JANINE L. BROWN,* KRISTINE D. DAHL,t AND PRABIR K. CHAKRABORTY*

Transcription

1 Journal of Anthology, Vol. 12, No. 4, July/August 1991 Copyright #{176} American Society of Andrology Effects of Follicular Fluid Administration on Serum Bioactive and Immunoreactive FSH Concentrations and Compensatory Testosterone Secretion in Hemicastrated Adult Rats JANINE L. BROWN,* KRISTINE D. DAHL,t AND PRABIR K. CHAKRABORTY* From the *Department of Obstetrics and Gynecology, Un(formed Services University of the Health Sciences, Bethesda, Maryland, and the tdepartment of Medicine, Veterans Administration Medical Center and Department of Medicine and Population Center for Research in Reproduction, University of Washington, Seattle, Washington. ABSTRACT: In adult rats, removal of one testis (hemicastration) results in an elevation of serum follicle-stimulating hormone (FSH) concentrations and a compensation in testosterone secretion by the remaining testis without a corresponding increase in testis size. To determine whether changes in FSH secretion and compensatory androgen production are related, serum testosterone concentrations were measured after inhibinrich porcine follicular fluid was administered twice daily for 4 days to block the hemicastration-induced rise in FSH. Both serum immunoreactive FSH (immuno-fsh) and bioactive FSH (bio-fsh) concentrations were increased 4 days after hemicastration. The significant increase in serum immuno-fsh in hemicastrated animals was prevented by follicular fluid administration, whereas the serum bio-fsh activity and biologic to immunologic (B/I) ratios were increased in follicular fluidtreated animals. The follicular fluid-induced reduction in serum immuno-fsh had no effect on serum testosterone secretion in hemicastrated rats. Serum inhibin concentrations were reduced 27% in hemicastrated rats compared with intact controls, while administration of exogenous follicular fluid increased serum inhibin concentrations. An elevation in serum immuno-fsh secretion after hemicastration apparently is not required for the compensatory testosterone response. However, the observation of increased bio-fsh in hemicastrated and follicular fluidtreated animals raises questions about the importance of FSH quality (bioactivity), rather than quantity, for controlling testicular steroidogenic activity. Key words: Rat, hemicastration, follicle-stimulating hormone (FSH), testosterone, inhibin, follicular fluid, bioactivity. J Androl 1991 ;12: U nilateral castration of adult rats results in compensatory testosterone production by the remaining testis within hours after surgery (Frankel and Mock, 1982; Frankel and Wright, 1982). This rapid response to removal of one testis is the result of a doubling of testosterone output by the remaining gonad (Mock and Frankel, 1982; Frankel et al, 1989) and is not related to changes in testis size (Cunningham et al, 1978; Frankel and Wright, 1982). The mechanisms responsible for the acute testosterone response to hemicastration are unknown. Although luteinizing hormone (LII) is an important regulator of testosterone production, there are no apparent changes in basal or gonadotropinreleasing hormone (GnRH)-stimulated LH secretion or LH pulsatility in the hemicastrated rat (Cunningham et al, 1978; Mock and Frankel, 1982; Frankel and Mock, 1982; Frankel This work was supported in part by USUHS grant No. C8527 to Dr. Brown. Correspondence to: Janine L. Brown, PhD, Department of Obstetrics and Gynecology, Uniformed Services University of the Health Sciences, 431 Jones Bridge Road, Bethesda, Maryland Received for publication December 5, 199; accepted for publication Februaiy5, and Wright, 1982). In addition, similar concentrations of testicular LH and follicle-stimulating hormone (FSH) receptors in intact and hemicastrated adult rats (Frankel et al, 1984; Brown and Chakraborty, 1991) suggest that gonadal sensitivity to circulating gonadotropins also is not altered. Increases in serum and pituitary concentrations of FSH are observed after hemicastration (Cunningham et al, 1978; Frankel and Wright, 1982; Brown and Chakraborty, 1991); however, the role of FSH in modulating steroid secretion in this experimental model has not been fully examined. Although some studies have failed to demonstrate any effect of FSH on testicular steroidogenic activity (for review, see Ewing and Zirkin, 1983), others show that FSH can stimulate testosterone production under certain conditions. For example, exposure of hypophysectomized rats to FSH increases the responsiveness of the testis to LH-stimulated testosterone release (Odell et al, 1973; Ketelslegers et al, 1978). In addition, Keteislegers et al (1978) found a correlation between rising FSH levels and testosterone secretion in the developing rat. Finally, FSH can stimulate the production of Sertoli cell proteins that, in turn, stimulate Leydig cell testosterone secretion in vitro (Benahmed et al, 1987). The increase in serum FSH after hemicastration is 221

