Uterus In Pigs Research Articles

A Novel Tissue Explant Culture Model For Testing of Candidate Treatments for Inflammation of the Uterus in Pigs.

Abstract Background: Endometritis is an inflammatory condition of the uterus leading to complications with reproductive organs, miscarriage and infertility. The first-line treatment of endometritis in pigs are antibiotics, which do not always resolve inflammation and contribute to antibiotic resistance. There is an urgent need for the development of alternative treatments for endometritis. Here we propose and validate the first porcine tissue explant model for uterine inflammation and test naturally derived compounds for their effectiveness as anti-inflammatory therapies in porcine endometrial tissue. Methods: Uterine endometrial explants were harvested from uteri of sows sent to slaughter. Tissue biopsies were collected at follicular and luteal phases of the oestrous cycle. Biopsies were washed and these tissue ‘explants’ were cultured. Explants were treated at 24 hr with oxytocin (to stimulate PGF 2αproduction) or LPS (to mimic infection and induce inflammation. Explants were retained for RT-PCR analysis. Supernatants were harvested and concentrations of markers were measured by ELISA and Luminex. Results: The data generated thus far show a dose dependent response of IL-6 secretion to LPS stimulation, which demonstrates the validity of our model. Inflammation can be stimulated in an in vitrosetting to mimic porcine endometritis and we present data to show the change in cytokine levels in response to treatment with naturally derived compounds. Conclusions: This data shows that inflammation can be stimulated in an in vitrosetting to mimic porcine endometritis This model is a tool for testing of novel compounds and their potential anti-inflammatory effects on the porcine endometrium. Supported by an Innovate UK Grant (Project Reference: 98015) (https://gtr.ukri.org/projects?ref=98015)

Small RNA-seq analysis of extracellular vesicles from porcine uterine flushing fluids during peri-implantation

The extracellular vesicles (EVs) of uterine flushing fluids (UFs) mediate intrauterine communication between conceptus and uterus in pigs. The small RNAs of UFs-EVs are widely recognized as important factors that influence embryonic implantation. However, small RNAs expression profiles of porcine UFs-EVs during peri-implantation are still unknown. In this study, cup-shaped EVs of porcine UFs on days 10 (D10), 13 (D13) and 18 (D18) of pregnancy were isolated and characterized. The expression of small RNAs in these EVs was comprehensively profiled through sequencing. A total of 152 known microRNAs (miRNAs), 43 novel miRNAs, 6248 known Piwi-interacting RNAs (piRNAs) and 110 novel piRNAs were identified. Among these small RNAs, RT-qRCR results indicated that ssc-let-7f-5p, ssc-let-7i-5p and ssc-let-7g were differentially expressed during the three stages. Bioinformatics analysis showed that the miRNAs differentially expressed in the three comparisons (D10 vs D13, D13 vs D18 and D10 vs D18) were involved in important processes and pathways related to immunization, endometrial receptivity and embryo development, which play important roles in embryonic implantation. Our results reveal that EVs from porcine UFs contain various small RNAs with potentially vital effects on implantation. This research also provides resources for studies of miRNAs and piRNAs in the cross-talk between embryo and endometrium.

Histomorphological and Histochemical Study of the Uterus of the Adult Guinea Pigs (Cavica porcellus)

Objectives: This study was carried out to identify the histomorphological and histochemical aspects of the uterus in the adult guinea pig (Cavica porcellus). To conduct such project, fourteen adult guinea pigs at their diestrous period were bought from the local breeders directly. Methods: Animals were euthanized, dissected, and subsequently uteruses specimens were collected and fixed by 10% neutral buffered formalin and some specimens were fixed in Bouin’s solution for subsequent histochemical staining. Then after specimens were subjected to routine processes such as dehydration, clearing, embedding, and block preparation. Sections of 6 µm were prepared and stained with haematoxylin–eosin, Masson’s Trichrome, Alcian blue (pH 2.5), and Periodic acid shiff stains. Findings: Gross findings of the uteruses revealed duplex uterus in the guinea pigs. It possessed two separate uterine horns united caudally to form the body, suspended by mesometrium in the abdominal cavity. Caudally, body formed the cervix, which was short and projected into the vaginal cavity forming portio vaginalis uteri with one cervical orifice.Microscopic findings revealed abrupt changes in the wall of the isthmus into the uterine horn at the utero-tubal junction. The wall of the uterus showed endometrium, vascular layer, myometrium, and adventitia or serosa. Lining epithelium was simple columnar which was similarly lined the endometrial glands. Myometrium was consisted of inner circular and outer longitudinal bundles of smooth muscle fibers. In the body, the lining epithelium was replaced gradually by simple mucous columnar epithelium. In addition to that the mucosa was thrown into long folds leaving small lumen at the cervix called os cervix. Such changes were associated with endometrial glands gradual diminishing till the cervix. Histochemically, the non-ciliated columnar cells present in the body and cervix were stained positively with AB (pH 2.5) and PAS stains. The reaction with AB (pH 2.5) was more intense compared with the staining with PAS so that it indicated that mucin was more acidic than neutral in nature.Keywords: Guinea Pig, Uterus, Diestrous, Histochemistry

