Immunohistochemical localization of epidermal growth factor, transforming growth factor-alpha and growth factor-beta s in the caprine peri-implantation period

Control over the action of steroid hormones in the uterus and conceptus during the initial period of gestation appears to be regulated locally by growth factors. This study involved immunohistochemical detection of epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and transforming growth factor-beta s (TGF-beta s), to determine their role in the caprine peri-implantation period. Epidermal growth factor was expressed in the luminal and glandular endometrial epithelium of goats on all days studied (Days 22 to 30 post coitum), but it was not detected in trophoblastic cells or in other embryonic structures. Between Days 22 and 30 post coitum, TGF-alpha was detected in the epithelial cells and superficial stroma of the uterus and in the trophoendodermic cells of the embryo. Transforming growth factor-beta s expression, observed in the endometrium, embryo and extraembryonic membranes on Day 22 post coitum, decreased by Day 24 post coitum and disappeared in the embryo by Day 30 post coitum, while remaining in the other structures. The presence of these growth factors during the peri-implantation period in the goat suggests their participation in proliferation and differentiation phenomena which occur during implantation and embryonic development.     

Binding of epidermal growth factor and transforming growth factor-alpha in mammalian preimplantation embryos

Preimplantation embryos of the pig (Days 11 to 15), cow (Days 14 to 16), sheep (Day 14) and pony (Day 16) bind epidermal growth factor (EGF) specifically. Binding was not detected in embryos of the rabbit at Day 5 or 6 or the hamster at Day 3. Transforming growth factor-alpha displaced [(125)I] EGF in pig, cow and pony embryos almost as much as unlabeled EGF. The binding affinities of EGF ranged from 12 to 233 pM in pig and cow embryos. The range of species and binding features indicate that the EGF family may play a significant role in mammalian preimplantation development.     

Measurement of early pregnancy factor activity for monitoring the viability of the equine embryo

The viability of embryos before flushing from donor mares (n = 5) and after transfer to recipient mares (n = 7) was monitored in mare serum by detecting early pregnancy factor (EPF) using the rosette inhibition test (RIT). The EPF activity was measured in donor mares before and after natural mating at natural estrus; after ovulation on Days 2, 5 and 8; and after embryo flushing (Day 8) on Days 8, 9, 10 and 13 after ovulation. The collected embryos were transferred immediately after flushing. The EPF activity in recipient mares were measured on the day of transfer and after embryo transfer on Days 1, 2, 3 and 5. Pregnancy was confirmed on Day 12 to 14 after embryo transfer. The mean EPF activity of donor mares was increased to the pregnant level (> an RI titer score of 10) on Day 2 after ovulation. Two days after flushing the embryos, the EPF activity of donor mares had decreased to the nonpregnant level. Among the 7 recipient mares, 3 mares were diagnosed pregnant on Day 12 after embryo transfer with ultrasound. The EPF activity of the pregnant recipient mares was increased above the minimum level observed in pregnant mares on Days 2 to 3 after transfer. However, among the nonpregnant recipient mares after embryo transfer, the EPF activity of 3 mares remained at the pregnant level only 2 to 3 d and then declined to the nonpregnant level. In one recipient mare, EPF activity did not reach the pregnant level throughout the sample collection. The results of this study indicated that equine EPF can be detected in serum of pregnant mares as early as Day 2 after ovulation. From our observation, we conclude that the measurement of EPF activity is useful for monitoring the in vivo viability of equine embryos and early detection of embryonic death.     

