PGF2α inhibits VEGF Expression in the Corpus Luteum in the Mid but not Early Luteal Phase of the Sheep Estrous Cycle

Vascular Endothelial Growth Factor (VEGF) is important for blood vessel development in a variety of tissues including an ovarian structure, the corpus luteum (CL). The CL is a transient endocrine gland that secretes progesterone, which prepares the uterus for pregnancy. In the absence of pregnancy, the corpus luteum regresses in response to endogenous prostaglandin (PG) F2α. Deficiencies in luteal function during early pregnancy may result in embryonic loss. Understanding of the mechanisms of luteal function could aid in the development of new methods to regulate fertility. Corpora lutea are susceptible to the luteolytic effects of PGF2αafter day 4 of the luteal phase; prior to that time the CL does not regress in response to treatment with PGF2α. The mechanisms responsible for the acquisition of responsiveness to PGF2α and the specific intracellular mediators of PGF2α induced luteolysis are unclear. The objectives of this research were to investigate if exogenous PGF2α affects VEGF mRNA expression in sheep corpora lutea and to determine if the effect is dependent upon whether the corpus luteum has not (early luteal phase CL) or has (mid-luteal phase CL) acquired luteolytic capacity. Mature female sheep (n=18) were randomly separated into two groups (early luteal phase, day 3, and mid-luteal phase, day 10). Each group was divided into two treatment groups, PGF2αtreated and saline-treated (control). From each animal, one CL was removed at 2h and a second CL was removed 24h after treatment. Total RNA was isolated from each CL, purified, and verified for integrity. Real-time Polymerase Chain Reaction (PCR) was used for relative quantification of VEGF mRNA. The real-time methods were optimized and validated using two endogenous reference genes; β-actin and L-19. The quantity of VEGF mRNA present in each sample was standardized using the reference genes and was expressed as fold change from the mean value for the control CL collected on day 3 at 2h. Luteal progesterone concentrations were determined using radioimmunoassay. Data were analyzed using analysis of variance for a split plot design with sheep as the main plot and CL as the sub-plot. Quantities of VEGF mRNA were similar among CL collected during the early versus mid-luteal phase in the control animals (p>0.05). PGF2α had no effect on VEGF mRNA levels in early luteal phase CL collected at 2h or 24h after injection (p>0.05). In contrast, on day 10 of the estrous cycle, PGF2α treatment resulted in a marked reduction of VEGF expression within 24h after injection (p=0.002); it is unclear if VEGF expression was changed during the first 2h after PGF2α administration at this stage of the cycle. There was no effect of PGF2α on luteal concentrations of progesterone during the early luteal phase (p>0.05). This is consistent with the expected lack of response at this stage. In contrast, PGF2α caused a decrease (p=0.03) in luteal progesterone in mid-luteal phase CL, demonstrating that these corpora lutea have acquired luteolytic capacity. Since the mid-cycle CL has acquired luteolytic capacity, it will regress in response to PGF2α. As such, the decline in the message for VEGF was associated with luteal regression. We conclude that inhibition of VEGF and subsequent vascular destabilization may be an important component of the luteolytic cascade invoked by PGF2α.We would like to thank The OARDC Research Enhancement Competitive Grants Program, The Ohio State Undergraduate Enrichment Grants Program, and William C. Hauk Memorial Scholarship Fund for funding this research. We would also like to thank Ann Ottobre, Laura Arbogast, Amanda Roberts, and Janelle Gaddis for their intellectual and technical contributions, Dr. Horacio Cárdenas for donating the L-19 primers used for real-time PCR, and Dr. Normand St-Pierre for statistical consultation

The Ultrastructure of the Corpus Luteum of the Goat

The ultra structure of luteal cells and the proportion of the different cell types in the functional corpus luteum of the goat were studied using the electron microscope. Two luteal cell types, large and small, were present in the corpus luteum of this species. The large more rounded luteal cell possessed numerous mitochondria and electron dense membrane- bound granules, extensive agranular endoplasmic reticulum and granular endoplasmic reticulum which at times appeared as stacks of closely packed cisternae. Few lipid droplets were present iri the luteal cell cytoplasma while whorled agranular endoplasmic reticulum was absent. Interspersed amongst the large luteal cells were smaller luteal cells with tapering cytoplasmic processes. These cells differed from the large luteal cells in that they possessed fewer mitochondria and electron dense membrane- bound granules. Occasional nuclei of the small luteal cells contained cytoplasmic inclusion bodies

