IU PMSG Research Articles

Hyperstimulation and a Gonadotropin-Releasing Hormone Agonist Modulate Ovarian Vascular Permeability by Altering Expression of the Tight Junction Protein Claudin-5

We investigated the mechanism by which a GnRH agonist (GnRHa) affects ovarian vascularity, vascular permeability, and expression of the tight junction protein claudin-5 in a rat model of ovarian hyperstimulation syndrome (OHSS). Hyperstimulated rats received excessive doses of pregnant mare serum gonadotropin (PMSG; 50 IU/d) for 4 consecutive days, from d 25 to 28 of life, followed by 25 IU human chorionic gonadotropin (hCG) on d 29. Control rats received 10 IU PMSG on d 27 of life, followed by 10 IU hCG on d 29. GnRHa (leuprolide 100 microg/kg.d) was administered to some hyperstimulated rats either on d 29 and 30 (short-term GnRHa treatment) or from d 25 to 30 (long-term GnRHa treatment). Ovarian vascular density (vessels per 10 mm(2)) and vessel endothelial area (percent) were assessed by immunohistochemical analysis of the distribution of von Willebrand factor, whereas vascular permeability was evaluated based on leakage of Evans blue. High doses of PMSG and hCG significantly increased ovarian weight, vascular permeability, vascular density, and the vessel endothelial area and significantly reduced expression of claudin-5 protein and mRNA. All of these effects were significantly and dose-dependently inhibited by administration of GnRHa. This suggests that reduced expression of claudin-5 plays a crucial role in the increased ovarian vascular permeability seen in OHSS and that its expression can be modulated by GnRHa treatment. Indeed, preventing redistribution of tight junction proteins in endothelial cells and the resultant loss of endothelial barrier architecture might be the key to protecting patients against massive extravascular fluid accumulation in cases of OHSS.

Effects of IGF-I and -II to Apoptosis Expression in Pre-Implantation Mouse Embryos

High levels of apoptosis and retarded development occur for mouse embryos fertilized in vitro compared to those in vivo. We reported that fertilization environment, in vivo or in vitro, could have influence on the expression of IGF-I or -II mRNA and embryonic apoptosis. These results suggested that higher endogenous growth factor expression should be beneficial for apoptosis suppression by autocrine pathway. Therefore, the objectives of the present study were 1) to investigate the apoptotic rates for embryos fertilized in vivo and in vitro and 2) to examine whether exogenous IGF-I and -II in the in vitro culture medium overcome apoptosis occurrence. The effects of exogenous IGF-I or/and -II in IVF and culture media were examined on embryo development and apoptosis expression. BD F1 mice, 5-6 weeks old, were superovulated by 5 IU PMSG followed 48 h later by 5 IU HCG injection. Some of them were paired overnight with males and others were not paired to perform IVF. The presence of a copulation plug was regarded as day 0 of pregnancy. Oocyte-cumulus complexes for IVF were collected from the oviduct 14-16 h after HCG injection. IVF was performed using sperm collected from 10-weeks aged males in a 20 ul of mMTF containing BSA (16 mg/ml) in the presence or absence of IGF-I and -II (10 ng/ml). Zygotes derived from IVF were cultured in a 20 ul of mMTF containing BSA (4 mg/ml) in which IGFs were supplemented at the same concentration(s) as IVF medium by day 5. Two-cell embryos fertilized in vivo were collected by flushing oviducts on day 1, and then cultured by day 5. Apoptotic cells of day 5 blastocysts were identified using TUNEL labeling and Hoechst counterstain to evaluate DNA fragmentation, total cell, and nuclear morphology, respectively. As shown in table, the fertilization rates were significantly higher in IGFs-treated groups than an untreated group (P < 0.05). Although the developments to the blastocyst stage in in vitro groups were lower than in vivo group, IGFs-treated groups were higher than an untreated group. However, no effect was observed between individual and combination of IGF-I and -II. Specially, total cell numbers and apoptosis occurrences obtained from IGFs-treated in vitro groups tended to be similar to those in in vivo group. Tabled 1 The embryos treated with IGFs showed higher fertilization rate and total cell number as well as lower apoptotic index than those not treated group. Although embryo development was low, the embryos in IGFs-treated groups showed similar total cell number and apoptotic index to those in in vivo group. This study suggests that IGFs should have influence on not only embryo development but also embryonic apoptosis. In conclusion, the addition of IGFs was hence overcome an apoptosis in embryos fertilized in vitro as those in vivo.

