Summary. The production of ovine chorionic somatomammotrophin (OCS) was demonstrated in the trophoblast from Days 16–17 of pregnancy. Concentrations in the placenta rose slowly until about Day 100 when there was a rapid increase to reach 70 ± 5 μg prolactin equivalent/g fresh placental tissue and 15 ± 2 mg/placenta on Day 120. After Day 140, the concentrations decreased. It is suggested that OCS may be luteotrophic and have an effect on fetal growth.
J. Martal and J. Djiane
Y. Heyman, Sylvaine Camous, J. Fèvre, Wided Méziou and J. Martal
Summary. One or two trophoblastic vesicles (0·4–2 mm diam.) from cow (Day 14) or ewe (Day 11–13) embryos without their disc were transferred, after culture for 24 h, into recipients. Each vesicle was transferred into the uterine horn ipsilateral to the CL by the cervical route in heifers and surgically in ewes on Day 12 of the oestrous cycle.
In cows, daily measurements of plasma progesterone concentrations and checks for return to oestrus showed that the CL was maintained in 8 out of 12 recipients. These 8 cows had 25- to 37-day cycles while 4 recipient heifers returned to oestrus normally. Three recipients with an extended cycle were slaughtered. The dissected uterus showed that trophoblastic vesicles had developed in the uterine horns.
In ewes, the serum progesterone curve, determined in each recipient, showed that the CL was maintained in 7 out of 12 recipients. These 7 ewes had 20- to 54-day cycles and the other 5 ewes had a normal cycle of 15–19 days comparable to that of 17·0 ± 0·5 days for the 6 control ewes. Whenever the CL was maintained, high blood progesterone levels were followed by rapid luteolysis. In cattle and sheep, therefore, a trophoblastic vesicle transferred into the uterus can develop in vivo, secreting the embryonic signals when there is no embryonic disc control and transforming the cyclic CL into a CL of pregnancy in about 60% of the cases. These results indicate that the early signals inhibiting luteolysis may be of trophoblastic origin and confirm that after normal embryo transfer some of the late returns to oestrus may be due to the development of trophoblastic tissue only in utero.
MA Driancourt, J Fevre, J Martal and KH Al-Gubory
Ovarian follicular growth and maturation and its control throughout pregnancy have not been described fully in sheep. Experiment 1 characterized the size and maturation (steroid production in vitro and aromatase activity) of ovarian follicles obtained at days 20, 50, 80 and 110 of pregnancy compared with those obtained at day 12 of the oestrous cycle. There was no difference in the number of small follicles (< 3 mm in diameter) between cyclic and pregnant ewes, regardless of the stage of pregnancy. There was a marked reduction (P < 0.01) in the number of medium follicles (3-5 mm) starting at day 80 of pregnancy. Large follicles (> 5 mm) were not detected at day 110 of pregnancy. In vitro testosterone output by follicles was constant throughout pregnancy. Oestradiol output remained steady until day 80, but decreased markedly at day 110 of pregnancy. This decrease was associated with a reduction in aromatase activity in follicles obtained at this stage. Experiment 2 examined the effect of administration of high concentrations of progesterone between day 100 and day 120 after mating on resumption of follicular growth in ewes that underwent Caesarean section at day 99 of pregnancy. In ewes that underwent Caesarean section, progesterone supplementation was successful in mimicking the profile found in pregnant ewes, but did not prevent re-initiation of follicular growth, as demonstrated by the presence of large follicles (> 5 mm) at day 120 after mating. Experiment 3 examined the effects of PGF(2alpha)-induced regression of the corpus luteum of day 100 of pregnancy on resumption of follicular growth. High concentrations of PGF(2alpha) (0.28 mg kg(-1) body weight) administrated at day 100 of pregnancy were required to initiate regression of the corpus luteum. At day 120 after mating, the mean (+/- SEM) diameter of the largest follicle in PGF(2alpha)-treated ewes (3.40 +/- 0.47 mm) was significantly greater (P < 0.05) than that in control pregnant ewes (2.52 +/- 0.34 mm). Experiment 4 examined the effect of removal of the fetus and of the corpus luteum at day 100 of pregnancy on resumption of ovulation. Removal of the corpus luteum by PGF(2alpha) treatment at the time of removal of the fetus resulted in earlier occurrence of short luteal phases (27.8 versus 40.6 days, PGF(2alpha)-treated versus non-treated) but did not alter the timing of the first normal luteal phases (41 days). In conclusion, the results from these experiments indicate that placental compounds play a major role in inhibiting follicular growth and maturation during late pregnancy in sheep.
N. Gharib-Hamrouche, N. Chêne, M. Guillomot and J. Martal
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 α (TGF-α), insulin-like growth factor-I (IGF-I) and ovine placental lactogen confirmed the specificity of EGF/TGF-α 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-α suggests that these factors could be involved in the regulation of embryonic development and fetal growth.
