This study investigated two hypotheses: 1) that consistent between-boar variation in frozen semen quality exists and is genetically determined, and 2) molecular markers linked to genes controlling semen freezability can be identified using amplified restriction fragment length polymorphism (AFLP) technology. Five ejaculates were collected from each of 129 boars. Semen was diluted into a commercial freezing buffer (700 mOsm/kg, 3% glycerol) and five straws (0.5 ml) per ejaculate were cryopreserved (to −5°C at 6°C/min, then −5°C to −80°C at 40°C/min). Semen was assessed for percentage of motile cells, motility characteristics (computer-aided semen analysis; CASA), plasma membrane integrity (SYBR-14 positive), and acrosome integrity (positive for fluorescein-labeled peanut agglutinin; PNA). Consistent between-boar variability was detected for postthaw sperm motility (P < 0.01), membrane integrity (P < 0.01), acrosome integrity (P < 0.01), and all CASA characteristics (P < 0.05). There was no significant difference between ejaculates (P > 0.05) or straws (P > 0.05) for any viability assessment. Multivariate pattern analysis of the viability data set highlighted three groups of boars producing spermatozoa with poor, average, and good postthaw recovery (42, 63, and 24 boars, respectively). DNA from Large White boars (n = 22) previously classified as good and poor freezers was screened for AFLP markers. Twenty-eight polymerase chain reaction primer combinations generated 2182 restriction fragment bands, of which 421 were polymorphic. Sixteen candidate genetic markers (P < 0.005) were identified by comparing the AFLP profile with semen freezability using logistic regression analysis. These findings support the hypothesis that there is a genetic basis for variation in postthaw semen quality between individuals, and that AFLP technology may be able to identify molecular markers linked to genes influencing this variation.

The factors controlling normal placental development are poorly understood. We have previously reported the presence of ovine placental growth hormone (oPGH) and growth hormone receptors in ovine placenta, and oPGH production by the trophectoderm and syncitium during the second month of pregnancy. To identify factors regulating oPGH production, we developed a perifusion system to measure oPGH and ovine placental lactogen (oPL) production by Day 45 ovine placental explants. The mRNAs for both hormones were quantitated by real-time polymerase chain reaction in explants collected after perifusion periods of up to 8 h. Ovine PGH and oPL were released into the medium at mean rates of 2.45 ± 0.2 and 353.6 ± 13.6 ng/g/h, respectively. Ovine placenta produces growth hormone-releasing hormone (GHRH), but addition of GHRH to the perifusion medium did not modify either oPGH or oPL production. In vivo, oPGH production occurs between Days 30 and 60 of pregnancy. Because modulation of the maternal diet during this period affects placental development, the potential regulation of oPGH and oPL production by glucose was evaluated. Glucose supplementation of the perifusion medium resulted in a concentration-dependent decrease in oPGH release after 4 h, but oPGH mRNA levels were not affected. Production of oPL was not affected by glucose. Thus, oPGH and oPL belong to the same growth hormone/prolactin family but are differentially regulated by glucose. Ovine PGH modulations should be taken into account in metabolic experiments performed during the first trimester of pregnancy in sheep.

The effects of β-mercaptoethanol (β-ME) on in vitro development under oxidative stress and cystine uptake of bovine embryos were investigated. Bovine 1-cell embryos obtained by in vitro fertilization were cultured in TCM-199 or synthetic oviductal fluid (SOF) in 20% O₂ supplemented with β-ME. Addition of β-ME significantly (P < 0.01) promoted embryo development when cultured in both TCM-199 and SOF under high levels of O₂, to almost the same rates when they were cultured in 5% O₂. To investigate whether the growth-promoting effect of β-ME was related to cystine uptake, which is an important amino acid for intracellular glutathione (GSH) synthesis, 1-cell, 8-cell, morula, and blastocyst stage embryos were incubated in cystine, cysteine-free TCM-199 containing radioisotope-labeled cystine supplemented with or without β-ME. It was found that cystine uptake was consistently low in each embryo stage incubated without β-ME. In contrast, addition of β-ME significantly (P < 0.05 to 0.0001) promoted cystine uptake in each stage of embryo development. This increase of cystine uptake by β-ME was significantly inhibited by supplementation of buthionine sulfoximine, a specific inhibitor of GSH biosynthesis (P < 0.0001). High-performance liquid chromatography (HPLC) analysis clearly revealed a decrease of cystine in culture medium after supplementation by β-ME, thereby forming another peak. HPLC analysis also showed the incorporated cystine by supplementation of β-ME was possibly metabolized for GSH synthesis in the embryos. These results indicate that β-ME has a protective effect in embryo development against oxidative stress and that the effect of β-ME is associated with the promotion of cystine uptake of low availability in embryos.

It is well established that the 43-kDa connexin (Cx43) is predominantly expressed by ovarian somatic cells, whereas the identity of the connexins contributed by the oocyte to form gap junctions with its neighboring cells is not fully elucidated. Our study aimed to examine oocytes for the expression and regulation of Cx43 throughout oogenesis. Growing and fully grown rat oocytes that were meiotically incompetent and competent, respectively, were examined. Fully grown oocytes were analyzed either before or after reinitiation of meiosis as well as at the second meiotic metaphase. Immunofluorescent analysis of zona pellucida-free oocytes using conventional and confocal microscopy demonstrated a characteristic pattern of punctuated staining of Cx43 on the oolema. Immunogold electron microscopy localized Cx43 to the oocyte surface and the microvillar processes. Reverse transcriptase-polymerase chain reaction and Western blot analysis revealed similar amounts of Cx43 gene and protein in oocytes of different developmental stages. However, a relative increase in the phosphorylated forms of the protein was observed in fully grown oocytes that had completed their maturation. Our findings demonstrate that rat oocytes express a developmentally regulated Cx43. They further suggest that homotypic gap junctions that consist of Cx43 may be present between rat oocytes and their adjacent cumulus cells.

During the period of attachment of the trophectoderm to the uterine lumenal surface in the pig, there is an increase in uterine blood flow and a localized hyperemic response induced by the developing conceptuses. The presence of tissue kallikrein in the porcine uterine lumen suggests that the kallikrein-kinin system may be functional during pregnancy in the pig. The objective of the present study was to determine the concentration of bradykinin within the uterine lumen during the estrous cycle and early pregnancy as well as endometrial gene expression and cellular localization of the bradykinin β₂ receptor. Concentration of bradykinin in uterine flushings was greatest during estrus (Day 0) and Days 12–18 of the estrous cycle. However, there was a 5- to 10-fold increase in bradykinin content in pregnant uterine flushings on Days 12–18 of pregnancy compared with the estrous cycle. Endometrial bradykinin β₂ receptor gene expression was greatest on Days 0, 12, 15, and 18 of the estrous cycle and pregnancy as gene expression decreased almost 6-fold on Days 5 and 10. Bradykinin β₂ receptors were detected in the endometrial surface and glandular epithelium with greatest intensity of staining observed on Days 0, 12, 15, and 18 of the estrous cycle and pregnancy. Results from the present study suggest that the kallikrein-kinin system plays a role in the establishment of pregnancy in the pig.

