D. J. Bolt

Changes in follicular estradiol-17β, 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.

Dopaminergic-like activity in toxic fescue alters prolactin but not growth hormone or thyroid stimulating hormone in ewes

Studies were conducted to determine the specificity and cause of altered pituitary hormone secretion when ewes ingest endophyte-infected (Acremonium coenophialum) GI-307 tall fescue (toxic fescue). Plasma concentrations of prolactin (PRL) but not growth hormone (GH) or thyroid stimulating hormone (TSH) in ewes grazing toxic fescue were significantly lower (P less than .01) than concentrations measured in ewes grazing orchardgrass (OG). Comparing hormone secretory responses of ewes grazing each grasstype, ewes on toxic fescue released less PRL following thyrotropin releasing hormone (TRH) challenge than ewes on OG. TSH responses to TRH were not affected by grasstype. At this dose of TRH, GH secretion was not significantly affected in either group of ewes. In a separate study, dopamine hydrochloride (DA) was infused into control ewes to define the effect of a pure dopamine agonist on basal and TRH-stimulated secretion of PRL, GH and TSH. DA depressed both basal and TRH-stimulated secretion of PRL without affecting the basal concentrations or responses of GH or TSH. Based on the assumption that the active agent in toxic fescue responsible for the observed hypoprolactinemia was a dopaminergic agonist, haloperidol (HAL), a DA receptor blocking drug, was administered to ewes grazing toxic fescue or OG. HAL evoked significant PRL secretion unaccompanied by any GH or TSH effect in both toxic fescue and OG ewes. Administration of HAL resulted in a gradual increase over 4 hr in PRL in toxic fescue ewes and prolonged the duration of the PRL response to TRH. No differences in circulating plasma concentrations of DA, epinephrine or norepinephrine were measured in ewes on troxic fescue or OG. Alterations in pituitary hormone secretion due to toxic factors in fescue were confined to PRL. Hormone secretory responses to TRH and HAL suggest that the effects on PRL are mediated through dopamine-like activity in toxic fescue.

In vitro synthesis of progesterone and prostaglandin F by luteal tissue and prostaglandin F by endometrial tissue from the pig.

Abstract The relationship between progesterone (P 4 ) synthesis in vitro by luteal tissue and prostaglandin F (PGF) synthesis in vitro by endometrium and luteal tissue from two stages of the cycle, Days 7 to 8 and 15 to 16, was determined. Luteal and endometrial tissues were collected from pigs in three experimental groups at two stages of the cycle: (A) 6 pigs on Days 7 to 8 with spontaneous, 5 to 6 day old corpora lutea (CL); (B) 5 pigs on Days 15 to 16 with spontaneous, 13 to 14 day old CL; and (C) 6 pigs on Days 15 to 16 with spontaneous, 13 to 14 day old CL and 5 to 6 day old CL induced by pregnant mares serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) injections. Pigs with spontaneous, 13 to 14 day old CL of the cycle and PMSG-HCG induced accessory, 5 to 6 day old CL were used so that P 4 and PGF synthesis in tissue from old and new CL could be compared in the same pig on Day 15 to 16 of the cycle. Tissues (100 mg minces) were incubated in 5 ml of Krebs Ringer solution in an atmosphere of 95% 0 2 :5% CO 2 for 2 hours at 0° C, 37° C, or 37° C with 1.3 x 10 −4 M indomethacin (IND). An aliquot of the incubation medium and an aliquot of the supernatant after homogenization of the tissue in the remaining medium of each flask was quantified for P 4 and PGF by radioimmunoassay. P 4 and PGF release into the medium and total accumulation of P 4 and PGF in the flasks indicated that de novo synthesis had occured at 37° C. Compared to tissue from 13 to 14 day old CL, tissue from 5 to 6 day old CL synthesized more P 4 per flask (53.9 vs 25.0 ng/mg tissue, P 4 into the medium (20.8 vs 8.8 ng/mg, P 4 synthesis by luteal tissue from 5 to 6 day old and 13 to 14 day old CL from pigs in group C was similar to P 4 synthesis by luteal tissue from pigs in group A and group B, respectively. Luteul PGF synthesis was not affected significantly by either the age of the CL or by PMSG-HCG treatment. For endometrial samples, the synthesis of PGF was not significantly different among pigs in groups A, B and C. If uterine PGF is involved in luteal regression in the pig, the sensitivity of the CL to PGF may be more important than an increase in PGF secretion during the late luteal phase of the estrous cycle.

