Transfer of an ovine metallothionein-ovine growth hormone fusion gene into swine.

An ovine metallothionein-1a (oMT1a)-ovine growth hormone (oGH) fusion gene was microinjected into 400 pig zygotes, the zygotes were transferred into recipient females, and 15 founder transgenic pigs were born. Of 12 transgenic pigs assayed, five expressed high levels of oGH (> 900 ng/mL plasma), one expressed low levels of oGH (10 to 30 ng/mL), and six did not express oGH. Dietary supplementation with 2,000 ppm of zinc for 6 d induced a 20-fold increase in plasma oGH in the transgenic pig with low expression but did not induce expression in the six transgenic pigs with no constitutive oGH expression. The average daily gain of five transgenic pigs with elevated oGH was similar to that of non-transgenic littermates during a 9-wk feeding trial (P = .52). The liver, kidney, adrenal, and thyroid weights were all significantly heavier for the oGH-expressing transgenic pigs than for non-transgenic littermates. Total carcass fat, longissimus muscle fat, subcutaneous backfat thickness, and loin eye area were lower and carcass protein and water content and beta R fiber area of longissimus muscle were higher in the transgenic pigs with elevated oGH than in their littermate controls (P < .05 for each). The data indicate that even though the oMT1a promoter was more inducible by zinc than was previously reported for the mouse MT promoter in swine, the former provided a higher level of oGH expression than the mouse MT promoter.

Lipid composition of carcass tissue from transgenic pigs expressing a bovine growth hormone gene.

Fatty acid profiles and cholesterol content of whole-carcass ground tissue were compared from 26 transgenic (T) pigs expressing a bovine growth hormone gene (bGH) to 26 sibling control (C) pigs. All pigs were fed a common diet and were slaughtered at five different live weights: 14, 28, 48, 68, and 92 kg. The left side of each intact carcass was ground and tissue samples were analyzed for lipid composition and cholesterol content. At 14-kg body weight, carcasses from bGH-T pigs contained 38% less fat, 44% less saturated fatty acids (SFA), 48% less monounsaturated fatty acids (MUFA), and 38% less polyunsaturated fatty acids (PUFA) than C pigs. At 28 kg, bGH-T pigs had 38% less total carcass fat, 42% less SFA, 46% less MUFA, and 24% less PUFA than C pigs. At 48-kg body weight, bGH-T pigs contained 48% less carcass fat, 55% less SFA, 59% less MUFA, and 22% less PUFA than C pigs. At 68 kg, bGH-T pigs had 78% less carcass fat, 78% less SFA, 79% less MUFA, and 53% less PUFA than C pigs. At 92 kg, carcasses from bGH-T pigs contained 85% less carcass fat, 85% less SFA, 91% less MUFA, and 66% less PUFA than those from C pigs. Cholesterol content was not different between bGH-T pigs and C pigs at any of the carcass weights. The trend was for cholesterol content to decrease from the 14- to 92-kg weight group. These results suggest a dilution effect of carcass fat and fatty acids in carcass tissue from bGH-T pigs with increasing live weight.

Expression of a bovine growth hormone transgene inhibits pregnant mare's serum gonadotropin-induced follicle maturation in prepuberal gilts.

Prepuberal gilts were injected with PMSG to determine whether expression of a bovine growth hormone (bGH) transgene inhibited preovulatory maturation of ovarian follicles. Seven transgenic (TG) gilts of line 3706, which expresses a mouse metallothionein-bGH transgene, and eight nontransgenic, control (C) gilts (128 to 147 d old) were injected with PMSG, 12.5 IU/kg BW, 72 h before necropsy. Surface ovarian follicles > or = 1 mm in diameter were counted, measured for diameter, and aspirated for fluid. Follicles were classified morphologically as healthy or atretic and those with follicular fluid estradiol-17 beta > or = 100 ng/mL were classified as estrogenactive (EA). The number of follicles per gilt was 64.3 +/- 6.1 (mean +/- SEM) and did not differ significantly between bGH-TG and C gilts. The PMSG treatment induced growth of large (> 5 mm) follicles in both bGH-TG and C gilts. However, compared with C gilts, bGH-TG gilts had fewer (P < .05) large follicles (5.9 +/- 1.5 vs 18.3 +/- 5.4), a lower proportion of EA large follicles (35 +/- 12.5 vs 69 +/- 13.2%), and in large follicles less (P < .05) estradiol-17 beta (86 +/- 17 vs 350 +/- 69 ng/mL) and androstenedione (300 +/- 33 vs 1,283 +/- 221 ng/mL). Follicular fluid progesterone and inhibin did not differ significantly between bGH-TG and C gilts. The incidence of atresia among small and medium follicles did not differ significantly between bGH-TG and C gilts.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of an homologous radioimmunoassay for chicken follicle-stimulating hormone and measurement of plasma FSH during the ovulatory cycle.

