[show abstract][hide abstract] ABSTRACT: Our previous report showed that supernatants of Lactobacillus acidophilus (LS) cultures possessed chemotactic and angiogenic properties. Specifically, LS stimulated gene expression and the secretion of tumor necrosis factor-alpha (TNF-alpha), the proliferation of immune cells in vitro, and blood vessel formation. Chemotaxis and proliferation of inflammatory cells in vivo were also stimulated by LS. In the current study, we hypothesized that LS stimulates the growth and development of other rapidly dividing cells, including embryonic cells. The stimulatory effects of LS on a neuroblastoma cell line (Neuro-2a), chicken embryos, and bovine embryos were examined. The addition of LS to Neuro-2a cultures caused a proliferation of cells in a concentration-dependent manner. Pretreatment of LS at 56 degrees C for 30 mins did not affect its stimulatory activity. The administration of LS to the chorioallantoic membrane (CAM) of chicken-embryonated eggs for 1-2 days resulted in extensive thickening of the membrane. The thickening was due to the influx and proliferation of fibroblasts and inflammatory cells, the accumulation of loose connective tissue composed primarily of mucopolysaccharides, and/or the formation of blood vessels. Stimulatory effects of LS on bovine embryos were also observed. The treatment with LS significantly promoted the development of zygotes to the four-cell stage and from the four-cell stage to blastocysts. These results have confirmed our hypothesis that LS exerts a stimulatory effect on the cells of embryonic stages including neuroblastoma cells, the CAM of chicken embryos, and bovine embryos from zygotes to blastocysts.
Experimental Biology and Medicine 08/2005; 230(7):494-500. · 2.80 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of this study was to determine, with a bovine model, the appropriate interval for xenografted adult and newborn ovarian tissue to develop gonadotropin-responsive follicles.
Academic research laboratory.
Male non-obese diabetic (NOD) severe combined immunodeficient (SCID) mice (n = 20) were hosts of bovine ovarian tissue. Two dairy calves and one adult beef cow were donors of ovarian tissue.
Newborn and adult bovine ovarian cortical pieces were transplanted to the SC space of intact male NOD SCID mice. Grafts were recovered after euthanasia at intervals after transplantation.
Microscopic examination of histologic sections to determine proportions of growing follicles.
There was an increase in the proportion of primary and secondary follicles on day 55 after surgery for the cow and on day 124 after surgery for calf tissue compared with nongrafted and xenografted ovarian tissues recovered at previous intervals. These observed increases were accompanied by decreases in proportions of primordial follicles. Results suggest a sudden increase in the proportion of primary and secondary follicles due to progressive development of primordial follicles.
In the NOD SCID mouse, bovine follicles survived xenotransplantation and underwent development. A longer interval was required for ovarian follicular development in calf tissues compared with that in adult cow ovarian tissues after xenotransplantation.
[show abstract][hide abstract] ABSTRACT: To study the response of human ovarian xenografts to transplantation into different sites and in different host conditions.
Academic research laboratory.
Donated ovarian tissue from two young women.
Human ovarian cortical pieces were transplanted either under the kidney capsule or to the subcutaneous space of intact or castrated male nonobese diabetic (NOD) severe combined immune-deficient (SCID) mice. Grafts were recovered after euthanasia.
Microscopic examination of histologic sections to determine proportions of growing follicles, and serum estradiol concentration measurements.
Six months after transplantation, ovarian grafts transplanted under the kidney capsule of intact male mice had significantly higher proportions of growing follicles compared with those recovered from the castrated/kidney capsule and intact/subcutaneous groups. However, no difference was detected between the intact/kidney capsule and the castrated/subcutaneous groups. Mean estradiol concentrations in serum were nonsignificantly increased in mice with ovarian grafts compared with those in mice without a graft.
Follicular development in xenotransplanted human ovarian tissue is influenced by the site of transplantation and the condition of the host.
