B M Adams

Effect of duration of infusion of stress-like concentrations of cortisol on follicular development and the preovulatory surge of LH in sheep.

Stress-like levels of cortisol suppress follicular growth and development and block or delay the preovulatory surge of LH when cortisol is continuously administered during the late luteal and early follicular phases of the ovine oestrous cycle. We postulated that cortisol infusion of shorter duration would have a similar effect. To test this hypothesis the oestrous cycles of mature ewes were synchronized using progestin-treated vaginal pessaries. Ewes were randomly assigned to one of four treatment groups. Animals received cortisol (0.1mg/kg/h; n=8) or vehicle alone (n=8) beginning 5 days before, and continuing for 5 days after, pessary removal (PR). Additional groups received cortisol only during the 5 days period before (n=7), or the 5 days period after (n=8), PR. Continuous delivery of cortisol established stable serum concentrations of cortisol of 72.0+/-2.5ng/ml within 6h of initiation of infusion. Serum concentrations of oestradiol increased progressively during the period after PR in control animals receiving vehicle alone and the preovulatory surge of LH was evident in all control animals (eight of eight) 55.5+/-5.0h after PR. In contrast, follicular development and the preovulatory surge of LH were evident during the period of cortisol infusion in only one of eight animals receiving stress-like levels of cortisol over the entire 10-day infusion period. Similarly, neither follicular development nor surge-like secretion of LH were evident during the infusion period in animals (zero of eight) receiving cortisol during the 5-day period after PR. This cortisol-dependent suppression of ovarian activity in sheep receiving stress-like levels of cortisol during the 5 days after PR was temporary and follicular development, the ovulatory surge of LH, and subsequent luteal function were evident in six of eight ewes after cessation of cortisol delivery. Similarly, follicular development and the preovulatory surge of LH were noted within 5 days after PR in four of seven ewes receiving cortisol only during the 5-day period prior to PR. Collectively, these data indicate that stress-like levels of cortisol reduce fertility of sheep by suppressing follicular development and the preovulatory surge of LH. Additionally, cortisol delivery during the follicular phase has a more profound suppressive effect on follicular development than cortisol administration during the luteal phase.

Effect of stress-like concentrations of cortisol on the feedback potency of oestradiol in orchidectomized sheep

The effect of stress-like concentrations of cortisol on oestradiol-induced change in LH secretion and GnRH receptor expression was evaluated in orchidectomized sheep (wethers). Twenty-four wethers were assigned at random to one of the four treatment groups in a 2x2 factorial design (n=6 wethers/group). Wethers received cortisol (90 microg/kg/h; groups 2 and 4) or a comparable volume of cortisol delivery vehicle (groups 1 and 3) by continuous infusion for 48 h. During the final 24 h of infusion, wethers received oestradiol (6 ng/kg/h; groups 3 and 4) or oestradiol delivery vehicle (groups 1 and 2). The pattern of LH secretion was assessed during a 3-h period of intensive blood collection beginning 21 h after initiation of oestradiol infusion. Although neither cortisol nor oestradiol alone affected (P>0.05) mean serum concentration of LH or LH pulse frequency, serum LH and the frequency of secretory episodes of LH were significantly reduced (P<0.05) in wethers receiving cortisol and oestradiol in combination. Anterior pituitary tissue was collected at the end of the infusion period. Oestradiol increased (P<0.05) tissue concentrations of GnRH receptor and GnRH receptor mRNA. Although cortisol alone did not affect (P>0.05) basal concentrations of receptor or receptor mRNA, the magnitude of oestradiol-induced increase in GnRH receptor and GnRH receptor mRNA was significantly reduced in wethers receiving cortisol and oestradiol concurrently. Conversely, steady-state concentrations of mRNA encoding the LHbeta and FSHbeta subunits were increased (P<0.05) in wethers receiving cortisol. These observations demonstrate that stress-like concentrations of cortisol act in concert with oestradiol to suppress LH secretion. In addition, cortisol blocks oestradiol-dependent increase in pituitary tissue concentrations of GnRH receptor and GnRH receptor mRNA.

