Progress 01/01/06 to 12/31/06
Outputs
That estradiol (E2) increases the number of GnRH receptors (GnRHR) on gonadotropes is well established. E2-induced increase in GnRHR requires both mRNA transcription and protein synthesis. Classically, E2 crosses the plasma membrane of a cell, binds to the ER in the cytoplasm, translocates to the nucleus and binds to an estrogen response element (ERE) on the target genes. However, an ERE has yet to be identified in the GnRHR gene. Recently, we demonstrated effects of E2 on pituitary function that are mediated at the plasma membrane. Based on the lack of an identifiable ERE in the GnRHR gene, we hypothesized that E2 stimulates an increases GnRHR by a membrane mediated mechanism. To test this hypothesis, dissociated ovine pituitary cells were treated with various doses of E2 or the membrane impermeable E2 (E2-BSA; 0.1 pM - 10 nM) for 12 h. We conclude that E2-BSA stimulated an increase in the number of GnRHR comparable to E2 and that stimulation of GnRHR was mediated by
a membrane action. Classically, progesterone has been thought to act only through a genomic pathway involving binding to nuclear receptors (PR). However, there is increasing evidence for rapid, nongenomic effects of progesterone, and thus it is likely that a membrane PR (mPR) may cause these events. We isolated and characterized an ovine mPR distinct from the nuclear PR. The mPR is a 350 amino acid protein having seven transmembrane domains typical of G-protein-coupled receptors and is expressed in the hypothalamus, pituitary, uterus, ovary and corpus luteum. In CHO cells that overexpress mPR, the mPR was uniquely localized to the endoplasmic reticulum (er) and not the plasma membrane. Given the localization in the er, we hypothesized that ligand stimulation of this receptor would increase intracellular Ca mobilization. Addition of progesterone to CHO cells expressing mPR caused a rapid increase in free intracellular Ca concentrations (P < 0.05). The increase in intracellular Ca
appears to be specific to progestins since treatment with estradiol, testosterone or cortisol did not evoke an increase in intracellular Ca. Iinsulin-like growth factors (IGFs) may be altered during development of intrauterine growth restriction (IUGR). We examined placental mRNA concentrations of components of the IGF system at 55 and 90 days gestational age (dGA); term 147 dGA) in a hyperthermia (HT)-induced sheep model of placental insufficiency-IUGR. Maternal insulin and IGF-I were constant at 55 and 90 dGA and not affected by treatment. Umbilical vein insulin tended to be reduced at 90 dGA following HT. Caruncle IGF-I mRNA was increased at 90 dGA in HT placentae (p<0.05), while cotyledon concentrations were constant over gestation and unaltered by treatment. In control cotyledons, IGF-II mRNA concentration increased (p<0.01) and IGFBP-3 decreased (p<0.01) between 55 and 90 dGA. Cotyledon IGF-II and caruncle IGFBP-4 mRNA were elevated at 55 dGA in HT placentae (p<0.01 and p<0.05
respectively). Changes in placental IGF expression in early and mid gestation may predispose the pregnancy to placental insufficiency, resulting in inadequate substrate supply for the developing fetus.
Impacts
Classically, steroid hormones have been thought to act only by acitvationg nuclear receptors in cells and altering expression of individual genes in tissue expressing those receptors. Virtually all pharmacological treatments utilizing steroid hormones have been developed based on this premise. However, it is becoming increasingly clear that steroid hormones also exert effects not only at the nuclues, but also via receptors located in cellular membranes. It is essential to better define the mechanisms by which steroid hormones act via this membrane receptors so that new, more efficacious treatment regimens may be developed. This information may well lead to an entirely different means for estrous synchronization schemes, methods to maintain pregnancy and induce parturtion, and for regulating fertility in domestic livestock.Studies on IUGR fetuses provide new information regarding the importance of maintaining a healthy intrauterine environment for the production of
normal offspring. Alterations in blood flow to the pregnant uterus can have dramatic effects on normal fetal development, impacts that will likely alter the growth potential of these fetuses for many months after they are born. Identification of a novel mPR may well provide an explanation as to how progesterone can induce, heretofore unexplained, rapid intracellular effects. This may lead to development of a new class of progestins that may be much more specific for inducing a particular effect (ie. inhibition of LH secretion) than those currently available.
Publications
- Erickson Hagen, A.S., R.J. Orbus, R.B. Wilkening, T.R.H. Regnault and R.V. Anthony. 2005. Placental expression of angiopoietin-1, angiopoietin-2 and Tie-2 during placental development in an ovine model of placental insufficiency-fetal growth restriction. Ped. Res. 58:1228-1232.
- Rispoli, L.A. and Nett, T.M. 2005. Pituitary gonadotropin-releasing hormone (GnRH) receptor: Structure, distribution and regulation of expression. Anim. Reprod. Sci. 88:57:74.
- Arreguin-Arevalo, A. and Nett, T.M. 2005. Nongenomic action of 17-estradiol as the mechanism underlying the acute suppression of secretion of luteinizing hormone. Biol. Reprod. 73:115-122.
- Arreguin-Arevalo, J.A. and Nett, T.M. 2006. A nongenomic action of 17-estradiol as the mechanism underlying the acute suppression of secretion of luteinizing hormone in ovariectomized ewes.. Biol. Reprod. 74:202-208.
- Ashley RL, Clay CM, Farmerie TA, Niswender GD, Nett TM. 2006. Cloning and characterization of an ovine intracellular seven transmembrane receptor for progesterone that mediates calcium mobilization. Endocrinology 147:4151-4159.
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Progress 01/01/05 to 12/31/05
Outputs
A study was performed to determine if activation of estrogen receptor (ER)alpha(A), ER beta (B) or both are required to: 1)acutely inhibit secretion of luteinizing hormone(LH), 2) induce a pre-ovulatory like surge of LH, and 3)decrease secretion of FSH. Ovariectomized ewes (n = 6) were administered (IM) 25micrograms estradiol (E2) or five times more (based on relative binding activity) PPT (an ER-A specific agonist), DPN (an ER-B specific agonist), or PPT + DPN. Compared with (E2)-treated ewes, the decrease in LH secretion occurred at the same time, but the beginning of the increase in LH secretion occurred earlier (mean +/- sem; 5.8 +/- 0.8 h vs. 10.8 +/- 0.8 h; P < 0.01) in DPN treated ewes, later (20.8 +/- 0.7 h vs.10.8 +/- 0.8 h; P < 0.01) in PPT treated ewes, and at similar interval (10.8 +/- 0.8 h vs. 12 +/- 0.4 h; P > 0.6) in PPT/DPN treated ewes. PPT alone or combined with DPN decreased (P < 0.05) secretion of FSH similar to that observed after E2 treatment;
however, unlike the E2-treated ewes, levels of FSH were suppressed much longer. A study was performed to evaluate systemic blood pressure (BP) normal in intrauterine growth restricted (IUGR) fetal lambs with elevated umbilical artery (UmA) Doppler indices. Five pregnant ewes were exposed to hyperthermic conditions for 80 days beginning at 40 days gestation (dGA) to induce IUGR. They were then placed in ambient conditions with 6 additional ewes that served as controls Compared with control pregnancies, the IUGR pregnancies showed: (1) reduced fetal and placental weights, (2) elevated systemic BP, (3) reduced umbilical blood flow (UBF), (4) elevated UmA and aortic Doppler velocimetry indices, (5) increased resistance per 100 g placenta, and (6) decreased UmA oxygenation and increased lactic academia. The UmA Doppler index of resistance (systolic/diastolic ratio) correlated strongly with calculated resistance (R2 = 0.7). Doppler indices also correlated with systemic BP (R2 =0.5). In
conclusion, ovine IUGR fetuses with high UmA Doppler indices have elevated systemic BPs. UmA Doppler indices of resistance correlate well with (1) fetal systemic BPs and (2) resistance as calculated by pressure/flow. This whole animal study shows that IUGR fetuses are hypertensive and that increased UmA Doppler resistance indices are consistent with a fetal-placental hypertensive state. There is increasing evidence for rapid, nongenomic progesterone (P4) effects in a variety of tissues in mammals; however the receptor responsible for these actions is yet to be characterized. The likelihood of a membrane progesterone receptor (mPR) causing these events is quite plausible. Therefore, the objective of this study was to isolate and characterize a putative ovine mPR distinct from the intracellular progesterone receptor (PR). An ovine mPR possessing seven transmembrane domains, characteristic of a G-protein coupled receptor (GPCR) was identified.. Tissues expressing the mPR include the
hypothalamus, pituitary, uterus, ovary and corpus luteum. The putative transcript was not found in liver, lung, spleen, kidney, heart, skeletal muscle, caruncle, or cotyledon. This is the first reported mPR transcript in mammals.
Impacts
We propose that both ER-A and ER-B are involved in both the inhibitory and stimulatory actions of E2 on secretion of LH and they interact to synchronize the beginning of the ovulatory surge. In contrast, only ER-A mediates the inhibition of FSH, probably by acting directly on the pituitary. A better understanding of how ER subtypes function will provide novel information that may be useful for developing new, more efficacious regimens for estrous synchronization or methods of suppressing estrous in feedlot animals. Studies on IUGR fetuses provide new information regarding the importance of maintaining a healthy intrauterine environment for the production of normal offspring. Alterations in blood flow to the pregnant uterus can have dramatic effects on normal fetal development, impacts that will likely alter the growth potential of these fetuses for many months after they are born. Identification of a novel mPR may well provide an explanation as to how progesterone can
induce, heretofore unexplained, rapid intracellular effects. This may lead to development of a new class of progestins that may be much more specific for inducing a particular effect (ie. inhibition of LH secretion) than those currently available.
