Although contemporary methods of physically separating X from Y chromosome-bearing spermatozoa are now very efficient, overall fertility rates following the use of sex-sorted sperm are not as impressive, in spite of many attempts to improve them. At the same time, there are suggestions from evolutionary biology, and from sex allocation theory in particular, that there may need to be a modification to the chance theory of sex determination in mammals. This is because it now appears that the mammalian female could have some influence on the sex of her offspring, and furthermore, that this influence could be preconceptual. If so, this could go some way towards accounting for the putative inefficiencies in fertilization following insemination with sex-sorted sperm.

The present study investigated the presence and location of fluorescent microspheres having the size of mouse hepatitis virus (MHV) and of mouse minute virus (MMV) in the zona pellucida (ZP) of in vivo-produced murine embryos, the transmission of these viruses by embryos during embryo transfer, and the time of seroconversion of recipients and pups. To this end, fertilized oocytes and morulae were exposed to different concentrations of MMVp for 16 h, while 2-cell embryos and blastocysts were coincubated for 1 h. In addition, morulae were exposed to MHV-A59 for 16 h. One group of embryos was washed, and the remaining embryos remained unwashed before embryo transfer. Serological analyses were performed by means of ELISA to detect antibodies to MHV or MMV in recipients and in progeny on Days 14, 21, 28, 42, and 63 and on Days 42, 63, 84, 112, 133, and 154, respectively, after embryo transfer. Coincubation with a minimum of 10⁵/ml of fluorescent microspheres showed that particles with a diameter of 20 nm but not 100 nm crossed the ZP of murine blastocysts. Washing generally led to a 10-fold to 100-fold reduction of MMVp. Washed MMV-exposed but not MHV-exposed embryos led to the production of antibodies independent of embryonic stage and time of virus exposure. Recipients receiving embryos exposed to a minimum of 10⁷ mean tissue culture infective dose (TCID₅₀)/ml of MHV-A59 and 10² TCID₅₀/ml of MMVp seroconverted by Day 42 after embryo transfer. The results indicate that MMV but not MHV can be transmitted to recipients even after washing embryos 10 times before embryo transfer.

The aryl hydrocarbon receptor (AHR) mediates the effects of many endocrine disruptors and contributes to the loss of fertility in polluted environments. While previous work has focused on mechanisms of short-term endocrine disruption and ovotoxicity in response to AHR ligands, we have shown recently that chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces premature reproductive senescence in female rats without depletion of ovarian follicular reserves. In the current study, premature reproductive senescence was induced using a range of low-dose exposure to TCDD (0, 1, 5, 50, and 200 ng kg⁻¹ wk⁻¹) beginning in utero and continuing until the transition to reproductive senescence. Doses of 50 and 200 ng TCDD kg⁻¹ wk⁻¹ delayed the age at vaginal opening and accelerated the loss of normal reproductive cyclicity with age without depletion of follicular reserves. Serum estradiol concentrations were decreased in a dose-dependent fashion (≥5 ng kg⁻¹ wk⁻¹) across the estrous cycle in perisenescent rats still displaying normal cyclic vaginal cytology. Serum FSH, LH, and progesterone profiles were unchanged by TCDD. The loss of reproductive cyclicity following chronic exposure to TCDD was not accompanied by decreased responsiveness to GnRH. Ovarian endocrine disruption is the predominant functional change preceding the premature reproductive senescence induced by chronic exposure to low doses of the AHR-specific ligand TCDD.

