Biomedical subjects

P G Knight

Publications and source records attributed to P G Knight.

At least 19 recordsLinked to original sources

PubMed2003

The reproductive response of rams to thyroidectomy: mediation by impaired inhibin feedback rather than a change in LH pulsatility.

A series of experiments was conducted to examine the mechanism by which removal of the thyroid glands in seasonally suppressed rams brings about rapid testicular growth. In the first experiment, thyroidectomy at the nadir of the testicular cycle (late winter) initiated testis growth without any detectable change in the extent of spermatogenesis compared with sham-operated controls. The serum concentration of FSH, but not LH, was also markedly increased by thyroidectomy. In the second experiment, serum FSH concentration was again increased by thyroidectomy in late winter but there was no effect of thyroidectomy on LH concentration, LH pulses (measured in frequent blood samples) or testosterone concentration. Furthermore, there was no evidence of a change in central dopaminergic inhibition of GnRH, as measured by the pulsatile LH response to an i.m. injection of the dopaminergic D(2) agonist bromocriptine or antagonist sulpiride. The rapid increase in FSH concentration occurred despite a markedly increased serum inhibin A concentration in thyroidectomized rams. Therefore, the efficacy of inhibin feedback was examined by testing the FSH-suppressive effect of an inhibin preparation (5 ml charcoal-stripped bovine follicular fluid i.v.) in long-term thyroidectomized and thyroid intact castrated rams. Bovine follicular fluid suppressed FSH concentrations in control rams as expected but in marked contrast, was completely without effect in thyroidectomized animals. In castrated rams, the FSH concentration was only marginally increased by thyroidectomy, indicating that there is a major component of the mediation of the effects of thyroidectomy that is testicular in origin. It was concluded that a reduction in the ability of endogenous inhibin to inhibit FSH release at the pituitary, rather than a hypothalamic mechanism, is the primary cause of the stimulation of testis growth by thyroidectomy.

Animals↗

PubMed2003

Ovarian follicle development in the laying hen is accompanied by divergent changes in inhibin A, inhibin B, activin A and follistatin production in granulosa and theca layers.

To study the potential involvement of inhibin A (inhA), inhibin B (inhB), activin A (actA) and follistatin (FS) in the recruitment of follicles into the preovulatory hierarchy, growing follicles (ranging from 1 mm to the largest designated F1) and the three most recent postovulatory follicles (POFs) were recovered from laying hens (n=11). With the exception of <4 mm follicles and POFs, follicle walls were dissected into separate granulosa (G) and theca (T) layers before extraction. Contents of inhA, inhB, actA and FS in tissue extracts were assayed using specific two-site ELISAs and results are expressed per mg DNA. InhB content of both G and T followed a similar developmental pattern, although the content was >4-fold higher in G than in T at all stages. InhB content was very low in follicles <4 mm but increased ~50-fold (P<0.0001) to peak in 7-9 mm follicles, before falling steadily as follicles entered and moved up the follicular hierarchy (40-fold; 8 mm vs F2). In stark contrast, inhA remained very low in prehierarchical follicles (< or =9 mm) but then increased progressively as follicles moved up the preovulatory hierarchy to peak in F1 (approximately 100-fold increase; P<0.0001); In F1 >97% of inhA was confined to the G layer whereas in 5-9 mm follicles inhA was only detected in the T layer. Both inhA and inhB contents of POFs were significantly reduced compared with F1. Follicular actA was mainly confined to the T layer although detectable levels were present in G from 9 mm; actA was low between 1 and 9 mm but increased sharply as follicles entered the preovulatory hierarchy (approximately 6-fold higher in F4; P<0.0001); levels then fell approximately 2-fold as the follicle progressed to F1. Like actA, FS predominated in the T although significant amounts were also present in the G of prehierarchical follicles (4-9 mm), in contrast to actA, which was absent from the G. The FS content of T rose approximately 3-fold from 6 mm to a plateau which was sustained until F1. In contrast, the FS content of G was greatest in prehierarchical follicles and fell approximately 4-fold in F4-F1 follicles. ActA and FS contents of POFs were reduced compared with F1. In vitro studies on follicle wall explants confirmed the striking divergence in the secretion of inhA and inhB during follicle development. These findings of marked stage-dependent differences in the expression of inhA, inhB, actA and FS proteins imply a significant functional role for these peptides in the recruitment and ordered progression of follicles within the avian ovary.