Publications
- Ashley, R., G. Niswender, C. Clay. and T. Nett. 2005. Identification of a putative ovine membrane progesterone receptor. Biology Reproduction 73(Suppl. 1):99.
- Breen, K.M., A.E. Oakley, E.R. Wagenmaker, L.A. Rispoli, T.M. Nett. and F.J. Karsch. 2005. Rapid action of cortisol to suppress pituitary responsiveness to GnRH occurs independent of decreased GnRH receptors. Biology Reproduction 73(Suppl. 1):112.
- Arreguin-Arevalo, A. and T. M. Nett. 2005. Differential modulation of gonadotropin secretion by selective ERalpha and ERbeta agonists in ovariectomized ewes. Biology Reproduction 73(Suppl. 1):203.
- Davis, T.L. and T.M. Nett. 2005. Estrogen receptor alpha and beta agonists increase GnRH receptor number in cultured ovine pituitary cells. Biology Reproduction 73(Suppl. 1):224.
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Progress 01/01/04 to 12/31/04
Outputs
Expression of periattachment factor (PF) within the conceptus of species other than cattle was examined, and it was determined that PF is also produced by sheep, pig and horse conceptuses during a window of early pregnancy, but mRNA for PF was undetectable in early human placenta and a variety of rodent tissues. Using a number of cell lines that produce PF, PF was shown to be a nuclear protein as hypothesized. Administration of growth hormone (GH) to pregnant ewes from day 35 to 55 of pregnancy, the time when GH is produced by the pregnant uterus, did not result in enhanced placental or fetal growth at either 55 or 135 d gestational age. Further, while maternal serum concentrations of GH and IGF-1 were elevated by approximately 10-fold, expression of IGF-1 and IGF-2 by the maternal and fetal placenta was not altered. However, IGFBP-1 expression was reduced in the maternal caruncle and elevated in the fetal cotyledon. Expression of IGFBP-2, -3 and -4 by the maternal
caruncle was not effected by GH treatment, but IGFBP-2 was significantly reduced in the fetal cotyledon and IGFBP-4 was elevated. Finally, it was shown that Sp1 and Sp3 were the transcription factors interacting with a cis-element within the proximal ovine placental lactogen (oPL) gene promoter that is responsible for the majority of trophoblast-specific activity of this promoter. Infusion of either estradiol (E2) or a membrane impermeable form of E2 (E2 conjugated to bovine serum albumin; EBSA) into ovariectomized ewes rapidly (within 30 minutes) suppressed secretion of luteinizing hormone (LH). Further, treatment of cultured ovine pituitary cells with either E2 or EBSA inhibited gonadotropin releasing hormone (GnRH)-stimulated LH secretion. Signaling pathways by which E2 inhibits LH secretion were examined in the L beta T2-1 cell line. GnRH stimulated LH secretion in L beta T2-1 cells 3.3-fold compared to controls (P< .0001). However, neither E2 nor EBSA were able to inhibit the
GnRH-induced LH secretion in L beta T2-1 cells. Unlike normal gonadotropes, L beta T2-1 cells express very low levels of estrogen receptor-alpha (ER alpha) but easily detectable levels of ER beta. It was hypothesized that if greater expression of ER alpha in L beta T2-1 cells were induced, then the acute effects of E2 on LH secretion observed in cultured ovine pituitary cells could be simulated. To test this hypothesis murine ER alpha cDNA was stably transfected into L beta T2-1 cells. In L beta T2-1 cells overexpressing ER alpha, both E2 and EBSA inhibited GnRH-induced LH secretion (91%, P<0.005 and 48%, P<0.08, respectively). Having developed a cell culture model for studying acute effects of E2 on gonadotropes, the effects of E2 on GnRH-induced receptor activation and subsequent initial signaling events were examined. Neither E2 nor EBSA interfered with GnRH stimulation of inositol 1,4,5 triphosphate formation, but the increase in intracellular calcium that normally occurs after
GnRH stimulation was blunted. Thus, the negative effect of E2 on GnRH-stimulated secretion of LH appears to be mediated via ER alpha not ER beta and result from a blunting of intracellular calcium following GnRH stimulation.
Impacts
Experiments conducted during the past year have examined mechanisms involved in pregnancy recognition in domestic species, mechanisms regulating synthesis and secretion of progesterone (the hormone responsible for maintaining pregnancy), and secretion of luteinizing hormone (the hormone responsible for ovulation). Increased knowledge in each of these areas is essential for developing better mechanisms to regulate reproductive potential (either to enhance or inhibit) in livestock or species that may compete with livestock for natural resources. Although not specifically discussed in the progress report, a method to inhibit fertility in deer for one year was tested and found to be 100% effective.
Publications
- Anthony, R.V., Scheaffer, A.N. and Rhodes, J. 2004. Development stage-specific prenatal hypoxia effects on the adolescent rat. J. Soc. Gynecol. Invest. 11(Suppl.):239A (abstract).
- Arevalo, J.A.A. 2004. A non-genomic action of 17beta-estradiol as the mechanism underlying acute suppression of secretion of LH in primary cultures of ovine pituitary cells and in ovariectomized ewes. Ph.D. Thesis, Colorado State University, Fort Collins, CO.
- Aron, E.A., McMahon, C.L., Scheaffer, A. and Anthony, R.V. 2004. Hypobaric-hypoxia induced expression of vascular endothelial growth factor (VEGF) in pregnant ewes. J. Soc. Gynecol. Invest. 11(Suppl.):93A (abstract).
- Jeckel, K.M., Limesand, S.W. and Anthony, R.V. 2004. Transcriptional regulation of the ovine placental lactogen gene proximal promoter. Biol. Reprod. 70 (Suppl. 1):251 (abstract).
- Ashley, R.L., Allen, M.A., Staley, A. and Nett, T.M. 2004. Effects of GnRH-PAP on gonadotropin secretion and testicular weight in rams. Biol. Reprod. 70 (Suppl. 1): 226-227.
- Baker, D.L., Wild, M.A., Connor, M.M., Ravivarapu, H.B., Dunn, R.L. and Nett, T.M. 2004. Gonadotropin releasing hormone agonist: A new approach to reversible contraception in female deer. J. Wildlife Dis. 40:712-723.
- Bogan, R.L., Pickering, M.A., Escudero, J.M., Ku, C-Y. and Niswender, G.D. 2004. Quantification of endozepine and StAR protein in the ovine corpus luteum throughout the luteal phase of the estrous cycle. Biol. Reprod. 70 (Suppl. 1): 268-269.
- Jozwik, M., Pietrzycki, B., Jozwik, M. and Anthony, R.V. 2004. Expression of enzymes regulating placental ammonia homeostasis in human intrauterine growth restriction. J. Soc. Gynecol. Invest. 11(Suppl.): 218A (abstract).
- Limesand, S.W., Jeckel, K.M. and Anthony, R.V. 2004. Pur alpha, a single-stranded DNA binding protein, augments placental lacogen gene transcription. Mol. Endocrinol. 18:447-457.
- Padmanabhan V., Lee, J.S., Anthony, R.V., Mohankumar, S. and Mohankumar, P.S. 2004. Fetal programming: prenatal testosterone excess leads to compromised cardiac development. The Endocrine Society 86th Annual Meeting and Abstracts, Abstract #P2-68, p. 322 (abstract).
- Rispoli, R.A. and Nett, T.M. 2004. Rapid effects of estradiol on gonadotropin-releasing hormone stimulated luteinizing hormone secretion in L beta T2-1 cells. Biol. Reprod. 70 (Suppl. 1): 165.
- Wright, C.D., Scheaffer, A.N. and Anthony, R.V. 2004. Expression of peri-attachment factor in ovine conceptus and adult tissues. Biol. Reprod. 70 (Suppl. 1):154 (abstract).
