Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations
1996 The prepubertal ontogeny of neuropeptides in the male Meishan pig brain Paul Lyle Pearson Iowa State University
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The prepubertal ontogeny of neuropeptides in the male Meishan pig brain
by
Paul Lyle Pearson
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of the
Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Department: Veterinary Anatomy iQterdepartmental Major: Molecular, Cellular, and Developmental Biology
Ap^ved: M^bers of the Committee: Signature was redacted for privacy. Signature was redacted for privacy. Ina Chargei^narge o^sMijoro^^Jiiajor WorkworK
Signature was redacted for privacy. Signature was redacted for privacy. For'or the Interdepartmental Major Signature was redacted for privacy. Signature was redacted for privacy. For"or the Myor Department
Signature was redacted for privacy. Signature was redacted for privacy. For the Graduate College
Iowa State University Ames, Iowa
1996
Copyright © Paul Lyle Pearson, 1996. All rights reserved. UMI Number; 9620983
Copyright 1996 by Pearson, Paul Lyle
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DEDICATION
This thesis is dedicated to my parents Nels Dale and Mae Phyllis Salmon Pearson. Without your love, support, guidance and understanding, I never would have made it this far.
I love you both so much. Dad, I will miss you always. iii
TABLE OF CONTENTS
GENERAL INTRODUCTION 1
Literatuie review 1 Dissertation organization 9
CHAPTER 1: THE PREPUBERTAL ONTOGENY OF NEUROPEPTIDE Y-LIKE IMMUNOREACTIVITY IN THE MALE MEISHAN PIG BRAIN 10
Sununary 10 Introduction 11 Materials and Methods 13 Results 17 Discussion 31 Acknowledgements 39 Literatuie Cited 39
CHAPTER 2: THE PREPUBERTAL ONTOGENY OF GALANIN-UKE IMMUNOREACTIVITY IN THE MALE MEISHAN PIG BRAIN 92
Summary 92 Introduction 93 Materials and Methods 94 Results 99 Discussion 105 Acknowledgements 110 Literature cited 110
CHAPTER 3: THE PREPUBERTAL ONTOGENY OF LUTEINIZING HORMONE-RELEASING HORMONE-LIKE IMMUNOREACTIVITY IN THE TELENCEPHALON AND DIENCEPHALON OF THE MALE MEISHAN PIG BRAIN 142
Summary 142 Introduction 143 Materials and Methods 145 Results 149 iv
Discussion 154 Acknowledgements 159 Literature cited 160
GENERAL SUMMARY AND DISCUSSION 186
General summary 186 General discussion 188
REFERENCES 199
ACKNOWLEDGEMENTS 219 V
ABSTRACT
This dissertation describes the immunohistochemical localization of galanin (GAL), neuropeptide Y (NPY) and luteinizing hormone-releasing hormone (LHRH) in the brain of the male Meishan pig throughout prepubertal development.
The first stody details the prepubertal ontogeny of NPY in the male Meishan pig brain. Neuropeptide Y-like immxmoreactivity (NPY-ER.) was seen in cell bodies and fibers throughout the brain in all ages stodied firom gestational day (g)30 through postoatal day
(pn)50. Cell bodies containing NPY-IR generally increase in nmnber and distribution in the brain prenatally, but decline in the early postnatal period, especially in the arcuate nucleus of the hypothalamus. This may indicate a fimctional maturation of food intake control pathways. The distribution of NPY-IR in fibers becomes more widespread throughout gestational development, showing a pattern by gllO that was characteristic of postnatal ages.
The second study describes the prepubertal ontogeny of galanin-like immunoreactivity
(GAL-IR). Galanin-IR in cell bodies and fibers was evident in the brain at all ages, especially in the hypothalamus. Throughout prenatal development cell bodies and fibers containing GAL-IR generally increased in number and distribution in the brain showing a pattern by pnl that was consistent through postnatal ages.
The third smdy investigated the prq)ubertal ontogeny of luteinizing hormone-releasing hormone-like immimoreactivity (IJEIRH-IR) in the telencephalon and diencephalon.
Luteinizing hormone-releasing hormone-ER in cell bodies and fibers was detected at g30 entering the brain along the terminal nerve and in the basal telencephalon. The number of immunoreactive cells increased and immunoreactive fibers were present in the median eminence at g50. Further increases were seen at g70 and g90, but changed little postnatally.
These results confirm the olfactory origin of LHRH cells in the pig and suggest a breed difference in the ontogeny of reproductive control systems in the pig.
Together these smdies detail the development of these peptides and support their respective roles in the control of growth and reproduction in the Meishan pig, a Chinese breed known for its superior reproductive characteristics but slow growth and obesity. 1
GENERAL INTRODUCTION
Literature review
The Meishau pig was brought to the United States from mainland China in 1989, and has been smdied primarily for its superior reproductive capabilities including precocial puberty and increased litter size (Harayama et al., 1991; Lunstra et al., 1992; Anderson et al., 1993; Ford and Youngs, 1993; Haley and Lee, 1993; Youngs et al., 1993, 1994;
Dlamini et al., 1995). The Meishan is also characterized by slow growth rate, low feed efficiency and abundant adipose tissue compared with traditional American and European breeds (Hays, 1980; Legault, 1985).
The developing pig undergoes gonadal differentiation at gestational day (g) 26
(Pellinemi, 1975), can be sexed by inspection of the external genitalia at g36 (Marrable,
1971) and sexual differentiation of hypothalamic control of LH release is thought to occur between g30 and g74 (Elsaesser and Parvizi, 1979). A period of elevated serum testosterone levels in the male fetus occurs between g35 and g40 (Ford et al., 1980). Cells containing
LH first appear in the porcine pimitary at g40 (Dacheux, 1984) or g45 (Danchin and Dubois,
1982) with the first FSH-containing cells appearing at g45 (Dacheux, 1984) or g60 (Danchin and Dubois, 1982). These studies indicate that the gonadotropic cells reach adult levels and
appearance by g80 (Danchin and Dubois, 1982) or g90 (Dacheux, 1984). Differentiation of
the capillary loops in the median eminence is not complete until after g60 (Danchin and
Dubois, 1982), and the fetal pig pituitary will secrete LH in response to electrical or 2 electrochemical stimulation of the hypothalamus at gSO, but not g60. In addition, the basal plasma LH levels double between g60 and g80 (Bruhn et al., 1983), suggesting that the relationship of the hypothalamus and pituitary is not complete in the pig imtil g70. The duration of gestation is approximately 114 days. Serum LH levels are 5- to 6-fold higher in the Meishan boar as compared to domestic breeds and peak at 3 weeks of age, decreasing until puberty in both Meishan and domestic breeds (Anderson, 1983; Lunstra et al., 1992).
The Meishan boar also has greater serum concentrations of both testosterone and FSH as compared to domestic boars (Lunstra et al., 1992). Noted for precocious puberty, the male
Meishan undergoes puberty at approximately postnatal day (pn) 75, producing viable sperm as early as pnl20. American and European breeds of pigs enter puberty at about pnl25 and produce viable sperm by pnl50-180 (Haryama et al., 1991). Thus, the Meishan pig may be an appropriate model for the study of biological phenomena such as obesi^, control of gonadotropic function and pubertal onset.
Neuropeptide Y
Neuropeptide Y (NPY) is a 36 amino acid amidated peptide originally isolated from porcine brain (Tatemoto et al., 1982). Neuropeptide Y is a member of the pancreatic polypeptide (PP) family and shows homology to both PP and peptide YY (Tatemoto et al.,
1982). Neuropeptide Y has been implicated in a number of biological processes, including circadian rhythm entrainment (Albers and Ferris, 1984), somatotropic and gonadotropic axis control (Kalra and Crowley, 1984; McDonald et al., 1985; Rettori et al., 1990; Kalra and
Crowley, 1992; Catzeflis et al., 1993), puberty onset (Sutton et al., 1988; Minami et al.,
1990; Minami et al, 1992; Fraley and Kuenzel, 1993; Gruaz et al., 1993), inhibition of 3 sexual behavior (Clark et al., 1985; Poggioli et al., 1990) and control of food intake (Clark et al., 1984; Levine and Morley, 1984; Stanley and Leibowitz, 1984). Neuropeptide Y has a widespread distribution in the central nervous system (CNS) and peripheral nervous system
(PNS) of a variety of species, including the ground squirrel (Reuss et al., 1990), golden hamster (Sabatino et al., 1987), rat (Allen et al., 1983; Chronwall et al., 1985; DeCJuidt and
Emson, 1986), cat (Hu et al., 1987), pig (Busch-Sorensen, et al., 1989), squirrel monkey
(Smith et al., 1985) and human (Adrian et al., 1983; Pelletier et al., 1984). In the rat CNS, the highest concentrations of NPY are found in the hypothalamus, pituitary, and pineal gland. Moderate concentrations are found in the olfactory bulb, septum, striatum, thalamus, and pons (Chronwall et al., 1985). The distribution of NPY in the pig CNS as described by radioimmunoassay (RIA), follows closely that described in rat (Busch-Sorensen et al., 1989).
Binding sites for NPY exist in the CNS as well, and these have been described for the rat
(Martel et al., 1986; Dumont et al., 1993) and pig (Busch-Sorensen et al., 1989).
Neuropeptide Y binding is present in several brain regions, including high levels of binding in the hippocampus, septum, thalamus, cortex, and area postrema. Moderate levels of bindmg are seen in the paraventricular nucleus of the hypothalamus (PVN) and premammillary nuclei, and low levels of binding are observed in other hypothalamic nuclei
(Martel et al., 1986).
Intracerebroventricular (ICV; Clark et al., 1984), as well as intrahypothalamic
(Stanley and Leibowitz, 1984), injection of NPY has been shown to increase food intake in the rat. Neuropeptide Y injection preferentially increases carbohydrate intake in the rat, with a slight inhibition of protein intake (reviewed in Leibowitz, 1992). Neuropeptide Y 4 administered by ICV injection increases food intake in pigs as well (Parrott et al., 1986).
Neuropeptide Y and NPY binding sites are present in the rat PVN and the perifomical hypothalanms (Martel et al., 1986), where intrahypothalamic injection is most effective
(Stanley et al., 1993). Additionally, injection of anti-NPY antibodies into the PVN decreases food intake in rats (Shibasaki et al., 1993). Innervation of the PVN by NPY fibers comes primarily from the hypothalamic arcuate nucleus (ARC) (Bai et al., 1985), but also includes
NPYergic and catecholaminergic projections from brainstem sites (A6 and Al cell groups;
Sahu et al., 1988; reviewed in Leibowitz, 1989). Overexpression of NPY in the arcuato- paraventricular system has been linked to the hyperphagia and obesity seen in the Zucker fatty rat (Sanacora et al., 1990, Schwartz et al., 1991; Beck et al., 1993), Wistar fatty rat
(Abe et al., 1991) and obese mouse (Wilding et al, 1993).
Neuropeptide Y exerts a negative influence on growth hormone (GH) secretion, as well. Plasma GH levels were decreased in rats by ICV injection of NPY (McDonald et al.,
1985), while chronic infusion of NPY caused reduction of pituitary GH content in intact rats
(Catzeflis et al., 1993). In addition, NPY axons synapse on somatostatin neurons in the anterior periventricular hypothalamus (Hisano et al., 1990), and NPY causes release of somatostatin from median eminence (ME) fragments in vitro (Rettori et al., 1990).
Galanin
Galanin is a 29 amino acid amidated peptide originally isolated from porcine intestinal extracts (Tatemoto et al., 1983). Since its discovery, mmierous fimctions have been ascribed to GAL, including stimulation of GH secretion (Ottlecz et al., 1986; Murakami et al., 1990) and stimulation of food intake behavior (Kyrkouli et al., 1986, 1990). Galanin has been 5 localized in both the CNS and PNS of a variety of species including the rat (Rokaeus et al.,
1984; Ch'ng et al., 1985; Skofitsch and Jacobowitz, 1985; Melander et al., 1986b;
Michener, et al., 1990), pig (Ch'ng et al., 1985; Michener, et al. 1990), Brazilian opossum
(Elmquist et al., 1992), and human (Michener et al., 1990). In the rat CNS, GAL has a
wide distribution, and is present in high concentration in the hypothalamus, limbic system,
nucleus of the solitary tract, locus coeruleus (LC), and pituitary (Ch'ng et al., 1985;
Skofitsch and Jacobowitz, 1985; Michener et al., 1990) Galanin binding sites are widespread
in the rat CNS as well, including the cortex, thalamus, hypothalamus, limbic system,
hindbrain, and spinal cord (Skofitsch et al., 1986). Galanin and GAL message-associated
peptide immimoreactivity has been reported in the porcine CNS (Ch'ng et al., 1985;
Bretherton-Watt, 1990), and parallels the distribution seen in the rat.
Galanin cell bodies are localized in the PVN, and GAL fibers arising from A6
catecholaminergic cells in the LC and Al catecholaminergic cells in the medulla innervate
the PVN in the rat (Melander et al., 1986c). Galanin binding sites are located in the PVN
(Melander et al., 1986a). Galanin ifljected into the PVN, but not into other hypothalamic
areas, stimulates food intake in satiated rats (Kyrkouli, et al. 1986, 1990). Galanin
administration into the PVN preferentially increases fat intake (Tempel et al., 1988) and
GAL levels can be correlated to spontaneous fat intake and increased body weight (reviewed
in Leibowitz, 1992). Sites in the ventromedial hypothalamus and lateral hypothalamus may
be involved in GAL stimulation of food intake in fasted or free-feeding rats as well (Schick
et al. 1993).
In addition to its role in control of food intake, GAL is also involved in the control 6 of GH secretion (Hulting et al., 1991). Galanin administration, either centrally or peripherally, increases plasma GH levels in the rat, presumably through stimulating GHRH
(Murakami et al., 1989). Galanin fibers innervate SS neurons in the hypothalamic periventricular nucleus of the rat hypothalamus (Liposits et al., 1993) as well, and thus may further modulate GH secretion through SS.
Luteinizing hormone-releasing hormone
Luteinizing hormone-releasing hormone (LHRH), also called gonadotropin releasing hormone, is a decapeptide originally isolated from porcine tissue (Matsuo et al., 1971).
Luteinizing hormone-releasing hormone is secreted from the brain at the median eminence and via the hypothalamo-hypophyseal portal blood causes the release of both luteinizing hormone (LJI) and follicle-stimulating hormone (FSH) from the anterior pituitary gland
(reviewed in Kalra, 1993; Jemies and Conn, 1994). The secretion of LHRH is controUed by a number of factors including circulating steroid levels and the neuropeptides GAL and
NPY (Kalra and Crowley, 1984; Rodriguez-Sierra et al., 1987; Sahu et al., 1987; Kineman et al., 1988; Ottlecz et al., 1988; Lopez and Negro-Vilar, 1990; Lopez et al., 1991; BCalra,
1993; Lopez et al., 1993; Xu et al., 1993; Sahu et al., 1994). Luteinizing hormone- releasing hormone is also involved in the regulation of reproductive behaviors such as lordosis (Moss and McCann, 1973; Pfaff, 1973).
The immunohistochemical distribution and development of the LHRH system in the
brain has been investigated in a number of species including the chick (Sullivan and
Silverman, 1993), Brazilian opossum (Schwanzel-Fukuda et al., 1988), rat (Joseph et al.,
1981; Shivers etal., 1983; Merchenthaler et al., 1984; Schwanzel-Fukuda, 1985; Silverman 7 et al., 1987; Jennes, 1989; Merchenthaler et al., 1990; Merchenthaler et al., 1991;
Merchenlhaler et al., 1993; Jennes and Conn, 1994), hamster (Jennes and Stumpf, 1980;
Buchanan etal., 1993), mouse (Gross and Baker, 1977; Schwanzel-Fukuda and Pfaff, 1989); guinea pig (Silverman, 1976; Schwanzel-Fukuda et al., 1981), sheep (Caldani et al., 1988), pig (Danchin and Dubois, 1982; Polkowska et al., 1985; Meijer et al., 1986; Kineman et al.,
1988; Kumar etal., 1991; Leshinetal., 1991; Calkaetal., 1993), macaque (Ronnekleiv and
Resko, 1990) and human (Barry, 1976). In rodents, LHRH neurons are bom early in gestation in the olfactory epithelium and migrate into the brain via the terminal nerve. These cells then disperse to locations in the septum, diagonal band, organum vasculosum of the lamina terminalis (OVLT), preoptic area and hypothalamus (Gross and Baker, 1977;
Schwanzel-Fukuda etal., 1981; Merchenthaler etal., 1984; Schwanzel-Fukuda etal., 1985;
Jennes, 1989; Schwanzel-Fukuda and Pfaff, 1989; Merchenthaler et al., 1993; Jennes and
Conn, 1994). Previous studies have shown that 50% or more of all LHRH-immunoreactive cells project to the median eminence (Silverman, 1987; Merchenthaler et al., 1990), where
LHRH can be secreted into the portal system to affect anterior pituitary secretion.
Regulation of gonadotropic hormone secretion by NPY, GAL and LHRH
As mentioned, NPY is involved in the control of gonadotropic functions. In castrated animals, NPY inhibits LH secretion, but in ovariectomized (ovx) steroid-primed animals,
NPY stimulates LH secretion (Sahu et al., 1987). Blockade of NPY with centrally administrated anti-NPY abolished the proestrus LH surge in ovx, primed rats (Wehrenberg et al., 1989), while chronic central administration of NPY inhibited the gonadotropic axis in intact rats, including suppression of ovulation (Catzeflis et al., 1993). Neuropeptide Y 8 is stimulatory to LHRH secretion (Crowley and Kalra, 1987) and is thought to be a regulator of the preovulatory LHRH surge in the female rat (reviewed in Kalra and Crowley, 1992) and modulate pituitary responsiveness to LHRH (Parker et al., 1991). Neuropeptide Y- containing axons may also contact LHRH neurons in the septum-preoptic area and exert an
influence on LHRH secretion at that level as well (Tsuruo et al., 1990)
Galanin also modulates reproductive fimction in the mammalian brain. Galanin is
stimulatory to LH secretion (Sahu et al., 1987; Sahu et al., 1994) in female rats and LHRH
secretion from in vitro preparations of ARC-ME fragments in the male rat (Lopez and
Negro-Vilar, 1990). Previous smdies showed no effect of GAL on plasma LH levels in the male rat, however, one smdy utilized porcine GAL instead of rat GAL (Ottlecz et al., 1988), and the other stody did not specify which synthetic GAL peptide was used (Melander et al.,
1987). Galanin is co-localized with LHRH in the OVLT and diagonal band of Broca in the rat (Merchenthaler et al., 1990). This co-localization is sexually dimorphic, with a higher rate of co-localization in females (65% vs. 20% in males; Merchenthaler et al., 1991).
Galanin is thought to be co-released with LHRH (Lopez et al., 1991) and potentiate LH
release from the pituitary (reviewed in BCaka, 1993). The roles that GAL and NPY play in relation to LHRH in the preovulatory LH surge in the female rat have been studied extensively, but are yet unclear. The role of these peptides in the control of gonadotropic
hormone secretion in the male have yet to be elucidated.
Neuropeptide Y is also involved in the regulation of pubertal onset. Single injections
of NPY can cause precocial puberty in the rat (Minami et al., 1992) and chick (Fraley and
Kuenzel, 1993), whereas prolonged ICV infusion of NPY delays pubertal onset (Gruaz et 9 al., 1993). Neuropeptide Y levels increased until puberty in the female rat (Sutton et al.,
1988). Galanin content in LHRH neurons increases at puberty as well (Rossmanith, et al.,
1994). As mentioned previously, NPY axons innervate LHRH neurons in the rat (Tsuruo,
1990) suggesting that the interaction between these three peptides may play a role in pubertal onset, along with the regulation of gonadotropic hormone secretion.
Dissertation oi^anization
The present dissertation consists of three papers, of which the first two have been accepted for publication in Developmental Brain Research (1995). The third paper has also been submitted to Developmental Brain Research for publication. The papers are preceded by a general introduction and followed by a composite summary and general discussion. The literature cited in the general introduction and general discussion is listed after the general discussion section. All of the experimental and research work presented in this dissertation was carried out by myself in the laboratory and under the guidance of Dr. Carol D.
Jacobson. 10
CHAPTER 1. THE PREPUBERTAL ONTOGENY OF NEUROPEPTIDE Y-UKE IMMUNOREACTIVITY IN THE MALE MEISHAN PIG BRAIN
A paper accepted for publication in Developmental Brain Research (1995)
Paul L. Pearson, Lloyd L. Anderson, and Carol D. Jacobson
SUMMARY
Neuropeptide Y (NPY) is widely distributed in the mammalian brain and is involved
in numerous fimctions including the control of feeding, growth and reproduction. Therefore,
NPY may be an important peptide to study in agricultural species. This study describes the
immunohistochemical localization of NPY throughout prepubertal development in the
Meishan pig, a Chinese breed known for its superior reproductive characteristics.
Brains of animals from gestational day (g) 30 through postnatal day (pn) 50 (duration
of pregnancy averaged 114 days) were processed using a standard immimohistochemical
technique utilizing a commercially available rabbit anti-porcine NPY antibody. Neuropeptide
Y-like immunoreactivity (NPY-IR) in cell bodies and fibers is evident in many areas of the
brain at g30, including the basal telencephalon, hypothalamus, mesencephalon, pons, and
medulla. Throughout prenatal development, cell bodies containing NPY-IR generally
increase in number and distribution in the brain. During postnatal development, the number
of cell bodies displaying NPY-IR decreases. The arcuate nucleus of the hypothalamus,
shows a dramatic reduction in the number of immimoreactive cell bodies between pnl (day 11 of birth) and pn20, just before weaning. The distribution of NPY-IR in fibers becomes more widespread throughout gestational development, showing a pattern by gllO that was characteristic of postnatal ages. The intensity of NPY-IR in fibers also increases throughout gestation. Some additional increases in immunoreactivity occur postnatally, especially in the periventricular hypothalamus and the hippocampus. Other brain areas like the caudate nucleus and putamen show decreases in immunoreactivity postnatally. The distribution of
NPY-IR in cell bodies and fibers is similar to that seen in other species, including the rat, and supports the hypothesis that NPY participates in controlling feeding, growth and reproduction in the pig.
INTRODUCTION
Neuropeptide Y (NPY) is a 36 amino acid amidated peptide originally isolated from porcine brain [81]. Neuropeptide Y is a member of the pancreatic polypeptide (PP) family and shows homology to both PP and peptide YY [81]. Neuropeptide Y has been implicated in a number of biological processes, including circadian rhythm entrainment [3], somatotropic and gonadotropic axis control [16,42,43,52,64], puberty onset
[32,33,54,55,79], inhibition of sexual behavior [22,63] and control of food intake
[21,49,77]. Neuropeptide Y has a widespread distribution in the central nervous system
(CNS) of a variety of species, including the platyfish [17], ground squirrel [65], rat
[5,19,24], cat [40], pig [14], squirrel monkey [76] and himian [2,61]. The ontogeny of
NPY-containing systems has been smdied extensively in the rat [4,31,41,88]. However, developmental studies of NPY in the brain in other species include only the platyfish [17], 12 human hippocampus [50] and visual system of the cat [37,84] and pigeon [9].
In the rat, the highest concentrations of NPY are found in the hypothalamus, pituitary and pineal glands. Moderate concentrations are found in the olfactory bulb, septum, striatum, fhalanniis and pons [19]. The distribution of NPY in the pig CNS as determined by radioimmunoassay (RIA) [14], follows closely to that described in the rat. Despite being originally isolated from porcine brain tissue [81], smdies examining the immunohistochemical localization of NPY in the porcine brain have been limited to the hippocampus [39] and organum vasculosum of the lamina tenninalis [15],
Intracerebroventricular (ICV) [21,49], as well as intrahypothalamic [77,78] injection of NPY has been shown to increase food intake in the rat. Neuropeptide Y injection preferentially increases carbohydrate intake in the rat, with a slight inhibition of protein intake [reviewed in 47]. Neuropeptide Y administered by ICV injection also increases food intake in pigs [57]. Overexpression of NPY in the arcuato-paraventricular system has been linked to the hyperphagia and obesity seen in the Zucker fatty rat [11,70,73], Wistar fatty rat [1] and obese mouse [87].
The Meishan pig was brought to the United States from mainland China in 1989, and has been stodied primarily for its superior reproductive capabilities [7,30,34,89,90]. The pig undergoes gonadal differentiation at gestational day (g) 26 [62], can be sexed by inspection of the external genitalia at g36 [51] and sexual differentiation of luteinizing hormone (LH) release control is thought to occur between g30 and g74 [28]. Pigs are bom after a gestational period of approximately 114 days. Noted for precocial puberty, the male
Meishan imdergoes puberty at approximately postnatal day (pn) 75, producing viable sperm 13 as early as piil20 [35]. Due to the variety of functions that NPY has related to the control of growth and reproduction, it may be an important peptide to stody in agricultural species.
Thus, we have studied the immunohistochemical locali2ation of NPY throughout prepubertal development in the Meishan pig.
MATERIALS AND METHODS
Animals
Purebred Meishan pigs were obtained from the breeding population maintained at the
Iowa State University Animal Reproduction Farm. The animals and procedures used were in accordance with the guidelines and approval of the Iowa State University committee on animal care. Boars were allowed access only to sows in estrus and the date of breeding was designated as gO. The day of birth was designated as pnl. At least four intact male animals from at least two different litters were used at each time point in this stody (gSO, g50, g70, g90, gllO, pnl, pnlO, pn20, and pnSO). At g30, the gender of the animals could not be determined by external visual inspection, and were thus considered sexually undifferentiated.
Pigs euthanized at pn50 in this stody were weaned at pn21 and given ad libitum access to a standard com/soybean diet imtil the date of termination.
Tissue preparation
Prenatal animals were obtained by rapid electrocution and exsanguination of the sow, followed by removal of the fetuses from the gravid uterus. At g30, fetal heads were removed and post-fixed 48 hours in 4% paraformaldehyde (pf). At g50, fetuses were perfused transcardially with ice-cold 4% pf, the heads removed, the brain exposed, and 14 postfixed 48 hours in 4% pf. At g70-110, the fetuses were perfused transcardially with
0.9% NaCl followed by ice-cold 4% pf, the brains removed and postfixed 24-48 hours in
4% pf. At all postnatal ages, animak were euthanized with a lethal intraperitoneal injection of sodium pentobarbital, perfused transcardially with 0.9% NaCl followed by ice-cold 4% pf. Brains were removed, blocked, and post-fixed 24-48 hours in 4% pf.
Following post-fixation, all brains were sunk in 30% sucrose at 4°C and sectioned on a cryostat (Reichert-Jung). For g30-g70, brains were cut at 20 fim. and thaw-mounted onto poly-L-lysine coated slides, dried overnight at 35°C, and stored at 4°C until processed for immimohistochemistry. At all other ages, brains were cut at 45/xm into wells containing cryoprotectant solution [86], and stored at -20''C until processed for immunohistochemistry.
At aU ages, brains were sectioned in the coronal plane. At g30, additional heads were sectioned in the horizontal or sagittal planes. Brains of gSO and older animals were sectioned from the genu of the corpus callosum (rostral) to the spinomeduUary junction (caudal). In g90 and older animals the cerebellum was removed prior to sectioning and was not processed.
Immunohistochemistry
The protocol utilized for immunohistochemistry was a modification of that reported previously from our laboratory for the Brazilian opossum brain [26,60]. Tissue mounted on slides was washed with 50 mM potassiiun phosphate buffered NaCl (KPBS), incubated with
0.3% H2O2 to remove endogenous peroxidase activity, exposed to 5% nonfat dry powdered milk (milk) and 0.4% Triton X-100 (Sigma) as a blocking agent, then incubated in rabbit anti-porcine NPY (Incstar; 1:5000) for 20 hours at room temperature in a humidified 15 chamber. Following adequate washing, the tissue sections were incubated in goat anti-rabbit
IgG (Kiricegaard & Perry Laboratories; 2/ig/ml) in diluent (KPBS containing 5% milk and
0.25% Triton X-100) for 2 hours at room temperature, rinsed, and reacted with streptavidin- peroxidase (Kirkegaard & Perry Laboratories; 2/ig/ml) in diluent for 1 hour at room temperature. After washing, the sections were stained by reacting them with 0.04% 3,3'- diaminobenzidine solution (Sigma), 2.5% nickel sulfate (Sigma), and 0.01% H2O2, dissolved in .IM sodiimi acetate. After staining for 7-12 minutes, the reaction was terminated by r^o consecutive rinses in 0.9% NaCl. Sections were counterstained with neutral red (Fisher
Scientific) and then dehydrated in graded alcohols, cleared in xylene, coverslipped with
Permount (Fisher Scientific) mounting media, and analyzed with a light microscope.
Tissue cut into wells and stored in cryoprotectant was processed for immimohistochemistry by a free-floating tissue technique utilizing plastic weigh boats with screens, as previously described for adult opossum tissue [26]. In addition, the following modifications from the protocol above were made. Tissue sections were incubated in 1 %
H2O2 to block endogenous peroxidase activity. Incubation in primary antibody solution was conducted at 4°C on an orbital shaker for 20 hours. Incubations in secondary antibody solution and streptavidin-peroxidase were also conducted on an orbital shaker. Following staining, tissue sections were mounted onto gelatin-coated slides, air-dried overnight, then counterstained and processed as above.
Section Intervals
Due to the size difference during brain development, brains were sectioned and collected in sets. The number of sets and approximate interval of sections processed for 16 immunohistochemistry for each age is listed in Table 1. One set of sections for each brain was processed for every animal at every age. In addition, a second set was processed for two brains at g90.
Controls
Negative controls were included in each run, which consisted of omission of the primary antiserum from the diluent. No staining was observed in any negative control section (not shown). Preabsorption controls were conducted and consisted of incubation of the primaiy antiserum with 15^M porcine NPY (Peninsula) for 20 hours prior to use. All staining in a gllO brain was abolished by this procedure (not shown). Incubation of ±e primary antiserum with 5/ig/ml recombinant protein A (Calbiochem-Novabiochem
Corporation) prior to immunohistochemical staining was conducted as a proposed control for nonspecific staining of magnocellular neurons [82]. This procedure failed to block immunohistochemical staining of cell bodies in the gllO brain (not shown).
Analysis of Tissue
Sections were examined with the light microscope and brain regions containing NPY-
IR were identified. Structures in the brain during early development were compared to atlases of developing mouse brain [72] and rat brain [59]. Structures in other ages were compared to the rat atlas [58], cat atlas [12] and previous descriptions of the pig brain
[69,80]. Use of neuroanatomical terminology and abbreviations followed Paxinos et al. [59] for g30 and g50 brains and Paxinos and Watson [58] for older animals, except in cases where subnuclear groups or areas defined in the rat could not be identified from counterstained tissue of the pig brain. Nuclear groups which could not be defined by cellular 17 characteristics or by location due to absence of landmarks during development were denoted as 'presumptive' nuclei. Abbreviations of nuclear groups contained in tables and figures are contained in Table 2. The number of cell bodies displaying immunoreactivity was described as few (less than 10) or numerous (more than 10) cell bodies in a brain area or nucleus in the processed sections. Immunoreactivi^ not contained in cell bodies was subjectively classified as a low, medium or high densiQr of immunoreactivity in an area or nucleus as shown in Figure 1. Although immunoreactivity not contained in cell bodies may include nerve fibers or terminals, these cannot be readily distinguished in all cases, especially in coronal sections where fibers may be running perpendicular to the plane of section. Thus, immunoreactivity outside of cell bodies was described as 'fibers' in all cases, imless specifically noted.
RESULTS
General observations
At g30, the Meishan pig brain has differentiated to the five vesicle stage, the midbrain is large and flexed and the cerebellar swelling is detectable as in the domestic pig
[51]. At this age, the Meishan pig brain is similar in shape and complexity to that of an embryonic day (E) 14 rat brain [59]. At g50, the cortical surfaces are still smooth, but the cerebrum has enlarged considerably. Mature cortical laminae are not yet distinguishable, but the developing cortical layers are similar to that seen in the E19 rat. The overall development and shape of the g50 pig brain is very similar to that of the E19 rat [59]. At g70, the cerebral cortex is corrugated and the six cortical laminae can be distinguished. The 18 pig brain has assumed a more mature proportion by this stage in developmem [51]. By g90, the overall shape and proportion of the brain and the convolution of the cortex resembled that of the older animals in this smdy. However, brain tissue taken from g90 pigs was fragile in comparison to that of older animals. Brains from ages gll0-pn50 difrered apparently only in relative size.
Immunoreactivity for NPY in cell bodies and fibers was detected at every age studied.
