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Neutrophil infiltration into the porcine endometrium is thought to be a specific feature during the follicular phase of the estrous cycle. To specify the localization and distribution of neutrophil granulocytes at different stages of the estrous cycle, porcine uterine samples were evaluated by immunohistochemical methods using anti-bovine lactoferrin (LF) antibody. Additionally, blood samples were collected from 30 pigs at different stages of the estrous cycle with a special focus on peri-estrous phase. Manual 100-cell differential counts were performed on routinely stained blood smears and evaluated statistically. Finally, the expression of granulocyte-colony-stimulating factor (G-CSF) and heat shock protein 27 (HSP 27), which are known to influence activation of the neutrophilic granulocytic lineage, was analyzed in porcine uteri using immunohistochemistry. Results show that LF is expressed regularly in the cytoplasm of neutrophil granulocytes. An increasing infiltration of subepithelial neutrophils was detected in the follicular phase. The highest number of intra- and subepithelial LF-positive cells was found on d 2 of the estrous cycle. Maximum level was followed by a strong decrease on d 3. Blood analysis revealed that the percentage of neutrophil granulocytes was significantly lower at d 2 (26.2+/-11.1%) than d 1 (42.1+/-11%) of the estrous cycle. HSP 27 staining was predominantly localized to luminal epithelium (LE) and glandular epithelium (GE) depending on stage of the estrous cycle. Strong immunostaining of HSP 27 is only found in LE during estrus. At d 2 of the estrous cycle, HSP 27 immunoreactivity in LE and superficial GE is reduced but moderate staining is found in deep GE. G-CSF immunostaining is uniformly not detected in endometrial cells of cyclic pigs. In conclusion, there is a clinically relevant relationship between neutrophil count in the blood and neutrophil infiltrate in the endometrium of the pig during the estrous cycle. This association may reflect the possibility of translocation of neutrophils from the blood to the endometrium up to d 2 of the estrous cycle. Additionally, HSP 27 could be a good candidate involved in migration and/or function of neutrophils within the porcine endometrium., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Previously it was shown that growth hormone (GH) and its receptor (GH-R) are involved in growth-promoting events during early embryonic development. However, it is still unknown if GH-induced GH-R signalling may support other functions within the oviduct. The purpose of our study was to analyse GH-R expression and localization in the porcine oviduct during different stages of the oestrus cycle and pregnancy (days 2-3 post inseminationem to days 65-71). As shown by reverse transcription polymerase chain reaction (RT-PCR), GH-R is expressed in the porcine oviduct during all stages of the oestrus cycle and pregnancy, respectively. Additionally, GH-R mRNA was detected in porcine conceptuses collected at day 18 of pregnancy. Using immunohistochemistry, GH-R was exclusively localized to the epithelium of the porcine oviduct throughout all segments examined. Localization of GH-R was mainly observed in the cytoplasm of ciliated epithelial cells. Generally, the number of GH-R-positive cells was elevated in the periovulatory phase of the oestrus cycle. Except for the isthmic epithelium, staining intensity of GH-R-positive cells was highest at oestrus and markedly reduced at met- and dioestrous stages. In infundibular and ampullar segments, percentage of GH-R-positive cells was significantly higher at days 2-3 post inseminationem compared to days 65-71 of pregnancy. Furthermore, in porcine conceptuses on day 18 of pregnancy GH-R protein expression was almost exclusively localized to trophectoderm. Our data suggest that GH-R mRNA and protein expression in the porcine oviduct throughout the oestrus cycle and pregnancy may suggest other activities of GH not described previously. Specifically, autocrine or paracrine GH-induced GH-R signalling may be linked to ciliated cell homeostasis of the porcine oviduct. Additionally, our results indicate that GH-R expression in the pig trophectoderm may be responsible for trophoblastic elongation.

