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Mammalian eggs are surrounded by an extracellular matrix called the zona pellucida (ZP). This envelope participates in processes such as acrosome reaction induction, sperm binding, protection of the oviductal embryo, and may be involved in speciation. In eutherian mammals, this coat is formed of three or four glycoproteins (ZP1-ZP4). While Mus musculus has been used as a model to study the ZP for more than 35 years, surprisingly, it is the only eutherian species in which the ZP is formed of three glycoproteins Zp1, Zp2, and Zp3, Zp4 being a pseudogene. Zp4 was lost in the Mus lineage after it diverged from Rattus, although it is not known when precisely this loss occurred. In this work, the status of Zp4 in several murine rodents was tested by phylogenetic, molecular, and proteomic analyses. Additionally, assays of cross in vitro fertilization between three and four ZP rodents were performed to test the effect of the presence of Zp4 in murine ZP and its possible involvement in reproductive isolation. Our results showed that Zp4 pseudogenization is restricted to the subgenus Mus, which diverged around 6 MYA. Heterologous in vitro fertilization assays demonstrate that a ZP formed of four glycoproteins is not a barrier for the spermatozoa of species with a ZP formed of three glycoproteins. This study identifies the existence of several mouse species with four ZPs that can be considered suitable for use as an experimental animal model to understand the structural and functional roles of the four ZP proteins in other species, including human.

Liver growth factor induces testicular regeneration in EDS-treated rats and increases protein levels of class B scavenger receptors. Am J Physiol Endocrinol Metab 308 E111-E121, 2015. First published November 11, 2014; doi10.1152/ajpendo.00329.2014.-The aim of the present work was to determine the effects of liver growth factor (LGF) on the regeneration process of rat testes after chemical castration induced by ethane dimethanesulfonate (EDS) by analyzing some of the most relevant proteins involved in cholesterol metabolism, such as hormone sensitive lipase (HSL), 3(3-hydroxysteroid dehydrogenase (3(3-HSD), scavenger receptor SR-BI, and other components of the SR family that could contribute to the recovery of steroidogenesis and spermatogenesis in the testis. Sixty male rats were randomized to nontreated (controls) and LGF-treated, EDS-treated, and EDS + LGF-treated groups. Testes were obtained on days 10 (T1), 21 (T2), and 35 (T3) after EDS treatment, embedded in paraffin, and analyzed by immunohistochemistry and Western blot. LGF improved the recovery of the seminiferous epithelia, the appearance of the mature pattern of Leydig cell interstitial distribution, and the expression of mature SR-BI. Moreover, LGF treatment resulted in partial recovery of HSL expression in Leydig cells and spermatogonia. No changes in serum testosterone were observed in control or LGF-treated rats, but in EDS-castrated animals LGF treatment induced a progressive increase in serum testosterone levels and 3(3-HSD expression. Based on the pivotal role of SR-BI in the uptake of cholesteryl esters from HDL, it is suggested that the observed effects of LGF would facilitate the provision of cholesterol for sperm cell growth and Leydig cell recovery. © 2015 American Physiological Society. All rights reserved.

Asynchronous embryo transfer (ET) is a common assisted reproduction technique used in several species, but its biological effects on postnatal and early development remain unknown. The aim of this study was to determine whether asynchronous ET produces long-term effects in mice. Postnatal development, animal weight, systolic blood pressure (SBP), relative organ weight (liver, spleen, kidneys, heart, lungs, brain, and testicles), and behavior (assessed in open-field and elevated plus maze tests) were assessed in CD1 mice produced by different ET procedures 1) the transfer of Day 3.5 (D3.5) blastocysts to the uterus (BL-UT); 2) the transfer of D3.5 blastocysts to the oviduct (BL-OV); or 3) the transfer of D0.5 zygotes to the oviduct (ZOV). In vivo conceived animals served as controls (CT). The transfer of blastocysts to the uterus or zygotes to the oviduct was defined as synchronous, and transfer of blastocysts to the oviduct was defined as asynchronous. Both synchronous and asynchronous ET resulted in increased weight at birth that normalized thereafter with the exception of asynchronous ET females. In this group, female BL-OV, a clear lower body weight was recorded along postnatal life when compared with controls (P < 0.05). No effects on animal weight were produced during postnatal development in the synchronous ET groups (BL-UT, ZOV, and CT). Both synchronous and asynchronous ET had impacts on adult (Wk 30) organ weight. SBP was modified in animals derived from blastocyst but not zygote ET. Effects on behavior (anxiety in the plus maze) were only detected in the BL-UT group (P < 0.05). Our findings indicate that zygotes are less sensitive than blastocysts to ET and that both synchronous and asynchronous blastocyst ET may have long-term consequences on health, with possible impacts on weight, arterial pressure, relative organ weight, and behavior. © 2015 by the Society for the Study of Reproduction, Inc.