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Progress 01/01/03 to 12/31/03
Outputs
We showed that estradiol (E) and E conjugated to bovine serum albumin (EBSA) to prevent its entry into cells inhibited gonadotropin-releasing hormone (GnRH)-induced secretion of luteinizing hormone (LH) from ovine pituitary cells. We also showed that other steroid hormones do not inhibit GnRH-induced LH secretion from cultured ovine pituitary cells. The other steroids examined were progesterone, testosterone, cortisol, and estradiol-17alpha (a nonbiologically active isomer of E). From these data, we concluded that the effect of E was specific and not being mediated by a general effect of steroids on the cell membrane. A second set of experiments was performed to ascertain if bioluminescence resonance energy transfer (BRET) which allow 96 simultaneous determinations and can be repeated over time was a suitable analytical procedure to study activation of second messenger pathways, which stimulate or inhibit steroidogenesis, influence the interaction of steroidogenic
acute regulatory protein and peripheral type benzodiazepine receptors in mitochondrial membranes. It was shown that the number of luteal peripheral type benzodiazepine receptors increase as the corpus luteum develops and is highly correlated with serum concentrations of progesterone throughout the estrous cycle. Likewise, the number of receptors in large steroidogenic luteal cells which secrete high levels of progesterone in a constituitive fashion is greater than the number in small luteal cells. In another experiment BRET was used to determine if E and membrane estrogen receptor (ER) prevent microaggregation of GnRH receptors (GnRHR). The experiment required a pure population of gonadotropes. Since gonadotropes comprise only about 10% of pituitary cells, it was necessary to utilize an immortalized cell line developed from a mouse gonadotrope tumor. The only cell line available that secretes LH in response to GnRH is the LbetaT2 cell line. Unfortunately, this cell line does not
express ER alpha (ERa) which is necessary for rapid inhibition of GnRH-induced LH secretion. Therefore, we cloned ERa into this cell line to produce cells that would recapitulate the mature gonadotropes. Conditions to perform BRET experiments were optimized. We examined the acute effect of estradiol on GnRHR microaggregation, the initial event following GnRH binding to it's receptor necessary for subsequent biochemical events. Simultaneously we began to examine inositol (1,4,5)-triphosphate (IP3) accumulation after treatment of LbetaT2 and LbetaT2-ERa cells with E2 and GnRH using an established radioreceptor assay. The microaggregation of GnRHR initiates the activation of G proteins which results in the conversion of membrane phospholipid phosphatidylinositol-4,5-bisphosphate into diacylglycerol and IP3. We measured the accretion of IP3 as an indicator for GnRHR stimulation of G proteins. We demonstrated that neither E2 nor EBSA interfered with GnRH-induced IP3 formation in LbetaT2
and LbetaT2-ERa cells. We inferred from this data that the acute suppression of LH secretion does not seem to involve the initial signaling pathway activated by GnRH, including GnRHR self-association.
Impacts
These studies demonstrated that a new technique bioluminescence resonance energy transfer (BRET) can be used to study second messenger activation in reproductive tissues from livestock. This technique allows investigators to examine interactions of molecules at the subcellular level and opens new avenues of research for designing regimens to control function of cells involved in reproductive processes. Such insights will be useful in developing the next generation of techniques that may be used for estrous synchronization, fertility regulation and pregnancy maintenance in our domestic livestock.
Publications
- Pickering MM, Escudero, JM, Escudero KW, Slough TL, and Niswender GD, 2003. Peripheral-type benzodiazepine receptors in ovine luteal tissue. Biol Reprod 68 (Suppl. 1): 143 (abst. 76).
- Pickering, M.M. Quantification of peripheral-type benzodiazepine receptors and steroidogenic acute regulatory protein in ovine luteal tissue. M.S. Dissertation, Colorado State University, 2004
- Arreguin-Arevalo JA and Nett TM. 2003. Acute nongenomic action of estradiol on LH secretion in ovariectomized ewes. Biology of Reproduction 68 Supp. 1: 222-223.
- Nett TM and Arreguin-Arevalo JA. 2003. Acute, presumably non-genomic action of estradiol on LH secretion in ovariectomized ewes. 3rd International Meeting: Rapid Responses to Steroid Hormones, Florence, Italy. p. 40 Rispoli L, Arreguin-Arevalo JA and Nett TM. 2003. Characterization of estradiol's acute effects on gonadotropin-releasing hormone (GnRH) receptor activation. 3rd International Meeting: Rapid Responses to Steroid Hormones, Florence, Italy. p. 39
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Progress 01/01/02 to 12/31/02
Outputs
The objective was to test the following two hypotheses: 1) Pregnant ewes subjected to hypobaric-hypoxia from day 40 to 50 of gestation will exhibit greater placental expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 (Hif-1 and Hif-2) at 50 days of gestation; and 2) Chronic hypobaric-hypoxic exposure (40 to 90 days of gestation) will result in reduced placental and fetal development and 135 days of gestation. While fetal and placental weights did not differ following 10 days of hypobaric-hypoxia, fetal lung weights were reduced and the right ventricular weight was increased significantly, suggesting acute pulmonary hypertension. In contrast to our hypothesis, fetal placental expression of VEGF, Hif-1 and Hif-2 were not effected by 10 days of hypobaric-hypoxia, rather maternal uterine (caruncular) expression of VEGF, Hif-1 and Hif-2 was reduced. Additionally, glucose transporter-1 (GLUT-1) expression was reduced in both maternal and
fetal tissues. At 135 days of gestation, there was no effect of hypobaric-hypoxia exposure from days 40 to 90 of gestation on placental and fetal weights. It is interesting to note that day 135 fetal lung exhibited significantly reduced concentrations of surfactant A, as a result of hypobaric-hypoxia during early to mid gestation. Collectively these results indicate that chronic hypobaric-hypoxia may be detrimental because of the eventual down-regulation of hypoxia-responsive genes, and that there may be long-lasting effects of early to mid gestation hypoxia on fetal lung development and function. Growth/Differentiation Factor-9 (GDF9) and Bone Morphogentic Protein 15 (BMP15) are newly discovered intra-ovarian growth factors that are essential for the normal development of ovarian follicles. Within the ovary, production and secretion of these novel growth factors is limited to oocytes. The purpose of this study was to answer two questions: 1) Can immunization against GDF9 or BMP15 be
used as a means to increase ovulation rate in ewes?; and 2) Does immunization against GDF9 or BMP15 affect fertility? To answer these questions, 8 normally cycling ewes were randomly assigned to each of 5 treatment groups: Group 1 - KLH immunized control; Group 2 - KLH-GDF9 peptide fragment; Group 3 - KLH-BMP15 peptide fragment; Group 4 - KLH-GDF9 full length protein; and Group 5 - KLH-BMP15 full length protein. Four weeks after initial immunizations, all ewes received booster injections of the appropriate antigen. To determine the effects of immunization on ovulation rate and fertility, all ewes were estrous-synchronized (8 weeks following initial immunization) and the number of corpora lutea on d10 of the cycle was determined by laparoscopy. During the next cycle, all ewes were bred to fertile rams. On d17 post-breeding ewes were euthanized and embryos flushed from uteri and evaluated. Preliminary findings indicate that immunization of ewes with either GDF9 or BMP15 is an effective
means to increase ovulation rate, and hence fertility. In the control group, a total of 16 corpora lutea were produced, whereas the number of corpora lutea in the treated groups ranged from 23-27.
Impacts
Many livestock in the western U.S. are exposed to high altitude during early to mid gestation. Results from this study indicate that such exposure may have a lasting effect on lung development and function. The negative effects appear to be the result of changes in gene activity. This means that some animals are likely to be resistant to genetic effects of exposure to high altitude (hypobaric-hypoxia) and selection for resistant animals (possibly via genetic testing) is a strategy to eliminate this problem from livestock in the western U.S. Production of viable oocytes is a limiting factor to rapidly increasing the frequency of genes from valuable females. Identification of factors that stimulate oocyte development is essential to increasing oocyte production. GDF-9 and BMP-15 are such factors, and regulating their production gives producers another mechanism for increasing the number of oocytes that can be harvested from genetically valuable females.
Publications
- Arreguin-Arevalo, J.A., and T.M. Nett, 2002. Acute inhibitory action of estradiol on GnRH-induced LH secretion in cultured ovine pituitary cells. Biol. Reprod. 66 (Suppl. 1):121 (abstr. 59).
- Nett, T.M., A.M. Turzillo, M. Baratta and L.A. Rispoli. 2002. Pituitary effects of steroid hormones on secretion of follicle-stimulating hormone and luteinizing hormone. Dom Anim Endocrinol 23:33-42.
- Baker, D.L., M.A. Wild, M.M. Conner, H.B. Ravivarapu, R.L. Dunn and T.M. Nett. 2002. Effects of GnRH agonist (leuprolide) on reproduction and behaviour in female wapiti (Cervus elaphus nelsoni). Reproduction, Suppl. 60:155-167.
- Yang, W.-H., M. Wieczorck, M.C. Allen and T.M. Nett. 2003. Cytotoxic activity of GnRH-PAP conjugates in cell lines expressing GnRH receptors. Endocrinology, in press.
- Niswender, G.D., 2002. Molecular control of luteal secretion of progesterone. Reproduction 123:333-339.
- Griffeth, R.J., Nett, T.M., Burns, P.D., Escudero, J.M., Inskeep, E.K., and Niswender, G.D., 2002. Is luteal production of PGF2a required for luteolysis? Biol. Reprod. 66 (Suppl. 1): 287 (abst. 465).
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Progress 01/01/01 to 12/31/01
Outputs
The objective was to: investigate the transcriptional control of the ovine growth hormone GH2-N gene, that allows it to be transcribed within the placenta during a window of early pregnancy. A GH2-N gene promoter/reporter construct was generated that contained 961 bp of 5'-flanking sequence, and used it in transient transfection assays in pituitary-derived cells (GH3), trophoblast-derived cells (BeWo and Rchol-1), and primary cultures of fibroblast cells derived from maternal caruncles or fetal cotyledons at 44 days post coitus. The reporter construct provided significant stimulation in GH3 cells as well as both sources of placental fibroblasts, but was inactive in the trophoblast-derived cells. To help verify that the primary cell cultures that were transfected originated from fibroblasts, these cells were plated on microscope slides and immunostained with anti-vementin antisera. The cells were stained positively for vimentin, supporting their fibroblast phenotype.
