Search

To understand the biology and evolution of ruminants, the cattle genome was sequenced to about sevenfold coverage. The cattle genome contains a minimum of 22,000 genes, with a core set of 14,345 orthologs shared among seven mammalian species of which 1217 are absent or undetected in noneutherian (marsupial or monotreme) genomes. Cattle-specific evolutionary breakpoint regions in chromosomes have a higher density of segmental duplications, enrichment of repetitive elements, and species-specific variations in genes associated with lactation and immune responsiveness. Genes involved in metabolism are generally highly conserved, although five metabolic genes are deleted or extensively diverged from their human orthologs. The cattle genome sequence thus provides a resource for understanding mammalian evolution and accelerating livestock genetic improvement for milk and meat production.

The imprints of domestication and breed development on the genomes of livestock likely differ from those of companion animals. A deep draft sequence assembly of shotgun reads from a single Hereford female and comparative sequences sampled from six additional breeds were used to develop probes to interrogate 37,470 single-nucleotide polymorphisms (SNPs) in 497 cattle from 19 geographically and biologically diverse breeds. These data show that cattle have undergone a rapid recent decrease in effective population size from a very large ancestral population, possibly due to bottlenecks associated with domestication, selection, and breed formation. Domestication and artificial selection appear to have left detectable signatures of selection within the cattle genome, yet the current levels of diversity within breeds are at least as great as exists within humans.

The National Research Initiative (NRI) Competitive Grants Program is the U.S. Department of Agriculture's major competitive grants program and is administered by the Cooperative State Research, Education, and Extension Service (CSREES). Since its inception in 1991, the NRI has funded competitive grants in the discipline of animal reproduction. Previously, this program provided funding for a broad range of projects encompassing almost every subdiscipline in reproductive biology of farm animals, including aquatic species important to the aquaculture industry. During fiscal year 2004, the NRI Animal Reproduction Program narrowed the focus of funding priorities to the topics of infertility, basic mechanisms regulating fertility, cryopreservation of gametes, reducing the postpartum interval to conception, and sterilization methods or development of monosex populations. In response to a directive to further narrow the focus of funding priorities for fiscal year 2005 and beyond, CSREES conducted a Stakeholder Workshop on Funding Priorities in Animal Reproduction at the 37th Annual Meeting of the Society for the Study of Reproduction in Vancouver, Canada. More than 75 stakeholder scientists from a cross section of federal, public, and private institutions from across the United States participated in the workshop and provided recommendations to CSREES for future NRI-funding priorities in Animal Reproduction. The recommendations provided by stakeholders included continuing efforts to focus funding priorities into fewer high-impact areas relevant to animal agriculture and aquaculture. Recommendations also included movement back toward subdisciplines of animal reproduction that cut across all applicable species. The three funding priorities that consistently emerged as recommendations from the workshop participants were 1) gonadal function and production of gametes, 2) pituitary-hypothalamic function, and 3) embryo and conceptus development, including interaction between the conceptus and uterus. These funding priorities were considered when preparing the fiscal year 2006 NRI Request for Applications.

An electronic workshop was conducted on 4 November-13 December 2002 to discuss current issues and needs in animal bioinformatics. The electronic (e-mail listserver) format was chosen to provide a relatively speedy process that is broad in scope, cost-efficient and easily accessible to all participants. Approximately 40 panelists with diverse species and discipline expertise communicated through the panel e-mail listserver. The panel included scientists from academia, industry and government, in the USA, Australia and the UK. A second 'stakeholder' e-mail listserver was used to obtain input from a broad audience with general interests in animal genomics. The objectives of the electronic workshop were: (a) to define priorities for animal genome database development; and (b) to recommend ways in which the USDA could provide leadership in the area of animal genome database development. E-mail messages from panelists and stakeholders are archived at http://genome.cvm.umn.edu/bioinfo/. Priorities defined for animal genome database development included: (a) data repository; (b) tools for genome analysis; (c) annotation; (d) practical application of genomic data; and (e) a biological framework for DNA sequence. A stable source of funding, such as the USDA Agricultural Research Service (ARS), was recommended to support maintenance of data repositories and data curation. Continued support for competitive grants programs within the USDA Cooperative State Research, Education and Extension Service (CSREES) was recommended for tool development and hypothesis-driven research projects in genome analysis. Additional stakeholder input will be required to continuously refine priorities and maximize the use of limited resources for animal bioinformatics within the USDA.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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)

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.

