Effects of oocyte quality, semen donor and embryo co-culture system on the efficiency of blastocyst production in goats

The aim of the study was to determine whether the selection of immature oocytes by a combination of cumulus-oocyte-complexes (COCs) morphology and staining with brilliant cresyl blue (BCB) would be helpful in selecting developmentally competent oocytes, and thereby increase the efficiency of blastocyst production from ovarian oocytes of FSH-primed, adult goats. In a second experiment the interaction between oocyte quality and semen donor was assessed. In a third experiment the usefulness of Vero cells for co-culture with goat embryos was investigated. In the pool of morphologically normal COCs recovered from ovaries following slicing (21.9+/-11.0), the mean rate of COCs classified as BCB+ was 85.6%, and the BCB- was approximately 11%. Oocytes classified as grade 1 and BCB+ exhibited the highest developmental competence (P<0.001) after in vitro maturation and fertilization compared with oocytes of grade 1 BCB- and grade 2 BCB+ or BCB-. There were no significant differences in developmental competence in grade 2 oocytes, regardless of BCB coloration. No significant differences in embryo cleavage and blastocyst formation rates among three bucks were observed when morphologically normal, BCB+ oocytes were used. For all tested bucks, differences in embryo production efficiency were related only to the oocyte quality. Similar blastocyst rates were developed from embryos co-cultured with goat oviduct epithelial cells (34.3%) and with Vero cells (33.3%). These results show that the most important criterion for selection of COCs before maturation is the visual assessment of morphological features. Staining with BCB of COCs recovered from adult goats does not enhance efficiency of selection of developmentally competent oocytes for IVF.     

小鼠植入前胚胎致密化与囊胚形成的分子调控

哺乳动物胚胎植入前的发育中致密化和囊胚形成分别标志着第一次、第二次细胞分化（即细胞命运决定）的起始,是胚胎正常发育的必要条件。因此对影响致密化和囊胚形成的蛋白及调节因子的研究尤为重要。本文探讨了与致密化相关的细胞黏附蛋白、连接蛋白、细胞骨架等分子和囊胚形成相关的紧密连接蛋白、钠钾三磷酸腺苷激酶等分子的一系列调控,以及致密化和囊胚形成在细胞命运决定中的重要作用。     

Effects of medium calcium, and agents affecting cytoskeletal function, on cellular volume and morphology in liver tissue in vitro

The possible role of an exocytotic, vesicular mechanism in cellular volume regulation under iso-osmotic conditions has been studied in slices of rat liver. The effects of incubation conditions and agents affecting the actin cytoskeleton were examined for changes of water, ionic composition, and ultrastructure. Slices were pre-incubated at 1°C in an iso-osmotic buffered medium to induce swelling. Upon restoration to 37°C in the same medium, tissue lost water. The Na+-K+ adenosine triphosphatase (ATPase) inhibitor ouabain inhibited water extrusion of about 50%, an effect that was accompanied by the formation of characteristic vesicles in the cytoplasmic region between the Golgi apparatus and the bile canaliculi. Water extrusion in the presence of ouabain was partially inhibited by trifluoroperazine and completely inhibited when the medium was free of Ca2+. In the presence of ouabain, brefeldin A caused a small reduction of water extrusion, whereas phalloidin and cytochalasins A, D, or E caused a marked inhibition. In these conditions there was a marked increase in size and number of cytoplasmic vesicles and a more widespread distribution of them within the cells, lacking the more specific orientation to the Golgi and canalicular regions that was seen in the presence of ouabain alone. Water extrusion was inhibited by phalloidin and cytochalasins in the absence of ouabain. In conclusion, our results are consistent with the hypothesis that iso-osmotic expulsion of water from hepatocytes can proceed partly through an accumulation of water in cytoplasmic vesicles, followed by exocytosis. This mechanism does not depend on Na+-K+ ATPase activity.     

Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion

The morphology and cytoskeletal structure of fibroblasts, endothelial cells, and neutrophils are documented for cells cultured on surfaces with stiffness ranging from 2 to 55,000 Pa that have been laminated with fibronectin or collagen as adhesive ligand. When grown in sparse culture with no cell-cell contacts, fibroblasts and endothelial cells show an abrupt change in spread area that occurs at a stiffness range around 3,000 Pa. No actin stress fibers are seen in fibroblasts on soft surfaces, and the appearance of stress fibers is abrupt and complete at a stiffness range coincident with that at which they spread. Upregulation of alpha5 integrin also occurs in the same stiffness range, but exogenous expression of alpha5 integrin is not sufficient to cause cell spreading on soft surfaces. Neutrophils, in contrast, show no dependence of either resting shape or ability to spread after activation when cultured on surfaces as soft as 2 Pa compared to glass. The shape and cytoskeletal differences evident in single cells on soft compared to hard substrates are eliminated when fibroblasts or endothelial cells make cell-cell contact. These results support the hypothesis that mechanical factors impact different cell types in fundamentally different ways, and can trigger specific changes similar to those stimulated by soluble ligands.     

Effects of exogenous hexoses on bovine in vitro fertilized and cloned embryo development: Improved blastocyst formation after glucose replacement with fructose in a serum-free culture medium

To evaluate the embryotrophic role of three hexoses (glucose, fructose, and galactose), bovine embryos derived from somatic cell nuclear transfer (SCNT) or in vitro-fertilization (IVF) were cultured in a modified synthetic oviductal fluid (mSOF), which contained either glucose (1.5 or 5.6 mM), fructose (1.5 or 5.6 mM), or galactose (1.5 or 5.6 mM). Compared to 1.5 mM glucose, use of 1.5 mM fructose significantly enhanced blastocyst formation in both SCNT (23 vs. 33%) and IVF embryos (26 vs. 34%), while 5.6 mM fructose did not improve blastocyst formation. Using 1.5 mM galactose did not improve blastocyst formation in SCNT embryos (22 vs. 23%), whereas it significantly inhibited blastocyst formation in IVF embryos (26 vs. 0%). In both SCNT and IVF embryos, 5.6 mM glucose or galactose significantly inhibited embryo development. In a second experiment, in glucose-free mSOF, fructose at concentrations of 0.75, 1.5, 3.0, or 5.6 mM was able to support to morula (32-42 vs. 12%) and blastocyst formation (30-38 vs. 12%) compared to 0 mM fructose. In Experiment 3, addition of fructose (1.5, 3.0, or 5.6 mM) to mSOF containing 1.5 mM glucose did not further promote blastocyst formation in SCNT embryos compared with replacement with 1.5 mM fructose only. Replacement of glucose with 1.5 mM fructose significantly increased total blastomeres (143 vs. 123 cells) and trophectodermal (TE) cells (116 vs. 94 cells) and decreased inner cell mass (ICM) to TE cell ratio (0.24 vs. 0.31) in blastocysts, compared to 1.5 mM glucose. The combined addition of 1.5 mM fructose and glucose significantly increased ICM cell number (36.7 cells) and ICM/TE ratio (0.46). In conclusion, fructose might be a more efficient energy substrate than glucose for producing large number of transferable blastocysts derived from SCNT.     

Effects of oocyte quality, oxygen tension, embryo density, cumulus cells and energy substrates on cleavage and morula/blastocyst formation of bovine embryos

Various factors, such as quality of the oocyte, oxygen tension, embryo density, and kind of energy substrate during in vitro production of embryos may affect the rate of preimplantation embryo development. In the present study we used 12553 bovine oocytes aspirated from slaughterhouse ovaries to evaluate various culture conditions that would increase in vitro production of advanced stages of preimplantation embryos. The morphological quality of the oocyte based on the compactness and number of layers of cumulus cells had significant positive effects on the rates of in vitro maturation, fertilization and development to the morula and blastocyst stages. None of the corona-enclosed or nude oocytes progressed beyond the 8- to 16-cell stage. The level of oxygen (5 or 20%) did not affect the proportion of one-cell embryos undergoing cleavage or progressing to morula and blastocyst stages. The rate of development of one-cell embryos originating from inferior quality oocytes was significantly improved when cultured in groups of 40 instead of 20 embryos per 0.5 mL medium. In the presence of cumulus cells, glucose had beneficial effects on in vitro maturation and subsequent development of IVM-IVF zygotes. The presence of serum improved the rate of in vitro development of one-cell embryos. Minimum Essential Medium supplemented with energy substrates according to the findings of metabolic studies was less effective in supporting in vitro maturation and subsequent development than TCM-199. In conclusion, morphological grading of immature oocytes is an appropriate selection criterion for their developmental ability. Embryo yields from low quality oocytes can be increased by culturing them in large groups. Serum is not essential for in vitro generation of embryos but its addition improves rates of success.     

