Placental development during early pregnancy in sheep: effects of embryo origin on vascularization.

Utero-placental growth and vascular development are critical for pregnancy establishment that may be altered by various factors including assisted reproductive technologies (ART), nutrition, or others, leading to compromised pregnancy. We hypothesized that placental vascularization and expression of angiogenic factors are altered early in pregnancies after transfer of embryos created using selected ART methods. Pregnancies were achieved through natural mating (NAT), or transfer of embryos from NAT (NAT-ET), or IVF or in vitro activation (IVA). Placental tissues were collected on day 22 of pregnancy. In maternal caruncles (CAR), vascular cell proliferation was less (P<0.05) for IVA than other groups. Compared with NAT, density of blood vessels was less (P<0.05) for IVF and IVA in fetal membranes (FM) and for NAT-ET, IVF, and IVA in CAR. In FM, mRNA expression was decreased (P<0.01-0.08) in NAT-ET, IVF, and IVA compared with NAT for vascular endothelial growth factor (VEGF) and its receptor FLT1, placental growth factor (PGF), neuropilin 1 (NP1) and NP2, angiopoietin 1 (ANGPT1) and ANGPT2, endothelial nitric oxide synthase 3 (NOS3), hypoxia-inducible factor 1A (HIF1A), fibroblast growth factor 2 (FGF2), and its receptor FGFR2. In CAR, mRNA expression was decreased (P<0.01-0.05) in NAT-ET, IVF, and IVA compared with NAT for VEGF, FLT1, PGF, ANGPT1, and TEK. Decreased mRNA expression for 12 of 14 angiogenic factors across FM and CAR in NAT-ET, IVF, and IVA pregnancies was associated with reduced placental vascular development, which would lead to poor placental function and compromised fetal and placental growth and development.

Placental development during early pregnancy in sheep: cell proliferation, global methylation, and angiogenesis in the fetal placenta.

To characterize early fetal placental development, gravid uterine tissues were collected from pregnant ewes every other day from day 16 to 30 after mating. Determination of 1) cell proliferation was based on Ki67 protein immunodetection; 2) global methylation was based on 5-methyl-cytosine (5mC) expression and mRNA expression for DNA methyltransferases (DNMTs) 1, 3a, and 3b; and 3) vascular development was based on smooth muscle cell actin immunolocalization and on mRNA expression of several factors involved in the regulation of angiogenesis in fetal membranes (FMs). Throughout early pregnancy, the labeling index (proportion of proliferating cells) was very high (21%) and did not change. Expression of 5mC and mRNA for DNMT3b decreased, but mRNA for DNMT1 and 3a increased. Blood vessels were detected in FM on days 18-30 of pregnancy, and their number per tissue area did not change. The patterns of mRNA expression for placental growth factor, vascular endothelial growth factor, and their receptors FLT1 and KDR; angiopoietins 1 and 2 and their receptor TEK; endothelial nitric oxide synthase and the NO receptor GUCY13B; and hypoxia inducing factor 1 α changed in FM during early pregnancy. These data demonstrate high cellular proliferation rates, and changes in global methylation and mRNA expression of factors involved in the regulation of DNA methylation and angiogenesis in FM during early pregnancy. This description of cellular and molecular changes in FM during early pregnancy will provide the foundation for determining the basis of altered placental development in pregnancies compromised by environmental, genetic, or other factors.

Placental development during early pregnancy in sheep: vascular growth and expression of angiogenic factors in maternal placenta.

