The levels of lactogenic activity in the serum of rabbits during pregnancy and early lactation have been determined. The accuracy and sensitivity of the radioreceptor assay used were increased by several technical improvements. Lactogenic activity remained low throughout pregnancy and increased at parturition; the highest values were reached on day 5 of lactation. No lactogenic activity was detected in placental extracts of rabbits, suggesting that the lactogenic activity measured in the serum is solely of pituitary origin. These results are discussed in relation to the main stages of development of rabbit mammary glands.

Laboratoire de Physiologie de la Lactation, Institut National de la Recherche Agronomique, 78350-Jouy-en-Josas, France

(Received 23 December 1974)

Hormonal induction of milk synthesis by the ruminant mammary gland in organ culture has received little attention. M. A. Cannata, C. Delouis, C. Jeulin & R. Denamur (unpublished observation) demonstrated the lactogenic effect of insulin + cortisol + prolactin in vitro on ewe mammary tissue with lobulo-alveolar development at the time of explantation, but there are no data on the induction of milk secretion in bovine mammary tissue cultured on a synthetic medium. Therefore, we have studied the culture conditions necessary to maintain bovine mammary tissue and the combination of hormones needed to induce milk secretion.

Biopsy samples were obtained from 2-year-old French Friesian heifers at various times during the oestrous cycle, after i.m. injection of the tranquillizer, xylazine (Rompun, Bayer; 2% solution, 1·5 ml/100 kg body weight). Expiants (1 mm³) were

Abstract

The binding of radioiodinated rabbit (rb) prolactin (PRL) to rabbit mammary membranes is low and its affinity constant, 0·02 nm ⁻¹, calculated from heterologous inhibition assays, is about 300 times lower than that of ovine (o) PRL. Although the differences between homologous and heterologous binding are well documented in different species, the reasons for such differences are still unknown.

Here we show that the low affinity of rbPRL for the native receptor does not affect its in vitro bioactivity compared with that of oPRL. We also show that rbPRL displays high specific binding to the baculovirus-expressed recombinant receptor and further establish that its lower affinity for binding to the homologous receptor is due to its faster and more complete dissociation compared with that of oPRL. Hormone binding affinity for full-length and carboxy-terminal truncated rbPRL receptor mutants expressed in mammalian or in baculovirus-infected cells was not affected by partial truncation of the cytoplasmic domain of the receptor, whereas the affinity for oPRL increased and that for rbPRL decreased upon truncation of both the cytoplasmic and membrane domains. The affinity of rbPRL for the native receptor is two orders of magnitude lower than that for the recombinant receptor. Affinity cross-linking and binding experiments showed that this difference in affinities is not related to selective cleavage of the native microsomal receptor during the binding reaction; however, this difference may be related to cell context-dependent differences in the oligomerization state of the receptor.

Thus, obviously, the cloned receptor is alone sufficient for binding to rbPRL without requiring any receptor-associated protein. The lower affinity for rbPRL binding to its homologous receptor in comparison with higher affinity binding of oPRL to the same receptor is attributable to differences in their dissociation kinetics and in the conformational requirements of the receptor–hormone interaction site for binding to the two hormones.

Journal of Endocrinology (1997) 153, 207–219

Laboratoire de Physiologie des Poissons and *Laboratoire de Physiologie de la Lactation, I.N.R.A., 78350 Jouy en Josas, France

(Received 11 October 1976)

Fish prolactin bioassays lack specificity (Sage & Bern, 1972), have poor sensitivity, are difficult to use (Ensor & Ball, 1968; Clarke, 1973) and contradictory conclusions have been drawn with them. Nicoll & Bern (1968) obtained no lactogenic effect of teleost pituitary gland in the pigeon-crop and the rabbit mammary gland tests, but Chadwick (1966) demonstrated the occurrence of a mammotrophic effect. Other workers have used heterologous radioimmunoassay (RIA) systems (McKeown & Van Overbeeke, 1972); these RIA systems do not seem to cross-react specifically with fish prolactin (Nicoll, 1975). We present a new approach using rabbit mammary gland prolactin receptors.