1. A highly specific and sensitive homologous radioimmunoassay was developed for measurement of chicken follicle stimulating hormone (cFSH). 2. Mammalian gonadotropin releasing hormone (GnRH) significantly stimulated secretion of chicken luteinising hormone (cLH) but not cFSH when administered to 22 week non-laying hens. 3. Chicken GnRH-I did not affect circulating cFSH concentrations but significantly stimulated cLH secretion when administered to 3 week cockerels. 4. The plasma concentration of cFSH was low throughout the ovulatory cycle, but a significant decline in cFSH occurred prior to the pre-ovulatory LH surge and a significant increase occurred during the 3 hr prior to oviposition as LH levels decline.

Changes in follicular estradiol-17 beta, progesterone and inhibin immunoactivity in healthy and atretic follicles during preovulatory maturation in the pig.

Follicular hormones, growth and granulosa cell gonadotropin sensitive adenylate cyclase activity were determined in healthy and atretic follicles during preovulatory maturation in pigs. Ovaries were recovered at slaughter which was 1, 3, 5 or 7 d after the last administration of a progesterone agonist (altrenogest). Plasma FSH decreased (P < .05) by 64% between days 1 and 3 and remained low through day 5. The number of large (> 5 mm) follicles increased from 2.7 on day 1 to 14.8 on day 3 and did not differ significantly among days 3, 5 and 7. The number of small (1-2 mm) and medium (3-5 mm) follicles decreased (P < or = .05) by 82% between days 3 and 5. Follicles first became estrogen-active (EA) (> or = 100 ng of estradiol-17 beta/ml of follicular fluid) on day 3, with 14.3% of medium and 73.8% of large follicles being EA. About 30% of small and 13% of medium follicles were morphologically atretic on days 1 and 3. However, by day 5, the proportion of atretic small and medium follicles had increased (P < or = .05) to 100 and 59%, respectively. Follicular fluid inhibin immunoactivity and estradiol-17 beta were lower (P < or = .05) and progesterone was greater (P < or = .05) in atretic than healthy follicles. Granulosa cells from large follicles produced (P < or = .05) more cAMP than cells from healthy or atretic small/medium follicles. Compared to control or pFSH treatment, pLH increased cAMP production by granulosa cells from large follicles on all days and from small/medium follicles on days 1 and 5; pLH had no effect on granulosa cells from atretic follicles. Compared to control, pFSH increased cAMP production in granulosa cells from healthy small/medium follicles only on day 1; no effect was detected in granulosa cells from large or atretic follicles on any day. We conclude that decreased secretion of FSH increased loss and atresia among non-ovulatory follicles. Atretic follicles were marked by loss of granulosa cell gonadotropin-sensitive adenylate cyclase activity and by low concentrations of estradiol-17 beta.

Two-dimensional electrophoresis and densitometric analysis of solubilized bovine sperm plasma membrane proteins detected by silver staining and radioiodination.