Additionally, mRNA was isolated from the cotyledon and caruncle fibroblast cultures, and verified to contain oGH mRNA by RT-PCR and Southern hybridization. A new series of reporter constructs were generated, including: minus 961 to plus 54, minus 776 to plus 54, minus 587 to plus 54, minus 399 to plus 54 and minus 166 to plus 54. These have been used in transient transfection assays with both GH3 cells as well as the day 44 placental fibroblasts. All constructs were active, in both types of cells, indicating that expression in the utero-placental unit cannot be accounted for by these domains. Estradiol (E2) induces a biphasic action on secretion of luteinizing hormone (LH) that is characterized by an acute inhibition of secretion followed by the characteristic preovulatory surge of LH. An increasing body of evidence indicates that E2 regulates cell function not only by a classic genomic action, but also via a plasma membrane (non-genomic) mediated mechanism. Due to the rapidity of the
inhibition of LH secretion after administration of E2 to ewes, it was hypothesized the rapid negative feedback occurred via a non-genomic effect. To test this hypothesis, the effects of E2 and a membrane impermeable form of E2, E2-6-CMO conjugated to bovine serum albumin (E2-BSA, Steraloids Co.), on GnRH-induced LH secretion were compared. Primary cultures of ovine pituitary cells (three pituitaries, 4 wells per treatment, 2 x 105 cells per well) were co-incubated with or without 2 nM gonadotropin-releasing hormone (GnRH) and 0, 0.01, 0.1, 1, 10, and 100 nM E2 or E2-BSA. After 15 minutes incubation, culture media was aspirated and concentrations of LH were quantified by radioimmunoassay. Data were analyzed by ANOVA, and significant differences between treatments were evaluated with LSD adjusted by Tukey's test. Relative to GnRH treated cells, 10 to 100 nM E2 decreased (P < 0.01) GnRH-induced LH secretion. Similarly, 1 to 100 nM E2-BSA decreased (P < 0.01) GnRH-induced secreton of LH.
The short period required for the inhibitory action of E2 together with the fact that this action was mimicked by the membrane impermeable form of E2 suggests the inhibition occured at the plasma membrane level.
Impacts
Regulation of placental growth is the primary factor controlling placental size which is directly correlated with size of the newborn. This may impact both difficulty with delivery at the end of gestation and health of the newborn. Understanding factors that regulate the rate of placental growth may lead to new treatments for decreasing difficulties with parturition and improving survival of newborn livestock. Regulation of gonadotropin secretion by gonadal steroids is the basis for most regimens for synchronizing estrus and or ovulation in livestock. Recently, it has been found that membrane (as well as nuclear) receptors may participate in the steroidal regulation of gonadotropin secretion. A better understanding of the mechanisms by which this occurs may provide more efficacious regimens for estrous synchronization.
Publications
- Anthony, R.V., S.W. Limesand and K.M. Jeckel. 2001. Transcriptional regulation in the placenta during normal and compromised fetal growth. Biochem. Soc. Trans. 29:42-48
- Galan, H.L., T.R.H. Regnault, T.D. Le Cras, R.W. Tyson, R.V. Anthony, R.B. Wilkening, S.H. Abman. 2001. Cotyledon and binucleate cell nitric oxide synthase expression in an ovine model of fetal growth retardation. J. App. Physiol. 90:2420-2426.
- Limesand, S.W. and R.V. Anthony. 2001. Novel activator protein-2a splice-variants function as transactivators of the ovine placental lactogen gene. Eur. J. Biochem. 268:2390-2401.
- Phillips, I.D., R.V. Anthony, J.A. Owens, J.S. Robinson and I.C. McMillen. 2001. Restriction of fetal growth has a differential impact on fetal prolactin and prolactin receptor mRNA expression. J.Neuroendocrinolol. 13:175-181.
- Baratta, M., L.A. West, A.M. Turzillo and T.M. Nett. 2001. Activin modulates differential effects of estradiol on synthesis and secretion of FSH in ovine pituitary cells. Biol. Reprod. 64: 714-719.
- Hashizume, T., W.-H. Yang, C.M. Clay and T.M. Nett. 2001. Internalization kinetics of murine and ovine gonadotropin-releasing hormone receptors. Biol. Reprod. 64:898-903.
- Amy S. Erickson-Hagen. Placental angiopoietin 1, angiopoietin 2 and their common receptor, Tie 2, in normal and intrauterine growth-restricted pregnancies. M.S. Thesis, Colorado State University, December, 2001.
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Progress 01/01/00 to 12/31/00
Outputs
The gonadal peptide inhibin stimulates gonadotropiin-releasing hormone receptor (GnRHR) gene expression in primary cultures of ovine pituitary cells, however, it has been difficult to recapitulate this effect in vivo. Thus, a role for inhibin in affecting GnRHR gene expression remains an issue. In transgenic mice harboring a fusion gene consisting of 9100 bp of proximal promoter from the oGnRHR gene linked to luciferase, expression of luciferase is confined to pituitary, brain and gonads. These animals present a unique opportunity to address the transcriptional response of the oGnRHR gene promoter to inhibin in vivo. To remove gonadal and hypothalamic inputs, transgenic mice were castrated and passively immunized against GnRH. Charcoal stripped bovine follicular fluid (bFF) was used as a source of inhibin. Animals remained either untreated (castrate + anti-GnRH) or received 400 ul of bFF intraperitoneally every 12 hours for 2 days (castrate + anti-GnRH + bFF).
Pituitary expression of luciferase was 31-fold higher in animals receiving bFF. None of the treatments affected expression of luciferase in the brain. Serum concentrations of FSH were lower in bFF treated mice compared to controls. To determine if the response of the oGnRHR fusion gene to bFF was due to inhibin in bFF, recombinant human inhibin-A (rh-Inh) was used in a similar paradigm. Animals were castrated, passively immunized against GnRH and then remained either untreated (castrate + anti-GnRH) or received 100 ng/g body weight of rh-Inh IP every 12 hours for 2 days (castrate + anti-GnRH + rh-Inh). Pituitary expression of luciferase was higher in rh-Inh treated mice, however the magnitude of the induction (5-fold) was strikingly less than the 30-fold induction after bFF raising the possibility that transcriptional induction of the oGnRHR transgene by bFF may not be solely due to inhibin. To determine if passive immunization against inhibin would block bFF induction of luciferase
expression. Four experimental groups were examined: 1) castrate + anti-GnRH; 2) castrate + anti-GnRH + bFF; 3) castrate + anti-GnRH + anti-inhibin; 4) castrate + anti-GnRH + bFF + anti-inhibin. The inclusion of anti-inhibin attenuated but did not block bFF induction of transgene expression in the pituitary (16-fold for group 4 vs. group 1 as compared to 32-fold for group 2 vs. group 1). These data suggest that inhibin may only partially contribute to bFF induction of the oGnRHR gene promoter in transgenic mice. We cannot eliminate the possibility that the partial blockade of bFF induced luciferase expression by anti-inhibin was due to incomplete immunoneutralization. However, treatment with inhibin anti-sera did completely reverse the suppressive effects of bFF on serum concentrations of FSH. Thus, the dose of inhibin anti-serum was at least sufficient to eliminate the well established biological effects of inhibin on FSH secretion. In summary, 9100 bp of proximal promoter from the
oGnRHR gene is responsive to bFF in transgenic mice. This response may only be partially mediated by inhibin. An additional factor(s) in charcoal stripped bFF is capable of stimulating GnRHR gene expression.
Impacts
These studies increased our knowledge concerning the regulation of the receptor for gonadotropin-relasing hormone, the major regulator of reproduction in all mammals. New methods for modulating the activity of the gene for this receptor could lead to novel paradigms for regulating (to either increase or decrease) reproduction in domestic animals. Likewise, it may be possible to use this information to control species that may compete with livestock for habitat or that cause significant damage to crops throughout the U.S. (i.e. deer, elk, wild-horses). The economic impact of methods for controlling reproduction in both livestock and other species is immense (several billion dollars per year).
Publications
- Duval, D.L., A.R. Farris, C.C. Quirk, T.M. Nett, D.L. Hamernik and C.M. Clay. 2000. Responsiveness of the ovine gonadotropin releasing hormone receptor gene to estradiol-17beta and gonadotropin releasing hormone is not detectable in vitro but is revealed in transgenic mice. Endocrinology. 141:1001-1010.
- Farris, A.R., D.L. Duval, T.M. Nett and C.M. Clay. 2000. Inhibin partially mediates transcriptional activation of the ovine gonadotropin releasing hormone receptor (oGnRHR) gene by bovine follicular fluid (bFF) in transgenic mice. 82nd Ann. Mtg.Endocrine Society. (Abstract #543)
- Baratta, M., L.A. West, A.M. Turzillo and T.M. Nett. 2001. Activin modulates differential effects of estradiol on synthesis and secretion of follicle-stimulating hormone in ovine pituitary cells. Biol. Reprod. 64:714-719.
