Cloning by somatic nuclear transfer is an inefficient process in which some of the cloned animals die shortly after birth and display organ abnormalities. In an effort to determine the possible genetic causes of neonatal death and organ abnormalities, we used real-time quantitative reverse transcription-polymerase chain reaction to examine expression patterns of eight developmentally important genes (PCAF, Xist, FGFR2, PDGFRa, FGF10, BMP4, Hsp70.1, and VEGF) in six organs (heart, liver, spleen, lung, kidney, and brain) of both cloned bovines that died soon after birth (n = 9) and normal control calves produced by artificial insemination. In somatic cloning of cattle, fibroblasts have often been used for doner nuclei, and the effect of the age of the fibroblast donor cells on gene expression profiles was investigated. Aberrant expressions of seven genes were found in these clones. The majority of aberrantly expressed genes were common in clones derived from adult fibroblast (AF) and in clones derived from fetal fibroblast (FF) compared to controls, whereas some genes were dysregulated either in AF cell-derived or in FF cell-derived clones. For the studied genes, kidney was the organ least affected by gene dysregulation, and heart was the organ most affected, in which five genes were aberrant. Most dysregulations (12 of 19) were up-regulation, but PDGFRa only showed down-regulation. VEGF, BMP-4, PCAF, and Hsp70.1 were extremely dysregulated, whereas the other four genes had a low level of gene dysregulation. Our results suggest that the aberrant gene expression occurred in most tissues of cloned bovines that died soon after birth. For each specific gene, aberrant expression resulting from nuclear transfer was tissue-specific. Because these genes play important roles in embryo development and organogenesis, the aberrant transcription patterns detected in these clones may contribute to the defects of organs reported in neonatal death of clones.

Full-length cDNA for a mouse gene A2-macroglobulin induced by pregnancy (A2mp) was cloned from mesometrial decidua at Gestation Day 10. The 4622-base pair cDNA encodes a protein of 1473 AA with >70% sequence identity and all typical domains of other A2M-family members in humans and rodents, despite unique absence of hepatic expression. The bait region is most distinct and has the greatest sequence similarity with rat acute-phase A2m. Northern blotting, reverse transcription and real-time-PCR, and in situ hybridization studies using C57Bl/6 mice revealed uterine induction of A2mp during decidualization and strong, midgestational association with modifying spiral arteries. Ovaries, testes, lactating mammary glands, heart, and kidney were the only additional organs with A2mp expression that was localized to granulosa and cumulus cells in secondary follicles; primary seminiferous epithelium, including Sertoli cells, mammary alveolar, and ductal epithelium; cardiac endothelium; and renal collecting tubules, respectively. Infusion of native human A2M into pregnant alymphoid or interferon-gamma gene-ablated mice overcame blocks to pregnancy-induced spiral artery modification in these strains. Activated human A2M was also effective, suggesting mechanisms independent of proteinase inhibition. Identification of cytokines, growth factors, or other molecules bound to A2MP should provide new insights into decidualization, spiral artery modification, and cardiovascular adaptation to pregnancy.

The mechanism underlying spontaneous pacemaker potential in the uterus is not clearly understood. Several spontaneously active smooth muscles have interstitial cells of Cajal (ICCs) or ICC-like cells. We therefore examined cells from freshly dispersed uterine muscle strips (from pregnant human and rat myometrium) and in situ uterine preparations to determine the cell types present. Both preparations revealed numerous ICC-like cells; they were multipolar, with spider-like projections and enlarged central regions. These cells were readily distinguished from uterine myocytes by their morphology and ultrastructure, i.e., no myofilaments, numerous mitochondria, caveolae, and filaments. In addition, the ICC-like cells were noncontractile. These cells were negative to c-kit, a classic marker for ICCs. They stained positive for the intermediate filament, vimentin, a marker for cells of mesenchymal origin but not differentiated myocytes. The ICC-like cells had a more or less stable resting membrane potential of −58 ± 7 mV compared with smooth-muscle cells, −65 ± 13 mV, and produced outward current in response to voltage clamp pulses. However, in contrast with uterine myocytes, inward currents were not observed. This is the first description of ICC-like cells in myometrium and their role in the uterus is discussed, as possible inhibitors of intrinsic smooth-muscle activity.

We used differential display in combination with cDNA cloning to isolate a novel rat gene, designated as Spetex2, that has an open reading frame of 582 nucleotides, encoding a protein of 194 amino acids. Spetex2 mRNA was highly expressed in testis and spleen, and its expression in rat testis was developmentally up-regulated. In situ hybridization revealed that Spetex2 mRNA was predominantly expressed in haploid spermatids at steps 1–13 within the seminiferous epithelium. A BLAST search against rat genome databases at the National Center for Biotechnology Information revealed that the Spetex2 gene is composed of four exons and is mapped to at least 18 loci in a cluster on rat chromosome 15p16, indicating that the genes occur as a repeated tandem array over a long stretch of genomic DNA. By immunocytochemical analysis with confocal laser-scanning microscopy, SPETEX2 protein was detected as a dot-like distribution on the cell periphery of haploid spermatids (steps 1–13) but was not observed in other spermatogenic cells. On the basis of these data, we hypothesize that SPETEX2 might be correlated with cell differentiation of spermaytids in rat testis.

