Search

To more clearly understand the equine gonadotrope response to kisspeptin and gonadotropin releasing hormone (GnRH), peripheral LH and FSH were quantified in diestrous mares after treatment with either equine kisspeptide (eKp-10, 0.5 mg iv), GnRH (25 μg iv), or a combination thereof every 4 h for 3 days. The following observations were made: 1) a diminished LH and FSH response to eKp-10 and GnRH was observed by Day 3, but was not different by treatment, 2) a decrease in basal LH concentration was observed from Day 1 to Day 3 for the eKp-10, but not the GnRH treated mares, 3) there was no change in basal FSH with either treatment. Additionally, pre-treatment with GnRH antagonist (antide 1.0 mg iv) eliminated any measurable change in LH after eKp-10 (1.0 mg iv) treatment. Both GnRH and kisspeptin are Gα q/11 coupled receptors, therefore quantifying the rise in intracellular calcium following treatment with cognate ligand allows simultaneous assessment of receptor activation. Direct stimulation of equine primary pituitary cells with GnRH and/or eKp-10 demonstrates three distinct populations of pituitary cells: one population responded to both eKp-10 and GnRH, a second, independent population, responded to only eKp-10, and a third population responded only to GnRH. These populations were confirmed using co-immunofluorescence of hemipituitaries from mares in diestrus. Although the rise in peripheral LH concentration elicited by eKp-10 is dependent on GnRH, this work suggests that kisspeptin also has a specific and direct effect on the equine gonadotrope, independent of GnRH., (Copyright © 2020. Published by Elsevier Inc.)

Gonadotropes represent approximately 5-15% of the total endocrine cell population in the mammalian anterior pituitary. Therefore, assessing the effects of experimental manipulation on virtually any parameter of gonadotrope biology is difficult to detect and parse from background noise. In non-rodent species, applying techniques such as high-throughput ribonucleic acid (RNA) sequencing is problematic due to difficulty in isolating and analyzing individual endocrine cell populations. Herein, we exploited cell-specific properties inherent to the proximal promoter of the human glycoprotein hormone alpha subunit gene (CGA) to genetically target the expression of a fluorescent reporter (green fluorescent protein [GFP]) selectively to ovine gonadotropes. Dissociated ovine pituitary cells were cultured and infected with an adenoviral reporter vector (Ad-hαCGA-eGFP). We established efficient gene targeting by successfully enriching dispersed GFP-positive cells with flow cytometry. Confirming enrichment of gonadotropes specifically, we detected elevated levels of luteinizing hormone (LH) but not thyrotropin-stimulating hormone (TSH) in GFP-positive cell populations compared to GFP-negative populations. Subsequently, we used next-generation sequencing to obtain the transcriptional profile of GFP-positive ovine gonadotropes in the presence or absence of estradiol 17-beta (E2), a key modulator of gonadotrope function. Compared to non-sorted cells, enriched GFP-positive cells revealed a distinct transcriptional profile consistent with established patterns of gonadotrope gene expression. Importantly, we also detected nearly 200 E2-responsive genes in enriched gonadotropes, which were not apparent in parallel experiments on non-enriched cell populations. From these data, we conclude that CGA-targeted adenoviral gene transfer is an effective means for selectively labeling and enriching ovine gonadotropes suitable for investigation by numerous experimental approaches., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Problem: Current methods to quantify kisspeptin (KP) are limited. To this end, a radioimmunoassay (RIA) specific for KP was developed and validated. We hypothesized that use of a RIA would reveal multiple hypothalamic regions as targets of negative seasonal feedback of estradiol on KP production in sheep., Method of Study: Ovariectomized (OVX) ewes bearing a subcutaneous implant of estradiol were euthanized during the breeding season (BS) (n = 4) and non-breeding season (NBS) (n = 3). Coronal sections of preoptic area (POA), anterior hypothalamic area (AHA), and mediobasal hypothalamus (MBH) were collected, as well as the median eminence (ME), cortex, brain stem, and cerebellum. Amounts of KP and gonadotropin-releasing hormone (GnRH) in individual hypothalamic nuclei were quantified by radioimmunoassay., Results: Concentration and content of KP were lower during the NBS than the BS in the MBH (P < 0.01) and POA (P < 0.01). Levels of KP in tissue adjacent to the POA and MBH were much lower, and neither concentration nor content of KP differed between the BS and NBS. Kisspeptin was also detected in the cortex, brain stem, and cerebellum, but concentrations were not affected by season. In addition, concentration and content of GnRH in the POA, AHA, MBH, and ME were similar between seasons., Conclusion: Our RIA results indicate that in addition to the MBH, the POA and AHA appear to be involved in the seasonal negative feedback of estradiol on KP expression., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Wildlife and humans are increasingly competing for resources worldwide, and a diverse, innovative, and effective set of management tools is needed. Controlling abundance of wildlife species that are simultaneously protected, abundant, competitive for resources, and in conflict with some stakeholders but beloved by others, is a daunting challenge. Free-ranging horses (Equus caballus) present such a conundrum and managers struggle for effective tools for regulating their abundance. Controlling reproduction of female horses presents a potential alternative. During 2009-2017, we determined the long-term effectiveness of GnRH vaccine (GonaCon-Equine) both as a single immunization and subsequent reimmunization on reproduction and side effects in free-ranging horses. At a scheduled management roundup in 2009, we randomly assigned 57 adult mares to either a GonaCon-Equine treatment group (n = 29) or a saline control group (n = 28). In a second roundup in 2013, we administered a booster vaccination to these same mares. We used annual ground observations to estimate foaling proportions, social behaviors, body condition, and injection site reactions. We found this vaccine to be safe for pregnant females and neonates, with no overt deleterious behavioral side effects during the breeding season. The proportion of treated mares that foaled following a single vaccination was lower than that for control mares for the second (P = 0.03) and third (P = 0.08) post-treatment foaling seasons but was similar (P = 0.67) to untreated mares for the fourth season, demonstrating reversibility of the primary vaccine treatment. After two vaccinations, however, the proportion of females giving birth was lower (P <0.001) than that for control mares for three consecutive years and ranged from 0.0-0.16. The only detectable adverse side effect of vaccination was intramuscular swelling at the vaccination site. Regardless of vaccine treatment (primary/secondary), approximately 62% (34/55) of immunized mares revealed a visible reaction at the vaccine injection site. However, none of these mares displayed any evidence of lameness, altered gait or abnormal range of movement throughout the 8 years they were observed in this study. Our research suggests that practical application of this vaccine in feral horses will require an initial inoculation that may provide only modest suppression of fertility followed by reimmunization that together could result in greater reduction in population growth rates over time., Competing Interests: The authors have declared that no competing interests exist.

