| Title |
Investigators | Department | Objectives | Approach Keywords | Progress Reports | Impact Statements | Publications | |
Project * COL00220 | |
| Title | Reproductive Performance in Domestic Ruminants |
| Investigator(s) | Nett, TM; Sawyer, HR; Niswender, GD |
| Department | Biomedical Sciences |
| Objectives | 1. Investigate molecular, cellular, and endocrine mechanisms that limit or control reproductive efficiency in domestic ruminants. 2. Develop and improve assisted reproductive technologies to enhance sustainability of production systems for domestic ruminants. |
| Approach | The studies proposed for the next 5 year period will determine the factors that control LH and FSH receptor expression in the developing follicle. Members of the group will use electron microscopy, in situ hybridization, immunocytochemistry, and molecular biology to examine the mechanisms by which the intraovarian growth factors, GDF9 and BMP15, control the initiation of folliculogenesis. Finally, the potential role of reactive oxygen species (ROS) in follicle atresia will be examined in both in vitro and in vivo studies. Experiments will be conducted using whole animals, purified preparations of luteal cells or appropriate cell lines. Initially, indomethacin, an inhibitor of prostaglandin synthase (COX-2) will be administered via the ovarian artery to determine the necessity for luteal PGF2-alpha synthesis in the luteolytic process. If intra-luteal synthesis of PGF2-alpha is important for luteolysis, a similar experiment will be conducted using a 15-hydroxyprostaglandin dehydrogenase (PDGH) inhibitor (suphasalazine) to block intraluteal metabolism of PGF2-alpha in pregnant ewes and determine the effects on maternal recognition of pregnancy. If appropriate, the next logical experiment is to elucidate the mechanisms whereby the pregnant uterus induces luteal PDGH expression which likely involves interferon tau and MX protein. Further studies on the role of StAR, PBR and endosepine in cholesterol transport across mitochondrial membranes will be conducted using fluorescence energy transfer and glutathione S-transferase pull-down assays. Experiments will be conducted using whole animals, cultured ovine anterior pituitary cells, or cell lines that express estradiol receptors in their membranes. Initially, estradiol will be conjugated to a peptide to prevent it from crossing the membrane of cells. After demonstrating that the conjugate will bind to estrogen receptors on the surface of cells (and that it does not cross the cell membrane), the ability of the conjugate to alter LH and/or FSH secretion in ovariectomized ewes will be examined. Once it is established that the conjugate influences secretion of LH and/or FSH, how that effect is mediated at the cellular level will be determined. Three possibilities will be tested: 1) the conjugate may alter the ability of GnRH to interact with its receptor; this will be investigated by examining binding of radioiodinated GnRH to pitutiary cells after they have been treated with conjugate; 2) GnRH receptors must be able to dimerize in the plasma membrane after binding ligand in order for signal transduction to occur; it is possible that interaction of the conjugate with a membrane-associated estrogen receptor, alters membrane fluidity, thus preventing dimerization of GnRH receptors; we will measure changes in membrane fluidity; and 3) it is possible that the conjugate may alter intracellular signalling systems that stimulate secretion of LH and FSH; this will be examined by evaluating the effects of the conjugate on those signalling pathways known to be responsible for LH and FSH. |
| Keywords | reproductive efficiency, domestic ruminants, follicular growth, corpus luteum, luteinizing hormone, estradiol receptors, prostaglandin F2 alpha, progesterone |
| Progress Reports | |
| 1993 | 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. |
| 1995 | 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. We concluded: 1) GH is essential for follicles to remain responsive to gonadotropins; 2) in hypophysectomized ewes, failure of ovarian follicles to respond to exogenous FSH and LH is not due to a reduction in follicular receptors for FSH, LH or IGF-1. |
| 1996 | 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. |
| 1997 | 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. |
| 1998 | 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. |
| 1999 | 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. |
| 2000 | 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. |
| 2001 | 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. |
| 2002 | 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. |
| 2003 | 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. |
| 2004 | 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. |
| 2005 | 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. |
| Impact | |
| 1999 | 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. |
| 2000 | 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). |
