Birth rates for older fathers have increased 30% since 1980. When combined with the increased risk for genetic and multifactorial disorders in children conceived by older fathers, paternal age has become an important health issue for modern society. Laboratory research in this area has been minimal, perhaps because of significant experimental barriers, not the least of which is inadequate access to fresh, disease-free human testicular tissue. Regardless, progress has been made and intriguing models supported by experimental evidence have been proposed. The putative mechanisms range from reduced DNA repair activity, leading to increased mutagenesis, to positive selection of germ cells harboring specific disease-causing mutations. There remain many important venues for research in this increasingly relevant phenomenon that impacts future generations.

H19 is a maternally expressed, imprinted, noncoding RNA with tumor-suppressor activity. During mouse preimplantation development, H19 is primarily expressed in the trophectoderm cells. The purpose of this project was to determine allelic expression of H19 in pre- and peri-implantation mouse embryos. We were further interested in determining if loss of imprinted H19 expression during blastocyst development occurred as a result of superovulation and/or culture. Our last goal was to ascertain if differential H19 allelic expression occurred between the inner cell mass (ICM)-containing half and the primary trophoblast giant cell (PTGC)-containing half of the embryo. C57BL/6J^(Cast-7)xC57BL/6J F1 embryos were collected from the uterus at 84, 96, and 108 h following natural ovulation or superovulation. In vitro-cultured F1 embryos were harvested from the oviduct at the 2-cell stage and cultured in KSOM aa supplemented with amino acids or Whitten media and collected at the above-mentioned times. Allele-specific H19 expression in single embryos was determined by qRT-PCR followed by fluorescence resonance electron transfer or RT-PCR followed by restriction fragment length polymorphism and polyacrylamide gel electrophoresis (RFLP-PAGE). Peri-implantation embryos were microdissected into two sections, one containing the ICM and the other containing the PTGC. TaqMan probes for Dek, Pou5f1, Itga7, H19, and Igf2 were used to ascertain gene expression enrichment in each section. Allele-specific H19 expression in embryo sections was determined by RFLP-PAGE. We found that as embryos advance through preimplantation development they start expressing H19 in a biallelic manner and this phenomenon was observed in the cultured and the in vivo-developed embryos. The PTGC-containing half of the embryo had greater expression of H19 when compared to the ICM-containing half of the embryo, as determined by qRT-PCR. In conclusion, loss of imprinting of H19 occurs in the PTGC-containing section of peri-implantation mouse embryos. We speculate that this is part of a physiologic event at the time of implantation in the mouse.

Fertility rates have dramatically decreased in the last two decades, especially in men. It has been described that environmental factors as well as life habits may affect semen quality. In this paper we use artificial intelligence techniques in order to predict semen characteristics resulting from environmental factors, life habits, and health status, with these techniques constituting a possible decision support system that can help in the study of male fertility potential. A total of 123 young, healthy volunteers provided a semen sample that was analyzed according to the World Health Organization 2010 criteria. They also were asked to complete a validated questionnaire about life habits and health status. Sperm concentration and percentage of motile sperm were related to sociodemographic data, environmental factors, health status, and life habits in order to determine the predictive accuracy of a multilayer perceptron network, a type of artificial neural network. In conclusion, we have developed an artificial neural network that can predict the results of the semen analysis based on the data collected by the questionnaire. The semen parameter that is best predicted using this methodology is the sperm concentration. Although the accuracy for motility is slightly lower than that for concentration, it is possible to predict it with a significant degree of accuracy. This methodology can be a useful tool in early diagnosis of patients with seminal disorders or in the selection of candidates to become semen donors.

