Comparison between effects of 3-isobutyl-1-methylxanthine and FSH on gap junctional communication, LH-receptor expression, and meiotic maturation of cumulus-oocyte complexes in pigs.

We investigated cAMP content, gap junctional communications (GJCs) status, and LH-receptor (LH-R) expression in porcine cumulus-oocyte complexes (COCs) during in vitro maturation treated with the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) or with FSH. COCs were cultured for 20 hr (1st culture) in M199 containing 10% FBS (basic medium, BM group) or BM supplemented with FSH (FSH group) or IBMX (IBMX group). Each COC was then transferred into BM containing both FSH and LH and cultured for an additional 24 hr (2nd culture). The proportions of metaphase-II (M-II) oocytes at the end of the 2nd culture did not differ between the FSH (75.7%) and IBMX (68.2%) groups, whereas only 10.1% of oocytes in the BM group reached the M-II stage. During the 1st culture, the cAMP content of COCs and oocytes became significantly higher in the FSH and IBMX groups than in the BM group; the FSH group had a far greater increment than did the IBMX group. GJCs in the FSH and BM groups gradually closed with increasing duration of the 1st culture, whereas a significantly higher proportion of COCs in the IBMX group still had open GJCs than in the other two groups. Furthermore, LH-R mRNA expression significantly increased in both the FSH and IBMX groups compared with the BM group. These results suggest that inhibition of PDEs in porcine COCs make the oocyte ready for release from meiotic arrest, and that maintenance of a moderate cAMP content may prolong GJCs and stimulate LH-R expression.

Development to the blastocyst stage of porcine somatic cell nuclear transfer embryos reconstructed by the fusion of cumulus cells and cytoplasts prepared by gradient centrifugation.

The present study was designated to examine the possibility of producing somatic cell nuclear transfer (SCNT) embryos in pigs using oocyte cytoplasm fragments (OCFs), prepared by centrifugations, as recipient cytoplasts. In Experiment 1, in vitro matured oocytes were centrifuged at 13,000 x g for 3, 6, and 9 min to stratify the cytoplasm, and then the oocytes were freed from zona pellucida and recentrifuged at 5,000 x g for 4 sec in Percoll gradient solution to produce OCFs as the source of recipient cytoplasts. It was found that a long duration of the first centrifugation tends to produce large-sized OCFs after the second centrifugation. In Experiment 2, two or three cytoplasts without chromosomes were aggregated, and then they were fused with a cumulus cell to produce SCNT embryos. The results showed that 66.4 +/- 9.4% of the reconstructed embryos underwent premature chromosome condensation at 1 h after activation, and 85.2 +/- 7.1% and 61.6 +/- 7.0% of them had pseudopronuclei at 10 and 24 h after activation, respectively. In Experiment 3, when SCNT embryos reconstructed by the fusion of three cytoplasts and one cumulus cell, a significantly higher (p < 0.05) rate of reconstructed embryos developed to the blastocyst stage (10.6 +/- 1.8%) than that of reconstructed with two cytoplasts and one cumulus cell (5.2 +/- 1.5%). These results indicate that cytoplasts obtained by two centrifugations can support the remodeling of a transferred somatic nucleus, resulting in the development of the reconstructed porcine embryos to the blastocyst stage.

Production of inhibin A and inhibin B in boars: changes in testicular and circulating levels of dimeric inhibins and characterization of inhibin forms during testis growth.

We investigated the production of inhibin in boars from the infantile to pubertal periods by: (1) measurement of testicular and circulating levels of inhibin, (2) characterization of inhibin forms and (3) localization of inhibin alpha subunits in the testis. Total inhibin levels in the testis increased until 8 weeks of age but then declined to much lower values at 15 weeks. Testicular inhibin A and inhibin B were high until 8 weeks. Circulating levels of total inhibin and inhibin A were also high until 8 weeks, then declined from 10 weeks; inhibin B was not detected, because of low sensitivity of the inhibin B assay. Analyses of inhibin A and inhibin B levels in the eluted fractions obtained from testes after immunoaffinity chromatography and SDS-PAGE showed the presence of a peak of approximately 45 kDa until 10 weeks of age. As the boars aged, the levels of inhibin A and inhibin B increased in the molecular weight region of 29-31 kDa. The fractions corresponding to 29 and 30 kDa suppressed FSH release from rat pituitary cells, but the 45 kDa fraction had no FSH-suppressing activity. Total amounts of inhibin A isolated from the SDS gels were similar to those of inhibin B until 10 weeks of age, but were three times higher than those of inhibin B between 15 and 25 weeks. Further fractionation by reverse phase high-performance liquid chromatography revealed that the 29-31 kDa immunoreactive material was composed of mature forms of inhibin A and inhibin B, in addition to a 26 kDa alpha monomer. Immunohistochemistry indicated that positive immunostaining for the alpha subunits was observed in Sertoli cells from the infantile to pubertal periods. Elongated spermatids also showed positive signals at age 25 weeks. These results clearly indicated that: (1) the boar testis has the ability to produce inhibin A and inhibin B during the infantile period but inhibin A is the predominant form towards puberty and (2) the molecular weight forms of inhibin and the sites of production of inhibin change with testicular development.

