Biomedical subjects

J J Ireland

Publications and source records attributed to J J Ireland.

At least 19 recordsLinked to original sources

PubMed2006

Molecular evidence that growth of dominant follicles involves a reduction in follicle-stimulating hormone dependence and an increase in luteinizing hormone dependence in cattle.

The bovine dominant follicle (DF) model was used to identify molecular mechanisms potentially involved in initial growth of DF during the low FSH milieu of ovarian follicular waves. Follicular fluid and RNA from granulosa and theca cells were harvested from 10 individual DF obtained between 2 and 5.5 days after emergence of the first follicular wave of the estrous cycle. Follicular fluid was subjected to RIA to determine estradiol (E) and progesterone (P) concentrations and RNA to cDNA microarray analysis and (or) quantitative real-time PCR. Results showed that DF growth was associated with a decrease in intrafollicular E:P ratio and in mRNA for the FSH receptor, estrogen receptor 2 (ER beta), inhibin alpha, activin A receptor type I, and a proliferation (cyclin D2) and two proapoptotic factors (apoptosis regulatory protein Siva, Fas [TNFRSF6]-associated via death domain) in granulosa cells. In contrast, mRNAs for the LH receptor in granulosa cells and for two antiapoptotic factors (TGFB1-induced antiapoptotic factor 1, LAG1 longevity assurance homolog 4 [Saccharomyces cerevisiae]) and one proapoptotic factor (tumor necrosis factor [ligand] superfamily, member 8) were increased in theca cells. We conclude that the bovine DF provides a unique model to identify novel genes potentially involved in survival and apoptosis of follicular cells and, importantly, to determine the FSH-, estradiol-, and LH-target genes regulating its growth and function. Results provide new molecular evidence for the hypothesis that DF experience a reduction in FSH dependence but acquire increased LH dependence as they grow during the low FSH milieu of follicular waves.

Activin Receptors, Type I↗

PubMed2006

Suppressed expression of genes involved in transcription and translation in in vitro compared with in vivo cultured bovine embryos.

In vivo-derived bovine embryos are of higher quality than those derived in vitro. Many of the differences in quality can be related to culture environment-induced changes in mRNA abundance. The aim of this study was to identify a range of mRNA transcripts that are differentially expressed between bovine blastocysts derived from in vitro versus in vivo culture. Microarray (BOTL5) comparison between in vivo- and in vitro-cultured bovine blastocysts identified 384 genes and expressed sequence tags (ESTs) that were differentially expressed; 85% of these were down-regulated in in vitro cultured blastocysts, showing a much reduced overall level of mRNA expression in in vitro- compared with in vivo-cultured blastocysts. Relative expression of 16 out of 23 (70%) differentially expressed genes (according to P value) were verified in new pools of in vivo- and in vitro-cultured blastocysts, using quantitative real-time PCR. Most (10 out of 16) are involved in transcription and translation events, suggesting that the reason why in vitro-derived embryos are of inferior quality compared with in vivo-derived embryos is due to a deficiency of the machinery associated with transcription and translation.

Animals↗

PubMed2004

Evidence for autocrine or paracrine roles of alpha2-macroglobulin in regulation of estradiol production by granulosa cells and development of dominant follicles.

