All mammalian uteri contain endometrial glands that synthesize or transport and secrete substances essential for survival and development of the conceptus (embryo/fetus and associated extraembryonic membranes). In rodents, uterine secretory products of the endometrial glands are unequivocally required for establishment of uterine receptivity and conceptus implantation. Analyses of the ovine uterine gland knockout model support a primary role for endometrial glands and, by default, their secretions in peri-implantation conceptus survival and development. Uterine adenogenesis is the process whereby endometrial glands develop. In humans, this process begins in the fetus, continues postnatally, and is completed during puberty. In contrast, endometrial adenogenesis is primarily a postnatal event in sheep, pigs, and rodents. Typically, endometrial adenogenesis involves differentiation and budding of glandular epithelium from luminal epithelium, followed by invagination and extensive tubular coiling and branching morphogenesis throughout the uterine stroma to the myometrium. This process requires site-specific alterations in cell proliferation and extracellular matrix (ECM) remodeling as well as paracrine cell-cell and cell-ECM interactions that support the actions of specific hormones and growth factors. Studies of uterine development in neonatal ungulates implicate prolactin, estradiol-17β, and their receptors in mechanisms regulating endometrial adenogenesis. These same hormones appear to regulate endometrial gland morphogenesis in menstruating primates and humans during reconstruction of the functionalis from the basalis endometrium after menses. In sheep and pigs, extensive endometrial gland hyperplasia and hypertrophy occur during gestation, presumably to provide increasing histotrophic support for conceptus growth and development. In the rabbit, sheep, and pig, a servomechanism is proposed to regulate endometrial gland development and differentiated function during pregnancy that involves sequential actions of ovarian steroid hormones, pregnancy recognition signals, and lactogenic hormones from the pituitary or placenta. That disruption of uterine development during critical organizational periods can alter the functional capacity and embryotrophic potential of the adult uterus reinforces the importance of understanding the developmental biology of uterine glands. Unexplained high rates of peri-implantation embryonic loss in humans and livestock may reflect defects in endometrial gland morphogenesis due to genetic errors, epigenetic influences of endocrine disruptors, and pathological lesions.

It is generally accepted that spermatozoa become functionally mature during epididymal transit. The objective of this study was to determine whether the cellular location of equine PH-20 is modified during epididymal transit and, if so, the mechanism for such modification. Sperm were isolated from caput and cauda epididymal regions from stallions undergoing castration (n = 7) and used as whole sperm cell or subjected to nitrogen cavitation for isolation of plasma membrane proteins. Both caput and cauda sperm and sperm protein extracts were subjected to N-deglycosylation, O-deglycosylation, or trypsinization. The SDS-PAGE and Western blot analysis using a polyclonal anti-equine PH-20 IgG were performed in sperm extracts, and indirect immunofluorescence on whole sperm was also performed to determine the cellular distribution of plasma membrane PH-20 following similar treatments (deglycosylation or trypsinization). Hyaluronan substrate gel electrophoresis was performed to detect hyaluronidase activity in SDS-PAGE proteins. Western blots revealed significant differences in electrophoretic migration of PH-20 proteins from caput and cauda epididymal sperm. No effect was seen from deglycosylation treatments on the Western blot pattern; caput protein extracts exposed to trypsin showed the same band pattern as extracts from the cauda epididymis. N-deglycosylation resulted in the loss of hyaluronidase activity of sperm from both epididymal regions, whereas O-deglycosylation or trypsinization did not affect hyaluronidase activity. In caput epididymal sperm, the PH-20 protein is distributed over the entire sperm head; in cauda epididymal sperm, it is restricted to the postacrosomal region. No effect from deglycosylation on the cellular distribution of PH-20 was observed; however, treatment with trypsin changed the cellular distribution of PH-20 in caput sperm similar to that of the distribution of cauda sperm. These results suggest that PH-20 distribution during epididymal maturation is dependent on proteolytic trypsin-like mechanisms and, possibly, on complementary membrane-associated factors.

The 94-kDa ram epididymal fluid form of the sperm membrane-derived germinal angiotensin I-converting enzyme (ACE) was purified by chromatography, and some of its enzymatic properties were studied. For the artificial substrate furanacryloyl-l-phenylalanylglycylglycine (FAPGG), the enzyme exhibited a Michaelis constant (K_m) of 0.18 mM and a V_max of 34 μmoles/(min·mg) and for hippuryl-l-histidyl-l-leucine a K_m of 2.65 mM and a V_max of 163 μmoles/(min·mg) under the defined standard conditions (300 mM NaCl and 50 mM Tris; pH 7.5 and 8.3, respectively). The FAPGG hydrolysis was decreased by 82.5% and 67.5% by EDTA and dithioerythritol, respectively, and was totally inhibited by specific ACE inhibitors such as captopril, P-Glu-Trp-Pro-Arg-Pro-Glu-Ile-Pro-Pro, and lisinopril. Optimum activity for FAPGG was with pH 6.0, 50 mM chloride, and 500 μM zinc. Under the various conditions tested, bradykinin, angiotensin (Ang) I, Ang II, and LHRH were competitors for FAPGG. Bradykinin and angiotensin I were the best competitors. The enzyme cleaved Ang I into Ang II, and the optimal conditions were with pH 7.5 and 300 mM chloride. The relationship between the carboxypeptidase activity in seminal plasma and the prediction of fertility of young rams was also studied. These results indicated a correlation between sperm concentration and ACE activity in semen but showed no statistically significant correlation between such activity and fertility of the animal. Finally, we tested the role of ACE in fertilization; no difference in the in vitro fertilization rate was observed in the presence of 10⁻⁴ M captopril.

When Sertoli cells were cultured in vitro on Matrigel-coated bicameral units, the assembly of the inter-Sertoli tight junction (TJ) permeability barrier correlated with an induction of occludin expression. Inclusion of a 22-amino acid peptide, NH₂-GSQIYTICSQFYTPGGTGLYVD-COOH, corresponding to residues 209–230 in the second extracellular loop of rat occludin, at 0.2–4 μM into Sertoli cell cultures could perturb the assembly of Sertoli TJs dose-dependently and reversibly. This peptide apparently exerts its effects by interfering with the homotypic interactions of two occludin molecules between adjacent Sertoli cells at the sites of TJs, thereby disrupting TJs, which, in turn, causes a decline in transepithelial electrical resistance across the Sertoli cell epithelium. When similar experiments were performed using a 22-amino acid myotubularin peptide, NH₂-TKVNERYELCDTYPALLAVPAN-COOH (residues 156–177), no effects on the assembly of inter-Sertoli TJs in vitro were noted. When a single dose of this synthetic occludin peptide was administered to adult rats intratesticularly at 1.5–10 mg/testis, germ cells began to deplete from the seminiferous epithelium within 8–16 days. By 27 days, virtually all tubules were devoid of germ cells. This antispermatogenic effect was reversible, because germ cells progressively repopulated the epithelium thereafter. Treated testes were indistinguishable from normal or control testes by 68 days post-occludin peptide treatment when assessed using histological analysis. In contrast, control rats receiving either no treatment, vehicle alone, or a 22-amino acid synthetic peptide of myotubularin displayed no changes in the testicular morphology at all time points. The occludin peptide-induced germ cell depletion was also accompanied by a disruption of the blood-testis barrier (BTB) when assessed by micropuncture techniques quantifying [¹²⁵I]-BSA in rete testis fluid and seminiferous tubular fluid following i.v. administration of [¹²⁵I]-BSA through the jugular vein. These results illustrate that the occludin peptide-induced disruption of the BTB may possibly affect the underlying adherens junctions, which causes premature release of germ cells from the epithelium and reversible infertility.

