Patterns of plasma sex hormone-binding globulin, thyroxine and thyroxine-binding globulin in relation to reproductive state and hibernation in female little brown bats G. G.

Kwiecinski, D. A. Damassa and A. W. Gustafson

Department of Anatomy and Cellular Biology, Tufts University Schools of Medicine, Dental Medicine, Veterinary Medicine and the Sackler School of Graduate Biomedicai Sciences, 136 Harrison Avenue, Boston, Massachusetts 02111, U.S.A. (G. G Kwiecinski to whom requests for offprints should be addressed is now at Department of Biology, University of Scranton, Scranton, Pennsylvania 18510-4625, U.S.A.) received

12

April

1990

ABSTRACT

A sex hormone-binding globulin (SHBG), which bound both oestradiol and dihydrotestosterone, was studied in the plasma of adult female little brown bats throughout the annual reproductive cycle. This protein was present at low baseline levels from September to May inclusive, months which correspond to the periods of hibernation, ovulation and early pregnancy. During the second half of pregnancy in June, SHBG levels increased 15- to 30-fold and remained increased throughout lactation and anoestrus/pro\x=req-\ oestrus (July\p=n-\August). Although SHBG was increased during late pregnancy, the fact that levels were also high during and after lactation indicates that this protein is not specific to pregnancy. Plasma concentrations of thyroxine (T4) and the percentage binding of T4 to thyroxine-binding globulin (TBG) also

showed marked seasonal variations, with T4 levels exhibiting a biphasic seasonal pattern. A major peak in plasma concentrations of T4 occurred around the time of spring arousal from hibernation and subsequent ovulation, while a second peak of lesser magnitude was measured in August, corresponding to the time of pro-oestrus and the onset of mating. The percentage binding of T4 by TBG was increased during the summer months in parallel with the increase in SHBG concentrations. Electrophoretic analysis of plasma T4 binding revealed a single peak of TBG activity throughout most of the year; however, during the early lactational period TBG was resolved as a double peak, suggesting the presence of a molecular variant during this reproductive stage. Journal ofEndocrinology (1991) 128, 63\p=n-\70

INTRODUCTION

& Damassa, 1985, 1987). Interestingly, SHBG in bats, like the protein in primates, exhibits high affinity for both testosterone and oestradiol (Damassa et al. 1982; Kwiecinski et al. 1987), suggesting that SHBG may also play an important role in steroid action during the female reproductive cycle. In little brown bats, hibernation is superimposed upon the reproductive cycle, and effectively lengthens it by several months (for reviews see Wimsatt, 1960, 1969; Gustafson, 1979; Oxberry, 1979). Females of this species are mono-oestrus, with a new oestrous cycle beginning in late summer after termination of lactation. During this period (post-lactation), the ovaries manifest patterns of follicular growth and atresia. Subsequently, in August, mating begins and the number of vesicular follicles becomes reduced to

A

high-affinity sex hormone-binding globulin (SHBG), also referred to as sex steroid-binding pro¬

tein and testosterone-oestradiol-binding globulin, is present in the circulation of many mammals (Westphal, 1986), including members of at least two families of bats (Kwiecinski, Damassa, Gustafson & Armao, 1987). In little brown bats this protein is pres¬ ent in both sexes (Damassa, Gustafson & King, 1982; Gustafson & Damassa, 1984), and in males has been shown to undergo wide physiological variations dur¬ ing the annual reproductive cycle (Gustafson & Damassa, 1985). Evidence has also been presented that these seasonal changes in SHBG levels have important influences on androgen action (Gustafson

(pro-oestrus/oestrus). When the animals enter hibernation in autumn, there is a single surviving fol¬ licle containing an ovum that is maintained in a state of meiotic arrest. Although copulations may continue throughout the hibernation period (late September/ October to April), and spermatozoa are stored in the reproductive tracts of both sexes, ovulation and sub¬ sequent fertilization do not take place until after per¬ manent arousal from hibernation in spring. Thus, a period of delayed ovulation occurs in these females during hibernation. Pregnancy is 50-60 days in length and is followed by a period of lactation/ one

anoestrus.

