Intrinsic neurons in the mammalian ovary

Mammalian ovarian function is under endocrine and neural control. Although the extrinsic innervation of the ovary has been implicated in the control of both ovarian development and mature function, it is now clear that, from rats to humans, the ovary is endowed with a network of intrinsic neurons displaying diverse chemical phenotypes. This article describes the presence of these intrinsic neurons in the ovary of different mammalian species, and discusses the possible functions that they may have in the regulation of ovarian physiology.     

Role of the central and peripheral nervous system in the ovarian function

This review attempts to give a comprehensive overview of ovarian innervation, considering the whole nervous system and its different levels that may modify the ovarian function. The connection between the ovary and the central nervous system through the autonomic pathways, including the peripheral ganglia, is highlighted. The evidence obtained over the last years highlights the role of the superior ovarian nerve (SON) in the ovarian phenomena. Besides, the effect on the ovary of conventional neurotransmitters and others such as indolamines and peptides, which have been found in this organ, are discussed. Various reproductive diseases have been studied almost exclusively from the endocrine point of view. It is evident that a better knowledge about the role of the neural factors involved in the ovarian physiology may facilitate the understanding of some of these. A review of the concepts and an update of some experimental designs is made that permits clarifying several aspects of the relationship between the neural system and the ovary. At present, there is no doubt that the innervation of the ovary is involved in several physiological aspects of this gland function. However, the relationship of some levels of the nervous system and the ovary offer a wide avenue for future research.     

Localization of calretinin in the rat ovary and in relation to nerve cell bodies in dorsal root and paravertebral ganglia projecting to the ovary

Retrograde tracing with True Blue was combined with immunocytochemistry to determine the source of any calretinin-immunoreactive (CR-ir) nerves projecting to the rat ovary. In the ovary, a strong signal for calretinin immunoreactivity was localized in interstitial gland cells; however, no intraovarian CR-ir nerves could be demonstrated. When the superior ovarian nerve was isolated, cut, and True Blue applied to the proximal end, the fluorescent dye was retrogradely transported to a population of cells located in T-12, T-13, and L-1 dorsal root and paravertebral ganglia. There was virtually no dual labeling of cells in these ganglia with calretinin (< 0.009% dual labeling in dorsal root and <0.014% in paravertebral ganglia). However, greater than two-thirds of the True Blue-labeled cells were immediately adjacent to CR-ir cells in dorsal root ganglia. This arrangement is suggestive of a paracrine mechanism between CR-ir cells and cells projecting to the ovary. In paravertebral ganglia, 63% of cells projecting to the ovary were surrounded completely or partially by beaded CR-ir nerve fibers. The source of these fibers (sensory or preganglionic sympathetic) is unknown but hypothesized to be preganglionic. Collectively, these observations suggest a participatory role for calretinin in ovarian function, either directly via effects on the interstitial gland or indirectly by influencing neurons projecting to the ovary.     

Neurotrophic control of ovarian development

Substantial evidence now exists indicating that the neurotrophins, a family of growth factors required for the survival, development, and differentiation of various neuronal populations of the nervous system, are also important for the development of nonneuronal tissues. Such a function was first suggested by studies showing the presence of high-affinity neurotrophin receptors in a variety of nonneuronal tissues including those of the cardiovascular, endocrine, immune, and reproductive systems. Within the latter, the gonads appear to be a preferential site of neurotrophin action as suggested by the presence in the mammalian ovary of at least four of the five known neurotrophins and all of the neurotrophin receptors thus far identified. While the various functions that the neurotrophins may have in the ovary are still being elucidated, it is now clear that in addition to recruiting the ovarian innervation, they play a direct role in the regulation of two different maturational periods that are critical for the acquisition of female reproductive function: early follicular development and ovulation. Neurotrophins facilitate the development of newly formed follicles by promoting the initial differentiation and the subsequent growth of primordial follicles. These actions appear to be related to the ability of neurotrophins to sustain the proliferation of both mesenchymal and granulosa cells, and to induce the synthesis of follicle stimulating hormone (FSH) receptors. At the time of the first ovulation, neurotrophins contribute to the ovulatory cascade by increasing prostaglandin E(2) release, reducing gap junction communication, and inducing cell proliferation within the thecal compartment of preovulatory follicles.     

