Histological and Immunohistochemical Characterization of Vomeronasal Organ Aging in Mice

animals

Article Histological and Immunohistochemical Characterization of Vomeronasal Organ Aging in Mice

Violaine Mechin 1,* , Patrick Pageat 2, Eva Teruel 3 and Pietro Asproni 1

1 Tissue Biology and Chemical Communication Department, IRSEA, Institute of Research in Semiochemistry and Applied Ethology, 84400 Apt, France; [email protected] 2 Research and Education Board, IRSEA, Institute of Research in Semiochemistry and Applied Ethology, 84400 Apt, France; [email protected] 3 Statistics and Data Management Service, IRSEA, Institute of Research in Semiochemistry and Applied Ethology, 84400 Apt, France; [email protected] * Correspondence: [email protected]; Tel.: +33-4-90-75-57-00

Simple Summary: Chemical communication has been intensely studied and the importance of its role in animal life has been ascertained. Located in the nasal cavity, the vomeronasal organ is one of the main actors in charge of chemical reception. Alterations of this organ have proven to modify behavioral responses to semiochemical expositions. For all the other organs, a well-known origin of alteration is aging. The objective of this study was to analyze this effect on the vomeronasal organ condition and to determine the nature of these potential changes. This study demonstrates that this organ is significantly impacted by aging. In particular, old mice present strong signs of neuronal degeneration compared to adults. Abstract: The vomeronasal organ (VNO) plays a crucial role in animal behavior since it is responsible Citation: Mechin, V.; Pageat, P.; for semiochemical detection and, thus, for intra- and interspecific chemical communication, through Teruel, E.; Asproni, P. Histological the vomeronasal sensory epithelium (VNSE), composed of bipolar sensory neurons. This study and Immunohistochemical aimed to explore a well-recognized cause of neuronal degeneration, only rarely explored in this Characterization of Vomeronasal Organ Aging in Mice. Animals 2021, organ: aging. Murine VNOs were evaluated according to 3 age groups (3, 10, and 24 months) by 11, 1211. https://doi.org/10.3390/ histology to assess VNSE changes such as cellular degeneration or glycogen accumulation and by ani11051211 immunohistochemistry to explore nervous configuration, proliferation capability, and apoptosis with the expression of olfactory marker protein (OMP), Gαi2, Gαo, Ki-67, and cleaved caspase-3 Academic Editor: Pablo proteins. These markers were quantified as percentages of positive signal in the VNSE and statistical Sánchez Quinteiro analyses were performed. Cellular degeneration increased with age (p < 0.0001) as well as glycogen accumulation (p < 0.0001), Gαo expression (p < 0.0001), and the number of cleaved-caspase3 positive Received: 3 March 2021 cells (p = 0.0425), while OMP and Gαi2 expressions decreased with age (p = 0.0436 and p < 0.0001, Accepted: 21 April 2021 respectively). Ki67-positive cells were reduced, even if this difference was not statistically significant Published: 22 April 2021 (p = 0.9105). Due to the crucial role of VNO in animal life, this study opens the door to interesting perspectives about chemical communication efficiency in aging animals. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: vomeronasal organ; aging; pathology; histology; chemoreception published maps and institutional affil- iations.

1. Introduction The Jacobson organ, or vomeronasal organ (VNO), is a tubular structure bilater- Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. ally located in the nasal septum of most animals, such as amphibians, reptiles, and This article is an open access article mammalians [1–4]. It is composed of a vomeronasal sensory epithelium (VNSE) and distributed under the terms and a non-sensory epithelium (NSE) arranged around a lumen [3,4]. Animals possess a conditions of the Creative Commons variety of vomeronasal receptors, belonging to three families: formyl peptide recep- Attribution (CC BY) license (https:// tors (FPRs) [5], vomeronasal type 1 receptors (V1R), and vomeronasal type 2 receptors creativecommons.org/licenses/by/ (V2R) [6]. Vomeronasal receptors are the ones implicated in pheromone reception and thus 4.0/). in chemosensory responses [7–9]. V1R and V2R are respectively coupled to the Gαi2 and

Animals 2021, 11, 1211. https://doi.org/10.3390/ani11051211 https://www.mdpi.com/journal/animals Animals 2021, 11, 1211 2 of 12

Gαo proteins and assure the function of semiochemical reception to the VNO [6,10,11]. These receptors detect semiochemicals in the vomeronasal lumen and transmit the message to the accessory olfactory bulb (AOB), which enables the animal to adapt its response to the signal. Thanks to this function, the VNO plays a vital role in chemical cues reception, in animal behavior and, thus, in its life [12–14]. Previous studies have shown that an experimentally damaged VNO could not assure a normal chemical communication among conspecifics, contributing to the onset of deficits in maternal, sexual, and social behaviors [15–19]. Moreover, spontaneous VNO alterations have been shown to be able to influence behavior in cats and were correlated with intraspe- cific aggression [20]. A study on pigs showed that vomeronasalitis was also associated with an increase in social conflicts depending on the VNSE inflammation intensity. Many research studies have been conducted on aging in the central and peripheral nervous system, showing its evident impact on the brain and neuronal cell degeneration [21,22]. However, until today, only the influence of age on cell proliferation has been investigated in the VNO, showing that the cell number and epithelium density increased until 4 months, decreased between 4 and 8 months, and remained unchanged after [23,24]. An interesting study of Brann and colleagues showed the high capacity of the neuronal tissue of VNO reconstitution after a clinical injury, even in aged subjects, suggesting the preservation of dynamic stem cells during lifetime [25]. However, to the best of our knowledge, the nature of aging effects on the VNO neurons in normal conditions has not been already described. Therefore, this study aimed to characterize the biologic effects of aging on vomeronasal organ degeneration, in particular, in its sensorial part, as it is one of the most important tools for animal communication. To achieve this purpose, the VNSE condition was assessed by histochemical and immunohistochemical approaches on three age groups of mice. The olfactory marker protein (OMP) was explored since it is commonly used as a marker of olfactory and vomeronasal neurons [26,27]. Gαi2 and Gαo proteins were studied to characterize the vomeronasal neuron type (expressing V1R or V2R) [6,10,11]. Finally, IHC markers of apoptosis and proliferation were also investigated between age groups to explore cellular turnover in the aging VNSE.

