Identifying the future needs for long-term USDA efforts in agricultural animal genomics.

Agricultural animal research has been immensely successful over the past century in developing technology and methodologies that have dramatically enhanced production efficiency of the beef, dairy, swine, poultry, sheep, and aquaculture industries. In the past two decades, molecular biology has changed the face of agricultural animal research, primarily in the arena of genomics and the relatively new offshoot areas of functional genomics, proteomics, transcriptomics, metabolomics and metagenomics. Publication of genetic and physical genome maps in the past 15 years has given rise to the possibility of being able finally to understand the molecular nature of the genetic component of phenotypic variation. While quantitative geneticists have been remarkably successful in improving production traits, genomic technology holds potential for being able to lead to more accurate and rapid animal improvement, especially for phenotypic traits that are difficult to measure.Recently, the agricultural research community has been able to capitalize on the infrastructure built by the human genome project by sequencing two of the major livestock genomes (Gallus domesticus and Bos Taurus). The 2005 calendar year is truly unprecedented in the history of agricultural animal research since draft genome sequences were completed for chickens and cattle. In addition, sequencing the swine and equine genome was initiated in early 2006. We now have in place a powerful toolbox for understanding the genetic variation underlying economically important and complex phenotypes. Over the past few years, new challenges have emerged for animal agriculture. Enhancements in production efficiency have not come without some negative side effects on animal well-being and longevity in production environments, including losses in reproductive efficiency, increased stress susceptibility, increased animal waste issues, and increased susceptibility to animal metabolic and infectious diseases. When considered in concert with societal concerns in the areas of natural resource conservation and protection, animal welfare, and food safety, it is clear that publicly supported agricultural research must be focused on enhancing the functionality and well-being of livestock and poultry in environmentally neutral production systems in the future. Realizing the great potential for animal genomics to address these and other issues, a workshop was convened by the U. S. Department of Agriculture (USDA) in Washington, DC in September of 2004. The workshop was entitled "Charting the Road Map for Long Term USDA Efforts in Agricultural Animal Genomics". This paper summarizes the proceedings of the workshop and the resulting recommendations. The need for a cohesive, comprehensive long-term plan for all of USDA's research efforts in animal genomics was evident at the workshop, requiring further integration of the efforts of the USDA's Cooperative State Research, Education, and Extension Service (CSREES) and the USDA's Agricultural Research Service (ARS) to achieve the greatest return on investment.

Responsiveness of the ovine gonadotropin-releasing hormone receptor gene to estradiol and gonadotropin-releasing hormone is not detectable in vitro but is revealed in transgenic mice.

Although the ability of estradiol to enhance pituitary sensitivity to GnRH is established, the underlying mechanism(s) remain undefined. Herein, we find that approximately 9,100 bp of 5' flanking region from the ovine GnRH receptor (oGnRHR) gene is devoid of transcriptional activity in gonadotrope-derived cell lines and is not responsive to either estradiol or GnRH. In stark contrast, this same 9,100 bp promoter fragment directed tissue-specific expression of luciferase in multiple lines of transgenic mice. To test for hormonal regulation of the 9,100-bp promoter, ovariectomized transgenic females were treated with a GnRH antiserum alone or in combination with estradiol. Treatment with antiserum alone reduced pituitary expression of luciferase by 80%. Pituitary expression of luciferase in animals receiving both antiserum and estradiol was approximately 50-fold higher than animals receiving antiserum alone. The estradiol response of the -9,100-bp promoter was equally demonstrable in males. In addition, a GnRH analog (D-Ala-6-GnRH) that does not cross-react with the GnRH antiserum restored pituitary expression of luciferase in males passively immunized against GnRH to levels not different from castrate controls. Finally, treatment with both estradiol and D-Ala-6-GnRH increased pituitary expression of luciferase to a level greater than the sum of the individual treatments suggesting synergistic activation of the transgene by these two hormones. Thus, despite the complete absence of transcriptional activity and hormonal responsiveness in vitro, 9,100 bp of proximal promoter from the oGnRHR gene is capable of directing tissue-specific expression and is robustly responsive to both GnRH and estradiol in transgenic mice. To begin to refine the functional boundaries of the critical cis-acting elements, we next constructed transgenic mice harboring a transgene consisting of 2,700 bp of 5' flanking region from the oGnRHR gene fused to luciferase. As with the -9,100 bp promoter, expression of luciferase in the -2,700 lines was primarily confined to the pituitary gland, brain and testes. Furthermore, the passive immunization-hormonal replacement paradigms described above revealed both GnRH and estradiol responsiveness of the -2,700-bp promoter. Thus, 2,700 bp of proximal promoter from the oGnRHR gene is sufficient for tissue-specific expression as well as GnRH and estradiol responsiveness. Given the inability to recapitulate estradiol regulation of GnRHR gene expression in vitro, transgenic mice may represent one of the few viable avenues for ultimately defining the molecular mechanisms underlying estradiol regulation of GnRHR gene expression.