Activin A regulation of the expression of mRNA for the LH receptor, FSH receptor, and the inhibin α subunit as well as the effect of activin A on the secretion of progesterone were investigated in chicken granulosa cell cultures. Granulosa layers were isolated from the F₁ and F₃ F₄ follicles from five hens, pooled according to size, dispersed, and cultured for 48 h. In experiment 1 (n = 3 replications), granulosa cells were cultured with or without highly purified ovine (o) FSH at 50 ng/ml and in the presence of 0, 10, or 50 ng/ml of recombinant chicken activin A. Experiment 2 (n = 4 replications) followed the same protocol as experiment 1, except that oFSH was replaced with oLH. Results from these experiments showed that addition of activin A to the granulosa cell cultures had no effect on the expression of mRNA for the inhibin α subunit or the FSH receptor, but it did affect the expression of mRNA for the LH receptor. Treatment of F₃ F₄ granulosa cells with LH stimulated the expression of mRNA for the LH receptor; however, when LH was combined with either dose of activin A, this induction was prevented. The highest dose of activin A with or without LH resulted in decreased expression of the LH receptor compared to the untreated controls in the F₃ F₄ cell cultures. Progesterone secretion by the granulosa cells from both follicle sizes was not altered by activin A. In experiment 3 (n = 3 replications), the effect of activin A on the growth of granulosa cells was examined with the following treatments: 0, 10, or 50 ng/ml of activin A; 50 ng/ml of either oLH or oFSH; and oLH or oFSH combined with 10 ng/ml of activin A. The highest dose of activin reduced the rate of granulosa cell proliferation in both follicle types. Growth of F₁ and F₃ F₄ granulosa cells was stimulated by the addition of either gonadotropin, and the presence of 10 ng/ml of activin A with either gonadotropin did not alter this proliferation, except for the LH-treated F₃ F₄ granulosa cells, in which the increase in proliferation was prevented. The results suggest that activin A could act as a local factor that regulates follicular maturation by preventing excessive or untimely LH receptor expression.

In human fertilization, the sperm introduces the centrosome; the microtubule-organizing center and microtubules are organized within the inseminated egg from the sperm centrosome. These microtubules form a radial array, called the sperm aster, the functioning of which is essential to pronuclear movement for union of male and female genome. The sperm centrosomal function is considered to be necessary for the normal human fertilization process. Therefore, the dysfunction of sperm centrosome is a possible cause of human fertilization failure. However, little information is available regarding human sperm centrosomal function during fertilization in clinically assisted reproductive technology. To assess the human sperm centrosomal function, we examined sperm aster formation and pronuclear decondensation following intracytoplasmic sperm injection (ICSI) with human sperm into the bovine egg using a Piezo-driven pipette and ethanol activation of eggs. After human sperm incorporation into bovine egg, we observed that the sperm aster was organized from sperm centrosome, and that the sperm aster was enlarged as the sperm nuclei underwent pronuclear formation. The sperm aster formation rate at 6 h post-ICSI and the male pronuclear formation rate at 8–12 h post-ICSI were 60.0% and 83.3%, respectively. No difference of the sperm aster formation rate and the male pronuclear formation rate was observed between eggs activated with ethanol and eggs without artificial activation. We concluded that this heterologous Piezo-ICSI system into bovine egg can be a novel assay for human sperm centrosomal function, and it is possible to explicate a course of fertilization failure that was unknown until now.

Efficient transfer of glucose from the mother to the embryonic compartment is crucial to sustain the survival and normal development of the embryo in utero, because the embryo's production of this primary substrate for oxidative metabolism is minimal. In the present study, the temporal sequence of expression of the sodium-independent facilitative glucose transporter isoforms GLUTs 1, 3, 4, and 5 was investigated in the developing rat uteroembryonic unit between conception and Gestational Day 8 using immunohistochemistry. The GLUTs 1, 3, and 4 were expressed in the embryonic tissues after the start of implantation, being colocalized in the parietal endoderm, visceral endoderm, primary ectoderm, extraembryonic ectoderm, and the ectoplacental cone. In the uterus, a faint GLUT1 labeling emerged, but not until Gestational Day 3, in the luminal epithelium, endometrial stroma, and decidual cells. The intensity of GLUT1 staining increased in the latter population with progressing decidualization. Endometrial glands and myometrial smooth muscle cells stained neither for GLUT1 nor for GLUT3 until postimplantation. During all developmental stages examined, GLUT4 was visualized throughout the pregnant rat uterus, as was GLUT3 (with the above-mentioned exceptions). The density of GLUT5 was generally less than the sensitivity of the immunohistochemical detection method in all tissues investigated. In conclusion, the data point to a significant expression of the high-affinity glucose transporters GLUTs 1, 3, and 4 in the rat uteroembryonic unit, providing supportive evidence for an important role of facilitative glucose diffusion during peri-implantation development.

Erectile dysfunction in the aging male is caused, in part, by inadequate relaxation of the corpora cavernosal smooth musculature. Calcitonin gene-related peptide (CGRP), a peptide neurotrasmitter localized in the corpora cavernosa, is down-regulated in the aging rat penis. We examined the hypothesis that this reduction in CGRP may contribute to decreased cavernosal smooth muscle relaxation. Therefore, we sought to determine whether adenoviral-mediated gene transfer of prepro-CGRP (AdRSVCGRP) could enhance erectile responses in aged rats. We found a significant decrease in CGRP concentrations and in cAMP and cGMP levels in aged rat cavernosal tissue compared to younger rats. Aged rats also had significantly lower erectile function as determined by cavernosal nerve stimulation compared to younger rats. Five days after transfection with AdRSVCGRP, these aged rats had an approximately threefold increase in cavernosal CGRP levels compared to animals transfected with adenoviruses encoding nuclear-targeted β-galactosidase (AdRSVβgal). The AdRSVCGRP-transfected animals also demonstrated an increase in CGRP mRNA and immunohistochemical localization of CGRP in the smooth muscle of the corpora cavernosa. In addition, cAMP levels in the corpora cavernosa were significantly increased, whereas cGMP levels remained unchanged. Adenoviral transduction efficiency of β-galactosidase reporter gene was measured by chemiluminescence and was observed in cavernosal tissue 5 days after transfection with AdRSVβgal. More importantly, 5 days after administration of AdRSVCGRP, a significant increase was observed in the erectile response to cavernosal nerve stimulation in the aged rat, similar to the response observed in younger rats. These data suggest that in vivo adenoviral gene transfer of CGRP can physiologically improve erectile function in the aged rat.

