Seasonal reproduction in the mongoose, Herpestes auropunctatus

GENERAL

AND

Seasonal I. Androgen,

COMPARATIVE

ENDOCRINOLOGY

Reproduction Luteinizing

Department

in the Mongoose,

Hormone,

M.J.

44, 350-358 (1981)

Herpestes auropunctatus

and Follicle-Stimulating

Hormone

in the Male’

SOARES~AND J.C. HOFFMANN

of Anutomy and Reproductive University of Huwaii,

Biology, Honolulu,

Accepted November

John A. Burns School Huwuii 96822

of Medicine,

13, 1980

The mongoose is a small carnivore that exhibits a distinct seasonal rhythm in reproductive activity. Feral mongooses were trapped alive on the island of Oahu (Hawaii) between May 1977 and December 1979. The animals were autopsied shortly after capture and testes and prostate glands were weighed and serum collected for later measurement of androgen, LH, and FSH. The validations of radioimmunoassays for mongoose LH and FSH are described. Testes and prostate gland weights were significantly larger during the breeding season than during the inactive season. Serum androgen, LH, and FSH levels were significantly higher during the breeding season than during the inactive season. Significant annual variations were found in testes and prostate gland weights and serum androgen and FSH levels. Duri,ng testicular regression (August to September) changes in gonadotropin levels paralleled changes in serum androgen levels; however, during the recrudescence phase (December to February) gonadotropin levels appeared to rise much more rapidly than serum androgen levels. Possible factors involved in the regulation of the seasonal pattern in serum gonadotropin levels and testicular activity are discussed.

The mongoose, Herpestes auropuncdisplays a seasonal pattern in reproductive activity (Pearson and Baldwin, 1953; Gorman, 1976). In Hawaii the male mongoose is reproductively active from February to August and quiescent from September to December (Pearson and Baldwin, 1953). Thus far, our knowledge of reproduction in the mongoose is based upon inspection of male and female reproductive tracts and changes in organ weights. The purpose of this study was to investigate further the seasonal pattern of reproduction in the male mongoose. Serum androgen, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels were measured by radioimmunoassay throughout the annual cycle, The hormone measurements were made in order to evaluate their relationship with the breed-

tatus,

’ Presented in part at the 13th Annual Meeting of the Society for the Study of Reproduction, Ann Arbor, Mich., August 1980. ’ Present address: Thimann Laboratories, University of California, Santa Cruz, California 95064. 0016.6480/81/070350-09$01.00/O Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

ing and inactive seasons. Additionally, the validations of radioimmunoassays for the measurement of mongoose LH and FSH are presented. MATERIALS

AND METHODS Animals

The mongoose was trapped alive in Havahart No. 4 traps (Ossining, N. Y.) on the island of Oahu. Upon arrival at the laboratory, the mongooses were housed individually in metal cages measuring 38.5 x 46 X 26 cm and provided Purina cat chow and water ad libitum. Adult male mongooses were distinguished from immature males by the prominence of the cranial sutures and the length of the bacculum (Pearson and Baldwin, 1953). Trapping was conducted from May 1977 to December 1979 with most of the animals being trapped during 1979.

Autopsy

Procedure

Al! animals were autopsied within 48 hr of capture. The mongooses were anesthetized with ether, weighed, and exsanguinated via cardiac puncture. The blood was allowed to clot at room temperature and centrifuged, and serum was aliquoted and frozen at -40” until the hormone measurements were made. The prostate, anterior pituitary, thyroid, adrenal glands, and one testis were excised and weighed. The cauda

MALE

MONGOOSE:

portion of one epididymis was removed, minced in 20 ml of 0.9% saline, and the spermatozoa concentration estimated with the aid of a hemocytometer.

