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1.
Cryopreservation of stallion semen has not reached the level of efficiency and positive results described in other species. This is mainly due to the greater sensitivity of stallion sperm to the freezing process, showing higher rates of oxidative stress and plasma membrane damage, which trigger the activation of several cell damage pathways that ultimately culminate in DNA fragmentation and cell death. Therefore, finding molecules that improve the efficiency of this technique in stallion by preventing oxidative stress and cell damage is required. Thus, the aim of the present study was to evaluate the effect of adding three antioxidants (MnTBAP, NAC and FeTPPS) to the freezing medium on the quality and functional parameters of stallion sperm. Semen samples from three stallions frozen with the antioxidants were evaluated in two conditions: (a) adding the antioxidants before freezing, and (b) before and after freezing. Plasma membrane integrity, mitochondrial membrane potential, lipid peroxidation, intracellular ROS levels, membrane lipid disorder, DNA damage, sperm motility and binding to the zona pellucida were assessed. The results showed that MnTBAP was the antioxidant treatment that best controlled the oxidative stress process and post-thaw cell damage, showing higher plasma membrane integrity, mitochondrial membrane potential, sperm motility, number of spermatozoa bound to the zona pellucida of bovine oocytes and lower lipid disorder. Additionally, it was determined that a second post-thaw application of antioxidants is detrimental since induced higher cell damage and lower sperm motility, without showing any beneficial effect on the spermatozoa.  相似文献   

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Granulosa cells from pooled bovine follicles were cultured under chemically-defined (serum-free) conditions to study the effects of exogenous steroids and FSH on production of immunoactive (ia) inhibin, oestradiol and progesterone. Levels of ia-inhibin in media samples and cell lysates were measured by radioimmunoassay (RIA) using an antiserum raised against a synthetic fragment of human inhibin -subunit [hI(1–32)].

Cells secreted measurable amounts of ia-inhibin, oestradiol and progesterone for at least 7 d of culture, although intracellular levels of inhibin were very low, indicating that newly-synthesized ia-inhibin is rapidly released from the cells. Treatment with androstenedione (0.2μmol/l) or testosterone (0.2μmol/l) increased ia-inhibin secretion markedly; levels on Day 5 of culture were approximately 6-fold (P<0.005) higher than control values. In contrast, treatment with the non-aromatizable androgen dihydrotestosterone (DHT; 0.2μmol/l) resulted in only a one- to two-fold increase (P<0.05) over control values (Day 5). Addition of exogenous oestradiol (8nmol/l) markedly increased ia-inhibin secretion (8–9 fold on Day 5; P<0.05) compared with basal levels, whereas progesterone had no effect. Secretion of oestradiol, undetectable in the absence of exogenous androgens, rose daily in the presence of either androstenedione or testosterone, levels rising approximately 6-fold and 9-fold respectively over a 4-d treatment period. Progesterone secretion increased 2-fold over the culture period and was unaffected by any steroid treatment.

Treatment with ovine FSH (10ng/ml) alone stimulated secretion of progesterone over basal levels (3-fold higher on Day 6; P<0.005), but did not affect output of either ia-inhibin or oestradiol. However, exposure to FSH in the presence of androstenedione not only promoted a further 4-fold increase in progesterone output but also led to a dose-dependent suppression of both ia-inhibin (90% lower on Day 6; P<0.001) and oestradiol (80% lower on Day 6; P<0.001) secretion compared to cells treated with androstenedione alone.

These observations indicate that the secretion of ia-inhibin by bovine granulosa cells in culture is positively regulated by oestradiol, implying an autocrine/paracrine role for this hormone in control of ovarian inhibin production. The ability of aromatizable androgens to stimulate secretion of inhibin, coupled with the inability of the non-aromatizable androgen DHT to elicit such an effect, suggests that inhibin output is largely unaffected by androgens prior to their conversion to oestradiol. The absence of any change in output of ia-inhibin or oestradiol following treatment with exogenous progesterone argues against a local role for this steroid hormone in regulation of inhibin or oestradiol production in the bovine follicle. Finally, the observation that co-treatment with FSH and andostenedione not only stimulated progesterone output but also suppressed secretion of ia-inhibin and oestradiol, indicates a synergistic positive effect of FSH and androgens on cellular luteinization.  相似文献   


