The variable success of in vitro maturation: can we do better?

The efficiency of in vitro assisted reproductive technologies, consisting of the transfer of embryos obtained in vitro through in vitro maturation, in vitro fertilization and early embryo culture is still limited. The quality of the oocytes is pivotal for assisted reproductive efficiency and the maturation of the oocyte represents the first key limiting step of the in vitro embryo production system. At the time of removal from the antral follicles, the oocyte is still completing the final growth and differentiation steps, needed to provide the so-called developmental competence, i.e. the machinery required to sustain fertilization and embryo development. In mono-ovular species only one oocyte per cycle is available for procreation, therefore the current assisted reproduction techniques strive to overcome this natural boundary. However, the success is still limited and overall the effectiveness does not exceed the efficiency achieved in millions of years of mammalian evolution. One of the problems lies in the intrinsic heterogeneity of the oocytes that are subjected to in vitro maturation and in the lack of dedicated in vitro approaches to finalize the differentiation process. In this review we will try to overview some of the salient aspects of current practices by emphasizing the most critical and fundamental features in oocyte differentiation that should be carefully considered for improving current techniques

The variable success of in vitro maturation: can we do better?

The efficiency of in vitro assisted reproductive technologies, consisting of the transfer of embryos obtained in vitro through in vitro maturation, in vitro fertilization and early embryo culture is still limited. The quality of the oocytes is pivotal for assisted reproductive efficiency and the maturation of the oocyte represents the first key limiting step of the in vitro embryo production system. At the time of removal from the antral follicles, the oocyte is still completing the final growth and differentiation steps, needed to provide the so-called developmental competence, i.e. the machinery required to sustain fertilization and embryo development. In mono-ovular species only one oocyte per cycle is available for procreation, therefore the current assisted reproduction techniques strive to overcome this natural boundary. However, the success is still limited and overall the effectiveness does not exceed the efficiency achieved in millions of years of mammalian evolution. One of the problems lies in the intrinsic heterogeneity of the oocytes that are subjected to in vitro maturation and in the lack of dedicated in vitro approaches to finalize the differentiation process. In this review we will try to overview some of the salient aspects of current practices by emphasizing the most critical and fundamental features in oocyte differentiation that should be carefully considered for improving current techniques(AU)

Head morphometry and chromatin instability in normal boar spermatozoa and in spermatozoa with cytoplasmic droplets

In boar studs, morphological analyses are used to evaluate sperm quality and there by categorize ejaculates as either approved or rejected. Normally, morphological characteristics correlate with chromatin disorders, but studies to date have only considered the average of abnormalities; cells were not segregated as normal or abnormal. The aim of this study was to assess whether the presence of cytoplasmic droplets was associated with morphometric characteristics and chromatin instability of spermatozoa heads. Morphological analyses were performed on semen from 11 boars using phase contrast microscopy (200 cells per sample). Normal cells were differentiated from those with cytoplasmic droplets and both types were evaluated separately. Photomicrographs were acquired ofnormal spermatozoa (Group NOR, N = 1,207) as well as spermatozoa with proximal and distal cytoplasmic droplets (Group DROP, N = 725). Sperm-head morphometry and chromatin structure were evaluated using the toluidine blue technique. Spermatozoa heads in the DROP group were longer (8.37 ± 0.60 × 8.31 ± 0.53; P = 0.025), narrower (4.16 ± 0.21 × 4.19 ± 0.19; P = 0.03), And more symmetric on the sides (0.973 ± 0.012 × 0.971 ± 0.011; P = 0.007) than were spermatozoa heads of the NOR group. The DROP group also had a greater average ellipticity (0.335 ± 0.034 × 0.329 ± 0.031; P= 0.0004),a greater percentage of decondensed chromatin (2.71 ± 3.87 × 2.28 ± 1.38; P < 0.0008), and a greater chromatin heterogeneity (4.66 ± 1.40 × 4.40 ± 1.42;P < 0.0001). A greater frequency of semen collection results in a shorter period of cell maturation and this probably affected the degree of chromatin condensation and the cytoplasmic droplet migration, with concomitant effect onthe head morphometry measurements.

