Introduction: The existence of ovarian intrinsic neurons is well established. However, the morphology and chemical phenotype are not completely characterized and are even unknown for some species used in medical research. The purpose of this work was to determine the morphology and chemical phenotype of intrinsic neurons of the guinea pig ovary at two ages: neonates (0 days old) and sexually mature reproductive animals (90 days old). Materials and Methods: For the morphological analysis, we employed the modified Golgi-Cox impregnation technique. For the chemical phenotype, we used immunohistochemistry and the following antibodies; tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), transient receptor potential type 1 (TRPV1), neuron-specific nuclear protein (NeuN) and proto-oncogene product of the cFos gene (cFos). We also used enzyme histochemistry for NADPH-diaphorase detection. Results: The number of intrinsic neurons in the neonate ovary was low in comparison to the adult guinea pig ovary. The intrinsic neurons were located in the cortex and the ovarian medulla; some were isolated or clustered, forming ganglia, and others were interconnected and formed networks. The neurons were small, medium or large. In the cortex of neonate vs adult ovaries, the small and medium neurons comprised 23% vs 36% and 5.2% vs 11.6%, respectively. In the medulla, the percent of the same neurons was 10.1% vs 10.1% and 1.1% vs 2.2% in the neonate and adult, respectively. In both cortex and medulla < 1% were large neurons at two ages. Also, the neurons were rounded, fusiform or multipolar. In the cortex, they were 12.7% vs 20.9%, 14.9% vs 24.2% and 1.1% vs 3.0%, respectively. In the medulla, the percent of small vs medium neurons was 6% vs 7.1% and 4.1% vs 4.8% in the neonate and adult ovary, respectively, and <1% were large neurons at both ages. The chemical phenotypes were in the neonate and adult: TH/NeuN-positive neurons, 16.3% vs 26.5%; CGRP/NeuN, 13.5% vs 35.8%; TRPV1/NeuN, 10.2% vs 38.6%; and cFos/NeuN, 4.6% vs 5.4%, respectively.The percent of NADPHd-positive cells in the cortex was 9.5% vs 25.1% and 3.2% vs 62.2% in the medulla in the neonate and adult, respectively. Conclusion: Altogether, these data showed that the number of ovarian intrinsic neurons was low at birth and increased in the sexually mature reproductive guinea pig. The chemical phenotype was rich and peptidergic, catecholaminergic and nitrergic in nature and positive for cFos immunoreactivity. Therefore, intrinsic neurons can be chemical sensors inside of the gonad and transmit signal to the central nervous system.
It has been established that the mammalian ovary is supplied by sympathetic and sensory nerve fibers [
During the experimental part of this study, we followed the principles of animal care approved by the BUAP Animal Care Committee and the national laws on animal protection (Mexican Council for Animal Care, Norma Oficial Mexicana NOM-062-ZOO-1999). The local guidelines were also in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals of the USA. All efforts were made to minimize animal suffering and to reduce the number of animals used. The immature guinea pigs (Caviaporcellus) were obtained at birth from pregnant animals. The adult guinea pigs were housed in groups (3 animals per cage) under a dark-light cycle of 12 - 12 hours with a room temperature of 22 ± 2 degrees Celsius. Additionally, the animals had free access to food and potable water, which was supplemented with Vitamin C. We used thirty female guinea pigs, fifteen immature animals (at birth day, P0) and another fifteen sexually mature reproductive animals (90 days old, P90), randomly divided by ages into six groups (n = 5). The animals of the first and second group (P0 or P90, respectively) were used for ovary collection and modified Golgi-Cox impregnation. The ovaries of the animals of the third and fourth group were used for immunocytochemistry and evaluation of the co-expres- sion of CGRP/NeuN-, TRPV1/NeuN-, TH/NeuN-, and cFos/NeuN-positive cells, and the ovaries of the animals of the fifth and sixth groups were used for enzyme histochemistry and evaluation of NADPHd-positive cells. All experimental guinea pigs were sacrificed with CO2.
The ovaries were treated by the modified Golgi-Cox technique [
The following antibodies were used: rabbit polyclonal and mouse monoclonal anti-CGRP (Calcitonin Gene- Related Peptide, Life Science); mouse monoclonal anti-TRPV1 receptor (Transient Receptor Potential Vanilloid type 1, Millipore); anti-TH (Tyrosine Hydroxylase, Abcam); rabbit polyclonal anti-cFos (Santa Cruz Biotechnology); rabbit polyclonal anti-FOX3/NeuN (FOX3, Abcam); and secondary antibodies FITC (IgG-free from goat, Millipore) and Texas Red (IgG-free from goat, Millipore).
