Stephen P. Ford

Luteal maintenance during early pregnancy in the pig: Role for prostaglandin E2

We previously demonstrated that prostaglandin E2 (PGE) directly inhibits prostaglandin F2 alpha (PGF)-induced regression of individual pig corpora lutea (CL) in a dose dependent manner. The present experiments were conducted to 1) characterize and compare uterine secretion of PGE and PGF during the estrous cycle and early pregnancy and 2) evaluate the local effect of the conceptus on uterine prostaglandin secretion and associated CL function in unilaterally pregnant pigs. In Experiment 1, utero-ovarian venous blood samples were collected from two nonpregnant and two pregnant gilts at 3-h intervals from day 10 through 16 (first day of estrus or mating = day 0) for quantitation of uterine PGE and PGF secretion. In Experiment 2, gilts (n = 4) were made unilaterally pregnant on day 2, and utero-ovarian venous catheters were placed bilaterally to determine if differences in PGE and/or PGF secretion might account for the known luteotrophic/antiluteolytic effect of the gravid uterine horn on the CL of the ipsilateral ovary. During the estrous cycle (Experiment 1), pulsatile secretion of PGF increased markedly on day 13 and continued to increase through day 16. PGE secretion also increased from day 13 to 16 of the estrous cycle; however, concentrations of PGE remained at least 3-fold lower than those of PGF. In contrast to changes in non-mated gilts, prostaglandin secretion in mated gilts peaked earlier (day 11-12), with PGE predominating. Thereafter, both PGE and PGF secretion declined to basal levels where they remained through day 16 of pregnancy. During unilateral pregnancy (Experiment 2), PGF concentration in nongravid and gravid horns was similar (P > 0.8). In contrast, PGE concentrations were greater (P < 0.06) in utero-ovarian venous blood draining the gravid uterine horn. This increase in PGE was associated with enhanced CL function on the ipsilateral ovary as evidenced by an elevated progesterone content and concentration as well as increased CL weights. These data are consistent with a role for conceptus-associated increases in uterine PGE secretion in the local stimulation of luteal function during early pregnancy in the pig.

Relationship between placental vascular endothelial growth factor expression and placental/endometrial vascularity in the pig.

Abstract We investigated the temporal association between placental vascular endothelial growth factor (VEGF), a potent stimulator of angiogenesis and vascular permeability, and changes in placental/endometrial vascularity on selected days throughout gestation in the pig. Placental and endometrial tissues were collected from sows on Days 25 (n = 4), 36 (n =6), 44 (n =6), 70 (n =5), 90 (n =5), and 112 (n =7) of gestation. Cross sections of the placental/endometrial interface of each conceptus were used to estimate the number of blood vessels per unit area via image analysis and the intensity of VEGF staining via immunohistochemistry. Placental tissues were also collected on these days to evaluate VEGF mRNA expression. Placental VEGF mRNA expression and the numbers of blood vessels per unit area of placental and adjacent endometrial tissue were low and decreasing from Day 25 to Day 44, before increasing (P < 0.05) markedly and progressively through Day 112. These data are consistent with the marked increase in VEGF immunostaining in the chorionic and uterine luminal epithelium from early to late gestation. Further, these increases in placental VEGF mRNA were positively correlated with fetal weight (r = 0.73; P < 0.0001) and placental efficiency (fetal weight/placental weight ratio; r = 0.66, P < 0.0001). These data are consistent with a role for VEGF in increasing the number of blood vessels at the placental endometrial interface, resulting in an increased capacity for nutrient transfer from the maternal to the fetal compartment.