Ovine prostaglandin F2alpha receptor: steroid influence on steady-state levels of luteal mRNA.

Expression of the receptor for prostaglandin F2alpha (PGF2alpha) is decreased in the ovine corpus luteum during regression and increased in early pregnancy. This study was designed to evaluate the influence of progesterone and/or 17beta-estradiol (E2) on this regulation. Circulating progesterone (functional regression) and luteal PGF receptor mRNA decreased (p < 0.05) within 8 h of PGF2alpha-induced luteal regression in midluteal phase (day 10; d 10) ewes; however, internucleosomal DNA fragmentation (structural regression) was not yet increased. Additionally, luteal PGF receptor mRNA and circulating progesterone were greater (p < 0.05) in pregnant than in nonpregnant ewes on d 14, but not on d 12. Twelve hours following injection of d 10 ewes with E2, steady-state levels of mRNA for PGF receptor were decreased (p < 0.05), although circulating progesterone and DNA laddering were unchanged. Conversely, luteal mRNA for PGF receptor was increased (p < 0.05) by E2 treatment in hysterectomized ewes. These results provide evidence that (1) luteal PGF receptor expression parallels circulating progesterone levels during functional regression and in early pregnancy, but (2) expression of PGF receptor can be dissociated from alterations in circulating progesterone by injection with E2. Additionally, decreased PGF receptor expression initiated by E2 is uterine-dependent, whereas the direct luteal effect (hysterectomized ewes) of E2 is a stimulation of PGF receptor expression. These results collectively support the belief that the apparent downregulation of PGF receptor during luteal regression is associated with uterine-derived PGF2alpha and its intracellular effects rather than with alterations in ovarian steroid production.

Insulin-like growth factor-I, insulin-like growth factor-binding proteins, and gonadotropins in the hypothalamic-pituitary axis and serum of nutrient-restricted ewes.

Body condition scores (BCS) of ovariectomized estradiol-treated ewes were controlled to examine effects of suboptimum BCS on insulin-like growth factor (IGF)-I, IGF-binding proteins (IGFBPs), and LH in the anterior pituitary gland, hypophyseal stalk-median eminence (SME), and circulation. Serum LH increased in ewes with BCS (1 = emaciated, 9 = obese) > 3 (HIGH-BCS), but not in ewes with BCS

Accumulation of caspase-3 messenger ribonucleic acid and induction of caspase activity in the ovine corpus luteum following prostaglandin F2alpha treatment in vivo.