- Jennifer McCallum. 2000. Immunological detection of the GnRH receptor. Ph.D. Thesis. Animal Reproduction and Biotechnology Laboratory, Colorado State University 8/2000
- Amy R. Farris. 2000. Regulation of the ovine GnRH receptor gene by cAMP and inhibin. M.S. Thesis. Cell and Molecular Biology program. Colorado State University 6/2000
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Progress 01/01/99 to 12/31/99
Outputs
Early embryonic wastage in cattle and sheep can be as high as 30% resulting in marked decreases in reproductive efficiency. One strategy for dealing with this problem is to understand and then manipulate the luteolytic hormone prostaglandin F2-alpha (PGF2A). 15-Hydroxy-prostaglandin dehydrogenase (PGDH), an enzyme that degrades PGF2A, and mRNA of PGDH were measured in ovine corpora lutea (CL) on day 13 of pregnancy and day 13 of the estrous cycle. On day 13 of pregnancy, when CLs are resistant to the activity of PGF2A, PGDH protein and message levels were significantly higher than on day 13 of the estrous cycle when CLs are known to be sensitive to PGF2A. This strongly suggests that CL metabolism of PGF2A by PGDH may play a role in pregnancy recognition in ruminants. The isolation and cloning of a novel intraovarian growth factor, growth-differentiation factor-9 (GDF-9), has been followed by discovery that this factor is expressed at the very early primordial stage of
follicular development. On-going immunocytochemical studies from recombinant GDF-9 will help to further understand the role this protein plays in female reproduction and fertility. The factors that control fetal growth are poorly understood, but placental lactogen is believed to play important roles. This has been difficult to verify in that removing the source of placental lactogen, the placenta, is not compatible with continued pregnancy. In an effort to determine the role of placental lactogen, fetal fibroblast cell lines were transfected with a "knockout" vector that deleted the second and third exons of the ovine placental lactogen gene by homologous recombination. Following screening of these cell lines for integration, clonal lines with correctly disrupted alleles can be used to generate transgenic sheep pregnancies with enucleated oocytes. Such transgeneic pregnancies could be engineered such that placental lactogen would not be produced provided an in vivo model with which to
study the effects of placental lactogen deficiency.
Impacts
The studies on PGF2A (pregnancy recognition) could lead to determining factors, including genetics, which would lead to higher rates of pregnancy. Studies on GDF-9 (understanding control of ovulation) and studies on PGF2A could increase reproductive efficiency and production. Placental lactogen studies could control fetal growth, impacting birth weights and neonatal survival.
Publications
- Bodensteiner, K., Clay, C.M. and Sawyer, H.R. 1999. Expression of growth/differentiation factor-9 (GDF-9) in the ovaries of domestic ruminants. Fifth International Symposium: Reproduction in Domestic Ruminants. J. Reprod. Fert., Suppl. 54.
- Phillips, I.D., Anthony, R.V., Houghton, D.C., McMillen, I.C. 1999. Regulation of prolactin receptor mRNA levels in the sheep liver before birth: relative roles of the fetal hypothalamus, cortisol and the external photoperiod. Endocrinology 140:1966-1971.
- Regnault, T.R.H., Battaglia, F.C., Wilkening, R.B. and Anthony, R.V. 1999. Altered arterial concentrations of placental hormones during maximal placental growth in a model of placental insufficiency. J. Endocrinol. 162:433-442.
- Liang, R., Limesand, S.W., and Anthony, R.V. 1999. Structure and transcriptional regulation of the ovine placental lactogen gene. Eur. J. Biochem. 265:883-895.
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Progress 01/01/98 to 12/31/98
Outputs
Two series of experiments were conducted to examine the regulation of gonadotropin-releasing hormone receptors (GnRH-R) in the ovine anterior pituitary gland. In the first series, ovariectomized (OVX) ewes were given a continuous infusion of GnRH (10 micrograms/hr) to down-regulate GnRH-R or saline for 132 h. At 120 h half the animals in each group were administered 25 micrograms estradiol intramuscularly. We found that: 1) continuous infusion of GnRH decreased expression of the GnRH-R gene and this lead to a decrease in the number of GnRH receptors in the anterior pituitary gland; and 2) estradiol was able to override the negative effect of GnRH by stimulating expression of the GnRH-R gene and GnRH-R concentrations. Therefore, although the gonadotrope becomes refractory to homologous desensitization, the desensitization does not affect the cell's ability to respond to estradiol. The second series of experiments was conducted to determine if progesterone could prevent
the stimulatory effect of estradiol on GnRH receptor gene expression. Ewes were treated with either a low or a high dose of estradiol, or vehicle during the luteal phase of the estrous cycle. Steady state concentrations of GnRH-R were similar among all treatment groups. Thus, a second experiment was conducted to determine if progesterone was the ovarian factor responsible for preventing the estradiol-induced increase in GnRH-R gene expression. Ewes were OVX on day 5 of estrous cycle and administered progesterone to simulate luteal phase concentrations of progesterone. Three days later ewes were treated with either estradiol or vehicle. In this experiment, progesterone was unable to override the effects of estradiol on GnRH-R gene expression. Therefore, since the effects of estradiol on GnRH-R gene expression were prevented during the natural luteal phase, and since progesterone was unable to prevent the estradiol-induced increase in GnRH-R gene expression, it appears that some other
ovarian factor besides progesterone functions to inhibit the effects of estradiol on expression of the GnRH-R gene.
Impacts
(N/A)
Publications
- Turzillo AM, Clapper JA, Moss GE, Nett TM. 1998. Regulation of ovine GnRH receptor gene expression by progesterone and estradiol. J Reprod Fert 113:251-256.
- Turzillo AM, Nolan TE, Nett TM. 1998. Regulation of gonadotropin-releasing hormone (GnR) gene expression in sheep: Interaction of GnRH and estradiol. Endocrinology 139:4890-4894.
- Mann RJ, Keri RA, Nett TM, Nilson JH. 1998. Transgenic mice with chronically elevated LH are infertile due to anovulation, defects in uterine receptivity, and midgestation pregnancy failure. Proceedings of 80th Annual Meeting of the Endocrine Society, p.390.
- Ginal JA. 1998. Immunocytochemical analysis of gonadotropes and the regulation of gonadotropes by estradiol. Colorado State University.
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Progress 01/01/97 to 12/31/97
Outputs
Messenger ribonucleic acid (mRNA) for gonadotropin-releasing hormone receptor and subunits of luteinizing hormone (LH) and follicle- stimulating hormone (FSH) was measured in pituitary glands collected from normal cows (NORM) and cows selected for twin births (TWIN). In NORM cows, there was a 69% reduction in mRNA for FSH as ovulation approached. In contrast, there was no reduction in mRNA for FSH as ovulation approached in TWIN cows. Thus, selection for TWINs is associated with altered regulation of expression of the gene for FSH. These observations provide additional insight into possible mechansisms underlying ovarian follicular development in cattle selected for natural and twin births.
Impacts
(N/A)
Publications
- Vizcarra JA, Wetteman RP, Turzillo AM, Braden TD, Nett TM. 1997. Effect of GnRH pulse frequency on serum and pituitary concentrations of LH and FSH, GnRH receptors and messenger RNA for gonadotropin subunits in cows. Endocrinology 138:594-601.
- Phillips ID, Anthony RV, Butler TG, Ross JT, McMillen IC. 1997. Hepatic prolactin receptor gene expression increases in the sheep fetus before birth and after cortisol infusion. Endocrinology 138:1351-1354.
- Eckery DC, Moeller CL, Nett TM, Sawyer HR. 1997. Localization and quantification of binding sites for follicle-stimulating hormone, luteinizing hormone, growth hormone, and insulin-like growth factor I in sheep ovarian follicles. Biol. Reprod. 57:507-513.
- Holland MD, Hossner KL, Williams SE, Wallace CR, Niswender GD, Odde KG. 1997. Serum concentrations of insulin-like growth factors and placental lactogen during gestation in cattle. I. Fetal profiles. Dom. Anim. Endocrinol. 14:231-239.
- Hossner KL, Holland MD, Williams SE, Wallace CR, Niswender GD, Odde KG. 1997. Serum concentrations of insulin-like growth factors and placental lactogen during gestation in cattle. II. Maternal profiles. Dom. Anim. Endocrinol. 14:316-324.
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Progress 01/01/96 to 12/30/96
Outputs
The ability of preantral follicles (primordial/primary and secondary follicles 100 um) to grow in vitro was evaluated. Using BrdU-labeling to detect proliferating cells, it was evident that granulosa cells of preantral secondary follicles proliferated in vitro, even in the absence of supplemental hormones such as FSH, EGF, IGF-1 or estradiol. Although it was possible to maintain primordial/primary follicles in vitro for up to 30 days, granulosa cells failed to proliferate/differentiate, regardless of hormones added to the culture media. Bovine follicular fluid (bFF) was administered to ovariectomized ewes to assess the effects of inhibin on synthesis of GnRH receptors and FSH. Treatment with bFF (inhibin) suppressed secretion of FSH to <30% of controls and reduced amounts of mRNA for FSHB-subunit to <10% of controls; no effect on mRNA for GnRH receptor or numbers of GnRH receptors was noted. We conclude that circulating inhibin does not influence synthesis of GnRH
receptors in sheep. Treatment of hypophysectomized ewes with LH increased the amount of mRNA for GH receptor in luteal tissue. Treatment with GH stimulated expression of mRNA for IGF-1 in luteal tissue. In a separate study, Atosiban (an antagonist of oxytocin) prevented regression of the corpus luteum.