Activins↗

PubMed2003

Immunohistochemical localization of inhibin/activin alpha, betaA and betaB subunits and follistatin in bovine oocytes during in vitro maturation and fertilization.

The aim of this study was to evaluate the distribution of inhibin/activin alpha, beta(A) and beta(B) subunits and follistatin in immature oocytes and in matured oocytes before and after IVF. Denuded oocytes were submitted to a whole-mount immunofluorescence procedure. Specimens were imaged and fluorescent intensities quantified by scanning laser confocal microscopy. Immunoreactivity for inhibin alpha subunit (both alpha(C) and pro-alpha regions), abundant in the ooplasm of immature oocytes, decreased after maturation (a 68% and 88% decrease, respectively; P < 0.001), but increased after IVF by 2- and 5.7-fold, respectively (P < 0.01). Intense staining for beta(A) was detected in immature oocytes (predominantly in the outer ooplasm and zona pellucida) but after maturation and fertilization it was localized mainly in the zona pellucida, perivitelline space and oolemma. Immunoreactivity for beta(A) in the ooplasm decreased by 58% after maturation (P < 0.001) but increased again by 75% after fertilization (P < 0.01). Immunoreactivity for beta(B) was localized mainly in the zona pellucida and did not change after maturation. However, immunoreactivity for beta(B) was not detected in the zona pellucida after fertilization, but remained unchanged in unfertilized oocytes. Immunoreactivity for follistatin was detected in the ooplasm and zona pellucida of immature oocytes but decreased progressively in the ooplasm after maturation (a 63% decrease; P < 0.001) and did not change after IVF. Examination of partially denuded cumulus-oocyte complexes confirmed abundant expression of alpha(C), pro-alpha, beta(A) and follistatin immunoreactivity in cumulus cells, whereas beta(B) subunit staining was weak or absent in cumulus cells, but intense in the zona pellucida. In conclusion, the present study shows that qualitative and quantitative changes in the distribution of inhibin/activin subunits and follistatin accompany oocyte maturation and fertilization. The possibility, indicated by these observations, that activin A and activin B may play distinct roles in bovine oocyte maturation and fertilization warrants further study.

Animals↗

PubMed2002

Follicle wave growth in cattle.

Ovarian follicle growth in cattle culminates in the selection of a single dominant follicle which attains the ability for final maturation and ovulation once or twice during the luteal phase and at the end of the oestrous cycle, as well as during other reproductive states. This review will describe in detail the first follicle wave of the cycle leading to selection of the first wave dominant follicle, indicating the specific gonadotrophin dependencies of cohort and dominant follicles, and relating follicle fate to steroidogenesis. As a differential gonadotrophin response of growing antral follicles during the follies-stimulating hormone (FSH) decline may determine which follicle becomes selected, first wave follicles are also characterized in relation to intrafollicular growth factors, which may modify the gonadotrophin response, such as inhibins and members of the insulin-like growth factor (IGF) family. Subsequently, the follicular control of the transient FSH rise and decline so crucial to dominant follicle selection will be discussed. It is concluded that successful hormonal manipulation of follicle wave growth and dominant follicle selection will depend on our detailed understanding of the gonadotrophin requirements of differentiating wave follicles.

Animals↗

PubMed2002

Day 5 inhibin B levels in a treatment cycle are predictive of IVF outcome.