Predominance of supraspinal innervation of the left ovary

In our previous studies using the viral transneuronal tracing technique we demonstrated the spinal and supraspinal components of the ovarian innervation. Since increasing number of data indicate the presence of morphological and functional laterality in the control of gonadal functions, we aimed to investigate whether cerebral structures trans-synaptically involved in the innervation of the ovary exhibit asymmetry or not. In one of the studies the left or the right ovary was injected with the red fluorescent protein expressing pseudorabies virus and the number of infected "red" autofluorescent neurons from the right and the left ovary was compared. In another study in order to have distinct labeling of cell groups connected with the right- and left-sided ovary in the same animal, a dual viral labeling was applied. The left- and right-sided ovary were inoculated with genetically engineered pseudorabies virus expressing a red fluorescent protein or a green fluorescent protein gene. Viral infection of brain nuclei including the dorsal vagal nucleus, caudal raphe nuclei, A5 noradrenergic cell group, hypothalamic paraventricular nucleus, from the left ovary in each case was enhanced when compared with labeling from the right gonad. Data suggest a predominance in the supraspinal innervation of the left ovary.     

Structural changes and cell properties of human ovarian surface epithelium in ovarian pathophysiology

The surface epithelial cells of the ovary, which are modified peritoneal cells, form a single, focally pseudostratified layer. The Müllerian ducts differentiate after invagination of the coelomic mesothelium over the gonadal ridges during the 6th week of embryonic life. On the basis of the embryologically putative Müllerian potential of this epithelium, endometriosis can be explained by coelomic metaplasia from the peritoneum, including ovarian surface epithelium. Some pelvic endometriosis specimens have shown that epithelial cells on the ovary or pelvis are serially changed to endometriotic gland cells. Immunohistochemistry as well as scanning electron microscopy also reinforce the light-microscopical findings. A three-dimensional culture system demonstrated that human ovarian surface epithelial cells exhibited a glandular-stromal structure when they were cocultured with endometrial stromal cells in an estrogen-rich environment. Ovarian carcinomas in the epithelial-stromal category are thought to arise from the surface epithelium and its inclusions. The ovarian surface epithelium is physiologically involved in follicular rupture, oocyte release, and the subsequent repair of follicle wall during reproductive age. Simultaneously, ovulation may cause a loss of integrity of the surface epithelium, followed by accumulation of multiple mutations. The cortical invagination, surface stromal proliferation, and Müllerian differentiation of these cells are likely not to be an early step in the cancer development. However, the inclusion cysts are closely related with carcinogenesis because they are significantly more common in ovaries contralateral to those containing epithelial cancers than in control ovaries. As an in vitro study, ovarian carcinoma cell lines were established from simian virus 40 large T antigen-transformed human surface epithelial cells of the ovary. Further investigations of these cell lines may lead to insights into the preneoplastic and early stages of carcinomas. To clarify the pathogenesis of endometriosis and epithelial ovarian cancer, specifically designed studies of ovarian surface epithelium are required.     

The ovary of Lagostomus maximus (Mammalia, Rodentia): an analysis by confocal microscopy

Lagostomus maximus is a notable mammalian model for reproductive studies. Females have an extremely high ovulation rate, which is due to down-regulation of the follicular apoptosis pathway, which ensures a large pool of developing follicles. This large pool is supported by the convoluted anatomy of the mature ovary, whose germinal tissue is found in irregularly curved ridges throughout the cortex. Medullary tissue is restricted to a minimum. Lyso Tracker Red reconstruction under confocal laser scanning microscopy was used to recognize and measure all follicular stages from primordial to antral. Unlike most mammals in which early primordial follicles are just found in fetal life, the adult ovary shows regions packed with early primordial follicles. Follicle size ranged from 24 to 316 microm. We discuss the relationships of L. maximus follicles size with regard to other species of mammals and propose that the physiology of the adult viscacha ovary obeys to a neoteny process in the evolution of this species.     

