BAX is involved in regulating follicular growth, but is dispensable for follicle atresia in adult mouse ovaries

Mammalian females are endowed with a finite number of primordial follicles at birth or shortly thereafter. Immediately following the formation of the primordial follicle pool, cohorts of these follicles are recruited to begin growth, and this recruitment continues until the primordial follicle population is depleted. Once recruited, a follicle will either grow and ovulate or undergo atresia. Follicle atresia results from the apoptotic death of follicular cells. Members of the BCL-2 family of proteins are important regulators of apoptosis in most cells including in the ovary. Here, we tested the hypothesis that the proapoptotic BAX is an important regulator of follicle survival. We used a variety of histological and biochemical techniques to investigate the impact of Bax deletion on follicle growth and death. We observed that the Bax deletion results in delayed vaginal opening and altered follicular growth. Young adult Bax-deficient ovaries contained increased numbers of primordial follicles and a trend towards reduced numbers of growing follicles. Bax deficiency led to a reduction in average litter size, and also a reduction in the number of oocytes ovulated in response to exogenous gonadotropins. In contrast, Bax deficiency did not alter follicle atresia. In conclusion, BAX appears to be an important regulator of follicle growth, but is dispensable for follicle atresia in mice.     

Oocyte growth and follicular development in KIT-deficient Fas-knockout mice

In mammals, oocyte growth and follicular development are known to be regulated by KIT, a tyrosine kinase receptor. Fas is a member of the death receptor family inducing apoptosis. Here, we investigated germ cell survival, oocyte growth and follicular development in KIT-deficient (Wv/Wv:Fas+/+), Fas-deficient (+/+:Fas-/-), and both KIT- and Fas-deficient (Wv/Wv:Fas-/-) mice during fetal and postnatal periods. Further, the ovaries of these mice were transplanted in immunodeficient mice to compare oocyte growth and follicular development under a condition isolated from the extraovarian effects of KIT- and Fas-deficiency. Higher numbers of germ cells were found in the fetal and postnatal ovaries of Fas-deficient mice than in the same-aged wild-type mice. In KIT-deficient mice, ovaries at 13 days postcoitum (dpc) contained 1106+/-72 (n=3) germ cells, but the ovaries contained no oocytes after birth. Twenty-one days after transplantation of the ovaries at 13 dpc, no oocytes/germ cells were found. A higher number of germ cells (3843+/-108; n=3) were observed in the Wv/Wv:Fas-/- genotypes than in Wv/Wv:Fas+/+ mice at 13 dpc. Furthermore, Wv/Wv:Fas-/- mice contained 528+/-91 (n=3) oocytes at 2 days, and follicles developed to the antral stage at 14 days of age. After transplantation of fetal and neonatal ovaries from Wv/Wv:Fas-/- mice, increased numbers of growing oocytes and developing follicles were obtained compared with those in 14-day old ovaries in vivo. These results show that oocytes grow and follicles develop without KIT signaling, although KIT might be essential for the survival of germ cells/oocytes in mice.     

Seminal plasma regulates ovarian progesterone production, leukocyte recruitment and follicular cell responses in the pig

Seminal plasma (SP) acts to influence the uterine endometrium after mating, activating synthesis of embryotrophic cytokines and inflammatory changes that condition the tract for embryo implantation and establishing pregnancy. The objective of this study was to investigate in pigs whether the ovary might also be responsive to SP exposure. Prepubertal gilts were synchronised with exogenous gonadotrophins and received transcervical treatment with pooled boar SP or PBS; then the ovarian tissue was recovered at 34 h (preovulation) and on days 5 and 9 after treatment. The ovarian response was assessed by measuring ovulation rate, number and size of corpora lutea, ovarian leukocyte populations, progesterone production in vivo, as well as responses of retrieved granulosa cells cultured in vitro. In SP-treated gilts, leukocyte recruitment into the ovarian tissues was increased fourfold at 34 h, with macrophages comprising the most abundant cell lineage. There was no effect of SP on the number of oocytes ovulated; however, the weight of corpora lutea was increased in SP-treated gilts. SP also induced an increase in plasma progesterone content seen from day 5 to at least day 9 after treatment. In addition, granulosa cells and thecal tissue retrieved from preovulatory follicles of SP-treated gilts were more responsive in vitro to growth factor- and gonadotrophin-stimulated cell proliferation and progesterone synthesis. These results suggest that uterine exposure to SP influences immune cell trafficking in the ovary and enhances steroidogenesis in early pregnancy. The effects of SP on ovarian function potentially contribute to reproductive success in the pig.     