The present study examines the existence of clonogenic patterns in the proliferation and differentiation of spermatogonial stem cells in two species of non-human primates, the marmoset and the rhesus monkey. We developed a novel approach to detect proliferating spermatogonial clones in whole mounts of seminiferous tubules. Dual fluorescence labeling of bromodeoxyuridine and acrosin in conjunction with confocal microscopy allows the description of the clonogenic and spatial arrangement of proliferating spermatogonia at specific stages of the seminiferous epithelial cycle. Cross-sections of paraffin-embedded tissue were labeled by the same approach. For both monkey species we demonstrate the presence of proliferating spermatogonial clones of variable size at specific stages of the cycle of the seminiferous epithelium. Detailed analysis of the rhesus monkey reveals proliferating A_pale spermatogonia at stages VII and IX of the cycle of the seminiferous epithelium, and of proliferating B spermatogonia at stages II, IV, VI, and XII. Proliferating A_pale spermatogonia at stages VII and IX of the cycle are organized in pairs or quadruplets. B₁ spermatogonia appear as quadruplets or eight-cell clones, and B₂ spermatogonia as 8- or 16-cell clones. We conclude that spermatogenesis in the rhesus monkey is initiated by two divisions of duplets or quadruplets of A_pale spermatogonia: a first division at stage VII, after which the clones of A_pale spermatogonia separate, and a second division at stage IX, which leads to clones of B₁ spermatogonia as well as pairs and quadruplets of A_pale spermatogonia replenishing the seminiferous epithelium to maintain the original size of the A spermatogonial population.

We previously demonstrated that genetically linking one or more of the glycoprotein hormone-specific β subunit genes to the common α subunit resulted in single-chain analogues that were bioactive in vitro. The ability of such large structures to bind their cognate receptors with high affinity supported the hypothesis that extensive flexibility exists between the ligand and receptor to establish a functional complex. To further characterize the extent of this conformational flexibility, we engineered a single-chain analogue that consists of sequentially linked thyroid-stimulating hormone (TSH) β, follicle-stimulating hormone (FSH) β, and chorionic gonadotropin (CG) β subunits to the α subunit and expressed this chimera in transfected CHO (Chinese hamster ovary) cells. Because the four subunits are genetically linked and expressed as a single-chain, this analogue presumably lacks significant native structural features of the individual heterodimers. However, it exhibited FSH, CG, and TSH activities in vitro. Here, we test whether this nonnative structure would be stable in vivo and thus biologically active. Using a variety of bioassay protocols, we demonstrate that the analogue elicits multihormone activities when injected in vivo. First, treatment with the analogue caused increases in ovarian and uterine weights and resulted in elevated serum estradiol. Second, the analogue-stimulated ovarian follicle growth and pharmacologically rescued in vivo FSH deficiency similar to recombinant human FSH or equine CG (eCG) as confirmed by induction of aromatase in the ovaries of FSHβ knockout mice. Third, in a superovulation protocol, when primed with eCG, the analogue elicited a dose-dependent ovulatory response comparable with that by native heterodimeric human CG. Finally, the analogue-stimulated thyroxin production in hypothyroid mice similar to the pituitary-derived human TSH standard. Based on these data, we conclude that a single-chain tetradomain glycoprotein hormone analogue, despite its presumed altered conformation, is stable and biologically active in vivo. Our results establish the permissiveness and conformational plasticity with which the glycoprotein hormones are recognized in vivo by their target cell receptors.

Puromycin N-acetyl transferase gene (pac), of which the gene product catalyzes antibiotic puromycin (an effective inhibitor of protein synthesis), has been widely used as a dominant selection marker in embryonic stem (ES) cell-mediated transgenesis. The present study is the first to report on the usefulness of puromycin for production of enhanced green fluorescent protein (EGFP) transgenic piglets after somatic cell cloning and embryo transfer. Somatic cells isolated from porcine fetuses at 73 days of gestation were immediately electroporated with a transgene (pCAG-EGFPac) carrying both EGFP cDNA and pac. This procedure aims to avoid aging effects thought to be generated during cell culture. The recombinant cells were selected with puromycin at a low concentration (2 µg/ml), cultured for 7 days, and then screened for EGFP expression before somatic cell cloning. The manipulated embryos were transplanted into the oviducts of 14 foster mother sows. Four of the foster sows became pregnant and nine piglets were delivered. Of the nine piglets, eight died shortly after birth and one grew healthy after weaning. Results indicate that puromycin can be used for the selection of recombinant cells from noncultured cells, and moreover, may confer the production of genetically engineered newborns via nuclear transfer techniques in pigs.

We have applied a statistical protocol based on principal component analysis, clustering methods, and discriminant analysis for the identification of sperm subpopulations in computer-assisted sperm analysis (CASA) data. Samples were obtained from the cauda epididymis of 11 Iberian red deer and cryopreserved following a standard protocol. Motility by CASA was analyzed just after sperm recovery, just before freezing, and after thawing, and eight motility descriptors for each individual spermatozoon were recorded. Sperm viability and acrosomal status were also assessed. Subpopulation analysis was performed in four sequential steps: principal component analysis using the eight motility descriptors; nonhierarchical clustering analysis (k-means) using the first two principal components; hierarchical clustering analysis (UPGMA); and selection of the final number of clusters. Three clusters were obtained for each motility analysis: slow and nonlinear; rapid and linear; and rapid, high ALH, nonlinear. We detected variations in the clusters between treatments (initial, prefreezing and postthawed). Indeed, motility increased and linearity decreased in the prefreezing analysis. A discriminant analysis isolated three descriptors that were used again in the same statistical analysis, giving four clusters that resembled the pattern found in the first classification. We also performed a clustering analysis of the males according to prefreezing/postthawed variation of total motility, viability, and acrosomal status. The proportion of the linear subpopulations in the prefreezing treatment, in both clustering analyses, correlated positively with postthawed viability recovery. Our results show that clustering analysis of CASA data gives useful and practical information that is not obtained by conventional sperm analysis.