The number of cell bodies displaying NPY-IR, as well as the number of brain areas containing immimoreactive cell bodies generally increased throughout prenatal development, while the nimo^ber of cell bodies and the nimiber of brain areas containing immimoreactive cell bodies decreased postnatally. The intensity of NPY-IR (in fibers) in the brain, as well as the number of brain areas containing immunoreactivity generally increased during prenatal development, with some increases in immunoreactivi^ postnatally. Several brain areas showed a decrease in immunoreactivity postnatally.
Neuropeptide Y-like immunoreactivity in cell bodies
The localization of NPY-IR detected in cell bodies is shown for all ages studied in
Table 3. The following descriptions detail changes in localization or relative numbers of immunoreactive cell bodies between ages studied. g30
Immimoreactive cell bodies were present in several areas of the developing brain at this age. Numerous labelled cells were seen in the lateral basal telencephalon (Fig. 2A), bordering the internal capsule in the forming diencephalon (Fig. 2B), the intermediate zone of the forming anterior hypothalamus (Fig. 2C), the presumptive nuclei of the lateral 19 lemniscus (Fig. 3B), the ventrolateral medulla (presumptive Al/Cl cells; Fig 3C), and the dorsal medulla in the presumptive spinal trigeminal nucleus (Fig. 3D). Few labelled cells were seen in the forming frontal and parietal cortex, the internal zone of the forming tectum, and the lateral pons in the presumptive trigeminal nucleus. gSO
Numerous immunoreactive cell bodies were present in the gSO brain, in the presumptive olfactory tobercle (Fig. 4A) and piriform cortex, induseum griseum, arcuate nucleus (Fig. 11 A), nucleus of the optic tract, inferior coUiculus, the presumptive medial cerebellar nucleus, the presumptive Al/Cl cell group in the ventrolateral medulla, the intermediate reticular formation, the dorsal motor nucleus of the vagus, and the caudal spinal trigeminal nucleus. Numerous ceil bodies were also observed in the dorsal metencephalon, just lateral and ventral to the mesencephalic trigeminal nucleus. It is presumed that this is the developing locus coeruleus (Fig. 14A), although some of these cells may also be localized to the presumptive medial parabrachial nucleus or subcoeruleus. Few labelled cell bodies were observed in the intermediate layer of the cortex, caudate nucleus, putamen, anterior hypothalamus, internal zone of the rostral median eminence, principal sensory trigeminal nucleus, and in the presumptive A5 cell group of the ventrolateral pons.
At g70, there was an increase in the number of immunoreactive cell bodies present as well as a more widespread distribution in the pig brain. A greater number of cell bodies was observed in the lateral forebrain (surrounding the olfactory mbercle; Fig. 4B), within the piriform cortex, throughout the caudate nucleus and putamen, throughout the cerebral 20 cortex (all cortical layers, primarily in layers FV, V, and VI; Fig. 6A), the inferior colliculus, A5 cell group (Fig. 5C), Al/Cl cell group, and in the intermediate reticular formation. Labelled cell bodies were seen for the first time in the dorsal lateral septum, nucleus accumbens, zona incerta, lateral and dorsolateral hypothalamus along the stria medullaris thalamus, anterior cortical amygdala, and hippocampus at g70. Labelled cells in the hippocampus were present in the polymorph layer of the dentate gyrus, stratum oriens and stratum lucidimi. In addition, a few immtmoreactive cells were seen in the hippocampal molecular layer and the pyramidal cell layer of field 3 of Ammon's cortex. No cell bodies were detect in the anterior hypothalamus at g70. A slight decrease in immunoreactive cell numbers was evident in the dorsal motor nucleus of the vagus (Fig. 15A) as compared with g50. g90
Cell bodies displaying NPY-IR were more abundant in the g90 brain than that of the g70 brain, but showed a similar localization. Cell bodies showing NPY-IR were greater in number in rhinencephalic and telencephalic areas, including the olfactory tubercle, piriform cortex, throughout the cerebral cortex, caudate nucleus, putamen, septum, and the hippocampus (stratum oriens and pyramidal cell layer). An increase in cell numbers was observed in the lateral and dorsolateral hypothalamus along the stria medullaris thalamus, zona incerta, arcuate nucleus (Fig. IIC) and nucleus of the optic tract. Cell bodies were seen for the first time at g90 in the anterior commissure, anterior amygdaloid area, basolateral amygdaloid nucleus, basomedial amygdaloid nucleus, lateral amygdaloid nucleus, and bed nucleus of the stria terminalis. Less immunoreactive cell bodies were observed in 21 the locus coeruleus at g90 (Fig. 14C). Very few immunoreactive cell bodies were observed in the A5 cell group. gllO
At gllO there is a slight increase in immunoreactive cell bodies in the bed nucleus of the stria terminalis and amygdala. A few immunoreactive cells were observed in the globus pallidus for the first time. A decrease in immunoreactive cell numbers was seen in the zona incerta and in the stratum oriens of the hippocampus. Fewer immunoreactive cell bodies were seen in the inferior colliculus and were primarily localized to the dorsal portion
(Fig. 13B). Only a few weakly immunoreactive cells were present in the A5 cell group in the metencephalon or the intermediate reticular formation of the medulla. No immunoreactive cell bodies were seen in the polymorph layer of the dentate gyrus, the nucleus of the optic tract, locus coeruleus, principal nucleus of the trigeminal nerve, or the dorsal motor nucleus of the vagus. pnl
At pnl, only a few immunoreactive cell bodies were present in the lateral hypothalamus and zona incerta. A few immunoreactive cells were present in the lateral hypothalamus along the stria medullaris thalamus and in the pyramidal cell layer of fields
1 and 2 of Ammon's cortex. Only a few weakly immunoreactive cell bodies were observed in the inferior colliculus (Fig. 13C), and no cell bodies were detected in the A5 cell group. pnlO-pnSO
In the arcuate nucleus, the number of immunoreactive cells was dramatically reduced at pnlO (Fig. 12B). Only a few immunoreactive cell bodies were detected at pn20 (Fig. 22 12C), and no cell bodies were ^parent in the pn50 arcuate (Fig. 12D). There was a decrease in the number of immunoreactive cell bodies in the olfactory tubercle, piriform cortex, nucleus accumbens, caudate nucleus, putamen, and septum at pn20. A lower density of immimoreactive cell bodies was seen postnatally in the cerebral cortex. It is not clear if this observation is due to an actual decline in the number of inununoreactive cell bodies or overall growth of the cortex. A few immunoreactive cell bodies were detected in the lateral hypothalamus along the stria medullaris thalamus at all postnatal ages. A few weakly immunoreactive cells were detected in the stratum oriens, stratum radiatum and pyramidal cell layer of the hippocampus at these postnatal ages.
Neuropeptide Y-like immunoreactivity in fibers
The localization and density of NPY-IR in fibers for all ages studied is shown in
Table 4. The following description details differences or changes in immunoreactive staining between ages studied. g30
Immunoreactive staining was seen in regions throughout the brain of the g30 embryo.
A high density of NPY-IR was seen surrounding the forming internal capsule (Fig. 2B), nuclei of the lateral lemniscus (Fig. 3A), ventral mesencephalon at the mesencephalic flexure
(Fig. 3B), ventral tegmental area, rostral pons, ventrolateral medulla (Fig. 3C), the presumptive dorsal motor nucleus of the vagus and the presumptive spinal trigeminal nucleus
(Fig. 3D). A medium density of NPY-IR was seen in the basal telencephalon (Fig. 2A), anterior hypothalamus (Fig. 2C), overlying the forming optic chiasm, lateral hypothalamus
(Fig. 2D), mammillary area, lateral lemniscus, the presumptive pontine and medullary 23 reticular formation, and in fibers parallel to the brain surface in the lateral pons, ventrolateral pons and medulla. A low density of immunoreactivity was seen in the frontal and parietal cortex, the forming tectum, and the principal nucleus of the trigeminal nerve.
Immunoreactive fibers were observed to cross the midline in the mesencephalon in coronal and horizontal sections at g30 (Fig. 3B). Immunoreactivity could not be definitively described as being present in cell bodies or fibers in all cases in the g30 brain, especially in the presumptive nuclei of the lateral lenmiscus (Fig. 3A) and in the medulla (Fig. 3D). g50
Rhinencephalon
Mediimi to high densities of NPY-IR were present in fibers around the presumptive olfactory tobercle (Fig. 4A) and piriform cortex. A medixnn density of immunoreactivity was observed in the induseum griseum.
Telencephalon
A medium density of NPY-IR was seen in the intermediate cortical layer dorsal to the corpus callosum. A low density of immunoreactive fibers were present in the intermediate cortical layer throughout the cortex, anterior commissure, fornix, caudate nucleus, putamen, medial septum, diagonal band of Broca, bed nucleus of the stria terminalis, medial amygdala, anterior cortical amygdaloid nucleus, posterior cortical amygdaloid nucleus, and the fimbria of the hippocampus.
Diencephalon
Immunoreactivity for NPY in fibers was observed in mediimi to high dexisities in the arcuate nucleus (Fig. 11 A), and the lateral hypothalamus bordering the forming optic tract. 24 A low to medium density of NPY-IR was seen in the anterior hypothalamus, periventricular hypothalamus, presumptive paraventricular hypothalamic nucleus, dorsomedial hypothalamic nucleus, lateral hypothalamus, median eminence and ventral premammillary nucleus. A low density of NPY-IR was seen in the medial preoptic area, supraoptic nucleus, ventromedial hypothalamic nucleus, anterior medial thalamus, ventromedial thalamus, paraventricular thalamic area, medial habenula, mammillary nuclei, supramammillary nucleus, and posterior hypothalamus.
Mesencephalon
A mediiun density of NPY-IR was seen in the ventral tegmental area, dorsal raphe, and diffuse immunoreactive staining in the compact substantia nigra. A low to medium density of NPY-IR was seen in the interpeduncular nucleus, deep mesencephalic nucleus, the nuclei of the lateral lemniscus, central gray, and inferior colliculus. A low density of NPY-
IR was seen in the nucleus of the optic tract.
Metencephalon
A medium to high density of NPY-IR was seen in the dorsal metencephalon in the presumptive locus coeruleus (Fig. 14A). A low to mediirai density of NPY-IR was detected in the medial parabrachial nucleus, lateral parabrachial nucleus, A5 cell group, pontine raphe nucleus, pontine reticular formation, parvocellular reticular nucleus, raphe magnus, raphe pallidus, and the primary nucleus of the trigeminal nerve.
Myelencephalon
A mediima density of NPY-IR was seen in the dorsal motor nucleus of the vagus.
A low to medium density of NPY-IR was detected in the caudal spinal trigeminal nucleus. 25 intermediate reticular formation and the presumptive Al/Cl cell group. A low density of
NPY-IR was detected in the solitary nucleus, hypoglossal nucleus, and inferior olive. g70
Rhinencephalon
An increase in immunoreactivity is evident surrounding the olfactory tubercle (Fig.
46) and in the piriform cortex at g70, as well as in the induseimi griseimi. A medium density of immunoreactivity was observed in the islands of Calleja at this age.
Telencephalon
An increase in immunoreactivity is evident in most telencephalic areas at this age.
An increasing number of immunoreactive fibers was observed in all layers of the cortex at this age. A low density of fibers was seen in layers II and HI, with a medium density of fibers in layers I, IV, V, VI, (Fig. 6A) and in the cortical white matter. Radial fibers showing immunoreactivity were observed in the cortex at g70. The caudate nucleus and putamen showed an increased density of immimoreactive fibers at g70, with highest densities in the lateral putamen at rostral levels and in the caudal extent of the caudate. A few immunoreactive fibers invaded the globus pallidus at this age. An increase in immunoreactivity in the medial and lateral septal nuclei was evident at rostral levels. An increase in immimoreactivity was evident in the amygdala, as most amygdaloid nuclei had a medium density of fibers present. Immimoreactive fibers were present for the first time
in the hippocampus at g70. A mediimi density of fibers was seen in the molecular layer continuous with layer I of the cortex. A low density of fibers was seen in the polymorph
layer of the dentate gyrus, stratum oriens, stratum lucidum and stratum radiatum of 26
Amnion's cortex, and in the £unbria.
Diencephalon
An increase in density and extent of localization of NPY-IR in fibers was observed in the diencephalon at g70. Immunoreactive fibers were observed in most hypothalamic nuclei in increasing density. Immunoreactivity for NPY was observed in the highest density in the ventral arcuate nucleus (Fig. IIB). A mediimi to high density of NPY-IR was also present in the lateral hypothalamus bordering the optic tract (Fig. 5A). A medium density of NPY-IR, many in dorsal-ventral oriented fibers, was observed in the paraventricular nucleus of the hypothalamus (Fig. IOC). In addition, an increase in immimoreactivity was observed in the paraventricular thalamic area and midline thalanuc nuclei. A low density of NPY-IR was observed in thalamic nuclei just lateral to the midline.
Mesencephalon
An increase in immunoreactivity was also observed in the mesencephalon at g70.
The ventral mesencephalon had a medium density of NPY-IR in most areas including the ventral tegmental area, interpeduncular nucleus, and diffuse immunoreactive staining in the compact substantia nigra (Fig. 5B). A few immunoreactive fibers were seen in the reticular substantia nigra.
Metencephalon
A slight increase in immunoreactivity was observed in the dorsal tegmental nucleus.
An increase in immimoreactivity along the midline in the raphe pontis was also observed.
A slight decrease in the intensity of immunoreactivity was observed in the presxmiptive locus
coeruleus (Fig. 14B). 27
Myelerxephalon
A low density of NPY-IR was observed in the hypoglossal nucleus. g90
Rhinencephcdon
An increase in inununoreactivity was seen in islands of Calleja and induseum griseum.
Telencephalon
Immunoreactive fibers were present in the entire cerebral cortex at g90. A high density of NPY-IR was seen in cortical layer I, a medium to high density in layers IV, V,
VI and cortical white matter, and a medium density in layers n and m. All amygdaloid nuclei showed a medium density of immunoreactive fibers. An increase in immunoreactive density was observed in the hippocampus at g90. A high density of immunoreactive fibers was present in the molecular layer. A light to medium density of fibers was seen in the stratum radiatum.
Diencephalon
An increase in immunoreactive fibers was evident in the thalamus at g90. Fibers displaying NPY-IR were seen in low density in a number of medial and lateral thalamic nuclei. An increased density of NPY-IR was evident in the paraventricular thalamic area.
An increase in immimoreactivity was also apparent in the organum vasculosum of the lamina terminalis and paraventricular hypothalamic nucleus at g90. Inununoreactivity in the paraventricular hypothalamic nucleus appeared primarily as varicose fibers and punctate immunoreactive staining (Fig. lOD). 28
Mesencephalon
Fibers showing NPY-IR increased slightly in density in the central gray, retrorubral field, and ventral tegmental area.
Metencephalon
A medium density of immunoreactivity was observed medial to the facial motor nucleus.
M^elencephalon
An increase in NPY-IR was observed in the intermediate reticular formation, ventrolateral to the hypoglossal nucleus. gllO
Rhinencephalon
No differences were apparent between g90 and gllO.
Telencephalon
The density of fibers in cortical layers n and HI increased at gl 10 (Fig. 6B). A light to medium density of immunoreactivity was observed in the stratum lucidum of the hippocampus. A light density of immunoreactive fibers was also observed in the pyramidal cell layer of the hippocampus at gllO (Fig. 7A).
Diencephalon
A fiirther increase in immunoreactive staining in the thalamus was observed at gllO.
A low density of fibers were present in most thalamic nuclei. A high density of immunoreactive fibers was present throughout the paraventricular thalamic area. An increase in the density of staining was observed in the periventricular hypothalamus. A slight 29 decrease in immunoreactivity was observed in the lateral hypothalamiis and zona incerta at gllO.
Mesencephalon
An increase in immunoreactive fibers was evident in the central gray. A decrease in the densi^ of immunoreactivity was observed in the compact substantia nigra and in the ventral tegmental area.
Metencephalon
The mesencephalic trigeminal nucleus had a low density of immmioreactive staining at gllO. A light density of immunoreactivity was present in the locus coeruleus. No immimoreactiviQr was detected in the principal nucleus of the trigeminal nerve or oral spinal trigeminal nucleus.
Myelencephalon
A medium density of inmnmoreactivity was seen in the medial solitary nucleus at gllO. A slight decrease in immunoreactive density was observed in the caudal spinal trigeminal nucleus. Immunoreactive fibers were present in the intermediate reticular formation which followed a diagonal orientation from the lateral reticular formation to an area ventrolateral to the hypoglossal nucleus. pnl
The distribution and density of NPY-IR in fibers of pnl brains follow closely those seen in gllO brains. A slight increase in immunoreactivity was observed in the hippocampus at pnl. A medium density of fiber immunoreactivi^ was observed in the stratum lucidtmi, stratum radiatum of field 2 of Ammon's cortex, and the polymorph layer of the dentate 30 gyrus. A light to medium density of fiber immunoreactivity was observed in the stratum radiatum in fields 1 and 3 of Ammon's cortex. A slight increase in immunoreactivity was seen in the organum vasculosum of the lamina terminalis (Fig. 8B), periventricular preoptic area, external zone of the median eminence, ventromedial hypothalamic nucleus, paraventricular thalamic area, medial parabrachial nucleus, pontine reticular formation, the dorsal motor nucleus of the vagus (Fig. 15C) and the medial solitary nucleus. Very little or no immimoreactivity was present in the compact substantia nigra at pnl. pnlO
A slight increase in immunoreactivity is seen in the nucleus accumbens, pyramidal cell layer of the hippocampus (Fig. 7B), preoptic area (Fig. lOB), dorsomedial hypothalamic nucleus (Fig. 9D), retrorubral field and central gray. A high density of inamimoreactivity in varicose fibers and punctate immimoreactivity is present in the periventricular preoptic area (Fig. lOB), whereas the medial and lateral preoptic areas contain a medium density of immunoreactive fibers. There is a slight decrease in immunoreactivity in the caudate nucleus, putamen, anterior commissure, and ventral tegmental area. No immunoreactivity is seen in the compact or reticular substantia nigra or nucleus of the optic tract at pnlO. pii20-pii50
The distribution and density of NPY-IR in brains from ages pn20 and pn50 followed that seen in pnlO brains. An increase in immunoreactive density was seen in the hippocampus at pn20, with a light to medium density of fibers in the stratum oriens and a medium density of fibers in the pyramidal cell layer (pn50; Fig. 9C). A decrease in immimoreactive density was observed in the caudate nucleus, fornix and the arcuate nucleus 31 (Figs. 12C,D) at pn20 and pn50. A slightly higher density of staining in the medial solitary nucleiis and caudal spinal trigeminal nucleus was observed at pn50. Neuropeptide Y-ER. in the cerebral cortex at pnSO had a high density in all cortical layers with a medium densiQr in the cortical white matter.
DISCUSSION
Distribution and development of NPY-IR in cell bodies in the pig brain
Neuropeptide Y immunoreactivity was present early in development (g30) in cell bodies and was seen throughout prepubertal development. Transient immunostained cell populations were seen in the arcuate nucleus (gS0-pn20), zona incerta (g90-pnl), nucleus of the optic tract (g50-g90), inferior colliculiis (g50-gll0), locus coeruleus (g50-g90), A5 cell group (g50-g90) and dorsal motor nucleus of the vagus (g30-g90) of the pig brain. Of these, most notable was the rapid decline in inmiunoreactive cell bodies in the arcuate nucleus from pnl to pn20 (Fig. 12). Previous reports in the rat indicate a similar decline in the rat arcuate nucleus diiring the early postnatal period in the absence of colchicine treatment [41,88].
Despite the difference in gestational length, the decline in cell bodies displaying NPY-ER occurs at a similar time in both species, suggesting that changes in synthesis and/or transport of NPY in arcuate neurons occur shortly after birth. One possibility is that transport of synthesized peptide is more rapid at this time. This is supported by the visualization of
NPY-ER in arcuate cell bodies following colchicine treatment in adult animals
[19,24,40,65,67,76]. The disappearance of immunoreactive cell bodies in the early postnatal arcuate nucleus may represent a fimctional maturation of the arcuato-paraventricular axis 32 involved in food intake control. An increase in inununoreactive cholecystokinin, as
measured by RIA, in the hypothalamus and medulla of the Meishan pig occiurs between pnl
and pn20 [27], in parallel with the maturation of the gastrointestinal tract [8]. Taken
together, these results suggest a rapid development of food intake systems in the early postnatal period of development. However, programmed cell death, cessation of synthesis or technical considerations cannot be ruled out as possibilities for the postnatal decline of
inununoreactive cell bodies seen in the arcuate nucleus (Figs. 11, 12). It is possible, although unlikely, that the arcuate nucleus is not a major source of NPY in the postnatal
Meishan pig brain. However, a similar decline in inununoreactive cell bodies with
increasing age has been reported for other peptides as well [74,75]. Further smdy will be required to identify the explanation for this phenomenon.
Immimoreactive staining in cell bodies in the dorsal metencephalon at ages g50-g90
is presumed to be present in the cells of the developing locus coeruleus (Fig. 14). However, no rq)orts describing the porcine locus coeruleus were available for comparison with this study. The presence of immunoreactive cell bodies in the locus coeruleus is substantiated in the rat [19,24,31,38,88] but is absent in the adult squirrel monkey [76]. Neuropeptide
Y-containing neurons of the locus coeruleus have been shown to project to the spinal cord, cerebral cortex, and particularly the hypothalamus [38]. As in the arcuate nucleus, increased transport of peptide may be responsible for the disappearance of cell bodies containing NPY-
IR in the locus coeruleus and may correlate with increased NPY-IR observed in the hypothalamus in late gestation.
The use of antibodies generated in rabbit has been implicated in non-specific staining 33 of magnocellular neurons in pig tissue [53,85], presunaably by Fc stem-binding [82].
However, very little cytoplasmic staining was observed in either the supraoptic or paraventricular hypothalamic nuclei with the antibody used in this smdy. In addition, incubation of the primary antisera with protein A prior to use on these tissues as prescribed by Van der Beek et al. [82]., apparently caused non-specific staining of blood vessel endothelium (not shown). This unfavorable result led us to preclude the general use of protein A in this study. However, no apparent differences in staining of cell bodies were detected in gllO tissue stained with antiserum incubated with or without protein A (not shown).
Distribution and development of NPY-IR in fibers in the pig brain
The distribution and relative densities of NPY-IR in the pig brain correlate well with the distribution of NPY in the pig as described previously by RIA [14], as immimoreactive staining in the pn50 pig brain is most dense in the telencephalon (cortex and amygdala) and hypothalamus. Immimoreactivity for NPY in the hippocampus of the pig followed closely that seen in a previous study [39], with the highest density of immunoreactivity being present in the molecular layer.
Neuropeptide Y-IR was widespread in the early (g30) pig brain and was localized to several areas containing immunoreactivity in older animals, including the basal telencephalon
(Fig. 2A), hypothalamus (Fig. 2B,C), and medulla (Fig. 3C,D). The distribution of NPY-IR in fibers became more widespread throughout prenatal development, showing a characteristic
'mature' pattern by gllO. Also, the intensity of immunoreactivity in several areas increased dramatically at g50, g70 and g90. Of particular note is the dranwtic increase in 34 immunoreactive staining in the paraventricular hypothalamic nucleus between g70 and gSK)
(Fig. 10C,D). At gllO and postnatal ages any increases in immunoreactivity are less pronounced and limited primarily to increases in pimctate staining. The substantia nigra pars compacta, and ventral tegmental area showed decreases in immunoreactivity at g90 and gllO. Other braia areas such as the caudate nucleus, putamen, medial preoptic area, lateral hypothalamus, arcuate nucleus (Figs. 11,12) and zona incerta showed mild decreases in inununoreactive density in late gestation and/or early postnatal development. Whether these changes are due to changes in innervation, peptide content, or a 'dilution' of
immunoreactivity due to brain growth is not clear at the present time. A fiuther increase in
immimoreactive density is apparent by pnlO in the medial preoptic area (Fig. lOB). It is unknown what functional significance this increase may have, however, an increase in NPY concentration in the medial preoptic area was reported in the female rat between birth and
the time of puberty [79].
Comparison of NPY-IR in pig development with other species
The distribution of NPY-IR in cell bodies in the pig brain is very similar to that seen
in other species [19,24,40,41,65,67,76,88]. Differences in the anatomical localization and
number of immunoreactive cell bodies in the pig brain seen in this smdy may be due to the
use of colchicine in other studies [19,24,41,65,67,76], as the animals used in this study were
not treated with colchicine. However, species differences cannot be ruled out.
Iromunoreactive cell bodies in postnatal ages in this study were primarily limited to the
rhinencephalon, telencephalon and metencephalon. Comparison with a report of NPY-IR in
non-colchicine treated postnatal rats [88] reveals a similar distribution of cell bodies with the 35 following few exceptions. No immunoreactive cell bodies are seen in the central gray, locus coeruleus (Fig. 14D), or dorsal tegmentum in early postnatal pigs. Unlike the postnatal rat, inmiimoreactive cell bodies were observed in the dorsolateral hypothalamus along the stria meduUaris fhalamns and caudal spinal trigeminal nucleus. However, immtmoreactive cell bodies were reported in the adult colchicine-treated rat in the caudal spinal trigeminal nucleus
[19,24].
Neuropeptide Y-IR in the pig brain is similar in distribution and density to that seen in other species [19,24,40,41,67,75,76,88]. However, some differences exist. Very sparse
NPY-IR was localized to the pig suprachiasmatic nucleus, as opposed to a very dense localization in the adult golden hamster [67] and groimd squirrel [75], which may reflect differences in circadian rhythm fimction. In the prenatal pig, the compact substantia nigra had a medium density of immimoreactivity (Fig. 6A). There is no previous report of a similar localization in either the developing [88] or adult rat [19,24,88], or the adult squirrel monkey [76]. This may indicate a species difference in the development of this cell group.
To our knowledge, this is the first report of the development of NPY expression in the brain of a mammalian species with a prolonged gestational period. The overall development of the brain at g30 correlates well with the E14 rat [59], as does the general developmental distribution of NPY-IR [88]. However, the pig brain shows more inmnmoreactivity in the developing forebrain, especially in the basal telencephalon (Fig. 2A), surrounding the forming internal capsule (Fig. 2B), and in the forming hypothalamus (Fig.
2C,D). Further smdy will be necessary to determine the date of first detection of NPY-IR in the pig brain, as no ages earlier to g30 were investigated in this smdy. The overall 36 development of NPY-IR in the pig and rat brain [88] is similar. However, the timing of developmental changes in NPY-IR in the cerebral cortex differs in relation to the percentage of the gestational period completed. The characteristic widespread distribution of immunoreactive fibers and cell bodies throughout the cortex was observed at g70 in the pig brain (at approximately 60% of the gestational period) as opposed to E20 in the rat brain (at approximately 90% of the gestational period) [88]. The general pattern of immunoreactive cells and fibers being localized first in the ventral portion of the cortex is consistent between the two species, although the rostral to caudal development of NPY in the rat cortex [88] is not as evident in the pig, perhaps due to the time points selected for this smdy. The developmental pattern of NPY expression in the cortex may reflect the need for a more mature state of cortical development at birth in a precocial species such as the pig when compared to the rat whose young are relatively immature in sensory and motor function at birth.
Neuropeptide Y-IR in the pig in relation to other peptides
Neuropeptide Y-IR in the periventricular hypothalamus overlaps the localization of somatostatin cell bodies seen in the pig [48], and supports the potential role of NPY in modulating somatostatin release in the hypothalamus of the pig. Further work will be required to determine if NPY-IR is localized in synapses on somatostatin cells in pigs, however a previous report in the rat supports this possibility [36]. Somatostatin and NPY have also been co-localized in cell bodies in the cerebral cortex and rostral arcuate nucleus in the rat [18], but this has not been stodied in the pig brain. The distribution of growth hormone-releasing hormone cells in the pig brain previously reported [48] correlates with 37
NPY-IR in cell bodies in the arcuate nucleus in this study. It is not known whether these peptides are co-localized as in the rat [20].
The oiganum vasculosum of the lamina terminalis contains luteinizing hormone- releasing hormone (LHRH) cell bodies in the pig [15,23,44] and NPY-IR was seen in the organum vasculosum of the lamina terminalis in this smdy (Fig. 8) as in the prepubertal female pig [15], A previous study showed LHRH fibers in the pig median eminence at g60
[23]. Neuropeptide Y-IR was detected in the median eminence in this smdy as early as g50.
Although breed differences in development of these peptides have not been smdied, these results suggest that NPY is not only already present at the time that LHRH fibers reach the median eminence, but also prior to the differentiation of capillary loops in the median eminence which occurs by g70 in the pig [23]. These results ftuther support the role of
NPY in modulating LHRH secretion in the pig. The increase in NPY-IR in the median eminence during prenatal development correlates with the maturation of hypothalamic control of LH and follicle stimulating hormone secretion from the pituitary in the pig [13].
Immunoreactivity for neurophysin, a cleavage product of oxytocin and vasopressin synthesis [6], is localized in cell bodies of the pig hypothalamus at g60 in the supraoptic nucleus and at g87 in the paraventricular nucleus [25]. The distribution of NPY-IR seen in this study overlaps that for neurophysin inmnmoreactive cell bodies as well as vasopressin- and oxytocin-immunoreactive cell bodies [83] of the paraventriciilar nucleus. The dramatic increase in immunoreactivity in the paraventricular nucleus between g70 and g90 (Fig.
10C,D) suggests that NPY may have effects on the development and/or fimction of these cells. Neuropeptide Y injection into the paraventricular nucleus is known to increase 38 vasopressin secretion in the rat [45] and oxytocin secretion in the lactating rat [56].
The inununohistochemical distribution of phenylethanolamine N-methyltransferase
(PNMT) inununoreactivity in the ventrolateral medulla of the pig brain [66] is similar to the distribution of NPY-IR in cell bodies seen in this study. The possible existence of NPY in catecholamine-containing neurons of the pig has yet to be studied, but is supported by these results. Neiuropeptide Y is present in both epinephrine- and norepinephrine-containing neurons in the rat brainstem [29,71] that project to the paraventricular nucleus of the hypothalamus [71]. This NPYergic pathway may represent a major food intake control pathway in the mammalian brain [reviewed in 46,68].
Conclusions
In conclusion, the distribution and density of NPY-IR during development of the
Meishan pig brain is similar to that shown in the rat and other species. The early appearance of NPY in the pig brain supports a yet undefined developmental role for this peptide. The localization of NPY-IR in hypothalantiic structures that have been shown to influence feeding, growth and reproduction in several mammalian species supports the hypothesis that NPY is involved in controlling these parameters in the pig. As Meishan pigs show precocial puberty
[10,35] and NPY has been implicated in the onset of puberty [32,33,54,55,79], further smdy into the peripubertal and post-pubertal localization of NPY-IR in the Meishan is warranted.
Direct comparison of NPY localization or concentrations with domestic breeds attaining puberty at later ages may yield insight into the explanation for this phenomenon. 39 ACKNOWLEDGMENTS
The authors wish to thank Mahlon Shell of the ISU Animal Reproduction Farm,
Tricia Heine, Sang-Joon Cho, Jack J. Swanson, Lynne R. Ross and Javed Iqbal for their technical assistance. This work was supported in part by USDA Formula Fimds (C.D.J.) and an MCDB Student Research Fellowship (P.L.P.).
LITERATURE CITED
[1] Abe, M., Saito, M., Ikeda H. and Shimazu, T., Increased neuropeptide Y content
in the arcuato-paraventricular hypothalamic neuronal system in both insulin-dependent
and non-insulin-dependent diabetic rats. Brain Res., 539 (1991) 223-227.
[2] Adrian, T.E., Allen, J.M., Bloom, S.R., Ghatei, M.A., Rossor, M.N., Crow, T.J.,
Tatemoto, K. and Polak, J.M., Neuropeptide Y distribution in human brain. Nature,
306 (1983) 584-586.
[3] Albers, H.E. and Ferris, C.F., Neuropeptide Y: Role in light-dark entrainment of
hamster circadian rhythms, Neurosci. Lett., 50 (1984) 163-168.
[4] Allen, J.M., McGregor, G.P., Woodhams, P.L., Polak, J.M. and Bloom, S.R.,
Ontogeny of a novel peptide, neuropeptide Y (NPY) in rat brain. Brain Res., 303
(1984) 197-200.