| 2001 | 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. |
| 2002 | 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. |
| 2003 | 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. |
| 2004 | 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. |
| 2005 | 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 | |
| 1993 |
BELFIORE, C.J., HAWKINS, D.E. and NISWENDER, G.D. 1992. Regulation of the mRNA encoding cytochrome P450 side-chain cleavage (P450scc) in the ovine corpus luteum (CL). Proc. West. Sec. Am. Soc. Anim. Sci. 43:55-58. DIGREGORIO, G.B. 1993. Regulation of LH and FSH in the Ewe by Estradiol and Progesterone. Ph.D. Dissertation, Colorado State University. HAWKINS, D.E., BELFIORE, C.J., KILE, J.P. and NISWENDER, G.D. 1993. Regulation of mRNA encoding 3B-hydroxysteroid dehydrogenase/-5--4 isomerase (3B-HSD) in the ovine corpus luteum. Biol. Reprod. 48:1185-1190. KILE, J.P. and NETT, T.M. 1994. Differential secretion of FSH and LH from ovine pituitary cells following activation of protein kinase A, protein kinase C or increased intracellular calcium. Biol. Reprod. 50:49-54. MCGUIRE, W.J. 1993. Second Messenger Systems Mediating Prostaglandin F2a-Induced Luteal Regression. Ph.D. Dissertation, Colorado State University. WIEPZ, G.J., WILTBANK, M.C., KATER, S.B., NISWENDER, G.D. and SAWYER, H.R. 1993. PGE2 attenuates PGF2a-induced increases in free intracellular calcium in ovine large luteal cells. Prostaglandins 45:167-176. WILTBANK, M.C., BELFIORE, C.J. and NISWENDER, G.D. 1993. Steroidogenic enzyme activity after acute activation of protein kinase (PK) A and PKC in ovine small and large luteal cells. Mol. Cell. Endocrin. 97:1-7. |
| 1995 |
ECKERY, D.C. 1995. The role of somatotropin in ovarian follicular growth and development in sheep. Ph.D. Dissertation, Colorado State University GUY, M.K., JUENGEL, J.L., TANDESKI, T.R. AND NISWENDER, G.D. 1995. Steady-state concentrations of mRNA encoding the receptor for luteinizing hormone during the estrous cycle and following F2a treatment of ewes. Endocrine 3:585-58 JUENGEL, J.L., MEBERG, B.M., TURZILLO, A.M., NETT, T.M. AND NISWENDER, G.D. 1995. Hormonal regulation of messenger RNA encoding steroidogenic acute regulatory protein in ovine corpora lutea. Endocrinology 136:5423-5429 JUENGEL, J.L., NETT, T.M., TANDESKI, T.R., ECKERY, D.C., SAWYER, H.R. AND NISWENDER, G.D. 1995. Effects of luteinizing hormone and growth hormone on luteal development in hypophysectomized ewes. Endocrine 3:323-326 TURZILLO, A.M. AND NETT, T.M. 1995. Effects of estradiol on concentrations of gonadotropin-releasing hormone receptor messenger ribonucleic acid following removal of progesterone. Endocrine 3:765-768 TURZILLO, A.M., JUENGEL, J.M. AND NETT, T.M. 1995. Pulsatile gonadotropin-releasing hormone (GnRH increases concentrations of GnRH messenger ribonucleic acid and numbers of GnRH receptors during . . . Biol. Reprod. 53:418-423 |
| 1996 |
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 of luteinizin 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 |
| 1997 |
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 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 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 |
| 1998 |
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 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 |
| 1999 |
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 |
| 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 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. 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) Jennifer McCallum. 2000. Immunological detection of the GnRH receptor. Ph.D. Thesis. Animal Reproduction and Biotechnology Laboratory, Colorado State University 8/2000 |
| 2001 |
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. 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 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. 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. 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. 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. |
| 2002 |
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). 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. 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). 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. Niswender, G.D., 2002. Molecular control of luteal secretion of progesterone. Reproduction 123:333-339. 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. |
| 2003 |
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 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 |
| 2004 |
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). 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. 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). 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). |
| 2005 |
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. 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. 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. |