Transport of uterine PGF2 alpha to the ovaries by systemic circulation and local lymphovenous-arterial diffusion during luteolysis in sheep.

The theory of countercurrent vascular transfer of PGF2 alpha during luteolysis was examined. In the first experiment, pulmonary clearance of PGF2 alpha was determined to re-examine whether the total amount of PGF2 alpha was degraded in the lungs after one passage. Cardiac output was measured by the Fick method and PGF2 alpha by radio-immunoassay before and after vascular lung supply, using pulmonary catheterization and the interventional radiology method in ten anaesthetized ewes on day 16 of the oestrous cycle. Cardiac output remained stable (7156 +/- 439 ml min-1). Infusion of 5 iu oxytocin resulted in an increase in plasma PGF2 alpha concentrations at 30 min in the uterine vein and the pulmonary and femoral arteries (3811 +/- 806, 224 +/- 55 and 18 +/- 4 pg ml-1, respectively). The PGF2 alpha concentrations decreased exponentially and the half-time decreases were 27 (r = 0.99), 16 (r = 0.99) and 18 (r = 0.98) min, respectively. Pulmonary clearance of PGF2 alpha was estimated at 6338 +/- 451 ml min-1. In a second experiment, an arterio-arterial gradient of plasma PGF2 alpha concentrations was analysed between the proximal and distal segments of the ovarian artery to verify whether the total amount of PGF2 alpha flowing to the ovary was from the local venous-arterial countercurrent pathway. Surgical catheterization techniques were performed on 11 ewes on day 16 of the oestrous cycle. The ovarian arterial blood flow was measured by the implantable Doppler method (8 +/- 1 ml min-1). The maximum plasma PGF2 alpha concentrations in the femoral and distal ovarian arteries were 23 +/- 6 and 42 +/- 11 pg ml-1 (P < 0.05), respectively. Plasma PGF2 alpha decreased exponentially in the femoral artery and the half-time decrease was 26 min (r = 0.98), and in the distal ovarian artery close to the ovary PGF2 alpha decreased linearly and the half-time decrease was 108 min (r = 0.96). Consequently, the arterio-arterial diffusion gradient of PGF2 alpha concentrations was extended to 3 h. These experiments showed that the PGF2 alpha flow rate in the pulmonary artery was 42.275 +/- 10.793 micrograms per 150 min (n = 10) and the systemic arterial PGF2 alpha flow rate was 5.359 +/- 1.658 micrograms per 150 min (n = 10). Therefore, 12% of the PGF2 alpha was not oxidized by the lungs. The proximal ovarian PGF2 alpha flow rate was 6.909 +/- 2.341 ng per 150 min, while the distal flow rate was 21.003 +/- 5.703 ng per 150 min (n = 11). Thus, 33% of the PGF2 alpha was transported rapidly to the ovary via the systemic route, while 67% was transported by slow local countercurrent diffusion, which extended the duration of luteolytic activity to four times that of the PGF2 alpha surge. These results indicate both rapid systemic transport of PGF2 alpha to the ovaries and a slower buffer mechanism involving a local diffusion pathway, rather than a direct countercurrent system.

IFN-tau: a novel subtype I IFN1. Structural characteristics, non-ubiquitous expression, structure-function relationships, a pregnancy hormonal embryonic signal and cross-species therapeutic potentialities.