BACKGROUND: Day 5 serum inhibin B during IVF treatment has been investigated as a predictor of outcome. METHODS: A total of 54 women (< or = 39 years, normal menses and endocrine profiles) were treated with urinary gonadotrophins or recombinant FSH following pituitary down-regulation. Serum day 3 FSH in a preceding cycle was <8.5 IU/l. Plasma inhibin B, inhibin A and estradiol were determined after 4 days of gonadotrophin administration (day 5). RESULTS: Day 5 inhibin B was the most highly correlated with the number of mature follicles (>14 mm), oocytes retrieved and fertilized. Receiver operating characteristic analysis gave high accuracy for day 5 inhibin B in predicting ovarian response and indicated that a threshold of 400 pg/ml may be helpful in the decision as to whether to continue treatment. Women with <400 pg/ml (n = 16) had lower numbers of follicles, mature follicles, oocytes retrieved, fertilized and cleaved compared with those >400 pg/ml (n = 36) and this threshold gave a positive likelihood ratio of 30, 92.9% sensitivity, 95.0% specificity and 86.7% positive predictive value to detect poor ovarian response. Day 5 inhibin B was the best predictor of pregnancy (no live births and four cycles cancelled, low inhibin group; nine live births and no cancelled cycles, high inhibin group). CONCLUSIONS: Normogonadotrophic, normogonadal women with day 5 inhibin B <400 pg/ml in down-regulated cycles have a poor response to ovarian stimulation and are less likely to conceive compared with women with higher day 5 inhibin B.

Adult↗

PubMed2002

Modulation of sex hormone secretion in cows by acute infection with bovine viral diarrhoea virus.

Bovine viral diarrhoea virus (BVDV) is a major pathogen of cattle and is responsible for considerable reproductive loss. In this study, the in vivo responses in six multiparous cows were investigated after a non-cytopathogenic BVDV challenge (strain Pe 515; 5 x 10(6) tissue culture infective dose 50) given 9 days before a synchronized ovulation. Six similar cows challenged with non-infectious culture medium served as controls. The experimental noncytopathogenic BVDV infection was followed by a viraemia and leucopenia at days 5-9 after challenge, but no other clinical signs of infection were detected. However, the BVDV infection altered endocrine function. Mean LH pulse frequency immediately before CIDR withdrawal was lower (P < or = 0.05) in the BVDV-infected (2.17 +/- 0.34 pulses per 8 h) compared with the sham-infected (4.83 +/- 1.04 pulses per 8 h) animals. At day 3 after CIDR withdrawal, plasma oestradiol concentrations remained high (P < 0.05) in the infected cows (2.19 +/- 0.51 pg ml(-1)) compared with the sham-infected controls (0.72 +/- 0.29 pg ml(-1)). However, there was no difference in the peak oestradiol concentration (BVDV: 2.31 +/- 0.29 versus sham: 2.34 +/- 0.41 pg ml(-1)). In addition, non-cytopathogenic BVDV significantly (P < 0.05) increased the duration of the interval between ovulation and onset of the postovulatory progesterone increase (values 1.0 ng ml(-1)) (BVDV: 3.0 +/- 0.26 versus sham: 4.0 +/- 0.26 days). The viral infection also significantly (P < 0.01) decreased mean plasma progesterone concentrations between day 3 and day 11 after ovulation (BVDV: 2.59 +/- 0.32 versus sham: 4.13 +/- 0.27 ng ml(-1)). These data show that non-cytopathogenic BVDV viraemias during the follicular phase can modulate the secretion of gonadotrophins and sex steroids, in particular progesterone, during a synchronized oestrous cycle. Therefore, viraemias during the follicular phase may reduce the fertility of cattle by disrupting the capacity of the ovulatory follicle to form a competent corpus luteum, thereby compromising early embryo development and maternal recognition of pregnancy.

Acute Disease↗

PubMed2002

Modulatory effects of gonadotrophins and insulin-like growth factor on the secretion of inhibin A and progesterone by granulosa cells from chicken preovulatory (F1-F3) follicles.