A null mutation in the murine gene encoding steroid 5α-reductase type 1 (5αR1) leads to failure of normal parturition at term. This observation, together with the finding that mRNA levels of uterine 5αR1 increase significantly at term in normal pregnant animals, indicates that 5αR1 plays an important role in murine parturition. The current studies were conducted to elucidate the regulation of 5αR1 in uterine tissues of nonpregnant and pregnant animals. Nonpregnant, ovariectomized ICR mice were treated with vehicle (control), 17β-estradiol (E₂), progesterone (P₄ ), or E₂ P₄ for 3 days. Thereafter, uterine tissues were obtained for histology, quantification of 5αR1 specific activity, and Northern blot analysis of 5αR1 mRNA expression. The 5αR1 enzyme activity was significantly increased in animals treated with E₂ P₄. However, activity was much less in uterine tissues from E₂ P₄-treated animals than in uterine tissues from pregnant animals near term. To evaluate further the regulation of 5αR1 during gestation, mice underwent unilateral tubal ligation before timed matings. The 5αR1 activity increased eightfold in uterine tissues from the fetal horn from Gestational Days 12 to 18. This temporal pattern in 5αR1 activity paralleled marked increases in uterine diameter. Taken together, these studies indicate that expression of 5αR1 is regulated by E₂ P₄ in uterine tissues. Whereas E₂ alone is insufficient to induce enzyme activity, E₂ may be required to increase P₄ receptors and, thereby, mediate the effects of P₄ on 5αR1 gene expression. Further increases in enzyme activity during late gestation are mediated by fetal occupancy, possibly through stretch-induced increases in endometrial growth. Thus, like other genes involved in parturition, expression of 5αR1 is regulated by both hormonal and fetal-derived signaling pathways.

Bovine ovarian cumulus-oocyte complexes (COCs) are used for in vitro maturation and fertilization after selection by size and morphology, but their developmental potential remains low. Stereology could provide more objective criteria for selecting the most competent complexes, but its application is lacking in cattle. COCs from small (1–4 mm) antral follicles were aspirated from diestrous ovaries of Holstein-Friesian cows, fixed in glutaraldehyde, randomly embedded in glycol-methacrylate, and sectioned at 20 μm. The unbiased nucleator principle was used for estimating the mean volumes of complexes, oocytes, cumulus cells, and nuclei of oocytes and cumulus cells. The thickness of the zona pellucida and the relative numerical percentages of the several morphologic types of cumulus cells were also evaluated. The optical disector procedure was used for cumulus cell sampling. Volume estimation based on a real physical unique point did not differ from those based on a particular point among many or on a virtual central point, and the mean cumulus cell volume was estimated by using the single section bearing the unique reference point. Quantitative data showed that COCs appear heterogeneous for all studied parameters and that the cumulus mass contains three different cell populations.

Desert hedgehog (Dhh) is a cell-signaling molecule that was first discovered in Drosophila. A unique testicular phenotype has been described in neonatal and adult Dhh-null animals that includes anastomotic seminiferous tubules, pertitubular cell abnormalities, and absence of adult-type Leydig cells. In the present study, we addressed the developmental basis for the abnormalities previously described for the adult Dhh-null phenotype. The source of Dhh is the Sertoli cell, and receptors are localized on peritubular cells and possibly Leydig cells. The development of testes from Dhh-null mouse embryos was studied using light and electron microscopy at 11.5, 12.5, 13.5, and 16.5 days postcoitum (dpc) and was compared with that in control Dhh heterozygous and wild-type embryos. Dhh-null and control testes were generally similar during the period of early cord formation (11.5–12.5 dpc). By 13.5 dpc, the basal lamina delimiting the cords was lacking in some regions and disorganized in Dhh-null testes, and occasional germ cells were seen outside cords. At 16.5 dpc, these defects were more prominent and cord organization was less well defined than in controls. In addition, there were numerous extracordal germ cells, some of which were partially enclosed by a somatic cell of unknown identity. Numerous fibroblast-like cells, apparently secreting collagen and basal lamina, characterized the interstitium of the Dhh-null testis. These defects likely stem from abnormal peritubular stimulation due to the lack of Dhh, leading to the abnormalities seen in the developmental stages studied here and in the adult testis.

Selection of dominant follicles in cattle is associated with a deviation in growth rate between the dominant and largest subordinate follicle of a wave (diameter deviation). To determine whether acquisition of ovulatory capacity is temporally associated with diameter deviation, cows were challenged with purified LH at known times after a GnRH-induced LH surge (experiment 1) or at known follicular diameters (experiments 2 and 3). A 4-mg dose of LH induced ovulation in all cows when the largest follicle was ≥12 mm (16 of 16), in 17% (1 of 6) when it was 11 mm, and no ovulation when it was ≤10 mm (0 of 19). To determine the effect of LH dose on ovulatory capacity, follicular dynamics were monitored every 12 h, and cows received either 4 or 24 mg of LH when the largest follicle first achieved 10 mm in diameter (experiment 2). The proportion of cows ovulating was greater (P < 0.05) for the 24-mg (9 of 13; 69.2%) compared with the 4-mg (1 of 13; 7.7%) LH dose. To determine the effect of a higher LH dose on follicles near diameter deviation, follicular dynamics were monitored every 8 h, and cows received 40 mg of LH when the largest follicle first achieved 7.0, 8.5, or 10.0 mm (experiment 3). No cows with a follicle of 7 mm (0 of 9) or 8.5 mm (0 of 9) ovulated, compared with 80% (8 of 10) of cows with 10-mm follicles. Thus, follicles acquired ovulatory capacity at about 10 mm, corresponding to about 1 day after the start of follicular deviation, but they required a greater LH dose to induce ovulation compared with larger follicles. We speculate that acquisition of ovulatory capacity may involve an increased expression of LH receptors on granulosa cells of the dominant follicle and that this change may also be important for further growth of the dominant follicle.

The enzyme glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) in an NADPH-dependent manner. A specific antibody raised against recombinant rat GR was used to localize the protein in the female reproductive organs during the estrous cycle in the rat. In the ovary, the strongest reactivity to the antibody was observed in oocytes, followed by granulosa cells, corpus luteum, and interstitial cells. A strongly positive reaction was also observed mainly in the oviduct epithelia, uterine epithelia, and endometrial gland in the reproductive tract. Oviducts contained the highest GR activity. The GR activity of uterus during metestrus was about twice as high as that for other stages of the cycle. The levels of GR proteins in the tissues roughly matched the activities. The expression of the GR mRNA was highest during metestrus. Because GSH is known to increase gamete viability and the efficiency of fertility, GR, which is expressed in these tissues, is predicted to play a pivotal role in the reproduction process as a source of GSH.

Oxidative base (8-oxoguanine) damage, DNA fragmentation, and apoptosis occurred among ovarian surface epithelial cells within the formative site of ovulation in sheep. The incidence of 8-oxoguanine adducts in surviving antiapoptotic Bcl-2/base excision repair polymerase β-positive cells at the margins of ruptured follicles (which avoid the focal point of the ovulatory assault) was intermediate between apoptotic and outlying healthy epithelium. Cells containing perturbations to DNA expressed the tumor suppressor p53. Localized reactions of DNA injury and programmed cellular death were averted by ovulation blockade with indomethacin. Progesterone enhanced the biosynthesis of polymerase β in ovarian surface epithelial cells exposed in vitro to a sublethal concentration of H₂O₂. Ovulation is a putative etiological factor in common epithelial ovarian cancer. A genetically altered progenitor cell, with unrepaired DNA, but not committed to death, could give rise to a transformed phenotype that is hence propagated upon healing of the ovulatory wound; it appears that this incongruity is normally reconciled by up-regulation of the base excision repair pathway during the ensuing luteal phase.

