Effects of exogenous lactoferrin on characteristics and functions of bovine epididymal, ejaculated and frozen-thawed sperm.

The purpose of this study was to investigate effects of addition of lactoferrin on characteristics and functions of bovine epididymal, ejaculated, and frozen-thawed sperm. The addition of lactoferrin was significantly (p < .05) effective on increasing values of progressive motility, straightness, and linearity in caput epididymal sperm and values of motility in cauda epididymal sperm. When ejaculated sperm were incubated in capacitation medium, percentages of motile and progressively motile sperm decreased largely within the first period of 30 min, followed by only minor changes. However, the addition of lactoferrin significantly lessened the early decreases of these parameters and additionally promoted capacitation-dependent changes of chlortetracycline staining patterns (from F pattern to B pattern). In other experiments, when ejaculated sperm were exposed to oxidative stress with 100-µM H2 O2 , the addition of lactoferrin partially protected them from dysfunction of flagellar movement and loss of progressive movement. In final experiments with frozen-thawed samples incubated in the capacitation medium, the addition of lactoferrin effectively survived dying sperm and suppressed occurrence of sperm agglutination. These results may suggest biological and biotechnological potentials of lactoferrin for modulation of bovine sperm viability, motility, capacitation state, and preservation in vitro.

Evidence for existence of insulin-like factor 3 (INSL3) hormone-receptor system in the ovarian corpus luteum and extra-ovarian reproductive organs during pregnancy in goats.

Insulin-like factor 3 (INSL3), initially described as a male hormone, is expressed in female reproductive organs during the estrous cycle and pregnancy but its function has not yet been established. This study explores the function of INSL3 in pregnant Saanen goats by characterizing the expression dynamics of INSL3 and its receptor, relaxin family peptide receptor 2 (RXFP2) and by demonstrating specific INSL3 binding in reproductive organs, using molecular and immunological approaches and ligand-receptor interaction assays. We demonstrate that the corpus luteum (CL) acts as both a source and target of INSL3 in pregnant goats, while extra-ovarian reproductive organs serve as additional INSL3 targets. The expression of INSL3 and RXFP2 in the CL reached maximum levels in middle pregnancy, followed by a decrease in late pregnancy; in contrast, RXFP2 expression levels in extra-ovarian reproductive organs were higher in the mammary glands but lower in the uterus, cervix and placenta and did not significantly change during pregnancy. The functional RXFP2 enabling INSL3 to bind was identified as an ~ 85 kDa protein in both the CL and mammary glands and localized in large and small luteal cells in the CL and in tubuloalveolar and ductal epithelial cells in the mammary glands. Additionally, INSL3 also bound to multiple cell types expressing RXFP2 in the uterus, cervix and placenta in a hormone-specific and saturable manner. These results provide evidence that an active intra- and extra-ovarian INSL3 hormone-receptor system operates during pregnancy in goats.

Improvement in identification of pro-estrous mice by using a novel method of detecting vaginal mucous cells.

Accurate identification of the murine estrous cycle using vaginal exfoliative cytology is the initial and crucial step for controlled reproduction of this species. However, it is generally difficult to discriminate each stage of the cycle, and thus to select pro-estrous mice for mating. To increase the accuracy of identification of the pro-estrous stage, we re-evaluated the vaginal fold histology and modified the method of exfoliative cytology. Tissue fixation using methanol in Carnoy's solution but not paraformaldehyde, combined with Alcian blue staining but not the conventional Giemsa staining, resulted in better manifestation of mucosal cell layers in the vaginal epithelium just above the keratinized layer. This mucous layer in the fold histology was found to form specifically in the pro-estrous and late di-estrous stages, and the mucous cells exfoliated in smear samples only in the pro-estrous stage. This novel method was found, by a blinded test, to increase the rate of accurate identification of the pro-estrous stage compared to the conventional method (80% vs 50%). Consistent with this finding, the mating experiment with "pro-estrous" females selected by the novel method revealed a significantly higher success rate than that with the conventional method (78.0% vs 47.5%). Thus, our study demonstrates vaginal exfoliative mucous cells as a better potential marker to detect the "receptive" state of female mice that leads to an improved success rate of mating.

Efficacy of relaxin for cisplatin-induced testicular dysfunction and epididymal spermatotoxicity.