Uterine glands and their secretions are hypothesized to be essential for blastocyst implantation and decidualization in the uterus of rodents and humans. One factor solely expressed by uterine glands in mice is leukemia inhibitory factor (LIF), and Lif null mice are infertile because of defective blastocyst attachment to the uterine luminal epithelium (LE). Progesterone treatment of neonatal mice permanently ablates differentiation of uterine glands, resulting in an aglandular uterus in the adult. Progesterone-induced uterine gland knockout (PUGKO) mice were used to investigate the biological role of uterine glands in blastocyst implantation and stromal cell decidualization. As compared to controls, PUGKO mice cycled normally but were infertile. Histological assessment of PUGKO uteri on Days 5.5 and 8.5 postmating found a hatched blastocyst apposed to an intact LE without evidence of implantation or stromal cell decidualization. Expression of several implantation-related factors, including Lif and PTGS2, were altered in the PUGKO uterus, whereas expression of steroid hormone receptors and their regulated genes was not different. Artificial decidualization was observed in the uteri of control but not PUGKO mice. Further, intrauterine administration of LIF failed to promote artificial decidualization in the uterus of PUGKO mice. Thus, uterine glands and their secretions have important biological roles in blastocyst implantation and stromal cell decidualization in the uterus.

In mammalian oocytes, cAMP-dependent protein kinase (PKA) has critical functions in meiotic arrest and meiotic maturation. Although subcellular localization of PKA is regulated by A-kinase anchor proteins (AKAPs) and PKA compartmentalization is essential for PKA functions, the role of AKAPs in meiotic regulation has not been fully elucidated. In the present study, we performed far-Western blot analysis using porcine PRKAR2A for detection of AKAPs and found, to our knowledge, several novel signals in porcine oocytes. Among these signals, a 150-kDa AKAP showed the major expression and was the product of porcine AKAP1. Overexpression of AKAP1 changed the PKA localization and promoted meiotic resumption of porcine oocytes even in the presence of a high concentration of cAMP, which inhibits meiotic resumption by inducing high PKA activity. On the contrary, knockdown of AKAP1 showed inhibitory effects on meiotic resumption and oocyte maturation. In addition, the expression level of AKAP1 in porcine growing oocytes, which show meiotic incompetence and PKA mislocalization, was significantly lower than that in fully grown oocytes. However, AKAP1 insufficiency was not the primary cause of the meiotic incompetence of the growing oocytes. These results suggest that the regulation of PKA localization by AKAP1 may be involved in meiotic resumption and oocyte maturation but not in meiotic incompetence of porcine growing oocytes.

We demonstrated previously that a knockout (KO) of the lactate dehydrogenase type C (Ldhc) gene disrupted male fertility and caused a considerable reduction in sperm glucose consumption, ATP production, and motility. While that study used mice with a mixed genetic background, the present study used C57BL/6 (B6) and 129S6 (129) Ldhc KO mice. We found that B6 KO males were subfertile and 129 KO males were infertile. Sperm from 129 wild-type (WT) mice have a lower glycolytic rate than sperm from B6 WT mice, resulting in a greater reduction in ATP production in 129 KO sperm than in B6 KO sperm. The lower glycolytic rate in 129 sperm offered a novel opportunity to examine the role of mitochondrial respiration in sperm ATP production and motility. We observed that in media containing a mitochondrial substrate (pyruvate or lactate) as the sole energy source, ATP levels and progressive motility in 129 KO sperm were similar to those in 129 WT sperm. However, when glucose was added, lactate was unable to maintain ATP levels or progressive motility in 129 KO sperm. The rate of respiration (ZO₂) was high when 129 KO or WT sperm were incubated with lactate alone, but addition of glucose caused a reduction in ZO₂. These results indicate that in the absence of glucose, 129 sperm can produce ATP via oxidative phosphorylation, but in the presence of glucose, oxidative phosphorylation is suppressed and the sperm utilize aerobic glycolysis, a phenomenon known as the Crabtree effect.