4.
Follicle-stimulating hormone (FSH) plays a critical role in follicular growth and granulosa cell function; however, the mechanism by which the aggressive stimulation of FSH leads to poorer oocyte quality and embryo development potential is unclear. In this study, bovine ovarian granulosa cells (BGCs) were challenged with FSH doses (vehicle, 0.1, 1, 10 and 100 ng/ml) to investigate the effects of FSH on BGCs. The results indicated that the relative viability of BGCs was significantly increased in cells challenged with 1 ng/ml FSH, whereas the viability was significantly decreased with 100 ng/ml FSH treatment. The mRNA abundance of FSHR, CYP19, StAR and BAX was significantly upregulated with 1, 10 and 100 ng/ml of FSH, while the BCL-2 mRNA level was downregulated with higher concentrations of FSH (10 and 100 ng/ml). Furthermore, BGC autophagy was detected in cells treated with 10 and 100 ng/ml FSH by MDC staining, and the mRNA abundance of LC3, BECN1, BNIP3, ATG3 and ATG7 was upregulated with increasing FSH concentration. Meanwhile, the protein expression of LC3 was increased in cells treated with 10 and 100 ng/ml FSH. 1 and 10 ng/ml FSH significantly increased E2 production, whereas 10 and 100 ng/ml FSH significantly increased P4 production. FSH significantly inhibited the phosphorylation of AKT in cells treated with higher concentrations (1, 10 and 100 ng/ml), while activating mTOR phosphorylation at concentrations of 10 and 100 ng/ml of FSH. In summary, we can conclude that higher doses of FSH (10 and 100 ng/ml) induce BGC autophagy via the AKT/mTOR signalling pathway.  相似文献   

5.
Connexin 43 (Cx43)-mediated gap junctional communication in granulosa cells is crucial for germ line development and postnatal folliculogenesis. We previously showed that follicle-stimulating hormone (FSH) promoted phosphorylation of Cx43 in rat primary granulosa cells. We further identified Ser365, Ser368, Ser369, and Ser373 in the carboxy-terminal tail as the major sites of phosphorylation by FSH, and found that the phosphorylation of these residues was essential for channel activity. In this study, we investigated the protein kinase(s) responsible for FSH-induced phosphorylation. H89, a cyclic AMP-dependent protein kinase (PKA) inhibitor, inhibited FSH-induced phosphorylation both in vivo and in vitro, whereas PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor, had little effect on the phosphorylation level. Ca2+-dependent protein kinase (PKC) appeared to negatively regulate phosphorylation. Phosphopeptide mapping with or without H89 treatment indicated that PKA could be responsible for phosphorylation of the four serine residues. In addition, the purified catalytic subunit of PKA could phosphorylate the recombinant C-terminal region of Cx43, but not the variant in which all four serine residues were substituted with alanine. These results suggest that FSH positively regulates Cx43-mediated channel formation and activity through phosphorylation of specific sites by PKA.  相似文献   

6.
The carcasses and meat and fat properties of a group of five bulls fed a basal diet with an addition of 0.80 kg of dried pig excrements for 135 days were compared with a group of five control bulls fed the basal diet. The pre-mortal examination of the clinical state of the animals, the post-mortal veterinary inspection of the meat and internal organs, and the comparison of blood transaminase activity did not reveal any signs of health disorders ascribable to the experimental diet. The organoleptic examination and determination of the basic and some other technological characteristics and other properties of the meat and fat did not show any significant differences in quality to justify a decision by which the meat of the experimental bulls would be treated as less valuable or non-edible.  相似文献   

7.
The aim of our study was to examine effects of the length of semen equilibration as well as two freezing techniques on ram sperm post-thaw quality. The ejaculates of Wallachian sheep rams (n = 12) were collected by an electro-ejaculation, equilibrated in a Triladyl® (0, 2, 4, 6, and 8 h) containing glycerol and egg yolk and frozen by programmable freezing (PF) or manual freezing (MF). After thawing, sperm samples were subjected to the motility (computer-assisted sperm analysis [CASA]), viability (SYBR-14/PI), and fertilizing ability (FA) (in vitro penetration/fertilization test on bovine oocytes) assays. It was found that the equilibration of 6 h (E-6) ensured higher post-thaw sperm motility and progressive movement compared with other lengths tested, irrespective of a freezing technique. The E-6 sperm viability did not differ between PF and MF but was lower (P < 0.05) than control. Sperm FA (E-6) was similar in PF (60.44%) and MF (62%) but slightly lower than in fresh (72.8%). Our data demonstrate that the use of MF was comparable with PF, which can be applied in the field conditions without need in a piece of cost-expensive equipment, which can greatly benefit the gene bank of animal genetic resources.  相似文献   

