Mid-luteal Stage Research Articles

Epidermal Growth Factor and Its Receptor in Bovine Corpus Luteum during the Estrous Cycle.

The epidermal growth factor (EGF) concentration and its binding characteristics in bo-vine corpus luteum (CL) during the estrous cycle (early: Days 3-5, mid-: 8-12, late stage: 15-18) were determined. The EGF concentrations in corpus luteum tissue, evaluated by a radioreceptor assay using human placenta, were 3.0 ± 0.7 ng/g in early luteal phase, 43.9 ± 2.2 ng/g in mid-luteal phase and 18.6 ± 2.7 ng/g in late luteal phase. Specific EGF receptors were present in bovine corpus luteum of all luteal phases. TGFα, but not IGF-I, FGF and NGF competitively displaced EGF binding. The binding affinity of luteal EGF receptors differed between the luteal stages and decreased significantly from early (Kd=0.8 ± 0.04 nM) to mid-luteal stage (Kd=1.6 ± 0.34 nM, P<0.05). The binding capacity of luteal EGF receptor at different luteal stages were similar (early stage; Bmax=35.1 ± 5.7 fmol/mg protein, mid-luteal stage; Bmax=46.3 ± 4.0 fmol/mg protein, late stage; Bmax=35.1 ± 5.7 fmol/mg protein, all values mean ± SEM). These results suggest that EGF plays a physiological role for the regulation of luteal function during the estrous cycle.

Secretory and Morphological Changes in the Baboon (Papio Anubis) Uterus and Placenta during Early Pregnancy1

The study was undertaken to characterize changes in uterine endometrial secretory activity and morphology in the baboon between Days 18 and 32 of pregnancy. Proteins synthesized by the implantation site endometrium (RI) and non-implantation site endometrium (RIII) and developing placenta were analyzed by two-dimensional PAGE and fluorography. The electrophoretic mobility and localization of chorionic gonadotropin (CG) in the baboon placenta, and insulin-like growth factor binding protein-1 (IGFBP-1) and retinol binding protein (RBP) in the baboon uterus were confirmed by Western blot analysis and immunocytochemistry. Placental protein synthesis was primarily characterized by the presence of radiolabeled CG at Days 18 and 25, which decreased by Day 32. Peak CG synthetic activity, immunolocalized to the syncytiotrophoblast, occurred during the formation of tertiary villi. Also associated with the development of mature villi was a corresponding increase in a basic protein (M(r) 23,000). The overall secretory pattern of the baboon endometrium was similar to that observed during the midluteal stage; however, the synthetic activity was enhanced during pregnancy. Radiolabeled and immunoreactive IGFBP-1 and RBP were detectable in the Day 32 functionalis and basalis, respectively, and their synthesis was higher in RI compared to RIII. The immunolocalization of IGFBP-1 in RI appeared to be directly related to the degree of decidualization. In addition, a novel group of low molecular-weight (M(r) 27,000-30,000) isoelectrovariant proteins was secreted predominantly by the basalis at Days 18 and 25 of pregnancy and disappeared by Day 32 in all but one animal. The synthesis of this group of proteins did not show regional specificity. These studies indicate that the baboon endometrium undergoes morphological and secretory changes during pregnancy that are associated with the development of a functional placenta and decidualized endometrium. Furthermore, these studies suggest that the conceptus/placenta may mediate specific changes in IGFBP-1 and RBP expression at the implantation site.

Effects of basic fibroblast growth factor, transforming growth factor-beta and nerve growth factor on the secretory function of the bovine corpus luteum in vitro.

The effects were investigated of basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta) and nerve growth factor (NGF) on the release of progesterone and oxytocin from the bovine corpus luteum (CL) at different stages of the oestrous cycle. A microdialysis system (MDS) of CL and a cell culture system with a reduced number of endothelial cells were used. In the MDS of CL from the mid-luteal stage (days 8-12 of the oestrous cycle), infusion with bFGF (0.1, 1, 10 and 100 ng/ml), TGF-beta (0.1, 1 and 10 ng/ml) and NGF (0.1, 1, 10 and 100 ng/ml) for 30 min induced significant acute effects on the release of progesterone. Both bFGF and NGF stimulated the release of progesterone during peptide infusion, TGF-beta and also bFGF in the period thereafter. This stimulation was dose-dependent during and after the infusion only for bFGF. This response pattern was observed at all luteal stages for the three growth factors, but bFGF was more stimulatory at the early (days 5-7) and mid-luteal stages during and after peptide infusion. The release of oxytocin was stimulated by bFGF in a dose-dependent manner. At the highest dose, bFGF, TGF-beta and NGF stimulated the release of oxytocin throughout all three luteal stages. When luteal cells were cultured with growth factors, only TGF-beta showed a dose-dependent inhibition of both basal and LH-stimulated progesterone as well as oxytocin release (measured between 48 and 52 h of culture). NGF had an inhibitory effect only on the basal release of oxytocin. bFGF had no effect on the release of either hormone under continuous stimulation in cell culture. The results indicate that bFGF, TGF-beta and NGF act directly and acutely on the secretory function of bovine CL in the MDS but also have long-term effects as shown in cell culture. bFGF appears to be an important autocrine/paracrine regulator of CL function, since local expression of its mRNA, peptide synthesis and its mitogenic and non-mitogenic actions have now been confirmed. Endothelial cells from the CL have been identified as target cells for bFGF. Differences observed between the two systems might thus be attributed to the presence or absence of cell-to-cell contact and a reduced number of endothelial cells, as well as to the duration of peptide stimulation and medium changes every 24 h compared with the flow-through conditions in the MDS.