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)

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)

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.

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.

Our primary objective for this study was to determine whether steady-state amounts of alpha- and LH beta-subunit mRNAs in the anterior pituitary are altered during sexual maturation in the bovine female. A secondary objective was to determine whether 17 beta-estradiol (E2) alters amounts of LH subunit mRNAs before onset of puberty. Heifers (7 mo old) were assigned to one of three treatments: 1) ovariectomized (OVX, n = 16); 2) OVX and administered E2 (OVXE, n = 16); or 3) ovary-intact (INTACT, n = 20). Pituitaries were collected at an estimated 120 days before onset of puberty (prepuberty) or 25 days before onset of puberty (peripuberty). Six INTACT heifers were used to determine time of puberty during the experimental period, and their pituitaries were collected 40 h after administration of prostaglandin F2 alpha (postpubertal INTACT group). Relative amounts of mRNAs for LH subunits in each pituitary were determined by Northern analysis and scanning densitometry. Amounts of alpha- and LH beta-subunit mRNAs were lower in pituitaries of INTACT heifers and OVXE heifers, regardless of stage of sexual maturation, than in those of OVX heifers. Amounts of alpha-subunit mRNA were similar in OVXE and INTACT heifers regardless of stage of sexual maturation. Amounts of LH beta-subunit mRNA did not change during sexual maturation in heifers in the INTACT group. Concentrations of E2 were higher and LH beta-subunit mRNA were lower in heifers from the prepubertal OVXE group than in heifers in all other treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)

To determine the acute and chronic effects of estradiol on synthesis and secretion of LH and FSH, ovariectomized ewes were administered estradiol via silastic capsules for 0 h, 12 h, 1 day, 2 days, 4 days, 8 days, 16 days, or 32 days (n = 5/group). Concentrations of GnRH in the median eminence began to decrease within 12 h and were lower (p less than 0.05) than in control ewes from 1 to 4 days after estradiol administration was begun. Serum concentrations of LH were decreased relative to pretreatment control levels from 1 to 10 h, elevated during a preovulatory-like surge from 11 to 22 h, and then decreased and remained below 1 ng/ml for the duration of the experiment. Serum concentrations of FSH followed a pattern similar to those for LH except that the magnitude of change was smaller. Treatment with estradiol initially (12 h) reduced (p less than 0.05) quantities of mRNA for alpha-, LH beta-, and FSH beta-subunits, after which the quantities of mRNA for the subunits returned to near or above control levels by Day 2. After 8 days of treatment the amounts of mRNAs for gonadotropin subunits were again less (p less than 0.05) than those of controls, and they remained suppressed through Day 32. Pituitary concentrations of LH and FSH decreased (p less than 0.05) during the first day of treatment and remained suppressed for the duration of the experiment. Thus, estradiol had a triphasic effect on secretion of gonadotropins and steady-state levels of mRNA for the gonadotropin subunits, but not on pituitary content of gonadotropins.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic administration of estradiol inhibits transcription of the gene encoding the alpha-subunit of pituitary glycoprotein hormones. Here, we show, using transfection analyses and a filter binding assay, that 1500 basepairs of proximal 5' flanking sequence of the human alpha-subunit gene lack a functional estrogen response element when transfected into heterologous cell lines, and fail to bind estrogen receptor purified from calf uterus. Yet, this same region of the alpha-subunit gene confers estradiol responsiveness (transcriptional suppression) to the bacterial chloramphenicol acetyltransferase gene in transgenic mice. A smaller promoter fragment of the bovine alpha-subunit gene also confers responsiveness to estradiol in transgenic mice, suggesting that the same element may mediate the steroid responsiveness of both promoters. Furthermore, regulation by estradiol of the chimeric human or bovine alpha-chloramphenicol acetyltransferase genes is pituitary specific, underscoring the physiological significance of these studies. Based on these results, we conclude that estradiol regulates expression of the alpha-subunit gene in vivo through a mechanism that does not involve high affinity binding of estrogen receptor to the alpha-subunit gene. Whether this mechanism is manifest at the level of the pituitary or hypothalamus remains to be determined.