8.
Theca cells (TCs) play a crucial role in follicular development and atresia. TCs synthesize androgens that act as substrate for granulosa cells (GCs) aromatization to oestrogens needed for follicular growth. However, the effects of TCs in the form of conditioned medium on steroidogenesis in buffalo GCs remain unclear. In the present study, the impacts of TC-conditioned medium (TCCM) on oestrogen synthesis in buffalo GCs were examined. The results showed that TCs secreted principally testosterone, but almost no androstenedione or oestradiol into TCCM. TCs at passage 3 had a stronger secretion capacity of testosterone in TCCM. Furthermore, TCCM collected at 72 hr improved both the expression levels of oestrogen synthesis-related genes (CYP11A1, CYP19A1, 3β-HSD and 17β-HSD) and the secretion levels of estradiol in GCs. The treatment of 72 hr in TCCM promoted both the expression levels of oestrogen synthesis-related genes (CYP11A1, CYP19A1 and 3β-HSD) and the secretion levels of estradiol in GCs. Besides, TCCM that was collected at 72 hr and applied to GCs for 72 hr (72 & 72 hr) improved the sensitivity of buffalo GCs to FSH. This study indicates that TCCM (72 & 72 hr) enhances the steroidogenesis competence of GCs mainly through facilitating the responsiveness of GCs to FSH in buffalo.  相似文献   

9.
Previously, we histochemically examined the localization of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its receptors in porcine ovarian follicles, and demonstrated a marked reduction in the expression of TRAIL-decoy receptor-1 (DcRI) in granulosa cells of atretic follicles. In the present study, to confirm the inhibitory activity of DcR1 in granulosa cells, granulosa cells prepared from healthy follicles were treated with phosphatidylinositol-specific phospholipase C (PI-PLC) to cleave glycophospholipid anchor of DcR1 and to remove DcR1 from the cell surface, and then incubated with TRAIL. PI-PLC treatment increased the number of apoptotic cells induced by TRAIL. The present finding indicated the possibility that TRAIL and its receptors were involved in induction of apoptosis in granulosa cells during atresia, and that DcR1 plays an inhibitory role in granulosa cell apoptosis.  相似文献   

10.
In this study, we cryopreserved pig spermatozoa using carboxylated poly-L-lysine (CPLL) as the cryoprotectant to determine its efficacy. Pig spermatozoa were placed in a freezing extender containing 3% (v/v) glycerol and different CPLL concentrations. The motility indices of the spermatozoa cryopreserved with 0.25% (v/v) CPLL at 6 (59.3), 9 (53.7), and 12 (26.2) h after thawing were significantly higher (P < 0.01 or P < 0.05) than those of the spermatozoa cryopreserved without CPLL (53.7, 40.1, and 17.5 at 6, 9, and 12 h after thawing, respectively). The concentration of CPLL in the freezing extender did not affect the ability of frozen-thawed spermatozoa to fertilize oocytes in vitro. However, the blastocyst formation rate of embryos derived from spermatozoa cryopreserved with 0.25% CPLL (24.6%) was significantly higher (P < 0.01) than that of embryos derived from spermatozoa cryopreserved without CPLL (11.2%). The conception rate of the sows inseminated with spermatozoa cryopreserved with 0.25% CPLL (72.2%) was not significantly different from that of the sows inseminated with spermatozoa stored at 17°C (81.3%). However, the mean number of total piglets born to the former (10.0) was significantly lower (P < 0.05) than that of total piglets born to the latter (13.4). The results showed that CPLL in the freezing extender maintained the motility of frozen-thawed pig spermatozoa and improved the in vitro development of embryos produced by in vitro fertilization. In addition, we have demonstrated that piglets could be obtained with artificial insemination using spermatozoa cryopreserved with CPLL.  相似文献   

