Abstract. The effects of preincubating rat granulosa cells with FSH, LH, and Prl on subsequent Prl mediated progesterone secretion were investigated. Granulosa cells were isolated from ovarian follicles 50 h after injection of 5 IU PMSG and were then plated on poly-l-lysine coated coverslips in serum supplemented medium. Cells were preincubated for 24 h in the absence of hormones (control) or with the addition of either 0.25, 2.5, 25 ng/ml rat FSH or rat LH, or 1 μg/ml rat Prl. Following the preincubation period, cells were maintained for an additional 6 or 8 days in the presence or absence of 1 μg/ml Prl. When cells were preincubated with FSH or LH, only the two higher concentrations (2.5 and 25 ng/ml) stimulated significantly more progesterone secretion than control cultures during the 24 h preincubation period. For each series of preincubations, cells cultured for 6 or 8 days in the presence of Prl secreted significantly more progesterone at each day of culture than cells cultured without Prl. Cells preincubated and cultured with Prl secreted only 3–7-fold more progesterone than cells preincubated in control medium and then cultured with Prl. Preincubation with FSH or LH promoted a 20–45-fold increase in Prl mediated progesterone secretion compared to control preincubation cultures that also subsequently were cultured with Prl. The magnitude of Prl mediated progesterone secretion observed through 6 days of culturing was dose dependent on the preincubation concentration of FSH or LH. The establishment of an in vitro model system in which gonadotrophins enhance the responsiveness of granulosa cells to Prl in serum supplemented medium provides the opportunity for study of the regulatory mechanisms involved with the induction and maintenance of such responsiveness.
J. Steven Alexander and Thomas M. Crisp
C. M. G. Thomas and J. H. Veerkamp
Human term placenta tissue homogenates were subjected to differential centrifugation procedures. The composition of the subcellular fractions was monitored with a number of marker enzymes and the effectiveness of these enzyme systems was evaluated. The subcellular fractions were tested for their 17β-hydroxy dehydrogenase activity on testosterone, oestradiol and the synthetic substrate retrotestosterone.
Buffer medium composition showed a direct influence upon enzyme distribution patterns of all fractions during the same differential centrifugation procedure. All enzyme activities tested became less sedimentable when glycerol was present in the fractionation buffer. Glycerol stabilized soluble 17β-hydroxy dehydrogenase activity during fractionation. The activity of steroid-converting enzymes was inhibited by the presence of glycerol in the medium.
Subcellular distribution of marker enzymes did not sustain the presence of mitochondrial 17β-hydroxy dehydrogenase but related it to microsomal contamination. In general the proportion of 17β-hydroxy dehydrogenase activity in the particulate fractions showed a decrease in the substrate order retrotestosterone > testosterone > oestradiol which was independent from the buffer medium used.
Specific activities for both particle-bound and soluble 17β-hydroxy dehydrogenase increased in the substrate order retrotestosterone < testosterone < oestradiol. The particulate enzyme activity was maximal with NAD+ for the three substrates tested, but in the cytosol fraction NADP+ was the preferential co-enzyme only when oestradiol was used as the substrate.
J. Steven Alexander and Thomas M. Crisp
Abstract. The effects of preincubating rat granulosa cells with porcine LH, and its dissociated α and β subunits on subsequent Prl mediated progesterone secretion were examined. Granulosa cells were isolated from ovarian follicles 50 h after injection of 5 IU PMSG and were then cultured as monolayers in serum supplemented medium. Cells were preincubated for 24 h with medium alone (control) or with 0.1 or 1 μg/ml of LH, LHα, LHβ, or both LHα and LHβ. Cells were also preincubated with 50 ng/ml LHα and 50 ng/ml LHα that had been further purified with immunoaffinity chromatography. Following the preincubation period, cells were maintained an additional 6 days in the presence or absence of 1 μg/ml Prl. Preincubation with LH or its individual subunits markedly increased the responsiveness to Prl stimulation through 6 days of culture, whereas cells cultured in the absence of Prl secreted decreasing amounts of progesterone. Preincubation with the dissociated subunits increased Prl mediated progesterone secretion approximately two-thirds as much as preincubation with intact pLH. Immunopurified pLHα increased the responsiveness to Prl to the same degree as non-purified pLHα, suggesting that the stimulatory effects of the subunits are not due to contamination by intact LH. The magnitude of Prl mediated progesterone secretion was dependent upon the preincubation concentration of LH or its dissociated subunits in most, but not all treatments. The increased responsiveness to Prl suggests that in the proper endocrine environment, individual subunits of gonadotrophins may functionally interact with target cells.
