Chromosome segregation errors in female meiosis lead to aneuploidy in the resulting egg and embryo, making them one of the leading genetic causes of spontaneous abortions and developmental disabilities in humans. It is known that aneuploidy of meiotic origin increases dramatically as women age, and current evidence suggests that most errors occur in meiosis I. Several hypotheses regarding the cause of maternal age-related aneuploidy have been proposed, including recombination errors in early meiosis, a defective spindle assembly checkpoint in meiosis I, and deterioration of sister chromatid cohesion with age. This review discusses findings in each area, and focuses especially on recent studies suggesting that deterioration of cohesion with increasing maternal age is a leading cause of age-related aneuploidy.

The implantation process begins with attachment of the trophectoderm (TE) of the blastocyst to the maternal endometrial epithelium. Herein we have investigated the transcriptome of mural TE cells from 13 human blastocysts and compared these with those of human embryonic stem cell (hESC)-derived-TE (hESC_troph). The transcriptomes of hESC_troph at Days 8, 10, and 12 had the greatest consistency with TE. Among genes coding for secreted proteins of the TE of human blastocysts and of hESC_troph are several molecules known to be involved in the implantation process, as well as novel ones, such as CXCL12, HBEGF, inhibin A, DKK3, WNT5A, and follistatin. The similarities between the two lineages underscore some of the known mechanisms and offer discovery of new mechanisms and players in the process of the very early stages of human implantation. We propose that the hESC_troph is a viable functional model of human trophoblasts to study trophoblast-endometrial interactions. Furthermore, the data derived herein offer the promise of novel diagnostics and therapeutics aimed at practical challenges in human infertility and pregnancy disorders associated with abnormal embryonic implantation.

Physiological perturbations of bovine follicle-enclosed oocytes during the lengthy period of follicular development can lead to reduced oocyte developmental competence. It is suggested that heat stress-induced alterations in germinal vesicle (GV)-stage oocytes are further expressed in the transcriptional levels of genes involved in oocyte maturation and early embryonic development. Bovine oocytes were collected during cold (December–April) and hot (May–November) seasons, matured, fertilized, and cultured in vitro. The percentage of fertilized oocytes cleaving to the 2- to 4-cell stage was higher in the cold vs. hot season (89.0% ± 2.63% vs. 75% ± 2.63%, respectively; P < 0.05), as was the percentage of cleaved embryos further developing to blastocysts (26.6% ± 0.9% vs. 10.1% ± 1.8%, respectively; P < 0.05). Total RNA and poly(A) mRNA of oocytes and developing embryos were isolated and subjected to semiquantitative and real-time PCR for MOS, GDF9, and POU5F1 genes. In GV-stage oocytes, their mRNA levels did not differ between the seasons. However, following maturation, mRNA levels were higher in oocytes collected in the cold season (P < 0.05). In 4-cell-stage embryos, GDF9 and POU5F1 showed opposite mRNA patterns between seasons (higher and lower levels, respectively) in the hot season (P < 0.05). In both 8-cell-stage embryos and blastocysts, POU5F1 expression was lower during the hot season (P < 0.05). Exposing the ovarian pool of oocytes to environmental stress appears to impair maternal mRNA storage and/or the mechanism of transcription renewal, in turn affecting embryo gene expression before and after embryonic genome activation. Such impairment might partially explain the carry-over effect of summer heat stress on dairy cow conception rates.

Myosalpinx contractions are critical for oocyte transport along the oviduct. A specialized population of pacemaker cells—oviduct interstitial cells of Cajal—generate slow waves, the electrical events underlying myosalpinx contractions. The ionic basis of oviduct pacemaker activity is unknown. We examined the role of a new class of Ca² -activated Cl⁻ channels (CaCCs)—anoctamin 1, encoded by Tmem16a—in oviduct slow wave generation. RT-PCR revealed the transcriptional expression of Tmem16a-encoded CaCCs in the myosalpinx. Intracellular microelectrode recordings were performed in the presence of two pharmacologically distinct Cl⁻ channel antagonists, anthracene-9-carboxylic acid and niflumic acid. Both of these inhibitors caused membrane hyperpolarization, reduced the duration of slow waves, and ultimately inhibited pacemaker activity. Niflumic acid also inhibited propagating calcium waves within the myosalpinx. Slow waves were present at birth in wild-type and heterozygous oviducts but failed to develop by birth in mice homozygous for a null allele of Tmem16a (Tmem16a^{tm1Bdh/tm1Bdh}). These data suggest that Tmem16a-encoded CaCCs contribute to membrane potential and are responsible for the upstroke and plateau phases of oviduct slow waves.