Spatial Transcriptomic and miRNA Analyses Revealed Genes Involved in the Mesometrial-Biased Implantation in Pigs.

Implantation failure is a major cause of early embryonic loss. Normally, the conceptus attachment is initiated at mesometrial side of the uterus and then spread to the anti-mesometrial side in pigs, however, the mechanisms that direct the mesometrial-biased attachment are largely unknown. In this study, the histological features of the entire uterine cross-section from gestational days 12 (pre-attachment stage) and 15 (post-attachment stage) were investigated and the differences in histological features between the mesometrial and anti-mesometrial side of the uterus were observed. Then, transcriptomic and miRNA analyses were performed on mesometrial and anti-mesometrial endometrium obtained from gestational days 12 and 15, respectively. Differentially expressed genes (DEGs) and miRNAs (DE-miRs) that were common to both or unique to either of the two anatomical locations of uterus were identified, respectively, indicating that differences in molecular response to the implanting conceptus exist between the two anatomical locations. In addition, we detected DEGs and DE-miRs between the two anatomical locations on the two gestational days, respectively. Of these DEGs, a number of genes, such as chemokine and T cell surface marker genes, were found to be significantly up-regulated mesometrially. Furthermore, we detected the interaction of CXCR4, CXCL11 and miR-9 using dual luciferase reporter assay. Taken together, this study revealed genes and pathways that might play the role of creating a receptive microenvironment at the mesometrial side, which is required to guide a proper positioning of conceptus in the uterus in pigs.

The Activity and Localization of 3β-hydroxysteroid Dehydrogenase/Δ<sup>5</sup>-Δ<sup>4</sup> Isomerase and Release of Androstenedione and Progesterone by Uterine Tissues During Early Pregnancy and the Estrous Cycle in Pigs

Steroid hormones are produced by the porcine uterus. We hypothesized that the uterus in pigs possesses active 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) responsible for progesterone and androstenedione production, that uterine steroids may supplement the amount of steroid hormones produced by embryos and corpus luteum and that these steroids are necessary for maintenance of pregnancy. In this study, we examined 1) endometrial and myometrial expression of 3β-HSD mRNA, 2) uterine 3β-HSD protein activity and 3) in vitro production of A4 and P4 by uterine slices harvested from pigs on days 10 to 11, 12 to 13 and 15 to 16 of pregnancy and the estrous cycle. The expression of 3β-HSD and the presence and activity of 3β-HSD protein were different in the endometrium and the myometrium during the examined periods of pregnancy and the estrous cycle. Production of A4 by the endometrium and myometrium was highest on days 12 to 13 of pregnancy and the estrous cycle. Endometrial secretion of P4 did not differ in the course of early pregnancy and on the respective days of the estrous cycle. The gravid myometrium was the highest source of P4 in pregnant pigs on days 12 to 13. The release of P4 by the cyclic myometrium rose during the examined days of the estrous cycle. The steroidogenic activity of the uterus, as described in this study, may support early pregnancy or the luteal phase of the estrous cycle in pigs.

Expression of inter-alpha-trypsin inhibitor heavy chains in endometrium of cyclic and pregnant gilts

Attachment of the placenta to the uterus in pigs involves extracellular interaction between the expanding trophoblastic membrane and the thick glycocalyx present on the uterine epithelial microvilli. Formation of complexes between members of inter-alpha-trypsin inhibitor family may function in the maintenance of the extracellular matrix. This study investigated the change in the inter-alpha-trypsin inhibitor heavy chains (ITIH1, ITIH2, ITIH3 and ITIH4) during the oestrous cycle and early pregnancy in pigs. Gene expression of ITIH1, ITIH2, ITIH3 and ITIH4 was detected in the endometrium of cyclic and pregnant gilts; however, gene expression of ITIH was not altered throughout the oestrous cycle or early pregnancy. Western blot analysis with an ITIH antiserum identified the possible linkage forms of ITIH with the serine protease inhibitor, bikunin. Pregnancy altered the release of the various inter-alpha-inhibitor forms from the endometrium during the period of trophoblastic attachment. The results from this study indicate that the inter-alpha-trypsin inhibitor family plays an important role in maintenance of the uterine surface glycocalyx during placental attachment in pigs.