Metabolism of the gravid uterus, foetus and utero-placenta at several stages of gestation in cows

To quantify changes in rates of metabolism and nutrient uptake of gravid uteiine, foetal and utero-placental tissues throughout gestation, mature Hereford cows received surgery at 132 ± 0·6 ( n = 12), 176 ± 0·5 ( n = 8), 220 ±0·4 ( n = 11) and 245 ±1·5 ( n = 7) days after mating. Indwelling catheters were implanted into a uterine artery and vein of all cows. Foetal catheters also were implanted into an umbilical vein and foetal femoral artery and vein (days 176 and 220) or into a placental artery and two placental veins (days 132 and 245). Approximately 5 days after surgery, deuterium oxide was infused into a foetal femoral venous or placental venous catheter during a 3 h period to quantify uterine and umbilical blood flows by steady-state diffusion methods. Oxygen, glucose, lactate and α-amino acid nitrogen concentrations were determined for uterine and foetal blood samples collected during this procedure. Uterine blood flow increased 4·5-fold (2·92–13·181/min) and umbilical blood flow increased 21-fold (0·28–5·861–min) during the interval of gestation studied. The relative rate of increase of umbilical blood flow was about twice as great as that of uterine blood flow. Uterine arterial and umbilical venous concentrations as well as uterine arterial-venous and umbilical venous-arterial concentration differences in metabolites changed little with stage of gestation. However, because rates of blood flow increased, uptakes of O 2 , glucose and α-amino N by the gravid uterus and foetus increased as gestation advanced. The proportion of gravid uterine uptakes utilized by the foetus increased from day 137 to 226 for O 2 (24–58%) and from day 137 to 180 for glucose (4–19%), then remained relatively constant. The proportion of gravid uterine α-amino N uptake utilized by the foetus remained relatively constant and averaged 60%. A net secretion of lactate from the utero-placenta to the uterine and foetal circulations was observed and increased as gestation advanced. These data indicate that increased rates of uptake or secretion of metabolites by tissues of the gravid uterus can be explained primarily by increased rates of uterine and umbilical blood flows.

Effects of chronic environmental heat stress on blood flow and nutrient uptake of the gravid bovine uterus and foetus

To evaluate the effects of chronic environmental heat stress during mid-gestation on gravid uterine and foetal metabolism, mature Hereford cows were assigned to control ( n = 8) or heat stress ( n = 5) treatments beginning on day 100 of gestation. Uterine and umbilical blood flows were estimated by the steady-state diffusion procedure on day 169 ± 4 of gestation. Oxygen (O 2 ), glucose, lactate, α-amino nitrogen and urea nitrogen concentrations were determined for uterine and umbilical blood samples collected during this procedure. Foetuses and foetal fluids were collected on day 174±4. Uterine and umbilical blood flows were reduced and foetal weight also was less for heat-stressed than for control cows. In addition, foetal liver weight as a proportion of foetal weight and total foetal liver RNA and protein were less for heat-stressed cows. Uterine and umbilical arterial–venous concentration differences in metabolites were similar between the two groups. Uterine, foetal and utero-placental uptake or secretion rates of the metabolites measured in this study were reduced in the heat-stressed cows, primarily because of differences in blood flow. Thus, chronic heat stress during mid-gestation had an adverse effect on foetal development resulting, at least in part, from decreased uterine and umbilical blood flows, which led to a reduction in uterine, utero-placental and foetal nutrient uptake or secretion rates.

Differential expression of cyclooxygenase-2 around the time of elongation in the pig conceptus.

Alterations in uterine luminal fluid composition as a result of conceptus estradiol-17beta production are believed to play a significant role in the loss of 30-40% of potential pig conceptuses. Shortly after the initiation of conceptus estradiol-17beta synthesis and secretion, the conceptuses are transformed from 1cm spheres to 2-5cm tubular forms and finally to filamentous threads of variable length via a process known as elongation. We have attempted to characterize gene products whose expression is either initiated or terminated as the conceptus elongates. Using RNA fingerprinting, we determined that the inducible form of the rate-limiting enzyme in prostaglandin synthesis, cyclooxygenase-2, is expressed in the filamentous pig conceptus, but not in either the spherical or transitional morphologies. Furthermore, increased expression of cyclooxygenase-2 by the filamentous conceptus was associated with increases in the content of prostaglandins (particularly prostaglandin E(2)) found in uterine luminal fluid.