Ultrasonography for detection of early pregnancy following embryo transfer in unknown breed of Bos indicus cows

Real time, B mode ultrasound was used from Days 18 to 64 to detect and monitor the conceptus in 30 unknown breeds of Bos indicus recipients following embryo transfer. The embryonic vesicle was first visible within the uterus between Days 18 and 20 (mean +/- SD 18.5 +/- 0.7d) d). The embryo proper was detected at Day 19.5 +/- 0.7. The heartbeats were detected at Day 22.6 +/- 0.9. The average day of first detection of the allantois, the C - shaped embryo and the amnion was 23.1 +/- 0.8, 23.8 +/- 1.4 and 25.1 +/- 1.4 d, respectively. The fore limb, the limb buds, the spinal cord and the optic area were observed on Days 32.7 +/- 1.3, 32.9 +/- 1.3, 33.0 +/- 1.5 and 33.6 +/- 1.4, respectively. Fetal movements could be detected at Day 50.7 +/- 1.0. Ribs and vertebrae were detected on Day 60.9 +/- 1.7. The mean length of the embryo proper was 4.5 +/- 0.8 mm. on Day 19. At Day 60 it was 52.5 +/- 7.0 mm. The growth of the embryo proper increased steadily until Day 39, growing rapidly thereafter. Embryonic death was detected in one recipient on Day 32. The echongenic area started increasing and recipient appeared in estrus on Day 40. Sixteen recipients exhibited estrus on Days 22 to 24. An increase in intrauterine fluid was observed on Days 18 to 24.     

Effects of embryo size at transfer (whole versus demi) and early pregnancy progesterone supplementation on embryo growth and pregnancy-specific protein bovine concentrations in recipient dairy heifers

The objectives of this study were to evaluate embryonic size and survival, plasma progesterone (P4) and pregnancy-specific protein bovine (PSPB) concentrations in early pregnancies (n = 99) following the transfer of one whole (n = 66) or one demi (n = 33) embryo to recipient virgin dairy heifers. The experiment was designed to evaluate the fixed effects of embryo size at transfer (whole or demi embryo) on Day 7 of the estrous cycle (Day 0 = estrus) and P4 supplementation between Days 7 to 19 through an intravaginal device (yes or no) on plasma P4 and PSPB concentrations and on embryo measurements. Plasma P4 concentrations were measured by RIA on Days 0, 7, 14, 19, 21, 25, 35, 42, 49, 56 and 63 of pregnancy and, PSPB concentrations were measured by ELISA on Days 7, 21, 25, 35, 42, 49, 56 and 63. The presence of an embryonic vesicle was detected on Day 25, embryonic/fetal movements and heartbeat were evaluated on Days 42 and 63 and embryo measurements [crown-rump length (CRL) and width at mid body] were obtained on Day 42 through ultrasonography.In non-supplemented pregnancies, Day 42 whole embryos had higher (P < 0.05) CRL and width than demi embryos, but the difference averaged only 1 to 2 mm. In P4 supplemented pregnancies, whole and demi embryos attained a similar size on Day 42 of pregnancy. Embryo size at transfer, early exogenous P4 supplementation and their interactions had no effects (P > 0.05) on plasma P4 concentrations. However, the post-hoc LSD evaluation showed that plasma P4 concentrations on Day 25 were higher (P < 0.001) in whole than in demi embryo derived pregnancies and, that exogenous P4 supplementation increased (P < 0.05) plasma P4 concentrations on Day 19 of pregnancy. The plasma PSPB detection rate on Days 7 to 63 of pregnancy was similar in pregnancies resulting from the transfer of whole and demi embryos. From a total of 93 recipients remaining pregnant until Day 63, plasma PSPB was constantly undetectable on Day 7, was detected in 4% of Day 21 samples, 41% of Day 25, 95% of Day 35, 96% of Day 42, 99% of Day 49 and in 100% of samples of Days 56 and 63. Concentrations of PSPB increased (P < 0.05) from Days 21 to 42 and from Days 56 to 63, with a plateau between Days 42 to 56. Demi embryo pregnancies had higher (P < 0.05) plasma PSPB concentrations on Days 35 and 42 than whole embryo pregnancies. Progesterone supplementation had a positive effect (P < 0.01) on PSPB concentrations from Days 35 to 63. Concentrations of PSPB were similar in non-supplemented whole and demi embryo pregnancies from Days 7 to Day 63. In contrast, in supplemented recipients, demi embryo pregnancies had higher (P < 0.05) PSPB concentrations on Days 25 to 42 than whole embryo pregnancies. No significant correlation was found between P4 and PSPB concentrations or between the concentrations of these hormones and embryonic measurements on Day 42. In conclusion, demi embryos experienced a compensatory growth until Day 42 of pregnancy, attaining a similar size to that of whole embryos and originating conceptuses producing similar plasma PSPB concentrations to those of whole embryo derived conceptuses. Embryonic growth and conceptus secretion of PSPB were positively stimulated by early pregnancy exogenous P4 treatment.     