Impacts
(N/A)
Publications
- GIRMUS, R.L., A.M. DUNN, T.M. NETT, E. ESQUIVEL AND M.E. WISE. 1996. Estradiol up-regulation of progesterone binding is required for progesterone inhibition ofluteinizin ghormone release. Endocrine 4:43-58.
- JUENGEL, J.L., M.C. WILTBANK, B.M. MEBERG AND G.D. NISWENDER. 1996. Regulation of steady-state concentrations of mRNA encoding prostaglandin F2a receptor in ovine corpus luteum. Biol. Reprod. 54:1096-1102.
- TANDESKI, T.R., J.L. JUENGEL, T.M. NETT AND G.D. NISWENDER. 1996. Regulation of mRNA encoding low density lipoprotein receptor and high density lipoprotein binding protein in ovine corpora lutea. Reprod. Fert. Dev. 8:1107-1114.
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Progress 01/01/95 to 12/30/95
Outputs
To determine the mechanisms responsible for increased synthesis of gonadotropin-releasing hormone receptors (GnRH-R) during the preovulatory period, anestrus ewes were induced to ovulate and were subsequently treated with prostaglandin (PG)F2a on day 10 to initiate luteal regression. Since GnRH secretion is photoperiodically inhibited in anestrus ewes, some of the ewes were given GnRH at hourly intervals for 12h to stimulate a follicular phase frequency of GnRH secretion. There was no increase in amounts of mRNA for GnRH-R when serum concentrations of Progesterone (P) declined. However, hourly pulses of GnRH increased amounts of mRNA for GnRH-R approx. 4-fold. We concluded that increase in synthesis of GnRH-R during the preovulatory period coincides with increased frequency of GnRH pulses resulting from decrease in serum concentrations of P. We have cloned the cDNA for steroidogenic acute regulatory (STAR) protein, the protein that appears to control rate of
steroidogenesis in the ovary. LH and dibutyryl cAMP increase amounts of mRNA for this protein, whereas PGF2a induces a rapid, dramatic decrease in amounts of mRNA for STAR in ovine corpus luteum. We examined role of growth hormone (GH) in stimulating maturation of ovarian follicles in sheep.
Impacts
(N/A)
Publications
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Progress 01/01/93 to 12/30/93
Outputs
Attempts to utilize cDNA for the mouse gonadotropin-releasing hormone (GnRH) receptor to evaluate changes in the mRNA for the ovine GnRH receptor were unsuccessful. Therefore, we cloned the cDNA for ovine GnRH receptor and used it to examine the effects of estradiol and GnRH on steady-state levels of mRNA for the GnRH receptor in sheep. Removal of GnRH decreased and estradiol increased amounts of mRNA for and numbers of GnRH receptors in the pituitary gland. Since this receptor represents the site at which inputs from the nervous system are transformed into endocrine signals that control the reproductive system, information concerning regulation of the synthesis of this receptor is crucial to developing efficient methods to enhance or inhibit reproduction. Cows with highest concentration of serum progesterone have increased first service conception rates (up to 20% increase). Feeding a high lipid diet decreased the rate of clearance of progesterone from blood
resulting in increased serum concentrations of progesterone. This occurred rather than due to an increase in secretion of progesterone by the corpus luteum. Progesterone is the hormone responsible for maintaining pregnancy in all mammals. Therefore, increased knowledge concerning the mechanisms responsible for controlling its synthesis and secretion will provide the impetus for development of novel methods to maintain or terminate pregnancy in our domestic animals.
Impacts
(N/A)
Publications
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Progress 01/01/92 to 12/30/92
Outputs
Since number of Sertoli cells per testis appears to be the major factor limitingnumber of sperm produced by a testis, a study was conducted to identify factors responsible for differentiation of Sertoli cells during testicular development in bull calves. Availability of testosterone, rather than expression of androgen receptor appears to be the major factor limiting development of Sertoli cells. A second study involved changes in the corpus luteum of ewes. Changes in amount of mRNA for cholesterol side-chain cleavage cytochrome P450 did not change during luteolysis, but mRNA for 3B-hydroxysteroid dehydrogenase (3B-HSD) decreased with increased secretion of prostaglandin (PG) F2a during luteolysis. A third study determined if changes in number of receptors for PGF2a or PGE2 could be responsible for maintenance of the corpus luteum during early pregnancy in ewes. Decreased sensitivity of the corpus luteum to PGF2a during early pregnancy was not due to reduction in
number of receptors for PGF2a or to increased number of receptors for PGE2. A fourth study with ewes determined if second messenger systems that regulate synthesis and secretion of LH also control synthesis and secretion of FSH. Second messenger pathways responsible for synthesis and secretion of LH and FSH are similar, but secretion of FSH requires a greater stimulation of each second messenger pathway than secretion of LH.
Impacts
(N/A)
Publications
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Progress 01/01/91 to 12/30/91
Outputs
In a first series of experiments, it was determined that the protein kinase A (PK), PK-C and calcium second messenger system all released both luteinizing hormone and follicle-stimulating hormone (FSH) from ovine pituitary cells. However, LH release was more sensitive to PK-C and CA than was FSH. These observations may provide insight to the differential secretion of these gonadotropins. In a second series of experiments, methods were developed to quantitate in mRNA levels encoding for the receptor for LH and two major steroidogenic enzymes. The levels of mRNA were quantitated throughout the estrous cycle, the relative levels in small and large luteal cells were compared and the effects of PGF2` induced luteolysis were evaluated. The major funding was that mRNA for 3 beta hydroxysteroid dehydrogenase was dramatically reduced at the end of the estrous cycle and by PGF2` treatment. In additional studies, it was demonstrated that treatment of hypophysectomized ewes with
gonadotropic hormones could not stimulate ovulation while ovulation occurred in ewes which had been hypothalamic-pituitary stalk disconnected (HPD). Growth hormone levels were normal in HPD ewes. Current studies are evaluating the effects of growth hormone infusions on ovulation in hypophysectomized ewes.
Impacts
(N/A)
Publications
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Progress 01/01/90 to 12/30/90
Outputs
During late pregnancy, secretion of estradiol by the placenta inhibits synthesisof luteinizing hormone (LH) in the anterior pituitary gland. By the end of pregnancy, this causes a depletion in the amount of LH in the anterior pituitary gland to a point that an animal cannot have normal estrous cycles. If the depletion of LH during late pregnancy can be prevented, it is likely that reproductive activity could be re-initiated very soon after parturition. We administered enclomiphene (a compound reported to have anti-estrogenic activity) to animals with blood levels of estradiol similar to those observed during late pregnancy in an attempt to prevent the estradiol-induced decrease in the amount of LH in the anterior pituitary gland. Unfortunately, enclomiphene did not function as an anti-estrogen at the level of the pituitary gland. In fact, it acted similar to estradiol and further decreased the amount of LH in the anterior pituitary. Undernutrition also decreases the
amount of LH in the anterior pituitary gland of animals, and as a result may decrease their reproductive capacity. This effect of undernutrition can be reversed by administering gonadotropin-releasing hormone to animals which acts to restore synthesis and secretion of LH, and thereby, return reproductive capacity to normal.
Impacts
(N/A)
Publications
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Progress 01/01/89 to 12/30/89
Outputs
Estradiol increases the number of pituitary receptors for gonadotropin-releasinghormone (GnRH). The following experiments were performed to determine if this action of estradiol is via a classic genomic mechanism. Pituitary glands from ovariectomized ewes were dissociated into single cells and maintained in culture. Experiment 1. Number of GnRH-receptors increased linearly as the concentration of estradiol was increased from 1 pg/ml to 100 pg/ml. At 100 pg/ml estradiol there was difference3-fold increase in the number of GnRH-receptors compared to untreated controls (P<0.05). Experiment 2. There was a gradual 3.5-fold increase (P<0.05) in numbers of GnRH-receptors from 0 to 12 h of treatment after which numbers of receptors remained stable for at least the next 12 h. Experiment 3. Inhibition of protein synthesis with cycloheximide (25 mu g/ml) or mRNA synthesis with actinomycin D (10 mu g/ml) prevented the estradiol-induced increase in the number of
GnRH-receptors. These findings support the concept that estradiol does increase numbers of pituitary GnRH-receptors through mechanisms requiring RNA and protein synthesis. The number, affinity and distribution of receptors for ovine trophoblast protein-1 (oTP-1) in endometrium was characterized during the ovine estrous cycle and early pregnancy. Receptor concentrations and affinity did not differ from 4 different regions of the endometrium.
Impacts
(N/A)
Publications
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Progress 01/01/88 to 12/30/88
Outputs
Experiments were conducted to examine the mechanism(s) by which estradiol inhibits synthesis of luteinizing hormone (LH). Ovariectomized ewes were treated with estradiol for 0.5, 1, 2, 4, 8 or 16 days and pituitary content of LH, follicle-stimulating hormone (FSH) and their respective mRNAs were examined. Estradiol induced a triphasic affect on the secretion, synthesis and pituitary content of LH. Changes were first noted in secretion of LH, then synthesis of LH and finally pituitary content. In terms of secretion, estradiol first was inhibitory from 0 to 0.5 days of treatment, stimulatory from 0.5 to 1 day and inhibitory from 1 to 16 days. In terms of synthesis, mRNAs for the LHbeta-subunit, FSHbeta-subunit and alpha-subunit all decreased from 0 to 0.5 days of treatment, increased from 0.5 to 2 days, decreased from 2 to 4 days and remain low from 4 to 16 days. Pituitary content of LH decreased from 0 to 1 day of treatment, increased slightly from 1 to 8 days and
again decreased by 16 days. In a separate experiment, follicles expected to give rise to short-lived corpora lutea were collected from beef cows 3h after onset of estrus induced by weaning their calves at 35 days after parturition. These follicles were compared to follicles collected 3h after the onset of estrus in normally cycling cows. There were more granulosa cells (approximately 2X) in follicles expected to produce short-lived corpora lutea but these cells had fewer receptors (approximately 0.