Expression of the glycoprotein hormone alpha-subunit gene occurs in the pituitary of all mammals but in placenta of only primates and horses. In humans, two different elements, termed upstream regulatory element (URE) and cAMP response element (CRE), are required for placenta-specific expression of the alpha-subunit gene. The URE binds a protein unique to placenta whereas the CRE binds a ubiquitous protein. Comparative analysis of the promoter-regulatory region of the alpha-subunit gene from a number of mammals indicates that a functional URE has been retained and suggests the potential for placenta-specific expression. Indirect evidence also indicates that the URE-binding protein has been conserved, even in placenta from mammals that fail to express the alpha-subunit gene. Lack of expression of the alpha-subunit gene in placenta of rodents and cattle can be traced to a single nucleotide change that renders the CRE-like sequence of these genes incapable of binding the protein that confers responsiveness to cAMP. In contrast, although expression of the alpha-subunit gene occurs in horse placenta, the promoter-regulatory region lacks a functional CRE but appears to retain a functional URE. This suggests that either a different accessory element and cognate protein interacts with the horse URE to provide placenta-specific expression or that a completely different set of regulatory elements is required for placenta-specific expression in horses.

Expression of the glycoprotein hormone alpha-subunit gene occurs in the pituitaries of all mammals and in the placentas of primates and horses. In humans, tandem cAMP response elements (CREs), located in the proximal promoter-regulatory region of the alpha-subunit gene, act together with an adjacent upstream regulatory element to confer placenta-specific expression. Here, we report that the alpha-subunit genes of Old World Monkeys contain a single functional CRE. This suggests that tandem CREs are unique to higher primates and humans and are not absolutely required for placenta-specific expression. In contrast, the comparable promoter-regulatory region of the horse alpha-subunit gene lacks a functional CRE but appears to retain a functional upstream regulatory element. This suggests that acquisition of placenta-specific expression of the alpha-subunit gene occurred independently in these distantly related mammals. As a result, different combinations of cis-acting elements may explain why expression of the alpha-subunit gene only occurs in placenta of primates and horses.

cAMP stimulates transcription of the human glycoprotein hormone alpha-subunit gene in choriocarcinoma cells. Combined treatment with phorbol esters potentiates this effect. Tandem cAMP response elements (CREs) in the proximal 5'-flanking sequence mediate the effect of cAMP. In this report, we show that the CREs can also mediate the synergistic effect of phorbol esters. In addition to serving as an inducible cis-acting element, the two CREs act synergistically to increase basal transcription. We now provide direct evidence via equilibrium binding studies that tandem CREs bind their trans-acting factors cooperatively. However, the level of cooperativity is insufficient to explain the high degree of transcriptional synergism, suggesting that another element in the alpha-subunit promoter may be required for complete synergism. In support of this hypothesis, we show that synergism is drastically reduced when the CREs are removed from the context of their native promoter and linked to a heterologous promoter. Thus, amplification of the transcriptional signal mediated by the tandem CREs occurs through at least three distinct mechanisms. First, at the level of signal transduction by convergence of the A- and C-kinase pathways; second, through homotropic interactions of trans-acting factors binding to tandem CREs; and finally through heterotropic interaction of the two CREs with another, as yet, undefined cis-acting element(s) in the human alpha-subunit promoter.

Twenty-three crossbred beef cows between 4 and 5 yr of age were assigned at random to one of six treatments: (1) ovariectomized 4 d postpartum (OVX) with early weaning of calves 21 d postpartum (OVX-EW; n = 4), (2) OVX-EW and 17 beta-estradiol implants (OVX-E2-EW; n = 4), (3) OVX and normal nursing by calves throughout the experiment (OVX-NN; n = 3), (4) OVX-NN and 17 beta-estradiol implants (OVX-E2-NN; n = 4), (5) intact cows and early weaning of calves 21 d postpartum (EW), (6) intact cows and normal nursed (NN). Blood was collected at 15-min intervals over a 4-h period once weekly during the 12-wk postpartum period in the OVX cows. Early weaned intact cows exhibited estrus 23 d sooner (P less than .05) than normally nursed cows. A hormone level for each cow at each week was determined from the mean of the 17 samples collected over the 4 h period each week. There were no significant changes due to E2 treatment, for concentrations of LH, FSH or number of pulses during wk 1 through 3. However, during wk 4 through 12 the linear and quadratic contrasts of wk X estrogen X nursing were significant for serum LH, indicating there was no difference between the treatments for EW and NN without E2 but there was a large difference in the presence of E2. During nursing E2 suppressed serum LH below that of nonestrogen-treated cows while after weaning E2 stimulated LH release above that of nonestrogen-treated cows.(ABSTRACT TRUNCATED AT 250 WORDS)