[5] Allen, Y.S., Adrian, T.E., Allen, J.M., Tatemoto, K., Crow, T.J., Bloom, S.R. and
Polak, J.M., Neuropeptide Y distribution in rat brain. Science, 221 (1983) 877-879. 40 [6] Alstein, M. and Gainer, H., Differential biosynthesis and post translational processing
of vasopressin and oxytocin in rat brain during embryonic and postnatal development,
/. NeuroscL, 8 (1988) 3967-3977.
[7] Anderson, L.H., Christenson, L.K., Christenson, R.K. and Ford, S.P., Investigations
into the control of litter size in swine: II. Comparisons of morphological and
fimctional embryonic diversity between Chinese and American breeds, J. Anim. Sci.,
71 (1993) 1566-1571.
[8] Argenzio, R.A. Digestion, absorption, and metabolism. In: Swenson, M.J. (Ed.),
Duke's Physiology of Domestic Animals, Cornell University Press, Ithaca, New York,
1984, pp.262-277.
[9] Bagnoli, P., Fontanesi, G., Alesci, R. and Erichsen J.T., Distribution of
neuropeptide Y, substance P, and choline ace^letransferase in the developing visual
system of the pigeon and effects of unilateral retina removal, J. Comp. Neurol. ,318
(1992) 392-414.
[10] Bazer, F. W., Thatcher, W. W., Martinat-Botte, F. and Terqui, M., Sexual maturation
and morphological development of the reproductive tract in Large White and prolific
Chinese Meishan pigs, J. Reprod. Fen., 83 (1988) 723-728.
[11] Beck, B., Burlet, A., Bazin, R., Nicolas, J.-P. and Burlet, C., Elevated neuropeptide
Y in the arcuate nucleus of young obese Zucker rats may contribute to the
development of their overeating, J. Nutr., 123 (1993) 1168-1172. [12] Bennan, A.L. and E.G. Jones. The Thalamus and Basal Telencephalon of the Cat:
A CytoarcJntectonic Atlas with Stereotaxic Coordinates. Univ. of Wisconsin Press,
Madison, Wisconsin, 1982.
[13] Brubn, T., Parvizi, N. and Ellendorf, F., Ontogeny of hypothalamus-pituitary
fimction in the fetal pig: Gonadotropin release in response to electrical and
electrochemical stimulation of the hypothalamus, Endocrinology, 112 (1983) 639-644.
[14] Busch-Sorenson, M., Sheikh, S.P., O'Hare, M., Tortora, O., Schwartz, T.W. and
Gammeltoft, S., Regional distribution of neuropeptide Y and its receptor in the
porcine central nervous system, J. Neurochem., 52 (1989) 1545-1552.
[15] Calka, J., Majewski, M., Kaleczyc, J. and Lakomy, M., InMnunocytochemical
demonstration of neuropeptide Y and luteinizing hormone-releasing hormone-
immunoreactive structures in the organum vasculosum laminae tenninalis of juvenile
gilts, Neurosci. Lett., 156 (1993) 21-24.
[16] Catzeflis, C., Pierroz, D.D., Rohner-Jeanrenaud, F., Rivier, J.E., Sizonenko, P.C.
and Aubert, M.L., Neuropeptide Y administered chronically into the lateral ventricle
profoundly inhibits both the gonadotropic and the somatotropic axis in intact adult
female rats. Endocrinology, 132 (1993) 224-234.
[17] Cepriano, L.M. and Schreibman, M.P., The distribution of neuropeptide Y and
dynorphin immunoreactivity in the brain and pitoitary gland of the platyfish,
Xiphophoms maculatus, from birth to sexual maturity. Cell Tissue Res., 271 (1993)
87-92. [18] Chronwall, B.M., Chase, T.N. and O'Donohue, T.L., Coexistence of neuropeptide
Y and somatostatin in rat and human cortical and rat hypothalamic neurons, Neurosci.
Lett., 52 (1984) 213-217.
[19] Chronwall, B.M., DiMaggio, D.A., Massari, V.J., Pickel, V.M., Ruggiero, D.A.
and O'Donohue, T.L., The anatomy of neuropeptide-Y-containing neurons in rat
brain, Neuroscience, 15 (1985) 1159-1181.
[20] Ciofi, P., Croix, D. and Tramu, G., Coexistence of hGHRF and NPY
immunoreactivities in neurons of the arcuate nucleus of the rat, Neuroendocrinology,
45 (1987) 425^28.
[21] Clark, J.T., Kalra, P.S., Crowley, W.R. and Kaira, S.P., Neuropeptide Y and
human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology, 115
(1984) 427-429.
[22] Clark, J.T., Kalra, P.S. and Kalra, S.P., Neuropeptide Y stimulates feeding but
inhibits sexual behavior in rats. Endocrinology, 117 (1985) 2435-2442.
[23] Danchin, E. and Dubois, M.P., Immimocytological study of the chronology of
pituitary cytogenesis in domestic pig (Sus scrofa) with special reference to the
fimctioning of the hypothalamo-pitoitary-gonadal axis, Reprod. Nutr. Develop., 22
(1982) 135-151.
[24] De Quidt, M.E. and Emson, P.C., Distribution of neuropeptide Y-like
immimoreactivity in the rat central nervous system-II. Inmiunohistochemical analysis,
Neuroscience, 18 (1986) 545-618. [25] Ellis, H.K. and Watkins, W.B., Ontogeny of the pig hypothalamic neurosecretory
system with particular reference to the distribution of neurophysin. Cell Tissue Res.,
164 (1975) 543-557.
[26] Ebnquist, J.K., Fox, C.A., Ross, L.R. and Jacobson, C.D., Galanin-like
immunoreactivity in the adult and developing Brazilian opossum brain. Dev. Brain
Res., 67 (1992) 161-179.
[27] Elmquist, J.K., Ross, L.R., Hsu, W., Rothschild, M.F. and Jacobson, C.D.,
Cholecystokinin like immunoreactivity in the brains of young Meishan and Duroc
pigs, /. Anim. Breed. Genet., 110 (1993) 473-479.
[28] Elsaesser, F. and Parvizi, N., Estrogen feedback in the pig: Sexual differentiation and
the effect of prenatal testosterone treatment, Biol. Reprod., 20 (1979) 1187-1193.
[29] Everitt, B.J., Hokfelt, T., Terenius, L., Tatemoto, K., Mutt, V. and Goldstein, M.,
Differential co-existence of neuropeptide Y (NPY)-like immunoreactivity with
catecholamines in the central nervous system of the rat, Neuroscience, 11 (1984) 443-
462.
[30] Ford, S.P. and Yoimgs, C.R. Early embryonic development in prolific Meishan pigs,
J. Reprod. Fertil. SuppL, 48 (1993) 271-278.
[31] Foster, G.A., Schultzberg, M. and Goldstein, M., Differential and independent
manifestation within co-containing neurones of neuropeptide Y and tyrosine
hydroxylase during ontogeny of the rat central nervous system, Neurochem. Int., 6
(1984) 761-771. [32] Fraley, G.S. and Kuenzel, W.J. Precocious puberty in chicks (Gallus domesticus)
induced by central injections of neuropeptide Y, Life Sci., 52 (1993) 1649-1656.
[33] Gruaz, N.M., Pierroz, D.D., Rohner-Jeanrenaud, F., Sizonenko, P.C. and Aubert,
M.L., Evidence that neuropeptide Y could represent a neuroendocrine inhibitor of
sexual maturation in unfavorable metabolic conditions in the rat. Endocrinology, 133
(1993) 1891-1894.
[34] Haley, C.S. and Lee, G.J., Genetic basis of prolificacy in Meishan pigs, J. Reprod.
Fertil. Suppl, 48 (1993) 247-259.
[35] Harayama, H., Nanjo, I., Kanda, S. and Kato, S., Testicular development in Chinese
Meishan boars, Theriogenology, 36 (1991) 637-643.
[36] Hisano, S., Tsuruo, Y., Kagotani, Y., Daikolai, S. and Chihara, K.,
Immunohistochemical evidence for synaptic connections between neuropeptide Y-
containing axons and periventricular somatostatin neurons in the anterior
hypothalamus in rats. Brain Res., 520 (1990) 170-177.
[37] Hogan, D. and Bennan, N.E., The development of neuropeptide Y inraiunoreactive
neurons in cat visual cortical areas. Dev. Brain Res., 67 (1992) 343-69.
[38] Holets, V.R., Hokfelt, T., Rokaeus, A., Terenius, L. and Goldstein, M., Locus
coeruleus neurons in the rat containing neuropeptide Y, tyrosine hydroxylase or
galanin and their efferent projections to the spinal cord, cerebral cortex and
hypothalamus, Neuroscience, 24 (1988) 893-906. [39] Holm, I.E., Geneser, F.A. and Zimmer, J., Somatostatin- and neuropeptide Y-like
immimoreactivity in the dentate area, hippocampus, and subiculum of the domestic
pig, J. Comp. Neurol, 322 (1992) 390-408.
[40] Hu, H., Rao, J.K., Prasad, C. and Jayaraman, A., Localization of neuropeptide Y-
like immunoreactivity in the cat hypothalamus. Peptides, 8 (1987) 569-573.
[41] Kagotani, Y., Hashimoto, T., Tsuruo, Y., Kawano, H., Daikoku, S. and Chihara K.,
Development of the neuronal system containing neuropeptide Y in the rat
hypothalamus. Int. J. Devi. Neurosci., 7 (1989) 359-374.
[42] Kaba, S.P. and Crowley, W.R., Norepinephrine-like effects of neuropeptide Y on
LH release in the rat. Life Sci., 35 (1984) 1173-1176.
[43] Kaha, S.P. and Crowley, W.R., Neuropeptide Y: A novel neuroendocrine peptide
in the control of pituitary hormone secretion, and its relation to luteinizing hormone.
From. Neuroendocrinology, 13 (1992) 1-46.
[44] Kineman, R.D., Leshin, L.S., Crim, J.W., Rampacek, G.B. and Kraeling, R.R.,
Localization of luteinizing hormone-releasing hormone in the forebrain of the pig,
Biol. Reprod., 39 (1988) 665-672.
[45] Leibowitz, S.F., Sladek, C., Spencer, L. and Tempel, D., Neuropeptide Y,
epinephrine, and norepinephrine in the paraventricular nucleus: Stimulation of feeding
and the release of corticosterone, vasopressin and glucose. Brain Res. Bull., 21
(1988) 905-912.
[46] Leibowitz, S.F., Hypothalamic neuropeptide Y, galanin, and amines: Concepts of
coexistence in relation to feeding behavior, .4wz. N.Y. Acad. Sci, 575 (1989)221-233. 46 [47] Leibowitz, S.F., Neurochemical-neuroendocrine systems in the brain controlling
macronutrient intake and metabolism, TINS, 15 (1992) 491-497.
[48] Leshin, L.S., Barb, C.R., Kiser, T.E., Rampacek, G.B. and BCraeling, R.R., Growth
hormone-releasing hormone and somatostatin neurons within the porcine and bovine
hypothalamus, Neuroendocrinology, 59 (1994) 251-264.
[49] Levine, A.S. and Morley, J.E., Neuropeptide Y; A potent inducer of consunmiatory
behavior in rats. Peptides, 5 (1984) 1025-1029.
[50] Lotstra, F., Schif&nann, S.N. and Vanderhaeghen, J.J., Neuropeptide Y-containing
neurons in the human infant hippocampus. Brain Res., 478 (1989) 211-226.
[51] MditdblQ, The Embryonic Pig: A ChronologiccLl Account. Pitman, London,
1971.
[52] McDonald, J.K., Lumpkin, M.D., Samson, W.K. andMcCann, S.M., Neuropeptide
Y affects secretion of luteinizing hormone and growth hormone in ovariectomized
rats, Proc. Natl. Acad. Sci. USA, 82 (1985) 561-564.
[53] Meijer, J., Poot, P., Molenaar, G. and Goede, R., PAP complex: A pitfall in
immunocytochemistry of the pig hypothalamus, J. Neurosci. Methods, 17 (1986) 269-
274.
[54] Minami, S., Frautschy, S.A., Plotsky, P.M., Sutton, S.W. and Sarkar, D.P.,
Facilitatory role of neuropeptide Y on the onset of puberty: Effect of
immunoneutralization of neuropeptide Y on the release of luteinizing hormone and
luteinizing-hormone-releasing hormone, Neuroendocrinology, 52 (1990) 112-115. 47
[55] Minami, S. and Sarkar, D.P., Central administration of neuropeptide Y induces
precocious puberty in female rats, Neuroendocrinohgy, 56 (1992) 930-934.
[56] Parker, S.L. and Crowley, W.R., Central stimulation of oxytocin release in the
lactating rat: interaction of neuropeptide Y with alpha-l-adrenergic mechanisms,
Endocrinology, 132 (1993) 658-666.
[57] Parrott, R.F., Heavens, R.P. and Baldwin, B.A., Stimulation of feeding in the
satiated pig by intracerebroventricular injection of neuropeptide Y, Physiol. Behav.,
36 (1986) 523-525.
[58] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Second
Edition. Academic Press, San Diego, 1986.
[59] Paxinos, G., Ashwell, K.W.S. and Tork, I., Atlas of the Developing Rat Nervous
System, Second Edition. Academic Press, San Diego, 1994.
[60] Pearson, P.L., Ross, L.R. and Jacobson, C.D., Differential down-regulation of
estrogen receptor-like immunoreactivity by estradiol in the female Brazilian opossum
brain. Brain Res., 617 (1993) 171-175.
[61] Pelletier, G., Desy, L., Kerkerian, L. and Cote, J., Immunocytochemical localization
of neuropeptide Y (NPY) in the human hypothalamus. Cell Tissue Res., 238 (1984)
203-205.
[62] Pellinemi, L.J., Ultrastructure of the early ovary and testis in pig embryos. Am. J.
Anat., 144 (1975) 89-111. [63] Poggioli, R., Vergoni, A.-V., Manama, D., Giuliani, D. and Bertolini, A., NPY-
induced inhibition of male copulatory activity is a direct behavioral effect.
Neuropeptides J 16 (1990) 169-172.
[64] Rettori, V., Milenkovic, L., Aguila, M.C. and McCann, S.M., Physiologically
significant effect of netiropeptide Y to suppress growth hormone release by
stimulating somatostatin discharge. Endocrinology, 126 (1990) 2296-2301.
[65] Reuss, S., Hurlbut, E.G., Speh, J.C. and Moore, R.Y., Neuropeptide Y localization
in telencephalic and diencephalic structures of the ground squirrel brain. Am. J.
Anat., 188 (1990) 163-174.
[66] Ruggiero, D.A., Anwar, M. and Gootman, P.M., Presumptive adrenergic neurons
containing phenylethanolamine N-methyltransferase inouaiimoreactivity in the medulla
oblongata of neonatal swine, Brcdn Res., 583 (1992) 105-119.
[67] Sabatino, F.D., Mumane, J.M., Hofftnan, R.A. and McDonald, J.K., Distribution
of neuropeptide Y-like immunoreactivity in the hypothalamus of the adult golden
hamster, J. Comp. Neurol., 257 (1987) 93-104.
[68] Sahu, A., Kaka, S.P., Crowley, W.R. and BCalra, P.S., Evidence that NPY-
containing neurons in the brainstem project to selected hypothalamic nuclei:
Implication in feeding behavior. Brain Res., 457 (1988) 376-378.
[69] Salinas-Zeballos, M.-E., Zeballos, G.A. and Gootman, P.M., A stereotaxic atlas of
the developing swine {Sus scrofd) forebrain. In Tumbleson, M.E. (Ed.), Swine in
Biomedical Research, Volume 2, Plenum Press, New York, 1986. [70] Sanacora, G., Kershaw, M., Finkelstein, J.A. and White, J.D., Increased
hypothalamic content of preproneuropeptide Y messenger ribonucleic acid in
genetically obese Zucker rats and its regulation by food deprivation. Endocrinology,
127 (1990) 730-737.
[71] Sawchenko, P.E., Swanson, L.W., Grzanna, R., Howe, P.R.C., Bloom, S.R. and
Polak, J.M., Colocalization of neuropeptide Y immimoreactivity in brainstem
catecholaminergic neurons that project to the paraventricular nucleus of the
hypothalamus, J. Comp. Neurol, 241 (1985) 138-153.
[72] Schambra, U.B., Lauder, J.M., Silver, J. (Eds.). Atlas of the Prenatal Mouse Brain.
Academic Press, San Diego, 1992.
[73] Schwartz, M.W., Marks, J.L., Sipols, A.J., Baskin, D.G., Woods, S.C., Kahn, S.E.
and Porte Jr., D., Central insulin administration reduces neuropeptide Y mRNA
expression in the arcuate nucleus of food-deprived lean (Fa/Fa) but not obese (fa/fa)
Zucker rats. Endocrinology, 128 (1991) 2645-2647.
[74] Senba, E., Shiosaka, S., Hara, Y., Inagaki, S., Kawai, Y., Takatsuki, K., Sakanaka,
M., lida, H., Takagi, H., Minagawa, H. and Tohyama, M., Ontogeny of the leucine-
enkephalin neuron system of the rat: Immimohistochemical analysis. I. Lower
brainstem, J. Comp. Neurol., 205-(1982) 341-359.
[75] Shiosaka, S., Takatsuki, K., Sakanaka, M., Inagaki, S., Takagi, H., Senba, E.,
Kawai, Y., lida, H., Minagawa, H., Hara, Y., Matsuzaki, T. and Tohyama, M.,
Ontogeny of somatostatin-containing neuron system of the rat: Immunohistochemical
analysis. U. Forebrain and diencephalon, J. Comp. Neurol, 204 (1982) 211-224. 50
[76] Smith, Y., Parent, A., Kerkerian, L. and Pelletier, G., Distribution of neuropeptide
Y immimoreactivity in the basal forebrain and upper brainstem of the squirrel
monkey (Saimiri sciureus), J. Comp. Neurol., 236 (1985) 71-89.
[77] Stanley, B.G. and Leibowitz, S.F., Neuropeptide Y: Stimulation of feeding and
drinking by injection into the paraventricular nucleus, Life Sci., 35 (1984) 2635-2642.
[78] Stanley, B.G., Magdalin, W., Seirafi, A., Thomas, W.J., and Leibowitz, S.F., The
perifomical area: the major focus of (a) patchily distributed hypothalamic
neuropeptide Y-sensitive feeding system(s). Brain Res., 604 (1993) 304-317.
[79] Sutton, S.W., Mitsugi, N., Plotslg^, P.M. and Sarkar, D.K., Neuropeptide Y (NPY):
A possible role in the initiation of puberty. Endocrinology, 123 (1988) 2152-2154.
[80] Szetyn, S., Galert, D., Dynowski, J. and Hoczyk, W., The stereotaxic configuration
of hypothalamus nerve centres in the pig. Anaz. Anz. 147 (1980) 12-32.
[81] Tatemoto, K., Carlquist, M. and Mutt, V., Neuropeptide Y-a novel brain peptide
with structural similarities to peptide YY and pancreatic polypeptide. Nature, 296
(1982) 659-660.
[82] Van der Beek, E.M., Pool, C.W., Van Eerdenburg, F.J.C.M., Sluiter, A.A., Van
der Donk, H., Van den Hurk, R. and Weigant, V.M., Fc-mediated nonspecific
staining of porcine brain with rabbit antisera in immunocytochemistry is prevented
by pre-incubation of the sera with proteins A and G, J. Histochem. Cytochem., 40
(1992) 1731-1739. [83] Van Eerdenburg, FJ.C.M, Swaab, D.F. and Van Leeuwen, F.W., Distribution of
vasopressin and oxytocin cells and fibres in the hypothalamus of the domestic pig {Sus
scrofa), J. Comp. Neurol, 318 (1992) 138-146.
[84] Wahle, P. and Meyer, G., Morphology and quantitative changes of transient NPY-ir
neuronal populations during early postnatal development of the cat visual cortex, J.
Comp. Neurol, 261 (1987) 165-192.
[85] Watkins, W.B., Precautions in the application of immxmohistochemical techniques to
tissues of the pig hypothalamo-neurohypophysial system, J. Histochem. Cytochem.,
25 (1977) 61-68.
[86] Watson, Jr., R.E., Wiegand, S.J., Clough, R.W. and Hoffinan, G.E., Use of
cryoprotectant to maintain long-term peptide immunoreactiviQ^ and tissue morphology,
Peptides, 7 (1986) 155-159.
[87] Wilding, J.P.H., Gilbey, S.G., Bailey, C.J., Batt, R.A.L., Williams, G., Ghatei,
M.A. and Bloom, S.R., Increased neuropeptide-Y messenger ribonucleic acid
(mRNA) and decreased neurotensin mRNA in the hypothalamus of the obese (ob/ob)
mouse. Endocrinology, 132 (1993) 1939-1944.
[88] Woodhams, P.L., Allen, Y.S., McGovem, J., Allen, J.M., Bloom, S.R., Balazs, R.
and Polak, J.M., Immunohistochemical analysis of the early ontogeny of the
neuropeptide Y system in rat brain, Neuroscience, 15 (1985) 173-202. 52 [89] Youngs, C.R., Ford, S.P., McGinnes, L.K. and Anderson, L.H., Investigations into
the control of litter size in swine: 1. Comparative studies on in vitro development of
Meishan and Yorkshire preimplantation embryos, J. Anim. Sci., 71 (1993) 1561-
1565.
[90] Youngs, C.R., Christenson, L.K. and Ford, S.P., Investigations into the control of
litter size in swiae: HI. A reciprocal embryo transfer study of early conceptus
development, J. Anim. Sci., 72 (1994) 725-731. 53
TABLE 1 Number of sets of coronal sections of porcine brain collected and approximate interval of sections processed for immunohistochemistiy
AGE g30 m m m ellO-pnSO sets collected 2 4 6 12 18 interval (/tm) 20 60 100 495 765 54
TABLE 2 Abbreviations used in tables
12 hypoglossal nucleus MeA medial amygdaloid nucleus A1 A1 noradrenaline cells Med medial cerebellar nucleus AS AS noradrenaline cells MHb medial habenula AA anterior amygdaloid area ml molecular layer of the hippocampus ac anterior commissure MM mammillary area ACo anterior cortical amygdala MnR median raphe nucleus AH anterior hypothalamic area MPA medial preoptic area AM anteromedial thalamic nucleus MPB medial parabrachial nucleus ARC arcuate nucleus MS medial septum AV anteroventral thalamic nucleus nAc accumbens nucleus BL basolateral amygdaloid nucleus nC caudate nucleus BM basomedial amygdaloid nucleus OT nucleus of the optic tract BST bed nucleus, stria terminalis OVLT organum vasculosum of the lamina BTel basal telencephalon terminalis CI CI adrenaline cells PCo posterior cortical amygdaloid nucleus C2 C2 adrenaline cells pel pyramidal cell layer, hippocampus cc corpus callosum PCRt parvocellular reticular formation CeA central amygdaloid nucleus Pe periventricular hypothalamus CG central gray PH posterior hypothalamus Cli caudal linear raphe nucleus Pir piriform cortex CM centromedial thdamic nucleus PMV ventral premammillary nucleus Ctx cerebral cortex PnR pontine reticular formation Cu cuneate nucleus PoDG polymorph layer, dentate gyrus DBB diagonal band of Broca PrS principal trigeminal nucleus DM dorsomedial hypothalamic nucleus PrC precommissural nucleus DMX dorsal motor nucleus of the vagus Put putamen DpMe deep mesencephalic nucleus PV paraventricular thalamic nucleus DR doi^ raphe nucleus PVN paraventricular hypothalamic nucleus Dig dorsal tegmental nucleus PvPOA periventricular preoptic area f fornix Re reuniens thalamic nucleus fi fimbria Rh rhomboid thalamic nucleus GP globus pallidus RLi linear raphe nucleus Gr gracile nucleus RMg raphe magnus nucleus lAM interanteromedial thalamic nucleus RPa raphe pallidus nucleus IC inferior colliculus RPn raphe pontis nucleus ICap internal capsule RRF retrorubral field ICj islands of Calleja Rt reticular thalamic nucleus IF interfasicular nucleus SC superior colliculus IG induseum griseum SCN suprachiasmatic nucleus 55
10 inferior olive si stratum lucidum, hippocampus IPN interpeduncular nucleus sm stria medullaris thalamus IRt intermediate reticular formation Sol solitary nucleus LA lateral amygdaloid nucleus SON supraoptic nucleus LC locus coeruleus SNc compact substantia nigra LD laterodorsal tbalamic nucleus SNR reticular substantia nigra LH lateral hypothalamic area so stratum oriens, hippocampus 11 lateral lemniscus Sp5c spinal trigeminal nucleus, caudal LL nuclei of the lateral lemniscus Sp50 spinal trigeminal nucleus, oral UPB lateral parabrachial nucleus sr stratum radiatum, hippocampus LPO lateral preoptic area SuM supramammillary nucleus LRt lateral reticular formation tu olfactory tubercle LS lateral septum VL ventrolateral thalamic nucleus LSD lateral septum, dorsal VM ventromedial thalamic nucleus MD mediodorsal thalamic nucleus VMH ventromedial hypothalamic nucleiis MdD dorsal medullary reticular field VPL ventral posterolateral thalamic nucl. ME median eminence VTA ventral tegmental area Me5 sensory trigeminal nucleus 21 zomi incerta 56
TABLES Distribution of neuropeptide Y-like immunoreactivity in the developing porcine brain: Cell Bodies
AREA g30 g50 m g9Q gllO pnl pnlO pn20 pnSO
EMnencephalon IG^ NA^ + + ++ + + + + + + + + + + + + Pir NA + + ++ + + + + + + + + + + + + Tu NA + + ++ + + + + + + + + + + + +
Telencephalon
AA NA - - + + + + + + + + + + ac NA - - + + + + + + ACo NA - + + + + + + + BL NA - - + + + + + 4- + +
BM NA - - + + + + + + + + + + + +
BST NA - - + + + + + + + + + + + + BTel + + NA NA NA NA NA NA NA NA Ctx + + + + + + + + -f- + + + + + + + +
GP NA - - - + + + + + Hippocampus
ml NA - + pel NA - + + + + + + + PoDG NA - + + + + so NA - + + + + + + + + + -1-
sr NA - - - - - + + -
LA NA - - + + + + + + + + + +
LSD NA - + + + + + + + + 4- + + + +
MeA NA - - + + + + + MS NA - - + + + + + + + + + + + + nAc NA - + + + + + + + + + + + + + + nC NA + + + + + + + + + + + + + + + Put NA + + + + + + + + + + + + + + +
Diencephalon
AH + + + -
ARC NA + + + + + + + + + + + + -
ICap + + ------LH + + + + + + + + + + + + + ME NA + + + + + + + + + + + + + sm NA - + + + + + + + + + 57
ZL NA ++ ++ +
Mesencephalon IC NA ++++++++ - LL ++ OT NA ++ ++ ++ SNc NA - + + Tecttim + NA NA NA NA NA NA NA NA
Metencephalon
AS NA + + + -1- + + + - - - - LC NA + + + + + + - - - - - Med NA + -1- + + NA NA NA NA NA NA Pr5 + + + +
Myelencephalon Al/Cl + + + + + + + + + •f + + + + + + +
DMX + + + + -I- + - - - - - mt + + + + + + -I- + + + + + Sp5C + + + + + -1- + + + + + + + + + + + +
^-Abbreviations of brain areas can be found in table 2. ^-Symbols used in this table refer to the following: no cell bodies detected in area or nucleus studied; +, less than 10 cell bodies detected in area or nucleiis; ++, more than 10 cell bodies detected in area or nucleus; NA, area not applicable or not studied. Due to immaturity of brain structures, many brain areas cannot be defined at g30 and are thus listed as NA. 58
TABLE 4 Distribution of neuropeptide Y-like immunoreactivity in developing porcine brain: Fibers
AREA g30 g70 m gllO pnl pnlO pn20 pnSO
Rhinencephalon icy NA=^ M M M M M M M IG NA M H H H H H H H Pir NA M M H H H H H H Tu NA M H H H H H H H
Telencephalon AA NA M M M M M M M M ac NA L L L L/M L/M L L L ACo NA L M M M M M M M BL NA L M M M M M M M BM NA M M M M M M M M BST NA L L/M M M M M M M BTel M NA NA NA NA NA NA NA NA cc NA L L L L L L L L CeA NA L M M M M M M M Ctx L L UM. M/H Mm M/H M/H M/H M/H DBB NA L M M M M M M M f NA L L/M L/M LM L/M L/M L L GP NA - L L L L L L L Hippocampus fi NA L L L L L L L L ml NA - M H H H H H H pel NA - - - L L L/M M M PoDG NA - L L L M M M M si NA - L L \m M M M M so NA - L L L L L L/M L/M sr NA - L LM im M M M M LA NA L L M M M M M M LS NA L L/M L/M L/M L/M L/M L/M L/M MeA NA L M M M M M M M MS NA L M M M/H M/H M/H M/H M/H nAc NA L LM LM L/M L/M L/M L/M L/M nC NA L M/H M/H M/H M LM L/M L/M PCo NA L L/M M M M M M M Put NA L M/H M/H M M L/M UM. L/M Diencephalon AH M L/M M M M M M M M AM NA L L L L L L L L ARC NA M/H urs. M/H M/H M/H M/H M/H M/H AV NA - - L/M L/M L/M L/M L/M L/M CM NA - L L L L L L L DM NA L L/M M M M M/H M/H M/H lAM NA - L L L L L L L ICap H LD NA - - L M M M M M LH M L/M M/H M M M M M M LPO NA L L/M L/M UU L/M M M M MD NA L LM M M L L L L ME NA L/M L/M L/M L/M M M M M MHb NA L L L L L L L L MM M L L L L L L L L MPA - L M M LM L/M M M M OVLT NA L LM M M M/H M/H M/H M/H Pe NA L M M M/H M/H H H H PH - L L/M L/M L/M L/M L/M L/M L/M PMV NA L L/M M M M M M M PrC NA L L/M L L L L L L PV NA L M M/H M/H H H H H PVN NA L/M M H H H H H H PvPOA NA L M M My^ H H H H Re NA L L M M M M M M Rh NA L L L L L L L L Rt NA - L L L L L L L SCN NA - - L L L L L L sm NA L L M M M M M M SON NA L L L L L L L L SuM NA L M M M M M M M VL NA - L L L L L L L VM NA L L L L L L L L VMH NA L L/M M L/M M M M M VPL NA - - L L L L L L ZI NA L L M M M M M M
Mesencephalon CG NA L L L/M UM L/M L/M L/M L/M CLi NA - L L L L L L L DpMe NA L L L L L L L L DR NA M M M M M M M M 60
IC NA L L L L - - - - IF NA L M M L L L L L IPN NA L/M L/M L L L L L L U M LL H L ------
Me5 NA - - L L L L L L MoR NA L M M M M M M M OT NA L L L L L - - - RLi NA L L L L L L L L RRF NA L M M M M M/H M/H M/H SC NA - - - L L L L L SNc NA M M M L L - - -
SNR NA - L L L L - - - Tectum L NA NA NA NA NA NA NA NA VTA H L/M M/H M/H M/H M/H UM L/M L/M
Metencephalon A5 NA L L L L L L L L DTg NA L L/M M L L L L L LC NA M M/H M L L M M M LPB NA L/M L/M LM L/M L/M L/M UM L/M MPB NA L/M LM L/M L M M M M PCRt NA L L M M L L L L PnR M L L L L UM L/M UM L/M Pr5 L L L L - - - - - RMg NA L/M L/M L L L L L L RPa NA L/M L L L L L L L RPn NA L/M M L L L L L L Sp50 NA L L L
Myelencephalon 12 NA L L Al/Cl M L/M UU UM L/M L/M L/M L/M UM Cu NA L L L L L L L L DMX NA L L M M H H H H Gr NA L L L L L L L L 10 NA L L L L L L L L IRt M L/M L/M L/M L/M L/M UM L/M UM LRt NA L LAI UM L/M L/M UM L/M UM MdD NA L L L L L L L L Sol NA L L UM UM L/H UH L/H UH Sp5C H UM M M UM L L L UM 61 '-Abbreviations of brain areas can be found in Table 2. --Symbols used in the table refer to the following: no immunoreactivity detected; L, low density of immxmoreactivity detected; M, meditmi density of immunoreactivity detected; H, high density of immunoreactivity detected; and NA, area not applicable or not studied. Due to immaturity of brain structures, many brain areas cannot be defined at g30 and are thus listed as NA. figure 1. Examples of neuropeptide Y-like immunoreactivity in porcine brain classified as being either low, medium or high density. LOW
V y.-'
: MEDIUM i ^ # I. - -.-1 . • •
0.\ ' f
<' tjjjr HIGH figure 2. Neuropeptide Y-like immunoreactivity in the gestational day 30 pig brain. A-
Basal telencephalon, sagittal section; B-area surrounding the internal capsule, sagittal
section; C-anterior hypothalamus, coronal section; D-lateral hypothalamus, coronal
section. Magnification bars indicate 100^. Abbreviations: 3V-third ventricle, ICap-
intemal capsule. AG
AG
dBOl Figure 3. Neuropeptide Y-like immunoreactivity in the gestational day 30 pig brain. A- mesencephalon at the mesencephalic flexure, horizontal section; 6-Nuclei of the lateral lemniscus, sagittal section; C-ventrolateral medulla, sagittal section; and D-spinal trigeminal nucleus, sagittal section. Magnification bars indicate lOO^m. Abbreviations:
4V-fourth ventricle, ^-Sylvian aqueduct in A, arrowheads indicate dense counterstain in the ependyma in D.
