Increasing Follicle Size Research Articles

Expression levels of the selenium-uptake receptor LRP8, the antioxidant selenoprotein GPX1 and steroidogenic enzymes correlate in granulosa cells.

Reactive oxygen species (ROS) are a by-product of the activity of cytochrome P450 steroidogenic enzymes. Antioxidant enzymes protect against ROS damage. To identify if any particular antioxidant enzyme is used to protect against ROS produced by granulosa cells as follicles enlarge and produce oestradiol, we measured in the bovine granulosa cells the expression of two steroidogenic enzymes (CYP11A1, CYP19A1), important for progesterone and oestradiol production. We also measured the expression of the members (FDXR, FDX1, POR) of their electron transport chains (ETC). We measured antioxidant enzymes (GPXs 1-8, CAT, SODs 1 and 2, PRDXs 1-6, GSR, TXN, TXNRDs 1-3). Since selenium is an active component of GPXs, the selenium-uptake receptors (LRPs 2 and 8) were measured. Only the selenium-dependent GPX1 showed the same increase in expression as the steroidogenic enzymes did with increasing follicle size. GPX4 and PRDX2/6 decreased with follicle size, whereas SOD1/2, CAT, GSR, and TXNRD3 were lowest at the intermediate sizes. The other antioxidant enzymes were unchanged or expressed at low levels. The expression of the selenium-uptake receptor LRP8 also increased significantly with follicle size. Correlation analysis revealed statistically significant and strongly positive correlations of the steroidogenic enzymes and their ETCs with both GPX1 and LRP8. These results demonstrate a relationship between the expression of genes involved in steroidogenesis and selenium-containing antioxidant defence mechanisms. They suggest that during the late stages of folliculogenesis, granulosa cells are dependent on sufficient expression of GPX1 and the selenium transporter LRP8 to counteract increasing ROS levels caused by the production of steroid hormones. In the ovary, eggs are housed in follicles which contain the cells that produce oestrogen in the days leading up to ovulation of the egg. Oestrogen is produced by the action of enzymes. However, some of these enzymes also produce by-products called reactive oxygen species (ROS). These are harmful to eggs. Fortunately, cells have protective antioxidant enzymes that can neutralise ROS. This study was interested in which particular antioxidant enzyme(s) might be involved in neutralising the ROS in follicle cells. It was found that only one antioxidant enzyme, GPX1, appeared to be co-regulated with the enzymes that produce oestrogen and progesterone in the follicular cells. GPX1 contains the essential mineral selenium. In summary, this study has identified which antioxidant appears to be involved in neutralising ROS in the days leading to ovulation. It highlights the importance of selenium in the diet.

Effect of altrenogest treatment before weaning on reproductive performance and production efficiency in primiparous and multiparous sows

BackgroundMost sows will experience negative energy balance during lactation resulting in impaired follicular development. This study aimed to treat 28-day lactating sows with altrenogest (ALT) to suppress follicle enlargement during lactation, and to assess the estrus and reproductive performance post-weaning.MethodsIn this study, we conducted two trials. In trial 1, we monitored the follicular development of lactating sows including 10 primiparous sows and 10 multiparous sows during the whole lactation to confirm the ALT administration time. In trial 2, a total of 42 primiparous and 111 multiparous sows were allocated to three treatments: Ctrl (control group, n = 51): no treatment; TAI (timed artificial insemination group, n = 51): sows were injected with equine chorionic gonadotropin (eCG) after weaning 24 h and gonadotropin-releasing hormone (GnRH) when they expressed estrus; and AT-TAI (ALT treatment-timed artificial insemination group, n = 51): base on the process of TAI group, the sows were fed with 20 mg ALT per day before weaning 10 days. All sows were artificially inseminated twice at 12 h and 36 h after estrus. The follicle size changes and serum hormone levels were explored in this process.ResultsAlthough the follicle size of multiparous sows was larger than primiparous sows during the whole lactation (P < 0.05), similar change trends of follicle size were observed in primiparous and multiparous sows. Meanwhile, the FSH, LH and E2 levels of multiparous sows were higher than primiparous sows. The ALT treatment significantly inhibits the increase in follicle size (P < 0.05) and reduces the serum levels of FSH, LH and E2 (P > 0.05). Additionally, ALT treatment increases estrus concentration and the preovulatory follicle size (P < 0.05), meanwhile, it delays the weaning-to-estrus interval (WEI, P < 0.001). However, the estrus rate, pregnancy rate, total pigs born and born alive did not differ between treatments (P > 0.05).ConclusionsThere were significant differences in the size of follicles in the lactation between primiparous and multiparous sows. ALT treatment during the last ten days of lactation concentrated estrus expression leading to higher work efficiency of breeder in batch production, however, with no improvement in reproductive performance.