The aim of this study was to compare the actions and interactions of gonadotrophins (LH and FSH) and an analogue of insulin-like growth factor I (LR3-IGF-I) on the secretion of inhibin A, inhibin B and progesterone by cultured chicken granulosa cells derived from the three largest (F1--F3) follicles of the preovulatory hierarchy. Treatment with LH or FSH promoted marked dose-(P < 0.0001) and time- (P < 0.0001) dependent increases in both inhibin A and progesterone secretion, with the magnitude of response (< 15-fold compared with basal) increasing over time in culture. Concentrations of inhibin B were below the detection limit in all samples. Initially, F1 cells were more LH-responsive than were F3 cells in terms of progesterone secretion (P < 0.02) but this difference between follicles decreased over time in culture. In contrast, LH-induced inhibin A secretion tended to be highest from F3 cells, although this was not significant. Cells from F3 follicles were consistently more FSH-responsive than F1 cells in terms of both progesterone (P < 0.01) and inhibin A (P < 0.02) secretion. Initially, F1 cells were more responsive to LR3-IGF-I than were F3 cells in terms of progesterone secretion (P < 0.001) but were less responsive in terms of inhibin A secretion (P < 0.001). Again, these inter-follicle differences decreased over time in culture (not significant on day 3 of treatment). Co-treatment experiments showed that LR3-IGF-I enhanced both LH- and FSH-induced secretion of inhibin A and progesterone in a time- (P < 0.001) and follicle- (P < 0.001) dependent way. Initially, F1 cells showed highest LR3-IGF-I enhancement of LH-induced inhibin A and progesterone secretion; in contrast, F3 cells showed the highest LR3- IGF-1 enhancement of FSH-induced inhibin A and progesterone secretion. These inter-follicle differences persisted over time in the case of FSH-induced hormone responses but not in the case of LH-induced responses, even though the relative degree of LR3-IGF-I enhancement increased markedly over time. Collectively, these data support a positive role for IGF-I, presumably of thecal origin, as an amplifier of gonadotrophin action on granulosa cell inhibin A and progesterone production by preovulatory chicken follicles.

Animals↗

PubMed2002

Effects of ovarian follicle ablation on FSH, oestradiol and inhibin A concentrations and growth of other follicles in sheep.

The aim of this study was to examine the effect of removal of the largest follicle or all visible follicles during the first follicle wave on subsequent follicular growth, steroid, inhibin A and gonadotrophin secretion in sheep. On day 4.5 of a synchronized oestrous cycle, ewes (n = 18) were assigned to one of three groups which underwent either no treatment (control), ablation of the largest follicle (largest follicle aspirated and cauterized via laparotomy) or ablation of all follicles (all visible follicles ablated). Between day 0 and day 10 of the oestrous cycle, blood samples were collected every 8 h and ovaries were examined daily using transrectal ultrasonography. The lifespan of the second largest follicle (number of days > 3 mm in diameter) was longer (6.7 +/- 0.9 days; P < 0.05) and the maximum diameter tended to be greater (4.8 +/- 0.3 mm; P = 0.07) in ewes in which the largest follicle was ablated than in the control ewes (3.8 +/- 0.4 days; 4.2 +/- 0.3 mm). There was no difference in the day of emergence of the second follicular wave between groups (day 6.9 +/- 0.4). However, the peak of the transient increase in FSH concentrations after ablation was earlier (day 5.67 +/- 0.15; P < 0.05) in ewes in which all follicles were ablated than in control ewes (day 6.72 +/- 0.36); the timing in ewes that had only the largest follicle ablated was intermediate (day 6.11 +/- 0.28). Serum inhibin A concentrations were about three-fold lower (P < 0.05) in both follicle ablation groups than in the control group. The numbers of follicles 2-3 mm in diameter during the first 3 days of the second follicular wave were greater in 'ablated ewes' (both groups had 2.6 +/- 0.2 follicles day-1) than in control ewes (1.7 +/- 0.3 follicles day-1). It is concluded that: (i) transient increases in FSH concentrations precede the emergence of follicle waves; (ii) ablation of all follicles on day 4.5 after oestrus advanced the timing of the next peak in FSH concentrations and the numbers of small follicles associated with the development of the second follicular wave; and (iii) ablation of the largest follicle resulted in an increase in the lifespan of the second largest follicle, indicating a regulatory role of large dominant follicles over smaller subordinate follicles.

Analysis of Variance↗

PubMed2002

Differential effects of activin A on basal and gonadotrophin-induced secretion of inhibin A and progesterone by granulosa cells from preovulatory (F1-F3) chicken follicles.