Head morphometry and chromatin instability in normal boar spermatozoa and in spermatozoa with cytoplasmic droplets

In boar studs, morphological analyses are used to evaluate sperm quality and there by categorize ejaculates as either approved or rejected. Normally, morphological characteristics correlate with chromatin disorders, but studies to date have only considered the average of abnormalities; cells were not segregated as normal or abnormal. The aim of this study was to assess whether the presence of cytoplasmic droplets was associated with morphometric characteristics and chromatin instability of spermatozoa heads. Morphological analyses were performed on semen from 11 boars using phase contrast microscopy (200 cells per sample). Normal cells were differentiated from those with cytoplasmic droplets and both types were evaluated separately. Photomicrographs were acquired ofnormal spermatozoa (Group NOR, N = 1,207) as well as spermatozoa with proximal and distal cytoplasmic droplets (Group DROP, N = 725). Sperm-head morphometry and chromatin structure were evaluated using the toluidine blue technique. Spermatozoa heads in the DROP group were longer (8.37 ± 0.60 × 8.31 ± 0.53; P = 0.025), narrower (4.16 ± 0.21 × 4.19 ± 0.19; P = 0.03), And more symmetric on the sides (0.973 ± 0.012 × 0.971 ± 0.011; P = 0.007) than were spermatozoa heads of the NOR group. The DROP group also had a greater average ellipticity (0.335 ± 0.034 × 0.329 ± 0.031; P= 0.0004),a greater percentage of decondensed chromatin (2.71 ± 3.87 × 2.28 ± 1.38; P < 0.0008), and a greater chromatin heterogeneity (4.66 ± 1.40 × 4.40 ± 1.42;P < 0.0001). A greater frequency of semen collection results in a shorter period of cell maturation and this probably affected the degree of chromatin condensation and the cytoplasmic droplet migration, with concomitant effect onthe head morphometry measurements.(AU)

Azul de toluidina como método para avaliação da integridade da cromatina de espermatozoides canino extraídos do tecido testicular fresco / Toluidine blue as a method for assessment of chromatin integrity canine spermatozoa extracted from fresh testicular tissue

Conservation of gonadal tissue is important for maintenance of endangered wild species. However, thepreservation can lead to cell damage and loss. Thus, it is necessary testicular tissue evaluation before and afterstorage in order to determine damage. The evaluation of sperm chromatin integrity ensures a better assessmentof sperm quality. The toluidine blue is a simple and sensitive test to evaluate the sperm chromatin integrity fromseveral species, but there are not reports in the dog. The aim of this study was evaluating canine spermatozoaextracted from fresh testicular tissue of seven post-pubertal mongrel dogs by toluidine blue and spermmorphology. For statistical analysis, it was used normality test (Shapiro Wilk) and Wilcoxon test (P<0.05).Toluidine blue showed 83% condensed chromatin. Morphological analysis presented 69.34% normalspermatozoa. Toluidine blue was sensitive for the evaluation. However, it still needs some adjustments for amore accurate result for such specie.(AU)

Azul de toluidina como método para avaliação da integridade da cromatina de espermatozoides canino extraídos do tecido testicular fresco / Toluidine blue as a method for assessment of chromatin integrity canine spermatozoa extracted from fresh testicular tissue

Conservation of gonadal tissue is important for maintenance of endangered wild species. However, thepreservation can lead to cell damage and loss. Thus, it is necessary testicular tissue evaluation before and afterstorage in order to determine damage. The evaluation of sperm chromatin integrity ensures a better assessmentof sperm quality. The toluidine blue is a simple and sensitive test to evaluate the sperm chromatin integrity fromseveral species, but there are not reports in the dog. The aim of this study was evaluating canine spermatozoaextracted from fresh testicular tissue of seven post-pubertal mongrel dogs by toluidine blue and spermmorphology. For statistical analysis, it was used normality test (Shapiro Wilk) and Wilcoxon test (P<0.05).Toluidine blue showed 83% condensed chromatin. Morphological analysis presented 69.34% normalspermatozoa. Toluidine blue was sensitive for the evaluation. However, it still needs some adjustments for amore accurate result for such specie.