BACKGROUND: Cisplatin (CP) is an extremely effective anticancer agent widely used to treat various cancer types, however, the potential side effects include testicular dysfunction. This study was to investigate, using a rat model of CP-induced testicular dysfunction, the protective effects of relaxin (RLN) against oxidative stress, testicular function, histological damage, spermatogenesis, germ-cell apoptosis, and sperm output, and to explore the usefulness of RLN as a potential protective drug for use with CP in chemotherapeutic treatments. METHODS: Sprague-Dawley male rats were used, which were divided into three groups: sham control, CP, and CP + RLN. Porcine RLN (500 ng/h) or saline was infused for 5 days using an implanted osmotic mini-pump following intraperitoneal injection of CP (6 mg/kg). RLN dose was chosen based on previous studies showing that it resulted in serum relaxin levels comparable to those in rats at the middle of pregnancy. At 5 days after CP administration, samples were collected and assessment of testicular histopathology, germ-cell apoptosis, oxidative stress, lipid peroxidation, and sperm quality was performed as main measures. RESULTS: The testicular CP model showed reduced testis weight and significantly decreased spermatogenesis scores. Additionally, CP administration induced a 4.6-fold increase in the apoptotic index associated with a significant increase in oxidative stress and upregulation of pro-apoptotic Casp3 and downregulation of anti-apoptotic Bcl2 levels, resulting in a marked reduction in sperm concentration. However, RLN administration caused a significant reduction in CP-mediated damage by attenuating oxidative stress and cell apoptosis. RLN administration efficiently scavenged ROS via the activation of SOD, CAT, and GPx and upregulation of GSH to prevent lipid peroxidation and decreased apoptosis by altering Bcl2 and Casp3 expression, thereby reducing histopathological damage and restoring spermatogenesis. Furthermore, RLN ameliorated attenuated sperm motility in the cauda epididymis resulting from CP treatment. CONCLUSIONS: This study clearly indicates that RLN exerts a protective effect against CP-induced testicular damage through attenuation of oxidative stress and suppression of apoptosis. Our findings suggest RLN as a potentially efficacious drug for use with cisplatin chemotherapy in order to ameliorate CP-induced side effects and testicular injury adversely affecting spermatogenesis, sperm quality, and oxidative-stress parameters.

CONTEXTE: Le cis platine (CP) est un agent anticancéreux extrêmement efficace largement utilisé pour traiter divers types de cancer. Parmi les effets secondaires potentiels associés aux traitements par CP on compte le dysfonctionnement des testicules. Le propos de ce manuscrit est d'étudier, à l'aide d'un modèle rat de dysfonctionnement testiculaire induit par la prise de CP, l'action protectrice de la relaxine (RLN) contre les effets délétères dus au CP lesquels incluent le stress oxydant, la perte de fonction testiculaire, les dommages histologiques au testicule, l'apoptose des cellules germinales et la baisse de la qualité des spermatozoïdes. L'objectif est d'explorer l'utilité de la RLN comme médicament protecteur potentiel à utiliser avec le CP dans les traitements chimiothérapeutiques. MÉTHODES: Des rats mâles Sprague-Dawley ont été utilisés. Trois groupes : contrôle, CP, et CP + RLN ont été comparés. Après une injection intrapéritonéale de CP (6mg/kg), de la RLN porcine (500 ng/h) ou du sérum physiologique a été perfusé pendant 5 jours en utilisant une mini-pompe osmotique implantée. La dose de RLN a été choisie en fonction d'études antérieures qui avaient montré qu'elle entraînait des taux sériques de RLN comparables à ceux de rats en milieu de la gestation. Cinq jours après l'administration de la CP, des échantillons ont été prélevés afin d'évaluer l'histopathologie, l'apoptose des cellules germinales, le stress oxydant, la peroxydation des lipides et les paramètres spermatiques. RÉSULTATS: Le groupe CP a montré une réduction du poids des testicules et une diminution significative des scores de spermatogenèse. De plus, l'administration de CP a entraîné une augmentation de l'apoptose de 4,6 fois associée à une augmentation significative du stress oxydant, de la régulation à la hausse de la Caspase 3 pro-apoptotique et à la baisse de Bcl2 anti-apoptotique conduisant in fine à une réduction marquée de la concentration en spermatozoïdes. La RLN a ainsi significativement corrigée les effets négatifs du CP en atténuant le stress oxydant et l'apoptose. La RLN a permis d'éliminer efficacement les ROS via l'activation de la triade enzymatique anti-oxydante superoxyde dismutase (SOD)/catalase (CAT)/glutathion peroxydase (GPx) et via la régulation à la hausse du GSH prévenant ainsi la lipopéroxydation. La RLN a par ailleurs diminué les atteintes histopathologiques testiculaires préservant la spermatogenèse. En parallèle, la RLN a amélioré la mobilité spermatique des spermatozoïdes prélevés dans la queue de l'épididyme. CONCLUSIONS: Cette étude montre clairement que la RLN exerce un effet protecteur contre les lésions testiculaires par l'atténuation du stress oxydant et la suppression de l'apoptose induite par le CP. Nos résultats suggèrent que la RLN est un médicament potentiellement pertinent à utiliser afin de diminuer les effets secondaires induits par le CP sur la fonction testiculaire et sur les spermatozoïdes lors de la chimiothérapie cancéreuse.

