Ovarian Follicular Growth Research Articles

Effect of Therapeutic Ultrasound on Folliculogenesis, Angiogenesis and Apoptosis After Heterotopic Mouse Ovarian Transplantation

One of the challenges in ovarian transplantation is ischemia-reperfusion damage. When transitional tissue faces an acute and critical condition in terms of blood supply (immediately after organ transplantation), treatment with low-intensity pulsed ultrasound (LIPUS) seems to be very beneficial. The aim of this study was to evaluate the effects of ultrasound therapy on heterotopic transplanted mouse ovarian tissue. Adult female Naval Medical Research Institute mice were divided into three groups. In the experimental groups, the transplanted ovary was exposed 5 min daily to ultrasound with an intensity of 0.3 W/cm2, frequency of 3 MHz and pulse mode of 1:4. The grafted ovaries were assessed with the usual histology and immunohistochemistry techniques. Results indicate that more CD31 angiogenic factor was expressed in irradiated animals than in control animals, and ultrasound therapy resulted in better follicular preservation, especially after 14 d. In conclusion, therapeutic ultrasound may accelerate and increase re-angiogenesis and can help to promote ovarian follicular growth.

Proteomic Analysis of the Follicular Fluid of Tianzhu White Yak during Diestrus

The aim of this study was to identify differentially expressed proteins in the follicular fluid of Tianzhu white yak during diestrus. Follicles obtained from female yak were divided into four groups according to their diameter: 0–2, 2–4, 4–6 mm, and greater than 6 mm. The follicular fluid was directly aspirated from the follicles and mixed according to follicular size, and two-dimensional gel electrophoresis was carried out on the crude follicular fluid samples. Thirty-four differentially expressed spots were generated from these four sizes of follicles. Fourteen of these spots were analyzed by MALDI-TOF/TOF-MS and identified as: AS3MT, VDP, ANKRD6, C10orf107 protein, MRP4, MAPKAP1, AGO3, profilin-β-actin, SPT2 homolog, AGP, AR, RNF20, obscurin-like-1, and one unnamed protein. These proteins were first reported in follicular fluid, in addition to VDP and AGP. Based on existing knowledge of their function and patterns of expression, we hypothesize that most of these differentially expressed proteins play a role in ovarian follicular growth and development, dominant follicle selection, or follicular atresia and development of oocytes; however, the function of the other differentially expressed proteins in reproduction remains ambiguous.

Short term dietary propylene glycol supplementation affects circulating metabolic hormones, progesterone concentrations and follicular growth in dairy heifers

This study was designed to determine the effects of dietary propylene glycol (PG; Propypact®, DIFAGRI, France) on blood metabolites, metabolic and reproductive hormones and follicular growth in 10 dairy heifers. Treatments consisted of (1) 1.1kg of sugar beet pulp (Control), (2) 150g PG (PG150) and (3) 300g PG (PG300). Each heifer received the three treatments in different randomized orders. A standard hay/concentrate diet, formulated to allow a daily liveweight gain of 900g/day, was given at 8:00 and the dietary treatments were given at 16:00 from Days 1 to 13 of the oestrous cycle following induced oestrus (Day 0). Oestrus induction treatment consisted of a subcutaneous 3mg norgestomet implant inserted for 9 days combined with GnRH treatment (i.m.) at implant insertion. Two days before implant removal, 500µg cloprostenol was administered i.m. Blood samples were collected by jugular venipuncture every 2h for 24h on Days 0 and 13 to measure plasma insulin, glucose, β-hydroxybutyrate (BHB) and urea concentrations. Blood samples were also collected to measure insulin-like growth factor-1 (IGF-1), oestradiol, progesterone concentrations on Days 2, 6, 9 and 12 and AMH (Anti-Müllerian hormone) on Days 0, 2, 6, 9 and 12. On Days 2, 6, 9 and 12 ovarian follicular growth was evaluated; the total number of follicles and their diameters were recorded and classed (2–3mm, 4–7mm, and >8mm). Results were analysed by repeated-measures ANOVA. There were no treatment, day and interaction effects on average urea concentrations while there were some differences between Days 0 and 13 for insulin, glucose and BHB. Insulin and glucose concentrations were higher on Day 13 compared to Day 0 and the opposite was observed for BHB. There were treatment, time and interaction effects on glucose and BHB concentrations measured over 24h on Day 13; glucose concentrations were higher (P<0.05) at 4:00, 8:00, 12:00, 16:00 and 20:00h, whereas BHB concentrations were lower (P<0.05) at 20:00 and 22:00h in the PG300 group compared to the control and PG150 groups. There were treatment, day and interaction effects on IGF-1 and progesterone concentrations, and the number of small follicles. PG150 resulted in higher progesterone concentrations on Days 9 and 12, and more small follicles on Day 2 compared to Control. AMH concentrations were unaffected by day of oestrous cycle and dietary treatment. However a negative correlation was observed between pre-PG distribution insulin and AMH (r=−0.47, P<0.05). These results indicate that short-term dietary PG supplementation affects circulating concentrations of metabolites and metabolic hormones, increases progesterone concentrations and the number of small follicles. Propylene glycol supplementation may be effective in improving oocyte production when combined with hormonal treatments to stimulate follicular growth for superovulation or ovum-pick up.