alpha(2)-Macroglobulin (alpha(2)-M) inhibits proteinases and modulates the actions of growth factors and cytokines. Despite the key roles proteinases, growth factors, and cytokines have in folliculogenesis, the role of alpha(2)-M in follicular development is unknown. Our objectives were to: 1) determine whether granulosa cells produce alpha(2)-M and have alpha(2)-M receptors, 2) examine the effect of alpha(2)-M on estradiol production by granulosa cells, 3) establish whether amounts of alpha(2)-M and alpha(2)-M receptors were altered during dominant nonovulatory follicle development, and 4) examine alpha(2)-M's mechanism of action. The results demonstrated that bovine granulosa cells contain 5.2- and 15-kb mRNAs and 720- and 500-kDa proteins that correspond, respectively, to sizes of mRNAs and proteins for alpha(2)-M and the alpha(2)-M receptor. Treatment of granulosa cells with alpha(2)-M resulted in a specific dose-responsive increase in estradiol production. Cell viability, cell number, and the amount of aromatase in granulosa cells were not altered by alpha(2)-M. Treatment of granulosa cells with factors that bind alpha(2)-M or its receptor did not mimic alpha(2)-M action. Although intrafollicular amounts of alpha(2)-M remained unchanged, amounts of alpha(2)-M receptor in granulosa cells were strongly inversely associated with concentrations of estradiol in dominant and subordinate follicles. Based on these results, we concluded that alpha(2)-M may have autocrine or paracrine roles in granulosa cells potentially important for regulation of estradiol production and development of dominant follicles.

Animals↗

PubMed2004

Identification of genes involved in apoptosis and dominant follicle development during follicular waves in cattle.

We hypothesize that granulosa and theca cells from growing dominant follicles, with relatively high intrafollicular concentrations of estradiol, have a greater expression of genes involved in inhibiting apoptosis pathways and lower expression of genes involved in apoptosis pathways than growing subordinate follicles with lower estradiol concentrations. Using the well-characterized bovine dominant follicle model, we collected granulosa and theca cells from individual dominant and the largest subordinate follicle 3 days after initiation of a follicular wave in four animals. Based on ultrasound analysis, both follicle types were in the growth phase at the time of ovariectomy. However, dominant follicles were larger (9.8 +/- 1.0 versus 7.6 +/- 0.6 mm in diameter, P < 0.05) and had greater intrafollicular concentrations of estradiol (132.2 +/-3 8.5 versus 24.1 +/- 12.1 ng/ml, P < 0.05), compared with the largest subordinate follicles. We used bovine cDNA microarrays, which contained a total of 1400 genes, including a subset of 53 genes known to be involved in apoptosis pathways, to determine which apoptosis and marker genes from each of the four dominant versus subordinate follicles were potentially differentially expressed. Using a low stringency-screening criterion, 22 genes were identified. Quantitative real-time polymerase chain reaction confirmed that 16 of these genes were differentially expressed. Our novel results demonstrate that the high intrafollicular concentrations of estradiol in growing dominant follicles were positively associated with enhanced expression of mRNAs in granulosa cells for aromatase, LH receptor, estradiol receptor beta, DICE-1, and MCL-1, compared with granulosa cells from subordinate follicles (all survival-associated genes). In contrast, the relatively low intrafollicular concentrations of estradiol in growing subordinate follicles were positively associated with enhanced expression of mRNAs in granulosa cells for beta glycan, cyclo-oxygenase-1, tumor necrosis factor alpha, caspase-activated DNase, and DRAK-2, and in theca cells for beta glycan, caspase 13, P58(IPK), Apaf-1, BTG-3, and TS-BCLL, compared with granulosa or theca cells from dominant follicles (genes that are all associated with cell death and/or apoptosis). We suggest that that these genes may be candidate estradiol target genes and that they may be early markers for the final stages of follicle differentiation or initiation of apoptosis and thus selection of dominant follicles during follicular waves.

Animals↗

PubMed2003

Evidence for a negative intrafollicular role for inhibin in regulation of estradiol production by granulosa cells.