Impacts
(N/A)
Publications
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Progress 01/01/87 to 12/30/87
Outputs
There were no differences in the number of receptors for LH on days 4 or 7 when corpora lutea from the first postpartum ovulation were compared to corpora lutea from normally cycling ewes. However, by day 7 following the preovulatory surge of LH, first postpartum corpora lutea had approximately 3 times more receptors for PGF(2)alpha than did corpora lutea collected from normally cycling animals (P<0.05). These results are consistent with the hypothesis that the first corpus luteum formed after parturition is more sensitive to the luteolytic effects of PGF(2)alpha. Morphometrical analysis of the numbers of steroidogenic luteal cells indicated that the number of small luteal cells decreased prior to a loss of large luteal cells after a luteolytic dose of PGF(2)alpha. All receptors for PGF(2)alpha are present on large steroidogenic luteal cells which are thought to originate from cells of the granulosal layer in the follicle. Thus, it appeared likely that the
preovulatory follicle which ovulated to form the first postpartum corpus luteum might contain more granulosal cells than normal follicles. To investigate this hypothesis, calves were weaned at 30 days postpartum, and the cows were checked for estrus at 6 h intervals to detect first ovulation. Three h after estrus was detected, the ovary containing the largest follicle was removed. The same cows were used two cycles later to collected normal follicles.
Impacts
(N/A)
Publications
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Progress 01/01/86 to 12/30/86
Outputs
Estradiol has an inhibitory effect on secretion of luteinizing hormone (LH) during periods of anestrus. This effect is mediated via an inhibition of gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus. Therefore, a study was conducted to determine the effect of estradiol on immunoreactive GnRH in the neuronal system of anestrous ewes. From this study it was concluded that GnRH is synthesized even when estradiol suppresses secretion of GnRH and that the excess GnRH is stored in axons that normally do not contain GnRH. A second objective was to determine if corpora lutea (CL) from animals that have short estrous cycles were more sensitive to prostaglandin F(2)alpha (PGF(2)alpha) than CL from animals having normal estrous cycles. CL were collected 4 to 7 days after ovulation from ewes during a short estrous cycle after parturition and from normally cycling ewes. CL from ewes having short cycles secreted less LH in vitro (both basal and
LH-stimulated) than from normally cycling ewes (P<.01). This was not due to a difference in LH receptors between the two groups of CL. By day 7 postovulation there were 2.4 times more receptors for PGF(2)alpha in CL from ewes having short cycles than in CL from normally cycling ewes (P>.05). The increased number of rec ptors may increase the sensitivity of CL from ewes having short estrous cycles to PGF(2)alpha. Two experiments were conducted to develop methods to increase in vitro fertilization rates of bovine oocytes.
Impacts
(N/A)
Publications
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Progress 09/01/84 to 08/30/85
Outputs
Major progress has been made in three areas. In studies designed to determine the endocrine basis for anestrus, it was demonstrated in hypothalamic stalk-disconnected ewes that the secretion of luteinizing hormone (LH) by the anterior pituitary is dependent upon gonadotropin-releasing hormone from the hypothalamus. In contrast, basal secretion of follicle-stimulating-hormone, prolactin and growth hormone occurs in the abasence of hypothalamic input. The effects of estradiol on secretion of LH appear to be regulated primarily at the level of the hypothalamus. In a second series of experiments, it was demonstrated that treatment of normally cycling ewes with LH or human chorionic gonadotropin resulted ina reduction in the number of small (LH dependent?) luteal cells with a concommitant increase in the number of large (LH independent?) luteal cells. Since the large luteal cells, which have few, if any, receptors for LH are the primary source of luteal progesterone,
these results have important implications for the maintenance of pregnancy. Administration of LH tohypothalamic-stalls-disconnected ewes was adequate to maintain normal luteal function and cell numbers. It was concluded that LH is the major luteotropic hormone in ewes and that prolactin played no role in regulating luteal function during the estrous cycle. A final series of experiments was conducted to characterize the nature of the cytotoxic factor secreted from large luteal cells incubated with PGF(2 alpha) in vitro.
Impacts
(N/A)
Publications
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Progress 01/01/84 to 12/30/84
Outputs
Studies regarding changes in the concentration of luteinizing hormone in the adenohypophysis of pregnant and postpartum ewes have been completed. There is a 7-fold decrease in hypophyseal concentrations of LH from day 50 of gestation to day 2 postpartum followed by a 50-fold increase as the ewes begin to exhibit estrous cycles by 35 days postpartum. The reduced content of LH in the adenohypophysis was associated with a reduced capacity of this gland to synthesize LH not due to increased release of the hormone. The reduced capacity to synthesize LH appeared to be due to the long-term suppressive effects of estradiol on the RNA for the subunits of LH. In experiments designed to determine if secretion of progesterone was enhanced in pregnant ewes compared to cycling ewes during the first 12 days post-estrus, there was no evidence that the embryo has early steroidogenic effect. It was demonstrated that the presence of an embryo in the uterus of ewes results in a
protective effect against the luteolytic effects of PGF(2x). The protective action was for relative short time period (12-16 days post mating). Finally, data were obtained in vitro which demonstrated that PGE(2) could prevent the negative effects of PGF(2x) on the secretion of progesterone by cultured luteal cells.
Impacts
(N/A)
Publications
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Progress 01/01/83 to 12/30/83
Outputs
Oral antibiotics can cause intestinal malabsorption. Our past studies show thatmaximum recommended doses of chloramphenicol and neomycin, used to treat diarrhea in calves, cause intestinal mucosal damage and induce malabsorption. To study other antibiotics used to treat diarrhea, maximum recommended oral doses of ampicillin or tetracycline were fed to healthy colostrum fed calves for 5 days starting at 4 days old. Four of 6 calves developed diarrhea after 4-5 days of ampicillin or after 3-5 days of tetracycline. Non-diarrheic calves had increased mucus in the feces. Oral glucose and lactose tolerance tests (OGTT, OLTT) were performed prior to and on the 5th day of antibiotic dosing. In calves receiving ampicillin, absoption of glucose was reduced 50% in the OGTT and 63% in the OLTT compared to controls, and proximal jejunum lactase activity was decreased 30%. In calves receiving tetracycline, absorption of glucose was reduced 33% in the OGTT and 45% in the OLTT
compared to controls, and proximal jejunum lactase activity was decreased 73%. Cytochemical electron microscopy for Na-K dependent ATPase activity indicates that the ampicillin treated group had decreased reaction deposits in comparison to control; activity in the tetracycline treated group was similar to or slightly less than the controls.
Impacts
(N/A)
Publications
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Progress 01/01/82 to 12/30/82
Outputs
Studies were completed on the effects of oral chloramphenicol and neomycin on gastrointestinal function in normal neonatal calves using therapeutically recommended doses. Chloramphenicol causes profound malabsorption and diarrhea in 3 to 4 days following daily administration at 50 mg./kg. b.i.d. Morphologically, major changes were seen in the intestinal mucosa from the duodenum through the spiral colon. Throughout the small intestines there was a decrease in the number of villi, in villus height, and a decrease in cell size. At the ultrastructuaral level, significant changes in mitochondrial structure were apparent and there was a shortening and thinning of the brush border. Neomycin administered orally at a level of 100 mg./kg. divided into 4 doses causes a severe malabsorption diarrhea in 4 to 5 days. Morphological lesions, in general, were less severe with neomycin than with chloramphenicol. One major difference between the effects of these two antibiotics on
intestinal function was the appearance of large numbers of cryptospyridia in the intestinal tract following neomycin therapy. The relationship of their increased numbers to the malabsorption-diarrhea is not known at this time.
Impacts
(N/A)
Publications
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Progress 01/01/81 to 12/30/81
Outputs
GnRH injections at 2 hr intervals for 96 hr did not induce ovulation in postpartum cows. Similar treatment prevented ovulation in cows during the follicular phase of the cycle. This treatment regime may work in cows under good nutritional conditions but does not work in all cows. LRRS data indicates that estrogen (E2) is necessary for GnRH priming to occur and that the ovary itself is not necessary in prepuberal heifers. They also found that removal of the suckling stimulus results in an increase in follicular LH receptors and a similar increase occurs prior to ovulation in estrous cycling cows. P4 implants failed to affect short estrous cycles or efficacy of GnRH induced pulses of LH in producing ovulation. The number of LH receptors in the thecal and granulosal compartments of the follicle increase until a few hours before the LH peak and then decrease dramatically. PGF(2)Alpha is the hormonal signal for maintenance of the CL of pregnancy. Bovine parvovirus may
persist in cells of fetuses, a reliable and rapid method for detection is being sought. The development of endotoxic shock was associated with hyperglycemia and a concomitant hyperlactemia and acidosis. Gluconeogenesis was impaired in isolated hepatocytes. They also found that low therapeutic levels of neomycin or ampicillin for 5 days induced clinical diarrhea in calves. A striking enhancement of plaque formation was found in bovine fetal thyroid and brain cells infected with bovine coronavirus L9.