Radioimmunoassays Ah dilutions of samples and assay standards were made with Gel-PBS LO.01 M sodium phosphate, 0.14 M NaCl, 0.1% sodium azide, and 0.1% gelatin (Knox) at pH 7.41. Working dilutions for antisera were made with EDTA-PBS (0.05 M EDTA, 0.01 M sodium phosphate, 0.14 M NaCl, and 0.1% sodium azide at pH 7.4). The diluent for the gonadotropin antisera also contained 4% normal rabbit serum.

Androgen

Radioimmunoassay

Serum androgen levels were estimated by a radioimmunoassay employing a sheep antiserum to testosterone-l I- BSA (Niswender No. 250). The cross-reactivity of steroids other than testosterone (taken as 100%) is as follows: dihydrotestosterone, 78.4%; androstanediol, 9.5%: epitestosterone, 1.0%; estradiol, 0.3%; and dehydroepiandrosterone, progesterone, and corticosterone <0.04% (Morishige and Retake, 1978). [ 1,2,6,7,16,17-:%H]Testosterone (152.0 Cifmmol) was purchased from New England Nuclear. The labeled steroid was stored in absolute ethanol at -20” and repurified by thin-layer chromatograpby in chloroform:ethyl acetate:petroleum ether (50:45:5) when necessary (Nieschlag and Loriaux, 1972). Nonradioactive steroids were purchased from Steraloids and used without further purification. Two-hundred-microliter aliquots of sera were extracted in 15 voi of benzene:hexane (1:2, v/v) and assayed without further purification. Serial dilutions from IO@ to 12.5~1 serum eq were assayed for each male. The antiserum was used at a final dilution of 1:96,000 which provides approximately 50% binding of labeled testosterone. The standard curve consisted of duplicate testosterone standards ranging from 2.5 to 100 pg. The sensitivity of the assay (90% of buffer cor?rol) ranged from 2.5 to 5.0 pgitube and the within and between assay coefficients of variation were 11 and lo%, respectively. The testosterone antiserum (50 PI), labeled testosterone (50 ~1 of a l-r&ml stock solution), and cold testosterone standards or unknown serum extracts (200 ~1) were incubated from 6 to 16 hr at 4”. Separation of bound and free hormone was achieved by addition of 1 ml of an ice-cold dextran-coated charcoal suspension [0.25% Norit A Matheson, Coleman, and Bell. Norwood, Ohio) and 0.05% Dextran T-70 (Pharmacia)] to each assay tube. The tubes were vortexed for 2 set, incubated for 10 min at 4”, and centrifuged at 2OOOg. The supernatant was then decanted and the radioactivity measured in 4 mi of scintillation fluid j3000 ml toluene. 126 ml Liquifluor (New England Nuclear), and I00 rn: Bio-Solv (Beckman)] with a Packard

ANDROGEN,

L

351

Tri-Garb liquid scintillation spectrometer having a counting efficiency of SO%. Gel-PBS and labe!ed testosterone were extracted for each assay and used to assess background blank values and recovery, respectively. Biank values were consistently below the sensitivity of the assay. Entraction recovery averaged 92.8%. Final serum hormone concentrations were not adjusted for iosses incurred during the extraction procedure. Nonspecific binding never exceeded 5% of the total counts.