Biological activity of Luteinizing Hormone (LH) during the estrous cycle of mares

Abstract Equine luteinizing hormone (LH) contains a high proportion of carbohydrate (27%) and large compliment of sialic acid residues (6%), and anterior pituitary stores exhibit high physiochemical polymorphism. We initiated studies to determine whether polymorphism in the pituitary was reflected in secretory forms of LH as divergence between biopotency and immunoreactivity. Serum samples were collected daily from mares throughout the estrous cycle. In addition, the dynamics of LH secretion was monitored during intensive collection periods during estrus and the mid-luteal phase. Immunoreactive LH (iLH) was evaluated using conventional radioimmunoassay procedures. The biological activity of LH (bLH) was determined using a mouse interstitial cell-testosterone bioassay. The relative biopotency of equine LH (bLH/iLH,B/I) is significantly (P

Effect of phytoestrogens on basal and GnRH-induced gonadotropin secretion

Plant-derived estrogens (phytoestrogens, PEs), like endogenous estrogens, affect a diverse array of tissues, including the bone, uterus, mammary gland, and components of the neural and cardiovascular systems. We hypothesized that PEs act directly at pituitary loci to attenuate basal FSH secretion and increase gonadotrope sensitivity to GnRH. To examine the effect of PEs on basal secretion and total production of FSH, ovine pituitary cells were incubated with PEs for 48 h. Conditioned media and cell extract were collected and assayed for FSH. Estradiol (E₂) and some PEs significantly decreased basal secretion of FSH. The most potent PEs in this regard were coumestrol (CM), zearalenone (ZR), and genistein (GN). The specificity of PE-induced suppression of basal FSH was indicated by the absence of suppression in cells coincubated with PEs and an estrogen receptor (ER) blocker (ICI 182 780; ICI). Secretion of LH during stimulation by a GnRH agonist (GnRH-A) was used as a measure of gonadotrope responsiveness. Incubation of cells for 12 h with E₂, CM, ZR, GN, or daidzein (DZ) enhanced the magnitude and sensitivity of LH secretion during subsequent exposure to graded levels of a GnRH-A. The E₂- and PE-dependent augmentation of gonadotrope responsiveness was nearly fully blocked during coincubation with ICI. Collectively, these data demonstrate that selected PEs (CM, ZR, and GN), like E₂, decrease basal secretion of FSH, reduce total FSH production, and enhance GnRH-A-induced LH secretion in a manner that is dependent on the ER.

Single-chain human gonadotropin analogs induce follicle development in sheep.

The biopotency of single-chain analogs of human hFSH, human chorionic gonadotropin (hCG), and a dually active gonadotropin construct (FcCGbetaalpha) was examined. Sheep (bwt=61.4+/-1.1 kg; n=6 ewes/treatment) received a single injection (5 IU/kg, i.v.) of the hFSH analog (Fcalpha), the hCG analog (CGbetaalpha), FcCGbetaalpha, or Fcalpha and CGbetaalpha. Control animals received conditioned media. Ovulation was induced 3 days after analog administration using hCG (1000 IU, i.v.). Basal serum concentrations of estradiol (E(2)) were maintained in control animals. Neither Fcalpha nor CGbetaalpha alone induced significant E(2) production during the pre-hCG period. Conversely, serum concentrations of E(2) were increased (P<0.05) 2 days after administration of FcCGbetaalpha or Fcalpha+ CGbetaalpha. Although P(4) concentrations were maintained at basal levels in control animals, significant increase was noted in all other treatment groups during the post-hCG period. Final ovarian weight was significantly increased (P<0.05) in animals receiving Fcalpha, Fcalpha+ CGbetaalpha, or FcCGbetaalpha, but not CGbetaalpha alone. Most of the ovarian enlargement was attributed to the formation of corpora lutea. Collectively, these observations demonstrate that the single-chain analogs of the human gonadotropins are active in sheep. The construct with singular FSH activity supports follicle development but not E(2) production. Conversely, the construct that incorporates beta-domains from both CG and FSH has dual activity. The long-lived nature of the single-chain constructs suggests that these recombinant gonadotropins may be effective alternatives to pituitary- or placenta-derived gonadotropins in out-of-season breeding and/or superovulation protocols.

Dietary restriction reduces the rate of estradiol clearance in sheep (Ovis aries)

Three experiments were designed to test the effect of dietary restriction on clearance of 17beta-estradiol (E(2)) in sheep. A preliminary experiment examined the effect of a 4-d fast on the rate of E(2) clearance in wethers. The second experiment tested the hypothesis that either long-term restriction (7 wk) or a 5-d fast would increase steroid-binding capacity of serum by increasing the concentration of sex hormone-binding globulin (SHBG) in the blood of ovariectomized ewes. In Exp. 3, we hypothesized that nutrition-dependent regulation of E(2) clearance by the liver would result in divergence in biliary extraction of E(2) in fed and fasted wethers receiving comparable levels of exogenous E(2). A marked difference in E(2) clearance between fed and fasted wethers was noted in the preliminary study. Relative to ad libitumfed wethers, a 4-d fast decreased E(2) clearance by 52%. Serum concentrations of SHBG were increased in long-term energy-restricted and fasted ewes, relative to the concentration in maintenancefed ewes (P = 0.015). Furthermore, a 5-d fast nearly doubled serum steroid-binding capacity in wethers. The E(2) concentration in bile was 2 times greater in fasted than in fed wethers. This fasting-dependent increase in biliary E(2) may be reflective of the increased serum E(2) in fasted animals, because each 1 pg/mL increase in serum E(2) increased bile E(2) by 0.86 +/- 0.12 pg/mL, independent of nutrition (P = 0.002). Our results demonstrate that the rate of clearance of E(2) is decreased during nutritional restriction. Additionally, these data indicate that altered SHBG expression, enterohepatic recirculation, or both are involved in the decreased E(2) clearance during dietary restriction.

