Histomorphometry and uterine proteomics during the normal reproductive cycle in bitches

We aimed to evaluate the histomorphometry and proteomic profile of the canine uterus during all stages of the reproductive cycle. Eighteen healthy female dogs had their estrous cycle identified by clinical evaluation, vaginal cytology, and serum progesterone levels, which were allocated to the proestrus (n=5), estrus (n=5), diestrus (n=5), and anestrus (n=3) groups. All were submitted to elective ovariosalpingohysterectomy, and the uteri were collected for histomorphometric measurement (Image J software). For proteomic analysis, fragments of the uterine horns were subjected to protein measurement (Bradford method) and extraction by 2D electrophoresis (PDquest software). The results showed that the diestrus promoted greater values of thickness in the uterine structures (μm): uterine wall (2,223.8±229.8), endometrium (819.7±109.1), and myometrium (1,392.6±294.2). Uterus showed a protein profile with good reproducibility per phase (pI: 3.5–9.0; PM: 24–150 KDa), with 11 spots in all phases. Despite the greatest histomorphometric changes in the diestrus, we observed a greater number of spots in the estrus (253±45), followed by the proestrus (185±21), diestrus (113±39), and anestrus (80±21). This finding showed probable participation of these proteins in the uterine preparation for receiving gametes for fertilization. Our results showed greater uterine thickness in the diestrus, and greater protein secretion in the estrus, contributing to the prospection of identification of proteins responsible for the biological reproduction processes.


Introduction
The female reproductive organs are complete at birth, except the uterus, which differentiates into three histological structures in the postnatal period: the endometrium, myometrium, and perimeter (Holst, 2019). The bitch's uterus develops continuously from birth to 180 days of life (Ramos et al., 2015), developing until the stage of the beginning of the estrous cycle. At each estrous cycle, the uterus prepares to harbor the conceptus through the action of ovarian hormones (Holst, 2019;Vermeirsch et al., 2000), which estrogen stimulates intense mitosis of endometrial cells in the proliferative phase, and progesterone stimulates hypertrophy and hyperplasia of them in the secretory phase (Aplin et al., 2008).
Thus, substances synthesized and secreted by endometrial cells are essential for pregnancy and embryo survival (Holst, 2019), however, molecular changes in the glands and endometrial epithelium alter the composition of uterine secretions (Gao et al., 2009).
In this way, we aimed to evaluate and measure the female uterus by histomorphometry and describe the protein profile in all phases of the estrous cycle.

Research
This work is an experimental study of a quantitative nature (Pereira et al., 2018) for the purpose of evaluating and measuring the healthy uterus and respective protein profile during the estrous cycle of bitches.

Ethical Permission
The study protocol and all experimental procedures were approved by the Ethics Committee in Animal Experimentation (Comitê de Ética para Uso Animal -CEUA, State University of Ceará, Fortaleza, CE, Brazil), which protocol number is 5999508/2015.

Experimental Animals and Procedures
For the experimental study, 18 healthy and pubescent bitches, weighing between 10 kg and 45 kg (body score: 3-4), were used, and the majority of them were mongrel. All had a history of at least one previous pregnancy, were fed commercial granulated feed, and belonged to private owners. For the study, the estrous cycle was determined, followed by ovariosalpingohysterectomy (OSH) elective for later collection of samples for histomorphological and proteomic analysis.

Histomorphometric Analysis
For histomorphometric analysis, the collected uteri were washed with distilled water to remove excess adipose tissue and dissected to obtain fragments of uterine horns (0.5 cm in length from the region close to the bifurcation). The samples were immersed in a phosphate-formaldehyde buffer for 24 hours at room temperature, and processed for routine histological techniques (dehydration, clearing, and inclusion in paraffin). Paraffin blocks were cut (0.5 μm thick), stretched on glass slides, and placed in an oven, with subsequent staining in Hematoxylin-Eosin and fixation in 70% alcohol.
The total thickness of the uterine wall, endometrium, and myometrium was measured (µm) in three different fields for each histological sample.
Each sample was added with a rehydration solution (7 M urea, 2 M thiourea, 65 mM dithiothreitol, 0.5% free ampholytes, 0.5% CHAPS, and bromophenol blue), satisfactory for 250 μL. This solution was added to the hydration tray channels and incubated with 13 cm strips with a linear immobilized pH gradient of 3-10 (IPGs; GE Lifesciences, USA) for 18 hours.
The isoelectric focusing was performed on an ETTAN TM IPGphorII TM equipment (GE Lifesciences), with the following programming: 100 V, 250 V, 500 V, 8000 V (12750 Volts hour (Vh) and 8000 V (40000 Vh). The strips were kept for 15 minutes under gentle agitation in equilibrium buffer I (6 M urea, 50 mM Tris-HCl pH 8.8, 29.3% glycerol, 2% SDS, 1% dithiothreitol) and rebalanced for another 15 minutes in another substance similar to equilibrium buffer I, however, containing 2.5% iodoacetamide instead of dithiothreitol.
Afterward, the proteins were separated on 15% SDS-PAGE polyacrylamide gel (SE 600 Ruby -Amersham Biosciences, USA) with a current of 25 mA per gel and a maximum voltage of 500 V (Hoefer SE 600; GE LifeSciences). A molecular marker with the following molecular weights was added: 12 kDa, 17 kDa, 24 kDa, 31 kDa, 38 kDa, 52 kDa, 76 kDa, 102 kDa, 150 kDa, and 225 kDa (Amersham ECL Rainbow Molecular Weight Markers; GE LifeSciences, USA). The proteins were visualized using the colloidal Coomassie method and scanned for analysis by the PDquest software.