MiR-34a targets the inhibin beta B gene, promoting granulosa cell apoptosis in the porcine ovary

During ovarian follicular growth and development, only a few follicles actually ovulate. Recently, it was found that follicular atresia is triggered by granulosa cell apoptosis, but the molecular mechanism of follicular atresia was not understood. Using flow cytometry, we found that miR-34a promotes granulosa cell apoptosis in pig ovarian follicles. In addition, inhibin beta B was found to be a miR-34a target gene, based on luciferase reporter assays, quantitative RT-PCR and Western blotting. Taken together, our data indicate that miR-34a plays an important role in granulosa cell apoptosis by targeting the INHBB gene in the porcine ovary.

Effects of Vaccination against GDF9 and BMP15 on Fertility and Ovarian Function in the White-tailed Deer

The physiological mechanisms controlling ovarian follicular growth and ovulation involve a complex exchange of systemic signals and a localized exchange of molecules between the oocyte and surrounding somatic cells. It has been demonstrated that the oocyte itself plays an essential role in regulating these processes by secreting two key regulatory proteins: bone morphogenetic protein-15 (BMP15) and growth and differentiation factor-9 (GDF9). Natural mutations in their expression and vaccination against these growth factors have been shown to cause sterility in sheep. The aim of this 3-year study was to determine the effect of vaccination against GDF9 and BMP15 on fertility in female white-tailed deer. Does were randomly assigned to two groups (n = 10/group). Each doe received a primary vaccination followed by a booster 47 days later. After the first year, a subset of animals (n = 4-5/group) received an additional booster vaccination. Blood samples were collected at regular intervals to determine antibody titers and progesterone concentrations. Early pregnancy was assessed by ultrasound, and fawning was subsequently monitored. For the BMP15 group, over the 3 years 80%, 100%, and 75% of does fawned with fawning rates of 2.4, 3.5, and 3.3 fawns/doe, respectively. For the GDF9 group, 80%, 25%, and 25% of does fawned with fawning rates of 2.6, 1, and 2 fawns/doe, respectively. The fawning rate of untreated animals in the herd was 1.8 fawns/doe. Although all animals in the BMP15 group had high antibody titers, they were not made infertile, and they became more fecund. Most animals in the GDF9 group had high antibody titers, but fecundity was not affected the first year; however, they were made infertile in Years 2 and 3. Results from this study demonstrate that vaccination against GDF9 has potential to control fertility in deer. Further research will be required to determine the appropriate timing for administering the vaccine and the longevity of effect.

Follicular Fluid Soluble Receptor for Advanced Glycation End-Products (sRAGE): A Potential Indicator of Ovarian Reserve

The interaction of advanced glycation end-products (AGEs) with their cellular receptor (RAGE) is implicated in the pathogenesis of abnormal ovarian follicular growth. RAGE has a circulating secretory receptor form, soluble RAGE (sRAGE), which neutralizes the action of AGEs and might exert a protective role on the follicular environment. The objective of the study was to investigate whether serum or follicular fluid (FF) sRAGE levels are associated with markers of ovarian reserve. Serum anti-Mullerian hormone (AMH) and sRAGE protein levels were correlated in 31 reproductive-aged women. An additional 33 women who underwent oocyte retrieval for in vitro fertilization were enrolled. AMH and its receptor (AMHR-II) mRNA levels were quantified in cumulus granulosa cells and FF sRAGE and AMH protein levels were measured. Granulosa cell AMH and AMHR-II gene expression, serum and FF AMH and sRAGE protein concentration, and number of oocytes retrieved were measured. In the serum, sRAGE levels were negatively correlated with body mass index (BMI) (r = -0.5, P < .001) but not with age or serum AMH. The higher the FF sRAGE, the lower the number of international units of gonadotropin needed per cycle independent of age, BMI, or day 3 FSH level (r = -0.4, P = .04). After adjusting for age, BMI, day 3 FSH, and the total dose of gonadotropins, FF sRAGE predicted the number of oocytes retrieved (R(2) = 0.27, P = .045). FF sRAGE positively correlated with FF AMH levels (r = 0.5, P = .0085). RT-PCR results showed no correlation between the FF sRAGE and AMH or AMHR-II mRNA levels. These data support a relationship between FF sRAGE and measures of ovarian reserve. The pathological significance of the harmful inflammatory AGEs in follicular health clearly requires further investigation. Targeting AGEs might offer potential therapeutic options for the treatment of diminished ovarian response.