Intrafollicular concentrations of inhibin A and estradiol vary inversely during development of dominant follicles in cattle. Thus, we hypothesized that inhibin has a negative autocrine or paracrine effect on estradiol production by granulosa cells. To examine this hypothesis, a homologous model system was used to test the effects of bovine antibovine inhibin antibodies, bovine inhibin, and a peptide fragment of bovine inhibin (bINH) on capacity of granulosa cells isolated from individual estrogen-active or -inactive dominant or subordinate follicles to produce estradiol during short-term (18 h) serum-free culture. Immunoblot analysis of media demonstrated that granulosa cells basally produce different molecular weight forms of inhibin, similar to those in bovine follicular fluid. Immunoneutralization of endogenous inhibin in culture with different doses (12.5-1000 microg) of highly purified bovine antibovine inhibin antibodies increased estradiol production 2- to 15-fold, compared with controls. Preadsorption of the anti-inhibin antibodies with bINH precursors or bovine pro-alpha(C) suppressed the capacity of anti-inhibin antibodies to enhance estradiol production by granulosa cells, compared with controls. Treatment of granulosa cells with an immunoaffinity-purified preparation of bINH suppressed basal estradiol production 60%, compared with controls. In contrast, treatment of granulosa cells with the bINH peptide increased estradiol production 14-fold, compared with controls. Based on these results, we concluded that both anti-inhibin antibodies and bINH blocked the suppressive local effects of basally produced inhibin on estradiol production during culture of granulosa cells and that inhibin has a negative autocrine or paracrine effect on the in vitro capacity of granulosa cells isolated from dominant or subordinate follicles to produce estradiol.

Animals↗

PubMed2002

Development and validation of a short-term, serum-free culture system for bovine granulosa cells: evaluation of the effects of somatotropin and growth hormone-releasing factor on estradiol production.

The objective of this study was to develop and validate a short-term, serum-free culture system to determine whether recombinant bovine somatotropin (rbST) or recombinant bovine growth hormone-releasing factor (rbGRF) altered the estradiol-producing capacity of bovine granulosa cells isolated from dominant or subordinate follicles of the first follicular wave. Thus, ovaries were obtained at an abattoir from cows that were between d 2 to 5 or 6 to 10 of the estrous cycle. Three size classes of follicles were isolated from each cow's ovaries: small (2 to 5 mm in diameter), medium (6 to 14 mm), or the largest (6 to 19 mm). In vivo steroid-producing capacity of follicles was assessed by measuring concentration of estradiol, progesterone, androstenedione and 5alpha-dihydrotestosterone in each follicle. In vitro steroid-producing capacity was assessed by culturing granulosa cells from the different follicle sizes for 48 h in serum-free media with 19-OH androstenedione and measuring the estradiol and progesterone concentrations in media at the end of culture. The effect of different doses of FSH, rbST, or rbGRF on estradiol and progesterone production by granulosa cells from each follicle size class during d 2 to 5 or 6 to 10 was also evaluated. A high percentage (91.7%) of the largest follicles obtained on d 2 to 5 was estrogen-active (estradiol > progesterone) compared with other follicle classifications (d 2 to 5, small = 0%, medium = 13.8%; d 6 to 10, small = 0%, medium = 3.3%, largest = 33.3%). Estradiol was highest (P < 0.05) in the largest follicle on d 2 to 5 and correlated positively with follicle diameter. The pattern of in vitro production of estradiol by granulosa cells from the different follicle size classes reflected the original in vivo capacity of follicles to produce estradiol. However, only granulosa cells from the largest estrogen-active follicle on d 2 to 5 produced more estradiol than progesterone in vitro. Progesterone production by granulosa cells from all follicle classifications was increased by FSH, but FSH only enhanced estradiol production by granulosa cells from the largest estrogen-active follicles on d 2 to 5. Recombinant bST blocked the FSH-induced increase in estradiol by granulosa cells from the largest estrogen-active follicles on d 2 to 5, whereas rbGRF had no effect on steroid production. Based on these results, we concluded that short-term, serum-free culture of bovine granulosal cells obtained from first-wave follicles at an abattoir could be used to reflect reliably the original in vivo estradiol-producing capacity of granulosal cells, and that neither rbST nor rbGRF enhance basal or FSH-induced estradiol production by bovine granulosa cells from first-wave follicles.

Androstenedione↗

PubMed2002

Effects of oestradiol and progesterone on secretion of gonadotrophins and health of first wave follicles during the oestrous cycle of beef heifers.