Immunization with ovarian autoantigens leads to reduced fertility in mice following follicular dysfunction

Immunoproteomics using sera of women with ovarian autoimmune diseases such as primary ovarian insufficiency and IVF embryo transfer recruits led to identification of three proteins namely alpha actinin 4 (α-ACTN4), heat-shock 70 protein 5 (HSPA5), and actin beta (ACTB). This study deals with the establishment of a peptide ELISA for screening sera of antiovarian antibody (AOA)-positive patients and further delves into understanding the role of these three proteins in ovarian autoimmunity in a mouse model. Using in silico approach, antigenic peptides of these proteins were identified and used for peptide ELISA. ELISA results indicated that AOA-positive sera showed reactivity with only specific peptides. The functional significance of the dominant peptides was studied by active immunization of female mice with these peptides. All immunized mice generated high antibody titers and profound effect on ovaries with few primordial (2.4±0.1, 2.4±0.2, and 2±0.1), primary (2.4±0.5, 1.7±0.3, and 2.4±0.3), preantral (2.3±0.5, 3.4±0.3, and 2.9±0.3), antral (0.9±0.2, 1.6±0.8, and 2.3±0.6) follicles, and corpora lutea (2.8±0.8, 2.9±1.7, and 4.6±2.3), and increased number of atretic follicles (5.5±0.4, 4.9±1.8, and 7.5±1.0) in ACTN4-, HSPA5-, and ACTB-immunized mice compared with control animals (3.0±0.2, 3.5±0.6, 3±0.1, 3.6±0.2, 4.7±0.3, and 1.5±0.3) respectively. These mice when mated with fertile male mice showed an overall 25-43% reduction in fertility compared with controls. The data clearly suggest that the dominant antigenic epitopes of the three proteins play critical role in fertility and could possibly be the key autoimmune targets. These epitopes could be used to develop a more specific and sensitive diagnostic test for women with ovarian autoimmune diseases and to design therapy for disease management for reinstatement of ovarian function.     

Ovarian VEGF(165)b expression regulates follicular development, corpus luteum function and fertility

Angiogenesis and vascular regression are critical for the female ovulatory cycle. They enable progression and regression of follicular development, and corpora lutea formation and regression. Angiogenesis in the ovary occurs under the control of the vascular endothelial growth factor-A (VEGFA) family of proteins, which are generated as both pro-(VEGF(165)) and anti(VEGF(165)b)-angiogenic isoforms by alternative splicing. To determine the role of the VEGF(165)b isoforms in the ovulatory cycle, we measured VEGF(165)b expression in marmoset ovaries by immunohistochemistry and ELISA, and used transgenic mice over-expressing VEGF(165)b in the ovary. VEGF(165)b was expressed in the marmoset ovaries in granulosa cells and theca, and the balance of VEGF(165)b:VEGF(165) was regulated during luteogenesis. Mice over-expressing VEGF(165)b in the ovary were less fertile than wild-type littermates, had reduced secondary and tertiary follicles after mating, increased atretic follicles, fewer corpora lutea and generated fewer embryos in the oviduct after mating, and these were more likely not to retain the corona radiata. These results indicate that the balance of VEGFA isoforms controls follicle progression and luteogenesis, and that control of isoform expression may regulate fertility in mammals, including in primates.     