Correlation analysis of polyamine metabolism and reproductive hormone levels in goose ovarian follicles

To investigate the relationship between polyamine metabolism and reproductive hormones in ovarian follicles of Sichuan white geese, follicle polyamine content and reproductive hormone levels and gene expressions related to polyamine metabolism, steroidogenesis and steroid hormone receptors were detected by HPLC, ELISA and RT-qPCR. The results showed that the overall trend of spermidine and spermine levels increased first and then decreased as increasing follicle size, with the highest level in F3 and F5 follicles (P < 0.05). Putrescine and 17β-estradiol (E2) levels in hierarchical follicles were significantly lower than those in prehierarchical follicles (P < 0.05). Progesterone (P4) first increased and then decreased, with the highest level in the F5 follicle (P < 0.05). The expression levels of estrogen receptor 1 (ER1) showed an overall increase as increasing follicle size (except in F3 follicles), while estrogen receptor 2 (ER2) in hierarchical follicles was significantly lower than that in the prehierarchical follicles (P < 0.05). In addition, the overall expression level of progesterone receptor (PR) decreased, with no significant differences among F1, F2 and F3 follicles (P > 0.05). Yolk putrescine contents were positively correlated with yolk E2 concentrations and PR expression levels (P < 0.05), A significant positive correlation of spermidine levels with yolk P4 concentrations and PR expressions was also observed, as well as the spermine levels with yolk P4 concentrations (P < 0.05). In summary, polyamines were involved in the regulation of follicular development in geese, and this regulation played a role in affecting steroidogenesis and the expression of genes related to hormone receptors.

Expression of Angiopoietin 2 in ovarian follicles of crossbred Malabari goat

Angiopoietin-2 (Ang-2) is a local ovarian growth factor belonging to the angiopoietin/Tie (tyrosine kinase with Ig and EGF homology domains) signalling pathway, one of the main pathways involved in ovarian follicular angiogenesis. The main objective of this study is to assess the differential protein expression of Ang-2 in the developmental phases of ovarian follicles of crossbred Malabari goats employing an immunohistochemical technique. Ang-2 protein expression was found in all developmental phases of ovarian follicles, including primordial, primary, small, and large antral ovarian follicles. Ang-2 was found in the granulosa cells, theca interna cells, follicular fluid (granulosa cells in the FF), endothelial cells of capillaries and germinal epithelium of the goat ovaries. The Quantitative Method-Percentage Positivity of Ang-2 expression revealed that it was higher in the initial stages of ovarian follicular development and decreased with later stages of follicular development in cross-bred Malabari goats. Moreover, the protein expression of Ang2 was found to be higher in granulosa cells (GC) than in theca interna (TI) cells of antral follicles as evident from the higher staining intensity in GC cells compared to TI cells. It is therefore concluded that the Ang-2 protein expression in ovarian follicular cells decreased with an increase in follicle size and development and based on the combined scores for immunostaining (staining intensity and the number of positive staining cells), the Ang-2 was found to be expressed more in the granulosa cell layer compared to the theca interna cells of the antral follicle in crossbred Malabari goats.

P-483 Improving in vitro culture of isolated human ovarian primordial-primary follicles by adding adipose derived stem cells

Abstract Study question Could adipose derived stem cells (ADSc) secrete survival- and growth-promoting factors able to improve in vitro culture (IVC) of human isolated follicles? Summary answer Co-encapsulation of isolated human primordial-primary follicles with ADSc enhances follicular survival, growth and maturation after 7 days of IVC. What is known already The ability to obtain in vitro mature oocyte from earliest follicular stage may help to preserve fertility in female cancer patients. Unfortunately, human primordial follicles show low rates of growth in vitro. The only study that obtained mature human oocytes from primordial follicle used an IVC multi-step method with a success rate lower than 1% and abnormal polar body. Recently, several studies have shown that, in mice, ADSc promote survival, antrum formation and oocyte maturation in vitro by secreting factors important for follicular development. To our knowledge, no IVC studies on human ovarian follicles have been attempted using ADSc. Study design, size, duration Fragments of frozen-thawed human ovarian tissue (n = 5) have been mechanically and enzymatically digested to isolate primordial-primary follicles. Eight-to-ten isolated unilaminar follicles have been embedded in alginate-matrigel, half with 20.000 ADSc and half alone. Alginate beads were then cultured in vitro for 7 days. At day (D) 7, we compared isolated follicles co-cultured with ADSc to isolated follicles cultured alone. Participants/materials, setting, methods Every two days, IVC medium has been collected to perform hormonal (estradiol, progesteron, anti-Müllerian hormone, androgen, dehydroepiandrosterone) and growth factors measurement (VEGF, bFGF, IGF, HGF) by enzyme-linked immunosorbent assay (ELISA) to study follicle and ADSc interactions. Follicle diameter have been measured at D0 and at D7 of IVC. At D0 and at the end of the IVC (D7), follicle viability (live dead assay) and ultrastructure (transmission electron microscopy [TEM]) have been investigated. Main results and the role of chance A total of 373 unilaminar follicles were obtained after enzymatic isolation. At the end of IVC the percentage of viable follicles (with &amp;lt;10% of dead granulosa cells) was found to be significantly higher in follicles co-cultured for 7 days with ADSc (63.3%) compared to follicles in vitro cultured alone (45.5%) (p &amp;lt; 0.01). A significant increase in follicle size was observed in both groups after culture (p &amp;lt; 0.0001). This increase was statistically higher in follicles co-cultured with ADSc than follicles cultured alone (p &amp;lt; 0.005), reflecting better follicle growth in the follicle-ADSc group. Preliminary results by TEM show ADSc did not affect the ultrastructure of isolated follicles after 7 days of IVC. Limitations, reasons for caution As demonstrated by our results, ASCs appears to improve IVC conditions and boost follicle activation and survival preserving the follicle ultrastructure. Our study, however, did not investigate the underlying mechanisms behind this improved follicle growth and survival after IVC. Wider implications of the findings Co-encapsulation of human unilaminar follicles with ADSCs promotes early-stage ovarian follicle development and survival, possibly because of bidirectional interactions between follicles and ADSCs. This represents a promising first step for fertility preservation in female cancer patients who cannot benefit from cryopreserved ovarian tissue transplantation. Trial registration number not applicable

