Alteration in the Intrafollicular Thiol–Redox System in Infertile Women

REPRODUCTIONRESEARCH Pregnancy-related changes in connections from the cervix to forebrain and hypothalamus in mice Steven M Yellon, Lauren A Grisham, Genevieve M Rambau, Thomas J Lechuga and Michael A Kirby Departments of Physiology, Pediatrics, Obstetrics and Gynecology, Pathology and Human Anatomy, and the Center for Perinatal Biology, School of Medicine, Loma Linda University, Loma Linda, California 92354, USA Correspondence should be addressed to S M Yellon; Email: [email protected] Abstract The transneuronal tracer pseudorabies virus was used to test the hypothesis that connections from the cervix to the forebrain and hypothalamus are maintained with pregnancy. The virus was injected into the cervix of nonpregnant or pregnant mice, and, after 5 days, virus-labeled cells and fibers were found in specific forebrain regions and, most prominently, in portions of the hypothalamic paraventricular nucleus. With pregnancy, fewer neurons and fibers were evident in most brain regions compared to that in nonpregnant mice. In particular, little or no virus was found in the medial and ventral parvocellular subdivisions, anteroventral periventricular nucleus, or motor cortex in pregnant mice. By contrast, labeling of virus was sustained in the dorsal hypothalamus and suprachiasmatic nucleus in all groups. Based upon image analysis of digitized photomicrographs, the area with label in the rostral and medial parvocellular paraventricular nucleus and magnocellular subdivisions was significantly reduced in mice whose cervix was injected with virus during pregnancy than in nonpregnant mice. The findings indicate that connections from the cervix to brain regions that are involved in sensory input and integrative autonomic functions are reduced during pregnancy. The findings raise the possibility that remaining pathways from the cervix to the forebrain and hypothalamus may be important for control of pituitary neuroendocrine secretion, as well as for effector functions in the cervix as pregnancy nears term. Reproduction (2010) 140 155–164 Introduction cervix temporally correlates with processes that are hypothesized to be critical for remodeling of the cervix, Innervation of the cervix is important for a variety of including immigration of immune cells, degradation of physiological process related to reproduction and extracellular collagen matrix, and changes in bio- nociception. Afferent fibers in the cervix convey physical capabilities to allow sufficient stretch for birth sensory information to the thoracolumbar spinal cord (Leppert 1995, Mackler et al. 1999, Yellon et al. 2003, through the hypogastric, pelvic, and vagus nerves Buhimschi et al. 2004). Innervation is also important for (Ortega-Villalobos et al. 1990, Steinman et al. 1992, the birth process because remodeling of the cervix is Cueva-Rolon et al. 1996, Komisaruk et al. 1996, Papka forestalled and parturition disrupted after transection of et al. 1998, Lee & Erskine 2000, Puder & Papka 2001, the parasympathetic pelvic nerve (Higuchi et al. 1987, Wiesel et al. 2004). These nerves mediate important Mackay et al. 2009) or vagus nerve (Yellon et al. 2009), functions related to pain perception, neuroendocrine but not the sympathetic hypogastric nerve (Boyd et al. secretion of oxytocin, and the fetal expulsion. By 2009). The potential importance of innervation for contrast to the marked decrease in innervation of the the birth process is countered by a lack of information uterus with pregnancy (Wikland et al. 1984, Haase et al. from neural tract tracing studies of connections with the 1997, Klukovits et al.2002), the cervix is well cervix during pregnancy. innervated in individuals that are not pregnant, as well CN innervation of the uterine cervix has been as throughout pregnancy and near term in a variety of extensively studied in nonpregnant females. Areas of the species (Bryman et al. 1987, Tingaker et al. 2006). forebrain and hypothalamus receive input from the lower Moreover, in rodents, the greatest presence of nerve thoracic and upper lumbar spinal cord either directly or via fibers in the cervix is found by the day before birth relays in the brainstem (Burstein et al.1990, Lee & Erskine compared to that earlier in pregnancy or in nonpregnant 2000). These nuclei in the brainstem also project to the controls (Kirby et al. 2005, Yellon et al. 2008, Boyd et al. pelvic viscera through the vagus nerve (Ortega-Villalobos 2009). The increase in nerve fibers in the preterm et al. 1990, Hubscher & Berkley 1995). Recent evidence q 2010 Society for Reproduction and Fertility DOI: 10.1530/REP-10-0002 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/29/2021 04:48:13AM via free access 156 S M Yellon and others indicates that sensoryand