Seventy-five crossbred heifers (mean weight 304 kg) were stratified by weight and source and placed in eight pens. Four duplicate treatments were randomly assigned to pens as follows: (1) intact-control (n = 19), (2) intact + melengestrol acetate (MGA, n = 20), (3) hysterectomy (n = 17) and (4) ovariectomy (OVX, n = 19). All heifers were preconditioned for 2 mo before beginning the finishing study. Surgery was performed through a high lumbar incision approximately 3 wk before the finishing study began. Group 2 heifers were fed .4 mg X head-1 X d-1 MGA. Blood samples were collected at 28-d intervals throughout the 112-d finishing phase. Ovaries were collected at slaughter and carcass data were obtained 24 h after slaughter. On d 112, progesterone was higher (P less than .05) in hysterectomized heifers than in other treatment groups. Serum progesterone concentration was nondetectable (less than .35 ng/ml) in OVX and MGA heifers. There were no differences (P greater than .05) in feed efficiency or carcass characteristics among the four treatments. Hysterectomized and MGA heifers had similar (P greater than .05) average daily gains, which were higher (P less than .15) than control and OVX heifers. These data suggest that the elevated levels of progesterone from the maintained corpora lutea and the subsequent absence of estrous activity in the hysterectomized heifers are advantageous for promoting growth in feedlot heifers.

To evaluate the effect of progesterone on the synthesis and secretion of gonadotropins, ovariectomized ewes either were treated with progesterone (n = 5) for 3 wk or served as controls (n = 5) during the anestrous season. After treatment for 3 wk, blood samples were collected from progesterone-treated and ovariectomized ewes. After collection of blood samples, hypothalamic and hypophyseal tissues were collected from all ewes. Half of each pituitary was used to determine the content of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and the number of receptors for gonadotropin-releasing hormone (GnRH). The amounts of mRNA for LH beta subunit, FSH beta subunit, alpha subunit, growth hormone, and prolactin were measured in the other half of each pituitary. Treatment with progesterone reduced mean serum concentrations of LH (p less than 0.001) but ot FSH (p greater than 0.05). Further, progesterone decreased (p less than 0.05) the total number of pulses of LH. We were unable to detect pulsatile release of FSH. Hypothalamic content of GnRH, number of receptors for GnRH, pituitary content of gonadotropins and mRNA for LH beta subunit, FSH beta subunit, alpha subunit, growth hormone, and prolactin were not affected (p greater than 0.05) by treatment with progesterone. Thus, after treatment with progesterone, serum concentrations of LH (but not FSH) are decreased. This effect, however, is not due to a decrease in the steady-state amount of mRNA for LH beta or alpha subunits.