Previous work has shown that activin A is expressed selectively within the theca rather than the granulosa layer of preovulatory chicken follicles. In the present study, this finding was verified and the potential paracrine actions of activin A on basal and gonadotrophin-induced secretion of inhibin A, inhibin B and progesterone by granulosa cells from the three largest preovulatory follicles (F1-F3) were investigated. Treatment with activin A (0, 0.25, 2.5 and 25 ng ml(-1)) alone increased inhibin A secretion markedly in a follicle- and time-dependent manner, with the greatest response (up to 15-fold increase; P < 0.0001) in F1 follicles after 3 days of treatment. In contrast, activin A alone had no effect on progesterone output at any time. Cells from F3 follicles were more responsive to FSH than were F1 cells in terms of both inhibin A (P < 0.02) and progesterone (P < 0.01) secretion. Furthermore, activin A greatly enhanced FSH-induced secretion of both inhibin A (up to tenfold; P < 0.0001) and progesterone (up to sixfold; P < 0.0001). In terms of LH-induced inhibin A and progesterone secretion, cells from F1, F2 and F3 follicles showed similar responses. Co-treatment with activin A enhanced LH-induced secretion of inhibin A markedly (up to ninefold; P < 0.0001) but had only a marginal effect on LH-induced progesterone secretion (up to twofold; P < 0.001). The presence of activin receptor subtypes IA, IB, IIA and IIB in cultured granulosa cells from F1, F2 and F3 follicles was demonstrated using immunocytochemistry. These findings support the hypothesis that activin A secreted by the theca layers of avian preovulatory follicles exerts a local paracrine action on granulosa cells to modulate 'basal' inhibin A secretion and to upregulate gonadotrophin-induced secretion of both inhibin A and progesterone. However, the extent to which this local role of activin A contributes to the generation of the preovulatory LH-progesterone surge remains to be established.

Activin Receptors↗

PubMed2001

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.

Activins↗

PubMed2001

The effect of gonadotrophins with differing LH/FSH ratios on the secretion of the various species of inhibin in women receiving IVF.

We have measured secretory patterns of inhibin A, B, total alpha inhibin, pro-alphaC inhibin and oestradiol in women following pituitary suppression who were randomised into two groups to receive either urinary gonadotrophin (25:75 IU/ampoule of luteinizing hormone (LH) and follicle stimulating hormone (FSH; Normegon; n = 11) or recombinant (r)FSH (75 IU/ampoule of FSH alone, n = 16). The women were of similar age (approximately 33 years) and length of infertility (approximately 4 years) and had a normal endocrine evaluation. Plasma FSH, LH, oestradiol, inhibin A, B, pro-alphaC and total alpha inhibin were measured by immunoassay prior to and following gonadotrophin stimulation. Immunoactive FSH, LH and oestradiol blood concentrations following pituitary down regulation were similar in the two groups being <2.0, <3.6 IU/l and <82 pmol/l respectively. The units of FSH given (2230 versus 2764 IU; Normegon versus rFSH), duration of treatment (9.1 versus 9.4 days) and number of follicles of > or =14mm on the day of human chorionic gonadotrophin (HCG) administration (17 versus 14) were also similar. Inhibin A or B concentrations rose similarly during Normegon or rFSH administration, peaking at days 9-11. Total alpha and pro-alphaC inhibin concentrations were lower (P < 0.05) in the rFSH group during days 10 and 11 of treatment being 18.9 +/- 15.9 ng/ml (Normegon) and 4.6 +/- 2.8 ng/ml (rFSH) for total alpha inhibin and 8.5 +/- 6.8 ng/ml (Normegon) and 2.8 +/- 1.6 ng/ml (rFSH) for pro-alphaC inhibin on day 10. Overall, higher total alpha inhibin concentrations were associated with more mature follicles and oocytes, greater fertilization rates and better quality embryos. We conclude that inhibin A and B secretion was similar in both groups and is primarily controlled by FSH, whereas total alpha inhibin and pro-alphaC increased preferentially in the Normegon group over the rFSH group, indicating that they are, in part, stimulated by LH.

Adult↗

PubMed2001

Relationship between follicle size and gonadotrophin surge attenuating factor (GnSAF) bioactivity during spontaneous cycles in women.