2. Materials and Methods This study was carried out in strict accordance with the requirements of French and European Law on the protection of animals used for scientiﬁc purposes (2010/63/EU). The protocol was approved by the Committee C2EA125 on the Ethics of Animal Experiments of IRSEA (approval number: CE_2019_06_POVN).

2.1. Animals and Sampling Procedure This study was conducted on the VNOs from 41 mice (82 VNSEs) Mus musculus (pro- vided by Janvier Lab, Le Genest-Saint-Isle, France), split into three age groups, according to Flurkey’s classification [28]: G1: n = 11 mice 3 months old (adult), G2: 10 mice 10 months old (middle aged), G3: 20 mice 24 months old (old). Males and females were used in each group, belonging to two strains: the C57/6JRj (inbred) and the RjOrl:SWISS (outbred), to evaluate VNO changes in two commonly used genetic profiles. The animals’ repartition is described in Table S1 in Supplementary files. Animals were maintained in the IRSEA’s facilities, at a temperature of 22 ± 2 ◦C and 60 ± 20% humidity with a 12–12 h light/dark cycle, with food and water ad libitum. At the required age, they were humanely euthanized with an intraperitoneal injection of Dolethal® (100 mg/kg, Vetoquinol, Lure, France). The mice were then decapitated; their heads were immediately sampled and placed in a 10% formalin solution (pH 7.4) for one week to fix the tissues. Decalcification was performed by 48 h immersion in decalcifying solution (DDK, Milan, Italy). The noses were cut transversally to obtain 2–3 mm thin sections containing vomeronasal organ portions, processed, and paraffin-embedded according to Animals 2021, 11, 1211 3 of 12

routine histological methods. Sections of 3.5 µm thickness were made and dried overnight at 37 ◦C on SuperFrostPlus™ (Cat No. 10149870, Thermo Fisher Scientiﬁc, Illkirch, France) before being submitted to histological and immunohistochemical analyses.

2.2. Histochemical Analyses Hematoxylin and eosin staining (H&E, BioOptica, Milan, Italy) was performed to assess the general condition of the VNO. Each VNSE was blindly classiﬁed on a scale score according to the degree of neuronal degeneration as 0 = nonexistent, 1 = weak, 2 = moderate, and 3 = strong. A score of zero was attributed when no physical signs of degeneration and no aberrant cellular vacuolization was detected. If any initial signs of degeneration were present, such as diffused vacuolization or non-homogenous cellular repartition, the tissue was noted as weak, with a score of 1. A moderate reduction of tissue density and diffuse vacuolization were qualiﬁed as score of 2. Finally, the maximal score of 3 was attributed in case of strong diffuse signs of degeneration, reduction of tissue density, and strong over vacuolization. Periodic acid Schiff (PAS) staining (Cat No. C062AA, DiaPath SpA, Martinengo, Italy) was also performed following the manufacturer’s instructions to reveal glycogen accumulations. The images were analyzed with ImageJ software, using the “Color deconvolution” plug-In and the “H-PAS” vector. Red-purple color corresponding to the positive PAS staining and the VNSE surface were selected to obtain a percentage of PAS-positive areas in the selected VNSE surface.

2.3. Immunohistochemical Analyses IHC was performed to evaluate the presence of proteins involved in VNO functionality and in cellular turnover. After rehydration, tissues were pretreated with a microwave in 0.1 M citrate buffer pH 6 (Cat No. F/T0050, DiaPath SpA, Martinengo, Italy), 3 min and 30 s at 560 W, and 15 min at 210 W. Slides were then incubated with a peroxidase blocking solution (Cat No. ACA500, Scytek, Logan, UT, USA) for 30 min protected from light, followed by an UltraVision Protein Block incubation step (Cat No. TA-125-PBQ, Thermo Scientiﬁc, Carlsbad, CA, USA), for 10 min. The sections were then incubated for 1 h at room temperature with the primary antibodies anti-olfactory marker protein, anti-Gαo, anti-Gαi2, anti-cleaved caspase 3, or anti-Ki67, as described in Table1.

Table 1. List of antibodies used in this study.

Antibody Host Clonality Dilution Reference Supplier Anti-Olfactory Marker Protein (OMP) Rabbit Polyclonal 1:10,000 O7889 Sigma, Saint-Louis, MO, USA Anti-Gαi2 protein Rabbit Monoclonal 1:200 Ab157204 Abcam, Cambridge, UK Anti-Gαo protein Goat Polyclonal 1:200 Sc26769 Santa Cruz Biotechnologies, CA, USA Anti-Cleaved Caspase 3 Rabbit Polyclonal 1:1000 9661 Cell Signaling Technology, Danvers, MA, USA Anti-Ki-67 Rabbit Monoclonal 1:50 MA514520 Life Technologies, Carlsbad, CA, USA

After washing in TBS Tween 1%, a 10 min incubation was performed with a secondary biotinylated anti-species (UltraTek Anti-rabbit, Cat No. T/ABE125, or Anti-goat, Cat No. F/AGL125, ScyTek Laboratories, Logan, UT, USA), followed by a 10 min streptavidin- peroxidase incubation (Cat No: 12694067, Invitrogen, Carlsbad, CA, USA). Visualization was obtained with 3,30 diaminobenzidine tetrahydrochloride (ImmPACT® DAB Peroxidase Substrate, Cat No. SK4105, Vector Laboratories, Burlingame, CA, USA) and a hematoxylin counterstain. The sections were ﬁnally dehydrated through alcohols, cleared in xylene, and mounted. For each antibody, as negative controls, the primary antibodies were replaced with a nonimmune rabbit or goat serum, while for positive controls for Ki-67 and cleaved caspase 3, murine lymph node sections were included in each IHC run. Animals 2021, 11, 1211 4 of 12

The software ImageJ was used to measure IHC positivity of each antibody in the VNSEs. To quantify the OMP and G proteins, the plugin “Color deconvolution” was used in Image J to measure the positive pixels as it allows for extrapolation of the DAB staining in the manually selected VNSE area. The software automatically calculated the percentage of positivity on the VNSE surface. For Ki-67 and cleaved caspase 3, positive cells were manually counted on each section using the ImageJ plugin “cell counter”, then the percentage of positive cells among the total VNSE cells was calculated. IHC quantiﬁcation methods for each antibody are presented in Table2.