To investigate the role of GnRH in regulating the synthesis and secretion of gonadotropins, GnRH (250 ng/6 min every other hour for 7 days) or saline was administered to ovariectomized (OVX) ewes after hypothalamic-pituitary disconnection (HPD). Blood samples were collected from all HPD ewes on the day before and the day after HPD and on days 1 and 7 of GnRH or saline. At the end of day 7, anterior pituitary glands were removed for analysis of hormone, receptor, and mRNA content. The amount of mRNA for gonadotropins was lower (P less than 0.05) in saline-treated HPD ewes than in GnRH-treated HPD or OVX ewes. Administration of GnRH restored the amount of mRNA for FSH beta and alpha-subunits to levels similar (P greater than 0.05) to those measured in OVX ewes. The amount of mRNA for LH beta was higher (P less than 0.05) in GnRH-treated HPD ewes than in saline-treated HPD ewes, but lower (P less than 0.05) than that in OVX ewes. The pituitary content of LH and FSH was lower (P less than 0.05) in saline-treated HPD ewes than in OVX ewes. Administration of GnRH to HPD ewes maintained the ewes. Administration of GnRH to HPD ewes maintained the pituitary content of LH, but not FSH, compared to the pituitary gonadotropin content in OVX ewes. There were no differences (P greater than 0.05) in the amount of mRNA for GH or PRL or the pituitary content of these hormones among treatments. The number of hypophyseal receptors for GnRH was reduced in saline-treated HPD ewes (P less than 0.05) compared to that in OVX ewes and GnRH-treated HPD ewes. The number of hypophyseal receptors for 17 beta-estradiol was lower (P less than 0.05) in GnRH- and saline-treated HPD ewes than in OVX ewes. Serum LH concentrations were lower (P less than 0.05) after HPD than before HPD, but were restored to normal (P greater than 0.05) by GnRH replacement. Serum concentrations of FSH were lower (P less than 0.05) after HPD and were not affected by GnRH replacement. Serum PRL concentrations in all ewes were higher (P less than 0.05) after HPD than before HPD. Serum GH concentrations in all ewes were similar (P greater than 0.05) before and after HPD. Since synthesis and secretion of GH and PRL were not diminished after HPD, it was considered that the pituitary gland remained viable and functioned independently of hypothalamic input in OVX ewes after HPD.(ABSTRACT TRUNCATED AT 400 WORDS)

A transnasal, transsphenoidal surgical approach was used to perform hypothalamic pituitary disconnections (HPD) in ovariectomized (OVX) ewes to examine the role of the hypothalamus in regulating the synthesis of anterior pituitary hormones. Ewes were killed at 1-3 days (n = 6), 1 week (n = 5), or 1 month (n = 5) after HPD. Pituitary glands were removed, and hemisected for analysis of hormone or messenger RNA (mRNA) content. Blot hybridization using specific complementary DNA probes was used to quantify the concentration of mRNA for LH beta-subunit, alpha-subunit, GH, and PRL. Concentrations of mRNA for LH beta- and alpha-subunits were lower (P less than 0.01) at 1-3 days after HPD than in OVX ewes. At 1 week and 1 month after HPD, concentrations of mRNA for LH beta- and alpha-subunits were near the lower limit of detection of this assay system. In contrast, for 30 days after HPD, pituitary concentrations of mRNA for GH and PRL were not different (P greater than 0.05) from those in OVX ewes. At 1 week and 1 month after HPD, pituitary content of LH, FSH, and GH was lower (P less than 0.01) than in OXV ewes. Pituitary PRL content in all HPD ewes was lower (P less than 0.05) than in OVX ewes. In a separate group of five ewes that were bled daily for 30 days after HPD, serum concentrations of LH and FSH fell dramatically during the first 3 days after HPD. In contrast, serum concentrations of GH and PRL remained similar to pre-HPD concentrations for 30 days after HPD. Thus, hypothalamic stimulation is essential for maintaining the concentration of mRNA for LH beta- and alpha-subunits within the anterior pituitary gland. Without continued hypothalamic support, pituitary and serum concentrations of LH and FSH rapidly decline. In contrast, concentrations of mRNA for GH and PRL are maintained in the absence of hypothalamic input.

