Charcoal-treated Bovine Follicular Fluid Research Articles

Effects of the presence of a dominant follicle and exogenous oestradiol on the duration of the luteal phase of the bovine oestrous cycle.

The presence of a developing dominant follicle may be a factor in the control of the luteolytic cascade mechanism and the number of follicular waves during the bovine oestrous cycle. In this study, ovaries of all animals were examined once a day by transrectal ultrasonography. It was expected that heifers (n = 18) would have two follicular waves if the second wave occurred later than day 10 after oestrus (Expt 1) and that cows (n = 14) would have three waves if the second wave occurred on or before day 10 (Expt 2). The objective of Expt 1 was to determine if absence of a large follicle late in the luteal phase delays luteal regression in heifers that are expected to have two follicular waves. Nine heifers were injected i.v. with 10 ml charcoal-treated bovine follicular fluid three times a day for 4 days, starting on the day after initiation of the second follicular wave, to delay growth of the second wave dominant follicle. Nine heifers were injected with 0.9% NaCl as controls. The duration of the luteal phase (calculated as the number of days that serum progesterone was > 0.5 ng ml-1) was greater (P < 0.01) in the follicular fluid-treated group compared with the controls (18.7 versus 14.1 days). FSH and follicular growth were suppressed during the period of injection of follicular fluid (P < 0.01 and 0.03, respectively). The objective of Expt 2 was to determine the effect of increased oestradiol on the duration of the luteal phase in cows that were expected to have three follicular waves. Seven cows were injected i.m. three times a day for 4 days with 1 ml oestradiol (100 micrograms ml-1 in corn oil) and seven cows were similarly injected three times a day with 1 ml 0.9% NaCl (control) starting the day after cessation of growth of the second wave dominant follicle. Luteal phase duration was shorter in oestradiol-treated animals than in the controls (14.0 versus 19.0 days; P < 0.04). Serum oestradiol concentrations were higher in the oestradiol-treated group during the period of injection (P < 0.01). In summary, luteolysis was delayed when follicular growth was suppressed with follicular fluid (Expt 1). Exogenous oestradiol administration during the development of uterine oestradiol responsiveness initiated luteolysis earlier compared with control animals (Expt 2).

Effect of charcoal-treated bovine follicular fluid on the secretion of FSH in ovariectomized and intact prepubertal heifers

The aim of the experiment was to determine whether charcoal-treated bovine follicular fluid (bFF) removed from visible (< 22 mm) follicles altered the secretion of follicle-stimulating hormone (FSH) in intact and ovariectomized prepubertal heifers. After a 10-ml injection of bFF given at three consecutive 8-h intervals, secretion of FSH was depressed in approximately 44% of the ovariectomized heifers but there was no effect in intact heifers. When the bFF treatment ceased, there were no rebound effects on FSH concentrations above that of controls pretreatment levels. These results suggest that proteins from bFF act at the pituitary level to inhibit FSH secretion and, differently of the intact, the ovariectomized heifer is an adequate model to put this effect in evidence, particularly when the bFF have low FSH suppressing activity.

Level of nutrition modulates the dynamics of oestradiol feedback on plasma FSH in ovariectomized ewes.

The frequency of multiple ovulations in mature, cyclic ewes is strongly influenced by the level of nutrition, but it is difficult to demonstrate concurrent changes in plasma concentrations of gonadotropins. The failure to do so might be a consequence of rapid compensation by the homeostatic feedback mechanism linking secretion by the hypothalamus/pituitary gland and ovarian hormones. Most experimental models have examined the components of the homeostatic feedback system after steady state relationships had been established. We hypothesised that the effects of nutrition might be observed more readily if the system were disrupted and then examined while equilibrium was being re-established. This hypothesis was tested in three experiments in Merino ewes by allowing gonadotropin secretion to escape feedback for 5 days after ovariectomy and then replacing ovarian hormones and examining effects of feeding regimen on the return of plasma concentrations of FSH to baseline values. In all three experiments, oestrogen replacement caused plasma concentrations of FSH to decline more rapidly (P < 0.05) in ewes fed at 0.5x maintenance, than in ewes fed at 1.4x maintenance, with groups fed at maintenance being intermediate. No effect of diet was observed on plasma FSH concentrations in the absence of oestradiol, and neither progesterone nor charcoal-treated bovine follicular fluid influenced the effect of nutrition. Plasma concentrations of oestradiol were 9.8% lower on average (NS) in ewes fed above maintenance than in the sheep fed below maintenance over the three experiments, suggesting that there may have been a reduced clearance of oestradiol which contributed to the result. We conclude that feeding regimen affects the secretion or clearance of gonadotropins in mature ewes, as in the mature ram, and that this is one mechanism by which ovulation rate may be affected.