Metabolism of progesterone by hamster blastocysts and the ontogeny of progesterone metabolic capability

Progesterone (P) is required for the differentiation of reproductive tracts and maintenance of pregnancy. This study investigates whether the hamster blastocyst is capable of metabolizing P and, if so, at what stage of preimplantation development such capability becomes detectable. When the blastocysts collected from superovulated hamsters on Day 4 of pregnancy were cultured in 0.4 microM P medium, P metabolism was easily detectable at 1.25 h of culture and over half was metabolized by 7.5 h. Two major metabolites were generated: 5 alpha-pregnane-3,20-dione (or 5 alpha-dihydroprogesterone; 5 alpha-DHP) and 5 alpha-pregnane-3 beta-ol-20-one (or allopregnanolone; AP), about 90-95% and 5-10%, respectively. This indicates the activity of two enzymes: delta 4-5 alpha-reductase and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). The rate of P metabolism increased with P concentration (0.4-6.4 microM), indicating a high capacity of the enzymes. Studies of embryos collected on Days 1-3 showed that P metabolism was not detectable up to 0100 h of Day 3 (2-4-cell), but was detectable with two metabolites, 5 alpha-DHP and AP, at 1515 h of Day 3 (morula) and thereafter. This indicates that, by the morula stage, the hamster embryo has already acquired the enzymatic capability (5 alpha-reductase and 3 beta-HSD) to metabolize P. These results, together with our earlier finding of 17 beta-hydroxysteroid dehydrogenase activity in Days 1-4 embryos, suggest that hamster preimplantation embryos can metabolize both P and estrogens, thus possibly modulating local actions of these hormones and causing local effects in the reproductive tract.     

Effect of mouse epidermal growth factor on plasma concentrations of FSH, LH and progesterone and on oestrus, ovulation and ovulation rate in merino ewes

The 24 h i.v. infusion of Merino ewes with 60 or 100 microgram mouse epidermal growth factor (EGF)/kg body weight on Days 4, 9 or 14 of the oestrous cycle decreased the strength of wool attachment and caused marked changes in subsequent reproductive performance. In ovaries removed 2 days after EGF treatment all follicles greater than or equal to 0.6 mm diameter were atretic. After 7 days either a normal pattern of atresia or no atresia was evident while after 12 days the pattern of follicular atresia was similar to that in controls. Irrespective of stage of cycle EGF caused dose-dependent increases in plasma FSH concentrations that persisted for up to 14 days. Changes in plasma LH concentrations were generally similar after infusion on Days 4 and 14, but were smaller and shorter-lived after infusion on Day 9. Irrespective of dose, the infusion of EGF on Days 4 and 14 caused immediate luteolysis then the formation of a luteinized follicle in many ewes. Most ewes treated on Day 4 returned to oestrus between Days 17 and 21 with the same ovulation rate (1.3) as the controls. Of those infused on Day 14 oestrus occurred about a cycle length later than expected and their ovulation rate then (1.9) was also similar to that of the controls (1.7). Luteal function was not affected in ewes infused on Day 9, and most returned to oestrus between Days 17 and 20 with an ovulation rate of 3.2. Fertile rams were not placed with the ewes until after the differences in ovulation rate had been observed. Mating occurred generally 2-4 weeks after treatment, and there were no differences between EGF-treated and control ewes in fertility or fecundity. The results are interpreted as indicating that mouse EGF induces ovarian follicular atresia but has differential effects on luteal function according to the stage of the oestrous cycle at which it is given. As a consequence of these two effects, which lead to differential changes in gonadotrophin secretion, ovarian function may be temporarily impaired, little affected or improved.     