Leptin, the 16-kDa protein product of the obese gene, was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta. In the present work, we studied a possible effect of leptin on trophoblastic cell proliferation, survival, and apoptosis. Recombinant human leptin added to JEG-3 and BeWo choriocarcinoma cell lines showed a stimulatory effect on cell proliferation up to 3 and 2.4 times, respectively, measured by ³H-thymidine incorporation and cell counting. These effects were time and dose dependent. Maximal effect was achieved at 250 ng leptin/ml for JEG-3 cells and 50 ng leptin/ml for BeWo cells. Moreover, by inhibiting endogenous leptin expression with 2 μM of an antisense oligonucleotide (AS), cell proliferation was diminished. We analyzed cell population distribution during the different stages of cell cycle by fluorescence-activated cell sorting, and we found that leptin treatment displaced the cells towards a G2/M phase. We also found that leptin upregulated cyclin D1 expression, one of the key cell cycle-signaling proteins. Since proliferation and death processes are intimately related, the effect of leptin on cell apoptosis was investigated. Treatment with 2 μM leptin AS increased the number of apoptotic cells 60 times, as assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining, and the caspase-3 activity was increased more than 2 fold. This effect was prevented by the addition of 100 ng leptin/ml. In conclusion, we provide evidence that suggests that leptin is a trophic and mitogenic factor for trophoblastic cells by virtue of its inhibiting apoptosis and promoting proliferation.

Testicular germ cell transplantation into the seminiferous tubules is at present the only way to induce spermatogenesis from a given source of spermatogonial stem cells. Here we show an alternative method that harnesses the self-organizing ability of testicular somatic cells. The testicular cells of embryonic or neonatal mice or rats and of newborn pigs were dissociated into single cells. Each of them reorganized into a tubular structure following implantation into the subcutis of immunodeficient mice. When mouse germline stem (GS) cells derived from spermatogonial stem cells and expanded in culture were intermingled with testicular cells of rodents, they were integrated in the reconstituted tubules and differentiated beyond meiosis into spermatids. Normal offspring were produced by the microinjection of those spermatids into oocytes. This method could be applicable to various mammalian species and useful for producing functional gametes from GS cells in a xenoectopic environment.

The regulation of early follicular growth and development involves a complex interaction of autocrine, paracrine, and endocrine signals. The ability of these factors to regulate follicle growth may depend in part on the extent of vascular delivery to and perfusion of the ovary. Vascular endothelial growth factor A (VEGFA) is a major regulator of vascular physiology in the ovary. VEGFA is produced in numerous ovarian compartments and likely plays a role in the regulation of all phases of follicular growth, from preantral through preovulatory. The aim of the present study was to further evaluate the role of VEGF in early follicle growth by neutralization of endogenous VEGF or VEGF receptors. Adult mice were injected systemically and prepubertal mice were injected directly under the ovarian bursa with antibodies designed to neutralize VEGF or block interaction with its receptors in the ovary. Both systemic and intrabursal injections of VEGF antibody significantly reduced the number of primordial follicles within 1–3 days after administration without affecting primary or secondary follicle numbers. Primordial follicle numbers were not different from control levels by 30 days after VEGFA antibody administration. Administration of antibodies to the kinase domain receptor (KDR), but not the FMS-like tyrosine receptor (FLT1), for VEGF also resulted in a significant decrease in primordial follicles. These data suggest that VEGF plays a vital role in the maintenance and growth of the primordial follicle pool.

It is believed that a finite pool of primordial follicles is established during embryonic and neonatal life. At birth, the mouse ovary consists of clusters of interconnected oocytes surrounded by pregranulosa cells. Shortly after birth these structures, termed germ cell cysts or nests (GCN), break down to facilitate primordial follicle formation. Tumor necrosis factor alpha (TNF) is a widely expressed protein with myriad functions. TNF is expressed in the ovary and may regulate GCN breakdown in rats. We investigated whether it participates in GCN breakdown and follicle formation in mice by using an in vitro ovary culture system as well as mutant animal models. We found that TNF and both receptors (TNFRSF1A and TNFRSF1B) are expressed in neonatal mouse ovaries and that TNF promotes oocyte death in neonatal ovaries in vitro. However, deletion of either receptor did not affect follicle endowment, suggesting that TNF does not regulate GCN breakdown in vivo. Tnfrsf1b deletion led to an apparent acceleration of follicular growth and a concomitant expansion of the primordial follicle population. This expansion of the primordial follicle population does not appear to be due to decreased primordial follicle atresia, although this cannot be ruled out completely. This study demonstrates that mouse oocytes express both TNF receptors and are sensitive to TNF-induced death. Additionally, TNFRSF1B is demonstrated to be an important mediator of TNF function in the mouse ovary and an important regulator of folliculogenesis.