11.
为使甜橙嫩芽组织通过超低温保存处理后能顺利分化再生,本文对甜橙嫩芽后培养基进行了筛选。通过柑桔组织常用培养基DM,MS和MT的比较以及在基础培养基中添加不同浓度激素组合,观察了不同激素组合水平对经过超低温前处理甜橙嫩芽的组织培养效果。研究结果表明, 经过前处理的甜橙嫩芽在添加PPM 0.5mgIAA 0.25mgBA 50g蔗糖/L的BM培养基上的成活率可达76.7%,褐化率16.7%,且生长较快,长势良好,推荐为甜橙超低温保存后恢复生长的培养基。  相似文献   

12.
猪成纤维细胞冻存方法的研究   总被引:1,自引:0,他引:1  
试验以猪的成纤维细胞为研究对象,认为在-70℃情况下冻存至少7d不会影响其存活率(可达80%以上),且生长形态正常。同时利用血清饥饿法使成纤维细胞周期同步化,在-70℃冻存2d、4d和7d,细胞存涪率可达92.3%、87.2%和83.6%,甚至在该务件下冻存15d,细胞也能恢复正常的生长。  相似文献   

13.
This study was aimed at testing the hypothesis that insulin-like growth factor binding protein (IGFBP)-3 can modulate hormone-dependent differentiation of granulosa cells in vitro. Granulosa cells from small (1 to 5 mm) follicles were collected from cattle, cultured for 2 d in medium containing 10% fetal calf serum, washed, and then treated for an additional 2 d in serum-free medium with follicle-stimulating hormone (FSH) (50 ng/ml), recombinant human IGF-I (0, 1.3, 4.0, or 13.3 nM), or recombinant human IGFBP-3 (0 to 4.26 nM). In one series of experiments, IGFBP-3 (0.53 and 2.13 nM) inhibited (51% to 92% decreases; P < 0.05) progesterone and estradiol production induced by 1.3 nM of IGF-I, but did not influence (P > 0.10) granulosa cell numbers or steroidogenesis in the absence of IGF-I. Only 4.26 nM of IGFBP-3 inhibited (by 35%) the increase in granulosa cell numbers induced by 1.3 nM of IGF-I. In another series of experiments, 13.3 nM of IGF-I, but not 4.0 nM of IGF-I, was able to completely overcome the inhibitory effect of 4.26 nM of IGFBP-3 on estradiol production. The increase in cell numbers induced by 4.0 and 13.3 nM of IGF-I was attenuated (P < 0.001) by 4.26 nM of IGFBP-3. In a third series of experiments, IGFBP-3 inhibited 125I-IGF-I binding to granulosa cells. These results indicate that IGFBP-3 has a pronounced inhibitory effect on IGF-I action in cultured bovine granulosa cells, and that this inhibitory effect is likely attributable to IGFBP-3 binding/sequestering IGF-I. Thus, IGFBP-3 may play a significant role in regulating granulosa cell proliferation and steroidogenesis during follicular development in cattle.  相似文献   

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Cryopreservation of testicular tissue has become a part of gamete preservation in wild animal post-mortem. Using domestic cats as a model for wild felids, this study aimed to (i) investigate the effect of temperature for testicular tissue storage on sperm quality; (ii) compare efficiency of freezing protocols; and (iii) evaluate properties of cryoprotective agents to protect testicular sperm quality. A pair of testes from each cat (n = 9) was cut into four pieces. Three randomly selected pieces were allocated to be (i) fresh controls; (ii) stored at 4 °C for 24 h; and (iii) stored at room temperature (28 °C) for 24 h. After storage, the testicular tissue from each group was cut into 10 small pieces. One piece was assigned to be a control while the others were assigned to three freezing protocols; -80 °C (n = 3), vitrification (n = 3) or two-step freezing (kept above liquid nitrogen vapour for 10 min and submerged in liquid nitrogen) (n = 3). Each of three pieces was frozen using dimethyl sulphoxide (DMSO), ethylene glycol (EG) or DMSO combined with EG. Sperm membrane (SYBR-14/EthD-1) and DNA (acridine orange) integrity were evaluated before and after cryopreservation. The storage of testicular tissue at room temperature decreased the percentage of sperm with intact membrane in fresh tissue (59.5 ± 30.5 vs 87.9 ± 7.0%, p < 0.05). DNA integrity was decreased after 24-h storage either at 4 °C or room temperature (p < 0.05). The two-step freezing resulted in a higher percentage of sperm with intact plasma membrane than the other techniques. Dimethyl sulphoxide, EG and DMSO combined with EG provided similar protection for the sperm membrane and DNA from cryodamages. In conclusion, storage of testicular tissue at 4 °C is necessary to maintain sperm membrane integrity during transportation of tissue for cryopreservation in the freezing laboratory. The results provide information for male gamete rescue in felid particularly when they die unexpectedly in the field where freezing facilities are not well equipped.  相似文献   