Camel milk regulates T-cell proliferation to alleviate dextran sodium sulphate-induced colitis in mice

The incidence of inflammatory bowel disease (IBD) is increasing in China. Current clinical methods of treatment for IBD are accompanied by side effects, and thus, the search for effective therapeutic drugs with minimal adverse effects is necessary. In the present study, the effects of camel milk (CM) on dextran sodium sulphate (DSS)-induced acute and chronic colitis in a mouse model were investigated. The results showed CM effectively alleviated the injury induced by DSS to the colon mucosa and imbalance of immune cells in mice. However, treatment with CM significantly increased the body weight of mice and decreased the disease activity index (DAI), histopathological score, proliferation of Th17 cells and concentration of inflammatory cytokine IL-17. The results from the present study indicate CM possesses intestinal protective effects.     

Alterations in follicular dynamics and steroidogenic abilities induced by heat stress during follicular recruitment in goats

We investigated the changes in follicular dynamics and steroidogenic activity during heat stress in goats. Adult female goats were exposed to heat stress at 36 degrees C and 70% relative humidity for 48 h and then injected with prostaglandin (PG) F2alpha (the time of PGF2alpha injection was designated as 0 h). In experiment 1, every follicle greater than 2 mm in diameter was monitored by ultrasonography to investigate the follicular dynamics, and plasma concentrations of FSH, LH, progesterone, and oestradiol were measured from -48 h to 120 h. In experiment 2, the follicles were recovered from the goats at 48 h, and the concentration of oestradiol, the aromatase activity, and the LH receptor level in the follicles were determined. In control (non-heat-stressed) goats, ovulatory follicles were mainly recruited from -24 h to 0 h, whereas no follicles recruited during that period were ovulated in the heat-stressed goats. The timing of the recruitment of ovulatory follicles was delayed by heat stress by approximately 24 h. The plasma concentration of oestradiol in the heat-stressed goats was significantly lower from 36 to 54 h compared with the controls, although the concentrations of FSH and progesterone did not differ between the treatments. In addition, the concentration of oestradiol, the aromatase activity, and the LH receptor level in the follicles from heat-stressed goats were significantly lower compared with the controls. These results indicate that heat stress during follicular recruitment suppresses subsequent growth to ovulation, accompanied by decreased LH receptor level and oestradiol synthesis activity in the follicles.     

Involvement of follicular basement membrane and vascular endothelium in blood follicle barrier formation of mice revealed by 'in vivo cryotechnique'

The molecular sieve with size- and charge selectivity in ovarian follicles, the so-called blood-follicle barrier (BFB), was examined during follicular development under physiological conditions to reveal ovarian structures responsible for the BFB by using our 'in vivo cryotechnique' (IVCT). Mouse ovary specimens were prepared with different methods including IVCT, immersion, or perfusion chemical fixation and quick-freezing following resection or perfusion. Their paraffin sections or cryosections were stained with hematoxylin-eosin or immunostained for serum proteins with different molecular weights: albumin, immunoglobulin (Ig) G1 heavy chain, inter-alpha-trypsin inhibitor (I alpha I), fibrinogen, and IgM. Their immunoreactivity was better preserved with IVCT. The immunostaining for albumin was clearly observed in blood vessels, interstitium, and developing follicles, but that of IgG1, I alpha I, or fibrinogen was significantly decreased inside the follicles. IgM was immunohistochemically decreased throughout the interstitium outside blood vessels. The immunoreactivities of IgG1 and IgM, as compared with albumin, were clearly changed along follicular basement membranes and around vascular endothelial cells respectively. These findings indicate that BFB functions throughout follicular development, and the follicular basement membrane and the vascular endothelium could play some significant roles in the permselectivity for such soluble proteins with intermediate and high molecular weight respectively.     