Antral follicle development in cattle is initially FSH dependent and then LH dependent. The aim of the present study was to determine the effects of oestradiol- and progesterone-induced suppression of FSH and LH on growth and differentiation of first wave follicles. Cyclic heifers (n = 45, n = 6-10 per group) received the following i.m. injections or treatments beginning 30 h after oestrus: (i) saline (controls); (ii) 0.75 mg oestradiol benzoate (ODB); (iii) insertion of a progesterone-releasing intravaginal device (PRID) for 42 h (progesterone); (iv) 0.75 mg oestradiol benzoate plus PRID (ODB plus progesterone); (v) 0.75 mg ODB plus injection of 1 mg Ovagen(TM) at 33, 39 and 45 h after onset of oestrus (ODB plus FSH). In Expt 1, follicle development was monitored by ovarian ultrasonography once a day. In Expt 2, heifers were ovariectomized. Emergence of the first follicle wave and dominant follicle selection were delayed in ODB plus progesterone-treated heifers compared with controls. Interval to nadir FSH concentration was shorter in ODB-, progesterone- and ODB plus progesterone-treated heifers compared with controls. Frequency of LH pulses was unaffected in ODB- or ODB plus FSH-treated heifers, decreased in progesterone-treated heifers and further decreased in ODB plus progesterone-treated heifers. Intrafollicular oestradiol concentrations were lower in the largest follicle from ODB plus progesterone-treated heifers compared with control (66 h) heifers, but follicle diameter and concentrations of insulin-like growth factor binding proteins (IGFBPs) and inhibin forms were unaffected. Treatment with ODB decreased follicular oestradiol concentration in smaller follicles in the cohort. It is concluded that growing cohort follicles are uniformly responsive to increased FSH concentration but differentially responsive to suppressed FSH and LH release, which is consistent with an LH-mediated survival advantage of the largest follicle in the cohort before cessation of the growth of remaining follicles in the cohort occurs.

Animals↗

PubMed2001

Pre-translational regulation of neutrophil L-selectin in glucocorticoid-challenged cattle.

L-selectin (CD62L) gene expression in neutrophils is commonly referred to as "constitutive" because circulating neutrophils require a constant supply of this adhesion molecule for continuous trafficking into peripheral tissues. Under normal circumstances, marginating blood neutrophils and neutrophils that become activated for migration into infected tissues rapidly shed surface CD62L that is ligated to the vascular endothelium. However, this does not shut down CD62L gene expression because these cells continue to express surface CD62L. In contrast, glucocorticoid challenges resulting from stress and hormone injections result in gradual and chronic down-regulation of CD62L on the surface of blood neutrophils. Rather than being associated with migration, this type of CD62L down-regulation associates with pronounced neutrophilia and increased susceptibility to infections. Nothing is currently known about glucocorticoid regulation of CD62L expression in neutrophils. In other cell systems, however, this steroid hormone binds to cytoplasmic glucocorticoid receptors (GR) that influence expression of glucocorticoid-responsive genes at multiple pre-translational levels. Thus, the hypothesis of the present study was that glucocorticoid challenge suppresses CD62L mRNA expression in blood neutrophils. Suppressed CD62L gene expression might help explain the chronic down-regulation of surface CD62L in neutrophils and accompanying neutrophilia. The main objectives of the study were to monitor neutrophil CD62L mRNA abundance before and during subtle and severe glucocorticoid challenges and to determine if CD62L mRNA expression correlates with degree of glucocorticoid challenge. Parturient dairy cows and dexamethasone-treated steers were used as models of subtle and severe (respectively) glucocorticoid challenges. Data presented from both models support the hypothesis and show for the first time that glucocorticoids regulate neutrophil CD62L at a pre-translational level. Results also showed that inhibited CD62L mRNA expression correlated precisely with down-regulated surface expression of CD62L on neutrophils and peak neutrophilia during severe glucocorticoid challenge. Therefore, results of this study indicate that bovine neutrophils are highly sensitive to the blood environment, displaying full capacity to alter CD62L gene expression and trafficking patterns in response to changing glucocorticoid levels. This may serve animals well when heightened inflammatory responses begin to lead to tissue damage, but may be detrimental to overall health if animals are exposed to opportunistic pathogens while stressed or undergoing glucocorticoid therapy. Although this study did not elucidate how glucocorticoids inhibit neutrophil CD62L mRNA expression, presented data implicate GR as possibly being involved because neutrophils from cattle in both models expressed GR mRNA. Further in vitro studies using purified populations of neutrophils will be required to determine if GR is directly involved in glucocorticoid regulation of CD62L gene expression and, if so, at what level.