Intrafollicular Concentrations of the Oocyte-secreted Factors GDF9 and BMP15 Vary Inversely in Polycystic Ovaries.

ContextThe oocyte-secreted factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) play essential roles in follicle development and oocyte maturation, and aberrant regulation might contribute to the pathogenesis of polycystic ovary syndrome.ObjectiveAre there measurable differences in concentrations of GDF9, BMP15, and the GDF9/BMP15 heterodimer in small antral follicle fluids from women with and without polycystic ovaries (PCO)?Design and SettingFollicle fluids (n = 356) were collected from 4- to 11-mm follicles in unstimulated ovaries of 87 women undergoing ovarian tissue cryopreservation for fertility preservation.PatientsTwenty-seven women with PCO were identified and 60 women without PCO-like characteristics (non-PCO women) were matched according to age and follicle size.Main outcome measuresIntrafollicular concentrations of GDF9, BMP15, GDF9/BMP15 heterodimer, anti-Mullerian hormone (AMH), inhibin-A and -B, total inhibin, activin-B and -AB, and follistatin were measured using enzyme-linked immunosorbent assays.ResultsThe detectability of GDF9, BMP15, and the GDF9/BMP15 heterodimer were 100%, 94.4%, and 91.5%, respectively, and concentrations were significantly negatively correlated with increasing follicle size (P < 0.0001). GDF9 was significantly higher in women with PCO (PCO: 4230 ± 189 pg/mL [mean ± SEM], n = 188; non-PCO: 3498 ± 199 pg/mL, n = 168; P < 0.03), whereas BMP15 was lower in women with PCO (PCO: 431 ± 40 pg/mL, n = 125; non-PCO: 573 ± 55 pg/mL, n = 109; P = 0.10), leading to a significantly higher GDF9:BMP15 ratio in women with PCO (P < 0.01). Significant positive associations between BMP15 and AMH, activins, and inhibins in non-PCO women switched to negative associations in women with PCO.ConclusionsIntrafollicular concentrations of GDF9 and BMP15 varied inversely in women with PCO reflecting an aberrant endocrine environment. An increased GDF9:BMP15 ratio may be a new biomarker for PCO.

P–610 Optimal timing of ovulation triggering to achieve highest success rates in natural cycles – an analysis based on follicle size and estradiol concentration in NC-IVF

