ELSEVIER

C I R C A D I A N AND PULSATILE VARIATIONS IN P L A S M A LEVELS OF INHIBIN, FSH, LH AND TESTOSTERONE IN A D U L T M U R R A H

BUFFALO BULLS

V.D. Dixi t , Bal j i t Singh, P Singh, G.C. Georgie , M.M. Galhotra and V.P. Dixi t I

Depar tment o f Animal Product ion Phys io logy CCS Haryana Agr icul tura l Univers i ty

Hisar 125004, India

Rece ived for publ icat ion: September 9, 1997 Accepted: March 23, 1998

ABSTRACT

The present study investigated pulsatile and circadian variations in the circulatory levels of inhibin, gonadotrophins and testosterone. Six adult buffalo bulls (6 to7 yr of age) were fitted with indwelling jugular vein catheters, and blood samples were collected at 2-h intervals for a period of 24 h and then at 15-min interval for 5 h. Plasma concentrations of inhibin, FSH, LH and testosterone were determined by specific radioimmunoassays. Plasma inhibin levels in Murrah buffalo bulls ranged between 0.201 to 0.429 ng/mL, with a mean of 0.278±0.023 ng/mL. No inhibin pulses could be detected during the 15-min sampling interval. Plasma FSH levels ranged between 0.95 to 3.61 ng/mL, the mean concentration of FSH over 24 h was 1.66±0.25 ng/mL. A single FSH pulse was detected in 2 of 6 bulls. The LH levels in peripheral circulation ranged between 0.92 to 9.91 ng/mL, with a mean concentration of 3.33± 1.02 ng/mL. Pulsatility was detected in LH secretion with an average of 0.6 pulses/h. Plasma testosterone levels in 4 buffalo bulls ranged from 0.19 to 2.99 ng/ mL, the mean level over 24 h were 1.34+0.52 ng/mL. Testosterone levels in peripheral circulation followed the LH secretory pattern, with an average of 0.32 pulses/h. The results indicate parallelism in inhibin, FSH and LH, and testosterone secretory pattern. Divergence in LH and FSH secretory patterns in adult buffalo bulls might be due to the presence of appreciable amounts of peripheral inhibin. © 1998 by Elsevier Science Inc. Key words: inhibin, gonadotrophins, testosterone, buffalo, circadian

Acknowledgments This work was carried out under a World Bank Funded NARP II grant through the Indian Council of Agricultural Research, New Delhi. We thank Dr. K Taya, Laboratory of Veterinary Physiology, Tokyo University of Agriculture & Technology, Fuchu, Tokyo 183, Japan, for training in inhibin assay technique and providing RIA reagents. The RIA reagents for FSH and LH were suppl ied grat is by NHPP, NIDDK, NIH, Bethesda,MD, USA.

Correspondence and reprint requests.

Theriogenology 50:283-292, 1998 0093-691)(/98/$19.00 © 1998 by Elsevier Science Inc. PII S0093-691X(98)00136-8

284 Theriogenology

INTRODUCTION

The relationship between the plasma levels of gonadotrophins and testosterone have been well documented in prepubertal bulls (25) and pubertal (13). Changes in serum inhibin, FSH, LH and testosterone in prepubertal and pubertal bovine bulls (20) and seasonal variations in plasma inhibin in male monkeys (17) as well as peripheral inhibin, FSH and testosterone concentrations in seasonal cycles of rams (14) have been reported. Immunization against inhibin in rams (19) and prepubertal bulls (12) points to the involvement of inhibin in the regulation of FSH levels. There is a general consensus that in male domestic species inhibin also plays a vital role in preferential control of FSH secretion (23). The circadian and ultradian secretory patterns of gonadotrophins in all likelihood modulate not only the activity pattern of the glandular epithelium of the gonads but also the germinal epithelium. Therefore, these have profound effects on the functional reproductive status of the species in question. In Swamp buffalo bulls, circadian variations of plasma LH and testosterone (4) and plasma testosterone levels have been reported (2, 7). However, to the best of our knowledge no information is available on circadian and pulsatile secretory profiles of inhibin, FSH, LH and testosterone in adult Murrah buffalo bulls. The present investigation was, therefore, undertaken to elucidate the interrelationship between these hormones and to understand their pulsatile and circadian rhythmic secretion patterns in the buffalo species.