Although

the seasonal

the factors that

regulate SHBG during reproductive cycle are not completely

understood, recent studies in male bats have indicated

that thyroid hormones play a key role in the induction of increases in SHBG levels following arousal from hibernation (Damassa, Gustafson, Kwiecinski & Pratt, 1985; Kwiecinski, Damassa & Gustafson, 1986). The importance of thyroid hormones in the regulation of SHBG has also been suggested by studies in other species, including man, where SHBG concen¬ trations are increased in hyperthyroidism and decreased in hypothyroidism (Anderson, 1974; Yosha, Fay, Longcope & Braverman, 1984a; Yosha, Longcope & Braverman, 1984Z»; Caron, Bennet, Barousse et al. 1989). In the present study the control and function of SHBG in relation to reproductive state and hiber¬ nation in female little brown bats was investigated by examining the seasonal changes in plasma SHBG con¬ centrations, and determining whether seasonal changes in plasma thyroxine (T4) and T4 binding to

thyroxine-binding globulin (TBG) are temporally related to changes in SHBG during the annual cycle.

Reagents

[1,2,4,5,6,7-3H]Dihydrotestosterone ([TTJDHT; 116 Ci/mmol), [2,4,6,7-3H]oestradiol (93 Ci/mmol), [l,2,6,7-3H]testosterone (102Ci/mmol), [l,2,-3H]cortisol (43 Ci/mmol) and [l,2,6,7-3H]corticosterone (101 Ci/mmol)

purchased from New England U.S.A.). Radiolabelled l-T4 ([3',5'-125I]T4; >1200pCi/pg) was purchased from Amersham Inc. (Arlington Heights, IL, U.S.A.). DEAE-cellulose filter paper discs (DE-81, 2-3 cm) were purchased from Whatman (Clifton, NJ, U.S.A.). Bromcresyl green solution (American Monitor Albumin Assay) was purchased from Fisher Scientific (Pittsburgh, PA, U.S.A.). Prepackaged barbital buffer was purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.) and diluted to 60 mmol/1 (pH 8-6). were

Nuclear (Boston, MA,

Polyacrylamide gel electrophoresis (PAGE) Analytical steady-state PAGE was performed as pre¬ viously described for bat plasma (Damassa et al. 1982) in gels containing 5% (w/v) acrylamide and 0-17% (w/v) bisacrylamide. Charcoal-treated plasma samples were diluted in 10 mmol Tris-HCl buffer/1 (pH 7-4, 4 °C) and incubated with tritiated steroids (4 nmol/1) for 20 min at 25 °C and for 20 min at 4 °C. Aliquots of the incubation mixture were applied to gels containing radiolabelled steroid (2 nmol/1). Following electro¬ phoresis, gels were cut into 2 mm slices, and tritium activity was determined by scintillation counting (Beckman Model LS-8100, Beckman Instruments, Irvine, CA, U.S.A.). For steroid competition studies, diluted plasma samples were separated on gels con¬ taining radiolabelled steroid (2 nmol/1) in combi¬ nation with steroid. For

a

500-fold

excess

of

a

non-radioactive

binding studies under non-steady-state conditions, samples were separated on gels that did

MATERIALS AND METHODS

contain steroids. For localization of albumin, were run on parallel gels and stained with Coomassie blue or bromcresyl green.

Collection of animals and

Measurement of SHBG

not

samples plasma

Adult female little brown bats (Myotis lucifugus) were collected in New England from hibernacula (September to April) and maternity colonies (May to August). To minimize stress, animals collected from hibernacula were transported to the laboratory in tor¬ por (4 °C), whereas animals collected from maternity colonies were transported in darkened holding cages. Within 24 h, animals were killed with anaestheticgrade ether and blood samples obtained by intracardiac cannulation (Gustafson, 1976). Whole blood was transferred to heparin-coated microhaematocrit tubes and centrifuged at 13 000 g for 7 min. Plasma was removed, frozen in aliquots and stored at 20 °C. —

Plasma concentrations of SHBG were determined by the DEAE-cellulose filter assay of Mickelson & Petra (1974), as previously described for bat plasma (Damassa et al. 1982). Samples were assayed in dupli¬ cate with an efficiency for bat plasma of 70% and an interassay coefficient of variation (C.V.) of 12-1%. Measurement of T4 Concentrations of

were determined by a specific established and validated for bat radioimmunoassay plasma (Kwiecinski et al. 1986). This assay dissociates T4 from TBG in the presence of high pH and extracts T4 on Sephadex G-25 minicolumns. Extraction of

T4

total T4 by these minicolumns was greater than 95% (Kwiecinski et al. 1986) and was not influenced by variations in plasma T4 or TBG concentrations. The intra-assay C.V. was 4-9% and the interassay C.V. was

12- / .