We used okadaic acid (OA), a potent inhibitor of protein phosphatases 1 and 2A, to study the regulatory effects of protein phosphatases on mitogen-activated protein (MAP) kinase phosphorylation, morphological changes in the nucleus, and microtubule assembly during pig oocyte maturation and fertilization in vitro. When germinal vesicle (GV) stage oocytes were exposed to OA, MAP kinase phosphorylation was greatly accelerated, being fully activated at 10 min. However, MAP kinase was dephosphorylated by long-term (>20 h) exposure to OA. Correspondingly, premature chromosome condensation and GV breakdown were accelerated, whereas meiotic spindle assembly and meiotic progression beyond metaphase I stage were inhibited. OA also quickly reversed the inhibitory effects of butyrolactone I, a specific inhibitor of maturation-promoting factor (MPF), on MAP kinase phosphorylation and meiosis resumption. Treatment of metaphase II oocytes triggered metaphase II spindle elongation and disassembly as well as chromosome alignment disruption. OA treatment of fertilized eggs resulted in prompt phosphorylation of MAP kinase, disassembly of microtubules around the pronuclear area, chromatin condensation, and pronuclear membrane breakdown, but inhibited further cleavage. Our results suggest that inhibition of protein phosphatases promptly phosphorylates MAP kinase, induces premature chromosome condensation and meiosis resumption as well as pronucleus breakdown, but inhibits spindle organization and suppresses microtubule assembly by sperm centrosomes in pig oocytes and fertilized eggs.

Using reverse transcriptase-amplified fragment length polymorphism (RT-AFLP) analysis of differential mRNA expression and semiquantitative reverse transcriptase-polymerase chain reaction, we compared mRNA expression in bovine blastocysts from 4 sources, known to differ in quality in terms of their ability to withstand cryopreservation: 1) in vitro culture in synthetic oviduct fluid of in vitro-matured (IVM)/in vitro fertilized (IVF) zygotes; 2) in vitro culture in TCM-199 supplemented with granulosa cells (coculture) of IVM/IVF zygotes; 3) in vivo culture in the ewe oviduct of IVM/IVF zygotes; or 4) superovulation, artificial insemination, and nonsurgical embryo recovery. Total mRNA was isolated from pools of blastocysts and reverse transcription was performed. Triplicate reactions from each sample were displayed, and only consistent banding variations were recorded. Using AFLP-differential display assay, we found that cDNA banding patterns are highly conserved between the 4 groups of blastocysts studied; however, there was a difference of 7% in bands either missing or expressed across the groups. Fifty bands were reamplified, and a sequence comparison search revealed similarity of 14 isolated fragments to ribosomal and mitochondrial genes, 16 matched to described cDNA, and 20 corresponded to unknown sequences that may represent novel genes. The study of 7 differentially expressed mRNAs known to be involved in developmental process in the embryo suggests roles for apoptosis, oxidative stress, gap junctions, and differentiation in the determination of embryo quality. The aberrant transcription patterns detected in in vitro-produced bovine embryos compared with those produced in vivo may explain their reduced quality in terms of viability after cryopreservation.

Hormones influence uterine contractility through their effects on intracellular calcium. The regulation of intracellular calcium in uterine smooth muscle is achieved by several mechanisms and includes mobilization from intracellular stores by inositol 1,4,5-trisphosphate and ryanodine-sensitive channels. Cyclic ADP-ribose (cADPR), a metabolite of NAD , is known to mediate calcium release through ryanodine receptor channels. A cell surface glycoprotein, CD38, catalyzes the synthesis and breakdown of cADPR and thus possesses bifunctional enzymatic activity. The regulation of cADPR synthesis by ADP-ribosyl cyclase (cyclase) or degradation by cADP-ribose hydrolase (hydrolase) by hormones in the myometrium is poorly understood. We investigated the effects of estradiol-17β on CD38 expression and the synthesis and degradation of cADPR in myometrial smooth muscle obtained from ovariectomized rats. CD38 expression was studied by reverse transcription polymerase chain reaction and Western blot analyses. In uterine microsomal fractions, cyclase and hydrolase activities were measured using nicotinamide guanine dinucleotide and [³²P]cADPR as substrates, respectively. Microsomal proteins subfractionated by SDS-PAGE and gel filtration were used to determine the fractions containing cyclase and hydrolase activities. The results demonstrate that cyclase and hydrolase activities are associated with a single protein fraction, similar to CD38 in uteri from both ovariectomized and estradiol-treated rats, and estradiol-17β causes 1) increased CD38 mRNA and protein expression and 2) significantly enhanced cyclase but not hydrolase activity. The differential regulation of CD38 by estradiol-17β, resulting in increased cADPR synthesis, would have profound effects on calcium regulation and myometrial contractility.

The working hypothesis was that 17β-estradiol (E₂) negative feedback on the hypothalamic-pituitary axis in regulation of LH secretion decreases during peripuberty in heifers of 2 different genotypes. We investigated whether Bos indicus heifers had a period postpuberty, as compared with prepuberty, of greater E₂ inhibition of LH secretion at a time when heifers of this genotype have been reported to have a period of anestrus. Prepubertal heifers 9 mo of age of 2 genotypes (B. indicus and B. taurus) were assigned to 3 groups (6 animals/group) to either remain intact (control), be ovariectomized, or be ovariectomized and implanted with E₂. Variables evaluated from 10 to 28 mo of age were circulating concentrations of progesterone (P₄), presence of corpora lutea, and pulsatile pattern of LH release. Results confirmed that B. taurus heifers attained puberty at younger ages (P < 0.001) and at lower live weights (P = 0.015) than did B. indicus heifers (507 ± 37 days of age vs. 678 ± 7 days of age; 259 ± 14 kg vs. 312 ± 11 kg; respectively). There was cessation of E₂ inhibition of LH pulses coincident with the onset of puberty in heifers of both breed types but at a much younger age in B. taurus heifers. There was no evidence of enhanced negative feedback of E₂ on LH secretion subsequent to puberty in B. indicus heifers nor was there cessation of estrous cycles in control heifers of either breed type after puberty.

In at least 9 mammalian species, females are masculinized throughout life, but the benefits of this remain unclear despite decades of thorough study, in particular of the spotted hyaena (Crocuta crocuta) in which the phenomenon has been associated with a high fitness cost. Through examination of wild and captive fossas (Cryptoprocta ferox, Viverridae), androgen assays, and DNA typing for confirmation of gender, we made the first discovery of transient masculinization of a female mammal. Juvenile female fossas exhibited an enlarged, spinescent clitoris supported by an os clitoridis and a pigmented secretion on the underpart fur that in adults was confined to males. These features appeared to diminish with age. The majority of adult females lacked them, and os clitoridis length was inversely related to head-body length. No evidence was found to link this masculinization to elevated female androgen levels. Circulating concentrations of testosterone and androstenedione, but not dihydrotestosterone, were significantly lower in females than in males. No significant differences in testosterone, androstenedione, or dihydrotestosterone levels were found between juvenile (masculinized) and adult (nonmasculinized) females. There are several possible physiological mechanisms for this masculinization. None of the hypotheses so far proposed to explain the evolutionary basis of female masculinization in mammals are applicable to our findings. We present 2 new hypotheses for testing and development.

