Abnormal zonae pellucidae in mice lacking ZP1 result in early embryonic loss.

All vertebrates have an egg shell that surrounds ovulated eggs and plays critical roles in gamete recognition. This extracellular matrix is known as the zona pellucida in eutherian mammals and consists of three glycoproteins, ZP1, ZP2 and ZP3 in the mouse. To investigate the role of ZP1 in fertilization and early development, we have used targeted mutagenesis in embryonic stem cells to create mouse lines (Zp1(tm/tm)) lacking ZP1. Although a zona pellucida composed of ZP2 and ZP3 was formed around growing Zp1(tm/tm) oocytes, the matrix was more loosely organized than zonae around normal oocytes. In some Zp1 null follicles, this structural abnormality resulted in ectopic clusters of granulosa cells, lodged between the zona matrix and the oolemma, that perturbed normal folliculogenesis. Comparable numbers of eggs were ovulated from Zp1 null females and normal females following hormonal stimulation. However, after mating with males, fewer two-cell embryos were recovered from Zp1 null females, and their litters were significantly smaller than those produced by normal mice. Therefore, although mouse ZP1 is not essential for sperm binding or fertilization, it is required for the structural integrity of the zona pellucida to minimize precocious hatching and reduced fecundity.     

Defective zonae pellucidae in Zp2-null mice disrupt folliculogenesis, fertility and development

All vertebrate eggs are surrounded by an extracellular matrix. This matrix is known as the zona pellucida in mammals and is critically important for the survival of growing oocytes, successful fertilization and the passage of early embryos through the oviduct. The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2 and ZP3), each encoded by a single copy gene. Using targeted mutagenesis in embryonic stem cells, Zp2-null mouse lines have been established. ZP1 and ZP3 proteins continue to be synthesized and form a thin zona matrix in early follicles that is not sustained in pre-ovulatory follicles. The abnormal zona matrix does not affect initial folliculogenesis, but there is a significant decrease in the number of antral stage follicles in ovaries isolated from mice lacking a zona pellucida. Few eggs are detected in the oviduct after stimulation with gonadotropins, and no two-cell embryos are recovered after mating Zp2-null females with normal male mice. The structural defect is more severe than that observed in Zp1-null mice, which have decreased fecundity, but not quite as severe as that observed in Zp3-null mice, which never form a visible zona pellucida and are sterile. Although zona-free oocytes matured and fertilized in vitro can progress to the blastocyst stage, the developmental potential of blastocysts derived from either Zp2- or Zp3-null eggs appears compromised and, after transfer to foster mothers, live births have not been observed. Thus, in addition to its role in fertilization and protection of early embryos, these data are consistent with the zona pellucida maintaining interactions between granulosa cells and oocytes during folliculogenesis that are critical to maximize developmental competence of oocytes.     

Do age and extended culture affect the architecture of the zona pellucida of human oocytes and embryos?

Advanced female age and extended in vitro culture have both been implicated in zona pellucida (ZP) hardening and thickening. This study aimed to determine the influence of (i) the woman's age and (ii) prolonged in vitro culture of embryos on ZP thickness and density using non-invasive polarized light (LC-PolScope) microscopy. ZP thickness and density (measured as retardance) were determined in oocytes, embryos and blastocysts in women undergoing intracytoplasmic sperm injection (ICSI) in two age groups (older, > 38 years; younger, < or = 38 years). A total of 193 oocytes from 29 patients were studied. The younger group contained 100 oocytes and the older group 93 oocytes. The ZP was significantly thicker in metaphase II oocytes in the older group compared with the younger group (mean +/- SD: 24.1 +/- 2.5 microm vs 23.1 +/- 3.3 microm; p = 0.01) but ZP density was equal (2.8 +/- 0.7 nm). By day 2 of culture, embryos from the two groups had similar ZP thickness (22.2 +/- 2.2 microm vs 21.7 +/- 1.6 microm; p = 0.28) and density (2.9 +/- 0.7 nm vs 2.8 +/- 0.8 nm; p = 0.57). For the embryos cultured to blastocyst (older: n = 20; younger: n = 18) ZP thickness was similar in the two groups (19.2 +/- 2.7 microm vs 19.1 +/- 5.0 microm; p = 0.8) but thinner than on day 2. The older group had significantly denser ZP than the younger group (4.2 +/- 0.5 nm vs 3.3 +/- 1.0 nm, p < 0.01). Blastocysts from both groups had significantly denser ZP than their corresponding day 2 embryos (older: 4.2 +/- 0.5 nm vs 2.9 +/- 0.7 nm, p < 0.001; younger: 3.3 +/- 1.0 nm vs 2.8 +/- 0.8 nm, p = 0.013). It is concluded that there is little relationship between ZP thickness and its density as measured by polarized light microscopy. While ZP thickness decreases with extended embryo culturing, the density of the ZP increases. ZP density increases in both age groups with extended culture and, interestingly, more in embryos from older compared with younger women.     