Disruptions in the regulatory pathways controlling sex determination and differentiation can cause disorders of sex development, often compromising reproductive function. Although extensive efforts have been channeled into elucidating the regulatory mechanisms controlling the many aspects of sexual differentiation, the majority of disorders of sex development phenotypes are still unexplained at the molecular level. In this study, we have analyzed the potential involvement of Wnt5a in sexual development and show in mice that Wnt5a is male-specifically upregulated within testicular interstitial cells at the onset of gonad differentiation. Homozygous deletion of Wnt5a affected sexual development in male mice, causing testicular hypoplasia and bilateral cryptorchidism despite the Leydig cells producing factors such as Hsd3b1 and Insl3. Additionally, Wnt5a-null embryos of both sexes showed a significant reduction in gonadal germ cell numbers, which was caused by aberrant primordial germ cell migration along the hindgut endoderm prior to gonadal colonization. Our results indicate multiple roles for Wnt5a during mammalian reproductive development and help to clarify further the etiology of Robinow syndrome (OMIM 268310), a disease previously linked to the WNT5A pathway.

Sperm capacitation is required for fertilization and involves several ion permeability changes. Although Cl⁻ and HCO₃⁻ are essential for capacitation, the molecular entities responsible for their transport are not fully known. During mouse sperm capacitation, the intracellular concentration of Cl⁻ ([Cl⁻]_i) increases and membrane potential (Em) hyperpolarizes. As in noncapacitated sperm, the Cl⁻ equilibrium potential appears to be close to the cell resting Em, opening of Cl⁻ channels could not support the [Cl⁻]_i increase observed during capacitation. Alternatively, the [Cl⁻]_i increase might be mediated by anion exchangers. Among them, SLC26A3 and SLC26A6 are good candidates, since, in several cell types, they increase [Cl⁻]_i and interact with cystic fibrosis transmembrane conductance regulator (CFTR), a Cl⁻ channel present in mouse and human sperm. This interaction is known to be mediated and probably regulated by the Na /H regulatory factor-1 (official symbol, SLC9A3R1). Our RT-PCR, immunocytochemistry, Western blot, and immunoprecipitation data indicate that SLC26A3, SLC26A6, and SLC9A3R1 are expressed in mouse sperm, localize to the midpiece, and interact between each other and with CFTR. Moreover, we present evidence indicating that CFTR and SLC26A3 are involved in the [Cl⁻]_i increase induced by db-cAMP in noncapacitated sperm. Furthermore, we found that inhibitors of SLC26A3 (Tenidap and 5099) interfere with the Em changes that accompany capacitation. Together, these findings indicate that a CFTR/SLC26A3 functional interaction is important for mouse sperm capacitation.

In mammalian fertilization, sperm-zona pellucida binding is considered to be a critical aspect of gamete interaction. In this study, we examine the mouse sperm acrosomal matrix protein zona pellucida 3 receptor (ZP3R; formerly called sp56) because of our interest in defining the function of the acrosomal matrix, the particulate compartment within the sperm secretory acrosome. Using targeted deletion of the Zp3r gene by homologous recombination, we examined the fertility of nullizygous animals. Our experiments showed that males and females homozygous for the affected gene exhibited no differences in litter sizes compared to wild-type and heterozygous animals. Testis weights of nullizygous males were equivalent to those of wild-type and heterozygous males, and no differences in the number of sperm produced by mice of three genotypes were found. In vitro fertilization rates using cumulus-intact and cumulus-free oocytes were also equivalent. Examination of sperm-binding zonae of unfertilized eggs and the ability of the sperm to undergo acrosomal exocytosis in response to calcium ionophore A23187 displayed no differences between wild-type, heterozygous, and nullizygous mouse sperm. These results provide further evidence that either ZP3R is not involved in sperm-zona pellucida binding or this process might be functionally redundant, involving multiple proteins for gamete interactions.

