Reproducibility of semi-quantitative assessment of aortic valve calcification and valve motion on echocardiography: a small-scale study.

BACKGROUND: Aortic stenosis (AS) is the most common degenerative valve disease in high income countries. While hemodynamic metrics are commonly used to assess severity of stenosis, they are impacted by loading conditions and stroke volume and are often discordant. Anatomic valve assessments such as aortic valve calcification (AVC) and valve motion (VM) during transthoracic echocardiography (TTE) can offer clues to disease severity. The reliability of these semi-quantitatively assessed anatomic imaging parameters is unknown. METHODS: This is a retrospective study of semi-quantitative assessment of AVC and valve VM on TTE. TTEs representing a range of AS severities were identified. The degree of calcification of the aortic valve and the degree of restricted VM were assessed in standard fashion. AVC scores and valve motion were assessed by readers with varied training levels blinded to the severity of AS. Correlation and inter-reader reliability between readers were assessed. RESULTS: 420 assessments (210 each for AVC and VM) were collected for 35 TTEs. Correlation of AVC for imaging trainees (fellows and students, respectively), ranged from 0.49 (95% CI 0.18-0.70) to 0.62 (95% CI 0.36-0.79) and 0.58 (95% CI 0.30-0.76) to 0.54 (95% CI 0.25-0.74) for VM. Correlation of anatomic assessments between echocardiographer-assigned AVC grades was r = 0.76 (95% CI 0.57-0.87)). The correlation between echocardiographer-assigned assessment of VM was r = 0.73 (95% CI 0.53-0.86), p < 0.00001 for both. For echocardiographer AVC assessment, weighted kappa was 0.52 (0.32-0.72), valve motion weighted kappa was 0.60 (0.42-0.78). CONCLUSION: There was good inter-reader correlation between TTE-based semi-quantitative assessment of AVC and VM when assessed by board certified echocardiographers. There was modest inter-reader reliability of semi-quantitative assessments of AVC and VM between board certified echocardiographers. Inter-reader correlation and reliability between imaging trainees was lower. More reliable methods to assess TTE based anatomic assessments are needed in order to accurately track disease progression. CLINICAL TRIAL NUMBER: STUDY00003100.

Phenotypic relationships between docility and reproduction in Angus heifers.

The objective of this study was to elucidate the phenotypic relationships between docility and first-service AI conception rate in heifers. Data ( = 337) collected from 3 cooperator herds in Kansas at the start of synchronization protocol included exit velocity (EV), chute score (CS), fecal cortisol (FC), and blood serum cortisol (BC). Data were analyzed using logistic regression with 30-d pregnancy rate as the dependent variable. The model included the fixed effect of contemporary group and the covariates FC, BC, EV, CS, BW, and age. Correlation coefficients were calculated between all continuous traits. Pregnancy rate ranged from 34% to 60% between herds. Blood cortisol positively correlated with EV ( = 0.22, < 0.01), negatively correlated with age ( = -0.12, < 0.03), and tended to be negatively correlated with BW ( = -0.10, = 0.09). Exit velocity was positively correlated with CS ( = 0.24, < 0.01) and negatively correlated with BW ( = -0.15, < 0.01) and age ( = -0.12, < 0.03). Chute score negatively correlated with age ( = -0.14, < 0.01), and age and BW were moderately positively correlated ( = 0.42, < 0.01), as expected. Older, heavier animals generally had better temperament, as indicated by lower BC, EV, and CS. The power of our test could detect no significant predictors of 30-d pregnancy for the combined data from all ranches. When the data were divided by ranch, CS ( < 0.03) and BW ( < 0.01) were both significant predictors for 30-d pregnancy for ranch 1. The odds ratio estimate for CS has an inverse relationship with pregnancy, meaning that a 1-unit increase in average CS will reduce the probability of pregnancy at ranch 1 by 48.1%. Weight also has a negative impact on pregnancy because a 1-kg increase in BW will decrease the probability of pregnancy by 2.2%. Fertility is a complex trait that depends on many factors; our data suggest that docility is 1 factor that warrants further investigation.

The effect of intermittent hypoxic training via a hypoxic inhaler on physiological and performance measures in rowers: a pilot study.

