Immunocytochemical localization of neuropeptides in the hypothalamus of the Japanese long-fingered bat,Miniopterus schreibersii fuliginosus

Summary To elucidate the role of hypothalamic neuropeptides in regulation of reproductive phenomena of seasonally breeding feral mammals, we used Japanese long-fingered bats, Miniopterus schreibersii fuliginosus, for immunocytochemical study of distribution of the following neuropeptides in the hypothalamus: arginin vasopressin, oxytocin, luteinizing hormone-releasing hormone, somatostatin, corticotropin-releasing factor, and growth hormone-releasing factor. The size, shape and location of supraoptic, paraventricular, suprachiasmatic, and arcuate nuclei of the bat were determined. Arginin vasopressin-and oxytocin-immunoreactive magnocellular neurons were found in the supraoptic and paraventricular nuclei, where they exhibited separate distribution into two distinct groups. Parvocellular arginin vasopressin neurons occurred only in the suprachiasmatic nucleus. The hibernating bats exhibited slightly increased numbers of vasopressin and oxytocin neurons in the supraoptic and paraventricular nuclei. The pregnant bat displayed further increased numbers of vasopressin and oxytocin neurons in both nuclei. Somatostatin-immunoreactive neurons in the paraventricular nucleus were also immunopositive to anti-oxytocin serum, while those in the ventromedial and arcuate nuclei reacted solely to anti-somatostatin serum. They projected to the anterior median eminence and infundibular stalk. Luteinizing hormone-releasing hormone-immunoreactive perikarya were scattered throughout the basal hypothalamus, being particularly abundant in the arcuate nucleus. They were larger in size in hibernating bats than those in normal (non-pregnant) and pregnant females. They projected fibers mainly to the internal layer of the median eminence and infundibular stalk. A few luteinizing hormone-releasing hormone-reactive fibers were also observed in the organum vasculosum laminae terminalis, lateral habenular nuclei, pineal stalk, retroflexus fasciculus, and olfactory tubercle. Corticotropin releasing factor-immunoreactive perikarya were distributed in the paraventricular nucleus and medial preoptic area and projected into the external layer of the anterior median eminence, while growth hormone-releasing factor-immunoreactive perikarya occurred only in the arcuate nucleus and projected into the posterior part of the median eminence.     

Changes in progesterone concentrations in the Japanese long-fingered bat, Miniopterus schreibersii fuliginosus

In the Japanese long-fingered bat, when compared with the baseline values during non-pregnancy in the autumn, plasma progesterone concentrations were not significantly elevated during the delayed implantation stage that begins before the bats enter hibernation. However, progesterone concentrations were significantly lower during the delayed development stage that occurs during hibernation and rose significantly during the rapid embryogenesis that occurs after arousal from hibernation in the spring. Changes in the corpus luteum volume corresponded closely with those of plasma progesterone values. Maintenance of gravid females at 25 degrees C for 2 weeks in winter resulted in significant increases in the plasma progesterone concentration and the corpus luteum volume.     

Isolation and characterization of microsatellite loci in the long-fingered bat Miniopterus fuliginosus

We isolated and characterized 10 microsatellite loci in the long-fingered bat Miniopterus fuliginosus. These loci were tested on 48 individuals from Anhui Province of China, and all loci were highly polymorphic. The mean number of observed alleles per locus was 13.6 (range from six to 27). Observed and expected heterozygosity values ranged from 0.364 to 0.957, and from 0.676 to 0.951, respectively. After Bonferroni correction, four loci deviated significantly from Hardy-Weinberg equilibrium. No pairs of loci were in linkage disequilibrium. These polymorphic markers will be used to examine population structure and genetic diversity in this species.     