The primate endometrium is characterized in pregnancy by a tissue-specific population of CD56^bright natural killer (NK) cells. These cells are observed in human, rhesus, and other nonhuman primate decidua. However, other subsets of NK cells are present in the decidua and may play distinct roles in pregnancy. The purpose of this study was to define the surface marker phenotype of rhesus monkey decidual NK (dNK) cell subsets, and to address functional differences by profiling cytokine and chemokine secretion in contrast with decidual T cells and macrophages. Rhesus monkey decidual leukocytes were obtained from early pregnancy tissues, and were characterized by flow cytometry and multiplex assay of secreted factors. We concluded that the major NK cell population in rhesus early pregnancy decidua are CD56^bright CD16 NKp30⁻ decidual NK cells, with minor CD56^dim and CD56^neg dNK cells. Intracellular cytokine staining demonstrated that CD56^dim and not CD56^bright dNK cells are the primary interferon-gamma (IFNG) producers. In addition, the profile of other cytokines, chemokines, and growth factors secreted by these two dNK cell populations was generally similar, but distinct from that of peripheral blood NK cells. Finally, analysis of multiple pregnancies from eight dams revealed that the decidual immune cell profile is characteristic of an individual animal and is consistently maintained across successive pregnancies, suggesting that the uterine immune environment in pregnancy is carefully regulated in the rhesus monkey decidua.

Mammalian sperm gain their ability to fertilize the egg during transit through the epididymis and by interacting with proteins secreted by the epididymal epithelial cells. Certain members of the CRISP (cysteine-rich secretory protein) family form the major protein constituent of the luminal fluid in the mammalian epididymis. CRISP4 is the newest member of the CRISP family expressed predominantly in the epididymis. Its structure and expression pattern suggest a role in sperm maturation and/or sperm-egg interaction. To study the relevance of CRISP4 in reproduction, we have generated a Crisp4 iCre knock-in mouse model through insertion of the iCre recombinase coding cDNA into the Crisp4 locus. This allows using the mouse line both as a Crisp4 deficient model and as an epididymis-specific iCre-expressing mouse line applicable for the generation of conditional, epididymis-specific knockout mice. We show that the loss of CRISP4 leads to a deficiency of the spermatozoa to undergo progesterone-induced acrosome reaction and to a decreased fertilizing ability of the sperm in the in vitro fertilization conditions, although the mice remain fully fertile in normal mating. However, removal of the egg zona pellucida returned the fertilization potential of the CRISP4-deficient spermatozoa, and accordingly we detected a reduced number of Crisp4-deficient spermatozoa bound to oocytes as compared with the wild-type spermatozoa. We also demonstrate that iCre recombinase is expressed in a pattern similar to endogenous Crisp4 and is able to initiate the recombination event with its target sequences in vivo.

Intrauterine implantation of fertilized ova can be blocked by exposing recently inseminated females with an unfamiliar male. This selective pregnancy failure, designated as the Bruce effect (Bruce, Nature 1959; 184:105), is well studied in laboratory mice and has been confirmed in several other rodent species. However, no clear information exists concerning this phenomenon in the laboratory rat. The present study was conducted to investigate whether or not the Bruce effect exists in the rat. Females of two F1 hybrid strains (n_total = 354) with different MHC genotypes (F344BNF1, RT1^lv1/n, and LEWPVGF1, RT1^l/c) were mated with males of their own strain and subsequently exposed during the first 4 days postcoitus either to a male of the other hybrid strain or to an unfamiliar male of the same strain as the stud. The litter rate of each treatment group was determined. As a control, mated females of both strains were reexposed to the stud male to determine baseline litter rates. Female rats of both F1 hybrid strains showed a significantly lower litter rate when exposed to males of a different strain than their stud male, compared to the expected values of birth rates observed in control females (F344BNF1: P = 0.017; LEWPVGF1: P = 0.019). In contrast, there was no difference between expected and observed litter rates in females of both F1 hybrid strains after exposure to an unfamiliar male of the same strain as their stud. Our results demonstrate for the first time that the Bruce effect, well documented in mice, occurs in the Norway rat.

