Zhangfei在小鼠动情周期卵巢和子宫中的分布

探讨Zhangfei(ZF)在动情周期小鼠子宫和卵巢中的分布规律.通过阴道涂片方法确定昆明雌性小鼠的动情周期,利用免疫组化SP法检测了ZF在小鼠动情周期子宫和卵巢组织中的定位.ZF在各级卵泡的卵母细胞中均呈阳性分布,且主要定位于胞质中;次级卵泡和三级卵泡的颗粒细胞层中可见ZF的存在;成熟卵泡的颗粒细胞中ZF蛋白的阳性更...     

Angiogenesis in Developing Follicle and Corpus Luteum

Angiogenesis is a process of vascular growth that is mainly limited to the reproductive system in healthy adult animals. The development of new blood vessels in the ovary is essential to guarantee the necessary supply of nutrients and hormones to promote follicular growth and corpus luteum formation. In developing follicles, the pre-existing endothelial cells that form the vascular network in the theca layer markedly develop in response to the stimulus of several growth factors, mainly produced by granulosa cells, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The angiogenic factors also promote vessel permeability, thus favouring the antrum formation and the events inducing follicle rupture. After ovulation, newly formed blood vessels cross the basement membrane between theca and granulosa layers and continue a rapid growth to sustain corpus luteum development and function. The length of luteal vascular growth varies in cycling and pregnant animals and among species; both angiogenesis and subsequent angioregression are finely regulated by systemic and local factors. The control of angiogenic development in the ovary could be a useful tool to improve animal reproductive performances.     

The localization and expression of epidermal growth factor and epidermal growth factor receptor in bovine ovary during oestrous cycle

Epidermal growth factor (EGF) is one of the important regulatory factors of EGF family. EGF has been indicated to effectively inhibit the apoptosis of follicular cells, to promote the proliferation of granulosa cells and the maturation of oocytes, and to induce ovulation process via binding to epidermal growth factor receptor (EGFR). However, little is known about the distribution and expression of EGF and EGFR in cattle ovary especially during oestrous cycle. In this study, the localization and expression rule of EGF and EGFR in cattle ovaries of follicular phase and luteal phase at different time points in oestrous cycle were investigated by using IHC and real-time qPCR. The results showed that EGF and EGFR in cattle ovary were mainly expressed in granulosa cells, cumulus cells, oocytes, zona pellucida, follicular fluid and theca folliculi externa of follicles. The protein and mRNA expression of EGF/EGFR in follicles changed regularly with the follicular growth wave both in follicular and in luteal phase ovaries. In follicular phase ovaries, the protein expression of EGF and EGFR was higher in antral follicles than that of those in other follicles during follicular growth stage, and the mRNA expression of EGFR was also increased in stage of dominant follicle selection. However, in luteal phase ovaries, the growth of follicles was impeded during corpus luteum development under the action of progesterone secreted by granular lutein cell. The mRNA and protein expressions of EGF and EGFR in ovarian follicles during oestrous cycle indicate that they play a role in promoting follicular development in follicular growth waves and mediating the selection process of dominant follicles.     

Leptin Immunohistochemical Staining in the Porcine Ovary

This study aimed to investigate leptin immuno‐staining of the porcine ovary in different reproductive stages. Ovaries from 21 gilts were collected from slaughterhouses. The ovarian tissue sections were incubated with a polyclonal anti‐leptin as a primary antibody. The immuno‐staining in ovarian tissue compartments was calculated using imaging software. Leptin immuno‐staining was found in primordial, primary, preantral and antral follicles. Leptin immuno‐staining was expressed in the oocyte and granulosa and theca interna layers in both preantral and antral follicles. In the corpora lutea, leptin immuno‐staining was found in the cytoplasm of the luteal cells. The leptin immuno‐staining in the granulosa cell layer of preantral follicles did not differ compared to antral follicles (90.7 and 91.3%, respectively, P > 0.05). However, the leptin immuno‐staining in the theca interna layer of preantral follicles was lower than antral follicles (49.4 and 74.3%, respectively, P < 0.001). There was no difference in leptin immuno‐staining in the granulosa cell layer between follicular and luteal phases (92.4 and 89.7%, respectively, P > 0.05). However, the leptin immuno‐staining in the theca interna layer of follicular phase was greater than that in the luteal phase (72.7 and 51.0%, respectively, P < 0.001). These findings indicated that leptin exists in different compartments of the porcine ovary, including the oocyte, granulosa cells, theca interna cells, corpus luteum, blood vessel and smooth muscles. Therefore, this morphological study confirmed a close relationship between leptin and ovarian function in the pig.     

