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ABSTRACT

The structure of RNA encoding the mouse testis FSH receptor was studied using reverse transcription and the polymerase chain reaction. Four major bands were observed by ethidium bromide staining and by hybridization to an FSH-receptor cDNA probe. The largest of these bands was the expected size (779 bp) while the other bands were spaced approximately 70 bp apart. Using alternative primers, each of the products was shown to contain exons 1, 9 and 10. Exons 2-8 in the FSH receptor gene are between 68 and 77 bp in size, suggesting that these multiple products arise by alternate splicing of the region encoding the extracellular domain of the receptor. A similar pattern of splicing was observed in cDNA from the testes of hypogonadal mice, showing that this alternative splicing pattern is not gonadotrophin-dependent.

ABSTRACT

The testicular feminized (Tfm) mouse lacks functional androgen receptors and develops with a female external phenotype and internal testes. The testes of these animals contain normal, or close to normal, numbers of Leydig cells but secrete very low amounts of androgen due to a lack of 17α-hydroxylase activity. To determine whether this loss of activity is due to a lack of enzyme synthesis or a change in catalytic activity we have examined 17α-hydroxylase cytochrome P-450 (P-450_17α) protein and mRNA levels in the testes of Tfm mice.

Levels of P-450_17α protein were measured by immunoblotting, while mRNA was measured following reverse transcription (RT) and amplification by the polymerase chain reaction (PCR). Conditions for RT-PCR were determined which allowed semiquantification of P-450_17α mRNA relative to β-actin mRNA. In extracts of Tfm testes P-450_17α protein was undetectable using antiserum against porcine P-450_17α. In contrast, a protein of around 54 kDa was clearly detectable in extracts of control cryptorchid testes. Using RT-PCR, P-450_17α mRNA was detectable in both control and [ill] testes but, expressed in terms of β-actin mRNA, levels of P-450_17α mRNA in control testes were 40-fold higher than those in [ill] testes. If the total amount of RNA extracted from each testis is taken into account then P-450_17α mRNA levels per testis were up to 400-fold higher in control testes. These results show that the reduced level of 17α-hydroxylase activity in [ill] testes is related to reduced protein synthesis. Previous results have shown that androgens reduce P-450_17α mRNA levels in cultured Leydig cells. Results from this study suggest, however, that androgens are required to induce normal levels of P-450_17α mRNA in Leydig cells.

ABSTRACT

The cytochrome P450 aromatase (P450_arom) enzyme is required for bioconversion of androgen to oestrogen. In this study ovarian P450_arom mRNA and enzyme activity have been measured during development in normal mice and hypogonadal (hpg) mice which lack circulating gonadotrophins. A semi-quantitative reverse transcription-PCR (RTPCR) technique was used to measure cytochrome P450_arom mRNA levels and aromatase enzyme activity was measured directly. Using RT-PCR, P450_arom mRNA was detectable in the adult mouse ovary and also in the uterus, kidney, brain and skeletal muscle but not in cardiac smooth muscle. In the normal mouse, P450_arom mRNA was detectable in the ovary on the day of birth (day 1) and levels increased significantly up to day 15 with the most marked changes seen between days 1 and 5. Aromatase activity was also detectable at all ages in the ovary and increased significantly between days 1 and 7. In ovaries from [ill] mice, normal levels of P450_arom mRNA were present on day 1 but there was no significant change in P450_arom mRNA at later ages up to day 15. These results show that in the newborn mouse ovary, which contains only primordial follicles, there is a basal expression of P450_arom mRNA which is not gonadotrophindependent. After 1 day, however, gonadotrophins are required for normal expression of ovarian P450_arom and this coincides with development of primary and secondary follicles.

ABSTRACT

The cytochrome P450 enzyme 17α-hydroxylase (P450c17) is required for androgen synthesis and therefore regulates substrate supply for aromatization. In this study, changes in P450c17 activity and mRNA levels were measured during ovarian development in the normal mouse and in the hypogonadal (hpg) mouse which lacks circulating gonadotrophins. At birth, low levels of P450c17 activity and mRNA were detectable in normal ovaries. This basal level of expression did not change until after day 10 at which time both enzyme activity and mRNA levels increased by six- to eightfold. In the hpg mouse, levels of P450c17 mRNA were normal at birth but did not change significantly during subsequent development and were significantly less than normal by day 15. Results show that there is a low level of gonadotrophin-independent expression of P450c17 in the ovary at birth and that gonadotrophins are required for the subsequent increase in expression between days 10 and 15. In the ovary, P450c17 is expressed solely in the thecal/interstitial compartment and interstitial cells arise in the mouse ovary around day 11. Changes in P450c17 are likely, therefore, to be related to gonadotrophin-dependent development of the interstitial tissue in the mouse. Treatment of adult hpg mice with LH and FSH showed that both gonadotrophins can act to increase P450c17 activity. Since FSH acts only on the granulosa cell compartment of the ovary it is likely that FSH acts through a paracrine mechanism to regulate thecal/interstitial cell activity.