Evidence for the involvement of FXR signaling in ovarian granulosa cell function.

Farnesoid X receptor (FXR) is mainly present in enterohepatic tissues and regulates cholesterol, lipid, and glucose homeostasis in coordination with target genes such as SHP and FABP6. Although FXR has been revealed to be expressed in reproductive tissues, FXR function and expression levels in the ovary remain unknown. In this study, we investigated FXR expression in mouse ovaries and its target genes in ovarian granulosa cells. In situ hybridization and immunohistochemical staining showed that FXR was mainly distributed in secondary and tertiary follicles. The agonist-induced activation of FXR in cultured granulosa cells induced the expression of SHP and FABP6, while siRNA targeting of FXR decreased CYP19a1 and HSD17b1 expression. Upon examination of the roles of SHP and FABP6 in granulosa cells, we found that SHP overexpression significantly decreased StAR, CYP11a1, and HSD3b gene expression. In addition, siRNA targeting of FABP6 decreased CYP19a1 and HSD17b1 expression, while FABP6 overexpression increased CYP19a1 expression. In conclusion, the present study demonstrates the presence of FXR signaling in the ovary and reveals that FXR signaling may have a role in function of granulosa cells.

Functional insulin-like factor 3 (INSL3) hormone-receptor system in the testes and spermatozoa of domestic ruminants and its potential as a predictor of sire fertility.

Insulin-like factor 3 (INSL3) is essential for fetal testis descent, and has been implicated in the testicular and sperm functions in adult males; however, similar functions in domestic ruminants remain largely unknown. This study investigated the functional INSL3 hormone-receptor system in adult ruminant testes and spermatozoa, and explored its potential to diagnose the fertility of sires. Testes and spermatozoa were obtained from fertile bulls, rams and he-goats, whereas subfertile testes and spermatozoa were obtained only from bulls. As expected, INSL3 was visualized in Leydig cells, while we clearly demonstrated that the functional receptor, relaxin family peptide receptor 2 (RXFP2), enabling INSL3 to bind was identified in testicular germ cells and in the sperm equatorial segment of bulls, rams and he-goats. In comparison to fertile bulls, the percentage of INSL3- and RXFP2-expressing cells and their expression levels per cell were significantly reduced in the testes of subfertile bulls. In addition, the population of INSL3-binding spermatozoa was also significantly reduced in the semen of subfertile bulls. These results provide evidence for a functional INSL3 hormone-receptor system operating in ruminant testes and spermatozoa, and its potential to predict subfertility in sires.

Expression of insulin-like factor 3 hormone-receptor system in the reproductive organs of male goats.

Relaxin-like factor (RLF), generally known as insulin-like factor 3 (INSL3), is essential for testis descent during fetal development. However, its role in adult males is not fully understood. We investigate the function of INSL3 in male Saanen goats by identifying cell types expressing its receptor, relaxin/insulin-like family peptide receptor (RXFP)2 and by characterizing the developmental expression pattern of INSL3 and RXFP2 and the binding of INSL3 to target cells in the male reproductive system. A highly specific RXFP2 antibody that co-localizes with an anti-FLAG antibody in HEK-293 cells recognizes RXFP2-transcript-expressing cells in the testis. INSL3 and RXFP2 mRNA expression is upregulated in the testis, starting from puberty. INSL3 mRNA and protein expression has been detected in Leydig cells, whereas RXFP2 mRNA and protein localize to Leydig cells, to meiotic and post-meiotic germ cells and to the epithelium and smooth muscle of the cauda epididymis and vas deferens. INSL3 binds to all of these tissues and cell types, with the exception of Leydig cells, in a hormone-specific and saturable manner. These results provide evidence for a functional intra- and extra-testicular INSL3 ligand-receptor system in adult male goats.