Different circulating progesterone concentrations during synchronization of ovulation protocol did not affect ovarian follicular and pregnancy responses in seasonal anestrous buffalo cows

Three experiments were designed to evaluate the effect of different circulating progesterone (P4) concentrations during synchronization of ovulation protocol for timed artificial insemination of seasonal anestrous buffalo cows. In the first trial, ovariectomized cows were randomly allocated into one of three groups: using new P4 devices (G-New; n = 8), using devices previously used for 9 days (G-Used1x; n = 8), and using devices previously used for 18 days (G-Used2x; n = 8). The P4 device was maintained for 9 days, and the circulating P4 concentration was measured daily. The circulating P4 concentrations during the P4 device treatment were the lowest for G-Used2x (1.10 ± 0.04 ng/mL), intermediate for G-Used1x (1.52 ± 0.05 ng/mL), and the highest for G-New (2.47 ± 0.07 ng/mL; P = 0.001). In the second trial, 31 anestrous cows had their ovarian follicular dynamics evaluated after receiving the treatments described previously (G-New [n = 10], G-Used1x [n = 11], and G-Used2x [n = 10]). At insertion of the P4 device, cows were administered 2.0 mg of estradiol benzoate. Nine days later, the P4 device was removed and cows were administered 0.53 mg of cloprostenol sodium plus 400 IU of eCG. Forty-eight hours after P4 device removal, 10 μg of buserelin acetate was administered. There were no differences among the groups (G-New vs. G-Used1x vs. G-Used2x) in diameter of the largest follicle at P4 device removal (9.0 ± 0.8 vs. 10.1 ± 0.9 vs. 8.6 ± 0.8 mm; P = 0.35), in interval from P4 device removal to ovulation (77.1 ± 4.5 vs. 76.5 ± 4.7 vs. 74.0 ± 4.4 hours; P = 0.31), or in ovulation rate (80.0% vs. 81.8% vs. 60.0%; P = 0.51). In experiment 3, 350 anestrous cows were randomly assigned into one of the three treatments described previously (G-New, n = 111; G-Used1x, n = 121; G-Used2x, n = 118) and received a timed artificial insemination for 16 hours after buserelin treatment. The 30-day pregnancy rates did not differ among groups (55.9% vs. 55.4% vs. 48.3%; P = 0.39). Thus, the low circulating P4 concentrations released from a used P4 device efficiently control the ovarian follicular growth and had no detrimental effect on the pregnancy rates of the seasonal anestrous buffalo cows.

Morphological and quantitative evaluation of the ovarian recrudescence in Nile tilapia (Oreochromis niloticus) after spawning in captivity

The Nile tilapia is one of the most important fish species for aquaculture worldwide. Understanding their reproductive biology is essential for improving their aquaculture methods. The morphological and quantitative dynamics of ovarian recrudescence of Oreochromis niloticus was studied for 21 days postspawning. To accomplish this, breeding females were kept in controlled conditions and ovarian samples were collected weekly for histological, ultrastructural and morphometric analyses. Ovarian follicle morphology revealed an intense synthesis activity of the follicular cells, which actively contributed to formation of the zona radiata and oocyte development following spawning. Recently spawned ovaries contained follicles at all developmental stages, but they were predominantly early primary growth (∼ 42%) and full-grown follicles (∼ 20%). Remnants of spawning, postovulatory follicle complexes represented approximately 5% of the former ovarian follicles immediately after spawning, and less than 1% after 7 days. Atretic follicles accounted for approximately 2% of the follicles studied during the period. The stock of primary growth follicles was stable during ovarian recrudescence, indicating their availability for continuous recruitment. Only the frequency of full-grown follicles significantly increased in the ovaries during recrudescence, representing approximately 35% of the follicles 21 days postspawning. The diameters of all follicles were significantly different between the periods analyzed. The ovaries' morphological characteristics, the maintenance of young follicles stocks and the gradual and significant increase in the proportion and diameter of full-grown follicles showed a rapid ovarian recovery and follicular growth of O. niloticus, in 21 days at 29.5°C, necessary for the next spawning.