In fish, like in other vertebrates, luteinizing hormone (Lh) is an essential hormone for the completion of oocyte maturation. In salmonid fish (i.e., salmon and trout), oocyte maturation is induced by Lh through its stimulation of the production of the maturation-inducing steroid, 17alpha,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P). In mammals, several factors, including ovarian cytokines and growth factors, have been reported to contribute to the regulation of oocyte maturation. In fish, growing evidence suggests that tumor necrosis factor alpha (hereafter referred to as Tnf) could play multiple physiological roles in the control of ovarian function. In the present study, we have investigated the possible involvement of Tnf in the regulation of oocyte maturation in brown trout (Salmo trutta). Our results show that in vitro treatment of brown trout preovulatory follicles with coho salmon (Oncorhynchus kisutch) Lh (sLh) significantly increased oocyte maturation, as assessed by germinal vesicle breakdown (GVBD), and that this effect was blocked by TAPI-1 (an inhibitor of Tnf-converting enzyme or Tace/Adam17). Furthermore, treatment of preovulatory follicles with sLh increased the expression of tnf and tace/adam17 as well as the secretion of the Tnf protein. Importantly, recombinant trout Tnf (rtTnf) significantly increased GVBD in vitro. Our results also show that the stimulatory effects of rtTnf on oocyte maturation may be the result of the direct involvement of rtTnf in stimulating the production of the maturation-inducing steroid as evidenced, first, by the stimulatory effects of rtTnf on 17,20beta-P production in vitro and on the expression of cholesterol side-chain cleavage P450 cytochrome (p450scc) and 20beta-hydroxysteroid dehydrogenase/carbonyl reductase 1 (cbr1), the enzyme responsible for the production of 17,20beta-P, and, second, by the ability of TAPI-1 to block the stimulatory effects of sLh on 17,20beta-P production and cbr1 expression. Furthermore, sLh and rtTnf increased the expression of the Lh receptor (lhr) and decreased the expression of aromatase (cyp19a1), and TAPI-1 completely blocked the effects of sLh. These results strongly suggest that Tnf may contribute to the regulation of oocyte maturation by Lh in trout.

The cell apoptosis susceptibility (CAS) gene is a homolog of the yeast chromosome segregation (CSE1) gene, which functions in cell proliferation and apoptosis. In the present study, a homolog of CAS was cloned from Chinese shrimp Fenneropenaeus chinensis (FcCAS). The full-length FcCAS cDNA is 3534 bp and contains an open reading frame encoding 968 amino acids. The predicted tertiary FcCAS structure is highly similar to that of CSE1 from the yeast Saccharomyces cerevisiae. RT-PCR analysis showed that the FcCAS gene is expressed mainly in testis, ovary, stomach, lymphoid organs, gills, and hemocytes. RNA in situ hybridization showed that FcCAS transcripts were distributed mainly in the cytoplasm of oocytes. Western blot analysis showed that FcCAS could be detected only in testis and ovary, and its expression levels differed at different developmental stages of ovaries. Immunohistochemical analysis showed that FcCAS existed in both the cytoplasm and the nucleus, which suggested that FcCAS might function as a nuclear protein. No transcript was detected in the abnormally developed ovaries of triploid shrimp. Therefore, we inferred that the FcCAS gene might be one of the key genes that is closely related to ovary development in shrimp.

Poliovirus receptor (Pvr), erythrocyte protein band 4.1-like 3 (Epb4.1l3), regulator of G-protein signaling 11 (Rgs11), and oxytocin receptor (Oxtr) expression were quantified in in vitro- and in vivo-grown mouse follicles. The expression of all genes was increased during antral growth in in vitro-grown cumulus cells, whereas only Rgs11 and Oxtr were increased and Pvr and Epb4.1l3 were decreased in in vivo grown cumulus cells. In vivo mural granulosa cells showed the highest expression of Pvr, Rgs11, and Oxtr. The in vitro granulosa theca compartment responded to human chorionic gonadotropinduring early luteinization by either an upregulation (Pvr, Oxtr) or downregulation (Epb41l3, Rgs11). Oocytes expressed Epb4.1l3, not Rgs11, and Pvr only in in vitro-grown oocytes. Translation into protein was confirmed for Epb4.1l3 in in vitro-grown follicles and in vivo-grown cumulus-oocyte complexes. Protein 4.1B was present during antral growth in cumulus, granulosa cells, and oocytes. Hypothetical functions of Epb4.1l3 and Pvr involve cell adhesion regulation and Rgs11 could be involved in cAMP production in the follicle. Oxtr is known to be important during and after the ovulatory stimulus, but, as in bovine, was also regulated during folliculogenesis. High expression of Pvr and Epb4.1l3 with culture duration in cumulus cells might mark inappropriate differentiation into a mural granulosa-like cell type and function as negative follicle development marker. Rgs11 and Oxtr are both in vivo and in vitro upregulated in cumulus cells during antral follicle growth and might be considered positive markers for follicle development.