IFN-tau (IFN-tau) constitutes a new class of type I IFN which is not virus-inducible, unlike IFN-alpha and IFN-beta, but is constitutively produced by the trophectoderm of the ruminant conceptus during a very short period in early pregnancy. It plays a pivotal role in the mechanisms of maternal recognition of pregnancy in ruminants and it displays high antiviral and antiproliferative activities across species with a prominent lack of cytotoxicity at high concentrations in vitro in cell culture and possibly in vivo. It exhibits high antiretroviral activity against HIV and exhibits immunosuppressive activity in a multiple sclerosis model and reduces embryo and fetal mortality by stimulation of IL-10 production. In this review all the biochemical and para-hormonal properties of this novel IFN-tau are described in detail: structural characteristics of proteins and genes, trophoblast expression, regulation of its expression, structure of its gene promoter, its absence in human species and in non-ruminant animals, the evolution of the IFN-tau genes, its structure-function relationships with its three-dimensional structure, structural localization of biological activities, its lack of cytotoxicity and its receptor. Surprisingly, for an IFN, IFN-tau is also a pregnancy-embryonic signal with paracrine antiluteolytic activity. In order to maintain luteal progesterone secretion, IFN-tau inhibits PGF-2alpha pulsatile secretion and oxytocin uterine receptivity in early pregnancy. It is believed to suppress pulsatile release of endometrial PGF-2alpha by preventing oxytocin and estrogen receptor expression. Additionally, it directly regulates prostaglandin metabolism and possibly the PGE:PGF-2alpha ratio.

Recent developments and potentialities for reducing embryo mortality in ruminants: the role of IFN-tau and other cytokines in early pregnancy.

This review considers the potential reduction of embryo mortality in vitro and in vivo in ruminants. Data on cytokines provided by different fields of reproductive immunology and biology were collated. Because of the crucial importance of the local interactions between the embryo and its dam, the expression of growth-factor and cytokine genes was analysed in the embryo proper, trophoblast, oviduct and endometrium by reverse transcriptase polymerase chain reaction in sheep and in cattle during the pre- and periimplantation periods. Many deleterious cytokines, such as tumour necrosis factor-alpha, interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and beneficial cytokines, such as transforming growth factor-beta, leukaemia inhibiting factor, colony-stimulating factor-1 (CSF-1), granulocyte-macrophage CSF, IL-1, IL-3, IL-4, IL-6, IL-10 and IFN-tau appeared to be involved in embryo survival in ruminants and other species. Their administration is efficient in a murine experimental model (CBA/J x DBA/2) of embryonic and fetal mortality. For instance, recombinant ovine IFN-tau (roIFN-tau) injected at the moment of implantation drastically reduces embryonic mortality in this model. In ruminants, roIFN-tau and recombinant bovine IFN-tau are very efficient in maintaining progesterone luteal secretion in cyclic animals. The involvement of IFN-tau in the mechanisms of maternal pregnancy recognition are particularly detailed in relation to inhibition of 13,14 dihydro-15-keto-prostaglandin F2 alpha (PGFM) pulses and oxytocin uterine receptivity. A synthetic model of the anti-luteolytic effects of IFN-tau on the endometrial cell is proposed. Finally, the particular potential of serum pregnancy-specific proteins (PSPs: PSPB, PSP60, pregnancy-associated glycoprotein) for monitoring embryo survival, with examples given for cattle and sheep is underlined.

[Trophoblastic interferons and embryonal immune tolerance]. / Les interférons trophoblastiques et la tolérance immunologique embryonnaire.

Current evidence support the hypothesis that trophoblast interferons play a key role in preventing maternal immunologal rejection of the embryonic semi-allograft. The information of this review is divided in two sections. In the first section we described molecular and biological characteristics of type I (alpha, beta, omega, tau and spl) and type II (gamma) interferons. In the second section we emphasize studies on immunoendocrine functions of IFN-tau (oTP-1 or trophoblastins) in the network of cytokines and hormonal environment at the uterine embryonic interface.

Comparative expression of TGF-alpha and EGF genes in the ovine conceptus and uterine endometrium in the peri-implantation period.

