FSH acts through the Sertoli cell to ensure normal testicular development and function. To identify transcriptional mechanisms through which FSH acts in the testis, we have treated gonadotrophin-deficient hypogonadal (hpg) mice with recombinant FSH and measured changes in testicular transcript levels using microarrays and real-time PCR 12, 24 and 72 h after the start of treatment. Approximately 400 transcripts were significantly altered at each time point by FSH treatment. At 12 h, there was a clear increase in the levels of a number of known Sertoli cell transcripts (e.g. Fabp5, Lgals1, Tesc, Scara5, Aqp5). Additionally, levels of Leydig cell transcripts were also markedly increased (e.g. Ren1, Cyp17a1, Akr1b7, Star, Nr4a1). This was associated with a small but significant rise in testosterone at 24 and 72 h. At 24 h, androgen-dependent Sertoli cell transcripts were up-regulated (e.g. Rhox5, Drd4, Spinlw1, Tubb3 and Tsx) and this trend continued up to 72 h. By contrast with the somatic cells, only five germ cell transcripts (Dkkl1, Hdc, Pou5f1, Zfp541 and 1700021K02Rik) were altered by FSH within the time-course of the experiment. Analysis of canonical pathways showed that FSH induced a general decline in transcripts related to formation and regulation of tight junctions. Results show that FSH acts directly and indirectly to induce rapid changes in Sertoli cell and Leydig cell transcript levels in the hpg mouse but that effects on germ cell development must occur over a longer time-span.
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M H Abel, D Baban, S Lee, H M Charlton, and P J O'Shaughnessy
I. S. Scott, M. K. Bennett, A. E. Porter-Goff, C. J. Harrison, B. S. Cox, C. A. Grocock, P. J. O'Shaughnessy, R. N. Clayton, R. Craven, B. J. A. Furr, and H. M. Charlton
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 (But)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.