Isolation and characterization of highly polymorphic microsatellite loci in Schreibers’ long‐fingered bat,Miniopterus schreibersii (Chiroptera: Vespertilionidae)

Five polymorphic, dinucleotide microsatellite loci have been identified and characterized in Schreibers’ long‐fingered bat, Miniopterus schreibersii (Chiroptera: Vespertilionidae). These include three uninterrupted (CA)_n repeats, ranging in length from (CA)₁₇ to (CA)₁₉, one interrupted repeat (CA)₄(CG)₂(CA)₁₃, and one uninterrupted (GA)₂₇ repeat. All loci were highly polymorphic, with 17–20 alleles identified per locus. Observed heterozygosity levels (0.66–0.82) are lower than expected due to homozygote excess, probably caused by pooling of populations, resulting in population substructuring. All five polymorphic loci were also successfully amplified in the closely related M. fraterculus. Two pairs of primers additionally amplified polymorphic microsatellites in Chaerophon sp.     

Development of the main and accessory placentae in the Japanese long-fingered bat, Miniopterus schreibersii fuliginosus

The placentae of the Japanese long-fingered bat were characterized by their morphological and functional transition from the main placenta to the accessory placentae. The main placenta transformed from an endotheliodichorial to a haemodichorial (one layer of syncytiotrophoblast and one layer of cytotrophoblast cells) condition. Degeneration of the main placenta was accompanied by development of two accessory placentae. These developed on both sides (fetal side) of the main placenta, and subsequently converted from a haemodichorial (two layers of cytotrophoblast cells) to a haemomonochorial condition.     

Effect of cyproterone acetate on cells of the pars distalis of the adenohypophysis in the Beagle Bitch

Summary The effects of oral administration of 100 mg per kg per day cyproterone acetate (CPA) for four weeks on cells of the pars distalis, as revealed by the immunoperoxidase technique and chemical staining, were studied in the ovariectomized beagle bitch. For immunochemical staining antisera to the following hormones were used: canine GH, canine PRL, porcine ACTH, bovine TSH, bovine LH and human FSH¹. The most striking effects of the treatment were an overall increase in the relative proportion of GH cells and a marked morphological indication of high secretory activity in these cells. In contrast, PRL cells were not affected significantly. In all ovariectomized control bitches a marked atrophy of the cells stained for FSH (FSH cells) and hypertrophy of the cells shown to contain LH(LH cells) were observed. FSH cells became enlarged, while LH cells appeared reduced in size by administration of CPA. In some treated bitches ACTH/MSH cells showed atrophy and regressive changes, whereas TSH cells seemed to become enlarged and were more densely arranged. These structural responses indicate that, in addition to its partial antigonadotropic properties, CPA as a synthetic progesterone derivative may stimulate GH secretion and possibly suppress CRH-ACTH activity in the ovariectomized beagle bitch.Abbreviations of Hormones cited in this Paper ACTH Adrenocorticotropin - FSH Follicle Stimulating Hormone - GH Growth Hormone - LH Luteinizing Hormone - MSH Melanocyte Stimulating Hormone - PRL Prolactin - TSH Thyrotropin - CRH Corticotropin Releasing Hormone The authors are grateful to Dr. Christel Schoebel and Mrs. P. Kurth for carrying out the experimental work on animals, to Dr. H. Wiemann for the statistical analysis, to Mrs. B. Schilk, G. Soulioti and Miss U. Tüshaus for their excellent technical assistance, and to Dr. P. Guenzel for his advice and encouragementRecipient of a Research Scholarship from the Arabic Republic of Egypt     

Noradrenergic and cholinergic nerves in the uterus of the Japanese long-fingered bat,Miniopterus schreibersii fuliginosus,change with reproductive cycle

The pattern of uterine innervation by noradrenergic (NA) and acetylcholinesterase-positive (AChE) nerves in different reproductive stages of the adult Japanese long-fingered bats were investigated histochemically and immunohistochemically. In the non-pregnant bat, the uterine horn was supplied with abundant NA and AChE nerves. These two types of nerves were closely associated with the uterine arteries and myometrial smooth muscles. In the pregnant bat, NA and AChE nerves supplying the uterus did not degenerate much during hibernating period, but reduced markedly after arousal. In the postpartum bat, the density of nerves recovered progressively. The significant change in the innervation pattern of uterine NA and AChE nerves in the pregnant bats under and after hibernation, and in the postpartum bat must be considered in relation to the adrenergic and cholinergic controlling mechanisms on the uterine function that is matched for the unique reproductive cycle of this bat.     

