This review summarizes the results of research on gene transfer to the mammalian genital tract. Gene transfer experiments have been developed during the last 2 decades and have been applied using in vitro, ex vivo and in vivo procedures. (i) In vitro methods have been applied to the uterine epithelial cells with the principal purpose of analysing some pathological change occurring in the uterus. In the male tract, epididymal cell lines have been used to evaluate the expression of particular genes and the function of specific proteins. (ii) Ex vivo methods have been applied to both the uterus and the vas deferens in humans, and good transgene expression has been recorded. (iii) In vivo gene transfer in the female tract has been employed in the uterus and oviduct using gene injections or electroporation methods. The glandular epithelium of both organs can be transfected efficiently, and transfection efficiency depends on the hormonal stage of the animal. The best expression occurred during pseudopregnancy and meta-estrus periods, when high progesterone and low estradiol concentrations occur. In the male tract, in vivo methods have been applied to mouse vas deferens and epididymis. In both organs, patches of epithelial regions appeared to express the transgenes. Furthermore, the secretions of both organs were also modified using gene constructions that led to the expression of some secretory proteins. In summary, gene modifications in the epithelium of the mammalian reproductive tract have been successful employing different technologies. Further improvements in transfection efficiency would help provide new insights into the physiology of these reproductive organs. Furthermore, the use of these methods could also be used to modify the fertility of mammals.

Certain morphological changes at the subcellular level caused by the current techniques for in vitro embryo production seem to affect mitochondria. Many of these, including dysfunctional changes, have been associated with the presence of serum in the culture medium. Thus, the aim of the present work was to assess the mitochondrial dynamics occurring in embryos during the first 4 days of development, in order to analyze the most appropriate time for adding the serum. We used transmission electron microscopy (TEM) micrographs to calculate the embryo area occupied by the different morphological types of mitochondria, and analyzed them with Image Pro Plus analyzer. The results showed hooded mitochondria as the most representative type in 1- to 4-day-old embryos. Swollen, on-fusion, orthodox and vacuolated types were also present. When analyzed in embryos cultured without serum, the dynamics of the different mitochondrial types appeared to be similar, a fact that may provide evidence that the developmental changes control the mitochondrial dynamics, and that swollen mitochondria may not be completely inactive. In contrast, in culture medium supplemented with serum from estrous cows, we observed an increased area of hooded mitochondria by developmental day 4, a fact that may indicate an increased production of energy compared with previous days. According to these results, the bovine serum added to the culture medium seems not to be responsible for the functional changes in mitochondria.

Survivin is a novel member of the inhibitor of apoptosis gene family that bear baculoviral IAP repeats (BIRs), whose physiological roles in regulating meiotic cell cycle need to be determined. Confocal microscopy was employed to observe the localization of survivin in rat oocytes. At the germinal vesicle (GV) stage, survivin was mainly concentrated in the GV. At the prometaphase I (pro-MI) and metaphase I (MI) stage, survivin was mainly localized at the kinetochores, with a light staining detected on the chromosomes. After transition to anaphase I or telophase I stage, survivin migrated to the midbody, and signals on the kinetochores and chromosomes disappeared. At metaphase II (MII) stage, survivin became mainly localized at the kinetochores again. Microinjection of oocytes with anti-survivin antibodies at the beginning of the meiosis, thus blocking the normal function of survivin, resulted in abnormal spindle assembly, chromosome segregation and first polar body emission. These results suggest that survivin is involved in regulating the meiotic cell cycle in rat oocytes.

We investigated whether the small litter size in the 129 inbred mouse strain results from a reduction in oocyte fertilizability. Sensitivity of the zona pellucida to α-chymotrypsin was examined for oocytes collected at 14 h (shortly after ovulation), 17 h, and 20 h after hCG injection. Passage of spermatozoa through the zona pellucida (using an in vitro fertilization (IVF) technique) and the density of cortical granules were examined for oocytes collected at 14 and 17 h after hCG injection. The capability of the oolemma to fuse with the sperm plasma membrane was also evaluated by IVF using zona-free eggs. The zona pellucida became markedly resistant to the enzyme 17 h after hCG injection. IVF rates significantly decreased at this time. In addition, there was a significant reduction in the density of cortical granules. When zona-free oocytes were inseminated, high fertilization rates were obtained at both 17 and 14 h after hCG injection. These results indicate that accelerated modification of the zona pellucida primarily causes a decreased fertilizability of oocytes in 129 mice, resulting in the low reproductive performance of this strain.