Intermittent hypoxic training and discontinuous exposure to altitude were used to improve performance at sea level in elite rowers. Altitude was simulated with a newly patented device which allowed athletes to experience altitude by re-breathing oxygen-depleted expired air. Seven elite rowers (five females, two males) used inhalers for a 90-min supervised daily session (alternating 6 min on and 4 min off) for 3 weeks, while four female elite rowers used placebo devices in the same sessions. The inhalers were adjusted to produce a progressive decrease in oxygen saturation over the 3 weeks (initially 90%; finally 80%). Immediately before and 7-10 days after altitude exposure, the rowers performed an incremental lactate test to determine power output equivalent to 4 mM [BLa], a 500-m time trial and a 5000-m time trial, all on a rowing ergometer. Relative to the control group, the altitude group showed a slight improvement in mean power for the 5000-m time trial (0.6%, 90% likely limits +/-3.7%), and a substantial impairment in mean power for the 500-m trial (2.2%, +/-4.1%). Power at 4-mM lactate declined in both groups, but overall the altitude group improved by 0.4% (+/-3.5%) relative to control. The device represents a practical way to simulate altitude exposure, but it is unlikely to have major effects on performance of elite rowers.

Head injury and family carers: a pilot study to investigate an innovative community-based educational programme for family carers and patients.

PRIMARY OBJECTIVE: The incidence of head injury is increasing among younger people with more family members undertaking their life-long care. Many research studies have highlighted the emotional well-being of such family carers and their unmet needs, however only a few consider the formal help provided for carers. Using a longitudinal, mixed variable, within and between-subject design, this pilot study evaluated the impact of an educational programme for family carers and their head-injured relatives in reducing carer and patient psychological distress and improving their coping ability. MAIN OUTCOMES AND RESULTS: The study comprised experimental and control samples each with carer and patient groups. The experimental sample had eight sessions of educational input. All groups were assessed pre- and post-intervention and at 3 months follow-up. The patient sample was further assessed using cognitive measures. There was evidence of reduction in psychological distress in the experimental carer group following the educational input, but these results were not statistically significant. However, the experimental patient population at follow-up assessment showed statistically significant improvements. CONCLUSIONS: A larger scale multi-centre study with a longer follow-up period of assessment is required for the generalization of findings. The pilot study identifies points for consideration in a potential main study.

Anatomical and possible clinical relationships between the calcaneofibular ligament and peroneus brevis--a pilot study.

The authors have observed in their clinical practice patients presenting with chronic retromalleolar pain following lateral ankle injuries. It has been hypothesized that persistent retromalleolar pain following a supination sprain may be due to peroneus brevis (PB) tendon tears (Boruta et al. 1990). The aims of this study were to investigate whether an anatomical relationship exists between the calcaneofibular ligament (CFL) and PB, and if so, the significance of this relationship in the positions of supination sprain and talar tilt test. Seven out of eight cadaveric ankles demonstrated fibrous connecting tissue between the tendon of PB and CFL. Four of the eight ankles demonstrated PB tendon abnormalities. The presence of connecting tissue between CFL and PB suggests an anatomical basis for concomitant damage to the PB tendon with a supination sprain, thus supporting the hypothesis that there may be an anatomical basis for persistent retromalleolar pain subsequent to injury to the lateral ankle complex.

Reversible changes in protein phosphorylation during germinal vesicle breakdown and pronuclear formation in bovine oocytes in vitro.

This study examined the event of protein phosphorylation in bovine oocytes during germinal vesicle breakdown (GVBD) and formation of pronuclei following fertilisation in vitro. Immature oocytes were obtained from abattoir materials and cultured in vitro. The oocytes were labelled with [32P]orthophosphate at 3 h intervals from 0 to 12 h following maturation in culture or from 3 to 18 h following insemination. One-dimensional gel electrophoresis indicated that levels of protein phosphorylation are low prior to GVBD. However, the levels of protein phosphorylation at approximately 40 kDa, 27 kDa, 23 kDa and 18 kDa increased substantially following GVBD and then decreased gradually as maturation in culture progressed. In contrast, the levels of protein phosphorylation increased gradually in the oocytes following pronucleus formation. Further, two-dimensional gel electrophoresis indicated that the protein at approximately 18 kDa reversibly changed in the oocytes during maturation and fertilisation. These results indicate that the reversible changes of this phosphoprotein may be related to either cell cycle transition or pronucleus formation during maturation and fertilisation in bovine oocytes.

Responses of the estrous cycle in dairy cows exposed to electric and magnetic fields (60 Hz) during 8-h photoperiods.