Differential gene expression during elongation in the preimplantation pig embryo.

Summary: On day 12–13 of gestation, the preimplantation pig conceptus undergoes a dramatic morphologic change from an approximately 1‐cm sphere to a nearly 1‐m long thread. This transformation, referred to as elongation, occurs in just 12–24 h. Elongation is primarily the result of trophectodermal cell shape changes, as there is relatively little mitosis during this stage of development. Thus far, descriptions of elongation have been limited to histologic and immunofluorescent studies of cell morphology and gross biochemical evaluations. We hypothesized that the changes in trophectoderm morphology likely involves significant changes in gene expression. Therefore, we used RNA arbitrarily primed‐PCR (RAP‐PCR) to characterize potential differential gene expression by trophectodermal cells during pig conceptus elongation. We found that the porcine heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 was shown to be differentially expressed by trophectodermal cells during elongation. We suggest that regulated alternative splicing may contribute to the morphogenetic process of elongation. genesis 26:9–14, 2000. Published 2000 Wiley‐Liss, Inc.

Signaling between the Placenta and the Uterus Involving the Mitogen-Regulated Protein/Proliferins*

The aim of this investigation was to examine signaling between the placenta and uterus during pregnancy. To do this, we determined the tissue messenger RNA and protein levels of members of a glycopeptide hormone family known to stimulate the proliferation of uterine cells and related these levels to the growth of the uterus during pregnancy in the mouse. This hormone family is known as mitogen-regulated protein (MRP); alternatively proliferin (PLF). Three mrp/plf genes, plf1, mrp3 and mrp4, are expressed by the placenta with different developmental profiles. The major increase of about 4-fold in DNA content of the uterus occurs between days 9 and 14 when MRP/PLFs are present in the placenta. By contrast, the gestational changes in estradiol-17b levels in placental and uterine tissues and in circulation do not correlate with the period of uterine growth. The previously reported mitogenic activity of the MRP/PLFs and their gestational profiles suggest that one or more of these proteins stimulates uterine proliferation during gestation. Evidence is also presented that expression of MRP3 and/or PLF1, but not MRP4, is negatively regulated by feedback from the uterus. Our results are consistent with the hypothesis that MRP/PLFs stimulate uterine proliferation in vivo and that a uterine factor shuts off PLF1 and/or MRP3 synthesis in the latter half of gestation. (Endocrinology 140: 5239 ‐5249, 1999)

Role of Adrenergic Receptors in Mediating Estradiol-17β- Stimulated Increases in Uterine Blood Flow of Cows

This study was conducted to investigate the association between estradiol-17 beta (E2 beta) and periarterial alpha and beta-adrenergic receptors in altering blood flow to the bovine uterus. Three nonpregnant Hereford cows were ovariectomized, and an electromagnetic blood flow transducer was placed around the middle uterine artery supplying one uterine horn. A catheter was then placed into a branch of the same artery for infusion of drugs or vehicle. Beginning 3 wk after surgery, uterine blood flow (UBF) of each cow was recorded for 5 min at 20-min intervals throughout a 160-min period three times weekly for 6 wk. Vehicle (.9% NaCl), phentolamine (PHEN, 1 or 10 micrograms) or propranolol (PROP, 1 or 10 micrograms) was infused for 5 min beginning 20 min after the initial UBF measurements, while vehicle or E2 beta (1 microgram) was infused for 5 min beginning at 40 min. Within a week, the first and third 160-min periods contained the same treatment (vehicle or E2 beta), and the second period consisted of vehicle or E2 beta plus PHEN or PROP, allowing for direct comparison of periods 1 and 3 with period 2. Twenty minutes after infusion of E2 beta, UBF was elevated (26.8 +/- 5.4 ml/min; P less than .05) over baseline, reaching a maximal increase at 40 min before starting to decline by 120 min. Uterine blood flow increased (39.2 +/- 7.6 ml/min; P less than .01) during the 5-min infusion of PHEN, and thereafter the magnitude and pattern were similar to those resulting from E2 beta treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of PGF2α-induced luteolysis in early pregnant and estrogen-treated ‘pseudopregnant’ gilts

