Revista Cienfica, FCV-LUZ / Vol. XXXV Recibido: 06/12/2024 Aceptado:10/02/2025 Publicado: 04/04/2025 hps://doi.org/10.52973/rcfcv-e35586 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 11 The distribuon of four homeobox proteins in the bovine stomach compartments during the fetal period Distribución de cuatro proteínas homeobox en los comparmentos gástricos bovinos durante el período fetal Uğur Topaloğlu * , Mehmet Erdem-Akbalık , Hakan Sağsöz , Muzaffer Aydın-Ketani, Berna Güney-Saruhan , Nurşin Aydın Department of Histology and Embryology, Faculty of Veterinary Medicine, Dicle University, 21280, Diyarbakır, Türkiye *Corresponding author: ugur.topaloglu@dicle.edu.tr ABSTRACT Homeobox proteins undertake important roles in the determinaon of the cell identy during embryonic development, the differenaon of embryonic stem cells, morphogenesis, and the formaon and development of the mammalian gastrointesnal tract. Based on these data, this study was designed to determine the immunohistochemical localizaon and expression levels of HOXA10, HOXA11, HOXC6 and HOXB6, which are subunits of the homeobox proteins, in the rumen, reculum, omasum and abomasum during fetal development. For this purpose, fetuses obtained from private slaughterhouses and were grouped according to their crown-rump length (CRL) measurements, and the gestaonal trimester they belonged to, as follows: first trimester (69-89 days old/10 fetuses), second trimester (99-178 days old/10 fetuses) and third trimester (188-269 days old/10 fetuses). Gastric ssue samples taken from each group underwent roune histological processing and immunohistochemical staining. Immunohistochemical staining demonstrated that the HOXA10, HOXA11 and HOXC6 proteins were expressed at varying levels in the rumen, reculum, omasum and abomasum, and their expression was stronger in the epithelial and smooth muscle cells. On the other hand, while there was almost no expression of HOXB6 in the epithelial cells of the rumen during the second and third trimesters of gestaon, the remaining gastric components were immunonegave. Based on these findings, it was concluded that some homeobox proteins could have crical roles in the development, morphogenesis and histogenesis of fetal bovine gastric compartments, and thus, could contribute to the lifeme performance and producvity of cale in terms of milk and meat yields. Key words: bovine; fetus; homeobox proteins; stomach RESUMEN Las proteínas HOX/Hox, ene funciones importantes en la determinación de la idendad celular durante el desarrollo embrionario, la diferenciación y la morfogénesis de las células madre embrionarias, así como en la formación y desarrollo del tracto gastrointesnal de los mamíferos. Con base en estos datos, este estudio fue diseñado para determinar la localización inmunohistoquímica y los niveles de expresión de HOXA10, HOXA11, HOXC6 y HOXB6, que son subunidades de las proteínas homeobox, en el rumen, reculo, omaso y abomaso durante el desarrollo fetal. Para ello, los fetos obtenidos de mataderos privados se agruparon según sus medidas de longitud cráneo-anca (LCC) y el trimestre gestacional al que pertenecían, de la siguiente manera: primer trimestre (69-89 días de edad/10 fetos), segundo trimestre (99-178 días/10 fetos) y tercer trimestre (188-269 días/10 fetos). Las muestras de tejido gástrico tomadas de cada grupo se someeron a procesamiento histológico de runa y nción inmunohistoquímica. Como resultado de la nción, se idenficaron las proteínas HOXA10, HOXA11 y HOXC6; Se determinó que se expresaban con intensidades variables en el rumen, reculo, omaso y abomaso, y especialmente esta intensidad de expresión fue más fuerte en las células epiteliales y de la capa de músculo liso. Mientras que, la expresion de HOXB6 en el rumen fue casi inexistente en las células epiteliales a parr del segundo y tercer trimestre del gestacion, la inmunorreacción resultó negava en todas las secciones restantes del estómago. Como resultado de estos hallazgos; Sugirió que algunas proteínas homeobox pueden tener una importancia críca para el desarrollo, la morfogénesis y la histogénesis de los segmentos del estómago bovino fetal y, por lo tanto, contribuir al rendimiento y la producvidad del ganado durante toda la vida, especialmente en términos de producvidad de carne y leche. Palabras clave: Bovino; fetos; proteínas homeobox; estómago

The distribuon of proteins in the bovine during the fetal period / Topaloğlu et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION The digesve system of vertebrates is a unique structure that takes in and digests food, absorbs nutrients, and removes waste products from the body. Digesve system acvity is closely related to the morphology of the digesve system as well as to the feeding habits of the organism [1]. In this context, the ruminant stomach with four compartments, are of interest as they convert low quality feed into highly nutrious products. Thus, there is need for a beer understanding of the funconality, including the morphology, of the ruminant digesve system [2]. At parturion, the forestomach of the newborn, namely, the rumen, reculum and omasum are undeveloped and therefore nonfunconal. On the other hand, during the neonatal period, the abomasum, otherwise known as the true stomach, is well- developed and highly funconal. In this period, the abomasum, which is the fourth gastric compartment, constutes the largest part (almost 70%) of the ruminant digesve system. The immature metabolic digesve system of neonatal calves resembles that of a young monogastric animal in terms of funconality. Before calves enter the ruminaon period, their rumen is small and loose, and the ruminal papillae are yet in a primive state. During the phase of transion to ruminaon, the rumen grows 4- to 8-fold of its birth size yet sll does not acquire the rumen wall thickness characterisc of the adult stage. With the advance of age, the spaces between the ruminal papillae and recular cristae and the omasal laminae enlarge and become evident [3 , 4]. This developmental process of the ruminant stomach enables the rumen microorganisms to convert carbohydrates, proteins and other fermentable substances into volale fay acids, ammonium, methane, carbondioxide and microbial proteins. The ruminant forestomachs act as fermentaon chambers, and thereby play an important role in the bacterial digeson of cellulose. These compartments also display peristalc movements and contracons and enable the separaon of fluids and solids. Histologically, the wall of the gastric compartments referred to as the ruminant forestomachs are composed of four layers, namely, the tunica mucosa, tunica submucosa, tunica muscularis and tunica serosa. The tunica mucosa is composed of a lamina epithelialis of keranized strafied squamous epithelium, a lamina propria and a lamina muscularis. The rumen differs from the other gastric compartments in that the ruminal mucosa does not contain a lamina muscularis. In all gastric compartments, the tunica muscularis comprises an inner stratum of circularly arranged muscles and an outer stratum of longitudinally arranged muscles. The stomach with four compartments are encapsulated by a tunica serosa. On the other hand, the abomasum differs from the forestomachs as it resembles the stomach of monogastric species and the tunica mucosa is lined by a simple, glandular and prismac epithelium [5 , 6]. Homeobox genes encode the transcripon factors referred to as homeodomains, which funcon as DNA-binding domains of 60 amino acids. These proteins take part in the regulaon of several embryonic development processes such as axis formaon, limb development and organogenesis [7]. The HOX proteins, known as the subunits of homeobox proteins, have been well invesgated in the genome of two mammalian species, the mouse and the human. In total 39 Hox proteins have been idenfied in both species. These proteins are expressed as Hox for mice and HOX for humans and are clustered at four genomic loci under the names the Hox/HOX-A, B, C and D complexes, and each cluster is composed of 9-13 genes [8]. HOX/Hox proteins are major transcriponal regulators, which are involved in mulple processes ranging from embryogenesis to carcinogenesis [9]. These proteins have also been reported to affect the acquision of cell identy during embryonic development, embryonic stem cell differenaon, and morphogenesis as well as the skeletal and nervous systems. Furthermore, they have been demonstrated to play a key role in the formaon of the mammalian gastrointesnal tract, parcularly in the differenaon of the muscle layer and epithelium [10 , 11 , 12]. It has been shown that the HOXA10 and HOXA11 proteins play a crucial effect in the organogenesis, development and differenaon of the reproducve system [13]. While no literature report is available on the potenal role and distribuon of homeobox proteins in the normal digesve system, it has been reported that HOXA10 and HOXA11 are associated with the presence, development and metastasis of gastric tumors [14 , 15]. Similarly, HOXB6 and HOXC6 are known to have very important roles in organogenesis, embryogenesis and cell differenaon [12 , 16 , 17]. Furthermore, it has been suggested that these proteins could have various physiological roles in the formaon and development of tumors in certain organs (stomach, colon, lungs, prostate) and ssues [12 , 18 , 19]. The present study bears significance as it is the first invesgaon on the distribuon of homeobox proteins, known to have significant roles in several processes including embryogenesis, organogenesis, morphogenesis and carcinogenesis as well as in several systems including the skeletal and nervous systems, in the gastric compartments of the bovine fetus. This study was aimed at: a) the immunohistochemical demonstraon of the distribuon of the HOXA10, HOXA11, HOXC6 and HOXB6 proteins in the rumen, reculum, omasum and abomasum during fetal development, b) the determinaon of the potenal physiological roles of the selected homeobox proteins during the prenatal period, and their various funconal correlaons for the different trimesters of gestaon, c) the establishment of a beer understanding of the mechanisms underlying gastric development in humans and mammals, and the invesgaon of the potenal contribuon of homeobox proteins to the structural components of the gastric compartments. MATERIALS AND METHODS Supply of the study material and preparaon of the ssue samples The study material comprised of 30 clinically healthy bovine fetuses (Bos taurus), which belonged to different gestaonal trimesters and were obtained from private slaughterhouses. The ages of the fetuses were determined using the formula ‘’y= 54.6 cm + 2.46 (x) cm’’, based on the measurement of the crown-rump length (CRL). In this equaon, ‘’x’’ refers to the CRL and ‘’y’’ expresses the fetal age in days (d) [12]. Once their ages were determined, the fetuses were assigned to three groups, as follows, according to the gestaonal trimester they belonged to: group of fetuses belonging to the first trimester (69-89 d old/10 fetuses), group of fetuses belonging to the second trimester (99-178 d old/10 fetuses), and group of fetuses belonging to the third trimester (188-269 d old/10 fetuses). In view of the specific trimester the fetus belonged to, each fetus underwent ssue sampling from the gastric compartments 2 of 11