By targeted disruption of the lactate dehydrogenase c (Ldhc) gene, we demonstrated that spermatozoa require Ldhc for capacitation, motility, and fertilizing capacity. Ldhc expression is restricted to the developing germ cells that, however, are apparently not compromised by the lack of the LDHC isozyme. Because LDHC is abundant in spermatozoa that utilize aerobic glycolysis for energy requirements, its main function was presumed to be the interconversion of pyruvate to lactate with the concomitant oxidation/reduction of NADH to NAD . We found that sperm without LDHC were still able to convert lactate to pyruvate as mediated by LDHA that is tightly bound to the fibrous sheath. It was assumed that the level of glycolysis was insufficient to power motility and the subsequent fertilizing capacity of the mutated sperm. To investigate whether LDHC possesses certain unique characteristics essential for fertility, human LDHA was introduced as a transgene to Ldhc-null mice. We report here that the exogenous LDHA rescued the phenotype of the Ldhc-null males. Sperm from the LDHA transgenic males with the Ldhc deletion (LDHA /Ldhc^−/−) are motile, capable of protein tyrosine phosphorylation, and able to fertilize, thus restoring these properties to LDHC-null sperm. However, the lactate and ATP levels in the rescued sperm did not differ significantly from sperm lacking LDHC. We suggest that it is the localization of the transgene to the sperm cytosol that is mainly responsible for restoration of sperm function and fertility.

The Foxos are key effectors of the PI3K/Akt signaling pathway and regulate diverse physiologic processes. Two of these factors, Foxo1 and Foxo3, serve specific roles in reproduction in the mouse. Foxo3 is required for suppression of primordial follicle activation in females, while Foxo1 regulates spermatogonial stem cell maintenance in males. In the mouse ovary, Foxo1 is highly expressed in somatic cells (but not in oocytes), suggesting an important functional role for Foxo1 in these cells. Given that invertebrate model species such as Caenorhabditis elegans and Drosophila melanogaster harbor a single ancestral Foxo homolog, these observations suggest that gene duplication conferred a selective advantage by permitting the Foxos to adopt distinct roles in oogenesis and spermatogenesis. Our objective was to determine if the remarkably specific expression patterns of Foxo1 and Foxo3 in mouse gonads (and, by inference, Foxo function) are conserved in diverse mammalian species. Western blotting was used to validate isoform-specific antibodies in rodents, companion animals, farm animals, nonhuman primates, and humans. Following validation of each antibody, immunohistochemistry was performed to ascertain Foxo1 and Foxo3 gonadal expression patterns. While Foxo1 expression in spermatogonia and granulosa cells was conserved in each species evaluated, Foxo3 expression in oocytes was not. Our findings suggest that Foxo3 is not uniquely required for primordial follicle maintenance in nonrodent species and that other Foxos, particularly Foxo1, may contribute to oocyte maintenance in a functionally redundant manner.

ACRBP/sp32 is a binding protein specific for the precursor (pro-ACR) and intermediate forms of sperm serine protease ACR. In this study, we examined the expression pattern, localization, and possible role of mouse ACRBP in spermatogenic cells and epididymal sperm. Unlike other mammalian ACRBPs, two forms of Acrbp mRNA—wild-type Acrbp-W and variant Acrbp-V5 mRNAs—were generated by alternative splicing of Acrbp in the mouse. ACRBP-W was synthesized in pachytene spermatocytes and haploid spermatids and immediately processed into a mature protein, ACRBP-C, by removal of the N-terminal half. The intron 5-retaining splice variant mRNA produced a predominant form of ACRBP, ACRBP-V5, that was present in pachytene spermatocytes and round spermatids, but was absent in elongating spermatids. ACRBP-W and ACRBP-V5 were both colocalized with pro-ACR in the acrosomal granules of early round spermatids, whereas the sperm acrosome contained only ACRBP-C. Glutathione S-transferase pull-down assays revealed that ACRBP-V5 and ACRBP-C possess a different domain capable of binding each of two segments in the C-terminal region of pro-ACR. Moreover, autoactivation of pro-ACR was remarkably accelerated by the presence of ACRBP-C. These results suggest that ACRBP-V5 and ACRBP-C may function in the transport/packaging of pro-ACR into acrosomal granules during spermiogenesis and in the promotion of ACR release from the acrosome during acrosomal exocytosis, respectively.