Abstract Study question What is the best follicle size, estradiol (E2) serum concentration and endometrial thickness to trigger ovulation in natural cycles? Summary answer Optimal follicles size is 18–22mm but estrogen concentration also need to be considered to maximize oocyte maturity and to minimize premature LH surge. What is known already Timing of the ovulation triggering is essential in infertility treatments based on natural menstrual cycles such as optimized vaginal intercourse, intrauterine inseminations and thawing cycles without hormone replacement therapy. Common parameters to define the day of ovulation triggering are the follicle size and the estrogen concentration. However, data on follicle size and estrogen concentration are either derived from longitudinal evaluations of few ideal participants, are not very detailed or were studied in stimulated cycles. The model of Natural Cycle IVF (NC-IVF) which provides more detailed information has never been used to study this issue. Study design, size, duration Retrospective cross sectional analysis of monofollicular NC-IVF cycles. Follicle size, E2 and LH serum concentrations and endometrial thickness were evaluated on day –5 to 0 (day 0 = day of aspiration). Ovulation was triggered with 5.000IE HCG 36h before aspiration if follicle size was 14–22mm. Patients with irregular cycles, endometriosis &amp;gt;II°, cycles with azoospermia or cryptozoospermia and with inconsistent data were excluded. 606 cycles from 290 women were analysed from 2016 to 2019. Participants/materials, setting, methods Mean age of women undergoing NC-IVF was 35.8±4.0y, median 36y [IQ-range: 34;39]. Each woman performed mean 2.1±1.4, median: 2 [IQ-range: 1–3] NC-IVF cycles at an university based IVF center. All parameters were analysed inter and intraindividually and associations were adjusted for maturity of oocyte, zygote development rate, embryo score, implantation rate and live birth rate. Associations were adjusted for age, cause of infertility and number of previous transfers. Main results and the role of chance Follicle size, E2 concentration and endometrial thickness increased constantly over time. The increase was computed for each cycle without considering any correlation intra patient, revealing an increase of follicle size by 1.04±0.64mm, an increase of E2 concentration by 167.3±76.8pmol/L and endometrial thickness by 0.69±0.59mm per day. Based on a multivariate adjusted model with follicle size, E2 and their interaction, number of retrieved oocytes was associated with E2 concentration (aOR 1.80, 95% CI 1.05–3.11; p = 0.034). Maturity of oocytes was associated not only with E2 concentration (aOR 1.84, 95% CI 1.15–2.94; p = 0.010) but also with follicle size (aOR 1.24, 95% CI 1.01–1.53; p = 0.037) and so was also the interaction of both parameters (aOR 0.96, 95% CI 0.94–0.99; p = 0.017). LH surge was calculated to start in 25% of cases at an E2 level of 545 pmol/l, in 50% of cases at 907pmol/l and in 75% of cases at an E2 level of 1531pmol/l. Live birth rate in cycles with follicles size 14–17 mm was 2.2–3.5% per initiated cycle and in cycles with follicle size 18–22mm 8.5–12.5%. Limitations, reasons for caution Cross sectional studies provides less precise information than longitudinal studies. Follicle size and endometrial thickness were evaluated by several physicians possibly causing some imprecision. Wider implications of the findings: There is a trend towards natural treatment cycles. The study contribute to an optimisation of infertility treatments involving natural cycles. The study gives guidance about the number of days required after a follicle monitoring to reach the optimal time for triggering ovulation. Trial registration number Not applicable

Transcriptional and translational abundance of visfatin (NAMPT) in buffalo ovary during estrous cycle and its in vitro effect on steroidogenesis

Visfatin is a highly conserved adipokine protein having multiple biological effects, including regulation of reproduction. Evidence in recent years has shown a pivotal role of visfatin in ovarian functions. The present study was conducted to evaluate the mRNA and protein abundance of visfatin in ovarian follicles and corpora lutea (CL) during different stages of their development in the ovary of water buffalo (Bubalus bubalis) and to investigate the role of visfatin on estradiol (E2) and progesterone (P4) secretion. Ovarian follicles were categorized in to small (F1), medium (F2), large (F3), and preovulatory (F4) follicles, whereas the CL were categorized into early (CL1), mid (CL2), late (CL3), and regressing (CL4) CL stage. In follicles, the mRNA and protein abundance of visfatin increased with an increase in follicle size in granulosa cells (GCs) and theca interna (TI) cells. In CL, the transcript of visfatin was significantly (P < 0.05) higher in the late luteal phase (CL3) than that in other phases. The translational abundance of visfatin was significantly higher in the mid and late luteal phase. Visfatin was localized in the cytoplasm of GC and TI of ovarian follicles and small and large luteal cells of CL. GCs were cultured in vitro and treated at 0, 1, and 10 ng/mL visfatin either alone or in the presence of FSH (30 ng/mL) or IGF-I (10 ng/mL) for 48 h. The luteal cells were treated with visfatin at 0, 1, and 10 ng/mL dose for 48h. There was significant (P < 0.05) increase in estradiol (E2) secretion from GCs at 10 ng/mL dose of visfatin and visfatin (10 ng/mL) +IGF-I (10 ng/mL). Visfatin also increased (P < 0.05) progesterone (P4) secretion from cultured luteal cells at both 1 and 10 ng/mL dose. In GCs, visfatin in the presence of IGF-I increased the transcriptional abundance of cytochrome P45019A1 (CYP19A1), the gene for key enzyme aromatase. In luteal cells, the visfatin increased mRNA abundance of factors involved in progesterone synthesis viz. steroidogenic acute regulatory protein (StAR), cytochrome P45011A1 (CYP11A1), 3beta-hydroxysteroid dehydrogenase (HSD3B1). The present study provided evidence that visfatin is expressed in ovarian follicles and CL of buffalo ovary and visfatin has a stimulatory effect on estradiol and progesterone secretion in ovarian cells of water buffalo.

Histological Characteristic that Accompanying with Ovaryfolliculogenisis of Rabbits for Effectiveness Seeds of (Trigonellatibetana)

Objectives: Alternative plants Medicine has been considered one of the world’s most important solutions tofemale reproductive diseases. The goal of the current study however, was to evaluate the mains histologicalchanges that follow the use of seeds of (Trigonellatibetana) on ovary folliculogenisis.Method: Twelve’s female healthy rabbits, in animal house all experimental animals have been adapted.For this study, animals were taken distributed into two groups, control administrated by distal water (5 ml/kg b.w) and treatment group was administered by Trigonellatibetana extract at a dose of 2g/kgb.w/orallyadministration during 2 week for 3 time daily.Results: The present study finding the numerouschanges happened in the ovary, the mains variance has beennoticed increase the numbers of primordial and atretic follicles in treatment group and increase in size offollicles. Moreover, the theca interna was thickness and has a thicker layers of collagen than control group.Conclusions: Such results indicate that (Trigonellatibetana) was considered to be suitable for early fertilityperiod arrival.