Bovine sperm plasma membranes were extracted with deoxycholate and subjected to two-dimensional gel electrophoresis. Proteins were visualized either by silver staining or autoradiography using 125I. When 1.5-2.0 micrograms of protein extract was applied to the first-dimension get, 250 spots could be detected by autoradiography. Thirty micrograms of protein was required to obtain spot visualization using silver strain, revealing more than 500 spots on the gel. These data establish the limit under which two-dimensional electrophoresis can be used in conjunction with flow cytometric sorting of sperm for the analysis of possible sex-specific membrane differences. The necessity of orienting sperm as they past the laser in the cell sorting system reduces throughput; thus, the number of sperm sorted within a given time is limited. It is suggested that autoradiography will allow the flow cytometer to be used in a time-efficient manner. Longer sorting times would be required to obtain sufficient sample to analyze total protein composition.

Inhibin-like activity in plasma from ovariectomized ewes and serum albumin: pitfalls for radioimmunoassay.

Progress to understand mechanisms that regulate inhibin secretion and action in farm animals has been handicapped by the shortage of simple, accurate assay methods to quantify inhibin in circulation. RIA would seem to provide the needed quantitative capability, but results of the following studies using inhibin RIA procedures reveal reasons to interpret inhibin immunological potency estimates with caution. Two sets of inhibin RIA reagents and various assay buffers were used. Initially, inhibin immunoactivity was estimated with an antiserum to a 32 amino acid peptide fragment from the alpha subunit of porcine inhibin [pI alpha(1-32)] and tracer to the peptide with tyrosine added in position 0 to permit radioiodination, pI alpha(Tyr1-32). Later, an antiserum to pI alpha(1-29Tyr30) peptide and pI alpha(1-29Tyr30) tracer was evaluated as were several combinations of assay buffer and assay conditions. Both sets of assay reagents provided quantitative recovery of pI alpha(1-32) peptide from plasma, parallel response between the peptide and either ovine or bovine plasma, as well as adequate sensitivity to measure inhibin immunoactivity in 25 microliters of plasma. However, plasma from long-term ovariectomized female sheep, swine or cattle appeared to contain nearly as much inhibin immunoactivity as intact animals. To explore the possibility that the adrenals may produce sufficient inhibin to account for unexplained high levels of inhibin immunoactivity in plasma from ovariectomized animals, ewes on days 12 and 13 of the estrous cycle were injected with either corn oil (CONT) or large doses of an adrenal steroid agonist, dexamethasone (DEX), to alter adrenal function. Likewise, ewes were either ovariectomized (OVX) on day 12 or injected on days 12 and 13 with estradiol-17 beta plus progesterone (E2 + P4) to alter ovarian function. The plasma concentration of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) increased following ovariectomy (P less than .001), and LH decreased following ovarian steroids (P less than .001). Treatment with DEX did not change plasma gonadotropin values (P greater than .1). When plasma was assayed using pI alpha(1-32) reagents and an assay buffer consisting of gelatin/phosphate/Tween-20 (GelT20), inhibin immunoactivity was not affected by any of the four treatments (P greater than .1), even including ovariectomy. Re-assay of these same samples with an RIA procedure that used gelT20 assay buffer and pI alpha(1-29Tyr30) reagents produced good agreement with the previous assay (partial correlation P less than .0001), but there was no statistical evidence that ovariectomy or treatment with ovarian or adrenal steroids changed the level of immunoassayable inhibin in plasma.(ABSTRACT TRUNCATED AT 400 WORDS)

Transferrin- and albumin-directed expression of growth-related peptides in transgenic sheep.

Chimeric genes containing either the mouse transferrin (Trf) enhancer/promoter fused to the structural sequences encoding bovine growth hormone (GH) or the mouse albumin (Alb) enhancer/promoter fused to the gene for human growth hormone-releasing factor (GRF) were microinjected into sheep zygotes. A low percentage of resulting transgenic sheep chronically expressed the respective genes, resulting in elevated plasma concentrations of circulating GH or GRF, respectively. Growth hormone-releasing factor expression induced elevated plasma levels of endogenous GH production. In addition, elevated levels of circulating insulin-like growth factor-I were observed in the bovine GH-expressing Trf transgenic sheep. Growth of these founder transgenic sheep relative to controls were not enhanced. In part, this may be due to the development of the diabetic condition exhibited by both transgenic groups. These results demonstrate that the mouse Trf and Alb enhancer/promoters are active in sheep and suggest that alternate strategies for expressing growth-related genes may be required to modulate growth in sheep.