The impact of compartmental expression of steroidogenic enzymes and of changes in flux through delta5 and delta4 metabolism on sex steroid synthesis was investigated by rebuilding pathways using recombinant enzyme expression by infection of insect cells with recombinant baculovirus constructs . Human cytochromes 17alpha-hydroxylase/17,20-lyase (P450c17) and aromatase (P450arom), always coexpressed with their redox partner NADPH-P450 oxidoreductase (CPR) and 3beta-hydroxysteroid dehydrogenase/delta5-4 isomerase (3betaHSD; types 1 or 2), were compartmentally expressed in different cell populations or coexpressed together with pregnenolone (100 nM) as substrate. Estrone was compared among cell compartments expressing different enzyme combinations or in cells coexpressing all enzymes (experiment 1). Additionally, P450c17, 3betaHSD, and CPR were all coexpressed, and androstenedione was measured in cells with different 3betaHSD expression levels or activity using an inhibitor, trilostane (experiment 2). Steroids were measured by immunoassay and mass spectrometry. In experiment 1, partitioning of P450c17, P450arom, and 3betaHSD markedly decreased estrone synthesis in comparison to cells coexpressing enzymes in different combinations. However, partitioning P450arom with 3betaHSD from P450c17 in different cell populations resulted in more estrone than either of the other two-cell compartment models. In experiment 2 (cells coexpressing P450c17, 3betaHSD, and CPR), androstenedione secretion was (paradoxically) higher at lower levels of 3betaHSD, and partial inhibition of 3betaHSD by trilostane also increased androstenedione when 3betaHSD expression was high. We conclude 1) that tissue or cell-specific, partitioned expression of sex steroid synthesizing enzymes limits rather than maximizes estrogen synthesis and 2) that limiting metabolism by 3betaHSD can paradoxically promote androgen synthesis when 3betaHSD expression is high by promoting delta5-steroid flux.

Copper/zinc superoxide dismutase (CuZn-SOD, SOD1) is one of the major antioxidant enzymes, and is localized in the cytoplasm to scavenge superoxide . To investigate the physiological role of SOD1 in the ovaries, we analyzed the fertility of Sod1-deficient female mice. To evaluate their hormonal metabolism, we measured pituitary and ovarian hormone levels in the plasma of the mutant mice. Plasma follicle-stimulating hormone, luteinizing hormone, and estradiol were not altered in the mutant compared to the wild-type females, while the plasma progesterone level was significantly reduced in the mutant females. Furthermore, the mutant mice showed decreased progesterone secretion under the condition of superovulation. In a histochemical analysis, we observed a remarkable reduction in the corpus luteum area in the mutant ovaries without atrophic changes. The mutant mice also displayed enhanced superoxide generation in the region surrounding the corpora lutea, which was associated with increased apoptotic cells and suppressed vasculature. These results suggested that SOD1 deficiency dysregulated luteal formation because of increased superoxide generation in the ovary. In vitro fertilization experiments showed no abnormal fertilization of Sod1-deficient oocytes. In addition, when Sod1-deficient embryos were transferred into the oviducts of wild-type females, mutant embryos developed at a normal rate, indicating that SOD1 deficiency in embryos did not cause miscarriage in the uterus of wild-type females. These results indicated that increased intracellular ROS impaired luteal formation and progesterone production in the mutant females, thus suggesting that SOD1 plays a crucial role in both the luteal function and the maintenance of fertility in female mice.