Evidence for a strong paternal effect on human preimplantation embryo development and blastocyst formation

In human in vitro fertilization (I.V.F.), it was first assumed that all the embryos obtained had the same developmental potential whatever the quality of sperm. However, this has not been confirmed. We have used the coculture technique and determined the blastocyst formation rate in three groups of patients: group 1: patients with normal sperm count (>20 × 10⁶/ml), motility (>30%), and morphology (>50%); group 2: patients treated by I.V.F. with frozen donor sperm; group 3: patients with severely impaired sperm quality (<3 × 10⁶ forward motile and morphologically normal spermatozoa per ml). In group 1, we found a strong correlation between cleavage rate and blastocyst formation rate (P < 0.0001) with a blastocyst formation rate comprised between 40% and 50%. This was not true for the two other groups for which the overall number of blastocysts obtained and the number of patients having at least one blastocyst were severely reduced (P < 0.0001). These data are discussed in terms of DNA quality, timing of formation of the pronuclei, and delays in cell cycles at the time of genomic activation. These observations lead to a new approach to the study of fertilizing ability of poor quality sperm. It may help in the decision as to whether couples treated for male infertility should be excluded from I.V.F. protocols. © 1994 Wiley-Liss, Inc.     

Fertilization characteristics and in vitro embryo production with bovine sperm containing multiple nuclear vacuoles

An in vitro fertilization and culture system was used to determine the effect of multiple nuclear vacuoles in bovine spermatozoa on fertilization and early embryonic development. After swim-up, semen parameters were similar between 2 bulls except that 60% of spermatozoa from bull A contained multiple nuclear vacuoles, whereas no spermatozoa from bull B (control) contained vacuoles. In Experiment 1, in vitro–matured (IVM) oocytes were inseminated with frozen-thawed semen from the 2 bulls to determine the ability of vacuolated sperm to bind with the zona pellucida. The mean number of spermatozoa bound to the zona pellucida was less (P< 0.05) for bull A (85.7 ± 5.7; n = 112) than for bull B (108.9 ± 5.4; n = 130). In Experiment 2, the percentages of zonae penetrated by spermatozoa from bull A (151 of 201; 75%) and bull B (116 of 150; 77%) were not different. However, the percentages of vacuolated spermatozoa from bull A bound to (43%) and penetrating the zona pellucida (34%) were lower than those in the inseminate (60%). In Experiment 3, fertilization rates, as evidenced by the presence of two pronuclei, were not different for bull A (101 of 136; 74%) and bull B (89 of 115; 77%). In Experiment 4, there was no significant difference in percentage cleavage (72.1% versus 76%) and morulae (29.2% versus 34.8%) or blastocyst production (7.2% versus 8.4 %) for bulls A and B, respectively. Data suggest that spermatozoa with multiple nuclear vacuoles are defective in zona binding. However, vacuolated spermatozoa gaining access to the ooplasm apparantly participate in fertilization and early embryonic development. Mol. Reprod. Dev. 50:328–333, 1998. © 1998 Wiley-Liss, Inc.     