Structural aspects of the zona pellucida of in vitro-produced bovine embryos: a scanning electron and confocal laser scanning microscopic study

Structural aspects of the bovine zona pellucida (ZP) of in vitro-matured (IVM) oocytes and in vitro-produced (IVP) embryos were studied in two experiments to find a tentative explanation for the zona's barrier function against viral infection. In Experiment 1, the ultrastructure of the outer ZP surface was studied. The diameter (nm) and the number of the outer pores within an area of 5000 microm(2) of 10 IVM oocytes, 10 zygotes, 10 8-cell-stage embryos, and 10 morulae were evaluated by scanning electron microscopy. In oocytes and morulae, the ZP surface showed a rough and spongy appearance with numerous pores. In zygotes, the ZP surface was found to have a smooth, melted appearance with only a few pores. In 8-cell-stage embryos, both surface patterns were found. The mean number (per 5000 microm(2)) and the mean diameter of the outer pores were different between the four stages of development (P < 0.001): 1511 pores in oocytes, 1187 in zygotes, 1658 in 8-cell-stage embryos, and 3259 in morulae, with mean diameters of 182, 223, 203, and 155 nm, respectively. In Experiment 2, the continuity of the meshes (network of pores) towards the embryonic cells was examined by confocal laser scanning microscopy. Therefore, the passage through and the location in the ZP of fluorescent microspheres, with similar dimensions as bovine viral diarrhea virus (BVDV, 40-50 nm) and bovine herpesvirus-1 (BHV-1; 180-200 nm), were evaluated. For all stages, the smallest beads were detected halfway through the thickness of the ZP, whereas the beads with a size of 200 nm were found only within the outer-fourth part of the ZP. It can be concluded that the intact ZP of bovine IVM oocytes and IVP embryos are constructed in such a way that BVDV and BHV-1 should not be able to traverse the ZP and reach the embryonic cells. However, the risk exists that viral particles can be trapped in the outer layers of the ZP.     

Gene expression during mammalian oogenesis and early embryogenesis: quantification of three messenger RNAs abundant in fully grown mouse oocytes

Ribonuclease protection assays have been used to quantitatively assess changes in steady-state levels of specific mRNAs during oogenesis and early embryogenesis in mice. The mRNAs encode ZP3 (a glycoprotein that serves as a sperm receptor), LDH-B (heart-type lactate dehydrogenase), and MOM-1 (a protein of unknown function). MOM-1 and LDH-B are expressed in a variety of adult mouse tissues and midgestation embryos, whereas ZP3 expression is restricted completely to oocytes. All three mRNAs are expressed by growing mouse oocytes and accumulate to unusually high levels in fully grown oocytes as compared to somatic cells; 240,000, 200,000 and 74,000 copies mRNA per fully grown oocyte for ZP3, LDH-B and MOM-1, respectively. Steady-state levels of LDH-B and MOM-1 mRNA undergo a modest decline (approximately 20-40%) during ovulation when fully grown oocytes become unfertilized eggs and, in general, mirror the reported change in poly(A)+RNA levels during this period of development. On the other hand, the level of ZP3 mRNA declines dramatically (approximately 98%) during ovulation, from approximately 240,000 copies per oocyte to approximately 5000 copies per unfertilized egg, and ZP3 mRNA is undetectable in fertilized eggs (less than 1000 copies per fertilized egg). MOM-1 mRNA is expressed at relatively low levels in morulae (approximately 2000 copies per embryo) and blastocysts (approximately 5000 copies per embryo), whereas ZP3 mRNA remains undetectable (less than 1000 copies per embryo) at these stages of preimplantation development. These findings are discussed in the context of overall gene expression during oocyte growth, meiotic maturation and early embryogenesis in mice.     