There is a tight relationship between fertility and changes in cholesterol metabolism during spermatogenesis. In the testis, class B scavenger receptors (SR-B) SR-BI, SR-BII, and LIMP II mediate the selective uptake of cholesterol esters from HDL, which are hydrolyzed to unesterified cholesterol by hormone-sensitive lipase (HSL). HSL is critical because HSL knockout (KO) male mice are sterile. The aim of the present work was to determine the effects of the lack of HSL in testis on the expression of SR-B, lipid raft composition, and related cell signaling pathways. HSL-KO mouse testis presented altered spermatogenesis associated with decreased sperm counts, sperm motility, and infertility. In wild-type (WT) testis, HSL is expressed in elongated spermatids; SR-BI, in Leydig cells and spermatids; SR-BII, in spermatocytes and spermatids but not in Leydig cells; and LIMP II, in Sertoli and Leydig cells. HSL knockout male mice have increased expression of class B scavenger receptors, disrupted caveolin-1 localization in lipid raft plasma membrane microdomains, and activated phospho-ERK, phospho-AKT, and phospho-SRC in the testis, suggesting that class B scavenger receptors are involved in cholesterol ester uptake for steroidogenesis and spermatogenesis in the testis. Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc.

Background Interchange of genetically modified (GM) mice between laboratories using embryos provides several advantages. Not only is transport stress avoided, but also the health status of the recipient colony is not compromised. Embryos do not need to be shipped in frozen stage, which requires expensive packaging in addition to a certain degree of expertise in order to freeze and thaw them correctly. The aim of this study was to examine different storage conditions and their effect on embryo viability in order to establish the feasibility of practical, non-frozen conditions for embryo shipment. Methods Mouse morulae developed in vivo (collected from donors 2.5d post coitum) or in vitro (zygotes cultured until morulae stage) were stored, combining two different media (KSOMeq or KSOM-H) and temperatures (4 degrees C, 15 degrees C and 37 degrees C) throughout 24 or 48 hours. After storage in vitro viability was assessed determining percentage of development to blastocyst and total cell number. In vivo viability was determined based on the number of implantations and living fetuses after embryo transfer of stored embryos. The storage effect at the molecular level was assessed by studying a gene pool involved in early development by quantitative RT-PCR. Results In vivo-produced morulae stored for 24 hours did not show differences in development up to the blastocyst stage, regardless of the storage type. Even though a decrease in the total cell number in vivo was observed, embryo development after embryo transfer was not affected. All 24 hour storage conditions tested provided a similar number of implantations and fetuses at day 14 of pregnancy. Morulae obtained from in vitro embryo culture collected at the 1-cell stage showed a decreased ability to develop to blastocyst after 24 hours of storage at 15degrees C both in KSOMeq and KSOM-H. Concomitantly, a significant decrease of embryo implantation rates after transfer to recipients was also found. In order to further cha