Conceptus estrogen clearly plays a major role in luteal maintenance in the pig; however, other conceptus-derived substances or conceptus-induced uterine secretory products appear to have a local luteotrophic/anti-luteolytic effect on the corpora lutea (CL) and likely may play a key role in maternal recognition of pregnancy in the pig. The objective of these studies was to compare PGF2α-induced luteolysis in estrogen-treated ‘pseudopregnant’ gilts versus pregnant gilts during the period of maternal recognition of pregnancy. In Experiment 1, doses of PGF2α ranging from 1 to 100 μg were administered via intraluteal silastic implants to pseudopregnant gilts to determine the dose necessary to cause functional (progesterone) and structural (weight) luteal regression similar to that observed during the natural estrous cycle. Luteal sensitivity to this minimally effective luteolytic dose of PGF2α was then determined for both pseudopregnant and pregnant gilts in Experiment 2. Experiment 3 investigated whether Day 13 porcine conceptus tissue could directly prevent PGF2α-induced luteolysis at the level of the CL. The minimally effective luteolytic dose of PGF2α (100 μg) determined in the pseudopregnant pig caused a similar decline in progesterone concentration and weight of CL in pregnant gilts, suggesting that the susceptibility of CL of pregnant and pseudopregnant pigs to PGF2α is similar. However, luteal weight was greater (P<0.05) for the pregnant gilts than for pseudopregnant gilts, suggesting that estrogen treatment alone cannot mimic the conceptus effects on CL growth and development. Experiment 3 demonstrated that lyophilized Day 13 conceptus tissue implanted directly into individual CL could partially inhibit PGF2α-induced luteolysis, providing for the first time direct evidence that porcine conceptuses as early as Day 13 contain factors which can directly (i. e. at the level of the CL) prevent luteal regression.

Estrogen associated changes in uterine blood flow and maternal recognition of pregnancy in Chinese (Meishan) pigs

Our laboratory has recently demonstrated a reduced embryonic growth rate and estradiol-17β (E2β) content of preimplantation Meishan embryos compared with embryos of European breeds. Estrogen secretion by the preimplantation conceptus has been linked to both the transient increase in uterine blood flow (UBF), and maternal recognition of pregnancy (MRP) on Days 11–13 of gestation in European swine breeds. The objective of these experiments was to evaluate the temporal association between estrogen secretion by the preimplantation conceptus, increases in UBF and the onset of MRP in Meishan pigs. In Experiment 1, multiparous Meishan sows (N = 3) were observed for estrus once daily, and hand-mated at first observed estrus (Day 0) and 24 h later with a Meishan boar. On Day 2 postmating, sows were bilaterally instrumented with blood flow transducers and uterine venous catheters. UBF (ml min−1) was monitored and blood samples collected once daily from Days 6 to 30 postmating. UBF increased on Day 11 (P = 0.06) to peak on Day 13, then decreased on Days 14–17, before increasing progressively thereafter, coincident with increasing uterine E2β secretion. In Experiment 2, second cycle Meishan (N = 20) and Yorkshire (N = 18) gilts were hand-mated at first observed estrus (Day 0), and 24 h later, to boars of their own breed. Embryos were flushed from the gilts on Day 9, 11, 13 or 15 postmating. Gilts were again observed for estrus to determine treatment estrous cycle length (ECL). Treatment ECL was similar (P > 0.1) to presurgical ECL when embryos were collected from either Meishan or Yorkshire gilts on Days 9 and 11. ECL was extended in both breeds (P < 0.05) when embryos were recovered on Days 13 and 15.