P. Humblot, S. Camous, J. Martal, J. Charlery, N. Jeanguyot, M. Thibier and R. G. Sasser
Summary. Pregnancy-specific protein B (PSPB) and progesterone concentrations were determined by RIAs in venous plasma during early pregnancy after 177 artificial inseminations (AI) performed in 76 cows and 71 heifers. The females were bled at 24, 26, 30–35 days and ∼ 70 days (for non-returns to oestrus) after AI. In non-pregnant females without extended CL maintenance (progesterone < 1·5 ng/ml on Day 24) and or showing a normal time of return to oestrus (Group 1, N = 63), PSPB concentrations were undetectable whatever the stage after AI except in 2 cows. In pregnant animals (N = 83; Group 2) progesterone concentrations were > 10 ng/ml from Day 24 to the time of rectal palpation and PSPB concentrations rose continuously from 0·42 ± 0·07 (s.e.m.) ng/ml (Day 24) to 4·06 ± 0·3 ng/ml (time of rectal palpation). No coefficient of correlation between PSPB and progesterone concentrations was significant whatever the day of gestation studied. In cows with extended luteal function and subsequently found to be non-pregnant (late embryonic mortality) PSPB was undetectable (N = 21; Group 3) or detectable (N = 10; Group 4) at Days 24, 26 and/or 30–35 of pregnancy. At 24 and 26 days after AI progesterone concentrations were intermediate between those of Groups 1 and 2. At Day 24 females of Group 4 had higher progesterone concentrations than those of Group 3 (P < 0·05), but no differences between these two groups existed at subsequent stages after AI. Animals of Group 4 had lower PSBP concentrations than those of Group 2 between Days 24 and 30–35 (P < 0·025) but at the time of rectal palpation PSPB values fell to undetectable levels in all but 1 cow of Group 4. We conclude that (1) most pregnancy failures in cows are due to nonfertilization or early embryonic death and if AI is performed after 70 days post partum >95% of these females have no detectable PSPB concentrations; (2) peripheral progesterone concentrations are lower at Days 24–26 after AI in cows with late embryonic mortality than in pregnant cows; (3) only 30% of non-pregnant females with extended luteal function (late embryonic mortality) have detectable PSPB levels which are lower than in pregnant cows; and (4) in pregnant animals there is no correlation between PSPB and progesterone concentrations. This suggests that under physiological conditions PSPB has no major effect on progesterone production or vice versa.
Keywords: PSPB; progesterone; pregnancy; embryonic death; cow
K. H. Al-Gubory, M-A. Driancourt, M. Antoine, J. Martal and N. Neimer
Porcine and ovine follicular tissues were used to investigate, in vitro, the effect of charcoal-treated aqueous extract from ovine corpora lutea of pregnancy on aromatase activity as determined by the conversion of [3H]testosterone to oestradiol by follicular walls and measurement of 3H2O release. Extract (500 μg protein) prepared from corpora lutea of day 112 of pregnancy but not extract (500 μg) prepared from ovine fetal cotyledonary tissue obtained at a similar time significantly decreased (P < 0.02) aromatase activity of pig follicles in the absence of FSH. These results demonstrate that a non-steroidal factor in the corpora lutea of late pregnancy directly inhibits aromatase activity. When the effects of different doses (300, 600 or 1200 μg) of luteal extract from corpora lutea of day 100 of pregnancy on aromatase activity of pig follicles were studied, the dose by treatment (presence or absence of FSH) interaction was not significant. Luteal extract dose at 300 μg did not affect aromatase activity but a significant decrease in activity occurred at 600 μg of luteal extract (600 versus 300 μg, P < 0.02). There was no further significant increase in the inhibitory effect with 1200 μg luteal extract. When the effects of 600 μg luteal extract from corpora lutea of days 15, 75 or 100 of pregnancy on aromatase activity of pig follicles were studied, a significant (P < 0.05) stage of pregnancy effect was detected, but the stage of pregnancy by treatment (presence or absence of FSH) interaction was not significant. No effect was noted with day 15 or day 75 luteal extract. In contrast, aromatase activity in the presence of day 100 luteal extract was significantly reduced compared with that of control (P < 0.01) and day 15 luteal extract (P < 0.05). A significant (P < 0.05) stage of pregnancy effect was also observed on aromatase activity of sheep follicles. Aromatase activity of sheep follicles was significantly reduced in the presence of day 100 luteal extract compared with that of control (P < 0.05) and day 15 luteal extract (P < 0.02). These data suggest that the stimulus triggering the synthesis of the aromatase inhibitor appears after mid-pregnancy. The aromatase-inhibiting activity was lost from luteal extract of corpora lutea of day 100 of pregnancy after treatment with proteolytic enzymes, demonstrating the proteic nature of the aromatase inhibitor. These experiments provide evidence for the existence in ovine corpora lutea of late pregnancy of a non-steroidal factor that reduces follicular aromatase activity. We propose the term aromatase-inhibiting factor or AIF to describe this activity.
J. Martal, M.-C. Lacroix, C. Loudes, M. Saunier and S. Wintenberger-Torrès
Summary. Trophoblastin, an antiluteolytic component from the embryo, was identified in the ewe by the means of intrauterine injections of homogenates from trophoblasts at 14–16 days pregnancy. Homogenates from embryos and their membranes at 21–23 days pregnancy did not extend the life of the corpus luteum, suggesting that trophoblastin synthesis occurs for only a short period. The trophoblastin was thermolabile (80°C for 30 min) and inactivated by pronase. Treatment of ewes with oCS, hCG, and extracts of 120-day placentae did not affect the time of luteolysis. The protein appears to be insoluble at pH 7 or 8, but to dissolve readily at pH 9·6.