Rabbit relaxin: the influence of pregnancy and ovariectomy during pregnancy on the plasma profile.

A porcine relaxin radioimmunoassay was developed to evaluate the profile of immunoreactive relaxin in rabbit plasma. Relaxin was nondetectable in pseudopregnant (Days 1, 4, 5-8, 12, and 16), nonpregnant, and male rabbits. However, in pregnant rabbits, relaxin was detected during the peri-implantation period (Days 4-9). Peak concentrations were reached on Day 15 and were maintained until parturition (Day 32). Relaxin concentrations abruptly decreased on Day 1 postpartum to low but detectable concentrations that were unchanged during the first week postpartum. In contrast, progesterone concentrations peaked earlier (Day 13), decreased after Day 25, and were not detectable on Day 1 postpartum. The effect of ovariectomy on the profile of plasma relaxin was evaluated. Four pregnant rabbits were ovariectomized (Day 13) and treated with medroxyprogesterone acetate to maintain pregnancy. As in normal pregnant rabbits, relaxin was observed initially during the peri-implantation period (Days 4-9) and increased to peak concentrations by Day 16. These concentrations were maintained until parturition and abruptly decreased on Day 1 postpartum to low yet detectable concentrations during the first week postpartum. The concentrations of relaxin in the plasma of ovariectomized medroxyprogesterone-treated rabbits were not different from those in three sham controls. These results indicate that the ovary is not a significant source of relaxin in pregnant rabbits. The unique observation of the presence of relaxin during the peri-implantation period suggests that this hormone has a role in preparing the rabbit uterus for implantation. The continued presence of relaxin during the first week postpartum may represent residual hormone, or it may suggest a physiological role during the early postpartum period.     

Developmental expression of adenosine deaminase in placental tissues of the early postimplantation mouse embryo and uterine stroma

In this study, we have investigated the distribution of adenosine deaminase (ADA) in embryonic, extra-embryonic, and decidual tissues of the developing mouse embryo. ADA catalyzes a key step in purine metabolism converting adenosine to inosine. ADA specific activity (nmol/min/micrograms protein) was present at low levels in the embryo-decidual unit during the first 2 days of postimplantation development but then increased starting late on Day 6 of gestation (Day 0 plug). By Day 9, ADA specific activity was 80-fold higher than on Day 6. A histochemical staining method for ADA activity was applied to cryostat sections of the implantation site. The developmental increase localized primarily to the trophoblast/antimesometrial decidua interface between Days 7 and 9 of gestation, and decidua basalis and the metrial gland by Day 11. Immunofluorescent staining with sheep anti-mouse ADA antiserum confirmed the presence of ADA antigenicity in tissues forming the maternal/fetal interface. ADA specific activity was 19-fold higher in homogenates of the Day 11 decidua/parietal yolk sac than in the thymus, a tissue generally thought of as ADA-rich. High levels of ADA activity and immunoreactivity were also detected in the embryonal plasma during organogenesis, but the embryo proper showed only low levels. These results indicate that ADA is tightly regulated within tissues forming the maternal/fetal interface during early postimplantation stages of development.     