Table 2. Studied parameters and their quantiﬁcation.

Antibody Targeted Cells/Molecules Quantiﬁcation Method Anti-Olfactory Marker protein (OMP) Mature neurons quantiﬁcation % of positivity signal/VNSE surface Anti-Gαi2 protein V1R neurons % of positivity signal/VNSE surface Anti-Gαo protein V2R neurons % of positivity signal/VNSE surface Anti-Cleaved Caspase 3 Apoptosis Cell number/VNSE surface Anti-Ki-67 Proliferation activity Cell number/VNSE surface

2.4. Statistical Analyses Data analyses used SAS 9.4 software (Copyright© 2021–2012 by SAS Institute Inc., Cary, NC, USA). The significance threshold was classically fixed at 5%. The principal explanatory variable was the age of the mice (3, 10, or 24 months). Sex (M/F) and strains (inbred/outbred) were also collected to see if the effect of age on the VNO degeneration was different depending on the sex and/or strains. For that, age * sex and age * strains interactions were included in the models in addition to age, sex, and strains factors. Two VNSEs (from left and right parts of each VNO) were available from each mouse. Thus, when possible, each mouse was considered as a random factor in the models. For OMP expression, accumulated glycogen and Gαo protein, a general linear mixed model was applied. The assumptions of residual normality and homoscedasticity were verified. Post hoc multiple comparisons were studied using the Tukey–Kramer adjustment. For the Gαi2 protein, assumptions of normality and homoscedasticity were not verified. Other distributions were tested in order to try to apply a generalized linear mixed model, but no distribution matched with this variable. Additionally, data transformation did not lead to validation of the conditions of application. As a result, only an age effect was studied using the nonparametric test of Kruskal–Wallis. Wilcoxon Mann–Whitney tests were performed to compare age groups two by two. The Bonferroni correction was applied considering the multiplicity of the tests. For cellular degeneration (non-existent, weak, moderate, strong), it was not possible to include the mice as a random effect because the results were unstable using a multinomial mixed model. Consequently, a classical ordinal multinomial model was performed using the LOGISTIC procedure. Odds ratios were computed for multiple comparisons. For proliferation and apoptosis parameters, which were count data, mixed Poisson models were applied. The total number of cells was added to the models as an offset term to take into account the fact that this number was not the same for all VNOs studied. The dispersion of the models was studied by regarding the Pearson chi-square/DF statistic. Post hoc multiple comparisons were studied using the Tukey–Kramer adjustment.

3. Results For each parameter, no statistical differences were found comparing sex, strains, and sex * age or strain * age interactions. Statistical differences were only found when ages were compared. Descriptive data are shown in Table3. Animals 2021, 11, 1211 5 of 11

dispersion of the models was studied by regarding the Pearson chi-square/DF statistic. Post hoc multiple comparisons were studied using the Tukey–Kramer adjustment.

3. Results For each parameter, no statistical differences were found comparing sex, strains, and sex * age or strain * age interactions. Statistical differences were only found when ages Animals 2021, 11, 1211 were compared. 5 of 12 Descriptive data are shown in Table 3.

Table 3. Distribution according to the age of the histochemical and immunohistochemical parameters. Table 3. Distribution according to the age of the histochemical and immunohistochemical parameters. Studied Parameters 3 Months (n = 20) 10 Months (n = 22) 24 Months (n = 40) CellularStudied Degeneration Parameters 3 Months (n = 20) 10 Months (n = 22)n (%) 24Months (n = 40) Cellularnon-existent Degeneration 2 (10.0) n (%) 5 (22.7) 0 (0.0) non-existentweak 2 (10.0) 13 (65.0) 5 (22.7) 13 (59.1) 0 (0.0) 15 (37.5) moderateweak 13 (65.0) 5 (25.0) 13 (59.1) 4 (18.2) 15 (37.5) 17 (42.5) strongmoderate 5 (25.0) 0 (0.0) 4 (18.2) 0 (0.0) 17 (42.5) 8 (20.0) strong 0 (0.0) 0 (0.0)% area (stderr) 8 (20.0) % area (stderr) PASPAS positivity positivity 4.2 (0.5) 4.2 (0.5) 6.3 (0.4) 6.3 (0.4) 9.9 (0.4) 9.9 (0.4) OMPOMP positivity positivity 75.3 (1.6) 75.3 (1.6) 69.3 (2.2) 69.3 (2.2) 69.3 (1.1) 69.3 (1.1) GαGi2α i2positivity positivity 39.1 (2.2) 39.1 (2.2) 42.7 (1.6) 42.7 (1.6) 20.8 (2.1) 20.8 (2.1) GαGoα positivityo positivity 6.1 (0.5) 6.1 (0.5) 13.2 (0.5) 13.2 (0.5) 18.6 (0.9) 18.6 (0.9) % positive cells (stderr) % positive cells (stderr) Proliferative cells 0.4 (0.2) 0.3 (0.1) 0.1 (0.1) ProliferativeApoptotic cells 1.0 (0.2) 0.4 (0.2) 1.1 (0.2) 0.3 (0.1) 1.8 (0.2) 0.1 (0.1) Apoptotic cells 1.0 (0.2) 1.1 (0.2) 1.8 (0.2) 3.1. Cellular Degeneration 3.1. Cellular Degeneration Qualitative scores of cellular degenerations in VNSEs were studied according to age Qualitative scores of cellular degenerations in VNSEs were studied according to age groups. A significant effect of age was revealed (DF = 2; X2 = 18.7; p < 0.0001; multinomial groups. A significant effect of age was revealed (DF = 2; X2 = 18.7; p < 0.0001; multinomial model). A post hoc comparison showed significant differences between 3 and 24 months model). A post hoc comparison showed significant differences between 3 and 24 months (odds ratio (OR) = 0.17; p < 0.0001) and between 10 and 24 months (OR = 11.5; p = 0.005). (odds ratio (OR) = 0.17; p < 0.0001) and between 10 and 24 months (OR = 11.5; p = 0.005). The difference observed between the mice at ages 3 and 10 months was not significant The difference observed between the mice at ages 3 and 10 months was not significant (OR = 1.9; p = 0.3174) (Figure1). (OR = 1.9; p = 0.3174) (Figure 1).

