Reprod Sci Research Articles

Analysis of the proteins and peptides isolated from incubation medium of early equine conceptuses - preliminary results

reLH as reported (Meyers-Brown et al., Anim Reprod Sci 2011). A total of 31 embryoswere obtained from11mares in a 70 day period; of these, 21 embryos were used for ESC isolation. In order to isolate the ICM, blastocysts were processed by either immunosurgery (n1⁄4 8) or microsurgery (n 1⁄4 13). The ICM was cultured on inactivated mouse embryonic fibroblasts (MEF) in control (n1⁄4 9) or inhibited (n1⁄4 11) medium. Control culture medium consisted of DMEM/F12, 15% FBS, 2 mM glutamine, 1 % nonessential amino acids, 0.1 mM2-mercaptoethanol,1mMsodiumpyruvate, and0.4 mg/ mL human LIF. The inhibited medium was supplemented with 3 mM GSK3 inhibitor (CHIR99021), 0.5 mM MEK inhibitor (PD0325901), and 2.5 mM TGF inhibitor (A83-01). No difference in initial cell line establishment was observed between immunosurgery and microsurgery ICM isolation (6/8 and 12/13, respectively) or between control and inhibited medium (8/9 and 10/11). The resulting cell lines grew incoloniesof tightlypackedcellswith ahighnuclear to cytoplasmic ratio. Some of the lines have been cultured for up to seven passages and have been successfully cryopreserved. Immunofluorescence analysis indicates that some cells expressed the pluripotency marker OCT4; however, all the cells were positive for the trophectoderm (TE) marker CDX2. To clarify the interpretation of these results we performed immunostaining of whole equine embryos at different stages of development. At the morula stage OCT4 was highly expressed in all cells and no CDX2 staining was observed. At day 7, expanded blastocysts expressed CDX2 clearly localized to the TE while OCT4 was present in both ICM and TE cells, although at higher intensity in the ICM. At Day 9, expanded blastocyst presented OCT4 expression exclusively localized to the ICM and CDX2 to the TE. In summary, the use of recombinant gonadotropins was effective at providing a large number of equine embryos for use in ESC isolation. Also, marker characterization of in vivo-derived embryos indicates that equine pluripotent cells should be positive for OCT4 and negative for CDX2, suggesting that cell lines derived in our study are of the TE lineage. Further approaches to isolate pure populations of ICM cells and maintain the undifferentiated characteristics of these cells in vitro are in progress.

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Lower serum apelin levels in women with polycystic ovary syndrome

We tested differences in serum apelin levels between women with polycystic ovary syndrome (PCOS) and those with a healthy regular menstrual cycle, finding that apelin levels were higher in normal women and that apelin was positively correlated with apolipoprotein A levels.

536: Progesterone inhibits TNFα-induced apoptosis in fetal membranes by altering expression of pro- and anti-apoptotic proteins: implications for inflammation-mediated preterm birth

Progesterone supplementation prevents PTB in some high-risk women, but its mechanism of action is unknown. One third of PTB is associated with intraamniotic inflammation (IAI). We have previously shown that progesterone inhibits inflammatory cytokine (TNFα)-induced apoptosis in fetal membranes (Luo et al. Reprod Sci 2010;17:532). This study investigates the molecular mechanisms responsible for this progesterone-mediated inhibition.

Association of Zinc Deficiency and Depression in the Patients With End-stage Renal Disease on Hemodialysis

Depression is a common psychological symptom in patients undergoing chronic hemodialysis. In the general population, low serum zinc level is associated with major depression. The current study surveys the possible relationship between the prevalence of depression and plasma level of zinc in patients on hemodialysis (HD). A total of 135 patients with end-stage renal disease (ESRD) on HD were enrolled in the study. The severity of depression was assessed using Beck Depression Inventory (BDI). Plasma zinc level was measured from fasting samples. Mean age of the patients was 52.45 (standard deviation: 15.33) years. In all, 76% of the patients had some degree of depression according to BDI scoring system (BDI >14). The mean level of plasma zinc in the depressed patients was significantly lower than the rest of the patients (67.46 ± 29.7 vs. 85.26 + 40.05). Zinc deficiency may be a reversible cause which might contribute to the increased rate of depression in HD patients. This is the first study reporting the association of zinc deficiency with the presence of depression in HD patients; therefore, these findings need further investigations.