Ultrasonographic evaluation of the conceptus from days 10 to 60 of pregnancy in jennies

Daily ultrasound examinations were conducted from Days 10 to 60 (ovulation = Day 0) of pregnancy to monitor the conceptus in jennies (n = 12). The embryonic vesicle was first detected on Day 11.5 +/- 0.9 (mean +/- SD; range 10 to 13 d) and was mobile until movement ceased (fixation) on Day 15.5 +/- 1.4 (range, 13 to 18 d). The vesicle was spherical from Days 10 to 18 (mean growth rate, 3.2 mm/d), non spherical (irregular) with a reduced growth rate (0.5 mm/d) from Days 19 to 29, and then grew at a moderate rate (1.6 mm/d) up to Day 46. On average, detection of the embryo proper (consistently located on the ventral aspect of the yolk sac) and embryonic heartbeat were Days 20.7 +/- 1.2 and 23.5 +/- 1.3, respectively. Formation of the allantoic sac was first detected on Day 24.4 +/- 1.7 and was complete on Day 36.8 +/- 1.6. Descent of the fetus (and formation of the umbilical cord) began on Day 37.9 +/- 1.7 and was complete on Day 44.1 +/- 2.1. Crown-rump length averaged 3.7, 15.4, 22.7, 37.5 and 59.6 mm on Days 20, 30, 40, 50 and 60, respectively. In general, morphologic features and dates of occurrence were similar to those reported previously in the mare.     

Luteotrophic effect, growth and survival of whole versus half embryos and, their relationship with plasma progesterone concentrations of recipient dairy heifers

This prospective and randomised experiment was designed to compare the luteotrophic effect of whole versus half embryos and, to evaluate the relationship between the plasma progesterone (P4) profiles and the rates of early embryonic (from Days 7 to 25), late embryonic (Days 25-42) and foetal (Days 42-63) mortalities of whole and half embryo recipients. Within a single herd, 188 virgin, healthy, cyclic, reproductively sound, with adequate body condition score, Holstein dairy heifers were randomly allocated to receive one whole or one half embryo on Day 7 of the oestrous cycle (Day 0=estrus). In each embryo-transfer (ET) group, half of the recipients were treated with a CIDR (controlled internal drug releasing device) between Days 7 and 19. Pregnancy was evaluated by ultrasound on Days 25, 42 and 63 and plasma P4 profiles were obtained until Day 63 of pregnancy. CIDR-treated and untreated heifers had similar pregnancy rates on Days 25, 42 and 63 and, embryo size on Day 42 was also similar in treated and untreated recipients. Therefore, CIDR treatment failed to promote growth and survival of half and whole embryos. Half embryos presented a significantly higher rate of early and late embryonic mortality than whole embryos. In contrast, foetal mortality was similar in whole and half embryos and, this was coincidental to a similar embryo size on Day 42. Therefore, half embryos exhibited a compensatory growth until Day 42, irrespective of CIDR treatment, after which they presented a similar survival rate to that of whole embryos. Half embryo-derived pregnancies presented significantly lower plasma P4 concentrations on Day 25 than whole embryo-derived pregnancies, suggesting that this lower luteotrophic effect of half embryos could be related to their higher rate of late embryonic mortality. No significant relationship between the early luteal P4 concentrations and embryo survival was observed in whole and half embryo recipients. The first detectable luteotrophic effect of embryonic origin was observed on Day 14 and no detectable second luteotrophic effect was observed until Day 63 of pregnancy. Treatment with CIDR significantly increased plasma P4 concentrations during treatment but induced a significant decrease after removal of the device, suggesting that secretion of luteotropins was downregulated in the course of treatment.     

Changes in serum immunoreactive inhibin and follicle-stimulating hormone during gonadal development in male and female rats.

We have measured serum and ovarian immunoreactive inhibin alpha (irI alpha) and serum FSH in fetal and neonatal rats from 20 days of gestation until 40 days of age. For animals aged 10 days or older, serum measurements were made on intact and gonadectomized animals. Serum irI alpha was detectable in intact male and female rats at all ages studied. In females, irI alpha levels were low until Day 5 and then increased steadily to peak at Day 25. Thereafter they declined until Day 35 to reach levels typical of adult females. There was a significant decrease in irI alpha levels 24 h after ovariectomy at all ages. Serum FSH levels in females were low until Day 7, then increased rapidly to plateau from Days 10-15. The levels then declined until Day 25 and were generally unchanged after that time. There was a significant increase in FSH 24 h after ovariectomy in rats aged 20 days and older, and in younger rats by 48 h after ovariectomy. In male rats, serum irI alpha levels were significantly higher than females until Day 7. The levels increased at Day 7 and then remained relatively constant until Day 20, after which they declined to reach typical adult male levels. Serum irI alpha levels were significantly lower in males than females from Days 25-40. There was a significant decrease in serum irI alpha 24 h after castration at all ages studied. Serum FSH levels in males were low until Day 20, increased at Day 25, and thereafter remained relatively unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in vascular perfusion of the endometrium in association with changes in location of the embryonic vesicle in mares