Figure 1. AgeFigure effect 1. Age on cellular effect on degeneration cellular degeneration in mouse VNSEs in mouse (A– VNSEsC). H&E (A staining–C). H&E was staining used to was highlight used tohistological degeneration signs, such as vacuolization or structure alterations (Inserts: ×40 magnification of VNSE samples) at 3 months highlight histological degeneration signs, such as vacuolization or structure alterations (Inserts: ×40 (A), 10 months (B), and 24 months (C). (Objective × 20, Scale bar = 200 µm). magnification of VNSE samples) at 3 months (A), 10 months (B), and 24 months (C). (Objective × 20, Scale bar = 200 µm).3.2. Increased Quantity of Accumulated Glycogen with Age 3.2. Increased QuantityAccumulated of Accumulated glycogen Glycogen was with quantified Age with PAS staining, and significant variations Accumulatedwere glycogen observed was among quantified the age with groups PAS (DF staining, = 2; F and= 23.30; significant p < 0.0001; variations general linear mixed were observed amongmodel). theThe age statistical groups model (DF = 2;indicated F = 23.30; thatp < the 0.0001; observed general difference linear mixed showed increasing model). The statisticalPAS positivity model between indicated 3 months that the and observed 24 months difference (p < 0.0001) showed and increasing between 10 months and PAS positivity between24 months 3 months (p = 0.0004) and (Figure 24 months 2). (p < 0.0001) and between 10 months and 24 months (p = 0.0004) (Figure2).

3.3. OMP Expression Decreases with Age Regarding the presence of olfactory marker protein in the VNSE of mice, statistical analysis showed that age signiﬁcantly impacted the expression of this protein (DF = 2; F = 3.84; p = 0.0436; general linear mixed model). Post hoc multiple comparisons revealed a signiﬁcant difference between 3 and 24 months (p = 0.0418). No differences were observed between 3 and 10 months (p = 0.2087) or between 10 and 24 months (p = 0.9998) (Figure3). Animals 2021, 11, 1211 6 of 11

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Animals 2021, 11, 1211 6 of 12 Animals 2021, 11, 1211 6 of 11

Figure 2. Age effect on PAS positivity in mouse VNSEs (A). Glycogen presence in mouse VNSEs aged 3, 10, and 24 months with PAS staining. Data are shown as the mean ± SD, * p ≤ 0.1; ** p ≤ 0.01; *** p ≤ 0.001 as indicated (B–D). PAS staining Figurewas used 2. Age to effectstain onthe PAS glycogen positivity accumulations in mouse VNSEs a red-purple (A). Glycogen color at presence 3 months in mouse(B), 10 VNSEs months aged (C), 3, and 10, and24 months 24 months (D). withPathologic PAS staining. glycogen Data accumulati are shownon aswas the often mean located ± SD, in* pthe ≤ 0.1; vacuoles ** p ≤ in0.01; the *** VNSEs p ≤ 0.001 of 24-month-old as indicated (mice.B–D). (Objective PAS staining × 40, wasScale used bar to= 100 stain µm). the glycogen accumulations a red-purple color at 3 months (B), 10 months (C), and 24 months (D). Pathologic glycogen accumulation was often located in the vacuoles in the VNSEs of 24-month-old mice. (Objective × 40,

Scale bar = 100 µm). 3.3. OMP Expression Decreases with Age Figure 2. 2. AgeAge effect effect on on PAS PAS positivity positivityRegarding in in mouse mouse the VNSEs VNSEs presence ( (AA).). Glycogen Glycogen of olfactory presence presence marker in in mouse mouse protein VNSEs VNSEs in theaged aged VNSE 3, 3, 10, 10, and andof mice, 24 24 months months statistical with PAS staining. Data are3.3. shown OMP as Expression the mean ± Decreases SD, * p ≤ 0.1;with ** Age p ≤ 0.01; *** p ≤ 0.001 as indicated (B–D). PAS staining with PAS staining. Data are shownanalysis as theshowed mean ± thatSD, age * p ≤ significantly0.1; ** p ≤ 0.01; impacted *** p ≤ 0.001 the asexpression indicated ( Bof– Dthis). PAS protein staining (DF was = 2; F = was used to stain the glycogen Regardingaccumulations the a presence red-purple of color olfactory at 3 months marker (B protein), 10 months in the (C VNSE), and 24of monthsmice, statistical (D). used to stain the glycogen accumulations3.84; p = 0.0436; a red-purple general color linear at 3mixed months model). (B), 10 months Post hoc (C), multiple and 24 months comparisons (D). Pathologic revealed a Pathologic glycogen accumulatianalysison was showed often locatedthat age in significantlythe vacuoles in impacted the VNSEs the of expression 24-month-old of mice.this protein (Objective (DF × 40,= 2; F = glycogen accumulation wassignificant often located difference in the vacuoles between in the 3 VNSEsand 24 ofmonths 24-month-old (p = 0.0418). mice. (ObjectiveNo differences× 40, Scalewere bar observed = Scale bar = 100 µm). 3.84; p = 0.0436; general linear mixed model). Post hoc multiple comparisons revealed a 100 µm). between 3 and 10 months (p = 0.2087) or between 10 and 24 months (p = 0.9998) (Figure 3). significant difference between 3 and 24 months (p = 0.0418). No differences were observed 3.3. OMP Expression Decreases with Age between 3 and 10 months (p = 0.2087) or between 10 and 24 months (p = 0.9998) (Figure 3). Regarding the presence of olfactory marker protein in the VNSE of mice, statistical analysis showed that age significantly impacted the expression of this protein (DF = 2; F = 3.84; p = 0.0436; general linear mixed model). Post hoc multiple comparisons revealed a significant difference between 3 and 24 months (p = 0.0418). No differences were observed between 3 and 10 months (p = 0.2087) or between 10 and 24 months (p = 0.9998) (Figure 3).

