Elsevier

Brain Research

Volume 1315, 22 February 2010, Pages 25-32
Brain Research

Research Report
Pregnancy inhibits cell proliferation and neuroblast differentiation without neuronal damage in the hippocampal dentate gyrus in C57BL/6N mice

https://doi.org/10.1016/j.brainres.2009.12.029Get rights and content

Abstract

Neural changes occur in the dam during gestation, and brain size has been shown to decrease across pregnancy in humans as well as rodents. In this study, we monitored neuronal damage, cell proliferation and neuroblast differentiation in the hippocampal dentate gyrus (DG) at age-matched virgin control (17- to 18-week-old), gestation day (GD) 14.5, 16.5 and 18.5 (17- to 18-week-old dams), using NeuN for mature neurons, terminal deoxynucleotidyl dUTP nick-end labeling (TUNEL) and Fluoro-Jade B (F-J B) for neuronal death, Ki67 for cell proliferation and doublecortin (DCX) for neuroblast differentiation in C57BL/6 mice. There were no significant differences in NeuN-immunoreactive (+) neurons between the age-matched control and gestating groups. TUNEL or F-J B positive neurons were rarely detected in the DG in all the groups. Ki67+ cell proliferation was significantly decreased in the subgranular zone of the dentate gyrus (SZDG) at GD16.5. In addition, DCX+ neuroblasts with/without tertiary dendrites were decreased in the SZDG with gestation by GD16.5. However, in the GD18.5 group, the number of Ki67+ nuclei and DCX+ neuroblasts with/without tertiary dendrites was slightly increased compared to that observed at GD16.5. DCX protein levels were low at GD16.5, and thereafter slightly increased. These results suggest that cell proliferation and neuroblast differentiation in DG of the hippocampus is decreased during gestation.

Introduction

The hippocampus is involved in some forms of learning and memory (Miyagawa et al., 2007). It is relatively susceptible to exogenous stress and shows remarkable degree of plasticity against stress (Blaise et al., 2008, Howland and Wang, 2008). Accordingly, progenitor cells in the dentate gyrus (DG), a part of hippocampus, retain the ability to proliferate and differentiate into neurons in all mammals, including non-human and human primates, forming functional synaptic inputs similar to those by mature (Song et al., 2002, Ambrogini et al., 2004, Llorens-Martín et al., 2006, Ngwenya et al., 2008, Snyder et al., 2009). There are some markers to visualize newly produced cells. Especially, Ki67 is a marker for all proliferating cells except early G1 phase (Gerdes et al., 1983, Cattoretti et al., 1992), and doublecortin (DCX) for progenitors differentiating into neurons (Brown et al., 2003).

There are neuronal changes affecting the dam during gestation and postpartum period (Numan, 2007, Brusco et al., 2008). The brain size has been shown to decrease across pregnancy, returning to preconception size after delivery in humans (Oatridge et al., 2002) and rodents (Galea et al., 2000). During pregnancy, there are dramatic fluctuations of many steroid hormones (Cameron and Gould, 1994, Ormerod et al., 2003, Casolini et al., 2007). Estradiol and corticosterone are regulators of adult neurogenesis. Estradiol initially (within 4 h) increases and subsequently suppresses (within 48 h) cell proliferation via adrenal steroids (Ormerod et al., 2003), while corticosteroids strongly inhibit cell proliferation in the hippocampus of adult female rodent (Cameron and Gould, 1994). Furthermore, corticosteroids are transferred to the pups by lactating (Casolini et al., 2007). In particular, corticosterone levels are significantly changed along gestation (Dalle et al., 1978), which is required. It is required for the prolactin receptor gene expression in the mammary gland of the late pregnant mouse (Mizoguchi et al., 1997) and corticosterone levels fall from day 17 of gestation until birth in the maternal plasma, thereafter remaining stable (Dalle et al., 1978).

There are studies focusing on changes of neurogenesis during gestation and postpartum period (Banasr et al., 2001, Shingo et al., 2003, Furuta and Bridges, 2005, Rolls et al., 2008), but only upon one time point or 7–10 days after gestation. In the present study, we investigated on (i) cell death using terminal deoxynucleotidyl dUTP nick-end labeling (TUNEL) and Fluoro-jade B (F-J B) staining and NeuN immunohistochemistry, (ii) cell proliferation and (iii) neuroblast differentiation using immunohistochemistry for Ki67 and DCX, and western blot for DCX in the DG at late stages of gestation in C57BL/6N mice (GD 14.5, 16.5 and 18.5).

Section snippets

Effects of pregnancy on mature neurons

In the control group, NeuN-immunoreactive (+) mature neurons were well detected in the hippocampus including the DG (Figs. 1A and B). In the GD14.5, GD16.5 and GD18.5 groups, the number and the distribution pattern of NeuN+ neurons were similar to the control group (Figs. 1C–H).

Effects of pregnancy on neuronal death

In the age-matched control group, TUNEL or F-J B positive (+) cells were rarely observed in the DG (Figs. 2A and B). In GD14.5, GD16.5 and GD18.5 groups, only few TUNEL+ or F-J B+ cells were detected in the DG (Figs. 2

Discussion

We studied the effects of pregnancy on (i) mature neurons using NeuN immunohistochemistry, (ii) cell death using TUNEL and F-J B staining, and (iii) cell proliferation and (iv) neuroblast differentiation using Ki67 and DCX immunohistochemistry, respectively, in the hippocampal DG at various time points during gestation in C57BL/6N mice.

In the present study, we could not find any significant change in numbers of mature neurons in any of the subregions during gestation based on NeuN

Experimental animals

Eight-week-old male and female C57BL/6N mice were purchased from Orient Bio Inc. (Seongnam, South Korea) and mated. The next morning, the plug was observed and considered as gestation day 0.5 (GD0.5). Pregnant mice were housed in a conventional state under adequate temperature (23 °C) and humidity (60%) control with a 12-h light/12-h dark cycle, and free access to food and water. The procedures for handling and caring for animals adhered to the guidelines that are in compliance with the current

Acknowledgments

The authors would like to thank Mr. Seok Han, Mr. Seung Uk Lee and Ms. Hyun Sook Kim for their technical help in this study. This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0084089) and by a grant (20090K001290) from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Education, Science and Technology, the Republic of Korea.

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