Inhibition of delta-like ligand 4 induces luteal hypervascularization followed by functional and structural luteolysis in the primate ovary

Using specific inhibitors established that angiogenesis in the ovarian follicle and corpus luteum is driven by vascular endothelial growth factor. Recently, it has been demonstrated that the Notch ligand, delta-like ligand 4 (Dll4) negatively regulates vascular endothelial growth factor-mediated vessel sprouting and branching. To investigate the role of Dll4 in regulation of the ovarian vasculature, we administered a neutralizing antibody to Dll4 to marmosets at the periovulatory period. The vasculature was examined on luteal d 3 or d 10: angiogenesis was determined by incorporation of bromodeoxyuridine, staining for CD31 and cell death by staining for activated caspase-3. Ovulatory progesterone rises were monitored to determine effects of treatment on luteal function and time to recover normal cycles in a separate group of animals. Additionally, animals were treated in the follicular or midluteal phase to determine effects of Dll4 inhibition on follicular development and luteal function. Controls were treated with human IgG (Fc). Corpora lutea from marmosets treated during the periovulatory period exhibited increased angiogenesis and increased vascular density on luteal d 3, but plasma progesterone was significantly suppressed. By luteal d 10, corpora lutea in treated ovaries were significantly reduced in size, with involution of luteal cells, increased cell death, and suppressed plasma progesterone concentrations. In contrast, initiation of anti-Dll4 treatment during the midluteal phase produced only a slight suppression of progesterone for the remainder of the cycle. Moreover, Dll4 inhibition had no appreciable effect on follicular development. These results show that Dll4 has a specific and critical role in the development of the normal luteal vasculature

Immunolocalization of angiogenic growth factors in the ovine uterus during the oestrus cycle and in response to Steroids

The vascular changes associated with endometrial maturation in preparation for embryo implantation depend on numerous growth factors, known to regulate key angiogenic events. Primarily, the vascular endothelial growth factor (VEGF) family promotes vascular growth, whilst the angiopoietins maintain blood vessel integrity. The aim was to analyse protein levels of VEGFA ligand and receptors, Angiopoietin‐1 and 2 (ANG1/2) and endothelial cell receptor tyrosine kinase (TIE‐2) in the ovine endometrium in the follicular and luteal phases of the oestrus cycle and in response to ovarian steroids. VEGFA and its receptors were localized in both vascular cells and non‐vascular epithelium (glandular and luminal epithelium) and stroma cells. VEGFA and VEGFR2 proteins were elevated in vascular cells in follicular phase endometrium, compared to luteal phase, most significantly in response to oestradiol. VEGFR1 was expressed by epithelial cells and endothelial cells and was stimulated in response to oestradiol. In contrast, Ang‐1 and Ang‐2 proteins were elevated in luteal phase endometrium compared to follicular phase, and in response to progesterone, evident in vascular smooth muscle cells and glands which surround TIE‐2‐expressing blood vessels. Our findings indicate that VEGFA is stimulated by oestradiol, most predominantly in follicular phase endometrium, and Ang‐1 and 2 are stimulated by progesterone and were increased during the luteal phase of the oestrus cycle, during the time of vascular maturation

