Growth factors promote meiosis in mouse fetal ovaries in vitro

Mouse fetal ovaries were cultured to investigate germ cell development in the presence of a combination of the growth factors (GFs) stem cell factor, insulin-like growth factor-1 and leukaemia inhibitory factor. Ovaries were isolated from fetal mice at 13 and 14 days post-coitum (dpc) and cultured to the equivalent of 17 dpc. Culture conditions comprised minimal essential medium-a plus 5% fetal calf serum, with or without GFs. Oocytes were assessed using immunofluorescence to illustrate synaptonemal complexes and recombination foci. The proportions of pachytene cells in freshly isolated 13, 14 and 17 dpc ovaries were 0, 8 and 74% respectively. There was a significant (P < 0.0001) increase in the number of pachytene cells after 4 days culture with GFs, with 24% of germ cells from 13 dpc ovaries reaching pachytene. In contrast, no pachytene cells were detected in cultures of 13 dpc ovaries without GFs. After 3 days in culture with GFs, 38% of germ cells from 14 dpc ovaries were at pachytene compared with 19% without GFs. In conclusion, we have demonstrated positive effects of GFs upon oocyte formation by meiosis in vitro. The observed results could be explained by an increased survival of premeiotic oogonia entering meiosis, or by effects on oocytes already in early meiosis

The effects of age on the incidence of aneuploidy rates in spermatozoa of oligoasthenozoospermic patients and its relationship with ICSI outcome

The development of intracytoplasmic sperm injection (ICSI) for treatment of infertility as a result of severe male factor has improved the chances of achieving pregnancy in many infertile couples. However, concerns have been raised regarding the safety of this technique, because natural sperm selection is bypassed. In the present study, 25 oligoasthenozoospermic patients who were divided into two groups according to age: group A, 20-34 (n = 10) and group B, 35-50 (n = 15), were included. Pooling the data of the three semen parameters that were tested (volume, concentration and progressive motility) no statistically significant difference between the two age groups was found. A total of 50 883 decondensed spermatozoa was analysed using the dual and triple colour fluorescence in situ hybridization to estimate the rates of aneuploidy for chromosomes 13, 18, 21, X and Y in the two age groups. There was a significantly higher incidence of disomy for chromosome 21 compared to the other autosomes (chromosomes 13 and 18) in both age groups. The disomy rate of XY was significantly higher in the younger subject group (0.1%) compared to the older group (0.05%, p &lt; 0.05). Statistically significant differences in the mean number of clinical pregnancies and abortions were not observed between the two age groups. The aneuploidy rates for all the analysed chromosomes did not differ significantly, both between and within the two age groups, and as a result there seems to be no effect of male age on chromosome numbers in the spermatozoa and on the ICSI outcome. © 2007 The Authors

Crossover analysis using immunofluorescent detection of MLH1 foci in frozen-thawed testicular tissue

To date, the effects of freezing on spermatogenesis have not yet been fully investigated at a molecular level. Antibody localization studies have identified the MutL homolog 1 (MLH1) protein, a mis-match repair protein, at the prophase I stage of meiosis, which allows the detection of recombination foci during pachytene. This study investigated the effect of long-term testicular tissue cryopreservation on meiotic prophase I, identified by recombination foci frequency and synaptonemal complex (SC) integrity. Frozen - thawed testicular tissues from 12 males who had each fathered a child were used. Because vasectomy or reverse vasectomy procedures are rare in the locale of the investigation, it was not possible to obtain fresh testicular tissue and use the males as their own controls. Immunocytogenetic analysis of 612 spermatocytes at the pachytene stage was performed. The results indicated a mean number of MLH1 foci of 49.2 (SD ± 5.9), and no correlation was found between the freezing period, the MLH1 frequency and the SC integrity. The results suggest that freezing of testicular tissue taken post-puberty does not appear to be detrimental to the crossover process as identified by occurrence of MLH1 loci. © 2007 Published by Reproductive Healthcare Ltd

Oogenesis and cell death in human prenatal ovaries : what are the criteria for oocyte selection?

Prenatal oogenesis produces hundreds of thousands of oocytes, most of which are discarded through apoptosis before birth. Despite this large-scale selection, the survivors do not constitute a perfect population, and the factors at the cellular level that result in apoptosis or survival of any individual oocyte are largely unknown. What then are the selection criteria that determine the size and quality of the ovarian reserve in women? This review focuses on new data at the cellular level, on human prenatal oogenesis, offering clues about the importance of the timing of entry to meiotic prophase I by linking the stages and progress through MPI with the presence or absence of apoptotic markers. The characteristics and responsiveness of cultured human fetal ovarian tissue at different gestational ages to growth factor supplementation and the impact of meiotic abnormalities upon apoptotic markers are discussed. Future work will require the use of a tissue culture model of prenatal oogenesis in order to investigate the fate of individual live oocytes at different stages of development