LH (5 ,ug) and FSH (2.5 pg) were routineiy iodinated according to the Greenwood et a/. (1963) chioramine-T method as modified by Orth (1975). The specific activity of labeled LH ranged from 55 to 78 pCiI,tig and FSH ranged from 120 to I38 $Zi/pg as determined by the elution profile from Sephadex G-75 columns (6.6 x 14.5 cm). The labeled hormone was frozen unrii use, then thawed and repurified on cellulose CF-11 (Wha?man) columns (Catt and Dufau, 1975). Hor.mones for radioiodination were obtained from Dr. Leo E. Reichert, Jr. [ovine EH (LER-1374A) and ovine PSI-I (LER-1976A2)]. Na*?“l was obtained from Amersham. Reference standards were obtained froam tbe National Institute of Arthritis, Metabolism. and gestive Diseases (NIAMDD) [ovine LH {N~A~~~~“. oLH-S16) and ovine FSH (NIAM~~-oFS~-~3)~. A double-antibody procedure was used to separate bound from free hormone. Nonspecific binding -was consistently below 5% of the total counts for both assays. LN. Serum LH was measured in an ovine-ovine system described by Niswender el ui. (1968) with modifications that have increased its sensitivity (Hoffmann, 1973). Tine antiserum {Niswender No. 15) was used at a tinai dilution of 1:24O,OOOwhich provided 4O-50% binding of the radioiodisated LH. Serial dihrtions from 100 to 6.25 ~1 of serum were assayed for each sample. The s?andard curve consisted of duplicate NIAMDD-oLH-S16 standards ranging frcm 0.025 to 2.5 cg. The sensitivity of the assay ranged from 25 to 50 pgitube and the within and between assay coefficients of variation were 6 and 1l%, respectively. The standards or the unknown (200 ~1) were incubated with anti-ovine LH (50 ~1) for 24 hr fohowed by (50 pl of a I-ngiml stock solu:icun) and a 48 hr incubation. Separation of bound and free hormone was accomplished by incubating the reaction mixture with 100 pi of goat anti-rabbit gamma globulin (1:16) (Antibodies,. Inc.. Davis, California) for an additional 24 hr and a 2O-min centrifugation at ZOOOg.The supernatant was decanted and the pellet counted in a Packard auto-gamma spectrometer with a counting efficiency of 60%. All incubations were conducted at 4”. FSH. Serum FSH was measured with a hereralogous system. Anti-human FSH was obtained from the

352

SOARES AND HOFFMANN

NIAMDD (batch 5) and used at a final dilution of 1:24,000 which provided 30-40% binding of labeled FSH. Duplicate determinations were made at 200 and 25 ~1 for each serum sample. The standard curve consisted of duplicate NIAMDD-oFSH-13 standards ranging from 0.25 to 10.0 ng. The sensitivity of the assay ranged from 0.25 to 0.50 ngitube and the within and between assay coefficients of variation were 12 and 14%, respectively. The standards or the unknown (200 ~1) were incubated with NIAMDD anti-hFSH (50 yl) for 48 hr followed by addition of ‘*“I-oFSH (50 ~1 of a I-rig/ml stock solution) and another 48-hr incubation. Separation of bound and free hormone and measurement of radioactivity were identical to the procedure described for the LH radioimmunoassay.

Validation of Gonadotropin Radioimmunoassays The validity of using these assays for the measurement of mongoose LH and FSH was determined by assessing whether inhibition curves produced by the ovine reference standards. sera from mongooses, and semipurified anterior pituitary hormone preparations were parallel. The semipurified anterior pituitary hormone preparations were extracted from frozen mongoose anterior pituitaries according to Reichert’s procedure for the rat (Reichert, 1975). Specificity of the gonadotropin assays was tested by evaluating changes in serum LH and FSH levels following: (1) treatment of male mongooses with gonadotropin releasing hormone (GnRH; Boehringer-Mannheim) and (2) orchidectomy. GnRH (2 &animal, n = 5) was injected intravenously in a vehicle of phosphatebuffered saline, pH 7.0. Blood samples were collected via jugular venipuncture under light ether

anesthesia at the time of injection and at 15 min postinjection. A control group (n = 5) was injected with the vehicle alone. Serum was recovered and frozen for later measurement of serum LH and FSH. The postcastration. response of LH and FSH was assessed in six castrated males and six sham-operated males. The surgery and blood collections were performed under ether anesthesia. A blood sample was collected at the time of surgery and 10 days later. Serum was recovered and frozen for later measurement of LH, FSH, and androgen.

Statistical

Analysis

The effects of GnRH, orchidectomy, and season on serum hormone levels and organ weights were analyzed by Student’s f test. Annual variations in testis and prostate gland weights and serum hormone levels were evaluated by analysis of variance. Post hoc analyses were conducted with the Dunn multiple comparison test (Keppel, 1973).