[show abstract][hide abstract] ABSTRACT: Ovarian cortex cryopreservation and xenotransplantation into immunodeficient mice represents a potential means for female germplasm conservation and an immediate model for investigation of folliculogenesis. The objectives of this study were to: (1) assess follicle survival after cryopreservation and transplantation of cat ovarian tissue into non-obese diabetic severely combined immunodeficient (NOD SCID) mice; and (2) evaluate the effects of gonadotropin treatments on follicular development in the transplanted tissue. Slices from the cat ovarian cortex were frozen and after thawing, transplanted under each kidney capsule of castrated male NOD SCID mice (eight xenografts in four mice). Sixty-two days after surgery, mice were randomly assigned (two per group) to gonadotropin-treated (eCG and hCG 88 h later) or control (saline-treated) groups. Twenty-four hours after the last injection, ovarian tissue was recovered and processed for histology. Fresh ovarian tissue from the same original source was similarly processed. Follicles were counted, measured, and classified as primordial, primary, secondary, or antral. Immunoreactive proliferating cell nuclear antigen (PCNA) stain was used to assess follicle viability. Microscopic examination revealed no evidence of necrosis or fibrosis. The grafts were well-vascularized, with follicles at all stages of development. Numbers of follicles in the transplanted tissue were markedly reduced compared to fresh tissue, with approximately 10% of follicles surviving freezing and transplantation procedures. Growing follicles positive for PCNA were found in all xenografts. Gonadotropin treatment did not alter the proportion of resting to growing follicles or mean follicle diameter by comparison with controls from untreated mice. By contrast, luteinization, but not ovulation, of antral follicles was observed only in grafts from treated mice. In summary, frozen-thawed cat ovarian cortex tissue not only survived xenotransplantation, it also contained follicles able to grow to antral stages. Exogenous gonadotropin treatment in this model resulted in luteinization of antral follicles but enhancement of follicular growth and ovulation did not occur.
[show abstract][hide abstract] ABSTRACT: Hormonal stimulation following xenografting of ovarian tissue into immunodeficient mice promoted antral follicle development. Efficient retrieval of oocytes contained in these antral follicles is crucial in implementing a protocol involving xenotransplantation for fertility preservation of cancer patients. Using a bovine model, our objective was to recover oocytes from calf ovarian tissue transplanted into immunodeficient mice. Fresh pieces of ovarian cortex, 1-2 mm3, from calves were placed in subcutaneous spaces of male NOD SCID mice. Hormonal treatments were initiated 3 days after surgery. Control mice (n=3) received saline while treated mice (n=4/group) received daily intraperitoneal injections of either 4 IU of FSH plus 4 IU of LH, 4 IU of FSH or 1 IU of FSH for 14 days. Forty eight to 72 h after the last injection, mice were euthanized and ovarian grafts were recovered. Serial sections of hematoxylin and eosin stained grafts were used for classifying and counting follicles. Randomly selected unstained ovarian sections were immunostained for nuclear proliferation antigen to ascertain viability. Other grafts were used for oocyte retrieval. Recovered oocytes were stained with orcein to assess maturation. Recovery of grafts (%) was no different across treatment groups. Treatment with FSH plus LH enhanced follicular development, but did not improve oocyte recovery. Evidence of maturation was only observed after incubation in vitro. We conclude that oocytes can be retrieved from xenotransplanted bovine ovarian tissue.
Journal of Animal and Veterinary Advances. 01/2004;
[show abstract][hide abstract] ABSTRACT: Pronuclear formation, and the chromosomal constitution and developmental capacity of bovine zygotes formed by intracytoplasmic sperm injection with freeze-dried (lyophilized) spermatozoa were evaluated. Frozen-thawed spermatozoa were selected, freeze-dried, and stored at 4 degrees C until use. After 22-24 h of in vitro maturation oocytes were denuded and injected singly with a lyophilized spermatozoon. Injected oocytes were activated by treatment with 10 microM ionomycin (5 min) alone and in combination with 1.9 mM 6-dimethylaminopurine (DMAP) for 4 h. Ionomycin plus DMAP activation treatment resulted in a significantly higher proportion of sperm-injected oocytes with two pronuclei than was found after activation with ionomycin alone (74% vs. 56%; P < 0.03). The rates of cleavage, morula, and blastocyst development of sperm-injected oocytes treated with ionomycin plus DMAP were higher than after activation with ionomycin alone (63.3%, 34.2%, and 29.6% vs. 44.7%, 18.7%, and 10.6%, respectively; P < 0.05). Seventy-three percent of blastocysts produced with lyophilized sperm were diploid. These results demonstrate that in vitro-matured bovine oocytes can be fertilized with freeze-dried sperm cells, and that resultant zygotes can develop into karyotypically normal blastocysts.
