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B. Goxe, R. Salesse, J. J. Remy, N. Genty and J. Garnier

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.

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Bolander FF Jr, E Ginsburg and BK Vonderhaar

In a previous study, infection with the mouse mammary tumor virus (MMTV) was shown to increase the sensitivity of the mammary epithelium toward prolactin (PRL); furthermore, this effect could be mimicked by the binding of the MMTV envelope protein (gp52) to its cell receptor. The present work has investigated the possibility that gp52-induced changes in the PRL receptor (PRLR) were responsible for this phenomenon. In vitro, gp52 doubled the PRLR concentration in the plasmalemma of mammary epithelium without affecting the affinity. The origins of these PRLRs were twofold: first, gp52 stimulated PRLR mRNA nearly fivefold, suggesting that some of the receptors were newly synthesized. Second, there was a redistribution of PRLRs within the mammary cell: PRLRs were shifted from an internal pool to the plasma membrane. This relocation was very rapid, occurring within 30 min. There did not appear to be any contribution from alterations in PRLR degradation, since the half-life of PRLR was not affected by gp52. In summary, the MMTV increases the PRL sensitivity of mouse mammary epithelium by elevating PRLRs through both enhanced synthesis and recruitment from microsomes.

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Bolander FF Jr

In a previous study, the envelope protein (gp52) of the mouse mammary tumour virus (MMTV) was shown to facilitate mammary gland differentiation by increasing prolactin (PRL) receptors via increased receptor synthesis and via the redistribution of existing receptors from an internal pool. In this study, receptors for other hormones known to affect mammary gland metabolism were investigated. Epidermal growth factor (EGF) stimulates mammary epithelial growth and inhibits differentiation; its receptor is rapidly and dramatically down-regulated by gp52. This is accomplished by its internalization and by decreasing its half-life from 27 h to 2.4 h. Surprisingly, it also increased EGF receptor synthesis, although this effect was not great enough to overcome receptor down-regulation. In contrast, gp52 did not affect the distribution, half-life or synthesis of the insulin receptor. These results demonstrate that MMTV can enhance mammary differentiation by coordinately regulating several hormone receptors: specifically, it can increase the number of receptors for PRL, a differentiative hormone, while decreasing the number of receptors for EGF, a growth/anti-differentiative hormone.

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P H Bird, K A K Hendry, D C Shaw, C J Wilde and K R Nicholas

ABSTRACT

Changes in milk protein gene expression and specific prolactin binding were quantified in mammary tissue from the tammar wallaby (Macropus eugenii) at different stages of lactation. The transition from early (phase 2) lactation to late (phase 3) lactation was characterized by the induction of the gene for late lactation protein, a novel whey protein. During the same period, the levels of β-lactoglobulin and β-casein gene expression increased, whereas there was no change in the levels of expression of α-lactalbumin and α-casein genes. Prolactin binding in the mammary gland doubled during the latter half of phase 2 of lactation but declined significantly during the transition to phase 3 of lactation. These changes in prolactin binding resulted from changes in the number of receptors and not from a change in the affinity of the receptor for prolactin. Treatment of membranes with concanavalin A increased the number of prolactin-binding sites by 40% in membranes from phase 2 mammary tissue but decreased binding by 40% in membranes from phase 3 tissue, indicating that significant changes had occurred in the membranes of cells during this period. The tammar wallaby can secrete phase 2 and phase 3 milk from adjacent mammary glands (asynchronous concurrent lactation) and the developmental changes in milk protein gene expression and prolactin binding observed during lactation were reflected in these individual glands. Taken collectively, these findings suggest that mammary development and milk secretion in the tammar wallaby are regulated by both endocrine and local (intramammary) mechanisms.

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Y Kato, I Sato, T Ihara, K Tomizawa, J Mori, M Geshi, T Nagai, K Okuda, T Kato and S Ueda