2 222 Journal of Andrology. July/August 1991 most likely due to a reduction in inhibin output since this hormone exhibits FSH-suppressing activity (Scott and Burger, 1981; de Jong and Robertson, 1985). Follicular fluid is a rich source of inhibin (Mason et al, 1985), and its administration has been shown to selectively suppress serum FSH levels in male rats (Lorenzen et al, 1981; Jones et al, 1985). In this study, we determined whether administration of porcine follicular fluid to adult hemicastrated rats would block the rise in FSH secretion, and if it did, whether it affected the compensatory testosterone response. Materials and Methods intra-assay coefficients of variation of 7.4%, 6.1%, and 5.9% for the LH, FSH, and testosterone assays, respectively. Serum inhibin concentrations were measured using two different assay systems: 1) a heterologous RIA that used antiserum raised against bovine 31-kd inhibin (No. 1989) and iodinated bovine 31-kd inhibin as tracer (Robertson et al, 1988); and 2) an RIA developed against a synthetic porcine a(1-3) inhibin fragment (inhibin-a, N. Ling, Salk Institute, San Diego, CA) with iodinated inhibin-a as tracer (Brown and Chakraborty, 1991). Intra-assay coefficients of variation were 12.% and 7.4% for the intact and a-fragment assays, respectively. Bioactivity of FSH (bio-fsh) in rat serum was determined using the in vitro granulosa cell aromatase bioassay as described by DahI et al (1989) with RP-2 as the reference preparation. The intra-assay coefficient of variation was 1%. Animals and Injections Statistics After a 1-day adaptation period, adult (6 days old) male Sprague-Dawley rats were hemicastrated or sham-operated under methoxyflurane anesthesia (Metophane, Pitman-Moore, Inc., Washington Crossing, NJ). Hemicastration was performed through a mid-scrotal incision by exposing the left testis, ligating all testicular and epididymal vessels, and removing the testis and associated epididymis. For sham-operations, the scrotal skin was incised only. Incisions were closed with nonabsorbable sutures. Porcine follicular fluid (FF), provided by the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases (NIADDK), was centrifuged at 2, X g at 4#{176}C to remove cellular debris and then charcoal-stripped to remove more than 95% of the steroids. In brief, FF was stripped twice with 1 mg Norit-Aiml of fluid for 4 hours at 4#{176}C, centrifuged twice (2, x g), and filtered under positive pressure through a series of filters (8, 1.2,.8, and.45 i.m). Porcine follicular fluid was frozen at - 2#{176}C in 1 ml aliquots and thawed only once before use. Hemicastrated rats were injected intraperitoneally with 5 l FF (N = 2) or saline (N = 1). Control rats were injected with saline (N = 1) every 12 hours (7: AM and 7: PM) for 96 hours. To determine whether exogenous FSH would override any negative FF effects, 1 FF-treated hemicastrated animals were concurrently injected subcutaneously with 5 p.g ovine FSH (NIADDK-oFSH- 17). Initial injections were given at the time of surgery. Rats were killed by exsanguination under methoxyflurane anesthesia 6 hours after the last injection. Blood was kept on ice and centrifuged (25 x g for 2 minutes at 4#{176}C) within 4 hours of collection. Serum was then divided into two aliquots (one for radioimmunoassay and the other for bioassay) and frozen at - 2#{176}C. Hormone Assays Serum concentrations of immunoreactive (immuno-) LH and FSH were quantified using radioimmunoassay (RIA) kits for rat serum provided by the NIADDK. Both standard preparations were RP-2. Serum testosterone was analyzed using a human iodine- 125 RIA kit (Radio Systems Laboratories, Carson, CA) previously validated for use with rat serum (Brown and Chakraborty, 1988). For each hormone, all samples were measured in a single assay with Significant differences were determined by analysis of variance using a completely randomized design. Mean differences between treatment groups were determined using Duncan s New Multiple Range Test. All values are presented as the mean ± SEM. Results Serum concentrations of immuno- and bio-fsh, and a comparison of FSH B/I ratio are depicted in Figure 1. After hemicastration, serum immuno-fsh (Fig 1A) and bio-fsh (Fig 1B) concentrations increased 34% and 15% (P <.5), respectively, while