The animals from each experimental group were sacrificed with CO2 and perfused with intracardiac isotonic saline solution (NaCl, 0.9%) followed by paraformaldehyde (4%) in PBS, pH 7.4 (PF-PBS solution). The ovaries were removed and post-fixed in PF-PBS solution, embedded in paraffin and sectioned with a Leica SM2010R microtome (5 µm). The sections were treated with xylene and ethanol and washed with PBS solution. The sections were blocked with bovine albumin (IgG-free), treated with Triton X-100 and washed with PBS at 24 degrees Celsius. The samples were incubated with the anti-CGRP (1:500), anti-TRPV1 receptor (1:50), anti-TH (1:800), FOX3/NeuN (1:250) or cFos (1:500) antibody overnight at 4 degrees Celsius and then incubated with a secondary antibody (FITC) (IgG-free from goat; 1:250) or Texas Red (IgG-free from goat; 1:400) for 2 hours at room temperature. The specificity of the antibodies was confirmed in separate experiments with additional negative controls, including tissue sections incubated in the absence of primary antibody. All sections were mounted on microscope slides with mounting fluid (Millipore, USA) and observed under a Leica fluorescence micro- scope.
We analyzed ovary sections from five different animals with three repeat analyses for each gonad. The images were captured with a Leica-DFC325 camera, and the data were stored on the PC hardware. For five different ovaries, we counted the positive cells in 10 representative fields of each tissue section using the cell counter tool from NIH ImageJ software. From a qualitative point of view, the following three levels of fluorescence intensity were identified: light, medium and high. The cells were considered positive if they had a strong color signal and negative if they had a light or medium color signal. The final quality score was assessed using the software measure tool; the cells were positive when the measure was ≥50 arbitrary units, and the cells were negative when the measure was ≤49 arbitrary units.
For NADPH-d staining, whole mounts of the ovary serial sections (30 μm) through five ovary segments from different animals were used. The samples were incubated for one hour at 37 degrees Celsius with 1 mg/mL βNA- DPH-d (Sigma-Aldrich, USA), 0.2 mg/mL nitro-blue tetrazolium chloride (Sigma-Aldrich, USA), and 0.5% triton X-100 PBS in 10 mM malic acid. After incubation, the whole mounts were washed in 0.1 M PBS, and the sections were mounted with synthetic resin and examined by light microscope (Zeiss, Germany). In each experiment and for negative controls, we used samples with the same protocol in the absence of the βNADPH-d enzyme.
Given that the ovaries are different sizes because they were from animals at two different ages, immature (0 days old, P0) and sexual mature reproductive guinea pigs (90 days old, P90), we standardized the number of samples using 10 histological sections per ovary symmetrically distributed along the entire gonad. After that, the neuron bodies were identified using light microscopy in 5 fields for each histological section, and the images were captured with a Cannon S80, stored in PC hardware and analyzed with the software Zoom Browser program, EX.
Data represent the number of neurons identified in 50 microscopy fields examined from 10 sections and 5 fields per section per ovary. All data are expressed as the mean and SEM and were compared using the Student t-test. A probability of less than 5% was considered significant.
In neonate ovaries, the number of intrinsic neurons was low (10 ± 0.4) but increased 62% over neonates in sexually mature guinea pig ovaries (16.1 ± 0.5). These neurons were differentially distributed; in the cortex of the neonate ovary there were 7.7 ± 0.5 intrinsic neurons (28.9%), and in the cortex of the adult ovary there were 12.9 ± 0.4 neurons (48.5%) (*p < 0.05, Student T-test). In the medulla of the neonate, the number of neurons was 2.8 ± 0.2 (10.5%), and in medulla of the adult ovary there were 3.2 ± 0.3 neurons (12.1%) which was not statically different from the neonate (Student t-test).
Using the size of the cell body, the ovarian intrinsic neurons were classified as small [7 - 15 μm (33.1% vs 46.3% for the neonate and adult, respectively)], medium [15 - 16 μm (6.2% vs 13.6% for the neonate and adult, respectively)] or large [26 - 40 μm < 1% at both ages], located both in the cortex and in medulla of the ovary. In the ovarian cortex, the number of small neurons in the neonate ovary was 6.2 (23%) and in the adult ovary the number was 9.7 (36%), 0.6 times higher than the neonate ovary (
By describing the morphology of the cell body the ovarian neurons could be considered rounded, fusiform and multipolar (
In general, we found that fusiform and rounded neurons were isolated while other neurons came together forming compact ganglia (Figures 3(A)-(D)). The multipolar neurons were persistently placed in the ovarian cortex, and this observation reached statistical significance (
Distinct from neurons in neonate ovaries, the adult guinea pig ovary had neurons making networks (
In the neonate ovary, the number of NeuN-positive cells, a specific marker for neurons, was 5.1 (23.7%), and in the adult ovary there were 7.2 (33.5%), 0.4 times higher than in the neonate ovary, whereas the number of neurons that co-expressed two neuronal markers (TH/NeuN-positive cells) was 3.5 (16.3%) and 5.7 (26.5%). In the adult ovary, the number of TH/NeuN-positive neurons increased 0.6 times in comparison to the neonate ovary and was statistically significant (Figures 5(A)-(B)) (*p < 0.05, Student t-test).