The equine embryonic vesicle is mobile on Days 12-14 (Day 0 = ovulation), when it is approximately 9-15 mm in diameter. Movement from one uterine horn to another occurs, on average, approximately 0.5 times per hour. Mobility ceases (fixation) on Days 15-17. Transrectal color Doppler ultrasonography was used to study the relationship of embryo mobility (experiment 1) and fixation (experiment 2) to endometrial vascular perfusion. In experiment 1, mares were bred and examined daily from Day 1 to Day 16 and were assigned, retrospectively, to a group in which an embryo was detected (pregnant mares; n = 16) or not detected (n = 8) by Day 12. Endometrial vascularity (scored 1-4, for none to maximal, respectively) did not differ on Days 1-8 between groups or between the sides with and without the corpus luteum. Endometrial vascularity scores were higher (P < 0.05) on Days 12-16 in both horns of pregnant mares compared to mares with no embryo. In pregnant mares, the scores increased (P < 0.05) between Day 10 and Day 12 in the horn with the embryo and were higher (P < 0.05) than scores in the opposite horn on Days 12-15. In experiment 2, 14 pregnant mares were examined from Day 13 to 6 days after fixation. Endometrial vascularity scores and number of colored pixels per cross-section of endometrium were greater (P < 0.05) in the endometrium surrounding the fixed vesicle than in the middle portion of the horn of fixation. Results supported the hypothesis that transient changes in endometrial vascular perfusion accompany the embryonic vesicle as the vesicle changes location during embryo mobility.     

Toxoplasma gondii: blood and tissue kinetics during acute and chronic infections in mice

Acute lethal infections were obtained in mice by intraperitoneal (IP) injection of 10(2) or 10(4) tachyzoites of the virulent RH and C56 strains. Chronic infections were obtained by IP injection or peroral (PO) gavage of 20 cysts of the avirulent C strain. Mice were sacrificed at varying intervals after infection and parasite burdens were quantitated in blood, brain, and lungs using a tissue culture method. Acutely infected mice died within 6 to 10 days postinfection as a function of the strain and inoculum size. With either strain, tachyzoites were first detected in lungs on either Days 2 or 4 postinfection, according to the inoculum size, then in brain and blood at Days 4 or 6; parasitic loads remained constantly at a higher level in lungs than in brain until the date of death. Bradyzoites could only be detected in lungs, from Days 4 or 6 until death. In chronic infections, similar results were obtained for IP and PO infected mice. Both tachyzoites and bradyzoites were first detected in lungs and brain from Day 7 after infection; tachyzoites remained at a higher level in lungs than in brain until Day 10, then subsequently decreased in lungs. At Day 50, tachyzoites were not detectable in lungs, whereas bradyzoites remained at a constant level; in brain, both parasitic stages were detectable at a similar level throughout the follow-up period. These results indicate that infection with a virulent Toxoplasma strain is characterized by an early involvement of lungs, with pneumonia as the principal cause of death.(ABSTRACT TRUNCATED AT 250 WORDS)     

Change in duration of elevated concentrations of melatonin is the major factor in photoperiod response of the tammar, Macropus eugenii