Isolation and identification of F-spondin in the boar testis and its production during testis growth.

F-spondin/vascular smooth muscle cell growth-promoting factor (VSGP), purified from the follicular fluid of adult bovine ovaries, has been identified as a promoter of neuronal differentiation and vascular smooth muscle growth. The objectives of the present study were (1) to clarify whether F-spondin is also produced in the testis, which is ontogenically equivalent to the ovary, and (2) to examine whether production of this protein changes with testicular growth. To isolate F-spondin from the testis, testicular homogenates obtained from 8-week-old boars were sequentially subjected to heparin-Sepharose chromatography, diethylaminoethyl (DEAE)-Sepharose chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). The isolated protein had a molecular mass of approximately 110 kDa and was cross-reactive with anti-F-spondin antibody by Western blotting. The purified protein was further characterized by amino acid sequence analysis of its internal peptide. The sequence obtained was GEQCNIVPDN VD, and a homology search indicated that the purified protein is a homologue of rat, human, and bovine F-spondin. By fractionation of the same amounts of testis tissue obtained from 1-, 8-, 16-, and 40-week-old boars, we analyzed age-related production of F-spondin in the testis. Western blotting of the fractions obtained from RP-HPLC revealed the presence of a band at approximately 110 kDa, corresponding to F-spondin, in the testes obtained from boars between 1 and 16 weeks old, but this band was not detected at 40 weeks. These results clearly indicate that (1) the porcine testis produces F-spondin and that (2) production of this protein is evident in the immature porcine testis, but not the adult testis.

Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions.

BACKGROUND: Recent work has shown that glucose may induce cell injury through the action of free radicals generated by autooxidation or through hypoxanthine phosphoribosyltransferase inhibition. The effect of glucose during early in vitro culture (IVC) period of porcine embryos on their developmental competence, contents of reactive oxygen species (ROS) and glutathione (GSH), and the quality of the blastocysts yielded was examined. METHODS: In vitro matured and fertilized porcine oocytes were cultured for the first 2 days (Day 0 = day of fertilization) of IVC in NCSU-37 added with 1.5 to 20 mM glucose (Gluc-1.5 to -20 groups) or pyruvate and lactate (Pyr-Lac group). The embryos in all groups were cultured subsequently until Day 6 in NCSU-37 with 5.5 mM added glucose. The ROS and GSH level were measured at Day 1 and 2. DNA-fragmented nuclei and the total cell numbers in blastocyst were evaluated by TUNEL-staining at Day 6. RESULTS: Under 5% oxygen the blastocyst rates and total cell numbers in the blastocysts in all glucose groups were significantly lower than that in the Pyr-Lac group. Similar result in blastocyst rate was found under 20% oxygen (excluding the Gluc-10 group), but total cell numbers in the blastocysts was similar among the groups. At both oxygen tensions, the H2O2 levels of Day 1 embryos in all glucose groups were significantly higher than that in the Pyr-Lac group, while only the Gluc-1.5 group of Day 2 embryos showed a significantly higher H2O2 level than that in the Pyr-Lac group. The GSH contents of either Day 1 or Day 2 embryos developed under 5% oxygen were similar among the groups. Only the content of Day 2 embryos in 1.5 mM group was significantly lower than the embryos in the Pyr-Lac group under 20% oxygen. Total cell numbers in the blastocysts (except in the Gluc-20 group) were significantly lower in the embryos cultured under 20% oxygen than 5% oxygen. Only the Gluc-20 blastocysts developed under 5% oxygen showed significantly higher DNA fragmentation rate than those of Pyr-Lac blastocysts. CONCLUSION: These results show that a decrease in developmental ability of embryos cultured by use of glucose instead of pyruvate and lactate after the ferilization may be due to the rise in ROS generation in Day 1 embryos. Moreover, results from this study suggest that the concentration of glucose in the medium that can be used by the Day 1-2 embryos is limited to 3.5 mM and exposure to higher glucose concentrations does not improve embryo development.