The cDNA for the full-length porcine estrogen receptor β (ERβ) and an alternatively spliced transcript with a deletion of exon 5 (ERβδ5) was cloned from pig ovary. RNase protection assays revealed that ERβ mRNA was expressed in the preovulatory follicles and early, midluteal, and regressing corpora lutea (CL) of eCG ± hCG-primed gilts. ERβ and ERβδ5 transcripts were shown by semiquantitative reverse transcription polymerase chain reaction to be expressed at a ratio of approximately 2:1 in granulosa cells, small, medium, and large antral follicles, and midluteal phase corpora lutea of unprimed animals. Immunoreactive ERβ proteins corresponding to the size of in vitro translated ERβ and ERβδ5 were detected by immunoblot. Full-length ERβ was detected in granulosa, small, medium, and large antral follicles, and midluteal phase CL of unprimed animals. Putative ERβδ5 immunoreactive bands were abundant only in granulosa cell extracts. In COS-1 cells, transfected ERβδ5 had no effect on basal transcription of an estrogen-responsive reporter construct but did repress wild-type ERβ transactivation when cotransfected at 10-fold excess plasmid. No repression of ERα transactivation was observed. In primary granulosa cell cultures, transfected ERβδ5 plasmid did not inhibit basal reporter activation. ERβδ5 was shown by immunofluorescence to localize to the nucleus in transfected COS-1 cells. In vitro translated ERβδ5 proteins bound estrogen response elements in DNA in electrophoretic mobility shift assays, as indicated by supershift analysis. ERβ is abundant in porcine ovary, and a naturally occurring splice variant missing exon 5 may have biological function.

Semenogelin plays an important role in sperm clotting and is degraded into smaller fragments by prostate-specific antigen (PSA) during clot liquefaction. Semenogelin and its fragments inhibit sperm motility in vitro. We studied the expression of semenogelin I mRNA and its localization in various tissues of the male genital tract. We also studied semenogelin concentrations with respect to sperm parameters and the outcome of in vitro fertilization. Semenogelin protein was detected by immunohistochemical staining and semenogelin I mRNA was detected by Northern blot analysis in the seminal vesicles and ampullary part of the vas deferens, whereas specimens from the prostate, epididymis, testis, and the female genital tract were negative. Using monoclonal antibodies against semenogelin, an immunofluorometric assay was developed to measure semenogelin levels in seminal plasma and to evaluate possible correlations with sperm parameters and fertilization in vitro. No correlation was found between the semenogelin concentration and the volume of the ejaculate, sperm concentration, sperm motility, or in vitro fertilization rate. Semenogelin levels were positively correlated with the total protein concentration in seminal plasma, and there was an inverse correlation between the concentration of semenogelin and that of PSA. The levels of semenogelin appear to bear no relationship to the in vitro fertilization capacity of the spermatozoa.

In porcine embryos, nucleoli are first observed during the third postfertilization cell cycle, i.e., at the 4-cell stage. However, direct studies of the initiation of rRNA transcription have not been reported. This transcription was investigated in the present study by simultaneous visualization of the rRNA genes and the rRNA by fluorescent in situ hybridization using a porcine 28S rDNA probe and subsequent visualization of argyrophilic nucleolar proteins by silver staining of extracted and fixed nuclei from in vivo-derived porcine embryos (n = 229). Nucleologenesis was observed by transmission electron microscopy. In general, the 2-cell and 4-cell embryos fixed at 10 and 20 h postcleavage (hpc) showed no signs of rRNA transcription. Four small clusters of fluorescein isothiocyanate (FITC) labeling were visible in interphase nuclei, consistent with hybridization to the rRNA gene clusters only; there was no silver staining at the sites of the rRNA genes and nucleolus precursor bodies. From 30 hpc onwards, most 4-cell embryos had medium size to large clusters of FITC-labeled areas colocalized with silver staining of rRNA gene clusters and fibrillogranular nucleoli. These observations indicate that rRNA transcription had been initiated. These signs of rRNA synthesis could be blocked by actinomycin D, which is a strong inhibitor of RNA polymerase I. The rRNA transcription of porcine embryos is initiated between 20 and 30 hpc, corresponding to the end of the S-phase or the beginning of the G2 phase during the third cell cycle.

Factors influencing pig oocyte activation by electrical stimulation were evaluated by their effect on the development of parthenogenetic embryos to the blastocyst stage to establish an effective activation protocol for pig nuclear transfer. This evaluation included 1) a comparison of the effect of epidermal growth factor and amino acids in maturation medium, 2) an investigation of interactions among oocyte age, applied voltage field strength, electrical pulse number, and pulse duration, and 3) a karyotype analysis of the parthenogenetic blastocysts yielded by an optimized protocol based on an in vitro system of oocyte maturation and embryo culture. In the first study, addition of amino acids in maturation medium was beneficial for the developmental competence of activated oocytes. In the second study, the developmental response of activated oocytes was dependent on interactions between oocyte age at activation and applied voltage field strength, voltage field strength and pulse number, and pulse number and duration. The formation of parthenogenetic blastocysts was optimal when activation was at 44 h of maturation using three 80-μsec consecutive pulses of 1.0 kV/cm DC. Approximately 84% of parthenogenetic blastocysts yielded by this protocol were diploid, implying a potential for further in vivo development.

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.

One of the postulated main luteolytic actions of prostaglandin (PG) F_2α is to decrease ovarian blood flow. However, before Day 5 of the normal cycle, the corpus luteum (CL) is refractory to the luteolytic action of PGF_2α. Therefore, we aimed to determine in detail the real-time changes in intraluteal blood flow after PGF_2α injection at the early and middle stages of the estrous cycle in the cow. Normally cycling cows at Day 4 (early CL, n = 5) or Days 10–12 (mid CL, n = 5) of the estrous cycle (estrus = Day 0) were examined by transrectal color and pulsed Doppler ultrasonography to determine the blood flow area, the time-averaged maximum velocity (TAMXV), and the volume of the CL after an i.m. injection of a PGF_2α analogue. Ultrasonographic examinations were carried out just before PG injection (0 h) and then at 0.5, 1, 2, 4, 8, 12, 24, and 48 h after the injection. Blood samples were collected at each of these times for progesterone (P) determination. The ratio of the colored area to a sectional plane at the maximum diameter of the CL was used as a quantitative index of the changes in blood flow within the luteal tissue. Blood flow within the midcycle CL initially increased (P < 0.05) at 0.5–2 h, decreased at 4 h to the same levels observed at 0 h, and then further decreased to a lower level from 8 h (P < 0.05) to 48 h (P < 0.001). Plasma P concentrations decreased (P < 0.05) from 4.7 ± 0.5 ng/ml (0 h) to 0.6 ± 0.2 ng/ml (24 h). The TAMXV and CL volume decreased at 8 h (P < 0.05) and further decreased (P < 0.001) from 12 to 24 h after PG injection, indicating structural luteolysis. These changes were not detected in the early CL, in which luteolysis did not occur. In the early CL, the blood flow gradually increased in parallel with the CL volume, plasma P concentration, and TAMXV from Day 4 to Day 6. The present results indicate that PGF_2α induces an acute blood flow increase followed by a decrease in the midcycle CL but not in the early CL. This transitory increase may trigger the luteolytic cascade. The lack of intraluteal vascular response to PG injection in the early CL appears to be directly correlated with the ability to be resistant to PG.