Aspects of in vivo oocyte production,blastocyst development,and embryo transfer in the cat

A brief overview of the progress made during the past approximately 40 years on the development of methods for in vitro production of cat embryos and intra- and interspecies embryo transfer is described. The presentation is focused primarily on research done over the past 30 years at the Cincinnati Zoo (1980–1995) and at the Audubon Nature Institute, New Orleans (1996–present) beginning with original studies on determining optimal doses of porcine FSH for ovarian stimulation and uterine embryo recovery, cryopreservation, and transfer. A key early finding was the ability of cats to respond to multiple gonadotropin (porcine FSH) treatments by repeated stimulation of follicular development. With a ≥6-month interval between FSH treatments, over the past 15 years (1998–2013), we have done 1603 laparoscopic oocyte retrievals on 337 cats and recovered >38,000 mature oocytes (mean = 24.1 per laparoscopic oocyte retrieval). The limited information available on in vivo blastocyst development in the cat during the latter portion of the preimplantation period (approximately Days 8 to 12 after coitum or approximately Days 7 to 11 after ovulation) was assembled for the purpose of comparing and contrasting it with the growth, expansion, and zona functioning of in vitro-derived blastocysts. Also, results of transferring morulae and/or blastocysts into synchronous recipients are described to emphasize evidence that appears to allude to an essential role for an intact zona pellucida in successful implantation and subsequent development in the cat. Until 2003, our in vitro-derived embryos were transferred into the uterine horns of recipients to determine the feasibility of producing offspring from such primary methods as IVF, intracytoplasmic sperm injection, SCNT, and embryo cryopreservation. With the exception of SCNT embryos, pregnancy rates were satisfactory, but embryo survival rates were not. Subsequently, after finding that SCNT embryo survival rate could be improved using laparoscopic transfer of early cleavage stage embryos into the oviduct, we applied the technique to embryos derived using IVF with sex-sorted sperm, oocyte vitrification, and embryo cryopreservation. Overall, a pregnancy rate of 67% (14/21) has resulted. Most recently, with the oviductal embryo transfer technique, two litters of Black-Footed cat kittens have been born from intra- and interspecies transfer of cryopreserved embryos.     

Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality.

The aim of this study is to examine the effect of bovine oocyte maturation, fertilization or culture in vivo or in vitro on the proportion of oocytes reaching the blastocyst stage, and on blastocyst quality as measured by survival following vitrification. In Experiment 1, 4 groups of oocytes were used: (1) immature oocytes from 2-6 mm follicles; (2) immature oocytes from > 6 mm follicles; (3) immature oocytes recovered in vivo just before the LH surge; and (4) in vivo matured oocytes. Significantly more blastocysts developed from oocytes matured in vivo than those recovered just before the LH surge or than oocytes from 2-6 mm follicles. Results from > 6 mm follicles were intermediate. All blastocysts had low survival following vitrification. In Experiment 2, in vivo matured oocytes were either (1) fertilized in vitro or (2) fertilized in vivo by artificial insemination and the resulting presumptive zygotes recovered on day 1. Both groups were then cultured in vitro. In vivo fertilized oocytes had a significantly higher blastocyst yield than those fertilized in vitro. Blastocyst quality was similar between the groups. Both groups had low survival following vitrification. In Experiment 3a, presumptive zygotes produced by in vitro maturation (IVM)/fertilization (IVF) were cultured either in vitro in synthetic oviduct fluid, or in vivo in the ewe oviduct. In Experiment 3b, in vivo matured/in vivo fertilized zygotes were either surgically recovered on day 1 and cultured in vitro in synthetic oviduct fluid, or were nonsurgically recovered on day 7. There was no difference in blastocyst yields between groups of zygotes originating from the same source (in vivo or in vitro fertilization) irrespective of whether culture took place in vivo or in vitro. However, there was a dramatic effect on blastocyst quality with those blastocysts produced following in vivo culture surviving vitrification at significantly higher rates than their in vitro cultured counterparts. Collectively, these results indicate that the intrinsic quality of the oocyte is the main factor affecting blastocyst yields, while the conditions of embryo culture have a crucial role in determining blastocyst quality.     