the B/I ratio (Fig lc) tended to be higher (P <.1) in hemicastrated rats compared with intact controls. Contrarily, the significant increase in serum immuno-fsh in hemicastrated animals was completely blocked (P <.5) by FF administration. Interestingly, the serum bio-fsh activity and B/I ratios were increased (P <.5) approximately fourfold in FF-treated males compared with intact controls and were approximately 3% to 5% greater (P <.5) than those measured in hemicastrated rats. The increase in bio-fsh in FF-treated rats was not due to activity of the follicular fluid itself since the concentration of bio-fsh measured in the fluid was only 4.8 p.g/l. Concentrations of immuno-fsh in FSII-supplemented rats were not different (P >.5) from intact animals (Fig 1A). Since the NIADDK anti-rat FSH antiserum used in this study did not cross-react with up to 5 ng of ovine FSH, only endogenous immuno-fsh was detected in this assay. In contrast, the bioassay measured both endogenous rat bio- FSH and the exogenously administered ovine FSH (mean, 3278 ± 5 p.g/l; data not shown). Hemicastration reduced (P <.5) serum inhibin concentrations approximately 25% compared with intact controls in both assay systems (Fig 2). Administration of exogenous FF increased (P <.5) serum inhibin concentrations approximately fivefold over the levels observed in

3 Brown, Dahi, and Chakraborty. Follicular Fluid Treatment of Male Rats A A s 2 I in C E E I b B a.8 9o.6 16 Ic -o.4.2 fl *. tnthc HC HC +FF +FF +FSH C 8 FIG. 2. Concentrations (mean ± SEM) of serum inhibin measured with a heterologous AlA which used antiserum raised against bovine 31 kd inhibin (no. 1989) and iodinated bovine kd inhibin as tracer (A), and an RIA developed against a synthetic porcine a(1-3) inhibin fragment (inhibin-a) with iodinated inhibin-a as tracer (B) in: 7-day-old male intact rats administered saline IP (INT; N = 1) or hemicastrated rats administered saline IP (HC; N = 1), 5 l charcoal-stripped follicular fluid IP (HC + FF; N + 1), or FE IP and 5 tg ovine follicle-stimulating hormone SC (HC + FE + FSH; N = 1). All treatments were given twice daily (7: AM and 7: PM) for 4 days. For each variable, values bearing different superscripts are significantly different (P <.5). +FSH FIG. 1. Concentrations (mean ± SEM) of serum immuno-fsh (A), bio-fsh (B), and the FSH biologic to immunologic (B/I) ratio (C), in: 7-day-old male intact rats administered saline IP (INT; N = 1) or hemicastrated rats administered saline IP (HC; N = 1), 5 l charcoal-stripped follicular fluid IP (HC + FF; N = 1), or FE IP and 5 g ovine follicle-stimulating hormone SC (HC + FF + FSH; N = 1). All treatments were given twice daily (7: AM and 7: PM) for 4 days. For each variable, values bearing different superscripts are significantly different (P <.5). *Data are not presented because of significant cross-reactivity of the exogenously administered ovine FSH in the FSH bioassay. intact animals when measured in the intact inhibin assay (Fig 2A) but only returned inhibin concentrations to intact levels using the a-fragment assay (Fig 2B). The FF was analyzed in the inhibin RIAs and found to contain 3.5 U X 14/L and 3 p.g/l for the intact and a-fragment assays, respectively. Supplementation of FF-treated rats with ovine FSH did not alter serum inhibin concentrations (P >.5) compared with unsupplemented animals. Right testis weight in hemicastrated rats (1.7 ±.5 g) did not differ (P>.5) from that of intact animals (1.6 ±.6 g) 4 days after hemicastration. In addition, FF treatment either with (1.7 ±.4 g) or without (1.5 ±.14 g)

4 224 Journal of Andrology. July/August 1991 FSH supplementation had no effect (P >.5) on testis weight. Similarly, serum testosterone concentrations did not differ (P>.5) between intact (9.7 ± 3.1 nmol/l) and hemicastrated (7.6 ± 1.4 nmol/l) rats. These values were similar (P >.5) to those from hemicastration and FF animals treated either with (9. ±.7 nmol/l) or without (9. ± 1.4 nmol/l) exogenous FSH. Serum LH concentrations also were similar (P >.5) among all treatment groups with an overall mean of.51 ±.7.Lg/L. Discussion Serum testosterone concentrations in hemicastrated rats were the same as those measured in intact controls possessing twice the testicular mass. This compensatory androgen response has been shown to be due to a doubling in testosterone secretion from the remaining gonad rather than to an increase in testis size (Cunningham et al, 1978; Frankel and Wright, 1982; Mock and Frankel, 1982; Frankel et al, 1989). Although the mechanism responsible for the hemicastration response is not known, evidence suggests it may be governed