This study was designed to investigate whether a non-invasive birefringence parameter, determined using the Oosight™ imaging system, is useful for estimating the hardness of human zona pellucida (ZP). The value for retardance (R) × thickness (T), but not R or T alone, of ZP was positively correlated (r = 0.92, p < 0.0001) with its hardness estimated by the time required for a 0.1% protease solution to solubilize ZP at 37 °C. In a model experiment to induce ZP puncture by Fluorinert™ fluid microinjection (sham-hatching), the R × T value at the punctured site was positively correlated (r = 0.78, p < 0.01) with the hardness of the ZP as estimated by the maximum expansion rate. The R × T values of ZP in in vitro fertilization-derived embryos (21.6 ± 7.5) and intracytoplasmic sperm injection-derived embryos (20.8 ± 6.3) were significantly higher than that in unfertilized metaphase II oocytes (16.6 ± 6.1; p < 0.05). The R × T value after in vitro hatching of viable blastocysts (10.8 ± 6.2) was significantly lower than that of unexpanded morulae and early blastocysts (19.0 ± 4.0; p < 0.05), while the value of expanding blastocysts (15.3 ± 4.1) was intermediate. In conclusion, hardness of human ZP can be estimated non-invasively by birefringence-based microscopic observation.

This study evaluated the effects of reversible meiotic inhibition and different culture media (PZM3 or NCSU23) on production of porcine embryos by either in vitro fertilization (IVF) or parthenogenetic activation (PA). Oocytes from abattoir-derived ovaries were allocated into two groups for maturation: CHX (5 μg/ml cycloheximide for 10 h) or Control (no CHX). The percentage of metaphase II (MII) oocytes was determined at 36, 40 or 44 h of in vitro maturation. For IVF and PA, denuded oocytes were fertilized with purified sperm for 6 h or activated by electric stimuli. Zygotes were then subdivided into two culture groups: NCSU23 or PZM3. No effect of treatment with CHX and culture media was observed on cleavage (D3) and blastocyst (D7) rates in IVF and PA groups. There are no differences of quality or development rates between IVF-derived embryos cultured in NCSU23 or PZM3. However, we observed high quality PA embryos in PZM3 compared with NCSU23. Maturation arrest with CHX decreased the average blastocyst cell number in IVF while it was increased in PA embryos. As older oocytes are more effectively activated, CHX– blocked oocytes reached the mature stage faster than the control group. In conclusion, the CHX treatment for 10 h, followed by oocyte maturation for 40 h, is an efficient protocol to produce high quality parthenote embryos, especially when they are cultured in PZM3. However, this protocol is not satisfactory for IVF embryos production. In this case, a shorter maturation period could provide better embryo quality.

A little studied aspect of developmental arrest (DA) in ART is zygote arrest (ZA). Etiologically, blockage at the first cleavage stage includes molecular and chromosomal anomalies, some of which manifest morphologically. Given considerations on embryo culture, transfer and cryopreservation, optimal zygote selection is very important. The aim of this study was to ascertain whether zygote morphological features were indicators of increased ZA. In this study we performed a prospective, observational study of 2105 zygotes obtained from consecutive patients who were undergoing IVF/ICSI treatment, of which 43 (2%) suffered ZA. Morphological features observed under the inverted microscope were qualitatively categorized: pronuclear size, nucleolar precursor bodies (NPB) alignment, light and dark halos, polar body placement and fragmentation observed at 16–18 h post-insemination. We compared these features in blocked versus cleaved zygotes at 48 h and found significant correlations (p < 0.05) between ZA and three features: the absence of a light halo (p = 0.001), the absence of a dark halo (p < 0.005), and non-aligned NPB (p < 0.05). We can say that certain morphological features are indicators of significantly increased zygote arrest. These findings may be of utility for optimal zygote selection and culture strategies, especially in countries under restrictive conditions.