In vitro embryo production and embryo transfer in domestic and non-domestic cats

Over a 5-year interval, multiple laparoscopic oocyte retrievals were done in fishing cats (Prionailurus viverrinus), caracals (Caracal caracal) and domestic cats after ovarian stimulation with gonadotropins. From 21 retrievals in five fishing cats, 579 preovulatory oocytes (mean = 27.6) were recovered and 348 embryos were produced in vitro (mean = 16.6). A total of 452 preovulatory oocytes (mean = 25.1) were recovered from 18 of 24 retrievals in six caracals and 297 (mean = 16.6) embryos were produced. An additional 16 caracal embryos (19%) were produced after in vitro maturation of 83 oocytes, 59 of which came from six retrievals producing only immature oocytes. The presence of corpora lutea at oocyte retrieval occurred in each species (1) at a similar frequency (33%) and (2) more frequently during January through May (11 of 15 retrievals) than during the latter half of the year (4 of 30 retrievals). Of the 12 embryo transfer procedures done in fishing cats, one pregnancy (8%) was obtained and one live kitten born after the auto-transfer of 10 Day-6 embryos. In caracals, a total of 46 Day-4 or Day-5 embryos were auto-transferred to six recipients, one of which delivered two live kittens. Then, 109 caracal embryos were cryopreserved before thawing and transferring to nine recipients (mean = 12.1) on Days 5 or 6. From three pregnancies established (33%), a total of three kittens were born. Two to six gonadotropin treatments/oocyte retrievals were done in domestic cats during 1999 through 2003; an average of 24.9, 23.5, 22.0, 23.1, 23.5 and 40.9 oocytes (P > 0.05) were recovered at the first through the sixth treatment cycles from 138, 138, 97, 49, 22, and seven retrievals, respectively.     

Herpes simplex virus infection of human spermatozoa correlates with decreased frequency of blastocyst formation and frequency of embryo implantation during in vitro fertilization

We have conducted a comparative analysis of developing human embryos in the course of in vitro fertilization (IVF) as a method of sterility treatment of two groups of patients: herpes simplex virus (HSV) was detected in the fraction of motile sperm of male partners in group I (n = 28) and no HSV was found in sperm in group II (n = 103). We assessed number of fertilized ova, embryos during cleavage, and blastocysts as well as such parameters as frequency of implantation and frequency of pregnancy in IVF cycles. It was established that the presence of HSV in spermatozoa did not affect the efficiency of fertilization or cleavage of zygotes. At the same time, in cases of virus-infected male gametes, the frequency of blastocyst formation was two times less (p = 0.015), and the frequency of embryo implantation and pregnancy was, on average, five times lower (p = 0.01 and p = 0.016, respectively). Based on the obtained results, a conclusion was made about the negative influence of HSV-virus in male gametes at the early stages of embryo development and the frequency of achievement of pregnancy in respect to the methods of assisted reproductive technologies (ART).     

Proliferation and morphology of chick embryo cells cultured in the presence of horse serum and hemoglobin

Summary We have shown previously that hemoglobin greatly stimulates chick embryo cell proliferation in Eagle's minimal essential medium supplemented with horse serum. In the present study we compared the effects of horse serum plus 10 μM hemoglobin to those of fetal bovine serum on subcultures of chick embryo cells serially propagated at high cell densities. The cells became elongated in the presence of fetal bovine serum and their rate of proliferation progressively decreased, whereas they became polygonal in the presence of horse serum plus hemoglobin and proliferated well in successive cell passages. The polygonal cell obtained in the presence of horse serum plus hemoglobin rapidly elongated if cultured at low cell densities in the presence of fetal bovine serum, but, in contrast, elongated cells did not yield polygonal cells if cultured at low densities in the presence of horse serum plus hemoglobin. It is possible that the polygonal and elongated cells are undifferentiated cells and differentiating myogenic cells, respectively.     

Practical measures to improve in vitro blastocyst production in the bovine

Media, chemicals, sera and protocols used for in vitro production of bovine embryos are different from one laboratory to another. This paper describes some of the critical steps required to produce embryos in vitro, suggests quality control measures to lower variations in blastocyst yield and describes different tests which may be used when implementing new procedures in a routine production system.     