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico (rumen, reculum, omasum and abomasum). The ssue samples were fixed in 10% formol-alcohol soluon for 18 h. Next, they were dehydrated through a graded series of alcohol, starng from 80% alcohol. Subsequently, the dehydrated ssue samples were passed through methyl benzoate, benzole and benzole-paraplast series, and embedded in paraffin. Later the paraffin blocks were cut into 5-micrometer-thick serial secons on a rotary microtome (Leica RM-2125, Germany). The secons were mounted onto slides coated with APES (3 amino propyl triethoxysilan; Sigma–Aldrich Chemicals, St. Louis, MO, USA) for immunohistochemical staining. Immunohistochemical procedure The serial secons mounted onto the APES-coated slides underwent immunohistochemical (IHC) staining according to the streptavidin-peroxidase technique and using the Zymed Histostain Plus Bulk Kit (South San Francisco, CA, USA, cat no: 85- 9043). Firstly, the secons were allowed to dry, and they were deparaffinized twice in xylol, each me for 5 min, rehydrated in graded alcohols for 3 min in each grade, and washed in dislled water. Then, the endogenous peroxidase acvity of the ssue samples was quenched with a 3% H 2 O 2 soluon in methanol for 20 min. Subsequently, the secons were washed three mes, each me for 5 min, in phosphate-buffered saline (PBS, pH: 7.4; 0.01 M). This was followed by the incubaon of the preparaons in a citrate buffer (pH: 6) for 30 min at 95ºC and unl the temperature fell to room level for the designaon of the anbody-binding angenic sites. Later, to prevent the non-specific binding of the primary anbody, the secons were treated with a blocking soluon (Ultra V Blok, catalog: TA-125-UB, Thermo Scienfic) for 15 min. Once the soluon was discarded, the secons were incubated overnight at +4 o C with anbodies diluted at a concentraon of 1:100 (TABLE I). Aſter being washed in PBS for another 3 mes, each me for 5 min, at room temperature, the secons were incubated with bionylated secondary anbody (Bionylated Goat An-Polyvalent, catalog: TP-125-BN, Thermo Scienfic) for 20 min at room temperature. This was followed by another 3 washes in PBS, each me for 5 min, and the treatment of the secons with streptavidin peroxidase (Thermo Fisher Scienfic, catalog: TA-125-HR) for 20 min at room temperature. Finally, the secons were added 3.3 diaminobenzidine (DAB Substrate, Thermo Scienfic, catalog no: TA-125-HD) and were maintained as such for a period from 5 to 15 min, depending on the reacon speed. Aſter being washed in dislled water, the secons were applied Mayer’s hematoxylin for 2 min for nuclear staining. Later, the secons were washed under running tap water for 5 min, dehydrated through a graded series of alcohol, cleared in xylol and mounted in Entellan. The stained preparaons were examined and imaged under a Nikon Eclipse E400 research microscope (Nikon, Tokyo, Japan) with a DS-RI1 video camera (DS-U3, Nikon, Tokyo, Japan) aachment. TABLE I. Primary anbodies used for immunohistochemistry (IHC) Primer Anbodies Clonality / Isotype Host Reacvity Diluon Catalog number HOXA10 Polyclonal/IgG Rabbit Human, Mouse 1/100 St John’s Laboratory, model no: STJ193159 HOXA11 Polyclonal/IgG Rabbit Human 1/100 Invitrogen, PA5-57341 HOXC6 Polyclonal/IgG Rabbit Rat, Cat, Human 1/100 Invitrogen, PA5-41479 HOXB6 Polyclonal/IgG Goat Human, Mouse, Rat, Dog, Bovine, Pig 1/100 St John’s Laboratory, model no: STJ73348 Semiquantave evaluaon The intensity scoring of the immunostaining was performed semiquantavely. Intensity scores were determined based on the posive staining intensies of the cells as follows: 0 (Negave), 1 (Weak Immunoreacons), 2 (Moderate Immunoreacons), 3 (Strong Immunoreacons). Immunohistochemical assessment was performed by three independent blinded observers (UT, MEA & HS) and the mean scores of the three observers were calculated. Posive immunoreacons for HOXA10, HOXA11, HOXC6 and HOXB6 were idenfied in the high expression areas by scanning the rumen, reculum, omasum and abomasum secons at 40x, 100x, 200x and 400x magnificaon. Five randomly selected areas were evaluated per secon and the average of these individual results was taken as one value. Different components of the rumen, reculum, omasum and abomasum, including epithelial cells, stromal cells and smooth muscle cells, were assessed. Blood vessels were not evaluated in detail, but their general appearance in some secons was described. In this study, 1250 values were averaged for each ssue and 2010 values were averaged per animal. Stascal analysis Analyses were evaluated with SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). All values are shown as mean ± standard deviaon (SD). Data normality was calculated with the Shapiro- Wilk test. The nonparametric Kruskal-Wallis test was used to analyze whether there was any significant difference in the