The insulin-like factor 3 (INSL3)-receptor (RXFP2) network functions as a germ cell survival/anti-apoptotic factor in boar testes.

Relaxin-like factor, commonly known as insulin-like factor (INSL3), is essential for testis descent during fetal development; however, its function in the adult testis is still being elucidated. The study aimed to identify a relaxin family peptide receptor 2 (RXFP2)-specific antibody suitable for immunological approaches, analyze which testicular germ cell types express RXFP2, and clarify its expression dynamics in the boar testis. In addition, the function of INSL3-RXFP2 signaling on the germ cells was explored by neutralizing INSL3 using long-term active immunization. Samples were collected from Duroc boars, and a commercially available RXFP2-specific antibody directed against the human RXFP2 endodomain was identified by characterizing its specificity in HEK-293 cells expressing mouse RXFP2, and by demonstrating the suitability for analyzing RXFP2 expression in porcine tissues. RXFP2 mRNA and protein were both localized mainly in meiotic and post-meiotic germ cells, but not in Leydig cells. Functional RXFP2, which enables INSL3 to bind, was detected as an ∼85-kDa band, which increased in intensity from the pubertal stage onward. Interestingly, INSL3 immunization significantly reduced testis weight and induced a 4-fold increase in the frequency of apoptotic germ cells, which was associated with the up-regulation of pro-apoptotic caspase-3 (CASP3) and BAX, and the down-regulation of anti-apoptotic XIAP and BCL2, and a substantial reduction in sperm concentration. These results revealed that RXFP2 was expressed in boar meiotic and post-meiotic germ cells, where INSL3 neutralization led to increased germ cell apoptosis and reduced sperm output, suggesting that INSL3 acts as a survival/anti-apoptotic factor in maintaining sperm production.

Dynamics of insulin-like factor 3 and its receptor expression in boar testes.

Relaxin-like factor (RLF), now mainly known as insulin-like factor 3 (INSL3), is essential for testis descent during fetal development; however, its function in the adult testis is still being elucidated. As a major step toward understanding the as-yet-unknown function of INSL3 in boars, this study aimed to develop a time-resolved fluoroimmunoassay for boar INSL3, characterize the dynamics of INSL3 expression during development, and demonstrate the expression of the INSL3 hormone-receptor system in the testis. All samples were collected from Duroc boars. The sensitivity of the assay system established was 8.2 pg/well (164 pg/ml), and no cross-reactivity with other hormones, such as porcine relaxin, was observed. Circulating INSL3 was shown to increase progressively during development. INSL3 secreted from the Leydig cells was released not only into the blood circulation but also into the interstitial and seminiferous compartments in sufficient concentrations. A testicular fractionation study revealed that its receptor RXFP2 transcripts were expressed mainly in testicular germ cells. In addition, INSL3 bound to the germ cell membranes in a hormone-specific and saturable manner. These results reveal that INSL3 secreted into the interstitial compartment from the Leydig cells is transported into the seminiferous compartments, where its receptor RXFP2 is expressed mainly in the germ cells to which INSL3 binds, suggesting that INSL3 functions as a paracrine factor on seminiferous germ cells.

Incorporation of phosphatase inhibitor in culture prompts growth initiation of isolated non-growing oocytes.