Premature chromosome condensation (PCC) was believed to promote nuclear reprogramming and to facilitate cloning by somatic cell nuclear transfer (NT) in mammalian species. However, it is still uncertain whether PCC is necessary for the successful reprogramming of an introduced donor nucleus in cattle. In the present study, fused NT embryos were subjected to immediate activation (IA, simultaneous fusion and activation), delayed activation (DA, activation applied 4 h postfusion), and IA with aged oocytes (IAA, activation at the same oocyte age as group DA). The morphologic changes, such as nuclear swelling, the occurrence of PCC, and microtubule/aster formation, were analyzed in detail by laser-scanning confocal microscopy. When embryos were subjected to IA in both IA and IAA groups, the introduced nucleus gradually became swollen, and a pronuclear-like structure formed within the oocyte, but PCC was not observed. In contrast, delaying embryo activation resulted in 46.5%–91.2% of NT embryos exhibiting PCC. This PCC was observed beginning at 4 h postcell fusion and was shown as one, two, or multiple chromosomal complexes. Subsequently, a diversity of pronuclear-like structures existed in NT embryos, characterized as single, double, and multiple nuclei. In the oocytes exhibiting PCC, the assembled spindle structure was observed to be an interactive mass, closely associated with condensed chromosomes, but no aster had formed. Regardless of whether they were subjected to IA, IAA, or DA treatments, if the oocytes contained pronuclear-like structures, either one or two asters were observed in proximity to the nuclei. A significantly higher rate of development to blastocysts was achieved in embryos that were immediately activated (IA, 59.1%; IAA, 40.7%) than in those for which activation was delayed (14.2%). The development rate was higher in group IA than in group IAA, but it was not significant (P = 0.089). Following embryo transfer, there was no statistically significant difference in the pregnancy rates (Day 70) between two of the groups (group IA, 11.7%, n = 94 vs. group DA, 12.3%, n = 130; P > 0.05) or live term development (group IA, 4.3% vs. group DA, 4.6%; P > 0.05). Our study has demonstrated that the IA of bovine NT embryos results in embryos with increased competence for preimplantational development. Moreover, PCC was shown to be unnecessary for the reprogramming of a transplanted somatic genome in a cattle oocyte.

Arginine and ornithine are known to be important for various biological processes in the testis, but the delivery of extracellular cationic amino acids to the seminiferous tubule cells remains poorly understood. We investigated the activity and expression of cationic amino acid transporters in isolated rat Sertoli cells, peritubular cells, pachytene spermatocytes, and early spermatids. We assessed the l-arginine uptake kinetics, Na dependence of transport, profiles of cis inhibition of uptake by cationic and neutral amino acids, and sensitivity to trans stimulation of cationic amino acid transporters, and studied the expression of the genes encoding them by RT-PCR. Our data suggest that l-arginine is taken up by Sertoli cells and peritubular cells, principally via system y L (SLC3A2/SLC7A6) and system y (SLC7A1 and SLC7A2), with system B⁰ making a minor contribution. By contrast, system B⁰ , associated with system y L (SLC3A2/SLC7A7 and SLC7A6), made a major contribution to the transport of cationic amino acids in pachytene spermatocytes and early spermatids. Sertoli cells had higher rates of l-arginine transport than the other seminiferous tubule cells. This high efficiency of arginine transport in Sertoli cells and the properties of the y L system predominating in these cells strongly suggest that Sertoli cells play a key role in supplying germ cells with l-arginine and other cationic amino acids. Furthermore, whereas cytokines induce nitric oxide (NO) production in peritubular and Sertoli cells, little or no upregulation of arginine transport by cytokines was observed in these cells. Thus, NO synthesis does not depend on the stimulation of arginine transport in these somatic tubular cells.