Placental vascular development (angiogenesis) is critical for placental function and thus for normal embryonic/fetal growth and development. Specific environmental factors or use of assisted reproductive techniques may result in poor placental angiogenesis, which may contribute to embryonic losses and/or fetal growth retardation. Uterine tissues were collected on days 14, 16, 18, 20, 22, 24, 26, 28, and 30 after mating and on day 10 after estrus (nonpregnant controls) to determine vascular development and expression of several factors involved in the regulation of angiogenesis in the endometrium. Compared with controls, several measurements of endometrial vascularity increased (P<0.001) including vascular labeling index (LI; proportion of proliferating cells), the tissue area occupied by capillaries, area per capillary (capillary size), total capillary circumference per unit of tissue area, and expression of factor VIII (marker of endothelial cells), but capillary number decreased (P<0.001). Compared with controls, mRNA for placental growth factor, vascular endothelial growth factor receptors, angiopoietins (ANGPT) 1 and 2, ANGPT receptor TEK, endothelial nitric oxide synthase, and hypoxia-inducible factor 1alpha increased (P<0.05) during early pregnancy. Vascular LI was positively correlated (P<0.05) with several measurements of vascularity and with mRNA expression of angiogenic factors. These data indicate that endometrial angiogenesis, manifested by increased vascularity and increased expression of several factors involved in the regulation of angiogenesis, is initiated very early in pregnancy. This more complete description of early placental angiogenesis may provide the foundation for determining whether placental vascular development is altered in compromised pregnancies.

Cellular proliferation and vascularization in ovine fetal ovaries: effects of undernutrition and selenium in maternal diet.

Sheep were fed a maintenance (M) diet with adequate (A) Se or high (H) Se concentration from 21 days before breeding to day 135 of pregnancy. From day 50 to day 135 of pregnancy (tissue collection day), a portion of the ewes from ASe and HSe groups were fed restricted (R; 60% of M) diet. Fetal ovarian sections were stained for: 1) the presence of proliferating cell nuclear antigen (a marker of proliferating cells) to determine the proportion of proliferating primordial follicles, or the labeling index (LI; percentage of proliferating cells) for primordial, primary, secondary and antral follicles, stromal tissues, and blood vessels; 2) factor VIII (a marker of endothelial cells) or 3) a presence of apoptotic cells/bodies. The number of proliferating primordial follicles and the LI of primordial follicles was decreased by R and/or HSe diets. The LI was similar for theca and granulosa cells, and for secondary or antral follicles, but was greater in secondary and antral than in primordial and primary follicles. R diet and/or Se affected the LI in all follicle types, in stromal tissues and blood vessels. A dense network of blood vessels was detected in the areas containing secondary to antral follicles, medulla, and hilus, but areas containing primordial follicles were poorly vascularized. The number of apoptotic cells was minimal. These results demonstrate that nutrient restriction and/or Se level in the maternal diet affected cellular proliferation in follicles, blood vessels, and stromal tissues in fetal ovaries. Thus, plane of nutrition and Se in the maternal diet may impact fetal ovarian development and function.

Morphology and function of cryopreserved whole ovine ovaries after heterotopic autotransplantation.

BACKGROUND: The objective of this study was to perform complex characterization of cryopreserved and then autotransplanted ovaries including determination of the ability to respond to in vivo follicle stimulating hormone (FSH)-treatment, fertilizability of retrieved oocytes, and morphology, vascularization, cellular proliferation and apoptosis in sheep. METHODS: Mature crossbred ewes were divided into two groups; an intact (control) group (n = 4), and autotransplanted group (n = 4) in which oophorectomy was performed laparoscopically and ovaries with intact vascular pedicles frozen, thawed and transplanted back into the same animal at a different site. Approximately five months after autotransplantation, estrus was synchronized, ewes were treated with FSH, and ovaries were collected. For all ovaries, number of visible follicles was determined, and collected cumulus oocyte complexes (COC) were matured and fertilized in vitro. Remaining ovarian tissues were fixed for evaluation of morphology, expression of factor VIII (marker of endothelial cells), vascular endothelial growth factor (VEGF; expressed by pericytes and smooth muscle cells), and smooth muscle cell actin (SMCA; marker of pericytes and smooth muscle cells), and cellular proliferation and apoptosis. Two fully functional ovaries were collected from each control ewe (total 8 ovaries). RESULTS: Out of eight autotransplanted ovaries, a total of two ovaries with developing follicles were found. Control ewes had 10.6 +/- 2.7 follicles/ovary, oocytes were in vitro fertilized and developed to the blastocyst stage. One autotransplanted ewe had 4 visible follicles from which 3 COC were collected, but none of them was fertilized. The morphology of autotransplanted and control ovaries was similar. In control and autotransplanted ovaries, primordial, primary, secondary, antral and preovulatory follicles were found along with fully functional vascularization which was manifested by expression of factor VIII, VEGF and SMCA. Proliferating cells were detected in follicles, and the rate of apoptosis was minimal in ovaries of control and autotransplanted ovaries. CONCLUSION: These data demonstrate successful autotransplantation of a portion of frozen/thawed ovaries manifested by restoration of selected ovarian function including in vitro maturation of collected oocytes, presence of follicles from several stages of folliculogenesis and blood vessels expressing specific markers of vascularization, and proliferation and apoptosis of ovarian cells. Thus, heterotopic autotransplantation of a whole frozen/thawed ovary allows for development of preovulatory follicles, oocyte growth, and for restoration of vascularization and cellular function. However, additional improvements are required to enhance the efficiency of autotransplantation of frozen/thawed ovaries to produce more oocytes.