Effect of bovine follicular fluid and follicle-stimulating hormone on follicular growth in unilaterally ovariectomized prepuberal heifers

A study was conducted to determine if charcoal-extracted follicular fluid inhibits FSH-induced follicular development in prepuberal heifers. Thirty-six prepuberal heifers were allotted by breed and weight to a 2 x 2 factorial experiment involving charcoal-extracted follicular fluid and FSH treatments. Heifers were unilaterally ovariectomized and injected (intravenously; 10 ml) every 8 h for 88 h with either charcoal-extracted follicular fluid or saline. Follicle-stimulating hormone (2 mg) or saline was injected (intramuscularly) every 8-h starting 24 h after initiation of charcoal-extracted follicular fluid to 88 h following unilateral ovariectomy. Plasma samples were collected at 8-h intervals from 48 h prior to unilateral ovariectomy to 96 h following unilateral ovariectomy when the remaining ovary was removed. Follicular fluid and total ovarian weight increased following FSH treatment. The increases were not inhibited by charcoal-extracted follicular fluid. Total number of surface follicles was similar among treatments. However, FSH induced a shift in follicular diameter from small (less than or equal to 3 mm) to medium (7 to 9 mm) or large (10 to 13 mm) follicles, which was unaffected by charcoal-extracted follicular fluid. Plasma concentration of FSH, but not LH, declined following charcoal-extracted follicular fluid administration. In summary, charcoal-extracted follicular fluid did not inhibit FSH-induced follicular development in prepuberal heifers when charcoal-extracted follicular fluid was administered at a dosage that reduced circulating concentration of FSH by approximately 40%.     

Diet‐induced obesity regulates the galanin‐mediated signaling cascade in the adipose tissue of mice

Galanin is a neuropeptide that regulates the food intake, neurogenesis, memory, and gut secretion. This study was conducted to evaluate the high‐fat diet (HFD)‐induced regulation of the galanin receptors (GalRs) and the associated signaling molecules in the adipose tissues of mice. Twenty C57BL/6J mice were given either an HFD or a normal diet for 12 wk. The results of the semiquantitative RT‐PCR analyses indicated that the HFD upregulated the expression of GalR1, GalR2, GalR3, resistance to audiogenic seizures, peroxisome proliferator‐activated receptorγ2, adipocyte protein 2, and protein kinase Cδ and downregulated the expression of peroxisome proliferative activated receptor γ coactivator 1α and uncoupling protein 1 in the adipose tissues. The immunoblot results showed that the protein levels of peroxisome proliferator‐activated receptorγ2 and adipocyte protein 2, and the phosphorylation of c‐Raf and extracellular signal‐regulated kinase 1/2 were increased, while the phosphorylation of cyclic adenosine monophosphate‐responsive element‐binding protein, which regulates peroxisome proliferative activated receptor γ coactivator 1α and uncoupling protein 1, was decreased in the epididymal adipose tissues of the HFD‐fed mice. These results suggest the possible association of the galanin‐mediated signaling pathways in the manifestation of the HFD‐induced activation of adipogenesis along with the suppression of thermogenesis in the adipose tissues of mice.     

Evaluation of the BAX gel and fluorometric systems for the detection of foodborne Salmonella

The present study compared the sensitivity of the BAX automated fluorometric and the recently discontinued BAX gel electrophoresis systems with a standard culture method to detect Salmonella in 333 high-moisture and 171 low-moisture foods. A total of 95 naturally contaminated foods, including 63 high-moisture and 32 low-moisture foods, were detected by the standard culture method. No contaminated samples were identified exclusively by the BAX systems. By means of the analytical protocol stipulated by the manufacturer, the BAX fluorometric system detected 36 (57.1%) and 29 (90.6%) of the contaminated high- and low-moisture foods, respectively. Similar results were obtained with the BAX gel electrophoresis system, which identified 40 (63.5%) and 26 (81.3%) of the contaminated high- and low-moisture foods. The rate of false-positive reactions with the BAX systems was low. Our results indicate that the low sensitivity of the BAX systems with high-moisture foods, notably raw meats and poultry products, was serovar-independent. The high levels of background microflora that commonly occur in raw meat and on fresh fruit and vegetable products, and the high successive dilutions of test materials for PCR analysis, suggestively undermined the sensitivity of the gel and the fluorometric BAX assays. The potential benefits of immunomagnetic separation of Salmonella in preenrichment cultures, of selective broth enrichment following preenrichment to markedly reduce levels of background microflora in PCR test materials, and the use of larger portions of test materials in PCR analyses should be investigated.     