To study the effects of exposure to extremely low frequency (ELF) electric and magnetic fields (EMF) on the estrous cycle of dairy cows under short-day photoperiod, 16 non-lactating, non-pregnant Holstein cows were exposed to a vertical electric field of 10 kV/m and a horizontal magnetic field of 30 microT for 16 h per day in a cross-over design consisting of two sequences. Each sequence included three periods, and each period corresponded to the duration of one estrous cycle. All animals were maintained under short photoperiod (8 h light/16 h dark) during the trial. Exposure to EMF had an impact on the duration of a complete estrous cycle (P<0.01) and on the duration of the luteal phase (P<0.01). The mean duration of one cycle was 19.5+/-0.4 for the control and 21.3+/-0.4 days for the exposed animals, respectively. The mean duration of the luteal phase was 15.4+/-0.4 days for the control and 17.2+/-0.4 days for the exposed group. The total area under the progesterone (P(4)) curve, the amplitude of the curve or the slope of the P(4) rise at the onset of the luteal phase were not affected by EMF exposure. Results indicate that exposure to EMF may increase the duration of the estrous cycle.

Seasonal variation in preovulatory events associated with synchronization of estrus in dwarf goats.

This experiment was conducted to define the temporal relationships among estrus, the LH surge and ovulation after estrus synchronization in dwarf goats and to assess the effect of season on these parameters. In November (breeding season), March (transition period) and July (non-breeding season), estrus was synchronized in 12 dwarf goats by means of intravaginal sponges containing 60 mg medroxyprogesterone acetate (MAP) for 10 d, coupled with 125 microg cloprostenol i.m. 48 h before sponge removal and 300 IU eCG i.m. at sponge removal. A different group of animals was used during each time period. Onset of estrus was monitored using two males, and blood samples for the measurement of plasma LH were collected at 2-h intervals from 24 to 60 h after sponge removal. Ovulation was confirmed by laparoscopy at 54 and 72 h after sponge removal. A seasonal shift was detected in the intervals to onset of estrus, LH surge, and ovulation after sponge removal (P<0.05), with sponge removal to onset of estrus being shorter (P<0.05) in November (25.0 +/- 1.56 h) and July (28.9 +/- 2.43 h) than in March (40.9 +/- 3.27 h). The intervals between onset of estrus and the LH surge and between the LH surge and ovulation were found to be constant throughout the different seasons. An optimal time for breeding, artificial insemination, oocyte and embryo recovery, and embryo transfer may be predicted using information gained from these studies.

Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes.

The developmental potential of caprine fetal fibroblast nuclei after in vitro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Cells were transfected with constructs containing the enhanced green fluorescent protein (eGFP) and neomycin resistance genes and were selected with G418. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% fetal calf serum for 4-8 days prior to use in NT. Immature oocytes were recovered by laparoscopic ovum pick-up and matured for 24 h prior to enucleation and NT. Reconstructed embryos were transferred as cleaved embryos into synchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 recipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultrasound. Of these, four recipients delivered five male kids (7.1% of embryos transferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase chain reaction, Southern blotting, and fluorescent in situ hybridization analyses. Nuclear transfer derivation from the donor cells was confirmed by single-strand confirmation polymorphism analysis. These results demonstrate that both in vitro-transfected and nontransfected caprine fetal fibroblasts can direct full-term development following NT.

Effect of centrifugation on early embryonic development and parthenogenetic activation of bovine oocytes matured in vitro.

This study examined the fertilization, early developmental competence and capacity for parthenogenetic activation of bovine oocytes matured in vitro after centrifugation. Immature oocytes were cultured in tissue culture medium 199 supplemented with 10% fetal bovine serum and 75 mIU mL(-1) FSH + LH at 5% CO2 to facilitate maturation. After culture for 24 or 30 h, the metaphase-II stage oocytes were centrifuged at 3000, 5000, 7000 or 10000g for 5 min before in vitro fertilization or parthenogenetic activation. Frozen-thawed bull semen was used for in vitro fertilization. For parthenogenetic activation, the oocytes were exposed to 20 microM calcium ionophore A23187 for 5 min at room temperature. Fertilization rates were not different between control and treatment groups (87.7% v. 74.6%, 73.4%, 75.9% and 76.4% respectively). Also, there were no differences in early embryonic development between control and treatment groups (rates of blastocyst formation were 21.1% v 20.2%, 28.8%, 31.2% and 24.1% respectively). When the oocytes were centrifuged at various speeds alone, the activation rate of oocytes was significantly higher (P < 0.05) in the 10000g treatment group compared with control (10.8% v 0.0%). There were no differences in the activation rates of oocytes between control and treatment groups at speeds up to 7000g (70.9% v. 71.9%, 78.3% and 77.2% respectively) after centrifugation and stimulation with Ca(2+)-ionophore. However, the activation rate of oocytes was significantly higher (P < 0.05) in the 10000g treatment group compared with control (70.9% v. 83.1%). In addition, the percentage of activated oocytes with diploid formation was significantly higher in the oocytes after centrifugation at 10000g and stimulation with calcium ionophore A23187 than in the control (18.4% v 7.1%). These results indicate that centrifugation of oocytes matured in vitro has no detrimental effect on fertilization and subsequent early embryonic development. They also indicate that the oocytes might be parthenogenetically activated after centrifugation and that high-speed centrifugation may induce activation of some oocytes. The results suggest that the optimal speed for centrifugation of bovine oocytes might be < or = 7000g to enhance the visibility of nuclear elements for further micromanipulation.