Methylation patterns of Brahma during spermatogenesis and oogenesis: potential implications

To compare methylation profiles and expression levels of Brahma at different stages of spermatogenesis, and to identify the methylation pattern during oogenesis, we analyzed gene expression and methylation patterns in murine germ cells at various developmental stages. The methylation levels of CpG islands within Brahma increased during spermatogenesis and decreased during oogenesis. This change in methylation pattern correlates with the change in expression of Brahma during spermatogenesis. As the degree of methylation increases, the expression decreases. The change in methylation is opposite during oogenesis, which suggests opposite expression levels.

Histologic and ultrastructural features of cryopreserved ovine ovarian tissue: deleterious effect of 1,2-propanediol applying different thawing protocols

Ovarian cortical fragments were frozen in presence of 1.5 M PROH, thawed at different temperatures (37 degrees, 30 degrees, or 4 degrees C) and submitted to histologic and ultrastructural analysis. Thawing at 30 degrees C minimized stromal and follicular damage when compared with 4 degrees and 37 degrees C.

154. EXTRACELLULAR CALRETICULIN ALTERS ENDOTHELIAL CELL AND TROPHOBLAST CELL FUNCTIONS IN VITRO CONSISTENT WITH PRE-ECLAMPSIA

Pre-eclampsia is a multisystem disorder of human pregnancy that involves abnormal placentation via insufficient trophoblast cell invasion of the maternal spiral arteries and widespread maternal endothelial cell dysfunction. Factors in plasma of pre-eclamptic women affect both trophoblast and endothelial cell functions during in vitro culture (1). The calcium-binding protein calreticulin is elevated in peripheral blood with pre-eclampsia compared to normotensive pregnancy (2). The aim of this study was to determine the effects of exogenous calreticulin at concentrations relevant to normotensive pregnancy (2 µg/mL) and to pre-eclampsia (5 µg/mL) on human trophoblast cell (HTR8) and microvascular endothelial cell (myometrial) numbers and migratory activity. Cell migration was measured by scratch assay; changes in cell number were measured by MTS assay (Promega). The results showed that calreticulin at 5µg/mL did not affect HTR8 cell number (control 68044+24542 cells, with calreticulin 72810 + 30673 cells, n = 3, P > 0.05) after 48 hours, but significantly inhibited migration of the cells by 48+11% compared to the control at 26 hours (n = 4, P < 0.02). Calreticulin at 5 µg/mL and under conditions that did not change cell number significantly increased migration of the myometrial endothelial cells by 39+7% (n = 4, P < 0.01) at 20 hours. Calreticulin at 5 µg/mL, however, significantly reduced endothelial cell numbers after 3–5 days (control 6213 + 1937 cells, with calreticulin 1937+728 cells, n = 6, P < 0.05). There was no significant change to the functions of either cell type with 2 µg/mL of calreticulin. In conclusion, exogenous calreticulin at a concentration consistent with that found in maternal blood with pre-eclampsia was shown to alter trophoblast and endothelial cell migratory activity and reduce endothelial cell numbers during in vitro culture. These results indicate that elevated circulating calreticulin may contribute to the cellular mechanisms that underlie the development of pre-eclampsia. (1) Harris et al, Reprod Sci, 2009, 16: 1082–90.(2) Gu et al, Molec Human Repro, 2008, 14: 309–15.

