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K. P. McNatty, D. A. Heath, S. Lun and N. L. Hudson

ABSTRACT

Granulosa cells from ovarian follicles (≥ 1 mm diameter) in Booroola ewes which are homozygous (FF) or heterozygous (F+) for the F gene have previously been shown to produce significantly more cAMP in response to FSH or LH than those from similar sized follicles in ewes without the F gene (++). The aim of these studies was to test whether these F gene-specific differences arose because of differences in cAMP-phosphodiesterase (cAMP-PDE) activity.

In the first study using 1 μmol cAMP/1 as substrate, no F gene-specific effects were noted in cAMP-PDE activity in granulosa cells from small (1–2·5 mm diameter, n = 4 per genotype) or large (≥ 3 mm diameter, n = 4 per genotype) follicles from FF, F+ or ++ ewes, despite F gene-specific effects in FSH (1 μg/ml)and LH (0·1 μg/ml)-induced cAMP accumulation in these same cell preparations. The overall mean levels of cAMP-PDE across all genotypes in cells from small and large follicles were 0·47± 0·04 (s.e.m., n = 12) and 0·28 ± 0·03 pmol cAMP/106 cells per min respectively; the mean PDE activity in cells from small follicles was significantly (P < 0·05) higher compared with that in cells from large follicles. In a second study, granulosa cells from each genotype were pooled over all follicle sizes (≥ 1 mm diameter, one pool per genotype) and the rates of cAMP hydrolysis tested over a range of substrate concentrations (0–16 μmol/l) but no genespecific differences with respect to the Michaelis constant and maximum velocity were noted. In a third study, the rates of FSH-induced cAMP accumulation by granulosa cells from small and large follicles of FF and ++ ewes were examined in the presence of a PDE inhibitor, 1-methyl-3-isobutylxanthine (0·2 mmol/l). In this study FSH (1 μg/ml) stimulated significantly more cAMP in cells from FF compared with ++ ewes in both small and large follicles. In cells from small follicles, the mean ± s.e.m. FSHstimulated cAMP levels were 4·7 ± 0·8 (n = 6) for FF ewes and 1·8 ± 0·4 (n = 8) pmol/106 cells per h for ++ ewes (P < 0·025). In large follicles, the cAMP levels were 17·0 ± 3·5 (n = 6) for FF ewes and 6·3 ± 0·5 pmol/106 cells per h for ++ ewes (P < 0·05).

Collectively, these data suggest that the F genespecific difference in cAMP synthesis is probably the result of an event(s) associated with its formation rather than its degradation.

Journal of Endocrinology (1989) 120, 287–293

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K. P. McNATTY, CAROLYN DOBSON, MARION GIBB, LINDA KIEBOOM and D. C. THURLEY

The temporal relationships between the levels of LH in peripheral plasma and in follicular fluid of ovarian follicles in anaesthetized sheep were investigated for a 10-h period after a single i.m. injection of LH releasing hormone (LH-RH; 100 μg). The ovarian secretion rates of oestradiol and androstenedione and the levels of these steroids accumulating in different sized follicles at varying time-intervals after the LH-RH injection were also compared.

The data show that the rates at which pituitary LH enters and leaves the intrafollicular fluid-filled spaces are substantially slower than those of peripheral blood. Two hours after LH-RH injection the levels of LH in plasma had increased from 1 to 200 ng/ml, whereas in the follicle the levels remained at approximately 2 ng/ml. Ten hours after the LH-RH injection, the levels of LH in plasma had returned to basal values (∼1·4 ng/ml) but in both small and large follicles the levels of LH (∼20 ng/ml) were comparable to those present in similar sized follicles 4 h earlier.

The data also indicate that more than 90% of the oestradiol produced by a large antral follicle (≥5 mm diameter) probably enters the bloodstream without first accumulating within the follicular antrum. Finally it is concluded that the clearance of the small amount of oestradiol which does accumulate in the follicular antrum is negligible compared with the clearance of this hormone from peripheral plasma.

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K. P. McNATTY, W. M. HUNTER, A. S. McNEILLY and R. S. SAWERS

SUMMARY

The concentrations of FSH, LH, prolactin, oestradiol and progesterone were measured in peripheral plasma and follicular fluid of women throughout the menstrual cycle. With the exception of prolactin, concentrations of pituitary and steroid hormones in follicular fluid correlated with those in peripheral plasma.

Follicle-stimulating hormone was present in a greater number of small follicles ( < 8 mm) during or just after the peaks of FSH in peripheral plasma. During the mid-follicular phase the concentration of both FSH and oestradiol in fluid from large follicles ( ≥ 8 mm) was high. During the late follicular phase the large follicles ( ≥ 8 mm) contained high amounts of progesterone in addition to oestradiol, low physiological levels of prolactin, and concentrations of LH and FSH about 30 and 60% respectively of those found in plasma. By contrast no large 'active' follicles ( ≥ 8 mm) were found during the luteal phase although many contained both LH and FSH. Luteinizing hormone was present in a proportion of small follicles ( < 8 mm) during the late follicular and early luteal but not at other stages of the menstrual cycle.

It is suggested that a precise sequence of hormonal changes occur within the microenvironment of the developing Graafian follicle; the order in which they occur may be of considerable importance for the growth of that follicle and secretory activity of the granulosa cells both before and after ovulation.

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K. P. McNatty, N. Hudson, M. Gibb, K. M. Henderson, S. Lun, D. Heath and G. W. Montgomery

ABSTRACT

The plasma concentrations of LH and prolactin and various parameters of ovarian function were examined in cows on known days of the oestrous cycle during May and June (autumn and winter) and during October (spring).