MATERIALS AND METHODS

Animals and Blood Sampling Schedule

Adult Murrah buffalo bulls (n=6) aged 7 to 8 yr were used in the study. The bulls were housed individually and were fed a balanced ration with free access to water. Bulls were fitted with indwelling jugular vein catheters 1 d prior to the start of blood sampling during the cool month of November. Maximum and minimum temperatures during the sampling hours were 28.5°C and 9.8°C, respectively, with 8.2 bright day light hours. Serial blood samples were collected for a period of 24-h at 2-h intervals to study the circadian circulatory profiles of the hormones. To assess pulsatile secretory patterns blood was collected at 15-min intervals for a period of 5-h between 1100 and 1600 h, into heparinized tube. The plasma separated and stored at -20°C until hormonal analysis.

Radioimmunoassay for Inhibin

Plasma inhibin levels were measured by a sensitive heterologus double antibody RIA(9) using purified bovine 32 kDa inhibin for iodination, rabbit anti inhibin antiserum (TNDH-1) and partially purified bovine inhibin as the reference

Theriogenology 285

preparation. The cross-reactivity of various inhibin-related peptides and other peptide hormones in the assay system have been previously reported (9). The sensitivity of the inhibin RIA was 3.9 pg/tube based on a 95% confidence limit of the zero standard. The intra- and inter-assay coefficients of variation were 7.2 and 14.6%, respectively (24).

Radioimmunoassay for LH and FSH

Plasma LH and FSH were estimated using a modified (11) heterologous double antibody RIA. Plasma LH levels were estimated using NIDDK oLH1 (AFP 7071B) for radioiodination, and cold standard and antiovine LH serum (NIDDK anti oLH 1 AFP192279) plasma FSH levels were determined using anti ovine FSH serum (NIDDK anti oFSH 1 AFP C528113) , N I D D K oFSH-1 AFP 75714 for radioiodination and NIDDK oFSH RP-1 as the reference standard. The sensitivity of the RIA for LH and FSH was 0.25 and 0.125 ng/tube, respectively. The intra-and inter-assay coefficients of variation for LH and FSH were 4.8 and 9.1% and 5.2 and 10.2%, respectively.

RIA for Testosterone

Testosterone concentration in plasma samples was determined by using direct solid phase 1251 RIA coat-a-count kit (Diagnostic Products Corp., Los Angeles, CA, USA. The kit had a broad reportable range of 4 to 1600 ng/dL.

An elevation in hormone concentration at any particular sampling over the 5-h period of 15-min intervals was considered to be a pulse when the value of two consecutive samples were greater than the mean of two previous samples (basal value) and the value of at least one of the peak samples exceeded the mean basal value by more than twice the coefficient of variation of the assay (3).

Statistical Analysis

Statistical analysis was performed on a PC using SPSS/PC+ studentware software a 2 way ANOVA was used for variable animals and times. Means were tested for differences using Duncan's multiple range test. The same software was used to investigate the interrelationships between variables investigated through correlation and regression analysis.

RESULTS

The mean 24-h variations in circulatory levels of inhibin, FSH, LH and testosterone are depicted in Figure 1 while pulsatile variations of these hormones are shown in Figure 2. There was a highly significant negative correlation between

286 Theriogenology

LH and time (P<0.01), and a significant slope of regression of LH on time, which indicated that there is a linear relationship between LH and time. The other correlation which was found to be of relatively high order and statistically significant (P<0.01) was between inhibin and FSH. As in the instance of correlation, the slope of regression of FSH on inhibin was significant. Other significant linear regressions were those between testosterone with time (P<0.01), inhibin (P<0.05), and LH (P<0.01).