In the neonate ovary, the number of NeuN-positive cells was 4.6 (14.1%), and in the adult guinea pig, it was 11.9 (36.6%). Therefore, the number of neurons that co-expressed two cell markers (CGRP/NeuN-positive cells) were 4.4 (13.5%) and 11.6 (35.8%), respectively, numbers that show an increase in neurons 1.6 times in comparison to neonate ovaries (Figures 6(A)-(B)) (*p < 0.05, Student-test).
In both the neonate and adult guinea pig ovaries, the NeuN-positive cells were 5 (11.6%) and 17.1 (39.6%), respectively. The number of neurons that co-expressed two cell markers (TRPV/NeuN-positive cells) was 4.4 (10.2%) in the neonate ovary and 16.7 (38.6%) in the adult guinea pig. In the adult, the number of TRPV1/ NeuN-positive neurons increased 2.8 times in comparison to the neonate ovary (Figures 7(A)-(B)) (*p < 0.05, Student t-test).
In both the neonate and adult ovary, the number of cFos-positive cells was similar, with 32.1 (38.6%) vs 33.6 (40.4%) positive cells, respectively. However, the number of NeuN-positive cells was 4.1 (4.9%) vs 5.1 (6.1%). Finally, the number of cells that co-expressed the two cell markers (NeuN/cFos) was 3.8 (4.6%) vs 4.5 (5.4%). No statistical significance was observed for any of the cases (
Neonate as adult guinea pig ovaries had NADPHd-positive neurons (Figures 9(a)-(b)). In the cortex there were
3.0 (9.5%) and 7.9 (25.1%) NADPHd-positive neurons for the neonate and adult, respectively, and in the medulla there was 1.0 (3.2%) and 19.6 (62.2%) positive cells, respectively (
This is the first report to indicate the distribution, morphology and chemical phenotype of intrinsic neurons in the guinea pig ovary. The high number of neurons found in the adult ovary in comparison to the neonate ovary, and the higher amount in the cortex in comparison to the ovarian medulla adds to the information on ovarian intrinsic innervation in mammals. Additionally, these results are in concordance with other studies previously reported in rats [
The small number of TH, CGRP and TRPV1 neurons in the neonate ovary (16.3%, 13.5% and 13.5%, respectively) that increased in the adult ovary (26.5%, 35.8% and 38.6%, respectively) correlates with the high rate of steroidogenesis and high vascular blood fluid displayed in sexually mature reproductive ovaries [
A novel result reported herein was the identification of multipolar neurons in the ovarian cortex, cells with several processes emerging from round or oval cell bodies. The multipolar ovarian neuron morphology was similar to Dogiel type II neurons described in the small intestine of several species [
The neuron networks occur in the adult ovary and not in the immature ovary of the guinea pig, suggesting that they are developed along with the maturation of the reproductive system. In the sexually mature ovary, neuron molding is possible because long-term 17β-estradiol treatment reduces the population of neurons in the sympathetic chain ganglia connected to the ovary [
The neuron networks and chemical phenotype of the intrinsic neurons in the guinea pig ovary suggest an in- tegrative role. The presence of TH-positive neurons are in agreement with numerous networks formed by catecholaminergic neurons in the primate ovary, which are highly developed at puberty [
The ovary contained round, fusiform and multipolar-shape neurons, isolated or grouped in ganglia, making networks in the adult ovary. The neurons had profuse chemical phenotypes (CGRP/NeuN-, TRPV1/NeuN-, TH/ NeuN- and NADPHd-positive) and active considering its cFos immunoreactivity, supporting the notion that ovarian intrinsic neurons can be sensors in the gonad and transmit signals to the central nervous system.
This work was supported by VIEP-BUAP grants 2011-12, and Ericka Barrientos was supported by the Consejo Nacional de Ciencia y Tecnología through the postgraduate program (CONACYT/418053). Additionally, we are grateful to Diego Luna for his English language editing.
The authors declare that they have no competing interests.
All authors read and approved the final manuscript.
Calcitonin Gene Relate Peptide (CGRP);
Transient Receptor Potential Vanilloid Type 1 (TRPV1);
Tyrosine Hydroxylase (TH);
NADPH-Diaphorase (NADPHd);
Proto-Oncogene Product of the cFos Gene (cFos);
Neuron-Specific Nuclear Protein (NeuN).