藏绵羊卵巢组织学及卵泡超微形态的观察

旨在通过观察藏绵羊卵泡、黄体的组织学特征及卵泡的超微形态,探讨其与生理功能的关系.本研究运用大体解剖、常规组织切片和H.E染色及透射电镜技术对藏绵羊卵巢卵泡和黄体的组织结构特点以及卵泡的超微形态进行观察和分析.结果发现,藏绵羊黄体期和卵泡期卵巢的宽度和厚度存在显著差异(P＜0.05),而重量和长度无显著差异(P＞0.0...     

Morphological study of the corpus luteum in the rat

The cyclic related growth and regression of the corpus luteum during four consecutive oestrous cycles of the regular four-day-cycling (ie, 16 days), virgin albino Wistar rat were followed up by light and electron microscopic investigation of the ovaries. After ovulation, follicular granulosa cells differentiated into luteal cells in the newly formed corpus luteum. Based on their specific histological characteristics, four various types of corpus luteum in each stage of the oestrous cycle could be identified. As soon as the luteal cells started to degenerate, the number of fibroblasts progressively increased and apoptotic degeneration of luteal cells was initiated and became most prominent during oestrus. Complete regression of the corpus luteum was seen after 15 days. This study shows a strictly organised pattern of luteal cell growth and degeneration in the corpus luteum of the regular four-day-cycling, virgin Wistar rat. The morphological alterations may be regulated by a hormonal fluctuation.     

Dynamics of apoptosis-related gene expression during follicular development in yak

The potential reproduction power of domestic animals is limited by a complicated follicular atresia process. P53, caspase-9 (Casp9), Bax, Bcl-2 and Fas play a crucial role in the ovarian mitochondrion-dependent apoptosis and death receptor pathway. In accordance with this study, the expression levels of Casp9, Bax, Bcl-2 and Fas were analysed in ovaries and oviducts of yak by immunohistochemistry (IHC). P53 and the above in ovarian granulosa cells (GCs) from atretic (3–6 mm) to healthy follicles (6–8 mm) and in oviducts were examined from the luteal phase to the follicular phase during the oestrous circle by Western blot (WB) and real-time PCR (RT-PCR). Results demonstrated that typical classic apoptotic factors Casp9, Bax, Bcl-2 and Fas were expressed in the cytoplasm and zonal pellucida of oocytes, primordial follicles, primary follicles, ovarian surface epithelium, ovarian GCs, granular lutein cells, surface epithelia in oviduct uterotubal junction and oviduct ampulla during the luteal phase. RT-PCR and WB revealed that P53 and Fas significantly increased in GCs of atretic follicles. P53 and Casp9 increased in oviduct epithelium during the luteal phase, but Fas was unchanged. A contrary tendency was noted in Bcl-2 and Bax expression. Overall, P53 and Fas play an essential role in inducing GC apoptosis, and Bax, Bcl-2, Casp9 and P53 are involved in oviduct epithelial regeneration in yak.     

犬不同生殖周期阶段卵巢的组织结构观察

为了探讨犬卵巢组织结构和生殖周期阶段的相关性,试验对犬不同生殖周期阶段卵巢的外观形态和组织结构进行观察。结果表明,犬卵泡期、黄体期和乏情期卵巢体积分别为812.63、1081.80和446.03 mm³,黄体期高于卵泡期和乏情期(P＜0.05),卵泡期高于乏情期(P＜0.05);卵泡期、黄体期和乏情期卵巢质量分别为0.89、1.14和0.71 g,卵泡期低于黄体期且高于乏情期,但3者之间不存在显著性差异(P＞0.05);卵泡期卵巢中可见较多次级卵泡和少量成熟卵泡,黄体期卵巢中可见部分次级卵泡和闭锁卵泡,并有大量黄体存在,乏情期卵巢中卵泡类型主要以原始卵泡为主。可见,犬卵巢形态及组织结构与所处生殖周期阶段有关。     

Cell‐specific Distribution of Progesterone Receptors in the Bovine Ovary

This study describes the localization of progesterone receptors (PR) in the bovine ovary. Ovaries were obtained from 11 non‐pregnant and two pregnant cows. Progesterone receptors were visualized by immunohistochemistry on paraffin sections. Nuclear staining for PR was observed in cells of the follicles, corpora lutea, theca layers, surface epithelium, tunica albuginea, and in superficial and deep stroma cells. No staining was noticed in apoptotic bodies of atretic follicles. Expression of PR in follicle cells indicates an intrafollicular role of progesterone. The higher expression in thecal cells compared with follicle cells indicates that thecal cells mediate some effects of progesterone on the follicular development. Superficial stroma cells showing high expression might have a similar influence on primordial and primary follicles. In general, luteal cells had a lower expression than follicle cells, which may be explained by the down‐regulatory effect of locally produced progesterone. The lower expression in luteal cells during pregnancy can be due to the longer life span of this corpus luteum and concomitant degeneration of its PR. The high and rather constant expression of PR in cells of the surface epithelium remains to be elucidated.     