Actins↗

PubMed2001

Alterations in intrafollicular regulatory factors and apoptosis during selection of follicles in the first follicular wave of the bovine estrous cycle.

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 > or = 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 > or = 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 (> or = 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.

Activins↗

PubMed2000

Identification of potential intrafollicular factors involved in selection of dominant follicles in heifers.

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 (approximately 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.

Activins↗

PubMed2000

Historical perspective of turnover of dominant follicles during the bovine estrous cycle: key concepts, studies, advancements, and terms.

This review chronicles the key concepts, studies, advancements and terms that have led to our current understanding of turnover of dominant follicles (growth and atresia) during the bovine estrous cycle. The "two-wave" concept of follicular development was first proposed in 1960, but remained controversial for the next 28 yr. The concept of the "dominant" follicle was adapted to cattle in 1987. By 1988, ultrasound analysis of individual follicles had demonstrated that heifers usually had two or three distinct waves of turnover of dominant follicles during an estrous cycle. From 1992 to 1993, it was established that a transient rise in serum concentrations of FSH initiated each follicular wave, and a decreased episodic secretion of LH was associated with loss of dominance and the end of a nonovulatory follicular wave. In the past decade, numerous intrafollicular growth factors, such as inhibins, activins, and insulin-like growth factors and their binding proteins, have been identified in follicular fluid of individual bovine follicles. In addition, in vitro studies demonstrate that these growth factors could have endocrine, autocrine, or paracrine actions that modify gonadotropin-stimulated follicular growth and differentiation. However, the precise role of intrafollicular growth factors in turnover of dominant follicles has not been defined. We concluded that two or three FSH-stimulated waves of follicular growth usually occur during the bovine estrous cycle, and each follicular wave culminates in development of a single nonovulatory or ovulatory dominant follicle.

Animals↗

PubMed1999

Effect of long-term immunization against inhibin on sperm output in bulls.

To determine the effect of neutralization of inhibin on sperm output, 12 Holstein bulls were paired by birth date and weight on Day 1 of age. Each bull was actively immunized against bovine inhibin alpha1-26 gly-tyr (bINH) conjugated to human alpha globulin (HAG, n = 6 bulls) or HAG alone (controls, n = 6) at 60 days of age; booster immunizations were administered at 90, 104, 124, 270, and 395 days of age. Body weights and scrotal circumferences were measured at the time of primary immunization and at 10 days after each booster. In addition, jugular blood was obtained at 60, 70, 100, 114, 134, 280, and 405 days of age, during the 3-wk sperm collection period, and during a 6-h blood-sampling period after sperm collection to determine bINH antibody titer and concentrations of FSH, LH, testosterone, and estradiol. Beginning at 405 days of age, sperm output was measured 3 days/wk for 3 wk with two successive ejaculates collected each day for a total of 18 ejaculates per bull. During Days 60-405 of age, the increase in titer of bINH antibodies, scrotal circumference, and serum concentration of FSH was greater (p < 0.01) for the bINH-immunized compared with control bulls. There were significant (p < 0.01) pair x treatment interactions for sperm output and serum FSH and LH concentrations. Specifically, bINH-immunized bulls for four of the six pairs had nearly 50% greater serum FSH concentrations and sperm output. For the remaining two pairs, sperm output was lower and FSH was either lower or only marginally higher in the bINH-immunized bulls compared with controls. Also, the control bulls for the two remaining pairs produced more sperm than all but one bINH-immunized bull, and had markedly higher serum LH concentrations than all other bulls. To summarize, enhancement of sperm output after immunization against inhibin depends on the subsequent increment in FSH concentrations. We conclude that inhibin suppresses spermatogenesis. Thus, methods to immunoneutralize inhibin may have merit as a therapeutic route to enhance sperm production in reproductively maturing bulls.