Equatorin is a sperm head equatorial protein, possibly involved in sperm-oocyte fusion (Toshimori et al., Biol Reprod 1998; 59:22–29). In the present work, we have shown that equatorin contained in the posterior acrosome is detectable only after spontaneous or induced acrosome reactions following fixation and permeabilization, but not in intact spermatozoa. The presence of protease inhibitors during sonication or ionophore treatments does not inhibit the exposure of the antigenic epitope. The zona-penetrated spermatozoa lying in the perivitelline space display equatorin, similar to those of the acrosome-reacted ones. After sperm-egg fusion during in vitro fertilization (IVF), the equatorin dissociates from the sperm head equatorial region and remains at the vicinity of the decondensing male pronuclei. During pronuclear apposition stage, it is pushed away from the pronuclei, possibly by the perinuclear microtubules. After first cleavage, equatorin is inherited by one of the proembryonic cells. The residual equatorin disappears after the second cleavage. Microinjected whole spermatozoa or sperm heads into the MII stage oocytes display equatorin similar to those of the perivitelline sperm. After activation, it dissociates from the sperm nuclei in a similar manner as during IVF. The mode of equatorin degeneration during fertilization is similar to those of the sperm tail components or mitochondria, but different from those of the membrane associated proteins.

The rat pregnancy-specific glycoprotein gene rnCGM3 is primarily expressed in the placenta. Previously, three DNase I footprinting sites (FPI, FPII, and FPIII) were identified in the rnCGM3 promoter region, a yeast one-hybrid screen was performed to identify the nuclear factors binding to the FPIII (5′-GCCTGGGAAAAAACTC-3′) element, and RBPJκ, a downstream effector of the Notch signaling pathway, was identified as one of the FPIII-binding factors. In the present study, the NFκB member p65 was identified as another FPIII-binding factor. Electrophoretic mobility shift assays showed that NFκB members, including p50 and p65, bound to the FPIII site. The core binding sequence in the FPIII element for p50 and p65 is GGGAAA, which overlaps with that for RBPJκ. Competition exists between p50 and RBPJκ for binding to the FPIII element. Transient expression analyses revealed that p65 significantly stimulated the expression of a reporter gene directed by the NFκB core sequence in the FPIII element. However, RBPJκ could block this stimulation. These results suggest that the regulation of rnCGM3 expression involves both NFκB and RBPJκ, and they are mutually exclusive in the FPIII element.

It is generally accepted that cyclic nucleotides are key signaling molecules in the control of oocyte meiotic resumption. Given the role of phosphodiesterases (PDEs) in cyclic nucleotide degradation, this study was undertaken to investigate the properties and regulation of PDEs expressed in rat oocytes. Cilostamide-sensitive PDE3 was the major activity detected in denuded oocytes, whereas no PDE3 activity could be detected in cumulus cells. Moreover, comparable levels of PDE3 activity were measured in cumulus-oocyte complexes (COCs) and in denuded oocytes. The oocyte PDE was recovered in the soluble fraction of the homogenate and immunoprecipitated with a specific PDE3A antibody. A significant and transient increase (P < 0.05) in PDE3 activity was measured in the oocytes after 30 min of culture (70 min after isolation) compared with immediately after collection (10 min after isolation). Conversely, no changes in activity were observed when denuded oocytes or cumulus cells were incubated for up to 130 min. Evaluation of oocyte maturation indicated that only 10% of oocytes had resumed meiosis at the peak of the PDE3 activity. A significant increase (P < 0.05) in PDE3 activity was measured in COCs when follicle-enclosed oocytes were cultured in the presence of hCG. Again, this increase preceded oocyte maturation. In conclusion, these data demonstrate that PDE3A is the major PDE form expressed in mammalian oocytes. PDE3A activity increases prior to resumption of meiosis in both spontaneous and gonadotropin-stimulated maturation. These findings strongly support the hypothesis that an increase in oocyte PDE3A activity is one of the intraoocyte mechanisms controlling resumption of meiosis in rat oocytes, at least in vitro.

The CRES protein is a member of the cystatin superfamily of cysteine protease inhibitors with restricted expression in stage-specific germ cells, proximal caput epididymidis, and anterior pituitary gonadotroph cells. To elucidate the molecular mechanisms regulating the highly restricted expression of the cres gene, we have sequenced 1.6 kilobases of mouse cres 5′ flanking sequence and performed studies to examine the cres gene promoter. Two putative CCAAT/enhancer binding protein (C/EBP) transcription factor binding motifs exist within the first 135 base pairs of cres promoter. Furthermore, our studies demonstrate that cres mRNA levels are dramatically reduced in the epididymides of C/EBPβ-deficient mice. These data suggest that the C/EBP family of transcription factors, in particular C/EBPβ, plays a role in the regulation of cres gene expression. In support of this finding, Northern blot analysis showed that C/EBPβ is the predominant C/EBP family member expressed in the LβT2 gonadotroph cell line and the proximal caput epididymidis. Also, gel shift and supershift assays demonstrated that C/EBPβ protein in nuclear extracts from LβT2 gonadotroph cells and epididymal cells bound to the two C/EBP sites in the cres promoter. Finally, to test the in vivo function of the C/EBP sites in cres gene expression, transfection studies were performed in LβT2 gonadotroph cells and two heterologous cell systems. These experiments showed a significant reduction of cres transactivation when either C/EBP sites were mutated, and no transC/EBP activation of the cres promoter when both C/EBP sites were mutated. Taken together, these studies demonstrate that the C/EBPβ transcription factor is necessary for high levels of cres gene expression in the proximal caput epididymidis and anterior pituitary gonadotroph cells.

We have recently shown that not only bradykinin, but also all components for the production of bradykinin, can be detected within the follicle of porcine ovaries. To elucidate the relevance of the intrafollicular bradykinin-producing system to its physiological role, we investigated the distribution of bradykinin receptor (B₂R) mRNA and the protein in porcine ovaries. A cDNA encoding porcine B₂R was first cloned from a porcine uterus cDNA library. The receptor mRNA was scarcely detected in the ovary by Northern blot analysis. Polymerase chain reaction analysis with total RNAs isolated from the ovary and from granulosa cells of small and large follicles demonstrated the ovarian expression of B₂R mRNA. The B₂R protein was detected by Western blot analysis in extracts of isolated granulosa cells. In situ hybridization of B₂R mRNA and immunohistochemical analysis of the protein revealed that the receptor is expressed in the theca and granulosa cells of all growing follicles. The effect of bradykinin on the expression of some matrix metalloproteinase (MMP) genes was examined using isolated granulosa cells. Bradykinin treatment induced MMP-3 and MMP-20 gene expression to an extreme degree. The expression of MT1-MMP was also affected by bradykinin treatment. These results suggest that MMPs play a role in follicle rupture during ovulation. The present study provides new information regarding the mechanisms of bradykinin-induced ovulation in porcine ovaries.