Using primary cell cultures of human endometrial stromal cells (ES cells), we investigated the role of phospholipase D (PLD) in 8-Br-cAMP-induced decidualization, which involves morphological and biological differentiation processes. When treated with 0.5 mM 8-Br-cAMP for 12 days, ES cells were transformed into a decidualized morphology and produced significant amounts of prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1). Simultaneously, the activity and expression levels of PLD1 increased. In addition, removal of 8-Br-cAMP from decidualized ES cells restored the undifferentiated state, and this was accompanied by decreases in PLD1 promoter activity and PLD1 expression. Overexpression of dominant negative (DN)-PLD1 inhibited the morphological changes induced by 0.5 mM 8-Br-cAMP, whereas PLD1 overexpression induced morphological changes in the absence of 0.5 mM 8-Br-cAMP treatment. Moreover, knockdown of PLD1 by siRNA and blockage of PLD by treatment with 0.3% 1-butanol decreased PRL/IGFBP1 mRNA expression, whereas PLD1 overexpression increased PRL/IGFBP1 mRNA expression. Treatment of ES cells with phosphatidic acid (PA) for 3 days induced PRL mRNA expression and morphological changes, which implies that PA is an end-product of PLD activation-induced decidualization. In addition, pretreatment of ES cells with mepacrine decreased PRL/IGFBP1 expression and inhibited morphological change, whereas pretreatment with propranolol caused no changes, as compared to cAMP-treated cells, which suggests that PA induces decidualization through phospholipase A2 (PLA2G1B). Taken together, these results suggest that PLD1 regulates 8-Br-cAMP-induced decidualization through PLA2G1B, and that PLD1 upregulation is essential for the decidualization of ES cells.

Morphometric methodologies were developed and applied to investigate the patterns of vascular development in maternal (caruncular; CAR) and fetal (cotyledonary; COT) sheep placentas throughout the last two thirds of gestation. We also examined the expression levels of the major angiogenic factors and their receptors in CAR and COT sheep placentas. Although the vascularity of the CAR tissues increased continuously from Day 50 through Day 140 of pregnancy, those of the COT tissues increased at about twice the instantaneous rate (i.e., the proportionate increase/day) of the CAR. For CAR, vascularity increased 2-fold from Day 50 through Day 140 via relatively small increases in capillary number and 2- to 3-fold increases in capillary diameter. For COT, the increased vascularity resulted from a 12-fold increase in capillary number associated with a concomitant 2-fold decrease in capillary diameter. This large increase in fetal placental capillary number, which was due to increased branching, resulted in 6-fold increases in total capillary cross-sectional area and total capillary surface, per unit of COT tissue. Different patterns of expression of the mRNAs for angiogenic factors and their receptors were observed for CAR and COT. The dilation-like angiogenesis of CAR was correlated with the expression of vascular endothelial growth factor receptor-1 (FLT1), angiopoietin-2 (ANGPT2), and soluble guanylate cyclase (GUCY1B3) mRNAs. The branching-like angiogenesis of COT was correlated with the expression of vascular endothelial growth factor (VEGF), FLT1, angiopoietin-1 (ANGPT1), ANGPT2, and FGF2 mRNAs. Monitoring the expression of angiogenic factors and correlating the levels with quantitative measures of vascularity enable one to model angiogenesis in a spatiotemporal fashion.