The distribution of transglutaminase in the rat oocytes and embryos

Transglutaminases (TGs) are calcium-dependent enzymes that catalyze the transamidation of glutamine residues of a protein substrate to form intermolecular isopeptide bonds. The zona pellucida (ZP) is an extracellular, glycoprotein matrix that surrounds the oocytes of all Eutherian mammals. We aimed to identify the immunoreactivity of tissue transglutaminase (tTG) and ultrastructural changes occuring in rat oocytes before and after fertilization. Female rats were stimulated to superovulate, then mated with males. Oocytes and embryos were collected and examined by immunohistochemistry and electron microscopy. Before fertilization, tTG was present only in the oolemma and the cortical cytoplasm. After fertilization, tTG reactivity increased in the ZP of the early zygote and the preimplantation embryos, but decreased in the cytoplasm and perivitelline space (PVS). After fertilization, the PVS ultrastructure became asymmetrical and large around the polar bodies with many cortical granule contents. In conclusion, tTG immunoreactivity was found to be spatially and temporarily heterogeneous in the rat oocytes and embryos, especially in the ZP.     

Zona pellucida solubility and cortical granule complements in human oocytes following assisted reproductive techniques

In this study the solubility to alpha-chymotrypsin of the zona pellucida (ZP) of human oocytes and polyploid embryos obtained during various clinical procedures of assisted fertilisation (IVF, ICSI, cyropreservation) was evaluated. The aim of the study was to determine whether changes in ZP solubility occur during such procedures and whether abnormal solubility could be likened to fertilisation failure. Correlation between ZP solubility and cortical granule (CG) density was also studied. The results showed that ZP solubility varied considerably among germinal vesicle or metaphase oocytes obtained from different subjects, but was essentially identical for the oocyte cohort obtained from individual women. On the basis of ZP solubility metaphase oocytes were subdivided into two classes: class I, average ZP dissolution time +/- SE = 24.1+/-0.9 min, n = 28; and class II, 46.7+/-2.0 min, n = 13. Prolonged ZP dissolution times of metaphase oocytes were significantly correlated with a low in vitro fertilisation rate in sibling oocytes. The zonae of fertilised eggs (polyploid embryos) showed long solubilisation times (IVF: 45.3+/-3.4 min, n = 18; ICSI: 48.9+/-2.7 min, n = 19). ZP solubility of oocytes that failed to fertilise was intermediate between that of class I metaphase oocytes and embryos (unfertilised IVF: 33.0+/-2.7 min, n = 13; unfertilised ICSI: 43.0+/-2.4 min, n = 9). A moderate spontaneous ZP hardening occurred when metaphase oocytes were cultured for 24 h. Finally, cryopreservation of unfertilised oocytes caused hardening of their ZP, with dissolution times that were comparable to those found in fertilised eggs (49.5+/-2.3 min, n = 10). In most cases, an inverse correlation was found between ZP dissolution time and CG density (longer solubilisation times corresponding to lower CG density). ZP hardening caused by cryopreservation, however, was not associated with a significant reduction in CG density in most of the oocytes examined.     

