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RESEARCH |
Department of Zoology and Entomology, University of Pretoria, Pretoria, South Africa, 1 Centre for Neuroscience Research, King’s College London, Guy’s Campus, London, UK, 2 Department of Chemical Pathology, Medical School, University of Cape Town, South Africa and 3 Department of Neurobiology, Institute of Experimental Medicine, Budapest, Hungary
Correspondence should be addressed to N C Bennett; Email: ncbennett{at}zoology.up.ac.za
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Abstract |
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Introduction |
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Damaraland mole-rats, Cryptomys damarensis, are eusocial, subterranean rodents that inhabit arid regions of south-western Africa. Breeding is limited to a single reproductive pair in colonies that may contain as many as 41 individuals (Jarvis & Bennett 1993). The non-reproductive females exhibit a socially induced infertility while in the colony, but start ovulating spontaneously in the absence of the breeding pair (Molteno & Bennett 2000). Anovulation in non-reproductive females is characterised by a reduced pituitary sensitivity to a single, exogenous GnRH challenge; given the autoregulatory action of GnRH on its receptors on the gonadotrophs, this may be due to an inhibition of GnRH release. The initiation of ovulation in non-reproductive females removed from the breeding pair is accompanied by a significantly greater plasma luteinising hormone (LH) response to exogenous GnRH administration. Breeding females exhibit significantly greater GnRH-stimulated LH secretion than non-reproductive females (Bennett et al. 1996, Molteno & Bennett 2000). Thus the social environment has a clear effect on the reproductive neuroendocrine system of non-reproductive females. The effect of the social environment on reproductive function does not appear to be comparable in non-reproductive males, their pituitary sensitivity to GnRH being no different from that of reproductive males (Molteno & Bennett 2000).
This study is the first to investigate the effect of the social environment on the GnRH system in a cooperatively breeding mammal, C. damarensis, by means of immunocytochemistry and radioimmunoassay. The study was designed to characterise the morphology and distribution of the GnRH-IR system in the brain of this species and to determine whether the inhibitory effects of social cues on fertility in non-reproductive female mole-rats are induced through changes in function and/or morphology of the GnRH system.
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Materials and Methods |
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Group 1
Reproductive females (RFs) were easily identifiable by their large teats and perforated vaginas. From a practical perspective, the social structure of the species (one reproductive female per colony) meant that a limited number of reproductive females were available and it was difficult to obtain large numbers of reproductive females in a specific reproductive phase.
Group 2
Non-reproductive females (NRFs) were housed in functionally complete, wild-caught colonies.
Group 3
Removed non-reproductive females (rNRFs) were maintained in the absence of the breeding pair. These reproductively non-functional colonies were maintained in the laboratory for 11 weeks prior to the females being killed. No sexual activity occurs in these colonies partly as a result of an inhibition to breed with kin.
For radioimmunoassay of brain GnRH content, forebrains were obtained from RFs and NRFs, and also from reproductive males (RMs) and non-reproductive males (NRMs).
GnRH immunohistochemistry
Perfusion and tissue processing
RFs, NRFs and rNRFs (n = 7 for each group) were anaesthetised using halothane followed by intramuscular injection of 30 mg ketamine/kg and 5 mg rompun/kg. Individuals were perfused through the aorta with approximately 150 ml 0.1 M PBS, followed by approximately 300 ml 4% paraformaldehyde in 0.1 M PBS. Solutions were filtered immediately prior to use. Heads were removed and immersed in 4% paraformaldehyde overnight; brains were removed and placed sequentially in 15 and 30% sucrose at 4 °C until they sank. They were then frozen on dry-ice and stored at -70 °C until sectioning. The brains were sectioned coronally at 30 µm using a cryostat. Every fifth section from the rostral junction of the hemispheres to the posterior hypothalamus was processed. After washing in 0.1 M PBS, free-floating sections were treated with 0.5% Triton X-100 (BDH Chemical Company, Poole, UK), to increase antibody penetration, and 0.02% hydrogen peroxide for 30 min to block endogenous peroxidase activity. Sections were placed in 2% normal donkey serum for 30 min to minimise non-specific binding of the antibodies. They were then incubated in mouse anti-GnRH antibody (1:1000; kindly supplied by Dr D Silversides) overnight and, after washing in 0.1 M PBS, in biotinylated goat anti-mouse IgG (1:1000; Vector Laboratories, Peterborough, UK) for 2 h at room temperature. Following treatment with Avidin Biotin Complex (Elite Kit, Vector Laboratories) for 90 min, the sections were washed in 0.1 M PBS and then in 0.05 M Tris buffer (Trizma 7.6, Sigma). A blue-black reaction product was formed by a 6 min incubation of sections in a solution containing nickel ammonium sulphate, 3'3-diaminobenzidine and hydrogen peroxide. Immunohistochemical controls included incubation of sections following immunoneutralization of the antibody with GnRH, or omission of either the primary or the secondary antibody; these procedures result in the loss of all immunoreactivity.