BACKGROUND: We have previously demonstrated that follicles < or =11 mm diameter from women undergoing IVF contain higher concentrations of gonadotrophin surge attenuating factor (GnSAF) bioactivity than large follicles from the same ovaries. METHODS: To determine whether this finding is relevant to spontaneous cycles, follicular fluid aspirated from 37 follicles between 3 and 25 mm in diameter from 14 pairs of ovaries from regularly cycling women undergoing total abdominal hysterectomy and bilateral salpingoophorectomy for benign gynaecological disease was pooled into size categories (3 + 4, 5 + 6, 7 + 8, 9 + 10, 11 + 12, 14 + 15, 18 and 25 mm). These pools were bioassayed for GnSAF and inhibin-A, inhibin-B and activin-A concentrations were determined. RESULTS: Follicles of 5 + 6 mm diameter contained the highest concentrations of GnSAF bioactivity (reducing GnRH-induced LH secretion to 38 +/- 8% of control, P < 0.001), while those of 25 mm diameter contained one quarter of this concentration (reducing GnRH-induced LH secretion to 72 +/- 2% of control, P < 0.05). GnSAF bioactivity was closely related to follicle size (r = -0.836, P < 0.01), but not to inhibin-A, inhibin-B or activin-A concentrations. CONCLUSIONS: The finding that small follicles contain high concentrations of GnSAF bioactivity, which fall as folliculogenesis progresses during spontaneous cycles, support the hypothesis that GnSAF has a role in preventing the premature onset of the LH surge in women.

Activins↗

PubMed2001

Changes in plasma inhibin A levels during sexual maturation in the female chicken and the effects of active immunization against inhibin alpha-subunit on reproductive hormone profiles and ovarian function.

Inhibins and activins are firmly implicated in the control of pituitary FSH secretion and ovarian follicular development in mammals. As in mammals, inhibin A and activin A are expressed in the preovulatory follicles of birds, and a defined ovulation cycle for inhibin A has recently been demonstrated in the laying hen. To investigate further the role of inhibin-related proteins in developing pullets, circulating concentrations of inhibin A, inhibin B, total immunoreactive inhibin alpha-subunit (ir-alpha), activin A, LH, FSH, and progesterone were measured from the juvenile state through to sexual maturity in 22 birds. In the 11 birds assigned to control groups, plasma inhibin A levels were low from 7 to 13 wk of age rising about threefold to a peak at Week 19 after which levels fell slightly to a plateau level characteristic of adult hens. Plasma inhibin A levels were negatively correlated with FSH (r = -0. 33; P: < 0.001) and positively correlated with progesterone (r = 0. 67; P: < 0.001) and ir-alpha (r = 0.53; P: < 0.001). Plasma ir-alpha levels were much higher than inhibin A levels although the relative differences varied with age. Plasma levels of inhibin B and activin A were below assay detection limits at all times. The remaining group of 11 birds was actively immunized (IMM) against a synthetic chicken inhibin alpha-subunit peptide (amino acids 1-26). The IMM generated circulating antibodies that bound native bovine inhibin A but altered neither plasma FSH nor progesterone levels relative to control birds at any stage of development nor the timing of first oviposition in week 19. Apart from a transient decline 1 wk after primary IMM, plasma LH concentrations did not differ from controls. Comparison of the numbers and size-class distribution of ovarian follicles at 29 wk showed an approximate twofold increase in the number of 8- to 9.9-mm-diameter follicles (control; 1.82 +/- 0.44 vs. IMM; 3.91 +/- 0.89; P: < 0.05), a size class that corresponds to follicles that have just joined the preovulatory hierarchy. The numbers of growing follicles in other size-classes and the sizes of hierarchical F(1)-F(7) follicles were not altered by IMM. However, the number of postovulatory follicles increased (control 3.73 +/- 0. 20 vs. IMM 5.55 +/- 0.28; P: < 0.01), and significantly more (P: < 0. 02) immunized hens laid two eggs within a 24-h period on at least one occasion (control 1 of 11 vs. IMM 9 of 11). The IMM increased (P: < 0.05) activin A content of F(1) and F(2) theca layers and decreased (P: < 0.05) activin A content in F(3) and F(4) granulosa layers, raising the possibility of a local intraovarian role of activin in mediating the response to IMM. These findings support a role for inhibin A in regulating the entry of follicles into the preovulatory hierarchy in the chicken, although further studies are required to establish the mechanism by which inhibin IMM increases the rate of follicle selection and ovulation without raising plasma FSH.

Activins↗

PubMed2001

Plasma inhibin A in heifers: relationship with follicle dynamics, gonadotropins, and steroids during the estrous cycle and after treatment with bovine follicular fluid.