Damage of zona pellucida reduces the developmental potential and quality of porcine circovirus type 2–infected oocytes after parthenogenetic activation

The present aimed to study if porcine circovirus type 2 (PCV2), which adhered to zona pellucida (ZP), was able to enter mature porcine oocytes with intact and damaged ZP. Four groups, including uninfected ZP-intact oocytes (UOZI), uninfected ZP-damaged oocytes (UOZD), PCV2-infected ZP-intact oocytes (POZI), and PCV2-infected ZP-damaged oocytes (POZD) were studied. The oocytes were incubated with 1 mL minimum essential medium, containing 3.1 × 10⁸ copies of PCV2 DNA for 1 hour. Mechanical procedure of the insertion by microneedle induced injuries to the ZP of porcine oocytes. At the blastocyst stage, the percentage of PCV2-infected embryos and the ratio of viral antigen-positive cells per embryo were determined by indirect immunofluorescence. To assess the effect of ZP injury on the developmental competence and quality of porcine PCV2-infected oocytes after parthenogenetic activation, blastocyst formation rates and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining were analyzed. Moreover, real-time polymerase chain reaction was used to evaluate the expression of genes related to apoptosis and pluripotency at different developmental stages. The results of indirect immunofluorescence showed that only POZD group presented PCV2-infected embryos and viral-positive cells. The blastocyst rate of POZD group dropped down to approximately half of POZI group's (7.1 ± 1.5 vs. 14.5 ± 3.3). At the blastocyst stage, ZP injury increased apoptotic index of PCV2-infected embryos. The relative expression levels of Caspase 3 were higher in POZD group than the ones in POZI group at the two- and four-cell stages (not statistically significant). Compared with the one in POZI group, the ratio of antiapoptotic Bcl-xl gene to proapoptotic Bax gene, an indicator of the ability to resist apoptosis, was lower in POZD group at the one-cell stage, but higher at the two- and four-cell stages. Expression levels of Oct4 and Nanog associated with pluripotency were lower in POZD group than the ones in POZI group at the morula stage (not statistically significant). Noteworthily, the expression of Nanog was significantly lower in POZD group versus POZI group (P < 0.05), whereas relative expression of Oct4 was significantly higher in the former at the blastocyst stage (P < 0.01). In conclusion, PCV2, which attached to ZP, was able to enter mature porcine oocytes with damaged ZP and subsequently reduced the developmental competence and quality of the oocytes after parthenogenetic activation.     

Bile duct ligation promotes covalent drug-protein adduct formation in plasma but not in liver of rats given zomepirac

Acyl glucuronides are reactive electrophilic metabolites of carboxylate drugs, capable of undergoing hydrolysis, rearrangement and covalent binding reactions with proteins in vivo. Such covalent drug-protein adducts may be prerequisites for certain idiosyncratic immune and toxic responses in susceptible individuals. The present study examined the effect of experimental cholestasis on the extent and pattern of formation of protein adducts in plasma and liver of rats given the non-steroidal antiinflammatory drug (NSAID) zomepirac (ZP). Groups of intact, bile-exteriorized and bile duct-ligated rats given a 50 mg/kg i.v. dose of ZP were studied for 24 hr. In intact rats, only 1.4% of the dose was recovered as the sum of ZP, ZP acyl glucuronide (ZAG) and its rearrangement isomers (iso-ZAG) in urine in 24 hr. In bile-exteriorized animals, 0.5% of the dose was recovered in urine in 24 hr, with 31.6% of the dose being recovered in bile (2.7% as ZP, 20.0% as ZAG and 8.9% as iso-ZAG). In the bile duct-ligated group, recovery of dose in 24 hr urine totalled 17.5% (1.7% as ZP, 6.7% as ZAG and 9.1% as iso-ZAG). ZAG and iso-ZAG were measurable in plasma only in the bile duct-ligated group, and covalent binding of ZP to plasma proteins was much higher (5-6 fold) than in intact or bile-exteriorized rats. Total adduct concentrations in liver were not significantly different among the three groups. Immunoblotting using a polyclonal ZP antiserum confirmed that serum albumin was a major target protein in plasma. The major ZP-modified bands in the livers of intact and bile-exteriorized rats were at about 110, 140 and 200 kDa. However, the bands at 110 and 140 kDa were much lower in the livers of bile duct-ligated rats. The results show that about 30% of ZP doses are normally excreted as ZAG and its isomers in bile, with only minor excretion in urine. Bile duct ligation shunts the glucuronide into blood (and urine), strongly promoting adduct formation with plasma proteins, and alters the pattern but not the total quantity of drug-modified proteins formed in the liver.     