Control of growth and differentiation during mammalian embryogenesis may be regulated by growth factors. With the use of a very sensitive method, the reverse transcription-polymerase chain reaction, expression of some growth factor transcripts was examined during ovine peri-implantation development. Transcripts for transforming growth factor-alpha (TGF-alpha) were found in day 15 to day 30 conceptuses, and in uterine tissues. Epidermal growth factor (EGF) was not detectable in any ovine stages or tissues studied. TGF-alpha could be the normal physiological ligand for the epidermal growth factor receptor present on the trophoblastic tissues, and its expression pattern suggests an autocrine and paracrine role in the growth and differentiation of ovine embryos.

[Cytokines and the corpus luteum]. / Cytokines et corps jaune.

This minireview present main findings concerning the contribution of cytokines to the regulation of some key processes of luteal functions. Data concerning the preovulatory follicles invasion by white blood cells and the migration of macrophages, granulocytes and T lymphocytes into corpus luteum suggest that local secretion of regulatory cytokines may be involved in regulating corpus luteum formation and demise as well its maintenance in early pregnancy. Several lines of evidence indicate that the pro-inflammatory cytokines such as IL-1, IL-6 and TNF-alpha modulate the steroidogenic changes which take place during luteinization. For instance, an inhibition of E2 biosynthesis is evidenced in granulosa cells in human or porcine species with IL-1, in rat with TNF-a and in bovine with IL-6. Moreover, IL-1 stimulates P4 production but to a much lower extent than LH, and PGE2 synthesis by rat thecal cells. The potential relevance of pro-inflammatory cytokines in the mechanisms controlling luteolysis is suggested by the ability of IL-1 and TNF-alpha to decrease both P4 production and the survival of bovine luteal cells. As opposed to ruminants, TNF-alpha has no effect in human luteal cells but potentiates the decrease of P4 secretion induced by IFN-gamma. Finally, data regarding the participation of trophoblast interferons in the mechanisms for maintaining the corpus luteum at the establishment of pregnancy are now available in ruminants. From these observations and others, we can consider that cytokines are involved in the regulation of the corpus luteum function.

Localization and characterization of EGF/TGF-alpha receptors on peri-implantation trophoblast in sheep.

Receptors for epidermal growth factor (EGF) have been identified on the ovine trophoblast as early as day 15 of gestation. A radioligand assay with 125I-labelled EGF was used to detect high and low affinity binding sites on the trophoblastic and placental membranes. The binding of 125I-labelled EGF was inhibited by increasing concentrations of unlabelled EGF. Competition studies with other peptide hormones including transforming growth factor alpha (TGF-alpha), insulin-like growth factor-I (IGF-I) and ovine placental lactogen confirmed the specificity of EGF/TGF-alpha for its receptor. Cross-linking experiments using disuccinimidyl suberate (DSS) revealed a radiolabelled band of relative molecular mass 170 kDa. Immunohistochemical localization of the receptors demonstrated their distribution on the epithelial layer cells. The presence of receptors for EGF/TGF-alpha suggests that these factors could be involved in the regulation of embryonic development and fetal growth.

Rabbit whey acidic protein concentration in milk, serum, mammary gland extract, and culture medium.

Rabbit whey acidic protein has been purified from whey using an AcA54 column. The purified whey acidic protein had an amino acid composition in agreement with the previously defined cDNA sequence. An antibody against whey acidic protein was raised in guinea pig. This antibody did not crossreact with mouse or cow milk or with rabbit alpha s1-casein and beta-casein. Whey acidic protein concentration was measured in rabbit milk using the antibody with a radioimmunoassay. The concentration of whey acidic protein in rabbit milk was 15 mg/ml, whereas the concentrations of alpha s1-casein and beta-casein were 16 and 45 mg/ml, respectively. The concentration of the three proteins was also evaluated in culture medium of rabbit primary mammary cells. The three proteins were induced by prolactin alone. Glucocorticoids amplified the prolactin effect on whey acidic protein more intensively than on caseins. The three proteins were present in mammary extract from virgin rabbit. The concentration of these proteins was lower at d 8 and 14 of pregnancy, and it was very high at d 25 of pregnancy. Whey acidic protein was undetectable in blood of virgin, weaned, and midpregnant females and of males. Whey acidic protein was present in blood of lactating rabbits, but alpha s1-casein and beta-casein were not detectably present in rabbit blood at the examined physiological states.