303. NUTRIENT INTAKE OF BOS INDICUS HEIFERS DURING EARLY AND MID-GESTATION CAUSES CHANGES TO PLASMA CONCENTRATIONS OF TRIIODOTHYRONINE (T3) AND THYROXINE (T4) OF THEIR PROGENY

Fetal and postnatal growth are mediated by thyroid hormones (TH). Maternal nutrient intake during gestation can program postnatal TH concentrations. This may have significant economic implications for beef cattle production. We investigated the effect of feeding beef heifers high (H = 240%) and low (L = 70%) levels of recommended daily crude protein intake during the first and second trimesters of gestation in a two-by-two factorial design on progeny (n = 68) plasma concentrations of free and total triiodothyronine (FT3 and TT3) and free and total thyroxine (FT4 and TT4) from birth until weaning at 6 mo of age. Exposure to low compared to high protein diets during the second trimester resulted in increased plasma FT3 concentrations relative to TT3 (P = 0.04) at birth. For male progeny, exposure to low compared to high protein diets during the first trimester resulted in greater plasma FT4 concentrations from birth until weaning (P = 0.02). Also for males from birth until weaning, LH had greater plasma TT3 concentrations than HH (P < 0.01). For female progeny, HH had greater plasma TT3 concentrations relative to TT4 than HL from birth until weaning (P = 0.03). Plasma concentrations of FT3 were positively associated with average daily gain relative to birth weight at 1 (r = 0.41; P < 0.01) and 3 mo FT3 (r = 0.41; P < 0.01). Heifer protein intake during the first and second trimesters of gestation has a permanent effect to progeny plasma TH concentrations and these changes are associated with the postnatal growth pathway. (1) Micke GC, Sullivan TM, Magalhaes RJS, Rolls PJ, Norman ST, Perry VEA. Heifer nutrition during early- and mid-pregnancy alters fetal growth trajectory and birth weight. Anim Reprod Sci. 2010; 117: 1–10.

201 ENDOSCOPE-GUIDED TRANSFER OF SPERM-INJECTED OOCYTES INTO THE OVIDUCTS OF ELAND AND BONGO ANTELOPES

We have previously established methods of gamete collection in Eland and Bongo antelopes; however, blastocyst development following IVF, intracytoplasmic sperm injection (ICSI), or somatic cell nuclear transfer (SCNT) has been sporadic, thus few developmentally competent embryos have been produced for uterine transfer. In the present study, we evaluated the possibility of oviductal transfer of presumptive zygotes. Estrous cycle synchronization and ovarian superstimulation were done as follows. Females were treated with altrenogest (0.11 g/os daily) for 7 days. The FSH Folltropin® was dissolved in 30% polyvinylpyrrolidone and administered, i.m. on Days 5 and 7 of altrenogest treatment at 266 mg and 134 mg, respectively. Prostaglandin F2α (Lutalyse®, 25 mg, i.m.) was administered on Day 7. Transvaginal ultrasound-guided oocyte retrieval was conducted on Day 9 as described previously (Wirtu G et al. 2009 Anim. Reprod Sci. 111, 160-172). Eleven oocytes were recovered from 2 Elands and 4 from 1 Bongo. One oocyte of each species was degenerate at the time of recovery. Oocytes were subjected to IVM for approximately 22 h, when Piezo drill-assisted ICSI was done using frozen-thawed Eland or Bongo spermatozoa. Oocytes were activated (5% ethanol, 5 min) and cultured overnight in CR1aa medium supplemented with BSA. Presumptive zygotes were subjected to endoscopic oviductal transfer at 21 to 24 h after ICSI. The oviductal transfer was adapted from the technique developed in domestic cattle (Wetscher F et al. 2005 Theriogenology 64, 30-40). Animals were sedated by i.m. administration of xylazine HCl and butorphanol tartrate and restrained in a hydraulic chute (Tamer®) that was used to squeeze and lift the females. An epidural block (5 mL of lidocaine) was induced after cleaning the rectum, perineal region, and the injection site. A uterine relaxant, isoxsuprine HCl (10 mg, i.v), was then administered. The Brem/Besenfelder set for laparoscopic bovine embryo transfer (Karl Storz Endoscope, Karl Storz GmbH & Co. KG, Tuttlingen, Germany) was used for the oviductal transfer. Briefly, a cannula (12.5 mm in diameter, 49.5 cm long) fitted with a blunt-tip obturator was introduced into the vagina and positioned, during transrectal manipulation, at the mid-dorsal aspect of the vaginal vault. The blunt-tip obturator was replaced with a sharp-tip obturator, which was used to puncture through the vaginal wall for entry into the pelvic cavity. The trocar was subsequently replaced with an inner sheath containing a telescope (5.5 mm in diameter, 54 cm long) and a pre-loaded transfer tubing. Visualization of the ovaries and oviducts required insufflation of the peritoneal cavity. Transfer was done into the oviduct ipsilateral to the recently ovulating ovary. Ten Eland oocytes had extruded the first polar body and 8 that survived ICSI were transferred into the right oviduct of an Eland female. Two Bongo oocytes had extruded the first polar body; both survived ICSI and were transferred to the right oviduct of a Bongo female. Pregnancy diagnosis is pending. This minimally invasive method of accessing the oviduct has the potential to advance the application of assisted reproductive technologies in large nondomestic ungulates.