In previous work, an EP2 prostanoid receptor (EP2R) agonist in vivo increased mRNA expression of luteal LH receptors (LHR), unoccupied and occupied luteal; LHR, and circulating progesterone, while an EP3R or FPR agonist decreased; mRNA expression of luteal LHR, unoccupied and occupied luteal LHR, and; circulating progesterone. An EP4R and lysophosphatidic acid (LPA) LPA2R and LPA3R agonists were reported to inhibit luteal function and sirtuins have been proposed to increase prostaglandin synthesis. The objectives were to determine; whether an EP4R, LPA2R, or LPA3R agonist affect ovine luteal function in vivo or; in vitro. In addition, whether sirtuin (SIRT)-1, 2, or 3; LPA2R or LPA3R; or EP1R, EP2R, EP3R, or EP4R agonists affect caruncular endometrial PGF 2α or PGE (PGE1+PGE2) secretion in vitro. Day-10 nonpregnant ewes received a single injection of Vehicle (N = 5); an LPA2R (N = 5); LPA3R (N = 6); or EP4R (N = 5) agonist given into the interstitial tissue of the ovarian vascular pedicle adjacent to the luteal-containing ovary to determine effects on circulating progesterone, mRNA expression of luteal LHR, and luteal unoccupied and occupied LHR. In addition, agonists for LPA2R, LPA3R, EP1R, EP2R, EP3R, or EP4R or SIRT-1, SIRT-2, or SIRT-3 activators were incubated with caruncular endometrial slices in vitro to determine their effect on caruncular endometrial PGF 2α , or PGE secretion. LPA2R, LPA3R, or an EP4R agonist in vivo did not affect (P ≥ 0.05) luteal weight, circulating progesterone, or occupied luteal LHR. However, an LPA2R or EP4R agonist, but; not LPA3R agonist, in vivo increased (P ≤ 0.05) mRNA expression of luteal LHR. An; LPA2R, LPA3R, or EP4R agonist increased (P ≤ 0.05) luteal unoccupied LHR, but; not occupied LHR. An LPA2R, LPA3R, or an EP4R agonist did not affect (P ≥ 0.05); luteal progesterone secretion in vitro. An LPA2R or LPA3R agonist did not affect (P ≥ 0.05) luteal PGF 2α , or PGE secretion in vitro. However, an EP4R agonist tended to decrease (P < 0.066) luteal PGF 2α secretion and increased (P ≤ 0.05) luteal PGE; secretion in vitro. EP1R, EP2R, EP3R, or an EP4R agonist did not affect (P ≥ 0.05); caruncular endometrial PGF 2α secretion in vitro. However, EP1R, EP3R, or an EP4R agonist increased caruncular endometrial PGE secretion in vitro, while two different EP2R agonists did not affect (P ≥ 0.05) caruncular endometrial PGE; secretion. A SIRT-1 activator, but not SIRT-2 or SIRT-3 activators, increased (P ≤ 0.05) caruncular endometrial PGE secretion, while sirtuin 1, 2, or 3 activators did not affect (P ≥ 0.05) caruncular endometrial PGF 2α secretion. In conclusion, receptors for EP4, LPA2, and LPA3 do not appear to be involved; in luteolysis, but EP4R and LPA2R might participate in preventing luteolysis by maintaining luteal mRNA expression for LHR and preventing loss of unoccupied luteal LHR. In addition, SIRT-1, EP1R, EP3R, and EP4R might be involved in; regulating caruncular endometrial PGE secretion, but not PGF 2α secretion., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The antiviral activity of interferon (IFN) increases in uterine vein serum (UVS) during early pregnancy in sheep. This antiviral activity in UVS collected on Day 15 of pregnancy is blocked by anti-IFN-tau (anti-IFNT) antibodies. Conceptus-derived IFNT was hypothesized to induce IFN-stimulated gene (ISG) expression in endometrium and extrauterine tissues during pregnancy. To test this hypothesis, blood was collected from ewes on Days 12-16 of the estrous cycle or pregnancy. Serum progesterone was >1.7 ng/ml in pregnant (P) and nonpregnant (NP) ewes until Day 13, then declined to <0.6 ng/ml by Day 15 in NP ewes. A validated IFNT radioimmunoassay detected IFNT in uterine flushings (UFs) on Days 13-16 and in UVS on Days 15-16 of pregnancy. IFNT detection in UF correlated with paracrine induction of ISGs in the endometrium and occurred prior to the inhibition of estrogen receptor 1 and oxytocin receptor expression in uterine epithelia on Day 14 of pregnancy. Induction of ISG mRNAs in corpus luteum (CL) and liver tissue occurred by Day 14 and in peripheral blood mononuclear cells by Day 15 in P ewes. Expression of mRNAs for IFN signal transducers and ISGs were greater in the CL of P than that of NP ewes on Day 14. It is concluded that: 1) paracrine actions of IFNT coincide with detection of IFNT in UF; 2) endocrine action of IFNT ensues through induction of ISGs in peripheral tissues; and 3) IFNT can be detected in UVS, but not until Days 15-16 of pregnancy, which may be limited by the sensitivity of the IFNT radioimmunoassay., (© 2015 by the Society for the Study of Reproduction, Inc.)

Previously, it was reported that intraluteal implants containing prostaglandin E1 or E2 (PGE1 and PGE2) in Angus or Brahman cows prevented luteolysis by preventing loss of mRNA expression for luteal LH receptors and luteal unoccupied and occupied LH receptors. In addition, intraluteal implants containing PGE1 or PGE2 upregulated mRNA expression for FP prostanoid receptors and downregulated mRNA expression for EP2 and EP4 prostanoid receptors. Luteal weight during the estrous cycle of Brahman cows was reported to be lesser than that of Angus cows but not during pregnancy. The objective of this experiment was to determine whether intraluteal implants containing PGE1 or PGE2 alter vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), angiopoietin-1 (ANG-1), and angiopoietin-2 (ANG-2) protein in Brahman or Angus cows. On Day 13 of the estrous cycle, Angus cows received no intraluteal implant and corpora lutea were retrieved, or Angus and Brahman cows received intraluteal silastic implants containing vehicle, PGE1, or PGE2 on Day 13 and corpora lutea were retrieved on Day 19. Corpora lutea slices were analyzed for VEGF, FGF-2, ANG-1, and ANG-2 angiogenic proteins via Western blot. Day-13 Angus cow luteal tissue served as preluteolytic controls. Data for VEGF were not affected (P > 0.05) by day, breed, or treatment. PGE1 or PGE2 increased (P < 0.05) FGF-2 in luteal tissue of Angus cows compared with Day-13 and Day-19 Angus controls but decreased (P < 0.05) FGF-2 in luteal tissue of Brahman cows when compared w Day-13 or Day-19 Angus controls. There was no effect (P > 0.05) of PGE1 or PGE2 on ANG-1 in Angus luteal tissue when compared with Day-13 or Day-19 controls, but ANG-1 was decreased (P < 0.05) by PGE1 or PGE2 in Brahman cows when compared with Day-19 Brahman controls. ANG-2 was increased (P < 0.05) on Day 19 in Angus Vehicle controls when compared with Day-13 Angus controls, which was prevented (P < 0.05) by PGE1 but not by PGE2 in Angus cows. There was no effect (P > 0.05) of PGE1 or PGE2 on ANG-2 in Brahman cows. PGE1 or PGE2 may alter cow luteal FGF-2, ANG-1, or ANG-2 but not VEGF to prevent luteolysis; however, species or breed differences may exist., (Published by Elsevier Inc.)

The objective of this study was to determine whether PGE1 or PGE2 prevents a premature luteolysis when oxytocin is given on Days 1 to 6 of the ovine estrous cycle. Oxytocin given into the jugular vein every 8 hours on Days 1 to 6 postestrus in ewes decreased (P ≤ 0.05) luteal weights on Day 8 postestrus. Plasma progesterone differed (P ≤ 0.05) among the treatment groups; toward the end of the experimental period, concentrations of circulating progesterone in the oxytocin-only treatment group decreased (P ≤ 0.05) when compared with the other treatment groups. Plasma progesterone concentrations in ewes receiving PGE1 or PGE1 + oxytocin were greater (P ≤ 0.05) than in vehicle controls or in ewes receiving PGE2 or PGE2 + oxytocin and was greater (P ≤ 0.05) in all treatment groups receiving PGE1 or PGE2 than in ewes treated only with oxytocin. Chronic intrauterine treatment with PGE1 or PGE2 also prevented (P ≤ 0.05) oxytocin decreases in luteal unoccupied and occupied LH receptors on Day 8 postestrus. Oxytocin given alone on Days 1 to 6 postestrus in ewes advanced (P ≤ 0.05) increases in PGF2α in inferior vena cava or uterine venous blood. PGE1 or PGE2 given alone did not affect (P ≥ 0.05) concentrations of PGF2α in inferior vena cava and uterine venous blood when compared with vehicle controls or oxytocin-induced PGF2α increases (P ≤ 0.05) in inferior vena cava or uterine venous blood. We concluded that PGE1 or PGE2 prevented oxytocin-induced premature luteolysis by preventing a loss of luteal unoccupied and occupied LH receptors., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The objective of this study was to determine whether prostaglandin E1 (PGE1) or prostaglandin E2 (PGE2) prevents premature luteolysis in ewes when progesterone is given during the first 6 days of the estrous cycle. Progesterone (3 mg in oil, im) given twice daily from Days 1 to 6 (estrus = Day 0) in ewes decreased (P < 0.05) luteal weights on Day 10 postestrus. Plasma progesterone concentrations differed (P < 0.05) among the treatment groups; toward the end of the experimental period, concentrations in jugular venous blood decreased (P < 0.05) compared with the other treatment groups. Plasma progesterone concentrations in ewes receiving PGE1 or PGE1 + progesterone were greater (P < 0.05) than in vehicle controls or in ewes receiving PGE2 or PGE2 or PGE2 + progesterone. Chronic intrauterine treatment with PGE1 or PGE2 prevented (P < 0.05) decreases in plasma progesterone concentrations, luteal weights, and the proportion of luteal unoccupied and occupied LH receptors on Day 10 postestrus in ewes given exogenous progesterone, but did not affect (P > 0.05) concentrations of PGF2α in inferior vena cava blood. Progesterone given on Days 1 to 6 in ewes advanced (P < 0.05) increases in PGF2α in inferior vena cava blood. We concluded that PGE1 or PGE2 prevented progesterone-induced premature luteolysis by suppressing loss of luteal LH receptors (both unoccupied and occupied)., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Kisspeptides (KiSS) are a recently discovered family of neuropeptides with a central role in regulating the onset of reproductive function in all animals studied to date. We have established biological and physiological evidence for KiSS signaling in the mare. The objective of the current study was to evaluate the physiological and behavioral responses of mares repeatedly given the equine-specific kisspeptpin decapeptide (eKp-10, YRWNSFGLRY-NH(2)) in an effort to shorten the interovulatory period. Administration of eKp-10 (0.5 mg iv every 4 h) to mares beginning on Day 16 postovulation (Group 2) or in estrus (Group 3) did not shorten the mean ± SEM interovulatory interval compared with untreated (Group 1) controls (21.9 ± 1.2, 22 ± 1.2, and 21.5 ± 1.5 days in Groups 1 to 3, respectively; N = 6 per group), nor was there a significant difference in follicle diameter before ovulation among groups, nor number of days treated with eKp-10 for Groups 2 and 3. Mean daily concentrations of FSH, the preovulatory LH surge (timing, mean, and peak concentrations), and mean progesterone concentrations from the newly formed CL were not significantly different among groups. The initiation of treatment was negatively correlated with sexual receptivity (scored 0 to 5: no interest to strong interest) and serum estradiol concentrations, indicating that eKp-10 can significantly disrupt normal sexual receptivity in the estrous mare. This effect on sexual receptivity was short-lived (< 72 h) and the overall change in sexual receptivity score was not significantly different between Groups 2 and 3 (-1.2 ± 0.5 and -1.4 ± 0.4, respectively). However, the day of the cycle that treatment was initiated significant affected the decline in sexual receptivity score, such that the later in the cycle that treatment was initiated, the greater the estimated decrease in sexual receptivity. In conclusion, the linear hypothalamic-pituitary mechanism for KiSS described in other species was not appropriate for the horse and administration of eKp-10 in the seasonally estrous mare may have been outside of the hormone's normal physiological context., (Copyright © 2012. Published by Elsevier Inc.)