RESULTS Validation of Gonadotropin Radioimmunoassays

Inhibition curves for the mongoose anterior pituitary hormone preparations and mongoose sera were parallel to inhibition curves for the respective ovine standards (Figs. 1 and 2). All mongoose LH and FSH values were thus expressed in terms of the ovine LH and FSH preparations, respectively. Injection of GnRH resulted in a

IOOJ !? 2 8 :: k 2 8 z’

80-

60-

40-

1

I

I I

.I

I IO

I 100

“cl or,ui

FIG. 1. Inhibition curves for NIAMDD-oLH-S16, a semipurified mongoose LH anterior pituitary preparation, and serum from castrated male mongooses in the radioimmunoassay for mongoose LH. Each point represents the mean of five determinations.

MALE

MONGOOSE:

ANDROGEN,

LH,

353

FS

$ 60I 8

40-

!5 5 ;

20-

o-

J

I IO

I I

I 100

ng orpI

FIG. 2. Inhibition curves for NIAMDD-oFSH-13, a semipurified mongoose preparation. and serum from castrated male mongooses in the radioimmunoassay Each point represents the mean of five determinations.

rapid elevation in serum LH (P < 0.05, Fig. 3) and a highly significant but less dramatic rise in serum FSH (P < 0.001, Fig. 3). This ntial gonadotropin response to is common (Lincoln, 1979) and very similar to findings in the dog (Reimers et

i1

change sig~i~ca~t~y as a function of season

2500 GnRH tn.51 2000

PBS (n=5) m

2000

1500-

m I 2 I500

IOOO-

? w E

IO00

E

ii

it?

2 5 i-

ii

i!

al., 1978). Castration res cant elevation in serum L (P < 0.01 for both, Fig. 4) and a decrease in seru prostate gland we Table 1).

not

Ei 5

FSH anterior pituitary for mongoose FSH.

8 500 -

5

l-

LH

FSH FIG. 3. Mean percentage change (,iSEM) in levels of LH and FSH from serum of male mongooses 15min after injection of GnRH or phosphate-buffered saline (PBS). GnRH significantly elevated LH (P < 0.05) and FSH (P i 0.001) levels.

i 1

500

8 3 a

sa u-l

FSH

FIG. 4. Mean percentage change (I-SEM) in ievels of LH and FSH from serum of male mongooses 10 days after castration or sham castration. Castration significantly elevated both LH and FSH leve!s :bJ < 0.01 for both).

354

SOARES AND HOFFMANN TABLE SERUM

ANDROGEN

LEVELS SHAM

AND

PROSTATE

CASTRATION

GLAND

IN THE

1 WEIGHTS

MALE

10 DAYS

MONGOOSE

FOLLOWING

(MEAN

Treatment

N

Androgen Wml)

Castration Sham castration

6 6

0.04 ?I o.ol”.* 1.88 T 0.85

k

CASTRATION

OR

SEM) Prostate gland (mg/lOO g body wt) 82.2 ” 7.7* 134.0 k 11.6

” Two samples were below the sensitivity of the assay and were not included in the calculations. * Values are significantly different from sham castrated values according to Student’s t test (P c: 0.01).

(Table 2). The anterior pituitary and adrenal glands were heavier during the breeding season than during the inactive season (P < 0.01 for both, Table 2). Epididymal sperm content was greater during the breeding season than during the inactive season (I’ < 0.01, Table 2). Testis and prostate gland weights were significantly greater during the breeding season than during the inactive season (I’ < 0.001 for both, Table 2). Serum androgen, LH, and FSH levels were significantly elevated during the breeding season as com-

pared with the inactive season (P < 0.001 for all, Table 2). According to analysis of variance, a significant annual variation was found in testis (P < 0.01, Fig. 5) and prostate gland (P < 0.001, Fig. 5) weights and in serum androgen (I’ < 0.01, Fig. 6) and FSH (P < 0.05, Fig. 8) levels. Annual changes in LH levels were not significant, probably due to the large variations during the breeding season (Fig. 7). Post hoc analyses indicated that some signs of testicular recrudescence (testis weights) were first evident in De-

TABLE SEASONAL

CHANGES

SPERMATOZOA

IN BODY

CONTENT.