Figure 4. Neuropeptide Y-like inununoreactiviQr surroundiug the olfactory tubercle. A- gestational day SO; B-gestational day 70; C-postnatal day SO. Magnificatioa bars indicate
200/im. Abbreviations: lo-lateral olfactory tract, Put-putamen. Put Figure 5. Neuropeptide Y-like immunoreactivity in the gestational day 70 pig brain. A-
Laieral hypothalamus; B-compact substantia nigra; C-AS noradrenaline cells in the metencephalon. Magnification bar indicates lOO^tm. All photomicrographs are at the same magnification level. Abbreviation: SNR-reticular substantia nigra. • Figure 6. Neuropeptide Y-like immunoreactiviQr in the developing parietal cerebral cortex. A-gestational day 70; B-gestational day 110, C-postnatal day 10. Magnification bar indicates lOO^tm. All photomicrographs are at the same magnification level. The
solid line at the top in each photomicrograph indicates the innennost extent of cortical
layer I. A* I Figure 7. Neuropeptide Y-like inununoreactivity in the developing hippocampus. A- gestational day 110; B-postnatal day 10; C-postnatal day 50. Magnification bar indicates lOO^on. All photomicrographs are at the same magnification level. Abbreviation: pcl- pyramidal cell layer. libl f, ','1 <
f ---V'>•-•»•-. rvt.-. • 1- 't. ». -
W - . A V I^ure 8. Neuropeptide Y-like immunoreactivi^ in the developing organum vasculosum of the laminae tenninalis. A-gestational day 90; B-postnatal day 1. Magnification bar indicates lOO/on. All photomicrographs are at the same magnification level. The solid line at the top in each photomicrograph indicates the midline of the brain. J •< Figure 9. Neuropeptide Y-like immunoreactivity in the developing pig brain. A- gestational day 110, basolateral amygdaloid nucleus; B-postnatal day 1, median eminence; C-postnatal day 10, bed nucleus of the stria terminalis, medial division; and
D-dorsomedial hypothalamic nucleus. Magnificationbars indicate lOO^im. Abbreviation:
3V-third ventricle.
Figure 10. Neuropeptide Y-like immunoreactivi^ in the developing pig brain. A- gestational day 70, medial preoptic area; B-postnatal day 10, medial preoptic area; C- gestational day 70, paraventricular hypothalamic nucleus; and D-gestational day 90, paraventricular hypothalamic nucleus. Magnification bar indicates lOO^on. All photomicrographs are at the same magnification level. The third ventricle is the space on the left in each photomicrograph.
Figure 11. Neuropeptide Y-like immunoreactivi^ ia the developing prenatal arcuate nucleus. A-gestational day 50; B-gestational day 70; C-gestational day 90; and D- gestational day 110. Magnification bar indicates lOO/un. All photomicrographs are at the same magnification level. The third ventricle is the space on the right in each photomicrograph.
Figure 12. Neuropeptide Y-like immunoreactiviQr in the developing postnatal arcuate nucleus. A-postnatal day 1; B-postnatal day 10; C-postnatal day 20; and D-postnatal day
SO. Magnification bar indicates 100/xm. All photomicrographs are at the same magnification level. The third ventricle is the space on the right in each photomicrograph.
Figure 13. Neuropeptide Y-like immunoreactivity in the developing inferior colliculus.
A-gestational day 90; B-gestational day 110; and C-postnatal day 1. Magnification bar indicates 200^. All photomicrographs are at the same magnification level. c \ Figure 14. Neuropeptide Y-iike inununoreactivity in the developing locus coeruleus. A~ gestational day 50; B-gestational day 70; C-gestational day 90; and D-postnatal day 1.
Magnification bar indicates lOOAtm. All photomicrographs are at the same magnification level. Arrowheads indicate densely counterstained and/or pigmented cell bodies of the mesencephalic trigeminal nucleus that are not immunoreactive. 0" o Figure 15. Neuropeptide Y-Iike inununoreactivity in the developing dorsal motor nucleus of the vagus. A-gestational day 70; B-gestational day 90; C-postnatal day 1; and D- postnatal day SO. MagniHcation bar indicates 100/xm. All photomicrographs are at the same magnification level. The fourth ventricle is the space at the left in each photomicrograph.
92
CHAPTER 2: THE PREPUBERTAL ONTOGENY OF GALANIN-LIKE IMMUNOREACnVITY IN THE MALE MHSHAN PIG BRAIN
A paper accepted for publication in Developmental Brain Research (1995)
Paul L. Pearson, Lloyd L. Anderson, and Carol D. Jacobson
SUMMARY
Galanin (GAL) is a neuropeptide found in the mammalian brain and is involved in numerous fimctions including the control of feeding, growth and reproduction, and therefore may be an important peptide to smdy in agricultural species. We describe the inununohistochemical localization of GAL throughout prepubertal development in the
Meishan pig, a Chinese breed known for its superior reproductive characteristics, but slow growth rate and abundant adipose tissue.
Brains of animals from gestational day (g) 30, 50, 70, 90 and 110 and postnatal day
(pn) 1, 10, 20 and 50 (duration of pregnancy averaged 114 days) were processed using a standard immimohistochemical technique utilizing a commercially available rabbit anti- porcine GAL antibody. Galanin-like immunoreactivity (GAL-IR) in cell bodies and fibers was evident in the brain at g30, primarily in the hypothalamus. Throughout prenatal development, cell bodies containing GAL-IR generally increased in number and distribution in the brain. During postnatal development, the number of cell bodies displaying GAL-IR decreased, particularly in hypothalamic areas. The distribution of GAL-IR in fibers became 93 more widespread throughout gestational development, showing a pattern by pnl that continued during later postnatal ages. The intensi^ of GAL-ER in fibers also increased throughout gestation. Some additional increases in immunoreactivity occurred postnatally, especially in the periventricular hypothalamus. The results of this smdy indicate that the distribution of GAL-IR in cell bodies and fibers in the Meishan pig brain was similar to that seen in other species, including the rat. These results support the hypothesis that GAL participates in the control of feeding, growth and reproduction in the pig.
INTRODUCTION
Galanin (GAL) is a 29 amino acid amidated peptide originally isolated from porcine
intestinal extracts by Tatemoto et al., 1983 [65]. Since its discovery, numerotis functions have been ascribed to GAL, including inhibition of insulin secretion [32], stimulation of food
intake behavior [28,29,66], and stimulating the secretion of growth hormone (GH) [46,48],
luteinizing hormone (LH) [55,56] and LH-releasing hormone (LHRH) [34]. Galanin has
been localized in both the central nervous system (CNS) and peripheral nervous system of
several species including human [18,20,27,45] and non-human primates [4,27], rat
[8,42,45,54,61,62,63], Brazilian opossum [12,13], and pig [6,8,45]. In the rat CNS, GAL
has a wide distribution, and is present in high concentration in the hypothalamus, limbic
system, nucleus of the solitary tract, locus coeruleus, and pituitary [8,45,62]. Galanin and
GAL message-associated peptide [6] immunoreactivity has been reported in the porcine CNS,
and parallels the distribution seen in the rat. However, there has been no description of the
immunohistochemical distribution of galanin in the porcine brain. The ontogeny of galanin 94 during development in the brain has been studied in fish [36], rat [61] and Brazilian opossum
[12,13], but has yet to be studied in a species with a long gestation period.
The Meishan pig was brought to the United States from mainland China in 1989, and has been studied primarily for its superior reproductive capabilities [1,11,16,21,71,72]. The
Meishan is also characterized by slow growth rate, low feed efficiency and abundant adipose tissue compared with traditional American and European breeds [23,30]. The developiug pig imdergoes gonadal differentiation at gestational day (g) 26 [S3], can be sexed by inspection of the external genitalia at g36 [38] and sexual differentiation of hypothalamic control of LH release is thought to occur between g30 and g74 [14]. The dmation of gestation is approximately 114 days. Noted for precocious puberty, the male Meishan undergoes puberty at approximately postnatal day (pn) 75, producing viable sperm as early as pnl20 [22]. American and European breeds of pigs enter puberty at about pnl25 and produce viable sperm by pnl50-180[2]. With respect to the role of GAL in the control of growth and reproduction, it may be an important peptide to study in agricultural species.
Thus, we have determined the immunohistochemical localization of GAL throughout prepubertal development in the male Meishan pig.
MATERIALS AND METHODS
Animals
Purebred Meishan pigs were obtained from the breeding population maintained at the
Iowa State University Animal Reproduction Farm. The animals and procedures used were in accordance with the guidelines and approval of the Iowa State University Committee on 95
Animal Caie. Boars were allowed access only to sows in estrus and the date of breeding was designated as gO. The day of birth was designated as pnl. Gestation averaged 114 days. At least four intact male animals from at least two different litters were used at each time point in this study (g30, g50, g70, g90, gllO, pnl, pnlO, pn20, and pn50). At g30, the gender of the animals could not be determined by external visual inspection, and were thus considered sexually undifferentiated. Pigs euthanized at pn50 in this study were weaned at pn21 and given ad libitum access to a standard com/soybean diet (18% protein) untU the date of termination.
Tissue preparation
Prenatal animals were obtained by rapid electrocution and exsanguination of the sow, followed by removal of the fetuses from the gravid uterus. At g30, fetal heads were removed and post-fixed 48 hours in 4% paraformaldehyde (pf). At g50, fetuses were perfused transcardially with ice-cold 4% pf, ±e heads removed, the brain exposed, and postfixed 48 hours in 4% pf. At g70-110, the fetuses were perfused transcardially with
0.9% NaCl followed by ice-cold 4% pf, the brains removed and postfixed 24-48 hours in
4% pf. At all postnatal ages, animals were euthanized with an intraperitoneal injection of a lethal dose of sodium pentobarbital, perfused transcardially with 0.9% NaCl followed by ice-cold 4% pf. Brains were removed, blocked, and post-fixed 24-48 hours in 4% pf.
Following post-fixation, all brains were sunk in 30% sucrose at 4°C and sectioned on a cryostat (Reichert-Jung). For g30-g70, brains were cut at 20 /im and thaw-moxmted onto poly-L-lysine coated slides, dried overnight at 35°C, and stored at 4''C imtil processed for immunohistochemistry. At all other ages, brains were cut at 45/im into wells containing 96 cryoprotectant solution [70], and stored at -20°C until processed for immunohistochemistry.
At all ages, brains were sectioned in the coronal plane. At g30, additional heads were sectioned in the horizontal or sagittal planes. Brains of gSO and older animals were sectioned from the genu of the corpus caUosum (rostral) to the spinomedullary junction (caudal). In g90 and older animals the cerebellimi was removed prior to sectioning and was not processed.
Dmnimobistochenustry
The protocol utilized for immunohistochenustry was modified from that reported previously from our laboratory for the Brazilian opossum brain [12,51] and pig brain [52].
Tissue mounted on slides was washed with 50mM potassium phosphate buffered saline
(KPBS), incubated with 0.3% H2O2 to remove endogenous peroxidase activity, exposed to
1.5% normal goat serum (NGS), 1% bovine serum albumin (BSA) and 0.4% Triton X-100
(Sigma) as a blocking agent. The tissue was then incubated in rabbit anti-porcine GAL
(Peninsula; 1:5000) in blocking serum containing 1 % NGS, 1 % BSA and 0.4% Triton X-100 for 20 hours at room temperature in a humidified chamber. Following adequate washing, the tissue sections were incubated in goat anti-rabbit IgG (Kirkegaard & Peny Laboratories;
2/ig/ml) in diluent (KPBS containing 5% nonfat powdered milk and 0.25% Triton X-100) for 2 hours at room temperature, rinsed, and reacted with streptavidin-peroxidase
(Kirkegaard & Perry Laboratories; 2|ig/ml) in diluent for 1 hour at room temperature. After washing, the sections were stained by reacting them with 0.04% 3,3'-diaminobenzidine solution (Sigma), 2.5% nickel sulfate (Sigma), and 0.01% H2O2, dissolved in O.IM sodium acetate. After staining for 7-12 minutes, the reaction was terminated by two consecutive 97 rinses in 0.9% NaCl. Sections were counterstained with neutral red (Fisher Scientific) and
then dehydrated in graded alcohols, cleared in xylene, coverslipped with Permount (Fisher
Scientific) mounting media, and analyzed with a light microscope.
Tissue cut into wells and stored in cryoprotectant was processed for
immunohistochemistry by a free-floating tissue technique utilizing plastic weigh boats with
screens, as previously described for adult opossum tissue [12]. In addition, the following
modifications from the previous protocol were made: 1) Tissue sections were incubated in
1% H2O2 to block endogenous peroxidase activity; 2) Incubation in primary antibody
solution was conducted at 4''C on an orbital shaker for 20 hours; 3) Incubations in secondary
antibody solution and streptavidin-peroxidase were also conducted on an orbital shaker; and
4) Following staining, tissue sections were mounted onto gelatin-coated slides, air-dried
overnight, then counterstained and processed as above.
Section Intervals
Due to the size differences during brain development, brains were sectioned and
collected in sets. The number of sets and approximate interval of sections processed for
iimmmohistochemistry for each age is listed in Table 1. One set of sections from each brain
was processed for every animal at every age.
Controls
Negative controls were included in each run and consisted of omission of the primary
antiserum from the diluent. No staining was observed in any negative control section (not
shown). Preabsorption controls were conducted and consisted of incubation of the primary
antiserum with ISfiM porcine GAL (Peninsula) at 4°C for 20 hours prior to use. All staining 98 in a gllO brain was abolished by this procedure (not shown), with the exception of magnocellular neurons of the paraventricular and supraoptic nuclei of the hypothalamus, as reported previously [12]. Incubation of the primary antiserum with 5;:g/ml recombinant protein A (Calbiochem-Novabiochem Corporation) prior to immunohistochemical staining was conducted as a proposed control for nonspecific staining of magnocellular neurons
[40,68,69]. This procedure blocked non-specific cytoplasmic staining of cells in the paraventricular and supraoptic nuclei of the hypothalamiis, but failed to block immunohistochemical staining of other immunoreactive cell bodies in adjacent sections in the pnlO brain (not shown).
Analysis of Tissue
Sections were examined with the light microscope and brain regions containing GAL-
IR were identified. Structures in the brain during early development were compared to atlases of developing mouse brain [58] and rat brain [50]. Other ages were compared to the rat atlas [49], cat atlas [5] and previous descriptions of the pig brain [57,64]. Use of neuroanatomical terminology and abbreviations followed Paxinos et al. [50] for g30 and g50 brains and Paxinos and Watson [49] for older animals, except in cases where subnuclear groups or areas defined in the rat could not be clearly identified from coimterstained tissue of the pig brain. Nuclear groups which could not be clearly defined by cellxilar characteristics or by location due to absence of landmarks during development were denoted as 'presumptive' nuclei. Abbreviations of nuclear groups contained in tables and figures are contained in Table 2. The number of cell bodies displaying immunoreactivity was described as few (less than 10) or nimierous (greater than 10) cell bodies in a brain area or nucleus in 99 the processed sections. Immunoreactivi^ not contained in cell bodies was subjectively classified as a low, medium or high density of inimunoreactivity in an area or nucletis as shown in Figure 1. Although immunoreactiviQr not contained in cell bodies may be contained in nerve fibers, terminals or other processes, these cannot be readily distinguished in all cases, especially in coronal sections where fibers may be running perpendicular to the plane of section. Thus, immunoreactivity not contained in cell bodies was described as
'fibers' in all cases, unless specifically noted. Qualitative differences in immunoreactive staining patterns between timepoints are described in the following section.
RESULTS
General observations
The general development of the pig brain has been discussed previously [38,52].
Galanin-like immunoreactivity in cell bodies and fibers was observed at all ages studied from g30 to pn50. Immunoreactive cell bodies generally increased in number and distribution in the brain during the prenatal period. During the postnatal period, however, the number of cell bodies displaying GAL-IR decreased in number, particularly in hypothalamic areas. The distribution of GAL-IR in fibers became more widespread and the density of inmiunoreactivity also increased during the prenatal period, showing a pattern by pnl consistent with later postnatal ages. Some additional increases in immunoreactivity occurred postnatally, especially in the periventricular hypothalamus. 100
Galcmin-Uke inanunoreactivity in cell bodies
The localization of galanin-like immunoreactivity in cell bodies throughout prepubertal development is summarized in Table 3. The following describes changes in localization or relative numbers of immunoreactive cell bodies between ages. g30
Cell bodies displaying immunoreactivity were observed at g30 in the diencephalon.
Numerous immimoreactive cells were seen in the forming hypothalamus, particularly in the anterior and lateral hypothalamus (Fig. 2). Few immunoreactive cells were seen in the preoptic area. gSO
Ntmierous cell bodies displaying GAL-IR were seen in the medial preoptic area, periventricular preoptic area, anterior periventricular hypothalamus, lateral hypothalamus, arcuate nucleus, dorsomedial hypothalamic nucleus and solitary nucleus. Few immunoreactive cell bodies were observed in the internal zone of the median eminence. g70
Numerous immunoreactive cells were observed in the ventral preoptic area at g70.
An increase in the number of immunoreactive cell bodies in the lateral hypothalamus was also observed at this stage.
g90
Numerous immimoreactive cells were observed in the bed nucleus of the stria
terminalis, medial amygdaloid nucleus, dorsolateral hypothalamus and basomedial amygdaloid nucleus. An increase in the number of immunoreactive cells was seen in the 101 arcuate nucleus at g90 (Fig. 6B) compared with earlier developmental stages. gllO
A small population of immunoreactive cells was observed in the posterior cortical amygdaloid nucleus at gllO. An increase in the number of immunoreactive cells in the medial amygdaloid nucleus was evident at this stage. pnl
Immunoreactive cells were observed in the zona incerta and in the dorsal endopiriform nucleus at pnl. An increase in immimoreactive cells was observed in the bed nucleus of the stria terminalis. A decrease in the number of immimoreactive cells was seen in the dorsomedial hypothalamic nucleiis (Fig. 5B) and arcuate nucleus (Fig. 6C) at pnl. pnlO
A slight decrease in the number of immunoreactive cells was observed in the lateral hypothalamus at pnlO. pn20
Immunoreactive cell bodies were observed in the Al/Cl cell group in the ventrolateral medulla at pn20. A uecrease in the number of immunoreactive cells was evident in the periventricular hypothalamic area, dorsomedial hypothalamic nucleus and lateral hypothalamus at pn20. pnSO
No apparent differences in the localization of cell bodies were observed at pn50 compared with earlier postnatal ages examined. 102
Galanin-like immunoreactivity in fibers
The lcx:alization of galaiun-like inununoieactivity in fibers tbrougliout prepubertal development is summarized in Table 4. The following describes changes in localization and relative densi^ of immunoreactive fibers among the gestational and postnatal ages studied. g30
A low to medium density of immunoreactive fibers was observed at g30 in the forming hypothalamus in the anterior, lateral, dorsomedial and posterior divisions (Fig. 2).
A low density of fibers was seen m the preoptic area, presumptive arcuate nucleus of the hypothalamus, and in the mesencephalon at the cephalic flexure. gSO
A low to high density of umnunoieactivity was seen in the median eminence at gSO, with the highest density present in the lateral portion (Fig. 4A). A medium density of immunoreactive fibers was present in the dorsomedial hypothalamic nucleus. A low to medium density of immimoreactivity was observed in the periventricular preoptic area and arcuate nucleus at g50. A low density of immunoreactivity was seen in the following areas: diagonal band of Broca, medial septum, lateral sqjtum, organum vasculosum of the laminae teiminalis, medial and lateral preoptic areas, anterior hypothalamus, periventricular hypothalamus, paraventricular hypothalamic nucleus, ventromedial hypothalamic nucleus, paraventricdiar thalamus, posterior hypothalamus, supramammillary nucleus, nucleus of the posterior commissure, ventral tegmental area, central gray, presumptive lateral parabrachial nucleus, presuniptive locus coeruleus, pontine reticular nucleus and solitaiy nucleus. 103 g70
A medium to high density of GAL-IR was observed in the spinal tract of the trigeminal nerve at g70. A low to medium density of punctate immunoreactivity was observed in the dorsal motor nucleus of the vagus. A low density of GAL-IR was seen in the olfactory mbercle, medial amygdala and mammillary bodies. An increase in GAL-IR was seen in a number of structures at g70 as compared with earlier stages of development.
The medial and periventricular preoptic areas displayed a medium density of GAL-IR, as did the lateral hypothalamus. A slight increase in immunoreactive density was also observed in the organum vasculosum of the laminae tenninalis, lateral preoptic area, paraventricular hypothalamic nucleus, paraventricular thalamic area and solitary nucleus. No immunoreactivity was observed in the pontine reticular formation at this stage of fetal development. g90
A low density of immunoreactivity was observed in the bed nucleus of the stria tenninalis, and suprachiasmatic nucleus for the first time at g90. linmunoreactive density increased in the organum vasculosum of the lamina terminalis, lateral preoptic area, anterior hypothalamus, arcuate nucleus (Fig. 66) and spinal tract of the trigeminal nerve, caudal part at g90. gllO
A low density of immunoreactivity was seen for the first time at gllO in the nucleus accumbens and area postrema. Immunoreactivity increased in the paraventriciilar hypothalamic nucleus, median eminence, medial amygdaloid nucleus and dorsal motor 104 nucleus of the vagus. A slight decrease in immunoreactive density was observed in the medial preoptic area at gllO.
pnl
A low density of immunoreactivity was observed in the islands of Calleja, supraoptic nucleus, dorsal raphe and medial parabrachial nucleiis at pnl. An increase in immunoreactivity was observed in the periventricular preoptic area, bed nucleus of the stria tenninalis, posterior hypothalamus, central gray and ventral tegmental area.
pnlO
An increase in immunoreactivity was observed at pnlO in the islands of Calleja, nucleus accumbens, organum vasculosum of the lamina tenninalis, periventricular hypothalamus (Fig. 3B), dorsomedial hypothalamic nucleus, dorsal raphe and locus coeruleus.
pii20
An increase in immunoreactivity was observed in the diagonal band of Broca, bed nucleus of the stria tenninalis, medial preoptic area, periventricular preoptic area and solitary nucleus at pn20.
pnSO
A low density of immunoreactivity was observed in the inferior colliculus at pn50.
An increase in immunoreactivity was seen in the nucleus accumbens and lateral parabrachial nucleus. Immunoreactivity decreased in the dorsal raphe by pn50. 105 DISCUSSION
Immunoreactivi^ for galanin in cell bodies and fibers was observed in the porcine brain at every age studied between g30 and pn50. The distribution and relative density of immunoieactive staining increased throughout prenatal development, exhibiting a pattern characteristic of later ages by pnl. Further increases in immunoreactive density were evident in some brain areas in the early postnatal period, especially in the areas immediately surrounding the third ventricle. Cell bodies displaying GAL-IR were visualized primarily in the preoptic area, hypothalamus (Figs. 2,5,6) and limbic (bed nucleus and amygdala) areas
(Fig. 3A,C), with the exception of the solitary nucleus (Fig. 7) and Al/Cl catecholamine cells. A dramatic increase in the number of brain areas containing GAL-immunoreactive cell bodies was evident by late gestation (g90), but immimoreactive cell numbers abruptly decreased in the early postnatal period, particularly in the hypothalamus. This decrease is similar to the early postnatal decline ia the number of neuropeptide Y-immunoreactive cell bodies in the pig brain [52], and other peptides in the rat [59,60] and may be indicative of a postnatal increase in transport of the peptide from the cell body. The number of GAL- immtmoreactive cell bodies in the hypothalamic arcuate nucleus decreased by pnl (Fig. 6C), but a population of immunoreactive cells were still detectable at pn50 (Fig. 6D). Very few cells containing neuropeptide Y-immunoreactivity were detectable at pn50 in the porcine arcuate nucleus [52].
No cell bodies displayed GAL-IR in the paraventricular hypothalamic nucleus, supraoptic nucleus, or locus coeruleus of the pig at any age smdied. Similarly, no GAL- immunoreactive cell bodies were found in the supraoptic nucleus or locus coeruleus of non- 106 colchicine-treated Cebus monkeys [27], whereas these nuclei contained robust populations of galanin-containing cells in the rat [24,42,61], human [27] and, with the exception of the locus coeruleus, the opossum [12], These differences may be due to the ages of pig studied, a species difference in localization or non-detection as a result of rapid transport of neuropeptide from the cell body. A previous study detected no galanin binding sites in the posterior pituitary of the pig [19], and retrograde tracing smdies in the rat indicated that the
GAL in the posterior pituitary originated mainly from the paraventricular and supraoptic nuclei [3]. These reports support a true species difference in localization and function of
GAL in the hypothalamo-neurohypophyseal system. The latter hypothesis of rapid transport is supported by the use of colchicine, an inhibitor of axonal transport, in previous studies of galanin localization in the rat [24,42,43,61]. However, colchicine treatment has been shown to increase galanin synthesis in the brain and even induce galanin expression in neuronal populations not normally expressing galanin [10], and thus was not used in this study. The localization of GAL messenger ribonucleic acid in the pig brain by in situ hybridization may be necessary to adequately address this question.
Galanin-IR was detected in the highest density in the preoptic area, hypothalamus, median eminence and brainstem. This observation agrees with radioimmunoassay data from the pig [6,8] and compares favorably to previous results in the rat [8,42,54,61] and Brazilian opossum [12].
Development of GAL-IR in relation to other species
The present smdy represents the first comprehensive immunohistochemical description of GAL-containing systems during prenatal development in the mammalian brain. The 107 results suggest that GAL-containing systems develop early in the pig brain, as GAL-IR was
detected in cell bodies and fibers in the hypothalamus as early as g30. This is supported by
a previous study which found GAL-IR in the pnl Brazilian opossum brain, prior to sexual
differentiation or the completion of neurogenesis [12]. Galanin-like immunoreactivity was
also present in the prenatal opossum brain at embryonic day 12, two days prior to birth [13].
However, GAL-IR was not detected in the brain of pnl rats in a previous stody [61]. This
may represent a fimdamental difference in development of GAL-containing systems io the
rat, or may be due to other factors, such as technical considerations or a transient expression
of GAL during embryonic development in the rat. Detectable levels of GAL were found by
radioimmimoassay at embryonic day 15 in the rat brain [17]. The presence of GAL-ER in
the brain as early as g30 in the pig, as well as the early expression of GAL in the opossum
[12,13], suggests that GAL may be playing a yet imdefined role during the development of
the brain.
Galanin-IR was detected almost exclusively in hypothalamic structures in the g30 pig
brain. The localization of GAL-IR cell bodies in the g30 pig brain was similar to that seen
in the pnl opossum [12]. However, GAL-IR in fibers was detected in the brainstem of the
opossum, whereas none was detected imtil g50 in the pig brain. This result suggests that the
expression of GAL in the hypothalamus may precede its expression in the brainstem, or may
reflect a species difference in the early ontogeny of GAL-IR in the brain.
Distribution of GAL in relation to other peptides in the pig brain
The distribution of GAL-IR in fibers in the porcine hypothalamus suggests that GAL
may play a role in the control of hypothalamic-hypophysial hormone secretion. Galanin is 108
involved in the control of GH secretion in the rat [7,25,37,46,48,67]. Galanin
administration, either centrally or peripherally, increases plasma GH levels in the rat [48],
presumably by stimulating the release of GH-releasing hormone (GHRH) [46]. Galanin
intravenously injected into rats not only increased blood concentrations of GH in a time- and dose-dependent manner, but also increased somatostatin (SS) messenger ribonucleic acid
(mSNA) in the hypothalamus and pituitary gland [7]. Thus, a GAL-induced increase in GH may increase SS synthesis in a classic feedback fashion. Conversely, injection of polyclonal
galanin antiserum decreased basal plasma GH concentrations and completely blocked the
ability of GH-releasing peptide (GHRP) to stimulate GH secretion [67]. These results
suggest that neuroendocrine mechanisms underlying the stimulatory effect of GHRP on GH
release involve hypothalamic and/or pituitary galanin. Galanin fibers also innervate SS
neurons in the periventricular nucleus of the rat hypothalamus [33], and thus may farther
modulate GH secretion through SS. Dense GAL-IR is detected in the periventricular
hypothalamus where SS neurons have been detected in the porcine brain [28]. It is not
known whether fibers displaying GAL-IR directly contact SS neurons as has been shown in
the rat, but this close apposition suggests that GAL may affect SS secretion in the pig also.
Additionally, cell bodies displaying GAL-IR are localized in the arcuate nucleus of the
hypothalamus, one site of GHRH neurons in the porcine brain [28]. Galanin and GHRH are
co-localized in cells in the rat [47] and monkey [41] arcuate nucleus. It is not known,
however, whether these peptides are co-localized in the pig. As GH administration to both
Chinese [39] and American breeds of pigs [9,15,35] improves production characteristics,
further study into the role of GAL in regulation of GH secretion in this species is warranted. 109 Galanin is stimulatoiy to LH secretion [34,55,56]. Galanin-IR in fibers was detected in the organum vasculosum of the lamina terminalis, preoptic area and anterior hypothalamus of the pig, where LHRH cell bodies are localized [23]. Thus, it is possible that galanin released from nerve terminals may affect LHSH cells directly. Also, in the rat, galanin is colocalized with LHRH in cells of the organum vasculosum of the lamina terminalis, diagonal band of Broca and medial preoptic area, with a higher rate of colocalization in females than seen in males [44]. It is not known whether a similar relationship is found in the pig, but cell bodies displaying GAL-IR were localized in areas displaying LHRH cells
[23]. Due to the dense GAL-IR detected in the median eminence, GAL may affect the release of hypophysiotrophic hormones directly at that site as well.
Conclusions
The present smdy describes the distribution and development of GAL-IR in the prepubertal Meishan pig brain. These results show a similar distribution to that of other species; however, the early detection of GAL-IR in the porcine brain suggests that GAL may play a yet undefined role during development of the brain. Galanin-IR was present in the porcine brain in areas that have been shown to participate in the control of food intake, growth and reproduction in other mammalian species, and thus may be an important peptide to study further in agricultural species. Because of marked differences in reproductive success and body composition between Chinese and American/European breeds of pigs, further studies conq)aring the relative expression of GAL between these breeds are warranted. 110 ACKNOWLEDGMENTS
The authors thanV Mahlon Shell of the Iowa State Universi^ Animal Reproduction
Fann, Tricia Heine, Sang-Joon Cho, Jack J. Swanson, Lynne R. Ross and Javed Iqbal for excellent technical assistance. This work was supported in part by USDA Fonnula Funds
(C.DJ.), an MCDB Student Research Fellowship (P.L.P.) and USDA, CSRS, NRICGP grant 93-37203-8965 (L.L.A. and C.D.J.).
LITERATURE CITED
[1] Anderson, L.H., Christenson, L.K., Christenson, R.K. and Ford, S.P.,
Investigations into the control of litter size in swine: H. Comparisons of
morphological and fimctional embryonic diversity between Chinese and American
breeds, J. Amm. Sci., 71 (1993) 1566-1571.
[2] Anderson, L.L., Reproductive cycles: Pigs. In: Hafez, E.S.E. (Ed.)
Reproduction in Farm Animals, 6th Edition, Lea. and Febiger, Philadelphia, 1993.
[3] Arai, R., Onteniente, B., Trembleau, A., Landry, M. and Calas, A.,
Hypothalamic galanin-immunoreactive neurons projecting to the posterior lobe of the
rat pituitary: A combined retrograde tracing and immunohistochemical smdy, J.
Camp Neurol., 299 (1990) 405^20.
[4] Beal, M.F., Gabriel, S.M., Swartz, K.J. and MacGarvey, U.M., Distribution of
galanin-like immunoreactivity in baboon bram. Peptides, 9 (1988) 847-851. [5] Beiman, A.L. and Jones, E.G., The Thalamus and Basal Telencephalon of the
Cat: A Cytoarchitectonic Atlas -with Stereotaxic Coordinates. Univ. Wisconsin Press,
Madison, Wisconsin, 1982.