Erratum

This study describes the localization of progesterone receptors (PR) in the bovine ovary. Ovaries were obtained from 11 non‐pregnant and two pregnant cows. Progesterone receptors were visualized by immunohistochemistry on paraffin sections. Nuclear staining for PR was observed in cells of the follicles, corpora lutea, theca layers, surface epithelium, tunica albuginea, and in superficial and deep stroma cells. No staining was noticed in apoptotic bodies of atretic follicles. Expression of PR in follicle cells indicates an intrafollicular role of progesterone. The higher expression in thecal cells compared with follicle cells indicates that thecal cells mediate some effects of progesterone on the follicular development. Superficial stroma cells showing high expression might have a similar influence on primordial and primary follicles. In general, luteal cells had a lower expression than follicle cells, which may be explained by the down‐regulatory effect of locally produced progesterone. The lower expression in luteal cells during pregnancy can be due to the longer life span of this corpus luteum and concomitant degeneration of its PR. The high and rather constant expression of PR in cells of the surface epithelium remains to be elucidated.     

Possible role of growth hormone, IGFs, and IGF-binding proteins in the regulation of ovarian function in large farm animals.

The aim of the study and short review was to present evidence that growth hormone (GH), locally produced insulin-like growth factors (IGFs), and IGF-binding proteins (IGFBPs) may have an important role in the control of ovarian function. There is clear evidence for a distinct GH-receptor mRNA expression and protein production in follicles (oocytes and granulosa-cumulus cells) and corpus luteum (CL). In hypophysectomized ewes, GH and LH are necessary for normal CL development. IGF-1 mRNA in the follicles is expressed in theca interstitial cells (TIC) and granulosa cells (GC) with already higher levels in the TIC before follicle selection. In contrast, IGF-2 is mainly expressed in the TIC. The IGFR-1 mRNA is expressed in both the TIC and GC, with increasing levels in GC during the final development of dominant follicles. IGF-1 is a very potent stimulator of progesterone and oxytocin release in GC. IGFBP-1, -2, -3, -4, -5, and -6 have been isolated from follicular fluid or ovarian tissue. Studies indicate that IGFBP expression and production in the developing follicle is dependent on both cell type and follicle size and is regulated by IGF-1 and gonadotropins. The highest expression of IGF-1 and IGFR-1 mRNA was demonstrated during the early luteal phase. Distinct receptors for IGF-1 and IGF-2 were present in CL membrane preparations at all stages investigated. Intense immunostaining for IGF-1 was observed mainly in bovine large and small luteal cells and in a limited number of endothelial cells. In contrast, IGF-2 protein was localized in perivascular fibroblast and pericytes of the capillaries. With the use of a microdialysis system, we found that in vitro and in vivo IGF-1, IGF-2, and GH stimulated the release of progesterone in cultures of luteal cells or intact tissues. In conclusion, there is clear evidence for a central role of the IGFs, IGFBPs, and GH in follicular development and CL function.     

Immunohistochemical Localization of Luteinizing Hormone Receptor in the Cyclic Gilt Ovary

Luteinizing hormone receptor (LHR) is a specific membrane receptor on the granulosa and theca cells that bind to luteinizing hormone (LH), resulting in androgen and progesterone production. Hence, the regulation of LHR expression is necessary for follicle maturation, ovulation and corpus luteum formation. We examined the immunolocalization of LHR in cyclic gilt ovaries. The ovaries were obtained from 21 gilts aged 326.0 ± 38.7 days and weighing 154.6 ± 15.7 kg. The ovarian tissues were incubated with rabbit anti‐LHR polyclonal antibody. The follicles were categorized as primordial, primary, preantral and antral follicles. Ovarian phase was categorized as either follicular or luteal phases. The immunolocalization of LHR was clearly expressed in primary, preantral and antral follicles. LHR immunostaining was detected in the cytoplasm of granulosa, theca interna and luteal cells. LHR immunostaining was evaluated using imaging software. LHR immunostaining in the theca interna cells in antral follicles was almost twice as intense as that in preantral follicles (65.4% versus 38.3%, P < 0.01). LHR immunostaining was higher in the follicular phase than in the luteal phase (58.6% versus 45.2%, P < 0.05). In conclusion, the expression of LHR in the theca interna cells of antral follicles in the follicular phase was higher than in the luteal phase. The expression of LHR in all types of the follicles indicates that LHR may impact follicular development from the primary follicle stage onwards.     