WEIGHT, AND

SERUM

2

ORGAN

WEIGHTS,

HORMONE

CAUDA

LEVELS

Breeding season Body weight (BW; g) Anterior pituitary (mg/lOO g BW) Thyroid gland (mgi100 g BW) Adrenal gland (mg/lOO g BW) Testis (mg/lOO g BW) Prostate gland (mg/lOO g BW) Cauda epididymal Androgen (&ml)

sperm content (X IO”)

692.8 k 31.6 (n = 74) 0.67 i 0.02 (n = 67) 8.9 t 0.2 (II = 74) 21.8 k 0.5 (n = 72) 283.1 -c 6.4 (n = 74) 135.2 i 1.7 (n = 74) 198.5 k 21.8 (n = 23) 3.1 + 0.5 (n = 74)

LH (@ml) FSH (&ml) * Significantly ** Significantly

8.7 (n 13.0 (n

k 1.6 = 67) t 1.7 = 56)

(MEAN

EPIDIDYMAI k

SEM) Inactive season 737.0 t (n = 0.57 + (n = 7.3 t (n = 16.7 t (PI = 145.5 -c (PI = 60.1 + (?I = 73.1 k (n = 0.14 t (n

2.8 (n 6.8 (n

15.1 41) 0.02* 41) 0.3 41) 0-V 41) 7.1** 41) 3.7** 41) 9.7* 30) 0.09**

= 38)

t = -c =

0.4”* 38) 0.5** 38)

different from values for the breeding season according to Student’s t test (P i 0.01). different from values for the breeding season according to Student’s t test (P
MALE

BREEDING

MONGOOSE:

ANDROGEN,

LH,

FSH

355

INACTIVE

L L.

MONTH

FIG. 5. Annual variations in testis and prostate gland weights. Each point on the figure represents the mean and the surrounding open area represents the SEM. The breeding and inactive seasons are indicated by tke regions with vertical lines and the stippled area, respectively. The numbers in parentheses denote the sample size for each month.

cember (P < 0.01); however, prostate gland and serum androgen levels displayed negligible recovery until February (initiation of the breeding season, P < 0.01 for both). During this period of testicular recrudescence (December to February) serum FSH levels were significantly elevated above levels measvred during the inactive season (19.3 + 3.6 vs 6.8 t 0.5 &ml, P < 0.01).

7. Annual variations in serum 5 legend for further details.

FIG.

Fig.

3-H levels

See

four distinct phases: (1) active or bree (February to August), (2) regression (August to September), (3) inactive (September to December), and (4) recrudesce cember to February). The active characterized by large testes and glands and elevated serum andr and FSH levels. Similar relation been reported for of gole el al., 1974; 1975a, b; Lincoln, Ford, 1976,1979; S 1976; Lincoln and

bather and Lunstra,

The annual reproductive cycle of the male mongoose can easily be divided into

FMAMJJASONDJ

FIG. 6. Annual See Fig. 5 legend

variations for further

in serum details.

androgen

levels.

8. Annual variations in serum 5 legend for further de?ails.

FIG.

Fig.

P;S%I levels.