Effects of Aloe vera on gap junctional intercellular communication and proliferation of human diabetic and nondiabetic skin fibroblasts.

OBJECTIVE: To evaluate the effects of Aloe vera on gap junctional intercellular communication (GJIC) and proliferation of human skin fibroblasts in the presence or absence of basic fibroblast growth factor (FGF-2). DESIGN: In vitro study using human type II diabetic and nondiabetic skin fibroblast cell lines. SETTING AND SUBJECTS: Diabetic (n = 4) and nondiabetic (n = 4) human skin fibroblast cell lines were purchased from Coriell Institute for Medical Research (Camden, NJ). The cells were cultured with or without Aloe vera extract in increasing concentrations (0%, 0.625%, 1.25%, 2.5%, 5%, 10%, and 20%; v/v) in culture medium and with or without FGF-2 (30 ng/mL). MEASUREMENTS: GJIC was evaluated after 48-hour incubation with treatments by laser cytometry. Cells were counted after 72-hour incubation with treatments by using a Coulter counter. RESULTS: The rate of GJIC was greater (p < 0.01) for diabetic than for nondiabetic fibroblasts (3.5 +/- 0.1 versus 3.0 +/- 0.1% per minute during the first 4 minutes after photobleaching). GJIC was increased ( p < 0.05) for diabetic fibroblasts in the presence of 2.5% and 5% of Aloe vera extract (4.2 +/- 0.1 and 4.0 +/- 0.2 versus 3.5 +/- 0.1% per minute for control, respectively). FGF-2 stimulated (p < 0.01) GJIC for diabetic (4.0 +/- 0.1 versus 3.5 +/- 0.1% per minute for control) and nondiabetic (3.5 +/- 0.1 versus 3.0 +/- 0.1% per minute for control) fibroblasts. Aloe vera extract did not affect GJIC of nondiabetic fibroblast cultured without FGF-2. However, Aloe vera extract decreased (p < 0.05) FGF-2 stimulatory effects on GJIC of diabetic and nondiabetic fibroblasts. Proliferation of diabetic fibroblasts was increased (p < 0.05) by 1.25% and 2.5% Aloe vera extract in medium. Proliferation of nondiabetic fibroblasts was not affected by Aloe vera extract. FGF-2 increased (p < 0.05) proliferation of nondiabetic fibroblasts and FGF-2 did not affect proliferation of diabetic fibroblasts. Aloe vera extract decreased (p < 0.05) FGF-2 stimulatory effects on proliferation of nondiabetic fibroblasts. CONCLUSIONS: These data demonstrate that Aloe vera has the ability to stimulate GJIC and proliferation of human skin fibroblasts in diabetes mellitus. Furthermore, these results indicate that Aloe vera contains a compound(s) that neutralizes, binds with FGF-2 receptor, or otherwise alters signaling pathways for FGF-2. By affecting both GJIC and proliferation of diabetic fibroblasts, Aloe vera may improve wound healing in diabetes mellitus.

Role of gap junctions in regulation of progesterone secretion by ovine luteal cells in vitro.