The 1-8 family (1-8U, 1-8D, Leu-13/9-27) of interferon (IFN)-inducible genes encodes proteins that are components of multimeric complexes involved with transduction of antiproliferative and homotypic adhesion signals. Human 1-8 family members are highly similar and are regulated by type 1 and type 2 IFNs. Because the bovine uterus is bathed in conceptus-derived IFNτ during early pregnancy, it was hypothesized that members of the 1-8 family were upregulated in the bovine uterus during early pregnancy. Oligonucleotide primers were designed based on human and rat 1-8U and Leu-13 cDNAs and used in reverse transcription polymerase chain reactions to amplify bovine cDNAs from endometrial RNA. The bovine 1-8U cDNA was sequenced, found to be 84% identical to the human 1-8U, and used to screen a bovine endometrial cDNA library to isolate the full-length 1-8U and Leu-13 cDNAs. The inferred amino acid sequences of bovine 1-8U and Leu-13 were 72% and 73% identical to their respective human counterparts. Bovine 1-8U and Leu-13 retain an amino acid motif that is conserved in other 1-8 family members and in some ubiquitin-conjugating enzymes (E2s). This motif is critical for function of E2s in covalently linking ubiquitin to targeted proteins. Northern blotting revealed that bovine endometrial 1-8U and Leu-13 mRNAs were upregulated on Day 15 of pregnancy (P < 0.0001) and continued to accumulate through Day 18 of pregnancy (P < 0.05) when compared with endometrium from nonpregnant cows. The bovine 1-8U and Leu-13 mRNAs were also upregulated (P < 0.05) by IFNτ (25 nM) within 3 h, continued to accumulate through 12 h, and reached a plateau at 12–24 h in cultured bovine endometrial cells. In situ hybridization revealed that mRNAs encoding 1-8 family members were heavily localized to glandular epithelium but also were present to a lesser extent in the luminal epithelium and stroma. The temporal upregulation of 1-8U and Leu-13 mRNAs by pregnancy and IFNτ and tissue distribution of these mRNAs paralleled closely that of the ubiquitin homolog, IFN-stimulated gene product 17. These IFN-induced proteins probably work together to prepare the endometrium for adhesion of the developing conceptus.

Mouse embryos at different stages of development were cocultured with human oviduct cells or cultured in the presence of oviduct-derived embryotrophic factor-1, -2, and -3 (ETF-1, -2, and -3) for various amounts of time within the preimplantation period. Cocultures that included the period from 48 to 72 h post-hCG stimulated cell division and increased the cell numbers in the inner cell mass (ICM) of the exposed blastocyst. Exposure of embryos to oviductal cells from 96 to 120 h post-hCG increased the cell number in the trophectoderm (TE), blastocyst size, hatching rate, attachment, and in vitro spreading of the blastocyst. ETF-1 and ETF-2 affected embryos between 48 and 72 h post-hCG by increasing the number of cells in the ICM. In contrast, ETF-3 had a more profound effect on embryos that were exposed from 96 to 120 h post-hCG, where it mostly affected the development of TE cells, leading to higher hatching rate. Human oviductal cells improved mouse embryo development partly by the production of high molecular weight embryotrophic factors. These factors had differential effects on mouse embryo development.

Previous studies have shown that 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity in rats is likely caused by acceleration of the normal rate of atresia (apoptosis). VCD-induced ovotoxicity is specific for small preantral follicles and is associated with increased activity of caspase cascades. The present study was designed to investigate the alteration of expression and distribution of several Bcl-2 family member proteins induced by dosing of VCD in rat small ovarian follicles. Female F344 rats were given a single dose of VCD (80 mg/kg, i.p., 1 day; a time when ovotoxicity is not initiated), or dosed daily for 15 days (80 mg/kg, i.p., 15 days; a time when significant ovotoxicity is underway). Four hours following the final dose, livers and ovaries were collected. Ovarian small (25–100 μm) and large (100–250 μm) preantral follicles were isolated, and subcellular fractions (cytosolic and mitochondrial) were prepared. Compared with controls, levels of the proapoptotic protein, Bad, were greater in both cytosolic and mitochondrial fractions of small preantral follicles collected from 15-day VCD-treated rats (cytosol, 1.97 ± 0.16; mitochondria, 2.20 ± 0.24, VCD/control, P < 0.05). After 15 days of daily VCD dosing, total cellular antiapoptotic Bcl-x_L protein levels were unaffected in small preantral follicles, but its distribution in mitochondrial and cytosolic components was altered (mitochondria, 0.635 ± 0.08; cytosol, 1.39 ± 0.14, VCD/control, P < 0.05). Likewise, VCD did not affect protein levels of proapoptotic Bax in small follicles on Day 15. However, consistent with a Bax-mediated mechanism of apoptosis, the relative ratio of Bax/Bcl-x_L in the mitochondrial fraction of small preantral follicles was significantly increased by VCD dosing (1.62 ± 0.21, VCD/control, P < 0.05). Immunofluorescence staining intensity evaluated by confocal microscopy visualized cytochrome c protein in the cytosolic compartment in granulosa cells of preantral follicles in various stages of development. Relative to controls, within the population of small preantral follicles, staining intensity was less (P < 0.05) and presumably more diffuse, specifically in stage 1 primary follicles from VCD-treated animals (15 days). VCD caused none of these effects in large preantral follicles or liver (not targeted by VCD). These data provide evidence that the apoptosis induced by VCD in ovarian small preantral follicles of rats is associated with increased expression of Bad protein, redistribution of Bcl-x_L protein and cytochrome c from the mitochondria to the cytosolic compartment, and an increase in the Bax/Bcl-x_L ratio in the mitochondria. These observations are consistent with the involvement of Bcl-2 gene family members in VCD-induced acceleration of atresia.

Brief exposure of rats to high doses of natural estrogens early in life results in permanent alterations of the prostate gland, which include differentiation defects, altered gene expression, and dysplasia with aging. Whether low-dose treatments can cause similar effects in the developing prostate remains controversial. The current project was designed to determine the dose-response relationship of the prostate gland to estradiol exposure during the developmentally critical neonatal period in the rat. Male Sprague-Dawley (SD) rats were treated on Days 1, 3, and 5 of life by s.c. injections of a 7-log range of doses (0.015 μg/kg to 15.0 mg/kg) of β-estradiol-3-benzoate (EB) in 25 μl of peanut oil (Arachis) as vehicle. In a separate block, neonatal Fisher 344 (F344) rats received 0.15, 15.0, or 1500.0 μg EB/kg. Rats were killed on Postnatal Day (PND) 35 or 90, and the prostates were microdissected, weighed, and frozen for immunohistochemistry. Preputial separation and hepatic testosterone hydroxlase activities were monitored and measured to determine the onset of puberty. On PND 35, there was an increase in prostate weights of SD rats treated with low doses of EB and a decrease in prostate weights of SD rats treated with high doses. The low-dose effect was entirely abolished by PND 90, and only high-dose suppression of organ sizes was found. The transient nature of the effect in low-dose animals suggests an advancement of puberty as the cause for increased reproductive organ weights on PND 35. F344 rats were more sensitive than SD rats to the suppressive effects of high doses of neonatal EB on PND 90. Despite this heightened responsiveness in the F344 rats, a low-dose estrogenic effect on adult prostate weights was not observed. Thus, in the rat model a sustained effect at low doses of natural estrogens is not present in the prostate glands.