In the mammalian testis, the binding of FSH to Sertoli cells activates the cAMP-dependent protein kinase A signaling pathway, resulting in the phosphorylation of the cAMP response element binding protein (CREB). Previous studies have also shown that CREB gene expression is activated by cAMP in Sertoli cells and that 2 cAMP response elements (CREs) that bind CREB and a neighboring Sp1 binding site are required for basal and cAMP-inducible CREB promoter activity. In contrast, CREB expression has been less well characterized in testis germ cells. We demonstrated that CREB and Sp1 are expressed in early germ cells only through the midpachytene stage of spermatogenesis. Furthermore, CREB promoter activity was induced over 70-fold by transient overexpression of Sp1 in SL2 cells, suggesting that Sp1 is an important regulator of CREB expression. Further studies of the CREB promoter revealed an additional regulatory element in the −130 region between the Sp1 and CREB transcription factor binding sites that is necessary for full promoter activity. Proteins expressed in Sertoli cells and germ cells bind specifically to the newly identified regulatory region. These studies suggest that proteins binding to Sp1 motifs and the −130 region are required to activate the CREB promoter.

The presence of the capacitative Ca² entry mechanism was investigated in porcine oocytes. In vitro-matured oocytes were treated with thapsigargin in Ca² -free medium for 3 h to deplete intracellular calcium stores. After restoring extracellular calcium, a large calcium influx was measured by using the calcium indicator dye fura-2, indicating capacitative Ca² entry. A similar divalent cation influx could also be detected with the Mn² -quench technique after inositol 1,4,5-triphosphate-induced Ca² release. In both cases, lanthanum, the Ca² permeable channel inhibitor, completely blocked the influx caused by store depletion. Heterologous expression of Drosophila trp in porcine oocytes enhanced the thapsigargin-induced Ca² influx. Polymerase chain reaction cloning using primers that were designed based on mouse and human trp sequences revealed that porcine oocytes contain a trp homologue. As in other cell types, the capacitative Ca² entry mechanism might help in refilling the intracellular stores after the release of Ca² from the stores. Further investigation is needed to determine whether the trp channel serves as the capacitative Ca² entry pathway in porcine oocytes or is simply activated by the endogenous capacitative Ca² entry mechanism and thus contributes to Ca² influx.

Dmrt1 is a recently described gene that is specifically expressed in the gonads and is required for postnatal testis differentiation. Here, we describe the transcriptional mechanisms regulating the Dmrt1 proximal promoter in testicular Sertoli cells. A genomic clone containing exon 1 of the rat Dmrt1 gene and more than 9 kilobases of 5′ flanking sequence was isolated and characterized. Several prominent transcriptional start sites were identified, with the major site located 102 bases from the translational start. The Dmrt1 5′ flanking region from −5000 to 74 was transcriptionally active in primary Sertoli cells, and deletion analysis of this fragment identified 2 major regions needed for full Dmrt1 promoter function. These regions were located between −3200 and −2000 base pairs (bp) and downstream of −150 bp relative to the major transcriptional start site. DNase I footprint analysis of the region downstream of −150 bp revealed 3 regions that are bound by proteins from Sertoli cell nuclear extracts. Site-directed mutagenesis of these regions identified 2 elements that activate the Dmrt1 promoter and 2 that repress it. The positive elements bind the transcription factors Sp1, Sp3, and Egr1, suggesting that these transcription factors play a critical role in Dmrt1 regulation in the testis.

Luteal tissue contains matrix metalloproteinases (MMPs) that cleave specific components of the extracellular matrix (ECM) and are inhibited by tissue inhibitors of metalloproteinases (TIMPs). We previously reported a decrease in luteal TIMP-1 within 15 min of prostaglandin F_2α (PGF_2α)-induced luteolysis. An increase in the MMP:TIMP ratio may promote ECM degradation and apoptosis, as observed in other tissues that undergo involution. The objectives of these experiments were to determine whether 1) PGF_2α affects expression of mRNA encoding fibrillar collagenases (MMP-1 and -13), gelatinases A and B (MMP-2 and -9), membrane type (mt)-1 MMP (MMP-14), stromelysin (MMP-3), and matrilysin (MMP-7), and 2) PGF_2α increases MMP activity during PGF_2α-induced luteolysis in sheep. Corpora lutea (n = 3–10/time point) were collected at 0, 15, and 30 min and 1, 2, 4, 6, 12, 24, and 48 h after PGF_2α administration. Northern blot analysis confirmed the presence of all MMPs except MMP-9. Expression of mRNA for the above MMPs (except MMP-2) increased significantly (P < 0.05) by 30 min, and all MMPs increased significantly (P < 0.05) by 6 h after PGF_2α administration. Expression of MMP-14 mRNA increased significantly (P < 0.05) by 15 min post-PGF_2α and remained elevated through 48 h. MMP activity in luteal homogenates (following proenzyme activation and inactivation of inhibitors) was increased significantly (P < 0.05) by 15 min and remained elevated through 48 h post-PGF_2α. MMP activity was localized (in situ zymography) to the pericellular area of various cell types in the 0-h group and was markedly increased by 30 min post-PGF_2α. MMP mRNA expression and activity were significantly increased following PGF_2α treatment. Increased MMP activity may promote ECM degradation during luteolysis.

In cattle, administration of retinol at the time of superovulation has been indirectly associated with enhanced developmental potential of the embryo. Vitamin A and its metabolites influence several developmental processes by interacting with 2 different types of nuclear receptors, retinoic acid receptors and retinoid X receptors (RXRs). Given the limited information available concerning the RXR-mediated retinoid signaling system, particularly in species other than rodents, this study was performed to gain insight into the potential role of retinoid signaling during preattachment embryo development in the cow. Bovine embryos were produced in vitro from oocytes harvested from abattoir ovaries and frozen in liquid nitrogen at the oocyte, 2-, 4-, 8-, and 16- to 20-cell, morula, blastocyst, and hatched blastocyst stages. Reverse transcription polymerase chain reaction (PCR) and whole mount in situ hybridization were utilized to investigate mRNA expression for RXRα, RXRβ, RXRγ, alcohol dehydrogenase I (ADH-I), retinaldehyde dehydrogenase 2 (RALDH2), peroxisome proliferator activated receptor gamma (PPARγ), and glyceraldehyde-3-phosphate dehydrogenase. Transcripts for RXRα, RXRβ, RALDH2, and PPARγ were detected in all stages beginning from the oocyte through to the hatched blastocyst. Whole mount in situ hybridization performed using digoxigenin-labeled antisense probes detected all 4 transcripts in both the inner cell mass and the trophectoderm of hatched blastocysts. PCR products obtained for ADH-I exhibited very low homology to known human and mouse sequences. Immunohistochemistry was performed using polyclonal anti-rabbit antibodies against RXRβ and PPARγ to investigate whether these embryonic mRNAs were translated to the mature protein. Strong immunostaining was observed for both RXRβ and PPARγ in the trophectoderm and inner cell mass cells of intact and hatched blastocysts. Messenger RNA was not detected at any stage for RXRγ. Expression of mRNA for RXRα, RXRβ, RALDH2, and PPARγ suggests that the early embryo may be competent to synthesize retinoic acid and regulate gene expression during preattachment development in vitro.