immunohistochemical staining intensity score for HOXA10, HOXA11, HOXC6 and HOXB6 of the luminal epithelium, stromal cells and smooth muscle cells of the bovine fetal stomach compartments during the different trimesters of gestaon or between these cell and ssue types. The Mann-Whitney U-test was used to detect differences in the staining score of each anbody among the cell types. Thereby, the final results were expressed as mean ± SD, and stascal significance was set at P<0.05. RESULTS AND DISCUSSION Histologically, bovine stomach secons consist of three layers called tunica mucosa, muscularis and serosa. The folds formed by the strafied squamous epithelium of the tunica mucosa towards the rumen are named as the ruminal papillae in the rumen, the recular crista in the reculum, and the omasal lamina in the omasum. It has been observed that these folds are short and present at varying numbers during the first trimester of gestaon and increase in both height and density over the course of gestaon as a result of cellular proliferaon and differenaon. The abomasum was observed to have a glandular mucosa lined by a simple columnar epithelium, beneath of which gastric glands developed as of the second gestaonal trimester. 3 of 11

The distribuon of proteins in the bovine during the fetal period / Topaloğlu et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Based on immunohistochemical stainings, it was determined that while posive immunoreacons of varying intensity were present in the fetal rumen, reculum, omasum and abomasum for HOXA10, HOXA11 and HOXC6 throughout gestaon, HOXB6 was excluded from the stascal evaluaon as it was only very weakly expressed in the luminal epithelial cells, and not expressed at all in the stromal cells and smooth muscle cells. Homeobox proteins undertake crical roles in mulple ssue and cell processes, including among others, development, apoptosis, differenaon, molity, receptor signaling and angiogenesis [20]. These proteins are also known to have very important funcons in the establishment of cell identy and cell posion during embryonic development. It has been reported that changes in these proteins could lead to major developmental defects in the formaon of body parts and cell identy [21]. Known to be posioned at the onset of mammalian development, Hox proteins are reported to be expressed in the ectoderm-derived layer (nervous system), mesoderm- derived layer (genitourinary system) and endoderm-derived layer (digesve system) and to take part in the development of these layers [22]. HOX/Hox proteins have also been described to be involved in the development of the skeletal and nervous systems and genital organs, as well as in the modelling of the mammalian gastrointesnal tract. It has been demonstrated that these proteins play a crical act in the differenaon of both the muscle layer and the epithelium of the gastrointesnal tract [11]. However, the presence and immunohistochemical distribuon of the HOXA10, HOXA11, HOXC6 and HOXB6 proteins in the fetal bovine gastric compartments have been demonstrated for the first me in the present research. Reports indicate that, in human fetuses, the HOXA10 and HOXA11 proteins are generally localized along the paramesonephric canal and contribute to embryogenesis as well as to the development and differenaon of the uterus [23]. Previous studies have demonstrated the expression of these proteins in the female genitals, skeleton and kidneys of (mouse) [16], the uterus of humans [24], (apes) [25], (raus) [26], and (swine) [27], the bovine placenta [12], (felis catus) testes [28], and bovine fetal liver [16], and have suggested their contribuon to the development of the normal physiological funcons of these organs. In a previous study on the healthy human gastric mucosa, it was determined that the weak expression of the HOXA10 protein increased in cases of gastric cancer and the differenaon of gastric cancer. Furthermore, the HOXA10 protein has also been reported to be expressed in the colon mucosa with normal metaplasia [29 , 30]. In another study, it was suggested that HOXA10, which enables the normal development and differenaon of hematopoiec stem cells, when expressed at an increased level increases the proliferaon of gastric cancer cells, strengthens the tumor and prevents apoptosis in mice [15]. On the other hand, HOXA11, known to have key roles in cell proliferaon and migraon, shows an expression level similar to that of HOXA10, which increases in cases of the malignant tumors of the mammary glands, ovaries, endometrium and lungs [31 , 32 , 33]. It has been shown that, in humans, while the expression of HOXA11 in the normal gastric mucosa is negave, it is posive and strong in cancerous gastric mucosa [34]. In another study on the human gastric mucosa, it was determined that the expression of the HOXA11 gene with DNA methylaon occurred at a higher level in cancerous gastric mucosa compared to the normal mucosa [35]. Based on these findings, it was observed that previous research on the distribuon of the HOXA10 and HOXA11 proteins in the stomach was rather limited and generally related to the development of gastric cancer in humans and mice. While these proteins have been reported to be present in the normal gastric mucosa of humans [29 , 30 , 34], literature review showed that there was no study available on their expression and funcons during normal gastric development in other species. In