In vitro folliculogenesis of primordial and early preantral follicles is necessary for increment of reproductive efficiency in domestic animals, humans and endangered species. Recent study in phosphatase and tensin homolog (Pten) -knockout mice has revealed that this phosphatase acts as an inhibitory factor in follicle activation of primordial pool with the resultant inhibition of oocyte growth. To test in vitro effect of a phosphatase inhibitor on growth initiation of isolated non-growing oocytes in neonatal ovaries, we applied a specific inhibitor (bpV (HOpic)) for PTEN in culturing system. Non-growing oocytes isolated from the ovaries of newborn BDF1 (C57BL/6 × DBA/2) pups were divided to four culture groups. Five days after culture, the oocytes in 14 µmol/l bpV only, 14 µmol/l bpV plus 100 ng/ml Kit Ligand (KL), and 100 ng/ml KL groups showed significantly (P<0.05) growth (19.3 ± 0.55, 25.8 ± 0.53 and 21.6 ± 0.29 µm, respectively) compared with that of the control (no additive) (16.9 ± 0.53 µm). In addition, western blotting in those groups showed enhanced expression of phosphorylated Akt. In conclusion, we clearly demonstrate that isolated non-growing oocytes develop in phosphatase inhibitor, especially to PTEN, incorporated culturing system, and show first as we know that oocytes with zona Pellucidae can be obtained in vitro from isolated non-growing oocytes.

The active form of goat insulin-like peptide 3 (INSL3) is a single-chain structure comprising three domains B-C-A, constitutively expressed and secreted by testicular Leydig cells.

Relaxin-like factor (RLF), also called insulin-like peptide 3 (INSL3), is a member of the insulin/relaxin gene family and is produced by testicular Leydig cells. While the understanding of its effects is growing, very little is known about the structural and functional properties of native INSL3. Here, we demonstrate that native INSL3 isolated from goat testes is a single-chain structure with full biological activity, and is constitutively expressed and secreted by Leydig cells. Using a series of chromatography steps, native INSL3 was highly purified as a single 12-kDa peak as revealed by SDS-PAGE. MS/MS analysis provided 81% sequence coverage and revealed a distinct single-chain structure consisting of the B-, C-, and A-domains deduced previously from the INSL3 cDNA sequence. Moreover, the N-terminal peptide was six amino acid residues longer than predicted. Native INSL3 exhibited full bioactivity in HEK-293 cells expressing the receptor for INSL3. Immunoelectron microscopy and Western blot analysis revealed that INSL3 was secreted by Leydig cells through the constitutive pathway into blood and body fluids. We conclude, therefore, that goat INSL3 is constitutively secreted from Leydig cells as a B-C-A single-chain structure with full biological activity.

The active form of goat insulin-like peptide 3 (INSL3) is a single-chain structure comprising three domains B-C-A, constitutively expressed and secreted by testicular Leydig cells.

Abstract Relaxin-like factor (RLF), also called insulin-like peptide 3 (INSL3), is a member of the insulin/relaxin gene family and is produced by testicular Leydig cells. While the understanding of its effects is accumulating, very little is known about the structural and functional properties of native INSL3. Here, we demonstrate that native INSL3 isolated from goat testes is a single-chain structure with full biological activity, and is constitutively expressed and secreted by Leydig cells. Using a series of chromatography steps, native INSL3 was highly purified as a single 12-kDa peak as revealed by SDS-PAGE. MS/MS analysis provided 72% sequence coverage and revealed a distinct single-chain structure consisting of the B-, C-, and A-domains deduced previously from the INSL3 cDNA sequence. Moreover, the N-terminal peptide was 6 amino acid residues longer than predicted. Native INSL3 exhibited full bioactivity in HEK-293 cells expressing the receptor for INSL3. Immunoelectron microscopy and Western blot analysis revealed that INSL3 was secreted by Leydig cells through the constitutive pathway into blood and body fluids. We conclude, therefore, that goat INSL3 is constitutively secreted from Leydig cells as a B-C-A single-chain structure with full biological activity.

Expression and localization of RLF/ INSL3 receptor RXFP2 in boar testes.

This study investigated the possibility of the presence of specific receptor for relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) in boar testes. While RLF/INSL3 was produced by Leydig cells in the boar testis, its own receptor RXFP2 was expressed mainly in meiotic and post-meiotic germ cells, but not in Leydig cells, suggesting the existence of RLF/INSL3-RXFP2 signaling in germ cells of boars.

Partial cDNA sequence of a relaxin-like factor (RLF) receptor, LGR8 and possible existence of the RLF ligand-receptor system in goat testes.