Polycystic ovary syndrome (PCOS) is characterized by ovarian enlargement, theca-interstitial hyperplasia, and increased androgen production by theca cells. Previously, our group has demonstrated that statins (competitive inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, a rate-limiting step of the mevalonate pathway) reduce proliferation of theca-interstitial cells in vitro and decrease serum androgen levels in women with PCOS. The present study evaluated the effect of simvastatin on rat ovarian theca-interstitial cell steroidogenesis. Because actions of statins may be due to reduced cholesterol availability and/or isoprenylation of proteins, the present study also investigated whether steroidogenesis was affected by cell- and mitochondrion-permeable 22-hydroxycholesterol, isoprenylation substrates (farnesyl-pyrophosphate [FPP] and geranylgeranyl-pyrophosphate [GGPP]), as well as selective inhibitors of farnesyltransferase (FTI) and geranylgeranyltransferase (GGTI). Theca-interstitial cells were cultured for 12, 24, and 48 h with or without simvastatin, GGPP, FPP, FTI, GGTI, and/or 22-hydroxycholesterol. Simvastatin decreased androgen levels in a time- and concentration-dependent fashion. This inhibitory effect correlated with a decrease in mRNA levels of Cyp17a1, the gene encoding the key enzyme regulating androgen biosynthesis. After 48 h, GGPP alone and FPP alone had no effect on Cyp17a1 mRNA expression; however, the inhibitory action of simvastatin was partly abrogated by both GGPP and FPP. The present findings indicate that statin-induced reduction of androgen levels is likely due, at least in part, to the inhibition of isoprenylation, resulting in decreased expression of CYP17A1.

Bidirectional signaling between oocytes and granulosa cells is required for normal folliculogenesis. Oocyte-secreted members of the transforming growth factor beta (TGFB) family, growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15) are well-known mediators of granulosa cell function. Deletion in granulosa cells of Smad4, the common SMAD mediating all canonical TGFB-related protein signals, results in infertility. Reciprocal signaling by granulosa cell-expressed TGFB family ligands, such as activin, to the oocyte during follicle development has been proposed but not tested in vivo using conditional knockout mice. Therefore, we generated two oocyte-specific conditional knockout models for the common SMAD, Smad4, using cre recombinase expression from either the zona pellucida 3 (Zp3) or Gdf9 promoter. Cre expression from the Gdf9 promoter occurs at a slightly earlier time point in follicle development than from Zp3. Deletion of Smad4 using Zp3cre had no effect on fertility, while deletion of Smad4 with Gdf9icre resulted in a slight, but significant, reduction in litter size. These mouse models suggest a novel, although minor, role for Smad4 in the oocyte restricted to the primordial follicle stage.

Insulin-like growth factor binding protein 1 (IGFBP1), the main secretory product of the decidualized endometrium of a pregnant woman, has previously been shown to interact with the alpha₅beta₁ integrin of extravillous trophoblast (EVT) cell surface to stimulate its migration in an IGF-independent manner. This migration stimulation has also been shown to require activation of extracellular signal regulated kinases 1 and 2 (ERK1/2; mitogen-activated protein kinase [MAPK] 3/1]) and focal adhesion kinase. The present study examined the roles of Rho GTPases RHOA, RHOC, RAC1, and CDC42 as well as RHO kinases ROCK1 and ROCK2 in IGFBP1-mediated migration of an immortalized EVT cell line HTR-8/SVneo. A nonselective RHO kinase inhibitor, Y27632, as well as siRNAs selective for ROCK1 and ROCK2 decreased the migration of these cells in a Transwell migration assay, and this inhibition could not be restored by IGFBP1. Clostridium difficile toxin B, which inhibits all the Rho GTPases, RAC inhibitor NSC23766, RAC1 siRNA, and CDC42 siRNA, decreased their basal migration, but none of these inhibitions except CDC42 siRNA-induced inhibition could be restored by IGFBP1. Clostridium botulinum C3 exoenzyme that inhibits RHOA, RHOB, and RHOC inhibited basal migration but not IGFBP1-induced migration. IGFBP1-induced activation of ERK1/2 (MAPK3/1), which did not require RHO proteins, might function as an alternate pathway for RHO action. However, selective siRNA-mediated downregulation of RHOA inhibited basal, but not IGFBP1-mediated, migration, whereas that of RHOC inhibited both basal and IGFBP1-mediated migration of these EVT cells. Therefore, RHO kinase, RHOC, and RAC1 are essential, but RHOA and CDC42 are not essential, for IGFBP1-induced EVT migration.