Gonadotropin-releasing hormone is intermittently released from the hypothalamus in consistent patterns from before birth to final maturation of the hypothalamic-pituitary-gonadal axis at puberty. Disruption of this signaling via GnRH vaccination during the neonatal period can alter reproduction at maturity. The objective of this study was to investigate the long-term effects of GnRH-antibody exposure on reproductive maturation and function in elk calves passively exposed to high concentrations of GnRH antibodies immediately after birth. Fifteen elk calves (eight males and seven females) born to females treated with GnRH vaccine or sham vaccine during midgestation were divided into two groups based on the concentration of serum GnRH antibodies measured during the neonatal period. Those with robust (>15 pmol (125)I-GnRH bound per mL of serum) titers (N = 10; four females and six males) were designated as the exposed group, whereas those with undetectable titers (N = 5; three females and two males) were the unexposed group. Onset of puberty, reproductive development, and endocrine function in antibody-exposed and unexposed male and female elk calves were compared. Neonatal exposure to high concentrations of GnRH antibodies had no effect on body weight (P = 0.968), endocrine profiles (P > 0.05), or gametogenesis in either sex. Likewise, there were no differences between groups in gross or histologic structure of the hypothalamus, pituitary, testes, or ovaries. Pituitary stimulation with a GnRH analog before the second potential reproductive season induced substantial LH secretion in all experimental elk. All females became pregnant during their second reproductive season and all males exhibited similar mature secondary sexual characteristics. There were no differences between exposure groups in hypothalamic GnRH content (P = 0.979), pituitary gonadotropin content (P > 0.05) or gonadal structure. We concluded that suppressing GnRH signaling through immunoneutralization during the neonatal period likely does not alter long-term reproductive function in this species., (Published by Elsevier Inc.)

The current dogma is that the differential regulation of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) synthesis and secretion is modulated by gonadotropin-releasing hormone (GnRH) pulse frequency and by changes in inhibins, activins, and follistatins both at the pituitary and at the peripheral level. To date no studies have looked at the overlapping function of these regulators in a combined setting. We tested the hypothesis that changes in GnRH pulse frequency alter the relative abundance of these regulators at the pituitary and peripheral levels in a manner consistent with changes in pituitary and circulating concentrations of FSH; that is, an increase in FSH will be accompanied by increased stimulatory input (activin) and/or reduced follistatin and inhibin. Ovariectomized ewes were subjected to a combination hypothalamic pituitary disconnection (HPD)-hypophyseal portal blood collection procedure. Hypophyseal portal and jugular blood samples were collected for a 6-h period from non-HPD ewes, HPD ewes, or HPD ewes administered GnRH hourly or every 3 h for 4 days. In the absence of endogenous hypothalamic and ovarian hormones that regulate gonadotropin secretion, 3-hourly pulses of GnRH increased pituitary content of FSH more than hourly GnRH, although these differences were not evident in the peripheral circulation. The results failed to support the hypothesis in that the preferential increase of pituitary content of FSH by the lower GnRH pulse frequency could be explained by changes in the pituitary content of inhibin A, follistatin, or activin B. Perhaps the effects of GnRH pulse frequency on FSH is due to changes in the balance of free versus bound amounts of these FSH regulatory proteins or to the involvement of other regulators not monitored in this study.

Previously, it was reported that chronic intra-uterine infusion of PGE(1) or PGE(2) every 4h inhibited luteolysis in ewes by altering luteal mRNA for luteinizing hormone (LH) receptors and unoccupied and occupied luteal LH receptors. However, estradiol-17β or PGE(2) given intra-uterine every 8h did not inhibit luteolysis in cows, but infusion of estradiol+PGE(2) inhibited luteolysis. In contrast, intra-luteal implants containing PGE(1) or PGE(2) in Angus or Brahman cows also inhibited the decline in circulating progesterone, mRNA for LH receptors, and loss of unoccupied and occupied receptors for LH to prevent luteolysis. The objective of this experiment was to determine how intra-luteal implants of PGE(1) or PGE(2) alter mRNA for prostanoid receptors and how this could influence luteolysis in Brahman or Angus cows. On day-13 Angus cows received no intra-luteal implant and corpora lutea were retrieved or Angus and Brahman cows received intra-luteal silastic implants containing Vehicle, PGE(1), or PGE(2) and corpora lutea were retrieved on day-19. Corpora lutea slices were analyzed for mRNA for prostanoid receptors (FP, EP1, EP2, EP3 (A-D), EP3A, EP3B, EP3C, EP3D, and EP4) by RT-PCR. Day-13 Angus cow luteal tissue served as pre-luteolytic controls. mRNA for FP receptors decreased in day-19 Vehicle controls compared to day-13 Vehicle controls regardless of breed. PGE(1) and PGE(2) up-regulated FP gene expression on day-19 compared to day-19 Vehicle controls regardless of breed. EP1 mRNA was not altered by any treatment. PGE(1) and PGE(2) down-regulated EP2 and EP4 mRNA compared to day-19 Vehicle controls regardless of breed. PGE(1) or PGE(2) up-regulated mRNA EP3B receptor subtype compared to day-19 Vehicle control cows regardless of breed. The similarities in relative gene expression profiles induced by PGE(1) and PGE(2) support their agonistic effects. We conclude that both PGE(1) and PGE(2) may prevent luteolysis by altering expression of mRNA for prostanoid receptors, which is correlated with changes in luteal mRNA for LH receptors reported previously in these same cows to prevent luteolysis., (Published by Elsevier Inc.)

Fertility control is a potential method for managing overabundant wildlife populations; however, current technology is limited by duration of treatment efficacy and unacceptable side effects. The objective of this study was to determine the efficacy of a single immunization with gonadotropin-releasing hormone (GnRH) vaccine to suppress reproductive function in pregnant female elk and to evaluate potential behavioral and pathological side effects of treatment. Eighteen captive adult female elk were randomly allocated to one of two experimental groups. Ten females were administered a conjugated and adjuvanted GnRH vaccine intramuscularly, and eight elk received an adjuvant sham vaccine without conjugated GnRH. We compared success of existing pregnancy, neonatal survival, subsequent fertility, reproductive behavior rates, and side effects of treatment between January 2006 and January 2010. The GnRH vaccination did not affect existing pregnancy or calf survival during the year that it was applied; however, it reduced the proportion of pregnant females for 3 yr. Male precopulatory behavior rates exhibited toward GnRH-vaccinated females tended to be greater than those directed at sham-vaccinated females during the second half of the breeding season, when GnRH vaccinates continued to be proceptive. Strong immune and inflammatory responses, including robust GnRH antibody concentrations in GnRH vaccinates, and sterile pyogranulomatous injection site abscesses in both groups, were consistent with vaccination. In conclusion, this GnRH vaccine resulted in prolonged, albeit reversible, impairment of fertility, and is associated with extended reproductive behaviors and partial suppression of hypothalamic-pituitary-gonadal axis function in captive female elk.

Estradiol-17beta (E2) is the major regulator of GnRH receptor (GnRHR) gene expression and number during the periovulatory period; however, the mechanisms underlying E2 regulation of the GNRHR gene remain undefined. Herein, we find that E2 conjugated to BSA (E2-BSA) mimics the stimulatory effect of E2 on GnRH binding in primary cultures of ovine pituitary cells. The time course for maximal GnRH analog binding was similar for both E2 and E2-BSA. The ability of E2 and E2-BSA to increase GnRH analog binding was blocked by the estrogen receptor (ER) antagonist ICI 182,780. Also, increased GnRH analog binding in response to E2 and the selective ESR1 agonist propylpyrazole triol was blocked by expression of a dominant-negative form of ESR1 (L540Q). Thus, membrane-associated ESR1 is the likely candidate for mediating E2 activation of the GNRHR gene. As cAMP response element binding protein (CREB) is an established target for E2 activation in gonadotrophs, we next explored a potential role for this protein as an intracellular mediator of the E2 signal. Consistent with this possibility, adenoviral-mediated expression of a dominant-negative form of CREB (A-CREB) completely abolished the ability of E2 to increase GnRH analog binding in primary cultures of ovine pituitary cells. Finally, the presence of membrane-associated E2 binding sites on ovine pituitary cells was demonstrated using a fluorescein isothiocyanate conjugate of E2-BSA. We suggest that E2 regulation of GnRHR number during the preovulatory period reflects a membrane site of action and may proceed through a nonclassical signaling mechanism, specifically a CREB-dependent pathway.