Evidence that endogenous relaxin promotes growth of the vagina and uterus during pregnancy in gilts.

Recently, it was demonstrated that endogenous relaxin promotes growth of the vagina during the second half of pregnancy in rats and that administration of porcine relaxin promotes growth of the uterus in nonpregnant or early pregnant gilts. This study examined the effects of circulating relaxin on growth of both the vagina and uterus during the last two thirds of the 114-day gestation period in gilts. Furthermore, this study employed an in vitro immunohistochemical localization technique to determine whether the vagina and uterus in pigs have specific relaxin-binding sites. Three groups of pregnant gilts were used: sham-ovariectomized controls (group C; n = 8), ovariectomized progesterone-treated (group OP; n = 6), and ovariectomized progesterone- plus relaxin-treated (group OPR; n = 7). Gilts were either sham ovariectomized or ovariectomized on day 40 of gestation. Hormone replacement therapy with progesterone (group OP), progesterone plus relaxin (group OPR), or hormone vehicles (group C) began on day 38 (progesterone) or day 40 (relaxin) and continued until day 110. On day 110, the vagina and uterus were collected, and wet weight, dry weight, and percent hydration were determined. Small pieces (2-3 cm3) of the vagina and uterus from groups C and OP were frozen and cryosectioned for the immunohistochemical localization of relaxin-binding sites. Relaxin promoted growth of both the vagina and uterus. The wet weights of both the vagina and uterus in relaxin-deficient gilts (group OP) were lower (P < 0.05) than those in controls (group C), and relaxin replacement therapy (group OPR) restored the wet weights of both tissues to values that did not differ from those in controls. The mean dry weights and percent hydrations in the vagina and uterus did not differ among treatments. Immunohistochemical localization studies in the vagina and uterus demonstrated that specific and saturable binding of relaxin was localized in the same cell types of both tissues, namely epithelial cells (luminal in vagina, and both luminal and glandular in uterus), smooth muscle cells (both circular and longitudinal in vagina, and myometrial in uterus), and cells associated with blood vessels. In conclusion, this study provides evidence that circulating relaxin promotes growth of both the vagina and uterus during pregnancy in the pig. Furthermore, this study provides evidence that both the vagina and uterus contain specific and saturable relaxin-binding sites in epithelial cells, smooth muscle cells, and cells associated with blood vessels. We conclude that these cells probably initiate relaxin's effects on the vagina and uterus of the pregnant pig.

Evidence That Endogenous Relaxin Promotes Growth of the Vagina and Uterus during Pregnancy in Gilts

Recently, it was demonstrated that endogenous relaxin promotes growth of the vagina during the second half of pregnancy in rats and that administration of porcine relaxin promotes growth of the uterus in nonpregnant or early pregnant gilts. This study examined the effects of circulating relaxin on growth of both the vagina and uterus during the last two thirds of the 114-day gestation period in gilts. Furthermore, this study employed an in vitro immunohistochemical localization technique to determine whether the vagina and uterus in pigs have specific relaxin-binding sites. Three groups of pregnant gilts were used: sham-ovariectomized controls (group C; n = 8), ovariectomized progesterone-treated (group OP; n = 6), and ovariectomized progesterone- plus relaxin-treated (group OPR; n = 7). Gilts were either sham ovariectomized or ovariectomized on day 40 of gestation. Hormone replacement therapy with progesterone (group OP), progesterone plus relaxin (group OPR), or hormone vehicles (group C) began on day 38 (progesterone) or day 40 (relaxin) and continued until day 110. On day 110, the vagina and uterus were collected, and wet weight, dry weight, and percent hydration were determined. Small pieces (2–3 cm3) of the vagina and uterus from groups C and OP were frozen and cryosectioned for the immunohistochemical localization of relaxin-binding sites. Relaxin promoted growth of both the vagina and uterus. The wet weights of both the vagina and uterus in relaxin-deficient gilts (group OP) were lower (P < 0.05) than those in controls (group C), and relaxin replacement therapy (group OPR) restored the wet weights of both tissues to values that did not differ from those in controls. The mean dry weights and percent hydrations in the vagina and uterus did not differ among treatments. Immunohistochemical localization studies in the vagina and uterus demonstrated that specific and saturable binding of relaxin was localized in the same cell types of both tissues, namely epithelial cells (luminal in vagina, and both luminal and glandular in uterus), smooth muscle cells (both circular and longitudinal in vagina, and myometrial in uterus), and cells associated with blood vessels. In conclusion, this study provides evidence that circulating relaxin promotes growth of both the vagina and uterus during pregnancy in the pig. Furthermore, this study provides evidence that both the vagina and uterus contain specific and saturable relaxin-binding sites in epithelial cells, smooth muscle cells, and cells associated with blood vessels. We conclude that these cells probably initiate relaxin’s effects on the vagina and uterus of the pregnant pig.