Biologically active recombinant porcine FSH (rec-pFSH) free from the cognate pituitary glycoprotein hormone LH was produced. It was synthesized by a baculovirus vector-insect cell system using two cDNAs encoding the glycoprotein alpha and FSH beta subunits. Its antigenicity was the same as that of pFSH prepared from the pituitary. Glycosylation of rec-pFSH was shown by tunicamycin treatment but the molecular mass of each subunit was lower than that of pituitary-derived FSH, because of the absence of trimming of terminal sugars in insect cells. Rec-pFSH was secreted into the culture medium at about 1 mg/l and purified in six fractions, because of the heterogeneity of the sugar group, by S-Sepharose and concanavalin A-Sepharose column chromatography. The biological activity of rec-pFSH was examined by measuring its effect on progesterone secretion from porcine granulosa cells and germinal vesicle breakdown (GVBD) of porcine oocytes. It showed adequate activity with respect to progesterone secretion, although some fractions rich in the sugar group showed lower activity than that of pituitary-derived FSH. It exhibited higher GVBD activity than that of pituitary-derived FSH at concentrations as low as 1 ng/ml. These results demonstrate that the baculovirus vector-insect cell system can provide biologically active rec-pFSH.

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S. Muttukrishna and P. G. Knight

ABSTRACT

Little information is available on the effects of activin and inhibin on the synthesis and secretion of pituitary gonadotrophins in species other than the rat. In this in-vitro study, ovine pituitary cell cultures derived from immature sheep were used to investigate the effects of recombinant human activin-A and native M r 32 000 bovine inhibin on basal and gonadotrophin-releasing hormone (GnRH)-induced release of FSH and LH. Residual cellular contents of FSH and LH were also determined, allowing total content/well to be calculated.

Activin-A promoted a dose-dependent increase in basal (+72%; P<0·001) and GnRH-induced (+25%; P<0·001) release of FSH as well as in the residual cell content (+114%; P<0·001) and total FSH content/well (+67%; P<0·001). Conversely, inhibin significantly (P<0·001) suppressed each aspect of FSH production examined, confirming that in sheep, as in rats, activin and inhibin exert opposing effects on pituitary FSH production.

In contrast to the rat, however, in which activin is reported to have no effect on LH secretion, exposure of sheep pituitary cells to activin-A promoted a dose-dependent suppression (−42%; P<0·001) of GnRH-induced LH release. This was associated with a corresponding increase (P<0·001) in residual cellular content of LH. Consistent with a previous report from this laboratory, inhibin had the opposite effect and significantly enhanced (+47%; P<0·001) GnRH-induced LH release. This was associated with a corresponding fall (P<0·001) in residual cellular content of LH. Neither activin-A nor inhibin significantly affected total LH content/well, indicating a lack of effect of either agent on LH biosynthesis.

Comparison of the dose-dependent effects of inhibin on FSH and LH production by cells cultured in the presence and absence of a fixed dose level of activin-A (5 ng/ml) revealed a significant (P<0·01) interaction between the two agents which appeared to be of a non-competitive nature.

These findings support the concept that both activin and inhibin participate in the gonadal feedback control of pituitary FSH secretion in the sheep. They also indicate that both agents are capable of modulating GnRH-induced LH secretion in this species.

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B. Dattatreyamurty, R. A. Smith, S.-B. Zhang, T. A. Santa-Coloma and L. E. Reichert Jr

ABSTRACT

A 240 kDa protein isolated from bovine calf testis has been shown to have properties characteristic of an FSH receptor. However, rat testis FSH receptor has, on the basis of cloning experiments, been found to have a much lower molecular mass of 75 kDa (peptide only). To examine this point, the size of the FSH receptor in membranes obtained from cultured Sertoli cells of immature rats was determined after polyacrylamide gel electrophoresis under non-reducing conditions, followed by transfer to polyvinylidene difluoride membranes and direct identification of the FSH receptor by ligand blot analysis utilizing radioiodinated human FSH. In this system, the rat Sertoli cell membrane FSH receptor also showed a molecular mass of 240 kDa. Bovine testis contains LH and FSH receptors. We compared the sizes of FSH and LH receptors present in the same bovine testis membrane preparation by ligand blot analysis. The FSH receptor again showed a molecular mass of 240 kDa, whereas the LH receptor showed a molecular mass of 90 kDa. The latter value is similar to that deduced by cloning techniques (75 kDa, peptide only). The evidence seems to suggest that, whereas the molecular mass deduced for the LH receptor on the basis of its cDNA is similar to that of the mature membrane receptor, the size of the FSH membrane receptor is considerably different from that deduced on the basis of its cDNA, presumably as a result of post-translational processing. The marked difference in size between mature FSH (240 kDa) and LH (90 kDa) receptors may reflect significant structural differences of importance with regard to mechanisms of signal transduction.

