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The spermatozoa delivered to the female genital tract need to swim towards the oocyte through viscous secretions. Once close to the oocyte, the spermatozoa are guided by a gradient of progesterone (P₄) and other unknown chemoattractants via a process known as chemotaxis. Using polyvinylpyrrolidone to establish the conditions of viscosity, we examined the response of mouse spermatozoa to P₄. Herein, we show that in low-viscous media, P₄ induces hyperactive-like motility whereby sperm show erratic trajectories and non-progressive movement. However, an opposite response is produced in viscous medium in that trajectories are linear and motility is more progressive and less erratic. Our observations provide a behavioural explanation for the chemotaxis of spermatozoa swimming under viscous conditions in a spatial gradient of the chemoattractant P₄. They also highlight the importance of using viscous solutions to mimic in vivo conditions when analysing sperm behaviour in response to any stimulus.

Reproduction (2016) 151 501–507

Sex chromosome transcripts can lead to a broad transcriptional sexual dimorphism in the absence of concomitant or previous exposure to sex hormones, especially when X-chromosome inactivation (XCI) is not complete. XCI timing has been suggested to differ greatly among species, and in bovine, most of the X-linked transcripts are upregulated in female blastocysts. To determine the timing of XCI, we analyzed in day 14 bovine embryos the sexual dimorphic transcription of seven X-linked genes known to be upregulated in female blastocysts (X24112, brain-expressed X-linked 2 (BEX2), ubiquitin-conjugating enzyme E2A (UBE2A), glucose-6-phosphate dehydrogenase (G6PD), brain-expressed X-linked 1 (BEX1), calpain 6 (CAPN6), and spermidine/spermine N-acetyltransferase 1 (SAT1)). The transcription of five genes whose expression differs between sexes at the blastocyst stage (DNMT3A, interferon tau (IFNT2), glutathione S-transferase mu 3 (GSTM3), progesterone receptor membrane component 1 (PGRMC1), and laminin alpha 1 (LAMA1)) and four genes related with sex determination (Wilms tumor 1 (WT1), gata binding protein 4 (GATA4), zinc finger protein multitype 2 (ZFPM2), and DMRT1) was also analyzed to determine the evolution of transcriptional sexual dimorphism. The expression level of five X-linked transcripts was effectively equalized among sexes suggesting that, in cattle, a substantial XCI occurs during the period between blastocyst hatching and initiation of elongation, although UBE2A and SAT1 displayed significant transcriptional differences. Similarly, sexual dimorphism was also reduced for autosomal genes with only DNMT3A and IFNT2 exhibiting sex-related differences. Among the genes potentially involved in sex determination, Wilms tumor 1 (WT1) was significantly upregulated in males and GATA4 in females, whereas no differences were observed for ZFPM2 and DMRT1. In conclusion, a major XCI occurred between the blastocyst and early elongation stages leading to a reduction in the transcriptional sexual dimorphism of autosomal genes, which makes the period the most susceptible to sex-specific embryo loss.

In adult tissues, sexual dimorphism is largely attributed to sex hormone effects, although there is increasing evidence for a major role of sex chromosome dosage. During preimplantation development, male and female embryos can display phenotypic differences that can only be attributed to the transcriptional differences resulting from their different sex chromosome complements. Thus, all expressed Y-linked genes and those X-linked genes that totally or partially escape X-chromosome inactivation at each specific developmental stage display transcriptional sexual dimorphism. Furthermore, these differentially expressed sex chromosome transcripts can regulate the transcription of autosomal genes, leading to a large transcriptional sexual dimorphism. The sex-dependent transcriptional differences may affect several molecular pathways such as glucose metabolism, DNA methylation and epigenetic regulation, and protein metabolism. These molecular differences may have developmental consequences, including sex-selective embryo loss and sex-specific epigenetic responses to environmental hazards, leading to long-term effects. This review discusses transcriptional sexual dimorphism in preimplantation embryos, its consequences on sex ratio biases and on the developmental origin of health and disease, and its significance for transcriptional studies and adult sexual dimorphism.