Background Some adult stem cells persist in adult tissue; however, we do not know how to stimulate stem cells in adults to heal injuries. Liver growth factor (LGF) is a biliprotein with hepatic mitogen activity. Its concentration increases markedly in the presence of any type of liver injury, and it shows in vivo therapeutic biological activity at extrahepatic sites.Methods We have analyzed the effect of LGF on the replenishment of germinal cells in the testes of mice injected with busulfan, a common cancer drug that also specifically affects germ line stem cells and spermatogonia. We determined the testicular and epididymal weight, spermatozoal concentration in the epididymis and sperm motility, and performed a histological analysis.Results Intraperitoneal administration of LGF was able to partially restore spermatogenesis, as well as sperm production and motility, in mice sterilized with busulfan. LGF treatment in busulfan-treated animals that have suffered a disruption of spermatogenesis can accelerate the reactivation of this process in most of the tubules, as shown in the histological analysis.Conclusions Our results suggest a potential use of LGF in the mobilization of testicular stem cells and in the restoration of spermatogenesis after busulfan-induced damage to the testicular germinal epithelium. © 2011 Pérez-Crespo et al; licensee BioMed Central Ltd.

Telomeres are repeated DNA regions that provide protection from enzymatic end-degradation and maintain chromosome stability during DNA replication. In most mammalian somatic cell types, telomeres shorten with each cell cycle. Telomerase, a reverse transcriptase that can elongate telomeres, adds telomeric repeats into chromosome ends, and is involved in maintaining telomere length in germ-line tissues, in adult stem cell, and in most immortal cancer cells. Telomere length is already determined at the moment of fertilization, and during preimplantation; and a detailed understanding of these telomeres elongation program could be important for studying embryo quality, and the origin of the embryonic stem cells; moreover, it has implications for the study of stem cell, regenerative medicine, ageing and cancer. Oocytes have shorter telomeres than somatic cells, but their telomeres lengthen extraordinarily during early cleavage development; however before blastocyst formation there are very low levels of expression of telomerase; even in telomerase-null mice, telomeres may elongate during preimplantation. Recent studies suggest that telomeres lengthen during early preimplantation use a recombination mechanism, and that from the blastocyst stage onwards, telomerase maintains the telomere length established by this recombinant mechanism. Telomere rebuilding subsequent to somatic cell nuclear transfer appears to vary according to species and type of donor cell used. It is speculated that the rate of telomere erosion and the incidence of chromosome abnormalities affect developmental potential of early embryos and may be potential predictors of developmental outcome. The purpose of this chapter is to review characteristic differences of telomere lengthening during the growing face of the oocyte and the spermatozoa, in early development of in vivo and in vitro (cloned, manipulated) produced embryos, in male and female embryos and in embryonic stem cells generated from these embr

El estrés térmico y la fragmentación del ADN espermático son dos factores muy influyentes en la fertilidad de los animales domésticos. El estrés térmico afecta tanto a la calidad embrionaria como a la calidad seminal. Sin embargo, en lo que respecta al desarrollo embrionario, no se conoce con precisión cómo afecta a los estadios tempranos de desarrollo y si los embriones de diferente sexo pueden responder de distinta forma al citado estrés. En cuanto a los gametos masculinos, se conoce muy poco cómo el estrés térmico puede afectar la calidad espermática, y las consecuencias que puede desencadenar en los embriones resultantes de la fecundación con estos gametos. Uno de los parámetros a tener en cuenta cuando se habla de calidad espermática es la fragmentación del ADN de los espermatozoides (una de las consecuencias del estrés térmico); no se conoce de forma precisa la relación existente entre este parámetro y la capacidad fecundante de los espermatozoides ni los efectos que para el desarrollo embrionario puede tener. En este trabajo se planteó, en primer lugar, conocer cuáles son las diferencias en la susceptibilidad al estrés térmico entre embriones de distinto sexo durante el desarrollo preimplantacional y determinar cuáles son los mecanismos moleculares responsables de las citadas diferencias; en segundo lugar, determinar cómo el estrés térmico puede alterar tanto el desarrollo embrionario preimplantacional (in vivo e in vitro) así como la espermatogénesis, utilizando como modelo experimental el ratón; y en tercer lugar, averiguar qué condiciones pueden favorecer un aumento en el porcentaje de espermatozoides con ADN fragmentado y las consecuencias sobre el desarrollo embrionario y fetal, ya sea tras la utilización de la monta natural o mediante una técnica de reproducción asistida, como es la inyección intracitoplasmática de espermatozoides (ICSI). Para abordar estos objetivos, se diseñaron tres ensayos. En el primer ensayo se analizó el efecto del estrés térmico