The relationship between follicle growth and plasma inhibin A, FSH, LH, estradiol (E), and progesterone was investigated during the normal bovine estrous cycle and after treatment with steroid-free bovine follicular fluid (bFF) to arrest follicle development. In the first study, four heifers were monitored over three prostaglandin (PG)-synchronized cycles. Blood was collected every 2-8 h, and ovaries were examined daily by ultrasonography. Inhibin A was measured using a modified enzyme-linked immunosorbent assay that employed a new monoclonal antibody against the alpha subunit of bovine inhibin. Plasma inhibin A ( approximately 50 pg/ml before luteolysis) rose steadily during the induced follicular phase (P < 0.05) to a peak ( approximately 125 pg/ml) coincident with the preovulatory E/LH/FSH surge. After ovulation, inhibin A fell sharply (P < 0.05) to a nadir ( approximately 55 pg/ml) coincident with the secondary FSH rise. During the next 3 days, inhibin A increased to approximately 90 pg/ml in association with growth of the new dominant follicle (DF). Plasma E also rose twofold during this period, whereas FSH fell by approximately 50%. Inhibin A was negatively correlated with FSH (r = -0.37, P < 0.001) and positively correlated with E (r = 0.49, P < 0.0001). Observations on eight cycles (two cycles/heifer), in which growth of the ovulatory DF was monitored from emergence to ovulation, showed that the first-wave DF (DF1) ovulated in three cycles and the second-wave DF (DF2) in five cycles. After PG, plasma inhibin A and E increased similarly in both groups, with concomitant falls in FSH. In the former group, the restricted ability of DF1 to secrete both inhibin A and E was restored after luteolysis. Results indicate that dynamic changes in the secretion of both E and inhibin A from the DF contribute to the fall in FSH during the follicular phase and to the generation and termination of the secondary FSH surge, both of which play a key role in follicle selection. In the second study, bFF (two dose levels) was administered to heifers (n = 3-4) for 60 h starting from the time of DF1 emergence. Both doses suppressed FSH (P < 0.05) and blocked DF1 growth to the same extent (P < 0.01), although inhibin A levels were only marginally raised by the lower dose (not significant compared to controls). The high bFF dose raised (P < 0.001) inhibin A to supraphysiological levels ( approximately 1 ng/ml). A large "rebound" rise in FSH occurred within 1 day of stopping both treatments, even though the inhibin A level in the high-dose bFF group was still approximately threefold higher than that in controls. This indicates that desensitization of gonadotropes to inhibin negative feedback is a contributory factor, together with reduced ovarian output of E, in generation of the post-bFF rebound in FSH.

Animals↗

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↗

PubMed2001

Interactions between follicle-stimulating hormone and growth factors in modulating secretion of steroids and inhibin-related peptides by nonluteinized bovine granulosa cells.

The aim was to investigate potential interactions between FSH and intraovarian growth factors in modulating secretion of inhibin A (inh A), activin A (act A), follistatin (FS), estradiol (E2), and progesterone (P4) by bovine granulosa cells cultured under conditions in which a nonluteinized FSH-responsive phenotype is maintained. Cells from 4- to 6-mm follicles were cultured in serum-free medium containing insulin (10 ng/ml) and androstenedione (10(-7) M), and effects of ovine FSH (0.037-3 ng/ml) were tested alone and in combination with insulin-like growth factors (IGF) (LR3 IGF-I analogue; 2-50 ng/ml) and epidermal growth factor (EGF; 0.1-10 ng/ml). Medium was changed every 48 h and cultures ended after 144 h, when cell number was determined. Between 48-96 h and 96-144 h, FSH promoted (P < 0.0001) increases in output of inh A (6-fold), act A (15-fold), FS (6-fold), and E2 (18-fold), with maximal responses (in parentheses) elicited by 0.33 ng/ml FSH during the final period. Higher FSH doses (1 and 3 ng/ml) gave reduced responses for each of the above hormones, whereas P(4) output was maximal (3-fold) at these doses. FSH promoted a slight increase in cell number ( approximately 1.7-fold; P < 0.001). LR3 IGF-I alone markedly increased (P < 0.0001) output of inh A (8-fold), act A (41-fold), FS (12-fold), and E2 (18-fold); this was accompanied by modest increases (P < 0.01) in P4 output ( approximately 2.5-fold) and cell number ( approximately 2-fold). Whereas FSH enhanced inh A, act A, FS, and E2 secretion evoked by lower doses of LR3 IGF-I, it suppressed (P < 0.001) the response to the highest dose. EGF alone promoted a 1.7-fold increase in cell number (P < 0.001) without affecting hormone release; however, it abolished (P < 0.001) FSH-induced secretion of inh A, act A, FS, and E2. Both FSH alone and LR3 IGF-I alone dose-dependently increased the act A:FS ratio ( approximately 3-fold; P < 0.005) and act A:inh A ratio (3-fold to 6-fold; P < 0.001), suggesting that both factors selectively raise activin "tone" and that this could be a key requirement for FSH and IGF-induction of follicular E2 production. This hypothesis was reinforced by the finding that addition of FS, to reduce the act A:FS ratio and sequester secreted activin, markedly suppressed (P < 0.001) FSH (3-fold)-, and LR3 IGF-I (2-fold)-induced E2 output.