[6] Bretherton-Watt, D., Kenny, M.J., Ghatei, M.A. and Bloom, S.R., The
distribution of galanin message-associated peptide-like immunoreactivi^ in the pig,
Regul. Pept., 27 (1990) 307-315.
[7] Brogan, R.S., Misitanol, V., Giustina, A., Coy, D., and Wehrenberg, W.B.,
Acute administration of three doses of galanin affects the growth axis in the rat
model. Endocrinology, 134 Suppl. 1 (1995) P3-109.
[8] Ch'ng, J.L., Christofides, N.D., Anand, P., Gibson, S.J., Allen, Y.S., Su, H.C.,
Tatemoto, K., Morrison, J.F., Polak, J.M. and Bloom, S.R., Distribution of
galanin immunoreactivity in the central nervous system and the responses of galanin
containing neuronal pathways to injury, Neuroscience, 16 (1985) 343-354.
[9] Chung, C.S., Etherton, T.D., and Wiggins, J.P., Stimulation of swine growth by
porcine growth hormone, J. Anim. Sci., 60 (1985) 118-130.
[10] Cortes, R., Ceccatelli, S., Schalling, M. and Hokfelt, T., Differential effects of
intracerebroventricular colchicine administration on the expression of mRNAs for
neuropeptides and neurotransmitter enzymes, with special emphasis on galanin: an
in situ hybridization smdy. Synapse, 6 (1990) 369-391.
[11] Dlamini, B.J., Li, Y., Klindt, J. and Anderson, L.L., Acute shifts in relaxin,
progesterone, prolactin and growth hormone secretion in Chinese Meishan gilts
during late pregnancy and after hysterectomy, J. Anim. Sci., (1995) in press. [12] Elmquist, J.K., Fox, C.A., Ross, L.R. and Jacobson, C.D., Galanin-like
immuiiOFeactivity in the adult and developing Bra2dlian opossum brain. Dev. Brain
Res., 67 (1992) 161-179.
[13] Elmquist, J.K., Iqbal, J., Ross, L.R., Swanson, J.J., and Jacobson, C.D.,
Characterization of galanin-like immimoreactivity in the prenatal Brazilian
opossum brain, Soc. Neurosci. Abstr., 19 (1993) 1730.
[14] Elsaesser, F. and Parvizi, N., Estrogen feedback in the pig: Sexual differentiation
and the effect of prenatal testosterone treatment, Biol. Reprod., 20 (1979) 1187-
1193.
[15] Etherton, T.D., Wiggins, J.P., Evock, C.M., Chimg, C.S., Rebhun, J.F.,
Walton, P.E., and Steele, N.C., Stimulation of pig growth performance by porcine
growth hormone: Determination of the dose-response relationship, /. Anim Sci., 64
(1987) 433-443.
[16] Ford, S.P. and Youngs, C.R. Early embryonic development in prolific Meishan
pigs, J. Reprod. Fertil. Suppl., 48 (1993) 271-278.
[17] Gabriel, S.M., Kaplan, L.M., Martin, J.B. and Koenig, J.I., Tissue-specific sex
differences in galanin-like immunoreactivity and galanin mRNA during
development in the rat. Peptides, 10 (1989) 369-374.
[18] Gai, W.P., Geffen, L.B., and Blessing, W.W., Galanin immunoreactive neurons
in the human hypothalamus: Colocalization with vasopressin-containing neiirons, J.
Camp. Neurol., 298 (1990) 265-280. 113 [19] Gaymann, W. and Falke, N., Galanin lacks binding sites in the porcine pituitary
and has no detectable effect on oxytocin and vasopressin release from rat
neurosecretory endings, Neurosci. Lett., 112 (1990) 114-119.
[20] Gentleman, S.M., Falkai, P., Bogerts, B., Herrero, M.T., Polak, J.M. and
Roberts, G.W., Distribution of galanin-like immunoreactivity in the human brain.
Brain Res., 5 (1989) 311-315.
[21] Haley, C.S. and Lee, G.J., Genetic basis of prolificacy in Meishan pigs, J.
Reprod. Fertil. SuppL, 48 (1993) 247-259.
[22] Harayama, H., Nanjo, I., Kanda, S. and Kato, S., Testicular development in
Chinese Meishan boars, Theriogenology, 36 (1991) 637-643.
[23] Hays, V.W., Swine production in animal agriculture in China. In: Hoefer, J.A.
and Tsuchitani P.J. (Eds.), Report 11 Submitted-Committee on Scholarly
Comnumication with the People's Republic of China, National Academic Press,
Washington, D.C., 1980, pp. 41-51.
[24] Holets, V.R., Hokfelt, T., Rokaeus, A., Terenius, L. and Goldstein, M., Locus
coeruleus neurons in the rat containing neuropeptide Y, tyrosine hydroxylase or
galanin and their efferent projections to the spinal cord, cerebral cortex and
hypothalamus, Neuroscience, 24 (1988) 893-906.
[25] Hxilting, A.L., Meister, B., Carlsson, L., Hilding, A. and Isaksson, O., On the
role of the peptide galanin in regulation of growth hormone secretion. Acta
Endocrinol. (Copenh), 121 (1991) 518-525. [26] Kineman, R.D., Leshin, L.S., Crim, J.W., Rampacek, G.B. and Kiaeling, R.R.,
Localization of luteinizing hormone-releasing hormone in the forebrain of the pig,
Biol. Reprod., 39 (1988) 665-672.
[27] Kordower, J.H., Le, H.K. and Mufson, E.J., Galanin immimoreactivity in the
primate central nervous system, J. Comp. Neurol, 319 (1992) 479-5(X).
[28] Kyrkouli, S.E., Stanley, B.G. and Leibowitz, S.F., Galanin: stimulation of
feeding induced by medial hypothalamic injection of this novel peptide, Eur. J.
Pharmacol, 122 (1986) 159-160.
[29] Kyrkouli, S.E., Stanley, B.G., Seirafi, B.D. and Leibowitz, S.F., Stimulation of
feeding by galanin: anatomical localization and behavioral specificity of this
peptide's effects in the brain. Peptides, 11 (1990) 995-1001.
[30] Legault, C., Seilier, P., Caritez, J.C., Dando, P., and Gruand, J.,
L'experimentation sur le porcchinois en France, n. Performances de production en
croisement avec les races europeenes. Genet Sel. EvoL, 17 (1985) 133-140.
[31] Leshin, L.S., Barb, C.R., Kiser, T.E., Rampacek, G.B. and Kraeling, R.R.,
Growth hormone-releasing hormone and somatostatin neurons within the porcine and
bovine hypothalamus, Neuroendocrinology, 59 (1994) 251-264.
[32] Lindskog, S., Dunning, B.E., Martensson, H., Ax'rajab, A., Taborsky, G.J. Jr.
and Ahren, B., Galanin of the homologous species inhibits insulin secretion in the rat
and pig. Acta Physiol. Scand., 139 (1990) 591-596. 115 [33] Liposits, Z., Merchenthaler, I., Reid, J.J. and Negro-VUar, A., Galanin
immunoreactive axons innervate somatostatin-synthesizing neurons in the anterior
periventricular nucleus of the rat. Endocrinology, 132 (1993) 917-923.
[34] Lopez, F.J. and Negro-Vilar, A., Galanin stimulates luteinizing hormone-releasing
hormone secretion from arcuate nucleus-median eminence fragments in vitro;
Involvement of an a-adrenergic mechanism. Endocrinology, 111 (1990) 2431-
2436.
[35] Machlin, J.L., Effect of porcine growth hormone on growth and carcass
composition of the pig, J. Anim. Sci., 35 (1972) 794-800.
[36] MagUulo-Cepriano, L., Schreibman, M.P. and Blimi, V., The distribution of
inmiimoreactive FMRF-amide, neurotensin, and galanin in the brain and pituitary
gland of three species of Xiphophorus from birth to sexual maturity. Gen. Comp.
Endocrinol., 92 (1993) 269-280.
[37] Maiter, D.M., Hooi, S.C., Koenig, J.I. and Martin, J.B., Galanin is a
physiological regulator of spontaneous pulsatile secretion of growth hormone in the
male rat. Endocrinology, 126 (1990) 1216-1222.
[38] Marrable, A. W., The Embryonic Pig: A Chronological Account. Pitman, London,
1971.
[39] McLaughlin, C.L., Baile, C.A., Shun-Zhang, Q., Lian-Chun, W., and Jin-Pu,
X., Responses of Beijing black hogs to porcine somatotropin, J. Anim. Sci., 67
(1989) 116-127. 116
[40] Meijer, J., Poot, P., Molenaar, G. and Goede, R., PAP complex: A pitfall in
immunocytochemistiy of the pig hypothalamus, J. Neurosci. Methods, 17 (1986) ®
274.
[41] Meister, B., Scanlon, M.F. and Hokfelt, T., Occurrence of galanin-like
immunoreactivity in growth hormone-releasing factor (GRF)-containing neurons of
the monkey (Macaca fasicularis) infundibular nucleus and median eminence,
Neurosci. Lett., 119 (1990) 136-139.
[42] Melander, T., Hokfelt, T. and Rokaeus, A., Distribution of galanin-like
immunoreactivity in the rat central nervous system, J. Comp. Neurol., 248 (1986)
475-517.
[43] Melander, T., Hokfelt, T., Rokaeus, A., Cuello, A.C., Oertel, W., Verhofstad,
A. and Goldstein, M., Coexistence of galanin-like immunoreactivity with
catecholamines, 5-hydroxytryptaniine, GABA, and neuropeptides in the rat CNS, J.
Neurosci., 6 (1986) 3640-3654.
[44] Merchenthaler, I., Lopez, F.J., Lennard, D.E. and Negro-Vilar, A., Sexual
differences in the distribution of neurons coexpressing galanin and luteinizing
hormone-releasing hormone in the rat brain. Endocrinology, 129 (1991) 1977-
1986.
[45] Michener, S.R., Aimone, L.D., Yaksh, T.L. and Go, V.L., Distribution of
galanin-like immunoreactivi^ in the pig, rat and human central nervous system.
Peptides, 11 (1990) 1217-1223. [46] Murakami, Y., Kato, Y., Shimatsu, A., Koshiyama, H., Hattori, N., Yanaihara,
N. and Imura, H., Possible mechanisms involved in growth hormone secretion
induced by galanin in the rat. Endocrinology, 12 (1990) 1224-1229.
[47] Niimi, M., Takahara, J., Sato, M. and Kawanishi, K., Immunohistochemical
identification of galanin and growth hormone-releasing factor-containing neurons
projecting to the median eminence of the rat, Neuroendocrinology, 51 (1990) 572-
575.
[48] Ottlecz, A., Samson, W.K. and McCann, S.M., Galanin: Evidence for a
hypothalamic site of action to release growth hormone. Peptides, 7 (1986) 51-53.
[49] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd
Edition. Academic Press, San Diego, 1986.
[50] Paxinos, G., Ashwell, K.W.S. and Tork, I., Alias of the Developing Rat Nervous
System, 2nd Edition. Academic Press, San Diego, 1994.
[51] Pearson, P.L., Ross, L.R. and Jacobson, C.D., Differential down-regulation of
estrogen receptor-like immunoreactivity by estradiol in the female Brazilian
opossum brain. Brain Res., 617 (1993) 171-175.
[52] Pearson, P.L., Anderson, L.L. and Jacobson, C.D., The prepubertal ontogeny of
neuropeptide Y-like immunoreactivity in the male Meishan pig brain. Dev. Brain.
Res., (1995) in press.
[53] Pellinemi, L.J., Ultrastructure of the early ovary and testis in pig embryos. Am.
J. Anat., 144 (1975) 89-111. [54J Rokaeus, A., Melander, T., Hokfelt, T., Lundberg, J.M., Tatemoto, K.,
Carlquist, M. and Mutt, V., A galanin-like pqptide in the central nervous system and
intestine of the rat, NeuroscL Lett., 47 (1984) 161-166.
[55] Sahu, A., Crowley, W.R., Tatemoto, K., Balasubramaniam, A., and Kalra, S.P.,
Effects of neuropeptide Y, NPY analog (Norleucine'^-NPY), galanin, and
neuropeptide K on LH release in ovariectomized (ovx) and ovx estrogen,
progesterone-treated rats. Peptides, 8 (1987) 921-926.
[56] Sahu, A., Xu, B. and Kalra, S.P., Role of galanin in stimiilation of pimitary
luteinizing hormone secretion as revealed by a specific receptor antagonist,
galantide. Endocrinology, 134 (1994) 529-536.
[57] Salinas-Zeballos, M.-E., Zeballos, G.A. and Gootman, P.M., A stereotaxic atlas
of the developing swine {Sus scrofa) forebrain. In: Tumbleson, M.E. (Ed.), Swine
in Biomedical Research, Volume 2, Plenum Press, New York, 1986.
[58] Schambra, U.B., Lauder, J.M., Silver, J. (Eds.). Atlas of the Prenatal Mouse
Brain. Academic Press, San Diego, 1992.
[59] Senba, E., Shiosaka, S., Hara, Y., Inagaki, S., Kawai, Y., Takatsuki, K.,
Sakanaka, M., lida, H., Takagi, H., Minagawa, H. and Tohyama, M., Ontogeny of
the leucine-enkephalin neuron system of the rat; Immunohistochemical analysis. I.
Lower brainstem, J. Comp. Neurol., 205 (1982) 341-359.
[60] Shiosaka, S., Takatsuki, K., Sakanaka, M., Inagaki, S., Takagi, H., Senba, E.,
Kawai, Y., lida, H., Minagawa, H., Hara, Y., Matsuzaki, T. and Tohyama, M.,
Ontogeny of somatostatin-containing neuron system of the rat: 119 Immunohistochemical analysis, n. Forebrain and diencephalon, J. Comp. Neurol.,
204 (1982) 211-224.
[61] Sizer, A.R., Rokaeus, A. and Foster, G.A., Analysis of the ontogeny of galanin
in the rat central nervous system by immunohistochemistry and radioinmnmoassay.
Int. J. Devi. Neuroscience, 8 (1990) 81-97.
[62] Skofitsch, G. and Jacobowitz, D.M., Immunohistochemical mapping of galanin-
like neurons in the rat central nervous system. Peptides, 6 (1985) 509-56.
[63] Skofitsch, G. and Jacobowitz, D.M., Quantitative distribution of galanin-like
immunoreactivity in the rat central nervous system. Peptides, 7 (1986) 609-613.
[64] Szetyn, S., Galert, D., Dynowski, J. and Hoczyk, W., The stereotaxic
configuration of hypothalamus nerve centres in the pig. Amt. Am.. 147 (1980) 12-32.
[65] Tatemoto, K., Rokaeus, A., Jomvall, H., McDonald, T.J. and Mutt, V., Galanin-
a novel biologically active peptide from porcine intestine, FEBS Lett., 164 (1983)
124-128.
[66] Tempel, D.L., Leibowitz, K.J. and Leibowitz, S.F., Effects of PVN galanin on
macronutrient selection. Peptides, 9 (1988) 309-314.
[67] Torsello, A., Locatelli, V., Deghenghi, R., Tolis, G. and Vrontakis, M.E.,
Galanin immunoneutralization inhibits hexarelin stimulation of GH secretion in the
rat. Endocrinology, 134 Suppl. 1 (1995) P3-110. [68] Van der Beek, E.M., Pool, C.W., Van Eerdenburg, F.J.C.M., Sluiter, A.A.,
Van der Donk, H., Van den Hurk, R. and Weigant, V.M., Fc-mediated nonspecific
staining of porcine brain with rabbit antisera in immunocytochemistry is prevented
by pre-incubation of the sera with proteins A and G, J. Histochem. Cytochem., 40
(1992) 1731-1739.
[69] Watkins, W.B., Precautions in the application of immunohistochemical techniques
to tissues of the pig hypothalamo-neurohypophysial system, J. Histochem. Cytochem.,
25 (1977) 61-68.
[70] Watson, Jr., R.E., Wiegand, S.J., Clough, R.W. and Hoffman, G.E., Use of
cryoprotectant to maintain long-term peptide immunoreactivity and tissue
morphology. Peptides, 7 (1986) 155-159.
[71] Youngs, C.R., Ford, S.P., McGinnes, L.K. and Anderson, L.H., Investigations
into the control of litter size in swine: I. Comparative smdies on in vitro development
of Meishan and Yorkshire preimplantation embryos, J. Anim. Sci., 71 (1993) 1561-
1565.
[72] Yoimgs, C.R., Christenson, L.K. and Ford, S.P., Investigations into the control
of litter size in swine: HI. A reciprocal embryo transfer smdy of early conceptus
development, J. Anim. Sci., 72 (1994) 725-731. 121
TABLE 1 Number of sets of coronal sections of porcine brain collected and approximate interval of sections processed for immunohistochemistry
AGE g30 g70 g90 ellO-pnSO sets coUected 2 4 6 12 18 interval (pm) 20 60 100 495 765 122
TABLE 2 Abbreviations of brain areas used in tables
A1 A1 noradrenaline cells AH anterior hypothalamic area AP area postrema ARC arcuate nucleus MPA medial preoptic area BM basomedial amygdaloid nucleus MPB medial parabrachial nucleus BST bed nucleus, stria terminalis MS medial septum CI CI adrenaline cells nAc accumbens nucleus CG central gray OVLT organum vasculosum of the lamina DBB diagonal band of Broca terminalis DEn dorsal endopiriform nucleus PCo posterior cortical amygdaloid nucleus DLH dorsolateral hypothalamic area PCom nucleus of the posterior commissure DM dorsomedial hypothalamic nucleus PCRt parvocellular reticular formation DMX dorsal motor nucleus of the vagus Pe periventricular hypothalamus DR dorsal raphe nucleus PH posterior hypothalamus IC inferior coUiculus PV paraventricular thalamic nucleus ICap internal capsule PVN paraventricular hypothal amic nucleus ICj islands of Calleja PvPOA periventricular preoptic area LC locus coeruleus SCN suprachiasmatic nucleus LH lateral hypothalamic area Sol solitary nucleus LPB lateral paiabrachial nucleus SON supraoptic nucleus LPO lateral preoptic area Sp5c spinal tract of the trigeminal nerve, LS lateral septum caudal part ME median eminence St solitary tract MeA medial amygdaloid nucleus SuM supramammillaiy nucleus MM mammillary area Tu olfactory mbercle VMH ventromedial hypothalamic nucleus VTA ventral tegmental area H zona incerta 123
TABLES Distribution of galanin-like immunoreactivi^ in the developing porcine brain: Cell Bodies
AREA g30 g50 g70 g90 gllO pnl pnlO pn20 pn50
Telencephalon
BST NA - - + + + + + + + + + + + +
BM NA - - + + + + + + + + + + + +
DEn NA - - - - + + + + + + + +
MeA NA - - + + + + + + + + + + + +
PCo NA - - - + + + + + + + + + +
Diencephalon AH + + + + + + -1- + + + +
ARC - + + + + + + + + + + + + + + + + DLH NA - - + + + + + + + + + + + +
DM - + + + -1- + + + + + + + + + + + + LH + + + + + + + + + + + + + + + + + +
ME NA + ------Pe NA + + + + + + + + + + + + + + + + MPA + + 4- + + + + + + + + + + + + + + pvPOA NA + + + + + + + + + + + + + + + + ZI NA - - - - + + + + + + + + Myelencephalon
Al/Cl NA ------+ + + + Sol NA + + + + + + + + + + + + + + + +
'-Abbreviations of brain areas can be found in table 2. ^-Symbols used in this table refer to the following: no cell bodies detected in area or nucleus studied; +, less than 10 cell bodies detected in area or nucleus; ++, more than 10 cell bodies detected in area or nucleus; NA, area not applicable or not studied. Due to immaturity of brain structures, many brain areas cannot be defined at g30 and are thus listed as NA. 124
TABLE1AX>UC t4 Distribution of galanin-like immunoieactivity in developing porcine brain: Fibers
AREA g30 g50 g70 g90 gllO pnl pnlO Dn20 pn50
Bhinencephalon ICji NA^ L L/M L/M L/M Tu NA - L L L L L L L
Telencephalon BST NA L L L/M L/M M M DBB NA L L L L L L L/M L/M LS NA L L L L L L/M L/M L/M MeA NA - L L L/M L/M L/M L/M L/M MS NA L L L L L L L L nAc NA - - - L L UM LM M
Diencephalon AH L/M L L L/M L/M L/M L/M L/M L/M ARC L LM L/M M/H M/H M/H M/H M/H M/H DM IVM M M M M M M/H M/H M/H LH L/M L/M M M L/M L/M L/M L/M L/M LPO NA L L L/M LM L/M L/M L/M LM ME NA L/H L/H L/H Mm M/H M/H MjOI M/H MM NA - L L L L L L L MPA L L M M LM L/M L/M M/H Mm OVLT NA L L/M M M M M/H M/H M/H Pe NA L M M M M M/H M/H M/H PH UU L L L L L/M L/M L/M L/M PV NA L M M M M M M M PVN NA L L/M L/M M M M M M PvPOA NA L/M M M M M/H M/H H H SCN NA - - L L L L L L SON NA - - - - L L L L SuM NA L L L L L L L L VMH NA L L L L L L L L
Mesencephalon CG NA L L L L L/M L/M L/M L/M DR NA - - - - L L/M L/M L IC NA ------L 125
PCom NA L L L L L L L L VTA L L L L L LM L/M L/M LAI
Metencephalon LC NA L L L L L/M M M M LPB NA L L L L L/M L/M L/M M/H MPB NA - - - - L L L L PCRt NA L L L L L L L L
Myelencephalon AP NA L L L L L DMX NA - L/M L/M M M M M M Sol NA L liM L/M L/M L/M L/M L/H L/H
Sp5 NA - M/H H H H H H H
'-Abbreviations of brain areas can be found in Table 2. ^-Symbols used in the table refer to the following: no inmmnoreactivity detected; L, low density of immunoreactivity detected; M, medium density of immunoreactivity detected; H, high density of immunoreactivity detected; and NA, area not applicable or not studied. Due to immaturity of brain structures, many brain areas cannot be de^ed at g30 and are thus listed as NA. Figure 1. Examples of galanin-like immunoreactivity in porcine brain classified as being either low, medium or high density. Low
Medium
High Figure 2. Galanin-like immunoieactivity in the gestational day 30 pig biain. A- hypothalamus, coronal section; B-hypotfaalamus, sagittal section. Magnification bar indicates 100/an. Both photomicrographs are at the same magnification level.
Abbreviations: 3V-third ventricle, c-caudal, r-rostral.
Figure 3. Galaoin-like iimnunoreactivity in the pig brain. A-bed nucleus of the stria terminalis, medial division, postnatal day (pn) 10; B-periventricular hypothalamus, pnlO;
C-medial amygdaloid nucleus, pnl. Magnification bar indicates lOO^m. All photomicrogr^hs aie at the same magnii^cation level. Abbreviation: 3V-third ventricle.
Figure 4. Galanin-like mununoreactivity in the developing median eminence. A- gestational day (g) 50; B-g70; C-g90; D-postnatal day 50. Magnification bars indicate
50^. All photomicrographs are from sections in the coronal plane. Abbreviations: 3V- third ventricle, ARC-arcuate nucleus, IZ-intemal zone.
Figure S. Galanin-like immunoreactivity in the developing dorsomedial hypothalamic nucleus. A-gestational day 90; B-postnatal day 1. Magnification bar indicates 100/mi.
Both photomicFOgraphs are at the same magnification level. The third ventricle is the space on the left of both photomicrographs. Arrowheads in A indicate dense counterstaining in the ependynia. figure 6. Galanin-like immunoreactivity in the developing arcuate nucleus. A-gestational day (g) 70; B-g90; C-postnatal day (pn) 1; D-pn50. Magnification bar indicates 100/an.
All photomicrographs are at the same magnification level. Abbreviation: 3V-thini ventricle. Arrowheads indicate dense counterstaining in the ependyma.
Figure 7. Galanin-like inunimoreactivity in the solitary nucleus and dorsal motor nucleus of the vagus, postnatal day 1. Magnification bar indicates 100/im. Abbreviations:
DMX-dorsal motor nucleus of the vagus, st-solitary tract. DMX Figure 8. Galanin-like immunoreactivity in the spinal tract of the trigeminal nerve, caudal part, postnatal day 10. Magnification bar indicates 100^. (ZZMf-
/T > ^1 V • f. 'S
'5 > ,> Jr-.' 142
CHAPTER 3: THE PREPUBERTAL ONTOGENY OF LUTEINIZING HORMONE-
RELEASING HORMONE-LIKE IMMUNOREACTTVITY IN THE TELENCEPHALON
AND DIENCEPHALON OF THE MALE MEISHAN PIG BRAIN
A paper submitted for publication in Developmental Brain Research (1995)
Paul L. Pearson, Lloyd L. Anderson, and Carol D. Jacobson
SUMMARY
Luteinizing hormone-releasing hormone (LHRH) is a decapeptide that regulates reproductive function and behaviors in mammalian species. Due to the importance of proper reproductive function and efficiency in agricultural species, we have investigated the prepubertal ontogeny of LHRH-like immunoreactiviQr (-IR) in the male Meishan pig. The
Meishan is a Chinese bteed known for reproductive traits including increased litter size and precocial puberty, but slow growth and obesity.
Brains of animals from gestational day (g) 30, 50, 70, 90 and 110 and postnatal day
(pn) 1, 10, 20 and 50 (duration of pregnancy averaged 114 days) were processed using a standard immunohistochemical technique utilizing a commercially available rabbit anti-LHRH antibody. Luteinizing hormone-releasing hormone-IR in cell bodies and fibers was detected 143 at g30 entering the brain via the terminal nerve and in the septal region of the basal telencephalon. The number of immunoreactive cells increased at g50 and cells were localized primarily to the septum, organum vasculosum of the lamina terminalis, preoptic area and lateral hypothalamus, whereas immunoreactive fibers were present throughout the septum and hypothalamus and had reached the median eminence. The density and distribution of immunoreactive fibers increased by g70 and g90, but did not change dramatically &om g90-p£i50. These results indicate that LHRH may be present in the
Meishan brain earlier during development and fibers containing LHRH-ER appear to reach the median eminence earlier than previously reported for the domestic pig. These results suggest a breed difference in the ontogeny of reproductive control systems in the pig. Future studies utilizing direct comparisons between domestic and Chinese breeds will be requked investigate the apparent differences in the ontogeny of LHRH-containing systems in the pig.
INTRODUCTION
Luteinizing hormone-releasing hormone (LHRH), also called gonadotropin releasing hormone, is a decapeptide originally isolated from porcine tissue [36]. Luteinizing hormone- releasing hormone is secreted from the brain at the median eminence and via the hypothalamo-hypophyseal portal blood causes the release of both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary gland [reviewed in 22,25].
The secretion of LHRH is controlled by several factors including circulating steroid hormone levels and the neuropeptides galanin (GAL) and neuropeptide Y (NPY)
[25,26,28,32,33,34,44,45,53,55,56,73]. Luteinizing hormone-releasing hormone is also 144 involved in tbe regulation of reproductive behaviors [43,51].
The inununohistochemical distribution and development of the LHRH system in the
brain has been investigated in a nimiber of species including the chick [66], Brazilian
opossum [59], mouse [16,61] rat [21,22,24,39,40,41,42,60,63,65], hamster [5,23], guinea
pig [62,64], sheep [8], pig [9,11,28,29,31,37,52], macaque [54] and human [3]. In rodents,
LHRH-producing neurons develop early in gestation in the olfactory epithelium and migrate
into the brain via the terminal nerve. These cells then disperse to locations in the septum,
diagonal band, organtmi vasculosum of the lamina terminalis (OVLT), preoptic area and
hypothalamus [16,21,22,39,60,61,62]. Previous smdies have shown that 50% or more of all LHRH-immunoreactive cells project to the median eminence [42,65], where LHRH can
be secreted into the portal system to affect anterior pituitary secretion.
Previous studies in domestic breeds of pigs have investigated the ontogeny of LHRH-
like immunoreactivity (LHRH-IR) during prenatal development [11,52]. These smdies have
indicated that LHRH-IR is present as early as gestational day (g)40 in the brain and
immunoreactive fibers project to the median eminence by g60, prior to the appearance of the
portal capillaries [11]. We have utilized the Meishan pig, a Chinese breed known for
superior reproductive characteristics [1,14,18,19,20,72,75] including precocial puberty
[19,35], but slow growth and poor feed efficiency [30], to study the prepubertal development
of LHRH-IR. 145 MATERIALS AND METHODS
Animals
Purebred Meishan pigs were obtained from the breeding population maintained at the
Iowa State University Animal Reproduction Farm. The animals and procedures used were in accordance with the guidelines and approval of the Iowa State University committee on animal care. Boars were allowed access only to sows in estrus and the date of breeding was designated as gO. The day of birth was designated as postnatal day (pn)l. Gestation averaged 114 days. At least four intact male animals from at least two different litters were used at each time point in this smdy (gSO, g70, g90, gllO, pnl, pnlO, pn20, and pn50). At g30, the earliest time point smdied, the gender of the animals could not be determined by external visual inspection, and thus four animals were used that were considered sexually undifferentiated. Pigs euthanized at pn50 in this study were weaned at pn21 and given ad libitum access to a standard com/soybean diet (18% protein) until the date of termination.
Tissue preparation
Prenatal fetuses were obtained by electrocution and immediate exsanguination of the sow, followed by removal of the fetuses from the gravid uterus. At g30, fetal heads were removed and post-fixed 48 hours in 4% paraformaldehyde (pf). At g50, fetuses were perfused transcardially with ice-cold 4% pf, the heads removed, the brain exposed, and postfixed 48 homrs in 4% pf. At g70-110, the femses were perfused transcardially with
0.9% NaCl followed by ice-cold 4% pf, the brains removed and postfixed 24-48 hours in
4% pf. At all postnatal ages, animals were euthanized with an intraperitoneal injection of a lethal dose of sodium pentobarbital, perfused transcardially with 0.9% NaCl followed by 146 ice-cold 47o pf. Brains were removed, blocked, and post-fixed 24-48 homx in 4% pf.
Following post-fixation, all brains were sunk in 30% sucrose at 4°C and sectioned on a cryostat (Reichert-Jung). For g30-g70, brains were cut at 20 ;^m and thaw-mounted onto poly-L-lysine coated slides, dried overnight at 35°C, and stored at 4®C until processed for immunohistochemistry. At all other ages, brains were cut at 45faji into wells containing cryoprotectant solution [71], and stored at -20°C until processed for immunohistochemistry.
At all ages, brains were sectioned in the coronal plane. At g30, additional heads were sectioned in the horizontal or sagittal planes. Brains of g50 and older animals were sectioned from the genu of the corpus callosimi (rostral) to the spinomedullary junction (caudal). In g90 and older animals the cerebellum was removed prior to sectioning and was not processed.
Immunohistochemistry
The protocol utilized for immunohistochemistry was modified from that reported previously firom our laboratory for the pig brain [48,49]. Tissue mounted on slides was washed with 50mM potassiimi phosphate buffered saline (KPBS), incubated with 1 % H2O2 to remove endogenous peroxidase activity, exposed to 1.5% normal goat serum (NGS), 1% bovine serum albtmiin (BSA) and 0.4% Triton X-100 (Sigma) as a blocking agent. The
tissue was then incubated in rabbit anti-LHRH (Incstar; 1:5000) in blocking serum containing
1% NGS, 1% BSA and 0.4% Triton X-100 for 20 hours at room temperature in a
humidified chamber. Following adequate washing, the tissue sections were incubated in goat
anti-rabbit IgG (Kirkegaard & Perry Laboratories; 2/ig/ml) in diluent (KPBS containing 5%
nonfat powdered milk and 0.25% Triton X-100) for 2 hours at room temperature, rinsed. 147 and red and reacted with streptavidin-peroxidase (Kirkegaard & Perry Laboratories; 2#4g/ml) in dilueiit diluent for 1 hour at room temperature. After washing, the sections were stained by reacting tbem wi them with 0.04% 3,3'-diaminobenzidine solution (Sigma), 2.5% nickel sulfate (Sigma), and
0.01% 0.01% H2O2, dissolved in O.IM sodium acetate. After staining for 15-20 minutes, the
reaction was terminated by two consecutive rinses in 0.9% NaCl. Sections were counter counterstained with neutral red (Fisher Scientific) and then dehydrated in graded alcohols, cleared in xylene, coverslipped with Permount (Fisher Scientific) mounting media, and
analyzed with a light microscope.