Our objectives were to compare the cellular and molecular effects of aggregating bovine embryonic vs. somatic cell nuclear transfer (ECNT vs. SCNT) embryos and to determine whether aggregation can improve cattle cloning efficiency. We reconstructed cloned embryos from: 1) morula-derived blastomeres, 2) six adult male ear skin fibroblast lines, 3) one fetal female lung fibroblast line (BFF), and 4) two transgenic clonal strains derived from BFF. Embryos were cultured either singularly (1X) or as aggregates of three (3X). In vitro-fertilized (IVF) 1X and 3X embryos served as controls. After aggregation, the in vitro development of ECNT but not that of SCNT or IVF embryos was strongly compromised. The inner cell mass (ICM), total cell (TC) numbers, and ICM:TC ratios significantly increased for all the aggregates. The relative concentration of the key embryonic transcript POU5F1 (or OCT4) did not correlate with these increases, remaining unchanged in the ECNT and IVF aggregates and decreasing significantly in the SCNT aggregates. Overall, the IVF and 3X ECNT but not the 1X ECNT embryos had significantly higher relative POU5F1 levels than the SCNT embryos. High POU5F1 levels correlated with high in vivo survival, while no such correlation was noted for the ICM:TC ratios. Development to weaning was more than doubled in the ECNT aggregates (10/51 or 20% vs. 7/85 or 8% for 3X vs. 1X, respectively; P < 0.05). In contrast, the SCNT and IVF controls showed no improvement in survival. These data reveal striking biological differences between embryonic and somatic clones in response to aggregation.

The transcription factor NF-kappaB modulates apoptotic machinery following activation by the IkappaB kinase (IKK) complex. Inhibiting activity of one of the catalytic subunits of the IKK complex, IKKbeta (also known as IKBKB and IKK2) severely inhibits NF-kappaB nuclear translocation in response to most stimuli, including ionizing radiation. Doubly floxed Ikkbeta^F/F mice (control) were compared to haplo-insufficient Ikkbeta^F/delta mice (NF-kappaB knockdown) to examine the in vivo apoptotic role of NF-kappaB in the testis. Although Ikkbeta^F/F control adult mice had spermatid head counts and testis and body weights similar to Ikkbeta^F/delta mice, cellular stress in the form of ionizing radiation elicited a differential phenotype. Lower body exposure to 5 Gy of ionizing radiation induced a greater NF-kappaB activation in Ikkbeta^F/F than in Ikkbeta^F/delta mice. In addition, exposure to ionizing radiation resulted in fewer apoptotic germ cells 3, 6, and 12 h after injury in NF-kappaB knockdown mice than in controls, concomitant with the reduced cleavage of caspases 3 and 9 at 3 h. There was also a reduction in total germ cells lost after radiation with NF-kappaB inhibition. Correspondingly, real-time RT-PCR showed a significant reduction in Cdnk1a (also known as p21) and Fasl expression 3 and 6 h, respectively, after irradiation in Ikkbeta^F/delta compared to control testes. These data indicate that, despite acting in an antiapoptotic manner in many tissue types, NF-kappaB is proapoptotic in modulating the germ cell response to ionizing radiation.

When females of the DDK inbred mouse strain are mated to males of other strains, 90–100% of the resulting embryos die during early embryonic development. This DDK syndrome lethality results from incompatibility between an ooplasmic DDK factor and a non-DDK paternal gene, which map to closely linked loci on chromosome 11. It has been proposed that the expression of the gene that encodes the ooplasmic factor is subject to allelic exclusion in oocytes. Previous studies have demonstrated the existence of recessive modifiers that increase lethality in the C57BL/6 and BALB/c strains. These modifiers are thought to skew the choice of allele undergoing allelic exclusion in the oocytes of heterozygous females. In the present study, we demonstrate the presence of modifiers in three Mus musculus domesticus wild-derived strains, PERA, PERC, and RBA. These modifiers completely rescued DDK syndrome lethality. We mapped the major locus that is responsible for rescue in PERA and PERC crosses to proximal chromosome 13 and named this locus Rmod1 (Rescue Modifier of the DDK Syndrome 1). Our experiments demonstrate that PERA or PERC alleles at Rmod1 rescue lethality independently of allelic exclusion. In addition, rescue of the lethal phenotype depends on the parental origin of the Rmod1 alleles; transmission through the dam leads to rescue, while transmission through the sire has no effect.