In Vivo Disruption of Leptin Receptor Function Impairs the Establishment and Viability of Endometriosis-Like Lesions

There has been an increased interest in the role of the metabolic regulator, leptin, in several reproductive processes including endometriosis. Leptin peritoneal fluid levels are elevated in women with endometriosis, and have been shown to be inversely proportional to disease extent, suggesting a potential role for leptin in disease establishment. Similar to other cytokines, the exact role of leptin in the establishment of ectopic lesions is incompletely understood. Unfortunately there are a limited number of in vivo models to test the function of specific cytokines in the development of ectopic lesions. Mutant mouse models have been used effectively to elucidate function of genes/proteins related to human disease. Thus, we sought to investigate the effect of leptin signaling disruption on the successful establishment and proliferation of ectopic endometrium derived from either wild type (WT) or leptin receptor mutant (Leprdb-nonfunctional receptor) mice and hosted by their WT and/or Leprdb sisters. Animal studies utilizing syngeneic WT and leptin receptor deficient mice. Female Leprdb and control mice 6-8 wks of age were ovariectomized and received a 8mm silastic 17β-estradiol (E2) implant. Uteri were removed 96 hrs later, incised longitudinally, and endometrium was excised, minced, and inserted into the peritoneal cavity through a dorsolateral incision. Leprdb and control mice received uterine tissue from the opposite genotype of the same litter, and were monitored for 14d. Animals were then euthanized, ectopic endometrial lesions were removed, fixed in 4% PFA, paraffin embedded, serial sectioned, and examined for histological evidence of viable glandular and stromal epithelial components (H&E), vascularization (VEGF), and proliferation (phospho-histone 3-PH3). Similarly, WT C57BL6 mice 6-8 wks of age underwent ovariectomy and received an E2 implant. Female siblings were primed with 10 IU PMSG. Their uteri were removed 41 hrs later, the endometrium was processed as previously described, and inserted into the peritoneal cavity. Treatment groups included: 60μM leptin-peptide receptor antagonist (LPA-2), 60μM nonfunctional scrambled peptide (LPA2-sc), DMSO 0.0005% (vehicle), and PBS (control). Treatments consisted of daily IP injections initiated 1d prior to tissue transfer and continued for 13d. Ectopic endometrial lesions were processed as previously described. Ectopic lesions of Leprdb mice (hosting control tissue) displayed a gross purulent, necrotic appearance and significantly fewer viable glands with disorganized and apoptotic epithelium compared to control hosts hosting Leprdb tissue. In the WT model, the histological appearance and IHC staining (PH3 and VEGF) of the native uterus was similar in all treatment groups. Ectopic lesions of the LPA-2 group were characterized by uniform pallor, reduced gross vascularization, minimal viable glandular epithelium and diffuse fibrosis, and no significant PH3 staining compared to the other groups. Disruption of leptin signaling in the peritoneum impairs the establishment and proliferation of endometriosis-like lesions in a syngeneic mouse model. The absence of a functional leptin receptor results in the failure of ectopic endometrium to proliferate. These results suggest that the functional leptin receptor of the host plays a significant role and is a necessary component for early lesion development.

Estrous synchronization during the natural breeding season in Anatolian black does

The efficiency of medroxyprogesterone acetate (MAP) and fluorogestone acetate (FGA) sponges with or without PGF&lt;sub&gt;2&amp;alpha;&lt;/sub&gt; (cloprostenol) for synchronizing estrous in non-lactating does was investigated during the natural breeding season. Does were treated for 11 days with 60 mg MAP (n = 38) or 40 mg FGA (n = 32) sponges. All does also received intramuscular injections of 500 IU PMSG. In addition, 19 and 14 of the does synchronized with MAP and FGA respectively, were injected with 125 &amp;mu;g cloprostenol and the remaining does from both groups were injected with 1.5 ml of sterile saline solution, 48 h prior the sponge removal. Cervical artificial insemination (AI) with diluted fresh semen was performed at a fixed time (36 and 48 h) following progestagen withdrawal. The different groups estrous response for the first 12 &amp;plusmn; 6 h and within 66 h, time to onset and duration of the induced estrous, and pregnancy rate was found to be 52.6%, 92.9%, 20.6 &amp;plusmn; 0.8 h, 29.7 &amp;plusmn; 1.3 h, and 70.0%, respectively. There were significant differences between groups FGA/PMSG/PGF&lt;sub&gt;2&amp;alpha;&lt;/sub&gt; and MAP/PMSG in terms of the duration of induced estrous (P &amp;lt; 0.05) and between groups FGA/PMSG/PGF&lt;sub&gt;2&amp;alpha;&lt;/sub&gt; and FGA/PMSG in terms of estrous response at the first 12 &amp;plusmn; 6 h (P &amp;lt; 0.05). These results indicate that, the use of MAP/PMSG and FGA/PMSG intravaginal progestagen treatments with or without cloprostenol are equally efficient in synchronizing estrous in non-lactating hair goats during the natural breeding season.

