[In vitro spermatogenesis new horizon to restore fertility?]. / Spermatogenèse in vitro nouvel horizon pour restaurer la fertilité ?

The survival of the young boy after cancer has considerably progressed in recent years due to the efficiency of chemo/radiotherapy against the tumor cells. However, this treatment causes adverse effects on healthy tissues, including fertility. Freezing testicular tissue before highly gonadotoxic treatment is a prerequisite for preserving fertility in prepubertal boys that do not produce sperm yet. But which strategy proposes to restore fertility from frozen-thawed testicular tissue? One potential solution would be to consider an in vitro maturation of spermatogonial stem cells. In this article we trace the chronological development of in vitro spermatogenesis that resulted in mouse sperm production in vitro and give an overview of new challenges for the future.

The feasibility of fertility preservation in adolescents with Klinefelter syndrome.

STUDY QUESTION: Is fertility preservation feasible after the onset of puberty in adolescents with Klinefelter syndrome (KS)? SUMMARY ANSWER: Fertility preservation counseling should be an integral part of the care of XXY adolescents. Frozen ejaculated or testicular spermatozoa and even frozen immature germ cells can give them the potential to conceive their genetic progeny. However, no biological or clinical parameters were predictive of mature or immature germ cell retrieval. WHAT IS KNOWN ALREADY: KS is the commonest sex chromosome disorder observed in azoospermic infertile males. Testicular sperm extraction success decreases with age and after testosterone therapy. Arguably, spermatozoa should be retrieved from KS males at the onset of puberty and before testosterone therapy to increase the chance of success. STUDY DESIGN, SIZE, DURATION: A retrospective study was performed in eight KS adolescents, aged between 15 and 17 years, who were referred for counseling about their future fertility to the center CECOS (Centre d'Etude et de Conservation des Oeufs et du Sperme humain) at Rouen University Hospital between October 2008 and December 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS: The patients were first seen with their parents and then separately. It was proposed to them that they should provide a semen sample, if this was azoospermic, two other semen samples spaced by 3 months were collected. If azoospermia was confirmed, a bilateral testicular biopsy was proposed for sperm retrieval and testicular tissue preservation. Each adolescent met the psychologist before undergoing testicular biopsy. Paraffin-embedded testicular tissue was evaluated after staining with hematoxylin-eosin and saffron and immunostaining using vimentin, anti-Müllerian hormone, androgen receptor and MAGE-A4 antibodies. Sertoli cell maturity, germ cell identification and lamina propria alteration were assessed on seminiferous tubules. MAIN RESULTS AND THE ROLE OF CHANCE: KS adolescents were not deeply concerned about their future fertility and only became involved in the process of fertility preservation after at least three medical consultations. The parents agreed immediately that fertility preservation should be attempted. Seven non-mosaic XXY adolescents presented with azoospermia and one XXY/XY adolescent had oligozoospermia. Increased plasma levels of FSH and LH as well as bilateral testicular hypotrophy were observed in all patients. The XXY/XY adolescent banked four semen samples before testosterone replacement therapy. Two patients refused testicular biopsy. Five patients accepted a bilateral testicular biopsy. Spermatozoa were retrieved in one patient, elongated spermatids and spermatocytes I in a second patient. LIMITATIONS, REASONS FOR CAUTION: The number of patients enrolled in our study was low because the diagnosis of KS is only rarely made before or at the onset of puberty. Most XXY males are diagnosed in adulthood within the context of male infertility. WIDER IMPLICATIONS OF THE FINDINGS: Spermatozoa can be retrieved in semen sample and in testicular tissue of adolescent Klinefelter patients. Furthermore, the testis may also harbor spermatogonia and incompletely differentiated germ cells. However, the physician should discuss with the patient and his parents over a period of several months before collecting a semen sample and performing bilateral testicular biopsy. Fertility preservation might best be proposed to adolescent Klinefelter patients just after the onset of puberty when it is possible to collect a semen sample and when the patient is able to consider alternative options to achieve fatherhood and also to accept the failure of spermatozoa or immature germ cell retrieval.

Multiple limb compartment syndromes in a recruit with sickle cell trait.

Sickle cell trait, as opposed to the disease, is a rare condition with fewer medical complications. We present a case of a 24 year old Army recruit, who required multiple fasciotomies for limb compartment syndrome, associated with sickle cell trait. We discuss the management, complications and screening programmes of the condition and make suggestions for the training of sickle cell trait personnel in planning a career in the Armed Forces.