In Exp. 1, 10 quiescent non-lactating tammars were exposed to 15L:9D (Days -41 to -1), 24L:0D (Days 0 to 14), 15L:9D (Days 15 to 34) and then to ambient increasing daylength from 13L:11D on Day 35. From Days 0 to 22 they received a s.c. injection of melatonin (400 ng/kg, N -5) on the arachis oil vehicle (N = 5) in the evening (19:30 h) 2.5 h before dark. Exposure to 24L:0D abolished the nocturnal plasma melatonin rise but this was reinstated by subsequent exposure to 15L:9D. Of 5 melatonin-treated tammars, 4 gave birth on Day 45, so had failed to respond to the melatonin injection alone but reactivated when this was combined with the endogenous melatonin rise during exposure to 15L:9D. Of 5 control tammars, 4 remained quiescent until reactivated by the decrease in daylength to 13L:11D, and gave birth significantly later (Day 63.7 +/- 2.2, mean +/- s.e.m., P less than 0.05). In Exp. 2, 6 tammars were exposed to 15L:9D (Days -15 to -1) and then to 12L:12D (Days 0 to 15) by extending the dark phase by 3 h in the morning. This extended the nocturnal melatonin rise by 2-3 h in the morning and all 6 tammars gave birth on Day 31.2 +/- 1.0. A transient pulse of peripheral plasma prolactin (81.5 +/- 31.0 ng/ml) was detected at dawn during 15L:9D in all 6 tammars but was not observed in any of them 5 days after exposure to 12L:12D. Together these results do not support the time of day hypothesis but indicate that increase in duration of the nocturnal melatonin rise mediates the effects of decreased daylength on reactivation of the corpus luteum, and that the first detectable result of this may be the abolition of a transient prolactin pulse at the end of the dark phase.     

Insulin-like growth factor-I expression during early conceptus development in the pig

Porcine conceptuses (embryo and associated membranes) in utero undergo developmental morphological transformations coincident with structural and biochemical changes in the uterine endometrium during early gestation. To elucidate a possible role for insulin-like growth factor-I (IGF-I) in these events, porcine endometrial (Days 8, 10, 11, 12, 14, and 30) and conceptus (Days 12, 14, and 16) tissues were characterized for the presence of IGF-I peptide and mRNAs. The corresponding uterine luminal fluids (ULF) at these stages of pregnancy were also analyzed for immunoreactive IGF-I concentration. ULF IGF-I was lowest on Day 8, highest on Day 12, and declined by Day 14. In contrast, endometrial tissue IGF-I content remained constant during this period. Conceptus tissues contained less IGF-I than endometrial tissues; however, conceptus IGF-I values were maximum on Day 12 coincident with peak values for ULF IGF-I. Dot-blot hybridization analyses revealed temporal variation in steady-state levels of IGF-I mRNAs in endometrium. Highest levels of endometrial IGF-I mRNA were detected on Day 12 and were about 4-fold greater than on Day 30 of pregnancy. IGF-I mRNA expression in conceptus tissues on Days 12, 14, and 16 was the same and was significantly less than that in endometrium on Day 12. These results demonstrate the temporal variation of IGF-I mRNA abundance in uterine endometrium and of immunoreactive IGF-I in ULF and in conceptus tissues, with the developmental processes occurring in the conceptuses at early pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the epidermal growth factor receptor in preimplantation pig conceptuses.

Embryos recovered from sows on Days 9-13 of pregnancy (Day 0 = first day of estrus) exhibited saturable and time-dependent specific binding of 125I-epidermal growth factor (EGF). The specific binding (pg/mg protein) was greater (P less than 0.001) for Day 13 elongated conceptuses than for conceptuses of earlier stages. Scatchard analyses showed two classes of binding sites (Kd = 7.0 +/- 2.6 x 10(-11) M, Bmax = 6.2 +/- 1.4 fmol/mg protein and Kd = 3.4 +/- 0.2 x 10(-8) M, Bmax = 420 +/- 80 fmol/mg protein). The EGF receptor in Day 13 conceptus membranes is a 170-kDa protein and was phosphorylated in the presence of EGF and adenosine triphosphate. EGF stimulated protein tyrosine kinase activity about 1.6-fold over basal levels. The results show that the preimplantation pig conceptus possesses EGF-binding sites with the properties of functional EGF-receptors.     

