不同保存条件对绵羊卵巢组织卵泡存活的影响

研究旨在通过组织学方法评估卵巢保存形式、温度、时间、保存液对绵羊卵巢组织短期保存以及培养后卵泡存活的影响。结果表明:碎块或整个卵巢组织39℃保存6 h或12 h后,正常卵泡比例与新鲜组织和其他组比较显著下降(P0.05)。经过6 d的组织培养后,在生理盐水中4、20℃保存6、12 h和39℃保存2 h以及在MEM中39℃保存2 h的组织碎块中正常卵泡比例显著低于整个卵巢保存后组织中正常卵泡比例(P0.05)。在MEM中4℃保存6 h或12 h,培养后卵泡存活率显著高于相同条件保存于生理盐水中的组织(P0.05)。此外保存12 h培养后正常卵泡比例显著低于保存2 h处理组(P0.05)。表明在生理盐水和MEM中整个卵巢4、20℃保存2~6 h,或者卵巢碎块20℃保存在MEM中2~6 h都可以用于卵巢体外培养的研究。     

卵巢组织玻璃化冷冻研究进展

卵巢组织玻璃化冷冻可替代直接冷冻卵母细胞或胚胎。玻璃化冷冻卵巢组织在辅助生殖上具有优越性。它无需控制供体的生殖周期 ,也无需取出卵泡。同样这一技术可用于保存濒危动物或受意外伤害的人或动物的卵母细胞 ;可为性成熟前失去生殖能力的动物或人提供生殖保险以及增加卵母细胞的来源 ;可用于建立生殖细胞 (卵母细胞 )的冷冻库。而传统的冷冻技术存在很多弊端。文章综述了玻璃化冷冻卵巢组织的研究背景和现状 ,并指出了其广阔的应用前景     

卵巢组织玻璃化冷冻保存的研究进展

就目前卵巢组织玻璃化冷冻保存的发展状况及影响卵巢冻融成功的因素等几个方面进行综述,并对卵巢组织冷冻保存所存在的问题及今后研究方向提出了自己的看法。     

Correlation Between Sexual Steroids and Angiogenin Secretion in Bovine Ovarian Follicles

Veterinary Research Communications -     

水牛小腔卵泡分离方法的初步研究

本研究比较了不同的分离方法和酶作用的不同时间,以及不同的卵巢表面状态等因素对水牛小腔卵泡分离效果的影响,以期建立有效的水牛小腔卵泡分离体系。结果发现,采用机械与酶结合的方法分离水牛小腔卵泡时,平均每个卵巢获得5.64个小腔卵泡,显著高于机械法分离平均每个卵巢获得的3.12个小腔卵泡数(P＜0.05);当胶原蛋白酶作用12或15 min,每个卵巢平均获得的小腔卵泡数均显著高于5和10 min处理组(P＜0.05),两组之间没有显著差异(P＞0.05),但当消化时间延长到20 min时,卵泡膜被酶消化而破裂,没法分离到小腔卵泡;从表面小于2 mm卵泡且无黄体的卵巢分离获得的小腔卵泡平均数显著高于表面无可见卵泡且有黄体的卵巢组的平均数(7.50和2.25,P＜0.05)。以上结果表明,选用表面无小于2 mm卵泡且无黄体的卵巢,采用机械与酶结合的方法,酶作用时间为12~15 min分离水牛的小腔卵泡,可获得的小腔卵泡数量最多。     

Long‐Term Effect of Deslorelin Implant on Ovarian Pre‐Antral Follicles and Uterine Histology in Female Rats