Environmental contaminants with estrogenic properties have been cause for heightened concern about their possible role in inducing adverse health effects. Brief exposure of rodents to high doses of natural estrogens early in life results in permanent alterations of the male reproductive tissues, but the question of whether environmentally relevant doses can cause the same effects remains controversial. The current project was designed to determine the dose-response relationship between neonatal estradiol exposure and the development of the male reproductive tract in the rat. Neonatal male Sprague-Dawley (SD) and Fisher 344 (F344) rats were exposed to β-estradiol-3-benzoate (EB) at concentrations ranging from 0.015 μg/kg body weight (BW) to 15.0 mg/kg BW and 0.15 μg/kg BW to 1.5 mg/kg BW, respectively. Results showed an inverted U-shaped dose-response profile for testis and epididymis weights in 35-day-old SD rats, with increased organ sizes at the low-dose end of the treatment. This effect was transient and was not sustained into adulthood. Increased hepatic testosterone hydroxylase activities in low-dose animals suggest an advancement of puberty as the cause for increased reproductive organ weights. On postnatal day (PND) 90, a stimulatory low-dose response to EB was present in SD rat testicular and epididymal weights, however at one order of magnitude lower dose than that seen on PND 35, suggesting a separate effect. All SD male reproductive tract organs and serum hormones showed a permanent inhibitory response to high doses of neonatal EB. F344 rats exhibited greater estrogen sensitivity on PND 90. Despite this heightened responsiveness, F344 rats did not exhibit a low-dose effect for any endpoint. These low-dose responses to estradiol are organ and strain specific.

The thermostability of the mammalian sperm genome was previously reported, but no live offspring after conception with heated spermatozoa had yet been obtained. In the present study, mouse spermatozoa were heated at 56°C for 30 min and microinjected into mouse oocytes. Fertilization did not occur unless activation was induced by incubation in a calcium-free medium containing strontium. Under these conditions fertilization and cleavage rates were comparable to those obtained after microinjection of control spermatozoa, but the developmental rate to the blastocyst stage was lower. When transferred to foster mothers, embryos derived from heated sperm developed into phenotypically normal offspring, which grew and reproduced normally. In the mouse, heated spermatozoa can therefore support full embryonic development after microinjection into oocytes.

The mouse lactate dehydrogenase c gene (mldhc) is transcribed only in cells of the germinal epithelium. Cloning and analysis of the mldhc promoter revealed that a 100-base pair fragment was able to drive testis-specific transcription in vitro and in transgenic mice. Several testis-specific genes are believed to be regulated at least in part through differential methylation of CpG dinucleotides. We investigated the possibility that transcriptional repression of the mldhc gene is mediated in somatic tissues by hypermethylation of CpG dinucleotides. The CpG dinucleotides within a fragment of the mldhc promoter containing a GC box and tandem activating transcription factor/cAMP-responsive element binding sites are hypermethylated in somatic tissues and hypomethylated in testis. Methylation of the activating transcription factor/cAMP-responsive elements altered the protein binding pattern observed in electrophoretic mobility shift assays using mouse liver but not testis nuclear extract. Furthermore, methylation of an extended mldhc promoter fragment driving lac Z silenced transcription from the promoter in a transient transfection assay. These data suggest that tissue-specific differential methylation plays a role in mldhc silencing in somatic tissues.

The use of nuclear transfer (NT) techniques to create transgenic offspring capable of producing valuable proteins may have a major impact on the pharmaceutical market. Our objective was to compare the in vivo developmental potential of NT embryos produced from the fusion of transgenic donor cells with cytoplasts prepared from either FSH-stimulated ovaries or nonstimulated abattoir-derived ovaries. Donor cells were prepared from a transgenic fetus carrying the gene for human antithrombin III as a marker and used within four to eight subpassages. Cells were serum deprived for 4 days prior to cytoplast transfer. Oocytes were enucleated by removing the metaphase plate using a DNA stain and epifluorescent illumination. Donor cells were fused to enucleated oocytes by electric pulse and then chemically activated. There was no difference in the number of transferable embryos produced from cytoplasts of FSH-stimulated ovaries or from the fusion of cytoplasts from abattoir ovaries, nor was there a difference in the number of pregnancies established per recipient with either treatment. All pregnancies from both groups culminated in the births of healthy female kids (five total). To our knowledge, this is the first report of cloned goats produced from NT using cytoplasts derived from abattoir ovaries.

Earlier studies have shown that the efferent ductules (ED) of the male mouse are a target for estrogen. The loss of estrogen receptor (ER) function through either knockout technology (αERKO mouse) or chemical interference (pure antagonist, ICI 182 780) results in a failure of a major function of the ED, the reabsorption of testicular fluids. The purpose of this study was to test the hypothesis that estrogen controls fluid (water) reabsorption in the ED by modulating ion transporters important for passive water movement through a leaky epithelium such as the ED. Northern blot analysis was used to detect the mRNA levels for key ion transporters in the following experimental groups: 1) wild-type (WT) control for the 14-day experiment, 2) ERα knockout (αERKO) control for the 14-day experiment, 3) WT treated with ICI 182 780 (ICI) for 14 days, 4) αERKO treated with ICI for 14 days, 5) WT control for the 35-day experiment, and 6) WT treated with ICI for 35 days. Estrogen differentially modulated the mRNA levels of key ion transporters. ERα mediated carbonic anhydrase II mRNA abundance, and there was a decrease in Na /H exchanger 3 mRNA levels in the αERKO that appeared to be a cellular effect and not a direct estrogen effect. The loss of ERα control resulted in an increase in mRNA abundance for the catalytic subunit of Na -K ATPase α1, whereas an increase in the mRNA abundance of the Cl⁻/HCO₃⁻ exchanger and the chloride channel cystic fibrosis transmembrane regulator was significantly ERβ mediated. Our results indicate for the first time that estrogen acting directly and indirectly through both ERα and ERβ probably modulates fluid reabsorption in the adult mouse ED by regulating the expression of ion transporters involved in the movement of Na and Cl⁻.

We evaluated the effects of follicular exposure in vitro to either of two mutually exclusive isoforms of FSH (least acidic and acid) on the subsequent capacity of oocytes for embryonic development. The effects of dose and follicle culture duration were examined. At the threshold dose (that required to produce antra) and at one subthreshold dose, the major difference between the two isoform fractions was the timing and effectiveness of acquisition of two-cell embryonic developmental capacity. With the least-acidic fraction, the highest rate of two-cell development (≈80%) occurred after 3 days of follicle culture only at the threshold dose (2.5 ng/ml). With the acid fraction, the highest two-cell rate (≈60%) occurred after 5 days of culture but at equivalent rates over a range of doses between 10 ng/ml and 100 ng/ml (threshold dose was 50 ng/ml). At threshold dose or below, the capacity for two-cell embryo production appeared not to be influenced by antral status for either isoform. At above threshold doses, the least-acidic fraction induced an increasing proportion of antral follicles with increasing dose, but this increase was associated with a progressive decrease in embryo production. This relationship was more extreme after longer culture and was due to degeneration of the cumulus-oocyte complex associated with apparently increased differentiation of the mural granulosa cells. The acid fraction was by comparison less bioactive and insensitive to overdosing. The broader isoform mix of the unfractionated FSH provided a measure of protection against overdosing characteristic of the acid fraction while retaining the capacity of the least-acidic fraction to induce antral formation at a low dose.