Previously, it was reported that chronic intra-uterine infusion of PGE(1) or PGE(2) every four hours inhibited luteolysis in ewes. However, estradiol-17β or PGE(2) given intra-uterine every 8h did not inhibit luteolysis in heifers, but infusion of estradiol+PGE(2) inhibited luteolysis in heifers. The objective of this experiment was to determine whether and how intra-luteal implants containing PGE(1) or PGE(2) prevent luteolysis in Angus or Brahman cows. On day-13 post-estrus, Angus cows received no intra-luteal implant and corpora lutea were retrieved or Angus and Brahman cows received intra-luteal silastic implants containing Vehicle, PGE(1), or PGE(2) and corpora lutea were retrieved on day-19. Coccygeal blood was collected daily for analysis for progesterone. Breed did not influence the effect of PGE(1) or PGE(2) on luteal mRNA for LH receptors or unoccupied or occupied luteal LH receptors did not differ (P>0.05) so the data were pooled. Luteal weights of Vehicle-treated Angus or Brahman cows from days-13-19 were lower (P<0.05) than those treated with intra-luteal implants containing PGE(1) or PGE(2). Day-13 Angus luteal weights were heavier (P<0.05) than Vehicle-treated Angus cows on day-19 and luteal weights of day-13 corpora lutea were similar (P>0.05) to Angus cows on day-19 treated with intra-luteal implants containing PGE(1) or PGE(2). Profiles of circulating progesterone in Angus or Brahman cows treated with intra-luteal implants containing PGE(1) or PGE(2) differed (P<0.05) from controls, but profiles of progesterone did not differ (P>0.05) between breeds or between cows treated with intra-luteal implants containing PGE(1) or PGE(2). Intra-luteal implants containing PGE(1) or PGE(2) prevented (P<0.05) loss of luteal mRNA for LH receptors and unoccupied or occupied receptors for LH compared to controls. It is concluded that PGE(1) or PGE(2) alone delays luteolysis regardless of breed. We also conclude that either PGE(1) or PGE(2) prevented luteolysis in cows by up-regulating expression of mRNA for LH receptors and by preventing loss of unoccupied and occupied LH receptors in luteal tissue., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Thirty to forty percent of ruminant pregnancies are lost during the first third of gestation due to inadequate progesterone secretion. During the estrous cycle, luteinizing hormone (LH) regulates progesterone secretion by small luteal cells (SLC). Loss of luteal progesterone secretion during the estrous cycle is increased via uterine secretion of prostaglandin F(2α) (PGF(2α)) starting on days 12-13 post-estrus in ewes with up to 4-6 pulses per day. Prostaglandin F(2α) is synthesized from arachidonic acid, which is released from phospholipids by phospholipase A2. Endocannabinoids are also derived from phospholipids and are associated with infertility. Endocannabinoid-induced infertility has been postulated to occur primarily via negative effects on implantation. Cannabinoid (CB) type 1 (CB1) or type 2 (CB2) receptor agonists and an inhibitor of the enzyme fatty acid amide hydrolase, which catabolizes endocannabinoids, decreased luteal progesterone, prostaglandin E (PGE), and prostaglandin F(2α) (PGF(2α)) secretion by the bovine corpus luteum in vitro by 30 percent. The objective of the experiment described herein was to determine whether CB1 or CB2 receptor agonists given in vivo affect circulating progesterone, luteal weights, luteal mRNA for LH receptors, and luteal occupied and unoccupied LH receptors during the estrous cycle of ewes. Treatments were: Vehicle, Methanandamide (CB1 agonist; METH), or 1-(4-chlorobenzoyl)-5-methoxy-1H-indole-3-acetic acid morpholineamide (CB2 agonist; IMMA). Ewes received randomized treatments on day 10 post-estrus. A single treatment (500 μg; N=5/treatment group) in a volume of 1 ml was given into the interstitial tissue of the ovarian vascular pedicle adjacent to the luteal-containing ovary. Jugular venous blood was collected at 0 h and every 6-48 h for the analysis of progesterone by radioimmunoassay (RIA). Corpora lutea were collected at 48 h, weighed, bisected, and frozen in liquid nitrogen until analysis of unoccupied and occupied LH receptors and mRNA for LH receptors. Profiles of jugular venous progesterone, luteal weights, luteal mRNA for LH receptors, and luteal occupied and unoccupied LH receptors were decreased (P≤0.05) by CB1 or CB2 receptor agonists when compared to Vehicle controls. Progesterone in 80 percent of CB1 or CB2 receptor agonist-treated ewes was decreased (P≤0.05) below 1 ng/ml by 48 h post-treatment. It is concluded that the stimulation of either CB1 or CB2 receptors in vivo affected negatively luteal progesterone secretion by decreasing luteal mRNA for LH receptors and also decreasing occupied and unoccupied receptors for LH on luteal membranes. The corpus luteum may be an important site for endocannabinoids to decrease fertility as well as negatively affect implantation, since progesterone is required for implantation., (Published by Elsevier Inc.)

Loss of progesterone secretion at the end of the estrous cycle is via uterine PGF(2alpha) secretion; however, uterine PGF(2alpha) is not decreased during early pregnancy in ewes to prevent luteolysis. Instead the embryo imparts resistance to PGF(2alpha)-induced luteolysis, which is via the 2-fold increase in prostaglandins E(1) and E(2) (PGE(1), PGE(2); PGE) in the endometrium during early pregnancy. Chronic intrauterine infusion of PGE(1) or PGE(2) prevents spontaneous or an estradiol-17beta, IUD, or PGF(2alpha)-induced luteolysis. Four PGE receptor subtypes (EP(1), EP(2), EP(3), and EP(4)) and an FP receptor specific for PGF(2alpha) have been identified. The objective of this experiment was to determine the effects of EP(1), EP(2), EP(3), or FP receptor agonists in vivo on luteal mRNA for LH receptors, occupied and unoccupied LH receptors, and circulating progesterone in ewes. Ewes received a single treatment of 17-phenyl-tri-Nor-PGE(2) (EP(1), EP(3)), butaprost (EP(2)), 19-(R)-OH-PGE(2) (EP(2)), sulprostone (EP(1), EP(3)), or PGF(2alpha) (FP) receptor agonists into the interstitial tissue of the ovarian vascular pedicle adjacent to the luteal-containing ovary. 17-Phenlyl-tri-Nor-PGE(2) had no effect (P> or =0.05) on any parameter analyzed. Butaprost and 19-(R)-OH-PGE(2) increased (P< or =0.05) mRNA for LH receptors, occupied and unoccupied LH receptors, and circulating progesterone. Both sulprostone and PGF(2alpha) decreased (P< or =0.05) mRNA for LH receptors, occupied and unoccupied LH receptors, and circulating progesterone. It is concluded that both EP(3) and FP receptors may be involved in luteolysis. In addition, EP(2) receptors may mediate prevention of luteolysis via regulation of luteal mRNA for LH receptors to prevent loss of occupied and unoccupied LH receptors and therefore to sustaining luteal function., (Published by Elsevier Inc.)

Background: We demonstrated that E2 conjugated to BSA (E2BSA) induces a rapid membrane-initiated inhibition of LH secretion followed hours later by a slight increase in LH secretion. Whether these actions of E2BSA are restricted to the pituitary gland and whether the membrane-initiated pathway of E2BSA contributes to the up-regulation of the number of GnRH receptors during the positive feedback effect of E2 were evaluated here. We have shown that the suppression of LH secretion induced by E2 and E2BSA is the result of a decreased responsiveness of the pituitary gland to GnRH. In this study we further tested the ability of E2BSA to decrease the responsiveness of the pituitary gland to GnRH under the paradigm of the preovulatory surge of LH induced by E2., Methods: For the first experiment GnRH and LH secretions were determined in samples of pituitary portal and jugular blood, respectively, in ewes treated with 12 mg E2BSA. In the second experiment, the number of GnRH receptors was quantified in ewes 12 h after administration of 25 micrograms E2 (the expected time for the increase in the number of GnRH receptors and the positive feedback effect of E2 in LH secretion) or 12 mg E2BSA. In the third experiment, the preovulatory-like surge of LH was characterized in ewes injected with 25 micrograms E2 alone or followed 8 h later (before the beginning of the LH surge) with 60 mg E2BSA., Results: a) the decrease in LH secretion induced by E2BSA was not accompanied by changes in the pulsatile pattern of GnRH, b) E2BSA increased the number of GnRH receptors, and c) the presence of E2BSA in E2-treated ewes delayed the onset, reduced the length, and decreased the amount of LH released during the preovulatory surge of LH., Conclusions: a) the rapid suppression of LH secretion induced by E2BSA is mediated only via a direct action on the pituitary gland, b) E2 acting via a membrane-initiated pathway contributes to increase the number of GnRH receptors and, c) administration of E2BSA near the beginning of the pre-ovulatory surge of LH delays and reduces the magnitude of the surge.