The objective of the present study was to investigate the inhibitory effects of long‐term deslorelin implant administration on the ovarian and uterine structures of female rats. A total of 16 non‐pregnant female rats were randomly assigned to two groups, each consisting of eight animals. Animals in the implant group (DESL) received subcutaneously (s.c.) a single deslorelin implant (4.7 mg), an analogue of GnRH, while no treatment was applied to the control group (CON). A single adult male rat was introduced into the cages of both the DESL and CON females after 6 weeks of implant administration. After 1 year of implant administration, all animals were killed and follicular structures and volumes of ovaries and uterus were examined using stereological methods. Stereological observations showed that the mean ovarian total volume of the DESL group (0.28 ± 0.07 cm3) was lower than that of the CON group (1.55 ± 0.23 cm3) (p < 0.001). On the other hand, the total number of pre‐antral follicles in the ovaries of DESL (555.32 ± 151.47) females were significantly lower than the control group (1162.96 ± 189.19) (p < 0.001). In the DESL group, the mean volumes of epithelium, endometrium, myometrium and total volume of the uterus were significantly (p < 0.001) lower than in the control groups. In conclusion, these findings indicate that the long‐term deslorelin implant (i) interferes with the normal cyclicity of female rats and (ii) affects the pre‐antral follicle population. Further studies will be required to determine the effects of long‐term deslorelin treatment on the pre‐antral follicle numbers and future fertility in other species.     

牛不同生长发育时期卵泡酶活性及定位

应用组织化学方法观察牛卵巢卵泡中葡萄糖-6-磷酸酶(G-6-P酶)和碱性磷酸酶(AP)在不同发育时期的活性变化及其在卵泡内的定位。旨在探讨这两种酶的活性同卵泡生长发育的关系。结果表明：不同发育时期的卵泡数量与AP和G-6-P酶活性阳性反应呈极显著正相关。其中酶活性阳性反应有腔卵泡数量多于腔前卵泡；但G-6-P酶活性阴性反应卵泡显著多于阳性卵泡，说明AP活性强于G-6-P酶。腔前卵泡酶活性位置主要在颗粒细胞和卵母细胞上，而有腔卵泡酶活性位置主要在内膜细胞上。     

Comparison of the Hormonal and Chemical Composition of the Fluid from Bovine Ovarian Follicles and Cysts

Contents Cystic ovaries and ovaries with pre-ovulatory follicles (POF) of cows were collected at the slaughter house and progesterone (P4), estradiol-l7β (E2), testosterone (T), IGF-I (selected samples only), glucose and lactate were determined in cystic and follicular fluid. According to the levels of P4 and E2, ovarian cysts were classified as luteinized cysts (LC; P4: 345.14 ng/ml, E2: 3.34 ng/ml), follicular cysts (FC; P4: 25.41 ng/rnl, E2 336.51 ng/ml) and mixed-function cysts (MfC; P4: 59.43ng/ml, E2: 76.62ng/ml). There were no differences in steroid hormone and IGF-I concentrations between FC and POF. In LC, glucose levels were lower and lactate levels were higher than in FC, MfC and POF, indicating a changed metabolic pattern. In polycystic ovaries, five times the combination LC X FC and three times the combination LC x MfC was found. From this, it was concluded that the final events leading to cyst formation are controlled by intrafollicular/intraovarian mechanisms rather than systemic mechanisms.     

无血清培养液对牛腔前卵泡体外发育的影响

本文研究了基础培养液中不添加犊牛血清(FCS)及添加不同比例FCS对牛腔前卵泡体外发育和激素分泌的影响。结果显示:添加FCS对腔前卵泡体外生长无明显促进作用,对腔前卵泡的体外发育、存活及激素分泌也影响不大。腔前卵泡在无血清条件下可持续生长、分泌性腺激素。     