Growth hormones. II. Structure-function relationships.

Localization of the somatotropic activity of growth hormones from several species and from different organs was attempted using different approaches. Sequences were compared in order to detect one or several regions with a common homology. The technique of peptide recombinants as well as chemical changes affecting some amino acids was applied to these hormones; the biological function in vivo of growth or binding to somatotropic receptors was then estimated. The few data available on biosynthetic molecules and secondary structures of natural growth hormones are reported. This study indicates the somatotropic function of particular sites.

In-vitro secretion of prolactin and growth hormone in the presence of melatonin by pituitary gland from mink kept under long or short days.

Mink anterior pituitaries were incubated in Medium 199 for up to 9 or 13 days. Biological activity of prolactin and GH was determined. Daily concentrations of prolactin and GH in the incubation medium were also measured by radioimmunoassay and radioreceptor assay. When females were kept under short days for several weeks before the experiment, a significant decrease in prolactin secretion by the anterior pituitary was observed as compared with that in females maintained under long days. In contrast, secretion of GH was not modified by the photoperiodic history of the animals. Pineal gland denervation by ablation of the superior cervical ganglia a few months before the experiment, or addition of melatonin to the incubation medium of anterior pituitaries from intact or ganglionectomized females, did not modify the secretion of prolactin and GH. The pituitary gland does not therefore seem to be a direct target site for melatonin in transducing the duration of daylength on the hypothalamo-pituitary axis.

[Trophoblastic proteins]. / Protéines trophoblastiques.

Many studies conducted on human or animal placenta (chorion) suggest that the trophoblast is not only a passive filter between maternal and foetal blood flow, but is also endowed with complex functions. Factors of trophoblast origin involved in the mechanism of pregnancy recognition or maintenance of the progesterone environment required for the embryo survival, are reviewed. The main proteins involved in pregnancy are reported in table 1. Emphasis is laid on early signals of pregnancy which are of practical interest in human clinical medicine and animal husbandry. Among them, human chorionic gonadotropin (hCG), protein SP1 ("Schwangerschaftsprotein" 1) and some pregnancy-associated plasma proteins such as the PAPP A are very useful in the diagnosis of pregnancy, abortion, foetal abnormality or tumor in the human species. The presence of trophoblastin (presently studied in our laboratory) of a pregnancy-specific protein B and of early pregnancy factor (EPF) attest the establishment of pregnancy in domestic animals and in other mammals. The biological properties of some hormones such as placental lactogens (PL) or chorionic somatomammotropins (CS), human placental growth hormone (hPGH) contribute to a better understanding of the gestation function. Many other factors participate in the foetal development, for example, proliferin. Some proteins can display immunosuppressive properties or be responsible for the immune tolerance between the mother and the foetus. Although many placental proteins have already been defined, their biological functions have not yet been elucidated.

Ovine chorionic somatomammotropin and foetal growth.

Ovine chorionic somatomammotropin (oCS) enhances the weight and bone growth of hypophysectomized rats. It acts as a bifunctional hormone, since it binds both to lactogenic and somatotropic receptors. Ovine foetus weight gain is closely related to oCS and oGH serum levels. oCS is able to stimulate somatomedins by foetal liver. Moreover, oCS specific receptors are present in some foetal tissues. So, all these facts involve oCS in foetal growth, whereas pituitary growth hormone intervenes in postnatal growth. A study of structure function relationships between growth hormones and placental hormones is exposed in order to localize somatotropic sites.

Relationships between structure and function of lactogenic hormones.

Lactogenic activity of several hormone derivatives obtained by chemical modifications of lysine residues was studied by radioreceptor assay. The relationships between structure and binding to lactogenic receptors are discussed taking into account lysine residue positions liable to be involved in the location of lactogenic function.