Consequences of transvaginal follicular puncture on well-being in cows

The purpose of this study was to evaluate the impact of repeated follicular puncture used in the ovum pick-up technique on the welfare of cows. The evaluation relies on the physiological measurement of stress, milk production criteria, immune status, and the histological examination of ovaries. Two groups of five Holstein cows were submitted to epidural anaesthesia and genital palpation with insertion of an intravaginal ultrasound probe for transvaginal puncture (the puncture was not performed in the control group). Animals were manipulated twice a week for 8 weeks (16 manipulation sessions). The blood cortisol concentrations increased after each session; however, the concentrations were the same in both the control and the punctured groups. Two adrenocorticotrophic hormone challenge tests, performed before the first session and after the last session, showed an unchanged adrenal sensitivity through repeated puncture sessions. The transvaginal puncture did not affect milk production, or blood and milk somatic cell counts. Ovariectomies were performed on another group of four Holstein cows at various intervals (0 to 30 days) after five similar puncture sessions. Histological examination of the ovaries 4 days after puncture revealed blood-filled follicles and haemorrhagic foci in ovarian stroma, but the examination 30 days after the last puncture session demonstrated very limited, if any, fibrosis. On the basis of the criteria chosen for this study, repeated transvaginal follicular puncture on its own does not impact adversely on the welfare of cows.     

The absence of corpus luteum formation alters the endocrine profile and affects follicular development during the first follicular wave in cattle

We previously established a bovine experimental model showing that the corpus luteum (CL) does not appear following aspiration of the preovulatory follicle before the onset of LH surge. Using this model, the present study aimed to determine the profile of follicular development and the endocrinological environment in the absence of CL with variable nadir circulating progesterone (P(4)) concentrations during the oestrous cycle in cattle. Luteolysis was induced in heifers and cows and they were assigned either to have the dominant follicle aspirated (CL-absent) or ovulation induced (CL-present). Ultrasound scanning to observe the diameter of each follicle and blood collection was performed from the day of follicular aspiration or ovulation and continued for 6 days. The CL-absent cattle maintained nadir circulating P(4) throughout the experimental period and showed a similar diameter between the largest and second largest follicle, resulting in co-dominant follicles. Oestradiol (E(2)) concentrations were greater in the CL-absent cows than in the CL-present cows at day -1, day 1 and day 2 from follicular deviation. The CL-absent cows had a higher basal concentration, area under the curve (AUC), pulse amplitude and pulse frequency of LH than the CL-present cows. After follicular deviation, the CL-absent cows showed a greater basal concentration, AUC and pulse amplitude of growth hormone (GH) than the CL-present cows. These results suggest that the absence of CL accompanying nadir circulating P(4) induces an enhancement of LH pulses, which involves the growth of the co-dominant follicles. Our results also suggest that circulating levels of P(4) and E(2) affect pulsatile GH secretion in cattle.     