To evaluate the role of gap junctions in the regulation of progesterone secretion, two experiments were conducted. In Experiment 1, luteal cells obtained on days 5, 10, and 15 were cultured overnight at densities of 50 x 10(3), 100 x 10(3), 300 x 10(3), and 600 x 10(3) cells/dish in medium containing: (1) no treatment (control), (2) LH, or (3) dbcAMP. In Experiment 2, luteal cells from days 5 and 10 of the estrous cycle were transfected with siRNA, which targeted the connexin (Cx) 43 gene. In Experiment 1, progesterone secretion, Cx43 mRNA expression, and the rates of gap junctional intercellular communication (GJIC), were affected by the day of the estrous cycle, cell density, and treatments (LH or dbcAMP). The changes in progesterone secretion were positively correlated with the changes in Cx43 mRNA expression and the rates of GJIC. Cx43 was detected on the luteal cell borders in every culture, and luteal cells expressed 3beta-hydroxysteroid dehydrogenase. In Experiment 2, two Cx43 gene-targeted sequences decreased Cx43 mRNA expression and progesterone production by luteal cells. The changes in Cx43 mRNA expression were positively correlated with changes in progesterone concentration in media. Thus, our data demonstrate a relationship between gap junctions and progesterone secretion that was supported by (1) the positive correlations between progesterone secretion and Cx43 mRNA expression and GJIC of luteal cells and (2) the inhibition of Cx43 mRNA expression by siRNA that resulted in decreased production of progesterone by luteal cells. This suggests that gap junctions may be involved in the regulation of steroidogenesis in the ovine corpus luteum.

Gap junctional connexin 37 is expressed in sheep ovaries.

The objective of current study was to evaluate the expression of Cx37 in ovarian follicles and in corpora lutea (CL) during the estrous cycle in sheep. Ovine Cx37 was cloned and characterized to design speciesspecific probe and primers. In Exp. 1, ovaries were collected on d 13, 14, 15, and 16 of the estrous cycle, or from FSH-induced ewes at 0, 2, 4, 8, 12, 24, and 48 h after hCG treatment on d 15 of the estrous cycle. In Exps. 2 and 3, CL were collected on d 5, 10, and 15 of the estrous cycle, or at 0, 4, 8, 12, and 24 h after prostaglandin F2alpha (PGF2alpha)-induced luteal regression on d 10 of the estrous cycle, respectively. Ovarian tissues (e.g., granulosa cells, theca cells, ovarian follicles, and /or CL) were used for Cx37 immunostaining followed by image analysis or for determination of Cx37 mRNA expression by real-time RT-PCR. We demonstrated that (1) Cx37 protein was expressed in granulosa and cumulus oocyte complex compartments, ovarian blood vessels, and on the luteal cell borders, (2) expression of Cx37 mRNA was greater in granulosa than in theca cells of preovulatory follicles, (3) Cx37 mRNA expression in granulosa but not theca cells was affected by hCG treatment, (4) Cx37 protein and mRNA expression were dependent on the stage of luteal development, and (5) Cx37 expression changed during PGF2alpha- induced luteal regression. Thus, Cx37 may play a role in follicular development and ovulation as well as in luteal tissue growth, differentiation, and regression.

Vascular composition, apoptosis, and expression of angiogenic factors in the corpus luteum during prostaglandin F2alpha-induced regression in sheep.