Cytological changes in the pars distalis of pituitary of the green frog,Rana esculenta L., during the reproductive cycle

Summary The cytological changes in the pars distalis of pituitary of male and female green frog, Rana esculenta during the reproductive cycle have been studied by examining the form, cytoplasmic granulation and numerical proportions of the different cell types. Some sex differences were recorded, particularly in relative total number of cells in a median sagittal section of the pars distalis. No extensive cytological changes occur during the hibernation period, but there are striking alterations at the time of spring awakening, during spawning months and during replenishment of the spent gonads i.e. during late summer and autumn months. Important changes are to be observed in the acidophils type one (A1) and basophils type two (B2) and three (B3). Changes in the A1 cells during breeding season have been speculated as being in support of the spawning activity of the animal. The B2 cells exhibit most profound changes which correlate precisely with the morphological changes in gonads, and these cells are considered as the FSH-gonadotrops. The functional significance of the B3 cells is discussed and it is suggested that they might be the source of an ICSH- or LH-like hormone.The work has been supported by grant from the National Research Council of Italy.The award of postdoctoral fellowship by the National Research Council of Italy is gratefully acknowledged.     

普通长翼蝠食性结构及其回声定位与体型特征

在普通长翼蝠(Miniopterus fuliginosus)的捕食区内用灯诱法和网捕法调查潜在食物(昆虫)种类; 用粪便分析法鉴定普通长翼蝠的食物组成,发现其主要捕食体型较大的鳞翅目和鞘翅目昆虫,体积百分比分别为55%和38%.普通长翼蝠具有相对狭长的翼,翼展比为6.94 ± 0.13;翼载为(9.85 ± 0.83)N/m2,相对较大.飞行状态下普通长翼蝠的回声定位叫声为调频下扫型,声脉冲时程为(1.45 ± 0.06)ms,脉冲间隔为(63.08 ± 21.55)ms,主频较低,为(44.50 ± 2.26)kHz.研究表明,普通长翼蝠的形态特征和回声定位特征与其捕食行为有着密切的联系.     

Effect of cyproterone acetate,d-norgestrel and progesterone on cells of the pars distalis of the adenohypophysis in the beagle bitch

Summary The effects of short-term (8 weeks) treatment with different doses of cyproterone acetate (CPA), d-norgestrel (d-N) and progesterone on cells of the pars distalis, as revealed by the immunoperoxidase technique, were studied in cycle-synchronized beagle bitches (first anoestrus). Pituitary glands from non-treated primiparous beagle bitches at the 6th and 9th week of pregnancy were also included. For immunochemical staining specific antisera to the following hormones were used: canine GH, canine PRL, porcine ACTH, bovine TSH, bovine LH and human FSH. Morphological features of high secretory activity in GH cells were evident even after the human oral contraceptive doses of CPA and d-N, and after a dose as low as 0.1 mg/kg/day subcutaneously (s.c.) of progesterone. In contrast, PRL cells did not show any significant treatment-related effects except in those animals which received the highest dose of d-N (0.5 mg/kg/day per os). In this group, as well as in all pregnant bitches, hyperplasia and hypertrophy of PRL cells were found. In the animals treated with the highest doses of CPA (4.0 mg/kg/day per os) and progesterone (42.5 mg/kg/day s.c.) as well as in pregnant bitches, ACTH/MSH and TSH cells showed marked atrophy and regressive changes. Similar morphological signs of depressed secretory activity were also observed in the cells shown to contain FSH and/or LH as a result of treatment with the highest dose of progesterone and at the 9th week of pregnancy. These structural responses indicate that quantitative and/or qualitative differences may exist between progesterone, the synthetic progesterone derivative CPA and the nortestosterone type progestagen d-N with regard to their effect on pituitary hormone secretion in the beagle bitch.Abbreviations ACTH Adrenocorticotropin - FSH Follicle Stimulating Hormone - GH Growth Hormone - LH Luteinizing Hormone - MSH Melanocyte Stimulating Hormone - PRL Prolactin - TSH Thyrotropin - CRH Corticotropin Releasing Hormone - TRH Thyrotropin Releasing Hormone The authors are grateful to Dr. Christel Schöbel and Mrs. P. Kurth for carrying out the experimental work on animals, to Mrs. B. Schilk and Miss U. Tüshaus for their excellent technical assistance, and to Dr. P. Günzel for his advice and encouragement     

