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

Efeito do líquido folicular bovino tratado com carvão ativado na secreção de FSH em novilhas pré-púberes intatas e ovariectomizadas

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

Efeito do líquido folicular bovino tratado com carvão ativado na secreção de FSH em novilhas pré-púberes intatas e ovariectomizadas

Rafael Herrera ALVAREZ¹; Juliana Rodrigues Pozzi ARCARO²; Maria Teresa Carvalho Pinto RIBELA²; Cibele Nunes PERONE²

Correspondence to:

Rafael Herrera Alvarez

Seção de Reprodução e Inseminação Artificial

Instituto de Zootecnia

Caixa Postal 60

13460-000 – Nova Odessa – SP

e-mail: [e-mail]

SUMMARY

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.

UNITERMS: Follicular fluid; FSH; Bovine.

INTRODUCTION

Charcoal-extracted bovine follicular fluid (bFF) has been shown to suppress FSH but not LH concentration when administered to ovariectomized (Ireland et al.⁶; Kiracofe et al.⁸ and intact (Lussier; Carruthers¹⁰; Turzillo; Fortune¹⁶; Wood et al.¹⁷ and Lussier et al.¹² heifers. In intact heifers, however, several reports (Johnson; Smith⁷ and Quirk; Fortune¹⁴) were unable to detect a significant bFF-induced suppression of FSH. The authors suggested that the inhibin activity in the follicular fluid injections may was insufficient to decrease FSH or that blood samples were not taken frequently enough to detect a decrease in FSH.

In the absence of available information concerning the optimal in-vivo model system for demonstration of bFF biological activity in cattle, the objective of this study is to determine the effect of charcoal-extracted bFF injections on circulating FSH concentrations in ovariectomized or intact prepubertal heifers.

MATERIAL AND METHOD

Bovine follicular fluid was aspirated from follicles (diameter < 22 mm) of ovaries collected from a local abattoir, centrifuged (700 g for 15 min at 4ºC) to remove cellular debris and frozen (-18ºC). After several collects, pooled bFF was thawed and incubated with 10 mg ml^-1 activated charcoal (Norit A, Sigma Chemical Company, St Louis MO) during 2 h at 4ºC, followed by sequential centrifugation (17000 g, 1 h at 4ºC) and filtration through sterile 45 µm nylon tissue. Charcoal treatment removed 99.8% of estradiol and progesterone steroids and had a protein content of 80 mg ml^-1 (Alvarez¹).

Six 10-months crossbred heifers (210 ± 30 kg) were used. Two months before, three of them were cirurgically ovariectomized by conventional method after lateral laparotomy. Heifers received four iv injections of 10 ml bFF at 8-h intervals. Blood samples were taken at 8-h intervals (or 4 h intervals on the period of bFF injections) 48 h before and 48 h after the first bFF injection by jugular venipuncture into 10 ml tubes under vacuum containing 143 USP heparin (Vacutainer; Beckton Dickinson, Crowley). After centrifugation at 700 g for 30 min, the plasma was stored at -20^oC until the assay. Plasma concentrations of FSH were measured using validated radioimmunoassay (Bolt; Rollins²). USDA -bFSH-1-2 (bFSH) served as both reference standard and tracer. bFSH was radioiodinated by a modification of the method of Greenwood et al.⁵. Briefly, 5 mg bFSH was dissolved in 20 ml 0.3M phosphate buffer pH 7.5 and reacted with 800 mCi ¹²⁵I (Amersham Corp., Arlington Heights, IL) and 0.8 mg chloramine T for 5 min This reaction was stopped by addition of 1mg sodium metabisulfite, chromatographed on Sephadex G100, and eluted with 0.05M PBS. The peak fraction of radioiodinated bFSH was diluted to 20000 cpm/100 ml in assay buffer (PBS containing 0.1 BSA and 0.1% sodium azide).

Unknowns and standards (100 ml) were pipetted into borosilicated glass tubes and incubated with 100 ml of rabbit anti-oFSH, NIDDK-oFSH-I, dilution 1:80000 (AFP-C5288113) and 100 ml (20000 cpm) bFSH tracer for 24 h at 4ºC. After this incubation, 100 ml rabbit anti-gamma globulin sheep serum (dilution 1:10) was added, and incubation continued for 2 h at room temperature. Tubes were washed with distilled water and centrifuged at 4ºC for 10 min at 3200 g. Supernatants were decanted, and radioactivity in each pellet was determined using a gamma counter. Intra and interassay coefficient of variation (CV) averaged 4.2 and 12.2%, respectively. Sensitivity of the bFSH was 1.4 ng ml^-1.

Since there were only three animals per treatment group, statistical analysis was restricted to within-group comparison, with each animal acting as its control. Student's paired t-test was used to compare the mean plasma FSH concentrations before (control pretreatment period), during and after administration of bFF.

RESULTS

No alterations in FSH concentrations were shown in intact heifers as an effect of bFF injections. Concentrations of FSH in ovariectomized heifers dropped (p<0.05) as early as 4 h after the first injection of bFF, reaching maximal suppression (43.9%; p<0.01) at the time of the second injection of bFF (Fig. 1).