Corpora lutea and blood samples were collected from superovulated ewes 0, 4, 8, 12 and 24 h after prostaglandin F(2alpha) (PGF) analog injection on day 10 of the estrous cycle. Changes in vascular cell and fibroblast composition, apoptosis and mRNA expression for several angiogenic factors in the corpus luteum (CL) were determined. While peripheral progesterone concentration decreased at 24 h after PGF injection, CL weight did not change. The area of positive BS-1 lectin staining (endothelial cell marker), smooth muscle cell actin (SMCA; pericyte and SMC marker), collagen type 1 (fibroblast marker), and the rate of cell death changed in luteal tissues after PGF treatment. In association with these cellular changes, mRNA for several angiogenic factors including vascular endothelial growth factor (VEGF) and receptors (Flt and KDR), basic fibroblast growth factor (FGF2) and receptor, angiopoietin (ANGPT) 1 and receptor Tie-2, endothelial nitric oxide synthase (NOS3), and angiotensin II receptor 1 (AT1) were altered. Changes in endothelial cell marker expression were positively correlated with changes in VEGF and NO systems. In addition, changes in mRNA expression for VEGF, Flt and KDR were positively correlated with changes in ANGPT2, Tie-2, and NOS3, indicating a functional relationship. This data demonstrates that after an initial increase, the endothelial component of the vascular bed decreases during PGF-induced luteal regression. However, SMCA expression remained high during luteal regression, potentially indicating a role of pericytes and vascular SMC in luteolysis, likely to regulate tissue remodeling and to maintain the integrity of larger blood vessels. Further, it appears that early regression may increase collagen type 1 production and/or expression by fibroblasts. Expression of angiogenic factors is influenced by PGF-induced luteolysis and may serve to maintain vascular structure in order to aid luteal regression.

Expression of gap junctional connexins 26, 32, and 43 mRNA in ovarian preovulatory follicles and corpora lutea in sheep.

The objective of the current study was to evaluate the expression of connexins (Cx)26, Cx32, and Cx43 mRNA in granulosa and theca cells during the peri-ovulatory period (experiment 1) and in the corpus luteum (CL) during the estrous cycle (experiment 2) and during prostaglandin F2alpha (PGF)-induced luteal regression (experiment 3) in FSH-treated ewes. In experiment 1, Cx26, Cx32, and Cx43 mRNA was expressed in granulosa and theca cells, and expression of Cx32 and Cx43 mRNA, but not Cx26, was greater (p<0.001) in granulosa than in theca cells throughout the peri-ovulatory period. Expression of Cx43 mRNA in granulosa and theca cells decreased (p<0.01) 24 h after hCG treatment. In experiment 2, expression of Cx26 mRNA in the CL tended to be greater (p<0.06) on day 10 than on days 5 or 15, but expression of Cx43 mRNA was greater (p<0.01) on day 5 than on days 10 and 15 of the estrous cycle. In experiment 3, expression of Cx26, but not Cx32 or Cx43 mRNA decreased (p<0.001) during PGF-induced luteal regression. In all 3 experiments, expression of Cx32 mRNA was much less than Cx26 and Cx43 mRNA. Moreover, Cx32 mRNA expression was unchanged during the peri-ovulatory period or during several stages of luteal development and PGF-induced regression of the CL. Thus, we have shown that the mRNA expression pattern of Cx26 and Cx43 changes during peri-ovulatory period and during several stages of the luteal development. This suggests that Cx26 and Cx43 play a role in ovarian tissue remodeling during the critical time around ovulation and throughout luteal tissue growth, differentiation, and regression in sheep.

Expression of connexin 43 and gap junctional intercellular communication in the cumulus-oocyte complex in sheep.

To evaluate the effects of FSH, LH and/or cAMP on expression of connexin 43 (Cx43) in the ovine cumulus-oocyte complex (COC) and gap junctional intercellular communication (GJIC) of cumulus cells, two experiments were carried out. In experiment 1, Cx43 was immunodetected in the COC, before or after maturation, obtained from non-treated or FSH-treated ewes. The expression of Cx43 in the COC was greater (P < 0.01) on day 16 than on day 15 of the estrous cycle. In vivo FSH treatment decreased (P < 0.02) Cx43 expression on day 16 but not on day 15 of the estrous cycle. In experiment 2, intact COCs or isolated cumulus cells obtained from small and large follicles from FSH-treated ewes were cultured with or without FSH, LH or cAMP agonist and evaluated for GJIC by laser cytometry. For large follicles, the basal rate of GJIC was greater (P < 0.01) for cumulus cells in intact COCs than for isolated cumulus cells. FSH increased (P < 0.04) GJIC in cumulus cells in intact COCs and tended to increase (P < 0.1) GJIC in isolated cumulus cells from small follicles but decreased (P < 0.01) GJIC in cumulus cells in intact COCs from large follicles. LH also increased (P < 0.01) GJIC in isolated cumulus cells from small follicles but decreased GJIC in intact COCs (P < 0.01) and isolated cumulus cells (P < 0.02) from large follicles. cAMP increased (P < 0.01) the GJIC in both intact COCs and cumulus cells from small and large follicles. These results indicate that day of estrous cycle, stage of maturation and duration of FSH treatment affect expression of Cx43 in ovine COCs. In intact COCs, GJIC in cumulus cells was enhanced, probably due to the presence of the oocyte. In addition, the effects of FSH and LH, but not cAMP, on GJIC of cumulus cells depended on the stage of follicular development and on the presence of the oocyte.

