Phil G. Knight

Activin A and inhibin A as possible endocrine markers for pre-eclampsia

BACKGROUND Inhibin A and activin A are produced by the placenta during human pregnancy. This study aimed to measure circulating concentrations of inhibin A, pro alpha C-containing inhibins, and activin A in the serum of women with pre-eclampsia and of healthy matched control pregnant women, and to establish the molecular-weight forms of circulating inhibin A and activin A in pre-eclampsia. METHODS In a retrospective cross-sectional study, blood samples were taken from 20 women in hospital with established pre-eclampsia, and from 20 control pregnant women attending antenatal clinics, who were matched for duration of gestation (pre-eclampsia mean 29.15 [SD 3.75] weeks; controls 29.30 [3.93] weeks), parity, and maternal age. Serum samples were analysed for inhibin A, inhibin B, pro alpha C, and activin A. Pooled samples of control (n = 3) and pre-eclampsia serum (n = 3) subsequently underwent fast protein liquid chromatographic analysis to assess the molecular-weight forms of inhibin A and activin A. Results are expressed as mean and SD for all variables measured. FINDINGS Serum concentrations of inhibin A, activin A, and pro alpha C were significantly higher in pre-eclampsia than in control normal pregnancy (inhibin A 3.05 [1.8] vs 0.36 [0.14] ng/mL, p < 0.001; activin A 38.08 [25.88] vs 3.95 [2.32] ng/mL, p < 0.001; pro alpha C-containing inhibins 2.2 [0.81] vs 0.71 [0.33] ng/mL, p < 0.001). Inhibin B concentrations in maternal serum were not increased. Molecular-weight forms of inhibin A (32 kDa) and activin A (> 100 kDa) were similar in pre-eclampsia and normal pregnancy. The mean concentrations of hCG were 59.05 [43.98] and 16.3 [8.72] ng/mL, respectively. INTERPRETATION Higher maternal serum concentrations of inhibin A, pro alpha C, and total activin A in pre-eclampsia than in control pregnancies could be helpful in the diagnosis of pre-eclampsia. These changes are interpreted as further evidence for trophoblast dysfunction in pre-eclampsia.

Numbers of Antral Follicles During Follicular Waves in Cattle: Evidence for High Variation Among Animals, Very High Repeatability in Individuals, and an Inverse Association with Serum Follicle-Stimulating Hormone Concentrations

Abstract The extent, causes, and physiological significance of the variation in number of follicles growing during ovarian follicular waves in human beings and cattle are unknown. Therefore, the present study examined the variability and repeatability in numbers of follicles 3 mm or greater in diameter during the follicular waves in bovine estrous cycles, and we determined if the variation in number of follicles during waves was associated with alterations in secretion of FSH, estradiol, inhibin, and insulin-like growth factor I (IGF-I). Dairy cattle were subjected to twice-daily ultrasound analysis to count total number of antral follicles 3 mm or greater in diameter throughout 138 different follicular waves. In another study, blood samples were taken at frequent intervals from cows that consistently had low or very high numbers of follicles during waves and were subjected to immunoassays. Results indicate the following: First, despite an approximately sevenfold variation in number of follicles during waves among animals and marked differences in age, stage of lactation, and season of the year, a very highly repeatable (0.95) number of follicles 3 mm or greater in diameter is maintained during the ovulatory and nonovulatory follicular waves of individuals. Second, variation in number of follicles 3 mm or greater in diameter during waves and the inverse association of number of follicles during waves with FSH are not directly explained by alterations in the patterns of secretion of estradiol, inhibin, or IGF-I. Third, ovarian ultrasound analysis can be used reliably by investigators to identify cattle that consistently have low or high numbers of follicles during waves, thus providing a novel experimental model to determine the causes and physiological significance of the high variation in antral follicle number during follicular waves among single-ovulating species, such as cattle or humans.