Maturation-associated changes in the rat zona pellucida

Rat follicular oocytes, arrested at prophase I, cannot be fertilized in vitro. This capacity is acquired following resumption of meiosis and a series of changes involving both the oocyte and the cumulus cells surrounding it. Oocytes exposed to sperm at different hours before ovulation show a gradual increase in the permeability of their zona pellucida (ZP). Our study examined whether the ZP, in response to the physiological stimulus for maturation and concomitant with the other oocyte--cumulus components, undergoes maturational changes. Two ZP characteristics were assessed, sensitivity to proteolysis and sperm binding. ZP surrounding oocytes and eggs were collected from five sources: 1) germinal vesicle (GV)-intact oocytes, 2) preovulatory eggs, 3) ovulated eggs isolated from oviducts of immature females, 4) fertilized eggs, 5) ovulated eggs isolated from oviducts of mature females. All ZP surrounding oocytes/eggs from groups 1-5 were dissolved by trypsin. When solubility by pronase and alpha-chymotrypsin was examined, a large variation between groups was found. All ZP from group 2 were dissolved by 0.001% pronase, compared to 0% solubility in group 4. Only 10% of the ZP surrounding GV-intact oocytes (group 1) were dissolved by this enzyme, compared to 82% in group 3. Solubility in 0.01% alpha-chymotrypsin showed a similar pattern. Capacitated sperm were incubated with eggs from groups 1 and 3. The number of sperm binding to ZP in group 3 was repeatedly higher than that in group 1. In both tests it was found that the ZP surrounding the mature eggs differ in their characteristics from ZP of GV-intact oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of eCG and FSH on ovarian response, recovery rate and number and quality of oocytes obtained by ovum pick-up in Holstein cows

The goal of the present study was to compare the ovarian response, oocyte yields per animal, and the morphological quality of oocytes collected by ultrasound guided follicular aspiration from Holstein cows treated either with FSH or eCG. Twenty four normal cyclic, German Holstein cows were randomly divided into two groups. Fourteen cows received 3000 IU eCG on day-4 prior to ovum pick-up (OPU) (day 0), 2 days later (day-2), 625 microg cloprostenol was administered. On day-1 GnRH was administered i.m. and 24h later OPU (day 0) was performed. In ten cows a total dose of 500 IU follicle stimulating hormone (Pluset) was administered intramuscularly in a constant dosage for 4 days with intervals of 12h, starting on day-5. Luteolysis was induced by application of 625 microg cloprostenol on day-2. On day-1 (24h after the last FSH treatment) GnRH was administered i.m. and 24h later OPU (day 0) was performed. Ovarian follicles were visualized on the ultrasound monitor, counted and recorded. All visible antral follicles were punctured. Recovered oocytes were graded morphologically based on the cumulus investment. Average follicle number in ovaries was higher in FSH group than eCG group (p<0.05). Oocyte yields per animal did not differ between FSH and eCG groups. The proportion of grade A oocytes was higher in the FSH group in the than eCG group (p<0.05). Likewise, rate of grade C oocytes in FSH group were lower than eCG group (p<0.05). In conclusion, these results suggest that ovarian response, follicle number in ovaries and oocyte quality are affected by the type of gonadotropin and FSH is better alternative than eCG for OPU treatment.     

