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Reproductive Endocrinology |
Division of Endocrinology (R.Q., W.H., P.-M.G.B.) and Department of Anatomy (W.H., C.T.), Royal Free Hospital School of Medicine, University College London; the Department of Otorhinolaryngology, Royal Free Hospital (W.G., R.E.Q.); the Department of Endocrinology, St. Bartholomew’s Hospital, and the Royal London School of Medicine and Dentistry (G.M.B.), London, United Kingdom
Address all correspondence and requests for reprints to: Dr. P.-M. G. Bouloux, Department of Endocrinology, Royal Free Hospital, Pond Street, London, United Kingdom NW3 2QG. E-mail pmb{at}rfhsm.ac.uk
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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In the present study, using an immunofluorescent, double immunostaining technique and confocal laser scanning microscopy, we observed GnRH-immunoreactive neurons in the hypothalamus of a 14-week-old human fetus. However, migration of GnRH neurons was not complete, and indeed, such cells were seen to be migrating along terminal nerve fascicles beneath the cribriform plate in a 16-week-old fetus. The same immunofluorescent technique demonstrated the presence of GnRH cells in biopsies of nasal mucosa obtained from three adults with Kallmann’s syndrome, one normosmic subject with hypogonadotropic hypogonadism, and a eugonadal male cadaver. These findings are consistent with two different interpretations: the nasal GnRH neurons may be vestigial, representing cells that failed to migrate during embryogenesis; alternatively, they may have been generated de novo later in life, a possibility consistent with the recognized plasticity of human postnatal olfactory neuroepithelium. They also reveal that subjects with the normosmic (i.e. non-Kallmann’s) form of GnRH deficiency are able to synthesize immunologically recognizable GnRH, implying that failure of GnRH synthesis is not responsible for this type of hypogonadotropic hypogonadism.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The diagnosis of IHH in adult life combines clinical findings of poor sexual development with biochemical evidence of low levels of gonadal steroids and gonadotropins. Olfactory testing will differentiate KS from normosmic IHH, although magnetic resonance imaging (MRI) may also be helpful, demonstrating abnormalities of olfactory anatomy in most, but not all, KS patients (5). In prepubertal children, the diagnosis of IHH poses difficulties. Not all investigators have found third generation gonadotropin assays to be useful in identifying gonadotropin-deficient subjects (6, 7), nor may olfactory testing be reliable in the very young (who may equally not tolerate MRI without sedation).
To address this diagnostic difficulty, we formulated the hypothesis that as a result of antenatal migration arrest, residual GnRH neurons would still be found in the upper nasal epithelium of KS patients, but not in normosmic IHH patients or eugonadal subjects. If this were verified, it might enable an early differential diagnosis of KS/IHH to be established with greater certainty. To test this hypothesis, upper nasal biopsy specimens from each of these three groups were examined for GnRH immunoreactivity.
This study thus had three principal aims: 1) to determine whether GnRH neurons persisted in adult human nasal epithelium; 2) to establish whether the distribution, number, or morphology of nasal GnRH cells in KS patients differed from those in normal subjects (such differences might be useful in diagnosing KS in early life); and 3) to establish whether immunoreactive GnRH was demonstrable in the nasal epithelium of a normosmic IHH subject (a positive finding would effectively exclude a gross transcriptional or translational failure of GnRH synthesis as the cause of this syndrome). A subsidiary area of study was to examine GnRH cell migration in early midtrimester human fetuses.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Subjects consisted of two KS males (one XKS, with a complete KAL deletion, and one sporadic case), a KS female, and a normosmic IHH male. All had biochemical evidence of isolated hypogonadotropic hypogonadism; anosmia was confirmed with the Smell Identification Test (Sensonics, Haddon Heights, NJ) (8), and MRI demonstrated dysgenesis of olfactory bulbs and/or sulci in the three KS patients (5). Olfactory epithelium was also obtained from a 64-yr-old male cadaver, who had died in hospital 13 h previously (GnRH is known to be stable up to and beyond 36 h postmortem) (9). He was phenotypically eugonadal, with normal olfactory bulbs and sulci at necropsy.
Biopsy of olfactory epithelium
In the case of living subjects, local ethical committee permission was obtained, and volunteers gave informed written consent (next of kin gave permission for postmortem examination of the cadaver). Nasal mucosa was prepared with an adrenaline/xylocaine spray to provide local anesthesia and induce vasoconstriction. A 7200A Hopkins 00 rod was introduced into the olfactory cleft, and using endoscopic cup forceps, mucosal biopsy specimens were obtained under direct vision, adjacent to the cribriform plate. Tissue was fixed in 4% paraformaldehyde for 2 h, washed, and dehydrated in 20% sucrose in HEPES buffer. The tissue was subsequently frozen, and 7-µm cryostat sections were cut.
Fetal tissue
Permission to use human fetal tissue was granted by the local ethical committee, and maternal consent was obtained (10) before termination of pregnancy. Gestational ages of 14 and 16 weeks, respectively, were estimated for the two fetuses obtained by transabdominal ultrasound examination and were confirmed after delivery using morphological criteria (11). After delivery, the fetal head was examined, and the cranial vault and meninges were incised before transfer to Bouin’s medium for 48-h fixation. The fixed brain, including olfactory bulbs, was then removed. Sagittal cuts were made in the midorbital planes, and axial cuts were made cranially, through the frontal lobes, and caudally, below the palate, with a coronal cut posteriorly through the brain stem. The resulting block of tissue was then transferred to 20% sucrose in HEPES buffer before freezing and cryostat sectioning.