Embryo transfer in mice is a crucial technique for generation of transgenic animals, rederivation of contaminated lines, and revitalization of cryopreserved strains, and it is a key component of assisted reproduction techniques. It is common practice to use females only once as surrogate mothers. However, their reuse for a second embryo transfer could provide hygienic and economic advantages and conform to the concept of the 3Rs (replace, reduce, refine). This investigation evaluated the potential for a second embryo transfer in terms of feasibility, reproductive results, and experimental burden for the animal. Virgin female ICR mice (age 8–16 wk) were used as recipients for the first embryo transfer. Immediately after weaning of the first litter, a second surgical embryo transfer was performed into the same oviduct. Virgin females of comparable age to the reused mothers served as controls and underwent the same procedure. The first surgery did not affect the success of the second embryo transfer. Histological sections showed excellent wound healing without relevant impairment of involved tissues. We observed no differences in pregnancy rates or litter sizes between the transfer groups. Most importantly, we found no change in behavior indicating reduced well-being and no increase of corticosterone metabolites in the feces of surrogate mothers reused for a second embryo transfer. We conclude that a second embryo transfer in mice is feasible with regard to reproductive and animal welfare aspects.

Primary Sertoli cells isolated from mouse testes survive when transplanted across immunological barriers and protect cotransplanted allogeneic and xenogeneic cells from rejection in rodent models. In contrast, the mouse Sertoli cell line (MSC-1) lacks immunoprotective properties associated with primary Sertoli cells. In this study, enriched primary Sertoli cells or MSC-1 cells were transplanted as allografts into the renal subcapsular area of naive BALB/c mice, and their survival in graft sites was compared. While Sertoli cells were detected within the grafts with 100% graft survival throughout the 20-day study, MSC-1 cells were rejected between 11 and 14 days, with 0% graft survival at 20 days posttransplantation. Nonetheless, the mechanism for primary Sertoli cell survival and immunoprotection remains unresolved. To identify immune factors or functional pathways potentially responsible for immune privilege, gene expression profiles of enriched primary Sertoli cells were compared with those of MSC-1 cells. Microarray analysis identified 2369 genes in enriched primary Sertoli cells that were differentially expressed at ±4-fold or higher levels than in MSC-1 cells. Ontological analyses identified multiple immune pathways, which were used to generate a list of 340 immune-related genes. Three functions were identified in primary Sertoli cells as potentially important for establishing immune privilege: suppression of inflammation by specific cytokines and prostanoid molecules, slowing of leukocyte migration by controlled cell junctions and actin polymerization, and inhibition of complement activation and membrane-associated cell lysis. These results increase our understanding of testicular immune privilege and, in the long-term, could lead to improvements in transplantation success.

FYN kinase is highly expressed in the testis and has been implicated in testis and sperm function, yet specific roles for this kinase in testis somatic and germ cells have not been defined. The purpose of the present investigation was to identify aspects of spermatogenesis, spermiation, or sperm fertilizing capacity that required FYN for normal reproductive function. Matings between Fyn-null males and wild-type females resulted in normal litter sizes, despite the fact that Fyn-null males exhibited reduced epididymal size and sperm count. Morphological analysis revealed a high frequency of abnormal sperm morphology among Fyn-null sperm, and artificial insemination competition studies demonstrated that Fyn-null sperm possessed reduced fertilizing capacity. Fyn-null sperm exhibited nearly normal motility during capacitation in vitro but reduced ability to undergo the acrosome reaction and fertilize oocytes. The typical pattern of capacitation-induced protein tyrosine phosphorylation was slightly modified in Fyn-null sperm, with reduced abundance of several minor phosphoproteins. These findings are consistent with a model in which FYN kinase plays an important role in proper shaping of the head and acrosome within the testis and possibly an additional role in the sperm acrosome reaction, events required for development of full fertilizing capacity in sperm.