Sperm binding, in vitro fertilization, and in vitro embryonic development of bovine oocytes fertilized with spermatozoa incubated with norepinephrine.

The final stages of sperm maturation, fertilization, and early embryonic development occur within the oviduct and are essential for successful reproduction in mammals. Norepinephrine was previously identified in native bovine oviductal fluid and its in vitro effects on bull sperm capacitation and the acrosome reaction have been determined. It was unknown how physiological concentrations of norepinephrine influence sperm binding, fertilization, and embryo development. Therefore, the objective of this study was to determine if pre-incubating bovine spermatozoa with physiological concentrations of norepinephrine prior to insemination of bovine oocytes would improve sperm-oocyte binding, fertilization, and embryonic development in vitro. Norepinephrine, in concentrations representing those measured in bovine oviductal fluid, was used to treat bovine spermatozoa prior to insemination. Spermatozoa incubated in norepinephrine were used to inseminate bovine oocytes matured in vitro, and oocytes were evaluated for sperm binding and fertilization. Additional experiments were conducted to evaluate how early in the co-incubation period oocytes were fertilized by spermatozoa pre-incubated with norepinephrine, and to test the developmental competence of those oocytes fertilized with norepinephrine-treated sperm. Sperm binding to the zona pellucida was reduced by pre-incubation with norepinephrine. Rates of fertilization and embryo development did not increase as a result of pre-incubating spermatozoa with norepinephrine, but as early as 4h after insemination, spermatozoa treated with 20 ng/ml norepinephrine fertilized more oocytes than spermatozoa incubated in medium alone. Interestingly, this concentration of norepinephrine was found to capacitate spermatozoa in previous studies. These data suggest that oocytes fertilized by spermatozoa incubated in 20 ng/ml norepinephrine fertilize earlier in vitro than sperm pre-incubated in medium alone, and provide additional support for the role of norepinephrine in sperm capacitation and the acrosome reaction.

Identification and immunohistochemical localization of a haptoglobin-like protein in the tissues and fluids of the bovine (Bos taurus) ovary and oviduct.

The objective of this study was to establish the identity of a 40 kDa bovine oviductal fluid protein as a haptoglobin-like protein and to evaluate the association of the haptoglobin-like protein with ovarian and oviductal tissues and fluids. An oviductal fluid protein band corresponding to a molecular mass of 40 kDa was excised and electroeluted from SDS-PAGE gels. Sequence analysis revealed an N-terminal region sharing 81% identity with the beta-subunit of bovine haptoglobin. The 40 kDa oviductal fluid protein crossreacted on immunoblots with antiserum against rabbit endometrial haptoglobin and with an anti-human haptoglobin polyclonal antibody. Two-dimensional PAGE revealed four protein variants ranging in pI from 7.7 to 8.6, which appeared identical, with respect to molecular weight, number of isoforms and pI, to bovine haptoglobin in acute phase serum. The haptoglobin-like protein was localized using immunohistochemistry to the lumina of blood vessels and to the extracellular matrix of ovarian and oviductal tissues. Immunostaining for the haptoglobin-like protein was also detected in the oviductal lumen, in the mucosa of the ampullary oviduct but not the isthmic oviduct, and in intermittent ampullary epithelial cells. Within the ovary, the haptoglobin-like protein was localized to the avascular granulosa cells and follicular fluid of antral follicles, but not in the theca cells or in preantral follicles of any developmental stage. It was concluded that the haptoglobin-like protein is a normal constituent of bovine ovarian and oviductal tissues and fluids, and it was hypothesized that the haptoglobin-like protein contributes to ovarian follicular development and oviductal function.

Identification of norepinephrine in bovine oviductal fluid by high performance liquid chromatography.

The final stages of sperm maturation, fertilization and early embryonic development take place in the microenvironment of the oviduct and are essential for successful reproduction in mammals. Although catecholamines have been shown to have beneficial effects on mammalian gametes in vitro, identification of catecholamines in native bovine oviductal fluid has not been studied. The objective of this research was to identify catecholamines in bovine oviductal fluid and to determine whether concentrations of catecholamines change with stage of the estrous cycle. Oviductal fluid was collected via indwelling oviductal cannulae and assayed for the presence of catecholamines by high performance liquid chromatography. Norepinephrine was the only catecholamine detected, in concentrations ranging from 0.828 ng/ml - 1117 ng/ml. The presence of norepinephrine in oviductal fluid corresponded to a period of time just prior to, during, and after ovulation, when serum progesterone levels were low. This was a consistent finding in ODF collected from normally cycling cows. Potential functions of norepinephrine in oviductal fluid include regulation of fluid formation, induction of capacitation and the acrosome reaction in spermatozoa, and cleavage of the early embryo.