In oviparous species, in addition to a full-length estrogen receptor alpha (ERα), another ERα isoform lacking the A domain and exhibiting a ligand-independent transactivation function has been consistently reported. Although both isoforms are expressed in the liver, their respective sites of expression in other potential target tissues are unknown. In contrast to the situation in Xenopus and chicken, the two isoforms of rainbow trout (Oncorhynchus mykiss) are generated from two classes of transcripts with different 5′ untranslated sequences issued from the same gene by alternative splicing and promoter usage. The aim of this study was to take advantage of the unique organization of the rainbow trout ERα gene to investigate the tissue distribution of these two messenger species along the reproductive axis of female trout. The S1 nuclease assay and in situ hybridization were used, with probes specific for each of the transcripts. Reverse transcription polymerase chain reaction (RT-PCR) with primers specific for each of the isoforms also was performed. The data indicated that the full-length ERα is expressed in liver, brain, pituitary, and ovary, whereas expression of the isoform with the truncated A domain is restricted to the liver, demonstrating a tissue-specific expression of these two ERα isoforms. The presence of a short liver-specific isoform in oviparous species suggests its role in the development and/or maintenance of the unique function of the liver in the vitellogenesis process.

The type of donor cell most suitable for producing cloned animals is one of the topics under debate in the field of nuclear transfer. To provide useful information to answer this question, G2/M- and G0/G1-stage fetal fibroblasts were used as donor cells for nuclear transfer. In vitro-matured oocytes derived from abattoir ovaries were used as recipient cytoplasts. In both groups, nuclear envelope breakdown and premature chromosome condensation were completed within 1–2 h after donor cells were injected into the cytoplasm of oocytes. Microtubules were organized around condensed chromosomes and formed a spindle within 1–1.5 h after activation. Decondensation of chromosomes could be seen within 2–4 h after activation. Reformation of the new nuclear envelope occurred 4–6 h after activation and was followed by nuclear swelling and formation of a pronucleus-like structure (PN) 8–12 h after activation. Most (80.6%) of the reconstructed oocytes derived from G2/M cells extruded polar body-like structures (PB). However, a much lower frequency of PB (21.7%) was observed in the reconstructed oocytes derived from G0/G1 donors. A variety of PN and PB combinations were observed in reconstructed oocytes derived from G2/M-stage donors, including 1PN 0PB, 1PN 1PB, 1PN 2PB, 2PN 0PB, 2PN 1PB, 2PN 2PB, and 3PN 1PB. Chromosomes of most embryos (10/13) derived from G2/M stage were diploid. The percentage of cleavage and blastocysts and the average nuclear number of blastocysts in the G2/M and G0/G1 groups were not different. These results demonstrate that the G2/M stage can be morphologically remodeled by cytoplasm of MII oocytes in pigs. To maintain normal ploidy, the extra chromosomes derived from G2/M-stage cells could be expelled by oocytes as a second polar body. G2/M-stage fibroblast nuclei could direct reconstructed embryos to develop to the blastocyst stage.

There is positive feedback pathway in the ovine large luteal cell, such that prostaglandin (PG) F_2α stimulation induces intraluteal PGF_2α production as the result of induction of one of the rate-limiting enzymes in PG production, cyclooxygenase-2 (Cox-2). The objective of the present study was to evaluate the intracellular effector systems and important DNA transcriptional element(s) involved in regulating the Cox-2 gene in ovine large luteal cells. In transient transfection assays, Cox-2 promoter was rapidly induced (4 h) by phorbol didecanoate (a protein kinase [PK] C activator), ionomycin, and cloprostenol (PGF_2α analogue), with a peak induction at 12 h. Cloprostenol-mediated promoter activation was not blocked by inhibition of various second messenger systems, including PKA, calcium calmodulin kinase II, or mitogen-activated protein kinases. However, myristoylated PKC pseudosubstrate peptide inhibited cloprostenol stimulation of Cox-2 promoter, indicating the critical role of PKC in this stimulation. The Cox-2 promoter could be reduced to 282 base pairs (bp) of the 5′ flanking sequence with retention of full inducibility by cloprostenol. Mutation of three critical cis-responsive elements within this 282-bp region (C/EBP, cAMP responsive element [CRE], and E-box) indicated that E-box was critical in both basal and cloprostenol-induced promoter activity. However, there was also significant but less dramatic inhibition of cloprostenol stimulation by mutation of C/EBP and CRE in the Cox-2 promoter, and mutation of all three elements eliminated cloprostenol induction of this promoter. Electrophoretic mobility shift assays of nuclear extracts from large luteal cells revealed that upstream stimulatory factor (USF)-1 and USF-2 bound to the E-box in Cox-2. Thus, PKC directly regulates transcription of the Cox-2 gene in large luteal cells by acting through DNA elements close to the putative transcriptional start point, particularly an E-box region at −50 bp.

The discovery of leptin has sparked a rapidly growing number of publications concerning the role of leptin in the regulation of body adiposity, feeding, and reproductive system in mammals. To date, there have been no reports on the presence of leptin-related peptide, and functional studies on the role of leptin remain limited in fishes. We investigated the effect of mouse recombinant leptin on basal and sea bream (sb) GnRH-induced LH release from dispersed pituitary cells obtained from male European sea bass (Dicentrarchus labrax) at different stages of sexual development. The potential interaction of leptin with the porcine neuropeptide Y (pNPY), known to play a dual role in feeding and reproduction in vertebrates, was also investigated. High doses of leptin (10^–8–10⁻⁶ M) and/or pNPY (0.1 and 1 nM) had different effects on LH release at various stages of sexual development. Porcine NPY alone was weakly effective on basal LH release, but it enhanced LH release induced by leptin (10⁻⁶ M) in late prepuberty but not in early postpuberty. Additive or inhibitory effects of leptin were observed on sbGnRH-induced LH release depending on sbGnRH dose and stage of sexual development. The direct action of leptin on LH release at the pituitary level in sea bass suggests that leptin is a regulator of the reproductive system in fishes.

The objective of this study was to assess fertilization and embryonic development following intracytoplasmic sperm injection (ICSI) of oocytes from porcine preantral follicles matured in vitro. Also, another aim was to describe actin filament distribution during fertilization and embryonic development of those oocytes after ICSI as one of the factors assessed. Preantral follicles isolated from prepubertal porcine ovaries were cultured in a system that supports follicular development. After in vitro maturation, the oocytes were fertilized by ICSI or conventional fertilization in vitro (IVF). Actin filaments of the fertilized oocytes and embryos produced by ICSI or IVF were stained by rhodamine-phalloidin and visualized by fluorescence microscopy. ICSI resulted in 64% fertilization of porcine preantral follicle oocytes matured in vitro. Of those, 51% of the fertilized oocytes cleaved and 21% developed to the blastocyst stage. No significant differences in percentages of oocyte fertilization, cleavage, and blastocyst formation were observed between ICSI and IVF (53%, 45% and 16%, respectively). Actin filament distribution during fertilization and embryonic development of ICSI- or IVF-fertilized oocytes from porcine preantral follicles was similar to that of oocytes derived from antral follicles and fertilized by standard IVF. These results indicate that oocytes from porcine preantral follicles matured in vitro following ICSI can undergo fertilization and subsequent embryonic development.

