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Brain Research Bulletin, Vol. 37, No. 6, pp. 657-662, 1995 Copyright © 1995 Elsevier Science Ltd Printed in the USA. All rights reserved

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Sex Differences in Cell Proliferation in the Ventricular Zone of Young Ring Doves

CHANGYING LING* AND MEI-FANG CHENG1-1

*Department of Brain & Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA 02139 tlnstitute of Animal Behavior, Rutgers-- The State University, 101 Warren Street, Newark, NJ 07102

[Received 11 January 1994; Accepted 20 December 1994]

ABSTRACT: Cell proliferation in the ventricular zone was studied in 3-month-oM ring doves, using [~l]-thymidine as the marker for DNA synlhmds and hence ceil division. We found sex differ- ences in the rate of ceil proMeration on the plane of preoptic area in these immature doves whose gonads were just under- gong changes.

KEY WORDS: Doves, Sex differences, Cell proliferation, Ventric- ular zone, DNA, PreopUc area.

INTRODUCTION

In birds, as in mammals, males and females exhibit numerous physiological and behavioral differences. Sexual dimorphism in discrete areas of the central nervous system (CNS) has been iden- tiffed in both mammals and birds [9,11,14,31,34]; these neural substrates presumably provide sexually dimorphic functional systems. The genesis of these structural differences is thought to be an organizational action of steroid hormones during a critical period of development. For example, androgen can promote neu- ral growth and suppress cell death in certain sexual dimorphic nuclei in both mammals and birds [10,15,21,22,29,30]. Demon- strated importance of hormone action in sexual dimorphism im- plies that the brain is born monomorphic, an assumption yet to be put to the test. With few exceptions, the birth of new neurons ceases at birth in higher vertebrates and hence does not lend itself to experimental observation on neuronal progenitors in male and female brains. Recent findings of adult neurogenesis in songbirds provide an exciting opportunity to address this question. With birds, cells are born in the lateral ventricular zone (LVZ), migrate and differentiate into functional neurons in the telencephalon [2,3,4,13,25]. Hormone action on sexual dimorphism would pre- dict that the LVZ of the bird system exhibits comparable levels of proliferative activity in both male and female. In the present study, we examine the rate of proliferative cells in young male and female ring doves. Ring doves (Streptopelia risoria) are monomorphic in appearance and have long postnatal develop- ment; birds develop fully functional gonads by 5 to 7 months old

To whom requests for reprints should be addressed.

but are not sexually competent until 9 months to 1 year [8]. We report here evidence for sex differences in proliferative activity in the lateral ventricular zone of 3-month-old ring doves, whose body weights were comparable to those of adult birds, but whose gonads just began rapid growth. We found evidence for sexual dimorphic brains in number and distribution patterns of new cells at the site where cells are born. We propose that these sex dif- ferences may be part of the mechanisms mediating sexual dif- ferentiation of the brain.

METHOD

Twenty-two (11 males, 11 females) 3-month-old ring doves were subjected to a single injection of [3H]-labeled thymidine, a marker for DNA synthesis and therefore for cell replication [1,4,17,23,24,36]. They were sacrificed following different sur- vival times ( lh n = 2F+2M; 8h n = 2F+2M; 24h n = 5F+5M; 48h n = 2F+2M). The entire brains were sectioned transversely at intervals of 6-micron; the sections were coated with Kodak NTB2 nuclear track emulsion and exposed at 4°(2 for 30 days [4,23], then counterstained with the fluorescent DNA dye Hoechst 3325, or cresyl violet for anatomical analysis [4]. Using a computer-microscope system [5], 10 series of entire brain sec- tions in each brain were screened for labeled cells (one 6-#m section was sampled every 300 #m, the 6th section is at the A- P level of anterior commissure, the reference plane). We analyzed the number of labeled cells in the ventricular zone, labeling in- tensity (the number of labeled silver grains over a nucleus), and the location of labeled cells in both hemisections.