Effect of Oxytocin,IBMX and dbcAMP on Rabbit Ovarian Follicles

Oxytocin (OT) and protein kinase A (PKA), a possible intracellular mediator of hormone action in the ovary, can be potent activators of ovarian functions and fertility. Nevertheless, action of OT on ovarian follicle atresia has not been studied yet. Only single administration of PKA activators [3‐isobutyl‐1‐methyl‐xanthine (IBMX) and dibutyryl cyclic adenosine monophosphate (dbcAMP)] on ovarian follicle atresia was studied previously. The aim of this study was to examine the effect of OT (single treatment per one reproductive cycle, multiple treatments for three cycles), IBMX and dbcAMP (multiple treatments) on folliculogenesis and follicular atresia in rabbit. The ovarian cycle in control females was induced only by gonadotropins. Experimental females received co‐administration of gonadotropins with OT, IBMX or dbcAMP (at 50 μg/female). All females were artificially inseminated. Single‐treated females were euthanized after 18–19 h. Multiple‐treated females were euthanized after the third reproductive cycle. Histological sections of the ovaries were prepared and evaluated by a light microscopy. The follicles were divided into four classes according to the structure of granulosa and theca cells as follows: none or small atresia, cystic atresia, obliterative atresia and atresia associated with luteinization. The ovaries from the control and experimental females, treated during one reproductive cycle or three cycles, were compared. Single OT co‐administration increased proportion of follicles with atresia associated with luteinization, but not other types of atresia. No influence of multiple OT co‐administration on follicular atresia was recorded. Multiple IBMX and dbcAMP co‐administration decreased the proportion of atretic follicles and increased the proportion of healthy follicles without atresia.     

Expression of Osteopontin (OPN) mRNA in Bovine Ovarian Follicles and Corpora Lutea

The matricellular protein osteopontin (OPN) plays a role in various physiological processes, including angiogenesis and tissue remodelling. As these processes are essential for the maintenance of ovarian physiology, the aim of the study was to investigate the expression of OPN (mRNA) in ovarian cells and to evaluate whether it can be regulated by gonadotrophins. Using conventional RT‐PCR and real‐time PCR, we have detected and quantified OPN mRNA as well as glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) mRNA expression in bovine granulosa, theca and luteal cells. In all cells examined, both genes were found in equal amounts and no striking variations in the expression could be observed between granulosa, theca and luteal cells. Furthermore, no effect on either OPN or GAPDH mRNA expression was evident after culturing ovarian cells in the presence of gonadotrophic hormones, although the cells were still highly responsive in terms of cAMP formation. Although neither variations between different cell types nor a regulation of OPN mRNA expression by gonadotrophic hormones could be detected, the high and unambiguous mRNA expression in steroidogenic cells suggests that OPN should be added to the growing list of intraovarian factors which may be involved in ovarian physiology.     

Distribution of Cytochrome P450-side Chain Cleavage in the Theca Interna Layers of Bovine Small Antral and Cystic Follicles

Cystic follicle is anovulatory follicular structure that is caused by an endocrine imbalance. The activity of cytochrome P450‐side chain cleavage (P450scc) is essential for the initiation of steroidogenesis in the follicle. The present study was designed to compare the frequency of cells containing P450scc between healthy and atretic small antral follicles, and among several types (I, II and III, classified based on the presence of granulosa layer) of cystic follicles. Paraffin sections of healthy (2–5 mm in diameter), atretic (2–5 mm) and cystic follicles (>25 mm) were immunohistochemically stained with rabbit polyclonal antibody to bovine P450scc. The P450scc‐positive cells were counted in four different regions of the follicles from the apical to the basal side. In small antral follicles and cystic follicles, P450scc‐positive cells were localized in the theca interna layers but not granulosa layers. The P450scc‐positive cell populations decreased in the late atretic follicles compared with the early and advanced atretic follicles at all the regions of follicle. Type III cystic follicles showed significantly lower frequencies of P450scc‐positive cells than those in the types I and II cystic follicles. These results suggest that in both small and cystic follicles in cows, total loss of granulosa cells may be associated with the reduction of frequency of P450scc‐positive cells in theca interna layer.     