Effects of Methallibure (ICI 33,828) on the pars distalis of pituitary,testis and thumb pad of the green frog,Rana esculenta L.

Summary The influence of ICI compound 33,828 (1--methylallylthiocarbamoyl-2-methylthiocarbamoylhydrazine = Methallibure) on the pars distalis of pituitary, testes and thumb pads was investigated in the intact adult male green frog, Rana esculenta. Methallibure affected the gonadotropic basophils (particularly the B2 cells) of the pars distalis, which showed varying degrees of degranulation and underwent a notable decrease in their percentage. Within the testis this compound caused the arrest of spermatogenesis. The most uniform effect of Methallibure was observed in the thumb pads, which invariably showed regression of the epidermis and glandular epithelium. No histological changes occurred in the thyroid and adrenal glands and the B1 and A1 cells of the pars distalis remained unchanged cytologically. It is concluded, in concordance with the available data, that Methallibure is a non-steroidal antigonadotropic compound. The important question about its mode of action has been brought into discussion.Work supported by the National Research Council of Italy, and the Population Council (Grant No. M70.082 C) of New York.     

The status of the corpus luteum during pregnancy in Miniopterus schreibersii (Chiroptera: Vespertilionidae) with emphasis on its role in developmental delay

Summary Developmental delay is correlated with torpor in the temperate zone bent-winged bat, Miniopterus schreibersii (latitude 37° S) as a period of pre-implantation delay (delayed implantation) followed by a short post-implantation delay (delayed development). During this time, the number of steroidogenic organelles in luteal cytoplasm is greatly reduced compared with normal embryogenesis, and granular endoplasmic reticulum is prominent. Nidation, which occurs while the animals are hibernating, is not accompanied by marked changes in luteal ultrastructure, although the number of lipid droplets decreases somewhat. Progesterone rises slightly but not significantly; however, a pre-nidation decrease in high 17-estradiol levels may play a role in implantation. Following implantation, the conceptus remains delayed at the blastocyst stage for several weeks. During this time the bats remain torpid and the only change in luteal cell ultrastructure is an increase in smooth endoplasmic reticulum as differentiation begins toward the trilaminar stage. At the end of developmental delay hypertrophy of the luteal cell begins and mitochondria and lipid droplets increase, markedly. By this time arousal from hibernation has occurred, placentation takes place, and normal development is underway. At placentation, smooth endoplasmic reticulum reaches its maximum in luteal cytoplasm; estrogen and progesterone levels peak about 6 weeks later. For the remaining 2 months of gestation, signs of luteolysis appear. These observations suggest that the corpus luteum of developmental delay, though suboptimally functional, is prolonged in its luteinization by the arrival of winter when the bats enter torpor. The capacity for maximal steroidogenesis is acquired at the end of winter, some weeks after implantation, when arousal occurs and normal development ensues.     

Isolation and characterization of microsatellite loci in the western long-fingered bat, Miniopterus magnater

We isolated and characterized 10 microsatellite loci in the western long-fingered bat, Miniopterus magnater. These loci were tested on 48 individuals from Anhui Province of China, and all loci were highly polymorphic. The mean number of observed alleles per locus was 13.6 (range from six to 27). Observed and expected heterozygosity values ranged from 0.364 to 0.957, and from 0.676 to 0.951, respectively. After Bonferroni correction, four loci deviated significantly from Hardy-Weinberg equilibrium. No pairs of loci were in linkage disequilibrium. These polymorphic markers will be used to examine population structure and genetic diversity in this species.     

