Fertility care in Low and Middle Income Countries - Commentary.

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Maintenance of Sertoli Cell Number and Function in Immature Human Testicular Tissues Exposed to Platinum-Based Chemotherapy-Implications for Fertility Restoration.

Background: Retrospective studies in adult survivors of childhood cancer show long-term impacts of exposure to alkylating chemotherapy on future fertility. We recently demonstrated germ cell loss in immature human testicular tissues following exposure to platinum-based chemotherapeutic drugs. This study investigated the effects of platinum-based chemotherapy exposure on the somatic Sertoli cell population in human fetal and pre-pubertal testicular tissues. Methods: Human fetal (n = 23; 14-22 gestational weeks) testicular tissue pieces were exposed to cisplatin (0.5 or 1.0 µg/ml) or vehicle for 24 h in vitro and analysed 24-240 h post-exposure or 12 weeks after xenografting. Human pre-pubertal (n = 10; 1-12 years) testicular tissue pieces were exposed to cisplatin (0.5 µg/ml), carboplatin (5 µg/ml) or vehicle for 24 h in vitro and analysed 24-240 h post-exposure; exposure to carboplatin at 10-times the concentration of cisplatin reflects the relative clinical doses given to patients. Immunohistochemistry was performed for SOX9 and anti-Müllerian hormone (AMH) expression and quantification was carried out to assess effects on Sertoli cell number and function respectively. AMH and inhibin B was measured in culture medium collected post-exposure to assess effects on Sertoli cell function. Results: Sertoli cell (SOX9+ve) number was maintained in cisplatin-exposed human fetal testicular tissues (7,647 ± 459 vs. 7,767 ± 498 cells/mm2; p > 0.05) at 240 h post-exposure. No effect on inhibin B (indicator of Sertoli cell function) production was observed at 96 h after cisplatin (0.5 and 1.0 µg/ml) exposure compared to control (21 ± 5 (0.5 µg/ml cisplatin) vs. 23 ± 7 (1.0 µg/ml cisplatin) vs. 25 ± 7 (control) ng/ml, p > 0.05). Xenografting of cisplatin-exposed (0.5 µg/ml) human fetal testicular tissues had no long-term effect on Sertoli cell number or function (percentage seminiferous area stained for SOX9 and AMH, respectively), compared with non-exposed tissues. Sertoli cell number was maintained in human pre-pubertal testicular tissues following exposure to either 0.5 µg/ml cisplatin (6,723 ± 1,647 cells/mm2) or 5 µg/ml carboplatin (7,502 ± 627 cells/mm2) compared to control (6,592 ± 1,545 cells/mm2). Conclusions: This study demonstrates maintenance of Sertoli cell number and function in immature human testicular tissues exposed to platinum-based chemotherapeutic agents. The maintenance of a functional Sertoli cell environment following chemotherapy exposure suggests that fertility restoration by spermatogonial stem cell (SSC) transplant may be possible in boys facing platinum-based cancer treatment.

Chemotherapy induced damage to spermatogonial stem cells in prepubertal mouse in vitro impairs long-term spermatogenesis.

Chemotherapy can affect testis development of young boys with cancer, reducing the chances of fatherhood in adulthood. Studies using experimental models are needed to determine the damage caused by individual chemotherapy drugs in order to predict the risk of infertility and direct patients towards appropriate fertility preservation options. Here, we investigated the individual role of two drugs, cisplatin and doxorubicin, using an in vitro culture model of prepubertal (postnatal day 5) mouse testis that supports induction and maintenance of full spermatogenesis. Twenty-four hour exposure with either drug at clinically-relevant doses (0.25, 0.5 or 0.75 µg/mL for cisplatin, or 0.01, 0.03 or 0.05 µg/mL for doxorubicin), induced an acute significant loss of spermatogonial stem cells (SSCs; PLZF+), proliferating SSCs (PLZF+BrdU+), total germ cells (MVH+), and spermatocytes (SCP3+) one week after chemotherapy exposure. By the time of the first (Week 4) and second (Week 8) waves of spermatogenesis, there was no longer any effect on SSC or proliferating SSC numbers in drug-exposed testis compared to untreated tissue: however, the populations of total germ cells and spermatocytes were still lower in the higher-dose cisplatin treated groups, along with a reduced frequency of round and elongated spermatids in both cisplatin- and doxorubicin-treated testis fragments. Overall, this study details a direct impairment of germ cell development following acute chemotherapy-induced damage during the prepubertal phase, most likely due to an effect on SSCs, using an in vitro culture system that successfully recapitulates key events of mouse spermatogenesis.