Although the molecular basis of sperm-oocyte interaction is unclear, recent studies have implicated two chaperone proteins, heat shock protein 1 (HSPD1; previously known as heat shock protein 60) and tumor rejection antigen gp96 (TRA1; previously known as endoplasmin), in the formation of a functional zona-receptor complex on the surface of mammalian spermatozoa. The current study was undertaken to investigate the expression of these chaperones during the ontogeny of male germ cells through spermatogenesis, epididymal sperm maturation, capacitation, and acrosomal exocytosis. In testicular sections, both HSPD1 and TRA1 were closely associated with the mitochondria of spermatogonia and primary spermatocytes. However, this labeling pattern disappeared from the male germ line during spermiogenesis to become undetectable in testicular spermatozoa. Subsequently, these chaperones could be detected in epididymal spermatozoa and in previously unreported “dense bodies” in the epididymal lumen. The latter appeared in the precise region of the epididymis (proximal corpus), where spermatozoa acquire the capacity to recognize and bind to the zona pellucida, implicating these structures in the functional remodeling of the sperm surface during epididymal maturation. Both HSPD1 and TRA1 were subsequently found to become coexpressed on the surface of live mouse spermatozoa following capacitation in vitro and were lost once these cells had undergone the acrosome reaction, as would be expected of cell surface molecules involved in sperm-egg interaction. These data reinforce the notion that these chaperones are intimately involved in the mechanisms by which mammalian spermatozoa both acquire and express their ability to recognize the zona pellucida.

Intervention in B7 (CD80/CD86)/B7-ligand (CD28/CTLA-4) pathways is an effective way of preventing unwanted immune responses, such as allograft rejection. Pregnancy maintenance represents maternal tolerance to the fetal allograft, which is accompanied by a type 2 helper cell (Th2) bias at the maternal-fetal interface. Here, the costimulatory signal of CD86 was selectively blocked, and that of CD80 was kept unimpaired by administration of anti-murine CD86 monoclonal antibody at the early gestational stage in abortion-prone CBA/J×DBA/2 matings and normal pregnant CBA/J×BALB/c matings. It was demonstrated that in vivo blockade of CD86 costimulation could suppress maternal immune attack to the fetus by shifting cytokines from Th1 predominance to Th2 bias at the maternal-fetal interface, and expanding peripheral CD4 CD25 regulatory T cells, which play an important role in the development and maintenance of maternal-fetal tolerance. Furthermore, the expression of CD28 and its ligands CD80/CD86 on peripheral lymphocytes was down-regulated, whereas that of CTLA-4 was up-regulated, which might facilitate the suppressive effect of CD4 CD25 regulatory T cells on the alloreactive T cells. The maternal-fetal immunotolerance induced by CD86 blockade decreased fetal resorption in CBA/J×DBA/2 matings, but did not affect normal pregnant CBA/J×BALB/c matings. These results suggest that selective blockade of CD86 costimulation leads to maternal immune tolerance to embryo antigen, and might contribute to a rational immunoregulatory regimen for recurrent spontaneous abortion.

In vivo capacitation of eutherian sperm cells coincides with changes in carbohydrate-dependent interaction with the oviduct epithelia (fucose-dependent for bovine). Heparin-like glycosaminoglycans (GAG) secreted by the oviduct compete for sperm-oviduct binding and are believed to release capacitated sperm cells from oviduct epithelia. A biochemical assay to quantify the specificity and dynamics of carbohydrate-mediated bovine sperm-oviduct binding is developed. Sperm apical plasma membranes (SPM) were purified by a factor eight and biotinylated carbohydrate probes were used for quantitative evaluation of carbohydrate binding. SPM of fresh sperm showed >12 times higher binding capacity for biotinylated fucose than for LewisA. SPM from fresh sperm also efficiently bound biotinylated fucoidan and mannan. Binding of biotinylated fucose could be inhibited by various mono- and oligosaccharides such as fucoidan, mannan, heparin, maltose, and, to a lesser extent, glucose (50% binding at 0.2 mM, 2 mM, 0.3 µg/ml, 15 mM, 50 mM, respectively). SPM from sperm cells that were in vitro capacitated for 4 h in bicarbonate-enriched media (either with or without 10µg/ml heparin) showed a 70–85% reduction in fucose binding. This was also achieved by follicular fluid or by GAG, both obtained from dominant follicles. Total follicular fluid was much more potent in competing with fucose for sperm binding than the isolated GAG moieties (50% competition at 0.02 µg/ml, 20 µg/ml based on number of GAG moieties, respectively). These results support the hypothesis that in vivo capacitation of sperm cells is regulated by carbohydrate moieties similar to those regulating sperm-oviduct adhesion.

