follicles; 2) IGF-I is associated with the growth and survival of the rapidly growing large preantral and antral follicles; 3) IGFBP-4 is an atretogenic candidate for mouse ovarian follicles; 4) ovulatory doses of PMSG/hCG up-regulate IGFBP-2 mRNA expression in the ovarian interstitium; and 5) transcripts of IGF type-I receptor and IGFBP-2 through -5, but not those of IGF-I are highly expressed in the mouse corpus luteum.Insulin growth factor I (IGF-I) appears necessary for the completion of follicular development in mice. However, little is known about changes in the IGF system components during follicular development and luteinization. This study determined the relation between gene expression of specific IGF system components and follicular growth, survival, or atresia in mice. Immature mice from three different strains (129, C57, and MF1), with or without gonadotropin treatment[ 2.5 IU PMSG/2.5 IU human CG (hCG)], were used. The strains were similar in all parameters measured. Apoptosis, as detected by in situ labeling of nicked DNA, preceded the appearance of morphological signs of atresia. In healthy follicles, IGF-I transcripts were low during the primary follicular stage but increased to a maximum in the late preantral and early antral stages (P < 0.001) irrespective of hormone treatment. Occasionally, IGF-I transcripts were also detected in apoptotic follicles but decreased (P < 0.05) as a function of atresia as ...

Impact of Growth Hormone Resistance on Female Reproductive Function: New Insights from Growth Hormone Receptor Knockout Mice

Abstract We examined multiple aspects of reproductive function in growth hormone receptor gene knockout (GHR-KO) and normal mice to clarify the role of growth hormone in female reproduction. In adult animals, estrous cycle duration was comparable in all mice housed individually but was significantly longer in group-housed GHR-KO females. Histological evaluation of ovaries of adult females at estrus showed that the numbers of preovulatory follicles and corpora lutea were significantly reduced in GHR-KO mice, as was the plasma estradiol level. The number of atretic preovulatory follicles was reduced in GHR gene-ablated animals. Although reverse transcription polymerase chain reaction analysis revealed reduced ovarian insulin-like growth factor I (IGF-I) mRNA expression in GHR-KO females, the expression of several steroidogenic enzyme mRNAs did not differ between groups. The numbers of active corpora lutea and uterine implantation sites were reduced in GHR-KO females at Day 7 of gestation. When young females were mated to normal males, latency to first mating and age of the female at first mating were significantly delayed in GHR-KO females, but maternal age at first conception was similar between groups. Significantly fewer virgin GHR-KO females exhibited pseudopregnancies when initially placed with vasectomized normal males than did normal female counterparts. Growth hormone resistance and IGF-I insufficiency negatively impacted 1) follicular development/ovulation rate, 2) sexual maturation, 3) production of and responsiveness to pheromonal signals, and 4) the ability of virgin females to respond to coitus by activation of luteal function. Although GHR-KO female mice are fertile, they exhibit quantitative deficits in various parameters of reproductive function.

Prolactin-secreting pituitary adenomas: Association with multiple endocrine neoplasia, type I☆

Abstract In the course of follow-up of two patients who presented with prolactin-secreting pituitary adenomas, we encountered an unemphasized sequel: the evolution of multiple extrapituitary neoplasms of endocrine-gland origin. Tumors were either present concurrently or evolved sequentially. The pedigree of one family was affected in an autosomal dominant manner with tumors of the pituitary, parathyroid glands and pancreatic islet cells, documenting with the most extensive kindred to date the association of prolactinoma with multiple endocrine neoplasia I. These cases, in conjunction with ultrastructural histopathology of three other multiple endocrine neoplasia I prolactinomas, and review of the literature, delineate the following considerations: (1) a prolactin-secreting pituitary adenoma may be the first manifestation of multiple endocrine neoplasia I, with concurrent or sequential tumor evolution; (2) the occurrence of endocrine tumors in these families is sporadic or conforms to a heritable pattern of autosomal dominance; (3) the ultrastructural morphology of prolactinomas in multiple endocrine neoplasia I cannot be used a priori to identify patients at risk for multiple endocrine neoplasia I from those with randomly occurring, isolated prolactinomas; (4) thus, we recommend serum calcium determinations and a detailed family history with inventory of endocrine systems in all patients with prolactinoma; (5) in identified families with the multiple endocrine neoplasia I syndrome, serum prolactin concentrations should be determined; and (6) the cost effectiveness of extensive biochemical screening for multiple endocrine neoplasia I in patients with prolactinoma without an incriminating family history will require further clarification.

Heterogeneity of insulin receptors on fat cell membranes

Abstract A highly purified and well characterized fat cell membrane preparation has been shown to retain the insulin binding characteristics of intact fat cells. Two major binding sites were identified, one a high affinity, low capacity site (K D = 5 × 10 −10 M) and the other a lower affinity, high capacity site (K D = 3 × 10 −9 M). Insulin binding to the membranes was prevented by insulin analogues (desoctapeptide and desalanine insulin) and proinsulin in direct relationship to their biological activities.