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L Gabou, M Boisnard, I Gourdou, H Jammes, J-P Dulor and J Djiane

ABSTRACT

cDNA clones coding for rabbit prolactin were isolated from a pituitary library using a rat prolactin RNA probe. One cDNA contained 873 bases including the entire coding sequence of rabbit prolactin, its signal peptide and the 5′ and 3′ untranslated regions of 44 and 145 nucleotides respectively. The deduced amino acid sequence of the cloned prolactin cDNA presented a 93–78% identity with mink, porcine and human prolactins. The prolactin gene transcription was investigated by RT-PCR analysis in several organs of midlactating New Zealand White rabbits. The ectopic transcription of the prolactin gene was examined in more detail in the mammary gland. A strong PCR signal was detected in the mammary gland of virgin does and was also observed during pregnancy and at the beginning of lactation. This PCR signal was very weak in mid-lactating and absent in post-weaning mammary gland.

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SF Wojcik, FL Schanbacher, LK McCauley, H Zhou, V Kartsogiannis, CC Capen and TJ Rosol

Parathyroid hormone-related protein (PTHrP) produced by the mammary gland has been postulated to have multiple functions in both the mother and neonate. In humans, alternative 3'-mRNA splicing and endoproteolytic processing result in multiple bioactive PTHrP peptides. Multiple PTHrP peptides also have been reported in bovine milk. To investigate the source of molecular heterogeneity of PTHrP in bovine milk, bovine PTHrP was cloned from a bovine brain cDNA library, sequenced and used to characterize the mammary PTHrP transcript. A 1065 bp clone (bP1) for bovine PTHrP was isolated from a brain cDNA library. The bP1 clone contained the entire coding sequence of PTHrP and 61 and 473 nucleotides of the 5'- and 3'-untranslated regions (UTRs) respectively. The predicted amino acid sequence of bovine PTHrP was 72-92% homologous to the sequences of chicken, rat, mouse, human, and canine PTHrP with the highest sequence divergence present in the C-terminal region of the peptide. The 5'- and 3'-UTRs of bovine brain PTHrP have a high degree of homology to exons 4 and 9 of human PTHrP respectively. PTHrP was expressed as a single 1200 nucleotide mRNA transcript in lactating bovine mammary tissue. RT-PCR using region-specific oligonucleotide primers derived from bP1 demonstrated that PTHrP mRNA transcripts in bovine brain and lactating mammary gland utilize the same 5'- and 3'-UTRs. Expression of PTHrP mRNA was localized to secretory and ductular epithelial cells within the lactating mammary gland, as detected using in situ hybridization. Expression of PTHrP mRNA was demonstrated in the mammary gland during late pregnancy and throughout lactation in cows.

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C Palmieri, S Saji, H Sakaguchi, G Cheng, A Sunters, MJ O'Hare, M Warner, JA Gustafsson, RC Coombes and EW Lam

Whilst oestrogen receptor (ER)-alpha and ERbeta have been shown to be important in the development of the mammary gland, the cell-specific expression pattern of these two receptors within the human breast is not clear. Although it is well established that in the developing rodent mammary gland stromal ERalpha mediates the secretion of growth factors which stimulate the proliferation of the ductal epithelium, the expression of ERalpha in human adult breast stromal fibroblasts is controversial, and the expression of ERbeta has not been properly defined. In the present study, we have evaluated the expression of ERalpha and ERbeta by immunohistochemistry in normal tissue samples, and in purified human breast fibroblasts by Western blotting, RT-PCR analysis and ligand-binding sucrose gradient assay. Our data clearly demonstrated that ERbeta variants, including ERbeta1, ERbeta2, ERbeta5, ERbetadelta and ERbetains, but not ERalpha, are expressed in human adult mammary fibroblasts. These results are supported by the findings that an ERbeta-selective ligand, BAG, but not the ERalpha high-affinity ligand oestradiol, can induce fibroblast growth factor-7 release and activate transcription from an oestrogen-responsive element promoter in these adult human mammary fibroblasts. Together, these observations revealed that, in the adult breast and in breast cancer, the proliferative signals derived from the stroma of adult mammary glands in response to oestrogen are not mediated by ERalpha and provide new insights into the nature of stromal-epithelial interactions in the adult mammary gland. In addition, the expression of these ERbeta variants in cells where there is no ERalpha suggested that these ERbeta splice forms may have functions other than that of modulating ERalpha activity.