Thyroid hormones permit the increase in response to estradiol negative feedback in ewes at the transition to anestrus. In this study, we tested whether the thyroid hormones are also required for steroid-independent seasonal changes in pulsatile LH secretion. In experiment 1, Suffolk ewes were ovariectomized and thyroidectomized (THX) or ovariectomized only (controls) in late November. LH pulse frequency and amplitude were measured for 4 h in December, April, May, June, and August. Pulse frequency was also measured in the presence of estradiol-containing implants during the breeding (December) and early anestrus (March) seasons. As expected, in the presence of estradiol, pulse frequency declined between December and March in control but not THX ewes. In the absence of estradiol, a seasonal decline in frequency and an increase in amplitude occurred in control ewes, concurrent with lengthening photoperiod. A similar trend was seen in THX ewes, but the seasonal changes were lower in magnitude and not significant. In experiment 2, the same protocol was used (pulse measurements in December, May, and June) with a larger THX group size (n = 7). Results were similar to those of experiment 1 for controls. In THX ewes, pulse frequency did not change over time and was significantly elevated relative to that of controls during the summer. Pulse amplitude in THX ewes tended to increase during summer and did not differ from pulse amplitudes in control ewes. These results demonstrate that thyroid hormones are required for steroid-independent cycles in LH pulse frequency; however, some seasonal changes in amplitude still occur in the absence of thyroid hormones. This finding contrasts with the changes in estradiol negative feedback at the transition to anestrus, which are entirely thyroid hormone dependent.

The transient synthesis and accumulation of hyaluronan (HA), an extracellular matrix component of cumulus cells, brings about expansion of cumulus-oocyte complexes (COCs) in preovulatory mammalian follicles. In this study, we investigated the mRNA expressions of hyaluronan synthase 2 (has2), hyaluronan synthase 3 (has3), and CD44, as well as the responsiveness to eCG and porcine follicular fluid (pFF) of these genes, in porcine COCs, oocytectomized complexes (OXCs), and oocytes during in vitro maturation. Immunolocalization of CD44 was also analyzed in COCs. After 12 h of culture, the area of cumulus expansion in medium 199 supplemented with both 10 IU/ml eCG and 10% (v/v) pFF was significantly greater than that in the medium supplemented with eCG or pFF. Oocytectomy reduced the expansion area in the group supplemented with eCG. In reverse transcription-polymerase chain reaction analysis, all transcripts were identified in COCs, but has3 transcript was not found in OXCs. Only has3 mRNA was detectable in oocytes, indicating that cumulus cells express has2 and CD44 mRNAs, and oocytes express has3 mRNA. The expression levels of has2 and CD44 mRNAs in COCs and OXCs increased in the presence of eCG and pFF after 24 h of culture, suggesting that these genes have a positive dependency on eCG and pFF. In contrast, the high level of has3 mRNA was detected in COCs cultured in the medium alone. Oocytectomy slightly reduced the expression level of has2 mRNA. On immunostaining for CD44, CD44 was expressed apparently in COCs cultured with eCG and pFF for 24 h. The positive staining was distributed on cytoplasm along the perimembrane of cumulus cells and at the junctions between cumulus cells and oocytes. CD44 was also localized on cytoplasm of some oocytes. These results indicate that 1) porcine oocytes promote eCG-dependent cumulus expansion and the expression of has2 mRNA in cumulus cells, but these are not essential for expansion of cumulus cells and the expression of has2 mRNA; 2) HAS2 is involved in HA synthesis during cumulus expansion, and eCG and pFF up-regulate its expression; 3) the expression profile of the has3 mRNA that is transcribed in oocytes is different from those of has2 and CD44 mRNA; and 4) CD44 may participate in the interaction between cumulus cells and oocytes.

Noninvasive, epitheliochorial placentation in the pig follows a prolonged preimplantation period characterized by migration, spacing and elongation of conceptuses, and secretion of estrogen for maternal recognition of pregnancy. Osteopontin (OPN) is an extracellular matrix protein that binds integrins to promote cell-cell attachment and communication. OPN appears to play a key role in conceptus implantation and maintenance of pregnancy in sheep; however, a role for OPN in the porcine uterus has not been established. Therefore, this study examined OPN expression and function in the porcine uterus and conceptus (embryo/fetus and associated extraembryonic membranes). Northern and slot blot hybridization detected an increase in endometrial OPN expression between Days 25 and 30, and levels remained elevated through Day 85 of pregnancy. In situ hybridization localized OPN mRNA to discrete regions of the uterine luminal epithelium (LE) on Day 15 of pregnancy and to the entire LE thereafter. Glandular epithelial (GE) expression of OPN mRNA was first detected on Day 35 of pregnancy and increased through Day 85. Both 70- and 45-kDa forms of OPN protein were detected in cyclic and pregnant endometrium by Western blotting. OPN protein was localized to the LE and GE by immunofluorescence; however, only the 70-kDa OPN was detected in uterine flushings. OPN protein was present along the entire uterine-placental interface after Day 30 of pregnancy. In addition, OPN mRNA and protein were localized to immune-like cells within the stratum compactum of the endometrium in both Day 9 cyclic and pregnant gilts. Incubation of OPN-coated microbeads with porcine trophectoderm and uterine luminal epithelial cells induced Arg-Gly-Asp (RGD)-dependent integrin activation and transmembrane accumulation of cytoskeletal molecules at the apical cell surface as assessed by immunofluorescence detection of talin or α-actinin as markers for focal adhesions. These results suggest that OPN, expressed by uterine epithelium and immune cells, may interact with receptors (i.e., integrins) on conceptus and uterus to promote conceptus development and signaling between these tissues as key contributors to attachment and placentation in the pig.