Alterations in Intrafollicular Regulatory Factors and Apoptosis During Selection of Follicles in the First Follicular Wave of the Bovine Estrous Cycle

Abstract Changes in follicular fluid (FF) concentrations of estradiol, inhibin forms, and insulin-like growth factor binding proteins (IGFBPs), percentage of apoptotic granulosa cells (%A), and follicular size for individual follicles in a growing cohort were determined throughout the first wave of follicular development during the bovine estrous cycle and related to FSH decline. Four groups of heifers (n = 31) were ovariectomized between Days 1.5 and 4.5 of the estrous cycle at 5 ± 1, 33 ± 2, 53 ± 1, and 84 ± 2 h after the periovulatory peak in FSH concentrations. Follicles ≥2.5 mm were dissected, measured, and FF aspirated. The five largest follicles were ranked based on their diameter (F1 to F5). Diameters of F1 to F5 were positively correlated with interval from FSH peak (r ≥ 0.6, P < 0.05). Five hours after the FSH peak, follicular diameter and FF concentrations of estradiol, inhibins, and IGFBPs were similar for F1 to F5. From 5 to 33 h, amounts of the six precursor inhibin forms (≥48 kDa) increased (P < 0.05) in F1 follicles. The IGFBPs in F1 follicles remained low at all time periods. At 33 h, amounts of IGFBP-4 and -5 were higher (P < 0.05) in F4 and F5 compared with F1 follicles. At 84 h, IGFBP-2, -4, and -5 were increased (P < 0.05) in F3, F4, and F5 compared with F1. At 5, 33, or 53 h, %A was not different between follicles in any size class. At 84 h %A was increased (P < 0.05) in follicles <6 mm in diameter. However, at that time, %A did not differ between the selected DF and the largest subordinate follicle. For individual heifers, the selected DF at 84 h was largest in size, highest in estradiol, and lowest in IGFBP-2 and -4. The F1 follicle had highest estradiol in 23 of 27 heifers irrespective of stage of the wave and lowest IGFBP-4 in 19 of 21 heifers from 33 h. We concluded that the earliest intrafollicular changes that differentiate a dominant-like follicle from the growing cohort are enhanced capacity to produce estradiol and maintenance of low levels of IGFBPs.

Identification of Potential Intrafollicular Factors Involved in Selection of Dominant Follicles in Heifers

Abstract A surgical procedure to aspirate follicular fluid concurrently from individual follicles from the same heifer was validated and used to determine if intrafollicular amounts of estradiol, progesterone, inhibins, activin-A, follistatins, and insulin-like growth factor binding proteins (IGFBP) differed for the future dominant compared with subordinate follicles during selection of the first wave dominant follicle. Heifers were subjected to surgery and aspiration of follicular fluid from the two or three largest follicles on Day 3 of the estrous cycle (∼1.5 days after emergence). Ultrasound was used to determine the fate of each aspirated follicle after surgery. At aspiration, diameter of the future dominant and largest subordinate follicle was similar in heifers. However, estradiol was higher, whereas IGFBP-4 was lower in the future dominant compared with the largest or next largest subordinate follicles. Also, the future dominant follicle in most cohorts had the highest estradiol and lowest IGFBP-4 compared with future subordinate follicles. We concluded that: IGFBP-4 and estradiol may have key roles in determining the physiological fate of follicles during selection of the first wave dominant follicle in heifers, and that both are reliable markers to predict which follicle in a growing cohort of 5- to 8.5-mm follicles becomes dominant.

Intra-ovarian roles of activins and inhibins

Granulosa cells are the main ovarian source of inhibins, activins and activin-binding protein (follistatin) while germ (oogonia, oocytes) and somatic (theca, granulosa, luteal) cells express activin receptors, signaling components and inhibin co-receptor (betaglycan). Activins are implicated in various intra-ovarian roles including germ cell survival and primordial follicle assembly; follicle growth from preantral to mid-antral stages; suppression of thecal androgen production; promotion of granulosa cell proliferation, FSHR and CYP19A1 expression; enhancement of oocyte developmental competence; retardation of follicle luteinization and/or atresia and involvement in luteolysis. Inhibins (primarily inhibin A) are produced in greatest amounts by preovulatory follicles (and corpus luteum in primates) and suppress FSH secretion through endocrine negative feedback. Together with follistatin, inhibins act locally to oppose auto-/paracrine activin (and BMP) signaling thus modulating many of the above processes. The balance between activin-inhibin shifts during follicle development with activin signalling prevailing at earlier stages but declining as inhibin and betaglycan expression rise.