The single-copy gene encoding the alpha subunit of glycoprotein hormones is expressed in the pituitaries of all mammals and in the placentas of only primates and horses. We have systematically analyzed the promoter-regulatory elements of the human and bovine alpha-subunit genes to elucidate the molecular mechanisms underlying their divergent patterns of tissue-specific expression. This analysis entailed the use of transient expression assays in a chorionic gonadotropin-secreting human choriocarcinoma cell line, protein-DNA binding assays, and expression of chimeric forms of human or bovine alpha subunit genes in transgenic mice. From the results, we conclude that placental expression of the human alpha-subunit gene requires a functional cyclic AMP response element (CRE) that is present as a tandem repeat in the promoter-regulatory region. In contrast, the promoter-regulatory region of the bovine alpha-subunit gene, as well as of the rat and mouse genes, was found to contain a single CRE homolog that differed from its human counterpart by a single nucleotide. This difference substantially reduced the binding affinity of the bovine CRE homolog for the nuclear protein that bound to the human alpha CRE and thereby rendered the bovine alpha-subunit promoter inactive in human choriocarcinoma cells. However, conversion of the bovine alpha CRE homolog to an authentic alpha CRE restored activity to the bovine alpha-subunit promoter in choriocarcinoma cells. Similarly, a human but not a bovine alpha transgene was expressed in placenta in transgenic mice. Thus, placenta-specific expression of the human alpha-subunit gene may be the consequence of the recent evolution of a functional CRE. Expression of the human alpha transgene in mouse placenta further suggests that evolution of placenta-specific trans-acting factors preceded the appearance of this element. Finally, in contrast to their divergent patterns of placental expression, both the human and bovine alpha-subunit transgenes were expressed in mouse pituitary, indicating differences in the composition of the enhancers required for pituitary- and placenta-specific expression.

This study was conducted to determine the effects of testosterone-propionate exposure during fetal development on sexual differentiation and growth rates in heifers. Ten pregnant cows were given subcutaneous injections of testosterone-propionate (250 mg/injection) every other day during d 40 to 60 of gestation. Four cows aborted after the end of testosterone treatment, while four heifers (androgenized females) and two bulls (androgenized males) were produced from the six remaining pregnant, testosterone-propionate treated cows. Calves from cows that did not receive exogenous hormone treatment were used as controls. At 8 mo of age, the androgenized heifers and control heifers and control steers were challenged with 1 mg estradiol-17 beta to induce a preovulatory luteinizing hormone (LH) surge. Two weeks later, pituitary responsiveness to exogenous luteinizing hormone releasing hormone (LHRH; 75 micrograms) was evaluated in androgenized heifers and in control heifers and control steers. To monitor growth rates, all animals were weighed at 28-d intervals from birth to 380 d of age. Androgenized females exhibited a partially masculinized phenotype as well as internal male reproductive structures. Treatment with estradiol-17 beta first depressed (P less than .05) serum LH concentrations in all animals, then induced (P less than .05) a preovulatory-like LH surge in control and androgenized females. Control steers did not (P greater than .05) exhibit a preovulatory-like LH surge following administration of estradiol-17 beta. Exogenous LHRH treatment stimulated peak LH concentrations (P less than .05) to a greater extent in control and androgenized females than in control steers.(ABSTRACT TRUNCATED AT 250 WORDS)

The amount of messenger RNA (mRNA) for luteinizing hormone beta-subunit (LH beta), follicle-stimulating hormone beta-subunit (FSH beta) and alpha-subunit was measured during estradiol-17 beta (E) positive feedback in ovariectomized (OVX) ewes. During the anestrous season, OVX ewes were given an i.m. injection of E (25 micrograms: n = 5) or oil (control; n = 4) and hourly blood samples were collected for 16 hr. After blood collection, ewes were killed and anterior pituitary glands were removed for analysis of hormone and mRNA content. Preovulatory-like increases in serum concentrations of LH and FSH were measured in E-treated OVX ewes. In two E-treated OVX ewes the serum concentrations of LH and FSH were still increasing, whereas in the remaining three E-treated OVX ewes, serum concentrations of LH were on the decreasing portion of the E-induced preovulatory-like surge. Pituitary content of LH was lower (P less than .10) in E-treated OVX ewes when serum concentrations of LH were decreasing than that measured in control ewes or E-treated OVX ewes in which serum concentrations were still increasing. Pituitary content of FSH and prolactin were similar (P greater than .05) among all groups. The amount of mRNA for LH beta-subunit was similar (P greater than .05) in ewes in which serum concentrations of LH were increasing and in control ewes, but was lower (P less than .05) in ewes with decreasing levels of LH. The amount of mRNA for FSH beta-subunit was lower (P less than .05) in all E-treated OVX ewes (independent of whether serum concentrations of FSH were increasing or decreasing) than that measured in control ewes. There was no difference (P greater than .05) in the amount of mRNA for alpha-subunit among any groups. Thus, amounts of mRNA for the beta-subunits of gonadotropins are reduced, while amounts of mRNA for alpha-subunit are unchanged during estradiol positive feedback in OVX ewes.