D. Wynford-Thomas and B. M. J. Stringer
This paper describes the effect of long-term goitrogen-administration on the mast cell population of the rat thyroid. Animals were treated with the goitrogen aminotriazole at a dose sufficient to block thyroid hormone synthesis completely, and compared after 80 days with a control group. Serum TSH was measured by radioimmunoassay, and mast cell number quantified from specially-stained tissue sections using appropriate stereological methods.
Goitrogen treatment led to a 9-fold increase in serum TSH and an 8-fold increase in thyroid weight. Mast cell number per unit volume of thyroid decreased, but total numbers per gland increased 4 to 5 fold. There was a significant fall in mean mast cell size.
The work demonstrates conclusively that mast cell hyperplasia occurs during goitre formation and provides a method for its quantitative assessment.
The possible mechanisms and significance of thyroid mast cell proliferation are discussed.
V. Fonseca, M. Thomas and G. Dusheiko
We measured thyrotropin receptor antibodies in serum obtained from 2 groups of patients participating in clinical trials of recombinant interferon-α 2b for viral hepatitis. Group I: Patients with hepatitis B (N=8), received interferon 5×106 units thrice weekly for 4 months. Group II: Patients with non-A, non-B hepatitis (N=16) were randomized to receive interferon in a dose of either 0.25×106 or 3×106 U thrice weekly for 6 months and then crossed over to receive the other dosage schedule for a further 6 months. None of the patients developed thyrotoxicosis. Thyrotropin receptor antibody activity was detectable within the "normal range" (<10 U/l) in 6 patients prior to treatment. In Group I, thyrotropin receptor antibodies became detectable in 6 patients on treatment, in 4 of whom it was 10 U/l. In Group II, thyrotropin receptor antibody activity was unchanged on low-dose interferon, but on the higher dose became detectable in 9 patients, in 7 of whom it was >10 U/l. We conclude that treatment with interferon is associated with the development of thyrotropin receptor antibodies in a large proportion of patients. It is possible that in some patients treated with higher doses of interferon the increase in thyrotropin receptor antibody activity may be sufficient to induce hyperthyroidism.
M. James Whitelaw, Thomas N. Foster and William H. Graham
Five prepubertal males ranging in age from 8–11 and whose estimated heights were all over 195 cm were treated by the induction of a premature puberty with testosterone oenanthate 200 mg twice monthly for 9 months in 4 and 5 months in one. During the first 8 months their linear growth increase ranged between 13.7 and 6.4 cm. There was also a corresponding sharp increase in weight of as much as 17.24 kg in one patient. The bone age advanced 51 months in a 9 year old boy, while in the eldest, age 11, the advance was only 24 months. There is a definite lag in the radiological evidence of epiphyseal acceleration for the first 3–4 months of anabolic therapy. After discontinuation of therapy, evidence of acceleration may persist for a like period. The final height of 4 boys is far under the estimated height on the Bayley table, the fifth is still not determined. Genital development and secondary sex characteristics were noted after the first month of therapy. The growth spurt induced by testosterone oenanthate duplicated that normally seen in the 12–14 year old boy. There is no psychic disturbance by the induction of puberty.
J. A. Thomas, M. Mawhinney and E. T. Knych Jr.
Single injections of testosterone (20 mg/kg) to castrated mice caused an early stimulation in TCA-extractable fructose. Later, and at somewhat higher doses, free fructose levels were observed to rise. The earliest detectable changes occurred about 12 h following injection.
Single injections of ethinyloestradiol to recently castrated mice (48 h) caused a stimulation of prostate gland TCA-extractable fructose 12 h after hormone administration. Free fructose in these same organs was generally reduced by the administration of this oestrogen.
Ethinyloestradiol given one hour prior to a fructose stimulating dose of testosterone produced essentially the same stimulatory response as ethinyloestradiol alone.