299 Treatment with prostaglandin F2α and an intravaginal progesterone insert in advance of gonadotropin-releasing hormone enhances response to estrus synchronization in mature beef cows

Abstract An experiment was designed to evaluate the effect of treatments in advance of gonadotropin-releasing hormone (GnRH; 100μg gonadorelin) administered at the start of estrus synchronization. We hypothesized that administration of prostaglandin F2α (PG; 500μg cloprostenol) followed by treatment with an intravaginal progesterone-releasing insert (CIDR; 1.38g progesterone) would result in increased follicle size at GnRH, thereby enhancing response to GnRH and overall response to estrus synchronization. Postpartum suckled beef cows (n = 183) in two locations were assigned to one of five treatments based on age, days postpartum, and body condition score. Treatments (Figure 1) were designed as a two-by-two factorial with a control (Treatment 1). Treatments 2–5 received a CIDR insert from Day -17 to Day -10, with (Treatments 2 and 3) or without (Treatments 4 and 5) administration of PG coincident with CIDR insertion on Day -17. Coincident with GnRH administration on Day -10, CIDR inserts were either removed (Treatments 2 and 4) or remained in place until Day -3 (Treatments 3 and 5). On Day -3, estrus detection aids were applied and a representative subset of cows in each treatment were fitted with estrus detection transmitters. Blood samples were collected on Days -27, -17, -10, -3, and 0 for determination of serum estradiol and/or progesterone concentrations via radioimmunoassay. For a representative subset of cows (n = 111), transrectal ovarian ultrasound was performed to assess ovarian follicle size and presence of corpora lutea on Days -17, -10, -3, and 0. Treatment with PG and CIDR in advance of GnRH resulted in improvements (P &amp;lt; 0.05) in several response variables, including follicle size on Day -17 and estrous response prior to AI. Results support the hypothesis that administration of PG and treatment with a CIDR for 7 d prior to GnRH improves likelihood of GnRH response and enhances response of mature cows to estrus synchronization.

Expression and localization of adiponectin and its receptors in ovarian follicles during different stages of development and the modulatory effect of adiponectin on steroid production in water buffalo

Adiponectin is an adipocyte-derived hormone regulating energy metabolism, insulin sensitivity and recently found to regulate reproduction. The current study was carried out to investigate gene and protein expression, immunolocalization of adiponectin and its receptors AdipoR1 and AdipoR2 in ovarian follicles of different developmental stages in water buffalo (Bubalus bubalis) and to investigate the effect of adiponectin on steroid production in cultured bubaline granulosa cells. qPCR, western blotting and immunohistochemistry were applied to demonstrate mRNA expression, protein expression and immunolocalization, respectively. The results indicate that adiponectin, AdipoR1 and AdipoR2 were present in granulosa cells (GC) and theca interna (TI) of ovarian follicles and the expression of adiponectin, AdipoR1, AdipoR2 in GC and AdipoR1 and AdipoR2 in TI increased with increase in follicle size (p<.05). Expression of adiponectin was high in small and medium size follicles in TI. The adiponectin and its receptors were immunolocalized in the cytoplasm of GC and TI cells. Further, in the in-vitro study, GCs were cultured and treated with recombinant adiponectin each at 0, 1 and 10µg/ml alone or with follicle stimulating hormone (FSH) at 30ng/ml) or Insulin-like growth factor I (IGF-I) at 10ng/ml for 48hr after obtaining 75%-80%s confluency. Adiponectin at 10µg/ml increased IGF-I-induced estradiol (E2 ) and progesterone (P4 ) secretion and FSH-induced E2 secretion from GC and also increased the abundance of factors involved in E2 and P4 production (cytochrome P45019A1 [CYP19A1] and 3-beta-hydroxysteroid dehydrogenase [3β-HSD]). In conclusion, this study provides novel evidence for the presence of adiponectin and its receptors in ovarian follicles and modulatory role of adiponectin on steroid production in buffalo.

Hormonal and cellular interactions in follicular steroid biosynthesis by the sheep ovary

Studies of isolated cell types from sheep follicles revealed several functional changes which occur during follicular maturation. Cyclic AMP production by granulosa cells from the smallest follicles studied (1-3 mm diameter) was stimulated by FSH but not by hCG, suggesting functional FSH receptors at this early stage of differentiation. Medium-sized follicles (4-6 mm) responded to both FSH and hCG. Granulosa cells were unable to synthesize androgens, but readily converted exogenous testosterone to oestradiol-17 beta. This conversion occurred to a limited extent in the cells from the smallest follicles, but was much greater in medium and large (greater than 6 mm) follicles. Oestradiol production by theca preparations from small follicles was barely detectable, but increased markedly with increasing follicle size. Androgen (androstenedione and testosterone) production by theca preparations was stimulated by hCG. This stimulation was short-lived, and levels declined to below control values after 6 h of culture. This decline could not be prevented by addition of cyclic AMP. The presence of granulosa cells with thecal preparations (i.e. follicle wall tissue) enhanced production of androgen by the theca, the effect being more marked for testosterone than for androstenedione. In-vivo studies in which granulosa cells and follicular fluid were removed during the preovulatory period suggested that granulosa cells and/or follicular fluid contributed to the oestradiol secreted into the ovarian vein during this period, but did not exclude a significant contribution by the theca as well.