Aging↗

PubMed1999

Effect of long-term infusion with recombinant growth hormone-releasing factor and recombinant bovine somatotropin on development and function of dominant follicles and corpora lutea in Holstein cows.

The objective of this study was to evaluate the effects of recombinant bovine growth hormone-releasing factor (rGRF) or recombinant bovine somatotropin (rbST) on growth and function of the first-wave dominant follicle and corpus luteum. Primiparous Holstein cows (117 d postpartum) were infused with 12 mg/d of rGRF (n = 10) or 29 mg/d of rbST (n = 10) for 63 d, and non-infused cows (n = 10) were controls. At slaughter on d 5 of an estrous cycle, blood and ovaries were collected and data from cows with a corpus luteum were analyzed (control, n = 8; rGRF, n = 5; rbST, n = 6). Treatment with rGRF or rbST increased somatotropin (ST) and IGF-I in serum similarly compared with controls. In contrast, rbST-treated cows had higher concentrations of ST in follicular fluid (FF) compared with rGRF-treated and control cows. In addition, rbST, but not rGRF, increased the number and decreased the size of estrogen-active follicles (EA; estradiol > progesterone concentrations in FF), increased the abundance of IGF binding proteins-2, -3, and -4 in FF from EA follicles, and increased the number but decreased the size of corpora lutea and decreased concentration of progesterone in serum compared with controls. Based on these results, we concluded that long-term infusion of rbST alters growth and function of the first-wave dominant follicle and the corpus luteum in cattle.

Animals↗

PubMed1998

Display of an inhibin epitope in a surface-exposed loop of the E. coli heat-labile enterotoxin B subunit.

In vitro gene manipulation was used to develop a novel chimeric antigen consisting of the non-toxic B subunit (EtxB) of an E. coli enterotoxin and the first 14 N-terminal amino acid residues of the carboxy-terminal portion of the alpha subunit of bovine inhibin (bINH1-14). Rabbits immunized subcutaneously (s.c.) or intravenously (i.v.) with EtxB::bINH1-14, with or without Freund's adjuvant, developed significant titres of antibodies that recognized an inhibin peptide fragment containing bINH1-14, native inhibins, and EtxB during separate enzyme-linked immunosorbent assay (ELISA). Passive immunization of mice with the rabbit anti-EtxB::bINH1-14 serum increased concentrations of follicle-stimulating hormone (FSH) in serum twofold compared with controls, whereas serum concentrations of luteinizing hormone (LH) were unaltered. Since FSH is the primary hormone from the pituitary gland that stimulates ovarian follicle growth and spermatogenesis, the results of this study demonstrate that EtxB::bINH1-14 has potential as antigen for development of inhibin-based fertility vaccines.

Amino Acid Sequence↗

PubMed1997

Expression of different molecular mass forms of inhibin in atretic and nonatretic follicles during the early luteal phase and altrenogest-synchronized follicular phase in pigs.