Catecholestrogens inhibit proliferation and DNA synthesis of porcine granulosa cells in vitro: comparison with estradiol, 5α-dihydrotestosterone, gonadotropins and catecholamines

Studies were conducted to assess the role of catecholestrogens on ovarian follicular growth using cultured porcine granulosa cells. Effects of the catecholestrogens, 2-hydroxyestradiol (2-OH-E2) and 2-methoxyestradiol (2-MeO-E2) were compared to those of estradiol (E2). Treatment with saturating concentrations of 2-OH-E2 caused a significantly greater decrease in cell numbers measured after 2 days of treatment than E2 treatment. The inhibitory effect of 2-OH-E2 was time and concentration dependent, not associated with a change in the viability of cells, and was partially reversible. The potency of 2-MeO-E2 to inhibit cell numbers was similar to or greater than that of 2-OH-E2. 2-MeO-E2 had a greater inhibitory effect on DNA synthesis, as measured by [3H]thymidine incorporation into trichloroacetic acid-precipitable macromolecules, than 2-OH-E2 or E2 in the absence or presence of insulin, epidermal growth factor or platelet-derived growth factor. Concurrent treatment with epinephrine significantly enhanced the inhibitory effect of 2-OH-E2 on granulosa cell DNA synthesis. Collectively, these studies indicate that catecholestrogens are more potent inhibitors of granulosa cell replication than E2 and 5 alpha-dihydrotestosterone (DHT), and that catecholamines may modulate the antimitotic activity of 2-OH-E2. These results support the hypothesis that catecholestrogens play a role in proliferation of granulosa cells during growth of ovarian follicles.

Regulation of ovarian function by catecholestrogens: Current concepts

Development of the ovarian follicle(s) destined for ovulation appears to be a process in which antral follicles undergo a recruitment, selection and subsequent dominance phase. Several intraovarian or autocrine/paracrine regulatory mechanisms have been evoked to explain these processes. One of these potential autocrine/paracrine regulators is a catecholestrogen, 2-hydroxy-estradiol (2-OH-E2). Evidence implicating 2-OH-E2 as an autocrine/paracrine regulator of follicular function is reviewed. Studies have shown 2-OH-E2 to be present in nanomolar concentrations in fluid of human and equine follicles. In addition, the enzyme responsible for converting estradiol (E2) into 2-OH-E2, estrogen 2-hydroxylase (E-2-H), is abundant in granulosa and thecal cells (but not corpora lutea) of porcine follicles. Moreover, activity of E-2-H increases during follicular development in pigs. In vitro, the actions of 2-OH-E2 have been compared to those of E2, gonadotropins, catecholamines, and androgens. Studies indicated that the maximal stimulatory effects of 2-OH-E2 on progesterone production were comparable to those of E2 and gonadotropins, and greater than androgens or catecholamines. The effect of 2-OH-E2 was found to be significantly additive to each of the other classes of compounds at maximally effective concentrations, suggesting that the mechanism of action of 2-OH-E2 was different. The mode of action of the several stimulators of progesterone biosynthesis were examined additionally with antihormones for E2, catecholamines, and androgens. In each instance, the hormonal antagonists were able to inhibit the action of the predicted class of effector compounds. However, with the exception of the antiandrogen, hydroxyflutamide, no effects of anti-hormones on the action of 2-OH-E2 were observed. In the aggregate, these studies suggested that the action of 2-OH-E2 is mediated by a mechanism discrete from those of the other classes of hormones examined to date, and that hydroxyflutamide exhibits both antiandrogen and anticatecholestrogen activity. 2-OH-E2 can also enhance the actions of other trophic hormones, epinephrine, LH and FSH, by enhancing hormone-stimulated cAMP production. This effect on epinephrine action appears to be due to a 2-OH-E2-stimulated increase in the density of beta-adrenergic receptors. Whether 2-OH-E2 stimulates an increase in the number of LH and FSH receptors remains to be determined. The precise locus of the stimulatory effect of 2-OH-E2 alone on steroidogenesis is unclear but preliminary data would suggest that 2-OH-E2 may be stimulating side-chain cleavage enzyme activity.(ABSTRACT TRUNCATED AT 400 WORDS)

β-2-adrenergic stimulation of ornithine decarboxylase activity in porcine granulosa cells in vitro

Abstract Epinephrine, norepinephrine, and isoproterenol produced dose-dependent stimulation of ornithine decarboxylase (EC 4.1.1.7) activity in isolated porcine granulosa cells maintained under defined conditions in vitro. β- but not α-receptor-blocking agents prevented enzyme stimulation by catecholamines. Application of preferential β-1 and β-2-receptor antagonists and agonists localized the epinephrine effect to β-2-adrenergic mediation. Epinephrine action was enhanced by the phosphodiesterase inhibitor, 1-methyl-3-isobutyl-xanthine, but not by saturating concentrations of the cyclic AMP analogue, 8-bromocyclic AMP, of follicle-stimulating hormone, or of prostaglandin E 2 . However, stimulation by epinephrine was additive to that of luteinizing hormone. Follicular fluid obtained from immature Graafian follicles contined concentrations of norepinephrine and epinephrine active in vitro. Thus, catecholamines may participate in the regulation of ornithine decarboxylase activity in the ovary. Catecholamine effects may be mediated by β-2-receptors linked to the adenylate cyclase system.