Caspase-3, a vertebrate homologue of the protein encoded by the Caenorhabditis elegans cell death gene, ced-3, induces apoptosis when overexpressed in eukaryotic cells. Since apoptosis occurs during corpus luteum (CL) regression in many species, including the ewe, these studies were conducted to 1) isolate a cDNA encoding ovine caspase-3, 2) measure steady state amounts of caspase-3 mRNA in the CL during luteolysis induced by prostaglandin F2alpha (PGF2alpha) and during the time of maternal recognition of pregnancy, and 3) measure changes in caspase activity during PGF2alpha-initiated luteal regression. Oligonucleotide primers corresponding to a human caspase-3 cDNA sequence were combined with total RNA from ovine CL in a reverse transcription-polymerase chain reaction-based procedure to amplify a 640-base pair partial cDNA with a nucleotide sequence 86% and 81% identical to the human and rat caspase-3 cDNAs, respectively. CL were collected from ewes at 0, 12, or 24 h after treatment with PGF2alpha on Day 10 of the estrous cycle and from nonpregnant and pregnant ewes on Day 12 or Day 14 of the cycle. Northern blot analysis of total cellular RNA from ovine CL and a radiolabeled ovine caspase-3 cRNA probe indicated the presence of a single mRNA transcript of approximately 2.5 kilobases. Levels of caspase-3 mRNA were approximately 3-fold higher (p < 0.05) in CL at 12 h and 24 h after PGF2alpha in comparison to those levels measured in matched CL from untreated ewes. There were no differences (p > 0.05) in amounts of caspase-3 mRNA in CL on Day 12 or Day 14 of the estrous cycle compared to Day 12 or Day 14 of pregnancy, respectively. Caspase activity in CL (measured by the ability of CL lysates to cleave an artificial caspase substrate) was also significantly (p < 0.05) increased in CL collected after treatment with PGF2alpha compared to CL collected from nontreated ewes. We conclude that physiological cell death during PGF2alpha-induced luteal regression in the ewe is mediated, at least in part, via increased expression and activity of the caspase family of pro-apoptotic proteases.

Estradiol and gonadotropin-releasing hormone (GnRH) interact to increase GnRH receptor expression in ovariectomized ewes after hypothalamic-pituitary disconnection.

Gonadotropin-releasing hormone (GnRH) receptor expression is regulated by estradiol and GnRH itself. The objective of this experiment was to determine the extent to which low levels of estradiol, similar to those observed during the transition from the luteal to the follicular phase of the estrous cycle, and GnRH interact to regulate expression of GnRH receptors and GnRH receptor mRNA. Ewes were ovariectomized (OVX) at least 2 wk prior to initiation of the experiment, and the pituitary gland was surgically disconnected from the hypothalamus to remove ovarian and hypothalamic inputs to the pituitary. Within 24 h after hypothalamic-pituitary disconnection, ewes received pulses of GnRH (250 ng/pulse) every 2 h for 6 d. At the end of 6 d, ewes were randomly assigned to treatments in a 2 x 2 factorial arrangement as follows: half of the animals received a single estradiol implant and half received an empty implant (placebo). At the same time, animals also received one of the following treatments: (1) saline or (2) GnRH (100 ng/pulse/2 h). Additionally, one group of ewes was ovariectomized, but not subjected to hypothalamic-pituitary disconnection (OVX controls). Blood samples were collected 15 min prior to each pulse of GnRH or saline and at 15-min intervals for 1 h after each pulse until tissues were collected and concentrations of luteinizing hormone (LH) were determined. Anterior pituitaries were collected 24 h after implant insertion to quantitate steady-state amounts of GnRH receptor mRNA and numbers of GnRH receptors. Mean LH was greatest in ovariectomized control ewes compared to all other treatments (p < 0.05). Mean LH and LH pulse amplitude in the placebo and GnRH-treated group most closely mimicked LH secretion in ovariectomized control animals. Mean LH and LH pulse amplitude were similar between both GnRH-treated groups (p < 0.05). Mean LH and LH pulse amplitude were significantly lower in all animals treated with saline compared to OVX controls (p < 0.05). Treatment with an estradiol implant and pulsatile GnRH increased (p < 0.05) relative amounts of GnRH receptor mRNA and the number of GnRH receptors compared to all other treatments. There were no differences in GnRH receptor expression between the remaining treatment groups (p > 0.05). Therefore, in OVX ewes after hypothalamic-pituitary disconnection, low levels of estradiol and GnRH are required to increase GnRH receptor mRNA and GnRH receptor numbers. Since we only observed an increase in GnRH receptor expression in the presence of both estradiol and GnRH, we conclude that there is a synergistic interaction between these two hormones in the regulation of GnRH receptor expression.

Regulation of amounts of mRNA for GnRH receptors by estradiol and progesterone in sheep.