[Background] Before ovulation, sperm-oviduct interaction mechanisms may act as checkpoint for the selection of fertilizing spermatozoa in mammals. Postovulatory mating does not allow the sperm to attach to the oviduct, and spermatozoa may only undergo some selection processes during the transport through the female reproductive tract and/or during the zona pellucida (ZP) binding/penetration., [Methods] We have induced DNA damage in spermatozoa by two treatments, (a) a scrotal heat treatment (42 degrees C, 30 min) and (b) irradiation with 137Cs gamma-rays (4 Gy, 1.25 Gy/min). The effects of the treatments were analyzed 21-25 days post heat stress or gamma-radiation. Postovulatory females mated either with treated or control males were sacrificed at Day 14 of pregnancy, and numbers of fetuses and resorptions were recorded., [Results] Both treatments decreased significantly implantation rates however, the proportion of fetuses/resorptions was only reduced in those females mated to males exposed to radiation, indicating a selection favoring fertilization of sperm with unfragmented DNA on the heat treatment group. To determine if DNA integrity is one of the keys of spermatozoa selection after postovulatory mating, we analyzed sperm DNA fragmentation by COMET assay in: a) sperm recovered from mouse epididymides; b) sperm recovered from three different regions of female uterine horns after mating; and c) sperm attached to the ZP after in vitro fertilization (IVF). Similar results were found for control and both treatments, COMET values decreased significantly during the transit from the uterine section close to the uterotubal junction to the oviduct, and in the spermatozoa attached to ZP. However, fertilization by IVF and intracytoplasmatic sperm injection (ICSI) showed that during sperm ZP-penetration, a stringent selection against fragmented-DNA sperm is carried out when the damage was induced by heat stress, but not when DNA fragmentation was induced by radiation., [Conclusion] Our results indicate that in postovulatory mating there is a preliminary general selection mechanism against spermatozoa with low motility and fragmented-DNA during the transport through the female reproductive tract and in the ZP binding, but the ability of the ZP to prevent fertilization by fragmented-DNA spermatozoa is achieved during sperm-ZP penetration, and depends on the source of damage.

El estrés térmico y la fragmentación del ADN espermático son dos factores muy influyentes en la fertilidad de los animales domésticos. El estrés térmico afecta tanto a la calidad embrionaria como a la calidad seminal. Sin embargo, en lo que respecta al desarrollo embrionario, no se conoce con precisión cómo afecta a los estadios tempranos de desarrollo y si los embriones de diferente sexo pueden responder de distinta forma al citado estrés. En cuanto a los gametos masculinos, se conoce muy poco cómo el estrés térmico puede afectar la calidad espermática, y las consecuencias que puede desencadenar en los embriones resultantes de la fecundación con estos gametos. Uno de los parámetros a tener en cuenta cuando se habla de calidad espermática es la fragmentación del ADN de los espermatozoides (una de las consecuencias del estrés térmico); no se conoce de forma precisa la relación existente entre este parámetro y la capacidad fecundante de los espermatozoides ni los efectos que para el desarrollo embrionario puede tener. En este trabajo se planteó, en primer lugar, conocer cuáles son las diferencias en la susceptibilidad al estrés térmico entre embriones de distinto sexo durante el desarrollo preimplantacional y determinar cuáles son los mecanismos moleculares responsables de las citadas diferencias; en segundo lugar, determinar cómo el estrés térmico puede alterar tanto el desarrollo embrionario preimplantacional (in vivo e in vitro) así como la espermatogénesis, utilizando como modelo experimental el ratón; y en tercer lugar, averiguar qué condiciones pueden favorecer un aumento en el porcentaje de espermatozoides con ADN fragmentado y las consecuencias sobre el desarrollo embrionario y fetal, ya sea tras la utilización de la monta natural o mediante una técnica de reproducción asistida, como es la inyección intracitoplasmática de espermatozoides (ICSI). Para abordar estos objetivos, se diseñaron tres ensayos. En el primer ensayo se analizó el efecto del estrés térmico