Early pregnancy detection and the hormonal characterization of embryonic-fetal mortality in fallow deer (Dama dama)

The objectives of this investigation were to 1) determine serum concentrations of progesterone (P4), estrone sulfate (E1S) and pregnancy-specific protein B (PSPB) from estrus synchronization through mid-gestation in the fallow doe (Dama dama) and 2) characterize the hormonal profiles of does whose embryos or fetuses died in utero. Ten fallow does were synchronized for 14 d with an intravaginal P4-releasing device (CIDR) and were naturally mated after CIDR removal. Blood samples were collected at CIDR insertion, CIDR removal and at intervals through Day 203 post-CIDR removal for analysis of P4, E1S and PSPB by radioimmunoassay (RIA). Ultrasonography was performed on Days 49 and 69 post-CIDR removal. Serum P4 at the time of CIDR insertion was 4.8 +/- 0.6 ng/ml, and at CIDR withdrawal it was 6.2 +/- 0.3 ng/ml. Concentrations of E1S and PSPB were nondetectable at CIDR insertion. Serum E1S was highest at Day 93, and PSPB was first detectable in pregnant does at Days 27 to 30 post-CIDR withdrawal. Ultrasonography on Day 49 revealed that 6 does were pregnant, 2 were not pregnant and 2 others were diagnosed originally as early pregnant. At Day 69, ultrasonography revealed that 6 does (60%) were pregnant and 4 (40%) were not. A comparison of the ultrasonographic and hormonal data indicated that the 2 does diagnosed as early pregnant on Day 49 had conceived but had lost the pregnancy. A third doe which was pregnant on Day 69 lost the fetus later in gestation. Hormonal profiles of does whose embryo or fetus had died were characterized by erratic P4 and E1S profiles, with PSPB becoming undetectable in the 3 does by Days 49, 65 and 80 post-CIDR removal. These data 1) demonstrate the timing for the collection of serum samples for determining early pregnancy in fallow does using 3 hormonal methods and 2) characterize the hormonal profiles of 3 fallow does with embryonic-fetal loss.     

Immunosuppressive activity associated with early pregnancy in the bovine

Immunosuppressive activity was assessed in uterine flushings (UF) and uterine vein serum and plasma from nonpregnant and early-pregnant cows, and in media from the short-term culture of Day 18 bovine embryos. The preparations were tested for their ability to inhibit [3H] thymidine (3H-TdR) incorporation into phytohemagglutinin-stimulated bovine lymphocytes. On Days 2-3 (called Day 3), Days 9-10 (called Day 10), and Days 17-19 (called Day 18) of the estrous cycle (estrus = Day 0) and pregnancy, untreated and superovulated cows were anesthesized and jugular vein and uterine vein blood was collected. The uteri were removed and flushed to obtain UF and embryos. Uterine flushings were concentrated and tested for immunosuppressive activity at 400 micrograms uterine protein/ml culture fluid. Uterine flushings from both Day 18 pregnant and Day 18 nonpregnant cows were immunosuppressive (8/8), whereas Day 10 UF were usually not immunosuppressive (7/10). Day 3 UF were usually stimulatory or only marginally suppressive (8/8). Uterine vein serum and plasma from Day 18 cows were not suppressive when compared to jugular vein serum or plasma from the same cow; neither were Day 18 uterine vein serum or plasma suppressive when compared to those same samples taken from Day 3 cows. Embryo culture media obtained from the 48-h culture of Day 18 embryos was consistently suppressive. The activity was lost after dialysis in 1000-Mr cut-off tubing, removed by charcoal, and reduced by protease digestion. These results suggest two mechanisms whereby the embryo could escape immune rejection: 1) the progesterone-induced secretion of a uterine immunosuppressive substance(s) and 2) the production by the embryo of a low molecular weight immunosuppressive substance(s).