Analysis
Distribution, morphology, size and number of immunoreactive GnRH perikarya in the forebrain extending from the rostral preoptic area to the posterior hypothalamus were determined and compared between RFs, NRFs and rNRFs. The number of processes deriving from all GnRH cells in every fifth section from the preoptic area of each female was determined. The size of the perikaryon of 20 randomly selected GnRH-IR cells in the preoptic area of each animal was established according to the method described by Robinson et al. (1997) using image analysis software (Scion Image Beta 3b, Frederick, Maryland, USA). The total number of GnRH cells was counted for each animal. Statistical comparisons were made using both non-parametric Kruskal–Wallis tests and parametric ANOVA.
To establish the area occupied by GnRH-IR fibres in the median eminence, computer-assisted image analysis was undertaken on matched sections from RFs and NRFs (three sections per animal; n = 5 animals for each group) using the thresholding and area measurement procedures in the ImageJ 1.26t program (W Rasband, National Institutes of Health, Bethesda, MD, USA). The mean tissue background value was established according to the grey scale (0 = black; 255 = white). The area covered by pixels with a grey scale value that fell below 80% of the background value was automatically measured.
GnRH measurement
GnRH extraction
Following decapitation, brains were removed from RFs, NRFs, RMs and NRMs (n = 10 for each group) and frozen on dry-ice. GnRH was extracted according to the method of King & Millar (1986). Prior to radioimmunoassay, whole brains were lyophilised and their dry weights determined. Lyophilised brains were minced with a sterile blade and processed individually in 5 ml 2 M acetic acid for 3 min using a polytron homogeniser. All steps were performed at 4 °C. The homogenates were centrifuged at 18 000 g for 60 min and the supernatant fluid was freeze-dried overnight. Individual brain extracts were reconstituted in 4 ml TF solution (2% triethylamine, 1.05% formic acid, 96.95% dH2O; pH 3.2). Extracts were sonicated for 3 min and centrifuged at 10 000 r.p.m. for 15 min. The supernatants were concentrated overnight using a Savant speed-vac concentrator connected to a freeze-drier and reconstituted in 1 ml gel-PBS buffer (PBS containing 0.1% gelatin) for radioimmunoassay.
GnRH radioimmunoassay
Concentrations of GnRH extracted from individual mole-rat brains were determined by radioimmunoassay based on the method described by King & Millar (1986). GnRH antiserum 678, raised in rabbits (J A King, Department of Chemical Pathology, University of Cape Town, South Africa), requires both the NH2- and COOH-termini for binding and cross-reacts with all seven known forms of GnRH (King et al. 1994). Synthetic mammalian GnRH (mGnRH) (R P Millar, Medical Research Council, Edinburgh, Scotland) was used as the standard and 125I-labelled mGnRH, iodinated using the chloramine-T method (Greenwood et al. 1963), was used as the tracer.
Serial doubling dilutions of mGnRH were prepared in gel-PBS over the range 0.48–1000 pg/ml. The 125I-labelled mGnRH tracer was used at approximately 15 000 c.p.m. per 100 µl in a total assay volume of 500 µl. The mGnRH standard preparations and samples of GnRH extract, assayed at two dilutions as a routine test for parallelism, were assayed in duplicate. NRM samples were assayed in crude form, and following purification through Sep-pak columns (Waters Associates) to determine whether purification was required. Following overnight incubation, bound and free peptides were separated using 200 ml Pharmacia Decanting Suspension (Kabi Phamacia Diagnostics, Uppsala, Sweden). After 30 min the reaction was terminated by adding 1 ml dH2O. Tubes were centrifuged at 3000 r.p.m. at 4 °C and the pellet containing the bound fraction was counted for 1 min on a gamma counter (Packard Riastar 5400, Meriden, CT, USA).