The technique used was a modification of the radioreceptor assay (RRA) for lactogenic hormones (Shiu, Kelly & Friesen, 1973). At the beginning of lactation the rabbit was injected with

The present study demonstrates that ovine placental lactogen (oPL) (ovine chorionic somatotrophin) may have an important role in the mammogenesis and/or lactogenesis of the ewe. Its effects were compared with that already described for ovine growth hormone (oGH). In the first experiment, 40 nulliparous ewes were induced to lactate by means of a 7 day (days 1-7) oestro-progestative treatment (E2+P4). The ewes from Group 1 (n=12) received no further treatment, while those of the other groups received either recombinant oGH (roGH, 28 micrograms/kg, i.m., twice daily, Group 2, n=12) or recombinant oPL (roPL, 79 micrograms/kg, i.m., twice daily, Group 3, n=12) from day 11 to 20. All ewes received 25 mg hydrocortisone acetate (HC) twice daily on days 18-20. Control Group 00 (n=2) received no steroid treatment at all, and the control Group 0 (n=2) received only the E2+P4 treatment. Thirteen ewes (three from each experimental group and the two of each control group) were slaughtered at the end of hormone treatments (day 21) before any milking stimulus. The 27 remaining ewes from Groups 1-3 were machine-milked and milk yields recorded daily from day 21 to 76. The E2+P4 treatment enhanced the plasma levels of oPRL, oGH and IGF-I between days 1 and 7 by 1.5, 2. 3 and 2.6 times respectively (P=0.002); roGH treatment induced a highly significant enhancement of IGF-I plasma levels from day 11 to 20, whereas a similar effect appeared for roPL-treated ewes only from day 17 to 20 (P<0.01). Eight weeks after the last exogenous hormone injections, milk yields of both roGH- and roPL-treated groups progressively rose to twice that of unsupplemented groups (P<0.001). The mammary DNA content on day 21 was higher for animals which received either oGH or oPL but, due to individual variations in so few samples (n=3), this difference was not significant. No beta-casein was measured in mammary tissue from control ewes, whereas steroid-treated ewes (E2+P4+HC) had higher casein concentrations regardless of subsequent hormonal treatment on days 11-20 (P<0.001). beta-Casein concentrations in mammary parenchyma of roGH-treated ewes did not differ from that of ewes which received only E2+P4+HC; roPL supplementation clearly enhanced expression of beta-casein (P<0.001). IGF-I stimulation by either roGH or roPL was more precisely examined during a second experiment, in which two twice-daily i.m. doses (58 or 116 micrograms/kg) of either roGH or roPL were administered to four groups of six ewes that were E2+P4 treated as those of Experiment 1. A control group (n=6) received no exogenous hormone from day 11 to 13. On day 13, hourly blood samples were taken from all ewes over 11 h. Both doses of roGH significantly stimulated IGF-I in a dose-dependent manner. The 58 micrograms/kg dose of roPL did not significantly stimulate IGF-I, but although being somewhat less efficient than the 58 micrograms/kg dose of roGH, the 116 micrograms/kg dose of roPL significantly stimulated IGF-I secretion (P<0. 001). These results suggest that mammogenesis and/or lactogenesis in the ewe is in part controlled by somatotrophic hormones such as oGH and oPL and that IGF-I could be one of the mediators of these hormones.