Figure 1

There was no significant elevation in plasma FSH concentration after the final bFF injections in both, intact and ovariectomized heifers.

DISCUSSION

Charcoal treatment of follicular fluid to remove steroids and the use of ovariectomized animals with or without steroid replacement has shown that follicular fluid contains a non-steroidal substance (inhibin), produced by the granulosa cells, which operates at the pituitary level to suppress FSH secretion (Campbell et al.³; Price¹³). The endocrine response to bFF observed in this study confirms previous results demonstrating in vivo the selective suppressive effect of charcoal-extracted bFF on FSH in ovariectomized female cattle (Ireland et al.⁶; Kiracofe et al.⁸).

The suppression of circulating FSH in this experiment corresponds to the 30-45% maximal decrease in the FSH concentration obtained in previous experiments in intact (Lussier and Carruther¹⁰; Wood et al.¹⁷; Lussier et al.¹²), ovariectomized (Ireland et al.⁶; Kiracofe et al.⁸) and in dispersed bovine pituitary cells (Lussier et al.¹¹).

Differently, the dose of bFF used was unable of suppressing plasma FSH below basal concentrations in intact heifers. This and other similar results (Johnson; Smith⁷ and Quirk; Fortune¹⁴) with mature intact heifers suggest a low content of inhibin in the bFF used. Quirk; Fortune¹⁴ suppressed plasma concentrations of FSH with 20 ml bFF injections twice daily while plasma FSH concentrations were not affected with 10 ml of bFF. Furthermore, charcoal extraction may remove inhibin as well as steroids from follicular fluid. Tsonis et al.¹⁵ reported that a 10-fold greater inhibin activity was present in ovine follicular fluid (oFF) treated with 1 mg charcoal ml^-1 of oFF compared with 10 mg charcoal ml^-1 of oFF. In the present study, 10 mg charcoal ml^-1 of bFF was used and may have reduced the inhibin activity of bFF. Consequently, the amount of injected bFF may have been insufficient of suppressing significantly plasma FSH concentrations in intact heifers. Moreover, since plasma FSH of ovariectomized heifers was significantly suppressed by 8 (Kiracofe et al.⁸) or 10 ml (present study) of bFF, there is the possibility that pituitary sensibility to bFF is higher in ovariectomized that intact prepubertal heifers

There was no significant elevation in plasma FSH concentration over those of control after the final bFF injections in both ovariectomized or intact heifers. Previous studies have shown that the transient increase in plasma FSH was not associated to previous suppression of FSH by the bFF (Johnson; Smith⁷; Quik; Fortune¹⁴ and Law et al.⁹) but was related to the presence of the ovaries, since a rebound effect was not reported following bFF-induced suppression of FSH in ovariectomized heifers (Ireland et al.⁶) or in ovariectomized ewes treated with bFF or pure bovine inhibin (Findlay et al.⁴). The absence of a transient hipersecretion of FSH of the intact heifers in the present study, may be attributed to insufficient inhibin or other not identified FSH-modulating compounds present in the follicular fluid (Law et al.⁹).

CONCLUSION

In conclusion, these results confirmed that the suppressive activity of bFF on FSH secretion is exerced at the pituitary level and, differently of intact, the ovariectomized heifer is an adequate model to put in evidence this effect, particularly when the bFF have low FSH suppressing activity.

ACKNOWLEDGMENTS

The authors wish to thank the USA National Hormone and Pituitary Program and NIDDKD for antisera and purified hormones used in the FSH assay.

RESUMO

O presente trabalho objetivou determinar o efeito do líquido folicular bovino tratado com carvão ativado (LFb) na secreção do hormônio folículo estimulante (FSH) de novilhas pré-púberes ovariectomizadas ou intatas. A aplicação de LFb (quatro injeções de 10 ml com intervalo de 8 horas) provocou uma queda de aproximadamente 44% na concentração plasmática de FSH nas novilhas ovariectomizadas, mas não teve efeito nas novilhas intatas. Não foi observada hipersecreção de FSH após o término da aplicação do LFb. Esses resultados sugerem que proteínas presentes no LFb atuam ao nível hipofisiário para inibir a secreção de FSH e, diferentemente das intatas, as novilhas ovariectomizadas constituem um modelo adequado para evidenciar esse efeito, particularmente quando o LFb possui reduzida atividade supressora do FSH.

UNITERMOS: Fluido folicular; FSH; Bovinos.