298. Dietary protein does not influence mitochondrial distribution in the 2-cell mouse embryo

Excess dietary protein can negatively influence fertility. The underlying mechanisms remain to be completely elucidated; however, variations in reproductive tract pH, and ammonium and urea concentrations have been implicated. Mouse embryos cultured in the presence of ammonium showed a shift in mitochondrial distribution away from the nucleus towards the cell cortex, suggestive of reduced mitochondrial activity, ATP production and embryo viability. In this study we determined the effect of dietary protein in vivo, on mitochondrial distribution in the 2-cell mouse embryo. Five-week-old Swiss female mice (n = 10) were fed low (9%), medium (14%) or high (25%) dietary protein for 3weeks; feed intake and body weight were recorded weekly. At 8 weeks of age mice were primed with 5 IU of PMSG, then 5 IU hCG 48 h later, and mated overnight with males of proven fertility. Forty hours post-hCG females were sacrificed, their oviducts collected and flushed with media. The total number of 2-cell embryos and oocytes retrieved were recorded. Active mitochondria were stained in the 2-cell embryos using Mitotracker Green (Molecular Probes), and were visualised using confocal microscopy. Density of perinuclear and cortical staining was determined in Photoshop 7.0, using an established method. Females fed the medium diet consumed significantly less and gained less weight than those fed the low or high diet (Table 1), despite similar final body weights (data not shown). Females fed the low diet tended to have a lower ovulation rate and fewer 2-cell embryos than females consuming the other diets (Table 1, P &gt; 0.05). There was no significant effect of dietary protein on the distribution of mitochondria between the perinuclear and cortical region of the embryo, which may be reflective of lower in vivo ammonium levels compared to those described in culture.

Tavşanlarda farklı süperovulatör ajanların oosit kalitesi ve in vitro fertilizasyonu üzerine etkisi

Summary: The aim of this study was to investigate the effect of two different superovulator agents on oocyte quality, quantity and in vitro fertilization of rabbits. In this study 16 female and 2 male New Zealand White rabbits were used. A group of female rabbits (n=8) was intramusculary injected with 75 IU PMSG 3 times with 24 hours intervals. Another group of female rabbits (n=8) was injected (intramuscular) with 7.5 IU FSH twice daily during three consecutive days. Twentyfour hours after the last hor-mon injection both groups of animals ovulation was induced by an intravenous injection of 100 IU hCG. Fifteen hours after hCG injection, oocytes were collected by flushing the oviducts. A total of 34 oocytes were obtained from PMSG applied group and 77 oocytes were collected from FSH applied group. Difference between groups for oocyte number was found significant (p 0.05). Detection of embryos reaching to 2-cell stage was approved as criterion for the assesment of

139 BOVINE AMNIOTIC FLUID FOR THE CULTURE OF TWO-CELL MURINE EMBRYOS

The objective of the experiment was to evaluate bovine amniotic fluid as an alternative medium for culture of two-cell murine embryos. Variability in embryo development rate and high purchase prices of medium supplements such as fetal bovine serum have prompted the search for a serum alternative. Amniotic fluid was collected postmortem from first-trimester bovine fetuses, pooled, heat inactivated at 56°C for 30 min and stored at −20°C until used. Two-cell mouse embryos were collected from (Balb/C × C57BL6) F1 females superovulated with 10 IU PMSG and 10 IU hCG. The experiment was performed to evaluate the effect of commercial fetal bovine serum (F4135, Sigma, South Africa) supplementation to amniotic fluid. Treatments consisted of (1) bovine amniotic fluid, (2) bovine amniotic fluid supplemented with 10% fetal bovine serum, (3) M16 (M7292, Sigma) supplemented with 10% fetal bovine serum, and (4) Medium 199 (M4530, Sigma) supplemented with 10% fetal bovine serum. The latter two media were controls. Twenty-four hours before use the culture media were supplemented with 1% antibiotic antimycotic solution (A5955, Sigma). Media were equilibrated for 24 hours at 37°C and 5% CO2 before use. Embryos were cultured in 50-μL droplets with oil overlay at 37°C in 5% CO2. A minimum of 5 and maximum of 10 embryos per droplet were allowed. Six replications per treatment were done giving a total of 292 embryos in treatment (1), 318 in treatment (2), 304 in treatment (3), and 303 in treatment (4). The embryos were monitored under an inverted microscope (Olympus model IX70) at 24-h intervals for 72 h for blastocyst formation. The differences between embryo growth in the different culture media were assessed by one-way analysis of variance. All culture media supported the development of mouse embryos to the hatched blastocyst stage. A higher (P &lt; 0.05) number of embryos hatched in M16 (64.6%) and Medium 199 (55.0%) supplemented with 10% fetal bovine serum than in frozen bovine amniotic fluid (12.2%) and frozen bovine amniotic fluid supplemented with 10% fetal bovine serum (17.8%). M16 was superior to all other treatments in supporting embryo development up to the morula stage. More than twice the number of embryos (94.9%) reached the morula stage in M16 than in frozen bovine amniotic fluid with (37.4%) or without (29.1%) serum supplementation. Bovine amniotic fluid obtained from postmortem first trimester fetuses supported the development of two-cell mouse embryos; however, embryo development in frozen fetal fluid was lower (P &lt; 0.05) than that obtained in the control media. Fetal bovine serum, when added to amniotic fluid, did not increase the development rate. It seems likely that freezing the amniotic fluid had an adverse effect on in vitro embryo development.