The criterion for a labeled cell is the same as those accepted in the literature, namely, the existence of 7 or more exposed silver grains over the nucleus, which is 10 or more times above back- ground [4,28]. In practice, none of the labeled cells in the present study contains less than 20 silver grains. Although incorporation of [3H]-thymidine also accompanies changes unrelated to cell proliferation such as the repeat of a particular DNA sequence, it is generally accepted that most instances of [3H]-thymidine la- beling is associated with cell proliferation, and only proliferating cells in S phase are likely to become labeled by a single [3H]-

657

658 LING AND CHENG

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A B FIG. 1. Morphology of ventricular cells in a 6 micron section stained with cresyl violet. (A) Most regions of the lateral ventricular zone consist of a single layer cells. (B) Anterior-ventral portion of the lateral ventricular zone consists of several layers cells, m:medial wall of LVZ, l:lateral wall of LVZ, arrows:newly proliferated labeled cells. Bar:50 #m.

thymidine injection [17,23,36]. Following a single intramuscular injection of [3H]-thymidine, [3H]-thymidine degrades quickly in the blood stream of ring doves, such that most of the labeling of cell nuclei occurs during the first 30 min postinjection, and al- most no trace of labeling thereafter [25]. Since the [3H]-thymi-

Anterlor-Postorlor level

FIG. 2. The distribution of [3H]-thymidine-labeled LVZ cells in coronal hemisections of a 3-month-old ring dove brain (#L3). Dots represent labeled LVZ cells in the lateral ventricle lh after receiving a single in- jection of [3H]-thymidine. The anterior-posterior (A-P) axis is numbered 1 to 9; for each section, left is medial, top is dorsal; and the interval between sections is 300 #m. The nucleus accumbens (Ac) emerges at A- P level 1, the lobus parolfactory (LPO) reaches its caudal edge and the nucleus preoptic anterior (POA) emerges at A-P level 3; the anterior commissure (AC) is at A-P level 6. The ventricles are shaded.

dine labeled cells can continue mitosis in vivo if the survival time is sufficiently long for a complete cell cycle, and since the number of labeled cells is doubled, whereas the number of la- beled silver grains over a nucleus decreases [23,36] during mi- tosis, the different survival times we used in the present study enables us to observe the time course of cell proliferation.

Localization of Labeled Cells

There are four ventricles in the brain of ring doves, that is, lateral, third, tectal, and fourth ventricles. The well defined regions surrounding ventricles are called ventricular zones. The highest density of [3H]-thymidine labeled cells was found in the lateral ventricular zone (LVZ); there were no labeled cells in the third, tectal, or fourth ventricular zones. However, there were labeled cells distributed throughout the brain parenchyma. The lateral ven- tricular zone consists of mostly a single layer of cells (Fig. 1A). However, along the anteroventral edge of LVZ (the anteroventral portion of the lateral wall of LVZ, Fig. 1B), multiple layers of epidermal cells can be seen surrounding the ventricle. These epi- dermal cells have small nuclei (the diameter is about 10 #m) con- taining heterochromatins. They were densely aggregated and were darker than surrounding cells in the section stained with cresyl violet (Figs. 1A & B). In the LVZ, about 88% of the labeled cells were located in the lateral wall in both sexes (number of labeled cells in the lateral wall of LVZ/total number of labeled cells in LVZ). The distribution of labeled cells was uneven extending from

SEX DIFFERENCES IN CELL PROLIFERATION 659

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the ventral to dorsal portions and from anterior to posterior edge along the lateral wall of the lateral ventricle with some densely labeled regions. For example, in the anterior-posterior (A-P) level 2 and 3, where the lobus parolfactory (LPO, avian basal ganglia) reaches its caudal portion, labeled cells were densely packed in the ventral portion of the LVZ (Fig. 2), whereas in the A-P level 8 and 9, there were more labeled cells along the dorsal portion than along the ventral portion of LVZ. From the anterior to the posterior axis, the mean number of labeled VZ tens/ram (length of LVZ) increased gradually from anterior sections, reaching a peak at the level where the LPO reaches its caudal edge and the nucleus preop- ticus anterior (PEA) emerges (A-P level 3 and 4), then declined sharply in sections thereafter (Fig. 3). The same pattern applied to all the groups, and there were no significant differences between two hemispheres.