Circulating Inhibin

Plasma inhibin levels in 6 adult Murrah buffalo bulls ranged from 0.201 to 0.429 ng/mL over the 24-h period, with an overall mean of 0.278+0.023 ng/mL. There was no significant variation (P<0.05) in inhibin levels during various sampling intervals. The fluctuations observed during the 15-rain intensive sampling intervals failed to fulfil the criteria of a pulse as defined by Bevers et al. (3).

Circulating FSH

Plasma FSH levels ranged from 0.95 to 3.61 ng/mL in 6 bulls. The mean concentration of FSH over 24-h was 1.66+0.25 ng/mL. The maximum concentration of FSH was observed at 0 I-h, which was significantly higher (P<0.05) than at 1500 and 1700 h of the day. The pulsatile secretory pattern was not observed in 4 of the 6 bulls, while in the remaining 2 bulls only a single pulse was observed over a 5-h sampling period (between 1100 to 1600-h).

Circulating LH

Plasma LH levels in these bulls ranged from 0.92 to 9.91 ng/mL. The maximum LH level over 24-h was observed at 0500 h which was significantly higher (P<0.05) than in 1500-h samples. The overall mean LH concentration over 24-h was 3.33±1.02 ng/mL, while the LH secretion into the peripheral circulation was observed to be pulsatile, with an average of 0.6 pulses/h.

Circulating Testosterone

Plasma testosterone levels were studied in 4 bulls because of the limited number of coat-a-count RIA kits available. Levels of testosterone in these bulls ranged between 0.19 and 2.99 ng/mL. During the course of 24 h two elevations of testosterone were noted, which followed the LH peaks. Overall mean testosterone levels for the 24-h period was 1.34±0.52 ng/mL. Pulsatility of testosterone was studied for 4-h, and secretion of testosterone was found to be pulsatile, with an average of 0.32 pulses/h.

Theriogenology 287

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DISCUSSION

The present study, to the best of our knowledge, is the first report on buffalo bull plasma inhibin profiles in relation to gonadotrophins and testosterone over a 24-h period and associated pulsatile changes in these hormones.

It has been observed that inhibin produced from Sertoli cells (8, 26, 27) seems to be secreted at a uniform rate into the peripheral circulation in adult buffalo bulls, without showing any circadian pattern or variations over different time periods of the day (P<0.05). Levels of inhibin were much lower and were less than half of the levels reported in female buffaloes (5, 22); similar findings have been reported in rats (1). It has been observed that there is a marked decline in inhibin levels after attainment of puberty in beef bulls (20) and in rams (26). Inhibin operates inhibitory regulation of FSH secretion in prepubertal bulls and adult bovine bulls (12, 18). In male rats of all ages depression of FSH levels has been observed (10) after treatment with exogenous inhibin-like activity. Age-related changes in inhibin levels in buffalo bulls have yet to be ascertained. However, the physiological relevance of inhibin during the post pubertal period cannot be underestimated, as appreciable amounts of inhibin were detected in the circulation. In adult buffalo bulls, inhibin secretion into the peripheral circulation was found to be nonpulsatile according to the criteria of Bevers et al. (3). We have not come across any report on pulsatile changes in peripheral inhibin levels in males of any species. In bovine bulls, it has been observed that inhibin production and its release into the circulation is not parallel (20), probably due to the presence of the blood-testis barrier, which precludes the entry of this high molecular weight glycoprotein hormone. It has been observed that inhibin release from testis via the basal and outer compartment of the seminiferous epithelium is drastically reduced at puberty (16). An FSH- induced increase in inhibin release by the testis is associated with an increase in the flow rate oftesticular lymph due to increased vascular permeability (28). Since FSH levels were also uniform and nonpulsatile in 4 of the 6 bulls, it may be a reason for the lack of pulsatility of inhibin in buffalo bulls. Secretion of FSH in domestic animals is nonpulsatile, while LH in this study was released in pulsatile manner as reported (23). Plasma FSH levels like inhibin did not show marked fluctuation during the 24-h period on the whole, a parallelism in their secretion was seen.