Ectopic testicular xenografting can be used to investigate spermatogenesis and as an alternative means for generating transgenic spermatozoa in many species. Improving the efficiency of spermatogenesis in xenografted testicular tissue will aid in the application of using this approach. The present study was conducted to evaluate age-related differences in the establishment of spermatogenesis in grafted testicular tissue from bulls between 2 and 16 wk of life. Testicular tissue was ectopically xenografted under the skin on the backs of castrated nude mice and subsequently evaluated for growth, testosterone production, and establishment of spermatogenesis 24 wk after grafting. The greatest weight increases occurred in donor tissue from calves of the ages 2, 4, and 8 wk compared with the ages of 12 and 16 wk. Recipient mouse serum testosterone concentration was at normal physiological levels 24 wk after grafting and no significant differences were detected between recipients grafted with testicular tissue from bull calves of different ages. The development of germ cells to elongated spermatids were observed in seminiferous tubules of grafts from donor calves of the ages 4, 8, 12, and 16 wk but not observed in grafts from 2-wk donors, which contained round spermatids as the most advanced germ cell stage. Grafts from 8-wk donors contained a significantly higher (10-fold) average percentage of seminiferous tubules with elongated spermatids than all other donor ages. These data demonstrate differences in the ability of testicular tissue from donor animals of different ages to establish spermatogenesis following ectopic testicular xenografting.

Bovine (bov) interferon-stimulated gene product 15 (ISG15) is produced in the endometrium in response to conceptus-secreted interferon (IFN)-τ. ISG15 conjugates to endometrial proteins through an enzymatic pathway that is similar to ubiquitinylation. Ubiquitin-activating enzyme 1-like protein (UBE1L) initiates enzymatic conjugation by forming a thioester bond with ISG15, thus preparing it for transfer to the next series of enzymes. The bovUBE1L has not been described. We hypothesized that bovUBE1L was induced by pregnancy and IFN-τ in the endometrium. A 110-kDa protein was purified from bovine endometrial (BEND) cells based on affinity with recombinant (r) glutathione S-transferase (GST)-ISG15. This protein was digested in gel with trypsin. Seven peptides were purified using HPLC, sequenced using liquid chromatography-mass spectroscopy-mass spectroscopy and found to share 43–100% identity with human UBE1L. The full-length bovUBE1L cDNA was isolated from a BEND cell cDNA library, sequenced, and found to share 83% identity with human UBE1L cDNA. Northern blot revealed two mRNAs that were detected in greater (P < 0.05) concentrations in endometrium from Day 17–21 pregnant versus nonpregnant cows. Western blots using antihuman UBE1L antibody revealed a similar pattern of pregnancy-associated expression of UBE1L protein in the uterus. The bovUBE1L mRNA was localized, using in situ hybridization, primarily to glandular and luminal epithelium, with more diffuse localization to stroma of the endometrium from pregnant cows. Because bovUBE1L was purified through its interaction with rGST-ISG15 and shares significant amino acid and cDNA sequence identity with human UBE1L, it is concluded that it mediates conjugation of ISG15 to uterine proteins in response to the developing and attaching conceptus.

This study was conducted to examine expression of centromere protein B (CENPB), spindle checkpoint protein MAD2 (mitotic arrest deficient protein), and antiapoptotic protein BCL2; activities of MAPK (mitogen-activated protein kinase) and mitochondria distribution in pig oocytes during aging, and their relationship with sister chromatid separation during meiosis II and embryo fragmentation and apoptosis after activation. After immature oocytes were cultured for 40–72 h, CENPB, MAD2, tubulin, BCL2, and MAPK in the oocytes were examined by immunoblotting. Spindles, chromosomes, kinetochores, and mitochondria were examined by immunofluorescence staining and apoptosis was examined by TUNEL assay. It was found that tubulin and CENPB was not changed during 40–72 h of culture. However, the expression of MAD2 and BCL2 and the activity of MAPK were gradually reduced during oocyte aging. The percentages of oocytes with normal spindle, chromosomes, and kinetochores were also reduced as oocyte aged from 9.5% at 40 h to 17.3%, 34.6%, and 42.9% at 48, 60, and 72 h, respectively. Aggregated mitochondria were found in the aged oocytes as compared with the uniform distribution in young oocytes. After activation, the proportions of oocytes with abnormal anaphase II were significantly increased in aged oocytes. More (P < 0.001) oocytes cultured for 60–72 h fragmented and showed apoptosis after activation as compared with the oocytes cultured for 40–48 h. This study indicates that aging reduces expression in spindle checkpoint protein and antiapoptosis protein and MAPK activity in pig oocytes. These events in turn cause abnormal sister chromatid segregation during meiosis II, embryo fragmentation, and apoptosis.