Received: 24/06/1996

Accepted: 04/12/1997

• ¹
  - ALVAREZ, R.H. Efeito da imunização passiva contra o líquido folicular bovino na taxa de ovulação e na produção de embriões de vacas superovuladas e não superovuladas Jaboticabal, 1995. 192p. Tese (Doutorado). Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista.
• ²
  - BOLT, D.J.; ROLLINS, R. Development and application of a radiommunoassay for bovine follicle-stimulating hormone. Journal of Animal Science, v.56, n.1, p.146-54, 1983.
• ³
  - CAMPBELL, B.K.; PICTON, H.M.; MANN, G.E.; MCNEILLY, A.S.; BAIRD, D.T. Effect of steroid - and inhibin-free ovine follicular fluid on ovarian follicles and ovarian hormone secretion. Journal of Reproduction and Fertility, v.93, n.1, p.81-96, 1991.
• ⁴
  - FINDLAY, J.K.; ROBERTSON, D.M.; CLARKE, I.J. Influence of dose and route of administration of bovine follicular fluid and the suppressive effect of purified bovine inhibin (Mr 31000) on plasma FSH concentrations in ovariectomized ewes. Journal of Reproduction and Fertility, v.80, n.2, p.455-61, 1987.
• ⁵
  - GREENWOOD, F.C.; HUNTER, W.M.; GLOVER, J.S. The preparation of¹³¹ I - labelled human growth hormone of high specific radioactivity. The Biochemical Journal, v.89, n.1, p.114-23, 1963.
• ⁶
  - IRELAND, J.J.; CURATO, A.D.; WILSON, J. Effect of charcoal-treated follicular fluid on secretion of LH and FSH in ovariectomized heifers. Journal of Animal Science, v.57, n.6, p.1512-6, 1983.
• ⁷
  - JOHNSON, S.K.; SMITH, M.F. Effect of charcoal-extracted, bovine follicular fluid on gonadotropin concentrations, the onset of estrus and luteal function in heifers. Journal of Animal Science, v.61, n.1, p.203-9, 1985.
• ⁸
  - KIRACOFE, G.H.; RAMIREZ-GODINEZ, J.A.; McGOWAN, R.D.; BOLT, D.J. Reduction of serum FSH in ovariectomized heifers with bovine follicular fluid. Journal of Animal Science, v.57, p.350, 1983.
• ⁹
  - LAW, A.S.; BAXTER, G.; LOGUE, D.N.; O’SHEA, T.; WEBB, R. Evidence for the action of bovine follicular fluid factor(s) other than inhibin in suppressing follicular development and delaying oestrus in heifers. Journal of Reproduction and Fertility, v.96, n.2, p.603-16, 1992.
• ¹⁰
  - LUSSIER, J.G.; CARRUTHERS, T.D. Endocrine and superovulatory responses in heifers pretreated with FSH or bovine follicular fluid. Theriogenology, v.31, n.4, p.779-94, 1989.
• ¹¹
  - LUSSIER, J.G.; CARRUTHERS, T.D.; MURPHY, B.D. Effect of bovine follicular fluid and partially purified bovine inhibin on FSH and LH release by bovine pituitary cells in culture. Reproduction Nutrition Development, v.33, n.2, p.109-19, 1993.
• ¹²
  - LUSSIER, J.G.; MATTON, P.; GUILBAULT, L.A.; GRASSO, F.; MAPLETOFT, R.J.; CARRUTHERS, T.D. Ovarian follicular development and endocrine responses in follicular-fluid-treated and hemi-ovariectomized heifers. Journal of Reproduction and Fertility, v.102, n.1, p.95-105, 1994.
• ¹³
  - PRICE, C.A. The control of FSH secretion in the large domestic species. Journal of Endocrinology, v.131, n.2, p.177-84, 1991.
• ¹⁴
  - QUIRK, S.M.; FORTUNE, J.E. Plasma concentrations of gonadotrophins, preovulatory follicular development and luteal function associated with bovine follicular fluid-induced delay of estrus in heifers. Journal of Reproduction and Fertility, v.76, n.2, p.609-21, 1986.
• ¹⁵
  - TSONIS, C.G.; QUIGG, H.; LEE, V.W.K.; LEVERSHA, L.; TROUNSON, A.O.; FINDLAY, J.K. Inhibin in individual ovine follicles in relation to diameter and atresia. Journal of Reproduction and Fertility, v.67, n.1, p.83-90, 1983.
• ¹⁶
  - TURZILLO, A.M.; FORTUNE, J.E. Suppression of the secondary FSH surge with bovine follicular fluid is associated with delayed ovarian follicular development in heifers. Journal of Reproduction and Fertility, v.89, n.2, p.643-53, 1990.
• ¹⁷
  - WOOD, S.C.; GLENCROSS, R.G.; BLEACH, E.C.; LOVELL, R.; BEARD, A.J.; KNIGHT, P.G. The ability of steroid-free bovine follicular fluid to suppress FSH secretion and delay ovulation persist in heifers actively immunized against inhibin. Journal of Endocrinology, v.136, n.1, p.137-48, 1993.

*

Research supported by Instituto de Zootecnia/CPA/SAA (Grant IZ-001/92).

¹ Seção de Reprodução e Inseminação Artificial do Instituto de Zootecnia - SP

² Departamento de Bioengenharia do Instituto de Pesquisas Energéticas e Nucleares - SP

Publication Dates

• Publication in this collection
  14 Aug 2000
• Date of issue
  1998

History

• Accepted
  04 Dec 1997
• Received
  24 June 1996