Infections of the uterus or mammary gland with Gram-negative bacteria cause infertility in cattle, not only during disease but also for some time afterward. Even though these infections are in organs distant from the ovary, metritis and mastitis perturb antral follicle development and function in vivo. Although granulosa cells from antral follicles express toll-like receptor 4 (TLR4), and detect and mount an inflammatory response to lipopolysaccharide (LPS) from Gram-negative bacteria, it is not known whether LPS impacts preantral follicle development. The present study tested the hypothesis that LPS perturbs the development of primordial ovarian follicles. Exposure of bovine ovarian cortex ex vivo to LPS reduced the primordial follicle pool associated with increased primordial follicle activation. Ovarian cortex culture supernatants accumulated the inflammatory mediators IL-1beta, IL-6, and IL-8 in an LPS concentration-dependent manner. In addition, LPS exposure modulated key intracellular regulators of follicle activation with loss of the primordial follicle PTEN and cytoplasmic translocation of FOXO3. Acute exposure of mice in vivo to LPS also reduced the primordial follicle pool associated with increased follicle atresia. The increased follicle atresia was TLR4-dependent as Tlr4-deficient mice were insensitive to LPS-mediated follicle atresia. However, LPS did not affect the diameter of individually cultured bovine secondary follicles or their enclosed oocytes. In conclusion, LPS reduced the primordial ovarian follicle pool in the bovine ovarian cortex ex vivo and in the murine ovary in vivo. These observations provide an insight into how bacterial infections distant from the ovary have long term effects on fertility.

Recently, a Chinese genomewide association study (GWAS) identified four autosomal single-nucleotide polymorphism (SNP) loci as being significantly associated with risk factors for nonobstructive azoospermia (NOA; P < 5 × 10⁻⁸). In the present study, we performed a replication study on two Japanese cohorts from different institutions in order to evaluate whether SNP loci are associated with NOA. The four SNPs (rs12097821, rs2477686, rs10842262, and rs6080550) reported in the Chinese GWAS were genotyped in 490 NOA patients and 1167 controls. To assess the significance of the associations between each of the four SNPs and NOA in the Japanese population, the association results for the two cohorts were combined by meta-analysis. In the meta-analysis, the combined per-allele odds ratios (ORs) for the four SNPs and their respective 95% confidence intervals (CIs) were as follows: rs12097821, OR = 1.10 (CI = 0.89–1.37); rs2477686, OR = 1.11 (CI = 0.87–1.43); rs10842262, OR = 1.11 (CI = 0.94–1.32); and rs6080550, OR = 0.96 (CI = 0.76–1.21). None of the SNPs was significantly associated with NOA (P > 0.05). However, three of four SNPs (rs12097821, rs2477686, and rs10842262) showed associations in the same direction in Japanese men as those reported in the Chinese GWAS. To determine whether the four SNPs are genetic risk factors for NOA, the effect sizes of NOA risk factors require further investigation using larger independent sets of case-control samples of populations, including Japanese and Chinese populations.

In males of seasonally breeding species, testes undergo a severe involution at the end of the breeding season, with a major volume decrease due to massive germ-cell depletion associated with photoperiod-dependent reduced levels of testosterone and gonadotropins. Although it has been repeatedly suggested that apoptosis is the principal effector of testicular regression in vertebrates, recent studies do not support this hypothesis in some mammals. The purpose of our work is to discover alternative mechanisms of testis regression in these species. In this paper, we have performed a morphological, hormonal, ultrastructural, molecular, and functional study of the mechanism of testicular regression and the role that cell junctions play in the cell-content dynamics of the testis of the Iberian mole, Talpa occidentalis, throughout the seasonal breeding cycle. Desquamation of live, nonapoptotic germ cells has been identified here as a new mechanism for seasonal testis involution in mammals, indicating that testis regression is regulated by modulating the expression and distribution of the cell-adhesion molecules in the seminiferous epithelium. During this process, which is mediated by low intratesticular testosterone levels, Sertoli cells lose their nursing and supporting function, as well as the impermeability of the blood-testis barrier. Our results contradict the current paradigm that apoptosis is the major testis regression effector in vertebrates, as it is clearly not true in all mammals. The new testis regression mechanism described here for the mole could then be generalized to other mammalian species. Available data from some previously studied mammals should be reevaluated.