Gap junctional intercellular communication of bovine granulosa and thecal cells from antral follicles: effects of luteinizing hormone and follicle-stimulating hormone.

Throughout each estrous cycle, the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are involved in regulation of folliculogenesis. We have shown that LH or FSH affect cellular interactions mediated by gap junctions in bovine granulosa and thecal cells in vitro. To evaluate further the hypothesis that gonadotropins influence gap junctional intercellular communication (GJIC) and expression of gap junctional proteins known as connexins (Cx), throughout antral follicle development, granulosa and thecal cells from large (>10 mm; n = 13), medium (5-10 mm; n = 20), and small (<5 mm; n = 27) follicles were cultured (n = 4 cultures per size) with or without LH, FSH, or LH + FSH for 24 h. GJIC was evaluated (n = 125-150 cells/treatment group) by using the fluorescent recovery after photobleaching technique and laser cytometry. Additionally, Cx43, Cx32, and Cx26 were detected in cultured cells by immunocytochemistry and Cx43 by Western immunoblot analysis. Finally, progesterone production by cultured cells was evaluated by radioimmunoassay. Across all follicles and treatments, GJIC was greater (p < 0.01) for granulosa than thecal cells (4.9 +/- 0.05 vs 3.8 +/- 0.04%/min). For granulosa cells of large and medium follicles, LH and/or FSH did not affect GJIC. For granulosa cells of small follicles, FSH increased (p < 0.05), but LH or LH + FSH had no effect on GJIC. For thecal cells of large follicles, LH increased (p < 0.01) GJIC, whereas FSH or LH + FSH had no effects. For thecal cells of medium and small follicles, LH and/or FSH did not affect GJIC. These results demonstrate that FSH influenced GJIC of granulosa cells from small, but not from medium or large, follicles, and LH influenced GJIC of thecal cells from large, but not from medium or small, follicles. Cx43 was present as punctate staining between granulosa or thecal cells from all cultures, indicating assembled gap junctions. LH + FSH increased (p < 0.05) expression of Cx43 only by thecal cells from large follicles. Cx32 was detected in the perinuclear cytoplasm of cultured granulosa or thecal cells, and in the cytoskeleton of a few cells per culture dish in all sizes of follicles. Cx26 was present in a regular pattern throughout the cytoplasm of granulosa or thecal cells in all sizes of follicles. For granulosa cells from large follicles, progesterone production was stimulated (p < 0.05) with LH or FSH alone but was unaffected by LH + FSH. For granulosa cells from medium and small follicles, progesterone production was unaffected by LH and/or FSH. For thecal cells from all sizes of follicles, LH, FSH, and LH + FSH stimulated (p < 0.05) production of progesterone. These data indicate that LH and FSH influence gap junction function and expression, which likely contributes to the development and maintenance of ovarian follicles.

Wound healing: the role of growth factors.

Wound healing is a complex biological process that requires cellular interactions between a variety of cells, including fibroblasts, myofibroblasts, smooth muscle cells, endothelial cells, keratinocytes and immune cells. These interactions are mediated by numerous factors such as growth factors, hormones, blood components and second messengers. Several growth factors that are released at the wound site are presumed to be necessary for wound healing. These include epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF) and vascular endothelial growth factor (VEGF). The clinical use of growth factors to stimulate the healing of wounds is currently being investigated. Several growth factors, including PDGF, FGF-2, IGF and KGF, have been used in clinical trials, and PDGF is currently approved for use in human medicine.