The Time Course of Cell Proliferation

As survival time increased from 1 h to 48 h, the mean number of labeled LVZ cells/per hemisection increased from 52 to 197,

and the mean number of labeled silver grains over a nucleus decreased from 128 to 67 (Fig. 4). The ratio of increased number of labeled LVZ cells does not match perfectly that of decreased number of labeled silver granules/cell. This is likely a result from miscounting the number of labeled silver granules, since it is very difficult to perform accurate counting when the number of silver granules exceed 100 granules. We also analyzed data based on the percentage of lightly labeled cells (which contains less than 100 labeled silver grains; percentage = number of lightly labeled cells/total number of labeled cells). The percentage of lightly labeled cells significantly increased with survival time. The per- centage of lightly labeled cells was 35.2, 46,7, 59.0, and 74.7, respectively, in birds which survived lh, 8h, 24h, and 48h after the [3H]-thymidine injection. As in the mean number of labeled cells, the changes in percentage of lightly labeled cells suggest that some labeled LVZ cells divided again between 1 h to 48 h after injection. Concurrently, the number of labeled cells in the outlying region of the lateral ventficular zone did not increase significantly in these groups indicating that few of the newly

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FIG. 4. The time course of cell proliferation in the lateral ventricular zone (A) The mean number of labeled LVZ cells increased with survival time (n = number of birds), 10 right hemisections from each bird were analyzed. (B) The mean number of labeled silver grains over a nucleus decreased with survival time:200 cells within the Anterior-Posterior level 3 were selected from each brain to count the number of labeled grains over the nucleus (n = number of birds).

proliferated cells migrated within 48 h after the [~H]-thymidine injection.

Sex Differences in Cell Proliferation in the Lateral Ventricular Zone

The pattern of sex differences in cell proliferation persists across different survival times. First, the mean number of labeled VZ cells was consistently higher in females than in males for all groups (Fig. 5A, n = 22 birds, p < 0.01: Two-way ANOVA Test, Abacuc Concepts. Inc.). The mean number of labeled cells/ mm (length of LVZ) was significantly higher in females than in males in the group that birds survived 24 h after the injection (Fig. 5B, n = 10, p < 0.05: ANOVA T-test, Abacuc Concepts. Inc.). Secondly, the number of labeled cells in the most densely labeled right and left hemisection (at the caudal edge of the LPO) was consistently higher in females than in males for all birds (Fig. 5C, n = 22 birds, p < 0.01 in a Two-way ANOVA Test, Abacuc Concepts. Inc.). Thirdly, the distribution pattern of la- beled cells along the A-P axis is different between male and female brain (Fig. 3). There were more labeled cells in the an- terior edge (in particular, the ventral portion), and fewer labeled cells in the posterior edge of the LVZ in females than in males.

Although the cell type ot these proliferating cells (labeled cells) in the LVZ has not yet been identified, recent studies give strong evidence that cells in the lateral ventricular zones during postnatal life give rise to neurogenesis [12,23,25,27,35,38]. In vitro, cells coming from the lateral ventricular zones of adult mammals and birds can produce both glia and neurons under suitable conditions [13,27,35,38]. In vivo, cells in the LVZ con- tinue to proliferate during postnatal life of songbirds. Many of these cells are radial glia [4]. Some of the daughter cells migrate from the LVZ to different target areas of the telencephalon (in the way similar to that during the embryonic stage) and become functional neurons later [12,27,32]. We have previously shown that in the ring dove, there is an age-related parallel change be- tween the number of labeled cells in the LVZ 24 h after [3H]- thymidine and the number of new neurons in the telencephalon as identified by neuron-specific antibodies (Neun and Map-2) 2 months after thymidine injections [25]. There is also an age-re-

lated parallel change in the distribution patterns of newly prolif- erated cells in the LVZ and new neurons in the telencephalon [25]. It is, therefore, likely that some of the newly proliferated cells in the LVZ are progenitors of neurons.