Reproductive performance of South African indigenous goats following oestrous synchronisation and AI

The reproductive performance following oestrous synchronisation and artificial insemination (AI) was evaluated during the natural breeding season (autumn) in 90 indigenous (Boer and Nguni) South African goats. All does were synchronised for 16 days with medroxyprogesterone acetate (MAP) followed by an IM injection of 300 IU PMSG at progestagen withdrawal. Cervical inseminations were performed at a fixed time (48 h and 60 h) with fresh diluted Boer goat semen. No significant differences between Boer and Nguni goats were recorded with respect to oestrous response (98% and 93%) and the onset of oestrus (29.1 ± 0.9 h and 30.7 ± 0.8 h), respectively. The duration of oestrus in the Nguni goats (29.9 ± 2.0 h) was significantly ( P < 0.01) shorter than in Boer goats (37.0 ± 2.1 h). Conception rates were 52% and 53%, litter size averaged 2.2 ± 0.2 and 2.0 ± 0.2 and the gestation period an average of 148.6 ± 0.9 days and 149.1 ± 0.8 days for Boer and Nguni goats, respectively, these differences being non-significant. Does with quadruplets had a significantly ( P < 0.05) shorter gestation length (142.7 ± 2.1 days) than does with singles, twins and triplets (150.0 ± 0.9 days, 148.8 ± 1.0 days and 150.0 ± 1.1 days, respectively). The mean kid birth weight was 2.7 ± 0.5 kg with males being (2.8 ± 0.1 kg) significantly ( P < 0.05) heavier than females (2.5 ± 0.1 kg) and crossbred kids (2.9 ± 0.1 kg) significantly ( P < 0.01) heavier than pure Boer goat kids (2.4 ± 0.1 kg). Kid birth weight decreased with an increase in litter size, resulting in all birth weights for singles, twins, triplets and quadruplets being significantly ( P < 0.01) different from each other (3.6 ± 0.4 kg, 3.1 ± 0.5 kg, 2.3 ± 0.6 kg and 1.6 ± 0.3 kg, respectively). The overall neonatal loss rate within 48 h postpartum was 22.2%, which increased significantly with an increase in litter size and was significantly ( P < 0.01) higher for Boer goats (34.2%) than for crossbred kids (12.7%). The results indicate that oestrous synchronisation with progestagen is efficient and the duration of the induced oestrus is shorter in Nguni does than in Boer goat does. The conception rates obtained following oestrous synchronisation and AI were, however, very low and this aspect needs more attention. Litter size reduces birth weight and survival rate of indigenous South African goat kids.

Effect of progestagen and PMSG on oestrous synchronization and fertility in Dorper ewes during the transition period

An oestrous synchronization study was conducted on 202 Dorper ewes kept under extensive veld conditions of South Africa, during the transition period from the natural breeding to the anoestrus season. Two types of intravaginal progestagen sponges, namely MAP (60 mg) ( n=102) and FGA (40 mg) ( n=100), three times of PMSG administration relative to sponge withdrawal (24 h before ( n=59), at ( n=56) or 24 h after ( n=57)) and two routes of PMSG administration (intramuscular ( n=87) or subcutaneous ( n=85)) were compared regarding synchronization efficiency (oestrous response, time to onset of oestrus and duration of oestrus) and fertility (pregnancy, lambing, and fecundity rates) following AI with 0.1 ml fresh diluted semen. There were no significant differences in terms of oestrous response, time to onset of oestrus and the duration of induced oestrus due to differences in the type of progestagen sponges or time and route of PMSG administration. The overall pregnancy, lambing, and fecundity rates were 72.3, 91.1, and 126.0%, respectively. There were no significant differences in pregnancy, lambing, and fecundity rates between ewes treated with MAP and FGA sponges (70.6, 85.3, and 120.8% versus 74.0, 97.0, and 131.1%, respectively). However, within treatment, pregnancy, lambing, and fecundity rates were significantly ( P<0.01) higher in ewes administered 300 IU PMSG 24 h prior to (78.0, 115.3, and 147.8%, respectively) or at sponge withdrawal (75.0, 94.6, and 126.2%, respectively), compared to those administered 24 h after sponge withdrawal (70.2, 73.7, and 105.0%, respectively) or those not injected with PMSG (60.0, 70.0, and 116.7%, respectively). Both MAP and FGA sponges could be used to synchronize oestrus in Dorper ewes during the transition period from breeding to anoestrus. Administration of 300 IU PMSG preferably 24 h prior to or at progestagen sponge withdrawal is essential to obtain better fertility rates at the induced oestrus and following AI. The subcutaneous administration of PMSG is preferable to intramuscular administration as it resulted in higher fertility rates and litter sizes.