Assay validation
The suitability of the assay for measuring GnRH from mole-rat brain extract was assessed by testing the parallelism between serial doubling dilutions of synthetic mGnRH standard and both crude extract and extract purified through Sep-pak columns (Waters Associates). Following logit-log transformation of the data, the slopes of the curves were tested for parallelism, using the Statistica computer package (Statsoft, Tulsa, OK, USA). The slopes of the three curves were not significantly different (ANOVA, F2–14 = 0.14, P > 0.05). Sep-pak purification did not improve the effectiveness of the assay to measure GnRH and yielded lower values than crude samples. The intra- and inter-assay coefficient of variations were 9.6 (n = 14) and 6.6% (n = 6) respectively. GnRH concentrations were analysed by two-way ANOVA with sex and reproductive status as independent factors (n = 9 for each group). Post-hoc analysis was carried out by Tukey’s Honest Significant Difference (HSD) test.
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Results |
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; Fig. 2a). Within the medial septal region, cells containing GnRH immunoreactivity are predominantly oriented with their long axis in the dorsoventral direction; more caudally, this axis is generally oriented mediolaterally and rostrocaudally. A high proportion of the total number of GnRH cell bodies is found from the rostrocaudal level of the medial septum and organum vasculosum of the lamina terminalis (OVLT) to the medial preoptic area (MPOA) (Fig. 1b and c). Dorsally these cells are located close to the midline; ventrally they are distributed more laterally (Fig. 1a–c). GnRH cells are also observed in the subfornical organ (Fig. 2c). Relatively few of the total number of GnRH cells are located in the mediobasal hypothalamus (MBH) caudal to the suprachiasmatic nuclei (Fig. 1f) (14.7% in RFs, 13.2% in NRFs and 15.6% in rNRFs). A few GnRH cells are found caudal to the MBH, reaching sites adjacent to the mammillary recess of the third ventricle (Fig. 2a).
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). GnRH cells totalling 341 in RFs, 378 in NRFs and 409 in rNRFs were analysed. In a small proportion of these cells distinct processes were not observed, probably due to the plane of sectioning; these were classified as non-polar (Fig 2d). Kruskal–Wallis analysis revealed that the mean number of cells per female that were unipolar (H = 0.2, P > 0.05, n = 21) or bipolar (H = 1.0, P > 0.05, n = 21) was not significantly different between the three groups (Fig. 3). Only two GnRH cells with multiple processes were observed in this study. The GnRH cell bodies and proximal processes possess a generally smooth contour (Fig. 2d and e); although immunoreactive processes were occasionally visible in the vicinity of GnRH dendrites, appositions between GnRH perikarya were not apparent.
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). Although GnRH fibres are apparent in the olfactory bulb, the major tract of fibres follows the septo-preoptico-infundibular continuum. A plexus of GnRH-IR fibres is also observed within the subfornical organ (SFO) (Fig. 2c). Fibres course in a dorsoventral direction from the medial septum and in a rostrocaudal direction along the ventral midline (Fig. 1a–c and Fig. 2b). A dense network of immunoreactive fibres is observed around and within the OVLT (Fig. 1b). This dense network of fibres continues primarily along the midline in a very dense aggregation ventral to the third ventricle (Fig. 1c). Caudal to this region evidence of an optic chiasm is minimal due to the vestigial state of the optic nerves in this species; nevertheless, immunoreactive fibres and perikarya are found within the membranous structure forming the floor of the third ventricle at this level (Fig. 1d). Further caudally, a smaller number of GnRH fibres can be seen beneath the third ventricle, medial to the suprachiasmatic nuclei (Fig. 1e). The general distribution of GnRH fibres extends into more lateral regions as they approach the rostral median eminence (ME) (Fig. 1f and g); caudal to this level the fibres form an extensive arching pattern from the borders of the third ventricle throughout the mediobasal regions, achieving a dense aggregation in the external zone of the rostral median eminence (Fig. 1h–j). Within the major part of the ME, GnRH fibres are principally located in its lateral margins (Fig. 1k–m). The internal zone of the ME in these animals is remarkably thick medially; it protrudes into the third ventricle and is surrounded by deep lateral recesses of that ventricle (Fig. 1k–l). The dense GnRH immunoreactivity in the external zone of the ME continues caudal to the pituitary stalk, as revealed in the parasagittal plane (Fig. 2b). As the pituitary stalk starts to form, dense areas of GnRH immunoreactivity are found around its immunonegative central core (Fig. 1n). The mean area (±S.E.M.) occupied by GnRH fibres in the median eminence was less (P < 0.01) in RFs (0.0178 ± 0.00159 mm2, n = 5) than in NRFs (0.0246 ± 00112 mm2, n = 5); examples illustrating this difference are presented for the RF (Fig. 1o) and NRF (Fig. 1n) conditions.
