激活素A对离体培养大鼠卵泡发育的影响

激活素A是一种由卵泡液中提取出来的蛋白,主要以旁分泌和自分泌途径调节卵泡颗粒细胞功能,而对卵泡发育的影响却不十分清楚。本实验目的旨在探讨激活素A对大鼠离体培养卵泡发育的影响。用胶原酶分离窦前卵泡并分别加入激活素A（140μg/L)、FSH（0．1IU/mL)以及卵泡抑素（FS）在DMEM培养液中进行离体卵泡培养4d,观察卵泡直径、雌二醇分泌的变化。结果表明：激活素A处理组和FSH＋激活素处理组的卵泡直径显著增大（P＜0．01）。FSH＋激活素A处理组雌二醇的分泌显著增加。FS可抑制激活素A对卵泡发育的影响。结果显示：激活素A可以促进未成年大鼠窦前卵泡的生长发育。     

Histological observation of the development of follicles and follicular atresia in immature rat ovaries

To clarify the development of follicular growth and atresia in the immature ovary, rats. ovaries and blood were removed at fixed points during the period from 0 to 35 days after birth (Day 0 to Day 35). The ovaries were immunohistochemically examined, and blood concentrations of serum follicle-stimulating hormone (FSH) and estrogen (E) were measured. We investigated how time-course changes in follicular cell proliferation, estrogen receptor beta (ERbeta), apoptosis, and FSH and E concentrations are connected with follicular growth and atresia. Apoptosis was found in the ova from Day 0 to Day 3. On Day 15, apoptosis occurred in some granulosa cell nuclei in some follicles, but BrdU uptake and the presence of cyclin D2 and ER beta could be observed in other granulosa cells. From Day 17, apoptosis increased in the follicular granulosa cells, and BrdU uptake and the presence of cyclin D2 and ERbeta were decreased. Follicular atresia continued, reaching a peak on Day 30. Serum FSH and E concentrations increased until Day 15, then markedly decreased after Day 17. The mechanism of apoptosis in the ova from Day 0 to 3 has not been clarified. However, the onset of follicular atresia was caused by apoptotic degeneration from Days 15 to 17. These results showed that the oocytes were selected by apoptosis at 2 points in the time-course of the maturation of the ovary.     

Aflatoxin B1-induced injury in monkeys

Short-term effects of small doses of aflatoxin (0.05 mg/kg of body weight twice a week) were studied in rhesus monkeys fed low-protein and high-protein diets. Animals on high- and low-protein diets without aflatoxin administration served as controls. There was no difference in weight gain, hemogram, serum protein levels, and liver function test results between aflatoxin-treated and control animals. However, morphological alterations in hepatocytes in the form of mild degenerative changes, focal areas of necrosis, and increased regenerative activity were seen in the aflatoxin-treated monkeys. Data on cell cycle analysis done by DNA content estimation through microfluorimetry on DNA-fluorescent, acriflavin-stained liver imprints revealed larger number of cells in the S and Gi2 phases in animals given aflatoxin. However, the changes in cell cycle, as well as in morphology, were more marked in the aflatoxin-treated, low-protein-diet group.     

Increase in transforming growth factor beta1 in ovarian follicular fluid following ovarian stimulation and in-vitro fertilization correlates to pregnancy

We have analysed the content of the growth and differentiation regulating peptide, transforming growth factor beta1 (TGFbeta1), in follicular fluid from patients undergoing in-vitro fertilization (IVF), and correlated concentrations of TGFbeta1 with the outcome of the IVF treatment and the concentrations of 17beta oestradiol in serum at ovum retrieval. A total of 88 women with infertility of >3 years duration and age <38 years participated in the study. During IVF treatment, follicular fluid and matched serum samples were collected at ovum retrieval and analysed for TGFbeta1, oestradiol, progesterone, follicle- stimulating hormone (FSH) and luteinizing hormone (LH) using radioimmunoassay and enzyme-linked immunosorbent assay. We found that the TGFbeta1 content in the follicular fluid at the time of oocyte retrieval correlated positively with subsequent pregnancy. In 29 women who became pregnant following IVF, follicular fluid TGFbeta1 values were significantly higher (P=0.005) than in 59 women where IVF was unsuccessful. In the pregnant group, TGFbeta1 values correlated positively with oestradiol at ovum retrieval. TGFbeta1 also correlated positively with the number of fertilized oocytes. TGFbeta1 may thus be important for successful human pre-embryo development, contribute to successful embryo implantation and development and may be necessary for the establishment of pregnancy.      