Endocrine changes, timing of ovulation, ovarian follicular growth and efficacy of a novel protocol (Estradoublesynch) for synchronization of ovulation and timed artificial insemination in Murrah buffaloes (Bubalus bubalis)

Experiments were conducted to investigate (1) ovarian follicular growth, timing of ovulation and associated endocrine changes (progesterone, estrogen, and LH) in cycling, and (2) efficacy in terms of pregnancy rate in cycling and anestrus Murrah buffaloes subjected to the Estradoublesynch protocol (prostaglandin F2α [PGF2α] 0, GnRH 2, PGF2α 9, estradiol benzoate, EB 10). Twelve cycling buffaloes were subjected to the Estradoublesynch protocol and observed for ovulation, follicle size, and endocrine changes after EB treatment. Ovulation occurred in 12 of 12 buffaloes (100%) at 48.5 ± 1.6 hours (range, 38.0–56.0) after EB treatment. Plasma LH, total estrogen, and progesterone concentrations were determined in intensive blood samples collected after EB treatment. Peak LH concentration of 34.2 ± 7.7 ng/mL (range, 17.8–178.5) occurred at 18.3 ± 0.8 hours (range, 14.0–22.0) after EB treatment. Peak total estrogen of 50.8 ± 6.9 pg/mL (range, 32.3–82.7) occurred 5.7 ± 1.0 hours (range, 2.0–14.0) after EB treatment. Follicle size was gradually increased from second PGF2α injection (9.7 ± 0.3 mm; range, 8.0–12.0) until ovulation was detected (12.9 ± 0.4 mm; range, 11.0–15.0). Fourteen cycling and 11 anestrus buffaloes were subjected to the Estradoublesynch protocol, with timed artificial insemination (TAI) 48 and 60 hours after EB treatment, and 58 cycling buffaloes were inseminated after spontaneous estrus (control group). Pregnancy rates were 62% for TAI of cycling buffaloes, 64% for anestrus buffaloes, and 34.5% for control group. Our observations demonstrated that the Estradoublesynch protocol followed by TAI satisfactory enhanced pregnancy rates in both cycling and anestrus buffaloes.

Acute follicular response to FSH in heifers downregulated long term with a GnRH agonist and with suppressed ovarian follicular growth

The objectives of this study were to (1) ascertain ovarian follicular status in heifers where the pituitary gonadotrope cells producing LH and FSH were downregulated by long-term treatment with a GnRH agonist and (2) characterize the acute superstimulation of follicular growth in response to FSH in heifers where the pituitary was downregulated with a GnRH agonist. At the start of the study, heifers (3 year old) were implanted with GnRH agonist (n = 20) or received no treatment (control, n = 5). After 6 months, follicular growth in heifers implanted with GnRH agonist was restricted to early antral follicles (2–4 mm). At this time, these heifers were allocated to four groups and follicular development was superstimulated with FSH as follows: group 1 (n = 5) and group 2 (n = 5), a conventional FSH protocol with injections over 4 days (Days 0–3) with Group 2 receiving two times the normal dose of FSH; group 3 (n = 5), a single injection of FSH in saline on Day 0; group 4 (n = 5), a single injection of FSH in slow-release polyvinylpyrrolidone on Day 0. Follicular growth in the control heifers (n = 5) was superstimulated using the conventional 4-day FSH protocol. On Day 5, heifers in groups 1 and 2 had similar (P > 0.05) numbers of follicles in the size categories 6 to 7 mm (12.8 ± 3.0 follicles) and 8 to 9 mm (6.5 ± 1.0) as control heifers (6–7 mm, 14.6 ± 2.2; 8–9 mm, 6.2 ± 2.2) and six of the former heifers had follicles 10 mm (3.2 ± 1.4). Also on Day 5, follicular growth for heifers in group 3 was restricted to 4 to 5 mm (11.6 ± 3.0) and 6 to 7 mm (7.6 ± 3.7), whereas four out of five heifers in group 4 had follicles of 6 to 7 mm (8.2 ± 2.3) and three heifers had follicles of 8 to 9 mm (4.6 ± 2.2) and 10 mm (2.2 ± 0.9). Injection of exogenous LH on Day 5 induced ovulation in 9 out of 10 heifers in groups 1 and 2, no heifers in group 3, and 2 of 5 heifers in group 4. Plasma concentrations of progesterone 6 days after ovulation were the same (P > 0.05) for heifers in groups 1 and 2 (8.9 ± 0.9 ng/mL) and control heifers (10.0 ± 0.9 ng/mL). This study has shown that heifers treated chronically with a GnRH agonist have suppressed ovarian follicular growth but are able to respond to acute superstimulation with FSH. Furthermore, follicles in heifers treated with a GnRH agonist undergo morphologically normal growth in response to FSH and can ovulate and develop into a CL. The long-term GnRH agonist–treated heifer provides a practical model for repeated ovarian follicular superstimulation, multiple ovulation, and embryo transfer, without the need to control stage of the estrous cycle.

Ovarian follicular growth and development in heifers during and subsequent to chronic treatment with a GnRH agonist

To investigate whether chronic treatment with a GnRH-agonist influences follicular growth and morphology during the gonadotrophin-independent phase and if normal ovarian function is restored after discontinuing agonist treatment.