Effects of accessory sex gland fluid on viability, capacitation, and the acrosome reaction of cauda epididymal bull spermatozoa.

The effect of accessory sex gland fluid (AGF) on viability and acrosomal integrity of spermatozoa was examined with cauda epididymal spermatozoa and AGF from the same Holstein bull (n = 6). Surgical cannulation of the vasa deferentia enabled the separate collection of cauda epididymal effluent and AGF from each bull. Cauda epididymal effluent was incubated with either AGF collected from the same bull or medium alone. Following coincubation, spermatozoa (5 x 10(7) sperm/mL) were incubated in medium alone or under capacitating conditions (10 microg/mL heparin) for 16 hours. Every 2 hours, an aliquot of spermatozoa was exposed to lysophosphatidylcholine (100 microg/mL) to induce the acrosome reaction in capacitated spermatozoa. Sperm motility decreased over time regardless of treatment. Overall, spermatozoa incubated in AGF had fewer acrosome-intact live spermatozoa than did those not incubated in AGF. Viability was significantly (P < .05) compromised over time when spermatozoa were exposed to AGF, compared with those not preincubated in AGF. Significantly more (P < .05) acrosome-reacted live spermatozoa were seen following exposure to heparin and lysophosphatidylcholine when spermatozoa were not preincubated in AGF. We conclude that exposure of spermatozoa to AGF accelerates cell death and that rapid removal of spermatozoa from seminal plasma is critical for maximal viability.

Human anti-porcine T cell response: blocking with anti-class I antibody leads to hyporesponsiveness and a switch in cytokine production.

Intervention in the molecular interactions that lead to an immune response is possible at various stages of Ag recognition and T cell activation. Perturbation of the interaction of the TCR with the MHC/peptide ligand complex is one approach that has shown promise for autoimmunity and graft rejection in blocking T cell-activated responses. In this study, we investigated the effect of altering the target MHC class I molecule by blocking with Abs. We established a system that analyzed the human T cell response against MHC class I+/class II- porcine stimulatory cell targets. The primary human response against porcine smooth muscle cells was CD8+ T cell dependent. In the presence of F(ab')2 fragments of the MHC class I-reactive Ab, PT-85, the proliferative response was inhibited and production of IL-2 and IFN-gamma was blocked. Moreover, in a secondary response, proliferation was reduced and type 1 cytokine levels were inhibited. In contrast, levels of IL-10 and IL-4 were sustained or slightly increased. These findings indicate that Ab against MHC class I blocked the recognition of porcine cells by the human CD8+ T cells and altered the cytokine secretion profile. Thus, a single treatment with PT-85 F(ab')2 directed against the MHC class I molecule provides an attractive approach to the induction of T cell tolerance that may provide long-term graft survival in porcine-to-human cell transplantation.

Influence of capacitation and fluids from the male and female genital tract on the zona binding ability of bull spermatozoa.

Before fertilization, inseminated spermatozoa acquire the ability to fertilize an egg, a phenomenon called capacitation. Bovine sperm capacitation is influenced by factors originating from both the male and female genital tract, and results in intracellular and membrane changes of the spermatozoa that facilitate the induction of the acrosome reaction. However, the effects of reproductive tract secretions and capacitation on the binding of spermatozoa to the zona pellucida have not been investigated. In this study, a sperm-egg binding assay was used to determine whether the ability of bull spermatozoa to bind to the zona pellucida was altered during in vitro capacitation by heparin or oviductal fluid, or by treatment of spermatozoa from the cauda epididymidis with accessory sex gland fluid. In addition, biotinylated solubilized zona pellucida proteins were used to visualize zona binding on spermatozoa. The ability of bull spermatozoa to bind to the zona pellucida was increased after both heparin and oviductal fluid induced in vitro capacitation. Exposure of spermatozoa from the cauda epididymidis to accessory sex gland fluid resulted in a direct increase in zona binding ability, followed by a further increase during capacitation in vitro. Binding of solubilized zona proteins was restricted to the acrosomal cap of bull spermatozoa. It is suggested that the observed increased ability of bull spermatozoa to bind to the zona pellucida enables optimal sperm-egg attachment, which also relates to the induction of the acrosome reaction by the zona pellucida. Thus, increased zona binding ability is likely to be an essential part of the process of capacitation.