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Progress 07/01/79 to 06/30/80
Outputs
Serum samples from both cattle and man were found to have antibodies against enteropathogenic bovine coronaviruses. These antibodies were detected by the use of a microimmunodiffusion test as well as by neutralization of infectivity. At least 7% of human donors to a blood bank had antibodies while over 25% of selected human clinical patients had antibodies. Bovine parvoviruses were identified as a cause of fetal disease and brain damage in cattle. Fetuses infected in the first and second trimester of gestation were most susceptible to parvoviral injury. Histological lesions of the cerebellum consisted of depopulation of the germinal external granule cell layer. Methods to propagate bovine parvoviruses in synchronized cultured bovine fetal spleen cells were developed. Virus induced nucleopathic changes and cell lysis indicated that these viruses are mitolytic. Data have been obtained which provide further evidence that prostaglandin (PG) E(2) in the signal from
the pregnant uterus which is responsible for maintenance of the corpus luteum of gestation. Studies performed in vitro indicated that the stimulatory effects of luteinizing hormone (LH; the hormone responsible for maintenance of the corpus luteum and progesterone secretion during the estrous cycle) on progesterone secretion were mediated via activation of adenylate cyclase in a pool or compartment which was biochemically dissociated from the pool of adenylate cyclase activated by PGE(2).
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Progress 01/01/78 to 12/30/78
Outputs
Experiments were conducted to decrease the anestrus period in sheep with melatonin. Other experiments concerned the site of action of the embryo on maintenance of luteal function specifically how pregnancy influences the synthesis of PG F(2)Alpha. Development of assays for assessing immune reactivity to virus-infected cells were started. These types of assay feed to be operational before one can optimally use the immune products resulting from viral infections of the gut or other portions of the body. The effect of bovine parvoviruses on fetuses of different developmental ages was investigated using viruses isolated from neonatal calves. Parasynchronous bovine fetal spleen (BFS) cells and immunofluorescence were used to trace progression of the infection. Accomplished in the past year have been (1) determination of developing intestinal enzyme activities in the neonatal calf intestine, and (2) preliminary experiments validating the methodology for determining the
effects of a corona virus on intestinal enzyme activities and transport properties in the neonatal calf. Insemination with contaminated semen results in at least a temporary infertility. Fertilization and the initial cleavage stages of development occur normally in such cases; the infertility is due to early embryonic death. In vitro fertilization of bovineoocytes.
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Progress 01/01/77 to 12/30/77
Outputs
The presence of vasectomized bulls increased pregnancy rates (P less than 0.05) with heifers inseminated artificially during a synchronized breeding period (Synchromate B). Temporary separation (48 hr) of calves from cows synchronized with Norgestomet implants reduced the average interval of estrus (36.6 vs. 42.6 hr, P less than 0.05). The average interval to estrus following removal of implants is less (P greater than 0.01)with a 9-day than with a 7-day Synchromate B treatment (33.6 vs. 45.6 hr). Eight prepubertal ewe lambs had two cannulas placed into the posterior vena cava one terminating just anterior to the entrance of the ovarian vein and the other just anterior to the entrance of the renal-adrenal vein. Precisely timed, paired samples were collected for 36 days and progesterone and cortisol were quantified in each sample. There was no significant difference in the level of progesterone or coritsol when values were compared between the paired samples.
Infertility of cows due to undernutrition is not caused by fertilization failure or abnormal levels of LH and progesterone. The zona pellucida prevented infection of mouse embryos with paramyxovirus but embryo infection and viral multiplication occurred in zona-free preimplantation embryos.--An antigen prepared by alkaline extraction of jejunal mucosa from calves infected with coronavirus produced 4 precipitin lines in the agar gel precipitation (AGP) test with coronavirus antiserum.
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Progress 01/01/76 to 12/30/76
Outputs
Changes in serum levels of reproductive hormones of the first post-partum estruswere found to be similar to those of the onset of puberty. Progesterone was elevated from 4.6 + or - 0.1 to 0.6 + or - 0.1 days prior to estrus. The numberof LH peaks in 15 min samples increased as estrus approached. Prolactin was high at parturition, dropped during the following 1 to 3 days and then increaseddramatically apparently due to the frequent sucking stimuli. Forced-breeding at 48 and 72 hours after removal of implants to control estrus (norgestomet B plus estradiol valerate) eliminated heat checking. Pregnancy rates were 53.5% in force-bred, 46.7% in cows bred at estrus, and 62.3% in control cows. These differences were not significant. A non-surgical method of embryo recovery was developed. Embryos from 15 of 18 cows (83%) inseminated without superovulation and 1 to 17 embryos of 9 superovulated cows were recovered. Non-surgical embryotransfer was employed with 100
recipient, but only 20 pregnancies resulted whichwas less than half the rate from surgical transfers. Bovine parvoviruses infected fetuses after direct fetal or dam inoculation. Three to 5-month fetuses had infection of adrenal glands, lung, liver, kidney, heart muscle, thymus and intestine.
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Progress 01/01/75 to 12/30/75
Outputs
Based on hormonal changes occurring prior to the onset of puberty in normal heifers, a hormonal treatment for inducing puberty in beef heifers was developed. It consists of injections of estradiol-17 B plus a synthetic progestin and an implant of this progestin on day 1. Four days after removal ofthe implant on day 9, heat was detected in 94%, 93%, and 79% of the heifers in three different trials. Approximately 60% of the heifers with induced heat became pregnant. This treatment proved equally effective in heifers that had not reached puberty due to age (less than 14 months old) or due to undernutrition (greater than 15 months old). Ultrastructural examination of thin sections of the intestinal tract of calves infected with coronavirus (LY-138) revealed infection of absorptive epithelial and goblet cells of the villi and of plasma cells, fibroblasts and endothelial cells in the lamina propria. The infected cells were at various stages of degeneration consisting
of cytoplasmic vacuolation, dilation of the rough endoplasmic reticulum, swelling and fragmentation of mitochondrial cristae, dissolution of membranes and cell lysis. The microvilli of the brush border of infected cells were shortened, sparse or absent and the ponocytotic system was reduced. The basal lamina of the villi was ocasionally denuded.--The cells degenerated relatively early in the course of viral multiplication.--Calves with naturally occurring and virus-induced diarrhea sequester lactate in peripheral tissues as diarrhea
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Progress 01/01/74 to 12/30/74
Outputs
Histologic lesions in intestinal chlamydial infections of calves were found to be characterized by bulbous intestinal villi, granulomas in the submucosa, supranuclear cytoplasmic inclusions in epithelial cells which desquamated. Crypts were distended and had plugs of cellular debris and Peyer's patches contained necrotic centers. Additional viral isolates previously obtained from enteric disease of calves were identified as bovine enteroviruses and as coronaviruses. Using immuno-electron-microscopy improved methods for viral identification, diagnosis, and separation of multiple mixed viral infections of calves were developed. -Chalamydial agents from cattle and sheep were found to be identical. Two major antigenic types were detected.
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Progress 01/01/73 to 12/30/73
Outputs
Work on the New Management system which embodies initial selection of cows on the basis of early calving coupled with estrous synchronization and enhanced nutrition during pregnancy is promising. A shorter breeding period and more pounds of calf weaned appears to be the result. Calving difficulty was analyzedas a function of both the bull and pelvic opening in the cow or heifer in the Angus-Hereford breeds. The analysis was based on data from 19 Hereford-Angus bulls and several hundred females. The interrelationships appear to be complex.Puberty induction and estrous synchronization research is promising. Metabolic hormone and substrate levels were determined in dairy cows in the pre- and postpartum period to elucidate a potential pre-ketotic state.
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Progress 07/01/71 to 06/30/72
Outputs
Further progress has been made in identifying and characterizing viral and chlamydial pathogens which interfere with reproduction. This includes infections, intrauterine in ewes, genital in bulls, as well as neonatal. In the latter study, particular attention was paid to mechanisms of action of the agents in inducing enteritis. Study of pathophysiologic alterations in calves infected with enteropathogenic viral agents has been continued centering on fluid, electrolyte and energy balance. A series of therapeutic regimes has been formulated to deal with varying degrees of sickness. Estrus synchronization and control of ovulation by administration of normal and synthetic hormones has been studied in cows and ewes.
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Progress 01/01/71 to 12/30/71
Outputs
Double infections of bovine enterovirus and virus of bovine diarrhea were studied using embryonic bovine tissue. The two viruses had different rates of tissue adsorption and their intracellular replication caused different morphological changes. Chlamydial agents increased colostral IgG and enhanced passive immunity as noted by a decrease in diarrheal severity without preventingagent multiplication in the intestinal tract. A complete balance study on waterand electrolyte losses during diarrhea was completed. Changes in water compartmentalization during diarrheal dehydration indicate that the major loss in acute diarrhea is vascular. A theoretical model of tritium hydrogen exchangeduring growth was developed on the basis of the balance studies.
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Progress 01/01/70 to 12/30/70
Outputs
This project was initiated and exploratory experiments were designed. These experiments are in progress but enough data have not yet been collected to give a competent report.
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