Plasma LH levels showed significant variation (P<0.05) during various times over the 24-h studied. The highest level was observed early in the morning at 0500-h and lowest at 1500-h. We also observed that libido and mounting activity by buffalo bulls is much better during the morning hours, thus semen is collected during these early hours of the day. Increased libido and mounting could be attributed to high testosterone levels in the early morning samples. So the possibility

290 Theriogenology

of sunlight and environmental temperature influencing LH release in this species cannot be ruled out. However, in bovine bulls daylight did not affect LH secretion in the circulation (13). In prepubertal bulls (25)a clear relationship between LH, FSH and testosterone has been suggested. In our present study, LH and FSH showed a marked divergence in their secretion into the circulation. In rams, fluctuation in levels of FSH could not be correlated with episodic secretory spikes of LH (15).

Plasma testosterone levels followed the LH secretory pattern throughout the 24-h period. In buffalo bulls, testosterone pulse followed the LH pulse but not with the regularity observed in rams (15) and bovine bulls (13, 25). In males androgens are involved in the negative regulation of 0~ and 8 LH mRNA and do not affect FSH B mRNA, which is under the control of inhibin (6). Accordingly, in buffalo bulls, no relationship could be drawn between plasma testosterone and FSH levels. In conclusion, the present results indicate that inhibin and FSH are secreted into the peripheral circulation in a parallel fashion, with testosterone consistently following the LH secretory pattern. Inhibin in adult buffalo bulls may be a cause for the observed divergence in LH and FSH secretion into the peripheral circulation.

A regression analysis of LH on time indicated that circadian variablility is an important aspect of the secretory pattern of LH in the male buffalo bull and that there was a linear relationship between inhibin - FSH and LH - testosterone patterns.

R E F E R E N C E S

1. Ackland JF, D'Agostino J, Ringstorm S J, Hostetler JP, Mann BG, Schwartz NB. Circulating radioimmunoassayable inhibin during periods of transient follicle stimulating hormone rise: Secondary surge and unilateral ovariectomy. Biol Reprod 1990; 43: 347-352.

2. A h m a d M, LatifM, Ahmad M, Qazi MH, Sahir N, Arslan M. Age-related changes in body weight, scrotal size and plasma testosterone levels in buffalo bulls (Bubalus bubalis) Theriogenology 1984; 22:651-656.

3. Bevers MM, Dieleman S J, VanTol HTM, Blankenstein DM, Vanden Broek J. Changes in pulsatile secretion pattern ofLH, FSH progesterone, androsteredione and oestradiol in cows after superovulation with PMSG. J Reprod Fertil 1989; 87: 745-754.

4. Chantaraprateep P, Kamonpatana M, Lohachit C, Kunawongkrit A, Verakul P, Bodhiprakash P, Ngramsuriyaroj C. Circadian variations of plasma LH and testosterone in adult swamp buffalo bulls. Theriogenology 1981; 15:161-171.

5. Dixit VD. Plasma Inhibin Levels in Relation to Gonadotrophins and Steroids in Superovulating Buffaloes and Adult Buffalo Bulls. M.V.Sc. thesis, Chaudhry Charan Singh Haryana Agricultural University, Hisar, India, 1996.

Theriogenology 291

6. Gharib SD, Wierman ME, Shupnik MA, Chin WW. Molecular biology of pituitary gonadotrophins. Endocrine Rev 1990; 11 : 177-195.

7. Gunarajasingam D, Rajamahendran R, Downey BR, Lague PC. Testosterone secretion in young and adult buffalo bulls. Theriogenology 1985, 24:185-195.

8. Gunsalus GI, Musto NA, Bardin CW. Bidirectional release of a sertoli cell product, androgen binding protein, into the blood and seminiferous tubuli. In: Steinberger A, Steinberger E (¢ds), Testicular Development, Structure and Function. New York: Raven Press Ltd., 1980; 291-297.

9. Hamada T, Watanab¢ G, Taya K, Sasamoto S, Hasegawa Y, Miyamoto K, Igarashi M. Radioimmunoassay of inhibin in various mammals. J Endocrinol 1989; 122: 697-704.