The stimulatory action of ethinyloestradiol is probably related to changes in membrane permeability in the target organ.
D. Wynford-Thomas, B. M. J. Stringer and E. D. Williams
This study was designed to investigate the changes in growth and function which occur in the rat thyroid during prolonged TSH stimulation.
Animals maintained on the goitrogen aminotriazole were sacrificed together with controls at frequent intervals over a period of 5 months. The levels of serum T3 and T4 and TSH were measured by radioimmunoassay. Functional activity was assessed by measurement of the thyroid/serum iodide ratio (T/S) and growth by measurement of thyroid weight, follicular cell number and follicular cell mitotic activity.
Serum T3 and T4 rapidly fell to undetectable levels within 2 weeks. The level of serum TSH rose to a stable 5-fold maximum after 4 weeks. The T/S ratio followed a closely similar pattern rising to a sustained 7-fold maximum. Thyroid weight and follicular cell number increased rapidly for the first few weeks but the growth rate declined progressively, falling almost to zero after 80 days. Mitotic activity rose dramatically to a 30-fold peak after 7 days but then declined almost to normal after 80 days, consistent with the observed change in cell number. The results thus demonstrate a clear dissociation between the functional and proliferative activity of the thyroid follicular cells during prolonged stimulation by a sustained elevation of serum TSH and point to the existence of specific growth regulating mechanisms which limit the mitotic response.
G. Schaison, P. Thomopoulos, D. Leguillouzic, G. Thomas and M. Moatti
Abstract. To investigate the respective role of triiodothyronine (T3) and thyroxine (T4) in the regulation of TSH secretion, we studied the action of sodium ipodate and propylthiouracil (PTU) in 11 athyreotic patients. The lT4 replacement dose was adjusted to obtain, in each patient, a normal basal TSH level and a normal TSH response to TRH. In the 5 ipodate-treated patients (single 6 g oral dose), the mean serum T3 level fell by 64% below the baseline value and serum rT3 rose 180% above the baseline. The free T4 index (FT4I) did not change whereas the mean serum TSH concentration increased 280% above baseline values. In the 6 PTU-treated patients (250 mg orally every 6 h for 10 days), serum T3 levels fell 33%, serum rT3 increased up to 82% and the FT4I did not change. The mean serum TSH concentration increased 68% above the baseline value. Thus, the mean percentage increase in serum TSH was less in PTU- than in ipodate-treated patients (68% vs 280%). Statistical analysis of the correlation between the serum T3 decrease (ΔT3) and the serum TSH (ΔTSH) increase demonstrated that for the same T3 diminution, the ipodate-treated group displayed higher increase of TSH than the PTU-treated patients. In the rat, PTU interferes with the 5'-deiodination of T4 in the liver and kidney but not in the pituitary, while ipodate appears to have the same effect in all tissues. If this holds true for human subjects, our data strongly suggest that circulating T4 (through its intrapituitary conversion to T3) shares with serum T3 the capacity to regulate TSH secretion in man.
D. Wynford-Thomas, B. M. J. Stringer and E. D. Williams
This study was designed to provide a reliable quantitative assessment of the circadian rhythm of mitotic activity in the follicular cell population of the rat thyroid, and the effect on this rhythm of prolonged stimulation by a raised level of circulating TSH, induced by goitrogen administration.
Mitotic activity in groups of control and goitrogen-treated rats was assessed by a stathmokinetic technique during four 4-h periods spaced equally through one 24-h cycle. Particular attention was paid to the method of sampling to eliminate systematic and minimise random errors, and to the assesment of rhythmicity which was carried out by an appropriate statistical method.
A highly significant circadian rhythm was found in control animals with a daytime peak (12.00 to 16.00 h). Goitrogen treatment led to a 5- to 6-fold increase in the mean but a loss of detectable rhythmicity.
The results show that the presence and timing of this circadian rhythm must be taken into account in future studies of thyroid growth, and they throw some light on the possible mechanisms of its control. Comparison of the rhythm with that of serum TSH reported previously raises the possibility of a dominant control by this hormone even in euthyroid animals and suggests that it may act on cells in the G2 and/or G1/G0 phases of the cell cycle.