Effect of bovine growth hormone gene expression, sex and age on plasma gonadotropins, estrone and testosterone in prepuberal pigs.

Chronic supraphysiological blood levels of growth hormone (GH) may retard sexual maturation in swine. Pigs used in this study included four founder transgenic pigs (two gilts and two boars) expressing a mouse transferrin (TF) promoter fused to a bovine (b) GH structural gene, 13 second- or third- generation transgenic pigs (seven gilts and six boars) expressing a mouse metallothionein (MT) promoter fused to a bGH structural gene and 16 control littermates (eight gilts and eight boars). Blood plasma levels of LH, FSH, estrone and testosterone were measured to determine whether expression of bGH genes altered secretion of hormones between 80 and 180 days of age. Presence of a bGH gene was detected by hybridization of DNA in dot blots of tail biopsies. Expression of a bGH gene was detected by radioimmunoassay of plasma bGH. In four TFbGH founder transgenic pigs bGH ranged from 164 to 1948 ng/ml; in one MTbGH transgenic boar of line 3104 bGH was 1211 ng/ml; and in 12 pigs of line 3706 bGH ranged from 25 to 190 ng/ml. Expression of bGH in transgenic pigs lowered (P = .0192) plasma LH with no significant differences between sexes, had no significant effect on plasma FSH and lowered plasma estrone (P = .0001) and testosterone (P = .0269) in boars (but not gilts). Plasma estrone and testosterone were higher (P = .0001) in boars than in gilts. Plasma FSH was higher (P = .0001) in gilts than boars and decreased (P = .0001) with advancing age in gilts but not in boars.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of feeding calcium soaps to early postpartum dairy cows on plasma prostaglandin F2 alpha, luteinizing hormone, and follicular growth.

Multiparous Holstein cows (n = 18) were fed a total mixed ration containing corn silage, corn grain, whole cottonseed, soybean meal, dried distillers grains, and chopped bermudagrass hay (control) or same diet plus Ca salts of long-chain fatty acids (2.2% of diet DM) for the first 60 d postpartum. Predicted energy balance was calculated from DM intake, milk yield and composition, and BW. On d 25 postpartum, cows were injected with 25 mg of prostaglandin F2 alpha and treated for 15 d with an intravaginal device containing 1.9 g progesterone. Profiles of 15-keto-13,14-dihydro-prostaglandin F2 alpha (d 1 to 21) and plasma triglycerides (d 7 to 60) were similar between groups. Average number of follicles, determined by ultrasonography prior to d 25, tended to differ between groups; controls had more 3- to 5-mm and fewer 6- to 9-mm follicles than the group of fat-fed cows. Basal, smoothed mean concentration, and average luteinizing hormone amplitude, determined by 10-min samples for 8 h on d 10, were not significantly different between groups. Increasing predicted energy balance was associated with increased pulse amplitude and diameter of the largest follicle on d 10. During the progesterone treatment period and the postprogesterone treatment estrous cycle, cows fed fat had greater numbers of 3- to 5-mm and greater than 15-mm follicles. In conclusion, feeding fat did not influence 15-keto-13,14-dihydro-prostaglandin F2 alpha or luteinizing hormone dynamics but did alter the average number of follicles within different size classes and the diameter of largest and second largest follicle after progesterone treatment.

Effects of gonadotropin treatment on ovarian follicle growth and granulosal cell aromatase activity in prepuberal gilts.