Sensory control of clutch size in the Common Swift Apus apus

Clutch size varies among individuals in most bird species. A widespread assumption is that such variation results from variable timing in the disruption of ovarian follicular growth that brings, with a few days' lag, egg‐laying to an end. Currently, there is empirical evidence that this is the case in Blue Tits Cyanistes caeruleus but not in Zebra Finches Taeniopygia guttata, in which the timing of follicular disruption has been shown to be invariant. Here, I investigate clutch size regulation of Common Swifts Apus apus. Using experimental egg removal, I show that, assuming the female gets enough egg contact, the determination of clutch size occurs at noon solar time on the day the first egg of a clutch is laid. In spite of individual variation in clutch size, there was no hint of any variability in the timing of clutch‐size determination. In this species therefore, the timing of the signal disrupting follicular growth appears invariant. The physiological mechanism that controls clutch size is discussed, including the existence of an endogenous circadian clock and potential zeitgeber, the developmental range of clutch size for the species, the sensory nature of the input that triggers follicular disruption, and the stress of laying extra eggs.

Effects of Feeding Dry Glycerol to Primiparous Holstein Dairy Cows on Follicular Development, Reproductive Performance and Metabolic Parameters Related to Fertility during the Early Post‐partum Period

This study examined the effects of dry glycerol supplementation on follicular growth, post-partum interval to first ovulation, concentration of serum metabolites and hormones related to fertility, body condition score (BCS) and body weight (BW) in primiparous Holstein dairy cows. Sixty primiparous Holstein dairy cows were randomly assigned to two groups (control: n=30 and glycerol supplemented: n=30). Dry glycerol (250g/day/cow) was fed as a top dressing to the common lactating total mixed ration (TMR) from parturition to 21days post-partum. Ovaries were examined four times using ultrasonography on days 13, 19, 25 and 36 post-partum to determine ovarian follicular growth. Concentration of serum metabolites and hormones was determined weekly. Body condition score was evaluated weekly from weeks 1 to 5 after parturition, and BWs were recorded three times on days 1, 11 and 21 during the experimental period. The cows fed dry glycerol had more large follicles (p<0.0001) and corpora lutea (CL) (p=0.02) compared with the control cows. Days to the first ovulation (p=0.06), days to first oestrus (p=0.05), services per conception (p=0.06) and days open (p=0.004) were positively affected by dry glycerol supplementation. Serum concentration of glucose and insulin was higher in dry glycerol-supplemented cows (p=0.1; p=0.06, respectively). Feeding glycerol had no effect on mean serum concentrations of β-hydroxybutyrate, non-esterified fatty acids and IGF-1 during the experimental period. However, significant differences were observed at concentration of BHBA and IGF-1 (p=0.02 and p=0.04, respectively) between two groups on day 21 after calving. The cows in the glycerol-fed group had higher serum progesterone concentrations on days 33 (p=0.007) and 36 (p=0.004) after calving. Supplemented cows had lower body condition loss during weeks 1-5 after calving compared with the control cows (0.34 vs 0.41 BCS). In week 13 post-partum, the proportion of cycling cows was 83.3 and 69.9% for those which received supplemented or non-supplemented diet, respectively. These results demonstrated that feeding dry glycerol as a glucogenic supply may be useful to improve negative energy balance and reproductive efficiency in young cows which calve with high requirement of energy.

Ultrasonographic and Endocrine Characterization of Follicular Waves in Llamas with a Special Reference to the Overlapping Phenomenon During Successive Waves

Ovarian follicular growth in non-mated llamas occurs in successive waves that generally superimpose their origin on the regression of the preceding wave (overlapping), originating prolonged sexual receptivity in the species. The aim of this study was to perform an ultrasonographic and endocrine characterization of individual and successive waves in non-mated llamas with a special interest on the overlapping phenomenon. Twelve llamas were examined daily by transrectal ultrasonography for at least two consecutive waves. In six females, blood samples were collected daily at the end of each examination. The development of the largest follicle (F) showed a wavelike pattern with a mean duration of 25days. All waves evaluated were partially overlapped on the preceding wave and emerged at a mean interval of 15.8±0.5days. This interwave interval determines a mean overlapping degree of 32% of the wave length. Similarly, mean plasma oestradiol-17β (E2 ) concentrations followed a wavelike pattern. However, E2 concentrations started to decline before the structural regression of the F was observed. Mean basal E2 concentrations remain higher than 10.9±0.6pmol/l. In conclusion, follicular activity in non-mated llamas is characterized by continuous emergence of successive waves that always overlap the preceding wave with variable degrees. E2 production during the follicular wave is shorter in duration than the morphological development of the F. Finally, the overlapping phenomenon maintains increased plasma E2 concentrations persistently and this could explain the prolonged periods of sexual receptivity registered in llamas.