Can antioxidants protect against chemotherapy in a rat spermatogonial stem cell line?

Boys administered chemotherapy to treat cancer are at risk of damage to their healthy testicular tissue, which can lead to infertility in adulthood. Researchers are therefore investigating treatments to protect the testis during cancer treatment. Here, cells originating from rat testicles were cultured for 4 days and exposed to chemotherapy drugs with or without antioxidants for the final 2 days. Antioxidants can reduce cellular damage by inactivating toxic compounds. Here, antioxidants such as melatonin or n-acetylcysteine were tested against chemotherapy agents cisplatin, doxorubicin, or vincristine. Cultures were repeated four times, with cell survival measured at the end of culture. The antioxidants were not damaging and partially protected against cisplatin, although not doxorubicin. Surprisingly, n-acetylcysteine enhanced vincristine-induced damage. The results suggest that using antioxidants to protect the testis could have either beneficial or harmful effects when given alongside different chemotherapy drugs: this is important, considering that patients are often treated with multiple drugs.

Cisplatin and carboplatin result in similar gonadotoxicity in immature human testis with implications for fertility preservation in childhood cancer.

BACKGROUND: Clinical studies indicate chemotherapy agents used in childhood cancer treatment regimens may impact future fertility. However, effects of individual agents on prepubertal human testis, necessary to identify later risk, have not been determined. The study aimed to investigate the impact of cisplatin, commonly used in childhood cancer, on immature (foetal and prepubertal) human testicular tissues. Comparison was made with carboplatin, which is used as an alternative to cisplatin in order to reduce toxicity in healthy tissues. METHODS: We developed an organotypic culture system combined with xenografting to determine the effect of clinically-relevant exposure to platinum-based chemotherapeutics on human testis. Human foetal and prepubertal testicular tissues were cultured and exposed to cisplatin, carboplatin or vehicle for 24 h, followed by 24-240 h in culture or long-term xenografting. Survival, proliferation and apoptosis of prepubertal germ stem cell populations (gonocytes and spermatogonia), critical for sperm production in adulthood, were quantified. RESULTS: Cisplatin exposure resulted in a significant reduction in the total number of germ cells (- 44%, p < 0.0001) in human foetal testis, which involved an initial loss of gonocytes followed by a significant reduction in spermatogonia. This coincided with a reduction (- 70%, p < 0.05) in germ cell proliferation. Cisplatin exposure resulted in similar effects on total germ cell number (including spermatogonial stem cells) in prepubertal human testicular tissues, demonstrating direct relevance to childhood cancer patients. Xenografting of cisplatin-exposed human foetal testicular tissue demonstrated that germ cell loss (- 42%, p < 0.01) persisted at 12 weeks. Comparison between exposures to human-relevant concentrations of cisplatin and carboplatin revealed a very similar degree of germ cell loss at 240 h post-exposure. CONCLUSIONS: This is the first demonstration of direct effects of chemotherapy exposure on germ cell populations in human foetal and prepubertal testis, demonstrating platinum-induced loss of all germ cell populations, and similar effects of cisplatin or carboplatin. Furthermore, these experimental approaches can be used to determine the effects of established and novel cancer therapies on the developing testis that will inform fertility counselling and development of strategies to preserve fertility in children with cancer.

In vitro administration of sodium arsenite in mouse prepubertal testis induces germ cell loss and apoptosis.