Number and size of cell bodies
A large variation in the number of GnRH cells in individual forebrains was observed from the confluence of the two hemispheres rostrally, to the posterior hypothalamus caudally (Fig. 4); this ranged from 76 to 173 in RFs (mean 124.57 ± 14.33), from 74 to 172 in NRFs (mean 130.43 ± 36.11) and from 79 to 234 in rNRFs (mean 134.0 ± 49.23). The number of GnRH cell bodies did not differ between the RF, NRF and rNRF groups in the whole forebrain (H2 = 0.12, P > 0.05, n = 21) or in the preoptic area (H2 = 0.27, P > 0.05, n = 21) or in the mediobasal hypothalamus (H2 = 0.56, P > 0.05, n = 21; Fig. 2). Given that only every fifth section was processed, the corrected mean total number of GnRH cell bodies per brain over all three groups of females was calculated as 648.33 ± 43.36. There was also a large variation in GnRH cell body size; this ranged from 40.6 to 154.2 µm2 (mean = 89.7 ± 2.0 µm2) in RFs, from 47.9 to 140.4 µm2 (mean = 86.0 ± 1.7µm2) in NRFs and from 36.4 to 168.2 µm2 in rNRFs (mean = 88.3 ± 1.9µm2); no significant differences were detected between the three groups (ANOVA, F = 0.97, P > 0.05, n = 420).
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Discussion |
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As found in all mammalian species studied to date, two main areas of dense GnRH-IR fibres, the OVLT and the ME, were observed in the mole-rat (Fig. 1b and Fig. 1j–m respectively). GnRH-IR fibres together with cell bodies were also found in another circumventricular organ, the SFO (Fig. 2c); this site has been reported to contain GnRH fibres with extrinsic cells of origin in rats (Witkin et al. 1982). The diameter of the GnRH cell body in C. damarensis is similar to that found in other small mammals, including white-footed mice (8 µm; Glass 1986) and rats (10 µm; Witkin et al. 1982), and smaller than that reported for sheep (15–20 µm; Lehman et al. 1986) or springboks (10–20 µm; Robinson et al. 1997). The mean total number of neurones calculated per Damaraland mole-rat (648 ± 43) is similar to that observed in the Syrian hamster (650–750; Jennes & Stumpf 1980) or the Djungarian hamster (300–400; Yellon & Newman 1991). In larger species including ungulates (Lehman et al. 1986, Robinson et al. 1997) and primates (Marshall & Goldsmith 1980, Silverman et al. 1982) these neurones number in the thousands.