Recently, F0 embryonic stem (ES) cell mice have been produced by injection of ES cells into eight-cell embryos using either laser- or piezo-assisted injection systems. To simplify the injection procedure, we have optimized the conventional blastocyst injection method, free of laser- or piezo-assisted micromanipulation systems, to produce F0 ES cell pups. To increase the efficiency of producing mice from ES cell injection into eight-cell and blastocyst stage embryos, we have tested 1) the effect of activating ES cell before injection, 2) the effect of in vitro culture in medium optimized for the survival of both ES cells and embryos, and 3) the effect of transferring the micromanipulated embryos into the oviduct versus into the uterus of CD1 foster mice. Two B6D2 hybrid ES cell lines were used for injection in a multifactorial analysis to evaluate the efficiency of producing live chimeric and F0 ES cell mice. Our results demonstrate that the activation of ES cells and the appropriate culture conditions are crucial parameters influencing the generation of F0 ES cell offspring. Transfer of blastocysts injected with ES cells into the oviduct of 0.5-day postcoitum pseudopregnant females increased the number of live animals with higher chimera proportion. Under these conditions, injections into eight-cell embryos produce a high number of F0 ES mice, and the conventional blastocyst injection method produces a lower number of F0 ES cell pups; however, the efficiency of production of chimeric mice with germline transmission was high. We have developed an economical and efficient technique for producing fully ES cell-derived F0 mice with full germline transmission that can be applied in many laboratories without the use of piezo or laser instruments. © 2009 by the Society for the Study of Reproduction, Inc.

Endogenous nucleases in mouse sperm can be activated by freeze-thawing the spermatozoa in media without cryoprotection and cleaving spermatozoa DNA. The role of sperm chromatin integrity during intracytoplasmic sperm injection (ICSI) is of critical importance. We analyzed in the B6D2 mouse the proportion of DNA-fragmented spermatozoa (DFS) produced by incubation in conditioned medium (CM) generated by freeze-thawing sperm in the absence of cryoprotection. We then examined the subsequent development, implantation, and offspring obtained after ICSI with incubated spermatozoa. When fresh sperm cells were incubated for 90 minutes in this CM, a significant increase in the amount of DFS was detected by the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay (27% vs 4.5% in fresh sperm). After ICSI of fresh and incubated spermatozoa, embryos were cultured in vitro to either the 2-cell or blastocyst stage before they were transferred into pseudopregnant CD1 females. On day 14, recipients were sacrificed, and implantation rates, estimated as the number of live fetuses plus resorptions, were determined. When ICSI was performed with sperm incubated in CM, no effects on fertilization, embryo cleavage, blastocyst rate, or blastocyst morphology were detected; however, the quality of the embryos was affected because the total implantation rate decreased significantly (P < .05) when 2-cell embryos or blastocysts were transferred. Independently of sperm pretreatment, in vitro cultures significantly affected the percentage of live fetuses present on day 14 of pregnancy. These results demonstrated that there are factors released from fragmented spermatozoa capable of inducing DNA fragmentation in intact sperm that may compromise, to some extent, birth rates after ICSI.

Genetic and environmental factors produce different levels of DNA damage in spermatozoa. Usually, DNA-fragmented spermatozoa (DFS) are used with intracytoplasmic sperm injection (ICSI) treatments in human reproduction, and use of DFS is still a matter of concern. The purpose of the present study was to investigate the long-term consequences on development and behavior of mice generated by ICSI with DFS. Using CD1 and B6D2F1 mouse strains, oocytes were injected with fresh spermatozoa or with frozen-thawed spermatozoa without cryoprotector. This treatment increased the percentage of TUNEL-positive spermatozoa, tail length as measured by comet assay, and loss of telomeres as measured by quantitative PCR. The ICSI-generated embryos were cultured for 24 h in KSOM, and 2-cell embryos were transferred into CD1 females. The DFS reduced both the rate of preimplantation embryo development and number of offspring. Immunofluorescence staining with an antibody against 5-methylcytosine showed a delay of 2 h on the active demethylation of male pronucleus in the embryos produced by ICSI. Moreover, ICSI affected gene transcription and methylation of some epigenetically regulated genes like imprinting, X-linked genes, and retrotransposon genes. At 3 and 12 mo of age, ICSI with DFS-produced animals and in vivo-fertilized controls were submitted to behavioral tests locomotor activity (open field), exploratory/anxiety behavior (elevated plus maze, open field), and spatial memory (free-choice exploration paradigm in Y maze). Females produced by ICSI showed increased anxiety, lack of habituation pattern, deficit in short-term spatial memory, and age-dependent hypolocomotion in the open-field test (P < 0.05). Postnatal weight gain of mice produced by ICSI with fresh or frozen sperm was higher than that of their control counterparts from 16 wk on (P < 0.01). Anatomopathological analysis of animals at 16 mo of age showed some large organs and an increase in pathologies (33% of CD1 females pr