Evidence that testosterone and follicular fluid do not interact in the control of FSH secretion in rams

The hypothesis that testosterone and inhibin interact in the control of FSH secretion in rams was tested. Adult rams were castrated and were simultaneously given testosterone implants and 3-times daily sc injections of 0, 0.4, 0.8 or 1.6 ml charcoal-treated bovine follicular fluid (bFF). After 1 wk, the implants were removed, and the bFF injections continued as before. Blood samples were taken daily for mean LH, FSH and testosterone concentrations, and every 10 min for 12 h in the presence and in the absence of testosterone for assessment of pulsatile LH release. The bFF specifically inhibited FSH secretion from rat pituitary cells in culture. In the presence of testosterone, there were no main effects of bFF on mean plasma FSH or LH concentrations, nor were these values different from their pre-treatment means (P>0.05). Treatment with bFF did not affect LH pulse frequency or amplitude, but the number of rams showing LH pulses was reduced in the 0.8 and 1.6-ml dose groups (P<0.05). Removal of testosterone increased (P<0.05) both gonadotropins. In the absence of testosterone, no main effect of bFF on mean LH or FSH concentrations was observed, although the 1.6-ml dose suppressed the postcastration rise of both LH and FSH. These data suggest that inhibin does not interact with testosterone and that a physiological level of testosterone is sufficient for the regulation of FSH secretion in adult rams.

Effects of bovine follicular fluid on gonadotrophin secretion in intact and chronically ovariectomized ewes before and after desensitization of pituitary gonadotrophs to gonadotrophin-releasing hormone.

Intact and chronically ovariectomized ewes were treated for 4 days with charcoal-treated bovine follicular fluid (FF) or charcoal-treated bovine serum during the late-anoestrous period, and the effects on basal and gonadotrophin-releasing hormone (GnRH)-induced secretion of LH and FSH observed. Subsequently, ewes received s.c. implants containing a sustained-release formulation of a potent GnRH agonist D-Ser(But)6-Azgly10-LHRH (ICI 118630) to desensitize pituitary gonadotrophs to hypothalamic stimulation, and the effects of bovine FF and bovine serum were re-assessed 2 weeks later. Chronic exposure (for 2-3 weeks) to ICI 118630 significantly reduced basal levels of LH and FSH in both intact and ovariectomized ewes and completely abolished both spontaneous LH pulses as well as exogenous GnRH-induced acute increases in plasma LH and FSH levels. Treatment with bovine FF significantly reduced plasma FSH levels, but not LH levels, in both intact and ovariectomized ewes before and after chronic exposure to ICI 118630. In intact ewes before exposure to ICI 118630, treatment with bovine FF actually enhanced pulsatile LH secretion and raised mean plasma LH levels by 240% (P less than 0.05). No such stimulatory effect of bovine FF on LH secretion was observed in intact ewes exposed to ICI 118630 or in ovariectomized ewes before or after exposure to ICI 118630, suggesting that the effect probably involved an alteration in ovarian steroid feedback affecting hypothalamic GnRH output. Treatment with bovine FF did not significantly affect the magnitude of GnRH-induced surges of LH or of FSH observed in either intact or ovariectomized ewes before exposure to ICI 118630.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma follicle stimulating hormone, ovulation rate and fertility in Merino ewes treated with bovine follicular fluid