Activins and Inhibins: Expression and Role in Normal and Pathological Canine Reproductive Organs: A review

Activins and inhibins are regulatory proteins of the reproductive function. Inhibins antagonise the activin signalling at different levels and are responsible for the negative feedback in the regulation of the release of pituitary follicle stimulating hormone (FSH), which, in turn, is promoted by locally produced activins. In the canine ovary, both peptides are expressed by developing follicles and corpora lutea. Activins may play a stimulatory role in follicular development, promoting the aromatase function; inhibins modulate these processes and suppress the hyperplasic/neoplastic stimuli. Activins are required for ovulation and corpus luteum formation, while inhibins stimulate progesterone synthesis. The exclusive production of alpha‐inhibin by granulosa cells allows the peptide to be used as marker to identify canine ovarian stromal tumours by immunohistochemistry. In the male, activins are powerful morphogenetic factors in the foetal testis. In the adult, they display a modulating action on spermatogenesis and Sertoli cell function. Inhibins, produced mainly by Leydig cells, promote testosterone secretion. Canine testicular tumours, such as Leydig, Sertoli and granulosa cell tumours (GCTs), may express inhibin subunits and produce high circulating levels of these glycoproteins. In the canine prostate, activins inhibit epithelium proliferation, antagonising androgen effects, but they are synthesised under androgenic stimulus.     

玉米赤霉烯酮中毒大鼠卵巢组织Bax和Bcl-2的表达

10周龄SD大鼠单剂量腹腔注射5mg／kg玉米赤霉烯酮玉米油溶液，分别于3、6、12、24、48h时剖杀取卵巢组织，检测不同时间卵巢组织Bax和Bcl-2的表达。结果显示，病理组织学观察发现卵巢组织出现不同程度损伤，卵泡颗粒细胞发生凋亡。免疫组化SP法试验组与对照组卵巢组织中均有Bax和Bcl-2的表达，并且随着时间的推进呈现动态变化。Bax在3、6、12h时表达量上调，表达量与对照组比较差异显著（P〈0．05），24、48h时表达量下降，与对照组比较差异不显著（P〉O．05）。3～48h卵巢组织中Bcl-2表达量与对照组比较差异不显著（P〉0．05）。结果表明，大鼠玉米赤霉烯酮中毒可引起卵巢组织的病变及颗粒细胞凋亡，且Bax在玉米赤霉烯酮中毒大鼠卵巢颗粒细胞凋亡中起着重要作用，Bcl-2的作用不明显。     

Expression of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in the ovarian follicles and corpora lutea of pregnant swine

The objective of the study was to demonstrate the presence of estrogen receptor alpha (ERalpha) and beta (ERbeta) protein and corresponding mRNA in porcine ovarian follicles and corpora lutea obtained on day 10, 18, 32, 50, 71 and 90 post coitum (p.c.) using immunohistochemistry, Western blot, and RT-PCR analysis. Immunohistochemistry showed that ERalpha protein was located in the granulosa cells of ovarian follicles and the strongest immunoreaction was observed on days 32 and 50 p.c. The ERbeta protein was found mainly in theca cells of follicles as well as in luteal cells. The most intense immunoreaction was observed on day 18 p.c. within theca cells, while in the corpus luteum (CL) the intensity of ERbeta staining gradually increased and remained elevated at mid and late pregnancy. In CL by day 50 p.c. immunoreaction for ERbeta was present only in small luteal cells, but starting from day 71 to 90 p.c. it was observed in both small and large luteal cells. Western blot analysis was performed and validated data obtained from immunohistochemistry. RT-PCR results indicated that ERalpha mRNA was expressed only in ovarian follicles of the pregnant swine, while that of ERbeta in both follicles and CL. The results suggest an autocrine/paracrine role of estrogens acting via both ERalpha and ERbeta in the regulation of the ovarian function during pregnancy and for the process of successful reproduction.     