Loss of luteal progesterone secretion at the end of the ovine estrous cycle is via uterine PGF(2)alpha secretion. However, uterine PGF(2)alpha secretion is not decreased during early pregnancy in ewes. Instead, the embryo imparts a resistance to PGF(2)alpha. Prostaglandins E (PGE; PGE(1)+PGE(2)) are increased in endometrium and uterine venous blood during early pregnancy in ewes to prevent luteolysis. Chronic intrauterine infusion of PGE(1) or PGE(2) prevents spontaneous or IUD, estradiol-17beta, or PGF(2)alpha-induced premature luteolysis in nonbred ewes. The objective was to determine whether chronic intrauterine infusion of PGE(1) or PGE(2) affected mRNA for LH receptors, occupied and unoccupied receptors for LH in luteal and caruncular endometrium, and luteal function. Ewes received Vehicle, PGE(1), or PGE(2) every 4h from days 10 to 16 of the estrous cycle via a cathether installed in the uterine lumen ipsilateral to the luteal-containing ovary. Jugular venous blood was collected daily for analysis of progesterone and uterine venous blood was collected on day-16 for analysis of PGF(2)alpha and PGE. Corpora lutea and caruncular endometrium were collected from day-10 preluteolytic control ewes and day-16 ewes treated with Vehicle, PGE(1) or PGE(2) for analysis of the mRNA for LH receptors and occupied and unoccupied receptors for LH. Luteal weights on day-16 in ewes treated with PGE(1) or PGE(2) and day-10 control ewes were similar (P>or=0.05), but were greater (PPGE(2)>Vehicle-treated ewes. Concentrations of PGF(2)alpha and PGE in uterine venous plasma on day-16 were similar (P>or=0.05) in the three treatment groups. Luteal mRNA for LH receptors and unoccupied and occupied LH receptors were similar (P>or=0.05) in day-10 control ewes and day-16 ewes treated with PGE(2) and were lower (P

Background: Classically, progesterone has been thought to act only through the well-known genomic pathway involving hormone binding to nuclear receptors and subsequent modulation of gene expression. However, there is increasing evidence for rapid, non-genomic effects of progesterone in a variety of mammalian tissues and it is possible that a membrane PR (mPR) is causing these events. We recently isolated and characterized an ovine mPR referred to as mPR-alpha, distinct from the nuclear PR. Based on predicted structural analysis, the ovine mPR-alpha possesses seven transmembrane domains typical of G protein-coupled receptors. Despite the homology to other reported mPRs, information pertaining to the steroid binding characteristics of the ovine mPR-alpha was lacking. Additionally, the ovine mPR-alpha transcript has been identified in the hypothalamus, pituitary, uterus, ovary and corpus luteum, yet changes in expression of the ovine mPR-alpha in these tissues were not known. Consequently, the purpose of this work was to determine the steroid binding characteristics of the ovine mPR-alpha and to investigate possible changes in expression of the ovine mPR-alpha in reproductive tissues throughout the estrous cycle., Methods: Binding studies were performed using crude membrane fractions from CHO cells expressing the mPR-alpha. Using quantitative Real-time PCR we determined the expression pattern of mRNA for the ovine mPR-alpha during the ovine estrous cycle in tissues known to express the mPR-alpha. Jugular blood samples were also collected and analyzed for serum concentrations of P4 to ensure ewes were at the appropriate stage of their cycle., Results: Only progesterone, 20alpha-hydroxyprogesterone and 17alpha-hydroxyprogesterone were able to displace binding of 3H-P4 (P < 0.001) to membrane fractions from CHO cells expressing ovine mPR-alpha. The average B-max and Kd values for three separate experiments were 624 +/- 119 fmol/micro gram protein and 122 +/- 50 nM, respectively. Significant changes in expression of mRNA for the mPR-alpha during the estrous cycle were noted in the corpus luteum and uterus., Conclusion: The mPR-alpha specifically binds progestins and its expression was correlated to progesterone secretion during the ovine estrous cycle. Results from the present studies suggest that mPR-alpha may have an important physiological role during the ovine estrous cycle.

Stress-like elevations in plasma glucocorticoids rapidly inhibit pulsatile LH secretion in ovariectomized sheep by reducing pituitary responsiveness to GnRH. This effect can be blocked by a nonspecific antagonist of the type II glucocorticoid receptor (GR) RU486. A series of experiments was conducted to strengthen the evidence for a mediatory role of the type II GR and to investigate the neuroendocrine site and cellular mechanism underlying this inhibitory effect of cortisol. First, we demonstrated that a specific agonist of the type II GR, dexamethasone, mimics the suppressive action of cortisol on pituitary responsiveness to GnRH pulses in ovariectomized ewes. This effect, which became evident within 30 min, documents mediation via the type II GR. We next determined that exposure of cultured ovine pituitary cells to cortisol reduced the LH response to pulse-like delivery of GnRH by 50% within 30 min, indicating a pituitary site of action. Finally, we tested the hypothesis that suppression of pituitary responsiveness to GnRH in ovariectomized ewes is due to reduced tissue concentrations of GnRH receptor. Although cortisol blunted the amplitude of GnRH-induced LH pulses within 1-2 h, the amount of GnRH receptor mRNA or protein was not affected over this time frame. Collectively, these observations provide evidence that cortisol acts via the type II GR within the pituitary gland to elicit a rapid decrease in responsiveness to GnRH, independent of changes in expression of the GnRH receptor.

The objectives of this study were: to determine if peripheral administration of the ovine C-terminal decapeptide (oKiSS-1) increases secretion of LH and FSH in ovariectomized (OVX) ewes; to evaluate the effect of ovarian steroid hormones on the predicted increase in gonadotropin secretion induced by oKiSS-1; to establish if the stimulatory effect of oKiSS-1 is due to a direct action on the hypothalamus or was mediated directly at the anterior pituitary gland. Intravenous injection of 3mg oKiSS-1 to OVX ewes (n=6) increased (P<0.01) basal and mean serum concentrations of LH. The amplitude and the area under the curve (AUC) of LH induced by oKiSS-1 was greater (P<0.01) than the highest pulse of LH detected before oKiSS-1. Pre-treatment with steroid hormones (1mg progesterone plus 50 microg estradiol-17beta for 3 days) in OVX ewes (n=6) reduced (P<0.1) the magnitude of the stimulatory effect of oKiSS-1 on LH secretion. In these animals, oKiSS-1 increased (P<0.1) basal and mean concentrations of LH, and the amplitude of the pulse of LH induced by oKiSS-1, but not the AUC (P>0.1). In our hands, oKiSS-1 failed to increase serum concentration of FSH at the dose used in this study. Administration of GnRH antiserum completely suppressed the secretory patterns of LH and prevented the increase in secretion of LH induced by oKiSS-1. We conclude that the rapid increase in secretion of LH induced by peripheral administration of oKiSS-1 is the result of a direct action on the hypothalamus.

The objectives of this study were to determine whether activation of estrogen receptor 1 (ESR1; also known as ERalpha), or estrogen receptor 2 (ESR2; also known as ERbeta), or both are required to: 1) acutely inhibit secretion of LH, 2) induce the preovulatory-like surge of LH, and 3) inhibit secretion of FSH in ovariectomized (OVX) ewes. OVX ewes (n = 6) were administered intramuscularly 25 micrograms estradiol (E2), 12 mg propylpyrazoletriol (PPT; a subtype-selective ESR1 agonist), 21 mg diaprylpropionitrile (DPN; a subtype-selective ESR2 agonist), or PPT + DPN. Like E2, administration of PPT, DPN, or combination of the two rapidly decreased (P < 0.05) secretion of LH. Each agonist induced a gradual, prolonged rise in secretion of LH after the initial inhibition, but neither agonist alone nor the combined agonists was able to induce a "normal" preovulatory-like surge of LH similar to that induced by E2. Compared with E2-treated ewes, the beginning of the increase in secretion of LH occurred earlier (P < 0.01) in DPN-treated ewes, later (P < 0.05) in PPT-treated ewes, and at a similar interval in ewes receiving the combined agonist treatment. Like E2, PPT decreased (P < 0.05) secretion of FSH, but the duration of suppression was much longer in PPT-treated ewes. DPN did not alter secretion of FSH in this study. Modulation of the number of GnRH receptors by PPT and DPN was examined in primary cultures of ovine pituitary cells. In our hands, both PPT and DPN increased the number of GnRH receptors, but the dose of DPN required to stimulate synthesis of GnRH receptors was 10 times higher than that of PPT. We conclude that in OVX ewes: 1) ESR1 and ESR2 mediate the negative feedback of E2 on secretion of LH at the level of the pituitary gland, 2) ESR1 and ESR2 do not synergize or antagonize the effects of each other; however, they do interact to synchronize the beginning of the stimulatory effect of E2 on secretion of LH, 3) ESR1 and ESR2 may mediate at least partially the positive feedback of E2 on LH secretion by increasing the number of GnRH receptors, and 4) only ESR1 appears to be involved in the negative feedback of E2 on secretion of FSH.