Expression of bone morphogenetic protein-15 in human oocyte and cumulus granulosa cells primed with recombinant follicle-stimulating hormone followed by human chorionic gonadotropin

Bone morphogenetic protein-15 is expressed in oocytes and cumulus granulosa cells. It may play an important role in the hCG-induced oocyte final maturation and corpus luteum (CL) formation.

Vitrification of immature mouse oocyte using stepwise equilibration before or after in vitro maturation

Immature mouse oocytes before or after in vitro maturation (IVM) were vitrified by the use of either conventional one-step or three-step equilibration. Although vitrification after IVM using the stepwise method does not affect the cleavage rate, the early embryonic development was profoundly impaired in both protocols.

Complete follicular development and recovery of ovarian function of frozen-thawed, autotransplanted caprine ovarian cortex

Frozen-thawed ovarian cortical fragments (1 mm(3)) were autotransplanted to the uterus of completely ovariectomized goats. The grafts developed preovulatory follicles, accompanied by estrous behavior and a rise in plasma E(2) levels, demonstrating successful cryopreservation and transplantation.

Glycolytic enzyme expression in human granulosa cells

To examine the differences in specific protein expression between mural and cumulus granulosa cells following 24-hour in vitro culture. Laboratory study. University Hospital. Human granulosa cells were collected at the time of egg collection during assisted reproduction. Cumulus cells associated with the oocyte were separated from mural cells from the periphery of the follicle before in vitro culture for 24 hours. Cells were then lysed and subjected to two-dimensional gel electrophoresis. Given that cumulus (cGC) and mural granulosa cells (mGC) differentiate from a single layer, it is likely that phenotypic differences between them may reflect specific molecular processes and structural adaptations. Computer-assisted analysis using dedicated software enabled the presence, absence, or relative volume of each individual protein spot to be estimated. Differentially expressed spots were identified using tandem mass spectrometry. The mean number of separate protein spots detected in mGC gels was 1,105 +/- 146, and in cGC it was 887 +/- 236, although there was no statistically significant difference between the two. Five enzymes of the glycolytic pathway were never expressed in cGC after 24 hours in vitro; these were triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase 1, and two isoforms of alpha enolase. These are the first data collected in humans consistent with a recent demonstration that isolated murine cGC cultured in vitro exhibit decreased expression of mRNA encoding glycolytic enzymes, and support the suggestion that some factor or factors secreted by the oocyte may be responsible for the maintenance of glycolysis in the adjacent cGC.

Features of follicle-stimulating hormone–stimulated follicles in a sheep model: keys to elucidate embryo failure in assisted reproductive technique cycles

To evaluate the individual functionality of gonadotropin-stimulated preovulatory follicles, for understanding embryo failure in assisted reproductive technique cycles, in a sheep model. Observational, model study. Public research unit. Fifteen adult Manchega ewes. Synchronization of the estrous cycle with intravaginal progestagens and ovarian stimulation with FSH; evaluation of reproductive activity, plasma sampling, ovarian ultrasonography, and ovariectomies. Determination of estrus behavior, plasma and intrafollicular concentrations of E(2) and inhibin A, number and size of ovarian follicles, and developmental competence of oocytes. These results support the usefulness of serial measurements of plasma inhibin A for assessment of follicular growth during the FSH treatment, rather than of E(2) assays commonly used. Functionality of FSH-stimulated preovulatory follicles is clearly disturbed, as confirmed by a negative correlation between follicular size and intrafollicular concentrations of inhibin A and E(2) in preovulatory follicles after individual dissection; moreover, the ability of their oocytes to resume meiosis was diminished. Functionality of follicles in controlled ovarian stimulation (COS), and developmental competence of their oocytes, is disturbed by the high doses of gonadotropin supplied and finally determined by follicular sizes at starting FSH treatment.

