Estudios de la región 5'UTR del gen FMR-1 en pacientes con falla ovárica prematura / Studies of the 5' - UTRTR region in the FMR-1 gene in patients withe Premature Ovarian Failure

La falla ovárica prematura (FOP) es un síndrome de patogénesis multicausal que afecta aproximadamente al 1% de las mujeres en edad reproductiva. Numerosos estudios asocian el estado de premutación (amplificación del número de tripletes CGG entre 50/55 y 200 repeticiones) en el gen FMR-1 y FOP. Alrededor de un 4% de las pacientes FOP presentan alelos con premutación. La amplificación del número de tripletes por encima de 200 repeticiones causa el Síndrome de Fragilidad del X (SFX). El objetivo del presente trabajo fue estudiar la región 5´ no codificante del gen en un grupo de pacientes FOP de Argentina. La región de interés se amplificó por PCR a partir de muestras de ADN de 100 pacientes FOP y 145 mujeres controles. Los alelos de las pacientes y controles fueron agrupados en 7 categorías de acuerdo al número de tripletes obtenidos. Se observó que el número de repeticiones más frecuente se encuentra en el rango de 26 a 30 tripletes, tanto en pacientes como en controles. En el grupo de pacientes FOP, 5/197 (2.6%) alelos no relacionados estudiados presentaron un número de tripletes CGG mayor a 50, mientras que sólo 1 de 290 (0.34%) para el grupo control. Todas las pacientes FOP con valores de tripletes CGG mayor a 50 presentaron amenorrea secundaria. Estos resultados están en concordancia con lo comunicado para otras poblaciones acerca de la existencia de una asociación entre la premutación del gen FMR-1 y el desarrollo de FOP. Asimismo, los resultados obtenidos refuerzan la importancia de la genotipificación del gen FMR-1 en las pacientes FOP, a los efectos de estimar el riesgo de su descendencia para el SFX.

Premature ovarian failure (POF) is a syndrome of multicausal pathogenesis that affects 1% of women before the age of 40. Several studies associate the premutation state (CGG repeats increased in number between 50/55 and 200) in the FMR-1 gene and POF. About 4% of POF women have alleles in the FMR-1 gene in the permutation range. An increase above 200 in the number of triplets in this gene causes the Fragile X Syndrome (FXS). The purpose of the present study was to analyze the 5´untranslated region of the FMR-1 gene in a group of patients from Argentina. The region of interest was amplified by PCR from DNA samples of 100 POF patients and 145 control women. Alleles from controls and patients were grouped in 7 categories according to the number of triplets obtained. We observed that the most frequent number of repeats ranged from 26 to 30 triplets, in both patient and control groups. In the POF group, 5 out of 197 (2.6%) not related alleles presented a number of CGG triplets higher than 50, while only 1 out of 290 (0.34%) was present in controls. All POF patients with a number of CGG repeats higher than 50 presented secondary amenorrhea. These results are in accordance with previous reports from other populations showing an association between the premutation state in the FMR-1 gene and POF development. In addition, these results reinforce the importance of genotyping POF patients to estimate the risk of their offspring for Fragile X Syndrome.

Acute hCG administration induces seminiferous tubule damage in the adult rat.

In order to study the early (or short time) effects of an hCG stimulation on the seminiferous tubules of the adult rat, a single dose of 100, 200 or 400 IU hCG was administered to eighty to ninety day old rats of the Sherman strain. The histological analysis revealed a tubular damage already noticeable six hours after the injection. This precocious lesion becomes more pronounced two to five days later, consisting of degeneration and hypocellularity of the germinal epithelium, margination of the chromatin in round spermatids and formation of multinuclear giant cells. This damage involves big areas of the testis, particularly of peripheral location. The analysis performed three months after the acute stimulation still showed tubular regressive changes, thus indicating an incomplete reversion of the damage. Changes in the hormonal environment of hCG treated rats were found. Serum testosterone significantly increased from 6 to 72 hours following a 200 IU hCG injection. A severe diminution of serum FSH levels and a significant increase of the serum estradiol were observed. Intratesticular administration of estradiol benzoate was able to reproduce the tubular damage in some animals, while restoration of FSH circulating levels by simultaneous administration of hCG and purified hFSH did not prevent the changes induced by hCG alone. These results suggest that the high intratesticular level of estradiol and not the lowering of serum FSH might be the mechanism responsible for the described testicular injury.