Immunocytochemical analysis of somatotrophs and mammotrophs in the pars distalis of postnatal dwarf (dw) mutant mice

Summary The postnatal differentiation of somatotrophs and mammotrophs in the pars distalis of normal and dwarf (dw/dw) mice was analyzed by means of immunocytochemistry at the electron-microscopic level. Thin sections of Araldite-embedded pituitaries from postnatal mice ranging in age from 2 days to 3–4 months were exposed to antigrowth hormone serum (anti-GH) or to anti-prolactin serum (anti-PRL) and were processed immunocytochemically by means of the colloidal-gold technique. In the normal adult pars distalis, somatotrophs reacted strongly with antiGH, and mammotrophs showed a positive anti-PRL reaction. In the dwarf adult, the granulated cells were unreactive with either anti-GH or anti-PRL. However, at 6 days postnatally, some cells in the dwarf pars distalis showed a positive reaction with anti-GH, though the reaction was not as strong as in the 6-day normal pars distalis. The 6-day dwarf was only faintly reactive with anti-PRL, and by 14 days the dwarf was unreactive with anti-GH as well as anti-PRL, in contrast to the strong positive reactivity to these two antisera in the normal 14-day pars distalis.Supported by USPHS grant HD12308     

A comparative in vitro study on LHRH responsiveness of LH cells of the pars tuberalis and pars distalis

Summary The aim of the present study was to test whether the luteinizing-hormone (LH) cells in the pars tuberalis (PT) of the rat and mouse respond to LH-releasing hormone (LHRH) as do those of the pars distalis. A part of the basal hypothalamus containing the pituitary stalk, median eminence and the pars tuberalis (H-PT), was dissected out and incubated in vitro.The LH-secreting capacity of the PT was investigated after removal of the pituitary body (i.e., partes distalis, intermedia and nervosa). First, some rat and mouse H-PT tissues were treated with synthetic LHRH (100ng/ml), while others were incubated without LHRH. After 24 h of incubation, variable amounts of LH release were detected in the medium. This LH discharge, however, was not LHRH-dependent but proportional to the number of PT LH cells that were immunohistochemically detected in each incubated tissue. Since there was marked individual variation in the number of LH cells in the PT, the LH levels in the incubation medium were next compared before and after LHRH treatment using the same H-PT of the rat. An effect of LHRH could not clearly be shown in this experiment.Finally, the cytological response of the PT to LHRH was investigated by incubating both the H-PT and pituitary body connected to the intact pituitary stalk. Immunohistochemical examination of LHRH-treated tissues after 24 h revealed that, in females of both rats and mice, hormone depletion occurred in LH cells of the pars distalis but not in those of the PT. These results indicate that although LH cells in the PT can release LH in vitro, their mode of hormone synthesis and/or discharge differs from that of LH cells in the pars distalis. Since there was a marked individual variation and small LH-secreting capacity by the PT tissue, it seems unlikely, at least in rats and mice, that LH of PT origin plays an important role in the normal physiological state.     

Immunohistochemical localization of FSH and LH in the pars distalis of vervet (Cercopithecus aethiops) and babbon (Papio hamadryas) pituitaries

Summary Antisera against oLH¹, oLH and hFSH were used to localize gonadotropic cells in the pars distalis of Cercopithecus aethiops and Papio hamadryas. Three separate cell types were observed for FSH and LH: 85% of immunohistochemically identified gonadotropic cells reacted to all the various antisera; 10% reacted with the anti-LH antibody only; and 5% with the anti-hFSH antibody only. Comparisons between adjacent serial sections treated with various antisera, other than anti-gonadotropic hormones, demonstrated that the gonadotropic cells of these monkeys did not respond to these antisera.