Leptin has been implicated in the regulation of feed intake, growth, and reproduction. The objective of this study was to determine if centrally administered leptin would affect feed intake and the secretion of growth hormone (GH) and luteinizing hormone (LH) in ewe lambs. Eighteen ewe lambs were ovariectomized and fitted with intracerebroventricular (i.c.v.) cannulae. Lambs were randomly assigned to receive either a maintenance diet (fed), or a diet that provided 38% of maintenance requirements (diet-restricted) for 14 weeks. Subsequently, recombinant ovine leptin or vehicle was continuously infused, via i.c.v. cannulae, in a linearly increasing dose for 8 days, reaching a maximum of 1.25 microg/kg per h. Feed intake was recorded on days -1 to 7. Blood was collected via jugular cannulae every 10 min for 4 h on days 0, 2, 4, 6 and 8 for the determination of serum leptin, insulin, LH and GH. Leptin suppressed feed intake in fed lambs on days 4 to 7 (P<0.001), but had no effect on feed intake in diet-restricted lambs (P>0.25). Fed lambs had greater serum concentrations of leptin than diet-restricted lambs (P=0.007). Also, although not different on day 0 (pretreatment), on day 8 serum leptin concentrations were greater in leptin-treated lambs than in saline-treated lambs (P=0.003). Insulin was lower in diet-restricted than in fed lambs (P=0.003), but was not affected by leptin treatment (P=0.82). LH pulse frequencies were lower in diet-restricted lambs than in fed lambs (P=0.038), but were not affected by leptin treatment (P=0.85). Mean serum GH was greater in diet-restricted than in fed lambs (P<0.01). In diet-restricted lambs treated with leptin or saline, mean GH did not differ on day 0, but increased in response to leptin treatment (P<0.006). Treatment of fed lambs with leptin did not affect serum GH (P>0.32). From this work, we propose that leptin represents an important functional link between adipose stores and hypothalamic function in ruminants. We demonstrate that leptin concentrations change in response to reduced nutritional status, and that leptin has the ability to regulate multiple physiological processes in lambs, including both feed intake and secretion of GH.

Abstract

To clone ovine placental lactogen (oPL) cDNA, total RNA from sheep placental cotyledon was reverse transcribed and the single-stranded cDNA was PCR-amplified with 5′ and 3′ primers containing, respectively, Ncol and PstI sites. The oPL cDNA fragment amplified between these two primers extended from A(−1) to the natural stop codon. The PCR product was gel-purified and subcloned into a Puc vector and the insert was sequenced on both strands, revealing several differences relative to the published sequence: S19N, S69N, D129E and R165Q. We assume that these differences can be accounted for by the high level of individual polymorphism, which has been described in detail for PLs of different species. The insert was subcloned into Ncol/PstI-digested pTrc99A procaryotic expression plasmid and protein expression was induced by isopropyl-1-thio-β-d-galactopyranoside. Because of low expression, oPL's cDNA was further subcloned into pET8 procaryotic expression plasmid. Its expression in E. coli strain BL21 transformed with this vector yielded 30–40 mg/l. The expressed protein, found in the inclusion bodies, was refolded into a monomer and purified on a Q-Sepharose column to homogeneity. Structural analysis using circular dichroism revealed a spectrum similar to that of human GH (hGH) thereby indicating proper refolding. Gel filtration and binding experiments, including real-time kinetic measurements using the surface plasmon resonance method revealed that oPL forms transient homodimeric complexes with extracellular domains of prolactin receptors from rabbit, rat and bovine and with hGH receptor. The purified oPL was biologically active in an Nb₂-11C cell proliferation bioassay, in its ability to stimulate β-casein synthesis in explants of ovine and rabbit mammary gland and fat synthesis in explants of bovine mammary gland, and in a proliferation assay using FDC-P1 cells transfected with rabbit or hGH receptors.

Journal of Endocrinology (1997) 152, 317–327

Caprine placental lactogen (cPL) cDNA was cloned by reverse transcription (RT)-PCR from total RNA of goat placenta. The PCR product encoding for the mature protein was gel purified, ligated to pGEM-T and finally subcloned into a pET8c prokaryotic expression vector. E. coli cells (BL-21) transformed with this vector overexpressed large amounts of cPL upon induction with Isopropyl-1-thio-beta-D-galactopyranoside. The expressed protein, found in the inclusion bodies, was refolded and purified to homogeneity on Q-Sepharose and SP-Sepharose columns, yielding two electrophoretically pure fractions (cPL-Q and cPL-S), composed of over 98% of monomeric protein of the expected molecular mass of approximately 23 kDa. Binding of cPL to the extracellular domain (ECD) of prolactin receptors (PRLR) from rat (r), rabbit (rb), and bovine (b), growth hormone receptors (GHR) from human (h) and rabbit, and binding to rabbit mammary gland membranes revealed similar binding profiles for cPL-Q, cPL-S and ovine (o)PL. Caprine PL was capable of forming 1:2 complexes with hGHR-ECD, rbGHR-ECD, rPRLR-ECD and rbPRLR-ECD whereas with bPRLR-ECD only a 1:1 complex was detected. The biological activity of both cPL fractions resulting from proper renaturation was further evidenced by their ability to stimulate proliferation of Nb2 cells, FDC-P1 cells transfected with rabbit or human GHRs and by stimulation of beta-casein synthesis in rabbit and ovine mammary gland acini cultures.