Cryopreservation of mammal embryos has been technically feasible for many years, but morphological injuries still persist in fish embryos during cryopreservation. Thus, the objective of the present study was to describe these freezing injuries in Piaractus mesopotamicus embryos. Two hundred and twenty-five embryos were collected at the post-gastrula stage and assigned into four treatments of sucrose at 8.5, 17.0, 25.0 or 34.0% plus 9.0% methanol. The control was prepared with distilled water only. The gradual decrease in the temperature was 0.5°C/min. After the seeding stage, the fish embryos were stored in liquid nitrogen at −33°C. Thereafter, they were thawed for evaluating per cent hatching, and the samples collected from every treatment were submitted to scanning electron microscopy for morphological analysis. The micrographic images showed that there was substantial alterations in embryo morphology under the highest concentrations of sucrose. These solutions did not prevent the formation of ice crystals, which lead to deformities and killed the embryos, but the observed reduced level of morphological structure in these embryos when treated with 17.0% sucrose plus 9.0% methanol is a compelling argument for additional studies.

Brycon gouldingi is an endemic species from Tocantins-Araguaia basin, used as a food source by riverine communities and relevant to aquaculture. Information about the initial morphology of B. gouldingi, a recently described species, is absent. In the present study, we analysed the fertilization and the embryonic development of this species based on light and scanning electron microscopy. After collection of adult specimens in Mortes River – Mato Grosso, Brazil, adaptation to captivity and induced spawning at Buriti Fishculture, Nova Mutum – Mato Grosso, Brazil, in December 2007 and January 2008, samples were collected at pre-defined periods from egg extrusion up to larval hatching, which occurred at 13.9 ± 0.06 h post-fertilization (hpf) in average. At the moment of extrusion, the eggs were slightly ovoid bearing a single micropyle per oocyte with a funnel-shaped micropyle canal and vestibule covered with longitudinal folds, typical of the genus Brycon. The embryonic development of B. gouldingi was characterized by six stages with distinct features: zygote (from fertilization up to formation of egg-cell); cleavage (cell divisions resulting in blastomeres, including the morula phase); blastula (several embryonic cells in a cup shape, without distinction of cell boundaries); gastrula (cell movement); histogenesis/organogenesis (formation of tissues and organs); and hatching (larval chorion rupture). Right after hatching, the larvae presented neither swimming abilities nor visual accuracy, and the digestive trait was undifferentiated. The present study is the first report on biological features of embryogenesis in B. gouldingi, providing relevant information to several approaches, mainly related to taxonomy, ecology, conservation and captive rearing of this new Brycon species.

Immature oocytes are arrested at prophase I of the meiotic process and maturation onset is indicated by oocyte nuclear disassembly (germinal vesicle breakdown or GVBD). Signaling pathways that elevate intracellular cyclic AMP (cAMP) may either prevent or induce oocyte maturation depending on the species. In some marine invertebrates and, in particular, in ascidian oocytes, cAMP triggers GVBD rather than blocking it. In this paper, we tested different cAMP elevators in fully grown oocytes at the germinal vesicle stage (GV) of the ascidian Ciona intestinalis. We demonstrated that through the activation of adenylate cyclase or the inhibition and phosphodiesterases the oocyte remained at the GV stage. This effect was reversible as the GV-arrested oocytes, rinsed and incubated in sea water, are able to undergo spontaneous maturation and extrusion of follicle cells. In addition, oocytes acquire the ability to be fertilized and start early development. However, morphology of follicle cells, embryos and larvae from in vitro matured oocytes showed different morphology from those derived from in vivo mature oocytes. The role and the transduction mechanism of cAMP in the regulation of oocyte maturation were discussed. Finally, we indicated a variation of biological mechanisms present in the ascidian species; moreover, we sustain evidence proving that tunicates share some biological mechanisms with vertebrates. This information provided new hints on the importance of ascidians in the evolution of chordates.