Prostaglandins and utero-ovarian relationships in the sheep

Previous work on the control of luteal function in the oestrous cycle of the sheep is assessed and discussed. The experimental work produced the following conclusions:-1. No difference was found between PGF₂α and PGE₂ content and synthesizing ability of the uterine caruncular and non-caruncular endometrial tissue; but the uterine myometrial tissue contained significantly less and produced less PGF₂α than did the endometrial tissues.2. A significantly higher PGF₂α content was found in the second half of oestrous cycle than in the first half3. No significant changes was found either in the content or synthesizing ability of PGE₂ and 6-oxo-PGF₁αof the different uterine tissue indicating that these compounds are probably not involved in luteolysis.4. Plasma concentrations of PGF₂α were episodic in nature and showed an increase at about day 12 and 13 of the oestrous cycle. The largest release of PGF₂α occurred on the day after progesterone secretion by the CL had ceased. These findings appear to support the involvement of PGF₂α in luteolysis but also suggest that a major role for PGF₂α at the end of the cycle may be to complete luteolysis and to prevent any functional recovery of the CL.5. A significant relationship was found between the ability of endometrial tissue to synthesis PGF₂α and PGE₂ and between the endometrial content of PGF₂α and PGE₂. This suggests that either PGE₂ production is a by-product of PGF₂α production or that it is the availability of a common pre cursor that controls the synthesis of the two prostaglandins. However the availability of arachidonic acid was not a limiting factor.6. It was found that the endometrial synthesizing ability for PGF2a was significantly increased 3 and 2 days before the onset of oestrus in sheep with an ovary adjacent to the uterine tissue sampled but not in those animals with the adjacent ovary re¬ moved. This indicates that the presence of an ovary adjacent to the uterine horn is necessary for the normal manifistation of the PGF₂α synthesizing ability of the endometrium and suggests that the ovary exerts a local influence over endometrial PGF2a synthesizing ability in the adjacent uterine tissue.7. No relationships was found between plasma concentrations of PGF₂α and its endometrial content and synthesizing ability, thus implying that the release of PGF₂α is under independent control.8. A significant relationship was found between the concentrations of PGF₂α and progesterone in the uterine venous blood. This was most demonstrable when PGF₂α levels were compared with the progesterone level half-hour previously. This suggests that increased PGF₂α occurs in response to an increase in progesterone secretion and PGF₂α may thus play an important role in retaining the progesterone secretion at optimum during the luteal phase of oestrous cycle.9. Contrary to expectations, the anastomosis of the utero-ovarian vein to the anterior mammary vein but leaving the ovary in situ was found not to interrupt the normal oestrous cycle. This suggests the involvement of another route in the transfer of PGF2a to the adjacent ovary in addition to therormal route through the counter-current mechanism of transfer from the uteroovarian vein to the ovarian artery.10. Significantly higher concentrations of PGF₂α were found in the oviducal vein and the ovarian vein as well as in the uterine vein when compared to peripheral levels. Thus the alternative route is probably via the oviducal vein and then by transfer from the ovarian vein to ovarian artery in the ovarian pedicle.11. Daily injection of progesterone to sheep was found to lead to accumulation of large amount of fluid rich in PGF₂α in the uterine lumen. Concentrations of PGF₂α higher than peripheral were also found in the uterine venous blood of these animals. These findings show that a high concentration of progesterone can cause PGF₂α release

Aspects of luteal function in pigs and sheep

Both the luteotrophic effects of estrogen and the relative lack of luteolytic effects of PGF[subscript]2[alpha] in cycling pigs might be interpreted to suggest that luteal function in this species differs markedly from that in other domestic animals. These aspects of porcine luteal function were studied to establish the basis for this premise. Additionally, the cellular effects of PGF[subscript]2[alpha] and the activation of protein kinase C on luteal cells was investigated using dispersed ovine luteal preparations. Dispersion of ovine CL results in luteal cell preparations suitable for cell culture experiments not obtainable by similar dispersion techniques;Experiment I. Estradiol-17[beta] containing implants were placed unilaterally inside 3 individual CL of one ovary on day 11 of the estrous cycle to evaluate the direct luteal effects of this hormone in pigs following recovery of ovaries on day 19. At the highest dose of estradiol, but not in other groups, CL were maintained bilaterally with an additional significant increase in luteal weight and progesterone content in those CL receiving estradiol implants over all others;Experiment II. Estrogen implants were placed unilaterally inside all CL on an ovary on day 11 of the cycle to evaluate the functional effects of estrogen exposure for 8 days on progesterone and PGF[subscript]2[alpha] concentrations in utero-ovarian venous (UOV) blood. Estrogen induced uniform, bilateral luteal maintenance, and markedly suppressed UOV PGF[subscript]2[alpha] concentration. Luteal function appeared to decline to day 15 then recovered in estrogen-treated gilts to preinjection levels on day 19 as indicated by UOV progesterone;Experiment III. An ovary was removed from gilts on day 8 of the estrous cycle, prior to PGF[subscript]2[alpha] or vehicle administration on day 9, and the final ovary was recovered on day 12. Progesterone was monitored in systemic blood and luteal weight and content of progesterone, protein and DNA were determined in luteal tissue before and after PGF[subscript]2[alpha] exposure;Experiment IV. The effects and interaction between PGF[subscript]2[alpha], phorbol ester and calcium ionophore were investigated in day 9 dispersed oveine luteal cells by monitoring progesterone in culture medium. Phorbol ester, calcium ionophore and PGF[subscript]2[alpha] were all inhibitory at higher concentrations but none were additive in their effects. (Abstract shortened with permission of author.