the present study, it was determined that the HOXA10 vand HOXA11 proteins induced immunoreacons in some cells and structures of the bovine gastric compartments throughout fetal development. Rumen The HOXA10 and HOXA11 proteins were observed to induce strong immunoreacons localized to the cytoplasm and nucleus of the rumen epithelial cells during the first trimester of gestaon (FIG. 1 A, B, a, b). The intensity of these immunoreacons decreased to a level below moderate for HOXA10 in the second trimester but increased to a moderate or moderate to strong level in the third trimester (FIG. 1 E, e, I, ı). The intensity of the immunoreacons for the HOXA11 protein over the course of gestaon was observed to be moderate or slightly stronger than moderate in the second trimester, and moderate or slightly weaker than moderate in the third trimester (FIG. 1 F, f, K, k). Furthermore, the immunoreacons for HOXC6, which were slightly stronger than moderate in the first trimester, showed slight weakening in the second and third trimesters and remained at the same level thereaſter (FIG. 1 C, c, G, g, L, l). FIGURE 1. Immunolocalisaon of HOXA10, HOXA11, HOXC6 and HOXB6 in fetal bovine rumen, in the first trimester of pregnancy (69 days) (A, B, C, D, a, b, c, d), second trimester (99 days) (E, F, G, H, e, f, g, h), third trimester (249 days) (I, K, L, M, I, k, l, m). E: Epithelium, Black arrowhead: Stroma cell, SM: Smooth Muscle layer, rp: ruminal papillae, bv; blood vessel. Scale Bar: 100 µm (A, B, C, D, E, F, G, H, I, K, L, M), magnificaon: 10X; 25 µm (a, b, c, d, e, f, g, h, ı, k, l, m), magnificaon: 40X. 4 of 11

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico The intensity of immunoreacons induced by HOXA10 and HOXA11 in the stromal cells was moderate or close to moderate in the first trimester but progressively weakened in the second and third trimesters. Moreover, immunoreacons for HOXC6 were very weak and almost absent in the stromal cells throughout gestaon. In the smooth muscle cells, the HOXA10 protein was observed to induce moderate immunoreacons throughout gestaon. The strong immunoreacons for HOXA11 in the first trimester weakened to a moderate level of intensity in the second trimester and to an intensity ranging from moderate to weak in the third trimester. On the other hand, the moderately intense immunoreacons for HOXC6 in the first trimester were observed to weaken to a level of almost disappearance in the second and third trimesters. Furthermore, some blood vessels presented with posive immunoreacons for HOXA10 during the second and third trimesters of gestaon, and for HOXA11 during the first and second trimesters (FIG. 1). FIGURE 2. Immunolocalisaon of HOXA10, HOXA11, HOXC6 and HOXB6 in fetal bovine reculum, in the first trimester of pregnancy (86 days) (A, B, C, D, a, b, c, d), second tri- mester (123 days) (E, F, G, H, e, f, g, h), third trimester (227 days) (I, K, L, M, ı, k, l, m). E: Epithelium, Black arrowhead: Stroma cell, SM: Smooth Muscle layer, cr: crista reculi, bv; blood vessel. Scale Bar: 100 µm (A, B, C, D, E, F, G, H, I, K, L, M), magnificaon: 10X; 50 µm (ı), magnificaon: 20X; 25 µm (a, b, c, d, e, f, g, h, k, l, m), magnificaon: 40X. Reculum During the first trimester, the HOXA10 and HOXC6 proteins were determined to produce strong immunoreacons localized to the nucleus and cytoplasm of the epithelial cells (FIG. 2A, a, C, c). While the intensity of these immunoreacons decreased to a moderate level for HOXA10 as gestaon advanced, it significantly and progressively decreased to a moderate and then to a weak level for HOXC6. On the other hand, immunoreacons for HOXA11 during the first and second trimesters increased from weak to moderate in intensity and maintained a moderate level throughout the third trimester of gestaon (FIG. 2 B, b, F, f, K, k). The immunoreacons produced by the HOXA11 and HOXC6 proteins in the stromal cells were observed to be very weak throughout gestaon. The immunoreacons, which were moderate for HOXA10 in the first trimester weakened as gestaon advanced. In smooth muscle cells, the HOXA10 protein induced strong immunoreacons during the first and third trimesters, and moderately intense immunoreacons in the second trimester. Immunoreacvity for HOXA11 was weak in the smooth muscle cells throughout gestaon. Moreover, HOXC6 immunoreacvity, which was close to moderate in the smooth muscle cells during the first trimester, were observed to become weak in the second and third trimesters. In some blood vessels, HOXA10 immunoreacvity was observed in the first trimester, while HOXA11 immunoreacvity was observed in the first and third trimesters. (FIG. 2). These immunoreacons, which were observed to be strong in the epithelial cells of the rumen, reculum, omasum and abomasum during the first trimester, showed stascally insignificant decrease and increase as gestaon advanced (P>0.05). Furthermore, in the fetal rumen and reculum the luminal epithelial cells displayed stronger HOXA10 and HOXA11 expressions compared to the stromal cells (P<0.05). Similarly, the expressions of HOXA10 and HOXA11 were stronger in the luminal epithelial cells, when compared to the smooth muscle cells, yet this difference was stascally insignificant. These findings obtained in the present study suggest that HOXA10 and HOXA11 may posively contribute to the proliferaon of the luminal epithelial and smooth muscle cells, and some of the stromal cells of the bovine gastric compartments throughout fetal development, as well as to the developmental regulaon and normal funconing of these organs. 5 of 11