Immunostaining and visualization
An immunofluorescent double immunostaining technique was used. Sections were incubated in 5% swine serum for 1 h and incubated overnight in a humid chamber with primary antisera raised against the ubiquitin-processing enzyme PGP-9.5 (12) (Ultraclone, Wellow, UK; rabbit polyclonal, 1:800 dilution) and against human GnRH (Sternberger Monoclonals, MD; mouse monoclonal, 1:500 dilution). The slides were then incubated for 90 min in the second layer immunofluorescent antibodies, goat antimouse fluorescein isothiocyanate and donkey antirabbit Texas red. Sections were viewed with both fluorescence (Vanox AH-2, Olympus, New Hyde Park, NY) and confocal scanning laser microscopy (MRC 600 with krypton-argon laser, Bio-Rad Laboratories, Richmond, CA). Dual channel usage of the confocal microscope allows examination of two immunoreactivities at the same time on the same section; a x20 Plan-Apo objective was used for image input with 10–15 single optical slices, of about 1 µm thickness, making up the final merged image. The confocal microscope GnRH immunoreactivity (GnRH+) is indicated by green (fluorescein isothiocyanate) fluorescence, and PGP-9.5 immunoreactivity (PGP+) is shown by (Texas) red fluorescence. Colocalization (GnRH+/PGP+) resulted in a yellow-green coloration as the green and red images were merged. Thus, where colocalization occurred, it indicated the presence of GnRH-synthesizing neuroendocrine cells.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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GnRH+/PGP+-immunoreactive neurons were
demonstrable in nasal epithelium obtained from all subjects and
appeared to be of normal morphology (Fig. 1
). However,
the small number of subjects sampled and the variation in both the
quantity and the quality of the biopsy specimens between subjects did
not permit us to comment objectively on cells numbers in each
subject.
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). However, as late as 16
weeks, GnRH+/PGP+-immunoreactive cells could
still be identified in the fila olfactoria close to the cribriform
plate, apparently still migrating (Fig. 3). However, in
neither fetus were GnRH neurons demonstrable within the epithelium of
the upper nasal cavity, contrasting with the findings of
Schwanzel-Fukuda and Pfaff in 19-week-old fetuses (4).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In humans, GnRH-immunoreactive cells are detectable within the medial olfactory placode in ganglia and fascicles of the terminal nerve at 42 (but not 28) days gestation; at 46 days, they have begun to enter the forebrain along with axons of the terminal nerve medial and caudal to the developing olfactory bulb (17), and by 19 weeks, migration is virtually complete (4). Extrapolating the results of previous animal work to humans suggests that migration might be completed between 13–16 weeks gestation (18). The human fetal work presented here confirms that GnRH-immunoreactive cells have reached the hypothalamus in significant numbers by 14 weeks gestation, but that the process of migration continues for at least another 2 weeks. Although no GnRH cells were seen in the olfactory epithelia of the two fetuses examined in this study, it is possible that a larger series might have demonstrated their presence in small numbers.
The presence of nasal GnRH neurons in a eugonadal adult was compatible with these cells being vestigial, the result of incomplete migration. However, the concept of fully differentiated GnRH-synthesizing neurons persisting unchanged from early gestation through to late adult life in a dynamic tissue with continuous cell turnover such as the olfactory epithelium remains perhaps less attractive than the following alternative interpretation. Terminal differentiation of olfactory epithelial stem cells into exclusively olfactory or GnRH-synthesizing neuron precursors may not occur; GnRH cells may thus continue to be generated postnatally. Indeed, such neuroendocrine cells might subserve a biological function. In the rat, GnRH axons and receptor are found throughout the rhinencephalon, in the hippocampus and pyriform cortex, olfactory bulb, and amygdala, raising the possibility that the peptide may modulate the conversion of incoming olfactory stimuli into endocrine signals and thus into reproductive behavior (19). GnRH messenger ribonucleic acid synthesis has been demonstrated outside the human central nervous system, including the placenta, testis, and ovary, where its potential regulatory role has likewise not been definitively established (20, 21, 22).
In KS, most of the olfactory neurons within the neuroepithelium are morphologically immature and axon numbers within individual olfactory fascicles are reduced, with the axons being swollen and degenerating. The overall appearance is similar to that in congenital or posttraumatic anosmia and in experimentally bulbectomized animals (23). In the absence of functioning olfactory bulbs, nasal GnRH cells in KS subjects may instead obtain trophic support from ganglia of the terminal nerve, which are known to persist within the nasal region (24). Detection of immunologically recognizable GnRH in a normosmic IHH subject is consistent with work by Crowley’s group demonstrating the absence of structural mutations of the GnRH gene in such subjects (25). Defective cellular synthesis of the peptide, therefore, does not appear to be a likely pathophysiological mechanism; perhaps GnRH-cell migration arrest occurs despite normal development of the olfactory apparatus.
Conclusions
GnRH-synthesizing cells persist in the upper nasal epithelium into adult life in normal human subjects and in both anosmic (KS) and normosmic IHH patients, thus ruling out mucosal biopsy as a technique for the early identification of KS. It is not clear whether such neurons are generated de novo postnatally and have a possible biological function or are merely vestigial. This presence of GnRH-synthesizing cells in a normosmic (i.e. non-KS) IHH subject implies that a cellular defect in GnRH synthesis is not responsible for this form of hypothalamic GnRH deficiency. Although GnRH-synthesizing neurons are well established in the human hypothalamus by 14 weeks gestation, migration of other GnRH cells along fascicles of cnIc axons is still in progress at 16 weeks.
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Acknowledgments |
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Received May 22, 1996.
Revised August 16, 1996.
Accepted September 4, 1996.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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