Anatomy and functions of brain neurosecretory cells in diptera

In the larval brain of dipteran insects, there are two medial and three lateral groups of neurons innervating the ring gland. One lateral group extends fibers to the corpus allatum. After metamorphosis, a large cluster of the medial group in the pars intercerebralis and two lateral groups in the pars lateralis innervate the retrocerebral complex and some neurons from the lateral group and a few from the medial group extend fibers to the corpus allatum in the adults. Neuropeptides such as insulin-like peptides, FMRFamide related peptides, Locusta-diuretic hormone, beta-pigment dispersing hormone, Manduca sexta-allatostatin, ovary ecdysteroidogenic hormone, and proctolin have been immunocytochemically revealed in medial groups in the pars intercerebralis, and FMRFamide related peptides, beta-pigment dispersing hormone, corazonin, and M. sexta-allatostatin in lateral groups in the pars lateralis of dipteran brains. In mosquitoes after the blood meal, ovary ecdysteroidogenic hormone from 2-3 pairs of medial neurosecretory cells is released at the corpus cardiacum to stimulate the ovaries to secrete ecdysteroid to cause ovarian development. In addition to ovarian development, removal and implantation experiments have shown that neurosecretory cells in the pars intercerebralis and pars lateralis are involved in control of reproductive diapause, cuticular tanning, sugar metabolism, and diures.     

Rhyncophorus palmarum L. (Linnaeus, 1758): A morphological and histological study of the female reproductive system

Rhyncophorus palmarum (Coleoptera) is a pest of great economic importance because of the damage caused in the plants of the Palmae family. This study showed that the female reproductive system of this beetle is composed of two ovaries of the telotrophic meroistic type invariably containing two ovarioles per ovary. Each ovariole is home to about 50 oocytes at different stages of maturation. The proximal region of the ovary is rather peculiar because it houses inside it a large quantity of bacilliform structures immersed in a glycolipoprotein substance, which has never before been described in the literature. The function of these structures is still unknown. However, it is suggested that they can function as a method of neutralizing the resistance of the plant because these insects are responsible for the transmission of the red ring disease to the palm plants. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Supraspinal connections of the ovary: structural and functional aspects

This review summarizes our recent studies using the viral transneuronal tracing technique to identify sites in the central nervous system (CNS) that are connected with the ovary. A neurotropic virus (pseudorabies virus) was injected into the ovary and various times after the inoculation the spinal cord and brain were examined for virus-infected neurons identified by immunocytochemistry. Such neurons could be detected in well-defined cell groups of the spinal cord (intermediolateral cell column), brain stem (vagal nuclei, area postrema, parapyramidal nucleus, caudal raphe nuclei, A1, A5, A7 noradrenergic cell groups, locus coeruleus, Barrington's nucleus, periaqueductal gray), hypothalamus (paraventricular nucleus, anterior hypothalamus, arcuate nucleus, zona incerta), and, at longer survival time, in some telencephalic structures (amygdala, bed nucleus of the stria terminalis). These findings provided the first neuromorphological evidence for the existence of a multisynaptic neuronal pathway between the brain and the ovary presumably involved in the neuronal control of the organ. The observations indicate that there is a significant overlap of CNS structures connected with the ovary, the testis, other organs and organ systems, suggesting similar neuronal circuitries of the autonomic nervous system innervating the different organs. The known descending neuronal connections between the CNS structures labeled from the ovary by the viral transneuronal tracing technique and the findings suggesting a pituitary independent interplay between certain cerebral structures such as the hypothalamus, the amygdala, and the ovary are also summarized in this review.     