Evidence exists to suggest detrimental effects of heat stress on male fertility. This study was designed to assess the effects of scrotal heat stress on mature and developing sperm in a mouse model. After receiving shock heat treatment (42°C for 30 min), mature spermatozoa were recovered from the epididymis hours (6) or Days (7, 14, 21, 28, 60) later, to determine the variables number of spermatozoa, sperm viability, motility and progressive motility, sperm DNA integrity as established by the TUNEL method, embryo implantation rate, and sex ratio of the fetuses conceived using the heat-exposed spermatozoa. Our results indicate that transient mild heat treatment does not affect in the same way the different types of male germ cells. Spermatocytes present within the testis at the time of heat stress resulted into a lower concentration of spermatozoa with reduced viability and low motility. Even though, DNA integrity of spermatozoa resulting from spermatocytes was also compromised by heat stress, the higher degree of DNA damage was found among spermatozoa resulting from spermatids present within the testis at the time of heat stress. At last, heat shock effect on spermatozoa present in the epididymis at the time of thermal stress resulted into a sex ratio distortion. These findings point to a higher sensitivity of spermatocytes to heat exposure and also suggest a different response of X and Y chromosome-bearing spermatozoa to heat stress that warrants further investigation. © 2007 Wiley-Liss, Inc.

Transgenes included in genomic-type constructs, such as yeast artificial chromosomes (YAC), P1-derived artificial chromosomes, or bacterial artificial chromosomes (BAC), are normally correctly expressed, according to the endogenous expression pattern of the homologous locus, because their large size usually ensures the inclusion of all regulatory elements required for proper gene expression. The use of these large genomic-type transgenes is therefore the method of choice to overcome most position effects, commonly associated with standard-type transgenes, and to guarantee the faithful transgene expression. However, in spite of the different methods available, including pronuclear microinjection and the use of embryonic stem cells as vehicles for genomic transgenes, the generation of transgenic animals with BACs and, particularly, with YACs can be demanding, because of the low efficiencies requiring extensive microinjection sessions and/or higher number of oocytes. Recently, we have explored the use of intracytoplasmic sperm injection (ICSI) into metaphase II oocytes as an alternative method for the generation of YAC transgenic mice. Our results suggest that the use of transgenic strategies based on ICSI significantly enhances the efficiency of YAC transgenesis by at least one order of magnitude.

Abnormalities of development potential arising from pre-implantation environment are not limited to in vitro culture (IVC) (for, i.e. in ruminants the large offspring syndrome produced by IVC), they may also be consequence of specific stress conditions experienced in vivo, like maternal diet, toxins, etc. A complex group of mechanisms (gene expression, epigenetic, metabolic, etc.) may operate to link early embryo environment with future health. Furthermore, during the pre-implantation period, in vitro produced male embryos have a higher metabolic rate, they grow faster than females, and they also have differential gene transcription of genes located in the Y-, X-, or in autosomal-chromosomes. As a consequence of these differences embryos may be affected differentially by natural or artificial environmental conditions, depending on their gender. It has been suggested that under some stress conditions male embryos are more vulnerable than females; however the biological fragility of male embryos is poorly understood. Evidences suggest that epigenetic differences produced by the presence of one or two X-chromosomes are the principal cause of the male and female pre-implantation differences, and we put forward the possible role of these early sex differences to control sex ratio of the offspring under different environmental conditions in Nature. By following the differences between male and female early embryos not only may be possible to manipulate sex ratio in farm animals, we can also gain further insight into aspects of early embryo development, X inactivation, and epigenetic and genetic processes related with early development that may have a long-term effect on the offspring. © 2006 The Authors.