Birth of piglets by in vitro fertilization of zona-free porcine oocytes

The present experiments were conducted to optimize in vitro fertilization conditions for zona pellucida-free (ZP-free) oocytes and their subsequent development. The results demonstrated that: (1) maximal fertilization efficiency was achieved at 200 spermatozoa per ZP-free oocyte. At this sperm dose, there were no significant differences in penetration rates and polyspermy rates from controls (zona-intact oocytes with 1000 spermatozoa/oocyte), indicating that ZPs of in vitro matured pig oocytes failed to block polyspermy during in vitro fertilization. (2) In vitro development of zygotes from ZP-free oocytes showed that there was no difference in cleavage rates. The blastocyst rate was slightly lower in the ZP-free group than the control. However, there was no difference in cell number per blastocyst between the control and the ZP-free group. (3) Examination of acrosome status by a specific fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) staining procedure revealed that frozen-thawed pig spermatozoa could undergo acrosome reaction and penetrate oocytes without induction by ZP. These data suggested that there are alternative mechanistic pathways for acrosome reaction induction during the fertilization process than the widely accepted sperm-zona receptor models. Finally, the viability of ZP-free derived embryos was demonstrated by full-term development and the delivery of healthy piglets following embryo transfer. In conclusion, the present experiments showed for the first time in farm animals, that normal embryos could be produced by in vitro fertilization of ZP-free oocytes in optimized conditions and that they could develop normally to full-term.     

The superovulation of synchronous adult rats using follicle-stimulating hormone delivered by continuous infusion

The estrous cycles of adult female rats were synchronized with an LHRH agonist on the morning of Day -4 (Day 0 = day of mating). On Day -2, animals received s.c. implants of continuous-infusion osmotic minipumps containing different doses of an FSH preparation (Folltropin) in combination with hCG at various ratios of hCG:FSH or were given single injections of eCG in doses ranging from 15 IU to 60 IU. Rats infused with the optimal dose (3.4 U/day) of FSH ovulated 44.1 +/- 5.4 oocytes/rat while rats treated with the most effective dose (60 IU) of eCG ovulated only 20.5 +/- 4.3 oocytes/rat on the morning of Day 1. The inclusion of hCG in pumps at ratios from 0.188:1 to 0.75:1 (hCG:FSH) had no significant effect on ovulation rate. The importance of synchronization of estrus in successful superovulation was demonstrated by the finding that only 70% of the unsynchronized animals ovulated (29.1 +/- 4.8 oocytes/rat) whereas 95% of the synchronized animals ovulated (51.0 +/- 3.6 oocytes/rat). Oocyte viabilities were assessed by determining fertilization rates and embryonic development in vivo following mating with fertile males. In rats superovulated by use of the FSH regimen, 92% (39.0 +/- 4.1) of the recovered embryos were 1-cell zygotes on Day 1, 89% (36.3 +/- 5.6) were at the 2-cell embryo stage of development on Day 2, and 88% (28.8 +/- 2.2) were at the morula and blastocyst stages on Day 5 following mating on Day 0. The high ovulation rates and oocyte viability in rats receiving infusions of Folltropin following estrus synchronization offer a reliable method for superovulation of adult rats.     

PYY(3-36) reduces food intake and body weight and improves insulin sensitivity in rodent models of diet-induced obesity

The gut hormone peptide YY (PYY) was recently proposed to comprise an endogenous satiety factor. We have studied acute anorectic functions of PYY(3-36) in mice and rats, as well as metabolic effects of chronic PYY(3-36) administration to diet-induced obese (DIO) mice and rats. A single intraperitoneal injection of PYY(3-36) inhibited food intake in mice, but not in rats. We next investigated the effects of increasing doses (100, 300, and 1,000 microg.kg-1.day-1) of PYY(3-36) administered subcutaneously via osmotic minipumps on food intake and body weight in DIO C57BL/6J mice. Whereas only the highest dose (1,000 microg.kg-1.day-1) of PYY(3-36) significantly reduced food intake over the first 3 days, body weight gain was dose dependently reduced, and on day 28 the group treated with 1,000 microg.kg-1.day-1 PYY(3-36) weighed approximately 10% less than the vehicle-treated group. Mesenteric, epididymal, retroperitoneal, and inguinal fat pad weight was dose dependently reduced. Subcutaneous administration of PYY(3-36) (250 and 1,000 microg.kg-1.day-1) for 28 days reduced body weight and improved glycemic control in glucose-intolerant DIO rats. Neither 250 nor 1,000 microg/kg PYY(3-36) elicited a conditioned taste aversion in male rats.     