See

356

SOARES

AND

Muduuli et al., 1979). The detection of elevated LH levels during the breeding season probably reflects an increase in the frequency of pulsatile LH release rather than an increase in the basal secretion of LH (Lincoln and Short, 1980). During the regression phase reproductive organ weights and serum androgen and gonadotropin levels are decreasing. The decline in reproductive function appears to be closely coupled with the decrease in daylength. Daylength has been demonstrated to be an important environmental cue regulating seasonal reproductive responses in other species (see Turek and Campbell, 1979; Lincoln and Short, 1980, for reviews). The importance of the reduction in daily lighting as a cue for regression of the mongoose reproductive system is presently unknown. Decreases in daylength accompanying testicular regression may act to synchronize changes in the hypothalamohypophyseal axis; however, initial restoration of the mongoose reproductive system does not require exposure to increasing daylength. Mongoose testis weights and serum gonadotropin levels are elevated prior to the winter solstice. Apparently the hypothalamohypophyseal axis becomes refractory to the inhibitory factors involved in the suppression of LH and FSH release. Similar responses to photoperiod have been demonstrated in the hamster (see Reiter, 1980 for review). Although testis weight and serum FSH and LH levels are elevated in the male mongoose during the recrudescence phase, minimal increases in serum androgen levels and prostate gland weights were detected until February (the onset of the breeding season). This finding is in contrast to endocrine changes during recrudescence in the hamster, in which serum LH and testosterone increase in synchrony (Matt and Stetson, 1979). The resumption of androgen secretion in the mongoose does not appear to be solely dependent upon circulating LH levels but may be related to changes in testicular responsiveness to LH.

HOFFMANN

In summary, we have demonstrated that androgen, LH, and FSH can be measured in the mongoose. Seasonal variations in serum levels of LH and FSH parallel changes in testicular activity. The only divergence from this relationship occurs during the recrudescence phase when gonadotropin levels rise much more rapidly than androgen levels. Mongoose reproduction in Hawaii is of interest in that marked seasonal changes in the reproductive system occur in an environment with relatively minor changes in photoperiod and temperature (Blumenstock and Price, 1974), stimuli which are believed to be important in the regulation of seasonal reproduction. ACKNOWLEDGMENTS The authors gratefully acknowledge Dr. Nicholas Palumbo and Roger Chang of the Research Animal Facility at the University of Hawaii for their care of the animals and Dr. B. Jane Rogers, Dr. Marita Nelson, Anne-Marie Cullin, and Salei’a Afele for valuable contributions during various stages of this study. Dr. Gordon D. Niswender supplied the antisera for the LH (No. 15) and androgen (No. 250) radioimmunoassays. Dr. Leo E. Reichert, Jr., supplied the LH and FSH used for iodination, the NlAMDD and Dr. A. Parlow supplied the reference standards for the LH and FSH radioimmunoassays and the antiserum for the FSH assay (anti-human FSH batch 5). We would also like to thank the Kaneohe Marine Corps Air Station, Waimea Falls Arboretum, and Mr. Nelson K. Rice of the State of Hawaii Division of Fish and Game for their assistance in obtaining mongooses. For secretarial assistance the authors express their thanks to Michele Ikeda and Mona Yamada. Our special thanks to Dr. Walter K. Morishige for his valuable advice during the course of this study.

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MALE

MONGOOSE:

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LH,

FSM

357

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Reimers. T. J., Phemisier. D., and Nisw=nder, 6. D. (1978). Radioimmunological measurement of FSH and prolactin in the dog. Biol .Repm!. 19, 6733679, Reiter,

R. J. (1980). The pineai and its hormones in the control of reproduction in mammals. Endoc+~e Rev. 1, 109-131,

Scbanbacher, B. D.. and Ford, J. 9. (1976). Season&i profiles of plasma luteinizing hormone. testosterone and estradio? in the ram. Endoc?irznk~p~ 99, 7522157. Schanbacher, periodtc

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SOARES AND HOFFMANN

358 Relationship

to serum hormones. Biol.

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Schanbacher, B. D., and Lunstra, D. D. (1976). Seasonal changes in sexual activity and serum levels

of LH and testosterone in Finnish Landacre and Suffolk rams. J. Anim. Sci. 43, 644-650. Turek, F. W., and Campbell, C. S. (1979). Photoperiodic regulation of neuroendocrine-gonadal activity. Biol. Reprod. 20, 32-50.