by extra-gonadal stimuli. For example, androgen compensation does not occur in hypophysectomized male rats (Frankel et al, 1984), and the response cannot be sustained in vitro (Frankel et al, 1989). Changes in LH secretion or gonadal sensitivity to gonadotropins also do not explain this phenomenon (Cunningham et al, 1978; Mock and Frankel, 1982; Frankel and Mock, 1982; Frankel and Wright, 1982; Brown and Chakraborty, 1991). Instead, an increase in FSH production is currently the only consistent endocrine response observed in hemicastrated rats (Cunningham et al, 1978; Frankel and Wright, 1982; Brown and Chakraborty, 1991). In this study, exogenous FF administration prevented the increase in immuno-fsh secretion. Despite a reduction in serum FSH concentrations, however, the compensatory testosterone response was unaffected. Furthermore, FSH supplementation of FF-treated hemicastrated rats did not enhance testosterone secretion. These data suggest that there is no apparent relationship between increased immuno-fsh secretion and testosterone compensation in adult hemicastrated rats; however, there was an increase in the biologic activity of FSH in hemicastrated rats that was similar to that reported for sheep (Brown et al, 199). In FFtreated rats, although serum immuno-fsh concentrations were decreased, the bioactivity of the released hormone was increased to an even greater extent than that measured in hemicastrated animals. It is not known whether the FFinduced increase in bio-fsh activity in vivo was due to inhibin or other, possibly unidentified, factors present in follicular fluid. We did show that the in vitro activity (as determined by granulosa cell bioassay) was not due directly to FF factors since the FSH bioactivity was only 5 i.g/l, which is much less than that measured in serum. In addition, we did not intend to conclusively determine the role of inhibin in regulating FSH bioactivity but rather to analyze how changes in circulating FSH affected the hemicastration response. These data suggest that increases in bio-fsh are related to compensatory androgen secretion and that changes in FSH quality, rather than quantity, may be driving the hemicastration response. Despite evidence that an increase in bio-fsh concentrations occurs at a time when testosterone secretion is elevated during sexual development in the rat (Ketelslegers et al, 1978; Dahl et al, 1989), no direct relationship between bio-fsh levels and testicular steroidogenic activity has been observed in the human (Tenover et al, 1987, 1988). Clearly, studies are needed to clarify the importance of FSH quality vs. quantity for regulating testicular steroidogenic activity. The increase in FSH secretion after hemicastration was possibly due to a reduction in serum inhibin, a hormone believed to regulate the secretion of this gonadotropin (Scott and Burger, 1981; de Jong and Robertson, 1985). The reduction in serum FSH after steroid-free FF administration has been related to the presence of inhibin in the fluid (Lorenzen et al, 1981; Jones et al, 1985; Mason et al, 1985). Recently, Rivier et al (1989) reported that inhibin concentrations were decreased after hemicastration in prepubertal (15-day-old) rats using a RIA generated against an a-inhibin fragment. However, they observed no consistent reduction in circulating inhibin levels in pubertal (45-dayold) rats and concluded that inhibin may be a more important regulator of FSH secretion in young, immature animals than in older ones (Rivier et al, 1988, 1989). We also observed no change in inhibin concentrations after hemicastration of aged rats (1 to 12 months) using a similar a-fragment assay, although the data were quite variable (Brown and Chakraborty, 1991). In the current study, a moderate reduction in serum inhibin concentrations was observed in adult rats using both the a-fragment and intact bovine inhibin RIAs. It is not clear at this time what is responsible for the discrepancies in inhibin data for adult rats. In this study, serum was used in both the a-fragment and intact inhibin assays. Although serum and plasma both result in displacement curves parallel to the a-fragment standard curve in that assay, serum generally is more active than plasma (Saito et al, 1989). However, the observation of a similar reduction in inhibin using both assays on the same serum samples suggests that these assays can be comparable. Contrarily, there was a large discrepancy between these two assays in the analysis of serum from FF-treated animals, with relative values being higher with the intact inhibin assay. It was demonstrated recently that the intact inhibin antiserum used in this study cross-reacts with an inhibin pro-a-c-subunit present in FF (Robertson et al, 1989). Although the authors of that study suggested that the