This experiment characterized changes in amounts and proportions of different molecular forms of inhibin in porcine follicular fluid as related to stage of follicular development. Thirty-seven follicles (2-4 per pig) were dissected from 12 pigs during early luteal phase of the estrous cycle on Days 5, 6, and 7 of the estrous cycle, whereas 34 follicles (2-4 per pig) were dissected from 11 pigs on Days 1, 3, 5, and 7 of a follicular phase synchronized by altrenogest. Follicles were designated atretic if incidence of apoptotic granulosa cells was > or = 10% as determined by DNA fluorescence flow cytometry. Porcine follicular fluid was fractionated on 12% SDS-PAGE gels under non-reducing conditions and electroblotted to Immobilin P membranes. Inhibin forms were detected by immunoblot analysis using a mink anti-bovine inhibin alpha C1-26 gly.tyr antiserum and quantified. Immunoblots detected seven bands corresponding to inhibin forms of 44, 49, 58, 69, 121, 227, and > 227 kDa in > 91% of porcine follicular fluid samples. Three additional forms of 27, 29, and 32 kDa were detectable in only 52%, 64%, and 48% of samples, respectively. Forms > or = 69 kDa represented 83% of total inhibin immunoblot activity. The 121-kDa form was most abundant, with 39% of the total immunoblot activity in nonatretic follicles. The proportions of individual forms and total immunoblot activity pooled over days did not differ between early luteal and follicular phase follicles. Total inhibin immunoblot activity was 59% less in atretic than in nonatretic follicles. Amounts of the 44-, 49-, 69-, 121-, and 227-kDa forms were 50-80% lower (p < or = 0.05) in atretic than in nonatretic follicles. Total inhibin immunoblot activity in nonatretic follicles decreased (p < or = 0.05) by 60% during the early luteal phase but did not change significantly during the follicular phase. In nonatretic follicles, the 121-kDa form decreased (p < 0.05) during the early luteal and follicular phases. During the early luteal phase, amounts of the other forms did not change, whereas during the follicular phase the 44-kDa form increased (p < 0.05) 10-fold. In atretic follicles, neither amount nor proportion of inhibin forms differed among days. We conclude that follicular production and/or intracellular processing of inhibin dimer and/or inhibin alpha subunits changes during different phases of follicular development, supporting the notion of physiological roles for these peptides.

Androstenedione↗

PubMed1997

Decline in serum follicle-stimulating hormone concentrations alters key intrafollicular growth factors involved in selection of the dominant follicle in heifers.

Declining FSH after a transient rise coincides with selection of a dominant follicle (DF) and atresia of the remaining cohort follicles (subordinates) in cattle. The objectives of this study were to determine 1) whether intrafollicular amounts of inhibins, activin-A, insulin-like growth factor I (IGF-I), and IGF-I-binding proteins (IGFBP) are altered during selection of the first-wave dominant follicle (DF1) and 2) whether these biochemical markers are FSH dependent. Beef heifers received six or eight 6-h injections of saline (controls) or eight 6-h injections of recombinant bovine FSH (1 mg/injection) at 38 to 42 h after estrus (Day 0). Daily ultrasound scanning was used to define selection of DF1. Controls (n = 6 per group) were ovariectomized 1) on Day 3 of the estrous cycle before DF1 selection (preselection follicles) and 2) after DF1 selection on Day 4.8 +/- 0.5. In controls, FSH declined between Days 2 and 3 and selection of DF1 occurred between Days 3 and 5. During this interval, intrafollicular estradiol concentrations increased > 5-fold in DF1, yet declined 4-fold in subordinates (p < 0.05). In DF1, total IGF-I increased 1.3-fold (p < 0.05), whereas the amounts of the 40- to 47-kDa and the 35-kDa IGFBP (ligand hybridization) decreased 2.4- and 2.5-fold, respectively (p < 0.05), compared to values in preselection follicles on Day 3; total dimeric inhibin-A decreased 1.8-fold (p < 0.05). In contrast, amounts of the 30- to 32-kDa IGFBP increased 12.4-fold (p < 0.05) in subordinates on Day 4.8 compared with preselection follicles on Day 3, while the amount of inhibins > 34 kDa decreased 4- to 9-fold (p < 0.05). In FSH-treated heifers, both selection of DF1 and atresia of subordinates were delayed by 2.2 days. Preselection follicles recovered on Day 4.9 +/- 0.1 from FSH-treated heifers were similar (p > 0.05) in almost all biochemical parameters to preselection follicles from control heifers; however, they differed markedly from both DF1 and subordinate follicles recovered from control heifers on Day 4.8 +/- 0.5. In conclusion, the decline in FSH beginning after Day 2 of the heifer estrous cycle causes differential alterations in FSH-dependent growth factors and hormones within the cohort of preselection follicles, simultaneously inducing growth and enhanced estradiol-producing capacity of the DF and atresia of subordinate follicles.