Cellular prion protein (PrP^C) has been well described as an essential partner of prion diseases due to the existence of a pathological conformation (PrP^Sc). Recently, it has also been demonstrated that PrP^C is an important element of the pluripotency and self-renewal matrix, with an increasing amount of evidence pointing in this direction. Here, we review the data that demonstrate its role in the transcriptional regulation of pluripotency, in the differentiation of stem cells into different lineages (e.g. muscle and neurons), in embryonic development, and its involvement in reproductive cells. Also highlighted are recent results from our laboratory that describe an important regulation by PrP^C of the major pluripotency gene Nanog. Together, these data support the appearance of new strategies to control stemness, which could represent an important advance in the field of regenerative medicine.

The role of the epididymis as a quality control organ in preventing infertile gametes entering the ejaculate has been extensively explored, and it has been suggested that a specific region of mammalian epididymis is able to phagocytose abnormal germ cells. This study examines whether the epithelium of certain zones of the mouse epididymis can act as a selection barrier by removing immature germ cells from the lumen by phagocytosis. To detect the presence of immature germ cells in the epididymis, we generated transgenic mice expressing enhanced green fluorescent protein under the deleted in azoospermia-like (mDazl) promoter to easily identify immature germ cells under fluorescence microscopy. Using this technique, we observed that during the first stage of spermatogenesis in prepuberal mice, a wave of immature germ cells is released into the epididymis and that the immature epididymis is not able to react to this abnormal situation. By contrast, when immature germ cells were artificially released into the epididymis in adult mice, a phagocytic response was observed. Phagosomes appeared inside principal cells of the epididymal epithelium and were observed to contain immature germ cells at different degradation stages in different zones of the epididymis, following the main wave of immature germ cells. In this paper, we describe how the epididymal epithelium controls sperm quality by clearing immature germ cells in response to their artificially induced massive shedding into the epididymal lumen. Our observations indicate that this phenomenon is not restricted to a given epididymis region and that phagocytic capacity is gradually acquired during epididymal development.

The steroid hormone progesterone (P₄) plays a key role in the reproductive events associated with pregnancy establishment and maintenance. High concentrations of circulating P₄ in the immediate post-conception period have been associated with an advancement of conceptus elongation, an associated increase in interferon-τ production and higher pregnancy rates in cattle. Using in vitro and in vivo models and ∼8500 bovine oocytes across six experiments, the aim of this study was to establish the route through which P₄ affects bovine embryo development in vitro and in vivo. mRNA for P₄ receptors was present at all stages of embryo development raising the possibility of a direct effect of P₄ on the embryo. Exposure to P₄ in vitro in the absence or presence of oviduct epithelial cells did not affect the proportion of embryos developing to the blastocyst stage, blastocyst cell number or the relative abundance of selected transcripts in the blastocyst. Furthermore, exposure to P₄ in vitro did not affect post-hatching elongation of the embryo following transfer to synchronized recipients and recovery on Day 14. By contrast, transfer of in vitro derived blastocysts to a uterine environment previously primed by elevated P₄ resulted in a fourfold increase in conceptus length on Day 14. These data provide clear evidence to support the hypothesis that P₄-induced changes in the uterine environment are responsible for the advancement in conceptus elongation reported previously in cattle and that, interestingly, the embryo does not need to be present during the period of high P₄ in order to exhibit advanced elongation.

Endocannabinoids are known to mediate practically all reproductive events in mammals; however, little is known about their role in oocyte maturation. Through RT-PCR and immunocytochemistry, this study confirms the presence of CB1 and CB2 cannabinoid receptors in bovine oocytes and shows how exposure to the exogenous cannabinoids HU-210 and THC during their in vitro maturation (IVM) activates the phosphorylation of AKT and ERK1/2 proteins associated with the resumption of meiosis. Although supplementation with HU-210 or THC during IVM did not increase blastocyst yields, the expression of interferon tau (IFNτ) and gap junction alpha-1 protein (GJA1) was enhanced at the blastocyst stage. Our data suggest that cannabinoid agonists may be useful IVM supplements as their presence during oocyte maturation upregulates the expression in blastocysts of key genes for embryo quality.