High levels of arsenic contamination in drinking water pose serious health risks in numerous countries. The documentation reporting arsenic toxicity on reproduction and development is increasing, with evidence of arsenic inducing fertility and developmental issues. Nonetheless, the impact of arsenic exposure on the development of the male reproductive system is not fully elucidated. In the present study, we have investigated the direct effects of arsenic on prepubertal mouse testis using an in vitro testicular organ culture system. Culture medium was supplemented with a range of concentrations of sodium arsenite, examining effects of low (0.5 and 1 µM) and high (10, 50, 100 µM) concentrations, in cultures of post-natal day 5 CD1 mouse testis. In vitro exposure of low arsenic concentrations (0.5 or 1 µM) for 6 days did not cause any change in the testicular morphology, germ cells density, or apoptotic marker cleaved caspase 3 (CC3) expression. In contrast, exposure of prepubertal testis to high arsenic concentrations (10, 50 or 100 µM) induced drastic changes: severe destruction of testicular morphology, with loss of seminiferous tubule integrity; a dose-dependent decrease in germ cell density, and a hundred-fold increase in CC3 expression after 50 µM arsenic exposure. In conclusion, high arsenic treatment induced a dose-dependent induction of apoptosis and germ cell loss in prepubertal mouse testis.

Comparative gonadotoxicity of the chemotherapy drugs cisplatin and carboplatin on prepubertal mouse gonads.

The treatment of childhood cancer with chemotherapy drugs can result in infertility in adulthood. Newer generations of drugs are developed to replace parent drugs, with the potential benefits of less toxic side effects. For platinum alkylating-like drugs, in contrast to the parent compound cisplatin, the newer-generation drug carboplatin is reported to have reduced toxicity in some respects, despite being administered at 5-15 times higher than the cisplatin dose. Whether carboplatin is also less toxic than cisplatin to the reproductive system is unknown. Here we compare the gonadotoxic impact of cisplatin and carboplatin on female and male mouse prepubertal gonads. In vitro cultured CD1 mouse ovaries or testis fragments were exposed to either cisplatin or carboplatin for 24 h on Day 2 of culture and analysed by Day 6. A dose response for each drug was determined for the ovary (0.5, 1 & 5 µg/ml cisplatin and 1, 5 & 10 µg/ml carboplatin) and the testis (0.01, 0.05 & 0.1 µg/ml cisplatin and 0.1, 0.5 & 1 µg/ml carboplatin). For the ovary, unhealthy follicles were evident from 1 µg/ml cisplatin (73% unhealthy, P = 0.001) and 5 µg/ml carboplatin (84% unhealthy, P = 0.001), with a concomitant reduction in follicle number (P = 0.001). For the testis, the proliferating germ cell population was significantly reduced from 0.05 µg/ml cisplatin (73% reduction, P = 0.001) and 0.5 µg/ml carboplatin (75% reduction, P = 0.001), with no significant impact on the Sertoli cell population. Overall, results from this in vitro animal model study indicate that, at patient equivalent concentrations, carboplatin is no less gonadotoxic than cisplatin.

Single and combined effects of cisplatin and doxorubicin on the human and mouse ovary in vitro.

Chemotherapy drugs are administered to patients using combination regimens, and as such the possibility of multiplicative effects between drugs need to be investigated. This study examines the individual and combined effects of the chemotherapy drugs cisplatin and doxorubicin on the human ovary. Although cisplatin and doxorubicin are known to affect female fertility, there is limited information about their direct effects on the human ovary, and none examining the possibility of combined, multiplicative effects of co-exposure to these drugs. Here, human ovarian biopsies were obtained from 14 women at the time of caesarean section, with 38 mouse ovaries also obtained from neonatal C57Bl/6J mice. Tissue was cultured for 6 days prior to analyses, with chemotherapy drugs added to culture medium on the second day of culture only. Treatment groups of a single (5 µg/mL human; 0.5 µg/mL mouse) or double (10 µg/mL human; 1.0 µg/mL mouse) dose of cisplatin, a single (1 µg/mL human; 0.05 µg/mL mouse) or double (2 µg/mL human; 0.01 µg/mL mouse) dose of doxorubicin or a combination of a single dose of both drugs together were compared to controls without drug exposure. Exposure to cisplatin or doxorubicin significantly decreased follicle health in human and mouse, supporting the suitability of the mouse as a model for the human ovary. There was also a significant reduction of mouse follicle number. Human ovarian stromal tissue exhibited increased apoptosis and decreased cell proliferation. Crucially, there was no evidence indicating the occurrence of multiplicative effects between cisplatin and doxorubicin.

Virulence gene profiles of rabbit enteropathogenic Escherichia coli strains isolated in Northern Italy.