Effect of time and dose of recombinant follicle stimulating hormone agonist on the superovulatory response of sheep

The objective of this study was to determine the superovulatory potential of a single-chain analog of human FSH (Fcα) when administered to ewes either 3 days before, or coincident with, simulated luteolysis (pessary removal [PR]). A total of 40 animals were randomly assigned to receive Fcα at doses of 0.62, 1.25, or 2.5 IU/kg of body weight (bwt) 3 days before PR or 0.31, 0.62, 1.25, or 2.5 IU/kg of bwt at PR. Control ewes received protein without FSH activity. Blood samples were collected during the periovulatory period and ovarian tissue was collected 11 days after PR. Ovulation rate did not differ from the control group in ewes receiving the smallest doses of Fcα (0.31 and 0.62 IU/kg). However, a significant superovulatory response was noted in sheep receiving Fcα at doses of 1.25 and 2.5 IU/kg and this response was comparable in animals receiving the largest dose levels of Fcα at, or 3 days before, PR. The interval between PR and the LH surge was significantly extended and the LH surges were less synchronous in animals receiving Fcα at PR when compared with animals receiving the potent FSH agonist 3 days before PR. Taken together, these data indicate that the human single-chain gonadotropin with FSH activity promotes superovulation in ewe lambs in the breeding season. A single injection of the recombinant gonadotropin 3 days before luteolysis synchronizes the LH surge. The use of the single-chain analog of FSH in assisted reproduction for domestic animals is likely to be of practical significance as an alternative to conventional gonadotropins in superovulation protocols in livestock species.

Effect of single-chain ovine gonadotropins with dual activity on ovarian function in sheep.

We examined the half-life and biological activity of two single-chain proteins that combined portions of ovine FSH and LH. We proposed the hypothesis that these chimeric proteins would display LH and FSH activities and would promote follicle maturation in ewes. Estrus activity was synchronized using progestogen-impregnated vaginal pessaries. To negate the impact of endogenous LH and FSH, animals received serum-containing antibodies against GNRH 1 day before pessary removal (PR). At PR sheep (five animals per group) received a single injection (10 IU/kg, i.v.) of either the ovine-based (oFcLcα) gonadotropin analog, an ovine-based analog containing oLHβ truncated at the carboxyl terminus (oFcL(ΔT)cα), or a human-based gonadotropin analog (hFcLcα). Control animals received a comparable amount of gonadotropin-free protein. Ovulation was induced 3 days after PR using human chorionic gonadotropin (1000 IU, i.v.). Ovaries were collected 11 days after PR. Neither estradiol (E2) or progesterone (P4) production, development of preovulatory follicles or corpora lutea (CL) were noted in control animals receiving gonadotropin-free protein. Significant increase in the synthesis of E2 and P4 was noted in sheep receiving the dually active gonadotropin analogs. The number of CLs present 11 days after PR was significantly increased in sheep receiving the chimeric glycoproteins compared with control animals. The magnitude of the secretory and ovarian responses did not differ between hFcLcα and oFcLcα or between oFcLcα and oFcL(ΔT)cα. Immunoactivity of LH and FSH was low in control animals, but was significantly elevated in sheep receiving the gonadotropin analogs. In conclusion, ovine-based gonadotropin analogs are functionally active in sheep and a single injection is adequate to induce the development of multiple ovulatory follicles.

Concentrations of bioactive and immunoactive luteinizing hormone in bovine anterior pituitary tissue

Tissue concentrations of LH as determined by radioimmunoassay (RIA) may not accurately measure actual changes that could occur in biological activity of the hormone. To examine this possibility, pituitary homogenates from 135 beef cows in various physiological states were analyzed for content of LH by both RIA and an in vitro bioassay. The ratio of biological/immunological active concentrations of LH remained constant (.52 +/- .02) even though tissue concentrations of immunoactive LH differed among groups. Tissue concentrations of bioactive LH were linearly related to, and highly correlated with (P less than .001), tissue concentrations of immunoactive LH. These data indicate that only a fraction of the immunoactive LH in the bovine pituitary is biologically active. However, this fraction does not vary with the reproductive status or plane of nutrition.