Transcription profile of the insulin-like growth factor signaling pathway during human ovarian follicular development.

The IGF signaling cascade exerts important regulatory functions in human ovarian folliculogenesis. The scope of this study was to evaluate the transcription profile of insulin-like growth factor (IGF) genes during human ovarian follicle development and to analyze follicle fluid levels of key IGF proteins. Gene expression profiling was performed with microarray gene analysis. The analysis was assessed from ovarian follicles and granulosa cells (GCs) obtained from isolated stage-specific human ovarian follicles, including preantral follicles, small antral follicles, and preovulatory follicles. Numerous genes involved in the IGF signaling pathway was evaluated and key genes were validated by qPCR from GCs. Protein levels of various IGF components of human follicular fluid (FF) were measured by ELISA and time-resolved immunofluorometric assays (TRIFMA). The gene expression levels of PAPPA, IGF2, IGF receptors and intracellular IGF-activated genes increased with increasing follicle size. This was especially prominent in the late preovulatory stage where IGF2 expression peaked. Protein levels of intact IGF binding protein-4 decreased significantly in FF from large preovulatory follicles compared with small antral follicles concomitant with higher protein levels of PAPP-A. The IGF modulators IGF-2 receptor, IGFBPs, stanniocalcins, and IGF-2 mRNA binding proteins were all observed to be expressed in the different follicle stages. This study confirms and highlights the importance of PAPP-A regulating bioactive IGF levels throughout folliculogenesis and especially for the high rate of granulosa cell proliferation and expression of key ovarian hormones important in the last part of the follicular phase of the menstrual cycle.

Docosahexaenoic acid (DHA) effects on proliferation and steroidogenesis of bovine granulosa cells

BackgroundDocosahexaenoic acid (DHA) is a n-3 polyunsaturated fatty acid (PUFA) belonging to a family of biologically active fatty acids (FA), which are known to have numerous health benefits. N-3 PUFAs affect reproduction in cattle, and notably directly affect follicular cells. In terms of reproduction in cattle, n-3 PUFA-enriched diets lead to increased follicle size or numbers.MethodsThe objective of the present study was to analyze the effects of DHA (1, 10, 20 and 50 μM) on proliferation and steroidogenesis (parametric and/or non parametric (permutational) ANOVA) of bovine granulosa cells in vitro and mechanisms of action through protein expression (Kruskal-Wallis) and signaling pathways (non parametric ANOVA) and to investigate whether DHA could exert part of its action through the free fatty acid receptor 4 (FFAR4).ResultsDHA (10 and 50 μM) increased granulosa cell proliferation and DHA 10 μM led to a corresponding increase in proliferating cell nuclear antigen (PCNA) expression level. DHA also increased progesterone secretion at 1, 20 and 50 μM, and estradiol secretion at 1, 10 and 20 μM. Consistent increases in protein levels were also reported for the steroidogenic enzymes, cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), and of the cholesterol transporter steroidogenic acute regulatory protein (StAR), which are necessary for production of progesterone or androstenedione. FFAR4 was expressed in all cellular types of bovine ovarian follicles, and in granulosa cells it was localized close to the cellular membrane. TUG-891 treatment (1 and 50 μM), a FFAR4 agonist, increased granulosa cell proliferation and MAPK14 phosphorylation in a similar way to that observed with DHA treatment. However, TUG-891 treatment (1, 10 and 50 μM) showed no effect on progesterone or estradiol secretion.ConclusionsThese data show that DHA stimulated proliferation and steroidogenesis of bovine granulosa cells and led to MAPK14 phosphorylation. FFAR4 involvement in DHA effects requires further investigation, even if our data might suggest FFAR4 role in DHA effects on granulosa cell proliferation. Other mechanisms of DHA action should be investigated as the steroidogenic effects seemed to be independent of FFAR4 activation.