13FOLLICLE TURNOVER DURING SYNCHRONIZATION TREATMENTS IN MEDITERRANEAN ITALIAN BUFFALOES (BUBALUS BUBALIS)

The utilization of synchronization protocols for ovulation and AI in the buffaloes has not gained a widespread use among breeders due to the usually low conception rates achieved. The tendency to enter into postpartum anestrus in the period of the year characterized by increasing day length further affects the manipulation of their reproductive efficiency. This study, carried out in the months of february to may, was designed to test the hypothesis that newly growing dominant follicles towards the end of synchronization protocols for ovulation, are more competent for establishing pregnancies following AI in cycling and non cycling buffaloes. Animals were checked by ultrasound (7.5MHz linear-array probe and SSD 500 Aloka monitor) for signs of ovarian activity and classified into cycling and not-cycling based on the presence of functional CLs and follicle turnover. Cycling buffalo heifers (CHE; n=30), cycling mixed parity buffaloes (CMP; n=14)and non-cycling mixed parity buffaloes (NCMP; n=17) were selected for a direct comparison between two synchronization protocols. CHE and CMP received two GnRH administrations at Day 0 and 9 and a luteolytic dose of PG2a at Day 7, followed by a single AI at 16h from last GnRH (‘Ovsynch’). NCMP group received a PRID implant for 9 days and 1,000 IU of PMSG at Day 7 followed by two AI at 72 and 96h from PRID removal (‘PRID+PMSG’). Ultrasound monitoring for CMP and CHE of animals was performed at day 0, 2, 4, 7, 9, and day of AI. In NCMP, ultrasound monitoring was continued also at 48, 72, 96, and 120h after PRID removal. Like superscripts (a,b) after % signs indicate significant difference. A follicle &amp;gt;9mm was present in 16/17 NCMP (94.1%a), 14/14 CMP (100%a), and 17/30 CHE (56.6%b; P&amp;lt;0.05) at the beginning of the synchronization protocol (day 0). Demise of the first large follicle recorded at day 0 and presence of a new large follicle at day 7 to 9 leading or not to ovulation, in the ovary ipsilateral or contralateral (‘follicle shift’) occurred in 15/17 (88.2%a), 11/14 (78.5%a), and 14/30 (46.6%b; P&amp;lt;0.05) for NCMP, CMP, and CHE respectively. Synchronized ovulations were recorded in 15/17 (88.2%a), 12/14 (85.7%a), and 26/30 (86.6%a; P&amp;gt;0.05) for NCMP, CMP, and CHE respectively. Conception rates (CR) by ultrasound examination at 25 to 30 days post-insemination were 12/17 (70.5%a), 6/14 (42.8%ab), and 11/30 (36.6%b; P&amp;lt;0.05) for NCMP, CMP, and CHE, respectively. Conceptions derived from follicle shift (either ipsilateral or contralateral) were 11/12 (91.6%a), 5/6 (83.3%a), and 7/11 (63.6%a; P&amp;gt;0.05) for NCMP, CMP, and CHE, respectively. In conclusion, both synchronization protocols in the three groups of buffaloes produced good ovulation and conception rates in the unfavourable period of the year. Most pregnancies resulted from fertilization of oocytes maturing in newly selected growing follicles toward the end of the synchronization protocol, suggesting a higher development competence compared to oocytes in large follicles already available at the beginning of the procedure, not regressing and leading to ovulation.

Interval between PMSG Priming and hCG Injection in Superovulation of the Mongolian Gerbil

To date, a practical method for inducing superovulation in the Mongolian gerbil has not been defined; therefore, in this study, we attempted to develop a flexible superovulation protocol for this species. Superovulation can be induced in the Mongolian gerbils by using PMSG with or without hCG. The injection schedule for PMSG (and hCG) has a high degree of flexibility, but the best protocol for embryo collection for reproductive biology is 20 IU PMSG followed by 20 IU hCG 54 hours later.

Mitochondrial aggregation patterns and activity in porcine oocytes and apoptosis in surrounding cumulus cells depends on the stage of pre-ovulatory maturation