During the embryonic stage, both in mammals and birds, new neurons are born in the ventricular zone and then migrate along the radial glia's fibers to different areas of the brain. It has been suggested that in both mammals and birds [6,23,32,36] the lo- cation of progenitors of neurons in the ventricular zone is related to the final location of new neurons. We propose that the con- centration and distribution pattern of proliferating cells in the LVZ may provide a clue for the concentration and distribution pattern of new neurons later in the adult brain. This naturally awaits verification in the further investigation.

Doves used in this experiment were raised in the indoor dove colony of the Institute of Animal Behavior, Rutgers University. In the colony, doves fledged at 25 days of age. They were then separated from their parents and were caged with other juvenile birds (about 6 birds in a cage). After laparotomy at 3 - 4 months- old, birds were caged separately by sex, so that they can see, and hear each other, but can not touch each other. We have previously shown that in ring doves, as in song birds, cell proliferation in the LVZ and neurogenesis persists throughout adult life but de- clines significantly with age [25,26]. The single largest change occurs from 3 months to 8 months of age, when gonads undergo rapid development [25]. Thus, the rate of cell proliferation ob- served in the present study may represent its peak activity in the postnatal development.

Although the precise role of sex differences in cell prolifera- tion in the ring dove remains to be determined, it is worth notic- ing that the difference took place on the planes of the preoptic nuclei, the regulatory sites of the reproductive system. We hy- pothesize that during the postnatal developmental period the sex biased proliferating ventricular cells may give rise to the sexually dimorphic distribution of neural substrates and thereby contribute to the development of sexually dimorphic behavioral and phys- iological systems. Alternatively, new cells in females may result in more glia cells, (radial glia) which can guide new neurons and provide the necessary survival environment of neurons, thereby, indirectly affecting the sculpture of the female brain.

SEX DIFFERENCES IN CELL PROLIFERATION 661

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FIG. 5. Sex differences in cell proliferation of the LVZ (A) The mean number of labeled LVZ cells per brain (n = number of birds). For each brain, 10 series sections of both hemispheres were analyzed. Females:dark bars; males:light bars. (B) The mean number of labeled cells/mm (length of LVZ) in the birds survived 24 h after the injection (n = number of birds). For each brain, 10 series sections of both hemispheres were analyzed. *Female vs. male is statistically significant at p < 0.05 level. (Two-factor ,MqOVA:Super ANOVA Program by Abacuc Concepts. Inc.) (C) The mean number of labeled LVZ cells in the most densely labeled coronal section (including both tight and left bemisections at the A-P level 3; n = number of birds). Females:dark bars; males:light bars.

Although there is evidence that gonadal hormones influence sexual dimorphism of the CNS during early development [9,15,21,28,29,31 [, recent studies have foreclosed any role of go- nadal hormone on proliferative activity [7,16]. This, however, does not preclude the role of gonadal hormones on the survival of new neurons. Indeed, there is some evidence that testosterone exerts such influence on new neurons in the song nuclei [29]. The possibility of sex differences in the rate of neural prolifera- tion has been discussed for at least a decade, and it has been demonstrated during the embryonic stage [18,19] that there are sex differences in [3H]-thymidine labeling neurons in the preoptic area of rats. The present study, however, presents the first evi- dence of sex differences in cell proliferation in the lateral ven- tricular zone during postnatal life of higher vertebrates. A recent unpublished study with songbirds suggested that the number of proliferating cells was higher in juvenile male zebra finches than in juvenile females [7]. If confirmed, this would add credence to the general finding of the present study.

ACKNOWLEDGEMENTS

We thank Drs. Fernando Nottebohm, Arturo Alvarez-BuyUa and Joan Morrell for the use of laboratory facilities, technical instructions, and helpful comments on earlier version.

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