The virulence gene profile of 26 rabbit enteropathogenic Escherichia coli strains, isolated from 17 colibacillosis outbreaks located in two regions of Northern Italy, was determined using an Echerichia coli virulence DNA microarray. All strains were classified according to their determined biotype, sero- and phylo-group. The distribution of virulence genes encoding for the Locus of enterocyte effacement (LEE), LEE type III secretion system (T3SS), non-LEE T3SS translocated proteins and adherence factors was also determined. All strains but one belonged to phylogroups A and B1. A prevalent association between the O103 serogroup with the rhamnose-negative phenotype (biotype 12 or 14) was found. The most prevalent LEE profile found in tested strains was ler/cesT/espA-1/espB-3/tir-1/eae(beta)/espD-2/escN/eprJ. All strains possessed either the adhesive factor rabbit-2 (afr/2) or the plasmid Rabbit adherence locus (ral) gene and 24 of them an additional individual or combined set of colonization factors efa1/lifA, lpfA and paa genes. Finally, the combined or single presence of a set of LEE and/or non-LEE effector proteins encoding genes, namely espG, cif, map and nle family genes, attested to the genetic potential of investigated strains to induce pathologic lesions to the host. The application of microarray-based technologies in assessing the genetic profile of rabbit E. coli is a reliable, cost-effective candidate for large scale investigations in monitoring programs aimed to survey the circulation of pathogenic strains within rabbit production units, their zoonotic genetic potential and to select E. coli strains eligible for vaccinal prophylaxis in fattening rabbit production.

How does chemotherapy treatment damage the prepubertal testis?

Chemotherapy treatment is a mainstay of anticancer regimens, significantly contributing to the recent increase in childhood cancer survival rates. Conventional cancer therapy targets not only malignant but also healthy cells resulting in side effects including infertility. For prepubertal boys, there are currently no fertility preservation strategies in use, although several potential methods are under investigation. Most of the current knowledge in relation to prepubertal gonadotoxicity has been deduced from adult studies; however, the prepubertal testis is relatively quiescent in comparison to the adult. This review provides an overview of research to date in humans and animals describing chemotherapy-induced prepubertal gonadotoxicity, focusing on direct gonadal damage. Testicular damage is dependent upon the agent, dosage, administration schedule and age/pubertal status at time of treatment. The chemotherapy agents investigated so far target the germ cell population activating apoptotic pathways and may also impair Sertoli cell function. Due to use of combined chemotherapy agents for patients, the impact of individual drugs is hard to define, however, use of in vivo and in vitro animal models can overcome this problem. Furthering our understanding of how chemotherapy agents target the prepubertal testis will provide clarity to patients on the gonadotoxicity of different drugs and aid in the development of cytoprotective agents.

Chemotherapy drugs cyclophosphamide, cisplatin and doxorubicin induce germ cell loss in an in vitro model of the prepubertal testis.

Long term survival rates for childhood cancers is steadily increasing, however cancer survivors can experience fertility problems as a consequence of chemotherapy treatment. This is particularly problematic for young boys, for whom no fertility preservation treatment is yet established. Here, we have determined the effects on prepubertal mouse testis of three commonly used chemotherapy drugs; cyclophosphamide (using its active metabolite phosphoramide mustard), cisplatin and doxorubicin, exposing testicular fragments to a clinically relevant range of concentrations in vitro. All three drugs induced a specific and highly significant loss of germ cells, including spermatogonial stem cells. In contrast, there was no significant effect on somatic cells, for either Sertoli or interstitial cells. Time course analysis of cleaved Caspase-3 expression showed a significant increase in apoptosis eight hours prior to a detectable decrease in germ cell numbers following exposure to phosphoramide mustard or cisplatin, although this pattern was not seen following doxorubicin-exposure. Moreover, analysis of DNA damage at 16 h showed increased γH2AX expression in response to all three drugs. Overall, results show that cisplatin, doxorubicin and cyclophosphamide all specifically induce loss of germ cells, including of spermatogonial stem cells, in the prepubertal mouse testis at concentrations relevant to human therapeutic exposures.

Pathotyping of diarrhoeagenic cattle Escherichia coli strains isolated in the Province of Tehran, Iran.