In this study, we evaluated the distribution and oxidative activity of mitochondria in ex vivo pre-ovulatory porcine oocytes using the fluorescence probe MitoTracker CMTM Ros Orange. Cumulus–oocyte complexes (COCs) were classified according to cumulus morphology and time from hCG administration. The meiotic configuration of the oocytes and the degree of apoptosis in the surrounding cumulus cells were also evaluated. Estrus was synchronized in 45 crossbred Landrace gilts by feeding altrenogest for 15 days and administering 1000 IU PMSG on Day 16. The LH peak was simulated by treatment with 500 IU hCG, given 80 h after PMSG. Endoscopic oocyte recovery was carried out 2 h before or 10, 22, or 34 h after hCG administration. Altogether 454 COCs were aspirated from follicles with a diameter of more than 5 mm. Cumulus morphology in the majority of COCs recovered 2 h before and 10 h after hCG was compact (60.4 and 52.7%, respectively; P<0.05). At 22 h after hCG, COC morphology changed significantly from 10 h dramatically: 74% of COCs had an expanded cumulus ( P<0.01). At 34 h after hCG, 100% of recovered COCs had an expanded cumulus. The percentage of oocytes with a mature meiotic configuration differed among COC morphologies and increased as the interval after hCG administration increased ( P<0.05). The type of mitochondrial distribution in the oocytes ( n=336) changed from homogeneous to heterogeneous as the interval after hCG administration increased ( P<0.01) and was associated with the cumulus morphology. Representative mitochondrial distributions were found as follows: −2 h: fine homogeneous in compact and dispersed COCs; 10 h: granulated homogeneous in compact and dispersed COCs; 22 h: granulated homogeneous in expanded COCs; and 34 h: granulated heterogeneous and clustered heterogeneous in expanded COCs ( P<0.01). The oxidative activity of mitochondria measured by fluorescence intensity (Em: 570 nm) per oocyte after Mitotracker CMTM Ros Orange labeling increased in the oocyte as the post-hCG interval increased ( P<0.01) and depended on the type of mitochondrial distribution. Lowest oxidative activity of mitochondria was found in oocytes with fine homogeneous distribution (253.1±9.4 μA). The oxidative activity increased (334.4±10.3 μA) in oocytes with granulated homogeneous distribution of mitochondria, and reached highest level in oocytes with granulated heterogeneous (400.9±13.0 μA) and clustered heterogeneous distributions (492.8±13.9 μA) ( P<0.01). Mitochondrial activity in oocytes coincided with apoptosis in surrounding cumulus cells which increased in a time-dependent manner during pre-ovulatory maturation in vivo ( P<0.01). These results indicate that there is a relationship between meiotic progression, cumulus expansion and mitochondrial redistribution and their oxidative activity during final pre-ovulatory maturation in pig oocytes. It appears that increased levels of mitochondrial activities in oocytes are correlated to increased levels of apoptosis in surrounding cumulus cells, in which mitochondria may play a role.

Identification and class assignment proteins expressed during mouse oocyte development using proteomics strategy

Identification and class assignment proteins expressed during mouse oocyte development using proteomics strategy

Relationship between apoptosis and IGF-II in mouse blastocyst fertilized in vivo and in vitro

Objective: Mouse embryos fertilized in vitro occurred at high levels of apoptosis and retarded development when compared to that in vivo. It was suggested that this might be due to lower levels of production or release of the putative growth factors by IVF zygotes. Recently, a number of studies have demonstrated that growth factors added to the culture medium may play a role in regulating levels of cell death in pre-implantation embryos. However, relationship between apoptosis and growth factors in vivo and in vitro fertilized embryos has not been reported. The objectives of the present study were 1) to examine a physiological relationship between the development and apoptosis of embryos fertilized in vivo and in vitro and 2) to investigate whether IGF-II expression influences an embryonic apoptosis. Design: Apoptosis and IGF-II expression were examined in the blastocysts fertilized in vivo and in vitro. Materials and Methods: B6/CBA F1 mice were superovulated by intraperitoneal injection of 7.5 IU PMSG followed 48 h later by 7.5 IU hCG injection. Half of mice were paired overnight with males and other half mice were not paired to induce in vitro fertilization (IVF). The presence of a copulation plug indicated day 0 of pregnancy. Oocytes for IVF were collected from the oviducts 14–16 h after of injection of hCG. IVF was performed using sperm collected from 10-weeks aged males. Zygotes derived from IVF were cultured in a 10 ul m-MTF containing BSA (4 mg/ml) until day 5. Oocytes fertilized in vivo were collected by flushing oviducts on day 1 and cultured to the blastocysts stage as group of fertilized in vitro. The quantity of apoptosis of blastocyst stage embryos was investigated with TUNEL assay. The expression of IGF-II mRNA in blastocysts of both groups was evaluated using RT-PCR. Results: The embryonic development was similar in both groups (98% in vivo: 482/492 vs. 92% in vitro: 355/384), while the hatching rate of embryos fertilized in vivo (64%: 318/492) was significantly higher compared with that of embryos fertilized in vitro (47%: 180/384). Total cell number of blastocyst stage embryos derived in vivo was 72.1 ± 5.5 and that in vitro was 66.6 ± 5.1. Apoptosis of in vitro group (6.9%) appeared higher compared with that of in vivo group (3.6 %). The expression of mRNA of IGF-II of in vivo group was detected greater than that of in vitro group. Conclusion: Apoptosis of blastocyst stage embryos derived in vivo was lower and their hatching rate and IGF-II mRNA appeared higher than those in vitro. These results suggested that the embryos fertilized in vivo and in vitro have a physiological difference, in addition, apoptosis play a role in embryo development. Also, higher IGF-II expression in blastocysts fertilized in vivo is beneficial for apoptosis suppression. Further studies are necessary to elucidate a relationship between the expression of IGF-II and an embryonic apoptosis.