The second messenger cAMP mediates its intracellular effects in spermatozoa through cAMP-dependent kinase (PKA, formally known as PRKACA). The intracellular organization of PKA in spermatozoa is controlled through its association with A-kinase-anchoring proteins (AKAPs). AKAP4 (A kinase [PRKA] anchor protein 4; also called fibrous sheath component 1 or AKAP 82) is sperm specific and the major fibrous sheath protein of the principal piece of the sperm flagellum. Presumably, AKAP4 recruits PKA to the fibrous sheath and facilitates local phosphorylation to regulate flagellar function. It is also proposed to act as a scaffolding protein for signaling proteins and proteins involved in metabolism. Akap4 gene knockout mice are infertile due to the lack of sperm motility. The fibrous sheath is disrupted in spermatozoa from mutant mice. In this article, we used Akap4 gene knockout mice to study the effect of fibrous sheath disruption on the presence, subcellular distribution, and/or activity changes of PKA catalytic and regulatory subunits, sperm flagellum proteins PP1γ2 (protein phosphatase 1, catalytic subunit, gamma isoform, formally known as PPP1CC), GSK-3 (glycogen synthase kinase-3), SP17 (sperm autoantigenic protein 17, formally known as SPA17), and other signaling proteins. There were no changes in the presence and subcellular distribution for PP1γ2, GSK-3, hsp90 (heat shock protein 1, alpha, formally known as HSPCA), sds22 (protein phosphatase 1, regulatory [inhibitor] subunit 7, formally known as PPP1R7), 14-3-3 protein (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein), and PKB (thymoma viral proto-oncogene, also known as AKT) in mutant mice. However, the subcellular distributions for PKA catalytic subunit and regulatory subunits, PI 3-kinase (phosphatidylinositol 3-kinase), and SP17 were disrupted in mutant mice. Furthermore, there was a significant change in the activity and phosphorylation of PP1γ2 in mutant compared with wild-type spermatozoa. These studies have identified potentially significant new roles for the fibrous sheath in regulating the activity and function of key signaling enzymes.

Melatonin regulation of reproductive functions in sheep is mediated by action in the premammillary hypothalamus (PMH). The aim of this study was to identify the high-affinity melatonin-receptor subtypes expressed in this structure. To achieve this, we used reverse transcription-polymerase chain reaction (RT-PCR) and developed in situ hybridization techniques (ISH). By using RT-PCR, we detected a band corresponding to the MTNR1A melatonin-receptor cDNA in the PMH as well as in the pars tuberalis (PT). On the opposite, MTNR1B melatonin-receptor transcripts were not detected using degenerate primers in any of the structures considered, confirming the lack of expression of this receptor subtype in sheep. The expression of MTNR1A mRNA was further confirmed in the PMH by ISH with a ³⁵S-labeled ovine MTNR1A riboprobe. We next investigated the variation in the expression of MTNR1A mRNA between the end of the day and the end of the night (absence and presence of melatonin, respectively). MTNR1A transcript expression was greater at the end of the night than at the end of the day in the PMH. In contrast, MTNR1A mRNA expression was lower at the end of the night than at the end of the day in the PT. No significant variation in the MTNR1A mRNA expression was observed in a more dorsal hypothalamic area. Overall, these results show that MTNR1A transcripts are expressed in the ovine PMH and that their expression follows a diurnal rhythm, which is different from the pattern of expression observed in the PT.

The effects of FSH, LH, and epidermal growth factor (EGF) on the dynamics of nuclear maturation and subsequent embryo development were examined in pig oocytes cultured either conventionally or after preincubation with cycloheximide (CHX). In conventional culture, FSH or EGF significantly increased the rate of attainment of metaphase II (MII) for both gilt (50.0% ± 4.2% and 54.8% ± 4.3%, respectively; control, 5.8% ± 1.8%; P < 0.001) and sow (87.6% ± 3.4% and 78.8% ± 3.9%, respectively; control, 7.8% ± 2.5%; P < 0.001) oocytes. Gilt oocytes treated with both FSH and EGF showed an additive response (93.7% ± 2.1%). Treatment with LH had no effect. Preincubation with CHX caused the majority (84–100%) of both gilt and sow oocytes to undergo germinal vesicle breakdown. Compared to those treated with LH and/or EGF (both >80%), fewer FSH-treated oocytes reached metaphase I (43.8% ± 5.3%, P < 0.001) by 14 h and MII (48.4% ± 5.9%, P < 0.001) by 24 h, although the majority (71%) did mature to MII by 36 h after removal of CHX. After in vitro fertilization, higher proportions of both CHX-pretreated and untreated, FSH-exposed oocytes cleaved (71.3% ± 2.9% and 75.3% ± 3.1%, respectively) compared with those not treated with FSH (37.7% ± 3.0% and 43.0% ± 2.9%, respectively; P < 0.001). Pretreatment with CHX significantly increased blastocyst yield for both FSH-treated (32.8% ± 2.0% and 10.3% ± 1.5%, respectively; P < 0.001) and untreated (16.7% ± 1.5% and 9.4% ± 1.2%, respectively; P < 0.001) oocytes. Polyspermy rates were unaffected. In conclusion, pig oocytes meiotically arrested by CHX before maturation retain and improve their developmental competence. FSH stimulates nuclear maturation but slows meiotic progression.

In mammals, the SRY/Sry gene on the Y chromosome is necessary and sufficient for a bipotential gonad to develop into a testis, regardless of its chromosomal sex. The SRY/Sry gene encodes a protein that belongs to a high-mobility-group (HMG) box protein family and that has been postulated to modulate the expression of genes necessary for male gonadal differentiation. Using a yeast two-hybrid screen, we identified a novel protein containing only a Krüppel-associated box (KRAB) domain, which is hereafter named KRAB-O (KRAB Only), as an SRY-interacting protein. The KRAB-O protein is encoded by an alternatively spliced transcript from the Zfp208 locus that also produces another transcript coding for a KRAB-zinc finger protein, ZFP208. The interaction of the mouse SRY with KRAB-O was further confirmed by glutathione S-transferase pull-down assay and coimmunoprecipitation in transfected COS7 cells. The KRAB-O interaction domain in both the human and mouse SRY was mapped to the bridge region outside the HMG box. Indirect immunofluorescence and confocal microscopy show that the mouse SRY colocalizes with KRAB-O in nuclear dots in transiently transfected COS7 cells and primary fetal mouse gonadal cells. Using similar approaches, we demonstrate that KRAB-O interacts directly with KAP1 (KRAB-associated protein 1), the obligatory corepressor for KRAB domain proteins. Furthermore, we show that the mouse SRY is associated indirectly with KAP1 and heterochromatin protein 1 (HP1) through its interaction with KRAB-O, suggesting that the mouse SRY could use the KRAB-KAP1-HP1 organized transcriptional regulatory complex to regulate its yet-to-be-identified downstream target genes.

Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM), two potent smooth-muscle relaxants, have been shown to cause uterine relaxation. Both CGRP- and AM-binding sites in the uterus increase during pregnancy and decrease at labor and postpartum. These changes in binding sites appear to be related to the changes in calcitonin receptor-like receptor (CRLR), receptor activity-modified protein 1 (RAMP₁), RAMP₂, and RAMP₃ mRNA levels. It is not clear, however, whether the changes in the receptor components occur in the myometrial cells and whether the steroid hormones can directly alter these receptor components in the muscle cells. In addition, the mechanism of CGRP and AM signaling in the rat myometrium is not well understood. Therefore, we examined the mRNA expression of CGRP- and AM-receptor components, G protein Gαs, CGRP, and AM stimulation of cAMP and cGMP, and the effects of progesterone on these parameters in the Eker rat uterine myometrial smooth-muscle cell line (ELT3). ELT3 cells expressed CGRP- and AM-receptor components CRLR, RAMP₁, RAMP₂, and RAMP₃. Expression of CRLR and RAMP₁ mRNA increased with progesterone treatment and decreased with estradiol-17β treatment. However, RAMP₂ and RAMP₃ mRNA expressions were unaltered by both progesterone and estradiol. Progesterone increased (P < 0.05) Gαs expression and augmented CGRP- and AM-induced increases in cAMP levels. In uterine smooth-muscle cells, the antagonist to Gαs protein NF449 decreased basal as well as CGRP- and AM-stimulated cAMP levels. None of the cell treatments affected cyclic GMP production. Our results suggest that the progesterone-stimulated increases in CGRP and AM receptors, Gαs protein levels, and cAMP generation in the myometrial cells may be responsible for increased uterine relaxation sensitivity to CGRP and AM during pregnancy.

In this study, we compared the long-term effects of neonatal exposure to diethylstilbestrol (DES, 0.0125–50 µg), tamoxifen (TAM, 0.0125–50 µg), and toremifene (TOR, 53 µg) on mammary gland development and differentiation. Allometric growth of the mammary ducts was stimulated by neonatal DES exposure (12.5 µg) and impaired by exposure to TAM (25 µg). Neonatal treatment with high doses of DES resulted in mammary ducts that displayed extensive dilatation and precocious lactogenesis in postpubertal, nulliparous females. Initiation of this precocious differentiation coincided with the absence of corpora lutea, increased levels of serum prolactin (PRL), and the induction of Prl mRNA expression within the mammary glands. Neonatal exposure to 1.25 µg TAM increased alveolar development in postpubertal, nulliparous females similar to that recorded in females treated with low doses of DES. Lower doses of TAM did not affect alveolar development, whereas branching morphogenesis and alveolar development were impaired by higher doses. Increased alveolar development in females exposed to 1.25 µg TAM was associated with elevated serum progesterone (P) and increased alveolar development in response to exogenous P. Taken together, our findings demonstrate that neonatal exposure to both DES and TAM exerts long-lasting effects on the proliferation and differentiation of the mammary glands in female BALB/c, primarily as the result of endocrine disruption

Sperm entering the epididymis gain progressive motility and fertilizing ability in a process termed maturation. The functional dependence of the epididymis on dihydrotestosterone (DHT) is well established, yet few studies have examined the consequences on the epididymis of inhibiting DHT formation. We have shown that inhibition of both isoforms of 5α-reductase (types 1 and 2), the enzyme that converts testosterone to DHT, has pronounced effects on epididymal gene expression. In the present study, we investigate whether inhibiting 5α-reductase has consequences on epididymal sperm maturation. Rats were treated with vehicle or 10 mg/kg/day PNU157706, a dual-type inhibitor, for 28 days. Fertility and several key facets of sperm maturation were analyzed. Changes in sperm motility were assessed by computer-assisted sperm analysis (CASA). Changes in sperm morphology were assessed by CASA and electron microscopy. The motility of spermatozoa from the cauda epididymidis of treated animals showed a significant decrease in both the percentage of motile and progressively motile sperm as well as altered motion parameters. The morphology of cauda epididymal spermatozoa was also adversely affected by the treatment; the most prominent effect was a markedly elevated proportion of sperm that retained their cytoplasmic droplet. Matings with treated males resulted in fewer successful pregnancies and a higher rate of preimplantation loss. Progeny outcome was unaffected. The compromised sperm motility and morphology likely contribute to the subfertility of inhibitor-treated rats. Our results indicate a role for dual 5α-reductase inhibitors in further studies of epididymal physiology and as a potential component of a male contraceptive.