OC10 A Negative Energy Balance but not Suckling Frequency (ad libitum, Twice or Once Daily) Modules Ovarian Resumption in Parda de Montaña Cattle Breed

In extensive systems, beef cows should be continuously with their calves to optimise pasture use but this practice can lengthen post‐partum interval (PPI). A study was conducted to determine the influence of suckling frequency on Parda de Montaña cow‐calf performance when cows are fed 70% energy requirements during lactation (outdoor winter conditions simulation). Thirty‐six autumn‐calving cows with similar body condition at calving (2.57) were assigned to three suckling systems [ad libitum (AL), twice (2D) or once daily (1D) for 30 min]. Blood samples were collected twice a week to analyse progesterone concentrations by RIA. Cows lost similar weight until start of mating period (day 52 post‐partum; −0.583, –0.513, –0.520 kg/day in AL, 2D and 1D). Standard milk yield was higher in AL than the rest (8.9, 6.2, 7.0 kg; p < 0.05), which was reflected on greater calf gain (0.895, 0.752, 0.676 kg/day; p < 0.05) and larger cow weight loss within 90 days post‐partum in this treatment (–0.345, –0.188, –0.083 kg/day; p < 0.05). Suckling system did not affect either PPI (69.6, 89.1, 65.5 days) or cows cycling within 90 days post‐partum (55, 46, 58%), which may compromise the target calving interval. In Parda de Montaña breed fed moderately pre‐calving and undernourished during lactation, restricted suckling did not favour ovarian activity resumption, but post‐partum subnutrition delayed about 40 days PPI observed in similar body condition at calving and calf management (Sanz et al., 2003; Anim Reprod Sci 79: 57–69)

Proteomic technology for the rapid and accurate diagnosis of intra-amniotic inflammation (IAI) in twin gestation

AMNIOTIC INFLAMMATION (IAI) IN TWIN GESTATION IRINA BUHIMSCHI, CATALIN BUHIMSCHI, MERT BAHTIYAR, ROB CHRISTNER, CARL P. WEINER, Yale University, Ob/Gyn & Reprod Sci, New Haven, Connecticut, Ciphergen Biosystems, Freemont, California, University of Maryland, Baltimore, Maryland OBJECTIVE: The diagnosis of IAI complicating preterm labor requires the timely decision of whether the fetus benefits more from early delivery versus continued growth in-utero. Proteomics has the power to identify a hostile fetal environment. The purpose of this study was to determine whether IAI can be accurately diagnosed in twin gestation using specific proteomic biomarkers. STUDY DESIGN: Amniotic fluid (AF) was retrieved by amniocentesis from 5 diamniotic, dichorionic twin pregnancies with either PPROM or preterm labor (GA range: 19-32 wk). Indigo carmine was instilled to assure both sacs were sampled. Three pregnancies were sampled again for clinical indications (4-7 d later). Delivery occurred within 20d of amniocentesis. AF was analyzed for glucose, cell counts, interleukin-6 (IL-6) and microbiologic cultures. A proteomic fingerprint was generated with the investigators blind to clinical outcome and a bioinformatic score was calculated. Chorioamnionitis was assessed histopathologically. RESULTS: A combination of 4 protein biomarkers identified on the hydrophobic ProteinChip array (H4) (Ciphergen Biosystems) with peaks located at 3,377.0 Da (defensin-2), 3,448.1 Da (defensin-1), 10,443.8 Da (calgranulin C) and 10,834.51 (calgranulin A) predicted IAI with 100% accuracy. Discordant inflammation was diagnosed with similar accuracy (Figure). The proteomics fingerprint predicted histological chorioamnionitis accurately in each instance when the amniocentesis-to-delivery interval was ! 24h (3/5). Results were available within 90 min. of beginning the assay. CONCLUSION: In twin gestation IAI begins as a localized process. Proteomic analysis of AF accurately identifies concordant and discordant inflammation. This technology has potential to alter clinical practice by providing an accurate diagnosis of inflammation which may enable targeted interventions.