The sperm adhesion molecule 1 (SPAM1 or PH-20) is an important sperm surface protein with a hyaluronidase activity and bifunctional roles in mammalian fertilization. Recently we reported that in the mouse, Spam1 is synthesized independently in the testis and the epididymis, where it is found in membranous vesicles in the principal cells of the epithelium in all three regions. Here we used mouse epididymal luminal fluid and cultured epididymal epithelial cells to demonstrate that epididymal Spam1 may be a secretory protein. Using a dual environment culture chamber system in which corpus or cauda epithelial cells are cocultured with their corresponding epididymal fibroblasts in medium supplemented with androgens and epidermal growth factor, we show that in 2- to 6-day cultures Spam1 can be detected immunocytochemically in the epithelial cells. The protein was also detected by Western blot analysis in extracts of the cultured cells and in their serum-free conditioned medium, as well as in luminal fluid from fresh caput, corpus, and caudal epididymis. Importantly, it was shown to have hyaluronidase activity, using hyaluronic acid substrate gel electrophoresis, and to be expressed in greater quantities in the corpus compared with the cauda and caput. The results not only confirm our previous finding that Spam1 is synthesized in the epididymis, but extend them by showing that it is released in the luminal fluid where it may effect posttesticular maturation and function of sperm. Results from transcript analysis indicate that epididymal and testicular Spam1 are under different transcriptional regulation.

The hypothesis for the present study is that the active immunization of female turkeys with inhibin (INH) would neutralize endogenous INH, and increase levels of circulating follicle stimulating hormone (FSH) and the number of preovulatory follicles, and subsequently enhance egg production. Two experiments were conducted with female turkeys in their first (30 wk of age) and second (62 wk of age) laying cycles. Treatment groups included control turkeys immunized with keyhole limpet hemocyanine (KLH) and experimental turkeys immunized with recombinant turkey inhibinα conjugated to KLH (rtINH), vasoactive intestinal peptide (VIP) conjugated to KLH or rtINH VIP. Egg production increased (P < 0.05) in VIP and rtINH VIP immunized birds, but not in rtINH immunized hens in comparison with a control group. A similar number of ovarian follicles, arranged in the follicular hierarchy of laying hens, was observed in all experimental groups. However, there was a larger number of nongraded yellow follicles in rtINH-immunized (62.5%) and rtINH VIP-immunized (73.5%) groups compared with that of controls, suggesting overstimulation by FSH. Anterior pituitary FSHβ subunit, LHβ subunit, and prolactin (PRL) mRNA contents were determined by Northern blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) in laying hens at the end of the experimental period. Hens immunized with rtINH showed increased FSHβ subunit mRNA content, but no change in the content of LHβ subunit or PRL mRNA. Hens immunized with VIP or rtINH VIP had significant increases in both pituitary LHβ subunit and FSHβ subunit mRNA contents, accompanied by a decline in PRL mRNA abundance. The magnitude of the increase in FSHβ subunit to INH immunoneutralization was greater in first-cycle hens than in second-cycle hens. These data suggest that active immunization of female turkeys with INH neutralizes endogenous INH and increases both circulating FSH and the number of preovulatory follicles. However, no significant increase in egg production was observed in INH-immunized hens. The data confirm previous reports that VIP immunoneutralization increases egg production in turkey hens and shows for the first time that it also increases FSHβ subunit and LHβ subunit gene expression.

In dorsal root ganglia (DRG) cell cultures, levels of calcitonin gene-related peptide (CGRP) are increased in the presence of ovarian hormones and nerve growth factor (NGF). In addition, injection of ovariectomized rats with ovarian hormones led to an increase in levels of two NGF receptors, TrkA and p75^NTR, in DRG. Thus, we hypothesized that increased levels of ovarian hormones during pregnancy may elevate the synthesis of CGRP and NGF receptors in the DRG. DRG harvested from rats on specific days of pregnancy, on Day 2 postpartum, and after ovariectomy were subjected to radioimmunoassay, Western blot analysis, and NGF immunoassay to determine levels of CGRP, TrkA and p75^NTR, and NGF, respectively. CGRP levels in rat DRG were significantly higher during pregnancy than at Day 2 postpartum or in ovariectomized rats. Levels of both TrkA and p75^NTR in DRG increased during pregnancy and remained elevated at Day 2 postpartum, but CGRP levels declined. Levels of NGF reached a statistically significant peak at Day 18 of gestation, and were not significantly reduced at Day 2 postpartum. Increased levels of ovarian steroid hormones during pregnancy may be involved in the synthesis of CGRP, however, the postpartum decreases in CGRP synthesis appear to be unrelated to NGF and its receptors.

Hyperactivated motility, a swimming pattern displayed by mammalian sperm in the oviduct around the time of ovulation, is essential to fertilization. Ca² has been shown to be crucial for the initiation and maintenance of hyperactivated motility. Nevertheless, how Ca² reaches the axoneme in the core of the flagellum to switch on hyperactivation is unknown. Ca² -releasing agents were used to determine whether an intracellular store provides Ca² to the axoneme. Hyperactivation was induced immediately in bull sperm by thapsigargin, caffeine, and thimerosal. The responses were dose-dependent and were induced in both capacitated and uncapacitated sperm. When external Ca² was buffered below 50 nM with 1,2-bis(2-aminophenoxy)ethane-NNN′,N′-tetraacetic acid, the response to caffeine was significantly reduced; however, the responses to thapsigargin and thimerosal were not affected. This indicates caffeine-induced hyperactivation depends on external Ca² influx, whereas hyperactivation by thapsigargin and thimerosal do not. Acrosome reactions were not induced by these treatments; therefore, an acrosomal store was probably not involved. Indirect immunofluorescence labeling showed type I inositol 1,4,5-trisphosphate receptors (IP₃R) in the acrosome and neck region, but no ryanodine receptors (RyR) were found using anti-RyR antibodies or BODIPY FL-X ryanodine. These data indicate that there is an IP₃R-gated Ca² store in the neck region of sperm that regulates hyperactivated motility.

The interplay between the fetus and mother may play a key role in the regulation of primate pregnancy and parturition. This study was designed to test the hypothesis that fetectomy alters maternal pituitary-adrenal function. Between 117 and 122 days of gestation (term = 167 days), six rhesus macaques underwent surgery for catheter implantation. At surgery the fetuses were removed while the membranes and placenta were left in situ. Six additional intact catheterized pregnant animals served as controls. Animals were maintained under a 12L:12D cycle with lights-on from 0700 to 1900 h. Beginning at least 1 wk after surgery, maternal arterial blood samples were collected at 3-h intervals for 24 h for hormone and catecholamine analysis. This sampling protocol was repeated at weekly intervals until cesarean delivery at 151–157 days of gestation. Following fetectomy, plasma ACTH, dehydroepiandrosterone sulfate (DHEAS), and cortisol levels were significantly lower (36%, 35%, and 44%, respectively) compared with control animals (P ;lt 0.05). Despite a significant reduction in overall levels, the rhythm in maternal plasma cortisol was maintained following fetectomy. Plasma dopamine and norepinephrine were also depressed (P ;lt 0.05), whereas epinephrine remained unaffected. Our data clearly demonstrate the role of the fetus in the regulation of the maternal pituitary-adrenal axis during gestation. This interaction plays a significant role in the regulation of maternal endocrine function that may influence the initiation of labor.