Occasional transsynaptic viral labeling in the central nervous system from the polycystic ovary induced by estradiol valerate

Increased density of catecholaminergic nerves in the human polycystic ovary has been observed. The aim of the present study was to investigate the distribution of transsynaptically virus-labeled neurons in the central nervous system from the rat polycystic ovary to see whether is it different or not from that of cycling control rats. To induce a polycystic ovary, a single injection of estradiol valerate was given to adult female rats and 30 days later a neurotropic virus was injected into the right ovary. Rats were sacrificed 72 or 96 hours after viral infection. Weight of the ovaries of the estradiol valerate-treated rats was significantly lower compared to controls, and the histology of the ovaries of the treated rats displayed severely atretic large antral follicles. There was almost no viral labeling in the central nervous system from the ovaries showing precystic morphology, in spite of the fact that such altered organs are rich in nerve fibres. It is assumed that presently unidentified factors in the precystic ovary, presumably related to the link between the immune and the nervous system, might be involved in the infectivity of the virus, and thus be responsible for the lack of viral labeling from such an ovary.     

Ovarian acetylcholine and muscarinic receptors: hints of a novel intrinsic ovarian regulatory system

More than two decades ago, the degrading enzyme of the neurotransmitter acetylcholine (ACH) was reported in nerve fibers of the rat ovary. Subsequently, it was assumed that ACH is a neurotransmitter of ovarian nerves, although the sole presence of the degrading enzyme, ACH-esterase, does not allow such a conclusion. That ACH may be involved in the complex regulation of ovarian functions, including hormone production, was indicated by studies using, for example, granulosa cells (GCs). The lack of detailed information about both source(s) and functions of ACH in the ovary prompted us to examine sites of ovarian ACH-synthesis and ACH-receptor-bearing target cells. We also started to identify functions of ACH in cultured human GCs. While ovarian innervation and recently described neuron-like cells of the ovary were not immunoreactive for the ACH-synthesizing enzyme, choline-acetyl transferase (CHAT), we found immunoreactivity in GCs of rodents and primates. Isolated human and rat GCs produced ACH and contained the vesicular ACH transporter (VACHT). These results indicate that endocrine GCs are an unexpected non-neuronal source of ACH in the ovary. Moreover, these cells and GCs in vivo contain ACH-receptors of the muscarinic subtype (MR), namely M1R and M5R. In contrast, oocytes express M3R. MR of human GCs are functional and cholinergic stimulation is linked to rapid increases in intracellular Ca(++) levels. M1/5R activation also led to increased cell proliferation of human GCs in vitro and this stimulatory effect was found to be associated with rapid disruption of gap junction communication. Ongoing studies begin to identify regulation of ion channels and altered gene expression as consequences of MR stimulation. Thus, our results outline first details of an unexpected intraovarian, non-neuronal cholinergic system, and suggest that it may be involved in the regulation of cell proliferation in the ovary.     

Ultrastructural characterization of apoptotic granulosa cells in caprine ovary

The unique phenomenon of cell proliferation and apoptosis is encountered in the ovarian follicles undergoing early stages of atresia. The aim of this study was to verify the morphological variations in these two physiologically distinct processes operating in antral follicles of caprine ovaries using histological and ultrastructural techniques. Histologically the degenerating granulosa cells were characterized by condensed cytoplasm, and nucleus fragmentation in hazy cytosol. The pyknotic nuclei of degenerating cells stained darkly with haematoxylin and giemsa while the cytoplasm was eosinophilic. Under electron microscopy, apoptosis was marked by asymmetrical shrinkage, vacuolization of cytoplasm, swollen and vacuolated mitochondria, increased irregularity and/or fragmentation of nucleus, chromatin condensation and finally, production of membrane enclosed nuclear fragments containing intracellular material, the apoptotic bodies. The parallel use of these two methods on caprine ovaries has enabled us to analyse the decline in the frequency of granulosa cells during follicular atresia due to apoptosis.     

Scanning electron microscopic changes in granulosa cells during follicular atresia in Caprine ovary

During this study, topographic changes in healthy and atretic granulosa cells have been investigated during follicular atresia in goat ovary. Under scanning electron microscopy atresia was marked by asymmetrical shrinkage and vacuolization of cytoplasm. The specific topographical alterations observed in atretic cells were loss of micro extensions, disruption of cell-cell interaction, and smooth-textured membrane with a number of uneven depressions and ruffles. Some portions of the cell membrane were marked by extensive shrinkage due to condensation of cytosol. Irregular membrane at occasions was studded with blunt microextensions. The findings of present investigation will help in understanding the cellular changes in granulosa cells during follicular atresia and will find applications in screening of follicles for in vitro culture, in vitro fertilization and Embryo transfer technology.     