Tissue cut into wells and stored in cryoprotectant was processed for iTnTniinr immunohistochemistry by a free-floating tissue technique utilizing plastic weigh boats with screens. screens, as previously described for pig tissue [48,49]. In addition, the following modiiica modifications from the previous protocol were made: 1) Incubation in primary antibody solution solution was conducted at 4°C for 20 hours; and 2) Following staining, tissue sections were mounted moimted onto gelatin-coated slides, air-dried overnight, then counterstained and processed as abov< as above.
SectHH) Section Intervals Due to the size differences during brain development, brains were sectioned and collectet collected ia sets. The number of sets and approximate interval of sections processed for unmuno immunohistochemistry for each age is listed in Table 1. One set of sections from each brain
WaSDFOC was processed for every animal at ages g30, g50, g70, pnlO, pn20 and pn50. Two sets were run Sinn run simultaneously for ages g90, gllO, and pnl. 148 Controls
Negative controls were included in each run and consisted of omission of the primary antiserum ficom the diluent. No staining was observed in any negative control section (not shown). Preabsorption controls were conducted and consisted of incubation of the primary antiserum with S.SfiM LHRH (Safolin, Co.) at 4®C for 20 hours prior to use. All staining in a g30, g50, g70 and pnl brain was abolished by this procedure (not shown). Incubation of the primary antiserum with 5/ig/mI recombinant protein A (Calbiochem-Novabiochem
Corporation) prior to immunohistochemical staining was conducted as a proposed control for nonspecific staining of magnocellular neurons [37,69,70]. This procedure blocked non specific cytoplasmic staining of cells in the paraventricular and supraoptic nuclei of the hypothalamus, but failed to block immunohistochemical staining of other immimoreactive cell bodies in adjacent sections in the pnl brain (not shown).
Analysis of Tissue
Sections containing the diencephalon and telencephalon were examined with the light microscope and brain regions containing LHRH-IR were identified. Structures in the brain during early development were compared to atlases of developing mouse brain [58] and rat brain [47]. Other ages were compared to the rat atlas [46], cat atlas [3] and previous descriptions of the pig brain [57,67]. Use of neuroanatomical terminology and abbreviations followed Paxinos et al. [47] for g30 and g50 brains and Paxinos and Watson [46] for older animals, except in cases where subnuclear groups or areas defined in the rat could not be clearly identified from coxmterstained tissue of the pig brain. Nuclear groups which could not be clearly defined by cellular characteristics or by location due to absence of landmarks 149 during development were denoted as 'presimiptive' nuclei. Abbreviations of nuclear groups contained in tables and figures are defined in Table 2. The number of cell bodies displaying immunoreactivity was described as few (less than 10) or numerous (greater than 10) cell bodies in a brain area or nucleus in the processed sections. Immunoreactivity that was not confined to cell bodies was subjectively classified as having a low, medixmi or high density in an area or nucleus as shown in Figure 1. Although such immunoreactivity not contained in cell bodies may be contained in nerve fibers, terminals or other processes, these cannot be readily distinguished in all cases, especially in coronal sections where fibers may be running perpendicular to the plane of section. Thus, immunoreactivity not contained in cell bodies was described as 'fibers' in all cases, unless specifically noted. (Qualitative differences in immunoreactive staining patterns between timepoints are described in the following section.
RESULTS
LHRH-like Immunoreactivity in Cell Bodies
The localization of LHRH-IR detected in cell bodies is shown for all ages smdied in
Table 3. The following descriptions detail changes in localization or relative numbers of inmiunoreactive cell bodies between the ages studied. g30
Numerous cell bodies displaying LHRH-IR were detected in the basal telencephalon of the g30 pig brain. Cell bodies were seen in midsagittal sections and in coronal sections and were localized to the medial surface of the brain in the preseptal area and along the 150 terminal nerve and tenninal ganglion (Fig. 2A). These cell bodies (along with associated inmimioreactive fibers) formed two arching pathways into the basal telencephalon (Fig. 2B), one running near the ventral surface of the brain and the other extending into the septal area.
Few to numerous immimoreactive cells were seen in the presumptive medial septum. Cell bodies displaying immunoreactivity at g30 were generally lightly stained. Immunoreactive cells were also detected in some animals in the olfactory epithelium near the basal telencephalon. gSO
Numerous immunoreactive cells were seen along the midline of the brain in the septal area, in the medial septum, OVLT (Fig. 3A), medial preoptic area, lateral preoptic area, and lateral hypothalamus. Few immunoreactive cells were seen in the dorsal lateral septum. gf70
Numerous immimostained cell bodies were seen for the first time in the median preoptic nucleus at g70. A few inmiiuioreactive cells were observed in the periventricular hypothalamus. An increase in the number immunoreactive cell bodies was seen in the medial preoptic area. A decrease in immimoreactive cell bodies was seen in the medial septum at g70. g90
A few immunoreactive cells were detected in the anterior hypothalamus for the first time at g90. One cell was seen in the anterior commissure and medial amygdala in one animal but was not Seen in the other animals. 151 gllO
A few immunoreactive cells were seen in the diagonal band of Broca at the level of the OVLT for the first time at gllO. One immmioreactive cell was seen in the dorsal hypothalamus in each of two animals and one immunoreactive cell was seen in the arcuate nucleus of one animal. The number of immunoreactive cells seen per section in the medial preoptic area, lateral preoptic area and lateral hypothalamus was lower than at g90. This decrease may result from brain growth between g90 and gl 10 rather than an actual drop in immunoreactive cell numbers. pnl
An immunoreactive cell was seen in the posterior hypothalamus in one animal at pnl.
An increase in the number of immunoreactive cells was seen in the diagonal band of Broca.
Only a few immunoreactive cells were detected in the lateral preoptic area at pnl. pnlO-pnSO
One immimoreactive cell was seen in the arcuate nucleus in one animal at pnlO and pn50. No other differences in cell body localization were observed from pnl0-pn50.
LHRH-like Immunoreactivity in Fibers
The localization of LHRH-ER in fibers is shown for all ages smdied in Table 4. The following descriptions detail changes in localization or relative density of immunoreactivity between the ages smdied. g30
A low to medium density of immunoreactive fibers was visualized in the basal telencephalon entering the brain along the terminal nerve (Fig. 2A). A low density of fibers 152 was seen in the piesunqitive medial septum and the basal telencephalon projecting towards the forming preoptic area (Fig. 2B). A low density of fibers was also observed in the forming internal capsule. g50
A medium density of immimoreactive fibers was seen along the midline of the brain in the septum and in the lateral hypothalamus. A low to medium density of fibers was seen in the medial septum, diagonal band of Broca, optic tract, in the area lateral to the median eminence and in the supramammillary nucleus. A low density of fibers was seen in the lateral septum (dorsal division), corpus callosum, OVLT (Fig. 3A), medial preoptic area, lateral preoptic area, optic chiasm, medial amygdala, anterior hypothalamus, dorsolateral hypothalamus, paraventricular thalamus, arcuate nucleus and posterior hypothalamus. A low density of immunoreactivity was also seen in die ventromedial nucleus and dorsomedial nucleus of the hypothalamus in one animal. A very low density of immimoreactive fibers was seen in the median eminence at gSO (Fig. SA). g70
A low to medium density of immunoreactivity was seen in the median preoptic nucleus and subfornical organ at g70. A low density of immunoreactivity was seen in the lateral septum (ventral division), fornix, periventricular preoptic area, periventricular hypothalamus, paraventricular hypothalamic nucleus, posterior cortical amygdala, and mammillary bodies at g70. An increase in immunoreactivity was observed in the diagonal band of Broca, OVLT (Fig. 3B), lateral preoptic area, medial preoptic area, anterior hypothalamus, and median eminence (Fig. 5B). A slight decrease in immimoreactivity was 153 seen in the optic tract and supramanunillary nucleus at g70. g90
A low density of immunoieactivity was detected in the supraoptic nucleus, suprachiasmatic nucleus, bed nucleus of the stria terminalis, dorsal hypothalamic area, reuniuns thalamic nucleus, and centromedial thalamic nucleus for the first time at g90. An increase in immunoreactive density was observed in the periventricular preoptic area, subfornical organ, periventricular hypothalamus, and median eminence. A slight decrease
in immunoreactivity was seen in the medial septum, midline of the brain in the septal area, diagonal band of Broca and posterior hypothalamus. Immunoreactive fibers in the ventral anterior hypothalamus were seen running parallel to the optic chiasm and could be followed from the lateral hypothalamic area to the midline of the brain (Fig. 4A). gllO
An increase in immunoreactive density was observed in the ventral anterior hypothalamus (Fig. 4B) and median emmence at gllO (Fig. 5C). A decrease in
immunoreactivi^ was observed along the midline of the brain in the septal area. No
immunoreactivity was observed in the corpus callosum or medial amygdaloid nucleus at gllO.
pnl
A slight increase in immunoreactivity was seen in the arcuate nucleus and OVLT
(Fig. 3D) at pnl. No immunoreactiviQr was seen in the centromedial thalamic nucleus or
reimiims thalamic nucleus at pnl. 154 pnl0-pn50
No differences in inununoreactivity were detected from pnl0-pn50.
DISCUSSION
LHRH-IR in the pig brain
Luteinizing hormone-releasing honnone-IR was detected in the Meishan pig brain in cell bodies and fibers at every age from g30-pn50. At g30, cells displaying LHRH-IR were seen entering the base of the brain in the basal telencephalon along the terminal nerve, and along the midline of the brain in the basal telencephalon. At g50, immunoreactive cells were seen in the septum, OVLT, preoptic area and hypothalamus and the distribution of immunoreactive cells expanded at g70 but remained virtually identical throughout prepubertal development. Immimoreactive fibers were detected at g30 in the basal telencephalon with some fibers extending toward the presumptive preoptic area. Immunoreactive fibers were seen throughout the septum, diagonal band of Broca, OVLT, preoptic area and hypothalamus at g50 and LHRH-IR in fibers was detected in the median eminence at this age. The density of immunoreactivity increased in the brain at g70 and g90, especially in the median eminence. Further increases in immunoreactivity were evident in the OVLT in the postnatal period. However, the distribution of immunoreactive fibers remained virtually identical from gll0-pn50. These results suggest that cells containing LHRH appear very early in gestation
in the pig (g30), migrate to their respective locations and innervate the median eminence by mid-gestation (g50-g70). These results further suggest that LHRH neurons are localized 10
days earlier in the brain of the Chinese Meishan breed than in the domestic pig, and that 155 LHRH-IR in fibers reaches the median eminence 10 days prior to that seen previously and before the differentiation of capillary loops in the median eminence [11], In addition, immunoreactive cells were also detected in the hypothalamus at g50, whereas they were limited to the paraolfactory and precommissural areas and OVLT in the previous study [11].
Another study of LHRH in the Landrace breed of pig did not detect immunoreactive cell bodies or fibers until g72 and the density of immunoreactive staining was less than the present stody at both g72 and gllO [52]. However, these smdies utilized paraffin-embedded tissue, which has been subsequently shown to reduce the amount of immunoreactive LHRH present [24]. This procedural difference or differences in immunohistochemical techniques may be responsible for the discrepancies seen between this study and previous reports.
However, these results extend the findings of the previous studies and confirm the olfactory origin and apparent migration into the brain of cells containing this peptide in the pig.
Direct comparison of tissue from domestic and Chinese pigs of the same gestational age should be made to confirm these results. This is the first report of the immunohistochemical distribution of LHRH-ER in the postnatal male pig brain. The distribution and density of
immunoreactivity in the postnatal male Meishan pig is similar to that reported in the sexually mature female domestic crossbred pig [28].
Comparison with other species
The distribution of UIRH immunoreactivity during prenatal development follows very
closely that seen in other species [7,16,21,54,59,61,62,66], with cells displaying LHRH-IR
being localized first in the terminal nerve and presumptive septal area. The arching pattern
of immunoreactive cells and fibers seen in the basal telencephalon at g30 in this smdy (Fig. 156 2B) is strikiiigly similar to that seen in the mouse [61]. Immunoreactive cells are then localized to the septum, diagonal band, OVLT/preoptic area and hypothalamus at later developmental ages. No immunoreactive cell bodies were detected at any age in the suprachiasmatic nucleus of the pig in contrast to that seen in the hamster [23], rat [39] and guinea pig [62,64], or in the supraoptic nucleus as in the Brazilian opossimi [59]. Only an occasional immunoreactive cell was observed in the hypothalamic arcuate nucleus in the pig, unlike the robust population observed in the guinea pig [62,64], mouse [16], sheep[8] and macaque [54]. This may be a species difference, as no immunoreactive cell bodies are reported in the arcuate nucleus of the Brazilian opossum [61], hamster [23] or rat
[21,22,39,63].
LHRH, NPY and GAL in the Meishan pig brain
Neuropeptide Y and GAL have been shown to participate in the regulation of LHRH secretion and responsiveness of the pituitary to LHRH
[25,26,27,32,33,34,44,45,53,55,56,73]. Inaddition, LHRH has been co-localized with GAL in cell bodies of the OVLT and diagonal band of Broca in the rat, primarily in females
[38,40,41]. Our previous smdies indicate that NPY-like immunoreactivity (-IR) and GAL-IR are both present in the median eminence of the Meishan brain at g50 [48,49], the same timepoint LHRH-IR was seen in the median eminence in this study. Luteinizing hormone- releasing hormone-IR is detected in cell bodies in the OVLT and preoptic area, both areas where cells containing NPY-IR and GAL-IR were localized [48,49]. It is not known whether
GAL and LHRH are co-localized in the pig as in the rat [38,40,41] or if NPY and LHRH are co-localized in the pig. 157 Our previous studies indicated an early postnatal decrease in inimunoreactive cell bodies, particularly in the hypothalamus, for both NPY and GAL [48,49]. No similar decrease was detected in this study, however, suggesting that the previous observations are probably not due to technical considerations regarding the age of tissue being processed.
Functional development of the gonadotropic axis in the pig
The pig undergoes gonadal differentiation at g26 [50], and a period of elevated serum testosterone levels in the male fetus occurs between g35 and g40 [12]. The results from this study indicate that LHRH producing cells are present in the Meishan brain as early as g30, with LHRH-IR in fibers first reaching the median eminence at g50. This is prior to the differentiation of capillary loops in that structure (g70) in the domestic pig [11]. Cells containing LH first appear in the porcine pituitary at g40 [10] or g45 [11] with the &st FSH- containing cells appearing at g45 [10] or g60 [11]. These studies indicate that the gonadotropic cells reach adult levels and appearance by g80 [11] or g90 [10]. The fetal pig pituitary will secrete LH in response to electrical or electrochemical stimulation of the hypothalamus at g80, but not g60. In addition, the basal plasma LH levels doubled between g60 and gSO [6]. Together, these results suggest that the hypothalamo-pituitary relationship is not established until g70 or gSO and that LHRH is not important in the differentiation or functioning of the pituitary at ages prior to that time period. It has been suggested, however, that LHRH cells outside of the brain (associated with the terminal ganglion) could secrete into the subarachnoid space to affect the pituitary before the portal vessels appear in the rat
[21,60]. Due to the early appearance (g30) of LHRH-IR in cell bodies outside of the brain seen in this study, it is possible that LHRH could affect the differentiation or function of the 158 pituitaiy in this manner in the pig as well. Luteinizing hormone and FSH receptors are present in the gonads as early as g28 in the pig, with levels peaking in males between g35 and gS8 and then declining until birth [15]. Taken together, these results reinforce the hypothesis that a parallel development of the gonads, pituitary gonadotropes and the gonadotropic control systems occurs in the brain, as has been suggested previously [11,17].
Development of the hypothalamo-pituitary-gonadal axis is markedly accelerated in the
Meishan pig, with onset of spermatogenesis at 9 weeks of age compared with 16 weeks in the Diuxx;, a domestic breed [13,35]. Breed differences exist in body sizei testis size, sperm per ejaculate and ejaculate volume [5]. During the first 50 days of postnatal development, circulating blood serum concentrations of LH are 5- to 6-fold greater in the Meishan boar compared with domestic breeds [2,35]. Serum LH levels peak at 3 weeks of age and decrease thereafter to onset of puberty, both in the Meishan and domestic breeds. Serum testosterone concentrations increase steadily from 1.0 ng/ml at birth to 8.0 ng/ml by onset of spermatogenesis at 9 weeks in Meishan boars whereas they remain at 1-2 ng/ml tintil puberty at 16 weeks in domestic boars. Of particular note is the 5-fold greater concentration of serum FSH in the Meishan compared with the Duroc [35], Serum FSH concentrations increase abruptly after castration in both breeds, with consistently greater hormone concentrations in the Meishan breed [72]. From the present study, it appears that the
LHRH-containing systems are well established at birth, with no apparent differences in
LHRH-ER correlating with the decline in serum UI levels at 3 weeks or the continual increase in serum testosterone levels. However, increases in NPY-IR were seen in the periventricular preoptic area, a site of cell bodies containing LHRH-IR, in the previous study 159 [48]. As fibers containing NPY-IR contact LHRH-immunoreactive neurons in the
OVLT/preoptic area in the rat [68], these changes may relate to increased function of gonadotropic control systems during prepubertal development of the pig.
Conclusions
This smdy represents the first description of LHRH-IR in the postnatal male pig brain and the first study of LHRH-IR in the Meishan pig, a breed known for its superior reproductive characteristics. This study indicates that LHRH-IR is present in cells and fibers in the Meishan brain as early as g30 and in fibers in the median eminence as early as g50.
These observations are both ten days earlier than previously reported for the domestic pig
[11]. These results suggest a breed difference in the development of the LHRH systems, but further investigation will be required to confirm this hypothesis. The distribution and density of LHRH-IR is the brain is very similar fi:om gllO through pnSO suggesting that the pathways controlling reproductive hormone secretion mature prenatally in the pig, changing little throughout prepubertal development. The distribution and densi^ of LHRH-IR is also very similar to that reported previously for the sexually mature female domestic pig [29].
The distribution and development of LHRH-IR in the Meishan pig is similar to other species and this smdy confirms the olfactory origin and apparent migration of LHRH-containing cells into the porcine brain.
ACKNOWLEDGMENTS
The authors thank Mahlon Shell of the Iowa State Universi^ Animal Reproduction
Farm, Tricia Heine, Sang-Joon Cho, Jack J. Swanson, Lynne R. Ross and Javed Iqbal for 160 excellent technical assistance. This work was supported in part by USDA Formula Funds
(C.DJ.), an MCDB Student Research Fellowship (P.L.P.) and USDA, CSRS, NRICGP grant 93-37103-8965 (L.L.A. and C.D.J.).
LITERATURE CITED
[1] Anderson, L.H., Christenson, L.K., Christenson, R.K. and Ford, S.P., Investigations
into the control of litter size in swine; n. Comparisons of morphological and
functional embryonic diversity between Chinese and American breeds, J. Anim. Sci.,
71 (1993) 1566-161L
[2] Anderson, L.L., Pigs. In: Hafez, E.S.E. (Ed.) Reproduction in Farm Ammals, 5th
Edition, Lea and Febiger, Philadelphia, 1987.
[3] Bany, J., Characterization and topography of LHRH neurons in the human brain,
Neurosci. Lett, 3 (1976) 287-291.
[4] Bennan, A.L. and Jones, E.G., The Thalamus and Basal Telencephalon of the Cat:
A Cytoarchitectonic Alias with Stereotaxic Coordinates. Univ. Wisconsin Press,
Madison, Wisconsin, 1982.
[5] Borg, K.E., Lunstra, D.D. and Christenson, R.K., Semen characteristics, testicular
size, and reproductive hormone concentrations in mature Duroc, Meishan, Fengjing,
and Minzhu boars, Biol. Reprod., 49 (1993) 515-521.
[6] Bruhn, T., Parvizi, N. and EUendorf, F., Ontogeny of hypothalamus-pituitary
fimction in the fetal pig: Gonadotropin release in response to electrical and
electrochemical stimulation of the hypothalamus. Endocrinology, 112 (1983) 639-644. 161 [7] Buchanan, K.L. and Yellon, S.M., Developmental smdy of GnRH neuronal
projections to the medial basal hypothalamus of the male Djungaiian hamster, J.
Comp Neurol, 333 (1993) 236-245.
[8] Caldani, M., Batailler, M., Thiery, J.C. and Dubois, M.P., LHRH-immimoreactive
structures in the sheep brain. Histochemistry, 89 (1988) 129-139.
[9] Calka, J., Majewski, M., Kaleczyc, J, and Lakomy, M., Immimocytochemical
demonstration of neuropeptide Y and luteinizing hormone-releasing honnone-
immunoreactive structures in the organum vasculosum laminae terminales of juvenile
gilts, Neurosci. Lett., 158 (1993) 21-24.
[10] Dacheux, F., Functional differentiation of the anterior pituitary cells in the fetal pig,
CeU Tissue Res., 235 (1984) 623-633.
[11] Danchin, E. and Dubois, M.P., Immunocytological study of the chronology of
pituitary cytogenesis in the domestic pig {Sus scrofa) with special reference to the
ftinctioning of the hypothalamo-pituitary-gonadal axis, Reprod. Nutr. Develop., 22
(1982) 135-151.
[12] Ford, J.J., Christenson, R.K. and Maurer, R.R., Serum testosterone concentrations
in embryonic and fetal pigs diiring sexual differentiation, Biol. Reprod., 23 (1980)
583-587.
[13] Ford, J.J., Wise, T., Lunstra, D.D. and Klindt, J., Follicle-stimulating hormone
secretion in purebred Meishan (MS) and white crossbred (WC) boars, J. Anim. Sci.,
72 (1994) 77 (abst. 163). [14] Ford, S.P. and Youngs, C.R., Early embryonic development in prolific Meishan
pigs, J. Reprod. Fertil. Suppl., 48 (1993) 271-278.
[15] Goxe, B., Prunier, A., Remy, J.J. and Salesse, R., Ontogeny of gonadal luteinizing
hormone and follicle-stimulating hormone receptors in the fetal pig and related
changes in gonadotropin and testosterone secretion, Biol. Reprod., 49 (1993) 609-616.
[16] Gross, D.S. and Baker, B.L., Immunohistochemical localization of gonadotropin-
releasing hormone (GnRH) in the fetal and early postaatal mouse brain. Am. J. Amt.,
148 (1977) 195-216.
[17] Gross, D.S. and Baker, B.L., Developmental correlation between hypothalamic
gonadotropin-releasing hormone and hypophysial luteinizing hormone. Am. J. Amt.,
154 (1979) 1-10.
[18] Haley, C.S. and Lee, GJ., Genetic basis of prolificacy in Meishan pigs, J. Reprod.
Fertil. Suppl, 48 (1993) 247-259.
[19] Harayama, H., Nanjo, I., Kanda, S. and Kato, S., Testicular development in Chinese
Meishan boars, Theriogenology, 36 (1991) 637-643.
[20] Hays, V.W., Swine production in animal agriculture in China. In: Hoefer, J. A. and
Tsuchitani P.J. (Eds.), Report 11 Submitted-Committee on Scholarly Communication
with the People's Republic of China, National Academic Press, Washington, D.C.,
1980, pp. 41-51.
[21] Jennes, L., Prenatal development of the gonadotropin-releasing hormone-containing
systems in rat brain. Brain Res., 482 (1989) 97-108. 163 [22] Jennes, L. and Conn, P.M., Gonadotropin-releasing hormone and its receptors in rat
brain, Fronr. NeuroendocrinoL, 15 (1994) 51-77.
[23] Jeones, L. and Stumpf, W.E., LHRH-systems in the brain of the golden hamster,
CeU Tissue Res., 209 (1980) 239-256.
[24] Joseph, S.A., Piekut, D.T. and Knigge, K.M., Immmiocytochemical localization of
luteinizing hormone releasing hormone (UlRH) in vibratome sectioned brain, J.
Histochem. Cytochem., 29 (1981) 247-254.
[25] Kalra, S.P., Mandatory neuropeptide-steroid signaling for the preovulatory luteinizing
hormone-releasing hormone discharge. Endocrine Rev., 14 (1993) 507-538.
[26] Kalra, S.P. and Crowley, W.R., Norepinephrine-like effects of neuropeptide Y on
LH release in the rat. Life ScL, 35 (1984) 1173-1176.
[27] Kalra, S.P. and Crowley, W.R., Neuropeptide Y: A novel neuroendocrine peptide
in the control of pituitary hormone secretion, and its relation to luteinizing hormone.
Front. NeuroendocrinoL, 13 (1992) 1-46.
[28] BCineman, R.D., Leshin, L.S., Crim, J.W., Rampacek, G.B. and Kraeling, R.R.,
Localization of luteinizing hormone-releasing hormone in the forebrain of the pig,
Biol. Reprod., 39 (1988) 665-672.
[29] Kumar, M.S.A., Becker, T. and Ebert, K., Distribution of substance P, GnRH, met-
enkephalin in the central nervous system of the pig, Brcdn Res. Bull., 26 (1991) 511-
514. [30] Legault, C., Sellier, P., Caritez, J.C., Dando, P., and Gruand, J., L'experimentation
sur le poiccbinois en France, n. Performances de production en croisement avec les
races europeenes, Genet Sel. EvoL, 17 (1985) 133-140.
[31] Leshin, L.S., Kineman, R.D., Crim, J.W., Rampacek, G.B. and Kraeling, R.R.,
Inununocytochemical localization of luteinizing hormone-releasing hormone within
the olfactory bulbs of pigs, Biol. Reprod., 44 (1991) 299-304.
[32] Lopez, F.J., Meade Jr., E.H. and Negro-Vilar, A., Endogenous galanin modulates
the gonadotropin and prolactin proestrous surges in the rat. Endocrinology, 132
(1993) 795-800.
[33] Lopez, F.J., Merchenthaler, L, Ching, M., Wisniewski, M.G. and Negro-Vilar, A.,
(jalanin; A hypothalamic-hypophysiotropic hormone modulating reproductive
functions, Proc. Natl. Acad. Sci. USA, 88 (1991) 4508-4512.
[34] Lopez, F.J. and Negro-Vilar, A., Galanin stimulates luteinizing hormone-releasing
hormone secretion firom arcuate nucleus-median eminence fragments in vitro;
involvement of an a-adrenergic mechanism. Endocrinology, 127 (1990) 2431-2436.
[35] Lunstra, D.D., Borg, K.E. and Klindt, J., Characterization of pubertal development
in the Meishan Chinese boar, J. Anim. Sci., 70 (1992) 267 (abst. 514).
[36] Matsuo, H., Baba, Y., Nair, R.M.G., Arimura, A. and Schally, A.V., Structure of
the porcine LH and FSH-releasing hormone. I. The proposed amino acid sequence,
Biochem. Biophys. Res. Comm., 43 (1971) 1334-1339. 165 [37] Meijer, J., Foot, P., Molenaar, G. and Goede, R., PAP complex: A pitfall in
immunocytochemistry of the pig hypothalamus, J. Neurosd. Methods, 17 (1986) 269-
274.
[38] Melander, T., Hokfelt, T., Rokaeus, A., Cuello, A.C., Oertel, W., Verhofstad, A.
and Goldstein, M., Coexistence of galanin-like immunoreactivity with
catecholamines, 5-hydroxytryptamine, GABA, and neuropeptides in the rat CNS, J.
Neurosd., 6 (1986) 3640-3654.
[39] Merchenthaler, I., Gores, T., Setalo, G., Petrusz, P. and Flerko, B., Gonadotropin-
releasing hormone (GnRH) neurons and pathways in the rat brain, Cell Tissue Res.,
237 (1984) 15-29.
[40] Merchenthaler, I., Lennard, D.E., Lopez, F.J. and Negro-Vilar, A., Neonatal
inq)rinting predetermines the sexually dimorphic, estrogen-dependent expression of
galanin in luteinizing hormone-releasing hormone neurons, Proc. Natl. Acad. Sd.
USA, 90 (1993) 10479-10483.
[41] Merchenthaler, I., Lopez, F.J., Lennard, D.E. and Negro-Vilar, A., Sexual
differences in the distribution of neurons coexpressing galanin and luteinizing
hormone-releasing hormone in the rat brain. Endocrinology, 129 (1991) 1977-1986.
[42] Merchenthaler, L, Setalo, G., Csontos, C., Petrusz, P., Rerko, B. and Negro-Vilar,
A., Combined retrograde tracing and immunocytochemical identification of
luteinizing hormone-releasing hormone- and somatostatin-contaimng neurons
projecting to the median eminence, Endocrinology, 125 (1990) 2812-2821. 166 [43] Moss, R.L. and McCann, S.M., Induction of mating behavior in rats by luteinizing
hormone-releasing factor. Science, 181 (1973) 177-179.
[44] Ottlecz, A., Snyder, G.D. and McCann, S.M., Regulatory role of galanin in control
of hypothalamic-anterior pituitary function, Proc. Ncal. Acad. Sci. USA, 85 (1988)
9861-9865.
[45] Parker, S.L., Kalra, S.P. and Crowley, W.R., Neuropeptide Y modulates the binding
of gonadotropin-releasing hormone (GnRH) analog to anterior pituitary GnRH
receptor sites, Endocrinology, 128 (1991) 2309-2316.
[46] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd Edition.
Academic Press, San Diego, 1986.
[47] Paxinos, G., Ashwell, K.W.S. and Tork, I., Atlas of the Developing Rat Nervous
System, 2nd Edition. Academic Press, San Diego, 1994.
[48] Pearson, P.L., Anderson, L.L. and Jacobson, C.D., The prepubertal ontogeny of
neuropeptide Y-Iike immunoreactivity in the male Meishan pig brain. Dev. Brain.
Res., (1995) in press.
[49] Pearson, P.L., Anderson, L.L. and Jacobson, C.D., The prepubertal ontogeny of
galanin-like immunoreactivity in the male Meishan pig brain. Dev. Brain. Res.,
(1995) in press.
[50] Pellinemi, L.J., Ultrastructure of the early ovary and testis in pig embryos. Am. J.
Anat., 144 (1975) 89-111.
[51] Pfaff, D.W., Luteinizing hormone-releasing factor potentiates lordosis behavior in
hypophysectomized ovariectomized female rats. Science, 182 (1973) 1148-1149. 167 [52] Polkowska, J., Liwska, J. and Dubois, M.P., Ontogeny of neuropeptidergic systems:
Luteinizing hoimone releasing hormone (LHRH); somatostatin (SRIF) and
neurophysin (NF) in the hypothalamus of the domestic pig by immimocytochemistry.
Folia Histochem. CytobioL, 23 (1985) 193-200.
[53] Rodriguez-Sierra, J.F., Jacobowitz, D.M. and Blake, C.A., Effects of neuropeptide
Y on LH, FSH and TSH release in male rats. Peptides, 8 (1987) 539-542.
[54] Ronnekleiv, O.K. and Resko, J.A., Ontogeny of gonadotropin-releasing hormone-
containing neurons in early fetal development of Rhesus macaques. Endocrinology,
126 (1990) 498-511.
[55] Sahu, A., Crowley, W.R., Tatemoto, K., Balasubramaniam, A., and Kalra, S.P.,
Effects of neuropeptide Y, NPY analog (Norleucine'^-NPY), galanin, and
neuropeptide K on LH release in ovariectomized (ovx) and ovx estrogen,
progesterone-treated rats. Peptides, 8 (1987) 921-926.
[56] Sahu, A., Xu, B. and Kalra, S.P., Role of galanin in stimulation of pituitary
luteinizing hormone secretion as revealed by a specific receptor antagonist, galantide.
Endocrinology, 134 (1994) 529-536.
[57] Salinas-Zeballos, M.-E., Zeballos, G.A. and Gootman, P.M., A stereotaxic atlas of
the developing swine {Sus scrofa) forebrain. In: Tumbleson, M.E. (Ed.), Swine in
Biomedical Research, Volume 2, Plenum Press, New York, 1986.
[58] Schambra, U.B., Lauder, J.M. and Silver, J. (Eds.). Alias of the Prenatal Mouse
Brain. Academic Press, San Diego, 1992. [59] Schwanzel-Fukuda, M., Fadem, B.H., Garcia, M.S. and Pfaff, D.W.,
Immunocytochemical localization of luteinizing hormone-releasing hormone (LHRH)
in the brain and nervus teiminalis of the adult and early neonatal gray short-tailed
opossum (Monodelphis domestica), J. Comp. Neurol., 276 (1988) 44-60.
[60] Schwanzel-Fukuda, M., Morrell, J.I. and Pfaff, D.W., Ontogenesis of neurons
producing luteinizing hormone-releasing hormone (LHRH) in the nervus tenninalis
of the rat, J. Comp. Neurol., 238 (1985) 348-364.