Expression of GnRH receptors increases prior to the onset of the preovulatory surge of LH in sheep. Two experiments were conducted to investigate the interactions of progesterone (P) and estradiol (E) on amounts of mRNA for GnRH receptors and the number of receptors for GnRH. The first study was designed as a 2 x 2 factorial arrangement of treatments to investigate effects of removal of P and the presence of E. Ewes that had been ovariectomized (OVX) for at least 4 wk received one silastic implant containing E and two silastic implants containing P for 6 d to mimic concentrations of these steroids during the luteal phase of the estrous cycle. Anterior pituitary glands were collected (n = 4 animals/group): 1. Prior to implant removal and 12 h after removal of: 2. P only. 3. E only. 4. P and E. Regardless of whether or not E was present, amounts of mRNA for GnRH receptors (P = 0.87) and number of GnRH receptors (P = 0.43) were not different within 12 h after removal of P. In the second experiment, ewes were OVX on d10-12 of the estrous cycle (d0 = estrus), and immediately received silastic implants containing E and P as described above. Anterior pituitary glands were collected on d12 of the estrous cycle (n = 5), prior to implant removal (n = 5), and from the remaining ewes 24 h after removal of P only (n = 7) or removal of P and E (n = 6). Relative amounts of mRNA for GnRH receptors and the number of GnRH receptors were similar (P > 0.05) on d12 of the estrous cycle and prior to implant removal. Removal of both P and E did not affect (P > 0.05) amounts of GnRH receptor mRNA or number of GnRH receptors. However, the removal of P and the presence of E increased (P < 0.05) amounts of mRNA for GnRH receptors, but did not affect (P > 0.05) the number of GnRH receptors. We conclude that increased amounts of GnRH receptor mRNA require the removal of P and the presence of E.

Estradiol increases relative amounts of insulin-like growth factor binding protein (IGFBP)-3 in serum and expression of IGFBP-2 in anterior pituitaries of ewes.

This study determined whether estradiol regulates insulin-like growth factor (IGF)-I and IGF binding proteins (IGFBPs) in the pituitary gland, hypophyseal stalk median eminence (SME), and circulation concomitantly with effects on LH. Ovariectomized ewes received an estradiol implant or no implant during the anestrous season and were slaughtered 80 days later. Estradiol suppressed serum LH to a greater extent during anestrus than after onset of the breeding season (Days 60 and 75). Amounts of mRNA for LHbeta subunit were decreased by estradiol, but mRNA for alpha and FSHbeta subunits were not affected. Estradiol increased serum IGF-I, IGFBP-3, and IGFBP-4 throughout the treatment period, but it did not influence other IGFBPs in serum. In response to estradiol, pituitary IGFBP-2 tended to increase and mRNA for IGFBP-2 increased twofold. Other IGFBPs in the pituitary gland were not influenced by estradiol. In the SME, IGFBP-2, IGFBP-5, and the 40-kDa IGFBP-3 were increased by estradiol. Thus, estradiol influences both the IGF and gonadotropin systems in sheep. Estradiol influences on gonadotroph function may be mediated by alterations in the IGF system.

The estradiol-induced luteinizing hormone surge in the ewe is not associated with increased gonadotropin-releasing hormone messenger ribonucleic acid levels.

This experiment was undertaken to determine whether the estrogen-induced LH and GnRH surge in the ewe is associated with activation of a specific subpopulation of neurons in the mid-brain of the ewe as indicated by a change in GnRH mRNA levels. Fifteen ovariectomized ewes were assigned to treatment groups 3-4 wk after ovariectomy. One group of ewes served as controls (n = 2); 50 microg estradiol-17beta (E2) was administered to the remaining ewes. Blood samples were collected from all ewes before treatment (2-h period at 10-min intervals) and continued at 30-min intervals until tissue was collected. At 6, 12, 18, and 24 h after E2 (n = 3 for each time point), brains were collected and processed for localization and measurement of GnRH mRNA by in situ hybridization histochemistry. Serum was analyzed for LH concentrations. Serum LH was pulsatile in controls and decreased at 6 h after E2, and by 12 h the LH surge was initiated. LH levels peaked at 18 h after E2 and returned to basal levels 24 h after E2 treatment. A cRNA probe corresponding to the GnRH-associated peptide region of ovine GnRH prepropeptide mRNA was used to identify GnRH mRNA. Associated with the onset and peak of the LH surge were decreased levels (p < 0.1) of GnRH mRNA in neurons of the preoptic area (POA). Neither the number nor mRNA content of GnRH neurons in the diagonal band of Broca, septal area, or medial basal hypothalamus (MBH) changed during the LH surge. In contrast to E2-induced increases in GnRH secretion during the LH surge, our data indicate that E2 decreases steady-state amounts of GnRH mRNA and that GnRH neurons in the POA are influenced to the greatest extent during the E2-induced GnRH surge.

