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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

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 (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.

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S A Gray, M A Mannan and P J O'Shaughnessy

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.

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L Couture, H Naharisoa, D Grebert, J-J Remy, E Pajot-Augy, V Bozon, T Haertle and R Salesse

ABSTRACT

The LH/hCG receptor is a G protein-coupled receptor with an N-terminal extracellular domain involved in hormone—receptor interaction. The recombinant porcine receptor, stably expressed in Chinese hamster ovary (CHO) cells, has the same characteristics (K d and cAMP production) as in Leydig cells. Six synthetic peptides derived from the receptor ectodomain and two polyclonal anti-peptide sera were tested in the homologous system porcine LH and porcine LH receptor. Their ability to inhibit hormone binding and signal transduction on CHO cells expressing the recombinant receptor was evaluated. Peptides 25–40 and 107–121 exhibited a high transduction inhibition as compared with hormone binding, peptides 21–36, 102–111, and 102–121 inhibited hormone binding more efficiently than signal transduction, and peptide 7–24 exhibited inhibition of both hormone binding and hormone-induced cAMP production. Immuno-globulins against peptides 21–36 and 102–111 inhibited both hormone binding and receptor activation suggesting that these sequences are located on the receptor surface.

The data suggest that multiple, discontinuous regions of the extracellular domain of porcine LH receptor are involved in hormone binding and signal transduction. Two minimum critical sequences, 21–24 and 102–107, are involved in hormone binding and vicinal segments may be implicated in signal transduction.

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R S Guenette, H B Corbeil, J Léger, K Wong, V Mézl, M Mooibroek and M Tenniswood

ABSTRACT

After weaning, the mammary gland ceases lactation and involutes. The wet weight of the gland decreases by 70% within 4 days of weaning. This involves significant tissue remodelling as the ducts regress and return to the resting state. The presence of apoptotic bodies in the luminal epithelial compartment 2 to 3 days after weaning provides clear evidence that a substantial proportion of the regression is attributable to the induction of active cell death (ACD) of the epithelial cells.

These changes in the architecture of the gland were found to be mirrored by changes in gene expression. The steady-state level of β-casein mRNA decreased rapidly after weaning from the high levels seen during lactation to undetectable levels by 8 days after weaning. The steady-state levels of expression of a number of genes associated with ACD, including TRPM-2, tissue transglutaminase (TGase) and poly(ADP-ribose) polymerase (PARP), increased transiently during this time-frame. The steady-state level of TRPM-2 mRNA increased 2 days after weaning, reaching a peak on day 4, and decreasing to undetectable levels by day 8 after weaning. The steady-state levels of two other mRNAs, TGase and PARP, showed very similar kinetics. In contrast, the mRNA for Hsp 27, which has been shown to be induced during prostate regression, was not significantly induced in the regressing mammary gland. In-situ hybridization demonstrated that the TRPM-2, TGase and PARP genes were expressed predominantly in the luminal epithelial cells of the ducts. These cells expressed β-casein mRNA during lactation, and underwent ACD after weaning.

While the ultrastructural changes in the mammary gland after weaning, and the induction of TRPM-2, TGase and PARP mRNAs, are reminiscent of apoptosis in the prostate, several features of the process are different. Most notably, the disruption of the secretory processes and the lack of increased expression of Hsp 27 in the regressing mammary gland suggest that there may be a number of important events in ACD that are not common to all cells.