Transforming growth factor-beta (TGF-beta) proteins are growth factors that have been shown to be involved in regulation of ovarian follicular development. Ovarian expression, activity and functional significance of TGF-beta1 and TGF-beta2 isoforms were extensively studied in most species. However, little is known about the biological role of TGF-beta3 previously shown to be expressed independently of the other two isoforms. Therefore, expression of TGF-beta3 mRNA and protein was evaluated by RT-PCR and immunohistochemistry, respectively, in porcine ovaries collected during different phases of the oestrus cycle. Results of RT-PCR analysis showed that TGF-beta3 mRNA is expressed throughout the oestrus cycle. The level of TGF-beta3 mRNA expression was found to be higher at metoestrus and dioestrus. Weak TGF-beta3 immunoreactivity was present in follicular epithelial cells and oocytes of preantral follicles in all stages examined. TGF-beta3 protein expression was exclusively present in theca interna cell layer of antral follicles, and was particularly prominent in large antral follicles. Immediately after ovulation, almost all theca cells outside of the granulosa cell layer were intensively stained with anti-TGF-beta3. Immunostaining of TGF-beta3 in theca lutein cells rapidly decreased during corpus luteum development. It is suggested that TGF-beta3 may play an important role in modulating theca cell function of pre- and postovulatory follicles of the pig.

Certain female reproductive tissues are known to express the non-neuronal cholinergic system. Using different experimental approaches, we tested the hypothesis that acetylcholine (ACh) in the porcine oviduct may also be derived from non-neuronal structures. Immunohistochemistry was performed to detect acetylcholine synthesizing enzyme choline acetyltransferase (ChAT) in different segments of the oviduct of cyclic and pregnant sows. Immunohistochemical experiments revealed strong immunoexpression of ChAT in the entire oviductal epithelium at metoestrus. Thereby, a particular pronounced staining was found in the supranuclear region of almost all epithelial cells. Immunostaining of ChAT decreased markedly during dioestrus and prooestrus stages, respectively. At prooestrus, ChAT immunoreactivity was confined to ciliated cells. Furthermore, we found elevated level of staining intensity of ChAT in the pregnant oviduct at day 13. Using the same ChAT antibody for Western blot analyses, we detected immunoreactive bands of MW 69,000 and 46,000 mainly in ampulla, while MW 58,000 and 30,000 forms were present mainly in infundibulum and isthmus. Furthermore ACh was detected by HPLC and fluorimetric methods in oviductal epithelium. In conclusion, we show expression of ChAT in oviductal epithelial cells at different stages of the oestrus cycle and pregnancy, indicating that these cells can synthesize ACh in a cycle-dependent manner. These results suggest as yet unexplored roles of epithelial ACh in the oviduct.

Although porcine uterus is known to contain active and inactive forms of epidermal growth factor receptor (EGF-R), the latter consist of the extracellular domain only; it is currently unknown whether different EGF-R isoforms are expressed in the porcine oviduct during estrous cycle. Therefore, we used two different monoclonal antibodies, one against the extracellular and the other against the cytoplasmic domain of the EGF-R, to investigate cycle-dependent and cell-type-specific expression of full-size and truncated receptor forms. At metestrus, the majority of epithelial cells of the oviduct were strongly immunopositive for both antibodies, indicating the presence of the full-size receptor. In diestrous and proestrous stages, we found a low level of cytoplasmic but no extracellular EGF-R staining in epithelial cells. While the staining intensity of cytoplasmic domain of the EGF-R was only faint or absent in muscular tissue and blood vessels throughout the estrous cycle, extracellular domain of the EGF-R exhibited a strong immunostaining of smooth muscle cells and vascular smooth muscle cells, especially in diestrous and proestrous stages. There was no significant difference between the oviductal ampulla and isthmus in either the intensity or the pattern of both cytoplasmic and extracellular EGF-R immunostaining. We conclude that the restricted presence of the functional full-size receptor to the epithelial layer indicates a specific role during early embryonic development, whereas truncated EGF-R forms may potentially regulate contractions and blood flow in the oviduct.