Plasma T4 binding

of [3H]DHT binding was specific in that it was eliminated by an excess of non-radioactive DHT, and was consequently identified as SHBG. Further studies revealed that this SHBG also exhibited specific bind¬ ing of [3H]oestradiol (Fig. lb) and of [3H]testosterone (data not shown) but did not bind corticosteroids.

peak

binding of T4 to plasma proteins was assessed by gel electrophoresis according to the method of Leopold, Wawschinek, Lind & Eber (1987). This procedure separates plasma equilibrated with trace amounts of [l25I]T4 using 0-9% agarose tube gels (5 40 mm). After electrophoresis, gels were cut into 2 mm slices and the radioactivity in each slice was determined by gamma spectroscopy (Gamma 4000, Beckman Instruments). Albumin was identified fol¬ lowing the addition of bromcresyl green to each gel slice. The identification of plasma TBG was aided by electrophoresis of human plasma in parallel with bat plasma. The percentage binding of [l25I]T4 to TBG, determined at 4°C, was calculated by dividing the amount of radiolabel activity in the TBG peak by the total amount of radiolabel in the gel. Since only trace The

agarose

of [l25I]T4 were used in these studies, the per¬ [125I]T4 to TBG reflects that of the For determination of the specificity endogenous T4. of T4 binding, plasma samples were incubated with trace amounts of [l25I]T4 plus various concentrations of non-radioactive hormone. A 2500-fold molar excess of non-radioactive T4 was found to saturate specific binding sites in undiluted plasma containing high TBG activity. amounts

centage binding of

Data

analysis

Data from various

sample groups are reported as Assay results were normalized by logarithmic transformation before statistical analyses. Samples which assayed below the limit of sensitivity were assigned values equal to the sensitivity limit. Differences between groups were assessed using one¬ way analysis of variance, followed by Newman-Keuls multiple comparison tests. Differences were regarded values were less than 005. as significant when means

±

s.e.m.

RESULTS

Identification of SHBG

Steady-state PAGE of plasma samples from female bats revealed a major peak and a minor peak of [3H]DHT binding activity (Fig. la). This plasma steroid binding present was identical to that pre¬ viously observed in the male (Damassa et al. 1982). The fast migrating (minor) peak corresponded to albumin in stained gels. The slow migrating (major)

20 10 Gel slice number

30

1. Steady-state binding of (a) [3H]dihydrotestoster¬ and (b) [3H]oestradiol to pro-oestrous female bat plasma after fractionation by polyacrylamide gel electrophoresis. The major peak of steroid binding in each pattern corres¬ ponds to the location of sex hormone-binding globulin. The solid bars on the abscissa indicate the migration of albumin in stained gels. Proteins migrated toward the anode at the figure one

right.

Seasonal patterns of plasma SHBG Concentrations of SHBG in the plasma of female bats are shown in Table 1. Values of SHBG throughout the period of delayed ovulation (i.e. hibernation) were low. SHBG levels remained low during the time around ovulation (April) and also during the first half of pregnancy (May). Concentrations of SHBG the latter half of preg¬ increased significantly increased throughout remained and nancy (June) lactation (June-July), post-lactation (July) and the pro-oestrus/oestrus period in August. By September, SHBG levels had returned to baseline values before the onset of hibernation.

during

1. Plasma concentrations of sex hormone-binding globulin (SHBG) and thyroxine (T4) and the percentage of thyroxine-binding globulin (TBG)-bound T4 in plasma from adult female little brown bats. Values are means ± s.e.m. for the number of animals in parentheses table

Group Delayed ovulation

(nmol 1)

Sept-Nov

9 + 2'

Feb-March

8±1« (10)

Periovulation

April

9 + 2"

(5)

(arousal) Pregnancy

May

10+1"

(4)

(1st half of hibernation)

Delayed ovulation (2nd half of hibernation)

(1st naif)

Pregnancy (2nd half) Lactation Post-lactation

Pro-oestrous/oestrous

(11)

2-2±0-5"b(9)

19-3 + 2-5«

4-6±0-9cd(12)

17-8 + 5-4bc(4)

160 + 6-2'

5-2±l-0*=d (3)

7-5 + 0-8»

(2)

(5)

43-8 + 3-3"

(3)

1-3 + 0-1'

June-July July August

256 + 33b(ll)

2-0 + 0-3"· (14) 3-6 + 0-5bc(15)

7-9+l-5dc(5)

53-5±4-3d (7) 62-9±3-8d (3) 56-8 ±2-8" (3) 22-6