Acute hCG administration induces seminiferous tubule damage in the adult rat.

In order to study the early (or short time) effects of an hCG stimulation on the seminiferous tubules of the adult rat, a single dose of 100, 200 or 400 IU hCG was administered to eighty to ninety day old rats of the Sherman strain. The histological analysis revealed a tubular damage already noticeable six hours after the injection. This precocious lesion becomes more pronounced two to five days later, consisting of degeneration and hypocellularity of the germinal epithelium, margination of the chromatin in round spermatids and formation of multinuclear giant cells. This damage involves big areas of the testis, particularly of peripheral location. The analysis performed three months after the acute stimulation still showed tubular regressive changes, thus indicating an incomplete reversion of the damage. Changes in the hormonal environment of hCG treated rats were found. Serum testosterone significantly increased from 6 to 72 hours following a 200 IU hCG injection. A severe diminution of serum FSH levels and a significant increase of the serum estradiol were observed. Intratesticular administration of estradiol benzoate was able to reproduce the tubular damage in some animals, while restoration of FSH circulating levels by simultaneous administration of hCG and purified hFSH did not prevent the changes induced by hCG alone. These results suggest that the high intratesticular level of estradiol and not the lowering of serum FSH might be the mechanism responsible for the described testicular injury.

Effects of induced hypoprolactinemia on testicular function during gonadal maturation in the rat.

We have evaluated the effects of hypoprolactinemia during gonadal maturation in the male rat. Intact 30-day-old rats were injected daily for 10 days with three different doses of bromocriptine (0.75, 1.5 or 3.0 mg/kg of body weight/day). At the end of the treatment period, the animals were sacrificed, serum was collected for prolactin (PRL), LH, and androgen measurements. Intratesticular testosterone and 5 alpha-androstanediol (androstanediol) were measured following celite column chromatography and a specific radioimmunoassay. In addition, the production of androgens by decapsulated testes and dispersed Leydig cells was also studied in vitro. Serum levels of PRL (9.4 +/- 1.9 ng/ml) were suppressed to undetectable levels in the three bromocriptine-treated groups, whereas LH levels were not altered. All three doses of bromocriptine markedly depressed serum testosterone (plus DHT) and androstanediol. Intra-testicular testosterone and androstanediol were diminished (25% and 35%, respectively, P less than 0.05) during hypoprolactinemia. Decapsulated testes and dispersed Leydig cells from bromocriptine-treated animals showed a significant reduction in the basal secretion of testosterone (plus DHT) and androstanediol, and in androgen responses to submaximal hCG stimulation. Maximal steroidogenic responses from bromocriptine-treated rats were similar to controls. The present findings show that, during puberty, bromocriptine influences testicular steroidogenesis, and these effects may be partly due to changes in PRL levels. A direct effect of this dopaminergic agonist on the male gonad cannot be completely ruled out.

Induced hypoprolactinemia and testicular steroidogenesis in man.

The effects of short-term hypoprolactinemia on the pituitary-gonadal axis were evaluated in a group of patients with untreated prostatic carcinoma. Each patient was studied prior to and during 7-day oral administrations of bromocriptine (2.5 mg q.i.d.). Serum LH, prolactin (PRL), androst-4-ene-3,17 dione (androstenedione), testosterone, and 5 alpha-androstane-3 alpha, 17 beta-diol (5 alpha-Diol) levels, as well as intra-testicular testosterone, dihydrotestosterone (DHT), 5 alpha-Diol and zinc (Zn) concentrations, were determined. Daily administration of bromocriptine caused a marked suppression of serum PRL (mean +/- SEM, 23.8 +/- 2.5 vs. 6.4 +/- 1.0 ng/ml) without concomitant changes in serum LH levels (mean +/- SEM, 8.3 +/- 1.6 vs. 8.9 +/- 2.1 ng/ml). Hypoprolactinemia induced a significant decrease (P less than 0.05) in the mean peripheral testosterone levels; but 5 alpha-Diol and androstenedione remained unchanged. However, in testicular tissues, bromocriptine treatment resulted in significant increases in mean concentrations of total androgens (P less than 0.001), testosterone (P less than 0.001) and DHT (P less than 0.02). Testicular levels of 5 alpha-Diol were not significantly altered. There was no change in Zn levels in basal conditions and during bromocriptine administration. These results indicate that short-term suppression of serum PRL levels in man affects basal testicular function without altering serum LH. However, a direct action of bromocriptine on the human gonad cannot be excluded.

Effect of prolactin on the steroidogenic response of rat luteal cells.

The role of prolactin on some ovarian functions was studied in collagenase-dispersed luteal cells obtained from PMSG/hCG-primed rats. The in vitro effect of ovine prolactin (oPrl) on luteal cell function was assayed. This hormone produced a dose-dependent increase of progesterone production and an additive effect on hCG stimulation. oPrl had no effect on cAMP production. Chronic effects of prolactin were studied in sulpiride (S), bromocriptine (Br) and oPrl-treated rats. Serum levels of prolactin were significantly higher in S-treated animals whereas Br administration rendered undetectable values. Serum progesterone was reduced in Br-treated animals and LH levels were similar in all groups studied. In vitro studies demonstrated a marked reduction of hCG stimulation of progesterone and cAMP production by luteal cells from hypoprolactinemic animals, while a significant increase was observed in hyperprolactinemic states. oPrl and S treatment significantly increased ovarian LH binding sites while a reduction was observed in Br-treated rats. These data suggest that luteal cell function is regulated by circulating levels of prolactin and that this hormone has some direct effect on the steroidogenic process.

Effects of prolactin, bromocriptine and sulpiride on estrogen and lactogenic receptors in the rat adrenal gland.

In prepubertal male rats, the injection of bromocriptine (Br) for 10 days caused an increase in adrenal weight (Br 0.75 mg/kg BW (Br I): 2.83%; Br 1.5 mg/kg BW (Br II): 12.1% and Br 3 mg/kg BW (Br III): 24.7%), and this effect was only significant at the highest dose. Sulpiride (S, 30 mg/kg BW/day) for 10 days produced a significant decrease in adrenal weight (18.6%), whereas ovine prolactin (oPRL) administered at doses of 0.5 or 5 mg/kg BW/day for 10 had no effect on this parameter. The action of these drugs on corticosterone serum levels was for Br III a 50.6% increase and for S a 29.2% decrease. Bromocriptine caused a significant increment of cytosolic available estrogen receptors C: 7.65 +/- 0.36 (SE); Br I: 10.2 +/- 0.36; Br II: 11.0 +/- 0.23 and Br III: 13.3 +/- 0.35) and total lactogenic receptors in the adrenal gland (C: 125.2 +/- 2.84; Br I: 203.8 +/- 4.43; Br II: 213.1 +/- 7.58 and Br III: 251.3 +/- 10.4), and this effect was dose-related. oPRL diminished adrenal estrogen receptors only at the highest dose used (C: 11.1 +/- 1.73; PRL 5: 8.2 +/- 0.75) as did S (C: 11.2 +/- 1.84 and S: 5.2 +/- 1.07); while the former originated a marked decrease in lactogenic adrenal binding sites at both doses (C: 198.7 +/- 12.2; PRL 0.5: 52.9 +/- 5.00 and PRL 5: 38.8 +/- 4.76), S also had a highly significant diminution over these receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of prolactin, bromocriptine and sulpiride on estrogen and lactogenic receptors in the rat adrenal gland.

In prepubertal male rats, the injection of bromocriptine (Br) for 10 days caused an increase in adrenal weight (Br 0.75 mg/kg BW (Br I): 2.83

; Br 1.5 mg/kg BW (Br II): 12.1

and Br 3 mg/kg BW (Br III): 24.7

), and this effect was only significant at the highest dose. Sulpiride (S, 30 mg/kg BW/day) for 10 days produced a significant decrease in adrenal weight (18.6

), whereas ovine prolactin (oPRL) administered at doses of 0.5 or 5 mg/kg BW/day for 10 had no effect on this parameter. The action of these drugs on corticosterone serum levels was for Br III a 50.6

increase and for S a 29.2

decrease. Bromocriptine caused a significant increment of cytosolic available estrogen receptors C: 7.65 +/- 0.36 (SE); Br I: 10.2 +/- 0.36; Br II: 11.0 +/- 0.23 and Br III: 13.3 +/- 0.35) and total lactogenic receptors in the adrenal gland (C: 125.2 +/- 2.84; Br I: 203.8 +/- 4.43; Br II: 213.1 +/- 7.58 and Br III: 251.3 +/- 10.4), and this effect was dose-related. oPRL diminished adrenal estrogen receptors only at the highest dose used (C: 11.1 +/- 1.73; PRL 5: 8.2 +/- 0.75) as did S (C: 11.2 +/- 1.84 and S: 5.2 +/- 1.07); while the former originated a marked decrease in lactogenic adrenal binding sites at both doses (C: 198.7 +/- 12.2; PRL 0.5: 52.9 +/- 5.00 and PRL 5: 38.8 +/- 4.76), S also had a highly significant diminution over these receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Ovarian dysfunction in streptozotocin-induced diabetic rats.

The effect of streptozotocin diabetes on some ovarian functions in adult rats was examined. Diabetic diestrus animals showed reduced ovary weight and lower circulating levels of progesterone. Scatchard plots of binding data derived from ovarian particulate fractions of normal and streptozotocin diabetic rats revealed the presence of one class of binding sites with high affinity for 125I-hCG. The apparent association constant of the hCG receptors of diabetic ovaries was comparable to that of normal gonads. However, a marked decrease (42%) in the number of hCG binding sites was found in diabetic animals. With isolated luteal cells similar results were obtained, and the administration of insulin to streptozotocin diabetic rats restored to normality the number of hCG binding sites. The maximal response of progesterone production by luteal cells from control ovaries was obtained with 10(-10) M hCG. A 100-fold higher concentration of hCG was required for the maximum stimulation of cAMP synthesis. The cAMP response of cells from diabetic rats was significantly higher than that of control cells. However, luteal cells from diabetic rats showed some loss of sensitivity in the synthesis of progesterone during incubation with hCG. Most of the alterations seen in diabetic female rats could be restored with insulin therapy, indicating that insulin plays an important role in the regulation and maintenance of normal reproductive functions. It is suggested that the diminution of the LH receptor population causes the disruption of normal luteal cell function. This fact could be responsible for some of the reproductive alterations in the diabetic female rat.

Development of the gonadotrophic resistant ovary syndrome in myasthenia gravis: suggestion of similar autoimmune mechanisms.

A woman with myasthenia gravis who developed hypergonadotrophic amenorrhoea was studied. This patient fulfilled all accepted criteria for the diagnosis of the gonadotrophin resistant ovary syndrome: high levels of serum LH and FSH by radioimmunoassay and urinary gonadotrophin excretion by bioassay, low serum oestradiol, lack of response to exogenous gonadotrophin and ovaries with multiple non-stimulated primordial follicles. The serum of this patient contained a substance which behaving like a gamma globulin, inhibited FSH specific binding to receptors in an in vitro system. This inhibitor was looked for in further patients with hypergonadotrophic amenorrhoea, other immunological diseases or myasthenia gravis with normal menses. Ovarian biopsy was not available in these patients. In all except one who also had the association of myasthenia gravis and hypergonadotrophic amenorrhoea, the inhibitor was not detected. It is suggested that development of antibodies directed against gonadotrophin receptors or a receptor related membrane domain may play a role in the mechanism of failure of gonadotrophin stimulation in some patients with the gonadotrophin resistant ovary syndrome.