Offspring telomere length (TL) has been correlated with paternal TL, but the mechanism for this parent of origin-specific inheritance remains unclear. The objective of this study has been to determine the role of spermatozoa TL in embryonic telomere lengthening by using two mouse models showing dimorphism in their spermatozoa TL: Mus musculus vs Mus spretus and old vs young Mus musculus. Mus spretu s spermatozoa displayed a shorter TL than Mus musculus. Hybrid offspring exhibited lower TL compared with Mus musculus starting at the two-cell stage, before the onset of telomerase expression. To analyze the role of spermatozoa telomeres in early telomere lengthening, we compared the TL in oocytes, zygotes, two-cell embryos and blastocysts produced by parthenogenesis or by fertilization with Mus musculus or Mus spretus spermatozoa. TL was significantly higher in spermatozoa compared with oocytes, and it increased significantly from the oocyte to the zygote stage in those embryos fertilized with Mus musculus spermatozoa, but not in those fertilized with Mus spretus spermatozoa or produced by parthenogenesis. A further increase was noted from the zygote to the two-cell stage in fertilized Mus musculus embryos, whereas hybrid embryos maintained the oocyte TL. Spermatozoa TL shortened with age in Mus musculus and the offspring from young males showed a significantly higher TL compared with that fathered by old males. These significant differences were already noticeable at the two-cell stage. These results suggest that spermatozoa telomeres act as a guide for telomerase-independent telomere lengthening resulting in differences in TL that persist after birth.

Free Spanish abstract: A Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/151/1/1/suppl/DC1.

Sperm-mediated gene transfer (SMGT) is a method for the production of transgenic animals based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA molecules and to transfer them into the oocyte at fertilization. Recombinase-A (RecA) protein-coated exogenous DNA has been used previously in pronuclear injection systems increasing integration into goat and pig genomes. However, there are no data regarding transgene expression after ICSI. Here, we set out to investigate whether the expression of transgenic DNA in porcine embryos is improved by recombinase-mediated DNA transfer and if it is possible to generate transgenic animals using this methodology. Different factors which could affect the performance of this transgenic methodology were analyzed by studying 1) the effect of the presence of exogenous DNA and RecA protein on boar sperm functionality; 2) the effect of recombinase RecA on in vitro enhanced green fluorescent protein (EGFP)-expressing embryos produced by ICSI or IVF; and 3) the efficiency of generation of transgenic piglets by RecA-mediated ICSI. Our results suggested that 1) the presence of exogenous DNA and RecA–DNA complexes at 5 μg/ml did not affect sperm functionality in terms of motility, viability, membrane lipid disorder, or reactive oxygen species generation; 2) EGFP-expressing embryos were obtained with a high efficiency using the SMGT–ICSI technique in combination with recombinase; however, the use of IVF system did not result in any fluorescent embryos; and 3) transgenic piglets were produced by this methodology. To our knowledge, this is the first time that transgenic pigs have been produced by ICSI-SGMT and a recombinase.

Elevated plasma nonesterified fatty acid (NEFA) concentrations are associated with negative energy balance and metabolic disorders such as obesity and type II diabetes. Such increased plasma NEFA concentrations induce changes in the microenvironment of the ovarian follicle, which can compromise oocyte competence. Exposing oocytes to elevated NEFA concentrations during maturation affects the gene expression and phenotype of the subsequent embryo, notably prompting a disrupted oxidative metabolism. We hypothesized that these changes in the embryo are a consequence of modified energy metabolism in the oocyte. To investigate this, bovine cumulus oocyte complexes were matured under elevated NEFA conditions, and energy metabolism-related gene expression, mitochondrial function, and ultrastructure evaluated. It was found that expression of genes related to REDOX maintenance was modified in NEFA-exposed oocytes, cumulus cells, and resultant blastocysts. Moreover, the expression of genes related to fatty acid synthesis in embryos that developed from NEFA-exposed oocytes was upregulated. From a functional perspective, inhibition of fatty acid β-oxidation in maturing oocytes exposed to elevated NEFA concentrations restored developmental competence. There were no clear differences in mitochondrial morphology or oxygen consumption between treatments, although there was a trend for a higher mitochondrial membrane potential in zygotes derived from NEFA-exposed oocytes. These data show that the degree of mitochondrial fatty acid β-oxidation has a decisive impact on the development of NEFA-exposed oocytes. Furthermore, the gene expression data suggest that the resulting embryos adapt through altered metabolic strategies, which might explain the aberrant energy metabolism previously observed in these embryos originating from NEFA-exposed maturing oocytes.