Influence of nitric oxide on in vitro growth, survival, steroidogenesis, and apoptosis of follicle stimulating hormone stimulated buffalo (Bubalus bubalis) preantral follicles

Effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on in vitro survival, growth, steroidogenesis, and apoptosis of buffalo preantral follicles (PFs) was investigated. PFs (200~250 µm) were isolated by micro-dissection and cultured in 0 (control), 10-3, 10-5, 10-7, and 10-9 M SNP. To examine the reversible effect of SNP, PFs were cultured with 10-5 M SNP + 1 mM Nω-nitro-L-arginine methyl ester (L-NAME) or 1.0 µg hemoglobin (Hb). The results showed that greater concentrations of SNP (10-3, 10-5, 10-7 M) inhibited (p < 0.05) FSH-induced survival, growth, antrum formation, estradiol production, and oocyte apoptosis in a dose-dependent manner. However, a lower dose of SNP (10-9 M) significantly stimulated (p < 0.05) the survival, growth, antrum formation, follicular oocyte maturation, and stimulated progesterone secretion compared to the control. A combination of SNP + L-NAME promoted the inhibitor effect of SNP while a SNP + Hb combination reversed this effect. Nitrate and nitrite concentrations in the culture medium increased (p < 0.05) in a dose-dependent manner according to SNP concentration in the culture medium. At higher concentrations, SNP had a cytotoxic effect leading to follicular oocyte apoptosis whereas lower concentrations have stimulatory effects. In conclusion, NO exerts a dual effect on its development of buffalo PFs depending on the concentration in the culture medium

Macrophage-derived vascular endothelial growth factor-A is integral to neuromuscular junction reinnervation after nerve injury

Functional recovery in the end target muscle is a determinant of outcome after peripheral nerve injury. The neuromuscular junction (NMJ) provides the interface between nerve and muscle and includes non-myelinating terminal Schwann cells (tSCs). After nerve injury, tSCs extend cytoplasmic processes between NMJs to guide axon growth and NMJ reinnervation. The mechanisms related to NMJ reinnervation are not known. We used multiple mouse models to investigate the mechanisms of NMJ reinnervation in both sexes, specifically whether macrophage-derived vascular endothelial growth factor-A (Vegf-A) is crucial to establishing NMJ reinnervation at the end target muscle. Both macrophage number and Vegf-A expression increased in end target muscles after nerve injury and repair. In mice with impaired recruitment of macrophages and monocytes

Luteal angiogenesis and its control

Angiogenesis, the formation of new blood vessels from pre-existing ones, is critical to luteal structure and function; In addition, it is a complex and tightly regulated process. Not only does rapid and extensive angiogenesis occur to provide the corpus luteum (CL) with an unusually high blood flow and support its high metabolic rate, but in the absence of pregnancy the luteal vasculature must rapidly regress to enable the next cycle of ovarian activity. This review describes a number of the key endogenous stimulatory and inhibitory factors, which act in a delicate balance to regulate luteal angiogenesis and ultimately luteal function. In vitro luteal angiogenesis cultures have demonstrated critical roles for fibroblast growth factor 2 (FGF2) in endothelial cell proliferation and sprouting, whilst other factors such as vascular endothelial growth factor (VEGFA) and platelet derived growth factor (PDGF) were important modulators in the control of luteal angiogenesis. Post-transcriptional regulation by small non-coding micro-RNAs, is also likely to play a central role in the regulation of luteal angiogenesis. Appropriate luteal angiogenesis requires the coordinated activity of numerous factors expressed by several cell types at different times and this review will also describe the role of perivascular pericytes and the importance of vascular maturation and stability. It is hoped that a better understanding of the critical processes underlying the transition from follicle to CL, and subsequent luteal development will benefit the management of luteal function in the future

Reduced Bone Mass and Muscle Strength in Male 5α-Reductase Type 1 Inactivated Mice