Relaxin-like factor (RLF), also known as insulin-like factor 3 (INSL3), is produced by testicular Leydig cells, but its specific receptor LGR8 (leucine-rich repeat family of G-protein-coupled receptor 8) has not been identified in goats. This study aimed to identify complementary DNA (cDNA) sequences of goat LGR8, and characterize the expression of both RLF and LGR8 in goat testes by RT-PCR and immunohistochemistry. Testes were collected from immature (3-month-old) and mature (24-month-old) Saanen goats, and partial cDNA sequences of the goat homologue of human LGR8 were identified. The sequence encoded a reduced peptide sequence of 167 amino acids, which corresponded to transmembrane regions 2 through 5, followed by the beginning of intracellular loop 3 of human LGR8. Expression of both LGR8 and RLF genes was drastically increased in mature testes compared with immature ones. Although RLF protein was restricted to Leydig cells, LGR8 protein was detected in both Leydig cells and seminiferous epithelial cells (possibly germ cells and Sertoli cells). These results reveal a possible existence of the RLF-LGR8 ligand-receptor system within the goat testis, suggesting that RLF may play a role in testicular function through LGR8 on Leydig cells and seminiferous epithelial cells in an autocrine and/or paracrine manner.

Evidence for expression of relaxin hormone-receptor system in the boar testis.

Although the physiological role of relaxin (RLN) in males remains largely unknown, there is limited evidence that the testis might be a candidate source and target of RLN in boars, as RLN transcripts are detected in the boar testis and it contains RLN-binding sites. To determine whether the boar testis acts as a source and target tissue of RLN, we characterised the expression pattern and cellular localisation of both RLN and its own receptor LGR7 (RXFP1) in boar testes during postnatal development by molecular and immunological approaches. Testes were collected from Duroc boars, and partial cDNA sequences of the boar homologue of human RXFP1 were identified. RLN expression increased through puberty onwards, while RXFP1 expression changed little during development. RLN mRNA and protein expression were restricted to the Leydig cells, whereas both Leydig cells and seminiferous epithelial cells expressed RXFP1 mRNA and protein. Interestingly, RLN was expressed in the testis as an 18 kDa form (the expected size of proRLN), but not as the 6 kDa mature form, during development because of a lack of the enzyme required for proRLN processing. In contrast, RXFP1 was detected at all stages as specific bands of 75 and 91-95 kDa (likely non-glycosylated and glycosylated RXFP1 respectively). Thus, we provide evidence for expression of RLN-RXFP1 ligand-receptor system in the boar testis, suggesting that the testis act as a source and possible target tissue of RLN.

Identification of boar testis as a source and target tissue of relaxin.

The expression and cellular localization of relaxin and its own receptor, LGR7/RXFP1, were demonstrated in the boar testis, where relaxin was produced by the Leydig cells as 18-kDa pro-relaxin and LGR7/RXFP1 was detected in both Leydig cells and seminiferous epithelial cells, suggesting that a functional relaxin-LGR7/RXFP1 hormone-receptor network operates within the boar testis.

Effect of cytochalasins B and D on the developmental competence of somatic cell nuclear transfer embryos in miniature pigs.

In many animals, cytochalasins have generally been used as cytoskeletal inhibitors for the diploid complement retention of somatic cell nuclear transfer (SCNT) embryos. However, limited information is available on the effects of cytochalasins on the in vitro development of SCNT embryos. Hence, we compared the effects of cytochalasin B (CB) and cytochalasin D (CD) on pseudo-polar body (pPB) extrusion, cortical actin filament (F-actin) distribution in porcine parthenogenetic oocytes and in vitro development of SCNT embryos that were reconstructed using foetal fibroblasts in the G0/G1 phase derived from miniature pigs. CB (7.5 microg/ml) and CD (2.5 microg/ml) treatments effectively inhibited pPB extrusion in SCNT embryos. CB (2.5 microg/ml) treatment could not inhibit pPB extrusion and insufficiently destabilized F-actin immediately following artificial activation. In parthenogenetic oocytes treated with 2.5 microg/ml CD, normal reorganization and uniform distribution of cortical F-actin at the cytoplasmic membrane were observed at 8 h after artificial activation; this finding was similar to that of control oocytes. In contrast, parthenogenetic oocytes treated with 7.5 microg/ml CB showed non-uniform distribution of F-actin at 8 h after artificial activation. On day 5 after in vitro cultivation, the blastocyst formation rate of SCNT embryos treated with 2.5 microg/ml CD was significantly higher than that of SCNT embryos treated with 2.5 and 7.5 microg/ml CB (p < 0.05). Hence, the present findings suggest that CD is more effective than CB as the cytoskeletal inhibitor for the production of SCNT embryos in miniature pigs.