The GV-stage is more effective for mouse ovum bank

Objective: Oocyte cryopreservation would be essential to establish the ovum bank for ovum donation in IVF-ET program. It is considered that the developmental state of oocyte is one of important factors for it. However, investigation on which maturation stage of the oocyte would be developmentally suitable for vitrification has not been tried. Therefore, this study was performed to compare survival rates and meiotic spindles of the oocytes vitrified at different developmental stages. Design: Two groups of mouse oocytes vitrified at different developmental stages, germinal vesicle (GV)-stage and in vitro matured MII-stage, were monitored for their survival rates and meiotic spindle by Pol-Scope. Materials and Methods: Immature oocytes (n=175) were collected from 4-weeks old mice 44–46 hours after 5 IU PMSG. The cumulus cells were removed with hyaluronidase.All oocytes at the GV-stage were randomly divided into two groups. One group of oocytes (n=101) was vitrified at GV-stage, whereas the other group of oocytes (n=62) was in vitro matured up to MII-stage and then vitrified. All the oocytes were vitrified using two-step method. pre-equilibration with 1.5 M ethylene glycol and 20% hFF in PBS for 1 min. vitrification with 5.5M ethylene glycol, 0.1 M sucrose and 20% hFF in PBS for 20 sec. Those oocytes were mounted on an EM grid. Thawing was performed at 0.5 M sucrose for 5 min. And then the oocytes in different treatment groups were examined for the survival rate and the meiotic spindles using Pol-Scope. The MII-stage oocytes were incubated in culture medium for at least 1 hour and observed the meiotic spindle with Pol-Scope. Results: The survival rate of oocytes vitrified at GV-stage was 84% (85/101). Of them, 82% (70/85) reached the MII stage, being similar to the maturation rate (84%: 62/84) of fresh oocytes at the GV-stage. All oocytes vitrified and survived at the GV-stage had had meiotic spindles when developed to the MII-stage. In comparison, the survival rate of oocytes cultured to the MII-stage and then vitrified showed 64% (40/62) and meiotic spindles were observed in 39 out of 40 oocytes. Further comparative studies regarding the fertility rate and the developmental capability of those oocytes in two groups are in progress. Conclusion: The present study suggests that the GV-stage of the oocytes is the better developmental status for the vitrification

Comparison of pregnancy rates with two estrus synchronization protocols in Italian Mediterranean Buffalo cows

The aim in this study was to compare two estrus synchronization protocols in buffaloes. Animals were divided into two groups: Group A ( n=111) received 100 μg GnRH on Day 0, 375 μg PGF 2α on Day 7 and 100 μg GnRH on Day 9 (Ovsynch); Group B ( n=117) received an intravaginal drug release device (PRID ®) containing 1.55 g progesterone and a capsule with 10 mg estradiol benzoate for 10 days and were treated with a luteolytic dose of PGF 2α and 1000 IU PMSG at the time of PRID ® withdrawal. Animals were inseminated twice 18 and 42 h after the second injection of GnRH (Group A) and 60 and 84 h after PGF 2α and PMSG injections (Group B). Progesterone (P 4) concentrations in milk samples collected 12 and 2 days before treatments were used to determine cyclic and non-cyclic buffaloes, and milk P 4 concentrations 10 days after Artificial insemination (AI) were used as an index of a functional corpus luteum. Cows were palpated per rectum at 40 and 90 days after AI to determine pregnancies. All previously non-cyclic animals in Group B had elevated P 4 (>120 pg/ml milk whey) on Day 10 after AI. Accordingly, a greater ( P<0.01) relative percentage of animals with elevated P 4 10 days after AI were observed in Group B (93.2%) than in Group A (81.1%). However, there was no difference in overall pregnancy rates between the two estrus synchronization protocols (Group A, 36.0%; Group B 28.2%). When only animals with elevated P 4 on Day 10 after AI were considered, pregnancy rate was higher ( P<0.05) for animals in Group A (44.4%) than Group B (30.3%). The findings indicated that treatment with PRID ® can induce ovulation in non-cyclic buffalo cows. However, synchronization of estrus with Ovsynch resulted in a higher pregnancy rate compared with synchronization with PRID ®, particularly in cyclic buffalo.

In vitro fertilisation of in vivo matured porcine oocytes obtained from prepuberal gilts at different time intervals after hCG injection.