Luteinizing hormone peak frequency and plasma prolactin concentrations were significantly higher in October than during the May–June period (LH, P<0·05; prolactin, P<0·01). The mean diameters of large healthy follicles (≥8 mm diameter) and the dominant oestrogen-secreting follicles were significantly larger (P<0·01 for both follicle types) and each follicle contained more granulosa cells (both P<0·01) in May–June than in October. The LH responsiveness of theca interna with respect to androstenedione production and the levels of aromatase activity in granulosa cells did not differ with time of year. The corpora lutea were heavier (P<0·05) and secreted more progesterone (P<0·01) in May–June than in October.

It is concluded that seasonal differences in ovarian activity exist in cows and that these differences are probably the consequence of seasonal differences in gonadotrophin secretion.

J. Endocr. (1984) 102, 189–198

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K. P. McNatty, N. L. Hudson, D. A. Heath, L. Shaw, L. Blay, L. Berry and S. Lun

ABSTRACT

This study in ewes examined the effects on ovarian function of a pulsatile regimen of ovine FSH (NIADDK-oFSH-17) administered over a 24- to 28-day period beginning on day 1 of the oestrous cycle (day 0 = oestrus). The FSH (1·66 μg or 5·00 μg) was administered i.v. over a 1-min interval once every hour throughout the treatment period. In other ewes ovine LH (NIDDK-oLH-23) was administered (10 μg once every 2 h) for 24–28 days together with oFSH (1·66 μg/h).

Compared with untreated controls (n = 19 ewes), FSH alone at both doses (n = 19 ewes/dose) as well as the FSH +LH treatment (n=10) led to significant increases in the plasma concentrations of FSH (P <0·01), ovarian weight (P <0·05) and ovulation rate (P <0·01) but there was no change in the mean weight of individual corpora lutea (CL). Exogenous FSH at the high but not the low dose alone or with LH stimulated a significant overall increase in plasma inhibin concentrations (P <0·05). The geometric mean (and 95% confidence limits) ovulation rates in the high FSH (i.e. 5·00 μg/h), low FSH (i.e. 1·66 μg/h), low FSH+LH, and control treatment groups were 15·3 (9·3, 24·8), 3·7 (2·1, 6·0), 3·7 (2·5, 5·8) and 1·4 (1·2, 1·7) respectively. The FSH or FSH+LH treatments did not alter the total numbers of antral follicles (≥1 mm diameter). However, the high but not the low FSH or low FSH + LH treatment led to significant increases in the mean numbers of large follicles (i.e. >4·5 mm diameter; P<0·01) and a higher proportion of non-atretic antral follicles.

Highly significant linear relationships were found between the mean plasma concentrations of FSH or inhibin and the ovulation rate (FSH: r=0·74, P<0·0001; inhibin: r=0·93, P<0·0001). Highly significant linear relationships were also found between the plasma concentrations of FSH or inhibin and the number of large follicles (i.e. >4·5 mm diameter; FSH, r=0·78, P<0·0001; inhibin, r=0·80, P<0·0001) and between the plasma concentrations of inhibin and the number of granulosa cells in large follicles (r=0·78, P<0·0001). After the high FSH but not the low FSH treatment there were significant increases in both FSH- and LH-induced responsiveness in granulosa cells with respect to cyclic AMP synthesis in vitro. In the high FSH treatment group, granulosa cells from 1–2·5 mm diameter follicles were responsive to LH whereas, in the low FSH or FSH + LH treatment groups and the controls, granulosa cells were not responsive to LH until the follicles were >4·5 mm in diameter. FSH or FSH+ LH treatment did not lead to increases in aromatase activity in granulosa cells (i.e. when expressed on a per cell basis) or to increases in oestradiol in follicular fluid.

Collectively these results show that chronic increases in plasma FSH concentrations influence, in a dose–responsive manner, the size distribution of antral follicles, the proportion of non-atretic follicles, the number of follicles with peak aromatase activity and the ovulation rate, without altering the total number of antral follicles, the granulosa cell composition of individual follicles or the sizes of individual CL. Exogenous FSH treatment at high but not low doses enhanced the sensitivities of granulosa cells to both FSH and LH in vitro. Increases in plasma FSH also led to higher concentrations of plasma inhibin as a consequence of an increase in the number of large follicles and thus the number of granulosa cells.

Journal of Endocrinology (1993) 138, 315–325

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Julia M Young, Jennifer L Juengel, Kenneth G Dodds, Mhairi Laird, Peter K Dearden, Alan S McNeilly, Kenneth P McNatty and Theresa Wilson

Bone morphogenetic proteins (BMPs) have been shown to influence the regulation of FSH synthesis and secretion at the level of the pituitary. Primary pituitary cells were harvested and cultured from Booroola ewes homozygous for a mutation in activin receptor-like kinase 6 (ALK6) also known as BMP receptor IB (BMPRIB), and from wild-type (WT) ewes to determine if the mutation caused alterations in FSH secretion in vitro. The cells were collected 24 h following induction of luteolysis and cultured for 72 h prior to being challenged for 24 h with BMP2, BMP4, BMP6, growth and differentiation factor-9 (GDF9), transforming growth factor-β 1, activin-A and GnRH. The levels of FSH and LH were measured by RIA and then compared with the untreated controls. Primary pituitary cell cultures from Booroola ewes secreted less FSH than WT cells in the presence of BMP2, BMP4 and BMP6. These BMPs did not affect the FSH stores within the cells, or the levels of LH released. GDF9 appeared to act in a BMP-like manner by suppressing FSH secretion. The ALK6 receptor however, was not found to co-localise with gonadotroph cells in either Booroola or WT pituitary tissues. These findings imply that the increased sensitivity of Booroola cells to BMP2, BMP4, BMP6 and GDF9 cannot be due to the direct action of the ALK6 mutant Booroola receptor in the cells that synthesise FSH.