The distribuon of proteins in the bovine during the fetal period / Topaloğlu et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico FIGURE 3. Immunolocalisaon of HOXA10, HOXA11, HOXC6 and HOXB6 in fetal bovine omasum, in the first trimester of pregnancy (82 days) (A, B, C, D, a, b, c, d), second tri- mester (178 days) (E, F, G, H, e, f, g, h), third trimester (257 days) (I, K, L, M, I, k, l, m). E: Epithelium, Black arrowhead: Stroma cell, SM: Smooth Muscle layer, lo: lamina omasi, bv; blood vessel. Scale Bar: 100 µm (A, B, C, D, E, F, G, H, I, K, L, M), magnificaon: 10X; 25 µm (a, b, c, d, e, f, g, h, ı, k, l, m), magnificaon: 40X. Omasum The HOXA10 and HOXA11 proteins induced strong immunoreacons in the omasal epithelium during the first trimester, which decreased to either a moderate or close to moderate level during the second and third trimesters (FIG. 3 A, a, E, e, I, I, B, b, F, f, K, k). However, in the first trimester, HOXC6 produced close to moderate immunoreacons, which were observed to be weaker during the second and third trimesters (FIG. 3 C, c, G, g, L, l). In the stromal cells, there was scarcely any immunoreacvity for HOXC6 and HOXA11 throughout all three trimesters. On the other hand, the weak immunoreacons observed for HOXA10 during the first and second trimesters were observed to be stronger and of close to moderate intensity in the third trimester. In the smooth muscle cells, during the first and second trimesters, the HOXA10 and HOXA11 proteins induced moderate and stronger than moderate immunoreacons, which increased in intensity for HOXA10 but decreased in intensity for HOXA11 in the third trimester. However, the immunoreacons, which ranged from weak to moderate for HOXC6 in the smooth muscle cells during the first trimester, decreased in intensity during the second and third trimesters to a constant weak level (FIG. 3). Abomasum In the abomasal epithelial cells, while the immunoreacons induced by the HOXA10 and HOXC6 proteins were either moderate or moderate to strong in the first trimester, those induced by HOXA11 were rather strong (FIG. 4 A, a, B, b, C, c). While HOXA10 produced strong immunoreacvity in the second and third trimesters, HOXA11 induced moderate immunoreacons in the second trimester and very strong immunoreacons in the third trimester (FIG. 4 E, e, F, f, I, I, K, k). The weaker than moderate immunoreacvity for HOXC6 during the second gestaonal trimester increased to a strong intensity in the last trimester (FIG. 4 G, g, L, l). FIGURE 4. Immunolocalisaon of HOXA10, HOXA11, HOXC6 and HOXB6 in fetal bovine abomasum, in the first trimester of pregnancy (73 days) (A, B, C, D, a, b, c, d), second trimester (106 days) (E, F, G, H, e, f, g, h), third trimester (269 days) (I, K, L, M, I, k, l, m). E: Epithelium, Black arrowhead: Stroma cell, SM: Smooth Muscle layer, gg: gastric gland, bv; blood vessel. Scale Bar: 100 µm (A, B, C, D, E, F, G, H, I, K, L, M), magnificaon: 10X; 50 µm (d, ı, k, l,), magnificaon: 20X; 25 µm (a, b, c, e, f, g, h, m), magnificaon: 40X. Moreover, the immunoreacons for HOXA10, HOXA11 and HOXC6 proteins in the epithelial cells were localized to the basal cytoplasm and nucleus during the third trimester. In the stromal cells, there was almost no immunoreacvity for the HOXA10 and HOXC6 proteins throughout gestaon. While HOXA11 immunoreacons displayed similarity to those for the HOXA10 and HOXC6 proteins during the first and second trimesters, they were observed to increase in intensity during the third trimester to a level stronger than moderate. In the smooth muscle cells, the HOXA10 protein was observed to induce close to moderate 6 of 11

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico immunoreacons during all three trimesters. Likewise, while HOXA11 produced moderate or slightly stronger than moderate immunoreacons during the first and second trimesters of gestaon, immunoreacvity was determined to reach a rather strong intensity level in the last trimester. However, the weak immunoreacvity observed for HOXC6 during the first and second trimesters increased to a moderate level of intensity in the third trimester. Furthermore, the gastric glands, which were observed to start to form and develop in the second trimester, displayed moderately intense immunoreacons for HOXA10, HOXA11 and HOXC6 proteins. These posive immunoreacons grew stronger for all three proteins in the third trimester. Moreover, the endothelial cells lining some blood vessels displayed posive immunoreacvity for the HOXA10 and HOXC6 proteins in the last trimester and for HOXA11 during both the second and third trimesters (FIG. 4). Furthermore, in the fetal omasum and abomasum, the luminal epithelial cells displayed stronger HOXA10 and HOXA11 expressions compared to the stromal cells (P<0.05). Furthermore, the expressions of the HOXA10 and HOXA11 proteins in some blood vessels of the rumen, reculum, omasum and abomasum during the different trimesters of gestaon suggest that these proteins could also be involved in the proliferaon of the vascular endothelial cells, as well as in angiogenesis. Moreover, it was concluded that these proteins may have crical roles in the development and physiological funconing of the gastric glands, which start