Gonadotropin secretion and development of ovarian follicles during oestrous cycles in heifers treated with luteinizing hormone releasing hormone antagonist.

The hypothesis tested was that reduced LHRH stimulation of the anterior pituitary would lead to attenuated development of ovarian follicles as a result of reduced gonadotropin secretion during oestrous cycles of cattle. Twenty heifers were randomly assigned to be treated ( n = 5/treatment) with an antagonist to LHRH (LHRH-Ant) 1) from Day 2 to 7 (Day 0 = behavioural oestrus), 2) Day 7 to 12, 3) Day 12 to 17, 4) or serve as untreated control animals. LHRH-Ant suppressed LH pulses of heifers in all treatment groups from treatment initiation through Day 17 as compared with untreated control heifers [Peters et al., 1994. Luteinizing hormone has a role in development of fully functional corpora lutea (CL) but is not required to maintain CL function in heifers. Biol. Reprod., 51 (1994) 1248-1254]. Circulating concentration of FSH from Day 8 to 10 of the oestrous cycle did not increase in heifers treated with LHRH-Ant from Day 2 to 7 or Day 7 to 12; however, there was increased (P < 0.05) FSH from Day 8 to 10 of the oestrous cycle in heifers treated with LHRH-Ant from Day 12 to 17 and control heifers. Compared with control heifers, heifers treated with LHRH-Ant from the Day 2 to 7 had suppressed (P < 0.05) size and persistence of the first and second dominant ovarian follicles. Heifers treated with LHRH-Ant from Day 7 to 12 had suppressed size (P < 0.05 and tended (P < 0.10) to have a shorter persistence of the second dominant ovarian follicle compared with control heifers. Heifers treated with LHRH-Ant from Day 12 to 17 had a similar (P > 0.10) size and persistence of dominant ovarian follicles but had reduced (P < 0.10) numbers of large follicles compared with control heifers. Heifers treated with LHRH-Ant from Day 2 to 7 had lower (P < 0.01) concentrations of 17 beta-oestradiol during the treatment period and tended (P < 0.10) to have lower concentrations of 17 beta-oestradiol from Day 7 to 12 of the oestrous cycle compared with control heifers. Heifers treated with LHRH-Ant from Day 7 to 12 or Day 12 to 17 had similar (P > 0.10) circulating LH concentrations of l7 beta-oestradiol compared with control heifers. Reduced LHRH stimulation of the pituitary from Day 2 to 12 of the oestrous cycle and the resulting modulation in circulating LH and FSH led to suppressed ovarian follicular development and oestradiol secretion. After Day 12 of the oestrous cycle, reduced LHRH stimulation of the anterior pituitary did not lead to altered ovarian follicular development to the extent as reduced LHRH stimulation before Day 12 although pulsatile release of LH was similarly suppressed by treatment with the LHRH-Ant.

Pituitary relationship of gonadotropins and messenger ribonucleic acid for gonadotropin subunits in white composite and Meishan boars.