Formation and early development of the corpus luteum in pigs.

Numerous corpora lutea form from the multiple follicles that ovulate during the oestrous cycle of pigs. Vascular elements invade the follicle from the theca compartment, first centripetally, and subsequently by lateral branching of centripetal veins and arteries. The vessels are the vehicle for dispersion of steroidogenic theca cells throughout the corpus luteum. Mitosis occurs in both the theca and granulosa layers before ovulation, and in luteal cells well into the luteal phase. Luteal cell proliferation undergoes gradual restriction as the corpus luteum matures, but the mechanisms of exit from the cell cycle are unknown. The extracellular ligands that direct luteinization and maintain the corpus luteum include LH, prolactin, insulin and insulin-like growth factors (IGFs). These ligands induce qualitative and quantitative changes in steroid output, with progesterone as the principal product. These changes upregulate the cholesterol synthetic pathways to increase substrate availability. The intracellular regulation of luteinization is complex. A model is presented in which LH stimulates arachidonic and lineoleic acid metabolism to produce ligands for the nuclear proteins of the peripheral peroxisome activator receptor family. These ligands have downstream effects on cell differentiation and exit from the cell cycle. Luteal function is maintained by interactions among ligands, cholesterol regulatory proteins and constitutively expressed and regulated transcription factors.

Porcine leptin receptor: molecular structure and expression in the ovary.

The porcine leptin receptor complementary DNA was cloned and sequenced and the leptin receptor gene expression evaluated in the porcine ovary. An open reading frame of 3498 nt cDNA was amplified from pig liver mRNA by RT-PCR. Sequence homology with the extracellular, transmembrane, and cytoplasmic domains of human, mouse, rat, sheep, and cow leptin receptors varied between 45% and 90%. Leptin receptor mRNA was present in porcine kidney, liver, spleen, lung, brain, testis, uterus, ovary, corpus luteum (CL), theca, and granulosa cells. The abundance of leptin receptor transcripts and protein varied during luteinization of granulosa cells in vitro and in the CL during the pig luteal phase. In the postovulatory CL, both mRNA and protein were low but detectable, maximal expression was observed in the midcycle CL, and lowest abundance occurred in regressed CL. Leptin receptor mRNA was present in granulosa cells at isolation and increased in abundance as the cells luteinized over 96 hr in culture. Leptin receptor protein was detectable after 12 hr of in vitro luteinization. We conclude that leptin receptor is expressed in granulosa and luteal cells, and varies during pig ovarian cell differentiation.

Activation of bovine oocytes following intracytoplasmic sperm injection (ICSI).