Activins↗

PubMed1996

Alterations in intrafollicular levels of different molecular mass forms of inhibin during development of follicular- and luteal-phase dominant follicles in heifers.

The experiment reported here characterized changes in serum concentrations of FSH, follicular fluid (FF) concentrations of immunoreactive alpha inhibins and dimeric inhibins, and different molecular forms of inhibin during development of dominant follicles in both luteal and follicular phases of the estrous cycle in heifers. Follicular status was determined daily by ultrasound examination of ovaries, and blood samples were taken at 4-6-h intervals. Heifers (5-6 per treatment) were ovariectomized on Days 0 (follicular phase, pre-LH surge), 1-Ov (post-LH surge, preovulation), 1 + Ov (postovulation), 3 (selection phase), 6 (dominance phase), and 12 (atretic phase) of the estrous cycle. Follicles were classified on the basis of FF estradiol (E) to progesterone (P) ratio and by ultrasound morphology. Concentrations of E, P, immunoreactive alpha inhibin, LH, and FSH were determined by RIA, and concentrations of dimeric inhibins were determined by a two-site dimeric-specific immunoradiometric assay (IRMA); the different molecular mass forms of inhibin were determined by immunoblot analysis. Serum FSH was transiently elevated during Days 0.5-1.5 and 8-10.5, in association with emergence of each new wave of follicular growth. The follicular-phase dominant follicle of heifers before the preovulatory LH/FSH surge had less immunoreactive alpha inhibin and 29-kDa inhibin but more 110-kDa inhibin compared with concentrations after the surge (p < 0.05). During growth of the dominant follicle between Days 3 and 6, there were no changes in either proportions or amounts of any inhibin forms (p > 0.05). However, while concentrations of dimeric inhibins decreased between Days 1 and 6, the concentration of immunoreactive alpha inhibin increased (p < 0.05). Atresia of the luteal-phase dominant follicle during Days 6-12 was associated with a decrease in amounts and proportions of the 110- and > 160-kDa forms, an increase in amounts and proportions of the 34-kDa form, and an increase in dimeric inhibin concentration (p < 0.05). Alterations in FF E concentrations were positively correlated with changes in amounts of the 77- and 110-kDa precursor forms of inhibin, but negatively correlated with 34-kDa inhibin. The results of this experiment demonstrate that 1) seven different molecular mass forms of inhibin were present in bovine FF during all stages of development of follicular- and luteal-phase dominant follicles examined, 2) amounts and proportions of most inhibin forms are altered differently in follicular- and luteal-phase dominant follicles, 3) FF inhibins and E are regulated by different factors, and 4) RIA, IRMA, and immunoblot measurements of inhibin in bovine FF produced divergent results.

Animals↗

PubMed1996

Development of a two-site solid-phase immunochemiluminescent assay for measurement of dimeric inhibin-A in human serum and other biological fluids.

Inhibin is a heterodimeric glycoprotein that inhibits the secretion of follitropin from the pituitary and has been isolated in two distinct forms composed of a common alpha subunit and either a beta A or beta B subunit. Utilizing paired monoclonal antibodies specific to the alpha and beta A subunit, we have developed an immunochemiluminescent assay for dimeric inhibin-A. The assay is capable of quantifying free and bound inhibin-A in human serum and follicular fluid. The limit of detection is 10 ng/L. Related proteins exhibit little cross-reactivity or interference. Recovery is excellent. Whereas samples from men and postmenopausal women are near the detection limit of the assay, inhibin-A is higher in the luteal than the follicular phase of normally cycling women, 20-fold higher during in vitro fertilization treatment, and approximately 200-fold greater in pregnancy. The assay measures inhibin-A in follicular fluid from a variety of other species.

Adult↗