The experimental objectives were to evaluate the influence of supplemental high-linoleate safflower seeds on fatty acid concentrations in plasma, medial basal hypothalamus, uterine tissues, and serum 13,14-dihydro-15-keto PGF(2)alpha metabolite (PGFM) in primiparous beef cows during early lactation. Beginning 1 d postpartum, 18 primiparous, crossbred beef cows (411 +/- 24.3 kg of BW) were fed foxtail millet hay at 1.68% of BW (DM basis) and either a low-fat supplement (control: 63.7% cracked corn; 33.4% safflower seed meal; and 2.9% liquid molasses; DM basis) at 0.35% of BW (n = 9) or a supplement (linoleate) containing 95.3% cracked high-linoleate (79% 18:2n-6) safflower seeds and 4.7% liquid molasses (DM basis) at 0.23% of BW (n = 9). Diets were formulated to be isonitrogenous and isocaloric. The linoleate diet contained 5.4% of DMI as fat vs. 1.2% for control. Beginning 1 d postpartum, cattle were bled every 3 d for collection of serum and plasma. Cattle were slaughtered at 37 +/- 3 d postpartum for collection of the medial basal hypothalamus, myometrium, endometrium, caruncular tissue, intercaruncular tissue, and oviduct. Feeding linoleate increased (P = 0.001) plasma concentrations of 18:2n-6, 18:2cis-9 trans-11 and total unsaturated fatty acids; however, 18:1trans-11 did not differ (P = 0.19) between treatments. Concentrations of 20:5n-3 in the medial basal hypothalamus tended (P = 0.10) to be greater for cattle fed linoleate. Concentrations of fatty acids in the oviduct were greater (P < 0.05) than in other uterine tissues. Cows fed linoleate had greater (P = 0.05) concentrations of 18:3n-3 in the endometrium and less (P = 0.06) 18:2cis-9 trans-11 in the myometrium than cows fed the control. Supplemental fat increased (dietary treatment x day postpartum, P = 0.01) concentrations of PGFM in serum more in linoleate than control cows from d 3 to 9 postpartum. Lipid supplementation early in the postpartum period altered the fatty acid composition of medial basal hypothalamus, uterine tissue, and serum concentrations of PGFM. The most novel observation was that the oviduct appeared to be the most sensitive tissue to additional dietary linoleic acid, which could potentially influence fertility.

Experiments were conducted to further our understanding of the cellular and molecular mechanisms that regulate luteal function in ewes. Inhibition of protein kinase A (PKA) reduced (P < 0.05) secretion of progesterone from both small and large steroidogenic luteal cells. In addition, the relative phosphorylation state of steriodogenic acute regulatory protein (StAR) was more than twice as high (P < 0.05) in large vs small luteal cells. Large steroidogenic luteal cells appear to contain constitutively active PKA and increased concentrations of phosphorylated StAR which play a role in the increased basal rate of secretion of progesterone. To determine if intraluteal secretion of prostaglandin (PG) F2alpha was required for luteolysis, ewes on day 10 of the estrous cycle received intraluteal implants of a biodegradable polymer containing 0, 1 or 10 mg of indomethacin, to prevent intraluteal synthesis of PGF2alpha. On day 18, luteal weights in ewes receiving 1 mg of indomethacin were greater (P < 0.05) than controls and those receiving 10 mg were greater (P < 0.05) than either of the other two groups. Concentrations of progesterone in serum were also increased (P < 0.05) from days 13 to 16 of the estrous cycle in ewes receiving 10 mg of indomethacin. Although not required for decreased production of progesterone at the end of the cycle, intraluteal secretion of PGF2alpha appears to be required for normal luteolysis. To ascertain if oxytocin mediates the indirect effects of PGF2alpha on small luteal cells, the effects of 0, 0.1, 1 or 10 mM oxytocin on intracellular concentrations of calcium were quantified. There was a dose-dependent increase (P < 0.05) in the number of small luteal cells responding to oxytocin. Thus, oxytocin induces increased calcium levels and perhaps apoptotic cell death in small luteal cells. Concentrations of progesterone, similar to those present in corpora lutea (approximately 30 microg/g), prevented the increased intracellular concentrations of calcium (P < 0.05) stimulated by oxytocin in small cells. In large luteal cells the response to progesterone was variable. There was no consistent effect of high quantities of estradiol, testosterone or cortisol in either cell type. It was concluded that normal luteal concentrations of progesterone prevent the oxytocin and perhaps the PGF2alpha-induced increase in the number of small and large luteal cells which respond to these hormones with increased intracellular concentrations of calcium. In summary, large ovine luteal cells produce high basal levels of progesterone, at least in part, due to a constituitively active form of PKA and an enhanced phosphorylation state of StAR. During luteolysis PGF2alpha of uterine origin reduces the secretion of progesterone from the corpus luteum, but intraluteal production of PGF2alpha is required for normal luteolysis. Binding of PGF2alpha to receptors on large luteal cells stimulates the secretion of oxytocin which appears to activate PKC and may also inhibit steroidogenesis in small luteal cells. PGF2alpha also activates COX-2 in large luteal cells which leads to secretion of PGF2alpha. Once intraluteal concentrations of progesterone have decreased, oxytocin binding to its receptors on small luteal cells also results in increased levels of intracellular calcium and presumably apoptosis. Increased secretion of PGF2alpha from large luteal cells activates calcium channels which likely results in apoptotic death of this cell type.

Classically, progesterone has been thought to act only through the well-known genomic pathway involving hormone binding to nuclear receptors (nPR) and subsequent modulation of gene expression. However, there is increasing evidence for rapid, nongenomic effects of progesterone in a variety of tissues in mammals, and it seems likely that a membrane PR (mPR) is causing these events. The objective of this study was to isolate and characterize an ovine mPR distinct from the nPR. A cDNA clone was isolated from ovine genomic DNA by PCR. The ovine mPR is a 350-amino acid protein that, based on computer hydrophobicity analysis, possesses seven transmembrane domains and is distinct from the nPR. Message for the ovine mPR was detected in hypothalamus, pituitary, uterus, ovary, and corpus luteum by RT-PCR. In CHO cells that overexpressed a mPR-green fluorescent protein fusion protein, the ovine mPR was localized to the endoplasmic reticulum and not the plasma membrane. Specific binding of 3H-progesterone to membrane fractions was demonstrated in CHO cells that expressed the ovine mPR but not in nontransfected cells. Furthermore, progesterone and 17 alpha-hydroxy-progesterone stimulated intracellular Ca2+ mobilization in CHO cells that expressed ovine mPR in Ca2+-free medium (P < 0.05) but not in CHO cells transfected with empty vector. This rise in intracellular Ca2+ is believed to be from the endoplasmic reticulum as intracellular Ca2+ mobilization is absent when mPR transfected cells are first treated with thapsigargin to deplete Ca2+ stores from the endoplasmic reticulum. Isolation, identification, tissue distribution, cellular localization, steroid binding, and a functional response for a unique intracellular mPR in the sheep are presented.

The mammalian type I GNRH receptor (GNRHR) is unique among G protein-coupled receptors (GPCRs) because of the absence of an intracellular C-terminus. Previously, we have found that the murine GNRHR is constitutively localized to low-density membrane microdomains termed lipid rafts. As such, association of the GNRHR with lipid rafts may reflect both a loss (C-terminus) and a gain (raft association address) of structural characteristics. To address this, we fused either the full-length C-terminus from the nonraft-associated LH receptor (LHCGR; GNRHR-LF) or a truncated (t631) LHCGR C-terminus to the GNRHR. These chimeric receptors are trafficked to the plasma membrane, bind ligand, and display increased agonist-induced receptor internalization, but they do not partition into lipid rafts. Thus, a heterologous C-terminus from a nonraft-associated GPCR redirects localization of the GNRHR to nonraft domains. In contrast to the murine GNRHR, the catfish GNRHR (cfGNRHR) possesses an intracellular C-terminus. We found that the cfGNRHR was localized to lipid rafts and that the cfGNRHR C-terminus did not alter raft localization of the mammalian receptor. Consistent with placement in different lipid microenvironments within the plasma membrane, fluorescence recovery after photobleaching revealed different lateral diffusion phenotypes of the raft-associated GNRHR and cfGNRHR versus the nonraft-associated GNRHR-LF fusion protein. We conclude that whereas an intracellular C-terminus is capable of redirecting the GNRHR to nonraft compartments, this is not a generalized feature of GPCR C-terminal tails. Thus, constitutive raft localization of the GNRHR is not simply a result of the loss of an intracellular C-terminus.