Evidence for oxytocin receptors in cultured bovine luteal cells.

Specific receptors for oxytocin (OT) on intact luteal cells are demonstrated. Cultured cells from bovine corpora lutea (CL) at different stages (Days 3-5, 8-12, and 15-18 of the estrous cycle) were examined for OT receptors by a radioreceptor assay using the 125I-labeled OT antagonist [d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH2(9)] -vasotocin. Binding specificity was demonstrated in displacement studies with various related peptides. Scatchard analysis revealed the presence of a binding site with an association constant of Ka = 2.6 x 10(9) M-1 and a capacity of 5.9 fmol/micrograms DNA. Additionally, in 50% of the experiments (n = 6) two different binding sites were observed. The Ka of the high-affinity site was 2.6 x 10(10) M-1; its capacity was 0.73 fmol/micrograms DNA. The low-affinity site had an apparent Ka of 4.9 x 10(8) M-1 and a capacity of 8.8 fmol/micrograms DNA. Observation of one versus two binding sites related neither to the assay conditions nor to the state of the individual CL used for the cell culture and therefore appeared to reflect individual variation within the OT receptor population. Significant binding of OT was observed at all luteal stages. OT binding was maximal at the mid-luteal stage (Days 8-12). We conclude that a direct action of OT on the bovine CL is mediated by the OT receptor, supporting the hypothesis that luteal OT plays an important physiological role in the regulation of progesterone release and/or other luteal functions in a paracrine or autocrine fashion.

Control of inhibin production by dispersed human luteal cells in vitro.

The production of inhibin in vitro by dispersed cells from early to mid (Days 16-19) and late stage (Day 23) human corpus luteum (CL) was examined, and the effects of human chorionic gonadotrophin (hCG), follicle stimulating hormone (FSH), oestradiol and testosterone on inhibin production were determined. Corpora lutea from five subjects in the early to mid luteal stage and three subjects in late luteal stage were dispersed with enzymes and the luteal cells cultured in medium supplemented with 5% calf serum and either FSH (1, 10 or 100 ng mL-1), oestradiol-17 beta (2.5, 5 or 10 micrograms mL-1) or testosterone (0.25, 1 or 5 micrograms mL-1) with or without hCG (1 I.U. mL-1). Cells were cultured for 1 to 3 days without changes of medium, and the concentrations of progesterone, oestradiol and immunoreactive inhibin in the medium were measured by radioimmunoassay. Cells from both types of CL produced inhibin in vitro under basal conditions, but only cells from early to mid CLs responded to hCG with a significant increase in inhibin production. Both progesterone and oestradiol production were stimulated by hCG in both groups of CL. Inhibin concentrations in the cell cultures declined with time in culture, particularly in the late CL group, whereas the concentration of steroids increased. Neither FSH, oestradiol nor testosterone significantly changed inhibin production in either CL group. It was concluded that inhibin production by human luteal cells in vitro is influenced by the age of the CL, and is dependent on LH (hCG) but not on FSH or sex steroids.

Superovulation in heifers using either pregnant mares serum gonadoptrophin or follicle stimulating hormone during the mid luteal stage of the estrous cycle