Growth differentiation factor 9 (GDF9) is a member of the transforming growth factor beta (TGFB) superfamily. As an oocyte-specific growth factor, GDF9 plays critical roles in controlling folliculogenesis in mammals. In the present study, we cloned a 2.1-kb cDNA of the zebrafish GDF9 homolog (Gdf9, gdf9), which shares ~60% homology with that of mammals in the mature region. RT-PCR analysis showed that zebrafish gdf9 expression was present only in the gonads and Northern blot analysis revealed a single transcript of about 2.0 kb in the ovary. Real-time RT-PCR analysis revealed that gdf9 expression was highest in primary growth (PG, stage I) follicles and gradually decreased during follicular development, with the lowest level being found in fully grown (FG) follicles. The expression of gdf9 was maintained through fertilization and early embryonic development until gastrulation, at which point the expression level dramatically decreased. Expression was barely detectable after the late gastrula stage. Within the follicle, gdf9 mRNA was localized exclusively in the oocytes, as demonstrated by RT-PCR of denuded oocytes and freshly isolated follicle layers as well as by in situ hybridization. Interestingly, when amplified for high numbers of cycles, the expression of gdf9 was detected in cultured zebrafish follicular cells that were free of oocytes. The expression of gdf9 was downregulated by hCG in both ovarian fragments and isolated follicles in dose- and time-dependent manners, and this inhibition appeared to be stage-dependent, with the strongest inhibition observed for the FG follicles and no effect seen for the PG follicles. This correlates well with the expression profile of the LH receptor (lhcgr) in zebrafish follicles. In conclusion, as an oocyte-derived growth factor, GDF9 is highly conserved across vertebrates. With its biological advantages, zebrafish provides an alternative model for studying gene function and regulation.

The roles of the leucine-rich repeat domain containing G protein-coupled receptor (GPCR) 4 (Lgr4), which is one of the orphan GPCRs, were analyzed with the Lgr4 hypomorphic mutant mouse line (Lgr4^Gt). This homozygous mutant had only one-tenth the normal transcription level; furthermore, 60% of them survived to adulthood. The homozygous male was infertile, showing morphologic abnormalities in both the testes and the epididymides. In the testes, luminal swelling, loss of germinal epithelium in the seminiferous tubules, and rete testis dilation were observed. Cauda epididymidis sperm were immotile. Rete testis dilation was due to a water reabsorption failure caused by a decreased expression of an estrogen receptor (ESR1) and SLC9A3 in the efferent ducts. Although we found differential regulation of ESR1 expression in the efferent ducts and the epididymis, the role of ESR1 in the epididymis remains unclear. The epididymis contained short and dilated tubules and completely lacked its initial segment. In the caput region, we observed multilamination and distortion of the basement membranes (BMs) with an accumulation of laminin. Rupture of swollen epididymal ducts was observed, leading to an invasion of macrophages into the lumen. Male infertility was probably due to the combination of a developmental defect of the epididymis and the rupture of the epithelium resulting in the immotile spermatozoa. These results indicate that Lgr4 has pivotal roles to play in the regulation of ESR1 expression, the control of duct elongation through BM remodeling, and the regional differentiation of the caput epididymidis.

The sperm from the testis acquires complete fertilizing ability and forward progressive motility following its transit through the epididymis. Acquisition of these characteristics results from the modification of the sperm proteome following interactions with epididymal secretions. In our attempts to identify epididymis-specific sperm plasma membrane proteins, a partial 2.83-kb clone was identified by immunoscreening a monkey epididymal cDNA library with an agglutinating monoclonal antibody raised against washed human spermatozoa. The sequence of the 2.83-kb clone exhibited homology to the region between 1 and 1097 bp of the homeobox gene, Hoxb2. This sequence was found to be species conserved, as revealed by RT-PCR analysis. To obtain a full-length clone of the sequence, 5′ RACE-PCR (rapid amplification of cDNA ends PCR) was carried out using rat epididymal RNA as the template. It resulted in a full-length 1.657-kb cDNA encoding a 32.9-kDa putative protein. The protein designated HOXBES2 exhibited homology to the conserved 61-amino acid homeodomain region of the HOXB2 homeoprotein. However, characteristic differences were noted in its amino and carboxyl termini compared with HOXB2. A putative 30-kDa protein was detected in the tissue extracts from adult rat epididymis and caudal spermatozoa, and a 37-kDa protein was detected in the rat embryo when probed with a polyclonal antibody against HOXB2 protein. Multiple tissue Western blot and immunohistochemical analysis further indicated its expression in the cytoplasm of the principal and basal epithelial cells, with maximal expression in the distal epididymal segments. Northern blot analysis detected a single ~2.5-kb transcript from the adult epididymis. Indirect immunofluorescence localized the protein to the acrosome, midpiece, and equatorial segments of rat caudal and ejaculated human and monkey spermatozoa, respectively. In conclusion, we have identified and characterized a novel epididymal homeoprotein different from HOXB2 protein and hereafter referred to as HOXBES2, (HOXB2 homeodomain containing epididymis-specific sperm protein) with a probable role in fertilization.