Observations on the morphology and histochemistry of the oviducts, uterus and placenta of the sheep

As has been mentioned in the Introduction, the literature on the reproductive tract of sheep is voluminous. The oestrous cycle and the cyclic changes in the reproduc¬ tive tract of the ewe have been studied by many investigators for different purposes. The study of the placenta of sheep also received much attention due to the unique position of the cotyledonazy placenta in the classification and evolution of the mammalian placentae.In this study the morphological and histochemical changes in the oviducts, uterus and placenta of sheep, during the oestrous cycle and pregnancy, have been further investigated and new histological and histochemical observations have been added. Use of the electron micro¬ scope, freeze-drying and freeze-substitution techniques were of great advantage in the present study.The material was taken from thirty-nine non-pregnant and fifty-four pregnant sheep of Scottish breedsj fourteen specimens of pregnant animals were of known history and the rest, of unknown history, were collected from the Edinburgh abattoir.The changes in the oviducts during the oestrous cycle have been described in the text and have more or less confirmed the findings of previous authors. The tubal epithelium was found to be in a phase of growth during pro-oestrum and by the onset of oestrous to be in a phase of secretory activity, which gradually diminished towards the end of the cycle.There were no marked changes in the oviducts during pregnancy and the author could find no reference to the morphology and the fine structure of the oviducts of pregnant sheep. In the present study, the fine structure and changes in the tubal epithelium of the pregnant ewe have been described; the epithelium was found to be quiescent and many cells were degenerating during the greater part of the pregnancy period. Only towards the end of gestation did any activity appear in the epithelial cells. The ultrastructure of the tubal epithelium revealed that it consisted of ciliated and non-ciliated columnar cells in alternating groups. It also showed that the ciliated cells are non-secretory and possibly have an absorptive function since they possess microvilli.The histochemical investigation showed that the active tubal epithelial cells are rich in carbohydrateprotein complex substances. They also contain ribonucleic acid, lipids and acid phosphatase. Alkaline phosphatase was found only at the free borders of the cells and iron was also demonstrable in traces along the cell borders. The cyclical variation in the quantity and distribution of these substances within the cells has been described in the text and the secretions of the oviducts were considered to be mainly carbohydrate-protein complexes, i.e. glyco¬ proteins or mucoproteins. The significance of the other chemical substances found in the tubal cells and the lumen of the oviduct was discussed.The uterine changes during the oestrous cycle were described. It has been observed that the cyclical changes in the uterus follow closely those of the oviducts. The endometrium showed a period of growth preceded by vascularisation during pro-oestrum, a period of secretory activity during metoestrum and early dioestrum, and, finally, a period of regression and infiltration of leucocytes towards the end of the cycle. The present observations have largely agreed with the findings of previous authors. In the endometrial stroma melanocytes were encountered in some of the specimens collected and were considered to be of no physiological value. Other lymphocyte-like cells containing important chemical substances were encountered in the subepithelial layer and around the uterine glands; these have been discussed in relation to similar cells in the gravid uterus.The uterine and glandular secretions have been studied histochemically and found to be rich in a variety of important chemical substances. The cells of the uterine and glandular epithelium showed the presence of ribonucleic acid, lipids, inorganic iron and acid phos¬ phatase; alkaline phosphatase is active only at the free borders of the cells. The histoohemical reaction at the free border of the uterine epithelium and in the lumen of the uterus and the ducts of the glands showed the presence of a carbohydrate-protein complex substance. Accordingly, a possible relationship between the phosphatases present at the free borders of the cells and the mucoid substance found outside the cells was suggested and discussed.The study of the morphology and histochemistry of the gravid uterus and placenta of the ewe was a major part of the present investigation. The changes in the gravid uterus and the development of the placenta have been described in the text at different stages of pregnancy. The condition of the