10.Hermans WP, vanLeeuwen ECM, Debets MHM, deJong FH. Involvement of inhibin in the regulation of follicle stimulating hormone concentration in prepubertal and adult, male and female rats. J Endocrinol 1980; 86:79 abstr.

11. Kaker ML, Razdan MN, Galhotra MM. Serum LH concentration in cyclic buffalo (Bubalus bubalis) J Reprod Fert 1980; 60: 419-424.

12.Kaneko H, Yoshida M, Hara Y, Taya K, Araki K, Watanabe G, Sasamoto S, Hasegawa Y. Involvement of inhibin in the regulation of FSH secretion in prepubertal bulls. J Endocrinol 1993; 137: 15-19.

13.Katongole CB, Naftolin F, Short RV. Relationship between blood levels of luteinizing hormone and testosterone in bulls and the effects of sexual stimulation. J Endocrinol 1971; 50: 457-466.

14. Lee VWK, Cumming IA, deKretser DM, Findlay JK, Hudson B, Keogh EJ. Regulation of gonadotrophin secretion in rams from birth to sexual maturity. 1. Plasma LH, FSH and testosterone levels. J Reprod Fertil 1976; 46:1-6.

15. Lincoln GA, Lincoln CE, McNeilly AS. Seasonal cycles in the blood plasma concentration of FSH, inhibin and testosterone and testicular size in rams of wild, feral and domesticated breeds of sheep. J Reprod Fertil 1990; 88: 623-633.

16. Maddock S, Sharpe RM The effect of sexual maturation and altered synthesis on the production and route of secretion of inhibin from testis. Endocrinology 1990; 126: 1541-1549.

17. Matsubayashi K, Watanabv G, Taya K, Katakai Y, Sasamoto S, Suzuki J, Nozaki M. Seasonal changes in plasma concentrations of immunoreactive inhibin and testicular activity in male Japanese monkeys. Biol Reprod 1991; 44: 822-826.

18. McGowan RD, Kiracofe GH, Bolt DJ. Depression of follicle stimulating hormone in bulls injected with follicular fluid. Theriogcnology 1988; 29: 979-986.

19. McKeown RM, Callaghan DO, Roche JF, Boland MP. Effect of immunization of rams against bovine inhibin ctl-26 on semen characteristics, scrotal size, FSH, LH and testosterone concentration. J Reprod Fertil 1997; 109: 237-245.

20. Miyamoto A, Umezu M, Ishii S, Furusawa T, Masaki J, Hasegawa Y, Ohta M. Serum inhibin, FSH, LH and testosterone levels and testicular inhibin content in beef bulls from birth to puberty. Anita Reprod Sci 1989; 20: 165-178.

292 Theriogenology

21. Niswender GD, Reichert LE Jr, Midgley AR Jr, Nalbandov AV. Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology 1969; 84: 1166-1173.

22. Palta P, Prakash BS, Madan ML. Peripheral inhibin levels during estrous cycle in Murrah buffalo (Bubalus bubalis). Theriogenology 1995; 123: 181-183.

23. Price CA. The control of FSH secretion in the larger domestic species. J Endocrinol 1991; 131: 177-184.

24. Rodbard D. Statistical quality control and routine data processing for radioimmunoassay and immunoradiometric assay. Clin Chem 1974; 20:1255-1270.

25. Schams D, Gombe S, Schallenberger E, Reinhardt V, Claus R. Relationship between short term variations of LH, FSH, prolactin and testosterone in peripheral plasma of prepubertal bulls. J Reprod Fertil 1978; 54: 145-148.

26. Schanbacher B. Radioimmunoassay of inhibin: serum response to unilateral and bilateral orchidectomy Endocrinology 1988; 123: 2323-2330.

27. Steinberger A, Steinberger E. Secretion of an FSH inhibiting factor by cultured sertoli cells. Endocrinology 1976; 99: 918-92.

28. Voglmayr JK, Jolley D, Vale W, Willoughby D, Moser A, So CK, Chen CL, Bardin CW. Effect of follicle stimulating hormone on inhibin release by different testicular compartments in the adult ram. Biol Reprod 1992; 47: 573-581.