Hormonal profiles and follicular growth in cycles with imminent ovarian hyperstimulation

Ovarian hyperstimulation syndrome is a common and serious complication of human menopausal gonadotrophin/human chorionic gonadotrophin treatment. We evaluated the changes in the pituitary and ovarian hormone profiles and ultrasonographic follicular regression in 12 patients in whom human menopausal gonadotrophin was discontinued due to 'imminent' ovarian hyperstimulation. Following discontinuation, three distinct periods were observed: (i) days 1-2, the levels of oestradiol, testosterone and prolactin, and the total number of follicles continued to rise; (ii) days 3-6, the levels of oestradiol, testosterone and prolactin declined sharply and the total number of follicles was reduced significantly, while the large and medium sized follicles continued to increase. Levels of follicle-stimulating hormone and luteinizing hormone gradually declined to reach their lowest levels by days 5-6 and then increased. (iii) Thereafter the number of follicles and steroid output declined to early follicular phase levels. We conclude that discontinuation of human menopausal gonadotrophin and withholding human chorionic gonadotrophin in cycles with laboratory signs of 'imminent' ovarian hyperstimulation syndrome, allows regression of the ovarian ultrasonographic finding and prevents the development of clinical symptoms. However, if rescue of the cycle is attempted, human chorionic gonadotrophin should be given during the first 4 days after discontinuation of stimulation.     

A differential effect of protein and calorie deficiency on ovarian follicular growth in the sow

Either protein or calorie deficiency alters the pre-antral growth of ovarian follicles in the sow. Protein deficiency depressed the rates of oocyte, oocyte nuclear and oocyte nucleolar growth in follicles composed of one or more layers of granulosa cells. Calorie deficiency depressed the growth of the follicle envelope but only in multilaminar follicles. It is concluded that the effect of protein deficiency on ovarian follicular growth differs significantly from that of calorie deficiency in this species. Neither treatment altered the maximum dimensions of the oocyte at the completion of the intra-ovarian growth phase (phase A).     

Prolactin and lipopolysaccharide treatment increased apoptosis and atresia in rat ovarian follicles.

Follicular atresia is associated with the presence of increased macrophages within the follicle. What is not known is whether, in the adult rat, macrophages are instrumental in inducing apoptosis and/or atresia or whether they are simply secondary to a hormonally mediated event. As prolactin is an immunoreactive hormone and stimulates the expression of monocyte chemoattractant, the present experiments compared the effects of prolactin treatment with that of an immune challenge with lipopolysaccharide (LPS) on the invasion of macrophages into the follicular and luteal compartments of the ovary and the occurrence of apoptosis/atresia in relation to macrophage invasion. Rats were treated for 3 days with either prolactin or LPS and ovaries obtained at pro-oestrus or oestrus. Prolactin and LPS increased the number of atretic vs. healthy follicles (P < 0.008, chi2) in pro-oestrus ovaries and increased the mean number of apoptotic cells and macrophages (P < 0.05 for some groups). Macrophages were typically observed in the thecal layer, apoptotic cells in the granulosa cell layer, although 84% follicles which had macrophages within the granulosa cell layer also contained relatively high numbers of apoptotic nuclei. Prolactin and LPS treatment in vivo reduced the progesterone response to follicle stimulating hormone (FSH) (P < 0.001) in cultures of ovarian dispersates but did not inhibit the response to forskolin. In contrast, prolactin or LPS added in vitro to the cultures inhibited the progesterone response to forskolin. Results show that both prolactin and LPS increase follicular apoptosis and atresia and reduce the progesterone response to FSH.     

Hydrolase cytochemistry during follicular atresia in mammals

Activity changes of thiamine pyrophosphatase (TPPase), acid phosphatase (aP), non-specific esterase (nE), acid beta-galactosidase (a beta Gal), beta-glucuronidase (beta-Gluc), and beta-D-N-acetylglucosaminidase (NAG) in follicles during atresia were investigated in the ovaries of mice, rats, Mongolian gerbils, hamsters, guines pigs, rabbits, cats, and pigs. Changes of hydrolase activity were highly enzyme dependent, species-specific and mostly confined to the granulosa. Decrease of TPPase activity and increase of lysosomal enzyme activities during atresia appeared to be true for all mammals. The start of activity changes in the time course of atresia depended on the occurrence of the enzyme in the growing granulosa. Continuous increase of lysosomal enzyme activity appeared in follicles where these enzymes could also be found in the growing granulosa. In contrast, when lysosomal enzyme activity was low or could not be detected in the growing granulosa, increased enzyme activity could only be observed at a time when degenerative processes have already progressed considerably. This distribution pattern suggests that hydrolytic enzymes in the granulosa cells as well as hydrolases of invading macrophages participate in this degenerative process. In some mammals, enzyme activity changes appeared in the cumulus oophorus for the first time in advanced stages of degeneration. In some mammals enzyme activity changes were dependent on the developmental stage of the follicle. This stage dependency argues for an interrelationship between activation of lysosomal enzymes and androgen metabolism.     