Effect of different doses of equine chorionic gonadotropin on follicular and luteal dynamics and P/AI of high-producing Holstein cows

The hypotheses of this study were (1) that the administration of 400IU eCG in a TAI protocol would increase ovarian follicular growth and diameter of the largest follicle (LF), volume of the CL, and produce an earlier rise on serum concentration of progesterone (P4) to ultimately improve P/AI compared to non-treated high-producing Holstein cows; and (2) that 600IU of eCG could enhance any potential effects of a greater gonadotropin treatment upon follicular and luteal size and function, improving P/AI. Cows were subjected to a protocol of synchronization of ovulation for timed artificial insemination (TAI): D0–P4 device insert and estradiol benzoate, D8–P4 device removal and PGF2α; Experiment 1, D10PM – GnRH plus TAI; and Experiment 2, D10AM – GnRH, D10PM – TAI. In Experiment 1, at P4 device removal, cows were assigned to one of the two treatments to receive none (n=232) or 400IU (n=232) of eCG. In Experiment 2, again at P4 device removal, cows were assigned to one of the three treatments to receive no eCG, (n=166) 400 (n=145) or 600IU (n=145) of eCG. Pregnancy was diagnosed 35 days after TAI. Ultrasonographic examination of both ovaries was done in a subset of cows in Experiments 1 [no eCG (n=27) and 400IU eCG (n=14)], and 2 [no eCG (n=15), 400IU eCG (n=14) and 600IU eCG (n=11)]. Exams were conducted at device removal (D8) and TAI (D10) to measure the diameter of the LF; then twice daily from D10 to 13, to determine time to ovulation and the maximum diameter of the LF; and then 3 (D14), 6 (D17), 9 (D20) and 12 (D23) days after presumed ovulation, concurrent with blood sampling, to measure the volume of the CL and serum concentration of P4. In both studies, eCG (400 or 600IU)-treated cows had similar diameter of the LF on D8 and D10, growth rate of the LF from Days 8 to 10, ovulation rate, time to ovulation, volume of the CL, serum concentration of P4 and P/AI as compared to control animals. Thus, adding either 400 or 600IU eCG to TAI protocols was inefficient to alter follicular and luteal dynamics and increase P/AI in high-producing dairy cows under the conditions of these experiments. The lack of positive effects of eCG in the present study might be explained by the small percentage of cows with poor body condition score and lesser incidence of anestrus.

Ovarian Follicular Dynamics, Ovarian Follicular Growth, Oocyte Yield, In vitro Embryo Production and Repeated Oocyte Pick Up in Thai Native Heifers Undergoing Superstimulation.

The objective of this study was to compare the effectiveness of the protocols for superstimulation of follicular growth in Thai native heifers. Heifers (n = 20) were randomly divided into four groups of five heifers/group. Heifers were given a single dose by i.m. administration of 100 mg Follicle Stimulating Hormone dissolved in polyvinylpyrrolidone (FSHp) at 24 h. Ovum pick up (OPU) occurred at 72 h (F24O72 protocol; Group 1) or 96 h (F24O96 protocol; Group 2), and at 36 h and OPU at 72 h (F36O72 protocol; Group 3) or 96 h (F36O96 protocol; Group 4) after follicular ablation. The dynamics of ovarian follicular growth were monitored by twice-daily ultrasonographic examinations. Blood sample collections were performed every 12 h after initiation of treatment for assessment of FSH, E2 and P4 profiles. All heifers were subjected to eight repeated sequential sessions of OPU. The follicular deviation commenced 24±5.32 h after follicular ablation in all groups. The circulatory FSH surged quickly from 24 to 36 h (>0.8 ng/ml) after follicular ablation and circulatory estrogen levels steadily increased from 36 h until OPU in all groups. At the end of the OPU sessions, the mean number of aspirated follicles/heifer/session in F36O72 protocol (Group 3) and F36O96 protocol (Group 4) were higher than in the two other groups (p<0.05). The number of cumulus-oocyte complexes (COCs), cleaved and day 8 blastocysts rates in the F36O72 protocol (Group 3) were higher than in the other groups (p<0.05). It can be concluded that a single dose i.m. administration of 100 mg FSHp at 36 h and OPU at 72 h after follicular ablation (F36O72 protocol; Group 3) was the most effective protocol for superstimulation of follicular growth for repeated OPU and subsequent in vitro embryo production in Thai native heifers.