to form as of the second trimester. While almost no immunoreacvity was detected for the HOXB6 protein in the ruminal epithelial cells during the second and third trimesters, immunoreacvity was negave for the remaining gastric compartments (FIG. 1-4 D, d, H, h, M, m). Previous studies have reported the involvement of the HOXC6 protein in mammary gland development and milk producon. Known to be expressed in normal ssues and cells, HOXC6 has also been determined to display excessively increased expression in cases of mammary, pulmonary and prostac cancers. Thus, this protein has been observed to be directly associated with the presence and development of cancer [36]. It has also been reported that, although not being expressed in the mucosal cells of the normal human stomach, the HOXC6 protein is expressed in the mucosal cells of cancerous gastric mucosal cells, suggesng the involvement of this protein in the pathogenesis of gastric cancer [20]. On the other hand, in another study, it was determined that HOXC6 was expressed in the nucleus and cytoplasm of normal human gastric mucosal cells, and that its expression increased in cases of gastric cancer [37]. Literature reviews showed that reports on the presence of HOXC6 in the normal stomach was limited to humans as species, and that these reports were mostly related to the presence and pathogenesis of cancer. In parallel with the report of [20], the present study demonstrated that the HOXC6 protein produced posive immunoreacons in the fetal gastric compartments. While these immunoreacons ranged from moderate to strong in the epithelial cells of the rumen, reculum, omasum and abomasum during the first trimester, over the course of gestaon their intensity increased in the abomasum but decreased in the other gastric compartments (P>0.05). The moderate HOXC6 immunoreacons detected in the smooth muscle cells of the rumen, reculum and omasum during the first trimester weakened in intensity over the course of gestaon. However, the weak HOXC6 immunoreacons detected in the abomasum during the first trimester were observed to increase to a moderate level of intensity as gestaon advanced (P>0.05). In the stromal cells, there was almost no immunoreacvity throughout gestaon, such that when compared to the epithelial and smooth muscle cells, the intensity of the immunoreacons was significantly lower (P<0.05). These findings explained in detail suggest that HOXC6 could have major roles in the regulaon of the development of the fetal bovine gastric compartments and the proliferaon and differenaon of the luminal epithelial and smooth muscle cells. Moreover, the periodic differences observed in the intensity of cellular immunoreacons suggested that the cellular effects of HOXC6 changed and that this protein could be included in the cellular structure, and thereby, affect physiological processes. Furthermore, HOXC6 was ascertained to induce immunoreacons in the abomasal gastric glands, the intensity of which increased over the course of gestaon. Based on this finding, it was concluded that HOXC6 could posively contribute to the development, growth and physiological funconing of these glands. Stascal findings for the different cell types and gestaonal periods The intensity scores for the expression of the HOXA10, HOXA11, HOXC6 and HOXB6 proteins in the fetal rumen, reculum, omasum and abomasum throughout gestaon are summarized in TABLE II. It was determined that, during all three trimesters, the expressions of HOXA10, HOXA11 and HOXC6 were stronger in the luminal epithelial cells of the fetal rumen, reculum, omasum and abomasum, compared to the stromal cells (P<0.05). Similarly, while the expressions of HOXA10, HOXA11 and HOXC6 were stronger in the luminal epithelial cells, compared to the smooth muscle cells, this difference was stascally insignificant (P>0.05) (TABLE II). The comparison of the gestaonal trimesters demonstrated that there was no difference between the first, second and third trimesters for the expressions of HOXA10, HOXA11 and HOXC6 (P>0.05) (TABLE II). 7 of 11

The distribuon of proteins in the bovine during the fetal period / Topaloğlu et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico TABLE II. Intensity scores for HOXA10, HOXA11, HOXC6 and HOXB6 expression in rumen, reculum, omasum and abomasum of bovine fetuses during pregnancy. Means ± SD. Immunohistochemical Parameter Pregnancy Trimesters Stomach Departments Histological Layers Luminal Epithelium Stromal Smooth Muscle HOXA10 1 st Rumen 2.80±0.42 a 2.0±0.0 b 1.90±0.32 b Reculum 2.70±0.48 2.0±0.47 2.60±0.52 Omasum 2.50±0.53 a 1.40±0.52 b 2.20±0.42 ab Abomasum 2.30±0.48 a 1.10±0.32 b 1.80±0.42 ab 2 nd Rumen 1.70±0.48 1.50±0.53 2.20±0.42 Reculum 2.30±0.48 a 1.30±0.48 b 2.30±0.48 a Omasum 1.80±0.42 1.50±0.53 2.20±0.42 Abomasum 2.60±0.52 a 1.50±0.53 b 1.90±0.32 ab 3 rd Rumen 2.40±0.52 a 1.30±0.48 b 2.20±0.42 ab Reculum 2.40±0.52 1.60±0.52 2.50±0.53 Omasum 1.90±0.32 1.80±0.42 2.60±0.52 Abomasum 2.50±0.53 a 0.90±0.32 b 2.20±0.42 a HOXA11 1 st Rumen 2.60±0.52 a 1.80±0.42 b 2.50±0.53 ab Reculum 1.50±0.53 0.80±0.42 1.20±0.42 Omasum 2.60±0.52 a 1.50±0.53 b 2.30±0.48 ab Abomasum 2.60±0.52 a 0.90±0.32 b 2.30±0.48 a 2 nd Rumen 2.20±0.42 1.70±0.48 2.10±0.32 Reculum 1.70±0.48 0.80±0.42 1.30±0.48 Omasum 2.40±0.52 a 1.20±0.42 b 2.20±0.42 a Abomasum 2.30±0.48 a 1.20±0.42 b 2.30±0.48 a 3 rd Rumen 1.80±.0.42 1.20±0.42 1.60±0.52 Reculum 2.10±0.32 a 0.70±0.48 b 1.20±0.42 ab Omasum 1.90±0.32 a 0.90±0.32 b 1.70±0.48 ab Abomasum 2.80±0.42 2.40±0.52 2.80±0.42 HOXC6 1 st Rumen 2.30±0.48 a 0.80±0.42 b 1.80±0.42 ab Reculum 2.50±0.53 a 1.20±0.42 b 1.80±0.42 ab Omasum 1.80±0.42 0.70±0.48 1.60±0.52 Abomasum 2.30±0.48 a 0.90±0.32 b 1.30±0.48 ab 2 nd Rumen 1.80±0.42 0.60±0.52 0.80±0.42 Reculum 1.50±0.53 0.70±0.48 1.30±0.48 Omasum 1.50±0.53 0.60±0.52 1.30±0.48 Abomasum 1.70±0.48 a 0.60±0.48 b 0.90±0.32 ab 3 rd Rumen 1.80±0.42 0.60±0.52 0.80±0.42 Reculum 1.80±0.42 a 0.70±0.48 b 1.40±0.52 ab Omasum 1.60±0.52 a 0.60±0.52 b 1.20±0.42 ab Abomasum 2.40±0.52 a 0.70±0.48 b 1.70±0.48 b 8 of 11