The BAX PCR assay for screening Listeria monocytogenes targets a partial putative gene lmo2234

The BAX PCR for screening Listeria monocytogenes is a commercial PCR assay for specifically targeting L. monocytogenes, a foodborne pathogen that can contaminate a variety of foods and cause a potentially fatal disease, listeriosis, among high-risk populations. The high specificity (> 98%) of this PCR assay is achieved by targeting a species-specific genomic region (approximately 400 bp) presumably found only in L. monocytogenes. In this study, the identity of the BAX PCR-targeted genomic region was determined by using PCR cloning, DNA sequencing, and basic local alignment search tool (BLAST) analysis of the amplicon sequences of an L. monocytogenes serotype 1/2a strain. BLAST analysis identified the BAX PCR amplicon (GenBank accession no. AY364605) as a 423-bp genomic region between nucleotides 224,409 and 224,831 in the genome of L. monocytogenes (serotype 1/2a strain EGD-e), including a 145-bp noncoding region and a 278-bp partial coding sequence of a putative gene, lmo2234. The translated amino acid sequence (92 amino acids) of this partial coding region is highly conserved between L. monocytogenes and Listeria innocua (93% homology). Reverse-position-specific BLAST analysis identified a conserved domain in Lmo2234 that was similar (95.3% aligned, E value = 9E-18) to the consensus amino acid sequence of sugar phosphate isomerases/epimerases (National Center for Biotechnology Information conserved domain database accession no. COG 1082.1, IolE), indicating that Lmo2234 might be involved in bacterial carbohydrate transport and metabolism.     

Ovarian follicular activity in lactating Holstein cows supplemented with monensin

The objective of this study was to determine effects of monensin on ovarian follicular development and reproductive performance in postpartum dairy cows. Forty-eight multiparous Holstein cows were randomly assigned to receive either a control total mixed ration (n = 24) or the same diet plus 22 mg of monensin/kg (n = 24) from 21 d before anticipated calving until cows were either confirmed pregnant or were >180 d postpartum. Monensin had no effect on development of the first dominant follicle postpartum or the numbers of class 1 (3 to 5 mm), 2 (6 to 9 mm), or 3 (10 to 15 mm) follicles. Control cows had more class 4 (>15 mm) follicles at 10 to 13 d postpartum than cows in the monensin group. The first dominant follicle postpartum ovulated, regressed, or became cystic unrelated to differences between diets. However, the first ovulation postpartum occurred earlier in monensin-fed cows than in the control group (27.2 +/- 2.1 d vs. 32.4 +/- 1.5 d), with no dietary effects on the diameter of the ovulating follicle. Similarly, treatments did not differ in the proportion of cows with 2 or 3 waves of ovarian follicular development per cycle, nor in the number of follicles of all classes during the breeding period. Times of ovulation following treatment with prostaglandin F2alpha were not different between dietary groups. Pregnancy rates after timed artificial insemination were similar between diets. Supplementation with monensin resulted in a shorter postpartum interval to first ovulation but did not affect other reproductive measures in healthy, lactating dairy cows.     

Purification of glycosaminoglycans from bovine follicular fluid

The follicular glycosaminoglycans, dermatan sulfate and heparan sulfate, are not available in sufficient quantities for detailed analytical studies or to use as reagents in cell cultures. The purpose of this study was to purify follicular glycosaminoglycans simultaneously from bovine follicular fluid. The use of a chloroform:methanol extraction of 10 ml of fluid resulted in recoveries of at least 90% of the glycosaminoglycans, otherwise an insoluble product resulted. Heparan sulfate and dermatan sulfate eluted at .11 and .46 M NaCl, respectively, when subjected to HPLC with an anion exchange column. Fluid from small follicles allowed to "age" in the ovaries exhibited marked reductions in the concentrations of glycosaminoglycans. Aging of the fluid from large follicles resulted in greater glycosaminoglycan concentrations and appearance of glycosaminoglycans reflecting other charge distributions. Known amounts of purified follicular fluid heparan sulfate and dermatan sulfate were applied to a gel filtration HPLC column, and corresponding integrated peak areas were plotted in relation to the glycosaminoglycan concentrations. Slopes of the lines for commercial chondroitin sulfate and follicular dermatan sulfate were not significantly different, but the slope of the line for follicular heparan sulfate was 13.5-fold greater than the slope for commercial heparin.