Natriuretic peptides improve the developmental competence of in vitro cultured porcine oocytes

BackgroundNatriuretic peptides (NPs), brain and C type NPs (BNP and CNP), were involved in the maintenance of porcine oocyte meiotic arrest. The present study investigated the effects of NPs on developmental competence of immature porcine oocytes with follicles of different sizes.MethodsFollicular fluid NP levels were examined by radioimmunoassay. The developmental competence of porcine oocytes was evaluated by cleavage and blastocyst developmental rates after in vitro fertilization (IVF) or parthenogenetic activation (PA) of cumulus oocyte complexes (COCs), which were recovered from follicle with different sizes. NP levels were examined and classified according to the cleavage potential after IVF with COCs released from these follicles.ResultsThe BNP and CNP concentrations were increased with follicular size in follicular fluid and sustained at the set ranges of 3.0 – 6.0 mm follicles compared to 6.1 – 8.0 mm follicles. The oocytes developed from 3.0 to 6.0 mm follicles demonstrated increased embryo cleavage and blastocyst ratios after IVF, with an increased follicle size (P < 0.05). Moreover, BNP and CNP significantly promoted the blastocyst developmental rates of 3.0 – 6.0 mm follicles, but could not improve the developmental competence of oocytes from 6.1 to 8.0 mm follicles due to low NP levels. The COCs from 3.0 to 4.0 mm follicles were pre-incubated in 100 ng/ml of BNP and CNP media for 20 h before regular in vitro maturation, which demonstrated 2 to 3 folds higher developmental competencies in both PA and IVF groups compared to respective controls (P < 0.01).ConclusionsThe effects of BNP and CNP supplementation in the pre-maturation culture media (PMC) on porcine developmental competence from COCs in follicles of different sizes were different and improved the developmental competence of porcine oocytes from small antral follicle in vitro.

Evidence that gene expression of ovarian follicular tight junction proteins is regulated in vivo and in vitro in cattle.

Tight junctions (TJ) are common paracellular sealing structures that control the transport of water, ions, and macromolecules across cell layers. Because the role of TJ in bovine follicular development is unknown, we investigated the developmental and hormonal regulation of the transmembrane TJ protein, occludin (OCLN), and the cytoplasmic TJ proteins, TJ protein 1 (TJP1) and cingulin (CGN) in bovine granulosa cells (GC) and theca cells (TC). For this purpose, bovine GC and TC were isolated from large (>8 mm) and/or small (1 to 5 mm) follicles and either extracted for real-time PCR (qPCR) or cultured in vitro. The abundances of both and mRNA were greater ( < 0.05) in TC than GC, whereas the mRNA abundance was greater ( < 0.05) in GC than TC. The abundance of mRNA in both GC and TC was greater ( < 0.05) in small follicles compared with large follicles, whereas the GC of large follicles had less ( < 0.05) mRNA abundance than the GC of small follicles. The abundance of mRNA in GC or TC did not differ ( > 0.10) among follicle sizes. In vitro treatment with various growth factors known to affect ovarian folliculogenesis indicated that , , and were hormonally regulated. Fibroblast growth factor 9 (FGF9) decreased ( < 0.05) the and mRNA abundances. Tumor necrosis factor α (TNFα) and vascular endothelial growth factor A (VEGFA) increased ( < 0.05) the mRNA abundance but decreased ( < 0.05) the mRNA abundance. Dexamethasone (DEX) increased ( < 0.05) and mRNA abundances. Epidermal growth factor (EGF) decreased ( < 0.05) and dihydrotestosterone (DHT) increased ( < 0.05) the abundances of , , and mRNA. We propose that the downregulation of OCLN and other TJ proteins during follicular development could reduce barrier function, thereby participating in increasing follicle size by allowing for an increase in the volume of follicular fluid as well as by allowing additional serum factors into the follicular fluid that potentially may directly impact GC functions. The results of the current study indicate the following in cattle: 1) gene expression of TJ proteins (i.e., , , and ) differs between GC and TC and changes with follicle size, and 2) autocrine, paracrine, and endocrine regulators, such as FGF9, EGF, DHT, TNFα, and glucocorticoids, modulate , , and mRNA abundance in TC in vitro.

Occludin expression and regulation in small follicles of the layer and broiler breeder hen

Synchronized yolk accumulation and follicle development are essential for egg production in oviparous species. In birds, yolk is incorporated into the oocyte by an avian specific yolk receptor (LR8), and it has been suggested that occludin (OCLN), a tight junction protein, mediates transfer of yolk material to the oocyte surface. OCLN may be a key regulator of yolk accumulation and follicle growth, however, the expression and regulation of OCLN in granulosa cells during various stages of follicle development is unknown. In the first experiment, we found that LR8 and OCLN mRNA were highest in small follicles within the ovary. In addition, OCLN decreased with increasing follicle size. OCLN mRNA was more abundant in the germinal disc region of the granulosa cell layer than the non-germinal disc region. In addition, we found epidermal growth factor (EGF) and activin B, decreased OCLN mRNA, while activin A increased OCLN. In the second experiment, restricted fed (RF) broiler breeder hens were randomly divided into two groups and one group remained on RF and the other was fed ad libitum (FF). OCLN expression in granulosa cells of 3–5mm follicles of FF hens was lower compared to RF hens and yolk weights were higher in the FF group, however, LR8 mRNA in small whole follicles (<3mm) did not differ between groups. In conclusion, the level of feed intake is related to or may directly regulate OCLN mRNA expression or may have an indirect effect through paracrine or autocrine factors in the ovary.