Figure 3. Age effect on olfactory marker protein positivity in mouse VNSEs (A). OMP expression in mouse VNSEs aged Figure 3. Age effect on olfactory marker protein positivity in mouse VNSEs (A). OMP expression in mouse VNSEs aged 3, 3, 10, and 24 months with immunohistochemical staining of the OMP protein. Data are shown as the mean ± SD, * p ≤ 0.05 10, and 24 months with immunohistochemical staining of the OMP protein. Data are shown as the mean ± SD, * p ≤ 0.05 Figure(B–D). 3. Immunohistochemical Age effect on olfactory staining marker of protein OMP withpositivity a DAB in ch mouseromogen VNSEs and (hematoxylinA). OMP expression counterstain in mouse at 3 months VNSEs (agedB), 10 3,(monthsB 10,–D and). Immunohistochemical ( 24C), months and 24 monthswith immunohistochemical (D staining). (Objective of OMP × 20, staining withScale abar DAB of = the200 chromogen OMP µm). protein. and Data hematoxylin are shown counterstain as the mean at ± 3SD, months * p ≤ 0.05 (B), (10B–D months). Immunohistochemical (C), and 24 months staining (D). (Objective of OMP× with20, Scalea DAB bar ch =romogen 200 µm). and hematoxylin counterstain at 3 months (B), 10 months (C), and 24 months (D3.4.). (Objective Gαi2 and ×G 20,αo ScaleProteins bar Modifications= 200 µm). 3.4. Gαi2 and Gαo Proteins Modiﬁcations Statistical analysis showed that the expressions of the Gαi2 and Gαo proteins are both Figure 3. Age effect on olfactory3.4. G markerαStatisticali2 and protein Gα analysiso Proteins positivity showed Modifications in mouse that VNSEs the expressions (A). OMP expression of the Gα ini2 mouse and G VNSEsαo proteins aged are highly dependent on aging, in an opposite manner. Indeed, the expression of Gαi2 protein 3, 10, and 24 months with immunohistochemicalbothStatistical highly dependent analysis staining showed on of aging, the th OMPat in the an protein. expressions opposite Data manner. are of shownthe G Indeed,α asi2 theand meanthe Gαo expression ± proteins SD, * p ≤ are0.05 of both Gαi2 decreased with age (DF = 2; Ki2 = 37.47; p < 0.0001; Kruskal–Wallis test) while the expression (B–D). Immunohistochemicalhighlyprotein staining dependent decreased of OMP onwith with aging, a DAB age in (DFch anromogen opposite = 2; Ki and2 manner.= hematoxylin 37.47; Indeed,p < 0.0001; counterstain the expression Kruskal–Wallis at 3 months of Gα ( test)i2B), protein 10 while of Gαo protein was increased (DF = 2; F = 58.68; p < 0.0001; general linear mixed model). months (C), and 24 monthsdecreased (theD). expression(Objective with × age of20, G Scale(DFαo = proteinbar 2; Ki= 2002 = was 37.47;µm). increased p < 0.0001; (DF Kruskal–Wallis = 2; F = 58.68; test)p < 0.0001;while the general expression linear Wilcoxon tests indicate that Gαi2 was significantly higher in the 3 months than the ofmixed Gαo protein model). was increased (DF = 2; F = 58.68; p < 0.0001; general linear mixed model). 3.4.24 months Gαi2 and group Gαo Proteins(p = 0.0003) Modifications and higher in the 10 months group than the 24 months group WilcoxonWilcoxon tests tests indicate indicate that that G Gααi2i2 was was significantly signiﬁcantly higher higher in in the the 3 3 months months than than the the (p = 0.0003) (Figure 4). 2424 months monthsStatistical groupgroup analysis (p (p == 0.0003) 0.0003) showed and and th higherat higher the expressionsin in the the 10 10 mont months of hsthe group groupGαi2 andthan than G the theαo 24 proteins 24 months months are group group both (highly(pp == 0.0003) 0.0003) dependent (Figure (Figure on 4).4). aging, in an opposite manner. Indeed, the expression of Gαi2 protein decreased with age (DF = 2; Ki2 = 37.47; p < 0.0001; Kruskal–Wallis test) while the expression of Gαo protein was increased (DF = 2; F = 58.68; p < 0.0001; general linear mixed model). Wilcoxon tests indicate that Gαi2 was significantly higher in the 3 months than the 24 months group (p = 0.0003) and higher in the 10 months group than the 24 months group (p = 0.0003) (Figure 4).

Figure 4. Gαi2 protein expression decreases with aging (A). Gαi2 expression in mouse VNSEs aged 3, 10, and 24 months with immunohistochemical staining of the Gαi2 protein. Data are shown as the median, ** p ≤ 0.01 (B–D). Immunohistochemical staining of Gαi2 with a DAB chromogen and hematoxylin counterstain at 3 months (B), 10 months (C), and 24 months (D). × µ (Objective 20, Scale bar = 200 m).

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Figure 4. Gαi2 protein expression decreases with aging (A). Gαi2 expression in mouse VNSEs aged 3, 10, and 24 months AnimalsFigurewith2021 immunohistochemical, 4.11 ,G 1211αi2 protein expression staining decreases of the G withαi2 protein.aging (A Data). Gα arei2 expression shown as thein mouse median, VNSEs ** p ≤ aged 0.01 (3,B –10,D). and Immunohisto- 24 months 7 of 12 Figurewithchemical immunohistochemical 4. G stainingαi2 protein of G expressionαi2 with staining a DABdecreases of thechromogen G withαi2 protein. aging and (hematoA Data). G αarei2xylin expressionshown counterstain as the in mousemedian, at 3 monthsVNSEs ** p ≤ aged0.01(B), 10( B3, – months10,D). and Immunohisto- 24(C ),months and 24 withchemicalmonths immunohistochemical ( stainingD). (Objective of Gα i2× 20, with staining Scale a DAB bar of = chromogenthe 200 G µm).αi2 protein. and hemato Data arexylin shown counterstain as the median, at 3 months ** p ≤ (0.01B), 10(B –monthsD). Immunohisto- (C), and 24 chemicalmonths ( Dstaining). (Objective of Gα ×i2 20, with Scale a DAB bar = chromogen 200 µm). and hematoxylin counterstain at 3 months (B), 10 months (C), and 24 months (D). (Objective × 20, Scale Forbar = G 200αo, µm). signiﬁcant differences were revealed between 3 and 10 months (p < 0.0001), For Gαo, significant differences were revealed between 3 and 10 months (p < 0.0001), 3 and 24 months (p < 0.0001), and 10 and 24 months (p = 0.0048) (Figure5). 3 andFor 24 G monthsαo, significant (p < 0.0001), differences and 10 wereand 24 revealed months between (p = 0.0048) 3 and (Figure 10 months 5). (p < 0.0001), For Gαo, significant differences were revealed between 3 and 10 months (p < 0.0001), 3 and 24 months (p < 0.0001), and 10 and 24 months (p = 0.0048) (Figure 5). 3 and 24 months (p < 0.0001), and 10 and 24 months (p = 0.0048) (Figure 5).

Figure 5.5. Gαo protein expression increases with aging (A). Gαo expression in mouse VNSEs aged 3, 10, and 24 monthsmonths Figurewith immunohistochemical 5. Gαo protein expression staining increases of the withGαo agingprotein. (A ).Data Gα oare expression shown as in the mouse mean VNSEs ± SD, aged** p ≤ 3, 0.01; 10, and*** p24 ≤ months0.001 as with immunohistochemical staining of the Gαo protein. Data are shown as the mean ± SD, ** p ≤ 0.01; *** p ≤ 0.001 as Figurewithindicated immunohistochemical 5. G (Bα–oD protein). Immunohistochemical expression staining increases of thestaining withGαo ofagingprotein. Gαo ( withA Data). G aα DABoare expression shown chromogen as in the mouse and mean hematoxylin VNSEs ± SD, **aged p counterstain ≤ 3,0.01; 10, ***and p at 24≤ 3 0.001months months as indicated (B–D). Immunohistochemical staining of Gαo with a DAB chromogen and hematoxylin counterstain at 3 months withindicated(B), 10immunohistochemical months (B–D). ( CImmunohistochemical), and 24 months staining (D of). staining(Objectivethe Gαo of protein. ×G 40,αo withScale Data a bar DAB are = 100 shownchromogen µm). as the and mean hematoxylin ± SD, ** pcounterstain ≤ 0.01; *** p at ≤ 3 0.001 months as indicated((BB),), 10 10 months months (B–D ).( (C CImmunohistochemical),), and and 24 24 months months ( (DD).). (Objective (Objectivestaining of × ×G 40,α40,o Scalewith Scale abar barDAB = =100 chromogen 100 µm).µm). and hematoxylin counterstain at 3 months (B), 10 months (C), and 24 months (D). (Objective × 40, Scale bar = 100 µm). 3.5. Proliferation and Apoptosis Factors 3.5. Proliferation and Apoptosis Factors 3.5. ProliferationAge did not and significantly Apoptosis Factors impact the expression of Ki67 protein in the VNSE of aging 3.5. ProliferationAge did not and signiﬁcantly Apoptosis Factors impact the expression of Ki67 protein in the VNSE of aging miceAge (DF did = 2; not F = significantly0.09; p = 0.9105; impact Poisson the expressi model) on(Figure of Ki67 6). protein in the VNSE of aging Age did not significantly impact the expression of Ki67 protein in the VNSE of aging micemice (DF (DF = = 2; 2; F F = = 0.09; 0.09; pp == 0.9105; 0.9105; Poisson Poisson model) model) (Figure (Figure 6).6). mice (DF = 2; F = 0.09; p = 0.9105; Poisson model) (Figure 6).

Figure 6. Aging has no significant effects on Ki67 protein expression in mouse VNSEs (A). Proliferative marker in mouse FigureFigureVNSEs 6. 6.aged AgingAging 3, 10,has has and no no significant24 signiﬁcant months obtainedeffects effects on on with Ki67 Ki67 immunohistochemical protein protein expression expression in in mousestaining mouse VNSEs VNSEs of the Ki67( (AA).). Proliferative Proliferativeprotein. Data markermarker are shown in in mouse mouse as the FigureVNSEs 6.aged Aging 3, 10, has and no 24significant months obtained effects on with Ki67 immunohistochemical protein expression in stainingmouse VNSEs of the Ki67 (A). protein.Proliferative Data marker are shown in mouse as the VNSEsmean ± agedSD (B 3,– 10,D). andImmunohistochemical 24 months obtained staining with immunohistochemical of Ki67 with a DAB stainingchromogen of the an Ki67d hematoxylin protein. Data counterstain are shown at as 3 VNSEsmeanmonths ± aged SD(B), ( 3,10B– 10, Dmonths). and Immunohistochemical 24 (C months), and 24 obtained months withstaining (D). immunohistochemical(Objective of Ki67 × with40, Scale a DAB bar staining =chromogen 100 µm).of the Ki67and hematoxylinprotein. Data counterstainare shown as at the 3 the mean ± SD (B–D). Immunohistochemical staining of Ki67 with a DAB chromogen and hematoxylin counterstain at meanmonths ± (SDB), (10B– monthsD). Immunohistochemical (C), and 24 months staining(D). (Objective of Ki67 × 40,with Scale a DAB bar =chromogen 100 µm). and hematoxylin counterstain at 3 months3 months (B), (B 10), 10months months (C (),C and), and 24 24months months (D ().D (Objective). (Objective × 40,× 40,Scale Scale bar bar = 100 = 100 µm).µm). On the converse, age had an impact on the expression of cleaved caspase 3 protein (DF On= 2; the F = converse, 3.42; p = age0.0425; had Poisson an impact model). on the Multiple expression comparisons of cleaved informed caspase 3us protein that a OnOn the the converse, converse, age age had had an an impact impact on on the the expression expression of of cleaved cleaved caspase caspase 3 3 protein protein (DFdifference = 2; F =was 3.42; obtained p = 0.0425; thanks Poisson to a trend model). between Multiple 3 and comparisons 24 months (informedp = 0.0748). us No that dif- a (DF(DF = = 2; 2; FF = = 3.42; 3.42; pp == 0.0425; 0.0425; Poisson Poisson model). model). Multiple Multiple comparisons comparisons informed informed us usthat that a differenceferences were was observedobtained betweenthanks to 3- a and trend 10-months between groups3 and 24 (p months= 0.9688) (p or = between0.0748). No 10- dif-and differencea difference was was obtained obtained thanks thanks to a to trend a trend between between 3 and 3 and 24 months 24 months (p = (0.0748).p = 0.0748). No dif- No ferences24-months were groups observed (p = 0.1538)between (Figure 3- and 7). 10-months groups (p = 0.9688) or between 10- and ferencesdifferences were were observed observed between between 3- and 3- 10-months and 10-months groups groups (p = 0.9688) (p = or 0.9688) between or between10- and 24-months groups (p = 0.1538) (Figure 7). 24-months10- and 24-months groups (p groups = 0.1538) (p = (Figure 0.1538) 7). (Figure 7).

Figure 7. Age effects on the apoptosis of mouse VNSEs (A). Cleaved caspase 3 expression in mouse VNSEs aged 3, 10, and Figure24 months 7. Age obtained effects onwith the immunohistoc apoptosis of mousehemical VNSEs staining (A ).of Cleaved this protein. caspase Data 3 expressionare shown asin themouse mean VNSEs ± SD, aged * p ≤ 3,0.1 10, (B and–D). Figure24FigureImmunohistochemical months 7. 7. AgeAge obtained effects effects withon on stainingthe theimmunohistoc apoptosis apoptosis of cleaved of of hemicalmouse mouse caspase VNSEs VNSEsstaining 3 was (A ( Aof ).used). Cleavedthis Cleaved to protein. reveal caspase caspase Datathe protein3 3 areexpression expression shown with as ain in DABthe mouse mouse mean chromogen VNSEs VNSEs ± SD, aged* aged pand ≤ 0.13, 3,hematox- 10, 10,(B –and andD). 24 months obtained with immunohistochemical staining of this protein. Data are shown as the mean ± SD, * p ≤ 0.1 (B–D). Immunohistochemical24ylin months counterstain obtained at with3 months staining immunohistochemical ( Bof), cleaved10 months caspase (C), staining and 3 was 24 ofusedmonths this to protein. reveal(D). (Objective the Data protein are × shown 40, with Scale asa DAB the bar mean =chromogen 100± µm).SD, *andp ≤ hematox-0.1 (B–D). Immunohistochemical staining of cleaved caspase 3 was used to reveal the protein with a DAB chromogen and hematox- ylinImmunohistochemical counterstain at 3 months staining (B of), 10 cleaved months caspase (C), and 3 was 24 usedmonths to reveal (D). (Objective the protein × with40, Scale a DAB bar chromogen = 100 µm). and hematoxylin ylin counterstain at 3 months (B), 10 months (C), and 24 months (D). (Objective × 40, Scale bar = 100 µm). counterstain at 3 months (B), 10 months (C), and 24 months (D). (Objective × 40, Scale bar = 100 µm).

Animals 2021, 11, 1211 8 of 12

For each parameter, sex and strain variables were statistically analyzed, and their interactions with aging were veriﬁed. No interactions were found in all cases, and no differences were observed according to these variables.

4. Discussion Our results showed that aging influences the vomeronasal organ structure. Moreover, this study allowed us to characterize the engendered effects and their delays in appearance. Our results indicated that the cellular degeneration seems to appear after the age of 10 months in mice, which is considered middle age in this species. In fact, our data demonstrated an increase of the degenerative changes with aging, between 3 and 24 months, and between 10 and 24 months. Previous studies [22,23] observed an increase of the VNO volume until the 4 months of age, followed by a decrease stabilized around the 8 months. Even if our study evaluated other kinds of alterations and involved only the VNSE, it seems possible to suggest that around 8–10 months of age, the aging process begins to alter the VNO’s structure. The observed vacuoles may result from water or lipid lysosomal accumulation, and these changes are frequently due to defective cellular metabolism [29]. It is well known that the histological sections can present artefacts that could influence the analysis of some degenerative signs. However, to reduce and to homogenize this risk, all the samples were submitted to the same processing procedure. The metabolism efficiency was also tested with PAS staining, a technique that allows the staining of glycogen, which is commonly present in low quantities in the nervous system to assure the vital energy required by neuronal activation [30,31]. An aberrant accumulation of this glucosidal polymer in neurons can drive early aging and neuronal degeneration and is frequently found in neurodegenerative diseases, such as Alzheimer, Parkinson, and dementia diseases [32,33]. In this study, glycogen accumulation appears from the age of 10 months, which could impact the neurons’ function, and, consequently, VNO function. Moreover, PAS positive staining was often located in the neurons’ vacuoles, suggesting a possible alteration of the cellular metabolism, as commonly occurs during normal aging [34,35]. As showed by the statistical analysis, olfactory marker protein was slightly but significantly decreased between adults and old mice. Since this protein is expressed by vomeronasal neurons, this finding could be due to the cellular degeneration revealed by the VNSE histological analysis, or to an age influence on the cellular protein synthesis and accumulation. OMP has a crucial role in neuronal functionality and nervous signal transmission [36], and thus a reduction of this protein could also suggest a VNO dysfunction. However, our results showed only a weak statistical difference, therefore, their interpretation should be taken with prudence. Moreover, further investigations are needed to deeper explore these changes and their possible implications. The Gαi2 protein, co-expressed on V1Rs in charge of the reception of volatile and steroidal molecules, has a major role in communication, and in maternal, sexual, and social pheromones’ detection [10,37,38]. Previous studies showed that when this protein was deleted or its expression decreased, the chemosensory behavior of animals was altered, leading to an increase of aggressive behaviors, and modified sexual behaviors [16,39,40]. This study reported that Gαi2 positivity was significantly decreased in the VNSE of aging mice. According to the previous literature, this phenomenon could lead to a reduction of the mouse’s communication capabilities, and thus to an increase in behavior disorders, such as aggression. The Gαo protein is co-expressed on V2Rs, the receptors of nonvolatile peptides, large proteins implicated in territorial marking and alarm pheromones, and plays a key role in social behavior [40]. Our statistical analysis seems to indicate a strong increase of this protein in VNSEs according to age, in contrary to the Gαi2 results. Since these data were quite surprising compared to the trends of the other markers included in this study, the authors’ opinion is that a compensatory mechanism can be presumed, as already proposed in the literature for other G proteins [41]. However, it is well known that V2Rs Animals 2021, 11, 1211 9 of 12

co-express the MHC class 1b molecules, in particular, the M10 family, which is linked to β2microglobuline (β2m) protein [42]. This protein significantly increases according to age in another murine peripheral sensory epithelium, such as the retina, as well as in various brain regions [43]. These findings could partially explain our observation on Gαo increase in old mice VNSE. Interestingly, the IHC stain of our samples was less intense than that observed in previous studies [44], probably because our protocol used a lower dilution, in order to obtain quantitative differences among samples, and not to assess the positivity or the negativity of the tissues. Consequently, this weak signal does not allow to exactly quantify the ratio between the two G-proteins in old mice. Finally, also considering the low percentages, these results should be interpreted with caution and corroborated with further studies. To explore if cellular turnover is maintained during aging, and if its alteration could be a factor in the onset of degenerative phenomena, we also investigated apoptotic and proliferative cellular activities, as some studies have already demonstrated that neuronal regeneration is preserved after surgical injuries during aging in mice [25]. Our results showed that the apoptotic marker was indeed higher in aged mice. The expression of proliferative protein seems to be decreased with aging, but no significant effect was revealed. Even if these results are not strong enough to conclude about the cellular turnover, they may suggest that the number of dead cells is not balanced by the proliferation of new cells, indicating a possible decrease of the regeneration capabilities of the aging VNSE, which could be one of the causes of the degenerative phenomena described in this article. The study of Brann and Firestein [25] showed that the VNSE proliferative activity remains robust in response to an experimental lesion even in 24 months-old mice, while this activity is slightly lower in the non-lesioned old VNO, suggesting that the increased proliferation is caused by the induced reparative process. Since the VNO shares some important characteristics with the olfactory mucosa [8], in particular, the histological pattern of the VNSE, it is interesting to compare our results to those reported in literature on the aging of the olfactory epithelium. Conversely to the VNO, the aging of the olfactory mucosa has been largely studied, in particular, in mice and humans. The most frequent changes observed in aging are the decrease of the thickness of the epithelium with a concurrent localized metaplasia, pigments deposits, and an important reduction of the neuroepithelium vascularization [45,46]. In our study, the main histological finding was the diffuse degeneration of the VNSE, which is less reported in olfactory neuroepithelium. Interestingly, among the causes that can lead to the aging of the olfactory mucosa, the decrease of the mitotic activity seems to play a crucial role, as well as the increase of the ratio of dead or dying neurons to the number of live cells [46]. As previously mentioned, our results do not allow to draw firm conclusion on cellular turnover in the aged VNSE, even if a dis-balance before newborn and apoptotic cells can be presumed. This study permits us to affirm that aging has a real impact on the neuronal cells of the VNO. Due to the role of this organ and of these sensory neurons, these changes may impact the mouse’s chemical communication capabilities, and, consequently, the animals’ behavior. Indeed, neuron functionality seems to be altered, as shown by the G proteins modification in natural conditions. However, these hypotheses should be confirmed by further studies evaluating the functionality of the VNO in old animals and their behavioral responses to semiochemicals’ stimuli. Considering the crucial role of the VNO in animals’ communication, these effects are supposed to directly impact also animals’ behavior and consequently, their life. To confirm these hypotheses, further studies are needed to investigate the age effect on the behavioral response after the exposition to semiochemical stimuli. Other investigations should be also led on other mammals in order to explore VNO aging and its impact in domestic and farm animals, to obtain more knowledge about their capabilities to communicate, and to adapt to their environment throughout the aging process. Animals 2021, 11, 1211 10 of 12

5. Conclusions In this study, we showed that aging has a strong impact on the neuronal cells of the vomeronasal organ, modifying their structure and their metabolism. Further studies are needed to evaluate if these changes may inﬂuence chemical communication capabilities in old animals and, consequently, to induce behavioral changes. To the best of our knowledge, this study is the ﬁrst to characterize the effects of aging on the vomeronasal neuronal cells of the mouse and in animals in general.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ani11051211/s1, Table S1: Animals’ repartition and raw data. Author Contributions: Conceptualization, V.M. and P.A.; methodology, V.M. and P.A.; validation, P.A.; formal analysis, E.T.; investigation, V.M.; resources, P.P.; data curation, E.T.; writing—original draft preparation, V.M.; writing—review and editing, P.A., P.P. and E.T.; visualization, V.M. and P.A.; supervision, P.A.; project administration, V.M.; funding acquisition, P.P. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The project, including this experimental procedure, was approved by the IRSEA’s (Research Institute in Semiochemistry and Applied Ethology) Ethics Committee (n◦ CE_2019_06_POVN). Data Availability Statement: The data presented in this study are available within the article in the supplementary files. Acknowledgments: We would like to thank the IRSEA’s Animal Health Service for taking good care of the mice, particularly the animal-keepers. We would like to thank the American Journal Experts team for their help in improving our manuscript and A. Cozzi for his suggestions. We are also grateful to IRSEA’s Ethical Committee for their comments and the approval of the protocol. Conflicts of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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