Transgene insertions in the mouse often cause mutations at chromosomal loci. Analysis of insertion mutations that cause male sterility may lead to the identification of novel molecular mechanisms implicated in male fertility. Here we show a line of transgenic mice with dominant inheritance of male sterility (DMS) that was found amid several lines that were normally fertile. Transgene-positive males from this line invariably were sterile, whereas transgenic females and transgene-negative male littermates were fertile. Histologic analysis and TUNEL staining for apoptotic cells in DMS testis showed spermatogenesis arrest at metaphase of meiosis I (M-I), accompanied by massive apoptosis of spermatocytes. Meiosis I arrest was incomplete, however, as small numbers of spermatids and spermatozoa were found. Both round spermatids and spermatozoa were evaluated for their permissiveness in the assisted reproductive technologies intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI). Surprisingly, ROSI but not ICSI gave live offspring, suggesting that mature sperm had deteriorated by the time of recovery from the epididymis. Mapping the transgene insertion by fluorescence in situ hybridization revealed a site on chromosome 14 D3-E1. Two candidate genes, GFRα2 and GnRH, that were previously mapped to that region and the functions of which in spermatogenesis are well established were not altered in DMS. As a consequence, positional cloning of the DMS locus will be essential to identify new molecules potentially involved in arrest at M-I. Furthermore, mice carrying this genetic trait might be useful for studies of assisted reproductive technologies and male contraceptives.

To identify novel human sperm membrane antigens, we analyzed two-dimensional gels of sperm extracts containing hydrophobic proteins that partitioned into Triton X-114. Four protein spots with isoelectric points (pIs) ranging from 4.5 to 5.5 and apparent molecular weights from 32 to 34 kDa were sequenced by mass spectrometry and found to contain common peptide sequences. Cloning the corresponding cDNA revealed that these protein spots were products of a single gene (SAMP32), encoding a protein of 32 kDa with a predicted pI of 4.57. SAMP32 has a potential transmembrane domain in the carboxyl terminus and is phosphorylated in vivo on serine 256. Northern blotting of eight human tissues and RNA dot blotting of 76 human tissues showed that SAMP32 expression was testis specific. SAMP32 contained an amino terminal domain homologous to the major malarial circumsporozoite surface protein and a domain similar to that of Krp1 from Schizosaccharomyces pombe in its carboxyl terminus. The SAMP32 locus consists of seven exons on chromosome 6q15-16.2. Antiserum against recombinant SAMP32 recognized protein spots originally cored from a two-dimensional gel. This antiserum strongly stained the equatorial segment and faintly stained the acrosome cap of ejaculated human spermatozoa by immunofluorescence. Immunoelectron microscopy showed that SAMP32 was associated with the inner acrosomal membrane in the principal and the equatorial segments of the sperm acrosome. By immunostaining enzyme-dissociated testicular cells, SAMP32 was localized to Golgi phase round spermatids and subsequent stages of acrosome biogenesis. Recombinant SAMP32 reacted with serum from an infertile man, suggesting that it is isoantigenic. Antibodies against recombinant SAMP32 inhibited both the binding and the fusion of human sperm to zona-free hamster eggs.

Platelet-derived growth factors (PDGFs) are paracrine factors with roles in mesenchymal-epithelial interactions during normal and pathologic processes. Previously, PDGF and its receptor (PDGFR) have been shown to be present in perinatal, peripubertal, and adult rat testes. The role of PDGF in embryonic testicular cord formation is not known. The hypothesis tested is that PDGFs and PDGFRs are expressed during cord formation and that inhibition of their action influences normal cord formation during embryonic testis development. Embryonic Day (E) 13 gonadal organ cultures were used. Organs were cultured for 3 days and treated daily with vehicle or a PDGFR-specific tyrosine phosphorylation inhibitor (i.e., the tyrphostin AG1295 or AG1296). Vehicle-treated testes formed normal cords, whereas tyrphostin-treated testes formed “swollen cords,” a phenomenon characterized by a significant decrease in the number of cords per testis area and increased cord diameter due to fusion of cords. Expression of PDGF and PDGFR in E13, E14, E16, Postnatal Day (P) 0, and P20 testes was examined. Messenger RNAs for PDGF-A and -B and PDGF α- and β-receptors were expressed in isolated testes during all developmental periods examined. Immunoreactivity for PDGF was present throughout the testicular compartment at E14, restricted primarily to testicular cords at E16, and present in cells of the testicular cords with a stronger immunoreactivity in certain interstitial cell types of P0 testis. PDGFR β-receptor immunoreactivity was primarily localized to the mesonephros of E14 organs and the testicular interstitium of E16 and P0 testes. Tyrphostins did not affect apoptotic cell number in the testis. PDGF had no effect on cell growth in P0 testis cultures. The results show that PDGFs and PDGFRs are expressed in embryonic testis during cord formation in a tissue-specific manner. Inhibition of PDGF actions does not inhibit cord formation but does alter normal cord development and morphology. The observations provide insight into the factors involved in male sex differentiation and embryonic testis development.

Fas antigen (Fas) is a cell surface receptor that triggers apoptosis in sensitive cells when bound to the Fas ligand (Fas L). The present study was undertaken to identify the presence of a Fas-Fas L system in bovine corpus luteum (CL) and to evaluate the regulation of Fas-mediated luteal cell death by leukocyte-derived cytokines. The reverse transcription-polymerase chain reaction showed higher levels of Fas mRNA expression in CL in the regressed luteal stage (Days 19–21) than in the other stages (P < 0.05). Bovine luteal cells from midcycle CL (Days 8–12) were exposed for 24 h to interferon γ (IFN; 50 ng/ml) and/or tumor necrosis factor α (TNF; 50 ng/ml). After 24 h of culture, the expression of Fas mRNA was detected in the cultured cells and was increased by IFN. Moreover, TNF augmented the stimulatory action of IFN, whereas TNF alone did not affect the expression of Fas mRNA. The effects of IFN and TNF on Fas-mediated cell death were also examined. Cells were exposed to IFN and/or TNF for 24 h and were further treated with IFN and/or TNF in the presence or absence of Fas L (100 ng/ml) for 24 h. Treatments of the cells with IFN alone and in combination with TNF resulted in killing of 30% and 50% of the cells (P < 0.05), respectively, whereas TNF alone did not have a cytotoxic effect on the cells. On the other hand, Fas L killed 60% of the cells treated with IFN (P < 0.01) and 85% of the cells treated with the combination of TNF and IFN (P < 0.01), respectively, whereas Fas L showed no effect on the viability of the luteal cells treated with or without TNF. Furthermore, shrunken nuclei and apoptotic bodies were observed in the cells treated with Fas L in the presence of TNF and IFN. The overall results suggest that a Fas-Fas L system is present in bovine CL and that leukocyte-derived TNF and IFN play important roles in Fas-mediated luteal cell death.