After injection of homogenates or extracts from 14–16-day-old trophoblasts, the initial CL were maintained for more than 1 month in most cyclic recipient ewes. Surgical removal of embryos at 21–23 days resulted in luteal maintenance for more than 1 month in over 50% of the operated animals. All the maintained CL were secretory although their average weight was about one-half of that CL of normal pregnancy, suggesting the existence of complementary luteotrophic placental factors. The uteri of most of these pseudopregnant ewes were distended with a clear, sterile fluid.
P. Bonnin, L. Huynh, R. L'Haridon, N. Chene and J. Martal
The theory of countercurrent vascular transfer of PGF2α during luteolysis was examined. In the first experiment, pulmonary clearance of PGF2α was determined to re-examine whether the total amount of PGF2α was degraded in the lungs after one passage. Cardiac output was measured by the Fick method and PGF2α by radioimmunoassay 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 PGF2α 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 PGF2α 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 PGF2α was estimated at 6338 ± 451 ml min−1. In a second experiment, an arterio–arterial gradient of plasma PGF2α concentrations was analysed between the proximal and distal segments of the ovarian artery to verify whether the total amount of PGF2α 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 PGF2α concentrations in the femoral and distal ovarian arteries were 23 ± 6 and 42 ± 11 pg ml−1 (P < 0.05), respectively. Plasma PGF2α 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 PGF2α decreased linearly and the half-time decrease was 108 min (r = 0.96). Consequently, the arterio–arterial diffusion gradient of PGF2α concentrations was extended to 3 h. These experiments showed that the PGF2α flow rate in the pulmonary artery was 42.275 ± 10.793 pg per 150 min (n = 10) and the systemic arterial PGF2α flow rate was 5.359 ± 1.658 pg per 150 min (n = 10). Therefore, 12% of the PGF2α was not oxidized by the lungs. The proximal ovarian PGF2α 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 PGF2α 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 PGF2α surge. These results indicate both rapid systemic transport of PGF2α to the ovaries and a slower buffer mechanism involving a local diffusion pathway, rather than a direct countercurrent system.
C Martin, L Pessemesse, M P de la Llosa-Hermier, J Martal, J Djiane and M Charlier
Our objective was to determine the effect of ovine interferon-τ (IFN-τ) on prolactin receptor (PRL-R) gene expression in the ovine endometrium. IFN-τ is an embryonic cytokine which, via its paracrine anti-luteolytic activity, plays a critical role in maternal recognition of pregnancy in ruminants. Using ribonuclease protection assay procedures, we compared endometrial PRL-R mRNA levels in ewes that were intrauterine injected with either 2 mg bovine serum albumin or 2 mg recombinant ovine IFN-τ on day 10 of the oestrous cycle (day 0 = day of oestrus). IFN treatment significantly increased the abundance of both the long and short forms of PRL-R mRNA in the ovine uterus, but had no effect on the long:short form ratio. In situ hybridization experiments revealed that the increase in abundance of PRL-R mRNA in the uterus was localized to the glandular compartment of the endometrium. In pregnant ewes, a similar increase in PRL-R mRNA abundance was found to occur in ovine endometrium on days 14–15 post conception. Collectively, these data provided strong evidence that IFN-τ modulates the level of lactogenic hormone receptor mRNA in the ovine uterus. Whether the effect of IFN-τ on PRL-R expression is mediated directly or influenced, at least in part, by progesterone remains to be elucidated.
C. La Bonnardière, F. Martinat-Botte, M. Terqui, F. Lefèvre, K. Zouari, J. Martal and F. W. Bazer
Summary. Antiviral activities present in uterine flushings from pregnant Large White, Large White 'hyperprolific', and prolific Meishan gilts, between Days 8 and 20 of gestation were compared. Flushings (20 ml) from all gilts between Days 14 and 20 were positive in an in-vitro interferon (IFN) assay using vesicular stomatitis virus as a challenge infection. Highest antiviral activities (of up to 400 000–1 200 000 total Units/flushing) were obtained at Day 16 of gestation, i.e. clearly after the beginning of attachment. There was no major difference between breeds although, at Day 14, flushings from Meishan gilts yielded significantly higher titres than those from the other two, suggesting a correlation with the previously described earlier trophoblast elongation in Meishan gilts. Conceptus cultures contained antiviral activity, with values very close to those obtained in vivo, but the difference between breeds was not significant. Cultures from Day 20 on contained very little antiviral activity. The antiviral activity was associated with a mixture of at least two IFNs, one of which was IFN-alpha like, and the other was serologically identified as an IFN-gamma, that is an 'immune IFN', previously found to be secreted only by T lymphocytes. This finding may have implications for our understanding of the immunology of early pregnancy.
Keywords: pig; conceptus; attachment; antiviral activity; immune interferon