Changes in circulating and testicular levels of inhibin A and B during postnatal development in bulls.

We investigated testicular and circulating levels of dimeric inhibins in Holstein bulls from the infantile to postpubertal periods (5 to 50 weeks of age) and examined the relationship between the profiles of circulating dimeric inhibins and FSH. Concentrations of total inhibin and inhibin B in the testis were highest at 4 to 5 weeks of age but decreased gradually as the bulls aged. Testicular inhibin A levels showed a gradual decline to a nadir at 15 to 26 weeks of age, but by 39 weeks, they were high again. The contents of total inhibin, inhibin A, and inhibin B per testis generally increased with age. Fractionation of testicular homogenates obtained from 15-week-old bulls by a combination of immunoaffinity chromatography and SDS-PAGE confirmed the presence of two major molecular weight forms (32 and 45 kDa) of dimeric inhibins in the testes. Circulating levels of total inhibin and inhibin A showed a significant increase in bulls at around 10 to 14 weeks of age compared to the levels between 5 and 7 weeks of age but decreased thereafter. However, immunoreactivity for inhibin B was not detected in the peripheral circulation, probably because of low sensitivity of the inhibin B assays. The concentrations of plasma FSH were high at 5 weeks of age but declined to lower levels between 11 and 40 weeks, and then increased from 41 weeks onward. There was no significant correlation between the plasma levels of FSH and inhibin A or total inhibin. The results clearly indicate that the bull testis produces inhibin A and B and secretes at least inhibin A into the circulation during postnatal development. However, the profile of circulating FSH in bulls shows no reciprocal relationship with the inhibin A or total inhibin profile during the postnatal period.

Diploid porcine parthenotes produced by inhibition of first polar body extrusion during in vitro maturation of follicular oocytes.

We investigated nuclear progression and in vitro embryonic development after parthenogenetic activation of porcine oocytes exposed to cytochalasin B (CB) during in vitro maturation (IVM). Nuclear progression was similar in control oocytes and oocytes matured in the presence of 1 microg/ml CB (IVM-CB group) by 37 h IVM; at this time the proportion of oocytes that had reached or passed through the anaphase-I stage did not differ significantly between the IVM-CB and the control groups (61.3 and 69.9% respectively; P < 0.05). After IVM for 37 h, no polar body extrusion was observed in the IVM-CB group. In these oocytes, the two lumps of homologous chromosomes remained in the ooplasm after their segregation and turned into two irregular sets of condensed chromosomes. By 41 h IVM, the double sets of chromosomes had reunited in 89.5% IVM-CB oocytes and formed a single large metaphase plate, whereas 68.8% of the control oocytes had reached the metaphase-II stage by this time. When IVM-CB oocytes cultured for 46 h were stimulated with an electrical pulse and subsequently cultured for 8 h without CB, 39.0% of them extruded a polar body and 82.9% of them had a female pronucleus. Chromosome analysis revealed that the majority of oocytes that extruded a polar body were diploid in both the control and the IVM-CB groups. However, the incidence of polyploidy in the IVM-CB group was higher than that in the control group (P < 0.05). In vitro development of diploid parthenotes in the control and the IVM-CB groups was similar in terms of blastocyst formation rates (45.8 and 42.8% respectively), number of blastomeres (39.9 and 44.4 respectively), the percentage of dead cells (4.3 and 2.9% respectively), and the frequency of apoptotic cells (7.3 and 6.3% respectively). Tetraploid embryos had a lower blastocyst formation rate (25.5%) and number of cells (26.2); however, the proportion of apoptotic nuclei (7.0%) was similar to that in diploid parthenotes. These results suggest that the proportion of homozygous and heterozygous genes does not affect in vitro embryo development to the blastocyst stage.