Activins↗

PubMed2001

Potential local regulatory functions of inhibins, activins and follistatin in the ovary.

The changing pattern of granulosa cell expression of inhibin/activin subunits and follistatin during follicle development and their differential regulation by extrinsic and intraovarian factors supports evidence from functional studies, mostly in vitro, that these proteins have important roles in folliculogenesis, oocyte maturation and corpus luteum function. Gonadal inhibins function as negative feedback hormones to regulate the synthesis and secretion of pituitary FSH, a key determinant of follicle development, but there is little supportive evidence for a peripheral endocrine role for ovary-derived activins or follistatin in this regard. However, activins and follistatin are expressed in numerous other tissues, including anterior pituitary, and they are firmly implicated as local intrapituitary regulators of FSH secretion. Intraovarian actions of granulosa cell-derived activins include the promotion of granulosa cell proliferation and upregulation of FSH receptors, P450arom, oestrogen synthesis, granulosa cell LH receptors and enhancement of oocyte maturation. Through its activin-binding role, follistatin can reverse each of these activin-induced responses. In addition to their endocrine feedback role, granulosa-derived inhibins can sensitize theca cells to LH, thereby enhancing the production of androgens, an essential requirement for follicular oestrogen synthesis. Activins can oppose this effect and suppress thecal androgen production. Granulosa cells overproduce inhibin a subunit precursor relative to betaA/betaB subunit precursors and evidence indicates that different parts of the inhibin a subunit precursor have intrinsic biological activities distinct from inhibin alphabetaA/B dimer, and serve as additional local modulators of follicle and corpus luteum function.

Activins↗

PubMed2001

Inter-relationships among gonadotrophins, reproductive steroids and inhibin in freemartin ewes.

Freemartins are sterile XX/XY chimaeras that occur as a result of placental fusion between male and female fetuses during early pregnancy. Freemartins occur predominantly in cattle, although the prevalence of ovine freemartinism is increasing. In this study, the reproductive endocrinology of ovine freemartins was compared with that of normal sheep. Freemartins had significantly (P < 0.001) higher basal concentrations of LH and FSH than did normal ewes or rams, although the response of LH to GnRH (10 microg) was similar in freemartins, ewes and rams. Resting concentrations of oestradiol were similar in freemartins and ewes and were increased in both after eCG administration. Testosterone concentrations were higher in freemartins than in ewes, but were unresponsive to GnRH or eCG. Administration of 62.5 mg progesterone or 25 lg oestradiol twice a day for 3 days suppressed LH concentrations to baseline values in freemartins, ewes and rams. In ewes, 500 microg oestradiol administered twice a day caused preovulatory surges in LH concentrations, but suppressed LH in freemartins to baseline values. Thus, LH secretion can potentially be regulated in freemartins by gonadal steroids. FSH concentrations in freemartins were not suppressed by doses of inhibin that were effective in ewes and rams. Therefore, freemartins behave in part like castrated animals, as they have high basal concentrations of LH and FSH, which can be stimulated by GnRH and suppressed by gonadal steroids. Conversely, inhibin does not suppress FSH concentrations in freemartins, and freemartins have circulating concentrations of steroids intermediate between those of castrated and normal animals.

Animals↗