Cellular ATP is mainly generated through mitochondrial oxidative phosphorylation, which is dependent on mitochondrial DNA (mtDNA). We have previously demonstrated the importance of oocyte mtDNA for porcine and human fertilization. However, the role of nuclear-encoded mitochondrial replication factors during oocyte and embryo development is not yet understood. We have analyzed two key factors, mitochondrial transcription factor A (TFAM) and polymerase gamma (POLG), to determine their role in oocyte and early embryo development. Competent and incompetent oocytes, as determined by brilliant cresyl blue (BCB) dye, were assessed intermittently during the maturation process for TFAM and POLG mRNA using real-time RT-PCR, for TFAM and POLG protein using immunocytochemistry, and for mtDNA copy number using real-time PCR. Analysis was also carried out following treatment of maturing oocytes with the mtDNA replication inhibitor, 2′,3′-dideoxycytidine (ddC). Following in vitro fertilization, preimplantation embryos were also analyzed. Despite increased levels of TFAM and POLG mRNA and protein at the four-cell stage, no increase in mtDNA copy number was observed in early preimplantation development. To compensate for this, mtDNA appeared to be replicated during oocyte maturation. However, significant differences in nuclear-encoded regulatory protein expression were observed between BCB and BCB⁻ oocytes and between untreated oocytes and those treated with ddC. These changes resulted in delayed mtDNA replication, which correlated to reduced fertilization and embryonic development. We therefore conclude that adherence to the regulation of the timing of mtDNA replication during oocyte maturation is essential for successful embryonic development.

Intracytoplasmic sperm injection (ICSI) of DNA-loaded sperm cells has been shown to be a valuable tool for the production of transgenic animals, especially when DNA constructs with submegabase magnitude are used. In order to optimize and to understand the mechanism of the ICSI-mediated transgenesis, we have evaluated the impact of transgene DNA concentration, transgene flanking with nuclear matrix attachment regions (MARs), and the use of recombinase A (RecA)-coated DNA on the efficiency of mouse transgenesis production by ICSI. Presented data include assays with three DNA constructs; an enhanced green fluorescent protein (EGFP) plasmid of 5.4 kb, this plasmid flanked with two MAR elements (2.3 Kb of the human beta-interferon domain boundaries), and a yeast artificial chromosome (YAC) construct of ~510 kb (the largest transgenic construct introduced by ICSI that we have seen reported). ICSI-mediated transgenesis was done in the B6D2 mouse strain using different concentrations for each construct. Analysis of generated data indicated that ICSI allows the use of higher DNA concentrations than the ones used for pronuclear microinjection, however, when a certain threshold is exceeded, embryo/fetal viability decrease dramatically. In addition, independently of the transgene concentration tested, transgene flanking with MAR sequences did not have a significant impact on the efficiency of this transgenesis method. Finally, we observed that although the overall efficiency of ICSI-mediated transgenesis with fresh spermatozoa and RecA-complexed DNA was similar to the one obtained with the common ICSI-mediated transgenesis approach with frozen-thawed spermatozoa and RecA free DNA, this method was not as efficient in maintaining a low frequency of founder animal mosaicism, suggesting that different mechanisms of transgene integration might result from each procedure.