An oligonucleotide DNA microarray targeting 348 virulence factors and genetic markers was used in the pathotyping, serotyping and phylogrouping of 51 Escherichia coli strains isolated from faecal samples. The samples were collected from diarrhoeic 1 to 30 days old calves located at 14 farms in the Tehran province, Iran. Positive microarray signals for genes encoding the Locus of Enterocyte E acement (LEE), the Type III Secretion System (TTSS), and the absence of EPEC adherence factor (EAF) permitted the pathotyping of 25 strains as atypical Enteropathogenic (aEPEC) or Enterohaemorrhagic Escherichia coli (EHEC). The lack of LEE and TTSS-associated genes distinguished the remaining 26 strains, which were classi ed as Extraintestinal pathogenic E. coli (ExPEC). Atypical EPEC belonged to phylogroup B1 and possessed a LEE pro le tir-1, eae(beta), espA-1, espB-3. The EHEC strains primarily belonged to the B1 phylogroup type-O26 and possessed either a LEE pro le tir-1, eae(beta), espA-1, espB-3, or a B1 type-O111, LEE tir-3, eae(gamma), espA-1, espB-2. ExPEC-typed strains generally harboured genes localised to the constant region of Colicin V plasmid (pColV), including increased serum survival factor (iss), complement resistance protein (traT), aerobactin operon (iucD), and the siderophore receptor (iroN). The microarray platform used in this study is well suited to accurately and rapidly type attaching and e acing E. coli (AEEC-types), thus providing a database for the meta-analysis of ExPEC-typed strains.

Irinotecan metabolite SN38 results in germ cell loss in the testis but not in the ovary of prepubertal mice.

STUDY QUESTION: Does the Irinotecan metabolite 7-ethyl-10-hydroxycamptothecan (SN38) damage the gonads of male and female prepubertal mice? SUMMARY ANSWER: The Irinotecan metabolite SN38 reduces germ cell numbers within the seminiferous tubules of mouse testes at concentrations that are relevant to cancer patients, while in contrast it has little if any effect on the female germ cell population. WHAT IS KNOWN ALREADY: Little is known about the role of the chemotherapeutic agent Irinotecan on female fertility, with only one article to date reporting menopausal symptoms in perimenopausal women treated with Irinotecan, while no data are available either on adult male fertility or on the impact of Irinotecan on the subsequent fertility of prepubertal cancer patients, female or male. STUDY DESIGN SIZE, DURATION: Male and female gonads were obtained from postnatal day 5 C57BL/6 mice and exposed in vitro to a range of concentrations of the Irinotecan metabolite SN38: 0.002, 0.01, 0.05, 0.1 or 1 µg ml-1 for the testis and 0.1, 1, 2.5 or 5 µg ml-1 for the ovary, with treated gonads compared to control gonads not exposed to SN38. SN38 was dissolved in 0.5% dimethyl sulfoxide, with controls exposed to the same concentration of diluent. The number of testis fragments used for each analysis ranged between 3 and 9 per treatment group, while the number of ovaries used for each analysis ranged between 4 and 12 per treatment group. PARTICIPANTS/MATERIALS, SETTING, METHODS: Neonatal mouse gonads were developed in vitro, with tissue analysed at the end of the 4-6 day culture period, following immunofluorescence or hematoxylin and eosin staining. Statistical analyses were performed using one-way ANOVA followed by Bonferroni post-hoc test for normally distributed data and Kruskal-Wallis test followed by Dunns post-test for non-parametric data. MAIN RESULTS AND THE ROLE OF CHANCE: Abnormal testis morphology was observed when tissues were exposed to SN38, with a smaller seminiferous tubule diameter at the highest concentration of SN38 (1 µg ml-1, p < 0.001 versus control) and increased number of Sertoli cell-only tubules at the two highest concentrations of SN38 (0.1 µg ml-1, p < 0.001; 1 µg ml-1, p < 0.0001, both versus control). Within seminiferous tubules, a dose response decrease was observed in both germ cell number (mouse vasa homologue (MVH)-positive cells) and in proliferating cell number (bromodeoxyuridine (BrdU)-positive cells), with significance reached at the two highest concentrations of SN38 (0.1 µg ml-1, p < 0.01 for both; 1 µg ml-1, p < 0.001-MVH, p < 0.01-BrdU; all versus control). No change was seen in protein expression of the apoptotic marker cleaved caspase 3. Double immunofluorescence showed that occasional proliferating germ cells were present in treated testes, even after exposure to the highest drug concentration. When prepubertal ovaries were treated with SN38, no effect was seen on germ cell number, apoptosis or cell proliferation, even after exposure to the highest drug concentrations. LIMITATIONS REASONS FOR CAUTION: As with any study using in vitro experiments with an experimental animal model, caution is required when extrapolating the present findings to humans. Differences between human and mouse spermatogonial development also need to be considered when assessing the effect of chemotherapeutic exposure. However, the prepubertal testes and ovaries used in the present studies contain germ cell populations that are representative of those found in prepubertal patients, and experimental tissues were exposed to drug concentrations within the range found in patient plasma. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate that the prepubertal mouse ovary is relatively insensitive to exposure to the Irinotecan metabolite SN38, while it induces a marked dose-dependent sensitivity in the testicular germ cell population. The study identifies the importance of further investigation to identify the risk of infertility in young male cancer patients treated with Irinotecan. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: Work supported by Medical Research Grant (MRC) grant G1002118 and Children with Cancer UK grant 15-198. The authors declare that there is no conflict of interest that could prejudice the impartiality of the present research.