The objective of this study was to investigate the preservation of spermatozoa in a simple medium without freezing and to examine the effects of the preserved sperm on fertilization and development after injection into mature mouse oocytes. Mouse spermatozoa were collected from two caudae epididymides of mature B6D2F1 males and stored under various conditions: 1) in KSOMaa medium (potassium simplex optimized medium with amino acids) supplemented with 0, 1, or 4 mg/ml BSA and held at room temperature (RT, 27°C); 2) in KSOMaa medium containing 4 mg/ml BSA (KSOM-BSA) and held at 4°C, RT, or 37°C (CO₂ incubator); 3) in KSOM-BSA with osmolarity ranging from 271 to 2000 mOsmol, adjusted by addition of NaCl and held at 4°C; and 4) a two-step preservation system consisting of storage in 800 mOsmol KSOM-BSA for 1 wk at RT followed by storage at −20°C. Preservation of mouse spermatozoa at 4°C in a medium with high osmolarity (700–1000 mOsmol) resulted in the highest frequency of live births after intracytoplasmic sperm injection (ICSI) into mature oocytes. The optimal conditions for preservation of mouse spermatozoa were 800 mOsmol KSOM containing 4 mg/ml BSA and a holding temperature of 4°C. More than 40% of oocytes injected with sperm heads stored under these conditions for 2 mo developed to the morula/blastocyst stage in vitro and 39% of the embryos developed to term after transfer to recipient mice. Our results also indicate that mouse spermatozoa can be stored in 800 mOsmol KSOM-BSA medium at RT for 1 wk and then at −20°C for up to 3 mo and retain their competence for ICSI. These new preservation methods permit extended conservation of viable spermatozoa that are capable of supporting normal embryonic development and the live birth of healthy offspring after ICSI.

Timing of artificial insemination (AI) in marsupials is critical because fertilization must occur before mucin coats the oocyte during passage through the oviduct. In this study, timing and the site of insemination were examined to develop AI in the tammar wallaby (Macropus eugenii). Birth and postpartum (p.p.) estrus was synchronized in 46 females. Epididymal spermatozoa (n = 4) or semen collected by electroejaculation (n = 42) were inseminated early (4–21 h p.p.) into the urogenital sinus (n = 7), the anterior vaginal culs de sac (n = 7), the uterus by transcervical catheter (n = 5), or the uterus by injection (intrauterine artificial insemination, IUAI) (n = 5). A further 16 females were inseminated late (19–48 h p.p.) by IUAI. All females were monitored for birth. A third group of six females was inseminated late (21–54 h p.p.) by IUAI and 0.4–6.6 h later, sperm had reached the oviduct in all animals. In total, an oocyte to which spermatozoa were attached was recovered and two young were born after IUAI using epididymal (n = 1) or electroejaculated (n = 2) spermatozoa, but no young resulted from insemination at other sites. Two females were successfully inseminated at 43 and 47 h p.p., later than most other animals, and the third was inseminated much earlier (18 h p.p.) but with highly motile spermatozoa. These young represent the first macropodids born by AI and the first marsupials conceived using epididymal spermatozoa.

We have examined the passage of maternal cells into the fetus during the gestation and postpartum in mice. Using enhanced green fluorescent protein (EGFP)-transgenic females, we showed that maternal cells frequently gain access to the fetus, mostly in syngeneic pregnancies, but also in allogeneic and outbred crosses. EGFP-transgenic cells, including B, T, and natural killer cells, can persist until adulthood, primarily in bone marrow and thymus. We then asked whether maternal cells, bearing antigens not inherited by the fetus, influence the development of fetal and neonatal B lymphocytes. We have used the B cell receptor 3-83 µ/δ transgenic mouse model, whose B cells recognize the major histocompatibility complex class I molecules H-2K^k and H-2K^b, with a high or moderate affinity, respectively. The fate of transgenic B cells in animals exposed to noninherited H-2K^k or H-2K^b maternal antigens (NIMA) during gestation and lactation was compared with those of nonexposed controls. In H-2K^k-exposed fetuses, NIMA-specific transgenic B cells are partially deleted during late gestation. Nondeleted cells have downmodulated their B cell receptor. In contrast, in NIMA H-2K^b-exposed neonates, transgenic B cells present an activated phenotype, including proliferation, upregulation of surface CD69, and preferential localization in the T cell zone of splenic follicles. This state of activation is still clearly detectable up to 3 wk of age. Thus, we show that fetal and neonatal B cell development is affected by maternal cells bearing antigens noninherited by the fetus and that this phenomenon is highly dependent on the affinity of the B cell receptor for the NIMA.

The prostate gland in the brushtail possum grows and regresses seasonally. It has similarities to the human prostate and may therefore provide a unique model for investigating prostatic hyperplasia. Oxytocin has been implicated in the regulation of prostate growth in eutherian mammals, and the initial aim of this study was to identify and localize the marsupial equivalent, mesotocin, and its receptor in the prostate of the brushtail possum. Seasonal changes in prostatic mesotocin concentrations and receptor localization were then assessed and related to prostate growth. Mesotocin and mesotocin receptor gene transcripts with high sequence homology to eutherian oxytocin/oxytocin receptors were demonstrated, and mesotocin, neurophysin, and the receptor were all localized predominantly in the epithelial cells of the glandular acini. Western blot analysis confirmed the presence of a single immunoreactive receptor protein of ∼60 M_r⁻³. Prostatic mesotocin concentrations were highest immediately before the increases in prostate weight associated with the autumn and spring breeding periods. At this time, mesotocin receptors were also present in the prostatic capsule in addition to those present in the glandular tissue. Mesotocin concentrations proceeded to decrease in association with the regression of prostate size toward the end of the breeding periods. No significant differences were present in serum testosterone or dihydrotestosterone throughout the year. The identification of mesotocin and its receptor in the possum prostate and the demonstration of seasonal changes in local mesotocin concentrations preceding changes in prostate size suggests that mesotocin may play a physiological role in regulating prostate growth and regression.

The process of spermatogenesis requires a delicate balance of proapoptotic and antiapoptotic signaling to maintain optimal sperm output. A major transcription factor known to regulate numerous apoptosis-related genes is NF-κB. Here we show that mono-(2-ethylhexyl) phthalate (MEHP, 1 g/kg) induces translocation of NF-κB subunits (p65, p50, and c-Rel) to germ cell nuclei in young rats (Postnatal Day 28) as early as 1 h after exposure. Immunohistochemistry of rat testes exposed to MEHP showed increased p50 and c-Rel presence in spermatocytes and spermatogonia. In addition, there was increased p65 nuclear positivity in Sertoli cells and germ cells after MEHP, while Rel-B localization was unchanged. These alterations correlated with increased nuclear NF-κB-binding activity after MEHP exposure, as shown by electrophoretic mobility shift assays of whole-testis nuclear protein extracts. The increased activity of this transcription factor was associated with a transient protection of the seminiferous epithelium manifested as a decreased number of germ cell apoptotic nuclei measured by TUNEL assay 6 h after MEHP exposure. These results suggest that NF-κB is involved in the testicular response to MEHP-induced injury.

The ovum mutant (Om) locus in mice affects early interactions between sperm and egg that in turn affect viability of embryos beyond the morula stage. Crosses of DDK females to males of many other inbred strains are 95% lethal around the morula stage, whereas reciprocal crosses are fully viable. Available data indicate that the early lethality is the result of an interaction between a factor in the ooplasm and the paternal genome. In this study, we examined whether this lethal interaction would likewise occur in cloned embryos produced by somatic cell nuclear transfer. We find that the Om effect is recapitulated but that the parental origin effect at the Om locus is no longer evident in cloned embryos.

The application of microarray technology to the study of mammalian organogenesis can provide greater insights into the steps necessary to elicit a functionally competent tissue. To this end, a temporal profile of gene expression was generated with the purpose of identifying changes in gene expression occurring within the developing male and female embryonic gonad. Gonad tissue was collected from mouse embryos at 11.5, 12.5, 14.5, 16.5, and 18.5 days postcoitum (dpc) and relative steady-state levels of mRNA were determined using the Affymetrix MGU74v2 microarray platform. Statistical analysis produced 3693 transcripts exhibiting differential expression during male and/or female gonad development. At 11.5 dpc, the gonad is morphologically indifferent, but at 12.5 dpc, transitions to a male or female phenotype are discernible by the appearance of testicular cords. A number of genes are expressed during this period and many share similar expression profiles in both sexes. As expected, the expression of two well-known sex determination genes, specifically Sry and Sox9, is unique to the testis. Beyond 12.5 dpc, differential gene expression becomes increasingly evident as the male and female tissue morphologically and physiologically diverges. This is evident by two unique waves of transcriptional activity occurring after 14.5 dpc in the male and female. With this study, a large number of transcripts comprising the murine transcriptome can be examined throughout male and female embryonic gonad development and allow for a more complete description of gonad differentiation and development.

Fox (Vulpes vulpes) sperm antigens were identified to assess them as a potential target for a contraceptive vaccine. We report here the cloning and sequencing of fSP13, a fox sperm protein of 97 kDa. The fSP13 protein was both auto- and iso-antigenic in foxes; it was recognized by sera of foxes immunized with fox sperm proteins and vasectomized foxes. The NH₂-terminal sequence of fSP13 was determined, and a piece of cDNA was amplified from testicular RNA by reverse transcription polymerase chain reaction. This piece was used to screen a cDNA library from fox testis by Southern blot. A sequence of 1662 base pairs was obtained, including a major open reading frame coding for 498 amino acid. Mass spectrometry analysis confirmed the position of the open reading frame and the presence of posttranscriptional modifications. Analysis of the predicted amino acid sequence revealed no apparent transmembrane regions. Comparison of the protein sequence with the Prosite database demonstrated the presence of four potential N-linked glycosylation sites. The fSP13 bears the closest amino acid similarity to two human sperm proteins: fibrousheathin 2 and testis-specific calcium binding protein 86-VII. The deduced 80 N-terminal amino acid sequence also presents similarity with the RIIα domain. By using a serum against fSP13, this antigen was localized on the principal piece of the fox spermatozoa. Northern blot analysis showed that fSP13 is specifically expressed in testis. The fSP13 is one of the first fox sperm antigens to be cloned and sequenced.