5 Brown, DahI, and Chakraborty. Follicular Fluid Treatment of Male Rats 225 cross-reacting proteins in FF probably are not released into the circulation in appreciable amounts, they cautioned against misinterpretation of data resulting from the analysis of inhibin in FF. Therefore, the high levels of inhibin in the FF-treated rats measured using the intact assay probably was the result of analyzing both inhibin and the a-subunit precursor. In summary, an elevation in serum immuno-fsh secretion after hemicastration of adult male rats apparently is not required for the compensatory testosterone response. However, an increase in bio-fsh in hemicastrated and FFtreated animals raises questions about the importance of FSH quality (bioactivity), rather than quantity, for controlling testicular steroidogenic activity. The mechanisms responsible for compensatory androgen secretion will not be realized until we have a more thorough understanding of how various putative intratesticular factors locally regulate androgen secretion (Sharpe et al, 1986; Jansz and Pomerantz, 1987; Saez et al, 1989). The ability of FSH to modulate testicular function through its action on intratesticular factors (Benahmed et al, 1987) also warrants further investigation. Acknowledgments The authors thank the NIADDK for the rat LH and FSH RIA kits, and A. Mitchell, M. Nelson, D. McGuinness, M. Stone, and A. Sarkissian for excellent technical support. References Benahmed M, Morera AM, Chauvin MA, Peretti ED, Revol A. FSH regulates testicular steroidogenesis through Sertoli cell factors. Ann N YAcad Sd. 1987;513: Brown JL. Chakraborty PK. Characterization of the effects of clomiphene citrate on reproductive physiology in male rats of various ages. Ada Endocrinol (Copenh). 1988; 118: Brown JL, Chakraborty PK. Comparison of compensatory pituitarytesticular responses to hemicastration between prepubertal and mature rats. JAndrol. 199l;12:l Brown JL, Schoenemann HM, Chakraborty PK, Stuart LD, DahI KD. Increased bioactivity of serum follicle-stimulating hormone, but not luteinizing hormone following hemicastration in ram lambs. Biol Reprod. l99;43: Cunningham OR, Tindall DJ, Huckins C, Means AR. Mechanisms for the testicular hypertrophy which follows hemicastration. Endocrinology 1978; 12: DahI KD, Jia XC, Hsueh AJW. Granulosa cell aromatase bioassay for follicle stimulating hormone. Methods Enzymol. I989;l68: de Jong FH, Robertson DM. Inhibin: 1985 update on action and purification. Mol Cell Endocrinol. 1985:42: Ewing LL, Zirkin B. Leydig cell structure and steroidogenic function. Recent Prog Horm Res. 1983:39: Frankel Al, Mock EJ. A study of the first eight hours in the stabilization of plasma testosterone concentration in the hemicastrated rat. J Endocrinol. 1982:92: Frankel Al, Wright WW. 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Possible involvement of inhibin in altered follicle-stimulating hormone (FSH) secretion during dissociated luteinizing hormone (LH) and FSH release: unilateral castration and experimental cryptorchidism. Biol Reprod. 1989;4l: Robertson DM. Hayward S. Irby D, Jacobsen I, Clarke L, McLachlan RI, de Kretser DM. Radioimmunoassay of rat serum inhibin: changes after PMSG stimulation and gonadectomy. Mol Cell Endocrinol. 1988;58: 1-8. Robertson DM, Giacometti M, Foulds LM, Lahnstein J, Ooss NH, Heam MTW, de Kretser DM. Isolation of inhibin a-subunit precursor proteins from bovine follicular fluid. Endocrinology. 1989;125: Saez JM, Avallet, Naville D, Perrard-Sapori MH, Chatelain PG. Sertoli- Leydig cell communications. Ann N Y Acad Sci. 1989;564: Saito 5, Roche PC, McCormick Di, Ryan Ri. Synthetic peptide segments of inhibin a-and 13-subunits: preparation and characterization of polyclonal antibodies. Endocrinology. 1989; 125: Scott RS. Burger HO. Mechanism of action of inhibin. Biol Reprod ;24: Sharpe RM, Kerr JB, Fraser HM, Bartlett JMS. Intratesticular factors and testosterone secretion. Effect of treatments that alter the level of testosterone within the testis. J Androl. l986;7:l Tenover JS, DahI KD, Hsueh AJW, LimP, Matsumoto AM, Bremner WJ. Serum bioactive and immunoreactive follicle-stimulating hormone levels and the response to clomiphene in healthy young and elderly men. J Clin Endocrinol Metab. 1987:64: Tenover JS, McLachlan RI, DahI KD, Burger HO, dekretser DM, Bremner Wi. Decreased serum inhibin levels in normal elderly men: evidence for a decline in Sertoli cell function with aging. J Clin Endocrinol Metab. 1988;67: