Molecules involved in the sperm interaction in the human uterine tube: a histochemical and immunohistochemical approach

European Journal of Histochemistry 2023; volume 67:3513 [page 118] [European Journal of Histochemistry 2023; 67:3513] Molecules involved in the sperm interaction in the human uterine tube: a histochemical and immunohistochemical approach David Cajas,1Emanuel Guajardo,2,3 Sergio Jara-Rosales,4,5 Claudio Nuñez,6Renato Vargas,6Victor Carriel,7 Antonio Campos,7Luis Milla,1Pedro Orihuela,2Carlos Godoy-Guzmán1,8 1Escuela de Medicina, Centro de Investigación Biomédica y Aplicada (CIBAP), Laboratorio de ingenieria de tejidos, Universidad de Santiago de Chile (USACH), Santiago, Chile 2Facultad de Química y Biología, Laboratorio de Inmunología de la Reproducción, Universidad de Santiago de Chile (USACH), Santiago, Chile 3Advanced Center for Chronic Diseases (ACCDiS), Facultad de Ciencias Químicas y Farmacéuticas and Facultad de Medicina, Universidad de Chile, Santiago, Chile 4Escuela de Obstetricia, Facultad de Ciencias para el Cuidado de la Salud, Universidad San Sebastián, Sede Los Leones, Santiago, Chile 5Programa de Doctorado en Enfermedades Crónicas, Universidad San Sebastián, Sede Los Leones, Chile 6Servicio de Ginecología y Obstetricia, Hospital San José, Santiago, Chile 7Department of Histology, Tissue Engineering Group, University of Granada, Spain; Instituto de Investigación Biosanitaria ibis.GRANADA, Granada, Spain 8Universidad de Santiago de Chile (USACH), Escuela de Medicina, Unidad de Histología, Santiago, Chile In humans, even where millions of spermatozoa are deposited upon ejaculation in the vagina, only a few thousand enter the uterine tube (UT). Sperm transiently adhere to the epithelial cells lining the isthmus reservoir, and this interaction is essential in coordinating the availability of functional spermatozoa for fertilization. The binding of spermatozoa to the UT epithelium (mucosa) occurs due to interactions between cell-adhesion molecules on the cell surfaces of both the sperm and the epithelial cell. However, in humans, there is little information about the molecules involved. The aim of this study was to perform a histological characterization of the UT focused on determining the tissue distribution and deposition of some molecules associated with cell adhesion (F-spondin, galectin-9, osteopontin, integrin αV/β3) and UT’s contractile activity (TNFα-R1, TNFαR2) in the follicular and luteal phases. Our results showed the presence of galectin-9, F-spondin, osteopontin, integrin αV/β3, TNFα-R1, and TNFα-R2in the epithelial cells in ampullar and isthmic segments during the menstrual cycle. Our results suggest that these molecules could form part of the sperm-UT interactions. Future studies will shed light on the specific role of each of the identified molecules. Key words: human uterine tube; epithelium; menstrual cycle; immunohistochemistry; cell-adhesion molecules. A B S T R A C T Correspondence: Prof. Dr. Carlos Godoy-Guzmán, Escuela de Medicina, Universidad de Santiago de Chile, Avda. Bdo. O'Higgins 3363, Correo 442, Santiago, Chile. Tel. +56.2.7183513. E-mail: [email protected] Contributions: CGG, conceptualization and supervision of the review; DC, EG, experiments performing; SJR, CN, RV, provision of patients; CGG, PAO, LAM, VC, AC, participated in planning the experiments and contributed to drafting the manuscript. All the authors read and approved the final version of the manuscript and agreed to be accountable for all aspects of the work. Conflict of interest: the authors have no conflict of interest to declare. Ethics approval and consent to participate: the Ethics and Biosafety Committee of the Servicio de Salud Metropolitano Norte and the Universidad de Santiago de Chile approved this study (No102/carta No17/2019). Informed consent was obtained from each participant in this study. 2023_2.qxp_Hrev_master 13/04/23 11:02 Pagina 118 Non-commercial use only Article Introduction The uterine tube (UT), also called the Fallopian tube (or oviduct in animals), is the tubular organ connecting the periovarian space with the uterus.1Considering the tube in all its length, one can distinguish the fimbria, the ampulla, the isthmus and the intramural segment.2Its two major segments, the ampulla and the isthmus, differ both in structure and function.2The previously held belief that the UT were merely a passive conduit for the transportation of gametes and embryos during reproduction has been revised.3Today, it is widely recognized that the UT play a crucial role in various reproductive processes, the maturation and transport of gametes, fertilization, the early development of the embryo, and the formation of a sperm reservoir.1,2,4-5 In humans, millions of sperm cells are deposited in the vagina during ejaculation, but only a few thousand reach the UT.5,7 Sperm have been observed to bind temporarily to the epithelial cells that line the caudal isthmus.7This interaction has been shown to extend the fertile lifespan of the sperm within the female reproductive tract.7In the isthmus region of the female reproductive tract, there exists a “functional spermatozoa reservoir” that serves to maintain a sufficient number of viable, potentially fertile sperm available for fertilization.7-9 The presence of a sperm reservoir in the isthmus region of the female reproductive tract serves to facilitate the selection of competent sperm.7,9 This process also modulates the capacitation of these sperm and ensures their release in controlled numbers, reducing the risk of polyspermy.7,9 The physical interactions that occur between spermatozoa and the epithelial cells lining the luminal surface of the UT may influence many aspects of sperm function.11-15 These interactions are mediated by diverse cell-adhesion molecules located on the surface of both cell types. In this context, studies conducted in animal models revealed the existence of several molecules involved in these cell-cell interactions such as sialic acid-rich glycoproteins,16 annexins,17 fucose,18 SBG,20 Galβ1-3GalNAc,19 galactose,20 mannose,21 osteopontin,22 and integrins,22 among others.7,23 Studies have indicated that the adhesion process plays a critical role in the selection of high-quality spermatozoa and the preservation of their fertile life.7,24-26 Several studies provide evidence that TNFα regulates the oviductal contractile activity.27-29 Muro et al.31 described that fluorescent spermatozoa moved back and forth together with peristaltic movement along the oviduct isthmus, suggesting that oviduct contractions may play a role in sperm migration. Thus, contractile activity in the isthmic could have a major influence on the binding or release between spermatozoa and epithelial cells, and migration through the UTs.2,5,7,31 Furthermore, oviductal motile cilia are essential for oocyte pickup but dispensable for sperm and embryo transport.32 The histological structure of the human UT has been studied;1,3,33 however, more profound knowledge of normal UTs is necessary for a better understanding of pathological conditions. Around 30% of the infertile women worldwide have an associated UT pathology.34,35 Studies have revealed that the interaction between human sperm and endosalpingeal tissue, which takes place in vitro, is disrupted in tissues obtained from women who have been diagnosed with endometriosis. This interaction is a vital component of the fertilization process and its disturbance in the presence of endometriosis has important implications for fertility and reproductive health.36,37 Nevertheless, there is little information about the molecules present in both normal and pathological UTs that could be involved in these important biological interactions. Therefore, a histological description of these molecules could be vital to better understand and treat female infertility. The aim of this study was to perform a histological characterization of the UT focused on determining the tissue distribution and deposition of some molecules associated with cell adhesion (F-spondin, galectin-9, osteopontin, integrin αV/β3) and UT’s contractile activity (TNFα-R1, TNFα-R2 ) in the follicular and luteal phases. Materials and Methods Human tissue collection The UTs were obtained exclusively from women undergoing surgical sterilization for reasons not related to this study. The tissues were collected in collaboration with the Servicio de Ginecología y Obstetricia of the Hospital San José, Santiago, Chile. The patients were fertile, aged 25 to 45 years, and voluntarily requested surgical sterilization. Table 1 summarizes the exclusion criteria. Menstrual cycle dating was determined using plasma levels of estradiol and progesterone together with the menstrual history. Seven women were in the follicular phase and three were in the luteal phase. The pieces of UTs removed by laparoscopy were ampullar and isthmic segments. The Ethics and Biosafety Committee of the Servicio de Salud Metropolitano Norte and the Universidad de Santiago de Chile approved this study (No102/carta No17/2019). Informed consent was obtained from each participant in this study. Histological evaluation UT samples, fixed in 10% neutral buffered formalin, were dehydrated in graded ethanol (70-100%) and embedded in paraffin for routine histology. Serial sections of 5 µm thickness were stained with hematoxylin/eosin for general histological assessment. Furthermore, an overview of the glycoproteins present in the UTs were identified by periodic acid-Schiff (PAS) histochemical stain (ScyTek Laboratories, Logan, UT, USA).38,39 The distribution of acid proteoglycans and mucopolysaccharides was evaluated by the Alcian blue (Panreac, Darmstadt, Germany) histochemical method at pH 2.5.1 Immunohistochemistry In this study, the slides were rehydrated and treated for immunohistochemistry following standardized procedures developed by our group.1,40 All steps were performed in a humid chamber to prevent dehydration of the sections. Antigen retrieval was performed with citrate sodium solution (10 mM, pH 6.0) for 20 min at 95°C. Each of the succeeding steps was followed by three rinses with PBS. Non-specific antibody reaction was blocked by incubating the slides in PBS-T buffer with Table 1. Exclusion criteria for patients in this work. 1. Use of hormonal contraceptive methods within three months before surgery 2. Endometriosis 3. Tubal disease 4. Pelvic inflammatory disease 5. Sexually transmitted infection (Chlamydia trachomatis and/or Neisseria gonorrhoeae) 6. Heavy alcohol usage and tobacco or drug abuse [European Journal of Histochemistry 2023; 67:3513] [page 119] 2023_2.qxp_Hrev_master 13/04/23 11:02 Pagina 119 Non-commercial use only Article 2.5% (w/v) normal horse serum for 2 h at 25°C, followed by incubation of the primary antibodies. Table 2 summarizes technical information of the antibodies used. Endogenous peroxidase activity was blocked after primary antibody incubation by using 3% (v/v) H2O2 (Panreac) in PBS for 30 min. After rinsing in PBS, the slides were incubated for 1 h at room temperature with specific biotinylated pan-specific universal secondary antibody and one hour with streptavidin-peroxidase complex (Table 2). The antigen-antibody reaction was visualized using either 3,3′-diaminobenzidine (DAB) peroxidase substrate kit SK-4105 (Vector Laboratories, Burlingame, CA, USA) or NovaRED substrate kit SK-4805 (Vector), followed by a slight contrast with Harris’ hematoxylin. These procedures were performed at the same time, using the same environmental conditions to ensure the reproducibility of the results. In addition, for each immunohistochemical reaction, negative technical controls were included by omitting the primary antibody and positive controls were used available (human placenta, human skin, between others). Results General histology The UT is a tubular organ composed of three layers: mucosa, muscular layer, and serosa (Figure 1 A,B). The contours of the lumen of UT show longitudinal folds of the mucosa, which are more pronounced in the ampulla than isthmus. The muscular layer of the isthmus is thicker than the muscular layer of the ampulla. During the menstrual cycle, in the epithelium of all regions, three distinct cell types, ciliated, nonciliated, and basal cells, were distinguished (Figure 1 C-D). Ciliated cells have a spherical-shaped nucleus. Nonciliated cells have elongated nuclei. Basal cells have hyperchromatic nuclei and very pale cytoplasm (Figure 1 D). The epithelium of the UT is simple columnar. The luminal portion of the epithelium and basal lamina reacted with PAS stain, confirming the presence of glycoproteins (Figure 1E). The apical surface of the epithelium was positive for Alcian blue staining, confirming the presence of acid mucopolysaccharides (Figure 1F). It was determined that there were no differences in the distribution of PAS and Alcian Blue staining between the isthmus and ampulla segments throughout the menstrual cycle. F-spondin F-spondin exhibited intense staining in the epithelial cells of the mucosa (Figure 2). The cytoplasm of ciliated cells and secretory cells are positive for F-spondin. This analysis confirms the presence of F-spondin in the tunica muscularis of blood vessels of different caliber. No immunostaining was observed in the muscular layer. No difference was found in the expression or distribution of F-spondin between the two segments (ampulla and isthmus) during the menstrual cycle. Galectin-9 The immunohistochemical analysis of galectin-9 showed a positive reaction in secretory cells and ciliated cells of the mucosa (Figure 3). This analysis confirms the presence of galectin-9 in the tunica intima and muscularis of blood vessels. No immunostaining was observed in the muscular layer. No difference was found in the expression or distribution of galectin-9 between the two segments (ampulla and isthmus) during the menstrual cycle. Osteopontin The analysis of osteopontin revealed positive immunostaining in the epithelial cells of the mucosa during the menstrual cycle (Figure 4). The analysis of blood vessels revealed a strong positive reaction in the tunica muscularis. There is no observable difference in osteopontin expression throughout the menstrual cycle or among the various segments of the UT (isthmus and ampulla). Integrin αV/β3 The analysis of integrin αV/β3revealed positive immunostaining in the epithelial cells of the mucosa during the menstrual cycle (Figure 5). In addition, a positive reaction was observed in the basal lamina of the mucosa’s epithelium. The analysis of blood vessels revealed a strong positive reaction in the tunica muscularis. No difference was found in the expression or distribution of integrin αV/β3between the two segments (ampulla and isthmus) during the menstrual cycle. TNFα-R2 TNFα-R2exhibited positive staining in the epithelial cells of the mucosa (Figure 6). The cytoplasm of ciliated cells and secretory cells are positive for TTNFα-R2. This analysis confirms the weak presence of TNFα-R2 in the tunica muscularis of blood vessels of different caliber. No immunostaining was observed in muscular layer. No differences were found in the expression or distribution of TNFα-R2between the two segments (ampulla and isthmus) during the menstrual cycle. TNFα-R2 The immunohistochemical analysis of TNFα-R2showed a positive reaction in secretory cells of mucosa (Figure 7). Although weak immunostaining was observed in ciliated cells. This analysis Table 2. Antibodies and conjugates used for immunohistochemical analysis. Antibody Origin Working dilution References Galectin-9 Rabbit polyclonal 1:75 Invitrogen, USA, product number PA5-32252 Osteopontin Rabbit polyclonal 1:100 Invitrogen, USA, product number PA5-13494 F-spondin Rabbit polyclonal 1:75 Santa Cruz Biotechnology, USA, product number sc-98924 Integrin αV/β3 Mouse monoclonal 1:10 Santa Cruz Biotechnology, USA, product number sc-7312 TNFα-R1 Mouse monoclonal 1:25 Santa Cruz Biotechnology, USA, product number sc-8436 TNFα-R2 Rabbit monoclonal 1:50 Santa Cruz Biotechnology, USA, product number sc-7862 Biotinylated Pan-specific Horse polyclonal RTU Vector Laboratories, USA, product number PK-7800 universal antibody (anti-mouse/rabbit/goat IgG) Streptavidin-peroxidase complex –––––––––––––––– RTU Vector Laboratories, USA, product number PK-7800 [page 120] [European Journal of Histochemistry 2023; 67:3513] 2023_2.qxp_Hrev_master 13/04/23 11:02 Pagina 120 Non-commercial use only Article confirms the presence of TNF-R2 in the tunica intima and muscular layer of the blood vessels. No immunostaining was observed in muscular layers. No differences were found in the expression or distribution of TNFα-R2between the two segments (ampulla and isthmus) during the menstrual cycle. The results for each of the individual structures have been summarized in Table 3. Figure 1. Histological analysis of UT cross section. A-D) Hematoxylin & Eosin stain. E) PAS stain. F) Alcian blue stain. Transversal section of isthmus (A) and ampulla (B). The UT is a tubular organ that connects the periovarian space with the uterus. A) Anatomical characteristics of UTs (inset); the ampulla has more mucosal folds than the isthmus. B) The muscular layer (m) of the isthmus is thicker than the muscular layer (m) of the ampulla. C) Shows a mucosal fold covered by a simple columnar epithelium (e) and its subjacent lamina propria (lp). D) Three different cell types were distinguished; ciliated (arrows), basal (arrowhead), and nonciliated cells (inset). E) The luminal portion of the epithelium (arrows) and basal lamina (arrowhead) reacted to PAS stain. F) The apical surface of the epithelium (arrows) was positive for Alcian blue staining. Muc, mucosa; M, muscular; S, serosa; I, isthmus; A, ampulla. Table 3. Molecules distribution in mucosa of UTs during menstrual cycle. Galectin-9 F-spondin Osteopontin Integrin αV/β3TNFα-R1TNFα-R2 Stage F L F L F L F L F L F L Mucosa Ciliated cells ± ± + + + + + + + + ± ± Secretory cells + + + + + + + + + + + + Basal lamina + + + + + + + + - - Lamina propria - - - - - - - - - - - Blood vessels + + + + + + + + ± ± + + F, Follicular phase; L, luteal phase; +, presence of the molecule; -, absence of the molecule; ±, weak presence of the molecule. [European Journal of Histochemistry 2023; 67:3513] [page 121] 2023_2.qxp_Hrev_master 13/04/23 11:02 Pagina 121 Non-commercial use only Article Discussion Sperm migration in the UTs depends on different factors:41 sperm motility and hyperactivation, peristaltic movements, and oviductal flow. A universal feature of sperm migration in the female genital tract of mammals is the remarkable reduction of the number of cells that reach the site of fertilization in comparison with the total number inseminated. The biological importance of this phenomenon is the prevention of polyspermy.42 Chang and Suarez43 showed that mouse spermatozoa can attach to and detach from the epithelium of the oviduct isthmus, suggesting that spermatozoa may bind and unbind several times as they migrate through the oviduct. In this context, the results of the present study demonstrated the presence and distribution of galectin-9, Fspondin, osteopontin, and integrin αV/β3in the human UTs during the follicular and luteal phases of the menstrual cycle. The importance of these molecules lies in their key roles during physical interactions between spermatozoa and UT epithelial cells.22,44-48 Epithelial-bound spermatozoa also contribute to the formation of an isthmic sperm reservoir, thought to be important to organize available functional spermatozoa for fertilization.8,9,36 Furthermore, several studies provide evidence that TNFα regulates the oviductal contractile activity, thus it could play a role in sperm migration.2729,49 In this sense, the deposition and distribution of TNFα-R1and TNFα-R2 observed in the epithelial cells of the UTs could suggest that the contractile activity in the UTs could eventually influence the interaction between the sperm and epithelial cells, and subsequent migration through the UTs.2,5,7,30,43 The molecular components that are responsible for mediating sperm-UT adhesion remain largely unknown. Although, it appears that various species, including pigs and cattle, may share some similar mechanisms.7,23 In general, adhesion is ensured by lectinlike molecules on the sperm rostral surface that can bind carbohydrates exposed on the apical membranes of oviductal cells in a species-specific manner.7,16 Reeve et al.37 postulated that the recognition between the amino acid sequence Arg-Gly-Asp (RGD) and integrin receptors may contribute to the interaction between sperm and the human endosalpinx in the isthmic region. Our histological and histochemical analyses showed that during the menstrual cycle, the epithelium of all regions is composed of three distinct cell types: ciliated, nonciliated, and basal cells. The apical surface of the epithelium was positive for Alcian blue staining, revealing the presence of acid mucopolysaccharides. Additionally, the PAS histochemical method revealed a positive stain in the apical surface and basal lamina of the epithelial cells. These observations suggest the synthesis of glycoprotein and acid mucopolysaccharides by epithelial cells. In addition, in a previous study,1the finding of versican and fibromodulin proteoglycan in the apical portion of the epithelium was reported. This proteoglycan, glycoprotein, and acid mucopolysaccharides distribution may be related to its cellular adhesion properties and probably to their potential interaction with gametes.7,49,50 Galectins are a class of β-galactoside binding proteins51 that play several roles, such as regulation of cell growth, immunomodulation, apoptosis, and cell adhesion.51-54 Popovic et al.55 suggest galectin-9 as a novel human epithelial endometrial marker for midand late-secretory and decidual phases. In this regard, galectin-9 Figure 2. Immunostaining of F-spondin (brown stain) in UT sections. Immunostaining is shown in brown (DAB colorimetric reaction), whereas cell nuclei were contrasted with Harris’ haematoxylin. A,C) Follicular phase. B,D) Luteal phase. A,B) Positive immunostaining in the ampulla’s epithelium was observed (arrows); note the positive marking for F-spondin around the blood vessels (inset). C,D) Positive immunoreaction in the isthmus’s epithelium was observed (arrows); note the positive marking for F-spondin around the blood vessels (inset). e, epithelium; lp, lamina propria; m, muscular layer. Figure 3. Immunostaining of galectin-9 (red stain) in UT sections. Immunostaining is shown in red (DAB colorimetric reaction), whereas cell nuclei were contrasted with Harris’ haematoxylin. A,C) Follicular phase. B,D) Luteal phase. A,B) Galectin9 was detected in the secretory cells and ciliated cells of the mucosa; note the positive marking for galectin-9 around the blood vessels (inset). C,D) Positive immunoreaction in the isthmus’s secretory cells was observed; weak immunostaining was observed in the ciliated cells; note the positive marking for galectin-9 around the blood vessels (inset). e, epithelium; lp, lamina propria; m, muscular layer. [page 122] [European Journal of Histochemistry 2023; 67:3513] 2023_2.qxp_Hrev_master 13/04/23 11:02 Pagina 122 Non-commercial use only Article is a possible candidate for supporting the binding between endometrial epithelial cells and blastocysts. Furthermore, it has been described that galectins can affect cell adhesion both as agonist, as well as antagonist.56 Galectins bind to cell adhesion molecules, such as fibronectin and laminin.57 Fibronectin has previously been detected in ejaculated spermatozoa and spermatogenic cells.58 Our results show a positive reaction in the secretory cells of UT’s epithelium during the menstrual cycle. These results could be related to a different role for galectin-9 in the UT mucosa compared to the endometrium. In fact, it has been described that exposure of sperm to Gal-1 resulted in glycan-dependent modulation of the acrosome reaction, a key event in the fertilization process.44 These studies, together with the presence of galectin-9 in the UT’s epithelium, are consistent with the hypothesis that galectin-9 could interact with sperm during the maturation and capacitation processes. On the other hand, the analysis of blood vessels using immunohistochemistry confirmed the presence of galectin-9. Aanhane et al.59 show that galectin-9 induced angiogenesis in the chick chorioallantoic membrane assay. In addition, O’Brien et al.60 found a significant increase in blood vessel formation in response to galectin-9 in the matrigel plug angiogenesis assay, a murine model of angiogenesis. It is suggested that the distribution of galectin-9 in blood vessels is associated with its angiogenic properties. Osteopontin is a highly phosphorylated glycophosphoprotein with acidic characteristics, rich in aspartic acid and N-terminal that includes an integrin-receptor binding zones.61,62 In addition, integrin αVβ1, αVβ5 and α9β1 act as receptors for osteopontin.63-65 The integrin αVβ3is primarily known to bind osteopontin via the RGD region.66 Integrin β1, β3 and β4 have been detected on the outer surface membrane and osteopontin in human spermatozoa.67-69 The literature indicates that multiple osteopontin isoforms may play different roles in fertilization, early embryo development, and placentation.22 Gabler et al.22 showed that differential presence of osteopontin isoforms and integrins in the bovine oviduct indicates that osteopontin-integrin interactions have functional roles in normal oviduct physiology. In the present study, the analysis of osteopontin revealed positive immunostaining in the epithelial cells of UTs mucosa during follicular and luteal phases. As a result, our findings suggest that osteopontin found in the UTs epithelium may bind to integrins found in human spermatozoa. Additionally, the analysis of blood vessels revealed a strong positive reaction in the tunica muscularis. In this context, experimental evidence suggests that osteopontin may affect angiogenesis by acting directly on endothelial cells.70,71 F-spondin is an extracellular matrix protein that participates in the outgrowth of the neural tissue as well as in the inhibition of angiogenesis in the floor plate and paranotochordal area of the developing embryo.72 In addition, F-spondin is expressed in the epithelial and stromal cells of mouse endometrium and Ishikawa cells, a human endometrial epithelial cell line.45,46 In this context, it has been described that 2-methoxyoestradiol impairs mouse embryo implantation via activation of F-spondin in the mice uterus.46 Curiously, F-spondin was identified as a new ovarian cancer marker.73 The immunohistochemical analysis of F-spondin in the human UTs demonstrated that this molecule was restricted to ciliated cells and secretory cells. Furthermore, F-spondin immunostaining was positive in the epithelial cells of UTs mucosa and correlated to the pattern of type αV/β3. In this regard, some studies Figure 4. Immunostaining of osteopontin (brown stain) in UT sections. Immunostaining is shown in brown (DAB colorimetric reaction), whereas cell nuclei were contrasted with Harris’ haematoxylin. A,C) Follicular phase. B,D) Luteal phase. A,B) Osteopontin was detected in the epithelial cells of the ampulla during the menstrual cycle; note the positive marking for osteopontin around the blood vessels (inset). C,D) Osteopontin was detected in the apical epithelial cells of the isthmus during the menstrual cycle; note the positive marking for osteopontin around the blood vessels (inset). e, epithelium; lp, lamina propria; m, muscular layer. Figure 5. Immunostaining of integrin αV/β3(brown stain) in UT sections. Immunostaining is shown in brown (DAB colorimetric reaction), whereas cell nuclei were contrasted with Harris’ haematoxylin. A,C) Follicular phase. B,D) Luteal phase. A,B) Integrin αV/β3was detected in the epithelial cells of the ampulla during the menstrual cycle; the analysis of blood vessels revealed a strong positive reaction in the tunica muscularis (inset); positive immunoreaction in the isthmus’s epithelium was observed (arrows); note the positive marking for integrin αV/β3around the blood vessels (inset). e, epithelium; lp, lamina propria; m, muscular layer. [European Journal of Histochemistry 2023; 67:3513] [page 123] 2023_2.qxp_Hrev_master 13/04/23 11:02 Pagina 123 Non-commercial use only Article reported that VSGP/F-spondin blockade αV/β3 on vascular endothelial cells.74 This finding led us to suggest that F-spondin could be related to the anti-implantation effect in UTs.45,46 Additionally, our study describes the presence of F-spondin in the tunica muscularis of blood vessels of different caliber. This expression may be related to its angiogenic properties because F-spondin is known to promote the growth of vascular smooth muscle cells.75,76 Integrins are alpha/beta heterodimeric adhesion glycoprotein receptors that regulate a wide variety of dynamic cellular processes such as cell migration, phagocytosis, growth, and development.77 Furthermore, they provide a physical transmembrane link between the extracellular environment and the cytoskeleton, and are capable of transducing bidirectional signals across the cell membrane.48 Additionally, αvβ3 is a receptor for proteins bearing an exposed Arg-Gly-Asp (RGD) tripeptide including vitronectin, fibronectin, fibrinogen, thrombospondin, osteopontin, von Willebrand factor, and some degraded laminins and collagens.47 Several authors, have indicated that the endometrial epithelial expression of the integrin αvβ3, correlates with receptivity to the presenting embryo in humans.78,79 Apparao et al.80 showed in adhesion assays using Ishikawa cells that αV/β3appears to be the primary receptor for osteopontin. These authors suggest that osteopontin and αV/β3may play complementary roles during the endometrial implantation process. Our findings show integrin αV/β3immunostaining in the epithelial surface cells of the mucosa during the menstrual cycle. Curiously, in this study, the association between αV/β3and osteopontin in the UTs epithelium suggests that αV/β3could play a role complementary to the interaction between spermatozoa and tubal epithelium. In addition, a positive reaction was observed in the basal lamina of mucosa’s epithelium. These results are in agreement with previous reports stating that αV/β3 is a component of focal contact.81 Analysis of blood vessels using immunohistochemistry confirmed the presence of αV/β3in the tunica muscularis. Our results are consistent with previous studies that reported that αV/β3 antagonists could inhibit angiogenesis during development, wound healing, retinal neovascularization, and in growing tumors.47 Tumor necrosis factor-α (TNF-α) is a cytokine associated to the immune response in the female genital tract.82 Several studies show that TNF-α could participate in reproductive functions, including fertilization, embryo development, and implantation.82-86 The TNFα gene is expressed in mouse oviductal epithelial cells and human oviductal fluid contains TNFα protein.87,88 Studies in human and animal models revealed that the embryo is capable of releasing TNFα.28,89 Furthermore, the stage-specific expressions of mRNA for TNFα, TNFα-R1, and TNFα-R2 were detected in the bovine oviductal epithelial cells by RT-PCR and suggested the possible involvement of TNFα in the control of cyclic oviductal contraction.27 Wijayagunawardane et al.27 provided evidence that TNFα stimulates prostaglandins secretion and that up-regulation of the TNFα system occurs in the cow oviduct during the periovulatory period. In turn, prostaglandins play a major role in the oviductal transport of gametes/embryo as they stimulate muscular activity of the oviduct.29,90 In this regard, contractile activity in the isthmus may have a significant influence on the distribution of spermatozoa relative to the time of ovulation.2,5 In the present study, the analysis of TNFα-R1and TNFα-R2 revealed positive immunostaining in the epithelial cells of the UTs mucosa during the menstrual cycle. These findings led us to suggest that contractile activity in Figure 6. Immunostaining of TNFα-R1(brown stain) in UT sections. Immunostaining is shown in brown (DAB colorimetric reaction), whereas cell nuclei were contrasted with Harris’ haematoxylin. A,C) Follicular phase. B,D) Luteal phase. A,B) TNFα-R1was detected in the epithelial cells of the ampulla during the menstrual cycle; note the weak immunostaining for TNFα-R1 around the blood vessels (inset). C,D) TNFα-R1was detected in the epithelial cells of the isthmus during the menstrual cycle; note the weak immunostaining for TNFα-R1 around the blood vessels (inset). e, epithelium; lp, lamina propria; m, muscular layer. Figure 7. Immunostaining of TNFα-R2(brown stain) in UT sections. Immunostaining is shown in brown (DAB colorimetric reaction), whereas cell nuclei were contrasted with Harris’ haematoxylin. A,C) Follicular phase. B,D) Luteal phase. A,B) TNFα-R2 was detected in secretory cells of mucosa; weak immunostaining was observed in ciliated cells; note the positive immunostaining for TNFα-R2around the blood vessels (inset). C,D) Positive immunoreaction in the isthmus’s secretory cells was observed; weak immunostaining was observed in ciliated cells; note the positive immunostaining for TNFα-R2around the blood vessels (inset). e, epithelium; lp, lamina propria; m, muscular layer. [page 124] [European Journal of Histochemistry 2023; 67:3513] 2023_2.qxp_Hrev_master 13/04/23 11:02 Pagina 124 Non-commercial use only Article the isthmic region could influence the binding or release of spermatozoa from epithelial cells and their migration through the UTs.2,5,7,42,43,82 Interestingly, TNFα-R1and TNFα-R2immunostainings were positive in the epithelial cells of UTs mucosa and correlated to the pattern of osteopontin. In this regard, some studies reported that TNFα strongly induces osteopontin expression.91,92 In conclusion, our comprehensive histological study demonstrates that UT epithelial cells produce galectin-9, F-spondin, osteopontin and αV/β3 integrin. In this sense, our results suggest that these molecules could form part of the sperm-UT interactions during the menstrual cycle. Additionally, an abundant distribution of TNFαR1 and TNFα-R2was observed at the epithelial level, which could be related to the contractile activity of the UTs. 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