Biology of Reproduction 08/2002; 67(2):409-15. · 4.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: The objective of this study was to improve sexed bovine embryo production with sorted sperm in chemically defined conditions by supplementing the IVF medium with db-cAMP. Cumulus-oocyte complexes (COCs) were matured for 18 h in supplemented TCM-199 and fertilized with X- or Y-bearing sperm in the pres- ence of heparin (10 µg/ml), db-cAMP (1 µM) or no treatment (control). Presumptive zygotes were cultured 54 h in g-SOF. From 72 to 144 h post- insemination (hpi) embryos were cultured in c-SOF+NEA and from 144 to 192 hpi embryos were placed in maturation medium without hormones. No significant differences were found among treatments for Y-sperm when compared to controls. A significant (P
[show abstract][hide abstract] ABSTRACT: In the present study, performance of 56 crossbred dual pur- pose heifers diagnosed as pregnant after direct transfer of in vitro produced embryos cultured in either media supplemented with serum (n = 23) or in a chemically defined media (n = 33) were compared. No differences were observed in the incidence of abortion (30.34% vs. 24.24%), dystocia (52.17% vs. 51.52%) and normal calving (17.39% vs. 24.24%) in pregnant heifers with embryos produced in either serum supplemented or chemi- cally defined media respectively (P > 0.05). Sex of calves affected significantly the rate of dystocia (males, 83.33% and fe- males, 50%; P < 0.05). The birthweight of calves was not af- fected (P > 0.05) neither by serum supplementation during in vi- tro culture (46.86 ± 2.04 kg for calves derived from embryos cul- tured in the serum supplemented media and 46.28 ± 1.42 kg, for calves derived from embryos cultured in the chemically defined media) nor by sex of calves (males, 47.20 ± 1.50 kg and fe- males, 45.45 ± 1.84 kg). The birthweigh of calves born dead or dying soon after birth was significantly (P < 0.05) higher (51.92 ± 1.76 kg) than that of survivors calves (43.88 ± 1.22 kg). Neither serum supplementation during in vitro culture, sex of calves nor dystocia affected the perinatal survival of calves. In conclusion, the presence of serum during in vitro culture did not affect the reproductive performance of dual purpose preg- nant heifers after direct transfer of in vitro produced embryos. Large offspring syndrome (LOS) as observed in this study was evidenced by high birthweigh of calves, high rate of abortions and dystocia.
[show abstract][hide abstract] ABSTRACT: Platelet-activating factor (PAF; 1-O-Alkyl-2-acetyl-sn-glycero- -3-phosphorylcholine) is a ubiquitous phospholipid that is im- plicated in the mediation of a wide variety of reproductive pro- cesses. To better understand the role of PAF in bovine repro- duction, it was designed experiments to: (a) determine whether bull spermatozoa express receptors for PAF and (b) study the effect of exogenous PAF on in vitro sperm physio- logy (i.e., capacitation, acrosome reaction, motility, and ferti- lizing ability). Bull sperm express PAF receptor as determined by two approaches: RT-PCR and immunofluorescence. However, exposure of spermatozoa to different concentra- tions of exogenous PAF (10-11-10-6 M) did not affect capacita- tion, acrosome reaction or motility. Consistent with these fin- dings, coculture of gametes in medium containing increasing concentrations of PAF (1 x 10-8 -8 x1 0 -6 M) did not improve in vitro fertilization outcome as measured by percentage of inse- minated oocytes reaching 2-cell stage 48 h after fertilization. In contrast, PAF at 8 x 10-6 M concentration significantly inhi- bited IVF. In conclusion, although bull sperm have PAF recep- tors, exposure of bull spermatozoa to exogenous PAF failed to enhance the sperm function parameters measured in this study. Additional studies are warranted to elucidate the biolo- gical role of PAF on bull spermatozoa.