Amount of mRNA and localization of vascular endothelial growth factor and its receptors in the ovarian follicle during estrous cycle of water buffalo (Bubalus bubalis)

The objective of the present study was to characterize the temporal patterns of gene expression for vascular endothelial growth factors (VEGF) and VEGF receptors during ovarian follicular growth, development and maturation in buffalo (Bubalus bubalis). Follicles were classified into four groups according to size and the concentration of estradiol-17β (E2) in follicular fluid (FF): Group I (small), 4–6mm diameter, E2>0.5ng/ml of FF; Group II (medium), 7–9mm, E2=0.5–5ng/ml; Group III (large), 10–13mm, E2=5–40ng/ml; Group IV(pre-ovulatory), >13mm, E2>180ng/ml). The mRNAs for FSH receptor (FSHR), LH receptor (LHR) and aromatase (CYP19A1) in theca interna and granulosa layers were also determined, further defining the maturational state of each group. The relative expression of VEGF isoforms (120, 164, and 188 amino acid forms), as determined by quantitative real-time PCR (qRT-PCR), increased during follicular development in both the granulosa (P<0.05) and theca layers. Relative amounts of VEGF receptors (VEGFR-1 and VEGFR-2) were least in granulosa cell (GC) and theca interna cell (TI) layers of Gp-I follicles. The amount of VEGFR-2 transcripts increased in the granulosa layer throughout development, reaching a maximum in Gp-IV follicles (P<0.05). The relative amount of VEGF isoforms and receptors in follicle lysates, as determined by western blotting, increased throughout follicular maturation to maximum amounts in pre-ovulatory follicles. Immunohistochemistry revealed a clear localization of VEGF isoforms and receptors in both steroidogenic cell types (GC and TI) and of VEGF receptors in the vascular endothelial cells of the thecal blood vessels. The most intense immunofluorescence was evident in pre-ovulatory follicles compared to other smaller follicles. These data provide evidence that the VEGF may contribute to the extensive capillary proliferation associated with the increase in size, selection, and maturation of the pre-ovulatory follicle. This may facilitate follicle maturation by enhancing the supply of nutrients, hormones, and other essential blood-borne signals to the follicle. VEGF may also promote maturation of follicles through recently recognized, non-angiogenic mechanisms.

Influence of equine growth hormone, insulin-like growth factor-I and its interaction with gonadotropins on in vitro maturation and cytoskeleton morphology in equine oocytes

In horses, successful in vitro fertilization procedures are limited by our inability to consistently mature equine oocytes by in vitro methods. Growth hormone (GH) is an important regulator of female reproduction in mammals, playing an important role in ovarian function, follicular growth and steroidogenesis. The objectives of this research were to investigate: the effects of equine growth hormone (eGH) and insulin-like growth factor-I (IGF-I) on the in vitro maturation (IVM) of equine oocytes, and the effects of eGH in addition to estradiol (E2), gonadotropins (FSH and LH) and fetal calf serum (FCS) on IVM. We also evaluated the cytoskeleton organization of equine oocytes after IVM with eGH. Equine oocytes were aspirated from follicles <30 mm in diameter and matured for 30 h at 38.5°C in air with 5% CO2. In experiment 1, selected cumulus–oocyte complexes (COCs) were randomly allocated as follows: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 200 ng/ml anti-IGF-I. In addition to these treatment groups, we also added 1 μg/ml E2, 5 IU/ml FSH, 10 IU/ml LH and 10% FCS in vitro (experiment 2). Oocytes were stained with markers for microtubules (anti-α-tubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and chromatin (TO-PRO3-iodide) and assessed via confocal microscopy. No difference was observed when eGH and IGF-I was added into our IVM system. However, following incubation with eGH alone (40%) and eGH, E2, gonadotropins and FCS (36.6%) oocytes were classified as mature v. 17.6% of oocytes in the control group (P < 0.05). Matured equine oocytes showed that a thin network of filaments concentrated within the oocyte cortex and microtubules at the metaphase spindle showed a symmetrical barrel-shaped structure, with chromosomes aligned along its midline. We conclude that the use of E2, gonadotropins and FCS in the presence of eGH increases the number of oocytes reaching oocyte competence.