In the pig, estrogens transiently produced by embryos and progestins of maternal origin target the uterine endometrium, causing alterations in gene expression and secretory activity, both of which are important for the initiation of embryo attachment. The potential direct embryotrophic roles of estrogens and progestins are, however, unknown. Here we report the cloning of porcine embryonic estrogen receptor-beta (ER-β) mRNA by reverse transcription-polymerase chain reaction (RT-PCR) using specific primer sets designed initially within conserved regions of human and bovine ER-β mRNAs, and subsequently within regions of identified porcine ER-β cDNA sequences. The ER-β mRNA has an open reading frame of 1578 nucleotides and encodes a 526 amino acid polypeptide that displays greater than 90% identity with other mammalian ER-β proteins. Northern and Western blot analyses using porcine filamentous embryos from Day 12 of pregnancy demonstrated the presence of multiple ER-β mRNA transcripts of approximately 9.5, 4.9, and 3.5 kilobases, and a ∼64-kDa protein corresponding in size to human ovarian granulosa cell ER-β, respectively. In Day 12 filamentous embryos, ER-β expression was immunolocalized to trophoblastic cell nuclei, coincident with that of proliferative cell nuclear antigen (PCNA). The developmental ontogeny of ER-β mRNA was evaluated in embryos of different morphologies (spherical, tubular, and filamentous) by semiquantitative RT-PCR, along with those for other steroid hormone receptors (ER-α and progesterone receptor) and known embryonic genes associated with cell differentiation (cytochrome P450 aromatase type III) and growth (cyclin D1). ER-β mRNA levels varied with embryo morphology (filamentous maximum at Day 12), coincident with that of cyclin D1. Progesterone receptor mRNA levels were maximal in tubular embryos, similar to that of P450 aromatase, whereas the expression of the ER-α gene was barely detectable and appeared constitutive for all developmental stages examined. Estradiol-17β treatment of Day 12 filamentous embryos in culture up-regulated ER-β and P450 aromatase (type III) mRNA levels, respectively, but decreased those of PCNA, and had no effect on cyclin D1 mRNA levels. These studies taken together suggest that embryonic ER-β likely mediates the autocrine functions of estrogens in the dynamic regulation of embryonic growth and development at periimplantation.

Recent studies have shown that microgravity induces changes at the cellular level, including apoptosis. However, it is unknown whether microgravity affects luteal cell function. This study was performed to assess whether microgravity conditions generated by clinostat rotation induce apoptosis and affect steroidogenesis by luteal cells. Luteal cells isolated from the corpora lutea of Day 8 pregnant rats were placed in equal numbers in slide flasks (chamber slides). One slide flask was placed in the clinostat and the other served as a stationary control. At 48 h in the clinostat, whereas the levels of progesterone and total cellular protein decreased, the number of shrunken cells increased. To determine whether apoptosis occurred in shrunken cells, Comet and TUNEL assays were performed. At 48 h, the percentage of apoptotic cells in the clinostat increased compared with that in the control. To investigate how the microgravity conditions induce apoptosis, the active mitochondria in luteal cells were detected with JC-1 dye. Cells in the control consisted of many active mitochondria, which were evenly distributed throughout the cell. In contrast, cells in the clinostat displayed fewer active mitochondria, which were distributed either to the outer edge of the cell or around the nucleus. These results suggest that mitochondrial dysfunction induced by clinostat rotation could lead to apoptosis in luteal cells and suppression of progesterone production.

Gastrointestinal motility is reduced and the incidence of functional gastrointestinal disorders is increased in pregnancy, possibly due to hormonal influences. This study aims to clarify whether the hormone relaxin, which attains high circulating levels during pregnancy and has a nitric oxide-mediated relaxant action on vascular and uterine smooth muscle, also reduces bowel motility and, if it does, whether nitric oxide is involved. Female mice in proestrous or estrous were treated for 18 h with relaxin (1 μg s.c.) or vehicle (controls). Isolated ileal preparations from both groups were used to record contractile activity, either basal or after acute administration of relaxin (5 × 10⁻⁸ M). Drugs inhibiting nitric oxide biosynthesis or neurotransmission were used in combination with relaxin. Expression of nitric oxide synthase isoforms by the ileum was assessed by immunocytochemistry and Western blot analysis. Relaxin caused a clear-cut decay of muscle tension and a reduction in amplitude of spontaneous contractions upon either chronic administration to mice or acute addition to isolated ileal preparations. These effects were significantly blunted by N^G-nitro-l-arginine, but not by the neural blockers we used. Moreover, relaxin increased the expression of nitric oxide synthases II and III, but not synthase I. Relaxin markedly inhibits ileal motility in mice by exerting a direct action on smooth muscle through the activation of intrinsic nitric oxide biosynthesis.

Formation of the tail in developing sperm is a complex process involving the organization of the axoneme, transport of periaxonemal proteins from the cytoplasm to the tail, and assembly of the outer dense fibers and fibrous sheath. Although detailed morphological descriptions of these events are available, the molecular mechanisms remain to be fully elucidated. We have isolated a new gene, named shippo 1, from a haploid germ cell-specific cDNA library of mouse testis, and also its human orthologue (h-shippo 1). The isolated cDNA is 1.2 kilobases long, carrying a 762-base pair open reading frame that encodes SHIPPO 1, a sperm protein predicted to consist of 254 amino acids. The amino acid sequence includes 6 Pro-Gly-Pro repeats, which are also present in the human orthologue protein (hSHIPPO 1) as well as in 2 other newly reported proteins of Drosophila melanogaster. Transcription of shippo 1 is exclusively observed in haploid germ cells. Antibody raised against SHIPPO 1 identified a testis-specific M_r 32 × 10⁻³ band in Western blot analysis. The protein was further localized in the flagella of the elongated spermatids and along the entire length of the tail in mature sperm. SHIPPO 1 in sperm is resistant to treatment with nonionic detergents and coextracted with the cytoskeletal core proteins of the mouse sperm tail.

Although the potential use of reproductive biotechnologies for safeguarding endangered wildlife species is undoubted, practical efforts have met with limited success to date. In those instances in which modern technologies have been adapted to rescuing rare or endangered species, procedures have been applied piecemeal, and no consistent breeding program based on reproductive biotechnologies has been undertaken. Here we describe for the first time the rescue of an endangered species, the European mouflon (Ovis orientalis musimon), by the application of an integrated package of reproductive biotechnologies. This genetic management extended from the initial collection of gametes, through the in vitro production of embryos and interspecific transfer, to the birth of healthy mouflon offspring. In addition, a genetic resource bank for the European mouflon was established, with cryopreserved sperm, embryos, and somatic cells.

Vascular development and its transformation are necessary for successful hemochorial placentation, and vascular endothelial growth factor (VEGF), angiopoietins, and their receptors may be involved in the molecular regulation of this process. To determine the potential role of these putative regulators in a widely studied primate, the common marmoset, we investigated their mRNA expression and protein location in the placenta throughout pregnancy using in situ hybridization, Northern blot analysis, and immunocytochemistry. VEGF was localized in decidual and cytotrophoblast cells, and its highest expression was found in the maternal decidua. The Flt receptor was exclusively detected in the syncytial trophoblast with increasing expression in placentae from 10 wk to term. Soluble Flt (sFlt) was also detectable by Northern blot analysis. KDR receptor expression was restricted to mesenchymal cells during early placentation and to the fetoplacental vasculature during later placentation. KDR expression increased throughout pregnancy. Angiopoietin-1 (Ang-1) was localized in the syncytial trophoblast, being highly expressed in the second half of gestation. Ang-2 mRNA localized exclusively to maternal endothelial cells, and was highly expressed in 10-wk placentae. The Tie-2 receptor was found in cytotrophoblast cells and in fetal and maternal vessels. High Tie-2 levels were detected in the wall of chorion vessels at 14-wk, 17-wk, and term placentae. These results suggest that the processes of trophoblast invasion, maternal vascular transformation, and fetoplacental vascular differentiation and development are regulated by the specific actions of angiogenic ligand-receptor pairs. Specifically, 1) VEGF/Flt and Ang-1/Tie-2 may promote trophoblast growth, 2) VEGF/KDR and Ang-1/Tie-2 may support fetoplacental vascular development and stabilization, 3) sFlt may balance VEGF actions, and 4) Ang-2/Tie-2 may remodel the maternal vasculature.