uterus before implantation of the blastocyst was found to be similar to that of early dioestrous, i.e. the endometrium was in the active phase. When the blastocyst came in contact with the endometrium, the uterine epithelium was destroyed at the sites of contact and before the end of the first month of gestation the blastocyst had increased enormously and the endometrium was denuded of its lining epithelium in most of the caruncular and intercaruncular areas.The destruction of the uterine epithelium and some of the endometrial stroma of the caruncles was considered to be due mainly to an ingrowth of dark staining binucleate cells of the trophoblast which migrated to the maternal side and possibly were aided by other chorionic cells. Thi3 destruction was believed to be caused by some process of digestion and absorption but not by true phagocytic action because the dark staining binucleate cells frequently seen nearest to or within the degenerating maternal tissues were apparently non-phagocytic.In the present study the formation of the foetal villi and the maternal crypts in the sheep's placentome have been described. It was found that the crypts developed before the primitive villi were formed. The histological evidence showed that the maternal tissues had degenerated in the form of tracks vertical to the surface of the caruncle, the degenerative tissues eventually disappeared and then folds of chorionic epithelium or primitive villi entered the evacuated ciypt3.The fine structure of the junctional zone of a fully differentiated placentome was examined under the electron microscope and it was found that the foetal and maternal lining cells possess apical microvilli which were interdigitating with each other except where debris and secretory granules had intervened. Intracytoplasmic inclusions were found in both foetal and maternal cell3j the former showed characteristic large vesicles near the microvilli while the latter showed characteristic electron dense granules.The histochemical reactions showed that glycogen granules were present in the walls of the foetal and maternal blood vessels. The columnar chorionic cells at the bases of the villi contained glycogen. They contained inorganic iron which is believed to be liberated by the phagocytosed red blood corpuscles. Lipid droplets were also found towards the bases of these cells. Acid and alkaline phosphatases, lipids and ribonucleoproteins were observed in greater quantities in the syncytial lining of the maternal crypts than in the chorionic epithelium.In the light of the present observations and the findings of other authors, the syncytial lining of the crypts being of foetal origin, the foetal-maternal relationship in the placentome of the ewe is believed to be a syndesmochorial one. The phosphatases and lipids in the binucleate cells and the syncytial lining were considered to be of vital importance in the transfusion of substances across the placental membrane.In the intercotyledonary areas of the sheep's placenta, the uterine epithelium was eroded in the places which were in contact with the blastocyst during early pregnancy excepting that around the glands and at the junctional zone between the caruncular and intercamncular areas and the tapering parts of the horns. The epithelium was believed to be destroyed by a similar process to that described in the caruncular areas. However, the uterine epithelium was restored in the intercotyledonary areas after the second month of pregnancy, and the foetal maternal relationship had eventually become an epitheliochorial one. The chorionic epithelium in apposition to the openings of the uterine glands wa3 thickened and invaginated giving rise to depressions known in the pig's placenta as "areolae". The uterine milk and debris were found in the spaces between the areolae and the openings of the glands.The uterine glands increased in length and complexity and they were of wide diameter and functional throughout pregnancy. The histochemical reactions showed that a copious flow of secretion was poured out by the glands. The epithelial and glandular cells contained ribonucleic acid, acid and alkaline phosphatase, lipids, and inorganic iron.lymphocyte-like cells containing intracytopla3iaic granules were observed within the uterine and glandular i epithelium of pregnant ewes. The granules were eosinophilic PAS positive, stainable by pyronim Y, i.e. containing ribonucleic acid, and showed alkaline phos¬ phatase activity. The similarity and relationship between these cells, the lymphocytes of the endometrial stroma and the plasma cells were discussed.Large round cells containing pigments, PAS positive material, phosphatases and ribonucleic acid, were observed in the endometrial stroma of pregnant ewes and these cells were considered to be macrophages of the endometrial connective tissue