Two experiments were conducted to compare amounts of mRNAs for gonadotropin subunits in two breeds of pigs that are genetically distinct with respect to litter size: the Chinese Meishan and the white composite (a composite of four European white breeds: Yorkshire, Landrace, large white, and Chester white). In experiment 1, pituitaries were collected from mature Meishan (n = 5) and white composite (n = 6) boars. In experiment 2, boars were assigned to three groups according to peripheral concentrations of FSH: white composite (approximately 100 ng/ml; n = 8), Meishan with low FSH (< 500 ng/ml; n = 7), and Meishan with high FSH (> 750 ng/ml; n = 7). Within each of the three groups, five males were castrated. Two months after castration, pituitaries were collected from castrates and from eight contemporary boars for comparison purposes. Concentrations of FSH and LH were measured in half of each pituitary by RIA. RNA was isolated from the other half of each pituitary, applied to Northern and slot blots, and hybridized with cDNA probes for bovine alpha, LH beta, or FSH beta subunits. In experiment 1, pituitary concentrations of FSH (micrograms FSH/mg pituitary) were greater (p < 0.05) in Meishan (40 +/- 7 micrograms/mg; mean +/- SE) than in white composite boars (19 +/- 4). Pituitary weight and content of FSH were greater (p < 0.01) in Meishan boars (335.1 +/- 18 mg and 13.5 +/- 18 mg/pituitary, respectively) than in white composite boars (270.9 +/- 12.0 and 5.0 +/- 1.2, respectively). Northern analysis indicated that sizes of gonadotropin subunit mRNAs were similar in pigs and cattle. Amounts of mRNA for alpha subunit and LH beta subunit were not different (p = 0.14 and 0.08, respectively) between breeds. Meishans, however, had greater (p < 0.01) amounts of mRNA for FSH beta subunit than white composites. In experiment 2, amounts of mRNA for alpha, LH beta, and FSH beta subunits were not different (p > 0.05) between castrated and intact animals. Meishan males with high FSH had greater (p < 0.05) amounts of mRNA for all three gonadotropin subunits than did white composite males. From these results we suggest that greater levels of gonadotropins in the circulation of Meishan boars result, in part, from increased amounts of mRNA for gonadotropin subunits as compared with those in the white composite breed of boars.

The proximal 350 bp of 5'-flanking sequence of the human α-subunit glycoprotein hormone gene functions in the pituitary gland, but not the placenta, in transgenic mice.

To understand better the minimal DNA sequence requirements for regulated expression of the human α-subunit glycoprotein hormone gene (Hα), two lines of transgenic mice were constructed that contained a fusion gene (Hα-350CAT) consisting of only 350 bp of 5'-flanking sequence of Hα linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). CAT activity was detectable in pituitary, but not in brain, heart, kidney, liver, lung, pancreas, or spleen in transgenic mice. Gonadectomy increased (p<0.05) CAT activity in the pituitaries of males (6.5±1.4% conversion/µg protein; mean ± SEM) and females (14.5±4.2) compared to intact males (1.2±0.3) and females (6.7±1.0). In addition, administration of a GnRH antagonist (antide; 60 µg/injection; one injection every other day) for 10 d to gonadectomized animals decreased (p<0.05) CAT activity in males (3.5±0.8) and females (2.9±0.5) compared to gonadectomized animals that received saline. Antide also reduced (p<0.05) serum concentrations of luteinizing hormone in gonadectomized males and females compared to gonadectomized animals that received saline. Surprisingly, CAT activity in the placenta of Hα-350CAT transgenic mice was not detectable (<3 SD above the mean of CAT activity in placenta from nontransgenic mice;n=77). Thus, expression of the human α-subunit promoter in the placenta of transgenic mice appears to require DNA sequences upstream of the proximal 350 bp of 5'-flanking sequence, whereas the proximal 350 bp of the human α-subunit gene contains sufficient DNA sequence to target pituitary-specific expression and confer responsiveness to gonadal hormones and GnRH.

Estradiol increases amounts of messenger ribonucleic acid for gonadotropin-releasing hormone receptors in sheep.

Two experiments were conducted simultaneously to investigate regulation of amounts of mRNA for GnRH receptors during the periovulatory period in sheep. In the first experiment, amounts of mRNA for GnRH receptors were measured before and after preovulatory surge of LH following regression of the CL by prostaglandin F2 alpha(PGF2 alpha). So that the time of the preovulatory surge of LH could be accurately predicted, ewes received two injections of PGF2 alpha on Day 14 of the estrous cycle. Anterior pituitary glands were collected from 5 control ewes on Day 14 of the estrous cycle (0 h after PGF2 alpha) and at 48, 72, and 96 h after PGF2 alpha (5 ewes per group). The second experiment was conducted to investigate the effects of 17 beta-estradiol on amounts of mRNA for GnRH receptors. On Day 14 of the estrous cycle, 20 ewes were ovariectomized (OVX); 15 of these ewes received estradiol implants when they were OVX (OVXEI). Sixteen hours after OVX, anterior pituitary glands were collected from 5 OVX and 5 OVXEI ewes, and the remaining OVXEI ewes received an i.m. injection of estradiol (25 micrograms in corn oil; OVXEI + E) to induce a preovulatory-like surge of LH. Anterior pituitary glands were collected from OVXEI + E ewes 18 or 54 h after injection of estradiol (n = 5 per group). Half of each anterior pituitary gland was used to measure the number of GnRH receptors. Poly(A)+ RNA was isolated from the remaining half of each anterior pituitary gland, applied to slot blots, and hybridized with a radioactive cDNA probe encoding the ovine GnRH receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular biology of gonadotrophins.