Charcoal-treated bovine follicular fluid (bFF) given as four 5-ml subcutaneous injections to 13 Merino-Border Leicester ewes around the time of natural luteolysis suppressed ( P<0.01) plasma levels of follicle stimulating hormone (FSH) [from 1.08 ± 0.05 to 0.41 ± 0.03, mean ± s.e.m. of log e ( ng+ 1) / ml plasma]. This was followed ( P < 0.01) by hypersecretion or a rebound of FSH (to 1.46 ± 0.11) lasting 32 h in 10 of the treated ewes, and then by a further fall (to 0.73 ± 0.03, P < 0.05) before the surge (1.21 ± 0.07, P < 0.05) associated with the preovulatory surge of luteinizing hormone (LH). Plasma FSH at 56–72 h before the LH surge (i.e., at the time of the FSH rebound) was correlated with the subsequent ovulation rate ( n=13, r= + 0.73, P < 0.01). Fewer ewes treated with four injections of 2 or 5 ml of bFF than control ewes (injected with bovine plasma) became pregnant (28 of 41 vs. 38 of 41, χ 2 = 4.05, P < 0.05), although plasma progesterone was similar at Day 11 in treated and control ewes. It is concluded that plasma FSH during such a rebound influences the subsequent ovulation rate in sheep.

Stimulation of steroid production in isolated rat Leydig cells by unknown factors in testicular fluid differs from the effects of LH or LH-releasing hormone.

After the addition of charcoal-treated testicular fluid to Leydig cells isolated from 22-day-old rats, pregnenolone production could be increased to a maximum of tenfold within 30 min in a dose-dependent manner. Testicular fluid, but not serum, further increased pregnenolone formation threefold when pregnenolone production by Leydig cells was stimulated by the addition of LH-releasing hormone (fourfold), LH (25-fold) and 22R-hydroxycholesterol (300-fold). The effect of testicular fluid on steroid production in the presence of 22R-hydroxycholesterol was not inhibited by cycloheximide whereas cycloheximide completely inhibited the effect of LH. It appears unlikely that steroids, lipoproteins or other plasma components constitute the stimulatory agents in testicular fluid. The biologically active principles may be locally produced factors with a molecular weight greater than 25,000. Similar biological activities could be shown in testicular lymph from boars but not in systemic lymph from boars nor in charcoal-treated bovine follicular fluid. The presumably locally produced factor(s) may amplify the effect of LH and can thus act as a local modulator(s).

Electrofocusing fractionation of follicle stimulating hormone in pituitary cell culture extracts from male and female rats

Three-day pituitary cell cultures from adult male and female rats were incubated for 4 h in the presence of 10 nM LHRH and the molecular heterogeneity of FSH was assessed in the media of LHRH-stimulated cells and in cell extracts from unstimulated cells using an electrofocusing technique. The pi distribution of FSH showed a high degree of similarity between cell media and cell extracts of each sex although differences were observed between sexes. Pituitary cell cultures from male rats were also incubated in the presence of 10 −8 M testosterone and 10 −8 M estradiol and the pI distribution of FSH from media after LHRH stimulation was determined. No significant differences in the pI profiles were observed. Incubation with charcoal-treated bovine follicular fluid (an inhibin source) resulted in a significant reduction in recovered FSH activity in the pH region 3.61-3.92 although this decrease did not markedly alter the pI profile of FSH. Close similarities were observed in the pI distribution of FSH of pituitary cell culture extracts and pituitary gland extracts from intact animals of both sexes, however, differences in pi distribution were noted in pituitary extracts in the male but not the female following gonadectomy. It is concluded that (1) stored FSH is released from the pituitary without major modification to its structure as assessed from its pI profile, (2) sex differences in the pI profile of FSH in pituitary extracts are retained in culture and following LHRH-stimulated release, (3) the pI distribution of FSH is not affected by testosterone or estradiol and only minimally by inhibin in short-term cultures. It is thus suggested that the failure to observe in vitro an effect of steroids on the pI profile of FSH is due either to the limited duration of the culture period employed or to the possibility that steroids exert their effect on the pituitary through an indirect mechanism.

Increase in ovulation rate after treatment of ewes with bovine follicular fluid in the luteal phase of the oestrous cycle.