autonomic connections from the This approach was used to test the hypothesis that neural thoracolumbar spinal cord to the murine cervix are connections between the brain and cervix are reduced reduced with pregnancy (Kirby et al. 2009). Fewer cells with pregnancy. The findings suggest a general reduction and fibers in the spinal cord that project to the cervix during in central pathways that innervate the cervix with pregnancy suggest that processing and transmission of pregnancy and raise the possibility that remaining information to the brain are reduced. However, connec- connections from specific locations may be important in tions between the cervix and the intermediolateral the process of parturition. column, a descending pathway in the autonomic region of the spinal cord, were sustained with pregnancy. Accordingly, the main objective of the present study was Results to determine the extent to which CN connections are Cell bodies and neuronal processes in the brain were maintained with the cervix during pregnancy. The retro- labeled following injection of pseudorabies virus into the grade transport of pseudorabies virus was used to identify cervix. Labeled neurons had a variety of morphologies, brain areas that are directly or indirectly connected to while fine fibers represented dendritic projections or thin primary neurons with terminal fibers in the cervix. This axonal projections (Fig. 1). These structures were well virus has served as a transneuronal tracer because it is preserved and counterstained, helping to identify rapidly taken up by nerve terminals, transported to the cell relevant neuroanatomical landmarks. Immunoreactive nucleus for replication, and, in the process, infect particles partially or completely filled neuron cell bodies other neurons that synapse on virus-containing neurons and their processes (Fig. 1 insets); isolated fibers with (Card et al.1993, Toth & Palkovits 1997, Enquist 2002). beaded varicosities were often evident. Clustering or Pe 3V AvPe 3V Pe ox RCh M1 LH opt cc LV Figure 1 Photomicrographs of cells and fibers in forebrain and hypothalamus after the cervix of mouse #8 was injected with pseudorabies virus on day 15 of pregnancy. Top panels are anteroventral periventricular area (AvPe) and periventricular region (Pe), medial preoptic area (MPA), and retrochiasmatic area (RCh). Bottom panels are lateral hypothalamus (LH) and sensorimotor cortex (M1). Other neuroanatomical structures are optic chiasm (ox), third ventricle (3V), corpus callosum (cc), and lateral ventricle (LV). Insets illustrate fine fibers that emanate from soma and diversityof somas that were labeled with virus. Scale bar is 50 mm for all panels and 25 mm for all insets. Reproduction (2010) 140 155–164 www.reproduction-online.org Downloaded from Bioscientifica.com at 09/29/2021 04:48:13AM via free access Brain innervation of cervix with pregnancy 157 Nonpregnant 3V AH SCh 3V opt Pregnant Figure 2 Photomicrographs of cells and fibers in AH forebrain and hypothalamus after the cervix of a 3V nonpregnant mouse (top panels) or day 15 pregnant mouse (bottom panels) was injected with pseudorabies virus. Left panels are from SCh the anterior hypothalamus (AH). Right panels 3V are of the suprachiasmatic nuclei (SCh). Other opt neuroanatomical structures are the third ventricle (3V) and optic tract (opt). Insets highlight morphology of cells and fibers labeled with virus. Scale bar is 50 mm for all panels and 25 mm for all insets. overlap of morphological features precluded an accurate Bilaterally, both parvocellular and magnocellular sub- count of labeled neurons. Virus-labeled cell bodies and divisions had clusters of virus-labeled neuron cell bodies fibers were scattered or isolated in the periventricular and fibers (Fig. 3). In nonpregnant mice, dense label was area and various hypothalamic nuclei in all mice, as well evident throughout the rostral–caudal expanse of the as in the medial preoptic area in most mice. In each of parvocellular paraventricular nucleus. The rostral para- these areas, immunoreactivity was more prevalent ventricular nucleus, corresponding to anterior and ventral within and over multiple sections in nonpregnant parvocellular subdivisions, contained many immuno- controls compared to that in mice that had been injected reactive cells and relatively large diameter fibers. Virus with virus into the cervix during pregnancy. Virus- label was also well distributed in the middle portion of the labeled cells and fibers were also present in the paraventricular nucleus, encompassing the medial par- anteroventral periventricular area and sparse or isolated vocellular, as well as lateral and medial magnocellular in the somatosensory and primary motor cortex in subdivisions. Caudally,

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