Androgens are important regulators of bone mass but the relative importance of testosterone (T) versus dihydrotestosterone (DHT) for the activation of the androgen receptor (AR) in bone is unknown. 5α-reductase is responsible for the irreversible conversion of T to the more potent AR activator DHT. There are two well established isoenzymes of 5α-reductase (type 1 and type 2), encoded by separate genes (Srd5a1 and Srd5a2). 5α-reductase type 2 is predominantly expressed in male reproductive tissues whereas 5α-reductase type 1 is highly expressed in liver and moderately expressed in several other tissues including bone. The aim of the present study was to investigate the role of 5α-reductase type 1 for bone mass using Srd5a1−/− mice. Four-month-old male Srd5a1−/− mice had reduced trabecular bone mineral density (−36%, p<0.05) and cortical bone mineral content (−15%, p<0.05) but unchanged serum androgen levels compared with wild type (WT) mice. The cortical bone dimensions were reduced in the male Srd5a1−/− mice as a result of a reduced cortical periosteal circumference compared with WT mice. T treatment increased the cortical periosteal circumference (p<0.05) in orchidectomized WT mice but not in orchidectomized Srd5a1−/− mice. Male Srd5a1−/− mice demonstrated a reduced forelimb muscle grip strength compared with WT mice (p<0.05). Female Srd5a1−/− mice had slightly increased cortical bone mass associated with elevated circulating levels of androgens. In conclusion, 5α-reductase type 1 inactivated male mice have reduced bone mass and forelimb muscle grip strength and we propose that these effects are due to lack of 5α-reductase type 1 expression in bone and muscle. In contrast, the increased cortical bone mass in female Srd5a1−/− mice, is an indirect effect mediated by elevated circulating androgen levels

Prostaglandin F2-alpha receptor (FPr) expression on porcine corpus luteum microvascular endothelial cells (pCL-MVECs)

<p>Abstract</p> <p>Background</p> <p>The corpus luteum (CL) is a transient endocrine gland and prostaglandin F2-alpha is considered to be the principal luteolysin in pigs. In this species, the in vivo administration of prostaglandin F2-alpha induces apoptosis in large vessels as early as 6 hours after administration. The presence of the prostaglandin F2-alpha receptor (FPr) on the microvascular endothelial cells (pCL-MVECs) of the porcine corpus luteum has not yet been defined. The aim of the study was to assess FPr expression in pCL-MVECs in the early and mid-luteal phases (EL-p, ML-p), and during pregnancy (P-p). Moreover, the effectiveness of prostaglandin F2-alpha treatment in inducing pCL-MVEC apoptosis was tested.</p> <p>Methods</p> <p>Porcine CLs were collected in the EL and ML phases and during P-p. All CLs from each animal were minced together and the homogenates underwent enzymatic digestion. The pCL-MVECs were then positively selected by an immunomagnetic separation protocol using Dynabeads coated with anti-CD31 monoclonal antibody and seeded in flasks in the presence of EGM 2-MV (Microvascular Endothelial Cell Medium-2). After 4 days of culture, the cells underwent additional immunomagnetic selection and were seeded in flasks until the confluent stage.</p> <p>PCR Real time, western blot and immunodetection assays were utilized to assess the presence of FPr on pCL-MVEC primary cultures. Furthermore, the influence of culture time (freshly isolated, cultured overnight and at confluence) and hormonal treatment (P4 and E2) on FPr expression in pCL-MVECs was also investigated. Apoptosis was detected by TUNEL assay of pCL-MVECs exposed to prostaglandin F2-alpha.</p> <p>Results</p> <p>We obtained primary cultures of pCL-MVECs from all animals. FPr mRNA and protein levels showed the highest value (ANOVA) in CL-MVECs derived from the early-luteal phase. Moreover, freshly isolated MVECs showed a higher FPr mRNA value than those cultured overnight and confluent cells (ANOVA). prostaglandin F2-alpha treatment failed to induce an apoptotic response in all the pCL-MVEC cultures.</p> <p>Conclusion</p> <p>Our data showing the presence of FPr on MVECs and the inability of prostaglandin F2-alpha to evoke an in vitro apoptotic response suggest that other molecules or mechanisms must be considered in order to explain the in vivo direct pro-apoptotic effect of prostaglandin F2-alpha at the endothelial level.</p