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ABSTRACT

Granulosa cells were prepared from small follicles (<3 mm) from the ovaries of 5-month-old gilts. They were cultured in plastic dishes coated with a synthetic adhesion peptide in a chemically defined medium supplemented with 2% serum substitute. After 3 days of culture, the cells reached confluence and expression of the LH receptor could be stimulated in a hormonally defined medium. LH receptor RNAs were estimated by autoradiography using Northern blots and dot blots of total cell RNA. LH receptor RNAs were hybridized with a homologous ³²P-labelled random-primed DNA probe. The LH receptor was measured using ¹²⁵I-labelled human chorionic gonadotrophin (hCG) as tracer.

Northern blots of LH receptor RNAs revealed a predominant signal of 4.4kb and two less-intense hybridization bands of 7.5 and 1.9kb. The 4.4kb band was used for quantification of LH receptor RNAs because it was the most intense and may be attributed to the full-length messenger RNA. In these conditions, after 72h stimulation, FSH (0.6 nm), insulin (5 μg/ml), oestradiol (30 nm) and deoxycorticosterone (0.3 nm) yielded high LH receptor RNA levels (eight times unstimulated cell level), while dibutyryl cyclic AMP (1 mm), cortisol (5.4 nm), thyroxine (100 nm) and epidermal growth factor (16pm) gave low LH receptor RNA levels (one to five times). However, the respective amounts of the receptor RNA did not give yield to the same proportion of LH receptor for every factor, indicating some post-transcriptional regulations.

The kinetic study of the production of the LH receptor obtained in a defined medium supplemented with FSH, oestradiol and insulin showed that the receptor appeared after 48 h of stimulation and reached a maximum of about 7000 receptors per cell at 72 h. The three hybridization bands on Northern blots evolved in parallel and appeared as early as 24 h. They were at maximal level from 24 to 48 h of stimulation. When the granulosa cells were pulse-treated for 2 h with cycloheximide (10 μg/ml), they exhibited a transient rise in LH receptor RNA content which was followed by a delayed receptor increase especially at 72 h of stimulation.

Taken together, these results indicate that the LH receptor in primary culture of granulosa cells seems to be regulated by different physiological factors both at the transcriptional and the translational levels.

ABSTRACT

Among all mammalian FSH receptors (FSH-R; including donkey (dk) FSH-R), only horse (hs) FSH-R does not bind hsLH/chorionic gonadotrophin (CG). In order to delineate the structural origin of hsFSH-R specificity precisely, we have cloned dkFSH-R cDNA from donkey testis mRNA by RT-PCR. Transiently expressed dkFSH-R endowed COS-7 cells with both hsLH/CG- and FSH-binding activity, as well as FSH-induced cAMP production.

The deduced dkFSH-R amino acid sequence shares 96% identity with the hsFSH-R: notably, in the hormone-binding domain, the specificity of hsFSH-R may be ascribed to only four divergent amino acids: Thr 173, Asp 202, Asn 268 and Pro 322. Interestingly, hsAsn 268 could bear an additional N-glycosylation. According to receptor negative specificity, these amino acids could be implicated in preventing LH/CG binding to FSH-R.

ABSTRACT

Overexpression of the porcine LH receptor (pLHR) ectodomain has been achieved using the baculovirusinsect cell system but mostly in an aggregated form with no secretion. In order to carry this out, new baculoviruses were selected to produce the pLHR ectodomain in insect Sf9 cells and caterpillars. In pLHR-P10–297 and pLHR-mel-319 baculoviruses, pLHR cDNA was under the control of the P10 promoter and the polyhedrin gene promoter respectively.

The constructs contained either the porcine signal peptide (pLHR-P10–297) or the insect signal peptide of melittin (pLHR-mol-319). Infected cells produced 1 × 10⁵−3 × 10⁵ receptors/cell 3 days after infection. The recombinant LH receptor ectodomains produced were secreted in a biologically active form and bound the hormone with high affinity. Infected caterpillars produced a larger amount of active pLHR ectodomain than insect cells. The products were not secreted into the haemolymph however.

Promoter and/or signal peptide modifications therefore enabled pLHR recombinant ectodomain secretion in a biologically active form, using the baculovirus—lepidopteran cell system. Moreover, moderate levels of expression seem to allow the production of biologically active ectodomain.

ABSTRACT

Porcine LH receptor ectodomain was overexpressed in insect cells and lepidopteran larvae using the recombinant baculovirus expression system. A low multiplicity of infection yielded the largest active production, of approximately 10⁷ receptors/cell or 3 μg active receptor/mg total protein in infected cells. The truncated ectodomain solubilized with Triton X-100 bound its ligand with a high affinity which was comparable with that of the native membrane receptor. Increasing the multiplicity of infection resulted in an optimum protein production of 0·6 mg receptor/mg total protein in infected cells. This receptor was largely inactive, probably trapped within aggregation pools. Active receptor could be recovered by dilution of the samples. No secretion of recombinant receptor was ever observed whatever the conditions of infection. Expression of the recombinant receptor in insect larvae was also tested. This low-cost system failed both to increase the amount of active receptor and to induce secretion into the haemolymph. Two methods remain for producing sizeable amounts of active receptor with this baculovirus/insect cell system. One relies on immunoaffinity purification of the active protein and requires large-scale production, and the other is based on the purification of overexpressed inactive receptor followed by renaturation.

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.

ABSTRACT

We have combined different techniques to analyse passages of five different rat spontaneous pituitary tumours (SMtTW) that were transplanted under the kidney capsule. These tumours were secreting prolactin (PRL), GH or both hormones. RIA, immunocytochemistry (ICC) and Western blot analysis were applied to characterize the hormone(s) stored (ICC and Western blot) and secreted (RIA). mRNA content was analysed by PCR, Northern blot analysis and in situ hybridization.

The data point not only to the reliability of the techniques used at both protein and RNA levels for each tumour studied but also to the complementarity of some techniques. For example, whereas Northern blot analysis demonstrates the presence and size of hormone mRNA, in situ hybridization indicates the percentage of cells expressing a given hormone mRNA and allows the presence of one population (or more) of cells in a given tumour to be identified.

Moreover, the tumours were compared with normal rat pituitary. Although the PRL and GH mRNAs were identical in size, the amount of mRNA was lower in the tumours. At the protein level, the PRL and GH variants exhibited a different pattern of expression in tumours compared with the normal rat pituitary.

The biological significance of these differences is discussed.