283ASSESSMENT OF CALVES PRODUCED BY IVP IN A SEMI-DEFINED MEDIUM

Heavy birth weight, increased calving difficulty, heart function defects, increased perinatal mortality and organ immaturity have been reported for calves produced from IVP embryos compared to those produced from MOET or AI (van Wagtendonk AM et al., 2000 Theriogenology 53, 575–597; Jacobsen H et al., 2002 Anim Reprod Sci 70, 1–11). In this study we examined birth weight (BWT), and blood chemistry at 1 day of age, gestation length and heart function at 7 days, and response to an ACTH challenge at 21 days of calves derived from IVP in a ‘semi-defined’ IVC system (Thompson JG et al., 2000 J. Reprod. Fertil. 118, 47–55) and of contemporary MOET or AI calves. Holstein Friesian (HF) 2- and 3-year-old recipients carrying single HF calves (101×IVP and 21×MOET) were monitored in this study. Within 1 day of birth the calves were weighed and a blood sample taken for analysis. At 7d, ultrasound measurement of the left ventricle diastolic diameter (LVEDd) and % ejection fraction (EF%) was determined. Each calf was then transported to a rearing unit. At 3 weeks of age, 30 IVP and 30 control AI calves of the same age were injected i.v. with Synacthen (synthetic ACTH, Ciba Corporation, 0.1μgkg−1 body weight). Blood samples were collected at −30, 0, 30, 60 and 90min (0min=time of injection) for cortisol measurements. There was no difference in BWT for MOET or IVP calves (40.9±4.7 v. 35.6±4.8kg, respectively). Moreover, gestation lengths (279 days v. 281 days) and calving assistance scores (1.3 v. 1.6) did not differ. Calf mortality at birth was higher for IVP calves (16%) than for MOET calves (5%). All but 7 surviving calves (6×IVP and 1×MOET) had high GGT levels at 1 day. Blood chemistry revealed no differences between the calf types, all measures being within normal ranges. For all calves, heart function analysis revealed no abnormalities with mean LVEDd=4.1±0.6cm and mean EF%=78.5±8.4%. All calves exhibited elevated cortisol following ACTH challenge. There was no difference between control and IVP calves for mean cortisol concentration at any time point (0min, 13.8±5.2; 30min, 46.6±9.8; 60min, 42.8±9.9; 90min, 28.1±8.9ngmL−1). These data suggest that, unlike calves produced in less defined culture systems, calves produced by IVP in a semi-defined culture system have birth weight and gestation lengths similar to those of MOET calves. Moreover, no abnormalities in organ (heart, adrenal) function were detected. However, of concern was the high number of unexplained deaths for IVP calves. This may be due to an overall lack of vigour in IVP calves that, in an unsupervised calving, results in calf death. More vigilence at calving may be needed to ensure calf survival. The authors thank Juliet Jensen, Waikato Hospital, for ultrasound measurements and David Stewart, Morrinsville Veterinary Services, for calf care. This study was funded by Vialactia Biosciences and FRST.

Unlike preeclampsia, gestational hypertension is not associated with increased neonatal and maternal morbidity except abruption: [formula omitted], P. Robertson, J. Perry x, M. Croughan-Minihane x. University of California, Dept Obstet. Gynecol. & Reprod Sci, San Francisco, CA

Unlike preeclampsia, gestational hypertension is not associated with increased neonatal and maternal morbidity except abruption: [formula omitted], P. Robertson, J. Perry x, M. Croughan-Minihane x. University of California, Dept Obstet. Gynecol. & Reprod Sci, San Francisco, CA

Molecular biology of human male infertility: links with aging, mitochondrial genetics, and oxidative stress?

Molecular biology of human male infertility: links with aging, mitochondrial genetics, and oxidative stress?