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico HOXB6 1 st Rumen N/W N N Reculum N/W N N Omasum N/W N N Abomasum N/W N N 2 nd Rumen N/W N N Reculum N/W N N Omasum N/W N N Abomasum N/W N N 3 rd Rumen W/N N N Reculum N/W N N Omasum N/W N N Abomasum N/W N N Different superscripts (a, b) in the same line indicate significant differences among epithelial, stroma and smooth muscle cells (P<0.05). Intensity scores (IS) of HOXA10, HOXA11 and HOXC6 immunostaining (0: no staining at high magnificaon, 1: immunostaining only visible at high magnificaon, 2: readily visible at low magnificaon, 3: strikingly posive at low power magni- ficaon. HOXB6 was excluded from stascal evaluaon because its expression was only in the epithelium and was weak. W; weak and N; negave Previous studies on HOXB6 have demonstrated that this protein induces immunoreacons in human hematopoiec progenitor-stem cells and affects the proliferaon and differenaon of murine hepac cells [9 , 38 , 39]. Furthermore, it has also been shown that HOXB6 is expressed during the oncogenic processes of some organs and ssues (esophagus and hepatocytes) and undertakes crical roles in the proliferaon, migraon and invasion of cells in these organs and ssues [34]. Previous studies on the digesve system have revealed that the intensity of the expression of the HOXB proteins progressively increases from the esophagus to the intesnes [40]. It has also been reported that HOXB6 plays a key role in the gastrointesnal system of humans and mice and is expressed in the normal human esophagus [41]. Moreover, it has been indicated that HOXB6 is expressed in the normal human gastric mucosa and that the expression of this protein increases in tumoral ssues. Thereby, a direct associaon has been established between HOXB6 and gastric cancer stages and tumor grades [42]. In a study on the development of the chicken stomach, it was determined that HOXB6 was expressed in the mucosa of the stomach and affected its development process [43]. In parallel with the findings of studies on the normal stomach of humans [42] and chickens [43], it has been reported that HOXB6 is also expressed in the bovine placenta [12] and, fetal bovine liver [16] and may contribute to physiological processes. However, in the present study, we determined that, excluding the rumen, HOXB6 was not expressed in any of the fetal bovine gastric compartments throughout gestaon. The very weak expression of HOXB6 in the rumen aſter the second trimester of gestaon suggests that this protein joins the structure of ruminal cells and affects the development of the rumen only aſter this period. Moreover, HOXB6 not being expressed in the other fetal bovine gastric compartments was aributed to the different structure and physiological funcons of the bovine rumen, reculum, omasum and abomasum, compared to the human and avian stomachs. CONCLUSION In conclusion, that the HOXA10, HOXA11 and HOXC6 proteins were present at varying levels in the epithelial, stromal and smooth muscle cells of the bovine gastric compartments throughout prenatal development. It was considered that HOXA10, HOXA11 and HOXC6 could have important physiological roles in the regulaon of the development of the bovine gastric compartments and the proliferaon and differenaon of the cells of these compartments. In view of the milk and meat yields of food-producing animals being directly related to stomach acvity, it was concluded that these proteins could have significant effects on the normal gastric development process, and thus, could directly contribute to the sustainability of animal producon. Furthermore, it can be suggested that homeobox proteins may play a role in the tumorigenesis of the bovine gastrointesnal system, and they could potenally be used as prognosc biomarkers in gastrointesnal tumors in future studies. Declaraon of Compeng Interest The authors declare no conflict of interest. ACKNOWLEDGMENTS This study is supported by Dicle University Scienfic Resear- ch Projects coordinatorship with the project number “VETERİ- NER.22.006”. BIBLIOGRAPHIC REFERENCES [1] Jones KR, John RE, Sundaram V. Morpho-histological studies of the gastrointesnal tract of the orange-rumped agou (Dasyprocta leporina Linnaeus, 1758), with special reference to morphometry and histometry. Animals (Basels). [Internet]. 2022; 12(19):2493. doi: hps://doi. org/g6fz3h [2] Hassan MAS, Karslı MA. The effects of some feed addives in nutrion of ruminant animals. IJVAR. [Internet]. 2022; 5(2):107-112. doi: hps://doi.org/pcsf [3] Scala G, Corona M, Maruccio L. Structural, histochemical and immunocytochemical study of the forestomach mucosa in domesc ruminants. Anat. Histol. Embryol. [Internet]. 2011; 40(1):47-54. doi: hps://doi.org/dpdxkt 9 of 11

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