Review: Studies on the reproductive and developmental biology of <I>Cichlasoma dimerus</I> (Percifomes,Cichlidae)

Many characteristics of the South American teleost fish Cichlasoma dimerus (body size, easy breeding, undemanding maintenance) make it amenable to laboratory studies. In the last years, many of the fundamental aspects of its reproductive and developmental biology have been addressed in our laboratory. Rather recently, the immunohistochemical localization of pituitary hormones involved in reproduction and in background color adaptation has been described in both adult and developing individuals, and the role of FSH in ovarian differentiation has been established. These findings have been correlated with mapping of some of their brain-derived controlling hormones. The latter include brain-derived gonadotropins which were shown to be active in vitro in the control of pituitary hormone secretions. The emerging picture shows C. dimerus as an interesting species in which many of their basic features have already been investigated and which conform a solid platform for comparative studies correlating neurohormones, pituitary hormones and behavior, from the molecular to the organismic level.     

A combination of stereological methods,biochemistry and electron microscopy for the investigation of drug treatment effects in experimental animals

Some chemotherapeutic agents used for breast cancer (BC) treatment can induce severe side effects in the ovarian tissue. The combination of cyclophosphamide and docetaxel (TC) is widely used for BC treatment; however, its late effects in the ovary are not completely understood. The main purpose of this study was to evaluate the structural and ultrastructural alterations in the ovarian stroma induced by TC treatment. Wistar rats were divided into two groups: a control group and a TC group. They were euthanized 5 months after the end of treatment, and their plasma and ovaries were collected. Important alterations were noted. The serum estradiol level was significantly reduced in the TC group compared with the control group. Additionally, the number of apoptotic nuclei was higher in the TC group. The role of the inflammatory response in the development of ovarian damage was investigated, and we found an increased number of mast cells and increased expression of TNF‐α in the TC group. The involvement of fibrosis was also investigated. The results showed that the TC group had increased expression levels of TGF‐β1, collagen type I (col‐I) and collagen type III (col‐III) compared with the control group. Ultrastructural analysis revealed the presence of collagen fibrils in the treated group and illustrated that the ovarian tissue architecture was more disorganized in this group than in the control group. The results from this study are important in the study of chemotherapy‐induced ovarian failure and provide further insight into the mechanisms involved in the development of this disease.     

The fertility tracks of pollen tube in the ovary of Solanum nigrum by three-dimensional reconstruction

In this paper, we present an enhanced method for automatically capturing a large number of consecutive paraffin sections using a microscope. Leveraging these microstructural images, we employed three-dimensional visualisation and reconstruction techniques to investigate the dispersal growth process of pollen tube bundles upon entering the ovary of Solanum nigrum. Additionally, we explored their behaviour within different ovules and examined the relationship between the germination rate of seeds and the fertilisation process. Our findings reveal that despite the abundance of Solanum nigrum seeds, only a fraction of them is capable of successful germination. The germination rate of seeds is closely related to whether fertilisation of the ovules and pollen tubes is completed. Due to the limited number of pollen tubes entering the ovary, only a portion of the ovules can be fertilised. The proportion of fertilised ovules positively correlates with the germination rate of the seeds. Through three-dimensional reconstruction, we observed a phenomenon of proximity during the pollination process, wherein ovules closer to the pollen tube bundles are more likely to be fertilised. Furthermore, fertilised ovules exhibited significant changes in morphology and embryo sac structure. The number of fertilised ovules directly impacts the germination rate of wild Solanum nigrum seeds. Although all Solanum nigrum ovules have the potential to develop into seeds, most seeds originating from unfertilised ovules are unable to germinate normally, resulting in an incomplete germination rate of seeds and preventing it from reaching 100%.