Vascular endothelial growth factor-A (VEGFA) is a hypoxia-inducible peptide essential for angiogenesis and targets nonvascular cells in a variety of tissues and cell types. The objective of the current study was to determine the function of VEGF during testis development in bulls. We used an explant tissue culture and treatment approach to test the hypothesis that VEGFA-164 could regulate the biological activity of bovine germ cells. We demonstrate that VEGFA, KDR, and FLT1 proteins are expressed in germ and somatic cells in the bovine testis. Treatment of bovine testis tissue with VEGFA in vitro resulted in significantly more germ cells following 5 days of culture when compared with controls. Quantitative real-time RT-PCR analysis determined that VEGF treatment stimulated an intracellular response that prevents germ cell death in bovine testis tissue explants, as indicated by increased expression of BCL2 relative to BAX and decreased expression of BNIP3 at 3, 6, and 24 h during culture. Blocking VEGF activity in vitro using antisera against KDR and VEGF significantly reduced the number of germ cells in VEGF-treated testis tissue to control levels at 120 h. Testis grafting provided in vivo evidence that bovine testis tissue treated with VEGFA for 5 days in culture contained significantly more differentiating germ cells compared with controls. These findings support the conclusion that VEGF supports germ cell survival and sperm production in bulls.
Sera from 143 rainbow trout Oncorhynchus mykiss surviving 1 and 5 exposures to infectious hematopoietic necrosis virus (IHNV) were analyzed for specific antibody against the virus with an enzyme linked immunoassay (ELISA), plaque neutralization test (PNT) and western blot. By the ELISA, 92 O/u of all survivors demonstrated antibodies against IHNV, while 82 % of then1 showed neutralizing antibodies against the virus by PNT. A correlation analysis (Kendall's Tau-b) performed on the ELISA values and PNT values indicated the results of the 2 assays correlated (p <0.05). Sera from fish which had survived 1 exposure (1 X ) to IHN had similar ELISA and PNT values compared to fish which had survived 5 exposures, but were rested in pathogen-free water for 80 d before bleeding (5 X R). Fish which had survived 5 exposures (5 X ) and bled 20 d after placement in pathogen-free water had a significantly different distribution of titer values compared to the other 2 groups. Sera from survivors were reacted in a western blot with the 5 IHNV proteins and probed with a monoclonal antibody recognizing trout immunoglobulin. The majority (74/143) of the trout sera gave no reaction in the western blot. In the groups of survivors which gave positive reactions, the proteins most frequently recognized \Yere the viral membrane protein [M11 by 1 X survivors (9/28) and the glycoprotein [G] by 5 X survivors (38/82). The group of 5 X R survivors also recognized G (10/33) most frequently.
A balance between self-renewal and differentiation of spermatogonial stem cells (SSCs) is required to maintain sperm production throughout male life. The seminiferous epithelium is organized into stages of spermatogenesis based on the complement of germ cell types within a tubular section of the testis. The stages exist in close physical proximity and foster diverse phases of germ cell development despite exposure to a similar endocrine milieu that supports coordinated spermatogenesis. The objective of the current study was to identify the population dynamics of SSCs in vivo. We hypothesized that SSC populations and their niches are specifically distributed across the mature seminiferous epithelium in the mouse testis. To test this hypothesis, we conducted stem cell transplantation of germ cells obtained from stage-specific clusters of seminiferous tubules representing areas of high responsiveness to follicle-stimulating hormone (IX–I), androgen (II–IV), and retinoid (V–VIII) signaling. Similarly, we analyzed the expression of genes linked with SSC activity in these groups of stages. No stage-specific differences in the colonization efficiency or the colony number were detected after SSC transplantation, indicating that SSCs are equally distributed across all stages of the seminiferous tubule. In contrast, SSCs obtained from donor stages IX–IV established larger donor-derived colonies due to increased colony expansion. SSCs originating from different stages have varying degrees of stem cell activity in vivo, a notion consistent with Gdnf, Ret, and Bcl6b expression data. These results support the conclusion of a stage-specific, microenvironment-regulating SSC self-renewal and suggest the presence of a transit-amplifying population of undifferentiated spermatogonia in vivo.
Somatic and germ cell maturation precedes the start of spermatogenesis and is coordinated, so efficient spermatogenesis will occur in the adults. The present study was conducted to evaluate endocrine regulation of germ and somatic cell homeostasis in the neonatal boar testis associated with the establishment of spermatogenesis. Testis tissue obtained from 3-, 5-, 7- and 14-day-old piglets were ectopically xenografted onto castrated, immunodeficient nude mice. Grafts were removed 22 weeks later and evaluated for growth and the establishment of spermatogenesis. Recipient mouse testosterone biosynthesis and follicle-stimulating hormone (FSH) concentrations were also assayed. Testis tissue graft growth was significantly greater in testis grafts from 3-day donor tissue when compared to all other ages; 5-, 7- and 14-day-old donor tissue weights were not significantly different at removal. Follicle-stimulating hormone concentrations in recipient mice supporting testis grafts from 5-, 7- and 14-day-old donor tissues did not differ and were similar to normal physiological levels in age-matched, intact nude mice. Serum FSH levels were significantly lower in recipient mice supporting testis grafts from 3-day-old donor tissue. Radioimmunoassay and biological assay indicated no differences in testosterone production by testis tissue grafts of varying donor age. Porcine testis tissue obtained from 3-, 5-, 7- and 14-day-old neonatal boars were all capable of producing round and elongate spermatids after 22 weeks of grafting, but testis grafts from 14-day-old donors had a significantly greater (eightfold) percentage of seminiferous tubules with spermatids compared to all other donor ages (p < 0.05). Cryopreservation did not affect the ability of testis tissue grafts to grow, produce testosterone or establish spermatogenesis when compared to controls (p < 0.05). Collectively, these data demonstrate intrinsic differences in the biological activity of germ and somatic cell populations during neonatal boar testis development associated with the establishment of spermatogenesis.
Ovarian response to follicle stimulating hormone (FSH) and luteinising hormone (LH) leads to the formation of a mature follicle that is eventually ovulated. FSH and LH are essential for this process because they direct changes in somatic cells associated with folliculogenesis by regulating the expression of multiple genes. We hypothesised that genes induced by FSH in rat Sertoli cells would also show hormonal regulation during rat folliculogenesis. The objective of this study was to determine the expression patterns of diacylglycerol O-acyltransferase 1 (Dgat1), nuclear receptor subfamily 4a1 (Nr4a1), an anti-proliferative gene (Btg2) and a protein tyrosine phosphatase (Ptp4a1) in the ovaries of pregnant mare serum gonadotrophin (PMSG)-treated and human chorionic gonadotrophin (hCG)-treated rats. Expression of Dgat1, Nr4a1 and Ptp4a1 was induced in ovaries 4 h post PMSG treatment. When rats were treated with hCG, Dgat1, Nr4a1 and Ptp4a1 expression was induced by 12 h. Expression of Nr4a1 protein increases 12-24 h after induction of gene expression. Nr4a1 protein was observed in the granulosa, theca and luteal cells post PMSG and hCG treatment. These findings should increase our knowledge of mechanisms regulating folliculogenesis and luteinisation and demonstrate the diverse proteins that are important in ovarian function.
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