Docetaxel induces moderate ovarian toxicity in mice, primarily affecting granulosa cells of early growing follicles.

Advances in cancer therapy have focused attention on the quality of life of cancer survivors. Since infertility is a major concern following chemotherapy, it is important to characterize the drug-specific damage to the reproductive system to help find appropriate protective strategies. This study investigates the damage on neonatal mouse ovary maintained in vitro for 6 days, and exposed for 24 h (on Day 2) to clinically relevant doses of Docetaxel (DOC; low: 0.1 µM, mid: 1 µM, high: 10 µM). Furthermore, the study explores the putative protective action exerted by Tri-iodothyronine (T3; 10(-7) M). At the end of culture, morphological analyses and follicle counts showed that DOC negatively impacts on early growing follicles, decreasing primary follicle number and severely affecting health at the transitional and primary stages. Poor follicle health was mainly due to effects on granulosa cells, indicating that the effects of DOC on oocytes were likely to be secondary to granulosa cell damage. DOC damages growing follicles specifically, with no direct effect on the primordial follicle reserve. Immunostaining and western blotting showed that DOC induces activation of intrinsic, type II apoptosis in ovarian somatic cells; increasing the levels of cleaved caspase 3, cleaved caspase 8, Bax and cleaved poly(ADP-ribose) polymerase, while also inducing movement of cytochrome C from mitochondria into the cytosol. T3 did not prevent the damage induced by the low dose of DOC. These results demonstrated that DOC induces a gonadotoxic effect on the mouse ovary through induction of somatic cell apoptosis, with no evidence of direct effects on the oocyte, and that the damaging effect is not mitigated by T3.

Cisplatin and doxorubicin induce distinct mechanisms of ovarian follicle loss; imatinib provides selective protection only against cisplatin.

PURPOSE: Chemotherapy treatment in premenopausal women has been linked to ovarian follicle loss and premature ovarian failure; the exact mechanism by which this occurs is uncertain. Here, two commonly used chemotherapeutic agents (cisplatin and doxorubicin) were added to a mouse ovary culture system, to compare the sequence of events that leads to germ cell loss. The ability of imatinib mesylate to protect the ovary against cisplatin or doxorubicin-induced ovarian damage was also examined. EXPERIMENTAL DESIGN: Newborn mouse ovaries were cultured for a total of six days, exposed to a chemotherapeutic agent on the second day: this allowed for the examination of the earliest stages of follicle development. Cleaved PARP and TUNEL were used to assess apoptosis following drug treatment. Imatinib was added to cultures with cisplatin and doxorubicin to determine any protective effect. RESULTS: Histological analysis of ovaries treated with cisplatin showed oocyte-specific damage; in comparison doxorubicin preferentially caused damage to the granulosa cells. Cleaved PARP expression significantly increased for cisplatin (16 fold, p<0.001) and doxorubicin (3 fold, p<0.01). TUNEL staining gave little evidence of primordial follicle damage with either drug. Imatinib had a significant protective effect against cisplatin-induced follicle damage (p<0.01) but not against doxorubicin treatment. CONCLUSION: Cisplatin and doxorubicin both induced ovarian damage, but in a markedly different pattern, with imatinib protecting the ovary against damage by cisplatin but not doxorubicin. Any treatment designed to block the effects of chemotherapeutic agents on the ovary may need to be specific to the drug(s) the patient is exposed to.