This experiment was conducted to compare the ability of USDA porcine FSH-B-1 (pFSH), USDA porcine LH-B-1 (pLH), and pregnant mare's serum gonadotropin (PMSG) to grow large follicles and induce granulosal cell aromatase activity in prepuberal gilts. Twenty-four gilts (164 d old) received one of four treatments by i.m. injection: 1) saline once, n = 8; 2) pFSH (8 micrograms/kg BW, nine times at 8-h intervals), n = 5; 3) pLH (2 micrograms/kg BW, nine times at 8-h intervals), n = 6; or 4) PMSG (15 IU/kg BW, once), n = 5. At slaughter, 72 h after the first injection, the ovaries to saline-treated gilts contained an average of 104 surface antral follicles 1 to 3 mm in diameter. compared to treatment with saline, pFSH increased (P less than .05) the number of follicles 46%, whereas pLH or PMSG decreased (P less than .05) the number by 70 and 84%, respectively. Compared with saline, treatment with PMSG or pLH induced growth of large follicles (7 to 9 mm) (10.8 and 4.8 follicles/gilt, respectively), increased plasma estrogen, increased granulosal cell aromatase activity, and decreased plasma FSH by 51 and 69%; treatment with pFSH had no significant effect on these traits. Results indicate that injected pFSH did not cause growth of large follicles or induce granulosal cell aromatase activity in prepuberal gilts. In contrast, LH initiated growth and increased granulosal cell aromatase activity in a small number of follicles and accelerated atresia among the remaining follicles.

Expression and performance in transgenic pigs.

Recent research clearly shows that fusion genes can be microinjected into a pronucleus of an ovum and integrate into the pig genome. Animals with such fusion genes are called 'transgenic'. The percentage of injected ova that developed into transgenic pigs varied among experiments from 0.31% to 1.73%. The percentage of transgenic pigs that expressed the fusion gene ranged from 17% to 100%. Eleven different regulatory sequences have been used for fusion genes transferred into pigs. Some of these regulatory sequences directed strong gene expression, but control over level of expression was inadequate. Other regulatory sequences directed weak expression, but imparted only brief spikes of induced expression. The predominant gene coding sequences transferred were for growth-related hormones. Elevation of growth hormone (GH) in expressing transgenic pigs enhanced plasma concentrations of insulin-like growth factor-I (IGF-I), insulin, and glucose, improved feed efficiency about 15%, and markedly reduced subcutaneous fat compared to nontransgenic siblings. Growth rate was enhanced in some transgenic GH pigs but not in others, possibly due to dietary limits. The 'over-expression' of GH was detrimental to the general health of most transgenic pigs. The most prevalent problems were lethargy, lameness, and gastric ulcers. Gilts that expressed foreign GH genes were anoestrous. Boars that expressed foreign GH genes lacked libido, but their semen was fertile when used by artificial insemination. Six different fusion genes have been transmitted from transgenic founders to progeny. Most of the transgenic pigs that produced progeny transmitted the fusion gene as an autosomal dominant trait to about half of their progeny. Regulatory sequences that will permit full control of gene expression must be developed before the full potential of gene transfer in pigs can be realized.

Integration, expression and germ-line transmission of growth-related genes in pigs.

We have produced transgenic pigs that harbour structural genes for bovine and human growth hormone (bGH and hGH) ligated to a mouse metallothionein-I (MT) promoter, human growth hormone-releasing factor (hGRF) ligated to the MT or mouse albumin (ALB) promoter, and human insulin-like growth factor-I (hIGF-I) ligated to MT promoter. From 0.31 to 1.03% of microinjected ova developed into transgenic pigs with the various fusion genes. Foreign GH was present in plasma of 61% of the MT-hGH and 89% of the MT-bGH transgenic pigs. Two of 7 pigs with MT-hGRF and all 3 ALB-hGRF transgenic pigs had high concentrations of GRF in their plasma, but plasma concentrations of porcine GH (pGH) were not higher in GRF transgenic pigs than in littermate control pigs. In contrast, plasma concentrations at birth ranged from 3 to 949 ng hGH/ml for MT-hGH transgenic pigs and 5 to 944 ng bGH/ml for MT-bGH transgenic pigs. Presence of the foreign GH depressed endogenous pGH to non-detectable levels. In MT-bGH transgenic pigs, plasma IGF-I was elevated more than 2-fold, plasma glucose was elevated about 30 mg/dl, and plasma insulin was 20-fold higher than in littermate or sibling control pigs. Two lines of pigs expressing the MT-bGH transgene gained 11.1% and 13.7% faster, and were 18% more efficient in converting feed to body weight gain than were sibling control pigs. Expression of the MT-bGH transgene caused a marked repartitioning of nutrients from subcutaneous fat into other carcass components, including muscle, skin, bone and certain organs. The persistent excess hGH or bGH in transgenic pigs was detrimental to general health; lameness, lethargy and gastric ulcers were the most prevalent problems. Gilts that expressed the hGH or bGH transgenes were anoestrous. Germ-line transmission was obtained in 4 of 5 expressing transgenic boars and 4 of 5 nonexpressing transgenic boars and gilts. From 2% to 73% of progeny inherited a transgene from founder transgenics. All transgenic progeny of MT-hGH, MT-bGH and MT-hGRF founder males expressed the transgene if their sire also expressed the gene. The concentration of bGH or hGH in plasma of transgenic progeny was similar to the concentration present in the founder transgenic.