[61] Schwanzel-Fukuda, M. and Pfaff, D.W., Origin of luteinizing hormone-releasing
hormone neurons. Nature, 338 (1989) 161-164.
[62] Schwanzel-Fukuda, M., Robinson, J.A. and Silverman, A.J., The fetal development
of the luteinizing hormone-releasing hormone (LHRH) neuronal systems of the guinea
pig brain, Brcdn Res. Bull., 7 (1981) 293-315.
[63] Shivers, B.D., Harlan, R.E., Morrell, J.I. and Pfaff, D.W., Immunocytochemical
localization of luteinizing hormone releasing hormone in male and female rat brains,
Neuroendocrinology, 36 (1983) 1-12.
[64] Silverman, A.J., Distribution of luteinizing hormone-releasiog hormone (LHRH) in
the guinea pig brain. Endocrinology, 99 (1976) 30-41.
[65] Silverman, A.J., Jhamandas, J. and Renaud, L.P., Localization of luteinizing
hormone-releasing hormone (LHRH) neurons that project to the median eminence,
/. NeuroscL, 7 (1987) 2312-2319.
[66] Sullivan, K. A. and Silverman, A. J., The ontogeny of gonadotropin-releasing hormone
neurons in the chick, Neuroendocrinology, 58 (1993) 597-608. 169
[67] Szetyn, S., Galert, D., Dynowski, J. and Hoczyk, W., The stereotaxic configuration
of hypothalamtis nerve centres in the pig. Amt. Anz. 147 (1980) 12-32.
[68] Tsuruo, Y., Kawano, H., Kagotani, Y., Hisano, S., Daikoku, S., Chihara, K.,
Zhang, T. and Yanaihara, N., Morphological evidence for neuronal regulation of
luteinizing hormone-releasing hormone-containing neurons by neuropeptide Y in the
rat septo-preoptic area, NeuroscienceLett., 110 (1990) 261-266.
[69] Van der Beek, E.M., Pool, C.W., Van Eerdenburg, F.J.C.M., Sluiter, A.A., Van
der Donk, H., Van den Hurk, R. and Weigant, V.M., Fc-mediated nonspecific
staining of porcme brain with rabbit antiseia in inmnmocytochemistry is prevented
by pre-incubation of the sera with proteins A and G, J. Histochem. Cytochem., 40
(1992) 1731-1739.
[70] Watkins, W.B., Precautions in the application of iinmunohistochemical techniques to
tissues of the pig hypothalamo-nemrohypophysial system, J. Histochem. Cytochem.,
25 (1977) 61-68.
[71] Watson, Jr., R.E., Wiegand, S.J., Clough, R.W. and Hoffinan, G.E., Use of
cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology,
Peptides, 1 (1986) 155-159.
[72] Wise, T., Lunstra, D.D. and Ford, J.J., Changes in serum testosterone and FSH in
crossbred (CX) and Meishan (MS) boars after castration and in response to steroidal
challenge, Biol. Reprod., 49 (1994) 67 (abst. 50). HO PS] Xu, B., Sahu, A., Crowley, W.R., Leranth, C., Horvath, T. and Kalra, S.P., Role
of neuropeptide-Y in episodic luteinizing hormone release in ovariectomized rats: An
excitatory component and opioid involvement. Endocrinology, 133 (1993) 747-754.
[74] Youngs, C.R., Ford, S.P., McGimies, L.K. and Anderson, L.H., Investigations into
the control of litter size in swine: I. Comparative smdies on in vitro development of
Meishan and Yorkshire preimplantation embryos, J. Aram. Sci., 71 (1993) 1561-
1565.
[75] Youngs, C.R., Christenson, L.K. and Ford, S.P., Investigations into the control of
litter size in swiue: m. A reciprocal embryo transfer smdy of early conceptus
development, J. Arum. Sci., 72 (1994) 725-731. 171
TABLE 1 Number of sets of coronal sections of porcine brain collected and approximate interval of sections processed for immunohistochemistry
AGE g30 gTO g90 gllO-pnl pnl0-pn50 sets collected 2 4 6 12 18 18 interval Otm) 20 60 100 270 405 765 172
TABLE 2 Abbreviations of brain areas used in tables
ac anterior commissure AH anterior hypothalamic area MnPO median preoptic nucleus ARC arcuate nucleus MPA medial preoptic area BM basomedial amygdaloid nucleus MS medial septum BST bed nucleus, stria tenninalis NT terminal nerve Btel basal telencephalon oc optic chiasm cc corpus callosum opt optic tract CM centromedial thalamic nucleus OVLT organum vasculosum of the lamina DA dorsal hypothalamic area terminalis DBB diagonal band of Broca PCo posterior cortical amygdaloid nucleus DLH dorsolateral hypothalamic area Pe periventricular hypothalamus DM dorsomedial hypothalamic nucleus PH posterior hypothalamus f fornix PV paraventricular thalamic nucleus ICap internal capsule PVN paraventricular hypothalamic nucleus LH lateral hypothalamic area PvPOA periventricular preoptic area LPA lateral preoptic area Re reunhins thalamic nucleus LSD lateral septum, dorsal division SCN suprachiasmatic nucleus LSV lateral septum, ventral division SFO subfornical organ ME median eminence SON supraoptic nucletis MeA medial amygdaloid nucleus SuM supramammillary nucleus mid midline, septal area VMH ventromedial hypothalamic nucleus MM mammillary area 173
TABLES Distribution of luteinizing hormone-releasing hormone-like immunoreactivity in the developing porcine brain: Cell Bodies
AREA g30 g50 gTO m alio pnl pnlO pn20 pn50
Telencephalon ac' NA^ - - +/------Btel + + NA NA NA NA NA NA NA NA DBB NA - - - + + + + + + -I- -h-l- LSD NA -i- - - MeA NA - - +/------mid + + + + + + -1- + + + + + + + + -I- MS + + -1- + + + -t- + -1- NT + + NA NA NA NA NA NA NA NA SFO NA - + + -I- + -1- + -1- -1-
Diencephalon AH - - - + -1- + + -1- -1- ARC NA - - - +/- - +/- - +/- DA - - - - +/- - - - - LH - + -I- + + + + 4--I- -I- + + -I- + + LPA NA + -I- + + + + -1- + + + + + MnPO NA - + + + -I- + -1- + + + -I- -1- + + + MPA - -1- + -I- + -I- + + 4-4- -1-4- + + OVLT - -1- + + + + + -t-H- + + + + + -I- + + Pe NA - + -1- + + + -1- + PH NA - - - - +/- +/- - -
'-Abbreviations of brain areas can be found in table 2. ^-Symbols used in this table refer to the following: -, no cell bodies detected in area or nucleus studied; +, less than 10 cell bodies detected in area or nucleus; more than 10 cell bodies detected in area or nucleus; NA, area not applicable or not smdied. Due to immaturity of brain structures, many brain areas cannot be defined at §30 and are thus listed as NA. 174
TABLE 4 Distribution of luteinizing hormone-releasing honnone-like inmiunoreactivity in developing porcine brain: Fibers
AREA g30 g50 gTO g90 gllO pnl pnlO pn20 pn50
Telencephalon BST' NA^ - - L L L L L L BTel L/M NA NA NA NA NA NA NA NA cc NA L L L DEB NA L M LM L/M L/M L/M UM L/M f NA - L L L L L L L LSD NA L L L L L L L L LSV NA - L L L L L L L MeA NA L L L - - - - - mid L M M L/M L/M L/M L/M L/M UM MS L L/M L/M L L L L L L NT L NA NA NA NA NA NA NA NA opt - L/M L L
PCo NA - L L L L L L L SFO NA - M M M M M M M
Diencephalon AH - L L/M L/M L/H L/H L/H L/H UH ARC NA L L L L L/M L/M L/M UM CM NA - - L L - - - - DA NA - - L L L L L L DUH NA L L L L L L L L DM NA -/L L L L L L L L
ICap L - LH - UU L/M L/M L/M L/M L/M UM L/M LPA NA L L/M UM L/M L/M L/M L/M UM ME NA L/M M M/H M/H M/H M/H M/H M/H MM NA - L L L L L L L MnPO NA - L/M L/M L/M LM L/M L/M UM MPA - L L/M L/M L/M UM L/M L/M UM oc - L L L L L L L L OVLT NA L M M M M/H M/H M/H M/H Pe NA - L L/M L/M L/M UM UM L/M PH - L L L L L L L L PV NA L L L L L L L L PVN NA - L L L L L L L 175
PvPOA NA - L LM IVM L/M L/M L/M L/M
Re NA - - L L - - - - SCN NA - - L L L L L L SON NA - - L L L L L L SuM NA L/M L L L L L L L VMH NA -/L L L L L L L L
'-Abbreviations of brain areas can be found in Table 2. ^-Symbols used in the table refer to the following: no inununoreactivity detected; L, low density of immunoreactivity detected; M, medium density of immunoreactivity detected; H, high density of immunoreactivity detected; and NA, area not applicable or not smdied. Due to immaturity of brain structures, many brain areas cannot be defined at g30 and are thus listed as NA. Figure 1. Examples of luteinizing hormone-releasii^ hormone-like immunoreactivity in the porcine brain classified as being either a low, medium or high density of immunnsfainiTig Low
Medium
•
ffigh Figure 2. Luteinizing hormone-releasing hormone-like immunoreactivity in the gestational day 30 pig brain. A-Immunoreactive cells and fibers at the base of the brain along the terminal nerve, coronal section. B-lmmunoreactive cells and fibers in the basal telencephalon arching toward the presumptive septal/preoptic area, sagittal section. The magnification bar indicates 50/xM and both photomicrographs are at the same magnification. The arrow indicates the medial portion of the photomicrograph in (A) and the caudal portion of the photomicrograph in (B). The arrowheads in (A) and (B) indicate the base of the brain. Abbreviation: vz-septal area, ventricular zone, gt-terminal ganglion. vz J
\ \
• V
gt
V figure 3. Luteinizing honnone-releasing hormone-like immunoreactivi^ in the organum
vasculosum of the lamina terminalis of the developing male Meishan pig brain. A- gestational day (g)SO, B-g70, C-g90 and D-postnatal day 1. The magmfication bar
indicates 100/tM. All photomicrographs are at the same magnification and were taken
from coronal sections. The line at the top of all photomicrographs indicates the midline
of the brain and the asterisk (*) denotes a blood vessel.
Figure 4. Luteinizing hormone-releasing hormone-like immunoreactivity in the ventral anterior hypothalamus of the developing pig brain, coronal section. A-gestational day
(g)90, B-gllO. The magnification bar indicates 100/tM and both photomicrographs are at the same magnification. The midline is to the right of both photomicrographs.
Abbreviation: oc-optic chiasm. / •
! / /' HguFe S. Luteinizing honnone-ieleasing hormone-like inununoreactivity in the median eminence of the developing pig brain. A-gestational day (g)50, B-g70, C-gllO. The magnification bars indicate SO/iM. The asterisk (*) indicates the third ventricle, the arrows indicate dense counterstain (non-immunoreactive) in the ependyma and/or pituitary and the arrowheads indicate light immunoreactive staining in the gSO median eminence.
186
GENERAL SUMMARY AND DISCUSSION
General summary
In the first study, we investigated the prepubertal ontogeny of NPY-IR in the male
Meishan pig brain. Neuropeptide Y-IR in cell bodies and fibers is evident in many areas of the brain at g30, including the basal telencephalon, hypothalamus, mesencephalon, pons, and medulla. Throughout prenatal development, cell bodies containing NPY-IR generally increase in number and distribution in the brain. During postnatal development the number of cell bodies displaying NPY-IR decreases. The arcuate nucleus of the hypothalamus shows a dramatic reduction in the number of immunoreactive cell bodies between pnl (day of birth) and pi]20, just before weaning. This dramatic reduction in the number of immmunoreactive cell bodies in the arcuate nucleus may indicate a functional maturation of food intake control pathways. The distribution of NPY-IR in fibers becomes more widespread throughout gestational development, showing a pattern by gllO that was characteristic of postnatal ages.
The intensity of NPY-IR in fibers also increases throughout gestation. Some additional increases in inamunoreactivity were observed postnatally, especially in the periventricular hypothalamus and the hippocampus. Other brain areas like the caudate nucleus and putamen showed decreases in immunoreactivity postnatally. These results showed that the distribution of NPY-IR in cell bodies and fibers is similar to that seen in other species, including the rat, and supports the hypothesis that NPY participates in controlling feeding, growth and reproduction in the pig.
In the second smdy, the prepubertal ontogeny of GAL-IR in the male Meishan 187 brain was investigated. Galanin-IR in cell bodies and fibers was evident in the brain at g30, primarily in the hypothalamus. Throughout prenatal development cell bodies containing GAL-IR generally increased in number and distribution in the brain. During postnatal development, the number of cell bodies displaying GAL-IR decreased, particularly in hypothalamic areas, however less drastically than that seen for cell bodies displaying NPY-IR. The distribution of GAL-IR in fibers became more widespread throughout gestational development, showing a pattern by pnl that was consistent through postnatal ages. The intensity of GAL-IR in fibers also increased throughout gestation.
Some additional increases in immunoreactivity occurred postnatally, especially in the periventricular hypothalamus. The results of this study indicate that the distribution of
GAL-IR in cell bodies and fibers in the Meishan pig brain was similar to that seen in other species, including the rat. These results support the hypothesis that GAL also participates in the control of feeding, growth and reproduction in the pig.
The third study investigated the prepubertal ontogeny of LHRH-IR in the telencephalon and diencephalon of the male Meishan pig brain. Luteinizing hormone- releasing hormone-IR in cell bodies and fibers was detected at g30 entering the brain along the terminal nerve and in the septal region of the basal telencephalon. The number of immunoreactive cells increased at g50 and cells were localized primarily to the septum, OVLT, preoptic area and lateral hypothalamus, whereas immunoreactive fibers were present throughout the septum and hypothalamus and had reached the median eminence. The density and distribution of immunoreactive fibers increased by g70 and g90, but did not change dramatically from g90-pn50. These results mdicate that LHRH 188 may be present in the Meishan brain earlier during development and fibers containing
LHRH-IR appear to reach the median eminence earlier than previously reported for the domestic pig. These results confirm the olfactory origin and apparent migration of
LHRH cells in the pig and suggest a breed difference in the ontogeny of reproductive control systems in the pig.
General discussion
As mentioned previously, NPY and GAL were both originally identified fi-om porcine
extracts in the early 1980's (Tatemoto et al., 1982; Tatemoto et al., 1983). The
neuroanatomical localization and development of these peptides has been well smdied in
other species, but not the pig. This is surprising, due to the central role these peptides play
in the control of growth and reproduction. As the pig is a species predominantly utilized for
human consumption, efficient growth, body characteristics and reproductive capacity are
paramount to producers. The information presented in this dissertation represents the first
comprehensive immunohistochemical studies investigating the localization and development
of NPY and GAL in the pig brain and extend previous investigations into the development
of LHRH-containing systems in the pig brain. These smdies utilize the Meishan pig, a
Chinese breed known for its superior reproductive characteristics (Harayama et al., 1991;
Anderson et al., 1993; Ford and Youngs, 1993; Youngs et al., 1993; Youngs et al., 1994;
Dlamini et al., 1995), but slow growth and poor feed efficiency (Hays, 1980; Legault,
1985), and detail the neuroanatomical distribution of these peptides, a necessity for future
explorations into the control of reproduction, growth and food intake in this species. 189 In these studies, brains from male Meishan pigs from g30-pn50 were used to study the prepubertal development of NPY, GAL and LHRH. At g30, the Meishan pig brain has diffeFentiated to the five vesicle stage, the midbrain is large and flexed and the cerebellar swelling is detectable as in the domestic pig (Marrable, 1971). At this age, the Meishan pig brain is similar in shape and complexly to that of an embryonic day (E) 14 rat brain
(Paxinos et al., 1994). At g50, the cortical surfaces are still smooth, but the cerebrum has enlarged considerably. Mature cortical laminae are not yet distinguishable, but the developing cortical layers are similar to that seen in the E19 rat. The overall development
and shape of the g50 pig brain is very similar to that of the E19 rat (Paxinos et al., 1994).
At g70, the cerebral cortex is corrugated and the six cortical laminae can be distinguished.
The pig brain has assumed a more mature proportion by this stage in development
(Marrable, 1971). By g90, the overall shape and proportion of the brain and the convolution
of the cortex resembled that of the older animals in this study. However, brain tissue taken
from g90 pigs was fragile in comparison to that of older animals. Brains from ages gllO-
pnSO differed apparently only in relative size.
The first smdy investigated the immunohistochemical localization of NPY throughout
prepubertal development in the male Meishan pig brain. Neuropeptide Y is a peptide
involved in the control of food intake (Clark et al., 1984; Levine and Morley, 1984; Stanley
and Leibowitz, 1984; Parrott et al., 1986), growth hormone secretion and gonadotropic
hormone secretion (Kalra and Crowley, 1984; McDonald et al., 1985; Rettori et al., 1990;
Kaka and Crowley, 1992; Catzeflis et al., 1993) and puberty onset (Sutton et al., 1988;
Minami et al., 1990; Minami et al, 1992; Fraley and Kuenzel, 1993; Gruaz et al., 1993). 190 Neuropeptide Y-IR was detected throughout the brain of the developing Meishan pig at all ages studied from g30-pn50. Cell bodies containing NPY-ER generally increase in number and distribution in the brain throughout prenatal development. During postnatal development, the number of cell bodies displaying NPY-IR decreases. The arcuate nucleus of the hypothalamus, shows a dramatic reduction in the number of immimoreactive cell bodies between pnl and pn20, just before weaning. The distribution of NPY-IR in fibers becomes more widespread diroughout gestational development, showing a pattern by g 110 that was characteristic of postnatal ages. The intensity of NPY-IR in fibers also increases throughout gestation. Some additional increases in immunoreactivity occur postoatally, especially in the periventricular hypothalamus and the hippocampus. Other brain areas like the caudate nucleus and putamen show decreases in immtmoreactivity postnatally.
At g30, the earliest timepoint stodied, NPY-IR was widespread throughout the relatively immature brain. Cell bodies containing NPY-IR were observed in the forming hypothalamus and brainstem, two primary sources of NPY in the adult brain (Chronwall et al., 1985; DeQuidt and Emson, 1986).
At g50, NPY-IR was more widespread throughout the brain. As the brain is more mature at this stage, NPY-IR in cells and fibers could be localized to discrete brain areas or nuclei. Cell bodies containing NPY-IR were detected in the ARC at this age. The ARC is a major source of NPY innervation of the ME and PVN in the adult animal (reviewed in
Leibowitz, 1992) and these cells may play an important role in food intake regulation.
Interestingly, NPY-IR was detected in the ME at this early age. Previous stodies in pig have shown that differentiation of portal capillaries in the ME is not complete until g70 (Danchin 191 and Dubois, 1982) and it is unlikely that pituitary secretion could be affected from the ME at this stage of development.
The distribution of NPY-ER in the g70 brain is even more extensive and the density of inmumoreactivi^ increases dramatically at this stage as well, especially in the hypothalamus. The innervation of the ME by fibers containing NPY-IR was more extensive at this age also, coinciding with the observation that hypothalamic control of pituitary function matures between g60 and g80 in the pig (Bruhn et al., 1983). A further dramatic increase in NPY-IR distribution and density was observed at g90. Most notable was the increase in NPY-IR in the PVN. A previous smdy in the pig indicated that neurophysin immunoreactivity was detected in the developing supraoptic nucleus at g60, but was not seen in the PVN until g87 (Ellis and Watkins, 1975), suggesting that cells in this nucleus may not acquire 'mature' characteristics, such as the expression of neurosecretory peptides, until this age. Whether the dense innervation of this nucleus by fibers containing NPY-IR seen at g90 in this study indicates a direct effect of NPY on the development of this nucleus or is merely coincidental is imknown. Further smdy will be required to ascertain the significance of this observation.
A further increase in the distribution and density of NPY-IR is observed in several brain areas at gllO, just prior to birth, especially in the periventricular hypothalamic area
and cerebral cortex. Other areas, such as the compact substantia nigra show a decline in
immunoreactivity. The general pattern of immunoreactive staining in the brain is consistent
from gll0-pn50, with increases or decreases in immunoFeactivity in specific areas.
Decreases in immunoreactivity may be due to 'dilution' of NPY-IR in fibers resulting from 192 brain growth rather than a true decrease in immunoreactive fibers. Areas like the hippocanq>us and medial preoptic area which show an increase in immunoreactivi^ in the postnatal period may be undergoing developmental changes in structure or function.
Immunoreactive content in the medial preoptic area increases until puberty in the female rat
(Sutton et al., 1988) and may relate to the onset of puberty. It is unknown if a similar phenomena occurs in the pig, but is worthy of study, particularly in comparisons of the
Meishan pig which undergoes precocial puberty in relation to American or European breeds
(Harayama et al., 1991)
A highly interesting result was the rapid decline in immunoreactive cell bodies in the
ARC in the early postnatal period (pnl-pn20). A similar result has been reported in the rat as well (Woodhams et al., 1985; Kagotani et al., 1989), despite the relative differences in maturity of the animal at birth. A previous study in our laboratory indicated an increase in inmiunoreactive cholecystokinin levels as measured by RIA in the hypothalamus and brainstem of the Meishan between pnl and pn20 (Elmquist et al., 1993b) also.
Cholecystokinin is another neuropeptide involved in the control of food intake (reviewed in
Gibbs and Smith, 1991). Taken together these results suggest that this observation may relate to a functional maturation of food intake control pathways. However, it is also possible that cells in the ARC die, cease synthesis of this peptide, or that technical considerations are responsible for this decline in immunoreactive cell bodies. As the PVN is heavily innervated by NPY cells in the ARC (reviewed in Leibowitz, 1992), and no decline in immunoreactivity is detected at these ages, the possibility that NPY cells die or cease synthesis is unlikely.
Immunoreactive cell bodies are detected in other brain areas in similar nimibers over this 193
dme period, suggesting that no general technical flaw is responsible for this observation.
Reports of NPY-IR in ARC cell bodies in postnatal animals from smdies (Chronwall et al.,
1985; Smithetal., 1985; DeQuidt andEmson, 1986; Huetal., 1987; Sabatinoetal., 1987;
Reuss et al., 1990) employing colchicine, an inhibitor of axonal transport (Cortes et al.,
1990), support the hypothesis that increased transport of this peptide from the cell bodies may be a plausible explanation for this observation. It is possible, however, that an unknown change in NPY synthesis or ARC cell characteristics may also be responsible for
this observation.
The overall development and distribution of NPY-IR in the pig brain is similar to that
seen in other species and supports the hypothesis that NPY participates in the control of
growth, reproduction and food intake in the pig brain.
In the second smdy, we investigated the prepubertal development of GAL-IR in the
male Meishan brain. As mentioned previously, GAL is involved in the control of food
intake (Kyrkouli et al., 1986,1990), growth hormone secretion (Murakami et al., 1989) and gonadotropic hormone secretion (Lopez et al., 1991, 1993). As seen in the first study,
GAL-IR in cells and fibers was detected in all ages from g30-pn50. Throughout prenatal
development, cell bodies containing GAL-IR generally increased in number and distribution
in the brain. During postnatal development, the number of cell bodies displaying GAL-IR
decreased, particularly in hypothalamic areas. The distribution of GAL-IR in fibers became
more widespread throughout gestational development, showing a pattern by pnl that
contiaued during later postnatal ages. The intensity of GAL-IR in fibers also increased
throughout gestation. Some additional increases in immunoreactiviQ^ occurred postnatally. 194 especially in the periventricular hypothalamus.
At g30, GAL-IR in cell bodies and fibers was detected in the brain, but was limited primarily to the developing hypothalamus. Galanin-IR was considerably less widespread throughout the brain at g30 than NPY-IR, suggesting that NPYergic systems may develop earlier than GALergic systems in the pig brain, but fiuther study on pigs at earlier gestational ages will be required to confirm this hypothesis.
Increases in the distribution and density of GAL-IR were evident at g50 in the
Meishan brain. Immimoreactive cell bodies were detected in the ARC at g50 and GAL-IR was evident in the ME, similar to that seen for NPY. Galanin-IR in cells and fibers was also observed for the first time in the braiostem at g50. A developmental stody of GAL in the
Brazilian opossum in our laboratory suggested that brainstem GALergic systems may develop prior to hypodialamic ^sterns (Ehnguist et al., 1992). The results of this smdy suggest that this is not the case in the pig. Further increases in immunoreactivity were observed in the g70, g90 and gllO prenatal pig brain, especially in telencephalic and diencephalic areas, reaching a characteristic pattern of immunoreactivity by pnl. Further increases in inmnmoreactivity were evident in the periventricular hypothalamus and periventricular preoptic area in the postnatal period. A decline in immunoreactive cell number in the ARC was also observed in this study in the early postnatal period (pnl and pnlO). However, a significant population of cell bodies containing GAL-IR were still observed at pn50. This observation further reinforces the possibility of a functional maturation of peptidergic pathways in the early postnatal period. The detection of immunoreactive ceil bodies at all ages in the ARC further argues against a technical reason for this observation. 195 The results of this study indicate that the distribution of GAL-IR in cell bodies and fibers in the Meishan pig brain was similar to that seen in other species, including the rat.
These results support the hypothesis that GAL participates in the control of feeding, growth and reproduction in the pig.
The early detection (g30) of NPY and GAL in the pig brain suggests a possible developmental role for these peptides. Other neuropeptides such as vasopressin, gastrin, bombesin and substance P have been shown to have trophic effects either in vitro or in vivo
(reviewed in Zachary et al., 1987). Previous smdies in our laboratory have shown GAL-IR and GAL binding sites present as early as pnl in the Brazilian opossum (Ehnquist et al.,
1993a). There no reports detailing the ontogeny of NPY or GAL receptors in the brain of the pig. Thus, it is not known whether these binding sites are present in the pig brain at the timepoints that these peptides are detected by immunohistochemistiy. It is not known at this time whether GAL or NPY may be playing a role in the morphogenesis or fimctioning of the developing brain. Further studies using fimction-blocking antibodies or receptor antagonists may yield evidence supporting a fimctional role for these peptides during development.
The third smdy investigated the prepubertal ontogeny of LHRH-ER. in the male
Meishan pig brain. As mentioned previously, LEIRH is a decapeptide involved in the control of reproductive behaviors (Moss and McCann, 1973; Pfaff, 1973) and secretion of LH and
FSH from the anterior pitoitary gland (reviewed in Jennes and Conn, 1994). As observed in the two previous studies, LHRH-IR was detected in cell bodies and fibers in all ages from g30-pn50. Luteinizing hormone-releasing hormone-IR in cell bodies and fibers was detected 196 at g30 in the olfactory epithelium, entering the brain via the terminal nerve and in the septal region of the basal telencephalon. This observation extends the results of previous smdies in the domestic pig (Danchin and Dubois, 1982) which did not detect LHRH-ER in cell bodies in the porcine brain until g40 and confirms the olfactoiy origin of LHRH cells in the pig, as seen in other species (Jennes and Stumpf, 1980; Schwanzel-Fukuda et al., 1981,
1985, 1988; Schwanzel-Fukuda and Pfaff, 1989).
The number of immimoreactive cells increased at g50 and cells were localized primarily to the septum, organum vasculosum of the lamina terminalis, preoptic area and lateral hypothalamiis, whereas immimoreactive fibers were present throughout the septum and hypothalamus and had reached the ME. Very few immunoreactive fibers are detected in the
ME at this age and it is likely that these are 'pioneering' projections to the ME and may represent the first fibers innervating this structure. Fibers containing LHRH-IR were not seen until g60 in the previous study in the domestic pig. As mentioned previously, the portal capillaries have not differentiated by this stage of development (Danchin and Dubois, 1982), and it is unlikely that LHRH-IR released in the ME could affect pituitary secretion at this stage in development. The observation of cell bodies in the brain and fibers in the ME of the Meishan ten days prior to that observed in the domestic pig suggest a breed difference in the development of reproductive control systems. This hypothesis is attractive, as the
Meishan undergoes puberty earlier than domestic pig breeds (Harayama et al., 1991).
However, the previous study utilized paraffin-embedded tissue, which has subsequently been shown to decrease the amount of immunoreactive LHRH present (Joseph et al., 1981).
Other factors including the timepoints of smdy chosen or the sensitivity of the respective 197 immunohistochemical techniques may explain the differing results obtained. Further study
directly comparing the developmental localization of LHRH-IR in the Meishan and domestic
breeds should be conducted to clarify this issue.
The density and distribution of immunoreactive fibers increased by g70 and g90, but
•>?-' did not change dramatically from g90-pnS0. This increase was particularly notable in the
ME and again corresponds with the previous observation that hypothalamic control of
pituitary function matures between g60 and g80 in the pig (Bruhn et al., 1983). These
results indicate that LHRH-containing systems establish their neuroanatomical pathways
during prenatal development and change litde throughout prepubertal development in the pig.
As mentioned previously, both NPY and GAL are stimulatory to LHRH secretion
(Sahu et al., 1987; Sahu et al., 1994). Neuropeptide Y-IR and GAL-IR was first detected
in the ME at g50, the same timepoint that LHRH-IR was detected in the ME. This finding iV suggests a concurrent development of the systems controlling gonadotropic hormone secretion
in the pig hypothalamo-hypophyseal system. As mentioned previously, NPY-IR, GAL-IR
and LHRH-IR increases in the ME at g70 and g90. As hypothalamic control of pituitary
fimction matures between g60 and g80 in the pig, with secretion mimicking that seen in the
adult animal by gl05 (Bruhn et al., 1983), it is possible that these peptides may all affect
pituitary secretion of LH and FSH at this early stage of development.
Cells containing both LHRH-IR and GAL-IR are present in high proportion in the rat
OVLT and diagonal band of Broca, particularly in the female (Merchenthaler et al., 1990,
1991). Double-label immunohistochemistry for GAL and LHRH has yet to be performed
in the Meishan pig, so it is not known if these peptides are colocalized in the pig. However, 198 cells containing GAL-IR or LHRH-ER are found in the preoptic area and hypothalamus, suggesting this possibility. To date there are no reports co-localizing NPY and GAL.
Neuropeptide Y-IR and GAL-IR are seen separately in cell bodies of the ARC of the
Meishan pig, but double-label inmiimohistochemistry has yet to be performed to investigate the possible co-localization of these peptides.
In sunmiaiy, we have described the prepubertal ontogeny of neuropeptides controlling food intake, growth and reproduction in the male Meishan pig brain. The results contained in this dissertation provide the first comprehensive immunohistochemical and developmental descriptions of NPY- and GAL-containing systems in the porcine brain and extend the previous developmental descriptions of IJIRH-containing systems in the pig. The spatial and temporal localization of these peptides support their respective roles in controlling somatotropic and gonadotropic axes in the pig. These smdies provide a long overdue characterization of these systems in the pig brain, and it is hoped that these smdies will lay the foundation for future explorations into the mechanisms controlling food intake, growth and reproduction in the pig. 199
REFERENCES
Abe, M., Saito, M., Ikeda H. and Shimazu, T., Increased neuropeptide Y content in the
arcuato-paraventricular hypothalamic neuronal system in both insulin-dependent
and non-insulin-dependent diabetic rats. Brain Res., 539 (1991) 223-227.
Adrian, T.E., Allen, J.M., Bloom, S.R., Ghatei, M.A., Rossor, M.N., Crow, T.J.,
Tatemoto, K. and Polak, J.M., Neuropeptide Y distribution in human brain.
Nature, 306 (1983) 584-586.
Albers, H.E. and Ferris, C.F., Neuropeptide Y: Role in light-dark entrainment of
hamster circadian rhythms, Neurosci. Lett., 50 (1984) 163-168.
Allen, Y.S., Adrian, T.E., Allen, J.M., Tatemoto, K., Crow, T.J., Bloom, S.R. and
Polak, J.M., Neuropeptide Y distribution in rat brain. Science, 221 (1983) 877-
879.
Anderson, L.H., Christenson, L.K., Christenson, R.K. and Ford, S.P., Investigations
into the control of litter size in swine: EE. Comparisons of morphological and
ftmctional embryonic diversity between Chinese and American breeds, J. Anim.
ScL, 71 (1993) 1566-1571.
Anderson, L.L., Pigs. In: Hafez, E.S.E. (Ed.) Reproduction in Farm Animals, 5th
Edition, Lea and Febiger, Philadelphia, 1987. Bai, F.L., Yamano, M., Shiotani, Y., Emson, P.C., Smith, A.D., Powell, J.F. and
Tohyama, M., An aicuato-paraventricular and -dorsomedial hypothalamic
neuropeptide Y-containing system which lacks noradrenaline in the rat. Brcdn
Res. 331 (1985) 172-175.