Administration of charcoal-treated bovine follicular fluid to Damline ewes twice daily (i.v.) from Days 1 to 11 of the luteal phase (Day 0 = oestrus) resulted in a delay in the onset of oestrous behaviour and a significant increase in ovulation rate following cloprostenol-induced luteolysis on Day 12. During follicular fluid treatment plasma levels of FSH in samples withdrawn just before injection of follicular fluid at 09:00 h (i.e. 16 h after previous injection of follicular fluid) were initially suppressed, but by Day 8 of treatment had returned to those of controls. However, the injection of follicular fluid at 09:00 h on Day 8 still caused a significant suppression of FSH as measured during a 6-h sampling period. Basal LH levels were higher throughout treatment due to a significant increase in amplitude and frequency of pulsatile secretion. After cloprostenol-induced luteal regression at the end of treatment on Day 12, plasma levels of FSH increased 4-fold over those of controls and remained higher until the preovulatory LH surge. While LH concentrations were initially higher relative to those of controls, there was no significant difference in the amount of LH released immediately before or during the preovulatory surge. These results suggest that the increase in ovulation rate observed during treatment with bovine follicular fluid is associated with the change in the pattern of gonadotrophin secretion in the luteal and follicular phases of the cycle.

Changes in FSH and the pulsatile secretion of LH during the delay in oestrus induced by treatment of ewes with bovine follicular fluid.

Treatment of Welsh Mountain ewes with i.v. injections of untreated or charcoal-treated bovine follicular fluid (BFF) after prostaglandin-induced luteal regression resulted in a significant delay in the onset of oestrous behaviour. Injections of BFF caused a significant decrease in plasma concentrations of FSH but levels were only suppressed below those in control ewes during the first 24 h of the 48-h period of treatment. At the end of BFF treatment there was a substantial increase in plasma FSH levels. In contrast, there were no significant effects of BFF treatment on basal plasma concentrations of LH or pulse frequency or amplitude of LH secretion. Corpus luteum function, as measured by plasma concentrations of progesterone, in BFF-treated ewes was normal. These results suggest that the delay in oestrus caused by treatment of ewes with BFF is due to a specific suppression of plasma levels of FSH while LH secretion remains unaffected.

Effect of charcoal-treated bovine follicular fluid on secretion of LH and FSH in ovariectomized heifers.

The objective of the experiment was to determine whether pools of charcoal-treated bovine follicular fluid (bFF) removed from large (greater than 10 mm) follicles during the preovulatory (d 18 to 1), postovulatory (d 2 to 6) or mid-cycle (d 7 to 17) stages of the bovine estrous cycle altered secretion of luteinizing hormone (LH) or follicle-stimulating hormone (FSH) in ovariectomized heifers. After a 20-ml injection of bFF given at three consecutive 6-h intervals, secretion of FSH was depressed from approximately 116 to 53 ng/ml by each pool of bFF. In contrast, bFF did not affect secretion of LH. Serum from an ovariectomized cow (control) had no effect on secretion of LH or FSH. We concluded that bFF contained a nonsteroidal component (perhaps inhibin) that suppressed FSH secretion in heifers.

Estimation of inhibin-like activity in spent medium from rat ovarian granulosa cells during long-term culture

Granulosa cells were isolated from antral follicles of intact, adult female rats by treatment with EGTA and hypertonic sucrose, and were kept in culture for 1, 15 or 37 days. The amount of inhibin-like activity in spent rat granulosa-cell media (rGCCM) was assessed by using a bioassay for inhibin (measurement of the unstimulated release of FSH and LH by pituitary cells in culture). After 1 day in culture, inhibin-like activity was detected in rGCCM of 0.016−0.8 × 10 5 cultured granulosa cells per dish. In the bioassay, maximal suppression of FSH occurred when 0.8 × 10 5 granulosa cells were plated. With this or a larger number of cells in long-term cultures, pooled rGCCM, collected up to 37 days after plating, suppressed levels of FSH (and not of LH) in the inhibin bioassay in parallel with the suppression found after addition of charcoal-treated bovine follicular fluid (bFF), which served as a standard. The amount of inhibin-like activity in rGCCM (relative to the amount present in bFF, which was given a potency of 1) ranged between 0.05 and 0.90 × 10 −3. Release of inhibin-like activity could not be estimated on day 4, and was low from days 29 to 37 in culture. Except after 1 day of culture, no substantial amounts of progesterone were detected in the media. It is concluded that granulosa cells collected from adult, intact female rats have and retain the capacity to secrete inhibin-like activity in vitro, under basal conditions in long-term cultures.