The biological activities of ovine (o) and bovine (b) placental lactogens (PLs) and their mutated analogues were compared using several binding and in vitro bioassays. In almost all cases, the biological activities of these analogues mediated through rat (r) prolactin receptor (PRLR) showed little or no change, despite a remarkable decrease in their capacity to bind to the extracellular domain of rPRLR and despite compromised stability of the 2:1 complexes. These results indicate that mutations impairing the ability of oPL or bPL to form stable complexes with lactogenic receptors do not necessarily lead to a decrease in the biological activity, because the transient existence of the homodimeric complex is still sufficient to initiate the signal transduction. In contrast, oPL and bPL analogues completely, or almost completely, lost their ability to activate homologous PRLRs, and some of them even acted as site-2 antagonists. To explain the difference between the activity transduced through homologous and that transduced through heterologous PRLRs, we propose the novel term 'minimal time of homodimer persistence'. This concept assumes that in order to initiate the signal transduction, the associated kinase JAK2 has to be transphosphorylated and this requires a 'minimal time' of homodimer existence. In the case of homologous interaction between ruminant PLs and homologous PRLRs, this 'minimal time' is met, though the interaction with homologous PRLRs has a shorter half-life than that with heterologous PRLRs. Therefore oPL or bPL are active in cells possessing both homologous and heterologous PRLRs. Mutations of oPL or bPL lead to reduced affinity and, consequently, the 'time of homodimer persistence' is shortened. Although in the case of heterologous interaction the 'minimal time' is still sufficient to initiate the biological activity, in homologous interactions, which are already weaker than heterologous interactions, further destabilization of the complex shortens its persistence to below the 'minimal time', leading to full or partial loss of biological activity.

Hyperprolactinemia and hyperleptinemia occur during gestation and lactation with marked hyperphagia associated with leptin resistance. Prolactin (PRL) induces the expression of orexigenic neuropeptide Y (NPY) through the activation of JAK-2/STAT-3 signaling pathway in hypothalamic paraventricular nucleus (PVN) leading to hyperphagia. PRL may also act through the inhibition of anorexigenic effect of leptin via induction of suppressor of cytokine signaling 3 (SOCS-3). This paper aimed to co-localize PRL (PRL-R) and leptin (ObRb) receptors in the hypothalamus of female rats and investigate the possible cross-desensitization between PRL-R and ObRb. We showed that: 1) PRL-R and ObRb are expressed in the PVN and co-localized in the same neurons; 2) in lactating females leptin failed to activate JAK-2/STAT-3 signaling pathway; 3) in Chinese Hamster Ovary (CHO) stably co-expressing PRL-R and ObRb, overexposure to PRL did not affect leptin signaling but totally abolished PRL-dependent STAT-5 phosphorylation. The overexposure to leptin produces similar results with strong alteration of leptin-dependent STAT-3 phosphorylation, whereas PRL-dependent STAT-5 was not affected; and 4) CHO-ObRb/PRL-R cells overexposure to leptin or PRL induces the expression of negative regulators SOCS-3 and PTP-1B. Thus, we conclude that these negative regulators affect specifically the inducer signaling pathway; for instance, SOCS-3 induced by PRL will affect PRL-R signaling but not ObRb signaling and vice versa. Finally, the lack of cross-desensitization between PURL-R and ObRb suggests that hyperphagia observed during gestation and lactation may be attributed to a direct effect of PRL on NPYexpression, and is most likely exacerbated by the physiological leptin resistance state.