Involvement of lysine residues in the binding of ovine prolactin and human growth hormone to lactogenic receptors.

The lactogenic activity (L.A.) of oPRL and hGH derivatives obtained by chemical modifications of lysine residues was studied by radioreceptor assay. Control treatment with borohydride had a slight effect on the L.A. of hGH but drastically reduced the oPRL activity; this latter was preserved in the presence of iodoacetamide. Methylation, ethylation, guanidination and acetimidination affected the L.A. of both hormones as a function of the degree of modification. The structure-binding relationships to the lactogenic receptors are discussed, suggesting that the lysine or arginine residues in homologous positions 42, 51, 73, 128, 146 of oPRL and 47, 50, 73, 128, 147 of hGH might be particularly involved.

Involvement of lysine residues in the binding of hGH and bGH to somatotropic receptors.

The biological activities of human (hGH) and bovine (bGH) growth hormone derivatives obtained by chemical modification of the lysine residues were studied by radioreceptor assays using rabbit liver homogenates for somatotropic activity (SA). Control treatment with BH4 had a very slight effect on the SA, whereas the methylation and ethylation drastically reduced the activity of both hormones. Guanidination of these hormones and even acetimidination at a lower rate are accompanied by a considerable loss of biological activity. These results show the involvement of lysine residues in the interaction of hGH and bGH with somatotropic receptors. The structure-function relationship of these molecules is discussed, suggesting that the lysine or arginine residues in positions 41, 64, 70 and 115 might be particularly implicated.

Involvement of lysine residues in the binding of ovine chorionic somatomammotropin to lactogenic and somatotropic receptors.

The biological activities of several ovine chorionic somatomammotropin (oCS) derivatives obtained by chemical modification of the lysine residues were studied by radioreceptor assays using rabbit mammary homogenates (lactogenic activity, L.A.) and liver homogenates (somatotropic activity, S.A.). Even if the control treatment with BH-4 markedly decreased the L.A., it was clear that methylation mainly affected the S.A. and that ethylation reduced both activities. Guanidination inactivated almost completely both activities and acetimidination at a very low degree (3 of 14 lysines) led to less than 50% of both activities. These results show the involvement of lysine residues in the interaction of oCS with lactogenic and somatotropic receptors.

Inhibitory effect of ovine and human placental lactogens on progesterone catabolism in luteinized rat ovaries in vitro.

Pseudopregnant rat ovaries, obtained by PMSG and hCG treatment, were incubated or perfused. Progesterone and 20 alpha-dihydroprogesterone (20 alpha-DHP) secretion and synthesis as well as 20 alpha-hydroxysteroid dehydrogenase (20 alpha-SDH) activities were determined by radioimmunoassays. Ovine and human chorionic somatomammotrophin (oCS; hCS) and ovine prolactin (10(-8) M) significantly (P less than 0.001) depressed 20 alpha-DHP ovarian contents (49, 64 and 69 per cent, respectively) and 20 alpha-DHP release (49, 55 and 55 per cent, respectively) in comparison with those of controls without hormones. The activity of 20 alpha-SDH was also inhibited by these three hormones (59, 68 and 56 per cent, respectively). The kinetics of 20 alpha-DHP release, studied by perfusion or incubation, showed a maximum inhibition from the first hour onwards with all lactogenic hormones. The fall in 20 alpha-DHP content of luteinized ovarian tissue coincided with a sharp rise in the progesterone/20 alpha-DHP ratio, from 0.5 in the controls to 1.5 after lactogenic hormone treatment. This study in vitro showed that two placental lactogens of sheep or human origin exhibit an inhibitory effect on 20 alpha-SDH activity analogous to that of prolactin. It is suggested that these placental hormones may be involved in the regulation of luteal and placental progesterone metabolism during pregnancy.