Changes in plasma follicle-stimulating hormone, luteinizing hormone, estrogen and progesterone during growth of ovulatory follicles in the pig.

This experiment was conducted to determine the changes in secretion of LH, FSH, estrogen and progesterone during follicle maturation. Ovaries were recovered from 11 non-treated (control) gilts, three on day 13, four on day 16, and four on day 19 of the estrous cycle, and from four altrenogest-treated gilts on day 19. Altrenogest, a progesterone agonist, was fed at a dose of 20 mg once daily from days 13 to 18 to block spontaneous follicle maturation. Gilts were bled daily from day 12 until slaughter. For control gilts, the number of follicles/gilt 1-6 mm in diameter decreased (P less than .05) from 93.5 on day 13 to 21.5 on day 19, and the number of large (greater than 6 mm) follicles increased (P less than .05) from 5.3 to 13.2. Altrenogest treatment blocked loss of small follicles and growth of large follicles between days 13 and 19. Plasma progesterone decreased (P less than .001) between days 12 and 16 in both control and altrenogest-treated gilts. Plasma FSH decreased (P less than .05) between days 12 and 16 only in control gilts. Plasma LH was not significantly affected by day or altrenogest treatment. Plasma estrogen increased (P less than .05) between days 15 and 19 only in control gilts. These results indicate that 1) no increased LH secretion was detected in conjunction with emergence of ovulatory follicles, and 2) atresia of nonovulatory follicles was associated with decreased secretion of FSH. Both atresia and decreasing FSH secretion began before estrogen concentration increased in the systemic circulation.

Genetic engineering of livestock.

Genetic engineering of livestock is expected to have a major effect on the agricultural industry. However, accurate assessment of the consequences of transgene expression is impossible without multigenerational studies. A systematic study of the beneficial and adverse consequences of long-term elevations in the plasma levels of bovine growth hormone (bGH) was conducted on two lines of transgenic pigs. Two successive generations of pigs expressing the bGH gene showed significant improvements in both daily weight gain and feed efficiency and exhibited changes in carcass composition that included a marked reduction in subcutaneous fat. However, long-term elevation of bGH was generally detrimental to health: the pigs had a high incidence of gastric ulcers, arthritis, cardiomegaly, dermatitis, and renal disease. The ability to produce pigs exhibiting only the beneficial, growth-promoting effects of growth hormone by a transgenic approach may require better control of transgene expression, a different genetic background, or a modified husbandry regimen.

Prolactin maintains relaxin and progesterone secretion by aging corpora lutea after hypophysial stalk transection or hypophysectomy in the pig.