Statistical analysis
Data of uterine measurements and number of spots were analyzed using a one-way ANOVA and compared with the Student't-test between the stages. A P value <0.05 was considered for statistical significance.

Results
In all phases of the normal estrous cycle, the endometrium presented irregular longitudinal folds, with the surface covered by a cubic or simple cylindrical epithelium, and a lamina propria of connective tissue and the endometrial glands (EG) were of the simple tubular type constituted by simple cubic epithelium or low cylindrical.
In the proliferative phases of proestrus ( Figure 1A) and estrus ( Figure 1B) in the histomorphometric analysis, the EG presented rectilinear morphology with a narrow flame and a slight depth in the endometrial tissue. In the secretory phase of diestrus ( Figure 1C), these EG increased in density and size, presenting a tortuous morphology with dilation of the flame to accommodate the endometrial secretion.
In the anestrus ( Figure 1D), both endometrial and EG extracts were reduced. In the myometrium, two thick smooth muscle layers were observedcircular internal and external longitudinalin all phases. However, the vascular extract showed large diameter vessels in the proestrus ( Figure 1A) and estrus ( Figure 1B), and of smaller caliber in the diestrus ( Figure 1C) and anestrus ( Figure 1D). In turn, the perimetry was visualized by covering the uterine wall externally with loose connective tissue.

Source: Authors
In the proteomic analysis, the canine uterus presented a protein profile of goo reproducibility per phase (range of pI: 3.5-9.0; PM: 24-150 KDa), with possible isoforms. During estrus, the largest number of spots was identified, followed by proestrus, diestrus, and anestrus (Table 1), and eleven spots were present in all phases evaluated. The number of sports in each estrous cycle is shown in Figure 1.

Discussion
Although histology is a widespread and accessible technique, the few uterine histomorphometric studies in bitches have addressed the phases of metestrus and anestrus (Galabova et al., 2003) or hormonal treatment conditions (Salinas, Miglino, & Del Sol, 2017), as well as in females submitted to elective castration (Monteiro et al., 2012;Camargo et al., 2019).
Uterine changes are influenced by estrogen and progesterone (Holst, 2019;Vermeirsch et al., 2000), and we demonstrated the histomorphometry of all phases for the first time in the canine species.
The thickness of the uterine wall and endometrium was reduced in the anestrus, however, they increased significantly and in increasing order from the proestrus, estrus to the diestrus, with myometrium also thick in this phase (Table 1). This change resulted from the growth of the mucosa, the accumulation of secretion and edema in the stroma, which occur in the diestrus (Holst, 2019).
In the proteomic study, the largest number of spots during estrus in our findings (Table 1) corresponded to the high mucus synthesis by EG, which is rich in mucin, ions, water, enzymes, peptides, antimicrobial compounds, and immunoglobulins (Agostinis et al., 2019), allowing the transit of sperm and protecting the uterus of ascending infections.
Despite the diestrus phase showed a significant increase in the uterine wall, morphological changes in the EG of endometrium and myometrium with increased diameter and size, and tortuous appearance, we observed that it was in the estrus that the greatest number of identified proteins occurred. This finding is possibly related to the participation of these proteins in preparing the uterus for the reception of gametes during fertilization. However, in bovine (Faulkner et al., 2013) and swine (Lee et al., 2013) species, the secretory or diestrus phase, under the action of progesterone, is reported as the phase with the highest number of proteins, differently from the canine species.
Although molecular dynamics studies are known in equine (Swegen et al., 2017), bovine (Faulkner et al., 2013), and swine (Lee et al., 2013), few studies have addressed the phases of the canine estrous cycle. It is essential to understand the proteomic profile in the proliferative phases, in which the uterus is adapted to receive sperm and, during the secretory phase, to receive the embryo. Because uterine proteomic studies in female dogs are restricted to the diestrus during the implantation period (Praderio et al., 2019), estrus (Fahiminiya et al., 2010), or proestrus (Reynaud et al., 2015), our study becomes be innovative and promising because it presents all phases of the estrous cycle.

Conclusion
In conclusion, we showed uterine histomorphometric and proteomic changes in all phases of the estrous cycle of healthy bitches, with greater thickness in uterine structures in diestrus, but greater number of spots in estrus, probably due to uterine preparation by proteins in estrus for fertilization. These findings are promising for prospects the identification of proteins responsible for biological processes during the estrous cycle. In this sense, studies using mass spectrometry can qualitatively identify the predominant protein groups in each phase of the cycle.