Addition of glutathione or thioredoxin to culture medium reduces intracellular redox status of porcine IVM/IVF embryos, resulting in improved development to the blastocyst stage.

The present series of experiments investigated the effect of a reducing environment created by addition of reduced glutathione (GSH) or thioredoxin (TRX) to in vitro culture medium on the developmental competence of in vitro produced porcine embryos, and their intracellular redox status. Porcine cumulus-oocyte complexes were collected from ovaries matured and fertilized in vitro. The putative zygotes were then cultured for 6 days in modified NCSU-37 medium with or without (control) GSH or TRX, and their developmental competence was evaluated. In addition, the intracellular redox status of the cultured embryos was compared quantitatively using an index based on the ratio of the intracellular GSH content relative to the intracellular H(2)O(2) level. The proportion of embryos that developed to the blastocyst stage was significantly increased when 0.5 or 1.0 microM GSH (29.6% or 30.4%, P < 0.05 or 0.01, respectively) or 1.0 mg/ml TRX (30.6%, P < 0.01) was added to the medium compared to that without any supplementation (control; 20.1%). The intracellular redox status of embryos at the 8- to 12-cell stage or the blastocyst stage in the group cultured in the presence of GSH or TRX was significantly reduced in comparison with the control (P < 0.05 to 0.001). Furthermore, administration of GSH or TRX enhanced the total cell number (from 48.3 to 49.2) and lowered the proportion of apoptotic cells (from 6.2% to 7.0%) in blastocysts compared with the control (cell number 39.3; apoptosis rate 11.1%, P < 0.05). These results suggest that GSH or TRX can improve the in vitro development of porcine embryos, while maintaining an intracellular reductive status.

Successful long term culture of immature porcine sertoli cells in the reconstructed testicular cell cord.

The ultimate goal of this study was to establish an in vitro system to produce sperms. To pursue this goal, immature porcine testicular cells were cultured in stereostructural form and cultured testicular cord was investigated morphologically. At 4 weeks of age, the seminiferous tubules of the porcine testes consisted of undifferentiated germ cells (gonocytes and undifferentiated spermatogonia) and immature Sertoli cells. The interstitial tissue was largely occupied by Leydig cells. The testes were enzymatically digested, and the dispersed cells were encapsulated with alginate either immediately or after freeze-thawing. The resulting testicular cell cords were cultured for up to 10 weeks. After 2 weeks of culture, Sertoli cells, which were identified by their inhibin-positive reaction in immunohistochemistry, and Leydig cells, which were identified by their morphological characteristics, were observed in the cords. Neither undifferentiated nor differentiated types of germ cells were detected. The number of cells in the cords progressively decreased during the culture period. In order to discover the fate of the Sertoli cells, the level of inhibin in the spent media was determined. Inhibin in the media was at a detectable level after 2 days of culture. The levels increased and peaked at 2 weeks. When frozen-thawed testicular cells were applied to the culture, the peak level was maintained for over 8 weeks, in contrast to the gradual decrease of inhibin level when fresh cells were cultured. These results indicate that the culture conditions can sustain the survival of Sertoli cells. Further improvement is required for proliferation and differentiation of germ cells.

Effects of gonadotrophin treatments on meiotic and developmental competence of oocytes in porcine primordial follicles following xenografting to nude mice.

Our objective was to improve the developmental ability of oocytes in porcine primordial follicles xenografted to nude mice, by treating the host mice with gonadotrophins to accelerate follicular growth. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the kidney capsules of ovariectomized nude mice. Gonadotrophin treatments were commenced around 60 days after vaginal cornification in the mice. Ovarian grafts were obtained 2 or 3 days after treatment with equine chorionic gonadotrophin (eCG-2 and eCG-3 groups), after porcine FSH infusion for 7 or 14 days, or after infusion of porcine FSH for 14 days with a single injection of estradiol antiserum (FSH-7, FSH-14 and FSH-14EA groups, respectively). Gonadotrophin treatments accelerated follicular growth within the xenografts compared with that in control mice given no gonadotrophins, consistent with higher (P < 0.05) circulating inhibin levels in the gonadotrophin-treated mice. In contrast, circulating mouse FSH levels were significantly (P < 0.05) depressed. We recovered large numbers of full-sized oocytes with meiotic competence to the mature stage from the eCG-3, FSH-7, and FSH-14EA, unlike in the control group. Moreover, 56% of matured oocytes with the first polar body (n = 39) were fertilized in vitro in the FSH-14EA group. After in vitro fertilization and subsequent culture for 7 days, one blastocyst was obtained from each of the eCG-3, FSH-7 and, FSH-14EA groups, whereas no blastocysts appeared in the other groups. Exogenous gonadotrophins--not mouse FSH--stimulated the growing follicles that had developed from the primordial follicles in the xenografts: the effects were incomplete but improved to some extent the meiotic and developmental abilities of the oocytes.