Several studies have investigated whether particular Y chromosome haplogroups are associated with spermatogenic failure in Japanese males; however, they produced differing results. In this study, to investigate the association of Y chromosome haplogroup with spermatogenic failure, we recruited 451 infertile patients and 730 fertile men from a Japanese population and typed their Y chromosome haplogroups. The infertile patients were suffering from varicocele, azoospermia, oligozoospermia, asthenozoospermia, obstructive azoospermia, karyotype abnormalities, microdeletions of the long arm of the Y chromosome, or other conditions that affect fertility. The frequency of haplogroup D2* was significantly higher (odds ratio = 2.28, 95% confidence interval = 1.44–3.61, P = 0.00034 using chi-square test) among the men with azoospermia than among the fertile men. None of the other Y haplogroups displayed associations with particular types of infertility. In conclusion, Y chromosome haplogroup D2* is associated with spermatogenic failure in Japanese males, suggesting that the Y chromosome lineage can have significant effects on spermatogenesis.

Gonadotropin-inhibitory hormone (GnIH) has been shown to inhibit reproduction in several species. GnIH suppresses gonadotropin synthesis/release at the hypothalamic and pituitary levels; however, increasing evidence suggests that GnIH has a putative function in the gonad. In this study, we demonstrated that GnIH receptors localize to the ovary and testis in goldfish. In situ hybridization illustrated that goldfish GnIHRs were localized exclusively to the oocytes before the cortical alveolus stage and to the interstitial tissue to the testis. Implantation of goldfish GnIH peptides did not affect the serum estradiol levels in female goldfish, but it did enhance the serum testosterone levels in males. Conversely, injecting goldfish GnIH peptides increased the expression of StAR and 3bHSD mRNA and decreased the expression of CYP19 mRNA significantly in the testis, but these genes remained unchanged in the ovary. In addition, goldfish GnIH peptides not only increased the expression of StAR and 3bHSD and decreased CYP19 mRNA, but they also increased the expression of FSHR and LHR mRNA in testicular cells. However, they did not affect the expression of these genes in ovarian cells in vitro. Thus, we suggest that GnIH may contribute to the sexual dimorphism of steroidogenesis in goldfish.

Intraovarian factors play important roles in coordinating germ cell and somatic cell growth in the ovary. Prior to the onset of gonadotropin stimulation and reproductive cyclicity, follicle development is dependent upon locally produced growth factors, such as the transforming growth factor beta family members inhibin, activin, and GDF9. In the absence of inhibin in prepubertal mice (Inha^−/−), there are marked alterations in preantral follicle growth, but no evidence of ovarian tumors characteristic of adult Inha-null mice. To ascertain the contribution of GDF9 to the Inha-null phenotype, we analyzed folliculogenesis in postnatal Inha Gdf9 double knockout mice. Deletion of Gdf9 from Inha^−/− rescues the initial growth defects found at early follicle stages in Inha^−/− ovaries, but surprisingly enhances the onset of pretumor lesions. The normalization of growth dynamics between granulosa cells and oocytes of Inha Gdf9 double knockout mice is also accompanied by a reduction in levels of the activin/inhibin beta B subunit, Inhbb, which is upregulated in Inha^−/− ovaries. However, at later ages, Inha Gdf9 double knockout ovaries are similar to Inha^−/− ovaries, and show upregulation of the activin/inhibin subunits and downregulation of the growth factor, kit ligand, thus resulting in a local environment that is growth-promoting for granulosa cells but growth-inhibitory for oocytes. These data suggest a sequential mechanism of action initiated by GDF9 in the Inha knockout mouse that promotes defective folliculogenesis. These studies thus provide a novel role for GDF9 in causing reproductive defects and suppressing tumor initiation in the Inha^−/− mouse model.