The Adverse Effect of Phytoestrogens on the Synthesis and Secretion of Ovarian Oxytocin in Cattle

The current investigations were undertaken to study the mechanism of the adverse effect of phytoestrogens on the function of bovine granulosa (follicles >1< cm in diameter) and luteal cells from day 1–5, 6–10, 11–15, 16–19 of the oestrous cycle. The cells were incubated with genistein, daidzein or coumestrol (each at the dose of 1 × 10^?6 m ). The viability and secretion of estradiol (E2), progesterone (P4) and oxytocin (OT) were measured after 72 h of incubation. Moreover, the expression of mRNA for neurophysin‐I/OT (NP‐I/OT; precursor of OT) and peptidyl‐glycine‐α‐amidating monooxygenase (PGA, an enzyme responsible for post‐translational OT synthesis) was determined after 8 h of treatment. None of the phytoestrogens used affected the viability of cells except for coumestrol. The increased secretion of E2 and P4 was only obtained by coumestrol (p < 0.05) from granulosa cells from follicles <1 cm in diameter and decreased from luteal cells on days 11–15 of the oestrous cycle, respectively. All three phytoestrogens stimulated (p < 0.05) OT secretion from granulosa and luteal cells in all stages of the oestrous cycle and the expression of NP‐I/OT mRNA in the both types of cells. The expression of mRNA for PGA was stimulated (p < 0.05) by daidzein and coumestrol in granulosa cells, and by genistein and coumestrol in luteal cells. In conclusion, our results demonstrate that these phytoestrogens can impair the ovary function in cattle by adversely affecting the synthesis of OT in follicles and in corpus luteum. However, their influence on the ovarian steroids secretion was less evident.     

Lectin histochemical pattern on the normal and cystic ovaries of sows

The lectin histochemical pattern (LHP) was characterized and compared in normal and cystic ovaries of sows. Six biotinylated lectins (PNA, SBA, WGA, RCA‐1, DBA and UEA‐1) were used on tissue sections. In the normal ovaries, the reaction to UEA‐1 and SBA was mild to moderate in mesothelial and endothelial cells. RCA‐1 staining was mild to moderate in theca interna of growing follicles, corpora luteum and mesothelium. In addition, this lectin presented strong reaction in endothelial cells, granulosa cells of atretic follicles, zona pellucida of growing follicles and plasma. DBA showed strong intensity in mesothelial and endothelial cells. There was mild to moderate reactivity to WGA in granulosa cells, corpus luteum and theca interna of follicles in development, and moderate in zona pellucida, in granulosa cells of atretic follicles and mesothelium. PNA staining was mild to moderate in oocytes and in the adventitia and media of medullary arteries. Changes in the LHP of the cystic ovaries were noted; however, there were no differences in these findings between the follicular and luteinized cysts. UEA‐1 reactivity in the cystic ovaries was moderately reduced in the mesothelial and endothelial cells, whereas there was mild reduction in the DBA staining in the granulosa cells. Reaction to RCA‐1 and WGA in the cysts also was decreased in theca interna, zona pellucida and granulosa cells of atretic follicles. Furthermore, endothelium and theca interna in the cystic ovaries presented mild reduction of marcation to SBA, whereas there was decreased reactivity to PNA in the oocytes and adventitia and media layers of the medullary arteries. The results of the current study show that cysts modify the LHP in swine ovaries. These changes of glycoconjugates in many ovarian structures could modify diverse process and may be one of the reasons for decreased fertility in sows.     

Expression of steroidogenic enzymes and synthesis of steroid hormones during development of ovarian follicles in prepubertal goats

Expression of mRNAs encoding cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17 -hydroxylase (P450c17), and cytochrome P450 aromatase (P450arom) were characterized by the RT-PCR technique and concentrations of progesterone (P4), testosterone (T0) and estradiol (E2) were measured by radioimmunoassay during follicular development of prepubertal goats. Synthesis of mRNAs encoding P450scc and P450c17 began in preantral follicles, but mRNA encoding P450arom was not detectable until early antral formation. While mRNA for P450scc was expressed in both theca and granulosa cells, mRNA for P450c17 was expressed only in theca cells while P450arom mRNA only in granulosa cells. In nonatretic follicles from prepubertal ovaries, the relative quantity of mRNA expression of all the three enzymes increased with follicle size; however, while the concentration of P4 and E2 increased, that of T0 decreased with follicle size. While expression of mRNA encoding P450scc was unaffected, that of P450c17 mRNA decreased to the lowest level and mRNA for P450arom became undetectable following atresia; accordingly, while the concentration of P4 increased in the atretic medium follicles, that of T0 and E2 decreased to the lowest level after atresia. While the adult follicular stage follicles showed a similar cytochrome expression as the nonatretic follicles of prepubertal goats, the former contained higher levels of E2 and P4 than the latter. The presence of corpus luteum in an ovary decreased expression of P450scc, significantly in large follicles while it increased concentration of P4. These findings indicated that (1) similar to other species, changes in follicular steroid production in goats were explained in large measure by changes in steroidogenic enzyme expression; (2) while mRNA expression was similar, activities of some of the steroidogenic enzymes may differ between sexually mature and immature goats.