Techniques for the superovulation of cattle involve administration of gonadotrophins at the mid-luteal phase of the estrous cycle, followed by prostaglandin F2α 48 hours later to induce luteolysis, estrus and ovulation. Two of the most common gonadotrophins used in cattle are pregnant mares serum gonadotrophin (PMSG) and follicle stimulating hormone (FSH). The objective of this experiment was to compare the superovulatory response and embryo yield in heifers following either gonadotrophin. Estrus was synchronized in 200 beef heifers using 500 μg cloprostenol (PG). These heifers were randomly allocated to receive either 24 mg pFSH (Folltropin, n=105) with 4.5, 3.5, 2.5 and 1.5 mg given twice daily for four days, or a single injection of 2000 IU PMSG (n=95) at mid-cycle. The PG was given at 48 (500 μg) and 60 hours (250μg) post gonadotrophin initiation. Heifers were artificially inseminated at estrus and slaughtered 7 days later, and their reproductive tracts were recovered. The numbers of corpora lutea (CL) and large follicles (≥10 mm) were determined. Following embryo recovery with phosphate buffered saline and 3% heat treated steer serum; the number and quality of embryos and the fertilization rate were determined. Significant differences between treatments were determined using t-tests and Chi-square tests. The mean (± SEM) number of CL for pFSH-treated heifers was 19.0±0.99, with 12.3±0.81 embryos collected. Both the numbers of CL (13.9±0.84) and embryos collected (8.1±0.60) were lower (P=0.001 and P=0.0001, respectively) in PMSG than in pFSH-treated heifers. There was a greater number (P=0.01) of freezable embryos (Grades 1 and 2; 4.4±0.45) in pFSH-treated heifers than in PMSG-treated heifers (3.0±0.32). There was a greater number (P=0.009) of degenerated embryos (Grades 4 and 5; 5.7±0.70) in the pFSH-treated heifers than in the PMSG-treated heifers (3.4±0.46). The number of transferable embryos (Grade 3; FSH-p, 2.1±0.24; PMSG, 1.5±0.23) and the number of unfertilized eggs (pFSH, 2.1±0.50; PMSG, 1.0±0.25) did not differ (P=0.09 and P=0.055, respectively) between the two treatments. Results of this study indicate that the numbers of CL, embryos recovered, and Grade 1 and 2 embryos were higher in the pFSH-treated heifers than the PMSG-treated heifers.

Effect of prostaglandin F 2α dosage and stage of estrous cycle on the estrous response and corpus luteum function in beef heifers

Ninety-five normal cyclic crossbred beef heifers were used to determine if the proportions of heifers showing estrus, intervals to estrus and corpus luteum (CL) function were influenced by PGF 2α dosage and (or) the stage of luteal phase when PGF 2α was administered. Heifers were assigned randomly to treatments in a 4 × 3 factorial arrangement. Treatments were 5, 10, 25 or 30 mg PGF 2α injected either in early (5 to 9 d), mid (10 to 14 d) or late (15 to 19 d) stages of the luteal phase. Jugular samples were taken at 0 h and at 8 h-intervals for 48 h and again at 60 h after PGF 2α treatment for progesterone assay. Heifers were observed for estrus continuously for 120 h PGF 2α treatment. The proportion of heifers showing estrus was dependent upon (P<0.05) both dosage of PGF 2α and stage of luteal phase. Heifers given 5 mg of PGF 2α showed estrus only if treated during the late stage, while those given 10 mg of PGF 2α showed a progressive increase of heifers in estrus as stage of luteal phase advanced. The proportion of heifers showing estrus after 25 and 30 mg of PGF 2α increased from 56% for the early stage to 100% for the mid and late stages. Interval to estrus in heifers showing estrus within 120 h after PGF 2α treatment did not differ (P>0.05) among dosages but tended (P=0.10) to be longer in heifers treated during the mid luteal stage (67 h) than in heifers treated in the two other stages (56 h). A greater proportion of heifers (P<0.05) showed estrus by 60 h after PGF 2α when treated during the early and late luteal stages (75.5%) than for heifers treated during the mid luteal stage (30.4%). Patterns of progesterone concentrations were influenced (P=0.08) by the three way interaction of dosage, stage and time. In heifers that showed estrus, rate of decline in progesterone tended (P=0.07) to be slower during the mid luteal stage than during the early and late stages. Progesterone did not drop below 1 ng/ml until 32 h in heifers treated during the mid luteal stage; whereas progesterone dropped below 1 ng/ml by 24 h in heifers treated during the early and late stages. These data may be useful in designing more efficient systems for using PGF 2α or its analogues in estrus synchronization of beef cattle.

The detection of oviduct-specific proteins in the baboon (Papio anubis).