Production of viable cloned miniature pig embryos using oocytes derived from domestic pig ovaries.

For production of viable somatic cell nuclear transferred (SCNT) miniature pig embryos, in vitro condition for controlling the quality of recipient oocytes derived from domestic pig ovaries should be evaluated. In the present study, to get information on optimal in vitro maturation (IVM) condition of oocytes, we investigated the effect of IVM duration of recipient oocytes on subsequent development of SCNT miniature pig embryos, the maturation-promoting factor (MPF) activity in recipient oocytes before and after SCNT, and the occurrence of premature chromosome condensation (PCC) and spindle morphologies of donor nuclei following SCNT. The optimal window of the IVM period in terms of in vitro developmental ability of SCNT embryos was determined to be 36-40 h after the start of IVM. The use of recipient oocytes matured for 36 and 40 h resulted in a high level of MPF activity before and after SCNT, and increased the occurrence of PCC in transferred nuclei compared to the use of oocytes matured for 44 and 52 h. The proportion of abnormal spindle-like structures increased as the IVM period was prolonged. In addition, SCNT embryos constructed from recipient cytoplasts obtained after 40 h of maturation by using fetal fibroblasts of miniature pigs were transferred to surrogate miniature pigs, and developed to full term. These results suggest that recipient oocytes matured for 36 h and 40 h effectively induce PCC with a normal cytoskeletal structure because of a high level of MPF activity; furthermore, the 40-h IVM period improves in vitro development of SCNT embryos to the blastocyst stage, resulting in the production of viable cloned miniature pigs.

Gene injections of vascular endothelial growth factor and growth differentiation factor-9 stimulate ovarian follicular development in immature female rats.

OBJECTIVE: To examine the effect of vascular endothelial growth factor (VEGF) and growth differentiation factor-9 (GDF-9) on follicular development of the ovaries in immature female rats. DESIGN: Superovulation and gene injection. SETTING: Animal reproduction laboratory in Tohoku University, Sendai, Japan. ANIMAL(S): Wister-Imamichi female rats. INTERVENTION(S): The ovulated oocytes from rats with injected VEGF and GDF-9 gene fragments were counted, and the ovaries removed from those rats were used in the histologic observation. MAIN OUTCOME MEASURE(S): Follicular dynamics and angiogenesis after VEGF and GDF-9 gene fragments injection. RESULT(S): A single injection of the VEGF gene led to the production of a large number of oocytes (approximately 110 oocytes) from an individual animal that was injected with the gene at 21 days after birth, and after mating most of the oocytes were fertilized. Direct ovarian injection of GDF-9 stimulated the development of medium-sized antral follicles. The number of ovulated oocytes after injection of the VEGF plus GDF-9 gene fragments was the same as with a single injection of the VEGF gene. CONCLUSION(S): A single injection of the VEGF or GDF-9 gene stimulated follicular development, and injection of both genes did increase the number of ovulated oocytes from individual animals. An exogenous gene fragments injection promoted the maximum potential of ovarian function in immature female rats.

Induction of estrus in pubertal miniature gilts.

Genetic engineering of miniature pigs has facilitated the development of numerous biomedical applications, such as xenotransplantation and animal models for human diseases. Manipulation of the estrus is one of the essential techniques for the generation of transgenic offspring. The purpose of the present study was to establish a useful method for induction of the estrus in miniature gilts. A total of 38 pubertal miniature gilts derived from 4 different strains were treated with exogenous gonadotropins. Estrus and ovulatory response were examined after treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as 200 IU PMSG and 100 IU hCG, 300 IU PMSG and 150 IU hCG, or 1,500 IU PMSG only, followed by 100, 150 or 750 IU hCG 72 h later, respectively. The optimal protocol was determined to be the combination treatment of 200 IU PMSG and 100 IU hCG followed by 100 IU hCG. The administration of 200 IU PMSG and 100 IU hCG was effective in inducing estrus regardless of the strain, although there was a strain difference in the ovulatory response. These results indicate that treatment with a low-dose combination of PMSG and hCG provides one of the simplest methods for induction of estrus and ovulation in pubertal miniature pigs.