Regulation of gonadotrophin synthesis involves a complex interaction between hypothalamic and gonadal hormones. Chronic administration of oestrogens and androgens to gonadectomized animals blocked the postcastration rise in amounts of mRNA encoding gonadotrophin subunits. Removal of endogenous GnRH decreased amounts of mRNA encoding gonadotrophin subunits. Pulsatile administration of GnRH to GnRH-deficient animals increased amounts of mRNA encoding gonadotrophin subunits. Studies using transgenic mice and transient transfection assays identified at least eight cis-acting DNA sequences in the proximal 350 bp of 5' flanking sequence of the human alpha subunit gene that directed expression to gonadotrophs or conferred responsiveness to oestrogens, androgens or GnRH. Unique DNA-binding proteins were also identified which directed expression of the human alpha subunit gene specifically to the pituitary. Pituitary cell lines that express bovine gonadotrophin subunit genes are not currently available; thus, relatively little is known about the molecular mechanisms that regulate expression of bovine gonadotrophin subunit genes. Recent studies with transgenic mice harbouring bovine alpha, LH beta, or FSH beta subunit transgenes revealed that DNA sequences important for gonadotroph-specific expression and hormonal regulation resided within the proximal 5' flanking sequences.

Luteinizing hormone has a role in development of fully functional corpora lutea (CL) but is not required to maintain CL function in heifers.

We tested the hypothesis that endogenous pulses of LH have a role in development and maintenance of CL during the estrous cycle of the bovine female. Twenty heifers were synchronized to estrus by treating two times with prostaglandin F2 alpha 11 days apart (Day 0 = behavioral estrus). Heifers were then randomly assigned to one of four treatments (n = 5/group). Heifers were treated with an antagonist to LHRH (LHRH-Ant; N-Ac-D-Nal[2]1,4Cl-D-Phe2,D-Pal[3]3,D-Cit6,D-Ala10- LHR H; 10 micrograms/kg body weight) or vehicle (5% mannitol) once every 24 h: 1) LHRH-Ant Days 2-7, 2) LHRH-Ant Days 7-12, 3) LHRH-Ant Days 12-17, 4) no LHRH-Ant (control). Blood samples were collected from the jugular vein twice daily on Days 0-24, and area under the profile of progesterone in circulation during the luteal phase of the estrous cycle was characterized from the start of each treatment period until the demise of CL or Day 24, whichever came first. Luteolysis was considered to have occurred when three consecutive samples contained less than 1 ng progesterone/ml plasma. Areas under the profile of progesterone in circulation during the luteal phase of the estrous cycle were compared to those of heifers from the control group for the same period. LHRH-Ant treatment diminished LH pulses in all treatment groups compared to control (p < 0.05). Treatment with LHRH-Ant on Days 2-7 diminished function of CL (3.72 +/- 0.93 vs. 7.36 +/- 1.02 units, respectively; p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Progesterone, 17 beta-estradiol, and opioid neuropeptides modulate pattern of luteinizing hormone in circulation of the cow.

The working hypothesis for this study was that 17 beta-estradiol (E2), progesterone (P4), and opioid neuropeptides modulate frequency of pulsatile release of LH during the luteal phase of the bovine estrous cycle. On Day 8 of the estrous cycle (Day 0 = estrus), 20 cows were ovariectomized (OVX) and immediately received one of three steroid replacement treatments: P4 alone (n = 6), E2 alone (n = 7), or P4 and E2 (n = 7). To characterize the pattern of LH in peripheral circulation, serial blood samples were collected (12-min intervals for 22 h) on the fifth day following OVX. LHRH was administered after the 22-h sample was taken. Naloxone (opioid neuropeptide antagonist; 0.5 mg/kg) was administered on the sixth day after OVX, and change of concentration of LH in peripheral circulation was monitored. Pulse frequency of LH and mean concentration of LH were lower (p < 0.01) in cows treated with P4 and E2 compared to cows treated with P4 alone. Moreover, cows treated with E2 alone had a greater (p < 0.01) frequency of LH pulses and mean concentration of LH compared to cows treated with P4 alone. The greatest amplitude (p < 0.01) of LH release after LHRH was in cows treated with E2 alone. The greatest percentage increase (p < 0.01) in LH after administration of naloxone occurred in cows treated with P4 alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional repression of the glycoprotein hormone alpha subunit gene by androgen may involve direct binding of androgen receptor to the proximal promoter.