Cromatina espermática: quebrando paradigmas / Sperm chromatin: breaking paradigms

Há algumas décadas, pensava-se que espermatozoides maduros possuíssem uma cromatina não funcional, inerte, sem a capacidade de transcrição. Acreditava-se que a matriz nuclear não existisse e que a presença de histonas no núcleo seria um erro no processo de compactação cromatínica, o que poderia interferir na fertilidade do macho. Hoje se sabe que, entre as estruturas toroidais, unidades básicas da cromatina espermática altamente compactada, há algumas poucas regiões que contêm sequências de nucleossomos, e estas geralmente estão anexadas a uma matriz nuclear proteica. Os espermatozoides possuem cromatina altamente organizada e são condutores metabolicamente funcionais do genoma masculino, carreando RNAs de diferentes tipos, os quais, tanto quanto os nucleossomos, são importantes sinalizadores epigenéticos paternos e, logo,influenciam o desenvolvimento embrionário inicial. Portanto, alterações cromatínicas podem não somente interferir no processo de fecundação, como principalmente no desenvolvimento embrionário, o que reforça a necessidade da análise da cromatina na avaliação de reprodutores machos.(AU)

Decades ago, it was thought thatspermatozoa had a chromatin nonfunctional, inert, without the abilityto transcription. It was believed in the absence of nuclear matrix and the presence of the core histones would be an error in the chromatin packaging, which could interfere with male fertility. Today we know that between thetoroidal structures, basic units of highly compacted sperm chromatin, there are a few regions containing nucleosome sequences, these usually being attached to a nuclear protein matrix. The sperm chromatin is highly organized and is metabolically functional carrier of male genome, carrying different RNA types, which, along with the nucleosomes are important paternal epigenetic signaling, and influencing early embryonic development. Therefore, chromatin alterations not only interfere with the fertilization process, but also influencethe embryonic development, which reinforces the need for chromatin analysis in the evaluation of breeding males.(AU)

Cromatina espermática: quebrando paradigmas / Sperm chromatin: breaking paradigms

Há algumas décadas, pensava-se que espermatozoides maduros possuíssem uma cromatina não funcional, inerte, sem a capacidade de transcrição. Acreditava-se que a matriz nuclear não existisse e que a presença de histonas no núcleo seria um erro no processo de compactação cromatínica, o que poderia interferir na fertilidade do macho. Hoje se sabe que, entre as estruturas toroidais, unidades básicas da cromatina espermática altamente compactada, há algumas poucas regiões que contêm sequências de nucleossomos, e estas geralmente estão anexadas a uma matriz nuclear proteica. Os espermatozoides possuem cromatina altamente organizada e são condutores metabolicamente funcionais do genoma masculino, carreando RNAs de diferentes tipos, os quais, tanto quanto os nucleossomos, são importantes sinalizadores epigenéticos paternos e, logo,influenciam o desenvolvimento embrionário inicial. Portanto, alterações cromatínicas podem não somente interferir no processo de fecundação, como principalmente no desenvolvimento embrionário, o que reforça a necessidade da análise da cromatina na avaliação de reprodutores machos.

Decades ago, it was thought thatspermatozoa had a chromatin nonfunctional, inert, without the abilityto transcription. It was believed in the absence of nuclear matrix and the presence of the core histones would be an error in the chromatin packaging, which could interfere with male fertility. Today we know that between thetoroidal structures, basic units of highly compacted sperm chromatin, there are a few regions containing nucleosome sequences, these usually being attached to a nuclear protein matrix. The sperm chromatin is highly organized and is metabolically functional carrier of male genome, carrying different RNA types, which, along with the nucleosomes are important paternal epigenetic signaling, and influencing early embryonic development. Therefore, chromatin alterations not only interfere with the fertilization process, but also influencethe embryonic development, which reinforces the need for chromatin analysis in the evaluation of breeding males.