Sexual behavior and contraceptive knowledge and use among adolescents in developing countries.

This article offers an overview of sexual behavior and contraceptive knowledge and use among adolescent women across a large number of developing countries. The results demonstrate that almost universally in sub-Saharan Africa and in the majority of countries in other regions, the gap between age at first sexual intercourse and age at first marriage has increased across age cohorts. The predominant pattern is one in which both age at marriage and age at first intercourse have risen, but the increase in age at marriage is greater, resulting in a widening gap. In most countries in sub-Saharan Africa, current contraceptive use is higher among sexually active, unmarried teens than it is among married teens, whereas in Latin America and the Caribbean, current-use levels are higher among married teens. The results also show that adolescents are unlikely to use a contraceptive the first time they have sex and are more likely than older women to experience a contraceptive failure.

PIP: This article presents an analysis of Demographic and Health Survey (DHS) data from developing countries that sheds light on the reproductive behavior of adolescent women. Topics include sex behavior and marriage; contraceptive knowledge/usage; contraceptive usage at first intercourse; and contraceptive failure, switching, and discontinuation. Tables illustrate 1) the percentage of all adolescents aged 15-19 who have ever had sex and who have ever married in 37 DHS countries; 2) the percentage of those aged 20-24 and of women aged 40-44 who first married and first had sex by age 18 in 36 DHS countries; 3) the percentage of women aged 15-19 and 20-49 who know any contraceptive method by marital and sexual activity status in 37 DHS countries; 4) the percentage of women aged 15-19 and 20-49 currently using contraception by marital status and sexual activity status in 43 DHS countries; 5) the percentage of people who used contraception at first intercourse by country, age, sex, and marital status in 6 countries; and 6) 12-month life-table gross discontinuation rates by country and age at start of use, by type of discontinuation. It is concluded that adolescent sex behavior and contraceptive knowledge/usage vary widely across and within regions, but that overall patterns show that the gap between first intercourse and marriage has increased. Levels of contraceptive knowledge are high, and usage is higher but less successful among unmarried than married adolescents. More research should be directed toward adolescent males.

Influence of bovine ampullary and isthmic oviductal fluid on sperm-egg binding and fertilization in vitro.

The objective of this study was to determine the effects of oviductal fluid on sperm binding and fertilization in vitro when either bovine oocytes or both spermatozoa and oocytes are incubated in fluid from different regions of the oviduct and at different stages of the oestrous cycle. Oocytes, or oocytes and spermatozoa, were incubated in nonluteal and luteal oviductal fluid obtained from the whole, isthmic or ampullary oviduct of dairy cows. Gametes were washed and coincubated in fertilization medium for 18 h, and then observed for sperm binding and penetration. More spermatozoa bound to oocytes that were preincubated in nonluteal oviductal fluid than to those that were incubated in luteal oviductal fluid; however, there were no differences in fertilization rates between the two treatments. More spermatozoa bound to oocytes incubated in isthmic than in ampullary oviductal fluid. However, if both gametes were incubated in oviductal fluid before their coincubation, binding was higher when both gametes were incubated in ampullary oviductal fluid. By reducing the time of coincubation of spermatozoa with the oocytes, differences in fertilization rates were seen among the various oviductal fluid treatments. At 14 and 16 h of coincubation, more oocytes were fertilized if spermatozoa were preincubated in isthmic oviductal fluid and oocytes were preincubated in ampullary oviductal fluid than if both gametes were incubated in whole or ampullary oviductal fluid. Because both sperm binding to the zona pellucida and fertilization rates were greater in treatments in which gametes were exposed to regional oviductal fluid than when they were incubated in whole oviductal fluid, it is concluded that oviductal fluid from different regions of the bovine oviduct may play different roles in facilitating fertilization.

Effect of bovine ampullary and isthmic oviductal fluid on motility, acrosome reaction and fertility of bull spermatozoa.