Problem: Biological effectiveness of targeted cytotoxins is dependent on their stability, circulating half-life, receptor binding ability, and cytotoxicity. The objective of this study was to compare stability of gonadotropin-releasing hormone (GnRH)-toxin conjugates made with disulfide linkers to those using a maleimidodibutyryl (mb) linkage., Method of Study: We developed a sandwich enzyme-linked immunoabsorbent assay recognizing both GnRH analog and cytotoxin to ensure the conjugate measured was intact. Anti-D-Leu(6)-GnRH was used for capture and anti-pokeweed antiviral protein (anti-PAP) or anti-RNase for quantification. Specificity was verified by lack of reactivity with ovine FSH and LH, PAP, RNase, and D-Lys(6)-GnRH., Results: Conjugates prepared using disulfide linkages were not stable in serum in vitro (half-lives <10 min), whereas mb conjugates had half-lives >2 hr. Clearance of mbGnRH-PAP from the circulation of sheep was rapid (t(1/2) <20 min)., Conclusion: The assays were found to be specific, sensitive and accurate for measurement of GnRH-toxin conjugates in vitro and in vivo.

The objective of the present study was to determine how rapidly estradiol (E2) was able to suppress the secretion of LH in ovariectomized (OVX) ewes and to evaluate the ability of conjugated forms of E2 (E2 conjugated to BSA [1,3,5(10)-estratrien-3,17beta-diol-6-one-6-carboxymethyloxime:BSA [E2-BSA] and a novel conjugate, E2 conjugated to a small peptide [E2-PEP]) to mimic the actions of E2 on secretion of LH and FSH. Animals (n = 5-6 per group) were given infusions for 4 h of 50 microg of E2 or equimolar concentrations of E2-BSA or E2-PEP. Treatments with E2, E2-BSA, and E2-PEP each induced an acute suppression of LH secretion (<20 min, P < 0.01). In contrast, E2, but not E2-BSA or E2-PEP, induced the characteristic preovulatory-like surge of LH (at 10 h after priming treatment) and decreased secretion of FSH (at 4 h after priming treatment). In conclusion, the acute inhibition of LH secretion induced by E2 in OVX ewes supports the concept of a nongenomic action as the mechanism underlying the sudden suppression in secretion of LH. In addition, the fact that conjugated forms of E2 mimicked only the acute suppression of secretion of LH, without inducing the putative genomic actions on secretion of LH or FSH (i.e., a preovulatory-like surge), suggests that the acute effect of E2 may be mediated via the plasma membrane.

Synthesis and secretion of prostaglandin F2alpha (PGF2alpha) is elevated following parturition and exerts divergent effects on the re-establishment of fertile estrous cycles in cows. The objective of these experiments was to determine if oil seed supplements differing in fatty acid composition differentially influence serum concentrations of the specific PGF2alpha metabolite, PGFM. Safflower seed supplements were formulated to provide 5% of dry-matter intake as fat. In Trial 1, 24 multiparous beef cows were individually fed control (beet pulp-soybean meal) or cracked high-linoleate safflower seed (78% 18:2n-6) supplements for 80 d postpartum. Linoleate supplemented cows had greater (P < 0.001) serum concentrations of PGFM than control cows. In Trial 2, primiparous beef cows (n = 36) were individually fed control (cracked corn-soybean meal), cracked high-linoleate (76% 18:2n-6) or -oleate (72% 18:1n-9) safflower seed supplements for 92 d postpartum. As in Trial 1, serum concentrations of PGFM were greater (P < or = 0.04) in linoleate than control or oleate supplemented cows. Serum concentrations of PGFM, however, did not differ (P = 0.40) among oleate and control supplemented cows. Although potential impacts on reproductive performance remain to be proven, dietary oil supplements high in linoleate, but not oleate, increased serum concentrations of PGFM compared to control supplements.

Practical application of fertility control technology in free-ranging wild ungulates often requires remote delivery of the contraceptive agent. The objective of this investigation was to evaluate the potential of remote delivery of leuprolide acetate for suppressing fertility in female elk (Cervus elaphus nelsoni). Fifteen captive adult female elk were randomly allocated to one of three experimental groups. Six elk were injected intramuscularly with a dart containing leuprolide, and the remaining nine elk received the same formulation without leuprolide. We determined pregnancy rates, suppression of luteinizing hormone (LH) and progesterone concentrations, and reversibility of treatments during 1 August 2002 to 3 September 2003. Leuprolide formulation caused a decrease in concentrations of LH and progesterone, temporary suppression of ovulation and steroidogenesis, and effective contraception (100%) for one breeding season. These results extend the practical application of this contraceptive agent to include dart delivery, where in the absence of such technology, wild elk must first be captured and restrained before treatment.

Recent studies profiling immediate early gene responses to GnRH in the LbetaT2 gonadotrope cell model revealed increased expression of numerous genes including activating transcription factor (ATF) 3. The present studies demonstrate similar results with GnRH administration in vivo in ovariectomized mice. In this model, ATF3 mRNA was markedly up-regulated at 20, 40, and 60 min after in vivo administration of a GnRH analog. In alphaT3-1 gonadotrope cells, ATF3 mRNA and protein were induced by GnRH in a manner consistent with in vivo observations. Pharmacological studies implicated a combined role for the activities of protein kinase C isozymes, ERK and c-Jun N-terminal kinase, in modulating ATF3 expression. The role of ATF3 was further investigated in the activation of the human glycoprotein hormone alpha-subunit gene promoter. GnRH induced the alpha-subunit promoter-luciferase reporter approximately 16-fold, and this induction was completely abolished with mutations in the dual cAMP response elements (CREs) or the combined inhibition of GnRH-induced ERK and c-Jun N-terminal kinase. GnRH induced recruitment of ATF3, c-Jun, and c-Fos to the dual CREs. Overexpression and specific knockdown of ATF3 by small inhibitory RNA implicate a functional role for ATF3 in mediating activation of the alpha-subunit gene promoter. These studies provide clear evidence that ATF3 is a key immediate early gene induced by GnRH administration in vivo and in the alphaT3-1 gonadotrope cell model. These studies support the conclusion that the dual CREs of the human alpha-subunit promoter are the target of GnRH-induced MAPK regulation through ATF3.

Reproduction in mammals is controlled by interactions between the hypothalamus, anterior pituitary and gonads. Interaction of GnRH with its cognate receptor is essential to regulating reproduction. Characterization of the structure, distribution and expression of GnRH receptors (GnRH-R) has furthered our understanding of the physiological consequences of GnRH stimulation of pituitary gonadotropes. Based on the putative topology of the amino acid sequence of the GnRH-R and point mutation studies, key elements of the GnRH-R have been identified to play a role in ligand recognition and binding, G-protein activation and internalization. Normally, reproductive function is mediated by GnRH-R expressed only on the membranes of pituitary gonadotropes. The density of GnRH-R on gonadotropes determines their ability to respond to GnRH. This density is highest just prior to ovulation and likely is important for complete expression of the pre-ovulatory surge of LH. Therefore, knowledge regarding what regulates the density of GnRH-R is essential to understanding changes in pituitary sensitivity to GnRH and ultimately, to expression of the LH surge. Regulation of GnRH-R gene expression is influenced by a multitude of factors including gonadal steroid hormones, inhibin, activin and perhaps most importantly GnRH itself.

The objective of the present study was to determine the ability of 17beta-estradiol (E(2)) and conjugated forms of E(2) (E(2) conjugated to BSA [E(2)-BSA] and a novel conjugate, E(2) conjugated to a small peptide [E(2)-PEP]) to prevent the GnRH-induced secretion of LH and to determine the role of estradiol receptors (ERs) and ER subtypes (ERalpha, also known as ESR1, and ERbeta, also known as ESR2) in the mediation of the acute action of E(2) in primary cultures of ovine pituitary cells. Preincubation of cells for 15 min with E(2), E(2)-BSA, or E(2)-PEP prevented the GnRH-induced secretion of LH (P < 0.01). Treatment of cells with nonestrogenic steroid hormones did not affect secretion of LH when given alone, nor did these steroids impair the E(2)-induced inhibition of LH secretion (P > 0.1). Likewise, treatment of cells with the ER-antagonists tamoxifen, hydroxytamoxifen, or ICI 182 780 did not affect (P > 0.1) secretion of LH when given alone but did prevent (P < 0.01) the inhibition by E(2) and the E(2)-conjugates on GnRH-induced secretion of LH. When cells were treated with subtype-selective ER agonists, the ERalpha agonist (propylpyrazole-triol), but not the ERbeta agonist (diarylpropionitrile), decreased (P < 0.01) the GnRH-induced secretion of LH. In conclusion, the rapidity by which E(2) prevented GnRH-induced release of LH in ovine pituitary cells suggests that this inhibition is mediated via a nongenomic action of E(2). The inhibition of GnRH-induced secretion of LH proved to be steroid specific and mediated by ERs. It may occur specifically through ERalpha. The fact that E(2)-BSA or E(2)-PEP mimicked the action of E(2) suggests that this effect was mediated by an ER associated with the plasma membrane.