Free access

Mihael Freamat and Stacia A Sower

The specificity of the vertebrate hypothalamic–pituitary–gonadal and hypothalamic–pituitary–thyroid axes is explained by the evolutionary refinement of the specificity of expression and selectivity of interaction between the glycoprotein hormones GpH (FSH, LH, and TSH) and their cognate receptors GpH-R (FSH-R, LH-R, and TSH-R). These two finely tuned signaling pathways evolved by gene duplication and functional divergence from an ancestral GpH/GpH-R pair. Comparative analysis of the protochordate and gnathostome endocrine systems suggests that this process took place prior or concomitantly with the emergence of the gnathostome lineage. Here, we report identification and characterization of a novel glycoprotein hormone receptor (lGpH-R II) in the Agnathan sea lamprey. This 781 residue protein was found ∼43% identical with mammalian TSH-R and FSH-R representative sequences, and similarly with these two classes of mammalian receptors it is assembled from ten exons. A synthetic ligand containing the lamprey glycoprotein hormone β-chain tethered upstream of a mammalian α-chain activated the lGpH-R II expressed in COS-7 cells but in a lesser extent than lGpH-R I. Molecular phylogenetic analysis of vertebrate GpH-R protein sequences suggests a closer relationship between lGpH-R II and gnathostome thyrotropin receptors. Overall, the presence and characteristics of the lamprey glycoprotein hormone receptors suggest existence of a primitive functionally overlapping glycoprotein hormone/glycoprotein hormone receptor system in this animal.

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H Khan, LG Jiang, GN Jayashree, TA Yarney and MR Sairam

To assess the functional significance of putative proteins encoded by alternately spliced mRNA of the sheep testicular FSH receptor, a short form cDNA comprising of the first four exons (117 residues mature protein) was engineered for expression in Escherichia coli. The expressed protein of molecular mass 15 kDa was purified to homogeneity and verified by reaction with an antibody against a synthetic peptide sequence unique to the amino (N)-terminal region FSH receptor. The purified FSH receptor domain protein bound 125I-labeled hFSH in a ligand blot on polyvinylidine difluoride membranes. Further analyses by slot blot revealed high affinity of the immobilized protein with significant reaction at 10 pmol. As the immobilized receptor protein also reacted with structurally related hormones (125I-labeled LH/125I-labeled human chorionic gonadotropin), we confirmed that interaction most probably occurred via the common alpha-subunit of these glycoprotein hormones. Our results reveal that this N-terminal portion of the FSH receptor contain(s) major site(s) for hormone recognition that could be mediated via the alpha-subunit. A rabbit antibody to the receptor inhibited FSH action in receptor bearing cells, revealing the utility of such recombinant FSH receptor protein(s) for modulation of hormone action.

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Antara A Banerjee, Madhavi Dupakuntla, Bhakti R Pathak and Smita D Mahale

The extracellular loop 2 (EL2) of FSH receptor (FSHR) plays a pivotal role in various events downstream of FSH stimulation. Because swapping the six FSHR-specific residues in EL2 (chimeric EL2M) with those from LH/choriogonadotropin receptor resulted in impaired internalization of FSH–FSHR complex and low FSH-induced cAMP production, six substitution mutants of EL2 were generated to ascertain the contribution of individual amino acids to the effects shown by chimeric EL2M. Results revealed that L501F mainly and I505V to a lesser extent contribute to the diminished receptor function in chimeric EL2M. HEK293 cells stably expressing WT and chimeric EL2M FSHR were generated to track the fate of the receptors post FSH induction. The chimeric EL2M FSHR stable clone showed weak internalization and cAMP response similar to transiently transfected cells. Furthermore, reduced FSH-induced ERK phosphorylation was also observed. The interaction of activated chimeric EL2M and L501F FSHR with β-arrestins was weak compared with WT FSHR, thus explaining the impaired internalization of chimeric EL2M and corroborating the indispensable role of EL2 in receptor function.