Increased mortality during early embryonic development after in-vitro fertilization of rat oocytes

Immature female rats (60-65 g) were injected with 4 i.u. PMSG on Day -2, and allocated to 3 groups. For Groups I and II, unmated donors were killed 67-69 h after PMSG injection, shortly after the expected time of ovulation. Oocytes were recovered from the oviducts and transferred immediately into the oviduct of mated recipients (Group I) whose ipsilateral ovary had been exposed by peeling back the bursa, preventing endogenous oocytes from entering the oviduct, or were fertilized in vitro (Group II) and were transferred 16-18 h later. Rats in Group III were allowed to mate and half were killed 6 h after mating. The fertilized oocytes were then incubated for 10-12 h until transfer. The remaining rats in Group III were killed 16-18 h after mating and fertilized oocytes were collected and transferred immediately. Recipient rats were killed on Days 2, 5, 8 and 20. Zygotes resulting from in-vitro fertilization (Group II) were as able as those fertilized in donors (Group III) or recipients (Group I) to develop to the 2-cell stage, but underwent significantly greater embryonic loss beyond this stage of development. There was a slower rate of development of such oocytes to the blastocyst stage (Day 5) and a lower mean weight of implantation sites (Day 8). Transfer of zygotes after in-vitro fertilization resulted in a loss of 35% of the embryos at the time of implantation. These results suggest that in-vitro fertilization of rat oocytes leads to defects in the embryos causing a delay in early embryo development and a large number of implantation losses.     

Cryopreservation of unfertilized mouse oocytes from inbred strains by ultrarapid freezing

Unfertilized mouse oocytes from inbred strains (BALB/c, C3H/He and C57BL/6) were frozen ultrarapidly by direct plunging into liquid nitrogen, immediately after exposure to a highly-concentrated solution (DAP 213: 2 M dimethyl sulphoxide, 1 M acetamide, and 3 M propylene glycol in PB 1), and were later thawed in a 37 degrees C waterbath. After thawing, 76.8-90.9% of recovered oocytes were morphologically normal. Following fertilization in vitro of cryopreserved oocytes, the proportion of 2-cell embryos 24 h after insemination ranged from 70.7% to 83.4%. Nearly all 2-cell embryos obtained from cryopreserved oocytes were transferred to the oviducts of pseudopregnant recipients and 31.0-43.0% of 2-cell embryos developed into normal young.     

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.     

R?le of histamine on plasma fibrinogen levels in rats with surgical injury (laparotomy)

The probable r?le of endogenous histamine in the increase of plasma fibrinogen in rats submitted to tissue injury (laparotomy) was studied. In laparotomized rats with 10 mg kg-1 day-1 of diphenhydramine (a H1-histamine receptor blocker) plasma fibrinogen decreased significantly as compared to the group of rats laparotomized only (P less than 0.02), reaching values similar to those observed in rats laparotomized with removal of the adrenal medulla or laparotomized with severing of splanchnic nerves. There is a significant difference between these latter groups and the normal noninjured group (P less than 0.01). Plasma fibrinogen did not modify (as compared with the uninjured group) in rats injected only with histamine (1 mg kg-1 day-1) or with diphenhydramine. Taking into account the results obtained and the mechanism of action of diphenhydramine, it would seen that endogenous histamine takes part in the increase of plasma fibrinogen in laparotomized rats, perhaps indirectly through stimulation of the adrenal medulla secretion.     

Effects of the administration of progesterone and adrenal medullectomy on the plasma fibrinogen levels in rats with surgical injury (laparotomy)

The probable r?le played by the adrenal medulla in the decrease of plasma fibrinogen due to the administration of progesterone (0.5 mg kg-1 day-1 during 72 h) in rats submitted to surgical injury (laparotomy) was studied. The results obtained lead to assume that the decrease of plasma fibrinogen observed in laparotomized rats injected with progesterone is indirectly produced through inhibition of the adrenal medulla. The action of progesterone on the plasma fibrinogen would be a pharmacological effect of the drug, since in doses of 0.10 mg kg-1 day-1 the decrease of the fibrinogen is not observed in laparotomized rats. The administration of progesterone in non injured rats does not modify the plasma fibrinogen as compared to the group of non injected rats.