Successful pig embryonic development in vitro outside a CO2 gas-regulated incubator: effects of pH and osmolality.

We investigated the effects of HEPES in the medium (to maintain pH) and paraffin oil covering the medium (to maintain osmolality) on the developmental ability of porcine embryos produced in vitro using tightly closed glass tubes in the absence of a CO2 gas-regulated incubator. Putative porcine zygotes obtained by in vitro fertilization (IVF) of in vitro-matured (IVM) oocytes (day of IVF=Day 0) were cultured in 5% CO2 gas-equilibrated NCSU-37 media containing pyruvate and lactate during Days 0-2, and glucose during Days 2-6, in open glass tubes in a CO2 incubator or tightly closed glass tubes without a CO2 incubator at 38.5 degrees C. The following four media were used: (1) medium covered with paraffin oil and supplemented with HEPES; (2) medium covered with paraffin oil but with no HEPES supplementation; (3) medium not covered with paraffin oil but supplemented with HEPES; (4) medium not covered with paraffin oil and with no HEPES supplementation. As a control group, zygotes were cultured in medium with neither paraffin oil coverage nor HEPES supplementation using a four-well dish in a CO2 gas-regulated incubator. After culture, the osmolality in each of the four closed conditions was maintained at approximately 285-286 mOsm, lower (P<0.05) than that in the control (291 mOsm). In the two HEPES-supplemented media groups in the closed-tube system, the pH was maintained at 7.5-7.7, and the blastocyst development rates (15.5% in non-oil covered and 18.5% in oil covered group) did not differ significantly from that of the control (20.2%), although the mean cell numbers in the blastocysts in the two closed-tube condition groups (28.2 and 33.0) were lower (P<0.05) than in the control (43.5). In contrast, the pH was higher in the two groups without HEPES supplementation (approximately 8.0) than the control (7.4; P<0.05), and the blastocyst development rates (10.9% in non-oil covered and 7.5% in oil covered group) or total cell numbers in the blastocyst (24.8 and 28.7) in the two non-HEPES groups were drastically decreased (P<0.05) compared to those in the control (20.2% and 43.5). These results suggested that maintenance of pH is important for successful in vitro porcine embryo culture under closed-air conditions, whereas the range of osmolality that suits embryo development is not limited to a small range. Furthermore, blastocyst production was possible in a glass tube without a CO2 incubator, although blastocyst quality was lower compared to those produced in an incubator.

Successful piglet production by IVF of oocytes matured in vitro using NCSU-37 supplemented with fetal bovine serum.

Recently, piglets have been obtained from in vitro-produced blastocysts by using in vitro maturation systems in which oocytes have been matured in North Carolina State University (NCSU) solution supplemented with porcine follicular fluid (PFF). However, PFF is not available commercially. To prepare PFF from the ovaries required time and effort and there is substantial variation in quality among batches. Furthermore, PFF is considered a potential source of infectious agents. We evaluated another commercially available potential protein source, fetal bovine serum (FBS), for in vitro maturation, to produce embryos and piglets. Cumulus-oocyte complexes were matured in NCSU-37 with PFF or with one of four batches of FBS. The proportions of oocytes with expanded cumulus cells were lower in all FBS batch groups (P < 0.05, 15-41%) than that in the PFF group (74%). The proportions of oocytes that matured were also lower in all FBS batch groups (P < 0.05, 26-41%) than in the PFF group (73%), irrespective of cumulus expansion. However, the proportions of oocytes that underwent germinal vesicle breakdown were almost the same in all groups (76-96%). After in vitro fertilization, the rate of sperm penetration into matured oocytes was higher in the PFF group (P < 0.05, 63%) than in one batch of FBS (22%) and removal of the compacted cumulus cells after maturation did not affect fertilization status (21%). Subsequent in vitro embryo culture of the PFF and FBS groups for 6 day resulted in similar rates of blastocyst formation (17 and 19%, respectively) and similar numbers of cells per blastocyst (43 and 46 cells, respectively). When blastocysts obtained from oocytes matured with FBS were transferred into two recipients, one became pregnant and farrowed seven piglets. Transfer of blastocysts obtained from oocytes matured with PFF into two other recipients resulted in one pregnancy and production of four piglets. These data suggested that porcine in vitro maturation in NCSU-37 supplemented with FBS reduced the maturational ability of oocytes, but once oocytes have matured, they have the same ability to develop to term after in vitro fertilization and embryo transfer as those matured with PFF.