ANALYSIS OF HOMA-IR, FOLLICLE SIZE, AND ESTRADIOL AFTER COMBINATION THERAPY OF METFORMIN AND CLOMIPHENE CITRATE IN POLYCYSTIC OVARY SYNDROME’S PATIENT

PCOS is an ovarian dysfunction syndrome which characterized by the present of unovulation, hyperandrogenism, and polycystic ovarian morphology. The main cause of PCOS is still unclear, but many studies report that genetic factor have a great role. There are also two big concept in PCOS, hyperandrogenism and insulin resistance. Previous research by Ayaz et al (2013) has shown that metformin was given if there has been a resistance of clomiphene citrate. This study was designed to provide metformin and clomiphene citrate combination from the early administration to determine the effectiveness of the combination therapy. The objective of this study is to analyze HOMA-IR value, follicle size, and estradiol concentration after metformin and clomiphene citrate treatment in PCOS patient. Design of this study was an observational longitudinal prospective method. There was only a group with a total sample size 19 women of productive age (18-40 years) diagnosed with PCOS. The pre and post combination therapy was observed for ten weeks. Metformin 500 mg three times daily for ten weeks and clomiphene citrate 100 mg in 3rd day until 7th day of menstruation for two cycles were given to the subjects. The result showed that HOMA-IR value decrease after 10 weeks in which value before therapy was 4.9 ± 2.4 and after therapy 3.7 ± 2.2 with the achievement 21.1% HOMA-IR under 2.5. Estradiol concentration after therapy also decreased from 64.8 ± 24.7 pg/ml to 37.9 ± 17.9 pg/ml in ten weeks this concentration remained the range of the follicular phase estradiol levels. On the other hand, follicle size increased from 4.7 ± 0.6 mm to 10.7 ± 2.2 mm after combination therapy of metformin and clomiphene citrate with the occurrence of ovulation 31.6% but none of the samples were successfully pregnant. It is concluded that women with PCOS, declined HOMA-IR, estradiol, and increased follicle size was associated with a good response to combination therapy of metformin and clomiphene citrate.

Differential expression of GDF-9 and BMP- 15 during follicular development in canine ovaries evaluated by flow cytometry

Growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) play important functions in follicular and oocyte development in many species. This study evaluated the dynamic expression of GDF-9 and BMP-15 in canine follicles cells using flow cytometry analysis. Follicular cells were removed from three sizes of antral follicles (small, medium and large) from ovaries of bitches throughout the estrus cycle. Cells were incubated with anti-human GDF-9 polyclonal and anti-mouse BMP-15 monoclonal antibodies. A size and complexity discriminatory gate was used for the cytometryc analysis in the initial dot plot and, additionally, a CD45 marker for leukocyte and propidium iodide (PI) were used for erythrocyte and debris discrimination. The evidence corroborated the presence of both proteins in canine follicle cells, but these proteins were not expressed equally during follicular development. The results analyzed by ANOVA showed that GDF-9 expression decreased (P<0.05) during follicular growth in anestrus and proestrous/estrous, but increased in diestrus (P<0.05). The expression levels of BMP-15 rose (P<0.05) from small to medium sizes in anestrous without changing at diestrus. Small antral follicles expressed the highest values of GDF-9 at anestrus while only BMP-15 showed higher value in small antral follicles at proestrous-estrus compared to diestrus and anestrus. Both proteins decreased in proestrous/estrous (P<0.05) with increasing follicle size, registering the lowest levels in large follicles. The flow cytometric assay was able to assess GDF-9 and BMP-15 expression in canine follicular cells, showing that these proteins were differentially expressed during follicular development, possibly related to the special features of canine reproduction.

Flow cytometric analysis of FSHR, BMRR1B, LHR and apoptosis in granulosa cells and ovulation rate in merino sheep

The aim of the present study was to determine the direct cause of the mutation-induced, increased ovulation rate in Booroola Merino (BB) sheep. Granulosa cells were removed from antral follicles before ovulation and post-ovulation from BB (n=5) and WT (n=12) Merino ewes. Direct immunofluorescence measurement of mature cell surface receptors using flow cytometry demonstrated a significant up-regulation of FSH receptor (FSHR), transforming growth factor beta type 1, bone morphogenetic protein receptor (BMPR1B), and LH receptor (LHR) in BB sheep. The increased density of FSHR and LHR provide novel evidence of a mechanism for increasing the number of follicles that are recruited during dominant follicle selection. The compounding increase in receptors with increasing follicle size maintained the multiple follicles and reduced the apoptosis, which contributed to a high ovulation rate in BB sheep. In addition, we report a mutation-independent mechanism of down-regulation to reduce receptor density of the leading dominant follicle in sheep. The suppression of receptor density coincides with the cessation of mitogenic growth and steroidogenic differentiation as part of the luteinization of the follicle. The BB mutation-induced attenuation of BMPR1B signaling led to an increased density of the FSHR and LHR and a concurrent reduction in apoptosis to increase the ovulation rate. The role of BMPs in receptor modulation is implicated in the development of multiple ovulations.