Porcine corpora lutea persist beyond 150 days in hysterectomized animals compared with about 114 days during normal pregnancy. To explore the mechanism(s) regulating the peak release of relaxin and secretion of progesterone by aging corpora lutea and to examine the direct effect of purified porcine (p) PRL on such corpora lutea, hypophysial stalk transection (HST), hypophysectomy (HYPOX) with or without PRL replacement, and sham operation control (SOC) were conducted on day 110 (estrus = day 0) on purebred Yorkshire gilts that were hysterectomized on days 6-8. The pPRL (0.5 mg every 6 h daily) or PBS (0.5 ml every 6 h daily) was given iv from days 110-120. HYPOX + pPRL, HYPOX + PBS, HST + PBS, and SOC + PBS formed four experimental groups. Peak relaxin concentrations in peripheral plasma (mean values ranged from 22-24 ng/ml) occurred on about day 113 for all groups [113.4 +/- 0.3 days (+/- SE)] regardless of the different surgical interventions. After peak release, relaxin decreased steadily in the HYPOX + PBS group, falling to less than 1.0 ng/ml by 6 days later, whereas relaxin in other groups remained elevated (approximately 7 ng/ml). In the HYPOX plus PBS group, progesterone decreased abruptly, remaining below 1 ng/ml from 1 week onward, lower (P less than 0.01) than that in controls (approximately 19 ng/ml); in the HYPOX + pPRL group, progesterone levels (approximately 17 ng/ml) remained similar (P greater than 0.05) to those in controls (approximately 19 ng/ml) and the HST + PBS group (approximately 15 ng/ml). These results clearly reveal that the pituitary gland plays no direct role in regulating the timed peak release of relaxin from aging corpora lutea in hysterectomized gilts and that the peak release of relaxin on about day 113 is preprogrammed and inherent within such aging luteal cells. This study provides strong evidence that purified pPRL maintains both relaxin and progesterone secretion as well as the morphology of aging corpora lutea for at least 10 days after hypophysectomy in hysterectomized gilts.

Expression of human or bovine growth hormone gene with a mouse metallothionein-1 promoter in transgenic swine alters the secretion of porcine growth hormone and insulin-like growth factor-I.

Endocrine profiles were examined in swine that had integrated and expressed a fusion gene consisting of mouse metallothionein-1 (MT) promoter fused to either a human (h) or bovine (b) GH structural gene. Eleven of 18 pigs that had integrated MT-hGH and eight of nine pigs that had integrated MT-bGH expressed the genes. The level of expression varied widely among pigs (14-4551 micrograms/l for MT-hGH and 23-1578 micrograms/l for MT-bGH). The level of expression varied over time within each pig with no general pattern. Concentrations of porcine GH (pGH) were lower in MT-hGH pigs that expressed the gene than in non-expressors or in littermate controls. Insulin-like growth factor-I (IGF-I) concentrations increased with age in all pigs and were raised threefold in pigs expressing either the MT-hGH or MT-bGH genes. Measurement of the foreign GH in samples taken at 15-min intervals failed to reveal any short-term fluctuations in concentration. Administration of hGH releasing factor (GRF) to pigs expressing MT-bGH resulted in attenuated release of pGH compared with that of contemporary controls. Concentrations of bGH did not change after GRF injection. Human and bovine GH expressed in transgenic pigs appear to be biologically active in that they induce IGF-I and suppress endogenous pGH secretion. The failure to find short-term fluctuations and the lack of response to GRF injections are consistent with a non-pituitary and non-GRF regulatable site of production.

Production of transgenic sheep with growth-regulating genes.

Pronuclei of fertilized sheep ova were injected with fusion genes consisting of the mouse metallothionein-I promotor/regulator ligated to either the structural gene for bovine growth hormone (mMTbGH) or to a minigene for human growth hormone-releasing factor (mMThGRF). From a total of 842 sheep ova injected with mMTbGH and transferred into recipient ewes, 47 lambs were born. Two of the lambs were transgenic with mMTbGH, and both had bGH mRNA present in liver, kidney, and gut. In one lamb, plasma growth hormone was as high as 700 ng/ml. From a total of 435 sheep ova injected with mMThGRF and transferred to recipients, 54 lambs were born and 9 fetuses were collected. Nine of the 63 had integrated the mMThGRF gene. One of the nine had high concentrations of immunoassayable hGRF in its plasma and high variable plasma concentrations of ovine growth hormone. The lamb that expressed the hGRF gene did not release GH in response to an hGRF challenge. Four of five fetal offspring of a nonexpressing mMThGRF transgenic ram also contained the mMThGRF gene and, like the sire, failed to express the gene as determined by either liver hGRF mRNA or by plasma hGRF. Growth of the single transgenic lamb expressing hGRF was similar to control lambs. These studies demonstrate efficient introduction of genes into the sheep genome and indicate that transgenes are expressed and heritable.

Relaxin and progesterone secretion as affected by luteinizing hormone and prolactin after hysterectomy in the pig.