In vitro and in vivo developmental ability of oocytes derived from porcine primordial follicles xenografted into nude mice.

We evaluated the developmental ability of oocytes in porcine primordial follicles xenografted into nude mice. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the kidney capsules of ovariectomized nude mice. Forty-nine to 89 days after grafting (mean +/- SEM, 66.9 +/- 1.9 days; n = 64), the host mice showed the presence of cornified epithelial cells in their vaginal smears for the first time. The mice were then treated with 4 IU of equine chorionic gonadotropin (eCG) 60 days after first detection of vaginal cornification. Oocytes were collected from the host mice 48 h after treatment with eCG, and then matured. The maturation rates, based on the incidence of first polar body, ranged from 25.1% to 42.5%. They were then fertilized in vitro and cultured in vitro for 6 days, or transferred into estrous-synchronized recipients and recovered after 6 days. On Day 6 of culture, 15.4% of the matured oocytes had cleaved to the 2- to 8-cell stage. However, neither the embryos cultured in vitro nor those transferred and recovered developed to advanced embryonic stages, such as morulae or blastocysts. This result suggests that the developmental ability of xenografted oocytes is insufficient, even after in vitro maturation. Further strategies, such as improvement of hormonal treatment for host mice, are required to enable oocytes in xenografted ovarian tissues to acquire the cytoplasmic maturation necessary for embryonic development.

Development to the blastocyst stage of immature pig oocytes arrested before the metaphase-II stage and fertilized in vitro.

In vitro fertilization (IVF) and embryonic development of mature and meiotically arrested porcine oocytes were compared in the present study. After in vitro maturation (IVM) of cumulus-oocyte complexes for 48 h, 75.4% of them extruded a visible polar body (PB). Most of the oocytes with a first polar body (PB+ group) were at the metaphase-II (M-II) stage (91.4%). Most of the oocytes without a visible polar body (PB- group) appeared to be arrested at the germinal vesicle (GV) (41.6%) and metaphase-I (M-I) (34.0%) stages. After IVF of oocytes (day of IVF=Day 0), there was no difference between PB+ and PB- groups in rates of sperm penetration, mono-spermy, however oocyte activation rate after penetration was greater in the PB+ than in the PB- group (P<0.05). On Day 2, there was no difference between rates of embryos cleaved at the 2-4 cell stages in PB+ and PB- groups (42.1+/-48.8% and 33.6+/-2.1%, respectively). On Day 4, the rate of PB+ embryos developing beyond the 4-cell stage was greater than that of PB- embryos (P<0.05, 31.7+/-3.9% and 14.1+/-1.5%, respectively), and PB+ embryos had more cells than the PB- embryos (P<0.05, 8.3+/-0.4 and 6.0+/-0.8 cells, respectively). On Day 6, a greater proportion of PB+ embryos developed to the blastocyst stage than did PB- embryos (P<0.05, 34.6+/-2.4% and 20.7+/-2.8%, respectively). However, when the GV oocytes of the PB- group were not included in recalculations, there was no difference in blastocyst rates between M-I arrested and M-II oocytes (35.3 and 34.6%, respectively). The number of blastomere nuclei in embryos obtained from the PB+ group (52.0+/-2.5) was greater than that from the PB- group (P<0.05, 29.1+/-2.8). The proportion of degenerated parts in the blastocysts, as determined by morphological appearance, was the same in the PB+ and PB- groups. Although the quality of PB+ embryos was enhanced as compared with that of the PB- group, the proportion of inner cell mass and trophectoderm cells in PB+ and PB- blastocysts did not differ (1:1.9 and 1:2.2, respectively). Chromosome analysis revealed that PB+ blastocysts had more diploidy (P<0.05, 69.7%) than did PB- blastocysts (44.0%), whereas PB- blastocysts had more triploid cells (P<0.05, 34.0%) than did PB+ oocytes (8.4%). These results indicate that pig oocytes arrested before the M-II stage (M-I oocytes) undergo cytoplasmic maturation during maturation culture and have the same ability to develop to blastocysts after IVF as M-II oocytes, but some of them resulted in degeneration or delayed development with poor embryo quality.