Barry, J. Characterization and topography of UHRH neurons in the human brain,
Neurosci. Lett, 3 (1976) 287-291.
Beck, B., Burlet, A., Bazin, R., Nicolas, J.-P. and Burlet, C., Elevated neuropeptide Y
in the arcuate nucleus of yoimg obese Zucker rats may contribute to the
development of their overeating, J. Nutr., 123 (1993) 1168-1172.
Bretherton-Watt, D., Kenny, M.J., Ghatei, M.A. and Bloom, S.R., The distribution of
galanin message-associated peptide-like immimoreactivity in the pig, Regid. Pept.,
27 (1990) 307-315.
Bruhn, T., Parvizi, N. and Ellendorf, F., Ontogeny of hypothalamus-pituitary function in
the fetal pig: Gonadotropin release in response to electrical and electrochemical
stimulation of the hypothalamus. Endocrinology, 112 (1983) 639-644.
Buchanan, K.L. and Yellon, S.M., Developmental study of GnRH neuronal projections
to the medial basal hypothalamus of the male Djungarian hamster, J. Comp
Neurol, 333 (1993) 236-245.
Busch-Sorenson, M., Sheikh, S.P., O'Hare, M., Tortora, O., Schwartz, T.W. and
Gammeltoft, S., Regional distribution of neuropeptide Y and its receptor in the
porcine central nervous system, /. Neurochem., 52 (1989) 1545-1552. Caldani, M., Batailler, M., Thiery, J.C. and Ehibois, M.P., LHRH-inmunoreactive
stmctuies in the sheep brain. Histochemistry, 89 (1988) 129-139.
Calka, J., Majewski, M., Kaleczyc, J. and Lakomy, M., Immunocytochemical
demonstration of neuropeptide Y and luteinizing hormone-ieleasing hormone-
immunoreactive structures in the organum vasculosum laminae tenninalis of
juvenile gilts, Neurosd. Lett., 156 (1993) 21-24.
Catzeflis, C., Pierroz, D.D., Rohner-Jeanrenaud, F., Rivier, J.E., Sizonenko, P.C. and
Aubert, M.L., Neuropeptide Y administered chronically into the lateral ventricle
profoimdly inhibits both the gonadotropic and the somatotropic axis in intact adult
female rats. Endocrinology, 132 (1993) 224-234.
Ch'ng, J.L., Christofides, N.D., Anand, P., Gibson, S.J., Allen, Y.S., Su, H.C.,
Tatemoto, K., Morrison, J.F., Polak, J.M. and Bloom, S.R., Distribution of
galanin inmiunoreactivity in the central nervous system and the responses of
galanin-containing neuronal pathways to injury, Neuroscience, 16 (1985) 343-354.
Chronwall, B.M., DiMaggio, D.A., Massari, VJ., Pickel, V.M., Ruggiero, D.A. and
O'Donohue, T.L., The anatomy of neuropeptide-Y-containing neurons in rat
brain, Neuroscience, 15 (1985) 1159-1181.
Clark, J.T., Kahra, P.S., Crowley, W.R. and Kaka, S.P., Neuropeptide Y and human
pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology, 115
(1984) 427-429.
Clark, J.T., Kalra, P.S. and Kalra, S.P., Neuropeptide Y stimulates feeding but inhibits
sexual behavior in rats. Endocrinology, 117 (1985) 2435-2442. Cortes, R., Ceccatelli, S., Schalling, M. and Hokfelt, T., Differential effects of
intracerebroventricular colchicine administration on the expression of mRNAs for
neuropeptides and neurotransmitter enzymes, with special emphasis on galanin: an
in situ hybridization stody. Synapse, 6 (1990) 369-391.
Crowley, W.R. and Kalra, S.P., Neuropeptide Y stimxilates the release of luteinizing
hormone-releasing hormone from medial basal hypothalamus in vitro: Modulation
by ovarian hormones, Neuroendocrinology, 46 (1987) 97-103.
Dacheux, F., Functional differentiation of the anterior pituitary cells in the fetal pig. Cell
Tissue Res., 235 (1984) 623-633.
Danchin, E. and Dubois, M.P., Immunocytological study of the chronology of pituitary
cytogenesis in domestic pig (Sus scrofa) with special reference to the fimctioning
of the hypothalamo-pituitaiy-gonadal axis, Reprod. Nutr. Develop., 22 (1982) 135-
151.
De Quidt, M.E. and Emson, P.C., Distribution of neuropeptide Y-like immxmoreactivity
in the rat central nervous system-II. Immunohistochemical analysis, Neuroscience,
18 (1986) 545-618.
Dlamini, B.J., Li, Y., Klindt, J. and Anderson, L.L., Acute shifts in relaxin,
progesterone, prolactin and growth hormone secretion in Chinese Meishan gilts
during late pregnancy and after hysterectomy, J. Anim. Set., (1995) in press.
Dumont, Y., Foumier, A., St. Pierre, S. and Quirion, R., Comparative characterization
and autoradiographic distribution of neuropeptide Y brain. J. Neurosci., 13
(1993) 73-86. Ellis, H.K. and Watkins, W.B., Ontogeny of the pig hypothalamic neurosecretory system
with particular reference to the distribution of neurophysin. Cell Tissue Res., 164
(1975) 543-557.
Ebnquist, J.K., Fox, C.A., Ross, L.R. and Jacobson, C.D., Galanin-like
immunoreactivity in the adult and developing Brazilian opossum brain. Dev. Brain
Res., 67 (1992) 161-179.
Ehnquist, J.K., Kao, A., Kuehl-Kovarik, M.C. and Jacobson, C.D., Developmental
profile of galanin binding sites in the mammalian brain, Mol. Cell. Neurosci., 4
(1993a) 354-365.
Ehnquist, J.K., Ross, L.R., Hsu, W., Rothschild, M.F. and Jacobson, C.D.,
Cholecystokinin like inmiunoreactivity in the brains of yoxmg Meishan and Duroc
pigs, J. Anim. Breed. Genet., 110 (1993b) 473-479.
Elsaesser, F. and Parvizi, N., Estrogen feedback in the pig: Sexual differentiation and the
effect of prenatal testosterone treatment, Biol. Reprod., 20 (1979) 1187-1193.
Ford, J.J., Christenson, R.K. and Maurer, R.R., Serum testosterone concentrations in
embryonic and fetal pigs during sexual differentiation, Biol. Reprod., 23 (1980)
583-587.
Ford, S.P. and Youi^s, C.R., Early embryonic development in prolific Meishan pigs, J.
Reprod. Fertil. Suppl., 48 (1993) 271-278.
Fraley, G.S. and Kuenzel, W.J. Precocious puberty in chicks [Gallus domesticus) induced
by central injections of neuropeptide Y, Life Sci., 52 (1993) 1649-1656. Gibbs, J. and G.P. Smith. Qiolecystokinin and satieQr; problems in brain-gut
interactions. In: Brain-Gia Interactions, Y. Tache and D. Wingate, Eds., Boca
Raton, FL: CRC, 1991, pp. 255-262.
Gross, D.S. and Baker, B.L., Immunohistochemical localization of gonadotropin-
releasing hormone (GnRH) in the fetal and early postnatal mouse brain. Am. J.
Anat., 148 (1977) 195-216.
Gruaz, N.M., Pierroz, D.D., Rohner-Jeanrenaud, F., Sizonenko, P.C. and Aubert,
M.L., Evidence that neuropeptide Y could represent a neuroendocrine inhibitor of
sexual maturation in unfavorable metabolic conditions in the rat. Endocrinology,
133 (1993) 1891-1894.
Haley, C.S. and Lee, G.J., Genetic basis of prolificacy in Meishan pigs, J. Reprod.
Fertil. SuppL, 48 (1993) 247-259.
Harayama, H., Nanjo, I., BCanda, S. and Kato, S., Testicular development in Chinese
Meishan boars, Theriogenology, 36 (1991) 637-643.
Hays, V.W., Swine production in animal agriculture in China. In: Hoefer, J. A. and
Tsuchitani P.J. (Eds.), Report 11 Submitted-Committee on Scholarly
Communication with the People's Republic of China, National Academic Press,
Washington, D.C., 1980, pp. 41-51.
Hisano, S., Tsuruo, Y., Kagotani, Y., Daikoku, S. and Chihara, K.,
Immunohistochemical evidence for synaptic connections between neuropeptide Y-
containing axons and periventricular somatostatin neurons in the anterior
hypothalamus in rats. Brain Res., 520 (1990) 170-177. 205 Hu, H., Rao, J.K., Prasad, C. and Jayaraman, A., Localization of neuropeptide Y-like
immunoieactivity in the cat hypothalamus. Peptides, 8 (1987) 569-573.
Hulting, A.L., Meister, B., Carlsson, L., Hilding, A. and Isaksson, O., On the role of
the peptide galanin in regulation of growth hormone secretion, Acta Endocrinol
(Copenh), 121 (1991) 518-525.
Jennes, L., Prenatal development of the gonadotropin-releasing hormone-containing
systems in rat brain. Brain Res., 482 (1989) 97-108.
Jennes, L. and Conn, P.M., Gonadotropin-releasing hormone and its receptors in rat
brain. Front. Neuroendocrinology, 15 (1994) 51-77.
Jennes, L. and Stumpf, W.E., LHRH-systems in the brain of the golden hamster. Cell
Tissue Res., 209 (1980) 239-256.
Joseph, S.A., I^ekut, D.T. and Knigge, K.M., Immunocytochemical localization of
luteinizing hormone releasing hormone (LHRH) in vibratome sectioned brain, J.
Histochem. Cytochem., 29 (1981) 247-254.
Kagotani, Y., Hashimoto, T., Tsuruo, Y., Kawano, H., Daikoku, S. and Chihara K.,
Development of the neuronal system containing neuropepude Y in the rat
hypothalamus, Ira. J. Devi. Neurosci., 7 (1989) 359-374.
Kalra, S.P., Mandatory neuropeptide-steroid signalling for the preovulatory luteinizing
hormone-releasing hormone discharge. Endocrine Rev., 14 (1993) 507-538.
Kalra, S.P. and Crowley, W.R., Norepinephrine-like effects of neuropeptide Y on LH
release in the rat, Life Sci., 35 (1984) 1173-1176. Kalra, S.P. and Crowley, W.R., Neuropeptide Y: A novel neuroendocrine peptide in the
control of pituitary hormone secretion, and its relation to luteinizing hormone.
Front. Neuroendocrinology, 13 (1992) 1-46.
IQneman, R.D., Leshin, L.S., Crim, J.W., Rampacek, G.B. and Kraeling, R.R.,
Localization of luteinizing hormone-releasing hormone in the forebrain of the pig,
Biol. Reprod., 39 (1988) 665-672.
Kumar, M.S.A., Becker, T. and Ebert, K., Distribution of substance P, GnRH, met-
enkephalin in the central nervous system of the pig. Brain Res. Bull., 26 (1991)
511-514.
Kyrkouli, S.E., Stanley, E.G. and Leibowitz, S.F., Galanin: stimulation of feeding
induced by medial hypothalamic injection of this novel peptide, Eur. J.
Pharmacol, 122 (1986) 159-160.
Kyrkouli, S.E., Stanley, E.G., Seirafi, B.D. and Leibowitz, S.F., Stimulation of feeding
by galanin: anatomical localization and behavioral specificity of this peptide's
effects in the brain. Peptides, 11 (1990) 995-1001.
Leibowitz, S.F., Sladek, C., Spencer, L. and Tempel, D., Neuropeptide Y, epinephrine,
and norepinephrine in the paraventricular nucleus; Stimulation of feeding and the
release of corticosterone, vasopressin and glucose. Brain Res. Bull., 21 (1988)
905-912. ion Leibowitz, S.F., Hypothalamic neuropeptide Y, galanin, and amines: Concepts of
coexistence in relation to feeding behavior. Am. N.Y. Acad. Sci., 575 (1989) 221-
233.
Leibowitz, S.F., Neurochemical-neuroendocrine systems m the brain controlling
macronutrient intake and metabolism. Trends Neurosci., 15 (1992) 491-497.
Legault, C., Sellier, P., Caritez, J.C., Dando, P., and Gruand, J., L'experimentation sur
le porcchinois en France. H. Performances de production en croisement avec les
races europeenes. Genet Sel. Evol., 17 (1985) 133-140.
Leshin, L.S., Barb, C.R., Kiser, T.E., Rampacek, G.B. and Kraeling, R.R., Growth
hormone-releasing hormone and somatostatin nemrons within the porcine and
bovine hypothalamus, Neuroendocrinology, 59 (1994) 251-264.
Leshin, L.S., Kineman, R.D., Crim, J.W., Rampacek, G.B. and Kraeling, R.R.,
Immunocytochemical localization of luteinizing hormone-releasing hormone within
the olfactory bulbs of pigs, Biol. Reprod., 44 (1991) 299-304.
Levine, A.S. and Morley, J.E., Neuropeptide Y: A potent inducer of consummatory
behavior in rats. Peptides, 5 (1984) 1025-1029.
Liposits, Z., Merchenthaler, L, Reid, J.J. and Negro-Vilar, A., Galanin-iimnunoreactive
axons innervate somatostatin-synthesizing neurons in the anterior periventricular
nucleus of the rat. Endocrinology, 132 (1993) 917-923. Lopez, F.J., Meade Jr., E.H. and Negro-Vilar, A., Endogenous galanin modulates the
gonadotropin and prolactin proestrous surges in the rat. Endocrinology, 132 (1993)
795-800.
Lopez, FJ., Merchenthaler, L, Ching, M., Wisniewski, M.G. and Negro-Vilar, A.,
Galanin; A hypothalamic-hypophysiotropic hormone modulating reproductive
fimctions, Proc. Natl. Acad. Sci. USA, 88 (1991) 4508-4512.
Lopez, F.J. and Negro-Vilar, A., Galanin stimulates luteinizing hormone-relying
hormone secretion from arcuate nucleus-median eminence fragments in vitro:
Involvement of an a-adrenergic mechanism. Endocrinology, 111 (1990) 2431-
2436.
Lunstra, D.D., Borg, K.E. and Klindt, J., Characterization of pubertal development in
the Meishan Chinese boar, J. Anim. Sci., 70 (1992) 267 (abst. 514).
Marrable, A.W., The Embryonic Pig: A Chronological Accoura. Pitman, London, 1971.
Martel, J.C., St. Pierre, S., and Quirion, R., Neuropeptide Y receptors in rat brain:
autoradiographic localization. Peptides, 7 (1986) 55-60.
Matsuo, H., Baba, Y., Nair, R.M.G., Arimura, A. and Schally, A.V., Structure of the
porcine Lil and FSH-releasing hormone. L The proposed amino acid sequence,
Biochem. Biophys. Res. Comm., 43 (1971) 1334-1339.
McDonald, J.K., Lumpkin, M.D., Samson, W.K. and McCann, S.M., Neuropeptide Y
affects secretion of luteinizing hormone and growth hormone in ovariectomized
rats, Proc. Natl. Acad. Sci. USA, 82 (1985) 561-564. 209 Meijer, J., Poot, P., Molenaar, G. and Goede, R., PAP complex: A pitfall in
immunocytochemistry of the pig bypotbalamus, J. Neurosci. Methods, 17 (1986)
269-274.
Melander, T., Hokfelt, T. and Rokaeus, A., Distribution of galaninlike inununoreactivity
in the rat central nervous system, J. Comp. Neurol., 248 (1986a) 475-517.
Melander, T., Hokfelt, T., Nilsson, S. and Brodin, E., Visualization of galanin binding
sites in the rat central nervous system. Eur. J. Pharmacol., 124 (1986b) 381-382.
Melander, T., Hokfelt, T, Rokaeus, A., Cuello, A.C., Oertel, W.H., Verhofstad, A. and
Goldstein, M., Coexistence of galanin-like immunoreactivity with catecholamines,
5-hydroxytryptamine, GABA and neuropeptides in the rat CNS, J. Neurosci., 6
(1986c) 3640-3654.
Melander, T., Fuxe, K., Harfstrand, A., Eneroth, P. and Hokfelt, T., Effects of
intraventricular injections of galanin on neiu'oendocrine functions in the male rat.
Possible involvement of hypothalamic catecholamine neuronal systems. Acta
Physiol. Scand., 131 (1987) 25-32.
Merchenthaler, I., Gores, T., Setalo, G., Petrusz, P. and Flerko, B., Gonadotropin-
releasing hormone (GnRH) neurons and pathways in the rat brain. Cell Tissue
Res., 237 (1984) 15-29.
Merchenthaler, I., Lennard, D.E., Lopez, F.J. and Negro-Vilar, A., Neonatal imprinting
predetermines the sexually dimorphic, estrogen-dependent expression of galanin in
luteinizing hormone-releasing hormone neurons, Proc. Natl. Acad. Sci. USA, 90
(1993) 10479-10483. Merchentbaler, L, Lopez, FJ., Lennard, D.E. and Negro-Vilar, A., Sexual differences
in the distribution of neurons coexpressing galanin and luteinizing hormone-
releasing hormone in the rat brain. Endocrinology, 129 (1991) 1977-1986.
Merchentbaler, I., Lopez, F.J. and Negro-Vilar, A., Colocolization of galanin and
luteinizing hormone-releasing hormone in a subset of preoptic hypothalamic
neurons: Anatomical and functional correlates, Proc. Natl. Acad. Sci., 87 (1990)
6326-6330.
Merchentbaler, I., Setalo, G., Csontos, C., Petrusz, P., Flerko, B. and Negro-Vilar, A.,
Combined retrograde tracing and immimocytochemical identification of luteinizing
hormone-releasing hormone- and somatostatin-containing neurons projecting to the
median eminence. Endocrinology, 125 (1990) 2812-2821.
Michener, S.R., Aimone, L.D., Yaksh, T.L. and Go, V.L., Distribution of galanin-like
immunoreactivity in the pig, rat and human central nervous system. Peptides, 11
(1990) 1217-1223.
Minami, S., Frautschy, S.A., Plotsky, P.M., Sutton, S.W. and Sarkar, D.P., Facilitatory
role of neuropeptide Y on the onset of puberty: Effect of immunoneutralization of
neuropeptide Y on the release of luteinizing hormone and luteinizing hormone-
releasing hormone, Neuroendocrinology, 52 (1990) 112-115.
Minami^ S. and Sarkar, D.P., Central administration of neuropeptide Y induces
precocious puberty m female rats, Neuroendocrinology, 56 (1992) 930-934.
Moss, R.L. and McCaim, S.M., Induction of mating behavior in rats by luteinizing
hormone-releasing factor. Science, 181 (1973) 177-179. 211 Murakami, Y., Kato, Y., Shimatsu, A., Koshiyama, H., Hattori, N., Yanaihara, N. and
Imura, H., Possible mechanisms involved in growth hormone secretion induced by
galanin in the rat. Endocrinology, 12 (1990) 1224-1229.
Ottlecz, A., Samson, W.K. and McCann, S.M., Galanin: Evidence for a hypothalamic
site of action to release growth hormone. Peptides, 7 (1986) 51-53.
Otdecz, A., Snyder, G.D. and McCann, S.M., Regulatory role of galanin in control of
hypothalamic-anterior pituitary fimction, Proc. Natl. Acad. Sci. USA, 85 (1988)
9861-9865.
Parker, S.L., Kalra, S.P. and Crowley, W.R., Neuropeptide Y modulates the binding of
a gonadotropin releasing-hormone (GnRH) analog to anterior pituitary GnRH
receptor sites. Endocrinology, 128 (1991) 2309-2316.
Parrott, R.F., Heavens, R.P. and Baldwin, B.A., Stimulation of feeding in the satiated
pig by intracerebroventricular injection of neuropeptide Y, Physiol. Behav., 36
(1986) 523-525.
Paxinos, G., Ashwell, K.W.S. and Tork, I., Atlas of the Developing Rat Nervous System,
Second Edition. Academic Press, San Diego, 1994.
Pelletier, G., Desy, L., Kerkerian, L. and Cote, J., Immimocytochemical localization of
neuropeptide Y (NPY) in the human hypothalamus. Cell Tissue Res., 238 (1984)
203-205.
Pellinemi, L.J., Ultrastructure of the early ovary and testis in pig embryos, Am. J. Anat.,
144 (1975) 89-111. 212 Pfaff, D.W., Luteinizing hormone-releasing factor potentiates lordosis behavior in
hypophysectomized ovariectomized female rats. Science, 182 (1973) 1148-1149.
Poggioli, R., Vergoni, A.-V., Marrama, D., Giuliani, D. and Bertolini, A., NPY-
induced inhibition of male copulatory activity is a direct behavioral effect.
Neuropeptides, 16 (1990) 169-172.
Polkowska, J., Liwska, J. and Dubois, M.P., Ontogeny of neuropeptidergic systems:
Luteinizing hormone releasing hormone (LHRH); somatostatin (SRIF) and
neiirophysin (NF) in the hypothalamus of the domestic pig by
immunocytochemistry. Folia Histochem. Cytobiol., 23 (1985) 193-200.
Rettori, V., Milenkovic, L., Aguila, M.C. and McCann, S.M., Physiologically
significant effect of neuropeptide Y to suppress growth hormone release by
stimulating somatostatin discharge. Endocrinology, 126 (1990) 2296-2301.
Reuss, S., Hurlbut, E.C., Speh, J.C. and Moore, R.Y., Neuropeptide Y localization in
telencephalic and diencephalic structures of the ground squirrel brain. Am. J.
Anax., 188 (1990) 163-174.
Rodriguez-Sierra, J.F., Jacobowitz, D.M. and Blake, C.A., Effects of neuropeptide Y on
LH, FSH and TSH release in male rats. Peptides, 8 (1987) 539-542.
Rokaeus, A., Melander, T., Hokfelt, T., Lundberg, J.M., Tatemoto, K., Carlquist, M.
and Mutt, V., A galanin-like peptide in the central nervous system and intestine of
the rat, Neurosci. Lett., A1 (1984) 161-166. Ronnekleiv, O.K. and Resko, J.A., Ontogeny of gonadotropin-releasing hormone-
containing neurons in early fetal development of Rhesus macaques.
Endocrinology, 126 (1990) 498-511.
Rossmanith, W.G., Maries, D.L., Clifton, D.K. and Steiner, R.A., Induction of galanin
gene e}q>ression in gonadotropin-releasing hormone neurons with puberty in the
rat. Endocrinology, 135 (1994) 1401-1408.
Sabatitno, F.D., Mumane, J.M., Hoffinan, R.A. and McDonald, J.K., Distribution of
neuropeptide Y-like immimoreactivity in the hypothalamus of the adult golden
hamster, J. Comp. Neurol., 257 (1987) 93-104.
Sahu, A., Crowley, W.R., Tatemoto, K., Balasubramaniam, A., and Kalra, S.P., Effects
of neuropeptide Y, NPY analog (Norleucine'^-NPY), galanin, and neuropeptide K
on LH release in ovariectomized (ovx) and ovx estrogen, progesterone-treated
rats. Peptides, 8 (1987) 921-926.
Sahu, A., Kalra, S.P., Crowley, W.R. and Kalra, P.S., Evidence that NPY-containing
neurons in the brainstem project to selected hypothalamic nuclei: implication in
feeding behavior. Brain Res., 457 (1988) 376-378.
Sahu, A., Xu, B. and Kalra, S.P., Role of galanin in stimulation of pituitary luteinizing
hormone secretion as revealed by a specific receptor antagonist, galantide,
Endocrinology, 134 (1994) 529-536. 214 Sanacora, G., Kershaw, M., Finkelstein, J.A. and White, J.D., Increased hypothalamic
content of preproneuropeptide Y messenger ribonucleic acid in genetically obese
Zucker rats and its regulation by food deprivation. Endocrinology, 127 (1990)
730-737.
Schick, R.R., Samsami, S., Zimmermann, J.P., Eberl, T., Endres, C., Schusdziarra, V.
and Classen, M., Effect of galanin on food intake in rats: involvement of lateral
and ventromedial hypothalamic sites. Am. J. Physiol., 264 (1993) R355-361.
Schwanzel-Fukuda, M., Fadem, B.H., Garcia, M.S. and Pfaff, D.W.,
Immunocytochemical localization of luteinizing hormone-releasing hormone
(LHRH) in the brain and nervus tenninalis of the adult and early neonatal gray
short-tailed opossum (Monodelphis domestica), J. Comp. Neurol., 276 (1988) 44-
60.
Schwanzel-Fukuda, M., Morrell, J.I. and Pfaff, D.W., Ontogenesis of neurons producing
luteinizing hormone-releasing hormone (LHRH) in the nervus tenninalis of the
rat, J. Camp. Neurol., 238 (1985) 348-364.
Schwanzel-Fukuda, M. and Pfaff, D.W., Origin of luteinizing hormone-releasing
hormone neurons. Nature, 338 (1989) 161-164.
Schwanzel-Fukuda, M., Robinson, J.A. and Silverman, A.J., The fetal development of
the luteinizing hormone-releasing hormone (LHRH) neuronal systems of the
guinea pig brain, Brain Res. Bull., 1 (1981) 293-315.
Schwartz, M.W., Marks, J.L., Sipols, A.J., Baskin, D.G., Woods, S.C., Kahn, S.E.
and Porte Jr., D., Central insulin administration reduces neuropeptide Y mRNA 215
expression in the arcuate nucleus of food-deprived lean (Fa/Fa) but not obese
(fa/fa) Zucker rats. Endocrinology, 128 (1991) 2645-2647.
Shibasald, T., Oda, T., Imaki, T., Ling, N. and Demura, H., Injection of anti-
neuropeptide y gamma-globulin into the hypothalamic paraventricular nucleus
decreases food intake in rats. Brain Res., 601 (1993) 313-316.
Shivers, B.D., Harlan, R.E., Morrell, J.I. and Pfaff, D.W., Immunocytochemical
localization of luteinizing hormone releasing hormone in male and female rat
brains, Neuroendocrinology, 36 (1983) 1-12.
Silvennan, A.J., Distribution of luteinizing hormone-releasing hormone (LHRH) in the
guinea pig brain, Endocrinology, 99 (1976) 30-41.
Silverman, A.J., Jhamandas, J. and Renaud, L.P., Localization of luteinizing hormone-
releasing hormone (LHRH) neurons that project to the median eminence, J.
NeuroscL, 7 (1987) 2312-2319.
Skofitsch, G. and Jacobowitz, D.M., Immunohistochemical mapping of galanin-like
neurons in the rat central nervous system, Peptides, 6 (1985) 509-556.
Skofitsch, G., Sills, M.A. and Jacobowitz, D.M., Autoradiographic distribution of '^I-
galanin binding sites in the rat central nervous system. Peptides, 1 (1986) 1029-
1042.
Smith, Y., Parent, A., Kerkerian, L. and Pelletier, G., Distribution of neuropeptide Y
immunoreactivity in the basal foiebrain and upper brainstem of the squirrel
monkey {Saindri sciureus), J. Comp. Neurol, 236 (1985) 71-89. 216 Stanley, B.G. and Leibowitz, S.F., Neuropeptide Y: Stimulation of feeding and drinking
by injection into the paraventricular nucleus. Life ScL, 35 (1984) 2635-2642.
Stanley, B.G., Magdalin, W., Seirafi, A., Thomas, W.J., and Leibowitz, S.F. The
perifomical area: the major focus of (a) patchily distributed hypothalamic
neuropeptide Y-sensitive feeding system(s), Brain Res., 604 (1993) 304-317.
Sullivan, K.A. and Silverman, A.J., The ontogeny of gonadotropin-releasing hormone
neurons in the chick, Neuroendocrinology, 58 (1993) 597-608.
Sutton, S.W., Mitsugi, N., Plotsky, P.M. and Sarkar, D.K., Neuropeptide Y (NPY): A
possible role in the initiation of puberty. Endocrinology, 123 (1988) 2152-2154.
Tatemoto, K., Carlquist, M. and Mutt, V., Neuropeptide Y-a novel brain peptide with
structural similarities to peptide YY and pancreatic polypeptide. Nature, 296
(1982) 659-660.
Tatemoto, K., Rokaeus, A., Jomvall, H., McDonald, T.J. and Mutt, V., Galanin-a novel
biologically active peptide from porcine intestine, FEBS Lett., 164 (1983) 124-
128.
Tempel, D.L., Leibowitz, K.J. and Leibowitz, S.F., Effects of PVN galanin on
macronutrient selection. Peptides, 9 (1988) 309-314.
Tsuruo, Y., Kawano, H., Kagotani, Y., Hisano, S., Daikoku, S., Chihara, K., Zhang,
T. and Yanaihara, N., Morphological evidence for neuronal regulation of
luteinizing hormone-releasing hormone-containing neurons by neuropeptide Y in
the rat septo-preoptic area, Neuroscience Lett., 110 (1990) 261-266. 217 Wehrenberg, W.B., Corder, R. and Galliard, R.C., A physiological role for neuropeptide
Y in regulating the estrogen/progesterone induced luteinizing hormone surge in
ovariectomized rats, Neuroendocrinology, 49 (1989) 680-682.
Wilding, Gilbey, S.G., Bailey, C.J., Batt, R.A.L., Williams, G., Ghatei, M.A.
and Bloom, S.R., Increased neiuropeptide-Y messenger ribonucleic acid (mRNA)
and decreased neurotensin mRNA in the hypothalamus of the obese (ob/ob)
mouse. Endocrinology, 132 (1993) 1939-1944.
Woodhams, P.L., Allen, Y.S., McGovem, J., AUen, J.M., Bloom, S.R., Balazs, R. and
Polak, J.M., Immunohistochemical analysis of the early ontogeny of the
neuropeptide Y system in rat brain, Neuroscience, 15 (1985) 173-202.
Xu, B., Sahu, A., Crowley, W.R., Leranth, C., Horvath, T. and Kalra, S.P., Role of
neuropeptide-Y in episodic luteinizing hormone release in ovariectomized rats: An
excitatory component and opioid involvement. Endocrinology, 133 (1993) 747-
754.
Youngs, C.R., Ford, S.P., McGinnes, L.K. and Anderson, L.H., Investigations into the
control of litter size in swine: I. Comparative studies on in vitro development of
Meishan and Yorkshire preimplantation embryos, J. Anim. ScL, 71 (1993) 1561-
1565.
Youngs, C.R., Christenson, L.K. and Ford, S.P., Investigations into the control of litter
size in swine: m. A reciprocal embryo transfer study of early conceptus
development, J. Anim. Sci., 72 (1994) 725-731. 218 Zachary, I., Woll, P.J. and Rozengurt, E., A role for neuropeptides in the control of cell
proliferation, Dev. Biol., 124 (1987) 295-308. 219
ACKNOWLEDGEMENTS
The information presented in the present dissertation was completed only with the help and support of many people. Thanks to Edwin M. Brogie for teaching me 'good' science and birdwatching and for your friendship and advice. I thank my fellow graduate smdents in the Jacobson laboratory. Dr. Charles Fox, Dr. Joel Elmquist, Dr. Cathy Kuehl-
Kovarik, Dr. Javed Iqbal, Jay Swanson, Dr. Tricia Heine, Sang-Joon Cho and Tracey
Pepper, technician Lynne Ross, and postdocs Dr. Shi Di and Dr. loana Sonea for their technical help, thoughtful discussions and criticism, as well as for making the laboratory a better place to be. Thank you to Margie Carter and Mahlon Shell for their expert assistance.
Thanks also to Vet Anatomy secretary Linda Kelley for all of her help dealing with the
'system', and to the best department chair in the University, Dr. Mary Helen Greer, for all of her help and support.
I tbanlc my committee members Dr. Don Sakaguchi, Dr. Srdija Jefdnija, Dr. Walter
Hsu and especially Dr. Lloyd Anderson for their assistance, support, and constructive criticism. The biggest thank you of all goes out to my major professor. Dr. Carol Jacobson, for her inspiration, encouragement, faith, guidance, support, criticism and especially her patience and understanding.
Last but not least, my sincerest and deepest thanks also go out to my family for your love, moral support and financial support (including rummy). I love you all. Ditto to all the friends (you know who you are) who stuck close by me through both the good and bad times on this long road, especially Phil Dykema. You made it better than I could have 220 hoped for. This thesis was fueled by the blues of Magic Slim and the Teardrops, Bugs
Henderson and the Shuffle Kings, the Chris Duarte Group and too many other artists to mention.
"If one advances confidently in the direction of his dreams, and endeavors to live the life which he has imagined, he will meet with a success unexpected in common hours." -Henry David Thoreau