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DF van de Wiel, PA van Rijn, RH Meloen and RJ Moormann

Superovulation treatment of cows can benefit from the application of very pure recombinant bovine FSH (rbFSH), which is produced in nonmammalian cells. rbFSH is completely free of LH, and therefore can possibly reduce the variability in the results of superovulation. Furthermore, it does not contain brain-tissue-derived proteins and, when produced under serum-free conditions, it is free of other mammalian substances or potentially infectious material. We have produced rbFSH in insect cells, with the ultimate aim of inducing superovulation in cattle. Sf21 insect cells were coinfected with two recombinant baculoviruses, containing the cDNAs of bovine FSH alpha- and beta-subunits respectively. High levels of production of bioactive rbFSH were obtained after cloning cDNA that contained a major part of the 3' untranslated region of the bFSH beta gene. Maximum production of rbFSH 1-5 micrograms/ml (as measured by immunoassay) was obtained 70-90 h after infection. The recombinant material was highly potent in two in vitro bioassays, giving biological activities of 13 IU/ml (Y1 cell rounding assay), 22 IU/ml (Y1 cell cAMP assay), and 23 IU/ml (bovine oocyte maturation inhibition assay), and had a lower but significant activity of 6 IU/ml in the rat Sertoli cell assay. rbFSH was purified by immunoaffinity chromatography, using a monoclonal antibody directed against the human FSH beta-subunit. The purified heterodimer appeared to be homogeneous by SDS-PAGE, whereas the free beta-subunit appeared as a doublet, possibly indicating differently glycosylated forms. Intact heterodimer and both subunits were further identified by western blot analysis, and showed apparent molecular masses of 20 kDa (alpha-subunit), 23 kDa (beta-subunit) and 32.5 kDa (heterodimer). This insect-cell-produced rbFSH did not bind to wheat germ agglutinin, thus indicating that glycosidic side-chains may not contain terminal sialic acid. The relevance of a large 3' untranslated region in bFSH beta cDNA to the level of production of rbFSH, and the possible implications of the pattern of glycosylation for the biological activity of the recombinant hormone are discussed.

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Zs Bösze, E Devinoy, C Puissant, M L Fontaine and L M Houdebine

ABSTRACT

The rabbit κ-casein cDNA was cloned and sequenced. One of the isolated clones included almost the entire 5′ end, while another clone corresponded to the 3′ end of the cDNA. No polyadenylation site was found and therefore this clone did not harbour the complete cDNA. The amino acid sequence of a full-length protein was deduced from the nucleotide sequence obtained for this partial cDNA. It revealed the presence of a chymosin cleavage site and five potential phosphorylation sites. Rabbit κ-casein was compared with those already described in other species. The rabbit sequence is closer to the ovine than to the mouse sequence. This result supports the idea that Lagomorpha are not closer to Rodentia than to Artiodactyla. The cDNA described above was used to study κ-casein gene expression in the rabbit mammary gland. This expression was induced primarily by prolactin in mammary gland organoids and was similar to αs1-casein gene expression in vivo. The κ-casein gene present in the casein gene locus is thus subject to the same regulation as the αs1-casein gene, although it has evolved from a fibrinogen gene.

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Josephine F Trott, Anke Schennink and Russell C Hovey

Prolactin (PRL) is required not only for maintenance of gestation in pigs but also for mammary gland development and subsequent lactogenesis. The actions of PRL are modulated by both long and short isoforms of the PRL receptor (PRLR), where short isoforms can interfere with the essential signaling function of the long isoform. Using 3′ RACE we have isolated a unique splice variant of the porcine PRLR (pPRLR) that contains a short intracellular domain of 38 aa that is encoded by splicing from exon 9 to a novel exon 11 located 17.5 kb downstream of exon 10 on chromosome 16. The short pPRLR was detected as a 42 kDa protein in membranes from porcine mammary glands. Functional analyses indicated that the short pPRLR functions as a dominant negative against the differentiative function of the long pPRLR and does not transduce a signal to the rat β-casein promoter. Differential abundance of long pPRLR and short pPRLR mRNA was established in a range of porcine tissues. The binding affinity of the short pPRLR for pPRL was lower (K d=3.7 nM) than the affinity of the long pPRLR (K d=1.6 nM) despite a fourfold higher level of binding sites for the short pPRLR. Our data raise the possibility that the short pPRLR in pigs may function independently from the long pPRLR, where the splicing strategy used to generate the short pPRLR likely evolved under different selection pressures to those acting on the long pPRLR.