Plasma concentrations of inhibin A in cattle with follicular cysts: relationships with turnover of follicular waves and plasma levels of gonadotropins and steroid hormones.

We investigated the profiles of circulating levels of inhibin A and total inhibin in beef cows with follicular cysts in relation to the patterns of follicular development and circulating gonadotropins and steroid hormones. Turnover of follicular waves was monitored in five cows every 2 days for 70 days from 10 days after detection of estrus without ovulation. The mean interwave intervals were 19.6 +/- 1.0 days (n = 18 waves with cysts from the five cows). Circulating levels of inhibin A were approximately 170 pg/ml before emergence of follicular waves with cysts and increased (P < 0.05) concomitantly with follicle emergence. High concentrations of inhibin A (greater than 300 pg/ml) were noted for 7 days during the growth phase of cystic follicles, but inhibin A levels decreased gradually when development of the cysts reached a plateau. This profile of inhibin A was similar to those of total inhibin and estradiol, but was inversely related to the changes in plasma FSH concentrations. LH pulse frequency and mean concentrations of LH in cows with cysts were higher than those observed in the luteal phase of normal cyclic cows. These results indicate that the capacity to secrete inhibin, as well as estradiol, is maintained in cystic follicles, the growth of which is extended by LH secretion at levels greater than those seen in the normal luteal phase. Inhibin A plays an important role in the extension of interwave intervals by suppressing recruitment of a new cohort of follicles.

Fine mapping of a region of rat chromosome 12 close to the aspermia (as) locus and comparison with the human orthologous regions.

The aspermia mutation of the rat exhibits male sterility caused by arrest of spermatogenesis, which is controlled by an autosomal single recessive gene (as). The as locus has been mapped on rat chromosome 12. We recently identified a causative mutation for the aspermia phenotype of the as homozygous rats in the gene encoding Fkbp6, a member of the immunophilins FK506 binding proteins. In this paper, we report the fine mapping of the as locus by linkage analysis combined with comparative mapping using rat, mouse, and human genomic sequences and expression analysis of genes located in the as region. We constructed a fine linkage map of the region of rat chromosome 12 close to the as locus by using 13 microsatellite markers and localized the as locus to a 1.0-cM interval. Comparison of the linkage map with physical maps of rat, mouse, and human refined the as critical region in a 2.2-Mb segment of the rat physical map between the D12Nas3 and D12Nas8 genes, which includes the Fkbp6 gene. A centromeric part of this segment corresponds to the region commonly deleted in Williams syndrome, a human complex developmental disorder, on human chromosome 7q11.23. The expression analysis of 23 genes located on the 2.2-Mb segments in various mouse tissues identified genes exclusively or strongly expressed in the testis.

Partial purification of mare early pregnancy factor.

PROBLEM: Early pregnancy factor (EPF) is an immunosuppressive protein detected in the serum in early pregnancy. We have already reported the development of the rosette inhibition test for mare EPF and have detected EPF in thoroughbreds and ponies. Here, we attempted to purify equine EPF from pregnant mare serum. METHODS OF STUDY: Mare EPF was purified by ultrafiltration and ion-exchange chromatography. Purified EPF was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting and a neutralization test. EPF activity was estimated as the rosette inhibition titer (RIT) by the rosette inhibition test. RESULTS: Purified EPF bound to carboxymethyl (CM) sepharose and did not adsorb to diethylaminoethyl (DEAE) sepharose. SDS-PAGE revealed that in the final purified fraction there were many proteins. In the immunoblotting analysis, a protein band of 25.8 kDa was detected as the pregnancy-specific band. Further, antibody gained from the 20 to 30 kDa protein band of the final purified fraction neutralized the RIT activity of pregnant mare serum. CONCLUSIONS: Mare EPF was detected in the final purified fraction and had a molecular weight of 25.8 kDa. EPF in the mare is similar to that obtained from the serum of pregnant cows.