Dynamics of follicular growth and atresia of large follicles during the ovarian cycle of the guinea pig: Fate of the degenerating follicles,a quantitative study

Background: A quantitative integrated study of healthy ovarian follicles of different sizes and their mitotic activity and of clearly defined atretic stages of involuting large growing follicles at different stages of the guinea pig ovarian cycle is not available in the literature. We considered that such a study would reveal new aspects of ovarian tissue dynamics and provide new information in an organ with a continuous phenotypic transformation of its cellular components. Methods: Ovaries from guinea pigs were removed on days 1 (opening of the vagina), 3, 6, 9, 13, and 16 of the cycle, and the following were measured in serial sections: (1) total number of healthy follicles falling into categories based on the volume occupied by granulosa cells, (2) total number of atretic follicles falling into clearly defined morphological stages of the degenerative and involutionary process affecting medium to large follicles, and (3) proportion of metaphase-arrested granulosa cells, after colcemid injection, in healthy follicles of different size categories. Results: Dynamic patterns of follicular growth and degeneration were revealed that permitted the following main conclusions and observations: (1) small to middle-size follicles can reach the maximal category mass of granulosa mass within 6–7 days, and the number of granulosa cells can increase 6–7-fold during this interval, (2) the cohort that gives rise to 2–6 preovulatory follicles and to the large follicles that will undergo atresia during each cycle varied from 68 to 108 follicles, (3) cell death starts in the granulosa cell layers of large follicles even when neighbouring cells maintain a high mitotic activity and it spreads rapidly; dead granulosa cells are cleare by nucleolysis and cytolysis in the absence of blood leucocytes or neovascularization, (4) foci of atresia are observed also in a few preovulatory follicles, (5) antral cavities of follicles with dead granulosa cells in the process of being lysed shrink and are filled within 2–3 days with large fibroblast-like cells arising from phenotypic transformation of inner layers of theca interna, with no evidence of mitotic activity or angiogenesis; the outer layers of theca interna involute, and by progressive atrophy and a process of cell death, minute nodular structures arise with remnants of the ovum and zona pellucida, and (6) a transient wave of degeneration affects a proportion of small and middle-size follicles during the metestrous period. This process does not resemble the morphological phenomenology of follicular involution, which affects only large follicles. Conclusions: This study contributes to a fuller understanding of the dynamics and time relationships of follicular growth and loss in the guinea pig ovary and provides new morphogenetic information on the atretic process. It would be valuable for the design of experiments on endocrine and paracrine interactions involved in follicular growth and atresia. © 1995 Wiley-Liss, Inc.     

Aflatoxin B1 and M1 contamination in cow feeds and milk from Iran

In this study, 288 milk samples were collected randomly from individual farms in Qazvin province from March to February 2012, Iran. Samples were analysed for Aflatoxin M1 (AFM1) by ELISA; also the Aflatoxin B1 (AFB1) contamination in animal feed was determined at the same time as the sampling of raw milk. One hundred and sixty-three samples (56.59%) were found to contain AFM1 at various levels; the presence of AFM1 was detected in a concentration ranging between 0.01 and 0.22 ppb. In 113 (69.32%) of the 163 milk samples, AFM1 levels were found to be higher than the maximum tolerable limit (0.05 ppb) accepted by the European union/Codex Alimentarius Commission. The mean AFM1 contamination levels in milk in summer and winter were 0.08 and 0.18 ppb, respectively. The AFB1 contamination level in the winter feed (2.27 ± 1.76) was higher than from summer (0.83 ± 0.60) (P < 0.05).     

The effects of vagotomy on compensatory ovarian hypertrophy and follicular activation after unilateral ovariectomy

Following unilateral ovariectomy in the rat, the remaining ovary undergoes rapid compensatory changes including an increase in the number of antral follicles (follicular activation) and an increase in ovarian weight (compensatory ovarian hypertrophy). The ovary is innervated by the vagus nerve (Burden et al., 1983). In the present study, the effects of right and left cervical vagotomy and abdominal vagotomy on follicular activation and compensatory ovarian hypertrophy in the remaining right or left ovary were compared 15 days after unilateral ovariectomy. Neither right nor left cervical vagotomy affected compensatory ovarian hypertrophy of the right or left ovaries but abdominal vagotomy depressed compensatory ovarian hypertrophy in both the right and left ovaries. Left cervical vagotomy did not inhibit follicular activation, but right cervical vagotomy prevented follicular activation in the right but not left ovary. Also, abdominal vagotomy inhibited follicular activation in the right but not the left ovary. In animals with both ovaries which were subjected to the left or right cervical vagotomy or abdominal vagotomy follicular counts in both right and left ovaries were similar. Collectively, these data indicate that the vagus nerve participates in follicular activation after unilateral ovariectomy. The data also indicate that the right ovary is more dependent on vagal influences for follicular activation than the left ovary.     