新生小鼠卵巢组织玻璃化冷冻保存与异体异位移植研究

开展小鼠新生胎儿卵巢组织冷冻保存与移植研究,为建立家畜卵巢组织冷冻保存与移植技术奠定基础。采用微滴法玻璃化冻存方法处理卵巢,卵巢复苏后移植到昆明系雄性小鼠受体肾囊下,19只雄性受体鼠接受了卵巢移植,每侧肾囊移植2枚,卵巢共移植1日龄小鼠卵巢38个。饲喂28 d有效回收移植卵巢的受体鼠为17只,受体总有效率分别为89.4%;有效回收移植卵巢30个,卵巢总回收率为78.9%。结果表明,小鼠早期卵巢经过冷冻、解冻并异体异位移植后,其原始卵泡能够重新启动生长发育。     

Comparison of Antral and Preantral Ovarian Follicle Populations Between Bos indicus and Bos indicus‐taurus Cows with High or Low Antral Follicles Counts

The objective was to compare populations of antral and pre‐antral ovarian follicles in Bos indicus and Bos indicus‐taurus cows with high and low antral follicle counts. Nelore (Bos indicus, n = 20) and Nelore X Angus (1/2 Bos indicus‐taurus, n = 20) cows were subjected to follicular aspiration without regard to the stage of their oestrous cycle (day of aspiration = D0) to remove all follicles ≥3 mm and induce growth of a new follicular wave. Ovaries were examined by ultrasonography on D4, D19, D34, D49 and D64, and antral follicles ≥3 mm were counted. Thereafter, cows were assigned to one of two groups: high or low antral follicular count (AFC, ≥30 and ≤15 antral follicles, respectively). After D64, ovaries were collected after slaughter and processed for histological evaluation. There was high repeatability in the numbers of antral follicles for all groups (range 0.77–0.96). The mean (±SD) numbers of antral follicles were 35 ± 9 (Bos indicus) and 38 ± 6 (Bos indicus‐taurus) for the high AFC group and 10 ± 3 (Bos indicus) and 12 ± 2 (Bos indicus‐taurus) follicles for the low AFC. The mean number of preantral follicles in the ovaries of Bos indicus‐taurus cows with high AFC (116 226 ± 83 156 follicles) was greater (p < 0.05) than that of Bos indicus cows (63 032 ± 58 705 follicles) with high AFC. However, there was no significant correlation between numbers of antral and preantral follicles.     

Vitrification of canine ovarian tissue using the Ovarian Tissue Cryosystem (OTC) device

The present study evaluated the effect of Ovarian Tissue Cryosystem (OTC) on follicular morphology and density, as well as on stromal cell density of vitrified canine ovarian tissue. Canine ovarian fragments collected from adult female dogs in stages of the random oestrous cycle were fixed (FC, fresh control) or vitrified (VIT) with an OTC device. After vitrification and warming, the fragments were fixed for histological analysis. Overall, the mean percentage of normal pre-antral follicles decreased after vitrification procedure (FC: 74.5% ± 1.6% vs. VIT: 52.05% ± 1.5%). Although the rates of normal primordial (71.1% ± 1.8%) and secondary (0.7% ± 0.4%) follicles vitrified showed a reduction (p < .05), vitrification using OTC showed considerable preservation of follicles, when compared to the fresh control (81.1% ± 1.5% and 2.3% ± 0.6%, respectively). The mean follicular density was maintained after vitrification (FC: 199.65 ± 12.8 vs. VIT: 199.68 ± 10.8), whereas the stromal cell density decreased in the VIT group. Based on the results, we recommend the use of OTC for vitrification of canine ovarian tissue.     

Localization of Estrogen Receptor Beta (ERß) mRNA within Different Bovine Ovarian Follicles