In the human and the mouse, intracytoplasmic sperm injection (ICSI) apparently triggers normal fertilization and may result in offspring. In the bovine, injection of spermatozoa must be accompanied by artificial methods of oocyte activation in order to achieve normal fertilization events (e.g., pronuclear formation). In this study, different methods of oocyte activation were tested following ICSI of in vitro-matured bovine oocytes. Bovine oocytes were centrifuged to facilitate sperm injection, and spermatozoa were pretreated with 5 mM dithiothreitol (DTT) to promote decondensation. Sperm-injected or sham-injected oocytes were activated with 5 microM ionomycin (A23187). Three hours after activation, oocytes with second polar bodies were selected and treated with 1.9 mM 6-dimethylaminopurine (DMAP). The cleavage rate of sperm-injected oocytes treated with ionomycin and DMAP was higher than with ionomycin alone (62 vs 27%, P < or = 0.05). Blastocysts (2 of 41 cleaved) were obtained only from the sperm-injected, ionomycin + DMAP-treated oocytes. Upon examination 16 h after ICSI, pronuclear formation was observed in 33 of 47 (70%) DMAP-treated oocytes. Two pronuclei were present in 18 of 33 (55%), while 1 and 3 pronuclei were seen in 8 of 33 (24%) and 7 of 33 (21%) oocytes, respectively. In sham-injected oocytes, pronuclear formation was observed in 15 of 38 (39%) with 9 (60%) having 2 pronuclei. Asa single calcium stimulation was insufficient and DMAP treatment could result in triploidy, activation by multiple calcium stimulations was tested. Three calcium stimulations (5 microM ionomycin) were given at 30-min intervals following ICSI. Two pronuclei were found in 12 of 41 (29%) injected oocytes. Increasing the concentration of ionomycin from 5 to 50 microM resulted in a higher rate of activation (41 vs 26%). The rate of metaphase III arrest was lower while the rate of pronuclear formation and cleavage development was higher in sperm-injected than sham-injected oocytes, suggesting that spermatozoa contribute to the activation process. Further improvements in oocyte activation following ICSI in the bovine are necessary.

Manipulative physiotherapists can reliably palpate nominated lumbar spinal levels.

Palpating a nominated spinal level is a prerequisite to more complex tasks such as palpating the level most likely to be the source of the patient's symptoms. The aim of this study was to investigate the reliability of physiotherapists with a post-graduate qualification in manipulation (manipulative physiotherapists) in palpating the lumbar spines of patients in a clinical setting. Three pairs of manipulative physiotherapists palpated the randomly-nominated lumbar spinal levels of 20 patients presenting to their practices for treatment of low-back pain. Each therapist marked the skin overlying the spinous process of the nominated spinal level with an ultraviolet pen and these marks were transcribed onto transparencies for analysis. The therapists obtained an overall weighted kappa of 0.92 indicating almost perfect agreement for locating the nominated spinal level. The results of this study indicate that manipulative physiotherapists can reliably palpate nominated lumbar spinal levels, suggesting further training in spinal therapy enhances the palpatory skills of physiotherapists in palpating nominated lumbar spinal levels.

A role for lipoxygenase metabolites of arachidonic acid in porcine ovulation.

Prostaglandins, products of arachidonic acid via the cyclooxygenase pathway, are essential to the porcine ovulatory process in that inhibition of their synthesis results in ovulation failure. Studies in the rat have shown that ovulation is also preceded by a rise in three ovarian hydroxyeicosatetraenoic acids, products of the lipoxygenase pathway, and inhibition of this pathway also inhibits ovulation. Experiments were designed, using a pregnant mare serum gonadotropin/human chorionic gonadotropin (hCG)-treated prepuberal gilt model, to measure pre-ovulatory changes in follicular fluid concentrations of 15-hydroxyeicosatetraenoic acid (15-HETE), and to compare the effects of indomethacin and nordihydroguaiaretic acid (NDGA) on ovulation in the pig and on 15-HETE and prostaglandin F2 alpha synthesis both in vivo and in vitro. Follicular fluid concentrations of 15-HETE were elevated significantly just prior to the expected time of ovulation (40 h after hCG). When indomethacin (10 mg) was injected into the ovarian stalk at 24 h after hCG, follicular fluid concentrations of both 15-HETE and prostaglandin F2 alpha were lower (P < 0.01) than controls at 40 h and ovulation rate was suppressed (P < 0.01). When NDGA (5 mg) was administered in the same manner, ovulation rate was suppressed (P < 0.01), but the levels of 15-HETE and prostaglandin F2 alpha were not altered. Synthesis of 15-HETE by cultured granulosa and theca interna cells was reduced by the presence of NDGA (1 mg/ml), whereas indomethacin (100 ng/ml) lowered 15-HETE production in theca interna cells only. These results clearly demonstrate that indomethacin can block the lipoxygenase as well as the cyclooxygenase pathways, depending on the dose used, and suggest that lipoxygenase metabolites of arachidonic acid are involved in the ovulatory process in the pig.

Connexin 43 gap junction protein expression during follicular development in the porcine ovary.