The goal of the present study was to find out the best interval after hCG injection in PMSG primed prepuberal gilts for retrieval of in vivo matured oocytes for in vitro fertilisation (IVF). Altogether 66 gilts were superovulated with 1500 IU PMSG and 500 IU hCG 72 h later. Ovum pick up was performed endoscopically 24, 28, 32 or 36 h after hCG and a total of 869 cumulus-oocyte-complexes (COCs) were aspirated from 1400 follicles. COCs were tested for quality, and an aliquot was immediately fixed and stained to determine meiotic configuration. The remaining COCs were fertilised in vitro using frozen-thawed epididymal semen. Quality and developmental stage of embryos were tested after IVF, and the number of nuclei was counted. At 24 to 32 h after hCG only few oocytes have entered the second meiotic cycle (18 to 25% vs. 58% at 36 h, p < 0.05). The overall cleavage rate was significantly influenced by insufficient maturation rate at the early collection times (14% at 24 h vs. 49% at 36 h). Additionally, when oocytes were collected 24 to 32 h vs. 36 h the cleavage rate based on mature oocytes was lower (26 vs. 62%, p < 0.05). Once embryonic development has been initiated, the further in vitro development to blastocyst stages did not differ between groups. However, the number of cells was lower at collection times 24 to 32 h as compared to 36 h after hCG (12 to 15 cells vs. 22 cells, p < 0.05). The results indicate that the time of COC collection affects the in vitro developmental competence up to the blastocyst stage and should not be performed earlier than 36 h after hCG treatment.

Embryo production and progesterone profiles in ewes superovulated with different hormonal treatments

The superovulatory response and embryo yield following hormonal treatments of Merino ewes during late spring and their estrous cycle were evaluated. Ewes ( n=17) were treated with progestagen-impregnated sponges and assigned to Group I (800 IU PMSG plus 11.5 mg FSH-p); Group II (1200 IU PMSG); Group III (1600 IU PMSG). Ewes were naturally mated and followed by laparotomy 6 days later. After laparotomy, ewes were injected with a prostaglandin analogue (PGF) and serum samples were obtained prior to surgery and then for 25 days to measure progesterone (P4) by radioimmunoassay. There were no differences among groups neither for estrous incidence (Group I: 83.3%; Group II: 83.3%; Group III: 100%), nor for the time interval to estrous onset (Group I: 26.4±2.4 h; Group II: 28.8±2.9 h; Group III: 24.0±3.8 h). Group I had more corpora lutea than Group II (14.2±1.2 and 6.2±0.8; P<0.05), and Group III was intermediate (11.0±3.0). There was a low incidence of persistent follicles in all treatments (Group I: 0.5±0.5; Group II: 0.6±0.4; Group III: 1.8±1.2). Number of collected ova were 9.0±2.6, 3.8±0.6 and 6.5±0.9 for Groups I, II and III, respectively. Significant differences in number of ova were detected between Groups I and II. Unfertilized ova did not differ among groups (Group I: 3.5±1.0; Group II: 2.8±0.8; Group III: 5.2±1.4; P>0.05). Embryos and high viability embryos were higher ( P<0.05) in Group I (5.2±1.9 and 4.8±2.0) than in Group II (1.0±0.5 and 1.0±0.5) or Group III (1.2±0.6 and 1.0±0.5). Total plasma progesterone (P4) and P4 per corpus luteum before PGF administration did not vary ( P>0.05) among groups (Group I: 71.0±14.7 and 4.9±0.7 nmol/l; Group II: 50.6±13.3 and 7.9±1.6 nmol/l; Group III: 90.4±42.6 and 6.8±1.8 nmol/l). There was a significant and positive correlation between P4 before PGF administration and number of corpora lutea ( r=0.76). No significant differences were detected among groups for: interval PGF to P4 <3.18 nmol/l (Group I: 2.7±0.3 days; Group II: 1.8±0.6 days; Group III: 2.2±0.5 days), cycle length (Group I: 18.3±1.4 days; Group II: 17.9±0.5 days; Group III: 16.8±0.9 days), duration of P4 levels <3.18 nmol/l (Group I: 11.3±1.9 days; Group II: 7.1±1.0 days; Group III: 7.2±2.4 days), duration of P4 levels ≥3.18 nmol/l (Group I: 7.0±1.3 days; Group II: 10.8±0.8 days; Group III: 9.5±1.7 days) and peak of P4 (Group I: 7.4±0.4 nmol/l; Group II: 10.8±1.6 nmol/l; Group III: 9.2±1.9 nmol/l). It was concluded that PMSG–FSH-p treatment was more efficient than PMSG alone for superovulation and embryo production in ewes while P4 profiles were similar among groups.

Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine.

The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.

Influences of gonadotropin stimulation on in vitro maturation and embryogenesis of denuded mouse oocytes

Influences of gonadotropin stimulation on in vitro maturation and embryogenesis of denuded mouse oocytes