Sheep (Ovis aries) Macrophage Migration Inhibitory Factor: molecular cloning, characterization, tissue distribution and expression in the ewe reproductive tract and in the placenta.

Macrophage Migration Inhibitory Factor (MIF) is a pivotal regulator of innate and acquired immunity affecting the response and behavior of macrophages and lymphocytes. However, a number of studies indicated wider physiological functions for this cytokine to include key-roles in reproductive biology. The present study was designed to clone the coding sequence of sheep MIF, to examine the characteristics of the protein in vitro, and to evaluate its expression in sheep tissues and in the ewe reproductive tract in vivo. Ovine MIF cDNA consisted of 348 nucleotides encoding a 115 amino acids protein with an estimated molecular mass of 12,343 Da and an isoelectric point of 7.68. Sheep MIF shared high amino acid identity with the other mammalian MIF family members and showed parallel functions to human MIF, displaying enzymatic oxoreductase activity and inducing monocyte transmigration. Expression studies detected a MIF transcript in all the sheep tissues examined. Among reproductive tissues, MIF mRNA and protein were detected in the ovary, oviduct, uterus and placenta. These results indicate that sheep MIF shares crucial features with other MIF family members and delineate its potential involvement in several aspects of ovine physiology.

Putative embryonic stem cell lines from pig embryos.

Embryonic stem cells (ES cells) were first established in the mouse, and they represent a population of pluripotent, undifferentiated cells derived from early embryos that is capable of proliferating without any limitation in an undifferentiated state. These cells retain the ability to differentiate in vitro or in vivo into derivates of all three germ layers, and when injected into blastocysts, they can participate in the formation of all tissues, including gonads (germ-line chimeras). It is possible to transfect them with a gene of interest, and the resulting transgenic cell lines can also be used for production of chimeras. Unfortunately, mammalian germ-line chimeras that can carry an inserted gene into their progeny have only been produced in the mouse. Logically, before application of stem cell therapies into a human medicine, it is necessary to verify the efficiency and safety of these methods with an acceptable animal model. The pig is currently used as a very convenient animal for pre-clinical applications, and therefore establishment of porcine ES cell lines is highly needed; unfortunately, no convincing ES cell lines have been produced in this species (and other domestic animals) to date. In this article, we discuss the recent advances in this field, especially oriented on possible reasons and obstacles why derivation of porcine ES cell lines is still unsuccessful.

Leukaemia inhibitory factor enhances sheep fertilization in vitro via an influence on the oocyte.

LIF is twice transiently expressed in the mouse uterus, first at the time of ovulation and again just prior to implantation, and studies have demonstrated a beneficial influence of this cytokine on embryo development in several species. We have investigated the effect of LIF on gametes in vitro, on the hypothesis that the ovulatory peak of LIF can exert an influence on gametes present within the oviduct. We also investigated the effect of LIF on in vitro fertilization and embryo development, in oocytes from adult sheep and from prepubertal lambs that lack the preovulatory hormone surge and that are unable to sustain early embryonic development. A higher rate of pronuclear-stage embryos derived from both, adult and prepubertal female, was obtained when in vitro fertilization was performed in the presence of LIF, and there was an improved cleavage of parthenogenetic embryos when incubated with LIF immediately following activation. In contrast, LIF was found to have no influence on the viability of ram semen. In vitro fertilized two-cell stage embryos from adult sheep and prepubertal lambs, cultured in defined medium enriched with LIF, both reached the blastocyst stage at similar rates to control embryos. However, LIF exerted a positive influence on the quality of the blastocysts as revealed by significantly higher number of ICM cells and total number of cells. Together, these data demonstrate that LIF exerts a beneficial effect on sheep oocytes and embryos in vitro, but only at stages concomitant with steroid hormones surges.