Plasma levels of relaxin and progesterone in hysterectomized and pregnant gilts were determined from days 100-120 to evaluate the effects of purified porcine (p) LH and pPRL on the secretory activity of the aging corpora lutea. Gilts were bred on the second observed estrus or were hysterectomized between 6-8 days after estrus (estrus = day 0) and were assigned randomly to one of three treatment groups; saline-treated control, im injections of pLH, and iv injections of pPRL from days 110-120. In control, pLH-treated, and pPRL-treated animals, average gestation lengths were 114 +/- 0.8, 116 +/- 1.9, and 115 +/- 0.5 days (+/- SE), respectively. The relaxin level in mated gilts on day 100 was less than 2 ng/ml; it began to increase after day 110 and peaked in control animals on day 113 (66 ng/ml), whereas in pLH- and pPRL-treated animals, prepartum peak values were greater (P less than 0.01) and occurred on days 113 (104 ng/ml) and 114 (117 ng/ml), respectively. Relaxin dropped to basal levels (less than 1 ng/ml) by day 115 in controls and by day 116 in both pLH- and pPRL-treated gilts. Although pLH and pPRL treatments markedly accentuated peak relaxin secretion, they did not significantly accelerate or delay parturition or delay the abrupt demise of the corpora lutea immediately postpartum. In hysterectomized gilts, relaxin began to increase after day 110, peaked in control animals on day 113 (27 ng/ml), and decreased abruptly thereafter to less than 4 ng/ml. In contrast, pLH caused an immediate release of relaxin on day 111 (23 ng/ml) and sustained elevated levels (P less than 0.01) of relaxin until day 118, but the original corpora lutea regressed. Relaxin in pPRL-treated animals increased steadily after day 110, reaching peak values by day 115 (29 ng/ml), and remained consistently elevated (P less than 0.01) until day 120. Progesterone secretion was maintained in the pPRL-treated hysterectomized gilts from days 110-120 by the original corpora lutea and with no luteinization of follicles or formation of new corpora lutea. It is evident from this study that administration of pPRL starting on day 110 enhanced and prolonged the preprogramed release of relaxin and maintained progesterone secretion by aging corpora lutea in hysterectomized animals until day 120.

Ovarian response to injections of charcoal-extracted porcine follicular fluid and porcine follicle-stimulating hormone in gilts fed a progesterone agonist (altrenogest).

This experiment was conducted to compare the negative effects of charcoal-extracted porcine follicular fluid (pFF) and the positive effects of purified porcine follicle-stimulating hormone (pFSH) on growth of follicles and on plasma hormone concentrations. Twenty gilts were fed altrenogest for 18 days (20 mg.day-1.gilt-1) to suppress spontaneous growth of large follicles (greater than 6 mm in diameter). Gilts, assigned at random to receive pFF and pFSH administered in a 2 x 2 factorial arrangement, were injected 9 times at 8-h intervals starting 48 h before the last feeding of altrenogest and ending 8 h before slaughter (24 h after the last feeding of altrenogest). Blood was collected periodically through vena cava catheters. Treatment groups and mean number of medium follicles (3 to 6 mm in diameter)/gilt at necropsy were 1) 20 ml of charcoal-extracted porcine serum i.v. + 4 ml saline i.m., 30.8; 2) 20 ml of pFF i.v. + saline i.m., 0.2; 3) serum i.v. + 8 micrograms of pFSH (USDA-pFSH-B1)/kg BW in saline i.m., 59.0; and 4) pFF i.v. + pFSH in saline i.m., 36.2. Injections of pFF decreased (p less than 0.01) and injections of pFSH increased the number of medium follicles, and the interaction of pFF and pFSH was not significant. Plasma FSH decreased (p less than 0.01) during pFF treatment of saline-injected gilts at a rate of 0.29 ng.ml-1.h-1. During pFSH treatment, plasma FSH increased (p less than 0.05) at statistically identical rates of 0.33 and 0.32 ng.ml-1.h-1 in serum- and pFF-injected gilts.(ABSTRACT TRUNCATED AT 250 WORDS)