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.

During the preimplantation period, in vitro cultured males have a higher metabolic rate, different gene expression, and grow faster than females. It has been suggested that under some stress conditions male embryos are more vulnerable than females; however, the biological fragility of male embryos is little understood. Since many forms of stress result in the overproduction of cellular reactive oxygen species (ROS), we addressed the hypothesis that the connection between female advantage during early developmental stages and heat stress involves ROS and differential gene expression of G6PD, an X-linked gene related to oxidative stress. We have found that after compaction, female heat-stressed embryos have less relative amounts of H2O2 than males, and female embryos survive better than males under in vivo or in vitro heat stress situations. In addition, in vitro produced female embryos grow slower than male embryos, have differential mRNA transcription of G6PD and also of some genes situated on autosomal-chromosomes (Sox, Bax, and Oct-4). Moreover, by inhibiting G6PD, all differences generated by oxidative stress between male and female embryos disappear. For the first time, we provide an experimental demonstration of a mechanism that explains why following exposure to heat stress-induced ROS, female preimplantation embryos are more resistant than males. © 2005 Wiley-Liss, Inc.

Background ICSI is a relatively new treatment for human male-related infertility, as well as an efficient method for the production of transgenic animals by injecting into the oocyte sperm previously incubated with foreign DNA. As semen samples collected in human infertility clinics are frequently contaminated with bacteria, one risk associated with the ICSI procedure is the injection of foreign, sperm-associated exogenous DNA into the oocyte, and the generation of transgenic offspring. Methods To analyse this possibility, ICSI was performed in mouse oocytes with frozen-thawed and Percoll-treated fresh sperm samples intentionally contaminated with plasmid EGFP-transformed E. coli bacteria or medium from which these bacteria were washed. Fertilized embryos were cultured in vitro until morula/blastocyst stage, transferred into pseudopregnant females, and at day 14, fetuses and reabsorptions were analysed by PCR for the genomic presence of integrated plasmid and/or bacterial DNA. Results Independently of the sperm pretreatment tested, transgenesis was produced. Conclusions Inadvertent transgenesis by conventional ICSI is a possibility that should not be neglected. Particular precautions, such as full bacteriological semen examinations and effective antibiotic semen pretreatments, should be taken in human infertility clinics, in order to exclude the possibility of accidental transgenesis. © The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

The long-term developmental and behavioral consequences of mammalian embryo culture are unknown. By altering the culture medium with the addition of FCS, we wanted to determine whether mouse embryos cultured under suboptimal conditions develop aberrant mRNA expression of imprinting genes at the blastocyst stage and whether fetal development, growth, and behavior of adult mice are affected. One-cell embryos obtained from superovulated female B6CBAF1 mice were cultured for 4 days in K+-modified simplex optimized medium in the presence of either 10% FCS or 1 g/liter BSA. After embryo transfer, born animals were submitted to several developmental and behavior tests. The mRNA expression of some imprinting genes was significantly affected in blastocysts cultured in the presence of FCS. Two of the eight measures of preweaning development and some specific measures of neuromotor development, such as the walking activity, were delayed in the group originated with FCS. After 34 weeks, the weight of female mice cultured in vitro in the presence of FCS was significantly higher than controls. In addition, the locomotion activity of mice was altered at 5 and 15 months. Anatomopathological and histological analysis of animals at 20 months of age showed some large organs and an increase in pathologies. We have found that mice derived from embryos cultured with FCS exhibited specific behavioral alterations in anxiety and displayed deficiencies in implicit memories. Our data indicate that long-term programming of postnatal development, growth, and physiology can be affected irreversibly during the preimplantation period of embryo development by suboptimal in vitro culture.