16.
A peptidyl-prolyl isomerase, Pin 1, has been shown to play a role in the regulation of cell cycle progression, both in vitro and in vivo. However, the involvement of Pin 1 during follicular development is not well understood. The aim of this study was first to investigate the expression of Pin 1 mRNA in the granulosa and theca cells of the follicle at different developmental stages of follicles in the bovine ovary, and second, to examine the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the expression of Pin 1 in the cultured bovine granulosa cells. Follicles were classified into four groups based on the diameter (dominant follicles >8.5mm in diameter, subordinate follicles <8.5mm in diameter) and the relative levels of E2 and progesterone (P4) (E2:P4>1, estrogen active; E2:P4<1, estrogen inactive): i.e. preovulatory dominant follicles (POFs); E2 active dominant follicles (EADs); E2 inactive dominant follicles (EIDs); small follicles (SFs). The expression of the Pin 1 gene was significantly increased in the granulosa cells of EADs as compared with those of other follicles, whereas its expression in theca cells did not differ among follicles at different developmental stages. The concentration of 5 ng/ml FSH alone and the combination of 1 ng/ml E2 and 5 ng/ml FSH stimulated the expression of the Pin 1 gene in bovine granulosa cells. Our data provide the first evidence that Pin 1 expression in the granulosa cells but not the theca cells changes during follicular development, and that FSH stimulate the expression of the Pin 1 gene. These results suggest that Pin 1 regulates the timing of cell proliferation and may act as an intracellular signal responder in the granulosa cells during bovine follicle development.  相似文献   

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Semen from Black Bengal bucks was collected to establish a cooling protocol (to −196°C) for buck semen preservation, and to study the effect of freezing on sperm motility and morphology. Semen was diluted with diluents (Triladyl & Tris) and cryoprotectants, filled into straws, sealed, cooled (to 5°C) and equilibrated. After dilution, motility ranged from 75.00% to 76.67% and from 73.33% to 80.00% in Triladyl and Tris diluents, respectively. Motility of sperm after cooling to 5°C in Triladyl and Tris diluents ranged from 65.00% to 66.67% and from 63.33% to 70.00%, respectively. After equilibration in straws, the semen was subjected to a freezing protocol in a computer-controlled biofreezer CL-3000 (cooling at 10°C per minute, from 5°C to −80°C) and plunged into liquid nitrogen. Sperm motility of re-thawed semen varied from 38.33% to 43.33% and from 6.00% to−6.67% in Triladyl and Tris diluents, respectively. Sperm morphology of re-thawed semen was studied and head damage or cryoinjury was found in 2–3% of sperm in Triladyl diluents and 3–6% in Tris diluents. Whether the differences of sperm motility and head damage reflect fertility after artificial insemination is yet unknown and needs to be studied further.  相似文献   

19.
探讨了前列腺素PGF2α类似物氯前列烯醇(Cloprostenol,CLO)对鸡等级前小黄卵泡颗粒细胞增殖的作用及其信号转导途径。颗粒细胞经16 h预培养后用CLO处理24 h,测定细胞的增殖情况。结果显示:0.1~10μg/L的CLO促进了颗粒细胞的增殖,10μg/L时刺激效果最为明显。蛋白激酶A(PKA)激活后颗粒细胞数量增加,PKA抑制剂H89抑制了CLO的促增殖作用。PKC的激活或抑制对CLO的促增殖作用无显著影响。这表明,CLO可通过PKA途径促进鸡等级前小黄卵泡颗粒细胞的增殖。  相似文献   

20.
试验研究了3种不同冷冻保护剂二甲基亚砜(DMSO)、乙二醇(EG)和甘油对羔羊肾原代细胞存活率的影响及复苏后的培养情况。结果表明,冷冻保护液Ⅰ极显著地高于冷冻保护液Ⅱ和Ⅲ的保护效果(P〈0.01);冷冻保护液Ⅲ显著地高于冷冻保护液Ⅱ(P〈0.05)。复苏后的羔羊肾原代细胞于体外培养时存在贴壁迟缓、前期生长速度较慢等现象。  相似文献   

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