Connexin 43, a member of the highly conserved connexin family of gap junction proteins, is expressed in the pig ovary. In other species, ovarian connexin 43 expression and phosphorylation are hormonally regulated. We characterized connexin 43 expression and phosphorylation in the ovaries of mature pigs during the estrous cycle and in prepubertal gilts during follicular development induced by eCG (750 IU)/hCG (500 IU; 72 h later). Ovarian connexin 43 protein expression and phosphorylation were examined by immunoblot analysis. Connexin 43 was localized to specific follicular cell types during development by immunofluorescence. While no change in total connexin 43 protein expression was seen during the cycle, connexin 43 phosphorylation was significantly higher (p < 0.05) during the late follicular stage of the cycle than during the early luteal and early to mid-follicular stages. In ovaries of eCG/hCG-primed prepubertal pigs, connexin 43 protein levels remained steady, while phosphorylation of the protein increased significantly at 72 h and 84 h after eCG treatment (p < 0.05), then declined to pretreatment levels by 96 h (24 h post-hCG administration). Immunoreactive connexin 43 was localized predominantly to granulosa cells of cyclic pigs and eCG/hCG-primed prepubertal gilts. Follicular connexin 43 was highest between 60 h and 84 h after eCG and declined after hCG administration. Connexin 43 was not detected in morphologically atretic follicles, stroma, or vascular tissue of the ovary. This is the first evidence that porcine ovarian connexin 43 phosphorylation is differentially regulated during follicular development. The results suggest that hormonally induced changes in connexin 43 phosphorylation may play a coordinating role in porcine follicular development.

Steroidogenic properties of a spontaneously established porcine granulosa cell line (PGC-2).

A spontaneously established porcine granulosa cell line (PGC-2) was cloned through the continuous culturing of primary granulosa cells collected from equine chorionic gonadotropin (eCG)-treated prepubertal gilts. This established cell line has undergone approximately 100 passages and shows contact-inhibition of growth. PGC-2 stained with a monoclonal antibody (mAb) directed against cytokeratin, indicating its epithelial nature, but not with a mAb directed against vimentin, suggesting that it is not fibroblast-derived. Immunoblotting revealed that PGC-2 expresses cadherin, an epithelial Ca+2-dependent cell adhesion molecule. The cells were dependent on serum for growth and had a doubling time of approximately 20 hr when cultured with 10% fetal bovine serum. The cell line was examined for the presence of FSH receptors, cAMP responses, and steroidogenic capabilities. The cell line lacks FSH receptors as assessed by radiolabelled-ligand binding, and no transcripts for FSH receptor were detected by Northern blotting of total cellular RNA. Neither FSH nor cholera toxin (0.5 ng/mL) stimulated increases in cAMP levels in these cells, whereas forskolin (10 microM) induced a fivefold increase in cAMP production. When a higher concentration of cholera toxin (300 ng/mL) was used, however, cAMP levels doubled by 2 hr. Despite a lack of responsiveness to purified of SH or oLH, the cells were capable of progesterone and estradiol production when provided with the appropriate substrates. We conclude that PGC-2 display properties that are similar to immature granulosa cells and may provide a suitable in vitro model for the study of granulosa cell function.

Ultrastructural changes in bovine oocytes cryopreserved by vitrification.

Oocytes recovered from abattoir-derived ovaries were exposed to a cryoprotectant solution (DAP213: 2 M DMSO, 1 M acetamide, 3 M propanediol, and 10% fetal calf serum in tissue culture medium 199) for less than 20 s and vitrified either at the germinal vesicle (GV) stage or after maturation in vitro (IVM). Survival was assessed by fertilization and culture in vitro to the blastocyst stage. To identify ultrastructural changes, some of the vitrified oocytes that were morphologically normal after thawing were immediately processed for transmission electron microscopy after DAP213 removal. Cleavage rates for vitrified IVM oocytes were 4.5 and 6.7% using one-step and three-step cryoprotectant dilution procedures, respectively. Four (3%) oocytes developed to the eight-cell stage with the three-step procedure, but none formed blastocysts. None of the GV oocytes cleaved, while 66.7% (78/117) of controls developed to the two-cell stage and 19.2% (15/78) of those became blastocysts. Vitrification induced profound ultrastructural modifications in microvilli, mitochondria, vesicle formation, and the ooplasm of GV oocytes, whereas these structures were generally better preserved in IVM oocytes. The integrity of cell organelles was relatively better maintained following the three-step than after the one-step procedure in both GV and IVM oocytes. Changes in the zona pellucida (ZP) of IVM oocytes due to vitrification were associated with fewer cortical granules in the ooplasm. Since previous work showed that short-term exposure to DAP213 did not cause ZP alterations in IVM oocytes, these findings suggest that ZP damage due to low temperatures may result from the premature release of cortical granules.