Relationship of length of uterus in prepubertal pigs and number of corpora lutea and fetuses at 30 days of gestation

Relationships of the length of the uterus at one reproductive stage to the length at other stages and the effect on potential litter size were determined. In Experiment 1, the length of the uterus was measured in situ at 20, 60, or 100 days of age at laparotomy with 20 gilts in each of the three age groups. Forty days after the initial measurement, the uterus was again measured in situ, gilts were ovariectomized and hysterectomized, and associations among uterine measurements at the two different stages were determined. Correlations between uterine length in situ and between uterine length and weight 40 days later were all greater than 0.75 ( P < 0.001). The length of one uterine horn increased from 13.9 cm at 20 days of age to 36.7 cm at 140 days of age ( P < 0.01). In Experiment 2, 66 gilts were unilaterally hysterectomized and ovariectomized (UHOX) at 150 days of age and the ovary was weighed. Length of the one horn was measured in 36 gilts. At 10 days after first estrus, the length of the remaining uterine horn was measured at laparotomy and corpora lutea were counted in 53 gilts. In 15 of the 53 gilts the remaining uterine horn was removed to obtain uterine weights. At the second or third estrus, 38 gilts were mated and at Day 30 of gestation, 31 gilts were pregnant. The gilts were killed, and the length of the uterus measured and corpora lutea (CL) and fetuses were counted. The length of one uterine horn at 150 days of age was 70 cm with a range of 47–110 cm. At 10 days after first estrus, length had increased to a mean of 141 cm with a range of 86–194 cm and at 30 days of gestation the mean was 244 cm with a range of 186–311 cm. There were 12.2 CL at first estrus, which was not different from 12.4 CL at the second or third estrus. The mean number of fetuses in one horn at 30 days of gestation was 9.5 with 77% prenatal survival. Length of uterine horn at 150 days of age was correlated with uterine horn length ( r = 0.56, P < 0.001) at 10 days after first estrus and number of live fetuses ( r=0.39, P<0.05) at 30 days of gestation. At Day 10 after first estrus, uterine length was not correlated with the number of CL, whereas at Day 30 of gestation, the number of CL and uterine length were correlated with the number of live fetuses in those gilts with below the mean number of live fetuses, but not in those gilts with above the mean of live fetuses. The number of live fetuses ( r=0.66, P < 0.001) and fetal survival ( r=0.63; P < 0.001) were correlated with uterine horn length in pregnant UHOX gilts. Length of the prepubertal uterus gives an indication of postpubertal length and the potential litter size in pigs.

Secretion of riboflavin by the porcine uterus.

A study of 10 mature crossbred, ovariectomized gilts was conducted to determine whether riboflavin secretion by porcine uteri is regulated by estrogen and progesterone. A second experiment was performed to determine whether secretion of riboflavin occurs in pregnant animals in a temporal and quantitative pattern similar to that in nonpregnant animals. In the hormonal induction experiment, it was found that animals receiving daily injections of estrogen for 5 days followed by a daily injection of a combination of estrogen and progesterone were able to secrete riboflavin in quantities comparable to those secreted by intact animals. Additionally, it was found that the secretion of riboflavin appeared to be related to onsetn of treatment with progesterone in that riboflavin secretion increased in a time-related pattern after the beginning of progesterone treatment. In a second experiment, pregnant animals secreted riboflavin in their uterine secretions in quantities and in a time pattern similar to those found in nonpregnant animals. Nonpregnant gilts in the 8th day of an estrous cycle were used for comparison to the pregnant animals in this experiment, and the quantity of riboflavin uterine secretions in the nonpregnant animals was similar to that found on day 8 in the pregnant animals. Taken together, these two experiments indicate that riboflavin is secreted under hormonal regulation by the uterus in both pregnant and nonpregnant pigs at a precisely controlled time in the progestational phase.