Vitellogenin (VTG) and apolipoprotein (APO) play a central role in animal reproduction and lipid circulation, respectively. Although previous studies have examined the structural and functional relationships of these large lipid transfer proteins (LLTPs) from an evolutionary perspective, the mechanism in generating these different families have not been addressed in invertebrates. In this study, the most comprehensive phylogenetic and genomic analysis of the LLTP superfamily genes is carried out. We propose the expansion and diversification of LLTPs in invertebrates are mediated via retrotransposon-mediated duplications, followed by either subfunctionalization or neofunctionalization in different lineages. In agreement with a previous hypothesis, our analysis suggests that all LLTPs originate from a series of duplications of a primitive yolk protein gene similar to VTG. Two early consecutive duplications of the yolk protein genes resulted in the formation of microsomal triglyceride transfer protein (MTP) and the APO gene ancestor. Gains and losses of domains and genes occurred in each of these families in different animal lineages, with MTP becoming truncated. MTP maintained only the components stabilizing the huge lipoprotein particle. Surprisingly, for the first time, two VTG-like protein families were found to independently arise in the lineages of insects. This work consolidates the reconstruction of the evolutionary roadmap of the LLTP superfamily and provides the first mechanistic explanation on the expansion of family members via retrotransposition in invertebrates.

In pigs, conceptus attachment to the uterine surface epithelium starts around Day 14 of pregnancy preceded by a pronounced vascularization at the implantation zones, initiating the epitheliochorial placentation. To characterize the complex transcriptome changes in the endometrium in the course of initial conceptus attachment, deep sequencing of endometrial RNA samples of pregnant animals (n = 4) and corresponding cyclic controls (n = 4) was performed using Illumina RNA-Seq. The obtained sequence reads were mapped to the porcine genome, and relative expression values were calculated for the analysis of differential gene expression. Statistical analysis revealed 1933 differentially expressed genes (false discovery rate 1%), 1229 with higher and 704 with lower mRNA concentration, in the samples from pregnant animals. Expression of selected genes was validated by the use of quantitative real-time RT-PCR. The RNA-Seq data were compared to results of a microarray study of bovine endometrium on Day 18 of pregnancy and additional related data sets. Bioinformatics analysis revealed for the genes with higher mRNA concentration in pregnant samples strong overrepresentation, particularly for immune-related functional terms but also for apoptosis and cell adhesion. Overrepresented terms for the genes with lower mRNA concentration in pregnant samples were related to extracellular region, ion transport, cell adhesion, and lipid and steroid metabolic process. In conclusion, RNA-Seq analysis revealed comprehensive transcriptome differences in porcine endometrium between Day 14 of pregnancy and corresponding cyclic endometrium and highlighted new processes and pathways probably involved in regulation of noninvasive implantation in the pig.

Severe prenatal undernutrition is usually associated with low birth weights in offspring and disorders including hypertension, obesity, and diabetes. Whether alterations in maternal nutrition insufficient to impair birth weight or prenatal growth impact the cardiovascular, stress, or metabolic systems is unknown. In addition, little is known about the effects of maternal dietary restriction on development of the reproductive system in mammals. Here, we use the bovine model, which has a gestational length and birth rate similar to humans, to show that offspring from nutritionally restricted dams (during the first trimester) were born with identical birth weights and had similar postnatal growth rates (to 95 wk of age), puberty, glucose metabolism, and responses to stress compared to offspring from control mothers. However, an increase in maternal testosterone concentrations was detected during dietary restriction, and these dams had offspring with a diminished ovarian reserve (as assessed by a reduction in antral follicle count, reduced concentrations of anti-Müllerian hormone, and increased follicle-stimulating hormone concentrations), enlarged aorta, and increased arterial blood pressure compared with controls. Our study links transient maternal undernutrition and enhanced maternal androgen production with a diminished ovarian reserve as well as potential suboptimal fertility, enlarged aortic trunk size, and enhanced blood pressure independent of alterations in birth weight, postnatal growth, or stress response and glucose tolerance. The implications are that relatively mild transient reductions in maternal nutrition during the first trimester of pregnancy (even those that do not affect gross development) should be avoided to ensure healthy development of reproductive and cardiovascular systems in offspring.

Photostimulation of retinal photoreceptors appears to inhibit reproductive activity in birds. In the present study, the involvement of serotonin and vasoactive intestinal peptide was investigated in relation to reproductive failure associated with retinal photostimulation. Hens at 23 wk of age were divided into six rooms equipped with individual cages. At 24 wk of age, three rooms were photostimulated (14L:10D) with white light (control). Three rooms had two parallel lighting systems, red (660 nm) and green (560 nm), which were both on during 6 h of the 14-h light period. Upon photostimulation, the red light was turned off after 6 h, and the green light was left on for a total of 14 h (Green). Five hens from each room served as controls, five hens were immunized against vasoactive intestinal peptide, and five hens received parachlorophenylalanine, an inhibitor of serotonin biosynthesis. Parachlorophenylalanine treatment increased reproductive performance and mRNA expression of GnRH-I, LH-beta and FSH-beta (P < 0.05) in the Green group to levels which did not differ from those of the White (control) group. Immunization against vasoactive intestinal peptide reduced plasma concentration and pituitary mRNA expression of prolactin but did not affect expression of gonadal axis genes. Collectively, the results suggest that retinal photostimulation inhibits the reproductive axis through serotonin and not through vasoactive intestinal peptide.

Oocyte cryopreservation is important for assisted reproductive technologies (ART). Although cryopreservation of metaphase II (MII) oocytes has been successfully used, MII oocytes are vulnerable to the damage inflicted by the freezing procedure. Cryopreservation of germinal vesicle stage oocytes (GV-oocytes) is an alternative choice; however, blastocyst development from GV-oocytes is limited largely due to the need for in vitro maturation (IVM). Herein, we evaluated the effects of l-carnitine (LC) supplementation during vitrification and thawing of mouse GV-oocytes, IVM, and embryo culture on preimplantation development after in vitro fertilization (IVF). We first compared the rate of embryonic development from the oocytes that had been collected at the GV stage from three mouse strains, (B6.DBA)F1, (B6.C3H)F1, and B6, and processed for IVM and IVF, as well as that from the oocytes matured in vivo, i.e. ovulated (IVO). Our results demonstrated that the rate of blastocyst development was the highest in the (B6.DBA)F1 strain and the lowest in the B6 strain. We then supplemented the IVM medium with 0.6 mg/ml LC. The rate of blastocyst development improved in the B6 but not in the (B6.DBA)F1 strain. Vitrification of GV-oocytes in the basic medium alone reduced the rate of blastocyst development in both of those mouse strains. LC supplementation to the IVM medium alone did not change the percentage of blastocyst development. However, LC supplementation to both vitrification and IVM media significantly improved blastocyst development to the levels comparable with those obtained from vitrified/thawed IVO oocytes in both of the (B6.DBA)F1 and B6 strains. We conclude that LC supplementation during vitrification is particularly efficient in improving the preimplantation development from the GV-oocytes that otherwise have lower developmental competence in culture.

Sperm utilize glycolysis to generate ATP required for motility, and several spermatogenic cell-specific glycolytic isozymes are associated with the fibrous sheath (FS) in the principal piece of the sperm flagellum. We used proteomics and molecular biology approaches to confirm earlier reports that a novel enolase is present in mouse sperm. We then found that a pan-enolase antibody, but not antibodies to ENO2 and ENO3, recognized a protein in the principal piece of the mouse sperm flagellum. Database analyses identified two previously uncharacterized enolase family-like candidate genes, 64306537H0Rik and Gm5506. Northern analysis indicated that 64306537H0Rik (renamed Eno4) was transcribed in testes of mice by Postnatal Day 12. To determine the role of ENO4, we generated mice using embryonic stem cells in which an Eno4 allele was disrupted by a gene trap containing a beta galactosidase (beta-gal) reporter (Eno4 ^/Gt). Expression of beta-gal occurred in the testis, and male mice homozygous for the gene trap allele (Eno4^Gt/Gt) were infertile. Epididymal sperm numbers were 2-fold lower and sperm motility was reduced substantially in Eno4^Gt/Gt mice compared to wild-type mice. Sperm from Eno4^Gt/Gt mice had a coiled flagellum and a disorganized FS. The Gm5506 gene encodes a protein identical to ENO1 and also is transcribed at a low level in testis. We conclude that ENO4 is required for normal assembly of the FS and provides most of the enolase activity in sperm and that Eno1 and/or Gm5506 may encode a minor portion of the enolase activity in sperm.