The possible role of natural cycle and modified natural cycle in IVF

1. Natural cycleThe first successful pregnancy and live birth resulting fromIVF were achieved during an unstimulated natural cycle(1). Soon thereafter, natural IVF was replaced by stimulatedIVF because of the very high cancellation rates of naturalcycles, and stimulated treatment became the standard inIVF. However, ovarian stimulation is not free from negativeconsequences and risks, including ovarian hyperstimulationsyndrome. (OHSS), which affects up to 5–10% of IVF cyclesand can be life-threatening (2). Multiple pregnancies occurin approximately 30% of pregnancies in patients who under-go the COH protocol and this phenomenon is related to in-creased risks of pregnancy loss, obstetrical complications,prematurity and neonatal morbidity with long-term damage.The long-term side effects however remain largely unknown.Ovarian cancer and gestational trophoblastic disease may beassociated with the chronic use of gonadotrophins (3). Nev-ertheless, the interest in natural IVF cycle treatment hasbeen renewed in recent years because of the increased effi-ciency of IVF technology. With the increasing awarenessof side effects of ovarian stimulation and better understand-ing of ovarian physiology in relation to ovarian folliculargrowth and maturation, IVF in natural cycles has gainedgreat attention and interest for both normal responder andpoor responder patients (4).1.1. AdvantagesNatural IVF cycles are simple, inexpensive and rapid andside effects associated with ovarian stimulation are elimi-nated. Natural cycle offers a more physiological, less drug-oriented, lower risk and more patient-friendly approach(4). Natural IVF cycles might be more efficient for obtainingideal embryos. It can be repeated on a monthly basis, andthe overall chances of success are therefore higher. Some pa-tients might prefer several successive natural IVF cycles in-stead of stimulated IVF cycles, which can only be repeatedonce a month (5). It has been reported that the clinical preg-nancy rate with stimulated IVF has reached approximately25–30% (6). However, repeated stimulated cycles shouldspan over several months to allow for the ovaries to recover.Further, if stimulated IVF cycles are repeated, the pregnancyrate will obviously decrease.1.2. DisadvantagesProblems related to natural IVF cycles include an increasedrisk of untimely LH surge and the possibility that no oocyteis retrieved and no embryo is available for transfer (5). Theefficacy of natural IVF is hampered by the high incidence ofoocyte retrieval failure (16.7–71.4%) and the relatively lowpregnancy rate per embryo transfer (ET) cycle (0–23.5%)(4). Natural IVF cycles resulted in an ongoing pregnancyrate of approximately 7% per started cycle and approxi-mately 16% per ET (7).1.3. Natural cycles in poor respondersPrevious studies found that the natural cycle works at leastas well as the COH in poor responder women who failedprevious ovarian hyperstimulation (8). In poor responder pa-tients, only very few follicles can be recruited and very fewoocytes can be retrieved after stimulation despite the highdose of gonadotropins administered or repeated stimulatedcycles performed and their management remains a challengein assisted reproduction. The majority of poor responderwomen had advanced reproductive age, diminished antral

Possible Involvement of Chemerin, a Novel Regulator in Follicular Growth and Steroidogenesis, in the Pathogenesis of Polycystic Ovarian Syndrome (PCOS).

Polycystic ovarian syndrome (PCOS) is a heterogeneous syndrome associated with follicle growth arrest, dysregulated sex hormone profile, hyperthecosis and insulin resistance. Chemerin is a novel adipokine and is associated with obesity and metabolic syndrome. Although obese women and PCOS subjects have elevated plasma chemerin levels, whether and how chemerin is involved in the regulation of follicular growth/steroidogenesis and pathogenesis of PCOS is unknown. Our objective is to better understand the complex regulatory mechanisms involved in the control of ovarian follicular growth and steroidogenesis and gain insights in their dysregulation in the pathogenesis of PCOS. We hypothesize that chemerin is a novel regulator of follicular growth and steroidogenesis and contributes to the pathogenesis of PCOS. Using an immature rat model (day13-15 for follicle culture and day 21-24 for granulosa cells culture) and a chronically androgenized rat model [dihydrotestosterone (DHT); daily release dose 83μg from day 21-105] which recapitulates the reproductive and metabolic phenotypes of human PCOS, we have examined the granulosa cell expression patterns of chemerin and its receptor CMKLR1 and their steroidogenic capability. Chemerin and CMKLR1 were expressed in granulosa cells from immature rats and negatively regulated by gonadotropin in vivo and in vitro. Serum and ovarian chemerin levels in DHT-treated rats were elevated, which was associated with arrested follicular growth and decreased expression of p450 side-chain cleavage enzyme (p450scc), aromatase and hydroxysteroid dehydrogenases. Recombinant chemerin inhibited FSH-induced estradiol secretion in granulosa cells from DHT-treated rats in vitro. In addition, chemerin suppressed basal and FSH-induced follicle growth and estradiol/progesterone production in a concentration-dependent manner in preantral follicle cultures. Moreover, chemerin suppressed FSH-induced p450scc/aromatase expression and progesterone/estradiol secretion in immature rat granulosa cells in vitro. Conclusion: This study demonstrated, for the first time, that chemerin is a novel negative regulator in FSH-induced follicular growth and steroidogenesis. Our findings support the notion that the dysregulation of chemerin expression and function contributes to pathogenesis of PCOS and infertility associated with compromised follicular growth and differentiation. This research is supported by a grant from the CIHR (B.K.T.), CIHR-QTNPR (Q.W.) and CIHR-REDIH (K.X.) graduate scholarships and a CIHR-QTNPR postdoctoral fellowship (J.Y.K.).