Functional significance of the signal transduction pathways Akt and Erk in ovarian follicles: in vitro and in vivo studies in cattle and sheep

BackgroundThe intracellular signalling mechanisms that regulate ovarian follicle development are unclear; however, we have recently shown differences in the Akt and Erk signalling pathways in dominant compared to subordinate follicles. The aim of this study was to investigate the effects of inhibiting Akt and Erk phosphorylation on IGF- and gonadotropin- stimulated granulosa and theca cell function in vitro, and on follicle development in vivo.MethodsBovine granulosa and theca cells were cultured for six days and stimulated with FSH and/or IGF, or LH in combination with PD98059 (Erk inhibitor) and/or LY294002 (Akt inhibitor) and their effect on cell number and hormone secretion (estradiol, activin-A, inhibin-A, follistatin, progesterone and androstenedione) determined. In addition, ovarian follicles were treated in vivo with PD98059 and/or LY294002 in ewes on Day 3 of the cycle and follicles were recovered 48 hours later.ResultsWe have shown that gonadotropin- and IGF-stimulated hormone production by granulosa and theca cells is reduced by treatment with PD98059 and LY294002 in vitro. Furthermore, treatment with PD98059 and LY294002 reduced follicle growth and oestradiol production in vivo.ConclusionThese results demonstrate an important functional role for the Akt and Erk signalling pathways in follicle function, growth and development.

Enzyme immunoassays for inhibins, activins and follistatins

In this short review, the authors summarise the inhibin, activin and follistatin assays developed by the Oxford group and collaborators, and some of the main purposes for which they have been applied. Over 500 research publications have used these assays. We also discuss new assays recently developed at the request of our collaborators for particular applications, and comment on outstanding assay problems.

Dysregulation of granulosal bone morphogenetic protein receptor 1B density is associated with reduced ovarian reserve and the age-related decline in human fertility

Reproductive ageing is linked to the depletion of ovarian primordial follicles, which causes an irreversible change to ovarian cellular function and the capacity to reproduce. The current study aimed to profile the expression of bone morphogenetic protein receptor, (BMPR1B) in 53 IVF patients exhibiting different degrees of primordial follicle depletion. The granulosa cell receptor density was measured in 403 follicles via flow cytometry. A decline in BMPR1B density occurred at the time of dominant follicle selection and during the terminal stage of folliculogenesis in the 23-30 y good ovarian reserve patients. The 40+ y poor ovarian reserve patients experienced a reversal of this pattern. The results demonstrate an association between age-induced depletion of the ovarian reserve and BMPR1B receptor density at the two critical time points of dominant follicle selection and pre-ovulatory follicle maturation. Dysregulation of BMP receptor signalling may inhibit the normal steroidogenic differentiation required for maturation in older patients.

The effect of ovarian reserve and receptor signalling on granulosa cell apoptosis during human follicle development

The poor oocyte quality in older women has previously been linked to the depletion of the ovarian reserve of primordial follicles and an increase in granulosal apoptosis. Granulosa cells were collected from 198 follicles and individually analysed by flow cytometry. In the young IVF patients, the level of apoptosis was inversely proportional to the expression of bone morphogenetic protein (BMPR1B) and follicle stimulating hormone (FSH) receptors. Conversely, in the older patients this relationship became dysregulated. In the older patients, at the time of preovulatory maturation, the reduced apoptosis reflects the poor mitogenic growth turnover rate of healthy follicles rather than the death rate in an atretic follicle. Restoring an optimum receptor density and down-regulation of receptors may improve oocyte quality and the pregnancy rate in older women.

Granulosa Cell Apoptosis in the Ovarian Follicle—A Changing View

Recent studies challenge the previous view that apoptosis within the granulosa cells of the maturing ovarian follicle is a reflection of aging and consequently a marker for poor quality of the contained oocyte. On the contrary, apoptosis within the granulosa cells is an integral part of normal development and has limited predictive capability regarding oocyte quality or the ensuing pregnancy rate in in vitro fertilization programs. This review article covers our revised understanding of the process of apoptosis within the ovarian follicle, its three phenotypes, the major signaling pathways underlying apoptosis as well as the associated mitochondrial pathways.