Aflatoxin B1 albumin adduct levels and cellular immune status in Ghanaians

Although aflatoxins (AFs) have been shown to be immune-suppressive agents in animals, the potential role of AFs in modifying the distribution and function of leukocyte subsets in humans has never been assessed. We examined the cellular immune status of 64 Ghanaians in relation to levels of aflatoxin B1 (AFB1)-albumin adducts in plasma. The percentages of leukocyte immunophenotypes in peripheral blood, CD4+ T cell proliferative response, CD4+ T(h) and CD8+ T cell cytokine profiles and monocyte phagocytic activity were measured using flow cytometry. NK cell cytotoxic function was determined by perforin and tumor necrosis factor-alpha expression in CD3-CD56+ NK cells. AFB1-albumin adducts levels ranged from 0.3325 to 2.2703 (mean = 0.9972 +/- 0.40, median = 0.9068) pmol mg(-1) albumin. Study participants with high AFB1 levels had significantly lower percentages of CD3+ and CD19+ cells that showed the CD69+ activation marker (CD3+CD69+ and CD19+CD69+) than participants with low AFB1 levels (P = 0.002 for both). Also, the percentages of CD8+ T cells that contained perforin or both perforin and granzyme A were significantly lower in participants with high AFB1 levels compared with those with low AFB1 (P = 0.012 for both). Low levels of CD3+CD69+ (r = -0.32, P = 0.016) and CD19+CD69+ (r = -0.334, P = 0.010) cells were significantly associated with high AFB1 levels using correlation analysis. By multivariate analysis, there were strong negative correlations between the percentages of these cells (CD3+CD69+: b = -0.574, P = 0.001, and CD19+CD69+: b = -0.330, P = 0.032) and AFB1 levels. These alterations in immunological parameters in participants with high AFB1 levels could result in impairments in cellular immunity that could decrease host resistance to infections.     

Distribution of androgen receptor in rat ovarian follicles undergoing atresia at the beginning of pregnancy

The immunohistochemical localisation of androgen receptor (AR) was investigated in a cohort of ovarian antral follicles developing, and subsequently undergoing atresia, in a hyperprolactinaemic milieu at the beginning of pregnancy in rats. Differentiation of the investigated follicles, observed during the first 5 days of pregnancy, was accompanied by a centripetal disappearance of androgen nuclear receptor in the granulosa layer, which did not include the cumulus oophorus complex and some antral granulosa cells. This pattern of decline resembled that typical of follicles maturing during the oestrous cycle but took longer to occur. The follicles did not ovulate and subsequently underwent atresia. The degeneration of some follicles was accompanied by a further loss of AR in the cumulus granulosa cells, but a strong positive AR immunoreaction persisted in the oocyte nucleoli. Some perinatal and early antral atretic follicles were found. In most cases their granulosa layers were AR-positive, although often only weakly. However, follicles with AR negative granulosa layers were also encountered. Nuclear immunolabelling for AR was positive in luteinized follicles. It can be concluded that follicular atresia involves changes in AR distribution which can be demonstrated as an abnormal depletion or persistence of AR.     

The effects of 'coasting' on follicular fluid concentrations of vascular endothelial growth factor in women at risk of developing ovarian hyperstimulation syndrome

BACKGROUND: The aim of this study was to assess the effect of withholding gonadotrophins (coasting) during controlled ovarian stimulation (COS) on individual follicle concentrations of follicular fluid vascular endothelial growth factor (VEGF) in women at high risk of developing ovarian hyperstimulation syndrome (OHSS). METHODS: Twenty-two women who had been coasted and 26 optimally responding women (control group) undergoing COS for IVF were studied. At the time of oocyte retrieval, the follicular fluid from four to six individual follicles of different sizes was collected for VEGF analysis. RESULTS: A total of 118 follicles was analysed in the coasted group and 137 in the control group. A negative correlation was observed between the follicle size and VEGF concentration (r = -0.18, P = 0.03) in the control group, which was not seen in the coasted group. Similarly, the correlation between oestradiol (E(2)) and VEGF (r = 0.4, P < 0.0001) observed in the control group was not apparent in the coasted group. Significantly lower concentrations of VEGF were seen in the follicular fluid of the coasted patients. CONCLUSIONS: It is clear that there are differences in follicular fluid VEGF concentrations between the two groups. It is possible that coasting alters the capacity of the granulosa cells to produce VEGF and/or their response to hCG and in this way acts to reduce the severity and incidence of severe OHSS.     

Synthesis and characterization of three Aflatoxin B1‐protein conjugates

Pure aflatoxin B, was obtained after extraction and chromatographic purification from cultures of A. flavus. Aflatoxin B, derivatives suitable for conjugate formation were synthesized. Intermediates and conjugates were characterized spectrophotometrically.