Leydig cells are the steroidogenic lineage of the mammalian testis that produces testosterone, a key hormone required throughout male fetal and adult life for virilization and spermatogenesis. Both fetal and adult Leydig cells arise from a progenitor population in the testis interstitium but are thought to be lineage-independent of one another. Genetic evidence indicates that Notch signaling is required during fetal life to maintain a balance between differentiated Leydig cells and their progenitors, but the elusive progenitor cell type and ligands involved have not been identified. In this study, we show that the Notch pathway signals through the ligand JAG1 in perivascular interstitial cells during fetal life. In the early postnatal testis, we show that circulating levels of testosterone directly affect Notch signaling, implicating a feedback role for systemic circulating factors in the regulation of progenitor cells. Between Postnatal Days 3 and 21, as fetal Leydig cells disappear from the testis and are replaced by adult Leydig cells, the perivascular population of interstitial cells active for Notch signaling declines, consistent with distinct regulation of adult Leydig progenitors.

We observed previously that after long-term suppression of luteinizing hormone (LH) and thus of Leydig cell steroidogenesis, restimulation of the Leydig cells by LH resulted in significantly higher testosterone production than by age-matched cells from control rats. These studies suggest that stimulation over time may elicit harmful effects on the steroidogenic machinery, perhaps through alteration of the intracellular oxidant-to-antioxidant balance. Herein we compared the effects of LH stimulation on stress response genes, formation of intracellular reactive oxygen species (ROS), and ROS-induced damage to ROS-susceptible macromolecules (DNA) in young and in aged cells. Microarray analysis indicated that LH stimulation resulted in significant increases in expression of genes associated with stress response and antiapoptotic pathways. Short-term LH treatment of primary Leydig cells isolated from young rats resulted in transiently increased ROS levels compared to controls. Aged Leydig cells also showed increased ROS soon after LH stimulation. However, in contrast to the young cells, ROS production peaked later and the time to recovery was increased. In both young and aged cells, treatment with LH resulted in increased levels of DNA damage but significantly more so in the aged cells. DNA damage levels in response to LH and the levels of intracellular ROS were highly correlated. Taken together, these results indicate that LH stimulation causes increased ROS production by young and aged Leydig cells and that while DNA damage occurs in cells of both ages, there is greater damage in the aged cells.

Embryo development is a complex and tightly controlled process. Nanoparticle injury can affect normal development and lead to malformation or miscarriage of the embryo. However, the risk that these nanoparticles may pose to reproduction is not clear. In this study, chitosan nanoparticles (CSNP) of near uniform size, in the range of 100 nm, were synthesized and confirmed by a particle size analyzer and transmission electron microscopy. Morulae-stage embryo exposure to CSNP during in vitro culture caused blastocyst complications that had either no cavity or a small cavity. Furthermore, CSNP-treated embryos showed lower expression of not only trophectoderm-associated genes but also pluripotent marker genes. When blastocysts developed in both media with and without CSNP were transferred to recipients, the percentage of blastocysts resulting in viable pups was significantly reduced. These detrimental effects are linked to the reduction of total cell numbers, enhanced apoptosis, and abnormal blastocoels forming at the blastocyst stage, indicating that CSNP treatment might have long-term adverse biological effects in view of pregnancy outcome.