Oviducts obtained from cycling baboons were first flushed with saline, then minced and cultured in the presence of L-[3H] leucine. The flushings, tissue culture media, and solubilized tissues were analyzed by electrophoresis for secretory products that were oviduct-specific. The fluorographs of one-dimensional 7.5% slab gels demonstrated that a macromolecule of Mr 130,000 was present in the tissue culture medium of oviducts obtained during the mid-to late follicular stages, and absent during the mid-to late luteal stages. Two-dimensional analysis revealed that the Mr 130,000 band consisted of one basic protein and two acidic proteins. The basic protein formed the major component of this band and was the protein either absent or greatly reduced in intensity during the luteal stage. A macromolecule with similar electrophoretic properties was present in silver-stained, two-dimensional gels of follicular stage flushings, and absent in both luteal stage flushings and follicular stage serum. Two other macromolecules (Mr approximately equal to 160,000 and 88,000) appeared to be secretory products that were oviduct-specific. While both were present at all stages of the menstrual cycle, the Mr 160,000 protein showed a midcycle intensification, and the Mr 88,000 protein showed a midluteal stage intensification. Thus, the synthetic ability of baboon oviduct tissue and macromolecular content of the oviduct lumen changed with the stage of the menstrual cycle. The major alteration was in the synthesis and secretion of the Mr 130,000 basic macro-molecule, which was the major protein present during the follicular stage of the cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of protein which binds progesterone from the bovine corpus luteum.

The nature of the proteins released together with progesterone from the bovine corpus luteum was investigated. Slices of bovine corpus luteum obtained at the mid-luteal stage of the oestrous cycle were incubated in vitro in the presence of bovine LH. Radioactive monitoring of compounds separated by gas-liquid chromatography showed that added [14C] acetate was converted to sterols and progesterone within the 2-h incubation period. Electrophoresis of the proteins recovered from the incubation medium showed the presence of a major band which migrated at the same rate as a progesterone-binding protein isolated from pooled corpora lutea. Densitometry revealed that the proteins in the incubation medium were enriched threefold in binding protein compared to those in an homogenate of corpus luteum. Equilibrium dialysis of the medium after addition of [3H] progesterone revealed the presence of progesterone-binding activity (association constant = 10(6)l/mol) analogous to that contained in the cytosol fraction from bovine corpus luteum. The finding that progesterone-binding protein is released concomitantly with the hormone has implications for the granule hypothesis of steroid secretion. The existence of an intracellular steroid-binding protein complex to maintain progesterone in high intragranular concentration would be predicted from this hypothesis.

Evidence for functional arterio--venous anastomoses in the ovaries of sheep.

Vessels with histological features characteristic of arterio--venous anastomoses were found in the stroma but not in CL. In 5 conscious ewes at the mid-luteal stage of the oestrous cycle, ovarian blood flow was significantly greater (P less than 0.025) with microspheres of 50 than of 15 micrometers diameter in ovaries without CL (0.23 +/- 0.04 (s.e.m.) and 0.11 +/- 0.02 ml/min, respectively), but not in ovaries with CL (4.42 +/- 0.86 and 3.73 +/- 0.70 ml/min, respectively). In 5 similar but anaesthetized ewes, the portion of each ovarian artery within the ovarian vascular pedicle was bypassed with re-entrant catheters through which microspheres were perfused. A greater proportion (P less than 0.01) of 50 than of 15 micrometers microspheres was retained in ovaries with (90 and 79%) or without (82 and 45%) a CL. It is concluded that functional arterio--venous anastomoses are present in sheep ovaries.

The effects of progesterone and oestrogen treatment in heifers on oestrous cycle control and plasma progesterone levels.

Data are presented on the effects of a short-term progesterone treatment for oestrous cycle control in cattle. Progesterone was administered by intravaginal sponge pessaries inserted for a 10-day period. Progesterone pessaries alone did not affect oestrous cycle length or corpus luteum function at either early (day 2) or midluteal (day 12) cycle stages. However, when progesterone (250 mg) and oestradiol benzoate (7-5 mg) were given intramuscularly on the day of pessary insertion corpus luteum development was inhibited in animals treated at day 2 and was regressed in animals at day 12. This combined oestrogen-progesterone treatment efficiently controlled oestrous cycle length.

Food rejection by female rhesus monkeys during the menstrual cycle and early pregnancy

Two hundred and two female rhesus monkeys were monitored for completion of meals and instances of food rejection during nonpregnant menstrual cycles and during pregnancy. Two prominent periods of increased food rejection were found. The first occurred midway through the menstrual cycle, around the expected time of ovulation. Incidence of food rejection during this periovulatory period was significantly greater than that observed during the early follicular or midluteal stages of the cycle. Food rejection also rose sharply during the third through the fifth weeks of pregnancy and then tapered off over the next month and a half. The feeding changes during early rhesus pregnancy are contrary to what has been described for rodents, but the pattern is consistent with the incidence of nausea and feeding changes reported for human pregnancies. The observations on rhesus females indicated a systematic relationship between levels of food intake and reproductive condition in primates. When feeding changes are compared to reports of endogenous estrogen and progesterone determinations, a positive correlation is noted between incidence of food rejection and levels of circulating estrogens.