The GnRH neurones in the Damaraland mole-rat are predominantly unipolar and bipolar, as found in many mammals including the rat (Witkin et al. 1982, Merchenthaler et al. 1984), white-footed mouse (Glass 1986), Syrian hamster (Jennes & Stumpf 1980) and mink (Ntoumi et al. 1992). In contrast, those found in the sheep (Lehman et al. 1986), springbok (Robinson et al. 1997) and rhesus monkey (Silverman et al. 1982) have been reported to have a more complex morphology. It should be noted that the number of bipolar neurones identified in the present study may have been underestimated; many of the apparently unipolar GnRH cells in the rat brain have been shown to be bipolar using silver–gold intensification of the immunocytochemical staining (Merchenthaler et al. 1984). In the male Djungarian hamster, differential changes in the incidence of these morphological subtypes have been observed during sexual maturation (Yellon & Newman 1991). Thus, the number of unipolar, but not bipolar cells, in the diagonal band of Broca (DBB) and medial preoptic area nearly doubles peripubertally; subsequently, between puberty and full adulthood, the number of bipolar cells in the DBB and lateral preoptic area also doubles. It has been speculated that change in the incidence of these cell types is related to differential GnRH storage and release during development (Yellon & Newman 1991). Nevertheless, in the present study, the number of unipolar and bipolar cells was not found to vary with reproductive status.
No significant differences in GnRH-IR neuroanatomy were found between reproductively active and inactive Damaraland mole-rats. Similar results were observed for seasonally anoestrous and reproductive sheep (Lehman et al. 1986) and for reproductive and bachelor male springboks (Robinson et al. 1997). These findings are in contrast to those for seasonally breeding female white-footed mice, in which the number of cell bodies and the optical density of their GnRH immunoreactivity increase in the preoptic area and anterior hypothalamus following exposure to an inhibitory, short photoperiod (Glass 1986). In the seasonally breeding Syrian hamster, Urbanski et al. (1991) did not find differences in the number of GnRH-IR neurones in individuals maintained under long or short day length regimens, but did find that the area of the cell body was larger in short-day maintained animals. This suggests that an alteration in the GnRH system, leading to an accumulation of GnRH in the cell bodies, may occur within some species in response to the inhibitory environmental cues that affect reproduction. Indeed, in the present study radioimmunoassay revealed significant differences in brain GnRH content between NRFs and RFs. The finding that the reproductively inhibited NRFs exhibit higher GnRH levels than RFs suggests that an inhibition of GnRH release may underlie the social suppression of reproductive functions in female C. damarensis. Evidence for a comparable process in the male members of this species was not obtained, supporting the hypothesis that NRMs exhibit reproductive inhibition that is largely behavioural (Bennett et al. 1996).
The question arises as to where the increased levels of GnRH accumulate. GnRH is transported from the site of synthesis in the cell body to the nerve terminals where it is released into the portal vessels in the ME. The results of the present study indicate no differences between the RF and NRF groups in the size or number of GnRH-IR cell bodies, but a significantly higher concentration of GnRH in the NRF group; these findings would be consistent with an accumulation of the peptide in the terminals rather than the cell bodies within the NRF group. This hypothesis is supported by the more intense level of GnRH immunoreactivity observed in the NRF group at the level of the median eminence and proximal pituitary stalk (e.g. Fig. 1n compared with Fig. 1o). A similar precedent exists, since a higher density of GnRH-IR fibres in the ME has been observed in seasonally breeding female white-footed mice maintained under an inhibitory, short photoperiod (Glass 1986). Furthermore, studies on musk shrews have shown that mating behaviour that fails to induce ovulation is associated with a higher level of median eminence GnRH than that found after successful reflex ovulation (Dellovade et al. 1995b).
The results of the present study are consistent with inhibition of GnRH release from the ME, and consequent accumulation of GnRH in the terminals, being one of the factors contributing to the suppression of ovarian function in socially suppressed female mole-rats. The GnRH system may also be inhibited at the level of gene transcription, mRNA translation or post-translational processing. Future research directions will involve the use of antibodies directed against pro-GnRH and together with in situ hybridization for GnRH mRNA we intend to gain further insights into the regulatory processes that mediate the effect of social environment on fertility in the Damaraland mole rat.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Anthony ELP, Weston PJ, Montivilo JA, Bruhn TO, Neel K & King JC 1989 Dynamic aspects of the LHRH system associated with ovulation in the little brown bat (Myotis lucifugus). Journal of Reproduction and Fertility 87 671–686.
Bennett NC, Faulkes CG & Molteno AJ 1996 Reproductive suppression in subordinate, non-breeding female Damaraland mole-rats: two components to a lifetime of socially induced infertility. Proceedings of the Royal Society of London Series B 263 1599–1603.[Medline]
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