Gonadotropin-releasing hormone (GnRH) is the hypothalamic factor that mediates reproductive competence. Intermittent GnRH secretion from the hypothalamus acts upon its receptor in the anterior pituitary to regulate the production and release of the gonadotropins, LH and FSH. LH and FSH then stimulate sex steroid hormone synthesis and gametogenesis in the gonads to ensure reproductive competence. The pituitary requires pulsatile stimulation by GnRH to synthesize and release the gonadotropins LH and FSH. Clinically, native GnRH is used in a pump delivery system to create an episodic delivery pattern to restore hormonal defects in patients with hypogonadotropic hypogonadism. Agonists of GnRH are delivered in a continuous mode to turn off reproductive function by inhibiting gonadotropin production, thus lowering sex steroid production, resulting in medical castration. They have been used in endocrine disorders such as precocious puberty, endometriosis and leiomyomata, but are also studied extensively in hormone-dependent malignancies. The detection of GnRH and its receptor in other tissues, including the breast, ovary, endometrium, placenta and prostate suggested that GnRH agonists and antagonists may also have direct actions at peripheral targets. This paper reviews the current data concerning differential control of GnRH and GnRH receptor expression and signaling in the hypothalamic-pituitary axis and extrapituitary tissues. Using these data as a backdrop, we then review the literature about the action of GnRH in cancer cells, the utility of GnRH analogs in various malignancies and then update the research in novel therapies targeted to the GnRH receptor in cancer cells to promote anti-proliferative effects and control of tumor burden.

Fertility control offers a potential alternative for controlling an abundance of wild ungulate populations where lethal methods are infeasible or unacceptable. A promising nonsteroidal, nonimmunologic approach to reversible contraception consists of agonist of gonadotropin-releasing hormone (GnRH). We evaluated the effects of the GnRH agonist, leuprolide, on reproduction, the suppression of luteinizing hormone (LH) and progesterone, blood parameters, and reproductive behavior in captive female mule deer (Odocoileus hemionus) during December 1999 through June 2001. Leuprolide, administered as a controlled release formulation (ATRIGEL), was 100% effective in preventing pregnancy for one breeding season. Infertility was achieved by suppressing LH levels, which prevented ovulation and the formation of corpus luteum. Treated females regained normal ovarian function and conceived the following breeding season. Leuprolide had no adverse effects on blood chemistry and hematology, body weight dynamics, or the general health of treated females. In contrast to our predictions, leuprolide did not suppress estrous behavior in female deer during the "normal" breeding period, nor did treated females return to normal ovarian function and exhibit reproductive behaviors during the post-breeding period. This prolonged-release leuprolide formulation offers an alternative approach to reversible contraception in female deer that overcomes some of the problems associated with existing technology.

Pokeweed antiviral protein (PAP) is a plant-derived, highly potent ribosome inactivating protein that causes inhibition of protein translation and rapid cell death. We and others have delivered this protein to various cell types, including cancer cells, using hormones to specifically target cells bearing the hormone receptor. Here, we compare binding and cytotoxicity of GnRH-PAP hormonotoxins prepared either by protein conjugation (GnRH-PAP conjugate) or through recombinant DNA technology (GnRH-PAP fusion). Although GnRH-PAP conjugate protein bound specifically to and caused cell death in cells bearing the gonadotropin-releasing hormone (GnRH) receptor, we could not detect binding or cytotoxicity using two different versions of the fusion protein in receptor-positive cells. We conclude that generation of an active GnRH-PAP fusion protein may not be feasible either because both ends of the GnRH molecule are required for receptor binding, but only the NH(2) terminus is free in the fusion protein and/or that more potent analogues of GnRH (inclusion of which is not feasible in the fusion protein) are needed for efficient targeting. In contrast, the GnRH-PAP conjugate shows promise as a novel anticancer agent, capable of targeting cancer cells expressing the GnRH receptor such as prostate, breast, ovarian, endometrial, and pancreatic cells. It may also be useful as a therapeutic agent to eliminate pituitary gonadotrophs, eliminating the need for chronic GnRH analogue administration to treat hormone-sensitive diseases.

Pokeweed antiviral protein (PAP) is a plant-derived, highly potent ribosome inactivating protein that causes inhibition of protein translation and rapid cell death. We have previously described potent cytotoxic activity of a GnRH (gonadotropin-releasing hormone) receptor-targeted conjugate protein (GnRH-PAP) and demonstrated that cytotoxicity depended on the number of GnRH receptors and the duration of exposure. Here, we demonstrate that the GnRH-PAP conjugate was cytotoxic to three different prostate cancer cell lines, supporting the feasibility of using such hormonotoxins as novel therapeutics for hormone-responsive cancers such as prostate cancer.

Steroidogenic factor-1 (SF-1) is a transcription factor originally characterized as a mediator of gene expression in steroidogenic tissues. Studies in SF-1 knockout mice revealed that SF-1 has additional roles at multiple levels of the hypothalamic-pituitary-gonadal axis, including regulation of gene expression in pituitary gonadotropes. Specific binding sites for SF-1 have been demonstrated in several pituitary genes with essential roles in gonadotropin synthesis, including alpha subunit, LHbeta subunit, and GnRH receptor. In studies aimed at identifying physiological factors controlling pituitary expression of SF-1, GnRH has been implicated as a co-regulator of SF-1 and gonadotropin subunit genes. In both rats and ewes, elevated endogenous secretion of GnRH following ovariectomy was associated with increased amounts of SF-1 mRNA in the anterior pituitary gland. Conversely, removal of GnRH input to the pituitary gland by hypothalamic-pituitary disconnection (HPD) in ovariectomized (OVX) ewes reduced SF-1 expression. Despite these changes, however, treatment of OVX ewes with GnRH following HPD only partially restored levels of SF-1 mRNA in the pituitary gland. Therefore, it is possible that regulation of SF-1 gene expression by GnRH during the estrous cycle may involve ovarian hormones or other hypothalamic factors. Additional studies are required to further define the physiological roles of SF-1 in regulation of the hypothalamic-pituitary-gonadal axis in domestic ruminants.

Pokeweed antiviral protein (PAP), a 29-kDa ribosome-inactivating protein isolated from the leaves of Phytolacca americana, has potent cytotoxic activity once it enters the cytoplasm of a cell. It is incapable of entering cells by itself. Therefore, our objective was to determine whether a GnRH analog could be used to deliver PAP specifically to cells expressing GnRH receptors. D-Lys(6)-GnRH-Pro(9)-ethylamide was conjugated to PAP (GnRH-PAP). Chinese hamster ovary cells stably transfected with cDNA for the murine GnRH receptor and a mouse gonadotroph tumor cell line that expresses endogenous GnRH receptors (alphaT3-1 cells) were used to evaluate the cytotoxic effects of GnRH-PAP. We also examined cytotoxicity of GnRH-PAP using human endometrial, breast, and prostate cancer cell lines. Treatment of GnRH receptor-positive cells with GnRH-PAP resulted in dose-dependent cytotoxicity. Cytotoxicity of GnRH-PAP was dependent on number of GnRH receptors (r(2) = 0.871, P < 0.05) and duration of exposure of GnRH-PAP to the cells. In contrast, GnRH-PAP was not cytotoxic to Chinese hamster ovary cells not harboring GnRH receptors. Moreover, the cytotoxic activity of GnRH-PAP could be inhibited by addition of excess GnRH analog. Neither PAP nor GnRH analog alone was cytotoxic. These results suggest that GnRH analogs can be used to specifically deliver toxin molecules to cells that express GnRH receptors. Thus, a new class of biomedicines that act as hormonotoxins against cells expressing GnRH receptors provides a novel approach for inhibiting reproduction and treating cancers that are dependent on reproductive hormones.

Following induction of ovulation with deslorelin acetate (Ovuplant), gonadotrophin concentrations are reduced in the subsequent cycle, leading to increased interovulatory intervals in some mares. This study determined whether implant removal after 2 days prevented the decrease in gonadotrophin concentrations and follicular growth during the ensuing cycle. Twenty-four mares were randomised equally into 3 groups. Group 1 ovulated spontaneously, Groups 2 and 3 received the deslorelin implant to induce ovulation. Two days after treatment, the implant was removed from Group 3. On Day 10 postovulation, FSH was lower (P = 0.009) in Group 2, but not different between Groups 1 and 3. Follicular diameter on Day 14 was less (P<0.05) in Group 2 (19.0 +/- 2.1 mm) than in Groups 1 and 3 (36.6 +/- 2.5 and 30.5 +/- 2.0 mm, respectively). Interovulatory interval was longer (P<0.05) for Group 2 (25.8 +/- 2.9 days) compared to Groups 1 and 3 (18.5 +/- 0.7 and 19.4 +/- 0.3 days, respectively). Removal of the deslorelin implant eliminated the decreased FSH secretion and the increased interovulatory interval associated with implant administration. Therefore, it is recommended that the implant be removed after ovulation is detected to prevent the occurrence of a prolonged interovulatory interval.