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.

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)

INSERTION OF GROWTH HORMONE GENES INTO PIG EMBRYOS

Recent research has clearly demonstrated that genes coding for growth hormone (GH) and growth hormone releasing factor (GRF) can be integrated into the genome of domestic swine by microinjection of the cloned genes into a pronucleus or nucleus of fertilized pig ova. The percentage of injected ova that developed into transgenic pigs varied from 0.30% to 1.69%. The proportion of transgenic pigs that expressed the gene varied from 17% to 100%. depending upon the composition of the fusion gene. The elevation of foreign GH in expressing transgenics stimulated the expected elevation in insulin-like growth factor (IGF-I). Enhanced growth rate has been reported for some transgenic GH pigs but not for others, with the difference possibly due primarily to dietary constraints and general health of the pigs. Expression of the GH genes has markedly reduced subcutaneous fat and improved the efficiency of converting feed into meat. The persistent excess GH in transgenic pigs was detrimental to general health; lameness, lethargy, and gastric ulcers were the most prevalent problems. Females that expressed foreign GH genes were anestrus. Most of the transgenic pigs that were reproductively sound transmitted the gene to a portion of their progeny.

Reduction by human chorionic gonadotropin of the luteolytic effect of prostaglandin F2α in ewes

Abstract The ability of human chorionic gonadotropin (HCG) to reduce the luteolytic effect of prostaglandin (PGF2α) was demonstrated in cycling ewes. As expected, treatment with 10 mg of PGF2α alone on Day 10 of the estrous cycle exerted a potent negative effect on the function and structure of corpus luteum (CL) as indicated by reduced plasma progesterone, CL progesterone, and CL weight. However, the identical PGF2α treatment failed to significantly reduce either luteal function or luteal weight when administered to ewes that were also treated with HCG on Days 9 and 10 of the estrous cycle. Treatment with HCG alone had a positive effect on CL as indicated by increased plasma progesterone, CL progesterone, and CL weight. Treatment with HCG did not render the CL totally insensitive to the negative effects of PGF2α because plasma progesterone was reduced when the dose of PGF2α was doubled. Whether CL regressed or continued to function after treatment with both HCG and PGF2α appeared to depend upon a balance between the positive and negative effects of the two hormones.

Gene insertion: Role and limitations of technique in farm animals as a key to growth

Structural genes for bovine (bGH) and human (hGH) growth hormones and human growth hormone releasing hormone (hGHRH) were ligated to the promoter for mouse metal-lothionein I and microinjected into the nuclei of embryos resulting in ‘transgenic’ pigs and sheep. Efficiency of microinjection was low (0.1 to 0.9% of injected embryos) resulting in transgenic young of which 60% actually produced the gene product. Concentration of bGH and hGH in plasma varied greatly among animals and was not related to number of copies of the gene. Growth rates were not enhanced in transgenic pigs with elevated growth hormone; however, subcutaneous fat content was dramatically reduced. Transgenic male pigs successfully transmitted the gene construct to their progeny; however, expressing transgenic females do not exhibit normal estrous cycles. Genes used in these experiments were not readily regulated by heavy metal supplementation. Lambs with hGHRH had a low rate of expression and did not exceed controls for gain or feed efficiency. A single expressing lamb was refractory to exogenous hGHRH challenge. Before the full potential of gene transfer can be realized for regulation of growth of farm animals, promoter/regulator sequences that will permit full control of gene expression must be found.

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)

A simple in vivo bioassay for inhibin-like activity using ovariectomized ewes

Long term ovariectomized ewes were used in a bioassay for inhibin-like activity. The concentration of FSH 6 to 7 hr after injection of follicular fluid (a rich source of inhibin), as a percentage of pretreatment, regressed on the log of the dose had a slope of -26.0 +/- 7.6 (5 replications, mean +/- SD) and an index of precision of .32 +/- .04. This system was rapid, relatively easy and specific for in vivo inhibin-like activity. This bioassay was also used to determine the relative potency of an affinity-purified fraction of follicular fluid.