Motility, acrosome reaction and oocyte fertilizing ability were assessed for bull spermatozoa after incubation in regional (isthmic or ampullary), bovine oviductal fluid, pooled by stage of the oestrous cycle. Oviductal fluids collected daily from isthmic and ampullary cannulae implanted in the same oviduct were divided into pools, representing two oestrous cycle stages, based on daily serum progesterone concentrations. Ejaculated bull spermatozoa were incubated for 0-6 h in each type of oviductal fluid. Incubation in isthmic oviductal fluid collected during the nonluteal stage, including oestrus and ovulation, decreased overall sperm motility (from 71.7% motile spermatozoa to 34.0%) and both path (78 microns s-1 versus 86-89 microns s-1) and progressive (74 microns s-1 versus 83-85 microns s-1) velocities of spermatozoa motion. Spermatozoa incubated in isthmic, non-luteal oviductal fluid had a higher rate and extent of sperm acrosome reaction (213% of control versus 136-161% of control by 2 h incubation) compared with spermatozoa incubated in other oviductal fluid types. However, incubation in nonluteal ampullary fluid increased the number of spermatozoa, which were both acrosome reacted and live, and able to fertilize bovine ova (88.7% fertilized versus 75-81%). Glycosaminoglycan concentrations were similar among types of oviductal fluid (0.77-0.88 mg ml-1). These findings indicate that oviductal fluid differentially affects sperm function, depending on the oviduct region and the stage of the oestrous cycle at which the fluid was obtained.

Porcine repeat element DNA: in situ detection of xenotransplanted cells.

Using a digoxygenin-labelled DNA probe derived from the porcine repeat element PRE-1, we have developed a protocol for the detection of transplanted porcine islets and hepatocytes against a background of murine host tissue. Analysis of this probe by Southern blotting indicated that PRE-1 hybridizes to pig genomic DNA but not to human or mouse DNA. On tissue sections, hybridizing probe was detected using alkaline phosphatase-conjugated antidigoxygenin antibody visualized with 5-bromo-4-chloro-3-indolyl-phosphate/4-nitro-blue tetrazolium chloride (BCIP/NBT) substrate. We have demonstrated sensitive and highly specific staining of porcine nuclei in fixed, paraffin embedded tissue sections, and have applied the technique to detect porcine pancreatic islets and hepatocytes transplanted into murine kidney and spleen. Application of this technique include detection of transplanted cells or organs across the variety of xenogeneic barriers.

Effect of follicular or oviductal fluids on movement characteristics of bovine sperm during capacitation in vitro.

Mammalian sperm exhibit characteristic motility changes associated with capacitation. Movement characteristics of bovine sperm incubated in noncapacitating (control, medium alone), capacitating (oviduct fluid, nonluteal, and luteal), or capacitating, acrosome reaction inducing (follicular fluid) conditions were investigated using a computer-assisted automated semen analysis system. Sperm were incubated up to 4 hours in a modified Tyrode's medium (control), 20 and 60% nonluteal (NL) or luteal (L) oviduct fluid (ODF), or 20 and 60% follicular fluid (FF). Relative to sperm incubated in control medium, motility of sperm treated with ODF or FF had increased linearity and vigorous motility. Sperm incubated in 60% ODF or FF showed a small decrease in mean trajectory/path straightness and velocity over time compared to 20% fluid treatments and control. Frequency distribution graphs were symmetric for 20% NL- and L-ODF treated sperm. However, 20% FF and 60% ODF and FF treatments had distributions skewed to the left, indicating smaller values for lateral head displacement (ALH) and curvilinear velocity (VCL). Median values for ALH and VCL were determined for control-treated sperm, and subtracted from individual sperm values for all treatments to estimate deviation from control, designated ALHc and VCLc. Three-dimensional plots of ALHc, VCLc and corresponding frequency indicated shifts in peak patterns for fluid-treated sperm compared to control sperm. Incubation in 20% ODF and FF resulted in peak shift for ALH and VCL values; yet, little change in peak position was observed in sperm incubated in 60% ODF and FF.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of a measles epidemic.

In January, February and March of 1989 an epidemic of rubeola occurred on the campus of Texas Tech University. A vaccination programme was initiated as soon as the epidemic was confirmed. Extensive case histories of all confirmed cases were collected by the Lubbock City Health Department and given an exhaustive statistical analysis by a group from the Department of Mathematics at Texas Tech University. The data and statistical analysis were used to formulate stochastic and deterministic models of the measles epidemic based on the standard SEIR model. The analysis and the simulations indicate that in order to prevent a measles outbreak on a university campus a high rate of immunity may be required (> 98 per cent). The assumptions in the models raise some interesting questions regarding social contacts which require further investigation.