The luminal epithelium of the porcine oviduct is composed of ciliated cells and secretory cells, but it is assumed for several species that under the control of steroid hormones secretory cells are able to be transformed into ciliated cells. In order to better understand such physiological changes during the different stages of the oestrous cycle, we evaluated epithelial cell proliferation together with oestrogen receptor alpha (ERalpha) expression of porcine ampullary oviducts. To identify the immunophenotype of proliferating cells, double immunohistochemistry was performed using anti-chromogranin A antibody (anti-CgA) as the second primary antibody. Anti-CgA, recently shown to be an immunocytochemical marker of ciliated cells of the cow, also labelled specifically the luminal surface of ciliated cells of the pig. Double labelling of sections with the monoclonal antibody MIB-1 against the proliferation-associated nuclear epitope Ki-67 and anti-CgA clearly demonstrates that MIB-1 was selectively localised in the nuclei of secretory cells. Proliferative activity was not observed in CgA-positive ciliated cells in all examined stages of the oestrous cycle. The percentage of Ki-67-positive epithelial cells was higher at pro-oestrus, compared with the other stages of the oestrous cycle. Furthermore, ERalpha immunoreactivity was exclusively detected in the nuclei of the epithelial cells, which were negative for CgA. We conclude, therefore, that oestrogen may induce the initial proliferation of secretory cells and promote the differentiation into ciliated cells., (Copyright 2004 Springer-Verlag)

The inositol 1,4,5-trisphosphate receptor type 2 (IP(3)R-2) is an intracellular Ca(2+) release channel responsible for mobilizing of Ca(2+) from intracellular storage sites and plays a key role in biological processes such as fertilization, cell differentiation, and growth. To study the cell-type-specific IP(3)R-2 expression in porcine ovaries during different phases of the oestrous cycle, we used reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. A total of 24 ovaries from gilts were collected in early luteal, mid-luteal, and follicular phases of the oestrous cycle. When amplified with the primers common to IP(3)R-2, a RT-PCR product of the expected size (approximately 388 bp) was clearly detected in the follicular and early luteal phase of the oestrous cycle, but there was no detectable PCR product in the corpus luteum of the mid-luteal phase of the oestrous cycle. Immunohistochemical studies showed that IP(3)R-2 protein is expressed in granulosa cells and theca cells of growing follicles. IP(3)R-2 immunostaining was first detected during the late pre-antral stage in granulosa and theca cells. Granulosa cell IP(3)R-2 expression increased from the pre-antral to mid-antral stage, but was strongly reduced in pre-ovulatory follicles. In the developing corpus luteum, intense IP(3)R-2 immunostaining was also present in luteal cells, but undetectable in mid-luteal corpora lutea. Furthermore, oocytes, atretic follicles and regressed corpora lutea were negative for IP(3)R-2. Our results indicate that the expression of the IP(3)R-2 protein was downregulated in terminally differentiated granulosa cells of pre-ovulatory follicles when granulosa cells lose follicle-stimulating hormone responsiveness. Therefore, we strongly suggest that IP(3)R-2 may play an important role in the initiation and propagation of intracellular Ca(2+) signals during follicular development of the pig.

The objectives of the study were to monitor the detailed pattern for mRNA expression (RT-PCR and RPA) of IGFs, IGFR-1, IGFBPs, GHR and localization of protein (immunohistochemistry) for IGF-1 and IGFR-1 in bovine follicle classes during final maturation and different corpus luteum (CL) stages during estrous cycle and during pregnancy. A relative high expression of IGF-1 in theca interna (TI) was observed before selection (E<0.5ng/mL). In GC, mRNA expression increased after selection. In contrast, IGF-2 was mainly expressed in the TI. The IGFR-1 mRNA was present in the TI and GC with increasing levels during final development. The expression results were confirmed by localization of IGF-1 and IGFR-1 proteins in GC and TI. There is clear evidence for the local expression of IGFBPs in TI and GC compartment with clear regulatory differences. In CL, the highest mRNA expression of IGF-1, IGF-2 and IGFR-1 was observed during early luteal phase, followed by a decrease, and then by a tendency of an increase during the mid and late luteal phases of the cyclic CL. This level remained low during pregnancy. Intense immunostaining for IGFR-1 in CL was observed mainly in large luteal cells. Evidence for a mRNA for all six IGFBPs were obtained with distinct differences for BP-3, -4 and -5. In conclusion, this comprehensive study gives clear evidence for an important role of the IGFs and IGFBPs in bovine follicular development and CL function. The relative amounts of IGFBPs may ultimately determine ovarian IGF action.