The involvement of protein kinase C (PKC) and arachidonic acid (AA) pathways were investigated in the GnRH regulation of oocyte meiosis and follicular testosterone production in the goldfish ovary. The results clearly demonstrate differences in the postreceptor mechanisms involving the stimulatory and inhibitory actions of GnRH peptides on basal and gonadotropin (GtH)-induced reinitiation of oocyte meiosis and steroidogenesis. In isolated goldfish follicles in vitro, the observed stimulatory effects of both salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II) on germinal vesicle breakdown were completely blocked by addition of PKC inhibitors, suggesting the involvement of PKC, presumably through activation of phospholipase C/diacylglycerol pathways in the GnRH-induced reinitiation of oocyte meiosis. Administration of an AA metabolism inhibitor, however, only blocked the stimulatory effect of sGnRH without affecting cGnRH-II-induced meiosis. As observed previously, in the presence of GtH, sGnRH was found to inhibit GtH-induced resumption of meiosis and testosterone production, whereas cGnRH-II was without effect. The inhibitory effect of sGnRH on GtH-induced meiosis and steroidogenesis was completely reversed by addition an AA metabolism inhibitor, whereas PKC inhibitors had no effect. These findings provide functional evidence in support of the novel hypothesis that goldfish ovarian follicles contain GnRH-receptor subtypes with different ligand selectivity mediating stimulatory and inhibitory actions of sGnRH and cGnRH in the goldfish ovary.

Evidence is increasing that complement components might play a role in fertilization. C1q, the first component of the classical complement cascade, has the ability to promote sperm agglutination in a capacitation-dependent manner as well as an effect on sperm-oolemma binding and fusion. We have previously detected gC1qR, the receptor for the globular head portion of C1q, on the surface of capacitated sperm. In this study, we examined the expression of gC1qR in both fresh and capacitated human spermatozoa. We performed immunoprecipitation for gC1qR and analyzed biotinylated sperm membrane by Western blot to illustrate an increase in receptor density after overnight capacitation. These results were confirmed by flow cytometric analysis of spermatozoa using fluorescein isothiocyanate-labeled monoclonal anti-gC1qR antibody. Confocal, indirect immunofluorescence microscopy revealed an increase in receptor expression over the rostral portion of the sperm head after capacitation. In addition, the ability of live spermatozoa to bind to monoclonal anti-gC1qR antibody-coated microtiter wells was also increased after capacitation. These results suggest that gC1qR may play a role in human fertilization.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide isolated from ovine hypothalamus. It has been demonstrated to be transiently expressed in preovulatory follicles and to positively affect several parameters correlated with the ovulatory process. The aim of the present study was to investigate whether PACAP influences the plasminogen/plasmin system in rat ovary. Plasminogen activators (PAs) are serine proteases, modulated by gonadotropins and several peptides in preovulatory follicles, that appear to be involved in ovulation. Granulosa cells obtained from immature eCG-treated rats were cultured for 24 h in the presence of increasing concentrations of PACAP and vasoactive intestinal peptide (VIP). A significant, dose-dependent increase in tissue-type PA (tPA) activity and decrease in urokinase-type (uPA) PA activity were observed in PACAP-treated cells. These effects were exerted at the mRNA level. The use of cycloheximide, a protein synthesis inhibitor, suggested that PACAP requires an intermediary protein to decrease uPA-mRNA, but not to induce tPA-mRNA. However, no significant modulation of PAs was observed in the presence of VIP. When granulosa cells were stimulated within the intact follicle (i.e., maintaining the three-dimensional structure and in the presence of the theca cell layers), both PACAP and VIP dose-dependently stimulated tPA. These data suggest that, in addition to the PACAP type I receptor present on granulosa cells, different subtypes of PACAP receptors are present in the different ovarian compartments.

A morphological classification of the immature cumulus-oocyte complex (COC), which grossly resembled the atresia grade of its follicle source, was used in bovine oocytes to determine 1) the developmental potential by either in vitro fertilization or parthenogenetic activation, 2) the calcium current activity by whole-cell voltage clamp technique, and 3) the intracytoplasmic calcium stores by microfluorimetric evaluation. The COC classification took into account some cumulus and ooplasm features, designated as follows: A) presence of a clear and compact cumulus and translucent ooplasm, B) dark and compact cumulus and dark ooplasm, and C) dark and expanded cumulus and dark ooplasm. We found no difference between in vitro fertilization and parthenogenetically activated oocytes in terms of cleavage rate and blastocyst production. Both protocols indicated a significant variability between the three compared COC categories. The B-COCs showed the highest embryo production efficiency as well as the greatest Ca² current activity, whereas A-COCs showed an opposite pattern. The C-COCs, mostly attributed to atretic and heavily atretic follicles, showed morphological characteristics between those of A- and B-COCs. Stores of Ca² were significantly greater in A-COCs than in B- and C-COCs in the case of immature oocytes, and greater in B-COCs than in C-and A-COCs in the case of in vitro-matured oocytes. These results demonstrate that in the bovine 1) the considered morphological criteria for oocyte classification are related to developmental competence, 2) plasma membrane Ca² current in the immature oocyte is related to developmental potential, and 3) calcium stores are related to morphological quality in immature oocytes and to developmental competence in mature oocytes.

We have identified KRP3, a novel kinesin-related protein expressed in the mammalian testis, and have examined the tissue distribution and subcellular localization of isoforms of this protein. Isolation of KRP3 clones, using the head domain identified in a previous PCR screen as probe, identified at least two KRP3 isoforms in the rat. We have isolated coding sequences of two highly related cDNAs from the rat testis that we have termed KRP3A and KRP3B (kinesin-related protein 3, A and B). Both cDNAs code for predicted polypeptides with the three-domain structure typical of kinesin superfamily members; namely a conserved motor domain, a region capable of forming a limited coiled-coil secondary structure, and a globular tail domain. Although almost identical in their head and stalk domains, these motors diverge in their tail domains. This group of motors is found in many tissues and cell types. The KRP3B motor contains DNA-binding motifs and an RCC1 (regulator of chromosome condensation 1) consensus sequence in its tail domain. Despite this similarity, KRP3B is not associated with the same structures as RCC1. Instead, KRP3 isoforms localize with the nuclei of developing spermatids, and their immunolocalization in the testis overlaps with that of the small GTPase Ran. Like Ran, KRP3 motors are associated in a polarized fashion with the nucleus of maturing spermatids at various stages of elongation. Our findings suggest a possible role for KRP3 motor isoforms in spermatid maturation mediated by possible interaction with the Ran GTPase.