Testicular androgens suppress the synthesis and secretion of the pituitary gonadotropins, in particular, luteinizing hormone. This suppressive effect includes transcription of both the common alpha subunit gene and the unique beta subunit genes. Herein, we demonstrate that 1500 base pairs (bp) of proximal 5'-flanking region derived from the human alpha subunit gene and a shorter 315-bp segment of the bovine alpha subunit gene confer negative regulation by androgen to the gene encoding bacterial chloramphenicol acetyltransferase in transgenic mice. Cotransfection assays with human androgen receptor indicated that the 1500-bp promoter region of the human alpha subunit gene also confers androgen regulation (transcriptional suppression) to reporter genes in both pituitary and placental cell lines. This raises the possibility of a role for DNA binding in suppression of alpha subunit transcription by activated androgen receptor. Consistent with this possibility, we have used a gel-mobility shift assay to detect several high affinity binding sites for androgen receptor located in the proximal promoter of the human alpha subunit gene. The strongest androgen receptor binding site is located at approximately -101 in the proximal 5'-flanking region. This steroid receptor binding site overlaps another binding site that defines one of several contiguous cis-acting regulatory elements required for basal transcriptional activity. Thus, binding of activated androgen receptor to this region may block the binding of a requisite trans-acting factor and lead to an attenuation in transcription. We conclude that this interaction, which occurs directly at the level of the pituitary, represents one of several physiological avenues through which androgens regulate gonadotropin gene expression.

Gonadotrope- and thyrotrope-specific expression of the human and bovine glycoprotein hormone alpha-subunit genes is regulated by distinct cis-acting elements.

The proximal 5'-flanking region of the alpha-subunit gene from humans and cattle confers pituitary-specific expression to heterologous reporter genes in transgenic mice. To investigate whether these promoter regions also contain the necessary regulatory elements for cell-specific expression and hormonal regulation, we used three independent lines of transgenic mice. Two lines of transgenic mice contained chimeric genes consisting of either 1.6 kilobasepairs (kbp) of human or 3 15 basepairs of bovine alpha-subunit proximal 5'-flanking sequence linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). A third line of transgenic mice contained the proximal 1.6 kbp of 5'-flanking sequence of the human alpha-subunit gene linked to the bacterial lacZ gene encoding beta-galactosidase (beta gal; H alpha beta gal transgenic mice). Hormonal replacement paradigms indicate that both human and bovine alpha CAT transgenes are regulated by GnRH, suggesting that their expression occurs in gonadotropes. Thus, the proximal 5'-flanking regions of both the human and bovine alpha-subunit genes must contain regulatory elements that confer both gonadotrope-specific expression and responsiveness to GnRH. In contrast to the human alpha-subunit promoter, the bovine alpha-subunit promoter lacks a functional cAMP response element, suggesting that transduction of both cell-specific and GnRH transcriptional signals occurs through cAMP response element-independent pathways. Thyrotropes also express the glycoprotein hormone alpha-subunit gene. Yet, hormone replacement paradigms with propylthiouracil and T3 were ineffective in altering CAT activity in the pituitary of human or bovine alpha CAT transgenic mice. Because a thyroid hormone response element has been localized to the proximal 5'-flanking region of the human alpha-subunit gene, these data suggest that the alpha CAT transgenes lack sufficient information to direct expression to thyrotropes. Direct evidence for this possibility was obtained through immunocytochemical studies performed on pituitaries from H alpha beta gal transgenic mice. beta-Galactosidase activity appeared in gonadotropes, but not thyrotropes. We conclude, therefore, that distinct and separable regulatory elements mediate the expression of the alpha-subunit gene in gonadotropes and thyrotropes.