In vivo and in vitro embryo production in goats

Assisted reproductive technologies (ART) such as artificial insemination (AI) and multiple ovulation and embryo transfer (MOET) have been used to increase reproductive efficiency and accelerate genetic gain. The principal limitations of MOET are due to variable female response to hormonal treatment, fertilization failures and premature regression of Corpora luteum. The in vitro production (IVP) of embryos offers the possibility of overcoming MOET limitations. The method of IVP of embryos involves three main steps: in vitro maturation of oocytes (IVM), in vitro fertilization of oocytes (IVF) with capacitated sperm and in vitro culture (IVC) of embryos up to blastocyst stage. Recovering oocytes from live selected females by laparoscopic ovum pick-up (LOPU) and breeding prepubertal females by juvenile in vitro embryo technology (JIVET) will allow a greater production of valuable goats. Also, IVP of goat embryos will provide an excellent source of embryos for basic research on development biology and for commercial applications of transgenic and cloning technologies. Different protocols of IVP of embryos have been used in goats. However oocyte quality is the main factor for embryos reaching blastocyst stage from IVM/IVF/IVC oocytes. One of the principal determinant factors in the results of blastocyst development is the age of the oocyte donor females. In goats, oocytes from prepubertal and adult females do not show differences in in vitro maturation and in vitro fertilization; however the percentage of oocytes reaching blastocyst stage ranges from 12 to 36% with oocytes from prepubertal and adult goats, respectively.     

In vitro production of bovine embryos in medium supplemented with a serum replacer: Effects on blastocyst development,cryotolerance and survival to term

In this study, we evaluated a serum replacer (SR; Knockout SR^®, Invitrogen) in our in vitro culture systems. We hypothesized that SR would benefit bovine embryo development, since SR supported survival of embryonic stem cells (which originate from embryos). Experiment 1 compared oocyte maturation with SR versus fetal bovine serum (FBS). Following fertilization, blastocyst development was lower for oocytes matured with SR (21.5 versus 34.1, P < 0.05). Experiment 2 evaluated SR for culturing embryos. Following fertilization, embryos were cultured for 3 days in KSOM, and then assigned to treatments: (1) KSOM static culture (KNM); (2) fresh KSOM (KD3); (3) KSOM + SR or (4) KSOM + FBS and cultured to Day 7 (fertilization = Day 0). Blastocyst development in FBS or SR was higher than either KNM or KD3 (48.2, 47.2, 32.7, and 35.5, respectively, P < 0.05). Experiment 3 evaluated cryosurvival of embryos cultured in the same manner as Experiment 2. On Day 7, embryos were vitrified and upon warming, embryos cultured in SR had greater 24 h survival rates (70.6%) than all other treatments (P < 0.05). Finally, Experiment 4 evaluated effects of SR on pregnancy rate and development to term. Culture in SR was not detrimental to pregnancy or calving rates (50 and 50%, respectively), and SR calves had normal birth weights (mean = 38.8 kg ± 1.5). In conclusion, the use of SR for maturation of oocytes was not beneficial; however, SR enhanced embryo culture by improving development in vitro, cryotolerance and survival, effectively replacing serum in culture.     

Stage-specific formation of the equine blastocyst capsule is instrumental to hatching and to embryonic survival in vivo

Early embryonic development in the horse is characterized by the formation of an unusual acellular glycoprotein "capsule" between the trophectoderm and the overlying zona pellucida. This structure is first detected between days 6 and 7 after ovulation and completely envelops the spherical conceptus until as late as day 23 of gestation. In the present study, a micromanipulator was used to remove the capsule from 15 embryos on day 6-7 after ovulation. None of these denuded embryos developed into ultrasonographically detectable pregnancies after surgical transfer into recipient mares whereas four of six control embryos handled and transferred similarly but without capsule removal developed normally, thereby demonstrating clearly a role for the capsule in embryonic survival. In addition, observation of the embryonic investments after embryo collection and during micromanipulation led to the hypothesis that hatching of the horse embryo from its zona pellucida is assisted by the force of the expanding capsule, which causes the attenuating zona to literally burst open.     