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Résumé Des anticorps anti-LH ovine, anti-LH ovine et anti-FSH humaine ont été utilisés pour localiser les cellules gonadotropes dans la pars distalis de l'hypophyse des Singes Cercopithecus aethiops et Papio hamadryas. Trois catégories cellulaires distinctes, réagissant avec des anticorps anti-hormones gonadotropes, ont été observées. 85% des cellules immunoréactives identifiées en tant que cellules gonadotropes réagissent simultanément avec les différents anticorps mentionnés; 10% des cellules gonadotropes réagissent seulement avec l'anticorps anti-oLH et 5% de ces cellules seulement avec l'anticorps anti-hFSH. La comparaison avec des coupes adjacentes traitées par divers anticorps autres que les anticorps anti-gonadotropines prouve que les cellules gonadotropes de ces Singes ne réagissent jamais simultanément avec l'un ou l'autre de ces anticorps.

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Abbreviations used in this Article oLH ovine luteinizing hormone - hFSH human follicle stimulating hormone - ACTH corticotropin - GH growth hormone - LPH lipotropin - TSH thyrotropin     

Immunocytochemical study of the endocrine pancreas in the grey kangaroo (Macropus fuliginosus)

Summary The endocrine pancreas of the grey kangaroo,Macropus fuliginosus, was investigated by means of immunocytochemistry using the PAP method on the same section at the light- and electron-microscopic levels. Semithin plastic sections were stained individually with primary antibodies for insulin, glucagon, somatostatin and pancreatic polypeptide (PP), and then photographed. Sections were osmicated, re-embedded in BEEM capsules, and ultrathin sections made and examined. The same labelled cells as in the semithin sections were localised in the thin sections, photographs taken and the morphology of secretory granules studied. The insulin cells were pleomorphic; their secretory granules displayed an electron-dense core surrounded by an empty halo. The glucagon cells possessed granules with an electron-dense core usually surrounded by a halo of less dense granular material. Somatostatin cells had larger, less dense secretory granules. The PP cells showed small, dense secretory granules. In order for an ultrastructural study to be considered reliable for the definite identification of endocrine cell types, it is essential that it be corroborted by correlated immunocytochemical data at the light-and electron-microscopic levels.     

Fine structure of the chromophobe in the pars distalis of the common snapping turtle,Chelydra serpentina

Summary Chromophobes of the pars distalis in young Chelydra serpentina have sparse cytoplasm with no specific granules; however, many cytoplasmic filaments are present. The chromophobes are connected to the other cell types by desmosomes, while different types of junctional specializations occur between adjacent chromophobes. Cytoplasmic filaments traverse the cytoplasm in a random manner and terminate on both the junctional complexes and the nuclear envelope. It is proposed that, in addition to providing a structural framework, the chromophobes may be involved in integrating cellular responses of the parenchyma to changes in the endocrine milieu.Supported in part by a General Research Support Grant RR05402, NIH to Dr. Tseng and a Human Development Grant HD-03484, NIH to Dr. Yntema.     

Morphometric evaluation of subcellular changes induced by in vivo TRH treatment in the pituitary gland of Rana perezi: Effects on prolactin and thyrotropic cells

Summary The effects of synthetic thyrotropin-releasing hormone on pituitary prolactin and thyrotropic cells were investigated in adult male Rana perezi (formerly Rana ridibunda) frogs. Animals were given daily injections of synthetic thyrotropin-releasing hormone into the dorsal lymph sac. Prolactin and thyrotropic cells were identified by the colloidal-gold method, using anti-human prolactin and anti-human--thyrotropin hormone as primary antisera. The stereological parameters of the rough endoplasmic reticulum, Golgi complex, and secretory granules of prolactin and thyrotropic cells were evaluated by ultrastructural morphometry (point-counting method). Thyrotropin-releasing hormone caused cytological changes in both cell-types which were consistent with increased synthesis and release of both prolactin and thryrotropin. These changes were still significant after 48 h treatment in the case of thyrotropic cells, while in prolactin cells the thyrotropin-releasing hormone increased the number of secretory granules. After 6 days, the cells resembled essentially those used as controls. These results indicate that thyrotropin-releasing hormone stimulates the synthesis and release of prolactin and thyrotropin, and that the response of each cell type to this hypothalamic stimulus follows a different time-course.This work has been supported by grants no. 2184-83 and PB 86-0095 from the Comisión Interministerial para la Ciencia y Tecnología, Spain