ABSTRACT

Hypogonadal (hpg) mutant mice, with a congenital deficiency of hypothalamic gonadotrophin-releasing hormone (GnRH), and testicular feminized (tfm) mice, which lack a functional androgen receptor, were used to study the effects of the potent GnRH agonist 'Zoladex' (ICI 118630; d-Ser (Bu^t)⁶, Azgly¹⁰-GnRH) on pituitary and gonadal function. Zoladex (0.5 mg) in a sustained-release lactide—glycolide copolymer depot was administered subcutaneously under anaesthesia and was left in place for 7 days, after which time the effects of the drug upon pituitary and serum gonadotrophin concentrations, glycoprotein hormone subunit mRNAs and testicular morphology were investigated.

At the pituitary level, Zoladex treatment resulted in a substantial reduction in LH content in normal males, and LH content was depressed in hpg mice even below the basal levels normally found in these mutants. Pituitary LH content in the Zoladex-treated animals was depressed in the tfm groups, but not to the same levels as those found in the normal and castrated normal mice. Zoladex treatment at the time of castration prevented the post-operative elevation in serum LH associated with castration alone. In the androgen-deficient tfm mouse, Zoladex did not depress the normally elevated serum LH levels. Serum LH in the hpg animals was, in all cases, below the limit of detection of the assay.

Pituitary FSH content was depressed into the hpg range in both the normal and castrated animals, but there was no further depression in the hpg mice. The pituitary content was reduced in the tfm mice, again the effects not being as dramatic as in the normal and castrated animals. Serum FSH content, as measured by radioimmunoassay, was depressed by 50% in normal mice; there was no reduction in the hpg mice, however.

With regard to pituitary gonadotrophic hormone gene expression, Zoladex administration to normal mice caused a dramatic reduction in LHβ mRNA content, to a level approximating that found in untreated hpg mice. The drug also depressed LHβ mRNA in the castrated group to the hpg range when given at the time of castration, whereas in untreated castrated mice there was a significant increase in LHβ mRNA. In the tfm mouse, which can be considered as a model for long-term failure of androgen feedback, Zoladex again induced a fall in LHβ mRNA, but not to the same extent as in the normal and normal castrated group. Zoladex had no effect on the already low levels of LHβ mRNA found in hpg mice.

Pituitary FSHβ mRNA levels were not significantly altered by Zoladex in any of the treatment groups, whereas the drug induced a substantial rise in the common α-subunit mRNA in normal and hpg mice, to a level equalling that found in castrated tfm mice. In the latter two groups, Zoladex treatment did not result in a further increase in α-subunit mRNA above that found after castration alone, or in the untreated tfm mutant.

Treatment for 7 days with Zoladex resulted in a significant increase in testis weight, with spermatogenesis advancing beyond the first meiotic division with many round spermatids found within the seminiferous tubules. However, the interstitial cells remained atrophic and there was evidence of seminal vesicle growth. Nevertheless, there was a small but significant increase in testicular androgen content. Administration of the agonist to hypophysectomized hpg mice did not stimulate testicular or seminal vesicle growth, suggesting that the drug does not stimulate steroidogenesis via a direct action upon the testis.

Overall, the pharmacological effects of the drug appear to have turned off the transcription of the LHβ gene, with a consequent reduction in LH synthesis and probably also secretion in the longer term. With FSHβ, gene transcription was apparently unchanged and, with a substantial increase in the common α-subunit message, it would appear that the pituitary gland of Zoladex-treated animals may be predominantly biased towards FSH secretion. Although the circulating FSH levels as measured by radioimmunoassay were unaltered by Zoladex, there are several reports that GnRH agonists increase serum levels of bioactive hormones, perhaps by altering glycosylation of the FSH dimer glycoprotein.