Influence of oocyte donor on in vitro embryo production in buffalo

The aim of this research was to estimate the variability between buffalo as oocyte donors. In Experiment 1, reproductive variables were retrospectively analyzed in buffalo (n = 40) that underwent repeated ovum pick up (OPU), over 16 puncture sessions (PS). The follicular recruitment among individuals and the relationship between follicular population and oocyte production were evaluated. In Experiment 2, eight buffalo underwent OPU for 28 PS and the oocytes were processed separately to correlate follicular and oocyte population at the first PS to blastocyst (BL) production. In Experiment 1, the average number of total follicles (TFL), small follicles (SFL), cumulus-oocyte complexes (COC) and Grade A + B COC recorded in each 4-PS period had great repeatability (r = 0.52, 0.54, 0.60 and 0.57, respectively). The average number of Grade A + B COC recovered during the subsequent 15 PS was positively correlated with the first PS number of TFL (r = 0.60; P < 0.001), SFL (r = 0.68; P < 0.001), COC (r = 0.48; P < 0.01) and Grade A + B COC (r = 0.40; P < 0.05). In Experiment 2, a large variability among animals was observed in blastocyst yields. When animals were grouped according to the BL yield, the greatest BL yield group had a greater (P < 0.05) number of TFL (8.3 ± 0.9 compared with 5.6 ± 0.7) and SFL (7.3 ± 0.3 compared with 3.8 ± 0.7) at the first PS than the lesser BL yield group. The average number of BL produced over the subsequent sessions was correlated with the number of TFL (r = 0.80; P < 0.05) and COC (r = 0.76; P < 0.05) observed at the first PS. These results demonstrated a donor influence on the oocyte and BL production, suggesting a preliminary screening to select the donors with greater potential.     

Calcyclin (S100A6) regulates pulmonary fibroblast proliferation,morphology, and cytoskeletal organization in vitro

Calcyclin (S100A6) is a member of the S100A family of calcium binding proteins. While the precise function of calcyclin is unknown, calcyclin expression is associated with cell proliferation and calcyclin is expressed in several types of cancer phenotypes. In the present study, the functional role of calcyclin was further elucidated in pulmonary fibroblasts. Antisense S100A6 RNA expression inhibited serum and mechanical strain-induced fibroblast proliferation. This attenuated proliferative response was accompanied by a flattened, spread cell morphology, and disruption of tropomyosin labeled microfilaments. Changes in cytoskeletal organization did not correspond with a decrease in tropomyosin levels. These observations suggest a role for calcyclin in modulating calcium dependent signaling events that regulate progression through the cell cycle. J. Cell. Biochem. 88: 848-854, 2003.     

Effects of superovulation, embryo recovery, culture system and embryo transfer on development of rabbit embryos in vivo and in vitro

Uninterrupted development of rabbit embryos in vivo was studied in 7 superovulated and 7 normally ovulating (GnRH-treated) does, while another 7 does were superovulated and 1-cell embryos were collected from them at 19 h after LH to compare development in vivo and in vitro. Embryos from the last group were either cultured in the presence or absence of rabbit oviduct epithelial cells for 65 h in Medium 199, or were immediately transferred to recipients. At 84 h after LH or GnRH, blastomere number, embryo volume and stage of development were assessed for all embryos. Intrazonal embryo volumes were significantly reduced in embryos recovered from superovulated donors. Superovulation also had a negative effect on embryo cell numbers. However, this reduction was more severe in embryos remaining in vivo in superovulated donors until 84 h after LH than it was in embryos transferred to nonsuperovulated recipients at the 1-cell stage (19 h after LH). The embryo recovery procedure apparently caused little harm to the embryos, except that the mucin layer on flushed and immediately transferred embryos was significantly thinner than that of embryos residing continuously in vivo. Co-culture with rabbit oviduct epithelial cells resulted in improved development in vitro, but this development was still significantly retarded compared with embryos developing in vivo.