The oestrogen receptor beta (ERß) is largely distributed in the ovary of many species but data for the bovine ovary are scare. Therefore, the expression of ERß mRNA in the different follicles of the bovine ovary was studied using in situ hybridization. Ovarian tissue sections of three cows with different plasma progesterone concentrations were used (cow 1: 3.50 ng/ml; cow 2: 1.00 ng/ml, cow 3: 0.35 ng/ml). A 602 bp fragment of ERß mRNA was cloned, sequenced and digoxigenin (DIG)‐labelled. Subsequently, in situ hybridization was performed by incubating the sections with the DIG‐labelled RNA anti‐sense probe. For the semi‐quantitative evaluation of ERß mRNA expression the ERß mRNA score (SER) was determined for the different follicular cell types using the formula: SER = 0.n0 + 1.n1+ 2.n2 + 3.n3 with n0, n1, n2, n3 indicating the percentage of cells exhibiting a staining intensity 0 (absent), 1 (weak), 2 (moderate) or 3 (strong), respectively. High ER mRNA levels were noticed in primordial and primary follicle cells, and suggest a role of ER mRNA in early folliculogenesis. A lower SER was observed in the granulosa cells of secondary and tertiary follicles. This significant difference in the SER of follicle cells during follicular growth may be associated with cell proliferation. In obliterative and cystic atretic follicles high SER were observed, although ERß mRNA levels in obliterative follicles showed much inter‐individual variation. This is suggestive for ERß mediated oestrogen action in atretic follicles. In the corpora lutea moderate ERß mRNA levels were noticed. Our findings are in accordance with studies in the ewe in which corpora lutea cells synthesize estrogen. These preliminary findings will be further elaborated in a higher number of cows to examine the role of ERß in the ovary throughout the oestrus cycle.     

Morphological and Apoptotic Comparison of Primordial and Primary Follicles in Cryopreserved Human Ovarian Tissue

There are few reports which were designed to compare the survival rate of human primary follicles with primordial follicles after cryopreservation. This study was designed to evaluate whether such a difference occurs. Human ovarian biopsies were cryopreserved using dimethylsulphoxide/sucrose as the cryoprotectants. Fresh and cryopreserved ovarian samples were evaluated for viability differences between the two types of follicles using the endpoints of histology, ultrastructure and DNA fragmentation. In comparison with fresh ovarian tissue (83.9% ± 10.0%), the percentage of morphologically normal primordial follicles was not significantly different in cryopreserved tissue (73.9% ± 17.2%). However, a lower percentage of primary follicles with normal morphology was seen in the cryopreserved group (43.3% ± 25.7% vs 74.8% ± 19.4% for the fresh group). Transmission electron microscopy revealed that the cryopreservation did not appear to affect the structural integrity of primordial follicles; however, varying ultrastructural damage to the cytoplasm was observed in the majority of the cryopreserved primary follicles. Using a DNA fragmentation assay, the percentage of apoptotic primordial and primary follicles in the unfrozen (26.3% and 20%) and frozen (23.3% and 25%) ovarian tissue was similar. A higher proportion of primary follicles, compared to primordial follicles, suffer histological damage after slow freezing.     

RT-PCR技术检测绵羊卵巢内Ghrelin的表达

为了明确Ghrelin在绵羊卵巢有腔卵泡上是否存在表达,本研究利用实时定量RT-PCR技术检测了绵羊卵巢有腔卵泡内的卵母细胞、卵丘细胞和壁层颗粒细胞的Ghrelin蛋白的表达量情况。结果揭示绵羊卵巢有腔卵泡内各类型细胞Ghrelin mRNA的相对表达量大致相同。绵羊卵巢有腔卵泡内各类型细胞Ghrelin蛋白的表达及Ghrelin mRNA的表达模式,尤其是卵母细胞中的表达,揭示这一新型分子在绵羊卵巢上具有潜在的调控作用。     

牛卵巢黄体状况与腔前卵泡采集数量的关系

根据牛离体卵巢黄体的不同状况，将31枚卵巢分为5种类型，并采用机构方法分离腔前卵泡，观察不同黄体状况卵巢与腔前卵泡采集数量的关系，结果表明，火山口型，圆锥型和蘑菇型3种大黄体的卵巢腔前卵泡采集量多，扁平片状和表面无黄体型卵巢腔前卵泡采集量较少，具有黄体状况的卵巢初级卵泡（Pm)采集数量多于无黄体类型卵巢，原始卵泡（Pf) 以3种黄体较大的卵巢采集数量最多，次级卵泡（Sc)则以黄体为火山口型卵巢为最多，说明卵巢黄体状况不同，其腔前卵泡采集数量有所不同。